Comparative resistome, mobilome, and microbial composition of retail chicken originated from conventional, organic, and antibiotic-free production systems.

The aim of this study was to investigate the microbial composition, and the profiles of antimicrobial resistance genes (ARGs, resistome) and mobile genetic elements (mobilome) of retail chicken carcasses originated from conventional intensive production systems (CO), certified antimicrobial-free intensive production systems (AF), and certified organic production systems with restricted antimicrobial use (OR). DNA samples were collected from 72 chicken carcasses according to a cross-sectional study design. Shot-gun metagenomics was performed by means of Illumina high throughput DNA sequencing followed by downstream bioinformatic analyses. Gammaproteobacteria was the most abundant bacterial class in all groups. Although CO, AF, and OR did not differ in terms of alpha- and beta-microbial diversity, the abundance of some taxa differed significantly across the groups, including spoilage-associated organisms such as Pseudomonas and Acinetobacter. The co-resistome comprised 29 ARGs shared by CO, AF and OR, including genes conferring resistance to beta-lactams (blaACT-8, 10, 13, 29; blaOXA-212;blaOXA-275 and ompA), aminoglycosides (aph(3')-IIIa, VI, VIa and spd), tetracyclines (tet KL (W/N/W and M), lincosamides (inu A,C) and fosfomycin (fosA). ARGs were significantly less abundant (P < 0.05) in chicken carcasses from AF and OR compared with CO. Regarding mobile genetic elements (MGEs), transposases accounted for 97.2% of the mapped genes. A higher abundance (P = 0.037) of MGEs was found in CO compared to OR. There were no significant differences in ARGs or MGEs diversity among groups according to the Simpson´s index. In summary, retail frozen chicken carcasses from AF and OR systems show similar ARGs, MGEs and microbiota profiles compared with CO, even though the abundance of ARGs and MGEs was higher in chicken carcasses from CO, probably due to a higher selective pressure.

Virulent potential of methicillin-resistant and methicillin-susceptible Staphylococcus pseudintermedius in dogs.

Staphylococcus pseudintermedius is a zoonotic pathogen responsible for several infectious diseases in pet animals, yet its pathogenic potential is not fully understood. Thus, this study aims to unravel the virulence profile of S. pseudintermedius from canine origin. Methicillin-resistant (MRSP) and methicillin-susceptible (MSSP) strains were isolated from different infection sites and their genotypic and phenotypic features were compared to determine the clinical implications of MRSP and MSSP strains. Bacterial identification was performed using MALDI-TOF and 16S-rDNA sequencing. In addition, we used multilocus sequence typing (MLST) for strains' sequence type (ST) determination and phylogenetic relationship. The strains were screened for toxin genes, including cytotoxins (lukS, lukF), exfoliative toxin (siet), enterotoxins (sea, seb, sec, secCanine, sel, sem, and seq) and toxic shock syndrome toxin (tst-1). In vitro phenotypic analyses assessing antimicrobial susceptibility profile, biofilm formation ability, and expression of extracellular matrix components were performed. The investigated S. pseudintermedius strains belong to 17 unique ST, most of which were classified as ST71. MSSP and MRSP strains shared siet, lukS, and lukF virulence markers. Our findings showed that some MSSP strains also harbored sel, seq, and sem enterotoxin genes, suggesting a more diverse virulence profile. All MRSP strains and 77% of MSSP strains were classified as multidrug resistant (MDR). Moreover, all investigated S. pseudintermedius strains showed strong biofilm formation ability. In summary, our findings highlight the wide spread of highly virulent and drug-resistant zoonotic S. pseudintermedius strains, being a potential concern for One Health issues.

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.

Critically Important Antimicrobial Resistance Trends in Salmonella Derby and Salmonella Typhimurium Isolated from the Pork Production Chain in Brazil: A 16-Year Period.

Knowledge about antimicrobial resistance in Salmonella is relevant due to its importance in foodborne diseases. We gathered data obtained over 16 years in the southern Brazilian swine production chain to evaluate the temporal evolution of halo for carbapenem, and the MIC for third-generation cephalosporins, fluoroquinolone, and polymyxin in 278 Salmonella Derby and Typhimurium isolates. All antimicrobial resistance assays were performed in accordance with EUCAST. To assess the diameter halo, we used a mixed linear model, and to assess the MIC, an accelerated failure time model for interval-censored data using an exponential distribution was used. The linear predictor of the models comprised fixed effects for matrix, serovar, and the interaction between year, serovar, and matrix. The observed halo diameter has decreased for ertapenem, regardless of serovars and matrices, and for the serovar Typhimurium it has decreased for three carbapenems. The MIC for ciprofloxacin and cefotaxime increased over 16 years for Typhimurium, and for Derby (food) it decreased. We did not find evidence that the MIC for colistin, ceftazidime, ciprofloxacin (Derby), or cefotaxime (food Typhimurium and animal Derby) has changed over time. This work gave an overview of antimicrobial resistance evolution from an epidemiological point of view and observed that using this approach can increase the sensitivity and timeliness of antimicrobial resistance surveillance.

Perception of poultry veterinarians on the use of antimicrobials and antimicrobial resistance in egg production.

This study aimed to describe the perception of veterinarians who work with commercial laying hens in the state of Rio Grande do Sul, Brazil, regarding the use of antibiotics and their possible impacts on animal, human, and environmental health. A descriptive epidemiological study was carried out through face-to-face or web conferencing interviews with the veterinarians that provide technical assistance at commercial laying hen operations. A standardized and structured questionnaire was developed based on the literature and expert opinion, which contained 1 opened and 40 closed questions. Conventional non-probabilistic sampling was used, based on an initial list of 15 veterinarians registered in the Poultry Production Association of Rio Grande do Sul, followed by the snowball technique. The acquisition of 26 contacts of veterinarians was accomplished, and 16 were interviewed. Through the answers obtained it was possible to verify that the interviewees' understanding regarding both the antibiotic resistance impact and the decision-making about the use of antibiotics seem to be linked to their practical experiences. Besides that, according to the veterinarians, farmers can acquire and administer the antimicrobials on their farms. Moreover, both farm storage and administration of lower doses of antibiotics than the recommended one could be contributing factors to resistant bacteria selection. Furthermore, controversially, the professionals believed that resistant bacteria can be transmitted to humans from eggs, but they said that there are no bacteria in eggs. Therefore, the veterinarians´ practices can be improved considering national and international guidelines on antimicrobial resistance to minimize the development of resistance. Finally, it is expected that the present results will contribute to a more complex discussion about antimicrobial resistance, helping to formulate public policies in the egg production industry.

