Humberto Barrios

RepA negatively autoregulates the transcription of the repABC operon of the Rhizobium etli symbiotic plasmid basic replicon

The basic replicon of Rhizobium etli CE3, like other members of the repABC plasmid family, is constituted by the repABC operon. RepC is essential for replication, and RepA and RepB play a role in plasmid segregation. It has been shown that deletion derivatives lacking the repAB genes have an increased copy number, indicating that these genes participate in the control of plasmid copy number. RepA is also a trans‐incompatibility factor. To understand the regulation of the repABC operon, in this paper: (i) the transcription start site of the repABC operon was determined; (ii) the promoter region was identified by site‐directed mutagenesis of the putative −35 and −10 hexameric elements; and (iii) RepA was recognized as a negative regulator of the transcription of the repABC operon.

Development of a Multiplex-PCR probe system for the proper identification of Klebsiella variicola

BackgroundKlebsiella variicola was very recently described as a new bacterial species and is very closely related to Klebsiella pneumoniae; in fact, K. variicola isolates were first identified as K. pneumoniae. Therefore, it might be the case that some isolates, which were initially classified as K. pneumoniae, are actually K. variicola. The aim of this study was to devise a multiplex-PCR probe that can differentiate isolates from these sister species.ResultThis work describes the development of a multiplex-PCR method to identify K. variicola. This development was based on sequencing a K. variicola clinical isolate (801) and comparing it to other K. variicola and K. pneumoniae genomes. The phylogenetic analysis showed that K. variicola isolates form a monophyletic group that is well differentiated from K. pneumoniae. Notably, the isolate K. pneumoniae 342 and K. pneumoniae KP5-1 might have been misclassified because in our analysis, both clustered with K. variicola isolates rather than with K. pneumoniae. The multiplex-PCR (M-PCR-1 to 3) probe system could identify K. variicola with high accuracy using the shared unique genes of K. variicola and K. pneumoniae genomes, respectively. M-PCR-1 was used to assay a collection of multidrug-resistant (503) and antimicrobial-sensitive (557) K. pneumoniae clinical isolates. We found K. variicola with a prevalence of 2.1% (23/1,060), of them a 56.5% (13/23) of the isolates were multidrug resistant, and 43.5% (10/23) of the isolates were antimicrobial sensitive. The phylogenetic analysis of rpoB of K. variicola-positive isolates identified by multiplex-PCR support the correct identification and differentiation of K. variicola from K. pneumoniae clinical isolates.ConclusionsThis multiplex-PCR provides the means to reliably identify and genotype K. variicola. This tool could be very helpful for clinical, epidemiological, and population genetics studies of this species. A low but significant prevalence of K. variicola isolates was found, implying that misclassification had occurred previously. We believe that our multiplex-PCR assay could be of paramount importance to understand the population dynamics of K. variicola in both clinical and environmental settings.

Draft Genome Sequence of the First Hypermucoviscous Klebsiella variicola Clinical Isolate

ABSTRACT An antibiotic-susceptible and hypermucoviscous clinical isolate of Klebsiella variicola (K. variicola 8917) was obtained from the sputum of an adult patient. This work reports the complete draft genome sequence of K. variicola 8917 with 103 contigs and an annotation that revealed a 5,686,491-bp circular chromosome containing a total of 5,621 coding DNA sequences, 65 tRNA genes, and an average G+C content of 56.98%.

Draft Genome Sequences of Klebsiella variicola Plant Isolates

ABSTRACT Three endophytic Klebsiella variicola isolates—T29A, 3, and 6A2, obtained from sugar cane stem, maize shoots, and banana leaves, respectively—were used for whole-genome sequencing. Here, we report the draft genome sequences of circular chromosomes and plasmids. The genomes contain plant colonization and cellulases genes. This study will help toward understanding the genomic basis of K. variicola interaction with plant hosts.

