[Epidemiological study and serotyping by multiple PCR of Listeria monocytogenes isolated from food matrices in Argentina]. / Estudio epidemiológico y serotipificación por PCR múltiple de Listeria monocytogenes aislada de matrices alimentarias en Argentina.

Listeria monocytogenes is an opportunistic foodborne pathogen. It can resist stress conditions by adapting through the production of biofilms, which represents a serious problem for the food industry. It is classified into 14 serotypes, although only four (1/2a, 1/2b, 1/2c, and 4b) account for 89.0-98.0% of listeriosis cases worldwide. The objective of this study was to detect and serotype L.monocytogenes isolated from different food matrices from processing plants in Argentina. In the period 2016-2021, 1832 samples (meat, ready-to-eat foods, ice cream, dairy foods, and frozen vegetables) were analyzed, of which 226 (12.34%) isolates compatible with L.monocytogenes were detected. At the same time, environmental and surface samplings were performed in processing plants for ready-to-eat foods, sausages and dairy products, where environmental contamination with L.monocytogenes was detected in numerous critical points of the process, yielding a positivity rate of 22.7%. The molecular analysis of serogroups was performed, where it was observed that serogroup IIb was the most frequent with 66.5% (n=107), and in descending order IIc with 22.3% (n=36), and IIa (n=9) and IVb (n=9) with 5.6%. The serogroup mostly isolated in environmental monitoring was IIb. This work highlights the importance of the detection and serotyping of L.monocytogenes for taking actionable measures and identifying outbreaks, and is the first study in Argentina to describe an extensive study in food matrices.

Genomic analysis of two Acinetobacter baumannii strains belonging to two different sequence types (ST172 and ST25).

OBJECTIVES: Acinetobacter baumannii is an opportunistic nosocomial pathogen that is the main focus of attention in clinical settings owing to its intrinsic ability to persist in the hospital environment and its capacity to acquire determinants of resistance and virulence. Here we present the genomic sequencing, molecular characterisation and genomic comparison of two A. baumannii strains belonging to two different sequence types (STs), one sporadic and one widely distributed in our region. METHODS: Whole-genome sequencing (WGS) of Ab42 and Ab376 was performed using Illumina MiSeq-I and the genomes were assembled with SPAdes. ARG-ANNOT, CARD-RGI, ISfinder, PHAST, PlasmidFinder, plasmidSPAdes and IslandViewer were used to analyse both genomes. RESULTS: Genome analysis revealed that Ab42 belongs to ST172, an uncommon ST, whilst Ab376 belongs to ST25, a widely distributed ST. Molecular characterisation showed the presence of two antibiotic resistance genes in Ab42 and nine in Ab376. No insertion sequences were detected in Ab42, however 22 were detected in Ab376. Moreover, two prophages were found in Ab42 and three in Ab376. In addition, a CRISPR-cas type I-Fb and two plasmids, one of which harboured an AbGRI1-like island, were found in Ab376. CONCLUSIONS: We present WGS analysis of twoA. baumannii strains belonging to two different STs. These findings allowed us to characterise a previously undescribed ST (ST172) and provide new insights to the widely studied ST25.

Combination of organic acids and low-dose gamma irradiation as antimicrobial treatment to inactivate Shiga toxin-producing Escherichia coli inoculated in beef trimmings: Lack of benefits in relation to single treatments.