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.

Antimicrobial resistance in commensal Escherichia coli and Enterococcus spp. isolated from pigs subjected to different antimicrobial administration protocols.

The antimicrobial resistance (AMR) in human and animal pathogens is a global concern, and antimicrobial use (AMU) is considered the most important driver for its increase. The aim of this study was to assess AMR in Escherichia coli and Enterococcus spp. in faecal samples of pigs subjected to four different AMU protocols from birth to finishing: G1, no in-feed antimicrobials; G2: a total average dose 6018 mg antimicrobials/pig; G3: a total average dose 8127 mg antimicrobials/pig; and G4: a total average dose 15,678 mg antimicrobials/pig. Faecal samples were collected at six time points and AMR was assessed in both bacteria. The microbiota composition was assessed by 16S rRNA sequencing. Minor differences on the microbiota profile was observed among groups, but a lower Firmicutes:Bacteroidetes ratio was noted in G4. Escherichia coli and Enterococcus spp. strains isolated from all groups showed a high level of multi-drug resistance (MDR). The amount of antimicrobials used was significantly positively associated with the probability of MDR in both bacteria. Approximately 43% of the variation in MIC90 for colistin could be explained by AMU, and a one-day increase in administration of colistin increased MIC90 by 0.05 µg mL-1. In conclusion, the results suggest that the higher the use of antimicrobials in farms, the higher the MDR frequency and resistance to the highest priority critically important antimicrobials for humans in commensal gut bacteria of pigs.

First reported genome of an mcr-9-mediated colistin-resistant Salmonella Typhimurium isolate from Brazilian livestock.

OBJECTIVES: To investigate the genetic context of colistin resistance in anmcr-9-harbouring Salmonella Typhimurium ST19 strain from swine in Brazil. METHODS: Minimum inhibitory concentrations (MIC) to colistin were determined by broth microdilution. Whole-genome sequencing was performed on an Illumina MiSeq system, followed by de novo genome assembly using SPAdes 1.13.1. The draft genome sequence was annotated in Prokka using KBase online server. Downstream analyses for resistome and plasmid detection were performed using online tools available at the Center for Genomic Epidemiology. The strain was typed in silico using MLST 2.0. Phylogenetic analysis involving 24 other genomes ofSalmonella Typhimurium ST19 and mcr-9-harbouring Salmonella Typhimurium isolated from humans, livestock and foodstuff in different regions was also performed. RESULTS: Assembly of the draft genome resulted in 5245 protein-coding sequences, 14 rRNAs, 83 tRNAs and a GC content of 51.81%. The strain was identified asSalmonella Typhimurium ST19 harbouring a 265.5-kb pN1566-2 plasmid carrying genes encoding resistance to colistin (mcr-9.1), aminoglycosides (aadA1), tetracycline [tet(C)] and sulfonamides (sul1). Our findings indicate that the Salmonella Typhimurium ST19 strain in this study showed low genetic variability compared with Salmonella Typhimurium ST19 isolated from swine and poultry in Brazil, and was less related to those reported in other countries. CONCLUSIONS: This is the first reported genome of a phenotypically colistin-resistantSalmonella Typhimurium harbouring the mcr-9 variant in Brazilian livestock. This genome will aid global investigations on epidemiological and evolutionary aspects of plasmid-mediated colistin resistance and the role of colistin-resistant Salmonella Typhimurium ST19 lineage as a zoonotic pathogen.

Investigation of Listeria monocytogenes, Salmonella enterica and Yersinia enterocolitica in pig carcasses in Southern Brazil / Pesquisa de Listeria monocytogenes, Salmonella enterica and Yersinia enterocolitica em carcaças de suínos no sul do Brasil

The intensification of pig production and advances in the sanitary control of herds profoundly changed the profile of risk attributed to pork consumption. In the actual scenario, most microorganisms related to macroscopic lesions observed in the post mortem inspection are not transmitted by food, while foodborne bacteria of importance to consumer health do not cause macroscopic lesions. In Brazil, the "Ministério da Agricultura, Pecuária e Abastecimento" requested a scientific opinion on the prioritizing of pathogens potentially transmitted by unprocessed pork. After conducting a qualitative risk assessment, only Salmonella enterica was classified as of high risk to consumers. The present study was part of the validation step of the risk assessment and aimed to investigate the frequency of S. enterica, Yersinia enterocolitica and Listeria monocytogenes and hygienic-sanitary indicators in pig carcasses of pigs rose under intensive production and slaughtered under the Federal Inspection System in three slaughterhouses located in Southern Brazil. Additionally, the antimicrobial resistance profile of the isolated pathogens was also investigated. A total of 378 carcasses were sampled by superficial sponges before the chilling step in three slaughterhouses. Samples were investigated for the presence of the three aforementioned pathogens and subjected to enumeration of Colony Formation Units (log CFU.cm-1) of total aerobic mesophiles (TAM) and Enterobacteriaceae. Salmonella strains were tested by disc diffusion test for resistance to eleven antimicrobials. There were significantly statistical differences (p<0.0001) on the median counts of both indicators between the slaughterhouses. The median of TAM was very close for Slaughterhouses A and B: 1.573 log CFU.cm-1 and 1.6014 log CFU.cm-1, respectively. While in Slaughterhouse C, a higher TAM median was detected (2.216 log CFU.cm-1). A similar profile was observed regarding to Enterobacteriaceae, and medians were calculated as follow: -0.426 log CFU.cm-1 in Slaughterhouse A; 0.2163 log CFU.cm-1 in B; and 0.633 log CFU.cm-1 in C. Regarding the pathogens investigated, L. monocytogenes was not detected and only one carcass from Slaughterhouse C was positive for Y. enterocolitica. Thus, the results suggest a very low prevalence of L. monocytogenes and Y. enterocolitica in the sampled population. A total of 65 (17.2%) carcasses were positive for S. enterica, with a difference in frequencies between slaughterhouses and slaughter days. The prevalence of Salmonella positive carcasses was higher in the Slaughterhouse C (25.4%; CI 95% 19-32%) in comparison with A (9.5%; CI 95% 9-14%) and B (18.3%; CI 95% 12-24%). There was no significantly statistical association between Enterobacteriaceae counts and Salmonella isolation on carcass surface (p=0.69). The slaughtering day, nested within the slaughterhouse, explains 31.3% of Salmonella prevalence variability. S. Typhimurium (38.1%) was the most prevalent, followed by S. Infantis (30.1%). Among the 61 Salmonella strains tested for resistance to antimicrobials, 18 (31.6%) were full-susceptible. No strain displayed resistance to azithromycin, ceftazidime, cefotaxime and meropenem. The highest resistance frequency was displayed to tetracycline (54.1%), followed by ampicillin (50.82%), nalidixic acid (42.62%) and chloramphenicol (42.62). Multi-resistance was detected in 52.54% of the, strains. In conclusion, S. enterica is more prevalent in pre-chill pig carcasses than Y. enterocolitica and L. monocytogenes and thus should be prioritized in monitoring and control programs at slaughter. Salmonella serovars varied among slaughterhouses and present significant differences in their resistance to antimicrobials. Slaughterhouses that present higher medians of TAM or Enterobacteriaceae in a monitoring period may have higher S. enterica prevalences as well. However, there is a high variation of S. enterica prevalence among slaughter days, which cannot be always related to the hygienic indicators counts observed on a given day.(AU)

