Quadruplex qPCR for detection and discrimination of C. Coli,C. fetus, and C. Jejuni from other Campylobacter species in chicken and sheep meat.

Campylobacter is gram-negative bacteria considered the predominant genera isolated from poultry samples and associated with gastroenteritis. Due to the problems in conventional cultural methods of time-consuming and technically demanding requirements, a rapid and feasible method for their identification and discrimination of the closely related spp. Including Campylobacter coli, Campylobacter fetus, and Campylobacter jejuni is needed. This study analyzes the chicken and sheep meats samples (n = 125) using culture and pre-enrichment-based Quadraplex real-time PCR by targeting OrfA, CstA, HipO, and 16 S rRNA genes of C. coli, C. fetus, C. jejuni and Campylobacter spp. Respectively. The analysis of 125 chicken and sheep meat samples by culture and real-time PCR showed high concordance between the results of the two methods. The present study show high prevalence of Campylobacter species (35% and 32% from chicken and meat respectively) of which C. jejuni were the most abundant. Reaction efficiencies were between 90 and 110%, and detect as low as 8.9 fg in C. jejuni. The need for quick detection and discrimination methods in sheep and chicken meat can be met using the described Quadraplex real-time PCR methodology.

Salmonella in Chicken and Pork Meat as a Likely Major Contributor to Foodborne Illness in Peru.

Nontyphoidal Salmonella is one of the major causes of self-limiting diarrheal disease and the most common foodborne pathogen worldwide. It is an important contributor to the burden of foodborne illness in South America, including Peru, where chicken and pork are important vehicles for Salmonella infection. Salmonella infections are underreported, particularly in low- and middle-income countries where concerted action tackling Salmonella along the chicken and pork chains, from primary production to retail, is urgently needed. To support and inform the implementation of new strategies to reduce Salmonella contamination of chicken and pork, this study describes the frequency and distribution of foodborne outbreaks attributed to Salmonella in Peru and evaluates the level of Salmonella in chicken and pork meat sold in markets of three regions of Peru. To that end, we analyzed historical reports of foodborne outbreaks, levels of Salmonella in chicken and pork sold in markets, and the number of mesophiles in the collected meat samples. As a result, the microbiological analysis reveals a widespread contamination of chicken (77.1%) and pork (26.8%) with Salmonella. It also pinpoints Salmonella as the causative agent in nearly half of the outbreaks (47.0%) where the potential origin is identified over a 11-year period with chicken, mayonnaise, and pork being the most likely food vehicles. These results suggest that Salmonella is a major contributor to foodborne illness in Peru and that the monitoring of mesophiles could be a good strategy for surveillance, generating data to support source attribution studies and ultimately evidence-informed policies.

Rapid and visual detection of Shiga-toxigenic Escherichia coli (STEC) in carabeef meat harnessing loop-mediated isothermal amplification (LAMP).

Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94 fg/µLand 25.8 fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6-12 h enrichment. The study highlights the importance of short (6-12 h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9 h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.

KPC-2-producing Enterobacterales from ready-to-eat food to hospitalized patients.

Foodstuffs are a well-documented source of multidrug-resistant bacteria, and hospitalized patients are usually susceptible to hospital infections owing to their immune status. Therefore, this study aimed to investigate the presence of beta-lactamase-producing Enterobacterales in ready-to-eat foods consumed by hospitalized patients. For this purpose, 51 vegetable and meat samples were collected over 2 months and analyzed. Enterobacterales isolates were identified and subjected to antimicrobial susceptibility testing, followed by beta-lactamase gene screening, pH tolerance assays, and whole-genome sequencing (WGS). Isolates harboring genes encoding extended-spectrum beta-lactamases, cephalosporinases, or carbapenemases were detected, and all isolates tolerated pH levels similar to those in the human gastrointestinal tract. The blaKPC-2 carriers were characterized by WGS and lineages closely related to those causing human infections were identified. These results showed that dietary intake is an alternative route for the transmission of antimicrobial-resistant bacteria, which must be considered when designing effective strategies for infection control.

Detection of Escherichia coli O157H7 and Campylobacter jejuni in Bovine Carcasses in Two Slaughterhouses in Bio-Bío District, Chile.

Escherichia coli O157:H7 (E. coli O157:H7) and Campylobacter jejuni (C. jejuni) are pathogenic microorganisms that can cause severe clinical symptoms in humans and are associated with bovine meat consumption. Specific monitoring for E. coli O157: H7 or C. jejuni in meat is not mandatory under Chilean regulations. In this study, we analyzed 544 samples for the detection of both microorganisms, obtained from 272 bovine carcasses (280 kg average) at two slaughterhouses in the Bio-Bío District, Chile. Sampling was carried out at post-shower of carcasses and after channel passage through the cold chamber. Eleven samples were found to be positive for E. coli O157:H7 (4.0%) using microbiological and biochemical detection techniques and were subjected to a multiplex PCR to detect fliC and rfbE genes. Six samples (2.2%) were also found to be positive for the pathogenicity genes stx1, stx2, and eaeA. Twenty-two carcasses (8.0%) were found to be positive for C. jejuni using microbiological and biochemical detection techniques, but no sample with amplified mapA gene was found.

