Evolutionary trajectories of beta-lactamase NDM and DLST cluster in <i>Pseudomonas aeruginosa</i>: finding the putative ancestor

Pseudomonas aeruginosa has different antibiotic resistance pathways, such as broad-spectrum lactamases and metallo-β-lactamases (MBL), penicillin-binding protein (PBP) alteration, and active efflux pumps. Polymerase chain reaction (PCR) and sequencing methods were applied for double-locus sequence typing (DLST) and New Delhi metallo-β-lactamase (NDM) typing. We deduced the evolutionary pathways for DLST and NDM genes of P. aeruginosa using phylogenetic network. Among the analyzed isolates, 62.50% of the P. aeruginosa isolates were phenotypically carbapenem resistance (CARBR) isolates. Characterization of isolates revealed that the prevalence of blaNDM, blaVIM, blaIMP, undetermined carbapenemase, and MexAB-OprM were 27.5%, 2%, 2.5%, 12.5%, and 15%, respectively. The three largest clusters found were DLST t20–105, DLST t32–39, and DLST t32–52. The network phylogenic tree revealed that DLST t26–46 was a hypothetical ancestor for other DLSTs, and NDM-1 was as a hypothetical ancestor for NDMs. The combination of the NDM and DLST phylogenic trees revealed that DLST t32–39 and DLST tN2-N3 with NDM-4 potentially derived from DLST t26–46 along with NDM-1. Similarly, DLST t5–91 with NDM-5 diversified from DLST tN2-N3 with NDM-4. This is the first study in which DLST and NDM evolutionary routes were performed to investigate the origin of P. aeruginosa isolates. Our study showed that the utilization of medical equipment common to two centers, staff members common to two centers, limitations in treatment options, and prescription of unnecessary high levels of meropenem are the main agents that generate new types of resistant bacteria and spread resistance among hospitals.</p

Additional file 4: of Carbonic anhydrase related protein expression in astrocytomas and oligodendroglial tumors

Table S4. Full results of comparative genomics prediction of TFBSs in CA11 promoter. (XLSX 105 kb

Additional file 3: of Carbonic anhydrase related protein expression in astrocytomas and oligodendroglial tumors

Table S3. Full results of comparative genomics prediction of TFBSs in CA8 promoter. (XLSX 82 kb

Additional file 1: of Carbonic anhydrase related protein expression in astrocytomas and oligodendroglial tumors

Table S1. Expression of CA8 CAGE peak, as derived from the FANTOM project. (XLSX 38 kb

Inactivation of <i>ca10a</i> and <i>ca10b</i> Genes Leads to Abnormal Embryonic Development and Alters Movement Pattern in Zebrafish

<div><p>Carbonic anhydrase related proteins (CARPs) X and XI are highly conserved across species and are predominantly expressed in neural tissues. The biological role of these proteins is still an enigma. Ray-finned fish have lost the <i>CA11</i> gene, but instead possess two co-orthologs of <i>CA10</i>. We analyzed the expression pattern of zebrafish <i>ca10a</i> and <i>ca10b</i> genes during embryonic development and in different adult tissues, and studied 61 CARP X/XI-like sequences to evaluate their phylogenetic relationship. Sequence analysis of zebrafish <i>ca10a</i> and <i>ca10b</i> reveals strongly predicted signal peptides, N-glycosylation sites, and a potential disulfide, all of which are conserved, suggesting that all of CARP X and XI are secretory proteins and potentially dimeric. RT-qPCR showed that zebrafish <i>ca10a</i> and <i>ca10b </i>genes are expressed in the brain and several other tissues throughout the development of zebrafish. Antisense morpholino mediated knockdown of <i>ca10a</i> and <i>ca10b</i> showed developmental delay with a high rate of mortality in larvae. Zebrafish morphants showed curved body, pericardial edema, and abnormalities in the head and eye, and there was increased apoptotic cell death in the brain region. Swim pattern showed abnormal movement in morphant zebrafish larvae compared to the wild type larvae. The developmental phenotypes of the <i>ca10a</i> and <i>ca10b</i> morphants were confirmed by inactivating these genes with the CRISPR/Cas9 system. In conclusion, we introduce a novel zebrafish model to investigate the mechanisms of CARP Xa and CARP Xb functions. Our data indicate that CARP Xa and CARP Xb have important roles in zebrafish development and suppression of <i>ca10a</i> and <i>ca10b</i> expression in zebrafish larvae leads to a movement disorder.</p></div

Phenotypes of zebrafish larvae knocked down with different concentrations of <i>ca10a</i> and <i>ca10b</i> antisense morpholinos.