A intensificação da produção de suínos e os avanços no controle sanitário dos rebanhos alterou de forma importante o perfil de risco do consumo de carne suína. No cenário atual, a maioria dos microrganismos causadores de lesões macroscópicas detectáveis na inspeção post mortem não são transmissíveis por alimentos, enquanto bactérias de importância como causadoras de doenças transmitidas por alimentos não causam lesões macroscópicas. No Brasil, o Ministério da Agricultura, Pecuária e Abastecimento solicitou uma opinião científica sobre a priorização de patógenos potencialmente transmitidos pela carne suína in natura. Após conduzir uma avaliação de risco qualitativa, apenas Salmonella enterica foi classificada como de alto risco para o consumidor. O presente estudo foi parte da etapa de validação da avaliação de risco e objetivou: investigar a frequência de S. enterica, Yersinia enterocolitica e Listeria. monocytogenes; e enumerar indicadores higiênico-sanitários em carcaças de suínos abatidos sob inspeção federal em frigoríficos dedicados ao abate de suínos sob sistema intensivo de criação no sul do Brasil. Além disso, o perfil de resistência a antimicrobianos dos patógenos isolados foi investigado. A superfície de um total de 378 carcaças foi amostrada por esponjas, na etapa de pré-resfriamento em três matadouros frigoríficos (A, B, C). As amostras foram investigadas quanto à presença dos três patógenos acima mencionados e quanto à enumeração de Unidades Formadoras de Colônia (log UFC.cm-1) de mesófilos aeróbios totais (MAT) e Enterobacteriaceae. As cepas isoladas de Salmonella foram testadas quanto à resistência a onze antimicrobianos pela técnica de disco difusão. As medianas de contagem de ambos os indicadores apresentaram diferença significativa (p<0,0001) entre matadouros-frigoríficos. A mediana de MAT foi bastante próxima para A e B (1,573 log UFC.cm-1 e 1,6014 log UFC.cm-1, respectivamente), enquanto em C uma mediana de MAT mais elevada foi determinada (2,216 log CFU.cm-1). Um perfil semelhante foi observado em relação a Enterobacteriaceae, sendo as medianas calculadas para A, B e C, respectivamente: -0,426 log CFU.cm-1; 0,2163 log UFC.cm-1; e 0,633 log UFC.cm-1. Em relação aos patógenos investigados, L. monocytogenes não foi detectada e apenas uma carcaça, do Matadouro C, foi positiva para Y. enterocolitica. Portanto, os resultados sugerem uma prevalência muito baixa desses patógenos na população amostrada. Em um total de 65 (17,2%) carcaças houve isolamento de S. enterica, com diferença nas frequências observadas entre matadouros e dias de abate. A prevalência de carcaças positivas para S. enterica foi maior no Matadouro C (25,4%; IC95% 19-32%) em comparação com A (9,5%; IC95% 9-14%) e B (18,3%; IC95% 12-24%). Não houve associação estatística entre o número de Enterobacteriaceae e o isolamento de S. enterica na superfície das carcaças (p=0,69). O dia de abate agrupado por frigorífico explica 31,3% da variação na prevalência de Salmonella. O sorovar mais frequente de S. enterica foi Typhimurium (38,1%) seguido de S. Infantis (30,1%). Entre as 61 cepas de S. enterica testadas quanto à resistência a antimicrobianos, 18 (31,6%) foram totalmente suscetíveis aos antimicrobianos testados. Nenhuma cepa apresentou resistência a azitromicina, ceftazidima, cefotaxima e meropenem. As maiores frequências de resistência foram demonstradas contra tetraciclina (54,1%), ampicilina (50,8%), ácido nalidíxico (42,62%) e cloranfenicol (42,62%). Em 52,54% das cepas foi detectada multi-resistência. Em conclusão, S. enterica é mais prevalente em carcaças suínas no pré-resfriamento do que Y. enterocolitica e L. monocytogenes. Portanto, S. enterica deve ser priorizada em programas de monitoramento e controle ao abate. Os sorovares de Salmonella variam entre matadouros e apresentam diferenças significativas na resistência a antimicrobianos. Matadouros de suínos que apresentam medianas de MAT e Enterobacteriaceae num período de monitoramento podem apresentar também prevalências mais de altas de presença de S. enterica. Entretanto, há uma alta variabilidade na frequência de S. enterica entre dias de abate, e nem sempre há relação entre essa frequência e a contagem de indicadores higiênico-sanitários determinados num determinado dia.(AU)

Listeria monocytogenes in sliced cheese and ham from retail markets in southern Brazil.