Quantitative risk assessments of Salmonella spp. in domestic pork in China.

Pork is one of the most commonly consumed meats, and its safety has always been a concern. Recently, safety incidents caused by chemical or biological contamination such as drug residues, heavy metals, and pathogenic microorganisms in pork have been reported, and the safety of pork is a cause for concern. Salmonella spp. is one of the important foodborne pathogens that threaten human health. Pork is a high-risk vector food for Salmonella spp. infection. The assessment of the safety risk of Salmonella spp. in pork is conducive to the prevention of related foodborne diseases. In this paper, risk assessment models for Salmonella spp. in meat were developed. The quantitative risk assessment model for Salmonella spp. based on the pork supply chain showed that the annual number of cases of salmonellosis due to pork consumption in China is approximately 27 per 10,000 males and 24 per 10,000 females. Sensitivity analysis showed that the main factors affecting the risk of Salmonella spp. in pork were the display temperature, display time, and Salmonella spp. contamination concentration in pork at the sale.

Low temperatures do not impair the bacterial plasmid conjugation on poultry meat.

Conjugation plays an important role in the dissemination of antimicrobial resistance genes. Besides, this process is influenced by many biotic and abiotic factors, especially temperature. This study aimed to investigate the effect of different conditions of temperature and storage (time and recipient) of poultry meat, intended for the final consumer, affect the plasmid transfer between pathogenic (harboring the IncB/O-plasmid) and non-pathogenic Escherichia coli organisms. The determination of minimal inhibitory concentrations (MIC) of ampicillin, cephalexin, cefotaxime, and ceftazidime was performed before and after the conjugation assay. It was possible to recover transconjugants in the poultry meat at all the treatments, also these bacteria showed a significant increase of the MIC for all antimicrobials tested. Our results show that a non-pathogenic E. coli can acquire an IncB/O-plasmid through a conjugation process in poultry meat, even stored at low temperatures. Once acquired, the resistance genes endanger public health especially when it is about critically and highly important antimicrobials to human medicine.

Bacterial microbiota shifts in vacuum-packed beef during storage at different temperatures: Impacts on blown pack spoilage.

We aimed to evaluate the bacterial growth and diversity in vacuum-packed beef bags stored at different temperatures and to monitor blown-pack spoilage. We used culture-based methods and high-throughput sequencing to study the development of the main bacterial groups naturally present in beef stored at 4 and 15 °C for 28 days. The growth of sulfite-reducing clostridium (SRC) was impaired in beef bags stored at 4 °C; significant differences among SRC counts were observed in beef bags stored at 4 and 15 °C on days 14, 21, and 28 (P = 0.001). Blown pack was observed in most beef bags stored at 15 °C, from day 14 to day 28, but not in beef bags stored at 4 °C. A storage temperature of 4 °C was able to maintain a stable bacterial microbiota (most prevalent: Photobacterium, Hafnia-Obesumbacterium, and Lactococcus). Remarkable changes in microbial abundance occurred at 15 °C from day 14 to day 28, with a predominance of strict anaerobes (Bacteroides) and the presence of Clostridium spp. The relative frequencies of strict anaerobes and Clostridium were statistically higher in the beef bags stored at 15 °C (P < 0.001 and P = 0.004, respectively). The temperature influenced the microbial counts and relative abundance of spoilage bacteria, leading to blown pack spoilage.

Shelf life estimation of refrigerated vacuum packed beef accounting for uncertainty.

This study estimates the shelf life of vacuum packed beef meat (three muscles: striploin (longissimus thoracis et lumborum, LTL), tenderloin (psoas major, PM) and outside chuck (trapezius thoracis, TT)) at refrigeration temperatures (0 °C-10 °C) based on modelling the growth of two relevant groups of spoilage microorganisms: lactic acid bacteria (LAB) and Enterobacteriaceae. The growth models were developed combining a two-step and a one-step approach. The primary modelling was used to identify the parameters affecting the growth kinetics, guiding the definition of secondary growth models. For LAB, the secondary model included the effect of temperature and initial pH on the specific growth rate. On the other hand, the model for Enterobacteriaceae incorporated the effect of temperature on the specific growth rate and the lag phase; as well as the effect of the initial pH on the specific growth rate, the lag phase and the initial microbial count. We did not observe any significant effect of the type of muscle on the growth kinetics. Once the equations were defined, the models were fitted to the complete dataset using a one-step approach. Model validation was carried out by cross-validation, mitigating the impact of an arbitrary division between training and validation sets. The models were used to estimate the shelf life of the product, based on the maximum admissible microbial concentration (7 log CFU/g for LAB, 5 log CFU/g for Enterobacteriaceae). Although LAB was the dominant microbiota, in several cases, both LAB and Enterobacteriaceae reached the critical concentration practically at the same time. Furthermore, in some scenarios, the end of shelf life would be determined by Enterobacteriaceae, pointing at the potential importance of non-dominant microorganisms for product spoilage. These results can aid in the implementation of effective control measures in the meat processing industry.

