tag:blogger.com,1999:blog-70400425346971180332024-03-05T01:25:43.339-08:00Selamat REzaReza Bocah Tengikhttp://www.blogger.com/profile/05494071928891050842noreply@blogger.comBlogger3125tag:blogger.com,1999:blog-7040042534697118033.post-61244452693446988092011-03-01T03:20:00.001-08:002011-03-01T03:20:13.628-08:00<table cellpadding="0" cellspacing="0"><tbody>
<tr style="vertical-align: top;"><td><div class="fm-citation"><div><div><span class="citation-abbreviation">Antimicrob Agents Chemother. </span><span class="citation-publication-date">2010 January; </span><span class="citation-volume">54</span><span class="citation-issue">(1)</span><span class="citation-flpages">: 45–51. </span></div><div><span class="fm-vol-iss-date">Published online 2009 November 2. </span><span class="fm-vol-iss-date"> </span><span class="fm-vol-iss-date">doi: <a class="ref-extlink" href="http://dx.crossref.org/10.1128%2FAAC.00427-09" target="pmc_ext">10.1128/AAC.00427-09</a>.</span></div></div></div></td><td class="fm-citation-ids"><div class="fm-citation-pmcid"><span class="fm-citation-ids-label">PMCID: </span><span>PMC2798517</span></div></td></tr>
</tbody></table><div class="fm-copyright"><a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/about/copyright.html">Copyright</a> © 2010, American Society for Microbiology</div><div class="fm-title">Molecular and Biochemical Characterization of the Natural Chromosome-Encoded Class A β-Lactamase from <em>Pseudomonas luteola</em><sup><a class="cite-reflink fn" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#fn1"><img alt="[down-pointing small open triangle]" border="0" src="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcents/x25BF.gif" title="" /></a></sup> </div><div class="contrib-group fm-author">Benoît Doublet,<sup>1,</sup><sup>2</sup> Frédéric Robin,<sup>3,</sup><sup>4</sup> Isabelle Casin,<sup>5</sup> Laëtitia Fabre,<sup>1,</sup><sup>6</sup> Anne Le Fleche,<sup>6</sup> Richard Bonnet,<sup>3,</sup><sup>4</sup> and François-Xavier Weill<sup>1,</sup><sup>6</sup><sup>*</sup></div><div class="fm-affl">Institut Pasteur, Laboratoire des Bactéries Pathogènes Entériques, Paris, France,<sup>1</sup> INRA, UR1282 Infectiologie Animale Santé Publique, Nouzilly, France,<sup>2</sup> CHU de Clermont-Ferrand, Laboratoire de bactériologie, Clermont-Ferrand, France,<sup>3</sup> Université Clermont 1, UFR de Médecine, Laboratoire de Bactériologie, JE2526 usc INRA2018, Clermont-Ferrand, France,<sup>4</sup> Hôpital St-Louis, Service de Microbiologie, Paris, France,<sup>5</sup> Institut Pasteur, Unité de Biodiversité des Bactéries Pathogènes Emergentes, Paris, France<sup>6</sup></div><div class="fm-footnote" id="cor1"><sup>*</sup>Corresponding author. Mailing address: Laboratoire des Bactéries Pathogènes Entériques, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris cedex 15, France. Phone: 33-(0)1 45 68 83 45. Fax: 33-(0)1 45 68 88 37. E-mail: <span class="e_id3024682"><a class="ext-reflink" href="mailto:fxweill@pasteur.fr">fxweill@pasteur.fr</a></span></div><div class="fm-pubdate">Received March 30, 2009; Revised May 22, 2009; Accepted October 23, 2009.</div><div class="sec" id="__abstractid3024768"><div class="head1 section-title" id="__abstractid3024768titletitle" style="text-transform: uppercase;"><div class="other-sections"><ul class="noext-menu"><li><a class="first-link" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#"> Other Sections▼</a><ul class="submenu head1"><li class="submenu-item current-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#" style="text-transform: uppercase;">Abstract</a></li>
<li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__secid3025178" style="text-transform: uppercase;">MATERIALS AND METHODS</a></li>
<li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__secid3025749" style="text-transform: uppercase;">RESULTS AND DISCUSSION</a></li>
<li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__ref-listid3026564" style="text-transform: uppercase;">REFERENCES</a></li>
</ul></li>
</ul></div><div>Abstract</div></div><div class="section-content" id="__abstractid3024768content"><div class="p p-first-last" id="__pid3024769"><em>Pseudomonas luteola</em> (formerly classified as CDC group Ve-1 and named <em>Chryseomonas luteola</em>) is an unusual pathogen implicated in rare but serious infections in humans. A novel β-lactamase gene, <em>bla</em><sub>LUT-1</sub>, was cloned from the whole-cell DNA of the <em>P. luteola</em> clinical isolate LAM, which had a weak narrow-spectrum β-lactam-resistant phenotype, and expressed in <em>Escherichia coli</em>. This gene encoded LUT-1, a 296-amino-acid Ambler class A β-lactamase with a pI of 6 and a theoretical molecular mass of 28.9 kDa. The catalytic efficiency of this enzyme was higher for cephalothin, cefuroxime, and cefotaxime than for penicillins. It was found to be 49% to 59% identical to other Ambler class A β-lactamases from <em>Burkholderia</em> sp. (PenA to PenL), <em>Ralstonia eutropha</em> (REUT), <em>Citrobacter sedlakii</em> (SED-1), <em>Serratia fonticola</em> (FONA and SFC-1), <em>Klebsiella</em> sp. (KPC and OXY), and CTX-M extended-spectrum β-lactamases. No gene homologous to the regulatory <em>ampR</em> genes of class A β-lactamases was found in the vicinity of the <em>bla</em><sub>LUT-1</sub> gene. The entire <em>bla</em><sub>LUT-1</sub> coding region was amplified by PCR and sequenced in five other genetically unrelated <em>P. luteola</em> strains (including the <em>P. luteola</em> type strain). A new variant of <em>bla</em><sub>LUT-1</sub> was found for each strain. These genes (named <em>bla</em><sub>LUT-2</sub> to <em>bla</em><sub>LUT-6</sub>) had nucleotide sequences 98.1 to 99.5% identical to that of <em>bla</em><sub>LUT-1</sub> and differing from this gene by two to four nonsynonymous single nucleotide polymorphisms. The <em>bla</em><sub>LUT</sub> gene was located on a 700- to 800-kb chromosomal I-CeuI fragment, the precise size of this fragment depending on the <em>P. luteola</em> strain.</div></div></div><div class="sec" id="__bodyid3024885"><div class="head1 section-title" id="__bodyid3024885titletitle" style="text-transform: uppercase;"><div class="other-sections"><ul class="noext-menu"><li><a class="first-link" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#"> Other Sections▼</a><ul class="submenu head1"><li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__abstractid3024768" style="text-transform: uppercase;">Abstract</a></li>
<li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__secid3025178" style="text-transform: uppercase;">MATERIALS AND METHODS</a></li>
<li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__secid3025749" style="text-transform: uppercase;">RESULTS AND DISCUSSION</a></li>
<li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__ref-listid3026564" style="text-transform: uppercase;">REFERENCES</a></li>
</ul></li>
</ul></div><div> </div></div><div class="section-content" id="__bodyid3024885content"><div class="p p-first" id="__pid3024886"><em>Pseudomonas luteola</em> (formely known as CDC group Ve-1 or <em>Chryseomonas luteola</em>) is a motile, strictly aerobic, gram-negative rod, producing a distinct yellow-orange pigment (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/9103607">4</a>). This organism is nonfermentative, oxidase negative, and catalase positive. <em>P. luteola</em> has been isolated from many sources in nature (water, soil, and damp environments) and is considered to be a saprophyte or commensal organism only rarely pathogenic to humans (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/16221303">11</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/10942647">19</a>). Clinical infections due to this microorganism have rarely been reported (fewer than 25 cases) and have mostly presented as septicemia, meningitis, peritonitis, endocarditis, and ulcer infections, usually in association with surgical operations or the use of catheters or prostheses (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/16221303">11</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/15071064">13</a>-<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/3667927">15</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/3384937">17</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/10942647">19</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/2041957">20</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/2041953">24</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/9934547">36</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/11858911">42</a>). It has been suggested that this organism is likely to become more frequent as a nosocomial pathogen (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/10942647">19</a>). The clinical isolates of <em>P. luteola</em> have generally been shown to be susceptible to extended-spectrum cephalosporins (ESC), aminoglycosides, and fluoroquinolones (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/16221303">11</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/2041957">20</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/9934547">36</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/11858911">42</a>). In most studies in which isolates were tested with a large panel of β-lactam antibiotics, resistance to original-spectrum and broad-spectrum cephalosporins was observed, whereas susceptibility to penicillins was variable (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#r5">5</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/15071064">13</a>-<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/3667927">15</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/3384937">17</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/2041957">20</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/9934547">36</a>). This β-lactam resistance phenotype suggests that this microbe may produce a natural β-lactamase.</div><div class="p" id="__pid3025152">We report here the cloning and sequencing of the <em>bla</em><sub>LUT-1</sub> gene, encoding the class A β-lactamase of the <em>P. luteola</em> clinical isolate LAM, which was isolated in January 2002 from a blood culture from a patient with an infected indwelling catheter. We investigated the biochemical characteristics of LUT-1. The presence, nucleotide diversity, and location of the <em>bla</em><sub>LUT</sub> gene were studied in five other genetically unrelated <em>P. luteola</em> strains.</div></div></div><div class="sec" id="__secid3025178"><div class="head1 section-title" id="__secid3025178titletitle" style="text-transform: uppercase;"><div class="other-sections"><ul class="noext-menu"><li><a class="first-link" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#"> Other Sections▼</a><ul class="submenu head1"><li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__abstractid3024768" style="text-transform: uppercase;">Abstract</a></li>
<li class="submenu-item current-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#" style="text-transform: uppercase;">MATERIALS AND METHODS</a></li>
<li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__secid3025749" style="text-transform: uppercase;">RESULTS AND DISCUSSION</a></li>
<li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__ref-listid3026564" style="text-transform: uppercase;">REFERENCES</a></li>
</ul></li>