The aims of this study were to evaluate the occurrence of Listeria monocytogenes and Salmonella spp. in sliced cheese and ham from retail markets in southern Brazil, as well as to perform molecular characterization and to assess the antimicrobial resistance profile of the isolates. Samples (n = 160) of sliced cheese and ham were collected at retail level from the city of Pelotas, Brazil. The isolation of L. monocytogenes and Salmonella spp. was performed and the isolates were confirmed by PCR, submitted to antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). Listeria monocytogenes was found in 9.4% (15/160) of the samples. All L. monocytogenes isolates were positive for the prs, inlA, inlC and inlJ genes. Salmonella spp. was not isolated. Regarding the antimicrobial susceptibility, one (6.6%) L. monocytogenes isolate was resistant to streptomycin and four (26.6%) to clindamycin. Macrorestriction analysis with ApaI and AscI enzymes yielded two major PFGE groups I and II. All L. monocytogenes isolates showed virulence genes, and some of them were resistant to clinically used antimicrobials, representing a risk to public health. Moreover, PFGE patterns with high similarity were visualized in L. monocytogenes isolates at different times, demonstrating adaptability of the pathogen at retail level in the region.

Antimicrobial susceptibility profile of historical and recent Brazilian pig isolates of Pasteurella multocida / Perfil de suscetibilidade a antimicrobianos de isolados históricos e contemporâneos de Pasteurella multocida de suínos no Brasil

Pasteurella (P.) multocida is the causative agent of pneumonic pasteurellosis in swine, which is commonly associated with the final stages of enzootic pneumonia or porcine respiratory disease complex. Although this syndrome is one of the most common and important diseases of pigs, data on antimicrobial susceptibility of P. multocida isolates are uncommon in Brazil. Therefore, the present study was carried out to determine and to compare antimicrobial susceptibility profile of Brazilian P. multocida isolated from pigs with lesions of pneumonia or pleuritis during two-time periods. Historical isolates (period of 1981 to 1997; n=44) and recent isolates (period of 2011 to 2012; n=50) were used to determine the MIC of amoxicillin, enrofloxacin, florfenicol and tetracycline by microbroth dilution. Florfenicol had the lowest level of resistance for both historical and recent isolates (0% and 6%, respectively), while tetracycline had the highest (20.5% and 34%, respectively). Multi-drug resistance (MDR) to amoxicillin/florfenicol/tetracycline was observed in 6% of recent isolates. There was a significant increase (p˂0.05) in resistance for amoxicillin and enrofloxacin in recent isolates compared with historic isolates (3.8% and 18%, respectively), most likely due to the selective pressure of antimicrobial usage to treat and prevent P. multocida infections. The results of this study showed an increase of isolates resistant to important drugs used in treatment of P. multocida infections in pigs, demonstrating the need for the implementation of rational use of antimicrobials in Brazilian swine industry.(AU)

Pasteurella (P.) multocida é o agente da pasteurelose pneumônica em suínos, a qual é comumente associada com o estágio final da pneumonia enzoótica suína ou complexo das doenças respiratórias dos suínos. Apesar de ser uma das doenças mais comuns e importantes na suinocultura, dados sobre suscetibilidade antimicrobiana de isolados de P. multocida são raros no Brasil. Dessa forma, o presente estudo foi realizado para determinar e comparar o perfil de suscetibilidade de isolados de P. multocida de suínos com lesões de pneumonia ou pleurite no Brasil durante dois períodos. Isolados históricos (período de 1981 a 1997; n=44) e contemporâneos (período de 2011 a 2012; n=50) foram usados para determinar a concentração inibitória mínima (CIM) de amoxicilina, enrofloxacina, florfenicol e tetraciclina através do teste de microdiluição em caldo. Florfenicol apresentou o menor nível de resistência para ambos os isolados históricos e contemporâneos (0% e 6%, respectivamente), enquanto que tetraciclina apresentou o maior nível de resistência (20.5% e 34%, respectivamente). Resistência a múltiplos antimicrobianos (amoxicilina, florfenicol e tetraciclina) foi observada em 6% dos isolados recentes. Foi observado aumento significativo (p˂0.05) na resistência a amoxicilina e enrofloxacina em isolados recentes comparado com isolados históricos (3.8% e 18%, respectivamente), provavelmente devido à pressão de seleção de antimicrobianos usados no tratamento e prevenção de infecções causadas por P. multocida. Os resultados deste trabalho demostraram o aumento de isolados resistentes a importantes drogas utilizadas no tratamento de infecções causadas por P. multocida em suínos, evidenciando a necessidade da implementação do uso racional de antimicrobianos na suinocultura brasileira.(AU)

Isolation of Salmonella Typhimurium, Listeria monocytogenes and coagulase-positive Staphylococcus from salami sold at street fairs in Porto Alegre, Brazil / Isolamento de Salmonella Typhimurium, Listeria monocytogenes e Staphylococcus coagulase positiva de salames comercializados em feiras livres de Porto Alegre, Brasil

Salami is a ready-to-eat (RTE) product frequently purchased at street fairs in Porto Alegre. Salmonella enterica, Listeria monocytogenes, and coagulase-positive Staphylococcus (CPS) are important causes of foodborne disease and can be transmitted through the consumption of RTE foods. The aim of this study was to evaluate the presence of these pathogens in salami sold at street fairs. Ninety salami samples from three commercial brands available at street fairs were analyzed by routine bacteriological methods for Salmonella spp. and Listeria spp., as well as enumeration of CPS. In addition, two samples from each commercial brand were analyzed for water activity (aw). Samples of brand A showed aw values (0.938 and 0.942) above those set by the legislation, while brand B (0.849 and 0.860) and brand C (0.826 and 0.854) were compliant. Microbiological analyses showed that 67.7% were negative to all investigated bacteria. Salmonella Typhimurium was isolated from 4.4% (4/90) of salami samples, all from commercial brand A. ­Listeria monocytogenes was detected in 3.3% (3/90) of samples, from commercial brands B and C. Moreover, 7.7% (7/90) of samples contained CPS populations non-compliant with legislation. Although the great majority of salami sold at street fairs of Porto Alegre was compliant with standards, S. enterica, L. monocytogenes, and CPS ≥ 5 × 103 cfu.g-1 could be found in this RTE product. Therefore, control measures in the processing industry and consumer's education about foodborne illness prevention should be maintained.(AU)