Peracetic acid application as an antimicrobial and its residual (HEDP): a holistic approach on the technological characteristics of chicken meat.

The most significant occurrence of food-borne diseases is due to Campylobacter and Salmonella contamination from chicken meat, and for this reason, strict regulations about strategies to improve the control of food pathogens are imposed by food safety authorities. Despite the efforts of poultry industry since the beginning of risk analysis and critical control point to reduce the burden of food-borne illness, technological barriers along the way are increasingly necessary to ensure safe food. The aim of this review was to carry out a scientific approach to the influence of peracetic acid (PAA) as an antimicrobial and its toxicological safety, in particular the stabilizer used in the formulation of PAA, 1-hydroxyethylidene 1,1-diphosphonic acid (HEDP), suggesting the possibility of researching the residual HEDP in meat, which would allow the approval of the PAA by the health authorities of several countries that still restrict it. This review also aims to ascertain the effectiveness of PAA, in different cuts and carcasses, by different application methods, comparing the effectiveness of this antimicrobial with other antimicrobials, and its exclusive or combined use, for the decontamination of poultry carcasses and raw parts. The literature results support the popularity of PAA as an effective intervention against pathogenic bacteria during poultry processing.

Prevalence and Antimicrobial Resistance of Salmonella spp. Isolated From Chilled Chicken Meat Commercialized at Retail in Federal District, Brazil.

Salmonella represents one of the most common foodborne pathogens, frequently associated with the contamination of poultry products, constituting a prominent worldwide public health concern. This study determined the prevalence and antimicrobial resistance of Salmonella spp. in chilled chicken meat (115 samples) commercialized at retail in the Federal District, Brazil. Microbiological tests were performed to screen for Salmonella spp. in the chicken meat samples, and the isolated strains were confirmed by the invA gene presence (PCR technique). The strains were evaluated for antimicrobial susceptibility by the disk diffusion technique (Kirby-Bauer method) and tested for the presence of the sul2, blaCTX, and tetB antimicrobial resistance genes. The Salmonella spp. prevalence in chilled chicken meat sold at retail in the Federal District, Brazil, was 46.1% (53 of 115 chicken meat samples analyzed had invA gene-positive strains). Seventy-eight strains of Salmonella spp. isolated from the 53 contaminated samples showed higher resistance to amoxicillin/clavulanic acid (83.3%), followed by sulfonamide (64.1%) and tetracycline (46.2%); 53.8% of the isolates were multidrug-resistant (MDR). The sul2 gene that confers resistance to sulfonamide was found in 53 strains (68.0%), the blaCTX gene that confers resistance to beta-lactams was identified in 39 strains (50.0%), and the tetB gene that confers resistance to tetracycline was identified in 29 strains (37.2%). The high percentage of Salmonella contamination in chicken meat can pose a risk to consumers' health due to the possibility of causing salmonellosis. In addition, many isolates were MDR and carried antimicrobial resistance genes. Public agencies can use these results to develop effective public health policies and strategies to ensure the safety of these food products.

Factors associated with microbiological contamination of chicken meat marketed in El Salvador. / Factores asociados con la contaminación microbiológica en la carne de pollo comercializada en El Salvador.

OBJECTIVE.: To determine the hygienic-sanitary factors associated with the microbiological contamination of chicken meat sold at the municipal markets of El Salvador. MATERIALS AND METHODS.: An analytical cross-sectional study was conducted in 33 municipal markets of the 14 departmental capitals of El Salvador. The sample consisted of 256 out of 456 possible market stalls. A sample of chicken meat was obtained from each market stall. The microbiological analysis was conducted at the National Public Health Laboratory. Frequencies, percentages, measures of central tendency and association were calculated with SPSS version 21. RESULTS.: Escherichia coli was found in 74% of the samples, Staphylococcus aureus in 24% and Salmonella spp. in 1%. The presence of Salmonella spp. was associated with not using hand sanitizer and not using towels for drying the hands. S. aureus was associated with the use of personal accessories and improper storage. The presence of S. aureus was associated with the lack of hand washing, not using a towel to dry the hands and not wearing an apron. CONCLUSION.: The hygienic-sanitary conditions of the handlers and the market stalls were associated with microbiological contamination of chicken meat marketed in El Salvador.

