Characterization of diarrheagenic Escherichia coli from different cattle production systems in Brazil.

Diarrheagenic E. coli (DEC) can cause severe diarrhea and is a public health concern worldwide. Cattle are an important reservoir for this group of pathogens, and once introduced into the abattoir environment, these microorganisms can contaminate consumer products. This study aimed to characterize the distribution of DEC [Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC)] from extensive and intensive cattle production systems in Brazil. Samples (n = 919) were collected from animal feces (n = 200), carcasses (n = 600), meat cuts (n = 90), employee feces (n = 9), and slaughterhouse water (n = 20). Virulence genes were detected by PCR in 10% of animal samples (94/919), with STEC (n = 81) as the higher prevalence, followed by EIEC (n = 8), and lastly EPEC (n = 5). Animals raised in an extensive system had a higher prevalence of STEC (average 48%, sd = 2.04) when compared to animals raised in an intensive system (23%, sd = 1.95) (Chi-square test, P < 0.001). From these animals, most STEC isolates only harbored stx2 (58%), and 7% were STEC LEE-positive isolates that were further identified as O157:H7. This study provides further evidence that cattle are potential sources of DEC, especially STEC, and that potentially pathogenic E. coli isolates are widely distributed in feces and carcasses during the slaughter process.

High prevalence of intermediate resistance to ciprofloxacin in Salmonella enterica isolated from a Brazilian poultry production chain, located in Minas Gerais state.

We characterized the distribution and diversity of antimicrobial-resistance Salmonella enterica isolated from a poultry production chain in Minas Gerais, Brazil, with special attention to ciprofloxacin and multidrug resistance (MDR). S. enterica (n = 96) of different serotypes and from different processing steps were subjected to broth dilution assay to estimate the minimum inhibitory concentration (MIC) for 12 antibiotics (8 classes) and screened using PCR for the presence of 17 antimicrobial-resistance genes. Isolates presented mainly resistance to ampicillin (11/96), and most presented intermediate resistance to ciprofloxacin (92/96). Roughly one-third (33/96) were resistant to streptomycin based on our interpretive criteria. Most strains resistant to streptomycin and ciprofloxacin were PCR-positive for aphA (51/96) and qnrB (94/96), respectively. Ciprofloxacin resistance was further investigated through high-resolution melting qPCR (HRM-qPCR) and sequencing of quinolone resistance-determining region (QRDR: gyrA, gyrB, parC, and parE). Minor differences were identified in melting temperatures (Tm), and a Thr57Sr mutation was observed in parC. MDR isolates harboring acrA and capable of expressing the AcrAB-TolC multidrug efflux pump were resistant to ethidium bromide at 0.4 mg/mL. The intermediate resistance to ciprofloxacin may be associated with qnrB, and the potential role of Thr57Ser mutation warrants further investigation. The high prevalence of antibiotic related genes and its association with the observed intermediary resistance to ciprofloxacin indicates the widespread of this hazard in the studied poultry production chain.

Occurrence and Distribution of Antibiotic-Resistant Staphylococcus aureus in a Brazilian Pork Production Chain.

ABSTRACT: Here we characterized the distribution and the antibiotic resistance of staphylococci from a Brazilian pork production chain. Samples (n = 1,114) from pig farms, pig lots, and slaughterhouses, located in two Brazilian states (Minas Gerais and Paraná), were subjected to coagulase-positive Staphylococcus enumeration. S. aureus isolates (n = 251) from this collection were further characterized for their resistance to oxacillin, cefoxitin, vancomycin, and tetracycline through phenotypic and molecular assays. Coagulase-positive Staphylococcus counts from pig farms were higher compared with other samples (P < 0.05). Other counts were relatively low but were present in all production stages. S. aureus isolates were commonly resistant to oxacillin and cefoxitin (54 of 73, 74.0%), qualifying them as methicillin-resistant S. aureus, but PCR assays indicated that few harbored the expected antimicrobial resistance genes (femB, mecA, and mecC). Lower frequencies of vancomycin and tetracycline resistance were found (6.8 to 37.0%). PCR sensitivity (34.5 to 86.7%) and specificity (26.6 to 85.0%) for detection of antibiotic resistance genes varied based on the assessed antibiotic. Antibiotic-resistant staphylococci are widely distributed in the Brazilian pork production chain, and methicillin-resistant S. aureus can become a potential health and economic impediment for the Brazilian pork industry.

Comparative genomics and antibiotic resistance of Yersinia enterocolitica obtained from a pork production chain and human clinical cases in Brazil.