</ul></div><div>MATERIALS AND METHODS</div></div><div class="section-content" id="__secid3025178content"><div class="sec sec-first" id="__secid3025185"><span class="head2">Bacterial strains and plasmids. </span> <div class="p p-first-last" id="__pid3025189">The bacterial strains and plasmids used in this study are described in Table <a class="fig-table-link table" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/table/t1/" style="text-decoration: none;"><span style="text-decoration: underline;">1</span></a>. <em>P. luteola</em> was identified with the API-20 NE system (bioMérieux, Marcy-l'Etoile, France) and by sequencing PCR-amplified <em>rrs</em> (16S rRNA gene) and <em>rpoB</em> (RNA polymerase beta subunit), as previously described (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/18280706">2</a>).</div><div class="canvas-table-ref-outer"><div class="canvas-table-ref-inner"><table border="0" cellpadding="0" cellspacing="0" class="thumb-caption" style="clear: both; width: 100%;"><tbody>
<tr align="left" valign="top"><td class="thumb-cell"><div class="thumb-ph" id="t1"><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/table/t1/"><img alt="TABLE 1." class="icon-reflink" src="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/table-icon.gif" style="border: 1px solid;" title="TABLE 1." /></a></div></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/table/t1/"><strong>TABLE 1.</strong></a><div class="figure-table-caption-in-article"><span>Bacterial strains and plasmids used in this study<sup><em>a</em></sup></span></div></div></td></tr>
</tbody></table></div></div></div><div class="sec" id="__secid3025227"><span class="head2">Antimicrobial susceptibility testing. </span> <div class="p p-first-last" id="__pid3025231">Antibiotic susceptibility was assessed by the disk diffusion method for 32 antimicrobial drugs (Bio-Rad, Marnes La Coquette, France), as previously described (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/15583311">43</a>). The MIC of each β-lactam antibiotic was determined by Etest (AB Biodisk, Solna, Sweden). Susceptibility was classified according to the guidelines of the Antibiogram Committee of the French Society for Microbiology (CA-SFM) (<span class="ext-reflink"><a class="ext-reflink " href="http://www.sfm.asso.fr/nouv/general.php?pa=2" target="pmc_ext">http://www.sfm.asso.fr/nouv/general.php?pa=2</a></span>). <em>Escherichia coli</em> ATCC 25922 was used as the control for disk diffusion analyses and MIC determinations.</div></div><div class="sec" id="__secid3025262"><span class="head2">Cloning experiments and analysis of recombinant plasmids. </span> <div class="p p-first" id="__pid3025266">Genomic DNA of the <em>P. luteola</em> strain LAM was partially digested with Sau3AI restriction enzyme, ligated into the BamHI-restricted phagemid pBK-CMV, and electroporated into <em>E. coli</em> strain DH10B, as previously described (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/19075063">34</a>). Antibiotic-resistant colonies were selected on Mueller-Hinton (MH) agar (Bio-Rad) containing kanamycin (30 μg/ml) and cefamandole (5 μg/ml).</div><div class="p" id="__pid3025290">The 1.5-kb cloned DNA fragment from the recombinant plasmid pBK-L3 was sequenced on both strands by Cogenics (Meylan, France). Analyses of nucleotide sequences and deduced amino acid sequences were performed with EditSeq and Megalign software (DNAstar, Madison, WI). The BLAST programs available from the NCBI were used for database searches (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/BLAST/" target="pmc_ext">http://www.ncbi.nlm.nih.gov/BLAST/</a></span>).</div><div class="p p-last" id="__pid3025305">We searched for a divergently transcribed regulator gene upstream for the <em>bla</em><sub>LUT-1</sub> gene in various recombinant plasmids shown by PCR to contain the longest DNA sequences upstream from the <em>bla</em><sub>LUT-1</sub> gene. The primers used were either T3 or T7 (binding to the multicloning site of pBK-CMV) and REG (5′-CTTTTGTGACTTGAGGAGATCGCA-3′) (binding 300 bp upstream from the ATG initiation codon of the LUT-1 gene). The amplification conditions were as described below, except that an annealing temperature of 47°C was used.</div></div><div class="sec" id="__secid3025325"><span class="head2">Isoelectric focusing and β-lactamase preparation. </span> <div class="p p-first" id="__pid3025329">Isoelectric focusing was performed with polyacrylamide gels containing ampholines with a pH range of 3.5 to 10, as previously described (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/10543745">7</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/7928805">38</a>). β-Lactamases of known pIs (in parentheses) were used as standards: TEM-1 (5.4), TEM-2 (5.6), TEM-6 (5.9), TEM-24 (6.5), and TEM-28 (6.1).</div><div class="p p-last" id="__pid3025356">LUT-1-producing <em>E. coli</em> DH5α(pBK-L3) was grown in 6 liters of 2× yeast-tryptone broth supplemented with 20 μg/ml kanamycin and 32 μg/ml amoxicillin for 18 h at 37°C. The β-lactamase LUT-1 was purified as previously described (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/11451684">8</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/16251281">37</a>), by ion-exchange chromatography with a Q Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden) and gel filtration chromatography with a Superose 12 column (Amersham Pharmacia Biotech). Total protein concentration was estimated with the Bio-Rad protein assay (Bio-Rad, Richmond, CA) with bovine serum albumin (Sigma Chemical Co., St. Louis, MO) used as the standard. The purity of LUT-1 extracts was estimated as previously described (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/11036023">9</a>), by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and staining with Coomassie blue R-250 (Sigma Chemical Co.).</div></div><div class="sec" id="__secid3025401"><span class="head2">Determination of kinetic constants for β-lactamase activity. </span> <div class="p p-first-last" id="__pid3025406">The kinetic constants <em>K<sub>m</sub></em> and <em>k</em><sub>cat</sub> for β-lactamases were obtained by a computerized microacidimetric method, as previously described (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/4198636">26</a>). The concentrations of the inhibitors (clavulanate and tazobactam) required to inhibit enzyme activity by 50% (IC<sub>50</sub>s) were determined with penicillin G (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/11036023">9</a>). Penicillin G (100 mM) was used as the reporter substrate for IC<sub>50</sub> monitoring. The kinetic constants were determined three times. The coefficients of variation did not exceed 15%.</div></div><div class="sec" id="__secid3025454"><span class="head2">PFGE typing. </span> <div class="p p-first-last" id="__pid3025458">The genetic diversity of <em>P. luteola</em> was assessed by pulsed-field gel electrophoresis (PFGE) of genomic DNA digested with XbaI or SpeI (Roche), as previously described (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/15583311">43</a>).</div></div><div class="sec" id="__secid3025475"><span class="head2">PCR amplification and sequencing of the <em>bla</em><sub>LUT</sub> gene in <em>P. luteola</em>. </span> <div class="p p-first-last" id="__pid3025490">The entire coding region of the <em>bla</em><sub>LUT</sub> gene of all <em>P. luteola</em> strains was amplified by PCR, using the primers UpPlut (5′-ACCGTCTAGGCTGCTACTTCA-3′) and LoPlut (5′-CCGCTGCGCATGAGCGTA-3′), binding 200 bp upstream from the ATG initiation codon of LUT-1 and 10 bp downstream from the stop codon, respectively. The PCR products (1,100 kb) were sequenced at the Plateforme de Génotypage des Pathogènes et Santé Publique, PF8 (Institut Pasteur).</div></div><div class="sec" id="__secid3025507"><span class="head2">Phylogenetic analysis of the amino acid sequences. </span> <div class="p p-first-last" id="__pid3025512">The ClustalW program (<span class="ext-reflink"><a class="ext-reflink " href="http://infobiogen.fr/" target="pmc_ext">http://infobiogen.fr</a></span>) was used to align the amino acid sequences obtained (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/7984417">40</a>). Phylogenetic analysis was carried out with the bioinformatics tool TOPALi v2.5 (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/18984599">28</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/14988107">29</a>). A phylogenic tree was constructed by the Bayesian method, as implemented in the MRBAYES program (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/11524383">22</a>). LUT-1 was compared with 37 class A β-lactamases. The consensus tree calculated by MRBAYES was imported into MEGA4 for the purposes of displaying and printing the tree (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/18417537">25</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/17488738">39</a>).</div></div><div class="sec" id="__secid3025591"><span class="head2">Resistance transfer determination. </span> <div class="p p-first-last" id="__pid3025595">Conjugation and transformation experiments were carried out on the <em>P. luteola</em> LAM isolate, as previously described (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/15583311">43</a>). The β-lactam antibiotic used for selection was cefotaxime with a final concentration of 0.25 μg/ml.</div></div><div class="sec" id="__secid3025613"><span class="head2">Chromosomal location of <em>bla</em><sub>LUT</sub> genes, as determined by PFGE-I-CeuI. </span> <div class="p p-first-last" id="__pid3025624">For determination of the chromosomal location of the <em>bla</em><sub>LUT-1</sub> gene, we digested agarose plugs, prepared as described previously, with the I-CeuI endonuclease (New England Biolabs, Beverly, MA) (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/7768842">27</a>). I-CeuI restriction fragments were subjected to Southern blot hybridization with a PCR-generated probe for the <em>bla</em><sub>LUT-1</sub> gene and with an <em>rrs</em> (16S rRNA gene) probe, as described above. Hybridization, labeling, and detection were performed according to the manufacturer's recommendations, using a nonradioactive enhanced chemiluminescence kit (ECL; GE Healthcare, United Kingdom).</div></div><div class="sec sec-last" id="__secid3025659"><span class="head2">Nucleotide sequence accession numbers. </span> <div class="p p-first-last" id="__pid3025663">The nucleotide sequences of the <em>bla</em><sub>LUT-1</sub> to <em>bla</em><sub>LUT-6</sub> genes of the <em>P. luteola</em> strains have been deposited in the GenBank database under accession numbers <span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=51832294" target="pmc_ext">AY695112</a></span>, <span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=224994880" target="pmc_ext">FJ750572</a></span>, <span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=224994882" target="pmc_ext">FJ750573</a></span>, <span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=224994884" target="pmc_ext">FJ750574</a></span>, <span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=224994886" target="pmc_ext">FJ750575</a></span>, and <span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=224994888" target="pmc_ext">FJ750576</a></span>, respectively.</div></div></div></div><div class="sec" id="__secid3025749"><div class="head1 section-title" id="__secid3025749titletitle" style="text-transform: uppercase;"><div class="other-sections"><ul class="noext-menu"><li><a class="first-link" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#"> Other Sections▼</a><ul class="submenu head1"><li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__abstractid3024768" style="text-transform: uppercase;">Abstract</a></li>
<li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__secid3025178" style="text-transform: uppercase;">MATERIALS AND METHODS</a></li>
<li class="submenu-item current-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#" style="text-transform: uppercase;">RESULTS AND DISCUSSION</a></li>