OBJETIVO.: Determinar los factores higiénico-sanitarios asociados a la contaminación microbiológica de la carne de pollo comercializada en los mercados municipales de El Salvador. MATERIALES Y MÉTODOS.: Se realizó un estudio transversal analítico en los 33 mercados municipales de las cabeceras departamentales de El Salvador. La muestra se calculó a partir de 456 puestos de venta, obteniendo un total de 256 puestos. Por cada puesto se obtuvo una muestra de carne de pollo. El análisis microbiológico se realizó en el Laboratorio Nacional de Salud Pública. Se calcularon frecuencias, porcentajes, medidas de tendencia central y de asociación utilizando SPSS versión 21. RESULTADOS.: En el 74% de las muestras se encontró Escherichia coli, en el 24%, Staphylococcus aureus y en el 16%, Salmonela spp. La presencia de Salmonella spp, estuvo asociada con el no uso de desinfectante para las manos y no utilizar toalla para secarse las manos. La presencia de E. coli estuvo asociada al uso de accesorios personales y la inadecuada temperatura de almacenamiento. Mientras que la presencia de S. aureus, estuvo asociada a la falta de lavado de manos, no utilizar toalla para secarse las manos y no utilizar delantal. CONCLUSIÓN.: Las condiciones higiénico-sanitarias de los manipuladores y de los puestos de venta están asociadas a la contaminación microbiológica en la carne de pollo comercializada en El Salvador.

Monitoring of Pig Slaughter Stages and Correlation in the Prevalence of Pathogens and Levels of Microorganisms That Indicate Microbiological Quality and Hygiene Using a Predictive Model.

Pig production is relevant to the Brazilian economy. Different stages of the raising and slaughtering process influence the microbiological quality of pig products and by-products. Microbiological analysis and hazard analysis and critical control points (HACCPs) are tools for monitoring microbiological quality indicator microorganisms. The construction of predictive models can assist the process of monitoring the microbiological quality of pig products. This study aimed to map the slaughter stages and develop a model to predict the absence or presence of Salmonella based on the process variables (distance from the farm to the slaughterhouse and aerobic mesophilic) and analyze their influence on contamination indicator microorganisms. A total of 810 samples were collected at nine stages of the slaughter process (bleeding, scalding, dehairing, singeing, washing, evisceration, inspection, final washing, and chilling). The binary class predictive model was used as a microbiological quality predictor at the slaughter stages. Salmonella was identified at all process stages, with lower contamination levels at the scalding and chilling stages, whereas the highest levels were found at the dehairing and bleeding stages. The predictive model revealed an accuracy of about 85% for Salmonella being a tool to monitor the microbiological quality of pig slaughter.

Characterization of ESBL-producing Escherichia spp. and report of an mcr-1 colistin-resistance Escherichia fergusonni strain from minced meat in Pamplona, Colombia.

Foods of animal origin are increasingly considered a source of extended spectrum ß-lactamase (ESBL) producing bacteria which can disseminate throughout the food chain and become a health concern for humans. This work aimed to evaluate the occurrence of ESBL-producing Escherichia coli in 100 retail minced meat samples taken in markets in Pamplona, Colombia. A total of 19 ESBL-producing isolates were obtained, 18 identified as E. coli and one as E. fergusonii. Fifteen isolates (78.9 %) carried blaCTX-M and blaTEM genes, one (5.2 %) blaSHV and blaTEM genes, one isolate (5.2 %) carried blaCTX-M and one (5.2 %) blaSHV alone. The majority of CTX-M-positive E. coli isolates carried the blaCTX-M-15 gene (13 isolates), being the blaCTX-M-9, blaCTX-M-2, and blaCTX-M-8 (one isolate each) also detected. Two SHV-positive isolates presented the blaSHV-5 and blaSHV-12 allele. The isolate identified as E. fergusonii was positive for blaCTX-M-65 gene and mcr-1 gene. Sixteen isolates (84.2 %) belonged to phylogroups A and B1 and grouped together in the phylogenetic tree obtained by MLST; phylogroups E and F were also detected. Transfer of ESBL resistance was demonstrated for the E. fergusonii isolate. Whole genome sequencing of this isolate revealed the presence of plasmids carrying additional resistance genes. This investigation showed the high prevalence of ESBL-producing E. coli in retail samples of minced meat. Also, the isolation of a strain of E. fergusonii is an additional concern, as some resistance genes are located in mobile elements, which can be transmitted to other bacteria. These evidences support the increasing public health concern considering the spreading of resistance genes through the food chain.

