ESBL-producing Escherichia coli and Klebsiella pneumoniae: The most prevalent clinical isolates obtained between 2005 and 2012 in Mexico.

OBJECTIVES: To identify the prevalence of ESBL genes in the principal group of Enterobacteriaceae causing nosocomial infections and to identify the phylogenetic group in Escherichia coli isolates. METHODS: There were collected 1084 ESBL-producing Enterobacteriaceae isolates during 2005-2012 from adult patients from 14 hospitals and corresponding to eight states and five regions (SE, S, N, W and NW) in Mexico. The CTX-M-(CTX-M-1 group), SHV-, TLA- and GES-type ESBLs genes were screened. The respective alleles were determined in the most of ESBLs genes. In E. coli isolates selected were used to identify the phylogenetic group. RESULTS: The ESBL-producing Escherichia coli and Klebsiella pneumoniae corresponded the most prevalent clinical isolates. CTX-M-type ESBLs genes were the most common, followed by SHV-type, GES-type and the ESBLs TLA-1 gene. The allelic frequency showed to CTX-M-15 ESBL the most prevalent, followed by the SHV-12, SHV-5 and GES-1, GES-19 in the GES family. Among ESBL-producing E. coli isolates the phylogenetic groups A and D were the most common ones. CONCLUSIONS: The present study showed an epidemiological change in terms of bacterial species, placing E. coli as the most frequently isolated bacteria among ESBL-producing Enterobacteriaceae in Mexico, followed by K. pneumoniae. This frequency is accompanied by a high frequency of ESBL CTX-M-15.

Molecular and microbiological report of a hospital outbreak of NDM-1-carrying Enterobacteriaceae in Mexico.

OBJECTIVES: To characterize the microbiological, molecular and epidemiological data of an outbreak of carbapenem-resistant Enterobacteriaceae (CRE) in a tertiary-care hospital in Mexico. METHODS: From September 2014 to July 2015, all CRE clinical isolates recovered during an outbreak in the Hospital Civil "Fray Antonio Alcalde" in Jalisco, Mexico were screened for antimicrobial susceptibility, carbapenemase production, carbapenemase-encoding genes, and plasmid profiles. Horizontal transfer of imipenem resistance; and clonal diversity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST); as well as biofilm production and the presence of 14 virulence genes were analyzed in selected isolates. RESULTS: Fifty-two carbapenem-resistant isolates corresponding to 5 species were detected, i.e., Klebsiella pneumoniae (n = 46), Enterobacter cloacae (n = 3), Escherichia coli (n = 1), Providencia rettgeri (n = 1) and Citrobacter freundii (n = 1) with carbapenemase encoding genes blaNDM-1 (n = 48), blaVIM (n = 3), blaIMP (n = 1) and blaKPC (n = 1) detected in these isolates. The blaNDM-1 gene was detected in plasmids from 130- to 170-kb in K. pneumoniae (n = 46); E. cloacae (n = 3), E. coli (n = 1) and P. rettgeri (n = 1). The transfer of plasmids harboring the blaNDM-1 gene was obtained in eight transconjugants. One plasmid restriction pattern was detected, with the blaNDM-1 identified in different restriction fragments. Predominant clone A of K. pneumoniae isolates archived 28/46 (60%) isolates and belongs to ST392. Besides, ST307, ST309, ST846, ST2399, and ST2400 were detected for K. pneumoniae; as well as E. cloacae ST182 and E. coli ST10. The fimA and uge genes were more likely to be identified in K. pneumoniae carbapenem-susceptible isolates (p = <0.001) and biofilm production was more liable to be observed in carbapenem-resistant isolates (p = <0.05). CONCLUSIONS: Four Enterobacteriaceae species harboring the blaNDM-1 gene were detected in a nosocomial outbreak in Mexico; horizontal transfer and strain transmission were demonstrated for the blaNDM-1 gene. Given the variation in the size of the plasmid harboring blaNDM-1, complex rearrangements must also be occurring.

Identification of beta-Lactamases and beta-Lactam-Related Proteins in Human Pathogenic Bacteria using a Computational Search Approach.