The aim of this study was to assess the efficacy of lactic acid (LA), caprylic acid (CA), high- (HDI) and low- (LDI) dose gamma irradiation and LDI combined with LA or CA on the inactivation of a pool of Shiga toxin-producing Escherichia coli (STEC) strains inoculated on beef trimmings. The three most efficacious treatments were selected to study their effect on meat quality parameters and sensory attributes. The inoculum included five native STEC serogroups (O26, O103, O111, O145 and O157). The treatments applied were 0.5% LA, 0.04% CA, 0.5 kGy LDI, 2 kGy HDI, LDI+LA and LDI+CA. Beef trimmings were divided into two groups; one was inoculated with high (7 log CFU/g) and the other with low (1 log CFU/g) level of inoculum. Efficacy was assessed by estimating log reduction and reduction of stx- and eae-positive samples after enrichment, respectively. Results showed that treatments with organic acids alone were not effective in reducing STEC populations. For high inoculum samples, the most effective treatment was HDI followed by LDI+LA and LDI alone or combined with CA. For low inoculum samples, the most effective treatment was HDI followed by LDI alone or combined with organic acids. Concerning meat quality parameters and sensory attributes, irradiation treatments (LDI and HDI) caused minimal changes, while LDI+LA modified them significantly compared with the control. Therefore, based on our results, no benefits were observed after combining organic acids with gamma irradiation.

Comprehensive evaluation and implementation of improvement actions in bovine abattoirs to reduce pathogens exposure.

The slaughter process plays an important role in animal welfare, meat quality, safety and public health through the meat production chain. In this study, we performed a three-stage evaluation: I) comprehensive evaluation, II) implementation of improvement actions and III) verification of the success of the actions implemented in three abattoirs from Argentina during 2016-2018. Risk was estimated using two checklists, quantified on a 1-100 scale and classified as high (1-40), moderate (41-70) and low (71-100). In stages I and III, Salmonella spp., E. coli O157:H7 and non-O157 STEC were detected and isolated in samples from carcasses (n = 252), the environment (n = 252); head meat (n = 21) and viscera washing and chilling water (n = 105). Carcass samples were analyzed for mesophilic aerobic organisms, coliforms and E. coli enumeration. Of 201 water samples taken, 42.0-75.6 % were non-potable quality. After the implementation of improvement actions in stage II (building, processes, systems for water purification and training), the estimation of risk of contamination was reduced from high to moderate in all three abattoirs, the count of indicator microorganisms decreased in two abattoirs, and the presence of pathogens significantly decreased. Salmonella spp. was not isolated from any of the samples collected in two abattoirs. Isolation of E. coli O157:H7 decreased in carcass and was not isolated from viscera washing and chilling water. Isolation of non-O157 STEC decreased in carcass but not in environmental samples. Finally, 75.0-95.0 % of water samples were of potable quality. Although this was only the first step in the process of change and improvement of abattoirs, the assessment of the situation and the proposal of solutions to correct deviations in a joint effort with the health authorities helped to implement a work model for enhancing food safety before meat reaches consumers.

Comparación de tres técnicas para subtipificación molecular de Listeria monocytogenes / Comparison of three molecular subtyping techniques for Listeria monocytogenes

Abstract Listeria monocytogenes is a foodborne pathogen. The recent alert for L. monocytogenes in vegetables from Argentina warns about the importance of reinforcing its isolation, characterization and subtyping in food, clinical and environmental samples. The aim of the present study was to compare the discriminatory power of enterobacterial repetitive interpower; genic consensus polymerase chain reaction (ERIC-PCR), automated ribotyping and pulsed-field gel electrophoresis (PFGE) to subtype strains of L. monocytogenes isolated from Argentine meat and environmental samples. Simpson's Diversity Index (DI) was calculated on the basis of based on the dendrograms obtained in the by cluster analysis, showing the following discriminatory power: ApaI-PFGE (0.980), AscI-PFGE (0.966), ribotyping (0.912), ERIC-PCR (0.886). The ID values between ApaI- and AscI-PFGE and between ribotyping and ERIC-PCR were not significantly different. Of the three techniques evaluated, PFGE showed the highest discriminatory power. However, the subtyping techniques should be accompanied by effective food monitoring strategies and reliable clinical and epidemiological studies.