Salame é um alimento pronto para o consumo frequentemente adquirido pela população em feiras livres de Porto Alegre. Salmonella enterica, Listeria monocytogenes e Staphylococcus coagulase positiva são importantes causas de doenças transmitidas por alimentos e podem ser veiculadas por alimentos prontos para o consumo. O objetivo desse estudo foi avaliar a presença desses patógenos em salames vendidos em feiras livres. Noventa amostras de salame pertencentes a três marcas comerciais foram analisados por métodos bacteriológicos de rotina quanto à presença de Salmonella spp. e Listeria spp., bem como enumeração de Staphylococcus coagulase positiva (SCP). Além disso, foi determinada a Atividade de Água (aw) de duas amostras de cada marca comercial. Amostras da marca A apresentaram valores de aw (0,938 e 0,942) acima do permitido na legislação, enquanto as amostras da marca B (0,849 e 0,860) e C (0,826 e 0,854) não violaram esse parâmetro. A análise microbiológica demonstrou que 67,7% das amostras foram negativas para todas as bactérias investigadas. Salmonella Typhimurium foi isolada de 4,4% (4/90) das amostras de salame, todas da marca comercial A. Listeria monocytogenes foi detectada em 3,3% (3/90) das amostras das marcas B e C. Além disso, 7,7% (7/90) das amostras apresentaram SCP acima do número permitido pela legislação. Apesar da grande maioria dos salames comercializados em feiras livres estarem de acordo com a legislação, S. enterica, L. monocytogenes e SCP ≥ 5 × 103 cfu.g-1 podem estar presentes nesse alimento pronto para o consumo. Dessa forma, o controle nas indústrias e a educação dos consumidores sobre a prevenção de doenças transmitidas por alimentos devem ser mantidos.(AU)

Bacillus cereus as the main casual agent of foodborne outbreaks in Southern Brazil: data from 11 years.

Foodborne diseases are a global concern. In Brazil, the most prevalent pathogen found in foodborne outbreaks is Salmonella sp. (14.4%), followed by Staphylococcus aureus (7.7%), Escherichia coli (6.5%), and Bacillus cereus (3.1%). With the aim to perform a regional detailed analysis of foodborne intoxication, we analyzed 253 outbreaks' profile reports to Food Surveillance team of the General Secretariat of Health Surveillance of Porto Alegre, Rio Grande do Sul State, between 2003 and 2013. In contrast to what was most notified in Brazil, in Porto Alegre the main outbreak agent identified was Bacillus cereus (32.2%) and, based on the patient symptoms, most cases were linked to enterotoxin production. The majority of the outbreaks were linked to the ingestion of food containing cereals or sauces poorly kept at environment temperature during the stock or preparation. We believe that, due to the compulsory use of pasteurized eggs in our city, Salmonella sp. outbreaks are less important here.

Bacillus cereus as the main casual agent of foodborne outbreaks in Southern Brazil: data from 11 years / Bacillus cereus como principal agente etiológico em surtos de intoxicação alimentar no Sul do Brasil: dados de 11 anos / Bacillus cereus como el principal agente causante de brotes de enfermedades alimentarias en el Sur de Brasil: datos de 11 años

Foodborne diseases are a global concern. In Brazil, the most prevalent pathogen found in foodborne outbreaks is Salmonella sp. (14.4%), followed by Staphylococcus aureus (7.7%), Escherichia coli (6.5%), and Bacillus cereus (3.1%). With the aim to perform a regional detailed analysis of foodborne intoxication, we analyzed 253 outbreaks' profile reports to Food Surveillance team of the General Secretariat of Health Surveillance of Porto Alegre, Rio Grande do Sul State, between 2003 and 2013. In contrast to what was most notified in Brazil, in Porto Alegre the main outbreak agent identified was Bacillus cereus (32.2%) and, based on the patient symptoms, most cases were linked to enterotoxin production. The majority of the outbreaks were linked to the ingestion of food containing cereals or sauces poorly kept at environment temperature during the stock or preparation. We believe that, due to the compulsory use of pasteurized eggs in our city, Salmonella sp. outbreaks are less important here.

As doenças de veiculação alimentar representam um problema de ordem global. No Brasil, o patógeno mais prevalente em surtos de intoxicação alimentar é a Salmonella sp. (14,4%), seguido pelo Staphylococcus aureus (7,7%), Escherichia coli (6,5%) e Bacillus cereus (3,1%). Com o objetivo de realizar uma análise regional detalhada da intoxicação alimentar, examinamos os relatórios de 253 surtos, notificados à equipe de Vigilância de Alimentos da Coordenadoria Geral de Vigilância em Saúde de Porto Alegre, Rio Grande do Sul, entre 2003 e 2013. Ao contrário do que é notificado no Brasil, em Porto Alegre, identificamos como principal agente etiológico, o Bacillus cereus (32,2%) e de acordo com os sintomas dos pacientes, a maioria dos casos esteve associada à produção de enterotoxina. A maioria dos surtos foram associados à ingestão de alimentos que continham cereais ou molhos mantidos à temperatura ambiente durante o armazenamento ou preparação. O uso compulsório de ovos pasteurizados em Porto Alegre pode explicar a relativa escassez de surtos de Salmonella sp. no município.

Las enfermedades transmitidas a través de la comida son una preocupación de carácter global. En Brasil, el patógeno encontrado de forma más prevalente en los brotes de epidemias alimentarias es la Salmonella sp. (14,4%), seguida por el Staphylococcus aureus (7,7%), Escherichia coli (6,5%), y el Bacillus cereus (3,1%). Con el fin de realizar un análisis regional detallado de los brotes de infecciones alimentarias, analizamos el perfil de 253 brotes, registrados por el servicio de Vigilancia Alimentaria de la Secretaría General de Vigilancia en Salud de Porto Alegre, Rio Grande do Sul, entre 2003 y 2013. En contraste con lo que fue más notificado en Brasil, en Porto Alegre el principal agente causante de brotes fue identificado como el Bacillus cereus (32.2%) y, basado en los síntomas del paciente, la mayoría de los casos estaban relacionados con la producción de la enterotoxina. Muchos de los brotes estaban relacionados con la ingesta de comida que contenía cereales o salsas mal conservadas a temperatura ambiente durante el almacenaje o la preparación. Creemos que, debido a la utilización obligatoria de huevos pasteurizados en nuestra ciudad, los brotes de Salmonella sp. son menos relevantes aquí.