<p>Phenotypes of zebrafish larvae knocked down with different concentrations of <i>ca10a</i> and <i>ca10b</i> antisense morpholinos.</p

Displacement patterns of the morphant and control fish.

<p>Representative displacement trajectories of the movement pattern are shown for <b>A)</b> larvae injected with <i>ca10a</i>-MO2, <b>B)</b> larvae injected with <i>ca10b-</i>MO2 <b>C)</b> larvae injected with RC-MOs and, <b>D)</b> wildtype <b>(</b>uninjected) larvae. Groups of 13 to 22 fish were video recorded in 90mm petri dishes over a 2 minute time period. ImageJ and MtrackJ plugin were used to track the paths of all fish [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134263#pone.0134263.ref039" target="_blank">39</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134263#pone.0134263.ref040" target="_blank">40</a>].</p

Silencing of <i>ca10a</i> and <i>ca10b</i> in zebrafish larvae.

<p><b>A)</b> Schematic presentation of matured <i>ca10a</i> mRNA showing the site of translational blocking with MO1 at the translation start site (arrow). <b>B)</b> Schematic structure of unprocessed mRNA for <i>ca10a</i> with a target region (horizontal bar) for a splice site blocking morpholino (MO2) which knocks down the exon eight. <b>C)</b> Schematic depiction of unprocessed mRNA for <i>ca10b</i> and target sites for splice site interfering morpholinos, MO1 and MO2 (black horizontal bars) and gRNA target regions (red horizontal bars). <b>D)</b> Gel electrophoresis showing RT-PCR analysis of <i>ca10a</i> morphant mRNA injected with MO2. <b>E)</b> RT-PCR gel image of <i>ca10b</i> morphant zebrafish mRNA injected with MO1 and MO2 targeting different exons. The images show the reduction in the length of the mRNAs (Lane 2 in <b>D</b> and Lane 3 and 4 in <b>E)</b> compared with wild type mRNAs of <i>ca10a</i> and <i>ca10b</i> in wild type fish (Lane 3 in <b>D</b> and lane 2 <b>E</b>). <b>F)</b> The efficiency of the CRISPR/Cas9 mediated mutagenesis in zebrafish embryos was evaluated with a T7 endonuclease assay. For both <i>ca10a</i> and <i>ca10b</i>, uninjected and gRNA control fish are shown and as well as two individual embryos with a mutated target site and a pool of 5–10 mutated embryos. Representative cleaved PCR products of the expected sizes are shown as arrow heads. Cleavage percentage was calculated from the band intensities of each lane.</p

Preliminary model of human CARP X.

<p>The model showing a disulfide bridge between conserved cysteines C60 and C223 (marked in red).</p

Knockdown of <i>ca10b</i> and <i>ca10b</i> genes results in abnormal movement pattern in morphant zebrafish larvae.

<p>The swim patterns of 5 dpf larvae injected with 100 μM antisense <i>ca10a-</i>MO2 or <i>ca10b-</i>MO2 were compared to the larvae injected with RC-MO and wildtype (uninjected) controls as described in the methods. (In addition to the swim patterns and body movement videos of 10 larvae from each group created with a Zeiss microscope for close observation as included in the supporting information). Distances traveled by 457 fish in the four groups are presented as boxplots as created with matplotlib [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134263#pone.0134263.ref043" target="_blank">43</a>]. The results of two sample Kolmogorov-Smirnov statistical analyses are included between bracket linked groups.</p