Caracterización de Escherichia coli productoras de toxina Shiga (STEC) recuperadas de canales bovinas uruguayas / Characterization of Shiga toxin-producing Escherichia coli (STEC) recovered from Uruguayan bovine carcasses

Escherichia coli productor de toxina Shiga (STEC) es un microorganismo zoonótico que coloniza el tracto intestinal de los animales y causa en el hombre diarrea de severidad variable y enfermedades graves, entre ellas, el síndrome urémico hemolítico post-entérico por STEC (SUH-STEC). El SUH-STEC integra el grupo de las microangiopatías trombóticas que afecta con mayor frecuencia a niños menores de 5 años. Es una enfermedad para la cual no existe tratamiento específico y, en los casos más graves, puede derivar en la muerte del paciente. América Latina tiene una situación endémica de casos de SUH-STEC, y la mayoría de ellos suceden en el sur del continente. En Uruguay se estima que ocurren entre 10 y 15 casos de SUH por año, con una tasa de incidencia de 0,5/100.000 habitantes y de 4-5/100.000 niños menores de 5 años. Hasta el momento no se han reportados brotes de origen común y en ningún caso de los estudiados se pudo establecer el/los alimentos implicados. El ganado bovino es reconocido como el principal reservorio de STEC, y el consumo de alimentos derivados contaminados, como una de las principales vías de transmisión a humanos. El contacto directo con animales portadores y su entorno, el contacto persona a persona, el consumo de productos lácteos o derivados, y de frutas y vegetales contaminados son otras fuentes importantes de contagio. La presencia de Escherichia coli productor de toxina Shiga (STEC) en alimentos cárnicos representa un riesgo para la salud pública, sobre todos en países como el nuestro, donde el consumo de carne es uno de los más altos del mundo; y al mismo tiempo genera pérdidas económicas en esta cadena productiva por rechazos fundamentados en la presencia de stx o STEC en productos cárnicos exportados a mercados exigentes.Varios factores de virulencia han sido descritos para estas bacterias, pero no se conocen cuáles son las características que definen con certeza a una cepa STEC con capacidad de producir enfermedad grave. El mecanismo de patogenicidad común, que juega un papel esencial en el desarrollo de SUH-STEC, y que define a este patotipo diarrogénico, es la producción de toxina Shiga (Stx). Diversos subtipos de Stx han sido descriptos y asociados a distintos riesgos de desarrollar enfermedades, siendo el Stx2a el mayormente asociado a casos de SUH-STEC. La adhesión al epitelio intestinal también ha sido reconocida como un Resumen 13 factor muy importante en la patogenia de estas bacterias, siendo clásicamente vinculada a la presencia y expresión del gen eae. Se han identificado más de 1200 serotipos de STEC y alrededor de 100 han sido asociados a enfermedad en humanos. Si bien se considera al serotipo O157:H7 stx+/eae+ como el más involucrado en brotes a nivel mundial, en la actualidad existen muchos casos de SUH causados por cepas de otros serotipos y con otros perfiles de virulencia (stx+/eae-). La distribución de estos serotipos es variable y depende de la región o país estudiado. Esta situación pone de manifiesto la necesidad de avanzar en el análisis y la determinación del potencial virulento de las cepas STEC locales, sobre todo aquellas recuperadas de animales utilizados para el abasto o de alimentos derivados de este origen. Los objetivos de la tesis fueron aportar al conocimiento local sobre la prevalencia de genes stx y STEC en carcasas bovinas listas para entrar en la línea de producción y conocer sus variaciones en función del tipo de animal faenado, tipo de frigorífico, época de la faena, aplicación de intervención y tipo de faena.También se estudiaron las características microbiológicas de un conjunto de cepas presentes en esta etapa de la cadena de producción de carne bovina, generando información sobre los serotipos STEC circulantes en el momento del estudio, su perfil de virulencia, genes de resistencia y así, poder estimar su potencial patogenicidad. En este trabajo se seleccionó para la toma de muestras la última etapa limpia del proceso de la faena para obtener carne bovina para consumo humano. Para esto, se analizaron 800 muestras de medias canales ubicadas en cámara de maduración y obtenidas en 37 establecimientos de todo del país, y se aislaron cultivos stx+ para su posterior caracterización. Las muestras fueron analizadas por PCR real time, y aquellas muestras stx+ se sembraron en medios sólidos en placa con el objetivo de aislar cepas de Escherichia coli positivas para los genes stx1 y/o stx2, para luego ser conservadas y caracterizadas. La prevalencia de genes stx (solos o asociados a eae) fue del 22,3% (IC95 19,5%-25,3%, n=179), mientras que la prevalencia de STEC fue del 11,3 % (IC95 9,3%-13,7%, n=90) (tomando como STEC positivas aquellas muestras donde se logró obtener al menos una colonia viable, indol positiva y con genes stx). Solo en 2 de las 800 muestras (0,25%, IC 95 0%-0,6%) se logró recuperar Escherichia coli O157:H7, ambas muestras pertenecientes al mismo establecimiento, en la misma fecha de muestreo y provenientes de medias canales distintas. A la hora de analizar los resultados de prevalencia de stx en función de las variables de muestreo, se realizaron pruebas de Chi2, test exacto de Fisher y regresión logística. Se determinó que época del sacrificio, tipo de faena y alimentación animal previa no están asociadas con la prevalencia de genes stx.La categoría animal, establecimiento de faena y presencia de pelos visibles se asociaron con mayor prevalencia de genes stx. En novillos y vaquillonas la prevalencia fue mayor que en el resto, representando un 43% y 34,4%, respectivamente. En frigoríficos de abasto la prevalencia de stx fue 6.7 veces mayor que en exportadores. El análisis multivariado mostró que las variables que inciden significativamente sobre la positividad para genes stx fueron: tipo de frigorífico (abasto), tipo de animal (vaquillona) y presencia de pelos visibles. Para caracterizar las cepas de STEC se utilizaron procedimientos microbiológicos clásicos, secuenciación completa del genoma ("Whole Genome Sequencing" - WGS) y criterios de riesgo de la FAO/OMS. Teniendo en cuenta los costos de la WGS se seleccionaron 50 de las STEC dentro de las 121 muestras con aislamientos recuperados para la caracterización. La selección se realizó de modo aleatorio contemplando cubrir todos los establecimientos positivos en los que se recuperaron cultivos STEC de modo que el muestreo fuese representativo. De las 50 cepas seleccionadas inicialmente, 39 correspondientes a 20 establecimientos fueron confirmadas como STEC por WGS. En este conjunto no detectamos cepas híbridas. Pertenecían a 20 grupos O diferentes y 13 tipos H diferentes. Solo se caracterizó una cepa O157:H7 y, dentro de las no-O157, prevalecieron los serotipos O130:H11 (n = 6), O174:H21/28 (n = 5) y O22:H8 (n = 5). Una cepa mostró resistencia in vitro a la tetraciclina y se detectaron genes de resistencia para doxiciclina, sulfonamida, estreptomicina y fosfomicina. Treinta y tres cepas (84,6%) portaban los genes para los subtipos stx2a, stx2c o stx2d. El gen eae se detectó solo en dos cepas STEC correspondientes a los serotipos O157:H7 (serotipo asociado a casos locales, regionales y mundiales de SUH-STEC) y O182:H25 (ST300).Los genes de virulencia más frecuentes encontrados fueron lpfA (n = 38), ompA (n = 39), ompT (n = 39), iss (n = 38) y terC (n = 39). Dentro del conjunto de STEC analizado, la mayoría (81,5%) pertenecía a los niveles de clasificación de riesgo 4 y 5 (menor riesgo) de la FAO/OMS. Además, se detectaron los serotipos STEC O22:H8, O113:H21, O130:H11 y O174:H21 pertenecientes al nivel de riesgo 2 asociados a diarrea, colitis hemorrágica o SUH. La única cepa O157:H7 Resumen 15 analizada pertenecía al Secuenciotipo (ST) 11. Treinta y ocho aislamientos pertenecieron al tipo B1 de Clermont, mientras que el O157:H7 se clasificó como E. Las STEC analizadas mostraron una alta diversidad genómica y contenían varios determinantes genéticos asociados con la virulencia, lo que subraya el importante papel de WGS para alcanzar una tipificación más completa. También se comparó entre los distintos aislados, las secuencias plasmídicas y de profagos presentes en el genoma. Al mismo tiempo, se identificaron los genes comunes a todos los aislados y los únicos en algunas cepas, determinando así el pangenoma del conjunto de cepas estudiadas. El análisis filogenético mediante WGS permitió determinar la relación entre los aislamientos, observándose agrupamientos en los árboles filogenéticos realizados (cgMLST y SNP) en función del serotipo y secuenciotipo. En este trabajo, no detectamos STEC no-O157 de los serogrupos (O26, O111, O145) previamente aislados de casos locales de SUH. Sin embargo, al interpretar estos hallazgos, se debe tener en cuenta el bajo número de aislamientos analizados y limitaciones metodológicas, así como algún sesgo en la selección de colonias realizada para conservar y estudiar. Los datos obtenidos sugieren que el ganado bovino constituye un reservorio local de serotipos STEC no-O157, incluyendo algunos asociados con enfermedades graves en seres humanos.Se necesitan otros estudios que incluyan más etapas de la cadena cárnica para evaluar de manera global su papel real en la propagación local de STEC.

