Alejandro Sánchez-Pérez

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.

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.

In vitro activity of tigecycline against extended-spectrum β-lactamase-producing Enterobacteriaceae and MRSA clinical isolates from Mexico: a multicentric study ☆ ☆☆

Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and methicillin resistant Staphylococcus aureus (MRSA) are important nosocomial pathogens. This study reports the in vitro activity of tigecycline against 573 and 482 ESBL-producing Enterobacteriaceae and MRSA isolates, respectively. More than 94% of all tested isolates were susceptible to tigecycline; MIC(90) found was 0.25 to 2 mg/L for ESBL-producing Enterobacteriaceae and was 0.125 mg/L for MRSA. Tigecycline demonstrated excellent in vitro activity against a wide spectrum of nosocomial pathogens.

Variability of the blaIMP-15-Containing Integrons, Highly Related to In95, on an Endemic Clone of Pseudomonas aeruginosa in Mexico

The acquisition of β-lactamases, such as class B metallo-β-lactamases, in Pseudomonas aeruginosa is detrimental to antimicrobial therapy in hospitalized patients. In Mexico, metallo-β-lactamase IMP-15 has been found to be encoded on the In95 class 1 integron in a major clone of P. aeruginosa. In this work, we describe the variability of this class 1 integron in an epidemic clone of carbapenem-resistant P. aeruginosa clinical isolates highly related to isolates previously described in Mexico.

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-

Molecular typing and characterization of macrolide, lincosamide and streptogramin resistance in Staphylococcus epidermidis strains isolated in a Mexican hospital

Staphylococcus epidermidis is a normal commensal of skin that has become a serious clinical problem because of the combination of increased use of intravascular devices and an increasing number of hospitalized immunocompromised patients. In addition, there is a lack of information pertaining to resistance to macrolide, lincosamide and streptogramin type B (MLS(B)) in developing countries, including Mexico. The aim of this study was to investigate the incidence of resistance to MLS(B) antibiotics in isolates of S. epidermidis obtained in the General Hospital of Acapulco in Mexico. Susceptibility to erythromycin, clindamycin and quinupristin-dalfopristin was tested by a diffusion test, and MICs to oxacillin, erythromycin and lincomycin were determined. Differentiation between MLS(B) phenotypes was performed by a double disc diffusion test. A total of 38 of the 47 strains of S. epidermidis isolated from nosocomial infections were resistant to oxacillin [meticillin-resistant S. epidermidis (MRSE)]. The phenotypes obtained were: 18 constitutive MLS(B), 3 inducible MLS(B), 6 macrolide streptogramin and 4 lincosamide; 7 strains were susceptible to MLS(B) antibiotics. The genes associated with resistance were detected by PCR. Genotyping showed a predominance of the ermA gene followed by genes ermC and msrA. The frequency of the genes detected varied slightly from results that have been reported in isolates from other countries. Clonal types were identified by PFGE and revealed the dissemination of two major clones of MRSE in the Mexican hospital. This is believed to be the first report in Mexico on the genes associated with the MLS(B) resistance phenotype in S. epidermidis, in addition to observing a wide distribution of clonal types in the General Hospital of Acapulco, Mexico.

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.

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.