Resumen Listeria monocytogenes es un patógeno alimentario. La reciente alerta por la presencia de L. monocytogenes en vegetales en Argentina advierte sobre la importancia de reforzar el aislamiento, la caracterización y la subtipificación de esta bacteria en muestras clínicas de alimentos y ambientales. El objetivo del presente estudio fue comparar el poder discriminatorio de enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), la ribotipificación automatizada y la pulsed-field gel electrophoresis (PFGE) para subtipificar cepas de L. monocytogenes aisladas de carne y de muestras ambientales en Argentina. El índice de diversidad (ID) de Simpson, calculado a partir de los dendrogramas obtenidos en el análisis de agrupamiento, mostró los siguientes resultados: Apal-PFGE (0,980), AscI-PFGE (0,966), riboti-pado (0,912), ERIC-PCR (0,886). Los valores obtenidos no fueron significativamente diferentes al comparar entre Apal- y AscI-PFGE, ni entre ribotipadoy ERIC-PCR. De las técnicas evaluadas, la PFGE presentó el mayor poder discriminatorio. Sin embargo, las técnicas de subtipificación deberían acompañarse de estrategias de control de los alimentos efectivas y de estudios clínicos y epidemiológicos confiables.

Comparison of six commercial systems for the detection of non-O157 STEC in meat and vegetables.

Shiga toxin-producing Escherichia coli (STEC) are important pathogens transmitted by food that may cause severe illness in human beings. Thus, systems for STEC detection in food should have increasingly higher sensitivity and specificity. Here we compared six commercial systems for non-O157 STEC detection in meat and vegetables and determined their sensitivity, specificity and repeatability. A total of 46 samples (meat n = 23; chard n = 23) were experimentally contaminated with strains O26:H11, O45:H-, O103:H2, O111:NM, O121:H19 and O145:NM isolated in Argentina. Strain detection was confirmed by isolation according to ISO 13136:2012. Detection of the stx and eae genes in meat samples was highly satisfactory with all commercial kits, but only five had 100% sensitivity and specificity in chard. Of four kits evaluated for serogroup detection, three had 100% sensitivity and specificity, and one had 93.7% sensitivity and 100% specificity. All kits were adequate to analyze meat but not vegetable samples, and were not therefore validated for the latter matrix. The challenge for microbiology laboratories is to identify the advantages and disadvantages of the available kits for STEC detection in food based on a clear knowledge of the particular needs of each laboratory.

Inactivation of Shiga toxin-producing Escherichia coli in fresh beef by electrolytically-generated hypochlorous acid, peroxyacetic acid, lactic acid and caprylic acid.

Several studies have been conducted to verify the decontamination potential of electrolytically-generated hypochlorous acid, peroxyacetic acid, lactic acid and caprylic acid against Shiga toxin-producing Escherichia coli (STEC) in beef products. However, there is no consensus regarding their effectiveness. The aim of this study was to compare these four treatments under the same conditions and establish a ranking according to their effectiveness to inactivate STEC in fresh beef. Samples were inoculated with two levels of inoculum and rinsed for 15 s in 100 mL of antimicrobial solution treatment. Caprylic acid was the most effective treatment, followed by lactic acid and peroxyacetic acid. Electrolytically-generated hypochlorous acid had no effect. Sensory analysis showed no significant differences either in flavor or in color between samples treated with caprylic acid and reference samples. Caprylic acid appears to be an effective and viable alternative to conventional interventions frequently used for meat product decontamination.

Endocarditis infecciosa por bacilos gram negativos no HACEK. Experiencia en un centro de alta complejidad de la República Argentina (1998-2016) / Infective endocarditis due to non-HACEK gram-negative bacilli in a Level III cardiovascular center in Argentina (1998-2016)

Los bacilos gram negativos (BGN) que no pertenecen al grupo HACEK son una causa infrecuente de endocarditis infecciosa. Los aspectos epidemiológicos, diagnósticos y pronósticos de esta entidad son poco conocidos y la experiencia aún es limitada. Nuestros objetivos fueron analizar las características clínicas y microbiológicas de las endocarditis infecciosas (EI) por BGN no HACEK diagnosticadas en un centro de alta complejidad de Argentina en el período 1998-2016 y conocer su evolución hospitalaria, a fin de compararlas con las EI debidas a otros microorganismos.