A Multicenter Study in Mexico Finds Acinetobacter baumannii Clinical Isolates Belonging to Clonal Complexes 636B (113B) and 92B Harboring OXA-72, OXA-239, and OXA-469

The prevalence of carbapenem-resistant Acinetobacter baumannii (CRAB) is increasing worldwide. There is not a regional study or national surveillance program in Mexico, but some recent reports include information from outbreaks at particular hospitals (1–4). This multicenter study described the sequence types (STs), clonal complexes (CCs), and carbapenem resistance mechanisms of A. baumannii clinical isolates with the carbapenem resistance phenotype from Mexican hospitals. A total of 192 carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates were collected from 16 hospitals between 2006 and 2013. Only one isolate per patient was included in the study. Identification of the isolates was performed by using the API20 NF (bioMérieux). The susceptibility of CRAB isolates to imipenem, meropenem, tigecycline, and colistin was determined by the microdilution method according to the CLSI recommendations (5) and EUCAST (www.eucast.org). All 192 CRAB isolates were resistant to imipenem and meropenem and susceptible to tigecycline and colistin. The genomic DNA macrorestriction profiles were determined by pulsed-field gel electrophoresis (PFGE) (6) and compared using the software package Gel Compare II (Applied Maths BVBA, Austin, TX, USA). In general, one differ-

Identification and Characterization of Imipenem-Resistant Klebsiella pneumoniae and Susceptible Klebsiella variicola Isolates Obtained from the Same Patient

Klebsiella variicola, a bacterium closely genetically related to Klebsiella pneumoniae, is commonly misidentified as K. pneumoniae by biochemical tests. To distinguish between the two bacteria, phylogenetic analysis of the rpoB gene and the identification of unique genes in both bacterial species by multiplex-polymerase chain reaction (PCR) provide the means to reliably identify and genotype K. variicola. In recent years, K. variicola has been described both as the cause of an intrahospital outbreak in a pediatric hospital, which resulted in sepsis in inpatients, and as a frequent cause of bloodstream infections. In the present study, K. pneumoniae and K. variicola were isolated from a unique patient displaying different antimicrobial susceptibility phenotypes and different genotypes of virulence determinants. Eight clinical isolates were obtained at different time intervals; all during a 5-month period. The isolates were identified as K. pneumoniae by an automated identification system. The clinical (biochemical test) and molecular (multiplex-PCR and rpoB gene) characterization identified imipenem resistance in the first six K. pneumoniae ST258 isolates, which encode the SHV-12 cephalosporinase and KPC-3 carbapenemase genes. The two last remaining isolates corresponded to susceptible K. variicola. The bacterial species showed a specific profile of virulence-associated determinants, specifically the fimA, fimH, and ecpRAB fimbrial-encoding genes identified only in K. pneumoniae isolates. However, the entb (enterobactin), mrkD (fimbrial adhesin), uge (epimerase), ureA (urease), and wabG (transferase) genes were shared between both bacterial species. Recent studies attribute a higher mortality rate to K. variicola than to K. pneumonia. This work highlights the identification of K. pneumoniae and the closely related K. variicola isolated from the same patient. The value of distinguishing between these two bacterial species is in their clinical significance, their different phenotypes and genotypes, and the fact that they can be isolated from the same patient.

Draft Genome Sequence of a Hypermucoviscous Extended-Spectrum-β-Lactamase-Producing Klebsiella quasipneumoniae subsp. similipneumoniae Clinical Isolate

ABSTRACT A clinical isolate of extended-spectrum-β-lactamase-producing Klebsiella quasipneumoniae subsp. similipneumoniae 06-219 with hypermucoviscosity phenotypes obtained from a urine culture of an adult patient was used for whole-genome sequencing. Here, we report the draft genome sequences of this strain, consisting of 53 contigs with an ~5.6-Mb genome size and an average G+C content of 57.36%. The annotation revealed 6,622 coding DNA sequences and 77 tRNA genes.