<li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__ref-listid3026564" style="text-transform: uppercase;">REFERENCES</a></li>
</ul></li>
</ul></div><div>RESULTS AND DISCUSSION</div></div><div class="section-content" id="__secid3025749content"><div class="sec sec-first" id="__secid3025753"><span class="head2">Antimicrobial susceptibility of <em>P. luteola</em>. </span> <div class="p p-first-last" id="__pid3025761">The disk diffusion method showed the five <em>P. luteola</em> isolates and the type strain to be resistant to cephalothin, cefamandole, cefoxitin (resistance or intermediate susceptibility), nalidixic acid, trimethoprim, and trimethoprim-sulfamethoxazole. All strains were susceptible to ciprofloxacin and aminoglycosides. Additional resistance to sulfonamides and chloramphenicol was observed only in the LAM isolate. The MICs of the β-lactams determined by Etest are shown in Table <a class="fig-table-link table" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/table/t2/" style="text-decoration: none;"><span style="text-decoration: underline;">2</span></a>. The addition of clavulanic acid slightly decreased (by a factor of 2 to 4) the MICs of amoxicillin, ticarcillin, and piperacillin. The MICs of the ESC and also aztreonam were slightly affected within the susceptible or intermediate range. Only the cefotaxime MICs of some <em>P. luteola</em> strains were in the resistant (>2-μg/ml) range according to the guidelines of the CA-SFM. By using the Etest ESBL cefepime/cefepime plus clavulanic acid strips, a deformation of the cefepime inhibition ellipse was observed for two strains with the highest MICs of cefepime (isolate 03-5093 and type strain CIP 102995<sup>T</sup>). The MICs of nalidixic acid and ciprofloxacin ranged from 16 to >256 μg/ml and from 0.032 to 0.25 μg/ml, respectively.</div><div class="canvas-table-ref-outer"><div class="canvas-table-ref-inner"><table border="0" cellpadding="0" cellspacing="0" class="thumb-caption" style="clear: both; width: 100%;"><tbody>
<tr align="left" valign="top"><td class="thumb-cell"><div class="thumb-ph" id="t2"><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/table/t2/"><img alt="TABLE 2." class="icon-reflink" src="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/table-icon.gif" style="border: 1px solid;" title="TABLE 2." /></a></div></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/table/t2/"><strong>TABLE 2.</strong></a><div class="figure-table-caption-in-article"><span>MICs of β-lactams for <em>P. luteola</em> and <em>E. coli</em> strains</span></div></div></td></tr>
</tbody></table></div></div></div><div class="sec" id="__secid3025795"><span class="head2">Cloning and sequence analysis of the <em>bla</em> gene from <em>P. luteola</em>. </span> <div class="p p-first" id="__pid3025807">Partially Sau3AI-digested DNA from the <em>P. luteola</em> clinical isolate LAM was inserted into the BamHI site of pBK-CMV. Ten <em>E. coli</em> DHB10B recombinant clones were obtained after selection on kanamycin and cefamandole (5 μg/ml). The inserts of the recombinant plasmids were between 1.5 and 3.6 kb in size. The pBK-L3 plasmid, which had a 1.5-kb insert, was selected for sequence analysis. An open reading frame (ORF) of 891 bp, preceded by a putative promoter region (339 bp), was identified and shown to encode a 296-amino-acid sequence. These nucleotide and amino acid sequences were absent from databases. However, the deduced protein had amino acid motifs typical of Ambler class A β-lactamases (<sup>70</sup>SXXK<sup>73</sup>, <sup>130</sup>SDN<sup>132</sup>, <sup>166</sup>EXXXN<sup>170</sup>, and <sup>234</sup>KTG<sup>236</sup>) (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/2039479">3</a>). We therefore named this putative novel class A β-lactamase and the corresponding gene LUT-1 and <em>bla</em><sub>LUT-1</sub>, respectively. We used the SignalP 3.0 server (available at: <span class="ext-reflink"><a class="ext-reflink " href="http://www.cbs.dtu.dk/services/SignalP/" target="pmc_ext">http://www.cbs.dtu.dk/services/SignalP/</a></span>) to determine whether this protein had a putative signal peptide. A putative cleavage site was identified between the 27th and 28th amino acids of the N-terminal region, giving a putative mature protein with a theoretical molecular mass of 28.9 kDa. A phylogenic study was carried out to assess the relationship between LUT-1 and its closest relatives and between this enzyme and members of the major lineages of class A β-lactamases (Fig. <a class="fig-table-link fig figpopup" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f1/" style="text-decoration: none;"><span style="position: relative; text-decoration: none;"><span class="figpopup-sensitive-area" style="left: -2.5em;">(Fig.1).</span></span><span style="text-decoration: underline;">1</span></a>). The predicted LUT-1 protein showed similarities to several other chromosome-encoded class A β-lactamases identified in beta- and gammaproteobacteria. Figure <a class="fig-table-link fig figpopup" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f2/" style="text-decoration: none;"><span style="position: relative; text-decoration: none;"><span class="figpopup-sensitive-area" style="left: -3em;">Figure2</span></span><span style="text-decoration: underline;">2</span></a> shows an alignment of the amino acid sequence of LUT-1 with representative members of the various branches of naturally occurring and acquired class A β-lactamases displaying similarity to LUT-1. The LUT-1 β-lactamase was 53 to 59% similar to the chromosomal β-lactamases of <em>Burkholderia cepacia</em> complex (Pen-A to Pen-L), 56% identical to that of <em>Ralstonia eutropha</em> (REUT), 54% identical to that of <em>Citrobacter sedlakii</em> (SED-1), 52% identical to that of <em>Serratia fonticola</em> (FONA-5), and 51% identical to that of <em>Klebsiella oxytoca</em> (OXY-5) (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/16048960">16</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/11451687">33</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/19075063">34</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/19015357">44</a>). LUT-1 also showed similarities with acquired β-lactamases such as KPC-7 from <em>Klebsiella pneumoniae</em> and SFC-1 from <em>S. fonticola</em> (class A carbapenemases) (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/15155245">21</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/19324295">30</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/19015357">44</a>). Interestingly, the LUT-1 β-lactamase was also 49 to 52% identical to members of the extended-spectrum β-lactamase (ESBL) CTX-M family (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/14693512">6</a>).</div><div class="canvas-figure-ref-outer screen-view"><div class="canvas-figure-ref-inner"><table border="0" cellpadding="0" cellspacing="0" class="thumb-caption" style="clear: both; width: 100%;"><tbody>
<tr align="left" valign="top"><td class="thumb-cell"><div class="thumb-ph" id="f1"><a class="icon-reflink figpopup" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f1/"><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img alt="FIG. 1." class="icon-reflink small-thumb" src="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/bin/zac0011086480001.gif" title="FIG. 1." /></div></div></a></div></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f1/"><strong>FIG. 1.</strong></a><div class="figure-table-caption-in-article"><span>Phylogeny of the amino acid sequences of LUT-1 and 37 other class A β-lactamases constructed with TOPALi v2.5 (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/18984599">28</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/14988107">29</a>). Branch lengths are drawn to scale and are proportional to the numbers of amino acid changes. The percentages at the branch</span><a class="side-caption" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f1/" style="font-size: 100%;"> (more ...)</a></div></div></td></tr>
</tbody></table></div></div><div class="canvas-figure-ref-outer print-view"><div class="canvas-figure-ref-inner"><div class="thumb-ph inline-block"><a class="icon-reflink figpopup" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f1/"><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img alt="FIG. 1." class="icon-reflink small-thumb" src="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/bin/zac0011086480001.gif" title="FIG. 1." /></div></div></a></div><div class="figure-table-caption-in-article"><div class="p"><div class="p"><strong>FIG. 1.</strong></div><div class="p"><div class="caption"> <div class="p"><div class="p" id="__pid3056397">Phylogeny of the amino acid sequences of LUT-1 and 37 other class A β-lactamases constructed with TOPALi v2.5 (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/18984599">28</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/14988107">29</a>). Branch lengths are drawn to scale and are proportional to the numbers of amino acid changes. The percentages at the branch points refer to the numbers of times that particular nodes were found in 10,000 bootstrap replications. The distance along the vertical axis has no significance. The amino acid identity of each β-lactamase to the amino acid sequence of LUT-1 from <em>P. luteola</em> is indicated in parentheses. The acquired β-lactamases of Gram-negative organisms (GenBank accession numbers are indicated in parentheses) are TEM-3 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=43810" target="pmc_ext">X64525</a></span>), SHV-2 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=972890" target="pmc_ext">L47119</a></span>), PSE-4 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=151527" target="pmc_ext">J05162</a></span>), PER-1 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=396144" target="pmc_ext">Z21957</a></span>), TLA-1 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPept&list_uids=5002292" target="pmc_ext">AAD37403</a></span>), VEB-1 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=4102199" target="pmc_ext">AF010416</a></span>), BES-1 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=7340223" target="pmc_ext">AF234999</a></span>), CTX-M-1 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=1524020" target="pmc_ext">X92506</a></span>), CTX-M-2 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=1524365" target="pmc_ext">X92507</a></span>), CTX-M-8 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=6137225" target="pmc_ext">AF189721</a></span>), CTX-M-25 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPept&list_uids=21637408" target="pmc_ext">AAM70498</a></span>), CTX-M-9 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPept&list_uids=11178696" target="pmc_ext">AAF05311</a></span>), GES-1 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=6752459" target="pmc_ext">AF156486</a></span>), KPC-1 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=10121874" target="pmc_ext">AF297554</a></span>), KPC-7 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=189916550" target="pmc_ext">EU729727</a></span>), PC-1 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=46626" target="pmc_ext">X04121</a></span>), SFO-1 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=4589727" target="pmc_ext">AB003148</a></span>), and SFC-1 (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=37992760" target="pmc_ext">AY354402</a></span>). The others are naturally occurring β-lactamases, such as CKO-1 from <em>Citrobacter koseri</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=19110886" target="pmc_ext">AF477396</a></span>); CME-1 from <em>Chryseobacterium meningosepticum</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=5851939" target="pmc_ext">AJ006275</a></span>); CEPA