Presence of extended-spectrum beta-lactamase (ESBL)- producing Enterobacteriaceae in bulk-tank milk of bovine dairy farms in Antioquia, Colombia / Presencia de Enterobacteriaceae productoras de betalactamasas de espectro extendido (BLEE) en tanques de leche de fincas lecheras de Antioquia, Colombia / Presença de Enterobacteriaceae produtoras de beta-lactamase de espectro estendido (BLEE) em amostras de leite de tanque de rebanhos de bovinos leiteiros em Antioquia, Colômbia

Abstract Background: antibiotic resistance is spreading worldwide. It is important to evaluate whether foods of animal origin constitute a reservoir of resistance genes. Objective: the aim of the present study was to assess the occurrence and characterize extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae from bulk-tank milk samples of dairy farms located in Entrerríos, Antioquia (Colombia). Methods: a total of 120 randomly selected raw milk samples (one bulk-tank milk sample per dairy farm) were collected between September and October, 2013. A commercial chromogenic agar was used for screening presumptive ESBL-E. Identification of genus and species of isolates was performed with a commercial biochemical identification kit. The ESBL production was confirmed using the double disc synergy test. An antimicrobial susceptibility test was performed by the agar disc diffusion method and the automatized broth microdilution method. The ESBL-positive isolates were analyzed for the presence of bla genes by polymerase chain reaction (PCR) and sequencing. For the exploration of risk factors, information on dairy farm management practices was recorded using a questionnaire and the associations of predictors and results were tested with a logistic regression analysis. Results: the ESBL-E were isolated from 3.3% (4/120; CI 95%: 3-3.5%) of the samples. Farm size was the only factor associated with the presence of ESBL-E (OR = 11.5; CI 95%: 1.14-115.54; p = 0.038). All isolates were resistant to several antibiotics and harbored blaCTX-M-96 (alternate name CTX-M-12a) enzymes. Conclusion: although apparent frequency of ESBL-E was low, the presence of these resistant bacteria in milk may constitute a public health risk and should be further investigated.

Resumen Antecedentes: la resistencia a antibióticos se está diseminando en el mundo. Es importante evaluar si los alimentos de origen animal constituyen un reservorio de genes de resistencia. Objetivo: evaluar la presentación y caracterizar las Enterobacteriaceae productoras de betalactamasas de espectro extendido (BLEE) a partir de muestras de leche de tanque de hatos lecheros localizados en Entrerríos, Antioquia (Colombia). Métodos: ciento veinte muestras de leche cruda (una muestra por tanque) de hatos seleccionados al azar fueron colectadas entre septiembre y octubre de 2013. Para el tamizaje de presuntiva BLEE se utilizó un agar cromogénico comercial. La identificación de los aislamientos se realizó utilizando un kit de identificación bioquímica comercial. La confirmación de la producción de BLEE fue confirmada con la prueba de sinergia de doble disco. La susceptibilidad antimicrobiana fue evaluada por el método de difusión de disco en agar y por el método de microdilución en caldo automatizado. Aislamientos positivos para BLEE fueron analizados para presencia de genes bla por PCR y secuenciación. Para la exploración de los factores de riesgo se registró la información sobre las prácticas de manejo del hato mediante cuestionario y se probó la asociación entre predictores y resultados mediante análisis de regresión logística. Resultados: las BLEE fueron aisladas de 3,3% (4/120; IC 95%: 3-3,5%) de las muestras de leche de tanque y el tamaño del hato fue el único factor que se encontró asociado con un incremento en su presencia (OR = 11,5; IC 95%: 1,14-115,54; p = 0.038). Todos los aislamientos fueron resistentes a varios antibióticos y albergaban enzimas blaCTX-M-96 (nombre alternativo CTX-M-12a) Conclusión: aunque se observó una baja frecuencia aparente de BLEE, la presencia de estas bacterias resistentes en los alimentos de origen animal puede constituir un riesgo para la salud pública y debe seguir siendo investigada.