Salmonella Derby from pig production chain over a 10-year period: antimicrobial resistance, biofilm formation, and genetic relatedness.

The aim of this study was to evaluate 140 Salmonella Derby isolates collected over a 10-year period from porcine origins (environment, pig carcass, lymph nodes, intestinal content, and pork) for their phenotypic and genotypic antimicrobial resistance, their ability to produce biofilm, and their genetic relatedness. The minimum inhibitory concentration (MIC) was determined using microdilution broth method and antimicrobial resistance genes were investigated by PCR. The quantification of biofilm formation was performed in sterile polystyrene microtiter plates. Genetic relatedness was determined by Xba-I macrorestriction analysis. The highest frequencies of non-wildtype (nWT) populations were observed against tetracycline (75.7%), streptomycin (70%), and colistin (11.4%), whereas wildtype populations were observed against ciprofloxacin, ceftazidime, and gentamicin. The resistance genes found were blaTEM (ampicillin), aadA variant (streptomycin/spectinomycin), tetA (tetracycline), and floR (florfenicol). On 96-well polystyrene microtiter plate, 68.6% of the isolates proved to be biofilm producers. Among 36 S. Derby isolates selected to PFGE analysis, 22 were clustered with 83.6% of similarity. Additionally, 27 isolates were clustered in 11 pulsotypes, which presented more than one strain with 100% of similarity. Most of S. Derby isolates were able to form biofilm and were classified as nWT or resistant to tetracycline, streptomycin, and colistin. PFGE allowed the identification of closely related S. Derby isolates that circulated in pig slaughterhouses and pork derived products along a decade.