from <em>Bacteroides fragilis</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=10048473" target="pmc_ext">U05888</a></span>); CBLA from <em>Bacteroides uniformis</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=436817" target="pmc_ext">L08472</a></span>); SALBG from <em>Streptomyces albus</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=153338" target="pmc_ext">M28303</a></span>); PenA from <em>Burkholderia multivorans</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPept&list_uids=2062565" target="pmc_ext">AAB53622</a></span>); PenB1 from <em>Burkholderia cenocepacia</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=214091188" target="pmc_ext">EU872211</a></span>); NMC-A from <em>Enterobacter cloacae</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=396086" target="pmc_ext">Z21956</a></span>); SME-1 from <em>Serratia marcescens</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=472863" target="pmc_ext">Z28968</a></span>); REUT from <em>Ralstonia eutropha</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPept&list_uids=73541373" target="pmc_ext">YP_295893</a></span>); YENT from <em>Yersinia enterocolitica</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=48813" target="pmc_ext">X57074</a></span>); SED-1 from <em>Citrobacter sedlakii</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=14486652" target="pmc_ext">AF321608</a></span>); ERP-1 from <em>Erwinia persicina</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPept&list_uids=20384876" target="pmc_ext">AAL86999</a></span>); PVUL from <em>Proteus vulgaris</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=511055" target="pmc_ext">X80128</a></span>); OXY-1, OXY-2, and OXY-5 from <em>Klebsiella oxytoca</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=456260" target="pmc_ext">Z30177</a></span>, <span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=1226009" target="pmc_ext">Z49084</a></span>, and <span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=60416702" target="pmc_ext">AJ871868</a></span>, respectively); FONA-5 from <em>Serratia fonticola</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=6468747" target="pmc_ext">AJ251243</a></span>); and L2 from <em>Stenotrophomonas maltophilia</em> (<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPept&list_uids=6580703" target="pmc_ext">CAB63490</a></span>).</div></div></div></div></div></div></div></div><div class="canvas-figure-ref-outer screen-view"><div class="canvas-figure-ref-inner"><table border="0" cellpadding="0" cellspacing="0" class="thumb-caption" style="clear: both; width: 100%;"><tbody>
<tr align="left" valign="top"><td class="thumb-cell"><div class="thumb-ph" id="f2"><a class="icon-reflink figpopup" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f2/"><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img alt="FIG. 2." class="icon-reflink small-thumb" src="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/bin/zac0011086480002.gif" title="FIG. 2." /></div></div></a></div></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f2/"><strong>FIG. 2.</strong></a><div class="figure-table-caption-in-article"><span>Alignment of the LUT-1 β-lactamase amino acid sequence from <em>P. luteola</em> with those of other class A β-lactamases. Naturally occurring β-lactamases: PenA from <em>Burkholderia multivorans</em> 249 (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/9371340">41</a>), PenB1 from <em>B. cenocepacia</em> (GenBank</span><a class="side-caption" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f2/" style="font-size: 100%;"> (more ...)</a></div></div></td></tr>
</tbody></table></div></div><div class="canvas-figure-ref-outer print-view"><div class="canvas-figure-ref-inner"><div class="thumb-ph inline-block"><a class="icon-reflink figpopup" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f2/"><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img alt="FIG. 2." class="icon-reflink small-thumb" src="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/bin/zac0011086480002.gif" title="FIG. 2." /></div></div></a></div><div class="figure-table-caption-in-article"><div class="p"><div class="p"><strong>FIG. 2.</strong></div><div class="p"><div class="caption"> <div class="p"><div class="p" id="__pid3056914">Alignment of the LUT-1 β-lactamase amino acid sequence from <em>P. luteola</em> with those of other class A β-lactamases. Naturally occurring β-lactamases: PenA from <em>Burkholderia multivorans</em> 249 (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/9371340">41</a>), PenB1 from <em>B. cenocepacia</em> (GenBank accession no. <span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=214091188" target="pmc_ext">EU872211</a></span>), REUT from <em>Ralstonia eutropha</em> (GenBank accession no.<span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPept&list_uids=123624975" target="pmc_ext">Q470Y4</a></span>), SED-1 from <em>Citrobacter sedlakii</em> (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/11451687">33</a>), FONA-5 from <em>Serratia fonticola</em> (GenBank accession no. <span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPept&list_uids=75471347" target="pmc_ext">Q9RIQ8</a></span>), and OXY-5 from <em>K. oxytoca</em> (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/16048960">16</a>). Acquired β-lactamases: SFC-1 from <em>Serratia fonticola</em> (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/15155245">21</a>), KPC-7 from <em>Klebsiella pneumoniae</em> (GenBank accession no. <span class="ext-reflink"><a class="ext-reflink " href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPept&list_uids=189916551" target="pmc_ext">ACE62798</a></span>), and CTX-M-2 extended-spectrum β-lactamase (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/14693512">6</a>). The positions of typical class A β-lactamase amino acids are indicated according to the standard numbering scheme of Ambler et al. (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/2039479">3</a>).</div></div></div></div></div></div></div></div><div class="p p-last" id="__pid3026028">We identified no putative Lys-R-type regulator upstream from the ORF encoding LUT-1 in pBK-L3. The upstream regions of the <em>bla</em><sub>LUT-1</sub> gene were amplified from nine other recombinant plasmids with the T3 and REG primers. The three plasmids containing the longest regions (750 bp, 600 bp, and 500 bp, respectively) were sequenced, but no regulator gene was identified. However, our results do not completely rule out the possibility that there is a regulator gene.</div></div><div class="sec" id="__secid3026041"><span class="head2">Properties of the LUT-1 β-lactamase. </span> <div class="p p-first" id="__pid3026046">The recombinant <em>E. coli</em>(pBK-L3) clone had a β-lactam resistance phenotype different from that of the parental strain <em>P. luteola</em> LAM (Table <a class="fig-table-link table" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/table/t2/" style="text-decoration: none;"><span style="text-decoration: underline;">2</span></a>). In <em>E. coli</em>, LUT-1 conferred resistance to amoxicillin and ticarcillin and an intermediate level of susceptibility to piperacillin and cephalothin. However, <em>E. coli</em>(pBK-L3) remained susceptible to cefoxitin, ESC, and imipenem. The β-lactamase inhibitors lowered the MICs of amoxicillin, ticarcillin, piperacillin, and cefotaxime by factors of 8 to 32. By using the Etest ESBL cefepime/cefepime plus clavulanic acid strips, a deformation of the cefepime inhibition ellipse was observed indicative of ESBLs. The discrepancy between <em>P. luteola</em> LAM and <em>E. coli</em>(pBK-L3) β-lactam resistance phenotypes may be due to the production of larger amounts of enzyme when the gene is expressed on a high-copy-number plasmid in <em>E. coli</em>. β-Lactam resistance phenotypes of the clinical <em>P. luteola</em> isolates (i.e., susceptibility to hydrolyzable penicillins) suggested a small amount of LUT. This hypothesis is strengthened by the kinetic parameters of LUT described below. Efflux mechanisms and/or differences in outer membrane permeability may also alter periplasmic β-lactam concentrations, thereby affecting apparent enzyme activity.</div><div class="p" id="__pid3026099">Both <em>P. luteola</em> isolate LAM and <em>E. coli</em>(pBK-L3) produced a single β-lactamase with an isoelectric point (pI) of approximately 6 (data not shown), consistent with the calculated theoretical pI. The purified LUT-1 protein appeared on SDS-polyacrylamide gels as a single band (≥98% pure) of approximately 29 kDa (data not shown). Kinetic parameters indicated that the LUT-1 enzyme had a broad substrate profile including penicillins and cephalosporins, such as cephalothin, cefuroxime, and cefotaxime in particular (Table <a class="fig-table-link table" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/table/t3/" style="text-decoration: none;"><span style="text-decoration: underline;">3</span></a>). As observed for CTX-M, LUT-1 displayed stronger hydrolytic activity against cefotaxime than against ceftazidime or aztreonam (<em>k</em><sub>cat</sub>, 452 versus 1.3 or 1.5 s<sup>−1</sup>, respectively). It was inhibited by low concentrations of clavulanic acid (IC<sub>50</sub>, 36 nM) and tazobactam (IC<sub>50</sub>, 40 nM). Given its susceptibility to clavulanate and its ability to cleave ESC, we can classify LUT-1 as a member of the 2e group in the functional classification of β-lactamases (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/7574506">10</a>). Particular residues of LUT-1 such as Ser104, Thr167, Thr237, and Arg276, identified as important positions for the action of other ESBLs (TEMs and CTX-Ms), may be involved in the activity of LUT-1 against oxyiminocephalosporins (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/15811373">12</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/9868772">18</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/8592985">23</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/18281307">31</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/7826350">32</a>, <a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/11709308">35</a>).</div><div class="canvas-table-ref-outer"><div class="canvas-table-ref-inner"><table border="0" cellpadding="0" cellspacing="0" class="thumb-caption" style="clear: both; width: 100%;"><tbody>
<tr align="left" valign="top"><td class="thumb-cell"><div class="thumb-ph" id="t3"><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/table/t3/"><img alt="TABLE 3." class="icon-reflink" src="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/table-icon.gif" style="border: 1px solid;" title="TABLE 3." /></a></div></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/table/t3/"><strong>TABLE 3.</strong></a><div class="figure-table-caption-in-article"><span>Substrate profile of the LUT-1 β-lactamase</span></div></div></td></tr>