Genotyping and antimicrobial resistance in Escherichia coli from pig carcasses / Genotipagem e resistência antimicrobiana de Escherichia coli em carcaças suínas

The increasing antimicrobial resistance observed worldwide in bacteria isolated from human and animals is a matter of extreme concern and has led to the monitoring of antimicrobial resistance in pathogenic and commensal bacteria. The aim of this study was to evaluate the antimicrobial resistance profile of Escherichia coli isolated from pig carcasses and to assess the occurrence of relevant resistance genes. A total of 319 E. coli isolates were tested for antimicrobial susceptibility against different antimicrobial agents. Moreover, the presence of extended-spectrum β-lactamase (ESBL) and inducible ampC-β-lactamase producers was investigated. Eighteen multi-resistant strains were chosen for resistance gene detection and PFGE characterization. The study showed that resistance to antimicrobials is widespread in E. coli isolated from pig carcasses, since 86.2% of the strains were resistant to at least one antimicrobial and 71.5% displayed multi-resistance profiles. No ampC-producing isolates were detected and only one ESBL-producing E. coli was identified. Genes strA (n=15), floR (n=14), aac(3)IVa (n=13), tetB (n=13), sul2 (n=12), tetA (n=11), aph(3)Ia (n=8) and sul3 (n=5) were detected by PCR. PFGE analysis of these multi-resistant E. coli strains showed less than 80% similarity among them. We conclude that antimicrobial multi-resistant E. coli strains are common on pig carcasses and present highly diverse genotypes and resistance phenotypes and genotypes.(AU)

O incremento de resistência frente aos antimicrobianos, observado em bactérias isoladas de humanos e animais, tem sido motivo de preocupação mundial e levado ao monitoramento dos perfis de resistência em bactérias patogênicas e comensais. O objetivo desse estudo foi avaliar o perfil de resistência em Escherichia coli isolada de carcaças suínas e descrever a ocorrência de alguns genes de resistência relevantes. Um total de 319 isolados de E. coli foi testado quanto à suscetibilidade frente a diversos antimicrobianos. A presença de isolados produtores de β-lactamases (ESBL) de espectro estendido e β-lactamase induzível do tipo ampC foi também investigada. Dezoito cepas multirresistentes foram escolhidas para investigação de genes de resistência e caracterização por macro-restrição (PFGE). Os resultados demonstraram que a resistência a antimicrobianos está disseminada, pois 86,2% dos isolados de E. coli foram resistentes ao menos a um antimicrobiano e 71,5% apresentaram perfil de multirresistência. Uma cepa de E. coli produtora de ESBL e nenhuma produtora de ampC induzível foram identificadas. Os genes strA (n=15); floR (n=14);aac(3)-IVa (n=13); tetB (n=13); sul2 (n=12); tetA (n=11); aph(3')Ia (n=8); sul3 (n=5) foram detectados por PCR. A análise de PFGE demonstrou que cepas de E. coli multirresistentes apresentaram similaridade inferior a 80% entre si. Concluiu-se que cepas multirresistentes de E. coli são frequentes em carcaças de suínos e apresentam uma alta diversidade genotípica, bem como de fenótipos e genes de resistência.(AU)

Caracterização anatomopatológica e bacteriológica em frangos de corte condenados totalmente por colibacilose sob Serviço de Inspeção Federal / Pathological and bacteriological characterization on broilers totally condemned due to colibacillosis under the control of the Federal Inspection Service

A colibacilose é a principal causa infecciosa de condenação total de carcaça em frangos de corte no sul do Brasil. Esse trabalho tem por objetivo determinar o grau de concordância entre a condenação total por colibacilose de frangos de corte abatidos em estabelecimento sob Serviço de Inspeção Federal (SIF) com o diagnóstico anatomopatológico e bacteriológico. O estudo foi realizado com 45 frangos de corte condenados totalmente por colibacilose (caso) e seus respectivos 45 controles (frangos sem lesões). Em todos os frangos condenados pelo SIF havia lesões macroscópicas e, nos controles não se observou. Através do teste Kappa-Cohen´s essas duas variáveis apresentaram concordância quase perfeita. As aves condenadas apresentaram lesões em fígado (27/45); em fígado e sacos aéreos (11/45); em fígado e coração (2/45); fígado, sacos aéreos e coração (2/45); fígado, sacos aéreos e oviduto (1/45); fígado, sacos aéreos, coração e tecido subcutâneo (1/45); e fígado, sacos aéreos, oviduto e baço (1/45). Observou-se concordância quase perfeita entre condenação e lesão hepática. Histologicamente, em 41 casos e 12 controles observaram-se lesões, sendo os mais frequentes hepatite necrosante aleatória, bronquite fibrino-heterofílica, pericardite aguda e traqueíte linfoplasmocitária. Nas aves com hepatite identificou-se E. coli, Enterococcus sp. e Streptococcus sp. (10/38) e, nas aves com bronquite ou broncopneumonia isolou-se Escherichia coli e Staphylococcus coagulase positiva (9/14). O PCR em tempo real e a imuno-histoquímica para Mycoplasma gallisepticum e M. synoviae foram negativos. Nos casos de condenação total por colibacilose o fígado foi o principal órgão acometido, portanto, o critério de condenação deveria ser revisto, sugerindo condenação por hepatite nesses casos, já que outras bactérias podem causar hepatite, como foi demonstrado nesse estudo.(AU)