Identification and bioinformatic analysis of invA gene of Salmonella in free range chicken.

Salmonella is a serious cause of the health issues in human and animal worldwide. Salmonella has been isolated from different biological samples and it considers as the key role in induction of inflammation of gastrointestinal tract which in turn cause diarrhoea in different species. To further understand the involvement of Salmonella in contaminating and infecting fresh eggs and meat of free-range chicken. This study aimed to establish the microbiological and molecular detections of Salmonella in the cloaca of the free-range chicken and to identify predicted biological functions using Kyoto Encyclopedia of Gene and Genomic (KEGG) pathways and protein-protein interaction. Cloacal swabs were collected from free range chicken raised in the local farm in Duhok city. The isolates were cultured and biochemical test performed using XLD and TSI, respectively. Molecular detection and functional annotation of invA gene was carried out using Conventional PCR and bioinformatics approaches. The present study found that Salmonella was detected in 36 out of 86 samples using microbiological methods. To confirm these findings, invA gene was utilised and 9 out of 36 Salmonella isolates have shown a positive signal of invA by agarose gel. In addition, bioinformatic analysis revealed that invA gene was mainly associated with bacterial secretion processes as well as their KEGG terms and Protein-Protein Interaction were involved in bacterial invasion and secretion pathways. These findings suggested that invA gene plays important role in regulating colonization and invasion processes of Salmonella within the gut host in the free range chicken.

Antimicrobial resistance profiles in Escherichia coli isolated from whole-chicken carcasses from conventional, antibiotic-free, and organic rearing systems / Perfil de resistência em Escherichia coli isoladas de carcaças de frango provenientes de sistemas de produção convencional, livre de antibióticos e orgânico

Antimicrobial resistance (AMR) is a growing concern in human and animal health. Public discussions on these issues have contributed to an increased demand for antibiotic-free food. Studies comparing the antimicrobial resistance profiles of bacteria in foodstuffs originating from farming systems with restrictions on the use of antimicrobials are scarce. This study aimed to assess the antimicrobial resistance profiles of generic Escherichia coli isolated from whole chickens originating from farming systems with and without restrictions on the use of antimicrobials. For this purpose, three groups of E. coli strains were formed: (GC) from chickens reared in conventional production systems, without restriction on the use of antimicrobials (n=72); (GL) from chickens reared in farming systems certified as free of any antibiotic use (n=72); and (GO) from chickens from an organic farming system (n=72). Whole chicken units were individually rinsed as recommended by ISO 17604:2015, and E. coli was isolated from the rinse suspension. To evaluate the resistance profile, E. coli strains were tested against 12 antimicrobials using broth microdilution or disk diffusion tests. Eighty strains (40.7%) were found to be fully susceptible to the tested antimicrobials, and 23.6% were multidrug resistant. The highest frequencies of resistance were observed to tetracycline (GC=37,5%; GL=34,7%; GO=25%) and trimethoprim (GC=27,8%; GL=34,7%; GO=22,2%). In the case of multidrug resistant strains, GC presented 32% (n=23) of strains with multidrug resistance characteristics whereas the GL and GO groups presented 22% (n=16) and 17% (n=12), respectively. As for the totally susceptible strains, a frequency of 56% of Tsus strains was observed in the organic group, whereas this frequency was 33% in the GC and GL groups. Using GC as a reference, the Poisson regression model showed a higher occurrence of fully susceptible E. coli strains, as well as lower frequencies of multidrug resistance and resistance to ampicillin and nalidixic acid in GO. The GL group exhibited the lowest frequency of ampicillin resistance. These observations suggest that the lower selection pressure for antimicrobial use in the farming system may be reflected in the resistance profile of bacteria present in foodstuffs purchased by consumers.(AU)