Outbreak Caused by blaOXA-72-Producing Acinetobacter baumannii ST417 Detected in Clinical and Environmental Isolates

We characterized an outbreak of imipenem-resistant Acinetobacter baumannii with clinical and environmental isolates from a tertiary care hospital in San Luis Potosi, Mexico. During a 4-month period, a total of 32 nonrepetitive imipenem-resistant clinical isolates of A. baumannii were collected. All isolates were susceptible to colistin and tigecycline and resistant to cefepime, ceftazidime, ceftriaxone, imipenem, and meropenem. Genotyping by pulsed-field gel electrophoresis showed a major clone (A). Multilocus sequence type (MLST) analysis was performed, revealing sequence type (ST) 417 (ST417) and 208 (ST208). The blaIMP-, blaVIM-, blaGIM-, blaSIM-, blaNDM-type, and blaOXA-type (blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, and blaOXA-58-like) genes were screened and showed that the blaOXA-51-like and blaOXA-24-like genes were present in all isolates. Sequencing and southern hybridization were performed, confirming the presence of the blaOXA-72 gene and its plasmid-borne nature. In addition, the blaOXA-72-XerC/XerD-like association was identified. These findings indicate that a clonal spread of blaOXA-72-producing A. baumannii ST417 had occurred throughout the hospital. The ST417 corresponded with a previous ST described in the United States.

Molecular characterization of ESBL-producing Escherichia coli isolates from hospital- and community-acquired infections in NW Mexico

We investigated the molecular characteristics of ESBL-producing E. coli (ESBL-PEc) isolates from two hospitals and community settings in Ciudad Obregon, Sonora, Mexico. Between 2011 and 2014, thirty-seven ESBL-PEc isolates were collected. The major encoded ESBL was the blaCTX-M-15 gene (97%); followed by 13.5% of the blaSHV-12 gene, and 5.5% encoded the blaTLA-1 gene. The PMQR gene aac(6´)-Ib-cr was detected in 97% of the isolates and the qnrB gene, in one isolate. The ESBL-PEc isolates corresponded to phylogenetic group B2, ST131. Our results highlight the dissemination of ESBL-PEc isolates in northwest Mexico (Ciudad Obregon, Sonora).

Genomic Footprinting Analyses of the Complex Bradyrhizobium japonicum fixRnifA Promoter Region

The B. japonicum Nif A protein is the central regulator for nitrogen fixation gene expression. This protein is directly regulated by oxygen, such that it can only activate nif and fix gene expression during microaerobiosis or in root-nodule bacteroid state. NifA activates transcription by binding to enhancer-like elements, located around 120bp from the transcription start, and melting the closed promoter complex to the active open form. NifA is encoded in the fixRnifA operon. This operon is positively autoregulated in anaerobiosis and, unexpectedly, it is also expressed in aerobic conditions. Deletion and mutagenesis analyses have demonstrated that a region around -68 is required for the aerobic expression. This region is the binding site for an unknown protein. Here, we report that this pattern of expression is due to the presence of two overlapping promoters: fixRp1, which is of the -24/-12 class recognized by the RNA polymerase-σ54 (Eσ54), and fixRp2, which shares homology with the -35 and -10 regions found in other putative B. japonicum housekeeping promoters, recognized by Eσ90. Primer extension analyses showed that fixRpl directs the synthesis of a transcript, denominated P1, that starts 12 nucleotides downstream of the -12 region. In addition to σ54, PI was dependent on NifA and low oxygen tension. Transcripts originating from fixRp2 started at two sites: one coincided with P1, while the most abundant, denominated P2, initiates just two nucleotides further downstream of P1. Expression from fixRp2 was dependent on the integrity of the upstream −68 promoter region but it was independent of σ54 and NifA. This promoter was expressed in aerobic and anaerobic conditions but was not expressed in bacteroids. Mutations in the -12 region for the σ54 promoter did not show any transcript because these mutations also disrupted the overlapping -10 region of the fixRp2 promoter. Conversely, mutations at the -24 region only affected the σ54-dependent P1 transcript, having no effect on the expression of P2. In the absence of σ54 the anaerobic expression from the fixRp2 promoter was enhanced, suggesting that in the wild type strain the two RNA polymerase holoenzymes compete for the binding to the same promoter region.