Colibacillosis is the main infectious cause of total carcass condemnation in broilers in southern Brazil. This study aims to determine the degree of agreement between the total carcass condemnation for colibacillosis in broilers slaughtered in establishments under Federal Inspection Service (SIF) with the pathological and bacteriological diagnosis. The study was conducted with 45 broilers totally condemned by colibacillosis (case) and theirs 45 respective controls (chickens without lesions). All broilers condemned had gross lesions and the controls had not. The Kappa-Cohen's test showed that these two variables had almost perfect agreement. Broilers condemned showed lesions in liver (27/45); liver and air sacs (11/45); liver and heart (2/45); liver, heart and air sacs (2/45); liver, air sacs and oviduct (1/45); liver, air sacs, heart and subcutaneous (1/45); and liver, air sacs, oviduct and spleen (1/45). There is almost perfect agreement between carcass condemnation and liver damage. Histologically, in 41 cases and 12 controls were observed lesions, the most frequent diagnoses were random necrotizing hepatitis, fibrinous-heterophilic bronchitis, acute pericarditis and lymphoplasmacytic tracheitis. In hepatitis cases was isolated Escherichia coli, Enterococcus sp. and Streptococcus sp. (10/38) and in bronchitis or bronchopneumonia E. coli and coagulase positive Staphylococcus (9/14). The polymerse chain reaction (PCR) and immunohistochemistry (IHC) tests for Mycoplasma gallisepticum and M. synoviae were negative. In cases of total carcass condemnation by Colibacillosis the liver was the main organ affected. Therefore, the condemnation criteria should be revised, suggesting conviction for hepatitis in these cases, because other bacteria can cause hepatitis, as demonstrated in this study.(AU)

Antimicrobial resistance profiles of Staphylococcus aureus clusters on small dairy farms in southern Brazil / Perfil de resistência a antimicrobianos de grupos clonais de Staphylococcus aureus isolados de pequenas propriedades leiteiras do sul do Brasil

In intensive dairy farming, persistent intramammary infection has been associated with specific Staphylococcus (S.) aureus strains, and these strains may be resistant to antimicrobials. The objective of this study was to evaluate the antimicrobial resistance phenotypes of S. aureus isolates and to assess the distribution and the persistence of clonal groups in small dairy herds of southern Brazil. Milk samples were collected from all lactating cows from 21 dairy farms over a two-year period, totaling 1,060 samples. S. aureus isolates were tested for susceptibility to thirteen antimicrobials using the disk diffusion method. The total DNA of the isolates was subjected to SmaI digestion followed by pulsed-field gel electrophoresis (PFGE). Banding patterns differing by ≤4 bands were considered members of a single PFGE cluster. The frequency of S. aureus isolation ranged from 3.45% to 70.59% among the 17 S. aureus-positive herds. Most S. aureus isolates (87.1%) were susceptible to all antimicrobials; resistance to penicillin (18.2%) was the most frequently observed. The 122 isolates subjected to macrorestriction analysis were classified into 30 PFGE-clusters. Among them, only 10 clusters were intermittent or persistent over the two-year period. The majority (93.6%) of isolates belonging to persistent and intermittent clusters were susceptible to all tested antimicrobials. S. aureus intramammary colonization in small dairy farms of southern Brazil is most frequently caused by sporadic PFGE clusters, although some persistent clusters can arise over time. Both sporadic and persistent isolates were highly susceptible to antimicrobials.(AU)

A infecção intramamária persistente em bovinos leiteiros tem sido associada com estirpes de Staphylococcus (S.) aureus específicos, os quais podem ser resistentes a antimicrobianos. Os objetivos deste estudo foram avaliar os fenótipos de resistência aos antimicrobianos de isolados de S. aureus e a distribuição e persistência de grupos clonais em pequenos rebanhos leiteiros do sul do Brasil. As amostras de leite foram coletadas de todas as vacas em lactação de 21 propriedades leiteiras, ao longo de um período de dois anos, perfazendo um total de 1.060 amostras. Isolados de S. aureus foram testados quanto à resistência frente a treze antimicrobianos, pelo método de disco-difusão. O DNA total dos isolados foi clivado com a enzima Smal e submetido a eletroforese em gel de campo pulsado (PFGE). Padrões de bandas diferentes por ≤4 bandas foram considerados como pertencentes ao mesmo grupo clonal. A freqüência de S. aureus variou de 3,45% até 70,59%, entre os 17 rebanhos com isolamento positivo de S. aureus. A maioria dos isolados de S. aureus (87,1%) foi suscetível a todos os antimicrobianos; resistência à penicilina (18,2%) foi a mais freqüentemente observada. Os 122 isolados submetidos à análise de macrorestrição foram classificados em 30 grupos clonais de PFGE. Entre eles, apenas dez grupos clonais foram intermitentes ou persistentes ao longo do período de dois anos. A maioria (93,6%) dos isolados pertencentes a grupos clonais persistentes e intermitentes foram suscetíveis a todos os antimicrobianos testados. Concluiu-se que a colonização intramamária em bovinos de pequenas propriedades leiteiras do Sul do Brasil é mais frequentemente causada por grupos clonais esporádicos de S. aureus, embora alguns grupos clonais persistentes possam ocorrer ao longo do tempo. Em ambos os grupos clonais os isolados foram majoritariamente suscetíveis a antimicrobianos.(AU)

Antimicrobial resistance and class 1 integron-associated gene cassettes in Salmonella enterica serovar Typhimurium isolated from pigs at slaughter and abattoir environment.

Forty-five multi-resistant Salmonella enterica subsp. enterica serovar (S.) Typhimurium isolates obtained at five pig abattoirs in Southern Brazil were characterized. Their relatedness was determined by XbaI-macrorestriction analysis. Resistance genes, integrons and plasmid-mediated quinolone resistance genes (PMQR) were investigated by PCR. Amplicons for the variable part of class 1 integrons and the quinolone resistance-determining regions (QRDR) were sequenced. Plasmids were characterized by conjugation assays and replicon typing. Eighteen XbaI-macrorestriction patterns and 19 plasmid profiles were seen. Resistance to ampicillin (blaTEM), chloramphenicol (catA1 and floR), streptomycin (strA-strB), streptomycin/spectinomycin (aadA variants), sulphonamides (sul1, sul2, sul3) and tetracyclines [tet(A) and tet(B)] were commonly found. A trimethoprim resistance gene, dfrA8, was identified on a 100-kb plasmid. Single substitutions in the QRDR of GyrA but no PMQR genes were found. Twenty-five isolates carried class 1 integrons with an aadA23 gene cassette or unusual class 1 integrons with a dfrA12-orfF-aadA27 gene cassette array. Both integrons were found on large conjugative plasmids. Salmonella plasmid-located virulence genes spvR, spvA, spvB, rck and pefA were found on an IncFIB resistance plasmid. Hybrid virulence-resistance plasmids or plasmids harbouring class 1 integrons may play a role in the maintenance and dissemination of antimicrobial resistance among S. Typhimurium in this pig production system.