</tbody></table></div></div><div class="p" id="__pid3026225">The level of hydrolysis of oxyiminocephalosporins observed, particularly for cefotaxime, was surprisingly high given their low MICs (Table <a class="fig-table-link table" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/table/t2/" style="text-decoration: none;"><span style="text-decoration: underline;">2</span></a>). However, low levels of expression in <em>P. luteola</em> LAM may be responsible for these low MICs, suggesting a possible chromosomal location for the <em>bla</em><sub>LUT</sub> gene. The presence of the natural promoter region (in silico analysis [data not shown]) may partly account for the low level of resistance observed in <em>E. coli</em>(pBK-L3).</div><div class="p p-last" id="__pid3026256">No biochemical characterization of the five variants of the LUT-1 β-lactamase (LUT-2 to LUT-6) identified on the basis of the <em>bla</em><sub>LUT</sub> gene sequences (see below) was performed. The effects of the amino acid substitutions on the hydrolytic profile of the protein therefore remain unclear.</div></div><div class="sec" id="__secid3026267"><span class="head2">Diversity of the <em>bla</em><sub>LUT</sub> gene in <em>P. luteola</em> strains. </span> <div class="p p-first-last" id="__pid3026282">We used PCR with the UpPlut and LoPlut primers to determine whether the <em>bla</em><sub>LUT-1</sub> gene was present in the six <em>P. luteola</em> strains and in type strains of <em>P. aeruginosa</em>, <em>B. cepacia</em>, <em>S. fonticola</em>, <em>Yersinia enterocolitica</em>, <em>C. sedlakii</em>, and <em>K. oxytoca</em>. All the <em>P. luteola</em> strains yielded a PCR product of the expected size (1,100 bp). No amplification was observed for the other species. However, the specificity of the PCR requires confirmation through testing in several other rare <em>Pseudomonas</em> species closely related to <em>P. luteola</em> in the phylogenetic tree (e.g., <em>P. anguilliseptica</em>, <em>P. pertucinogena</em>, and <em>P. lundensis</em>) (<a class="cite-reflink bibr popnode" href="https://www.ncbi.nlm.nih.gov/pubmed/15950132">1</a>). Sequencing of the PCR products on both strands of the DNA revealed that the six <em>bla</em><sub>LUT</sub> genes had nucleotide sequences 98.1 to 99.5% identical to that of the <em>bla</em><sub>LUT-1</sub> gene. Two to four nonsynonymous single nucleotide polymorphisms with respect to the LUT-1 β-lactamase were observed. These five variants of the <em>bla</em><sub>LUT-1</sub> gene were named <em>bla</em><sub>LUT-2</sub> to <em>bla</em><sub>LUT-6</sub> and deposited in the GenBank database.</div></div><div class="sec" id="__secid3026393"><span class="head2">Genomic diversity of <em>P. luteola</em> strains. </span> <div class="p p-first-last" id="__pid3026401">The genomic diversity of the six <em>P. luteola</em> strains was assessed by PFGE of XbaI- or SpeI-digested whole-cell DNA. The XbaI enzyme did not appear suitable for this species due to the presence of numerous compressed bands of small molecular size (in the range of 0 to 100 kb) (data not shown). However, the <em>P. luteola</em> strains were found to be genetically unrelated, as each strain harbored an SpeI profile differing from those of the other strains by at least 7 bands (Fig. <a class="fig-table-link fig figpopup" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f3/" style="text-decoration: none;"><span style="position: relative; text-decoration: none;"><span class="figpopup-sensitive-area" style="left: -2.5em;">(Fig.3</span></span><span style="text-decoration: underline;">3</span></a>).</div><div class="canvas-figure-ref-outer screen-view"><div class="canvas-figure-ref-inner"><table border="0" cellpadding="0" cellspacing="0" class="thumb-caption" style="clear: both; width: 100%;"><tbody>
<tr align="left" valign="top"><td class="thumb-cell"><div class="thumb-ph" id="f3"><a class="icon-reflink figpopup" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f3/"><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img alt="FIG. 3." class="icon-reflink small-thumb" src="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/bin/zac0011086480003.gif" title="FIG. 3." /></div></div></a></div></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f3/"><strong>FIG. 3.</strong></a><div class="figure-table-caption-in-article"><span>Pulsed-field gel electrophoresis of SpeI-digested genomic DNA from <em>P. luteola</em>. Lanes 1 and 8, XbaI-digested genomic DNA from <em>Salmonella enterica</em> serotype Braenderup H9812, used to provide molecular size markers (band sizes in kilobases); lane 2, clinical</span><a class="side-caption" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f3/" style="font-size: 100%;"> (more ...)</a></div></div></td></tr>
</tbody></table></div></div><div class="canvas-figure-ref-outer print-view"><div class="canvas-figure-ref-inner"><div class="thumb-ph inline-block"><a class="icon-reflink figpopup" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f3/"><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img alt="FIG. 3." class="icon-reflink small-thumb" src="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/bin/zac0011086480003.gif" title="FIG. 3." /></div></div></a></div><div class="figure-table-caption-in-article"><div class="p"><div class="p"><strong>FIG. 3.</strong></div><div class="p"><div class="caption"> <div class="p"><div class="p" id="__pid3057077">Pulsed-field gel electrophoresis of SpeI-digested genomic DNA from <em>P. luteola</em>. Lanes 1 and 8, XbaI-digested genomic DNA from <em>Salmonella enterica</em> serotype Braenderup H9812, used to provide molecular size markers (band sizes in kilobases); lane 2, clinical isolate LAM; lane 3, type strain CIP 1102995<sup>T</sup>; lane 4, clinical isolate 02-5971; lane 5, clinical isolate 03-5093; lane 6, clinical isolate 04-8684; lane 7, clinical isolate HEGP.</div></div></div></div></div></div></div></div></div><div class="sec sec-last" id="__secid3026425"><span class="head2">Chromosomal location of <em>bla</em><sub>LUT</sub> genes in <em>P. luteola</em>. </span> <div class="p p-first" id="__pid3026440">β-Lactam resistance could not be transferred by conjugation or by electroporation from the <em>P. luteola</em> LAM isolate to <em>E. coli</em> by using cefotaxime at 0.25 μg/ml for selection. These results suggested that the <em>bla</em><sub>LUT-1</sub> gene might be located on the chromosome. We tested this hypothesis, by digesting high-molecular-weight <em>P. luteola</em> DNAs embedded in agarose plugs with I-CeuI, separating the digestion products by PFGE and Southern blotting, and hybridizing them with a PCR-generated <em>bla</em><sub>LUT-1</sub> probe and an <em>rrs</em> (16S rRNA gene) probe. The <em>bla</em><sub>LUT</sub> genes were assigned to a single large I-CeuI fragment, between 700 and 800 kb in size, depending on the strain, which also hybridized with the <em>rrs</em> probe (Fig. <a class="fig-table-link fig figpopup" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f4/" style="text-decoration: none;"><span style="text-decoration: underline;">4B and C</span></a>). All the <em>P. luteola</em> strains harbored 6 chromosomal I-CeuI fragments which hybridized with the <em>rrs</em> probe (Fig. <a class="fig-table-link fig figpopup" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f4/" style="text-decoration: none;"><span style="position: relative; text-decoration: none;"><span class="figpopup-sensitive-area" style="left: -2.5em;">(Fig.4C).</span></span><span style="text-decoration: underline;">4C</span></a>). These results demonstrated a chromosomal location for <em>bla</em><sub>LUT</sub> genes.</div><div class="canvas-figure-ref-outer screen-view"><div class="canvas-figure-ref-inner"><table border="0" cellpadding="0" cellspacing="0" class="thumb-caption" style="clear: both; width: 100%;"><tbody>
<tr align="left" valign="top"><td class="thumb-cell"><div class="thumb-ph" id="f4"><a class="icon-reflink figpopup" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f4/"><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img alt="FIG. 4." class="icon-reflink small-thumb" src="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/bin/zac0011086480004.gif" title="FIG. 4." /></div></div></a></div></td><td class="caption-cell"><div class="caption-ph"><a class="side-caption" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f4/"><strong>FIG. 4.</strong></a><div class="figure-table-caption-in-article"><span>Chromosomal location of the <em>bla</em><sub>LUT-1</sub> gene. (A) PFGE separation of I-CeuI-digested DNA from <em>P. luteola</em>. Lane 1, clinical isolate LAM; lane 2, type strain CIP 1102995<sup>T</sup>; lane 3, clinical isolate 02-5971; lane 4, clinical isolate 03-5093; lane 5, clinical</span><a class="side-caption" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f4/" style="font-size: 100%;"> (more ...)</a></div></div></td></tr>
</tbody></table></div></div><div class="canvas-figure-ref-outer print-view"><div class="canvas-figure-ref-inner"><div class="thumb-ph inline-block"><a class="icon-reflink figpopup" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/figure/f4/"><div class="small-thumb-canvas"><div class="small-thumb-canvas-1"><img alt="FIG. 4." class="icon-reflink small-thumb" src="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/bin/zac0011086480004.gif" title="FIG. 4." /></div></div></a></div><div class="figure-table-caption-in-article"><div class="p"><div class="p"><strong>FIG. 4.</strong></div><div class="p"><div class="caption"> <div class="p"><div class="p" id="__pid3057111">Chromosomal location of the <em>bla</em><sub>LUT-1</sub> gene. (A) PFGE separation of I-CeuI-digested DNA from <em>P. luteola</em>. Lane 1, clinical isolate LAM; lane 2, type strain CIP 1102995<sup>T</sup>; lane 3, clinical isolate 02-5971; lane 4, clinical isolate 03-5093; lane 5, clinical isolate 04-8684; lane 6, clinical isolate HEGP; lanes M, XbaI-digested genomic DNA from <em>S. enterica</em> serotype Braenderup H9812 used to provide molecular size markers (band sizes in kilobases). (B) Southern blot hybridization with the <em>bla</em><sub>LUT-1</sub> probe. (C) Southern blot hybridization with the <em>rrs</em> (16S rRNA gene) probe. For panels B and C, lanes are as described for panel A.</div></div></div></div></div></div></div></div><div class="p p-last" id="__pid3026524">In conclusion, we have identified LUT-1, a class A β-lactamase naturally occurring in <em>P. luteola. P. luteola</em> has a weak narrow-spectrum β-lactam-resistant phenotype, but this environmental species may act as a reservoir of β-lactam resistance determinants. Provided that these findings are confirmed with a larger number of <em>P. luteola</em> (sensitivity) and <em>Pseudomonas</em> sp. (specificity) isolates, two practical applications of this study would be the use of the <em>bla</em><sub>LUT</sub> gene as a molecular identification marker for <em>P. luteola</em> species and the use of the DNA sequence microvariation of this gene as an alternative to PFGE for strain differentiation during investigations of outbreaks.</div></div></div></div><div class="sec" id="__ackid3026555"><div class="head1 section-title" id="__ackid3026555titletitle" style="text-transform: uppercase;"><div>Acknowledgments</div></div><div class="section-content" id="__ackid3026555content"><div class="sec"><div class="p" id="__pid3026556">B.D. was supported by an INRA postdoctoral fellowship.</div><div class="p" id="__pid3026559">We thank Isabelle Pogdlajen from Hôpital Européen Georges Pompidou for providing the HEGP strain and Bernadette Grandry and Tania Rybkine for expert technical assistance.</div></div></div></div><div class="sec" id="__articleid3049304footnotes"><div class="head1 section-title" id="__articleid3049304footnotestitletitle" style="text-transform: uppercase;"><div>Footnotes</div></div><div class="section-content" id="__articleid3049304footnotescontent"><div class="fm-footnote" id="fn1"><sup><img alt="[down-pointing small open triangle]" border="0" src="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcents/x25BF.gif" title="" /></sup>Published ahead of print on 2 November 2009.</div></div></div><div class="sec" id="__ref-listid3026564"><div class="head1 section-title" id="__ref-listid3026564titletitle" style="text-transform: uppercase;"><div class="other-sections"><ul class="noext-menu"><li><a class="first-link" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#"> Other Sections▼</a><ul class="submenu head1"><li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__abstractid3024768" style="text-transform: uppercase;">Abstract</a></li>
