RESUMEN
In the course of developing an assay to identify genes responsible for antibiotic resistance in gram-negative bacteria, it has been found that standard (not DNA-free) Taq DNA polymerases were contaminated with blaTEM gene fragments that varied in length and quantities. The complete blaTEM gene sequence was either absent or was detected in infinitesimal amounts. We developed an approach to avoid false-positive findings caused by contaminating blaTEM gene sequences in conventional polymerases. The method is based on selection of a target sequence to be detected within the blaTEM gene in such a way that the chosen sequence is amplified with primers incapable of amplifying contaminating DNA sequences of the polymerase.
Asunto(s)
Contaminación de ADN , ADN Bacteriano/genética , Reacción en Cadena de la Polimerasa/métodos , Polimerasa Taq/análisis , Cartilla de ADN/química , Escherichia coli/genética , Reacciones Falso PositivasRESUMEN
OBJECTIVES: Characterizing the molecular epidemiology of antibiotic resistance is crucial for a better understanding of the evolution and spread of resistance in Neisseria gonorrhoeae. Here, we examine the molecular epidemiology of penicillinase-producing N. gonorrhoeae (PPNG) isolates in France. METHODS: We investigated 176 PPNG isolates collected between 2010 and 2012 by the National Reference Centre in France. Genotyping was performed using the NG-MAST technique, blaTEM genes were Sanger-sequenced, and plasmids were characterized by PCR-typing. RESULTS: We revealed the existence of four major clusters representing about one-third of PPNG circulating in France. These clusters were related to ST1479 (18/176, 10.2%), to ST1582 (15/176, 8.5%), to ST8922 (10/176, 5.6%), and to ST1285 (9/176, 5.1%). Wild-type TEM-1 was identified in 151 (151/176, 85.8%) PPNG isolates, and TEM-1 variants were mostly represented by the M182T mutation (14/176, 8%), followed by P14S/L (8/176, 4.5%), G228S (2/176, 1.1%), and Q269K (1/176, 0.6%). The blaTEM genes were carried by African (157/176, 89.2%), Asian (13/176, 7.4%), and Toronto/Rio (6/176, 3.4%) plasmids. The M182T variants were found in various genetic backgrounds, whereas the P14S variants were disseminated clonally. The G228S and Q269K variants belong to one of the four major clusters of PPNG, which suggests a recent de novo emergence of these mutations. CONCLUSIONS: Our results show that approximately one-third of the penicillinase-producing N. gonorrhoeae isolates in France belong to one of four major clusters and that the spread of the different TEM variants is associated with distinct patterns of molecular epidemiology.
Asunto(s)
Gonorrea/epidemiología , Neisseria gonorrhoeae/genética , Penicilinasa/genética , Farmacorresistencia Bacteriana/genética , Francia/epidemiología , Gonorrea/tratamiento farmacológico , Humanos , Epidemiología Molecular , Filogenia , Reacción en Cadena de la PolimerasaRESUMEN
Escherichia coli is an important cause of hospital-acquired infections worldwide. Antimicrobial resistance leads to treatment failure of hospital infections caused by E. coli. Production of extended-spectrum ß-lactamases (ESBLs) is one of the major causes of antibiotic resistance in these bacteria. This study aimed to investigate the frequency of blaTEM and blaCTX-M genes in ESBL-producing E. coli strains isolated from clinical specimens of patients admitted to six hospitals in the north of Iran. A total of 160 E. coli strains were isolated from various clinical samples of hospitalised patients. Antibiotic resistance patterns were determined by the Kirby-Bauer disk diffusion method. The double-disk phenotypic confirmatory test was carried out amongst ß-lactam-resistant isolates to detect ESBL-producing strains. Plasmid DNA of ESBL-producing strains was extracted and subjected to PCR for detection of the blaTEM and blaCTX-M genes, and isolates were extensively verified by sequencing. The highest resistance rate was to amoxicillin; all E. coli isolates (100%) were susceptible to imipenem. Amongst the 160 clinical E. coli isolates, 83 (51.9%) were ESBL-positive, of which 27 (32.5%) and 72 (86.7%) were positive for blaTEM and blaCTX-M, respectively. This study is the first report of an ESBL phenotype disseminated in hospitals in the north of Iran. These findings showed that there was a direct relationship between the development of resistance to ß-lactam antibiotics and production of TEM and CTX-M enzymes.
Asunto(s)
Escherichia coli/genética , Plásmidos/genética , beta-Lactamasas/genética , Estudios Transversales , Escherichia coli/enzimología , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/microbiología , Humanos , Irán/epidemiologíaRESUMEN
The emergence of antimicrobial resistant bacteria is an important public health and environmental contamination issue. Antimicrobials of ß-lactam group accounts for approximately two thirds, by weight, of all antimicrobials administered to humans due to high clinical efficacy and low toxicity. This study explores ß-lactam resistance determinant gene (blaTEM) as emerging contaminant in Indo-Gangetic region using qPCR in molecular beacon format. Quantitative Microbial Risk Assessment (QMRA) approach was adopted to predict risk to human health associated with consumption/exposure of surface water, potable water and street foods contaminated with bacteria having blaTEM gene. It was observed that surface water and sediments of the river Ganga and Gomti showed high numbers of blaTEM gene copies and varied significantly (p<0.05) among the sampling locations. The potable water collected from drinking water facility and clinical settings exhibit significant number of blaTEM gene copies (13±0.44-10200±316 gene copies/100mL). It was observed that E.crassipes among aquatic flora encountered in both the rivers had high load of blaTEM gene copies. The information on prevalence of environmental reservoirs of blaTEM gene containing bacteria in Indo-Gangetic region and risk associated will be useful for formulating strategies to protect public from menace of clinical risks linked with antimicrobial resistant bacteria.
Asunto(s)
Microbiología del Agua , Contaminantes del Agua , Resistencia betalactámica/genética , beta-Lactamasas/genética , Agua Potable/microbiología , Sedimentos Geológicos/microbiología , Humanos , India , Pruebas de Sensibilidad Microbiana , Medición de Riesgo , Ríos/microbiología , beta-Lactamas/farmacologíaRESUMEN
OBJECTIVES: Production of extended-spectrum beta-lactamases (ESBLs) by enteric bacteria continues to be a major problem in hospitals and community. ESBLs producing bacteria cause many serious infections including urinary tract infections, peritonitis, cholangitis and intra-abdominal abscess. The aim of this study was to determine the prevalence of ESBLs producing Escherichia coli and Klebsiella pneumoniae bacteria isolated from clinical samples of patients attending Imam Reza and Ghaem University Hospitals, Mashhad, Northeast of Iran. MATERIALS AND METHODS: During 2009 and 2010, 82 strains of E. coli and 78 strains of K. pneumoniae were isolated from out-patients and hospitalized patients and they were examined by Oxoid combination disk test and PCR methods. RESULTS: We found that 43.9% of E. coli and 56.1% of K. pneumoniae produced ESBLs. The frequency of SHV and TEM among the ESBLs producing isolates were 14.4% and 20.6%, respectively. Ratios of ESBLs positive isolates from out-patients to hospitalized patients were 24/33. CONCLUSION: This study shows that the prevalence of ESBLs producing E. coli and K. pneumoniae is high in both study groups (out-patients and hospitalized patients). Therefore it seems that continuous surveillance is essential to monitor the ESBLs producing microorganisms in hospitals and community.