Jesus Silva-Sanchez

ISEcp1-Mediated Transposition of qnrB-Like Gene in Escherichia coli

ABSTRACT A novel QnrB-like plasmid-mediated resistance determinant, QnrB19, was identified from an Escherichia coli clinical isolate from Colombia. Its gene was associated with an ISEcp1-like insertion element that did not act as a promoter for its expression. Using an in vitro model of transposition, we showed that the ISEcp1-like element was able to mobilize the qnrB19 gene.

Metallo-β-Lactamase Gene blaIMP-15 in a Class 1 Integron, In95, from Pseudomonas aeruginosa Clinical Isolates from a Hospital in Mexico

ABSTRACT During 2003, 40 carbapenem-resistant Pseudomonas aeruginosa clinical isolates collected in a Mexican tertiary-care hospital were screened for metallo-β-lactamase production. Thirteen isolates produced IMP-15, and 12 had a single pulsed-field gel electrophoresis pattern. The blaIMP-15 gene cassette was inserted in a plasmid-borne integron with a unique array of gene cassettes and was named In95.

Molecular epidemiology and risk factors of bloodstream infections caused by extended-spectrum β-lactamase-producing Klebsiella pneumoniae: A case–control study

OBJECTIVES To study the prevalence, risk factors, outcome, and molecular epidemiology in patients with bacteremia caused by extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae (Kp) (cases), in comparison with patients with bacteremia caused by a susceptible Kp (controls). METHODS This was a retrospective case-control study including all episodes of Kp bacteremia for the period 1993 to 2002 at a referral hospital for adults in Mexico. ESBL production was tested for by E-test. All isolates were typed by pulsed field gel electrophoresis (PFGE). A subset of isolates underwent plasmid analysis, conjugal transfer of cefotaxime resistance to Escherichia coli J53-2, isoelectric focusing bioassay, colony-blot hybridization, PCR, and sequencing. RESULTS Of the 121 patients with bacteremia due to Kp included in the study, 17 (14.0%) had an ESBL-Kp isolate (cases). Multivariate analysis identified prior use of cephalosporins (OR 7.6, 95% CI 1.1-53.5; p=0.039) and stay in the intensive care unit (ICU; OR 5.6, 95% CI 1.1-27.9; p=0.033) as significant risk factors. No differences were observed in hospital stay or mortality after the event. Multi-drug resistance was more frequent in ESBL-Kp. There was no clonal predominance. A distinct beta-lactamase profile was identified, which included a combination of TEM-1 (pI 5.4) and SHV-5 (pI 8.2) in 13/17 ESBL-Kp isolates. Cefotaxime resistance was transferred by conjugation in 14/17 isolates with a >120-kb plasmid encoding ESBL. CONCLUSIONS The prevalence of ESBL-Kp was found to be lower than that previously reported in Latin America. ESBL-Kp bacteremia was not associated with a worse clinical outcome. We were able to identify a plasmid-mediated horizontal dissemination over the 10-year period.

Vejovine, a new antibiotic from the scorpion venom of Vaejovis mexicanus

Multidrug resistant bacterial infections are one of the most important health problems in recent years. Resistance to conventional antibiotics limits the therapeutic options causing increase rate in morbid-mortality in hospitals. Therefore, new antibacterial agents with new bacterial targets have been searched and found in many different sources, including scorpion venom and scorpion hemolymph. Here, we report a new anti-microbial peptide named Vejovine. This peptide was isolated from the venom of the Mexican scorpion Vaejovis mexicanus by two steps of reversed phase high performance liquid chromatography (RP-HPLC). It is composed of 47 amino acid residues with no cysteine residues in its sequence, with a molecular weight of 4873 Da. The chemical synthesis of Vejovine was performed by the solid phase method of Merrifield, using fluoren-9-ylmethoxycarbonyl (Fmoc)-amino acids. Both the native and synthetic peptides were shown to have essentially the same activity. Vejovine inhibits growth of clinical isolates of Gram-negative multidrug resistant (Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Acinetobacter baumanii) causing nosocomial infections with a minimum inhibitory concentration (MIC) of 4.4 μM up to 50 μM. This peptide has also hemolytic activity against human erythrocytes with a HC(50) value of 100 μM. A cDNA library of the venomous gland of this scorpion provided material for cloning the gene encoding Vejovine. This peptide is a new type of antibiotic, showing less than 50% similarity to other known scorpion peptides. Vejovine is a candidate to be used as a leading compound for future development of an effective peptide against multidrug resistant bacteria.

Molecular Analysis and Risk Factors for Escherichia coli Producing Extended-Spectrum β-Lactamase Bloodstream Infection in Hematological Malignancies

Introduction Patients with hematologic malignancies have greater risk-factors for primary bloodstream infections (BSI). Methods From 2004–2009, we analyzed bacteremia caused by extended-spectrum beta-lactamase Escherichia coli (ESBL-EC) (n = 100) and we compared with bacteremia caused by cephalosporin-susceptible E. coli (n = 100) in patients with hematologic malignancies. Objective To assess the clinical features, risk factors, and outcome of ESBL-EC BSI in patients with hematologic malignancies, and to study the molecular epidemiology of ESBL-EC isolates. Results The main diagnosis was acute leukemia in 115 patients (57.5%). Death-related E. coli infection was significantly increased with ESBL-EC (34% vs. control group, 19%; p = 0.03). Treatment for BSI was considered appropriate in 64 patients with ESBL-EC (mean survival, 245±345 days), and in 45 control patients this was 443±613 (p = 0.03). In patients not receiving appropriate antimicrobial treatment, survival was significantly decreased in cases compared with controls (26±122 vs. 276±442; p = 0.001). Fifty six of the ESBL-EC isolates were characterized by molecular analysis: 47 (84%) expressed CTX-M-15, two (3.6%) SHV, and seven (12.5%) did not correspond to either of these two ESBL enzymes. No TLA-1 enzyme was detected. Conclusions Patients who had been previously hospitalized and who received cephalosporins during the previous month, have an increased risk of ESBL-EC bacteremia. Mortality was significantly increased in patients with ESBL-EC BSI. A polyclonal trend was detected, which reflects non-cross transmission of multiresistant E.coli isolates.