Effect of slaughterhouse and day of sample on the probability of a pig carcass being Salmonella-positive according to the Enterobacteriaceae count in the largest Brazilian pork production region.

Sources of contamination of carcasses during slaughter include infected pigs as well as environmentally related sources. There are many microbial indicators that can be used in the processing of food to assess food hygiene and the safety of food processing. The presence of some microbial indicators can be viewed as a result of direct or indirect contamination of a food with fecal material. The presence of Enterobacteriaceae is often used as a hygiene indicator, as they are found both in the environment and in the intestine of warm-blooded animals. An association between Salmonella isolation and Enterobacteriaceae count (EC) on pre-chill carcasses has been described, however the impact of slaughterhouse and the day of sampling on the occurrence of Salmonella has not been previously investigated. To this end, mixed logistic regressions (MLRs) with random effects and fixed slopes were performed to assess the change in EC and its correlation with Salmonella occurrence using two data sets. The first describes the EC and Salmonella isolation in 60 pork carcasses in one slaughterhouse sampled at 11 different slaughter steps, including the carcass as a random effect. The second describes the EC and Salmonella isolation on 1150 pre-chill carcasses sampled in 13 slaughterhouses over 230 sampling days, and the model combined two random intercepts, slaughterhouse and date of sampling nested with slaughterhouse (day/slaughterhouse). Statistically significant associations (p<0.0001) between the log of the EC and Salmonella occurrence were found in all models. Nevertheless, although a strong association was found between Enterobacteriaceae and Salmonella contamination in pork carcasses, this association was not constant, given that there was a high variation in the probability of a carcass being positive for Salmonella according to the EC mainly between days of samples. The effect of the day of sampling on Salmonella prevalence was so large that the predictive value of the EC count for Salmonella isolation on a daily basis was compromised. It is possible that on some days batches with a high prevalence of Salmonella carriers shedding a high number of Salmonella were slaughtered. On these days, the potential for contamination/cross-contamination of carcasses will be so large that even hygienic slaughter, confirmed by the low EC on carcasses, will not be able to prevent the presence of Salmonella on some carcasses. The results of this study demonstrate that, despite the statistically significant association found, it may be difficult to predict when hygiene failure measured via EC actually indicates Salmonella contamination, and neither the inverse.

Virulence profiles of enterotoxigenic Escherichia coli isolated from piglets with post-weaning diarrhea and classification according to fecal consistency / Perfis de virulência de Escherichia coli enterotoxigênica isoladas de leitões desmamados com diarreia do Sul do Brasil e classificação das amostras de acordo com a consistência fecal

The aim of this study was to assess the frequency and association of virulence factors of Escherichia (E.) coli isolated from weaned piglets with diarrhea and to correlate it with fecal consistency. A total of 152 rectal swabs were collected from 25-40 day-old piglets with diarrhea, in farms of Southern Brazil. Phenotypical and molecular techniques were used for bacterial isolation, characterization and classification of enterotoxigenic E. coli (ETEC) pathotypes. Statistical analysis was carried out to determine the frequency of virulence factors and virotypes, of fimbriae F4, F5, F6, F18, F41 and toxins LT, STa, STb and STx2e. Out of 456 E. coli isolates, 287 (62.9%) samples showed significant growth of E. coli. Among them, 194 (67.6%) samples showed at least one virulence factor, indicating that ETEC is an important etiological agent of diarrhea in weaned piglets. Higher frequencies were found of fimbria F4 and F18 and enterotoxins LT, STa and STb. Significant association was found to F4, LT, STa and STb; between F18 and STa and STx2e; between F5 and LT, STa and STb. The most frequent virotypes were F18-STa, F4-LT-STa-STb, F4-STa, F4-LT-STb and F18-STa-STx2e. Beta-hemolysis was observed in 47.4% of samples and there was significant association between hemolytic samples and virulence factors F4, F18, STa and STx2e. Regarding fecal consistency, there was significant association of liquid feces and F4 fimbria, STa toxin and virotypes F4-STa and F4-F5-LT-STa-STb. Since there was significant association of ETEC and liquid feces in nursery piglets, it is important to prioritize the sampling of liquid feces for the diagnosis etiologic cause of diarrhea.

O objetivo deste estudo foi avaliar a frequência e associação de fatores de virulência de Escherichia (E.) coli isoladas de leitões desmamados com diarreia e correlacioná-la com consistência fecal. Suabes retais foram coletados em leitões com 25-40 dias de idade com sinal clínico de diarreia, em granjas do Sul do Brasil, totalizando 456 amostras. Foram utilizadas técnicas fenotípicas e moleculares para isolamento bacteriano, caracterização e classificação de patotipos de E. coli enterotoxigênica (ETEC). A análise estatística foi realizada para determinar a frequência de fatores de virulência e virotipos, de fímbrias F4, F5, F6, F18, F41 e toxinas LT, STa, STB e STx2e. Duzentas e oitenta e sete (62,9%) amostras apresentaram crescimento significativo de E. coli. Entre os quais, 194 (67,6%) amostras apresentaram pelo menos um fator de virulência, indicando que ETEC é um importante agente etiológico de diarreia em leitões desmamados. As frequências mais elevadas foram encontradas para as fímbrias F4 e F18 e enterotoxinas LT, STa e STb. Associação significativa foi encontrada para F4, LT, STa e STb; entre F18 e STa e STx2e; entre F5 e LT, STa e STb. Os virotipos mais frequentes foram F18-STa, F4-LT-STa-STb, F4-STa, F4-LT-STb e F18-STa-STx2e. Beta-hemólise foi observada em 47,4% das amostras e houve associação significativa entre amostras hemolíticas e fatores de virulência F4, F18, STa e STx2e. Quanto consistência fecal, houve associação significativa de fezes líquidas e fímbria F4, toxina STa e virotipos F4-STa e F4-F5-LT-STa-STb. A associação significativa da ETEC e fezes líquidas em leitões de creche, é importante para priorizar a amostragem de fezes com essa consistência para no diagnóstico etiológico da diarreia.