A systematic analysis of beta-lactamases based on comparative proteomics has not been performed thus far. In this report, we searched for the presence of beta-lactam-related proteins in 591 bacterial proteomes belonging to 52 species that are pathogenic to humans. The amino acid sequences for 19 different types of beta-lactamases (ACT, CARB, CifA, CMY, CTX, FOX, GES, GOB, IMP, IND, KPC, LEN, OKP, OXA, OXY, SHV, TEM, NDM, and VIM) were obtained from the ARG-ANNOT database and were used to construct 19 HMM profiles, which were used to identify potential beta-lactamases in the completely sequenced bacterial proteomes. A total of 2877 matches that included the word "beta-lactamase" and/or "penicillin" in the functional annotation and/or in any of its regions were obtained. These enzymes were mainly described as "penicillin-binding proteins," "beta-lactamases," and "metallo-beta-lactamases" and were observed in 47 of the 52 species studied. In addition, proteins classified as "beta-lactamases" were observed in 39 of the species included. A positive correlation between the number of beta-lactam-related proteins per species and the proteome size was observed (R 0.78, P < 0.00001). This correlation partially explains the high presence of beta-lactam-related proteins in large proteomes, such as Nocardia brasiliensis, Bacillus anthracis, and Mycobacterium tuberculosis, along with their absence in small proteomes, such as Chlamydia spp. and Mycoplasma spp. We detected only five types of beta-lactamases (TEM, SHV, CTX, IMP, and OXA) and other related proteins in particular species that corresponded with those reported in the literature. We additionally detected other potential species-specific beta-lactamases that have not yet been reported. In the future, better results will be achieved due to more accurate sequence annotations and a greater number of sequenced genomes.

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).

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.

Antimicrobial Activity and Stability of Short and Long Based Arachnid Synthetic Peptides in the Presence of Commercial Antibiotics.

Four antimicrobial peptides (AMPs) named Pin2[G], Pin2[14], P18K and FA1 were chemically synthesized and purified. The four peptides were evaluated in the presence of eight commercial antibiotics against four microorganisms of medical importance: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The commercial antibiotics used were amoxicillin, azithromycin, ceftriaxone, gentamicin, levofloxacin, sulfamethoxazole, trimethoprim and vancomycin. The best AMP against P. aeruginosa was the peptide FA1, and the best AMP against S. aureus was Pin2[G]. Both FA1 and Pin2[G] were efficient against E. coli, but they were not effective against K. pneumoniae. As K. pneumoniae was resistant to most of the commercial antibiotics, combinations of the AMPs FA1 and Pin2[G] were prepared with these antibiotics. According to the fractional inhibitory concentration (FIC) index, the best antimicrobial combinations were obtained with concomitant applications of mixtures of FA1 with levofloxacin and sulfamethoxazole. However, combinations of FA1 or Pin2[G] with other antibiotics showed that total inhibitory effect of the combinations were greater than the sum of the individual effects of either the antimicrobial peptide or the antibiotic. We also evaluated the stability of the AMPs. The AMP Pin2[G] manifested the best performance in saline buffer, in supernatants of bacterial growth and in human blood plasma. Nevertheless, all AMPs were cleaved using endoproteolytic enzymes. These data show advantages and disadvantages of AMPs for potential clinical treatments of bacterial infections, using them in conjunction with commercial antibiotics.

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 Sequences of Klebsiella variicola Plant Isolates.

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.

Draft Genome Sequence of the First Hypermucoviscous Klebsiella variicola Clinical Isolate.

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%.

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

BACKGROUND: Klebsiella 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. RESULT: This 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. CONCLUSIONS: This 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.

Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico.

The aim of this study was to determine the clinical characteristics, molecular epidemiology and biofilm production of Acinetobacter baumannii clinical isolates obtained from a tertiary-care hospital in Mexico. Clinical isolates of A. baumannii (n=152) isolated from 2007 to 2012 were included. Clonal diversity was analysed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antimicrobial susceptibility was determined using the broth microdilution method. IMP, VIM, NDM and OXA-type genes were screened by PCR. Biofilm production was analysed using the crystal violet method. Mortality attributable to A. baumannii infection was 14.5%. Fifty-four clones were detected, of which five predominated. MLST results showed three new sequence types and two reported sequence types. More than 86% of the isolates were resistant to ciprofloxacin, ceftazidime and cefotaxime. Furthermore, 50.7% and 35.5% of the isolates were resistant to imipenem and meropenem, respectively. Of the isolates evaluated, 28.3% and 25.7% were positive for the blaOXA-58 and blaOXA-72 genes, respectively. Biofilm production was associated with resistance to imipenem (P=0.002).

Widespread of ESBL- and carbapenemase GES-type genes on carbapenem-resistant Pseudomonas aeruginosa clinical isolates: a multicenter study in Mexican hospitals.