Characterization of Plasmid-Mediated Quinolone Resistance (PMQR) Genes in Extended-Spectrum β-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 β-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 blaCTX-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.

Extended-spectrum β-lactamase-producing enterobacteriaceae causing nosocomial infections in Mexico. A retrospective and multicenter study.

BACKGROUND AND AIMS Extended-spectrum β-lactamase (ESBLs) production is still the most frequent mechanism of resistance to cephalosporins in gram-negative bacteria. The aim of the study was to identify the types of ESBL-producing Enterobacteriaceae clinical isolates causing nosocomial infections in Mexico. METHODS ESBL production was performed using a disk diffusion method. The MIC for several antibiotics was performed by agar dilution on Mueller-Hinton. PFGE typing was carried out on all enterobacteria assayed. The β-lactamase pattern was obtained by IEF and bioassay. Genes of β-lactamases were amplified by PCR with specific primers and products were sequenced and analyzed using informatics programs. Plasmid isolation and conjugation experiments were carried out using standard methodologies. RESULTS There were 134 isolates of Enterobacteriaceae included from a retrospective and multicenter study that included eight Mexican hospitals from 1999 to 2005. The most prevalent species were K. pneumoniae (56%), Enterobacter cloacae (29%), and Escherichia coli (15%). Molecular analysis identified the underlying endemic and polyclonal spread of enterobacterials in each hospital. The most frequent ESBLs identified were SHV-type (84%), TLA-1 (11%), and CTX-M-15 (5%). Successful matings were detected in 68.4% (71/104) isolates. CONCLUSIONS ESBL-producer K. pneumoniae remains the most frequent bacterial species obtained in nosocomial infections. The SHV-type and TLA-1 ESBLs were disseminated in most hospitals analyzed and CTX-M-15 was emerging in one of the studied hospitals. This work highlights the proper use of antibiotics to avoid the selection of these types of multiresistant bacteria.

SHV-type extended-spectrum beta-lactamase (ESBL) are encoded in related plasmids from enterobacteria clinical isolates from Mexico

OBJECTIVE In this work we report the molecular characterization of beta-lactam antibiotics resistance conferred by genes contained in plasmids from enterobacteria producing extended-spectrum beta-lactamases (ESBL). MATERIAL AND METHODS Fourteen enterobacterial clinical isolates selected from a group of strains obtained from seven different hospitals in Mexico during 1990-1992 and 1996-1998 were analyzed at the Bacterial Resistance Laboratory (National Institute Public Health, Cuernavaca). Molecular characterization included PFGE, IEF of beta-lactamases, bacterial conjugation, PCR amplification and DNA sequencing, plasmid extraction and restriction. RESULTS Isolates were genetically unrelated. ESBL identified were SHV-2 (5/14) and SHV-5 (9/14) type. Cephalosporin-resistance was transferable in 9 of 14 (64%) clinical isolates with only one conjugative plasmid, DNA finger printing showed a similar band pattern in plasmids. CONCLUSIONS The dissemination of cephalosporin resistance was due to related plasmids carrying the ESBL genes.

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.

Microbiological indicators of water quality in the Xochimilco canals, Mexico City.

OBJECTIVE To quantify microbiology indicators of fecal contamination in the effluents of two waste water treatment plants and in samples collected in several canals in Xochimilco. MATERIAL AND METHODS A cross sectional study was performed. Ten sites, 5 from plant effluents and 5 from canals, were selected for sampling during November and December 2001. Fecal coliforms and enterococci were quantified by membrane filtration, male specific (F+) and somatic coliphages by double agar layer technique, and Cryptosporidium oocysts and Giardia cysts by concentration with Envirocheck filter followed by immunofluorescence microscopy quantification. The average of organisms counts from effluents and canal water were compared with t Student test. RESULTS Treated water discharge in canals showed a low count of Fecal Coliforms (average 40.4/100 ml), enterococci (average 58.8/100 ml) and Cryptosporidium oocysts (average 13.2/100 l), while coliphages and Giardia cyst rendered higher counts (average 1467.5/100 ml and 1199.8/100 l, respectively) suggesting the water treatment methods could fail to remove these agents. A significant lower count of Giardia cysts (average 45/100 l) and no Cryptosporidium oocysts were found in irrigation canals, which suggests a natural clearance of these pathogens. Strains of Escherichia coli isolated in one of the canals contaminated with sewage had antimicrobial multi-resistance that was transferred by conjugation suggesting that resistance is encoded in a plasmid potentially transferable to other pathogenic bacteria. CONCLUSIONS Cost effective and culturally acceptable waste treatment methods will require careful planning and consultation if they are to be adopted and mantained by local populations.