A resistência antimicrobiana (AMR) é uma preocupação crescente para a saúde humana e animal. A discussão pública dessas questões tem contribuído para o aumento da demanda por alimentos produzidos sem o uso de antibióticos. No entanto, estudos que comparem os perfis de resistência antimicrobiana de bactérias em alimentos oriundos de sistemas agrícolas com restrição ao uso de antimicrobianos ainda são escassos. O objetivo deste estudo foi avaliar o perfil de resistência antimicrobiana em Escherichia coli genérica isolada de carcaças de frangos inteiros oriundos de sistemas de criação com e sem restrições ao uso de antimicrobianos. Para tanto, três grupos de cepas de E. coli foram formados: (GC) isolados de carcaças de frangos criados no sistema convencional - sem restrição de uso de antimicrobianos (n=72); (GL) isolados de frangos de sistemas certificados sem uso de antimicrobianos (n=72); (GO) de frangos originados de produção orgânica (n=72). As unidades de frango inteiro foram submetidas à lavagem conforme recomendado pela ISO 17604: 2015 e E. coli foi isolada da suspensão de enxágue. Para avaliar o perfil de resistência, as cepas de E. coli foram testadas frente à 12 antimicrobianos pelos testes de microdiluição em caldo ou difusão em disco. Oitenta cepas (40,7%) foram totalmente suscetíveis aos antimicrobianos testados e 23,6% multirresistentes. As maiores frequências de resistência foram observadas frente a tetraciclina (GC=37,5%; GL=34,7%; GO=25%) e trimetoprima (GC=27,8%; GL=34,7%; GO=22,2%). No caso de cepas multirresistentes, GC apresentou 32% (n=23) das cepas com características de multirresistência enquanto os grupos GL e GO apresentaram 22% (n=16) e 17% (n=12), respectivamente. Quanto às cepas totalmente suscetíveis, foi observada uma frequência de 56% de cepas Tsus no grupo orgânico enquanto tal frequência foi de 33% nos grupos GC e GL. Utilizando GC como referência, o modelo de regressão de Poisson demonstrou maior ocorrência de cepas de E. coli totalmente suscetíveis, bem como menores frequências de multirresistência e resistência à ampicilina e ácido nalidíxico no GO. Em GL, apenas a frequência mais baixa de resistência à ampicilina pôde ser demonstrada. Essas observações sugerem que a menor pressão de seleção do uso de antimicrobianos no sistema de cultivo pode se refletir no perfil de resistência das bactérias presentes nos alimentos adquiridos pelo consumidor.(AU)

The Physicochemical, Microbiological, and Structural Changes in Beef Are Dependent on the Ultrasound System, Time, and One-Side Exposition.

The effect of high-intensity ultrasound (HIU) system (bath, 37 kHz and 90 W/cm2; or probe, 24 kHz and 400 W) and application time (25 or 50 min, one-side exposition) on the properties of bovine Longissimus lumborum after 7 d of storage at 4 °C was studied. The bath system significantly increased the lightness of the muscle, while other color parameters (a*, b*, hue, and chroma) were not different from the control. The water holding capacity and shear force decreased significantly (3.1-5% and 0.59-0.72 kgf, respectively) in sonicated meat independently of the system, favoring the tenderization of the muscle after storage. Microstructural changes observed in the HIU-exposed surface provided evidence of a higher area of interfibrillar spaces (1813 vs. 705 µm2 in the control), producing tenderization of the muscle, compared with the control. HIU significantly increased counts of total aerobic and coliform bacteria, especially after 50 min of ultrasonication. HIU also increased lactic acid bacterial counts in the bath system. Single-sided muscle exposition to ultrasound may produce sufficient significant changes in muscle properties, which could decrease long treatment times that would be needed for the exposition of both sides. HIU in bath systems increases tenderness by modifying meat ultrastructure, with no significant changes in physicochemical parameters. Nevertheless, microbiological quality may need to be considered during the process due to a slight increase in bacterial counts.

Effect of gaseous ozone application during chilling on microbial and quality attributes of pig carcasses.

Ozone application has been suggested as an additional measure to the slaughter animals under hygiene programs. In this study, we determined the efficacy of gaseous ozone applied to pig carcasses during chilling (16 h at 2-5°C). Forty carcasses were allocated to each treatment: control, without ozone application (T1) and 5 ppm gaseous ozone application (T2), divided in two 4-h periods. The carcasses were sampled before and after chilling. The average counts of total aerobic mesophilic (TAM) bacteria before chilling were not different (p = 0.55) between T1 and T2. In turn, after chilling, the ozone-treated carcasses had significantly reduced about 0.4 colony-forming units (CFU)/cm2 of TAM counts (p < 0.001) than the control carcasses. No significant reduction was observed in the number of carcasses positive for Listeria sp. and Escherichia coli after gaseous ozone treatment; while a tendency (p = 0.08) of lower number of Salmonella positive carcasses in T2 was observed. Common macrorestriction (pulsed-field gel electrophoresis) patterns of S. enterica were observed in the carcasses before and after chilling. Pork samples from treated and untreated carcasses with ozone showed no lipid oxidation or altered color and pH. The results indicate that the gaseous ozone in the tested protocol is effective in reducing TAM populations, but not effective in decreasing the number of carcasses positive for E. coli and Listeria sp. Regarding Salmonella, the tendency of positive carcasses reduction may encourage further studies by testing other protocols of gaseous ozone application inside the chilling chamber.