Non-HACEK Gram-negative bacilli are a rare cause of infective endocarditis. Epidemiological, diagnostic and prognostic aspects of this entity are little known, and there is limited experience. The aim of this study was to analyze the clinical, microbiological and in-hospital outcomes of non-HACEK Gram negative bacilli endocarditis and to compare them with those due to other microorganisms.

Comparison of three molecular subtyping techniques for Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen. The recent alert for L. monocytogenes in vegetables from Argentina warns about the importance of reinforcing its isolation, characterization and subtyping in food, clinical and environmental samples. The aim of the present study was to compare the discriminatory power of enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), automated ribotyping and pulsed-field gel electrophoresis (PFGE) to subtype strains of L. monocytogenes isolated from Argentine meat and environmental samples. Simpson's Diversity Index (DI) was calculated on the basis of based on the dendrograms obtained in the by cluster analysis, showing the following discriminatory power: ApaI-PFGE (0.980), AscI-PFGE (0.966), ribotyping (0.912), ERIC-PCR (0.886). The ID values between ApaI- and AscI-PFGE and between ribotyping and ERIC-PCR were not significantly different. Of the three techniques evaluated, PFGE showed the highest discriminatory power. However, the subtyping techniques should be accompanied by effective food monitoring strategies and reliable clinical and epidemiological studies.

[Infective endocarditis due to non-HACEK gram-negative bacilli in a Level III cardiovascular center in Argentina (1998-2016)]. / Endocarditis infecciosa por bacilos gram negativos no HACEK. Experiencia en un centro de alta complejidad de la República Argentina (1998-2016).

Non-HACEK Gram-negative bacilli are a rare cause of infective endocarditis. Epidemiological, diagnostic and prognostic aspects of this entity are little known, and there is limited experience. The aim of this study was to analyze the clinical, microbiological and in-hospital outcomes of non-HACEK Gram negative bacilli endocarditis and to compare them with those due to other microorganisms.

Evaluation of decontamination efficacy of commonly used antimicrobial interventions for beef carcasses against Shiga toxin-producing Escherichia coli.

In Argentina, Shiga toxin producing Escherichia coli (STEC) serogroups O157, O26, O103, O111, O145 and O121 are adulterant in ground beef. In other countries, the zero-tolerance approach to all STEC is implemented for chilled beef. Argentinean abattoirs are interested in implementing effective interventions against STEC on carcasses. Pre-rigor beef carcasses were used to determine whether nine antimicrobial strategies effectively reduced aerobic plate, coliform and E. coli counts and stx and eae gene prevalence. These strategies were: citric acid (2%; automated), acetic acid (2%; manual and automated), lactic acid (LA 2%; manual and automated), LA (3%; automated), electrolytically-generated hypochlorous acid (400 ppm; manual), hot water (82 °C; automated) and INSPEXX (0.2%; automated). Automated application of 2% LA after 30-60-min aeration and 3% LA at 55 °C were the most effective interventions. Automated application was more effective than manual application. Decontamination of beef carcasses through automated application of lactic acid and hot water would reduce public health risks associated with STEC contamination.

Isolation and characterization of non-O157 Shiga toxin-producing Escherichia coli from beef carcasses, cuts and trimmings of abattoirs in Argentina.