Resumo Antecedentes: a resistência aos antibióticos está se espalhando rapidamente no mundo inteiro. É importante avaliar se os alimentos de origem animal constituem um reservatório de genes de resistência. Objetivo: avaliar a ocorrência e caracterizar Enterobacteriaceae produtoras de betalactamases de espectro estendido (BLEE) de amostras de leite de tanques de rebanhos leiteiros. Métodos: um total de 120 amostras leite crua (uma amostra por leite do tanque) de explorações leiteiras selecionadas aleatoriamente no município de Entrerríos, Antioquia (Colombia), foram coletados emtre setembro e outubro de 2013. Para o rastreio das presumíveis BLEE foi utilizado um meio de ágar cromogénico comercial. A identificação das espécies isoladas foi realizada utilizando um kit comercial da identificação bioquímica. A produção de BLEE foi confirmada pelo teste de sinergia de duplo disco. Um teste de susceptibilidade antimicrobiana foi realizado pelo método de difusão em disco de ágar e método de microdiluição em caldo automatizado. Isolados BLEE positivos foram analisados para a presença dos genes bla por PCR e sequenciação. Para a exploração dos fatores de risco, as informações sobre as práticas de gestão do rebanho foram gravadas utilizando um questionário. As associações de preditores e os resultados foram testados por uma análise de regressão logística. Resultados: o BLEE foram isoladas de 3,3% (4/120; IC 95%: 3-3,5%) das amostras de leite do tanque. O tamanho do rebanho foi o único fator encontrado associado com um aumento na sua presença (OR = 11,5; IC 95%: 1,14-115,54; p = 0,038). Todos os isolados foram resistentes a vários antibióticos e albergava enzimas blaCTX-M-96 (nome alternativo CTX-H-12a). Conclusão: embora tenha sido observada uma baixa frequência aparente de BLEE, a presença dessas bactérias resistentes nos alimentos de origem animal pode constituir um risco para a saúde pública e deve permanecer sob pesquisa.

Emergence of CTX-M-12, PER-1 and OXA-30 beta-Lactamase-Producing Klebsiella pneumoniae / 대한임상미생물학회지

BACKGROUND: The aim of this study was to determine a nation-wide prevalence of Ambler class A and D extended-spectrum-lactamases (ESBL) in Klebsiella pneumoniae isolates in Korea. METHODS: During the period of April to May 2005, 189 isolates of K.pneumoniae were collected from 11 Korean hospitals. Antimicrobial susceptibilities to ceftazidime and cefotaxime were tested by the disk diffusion method, and ESBL production was determined by double-disk synergy test. Determinants of ceftazidime or cefotaxime-resistance were transferred to Escherichia coli J53 (azide-resistant) by transconjugation. Genotypes of class A and D ESBL genes were determined by PCR amplification and sequencing. RESULTS: One hundred-sixty isolates of K.pneumoniae showed positive results in double-disk synergy test. The most prevalent ESBL was SHV-12 (n=148). Also detected were genes encoding ESBLs including TEM-52 (n=1), SHV-2a (n=2), CTX-M-3 (n=15), CTX-M-9 (n=6), CTX-M-12 (n=2), CTX-M-14 (n=9), CTX-M-15 (n=1), PER-1 (n=1), GES-5 (n=3), and OXA-30 (n=2) beta-lactamases. CONCLUSION: With the emergence of CTX-M-12, PER-1, and OXA-30 beta-lactamases, the ESBLs in K.pneumoniae isolates are becoming more diverse in Korea.

Dissemination of CTX-M Type Extended-Spectrum beta-Lactamases and Emergence of CTX-M-12 in Escherichia coli / 대한진단검사의학회지

BACKGROUND: Clinical isolates of Escherichia coli were evaluated to determine the prevalence and genotypes of Ambler class A extended-spectrum beta -lactamases (ESBLs). METHODS: Clinical isolates of E. coli were collected from 12 hospitals from February through July, 2004. Antimicrobial susceptibility was tested by disk diffusion and agar dilution methods, and ESBLproduction was determined by double-disk synergy test. TEM, SHV, CTX-M, PER-1, VEB, IBC, GES, and TLA type ESBL genes were detected by PCR amplifications, and the PCR products were subjected to direct sequencing. RESULTS: The double-disk synergy test was positive in 90.9% (149 in 164) of the ceftazidime- or cefotaxime-resistant E. coli isolates. The most prevalent types of Ambler class A ESBLs in E. coliisolates were CTX-M-15 (n=53). CTX-M-14 (n=24), CTX-M-3 (n=9), CTX-M-9 (n=3), CTX-M-12 (n=3), SHV-2a (n=1), SHV-12 (n=5) and TEM-52 (n=3) were also found. CTX-M-12 ESBL had never been reported before in Korea. CONCLUSIONS: CTX-M type ESBL-producing E. coli isolates are spreading and CTX-M-12 is emerging in Korea.

Cloning and sequencing of the CTX-M-12 encoding gene of ?-lactamase / 中华传染病杂志

Objective To investigate the ESBL gene in a Klebsiella pneumoniae strain isolated from a patient in Huashan Hospital, Shanghai. Methods ESBL gene in the transconjugant from Klebsiella pneumoniae isolate was amplified by PCR. The PCR products were cloned into pHSG398 vector. The antibiotic susceptiblity and ?-lactamases activity of the clone strain were tested; The PCR product was sequenced and analyzed. Results The ESBL gene was identified as CTX-M-12 gene and CTX-M-12 is much more active against cefotaxime than against ceftazidine.Conclusions A clinical isolate of Klebsiella pneumoniae k99-442 isolated from a patient in Huashan Hospital, Shanghai produced ESBL type CTX-M-12 which caused this isolate resistant to most ?-lactams.