The present work describes a prevalence of 36.2% of carbapenemases IMP-, VIM-, and GES-type on 124 imipenem-resistant Pseudomonas aeruginosa clinical isolates. The ESBL GES-19 and carbapenemase GES-20 genes were the most prevalent (84.4%) ß-lactamases among imipenem-resistant P. aeruginosa clinical isolates in Mexico. These genes are chromosomal encoded on embedded class 1 integron arrays.

Characteristics of KPC-2-producing Klebsiella pneumoniae (ST258) clinical isolates from outbreaks in 2 Mexican medical centers.

The KPC-producing Klebsiella pneumoniae sequence type 258 (ST258) is an important pathogen widely spread in nosocomial infections. In this study, we identified the KPC-2-producing K. pneumoniae clinical isolates of 2 unrelated outbreaks that corresponded to pandemic strain ST258. The isolates showed high resistance to cephalosporins, carbapenems, quinolones, and colistin. The KPC-2-producing K. pneumoniae isolates were compared to the previously studied KPC-3-producing K. pneumoniae isolates from an outbreak in Mexico; they showed an unrelated pulsed-field gel electrophoresis fingerprinting pattern and a different plasmid profile. The KPC-2 carbapenemase gene was identified in two 230- and 270-kb non-conjugative plasmids; however, 1 isolate transferred the KPC-2 gene onto an 80-kb plasmid. These findings endorse the need of carrying out a continuous molecular epidemiological surveillance of carbapenem-resistant isolates in hospitals in Mexico.

Characterization of plasmid-mediated quinolone resistance (PMQR) genes in extended-spectrum ß-lactamase-producing Enterobacteriaceae pediatric clinical isolates in Mexico.

This work describes the characterization of plasmid-mediated quinolone-resistance (PMQR) genes from a multicenter study of ESBL-producing Enterobacteriaceae pediatric clinical isolates in Mexico. The PMQR gene-positive isolates were characterized with respect to ESBLs, and mutations in the GyrA and ParC proteins were determined. The phylogenetic relationship was established by PFGE and the transfer of PMQR genes was determined by mating assays. The prevalence of the PMQR genes was 32.1%, and the rate of qnr-positive isolates was 15.1%; 93.3% of the latter were qnrB and 6.4% were qnrA1. The distribution of isolates in terms of bacterial species was as follows: 23.5% (4/17) corresponded to E. cloacae, 13.7% (7/51) to K. pneumoniae, and 13.6% (6/44) to E. coli. In addition, the prevalence of aac(6')-Ib-cr and qepA was 15.1% and 1.7%, respectively. The molecular characteristics of qnr- and qepA-positive isolates pointed to extended-spectrum ß-lactamase (ESBL) CTX-M-15 as the most prevalent one (70.5%), and to SHV-12 in the case of aac(6')-Ib-cr-positive isolates. GyrA mutations at codons Ser-83 and Asp-87, and ParC mutations at codons Ser-80 were observed in 41.1% and 35.2% of the qnr-positive isolates, respectively. The analysis of the transconjugants revealed a co-transmission of bla(CTX-M-15) with the qnrB alleles. In general, the prevalence of PMQR genes (qnr and aac(6')-Ib-cr) presented in this work was much lower in the pediatric isolates, in comparison to the adult isolates in Mexico. Also, ESBL CTX-M-15 was the main ESBL identified in the pediatric isolates, whereas in the adult ones, ESBLs corresponded to the CTX-M and the SHV families. In comparison with other studies, among the PMQR-genes identified in this study, the qnrB-alleles and the aac(6')-Ib-cr gene were the most prevalent, whereas the qnrS1, qnrA1 and qnrB-like alleles were the most prevalent in China and Uruguay.

Molecular epidemiology of Escherichia coli O25b-ST131 isolates causing community-acquired UTIs in Mexico.

Escherichia coli is a common uropathogen causing community-acquired urinary tract infections (UTIs). Out of 4735 E. coli community-acquired UTIs, 10.2% were extended spectrum ß-lactamases (ESBL)-producing. The identified ESBL types were CTX-M-15 (96.4%), SHV-2a (3%), and TLA-1 (1%). Of the isolates, 94.6% tested positive for plasmid-mediated quinolone resistance (PMQR) genes (aac(6')-lb-cr [92.1%] and qepA1 [7%] and for qnr-determinants [3.5%]). E. coli O25b-ST131 was identified in 25% of the isolates that harbor a non-conjugative 160-kb plasmid (IncFIA) containing the CTX-M-15, and all of these isolates were found to contain PMQR genes. This work can be useful in modeling the potential impact that may have on community-acquired UTIs in Mexico.