Several foods contaminated with Shiga toxin-producing Escherichia coli (STEC) are associated with human diseases. Some countries have established microbiological criteria for non-O157 STEC, thus, the absence of serogroups O26, O45, O103, O104, O111, O121, and O145 in sprouts from the European Union or ground beef and beef trimmings from the United States is mandatory. While in Argentina screening for O26, O103, O111, O145 and O121 in ground beef, ready-to-eat food, sausages and vegetables is mandatory, other countries have zero-tolerance for all STEC in chilled beef. The aim of this study was to provide data on the prevalence of non-O157 STEC isolated from beef processed in eight Argentinean cattle slaughterhouses producing beef for export and local markets, and to know the non-O157 STEC profiles through strain characterization and genotypic analysis. Samples (n = 15,965) from 3,205 beef carcasses, 9,570 cuts and 3,190 trimmings collected between March and September 2014 were processed in pools of five samples each. Pools of samples (n = 3,193) from 641 carcasses, 1,914 cuts and 638 trimming were analyzed for non-O157 STEC isolation according to ISO/CEN 13136:2012. Of these, 37 pools of carcasses (5.8%), 111 pools of cuts (5.8%) and 45 pools of trimmings (7.0%) were positive for non-O157 STEC. STEC strains (n = 200) were isolated from 193 pools of samples. The most prevalent serotypes were O174:H21, O185:H7, O8:H19, O178:H19 and O130:H11, and the most prevalent genotypes were stx2c(vh-b) and stx2a/saa/ehxA. O103:H21 strain was eae-positive and one O178:H19 strain was aggR/aaiC-positive. The prevalence of non-O157 STEC in beef carcasses reported here was low. None of the non-O157 STEC strains isolated corresponded to the non-O157 STEC serotypes and virulence profiles isolated from human cases in Argentina in the same study period. The application of microbiological criteria for each foodstuff should be determined by risk analysis in order to have a stringent monitoring system. Likewise, zero-tolerance intervention measures should be applied in beef, together with GMP and HACCP. Further, collaborative efforts for risk assessment, management and communication are extremely important to improve the safety of foodstuffs.

Comprehensive Evaluation and Implementation of Improvement Actions in Butcher Shops.

Foodborne pathogens can cause acute and chronic diseases and produce a wide range of symptoms. Since the consumption of ground beef is a risk factor for infections with some bacterial pathogens, we performed a comprehensive evaluation of butcher shops, implemented improvement actions for both butcher shops and consumers, and verified the impact of those actions implemented. A comprehensive evaluation was made and risk was quantified on a 1-100 scale as high-risk (1-40), moderate-risk (41-70) or low-risk (71-100). A total of 172 raw ground beef and 672 environmental samples were collected from 86 butcher shops during the evaluation (2010-2011) and verification (2013) stages of the study. Ground beef samples were analyzed for mesophilic aerobic organisms, Escherichia coli and coagulase-positive Staphylococcus aureus enumeration. Salmonella spp., E. coli O157:H7, non-O157 Shiga toxin-producing E. coli (STEC), and Listeria monocytogenes were detected and isolated from all samples. Risk quantification resulted in 43 (50.0%) high-risk, 34 (39.5%) moderate-risk, and nine (10.5%) low-risk butcher shops. Training sessions for 498 handlers and 4,506 consumers were held. Re-evaluation by risk quantification and microbiological analyses resulted in 19 (22.1%) high-risk, 42 (48.8%) moderate-risk and 25 (29.1%) low-risk butcher shops. The count of indicator microorganisms decreased with respect to the 2010-2011 period. After the implementation of improvement actions, the presence of L. monocytogenes, E. coli O157:H7 and stx genes in ground beef decreased. Salmonella spp. was isolated from 10 (11.6%) ground beef samples, without detecting statistically significant differences between both study periods (evaluation and verification). The percentage of pathogens in environmental samples was reduced in the verification period (Salmonella spp., 1.5%; L. monocytogenes, 10.7%; E. coli O157:H7, 0.6%; non-O157 STEC, 6.8%). Risk quantification was useful to identify those relevant facts in butcher shops. The reduction of contamination in ground beef and the environment was possible after training handlers based on the problems identified in their own butcher shops. Our results confirm the feasibility of implementing a comprehensive risk management program in butcher shops, and the importance of information campaigns targeting consumers. Further collaborative efforts would be necessary to improve foodstuffs safety at retail level and at home.