<li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__secid3025178" style="text-transform: uppercase;">MATERIALS AND METHODS</a></li>
<li class="submenu-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#__secid3025749" style="text-transform: uppercase;">RESULTS AND DISCUSSION</a></li>
<li class="submenu-item current-item"><a class="" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798517/#" style="text-transform: uppercase;">REFERENCES</a></li>
</ul></li>
</ul></div><div>REFERENCES</div></div><div class="section-content" id="__ref-listid3026564content"><div class="sec"><div class="back-matter-section" id="reference-list"><div class="ref-cit-blk" id="r1"><span class="ref-label">1.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3026575"><strong>Ait Tayeb, L., E. Ageron, F. Grimont, and P. A. D. Grimont.</strong> 2005. Molecular phylogeny of the genus <em>Pseudomonas</em> based on <em>rpoB</em> sequences and application for the identification of isolates. <span class="ref-journal">Res. Microbiol.</span> <span class="ref-vol">156</span><strong>:</strong>763-773. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/15950132" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r2"><span class="ref-label">2.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3026622"><strong>Ait Tayeb, L., M. Lefevre, V. Passet, L. Diancourt, S. Brisse, and P. A. D. Grimont.</strong> 2008. Comparative phylogenies of <em>Burkholderia</em>, <em>Ralstonia</em>, <em>Comamonas</em>, <em>Brevundimonas</em>, and related organisms derived from <em>rpoB</em>, <em>gyrB</em> and <em>rrs</em> gene sequences. <span class="ref-journal">Res. Microbiol.</span> <span class="ref-vol">159</span><strong>:</strong>169-177. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/18280706" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r3"><span class="ref-label">3.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3026689"><strong>Ambler, R. P., A. F. W. Coulson, J. M. Frère, J. M. Ghuysen, B. Joris, M. Forsman, R. C. Levesque, G. Tiraby, and S. G. Waley.</strong> 1991. A standard numbering scheme for the class A β-lactamases. <span class="ref-journal">Biochem. J.</span> <span class="ref-vol">276</span><strong>:</strong>269-270. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1151176/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/2039479" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r4"><span class="ref-label">4.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3026728"><strong>Anzai, Y., Y. Kudo, and H. Oyaizu.</strong> 1997. The phylogeny of the genera <em>Chryseomonas</em>, <em>Flavimonas</em>, and <em>Pseudomonas</em> supports synonymy of these three genera. <span class="ref-journal">Int. J. Syst. Bacteriol.</span> <span class="ref-vol">47</span><strong>:</strong>249-251. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/9103607" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r5"><span class="ref-label">5.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3026779"><strong>Berger, S. A., Y. Siegman-Igra, J. Stadler, and A. Campus.</strong> 1983. Group VE-1 septicemia. <span class="ref-journal">J. Clin. Microbiol.</span> <span class="ref-vol">7</span><strong>:</strong>926-927.</span></span></div><div class="ref-cit-blk" id="r6"><span class="ref-label">6.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3026812"><strong>Bonnet, R.</strong> 2004. Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">48</span><strong>:</strong>1-14. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC310187/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/14693512" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r7"><span class="ref-label">7.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3026851"><strong>Bonnet, R., C. De Champs, D. Sirot, C. Chanal, R. Labia, and J. Sirot.</strong> 1999. Diversity of TEM mutants in <em>Proteus mirabilis</em>. <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">43</span><strong>:</strong>2671-2677. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC89541/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/10543745" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r8"><span class="ref-label">8.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3026894"><strong>Bonnet, R., C. Dutour, J. L. Sampaio, C. Chanal, D. Sirot, R. Labia, C. De Champs, and J. Sirot.</strong> 2001. Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp-240/Gly. <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">45</span><strong>:</strong>2269-2275. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC90641/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/11451684" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r9"><span class="ref-label">9.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3026933"><strong>Bonnet, R., J. L. Sampaio, C. Chanal, D. Sirot, C. De Champs, J. L. Viallard, R. Labia, and J. Sirot.</strong> 2000. A novel class A extended-spectrum β-lactamase (BES-1) in <em>Serratia marcescens</em> isolated in Brazil. <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">44</span><strong>:</strong>3061-3068. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC101603/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/11036023" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r10"><span class="ref-label">10.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3054919"><strong>Bush, K., G. A. Jacoby, and A. A. Medeiros.</strong> 1995. A functional classification scheme for β-lactamases and its correlation with molecular structure. <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">39</span><strong>:</strong>1211-1233. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC162717/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/7574506" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r11"><span class="ref-label">11.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3054958"><strong>Casalta, J.-P., P.-E. Fournier, G. Habib, A. Riberi, and D. Raoult.</strong> 2005. Prosthetic valve endocarditis caused by <em>Pseudomonas luteola</em>. <span class="ref-journal">BMC Infect. Dis.</span> <span class="ref-vol">5</span><strong>:</strong>82. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1274313/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/16221303" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r12"><span class="ref-label">12.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055001"><strong>Chen, Y., J. Delmas, J. Sirot, B. Shoichet, and R. Bonnet.</strong> 2005. Atomic resolution structures of CTX-M β-lactamases: extended spectrum activities from increased mobility and decreased stability. <span class="ref-journal">J. Mol. Biol.</span> <span class="ref-vol">348</span><strong>:</strong>349-362. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/15811373" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r13"><span class="ref-label">13.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055040"><strong>Chihab, W., A. S. Alaoui, and M. Amar.</strong> 2004. <em>Chryseomonas luteola</em> identified as the source of serious infections in a Moroccan university hospital. <span class="ref-journal">J. Clin. Microbiol.</span> <span class="ref-vol">42</span><strong>:</strong>1837-1839. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC387548/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/15071064" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r14"><span class="ref-label">14.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055083"><strong>Connor, B. J., R. T. Kopecky, P. A. Frymoyer, and B. A. Forbes.</strong> 1987. Recurrent <em>Pseudomonas luteola</em> (CDC group Ve-1) peritonitis in a patient undergoing continuous ambulatory peritoneal dialysis. <span class="ref-journal">J. Clin. Microbiol.</span> <span class="ref-vol">25</span><strong>:</strong>1113-1114. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC269148/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/3597754" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r15"><span class="ref-label">15.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055126"><strong>Engel, J. M., F. S. Alexander, and C. T. Pachucki.</strong> 1987. Bacteremia caused by CDC group Ve-1 in previously healthy patient with granulomatous hepatitis. <span class="ref-journal">J. Clin. Microbiol.</span> <span class="ref-vol">25</span><strong>:</strong>2023-2024. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC269395/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/3667927" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r16"><span class="ref-label">16.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055165"><strong>Fevre, C., M. Jbel, V. Passet, F.-X. Weill, P. A. D. Grimont, and S. Brisse.</strong> 2005. Six groups of the OXY β-lactamase evolved over millions of years in <em>Klebsiella oxytoca</em>. <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">49</span><strong>:</strong>3453-3462. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1196214/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/16048960" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r17"><span class="ref-label">17.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055208"><strong>Freney, J., W. Hansen, J. Etienne, F. Vandenesch, and J. Fleurette.</strong> 1988. Postoperative infant septicemia caused by <em>Pseudomonas luteola</em> (CDC group Ve-1) and <em>Pseudomonas oryzihabitans</em> (CDC group Ve-2). <span class="ref-journal">J. Clin. Microbiol.</span> <span class="ref-vol">26</span><strong>:</strong>1241-1243. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC266575/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/3384937" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r18"><span class="ref-label">18.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055255"><strong>Gazouli, M., N. J. Legakis, and L. S. Tzouvelekis.</strong> 1998. Effect of substitution of Asn for Arg-276 in the cefotaxime-hydrolyzing class A β-lactamase CTX-M-4. <span class="ref-journal">FEMS Microbiol. Lett.</span> <span class="ref-vol">169</span><strong>:</strong>289-293. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/9868772" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r19"><span class="ref-label">19.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055294"><strong>Ghosh, S. K.</strong> 2000. A rare infection caused by <em>Chryseomonas luteola</em>. <span class="ref-journal">J. Infect.</span> <span class="ref-vol">41</span><strong>:</strong>109-110. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/10942647" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r20"><span class="ref-label">20.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055336"><strong>Hawkins, R. E., R. A. Moriarty, D. E. Lewis, and E. C. Oldfield.</strong> 1991. Serious infections involving the CDC group Ve bacteria <em>Chryseomonas luteola</em> and <em>Flavimonas oryzihabitans</em>. <span class="ref-journal">Rev. Infect. Dis.</span> <span class="ref-vol">13</span><strong>:</strong>257-260. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/2041957" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r21"><span class="ref-label">21.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055383"><strong>Henriques, I., A. Moura, A. Alves, M. J. Saavedra, and A. Correia.</strong> 2004. Molecular characterization of a carbapenem-hydrolyzing class A β-lactamase, SFC-1, from <em>Serratia fonticola</em> UTAD54. <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">48</span><strong>:</strong>2321-2324. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC415594/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/15155245" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r22"><span class="ref-label">22.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055426"><strong>Huelsenbeck, J. P., and F. Ronquist.</strong> 2001. MRBAYES: Bayesian inference of phylogenetic trees. <span class="ref-journal">Bioinformatics</span> <span class="ref-vol">17</span><strong>:</strong>754-755. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/11524383" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r23"><span class="ref-label">23.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055465"><strong>Knox, J. R.</strong> 1995. Extended-spectrum and inhibitor-resistant TEM-type β-lactamases: mutations, specificity, and three-dimensional structure. <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">39</span><strong>:</strong>2593-2601. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC162995/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/8592985" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r24"><span class="ref-label">24.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055504"><strong>Kostman, J. R., F. Solomon, and T. Fekete.</strong> 1991. Infections with <em>Chryseomonas luteola</em> (CDC group Ve-1) and <em>Flavimonas oryzihabitans</em> (CDC group Ve-2) in neurosurgical patients. <span class="ref-journal">Rev. Infect. Dis.</span> <span class="ref-vol">13</span><strong>:</strong>233-236. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/2041953" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r25"><span class="ref-label">25.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055550"><strong>Kumar, S., M. Nei, J. Dudley, and K. Tamura.</strong> 2008. MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. <span class="ref-journal">Brief. Bioinform.</span> <span class="ref-vol">9</span><strong>:</strong>299-306. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2562624/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/18417537" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r26"><span class="ref-label">26.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055589"><strong>Labia, R., J. Andrillon, and F. Le Goffic.</strong> 1973. Computerized microacidimetric determination of β-lactamase Michaelis-Menten constants. <span class="ref-journal">FEBS Lett.</span> <span class="ref-vol">33</span><strong>:</strong>42-44. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/4198636" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r27"><span class="ref-label">27.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055628"><strong>Liu, S. L., and K. E. Sanderson.</strong> 1995. I-CeuI reveals conservation of the genome of independent strains of <em>Salmonella typhimurium</em>. <span class="ref-journal">J. Bacteriol.</span> <span class="ref-vol">177</span><strong>:</strong>3355-3357. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC177035/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/7768842" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r28"><span class="ref-label">28.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055671"><strong>Milne, I., D. Lindner, M. Bayer, D. Husmeier, G. McGuire, D. F. Marshall, and F. Wright.</strong> 2009. TOPALi v2: a rich graphical interface for evolutionary analyses of multiple alignments on HPC clusters and multi-core desktops. <span class="ref-journal">Bioinformatics</span> <span class="ref-vol">25</span><strong>:</strong>126-127. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2638937/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/18984599" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r29"><span class="ref-label">29.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055710"><strong>Milne, I., F. Wright, G. Rowe, D. F. Marshall, D. Husmeier, and G. McGuire.</strong> 2004. TOPALi: software for automatic identification of recombinant sequences within DNA multiple alignments. <span class="ref-journal">Bioinformatics</span> <span class="ref-vol">20</span><strong>:</strong>1806-1807. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/14988107" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r30"><span class="ref-label">30.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055750"><strong>Nordmann, P., G. Cuzon, and T. Naas.</strong> 2009. The real threat of <em>Klebsiella pneumoniae</em> carbapenemase-producing bacteria. <span class="ref-journal">Lancet Infect. Dis.</span> <span class="ref-vol">9</span><strong>:</strong>228-236. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/19324295" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r31"><span class="ref-label">31.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055793"><strong>Pérez-Llarena, F. J., M. Cartelle, S. Mallo, A. Beceiro, A. Pérez, R. Villanueva, A. Romero, R. Bonnet, and G. Bou.</strong> 2008. Structure-function studies of arginine at position 276 in CTX-M β-lactamases. <span class="ref-journal">J. Antimicrob. Chemother.</span> <span class="ref-vol">61</span><strong>:</strong>792-797. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/18281307" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r32"><span class="ref-label">32.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055833"><strong>Petit, A., L. Maveyraud, F. Lenfant, J. P. Samama, R. Labia, and J. M. Masson.</strong> 1995. Multiple substitutions at position 104 of β-lactamase TEM-1: assessing the role of this residue in substrate specificity. <span class="ref-journal">Biochem. J.</span> <span class="ref-vol">305</span><strong>:</strong>33-40. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1136426/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/7826350" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r33"><span class="ref-label">33.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055873"><strong>Petrella, S., D. Clermont, I. Casin, V. Jarlier, and W. Sougakoff.</strong> 2001. Novel class A β-lactamase from <em>Citrobacter sedlakii</em>: genetic diversity of β-lactamases within the <em>Citrobacter</em> genus. <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">45</span><strong>:</strong>2287-2298. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC90644/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/11451687" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r34"><span class="ref-label">34.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055920"><strong>Poirel, L., J.-M. Rodriguez-Martinez, P. Plésiat, and P. Nordmann.</strong> 2009. Naturally occurring class A β-lactamases from the <em>Burkholderia cepacia</em> complex. <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">53</span><strong>:</strong>876-882. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2650541/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/19075063" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r35"><span class="ref-label">35.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3055963"><strong>Poirel, L., T. Naas, I. Le Thomas, A. Karim, E. Bingen, and P. Nordmann.</strong> 2001. CTX-M-type extended-spectrum β-lactamase that hydrolyzes ceftazidime through a single amino acid substitution in the omega loop. <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">45</span><strong>:</strong>3355-3361. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC90837/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/11709308" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r36"><span class="ref-label">36.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3056003"><strong>Rastogi, S., and S. J. Sperber.</strong> 1998. Facial cellulitis and <em>Pseudomonas luteola</em> bacteremia in an otherwise healthy patient. <span class="ref-journal">Diagn. Microbiol. Infect. Dis.</span> <span class="ref-vol">32</span><strong>:</strong>303-305. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/9934547" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r37"><span class="ref-label">37.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3056045"><strong>Robin, F., J. Delmas, C. Chanal, D. Sirot, J. Sirot, and R. Bonnet.</strong> 2005. TEM-109 (CMT-5), a natural complex mutant of TEM-1 β-lactamase combining the amino acid substitution of TEM-6 and TEM-33 (IRT-5). <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">49</span><strong>:</strong>4443-4447. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1280126/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/16251281" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r38"><span class="ref-label">38.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3056085"><strong>Sirot, D., C. Chanal, C. Henquell, R. Labia, J. Sirot, and R. Cluzel.</strong> 1994. Clinical isolates of <em>Escherichia coli</em> producing multiple TEM-mutants resistant to β-lactamase inhibitors. <span class="ref-journal">J. Antimicrob. Chemother.</span> <span class="ref-vol">33</span><strong>:</strong>1117-1126. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/7928805" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r39"><span class="ref-label">39.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3056128"><strong>Tamura, K., J. Dudley, M. Nei, and S. Kumar.</strong> 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. <span class="ref-journal">Mol. Biol. Evol.</span> <span class="ref-vol">24</span><strong>:</strong>1596-1599. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/17488738" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r40"><span class="ref-label">40.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3056167"><strong>Thompson, J. D., D. G. Higgins, and T. J. Gibson.</strong> 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. <span class="ref-journal">Nucleic Acids Res.</span> <span class="ref-vol">22</span><strong>:</strong>4673-4680. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC308517/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/7984417" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r41"><span class="ref-label">41.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3056206"><strong>Trépanier, S., A. Prince, and A. Huletsky.</strong> 1997. Characterization of the <em>penA</em> and <em>penR</em> genes of <em>Burkholderia cepacia</em> 249, which encode the chromosomal class A penicillinase and its LysR-type transcriptional regulator. <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">41</span><strong>:</strong>2399-2405. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC164135/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/9371340" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r42"><span class="ref-label">42.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3056258"><strong>Tsakris, A., H. Hassapopoulou, L. Skoura, S. Pournaras, and J. Douboyas.</strong> 2002. Leg ulcer due to <em>Pseudomonas luteola</em> in a patient with sickle cell disease. <span class="ref-journal">Diagn. Microbiol. Infect. Dis.</span> <span class="ref-vol">42</span><strong>:</strong>141-143. <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/11858911" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r43"><span class="ref-label">43.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3056300"><strong>Weill, F. X., R. Lailler, K. Praud, A. Kérouanton, L. Fabre, A. Brisabois, P. A. D. Grimont, and A. Cloeckaert.</strong> 2004. Emergence of extended-spectrum-β-lactamase (CTX-M-9)-producing multiresistant strains of Salmonella enterica serotype Virchow in poultry and human in France. <span class="ref-journal">J. Clin. Microbiol.</span> <span class="ref-vol">42</span><strong>:</strong>5767-5773. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC535271/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/15583311" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r44"><span class="ref-label">44.</span> <span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3056341"><strong>Wolter, D. J., P. M. Kurpiel, N. Woodford, M.-F. I. Palepou, R. V. Goering, and N. D. Hanson.</strong> 2009. Phenotypic and enzymatic comparative analysis between the novel KPC variant, KPC-5, and its evolutionary variants, KPC-2 and KPC-4. <span class="ref-journal">Antimicrob. Agents Chemother.</span> <span class="ref-vol">53</span><strong>:</strong>557-562. <span style="white-space: nowrap;">[<a class="int-reflink" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2630594/">PMC free article</a>]</span> <span style="white-space: nowrap;">[<a class="ref-extlink" href="https://www.ncbi.nlm.nih.gov/pubmed/19015357" target="pmc_ext">PubMed</a>]</span></span></span></div><div class="ref-cit-blk" id="r44"><span class="ref-cit"><span class="mixed-citation" id="__mixed-citationid3056341"><span style="white-space: nowrap;"> </span></span></span></div></div></div></div></div>Reza Bocah Tengikhttp://www.blogger.com/profile/05494071928891050842noreply@blogger.com0tag:blogger.com,1999:blog-7040042534697118033.post-42587876928731331492011-02-21T23:52:00.001-08:002011-02-21T23:52:54.160-08:00<h3 class="post-title entry-title"> <a href="http://ranggaplaystation.blogspot.com/2009/03/cheat-mortal-kombat-shaolin-monk.html">Cheat Mortal Kombat Shaolin Monk</a> </h3><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiwgoGJZM6GfxUB-CoKPj-4z7me8qeilbnNE4xA_tgue-RHEGE6lKysBdtwiitdcG2o85INx5duajO-UqSBeiuGM_XO9U4aXzHbak-LKJcy_zWtMvCuJRrmPaJWz1opSQx2aqy_j8JmAybk/s1600-h/mortal.jpg"><img alt="" border="0" id="BLOGGER_PHOTO_ID_5317702986269973170" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiwgoGJZM6GfxUB-CoKPj-4z7me8qeilbnNE4xA_tgue-RHEGE6lKysBdtwiitdcG2o85INx5duajO-UqSBeiuGM_XO9U4aXzHbak-LKJcy_zWtMvCuJRrmPaJWz1opSQx2aqy_j8JmAybk/s320/mortal.jpg" style="display: block; height: 240px; margin: 0px auto 10px; text-align: center; width: 320px;" /></a><br />
<br />
<br />
<div>Unlock Scorpion for Story Mode ...<br />
Hold L2 and input SQUARE, UP, L1, R1, LEFT, RIGHT, SQUARE.<br />
<br />
Unlock Sub-Zero for Story Mode ...<br />
Hold L2 and input SQUARE, DOWN, UP, L1, L1, UP, SQUARE. Mortal Kombat: Shaolin Monks<br />
<br />
Kung Lao Fatalities<br />
Fatality 1: LEFT, UP, RIGHT, RIGHT, SQUARE<br />
Fatality 2: RIGHT, RIGHT, RIGHT, RIGHT, SQUARE<br />
Fatality 3: UP, UP, UP, RIGHT, SQUARE<br />
Fatality 4: UP, UP, UP, DOWN, SQUARE<br />
Fatality 5: LEFT, RIGHT, LEFT, DOWN, SQUARE<br />
Fatality 6: LEFT, RIGHT, LEFT, LEFT, SQUARE<br />
Fatality 7: UP, UP, LEFT, UP, SQUARE<br />
Fatality 8: UP, DOWN, UP, RIGHT, SQUARE<br />
Fatality 9: RIGHT, RIGHT, UP, UP, SQUARE<br />
Mutality 1: UP, RIGHT, DOWN, LEFT, LAUNCH<br />
Mutality 2: LEFT, RIGHT, RIGHT, LEFT, LAUNCH<br />
Brutality 1: LEFT, LEFT, UP, UP, POWER<br />
<br />
Liu-Kang Fatalities<br />
Fatality 1: DOWN, LEFT, RIGHT, UP, SQUARE<br />
Fatality 2: DOWN, LEFT, UP, RIGHT, SQUARE<br />
Fatality 3: LEFT, UP, UP, RIGHT, SQUARE<br />
Fatality 4: LEFT, RIGHT, DOWN, DOWN, SQUARE<br />
Fatality 5: UP, RIGHT, DOWN, LEFT, SQUARE<br />
Fatality 6: DOWN, RIGHT, LEFT, LEFT, SQUARE<br />
Fatality 7: LEFT, LEFT, LEFT, UP, SQUARE<br />
Fatality 8: RIGHT, UP, RIGHT, UP, SQUARE<br />
Fatality 9: RIGHT, LEFT, DOWN, DOWN, SQUARE<br />
Mutality 1: UP, DOWN, UP, DOWN, LAUNCH<br />
Mutality 2: LEFT, RIGHT, UP, UP, LAUNCH<br />
Brutality 1: RIGHT, UP, DOWN, DOWN, POWER<br />
<br />
Scorpion Fatalities<br />
Fatality 1: UP, UP, DOWN, DOWN, SQUARE<br />
Fatality 2: RIGHT, DOWN, RIGHT, RIGHT, SQUARE<br />
Multality 1: DOWN, DOWN, DOWN, UP, LAUNCH<br />
Brutality 1: RIGHT, LEFT, RIGHT, RIGHT, POWER<br />
<br />
Sub-Zero Fatalities<br />
Fatality 1: RIGHT, DOWN, RIGHT, RIGHT, SQUARE<br />
Fatality 2: LEFT, LEFT, DOWN, RIGHT, SQUARE<br />
Fatality 3: RIGHT, RIGHT, DOWN, RIGHT, SQUARE<br />
Multality 1: UP, UP, DOWN, UP, LAUNCH<br />
Brutality 1: UP, DOWN, LEFT, UP, POWER</div>Reza Bocah Tengikhttp://www.blogger.com/profile/05494071928891050842noreply@blogger.com0tag:blogger.com,1999:blog-7040042534697118033.post-72109907842771862612011-02-21T23:46:00.001-08:002011-02-21T23:46:43.520-08:00<div style="height: 3600px; overflow: auto; width: 100%;"><span style="font-family: Arial;">PES 2011 PS2 mendukung beberapa bahasa seperti seri PES Konami sebelumnya, diantaranya adalah bahasa Inggris, <span class="hps">Rusia</span>, Jerman, Swedia, dan Turki.<br />
</span> <span style="font-family: Arial;">Dan setiap bahasa memiliki file-file AFS tersendiri yang akan digunakan salasatunya oleh engine PES 2011 untuk menampilkan bahasa saat game tersebut dimainkan, dan file-file AFS bahasa tersebut diantaranya:</span><br />
<span style="font-family: Arial;"><br />
</span><br />
<blockquote><ul><li><span style="font-family: Arial;"><b>English</b> pada file <b>E_TEXT.AFS</b></span></li>
<li><span style="font-family: Arial;"><b>Russian</b> pada file <b>R_TEXT.AFS</b></span></li>
<li><span style="font-family: Arial;"><b>Deutsch</b> (Jerman) pada file <b>N_TEXT.AFS</b></span></li>
<li><span style="font-family: Arial;"><b>Swedish</b> pada file <b>T_TEXT.AFS</b></span></li>
<li><span style="font-family: Arial;"><b>Turkish </b>pada file <b>V_TEXT.AFS</b></span></li>
</ul></blockquote><span style="font-family: Arial;"><br />
</span><br />
<span style="font-family: Arial;">Dan tentunya saat kita memainkan game PES 2011, maka bahasa yang kemungkinan besar kita pilih atau gunakan adalah bahasa yang paling dimengerti dari beberapa bahasa tersebut, sementara bahasa yang lainnya kemungkinan besar tidak pernah kita gunakan dalam memainkan PES 2011.</span><br />
<span style="font-family: Arial;"><br />
</span><br />
<span style="font-family: Arial;">Karena disini kita hanya menggunakan bahasa yang paling dimengerti saja, maka file-file bahasa yang lain (yang tidak digunakan) kita hapus saja, agar data game PES 2011 bisa lebih kecil dari sebelumnya untuk kita instal nanti-nya di harddisk PS2.</span><br />
<span style="font-family: Arial;"><br />
</span><br />
<span style="font-family: Arial;">Dan sebagian besar gamer yang ada di Indonesia tentunya lebih mengerti bahasa Inggris ketimbang bahasa-bahasa lainnya yang didukung oleh PES 2011, maka alangka baiknya jika game PES 2011 yang kita punya untuk file-file bahasa yang tidak terpakai di hapus saja, seperti pada contoh gambar dibawah ini setelah PES 2011 hanya menggunakan file bahasa Inggris saja.</span><br />
<span style="font-family: Arial;"><br />
</span><br />
<div align="center"><img alt="" border="0" id="BLOGGER_PHOTO_ID_5562343072956084418" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg5lKx409ByWx81_x01uOZ8WaQsDrEchakmceX2c02kGWk_p5S5hzNz0_MIqwXDD4vICWaBvl0XwcPE5u_XNyG19g2jLgOOs4NDjReUufymFBXuR3ACTOJWNlUBJIld9s2xlhJ9brJvVpk/s320/1a.jpg" style="cursor: pointer; height: 249px; width: 320px;" /></div><div align="left"><span style="font-family: Arial;"><br />
</span></div><div align="left"><span style="font-family: Arial;">Setelah itu kita sesuaikan pada file <b> ROMLIST.DIR</b> agar hanya menggunakan file bahasa Inggris saja, dalam hal ini adalah <b>E_TEXT.AFS</b>.</span></div><div align="left"><span style="font-family: Arial;"><br />
</span></div><div align="left"><span style="font-family: Arial;">Buka <b>ROMLIST.DIR</b> di notepad, dan akan seperti pada contoh gambar dibawah ini.</span></div><div align="left"><span style="font-family: Arial;"><br />
</span></div><div align="center"><img alt="" border="0" id="BLOGGER_PHOTO_ID_5562344214541115090" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj3dyhjcRZkdcTf1clAaY_V7i96wUNB7T4UXTlPSuvVoBGWzXUUqayBpZObsM1oezqdjXHc0QsdAWEIqT5kyrbgLYLrxLQM0ZlmLXyUROQFM1RAN7j02S22Iw-cFVR2LBEYTUe6A3dUDmo/s320/2a.jpg" style="cursor: pointer; height: 197px; width: 320px;" /></div><div align="left"><span style="font-family: Arial;"><br />
</span></div><div align="left"><span style="font-family: Arial;">Seperti pada contoh gambar diatas yang diberi tanda merah adalah nama file bahasa PES 2011.</span></div><div align="left"><span style="font-family: Arial;"><br />
</span></div><div align="left"><span style="font-family: Arial;">Kemudian untuk semua nama file bahasanya kita rubah menjadi nama bahasa yang akan kita gunakan, yaitu <b>E_TEXT.AFS</b>.</span></div><div align="left"><span style="font-family: Arial;"><br />
</span></div><div align="left"><span style="font-family: Arial;">Dan setelah semua nama file bahasa disesuaikan, akan seperti pada contoh gambar dibawah ini.</span></div><div align="left"><span style="font-family: Arial;"><br />
</span></div><div align="center"><img alt="" border="0" id="BLOGGER_PHOTO_ID_5562344663143070562" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEicgJJ1mTleW1Dngbuokbn5Yl5yCAsnhzA7WFJwPobdmoPxFTYetYQDE1rA-JnyrEAC7HLpEeaH_weGW3EcDdF-KjP-X2ZjfKgyZSQhK_My_V03VaZJ69PsMWGiLh215nu069J2NF1pUjQ/s320/3a.jpg" style="cursor: pointer; height: 186px; width: 320px;" /></div><div align="left"><span style="font-family: Arial;"><br />
</span></div><div align="left"><span style="font-family: Arial;">Selanjutnya kita sesuaikan lagi untuk file elf-nya agar hanya menggunakan satu bahasa saja sekalipun user memilih untuk menggunakan bahasa yang lain pada awal menu PES 2011.</span></div><div align="left"><span style="font-family: Arial;"><br />
</span></div><div align="left"><span style="font-family: Arial;">Dan contoh ini pada file SLES_556.36 kita edit agar hanya menggunakan file E_TEXT.AFS sebagai bahasa yang digunakan oleh PES 2011. Dan untuk memudahkan teman-teman, file SLES_556.36 sudah saya editkan agar menggunakan satu bahasa saja yaitu bahasa Inggris. <a href="http://www.ziddu.com/download/13406776/PES2011_SLES_556.36.rar.html">Download</a> dan gantikan file SLES_556.36 yang sementara teman-teman edit.</span></div><div align="left"><span style="font-family: Arial;"><br />
</span></div><div align="left"><span style="font-family: Arial;">Setelah itu bikin kembali menjadi file ISO untuk selanjutnya kita instal ke harddisk PS2-nya, baik yang menggunakan harddisk external, maupun harddisk internal.</span></div><div align="left"><span style="font-family: Arial;">(<span style="font-style: italic;">cara membuat iso game dari file-file game sudah dijelaskan pada postingan sebelumnya</span>).</span></div><div align="left"><span style="font-family: Arial;"><br />
</span></div><div align="left"><span style="font-family: Arial;">Sebenarnya untuk memperkecil lagi data game PES 2011 masih bisa kita lakukan dengan cara memperkecil file video-nya (*.pss) dan mengurangi bitrate sound yang digunakan oleh PES 2011 dengan cara yang sudah dijelaskan pada beberapa postingan yang lalu yang berkaitan dengan ripping game.</span></div><div align="left"><span style="font-family: Arial;"><br />
</span></div><div align="left"><span style="font-family: Arial;">Namun penurunan kualitas video dan sound akan mempengaruhi kualitas tampilan video dan suara dari game PES 2011-nya sendiri.</span></div><div align="left"><span style="font-family: Arial;"><br />
</span></div><div align="left"><span style="font-family: Arial;">Selesai.</span></div></div><div style="background-color: transparent; border: medium none; color: black; overflow: hidden; text-align: left; text-decoration: none;"><br />
Read more: <a href="http://satriogame.blogspot.com/#ixzz1DvB2Hl3q" style="color: #003399;">Tip & Tricks PlayStation 2</a> <a href="http://satriogame.blogspot.com/#ixzz1DvB2Hl3q" style="color: #003399;">http://satriogame.blogspot.com/#ixzz1DvB2Hl3q</a></div>Reza Bocah Tengikhttp://www.blogger.com/profile/05494071928891050842noreply@blogger.com0