Previous work found a high similarity of macro-restriction patterns for isolates of Yersinia enterocolitica 4/O:3 obtained at a pork production chain from Minas Gerais, Brazil. Herein we aimed to determine the clonality and the antibiotic resistance profiles of a subset of these isolates (n = 23) and human clinical isolates (n = 3). Analysis based on whole genome sequencing (WGS) showed that the isolates were distributed into two major clades based on single nucleotide polymorphisms (SNP) with one isolate defining Clade A (isolate R31) and remaining isolates (n = 25, 96.2%) defining Clade B. Seven clonal groups were identified. The inclusion of isolate R31 as a distinct clonal group was due to the presence of several phage-related genes, allowing its characterization as serotype O:5 by WGS. Disk-diffusion assays (14 antibiotics) identified 13 multidrug resistant isolates (50.0%). Subsequent sequence analysis identified 17 different antibiotic resistance related genes. All isolates harbored blaA (y56 beta-lactamase), vatF, rosA, rosB and crp, while nine isolates harbored a high diversity of antibiotic resistance related genes (n = 13). The close genetic relationship among Y. enterocolitica obtained from a pork production chain and human clinical isolates in Brazil was confirmed, and we can highlight the role of swine in the potential transmission of an antibiotic-resistant clones of a pathogenic bio-serotype to humans, or the transmission of these resistant bacteria from people to animals. The role of veterinary antibiotic use in this process is unclear.

Persistence of Yersinia enterocolitica bio-serotype 4/O:3 in a pork production chain in Minas Gerais, Brazil.

Yersinia enterocolitica bio-serotype 4/O:3 was previously identified in a pork production chain in Brazil and the obtained isolates presented high identity by pulsed-field gel electrophoresis (PFGE, XbaI). For the current study, an additional 147 porcine samples (tonsils = 100, palate = 30, head meat = 17) were collected from the same pork production chain 2-years later and 14 (9.5%) tested positive for Y. enterocolitica. Isolates (n = 24, 1 to 2 per positive sample) were bio-serotype 4/O:3 and harbored virulence genes ail, inv, wbbU, virF, myfA, ystA, ymoA, hreP and sat, and the multidrug resistance related genes emrD, marC and yfhD. PFGE (XbaI) demonstrated no differences among isolates (100% similarity) and were identical to some Y. enterocolitica isolates (n = 13) obtained previously from the same pork chain. A second PFGE analysis (NotI) confirmed the high degree of similarity among isolates obtained over time, demonstrating the persistence of an apparent clonal Y. enterocolitica bio-serotype 4/O:3 in this particular pork production chain in Brazil.

Phenotypic and genotypic characterization of non-typhoidal Salmonella isolated from a Brazilian pork production chain.

Pork products are important sources of foodborne non-typhoidal Salmonella in Brazil where antibiotics are commonly used throughout the pork production process and this has the potential to selectively favor antibiotic-resistant strains. We characterized the genotypic and phenotypic diversity of S. enterica isolates (n = 41) that were isolated in Brazil. Isolates were collected from ten swine farms and one slaughterhouse. Whole-genome sequencing and in silico serotyping demonstrated that the S. enterica serovar Typhimurium was the most common serotype (n = 17), but eight additional servoars were identified. Isolates presented high similarity based on comparison of DNA sequences (minimum of 89.6%), and sequence variation grouped according to serotype. Eight multilocus sequence types were identified with ST19 being most common (n = 21). Several plasmids replicons were detected, with Col (RNAI) the most abundant (n = 30), followed by IncR (n = 22), IncI1 (n = 10) and IncA/C2 (n = 10). Minimum inhibitory concentration assays showed that the principle resistance phenotypes were for streptomycin (90.2%), tetracycline (87.8%), ampicillin (80.5%), chloramphenicol (70.7%) and ciprofloxacin (51.2%). Only two isolates were resistant to third-generation cephalosporins and no isolates were resistant to two tested carbapenems. Twenty-six unique antimicrobial-resistance genes were identified with blaTEM-1A and blaTEM-1B likely responsible for most beta-lactam resistance and floR responsible for most chloramphenicol resistance. Six strains were positive for mcr-1. At the time of collection, the sampled farms were adding ciprofloxacin to feed and this may have contributed to the high prevalence of resistance to this antibiotic. The high number of multidrug resistant Salmonella and the presence of multiple resistant genes and plasmids emphasize the diversity of Salmonella in the studied pork chain, specially from serotype Typhimurium.

Distribution, diversity, virulence genotypes and antibiotic resistance for Salmonella isolated from a Brazilian pork production chain.

Pigs infected with Salmonella are an important source of contamination at slaughterhouses. We characterized the distribution, virulence genotypes and antimicrobial-resistance phenotypes for Salmonella isolates that were collected from different stages of a pork production chain. Each of ten pig lots were sampled for feed (n = 10), water (n = 10), barn floor (n = 10), lairage floor (n = 10), mesenteric lymph nodes (n = 100), tonsils (n = 100), processing environment (n = 120), pork cuts (n = 40) and carcasses after bleeding (n = 100), after singeing (n = 100), after evisceration (n = 100), and after final rinsing (n = 100). Salmonella was isolated according to ISO 6579, and after confirmation the isolates were subjected to serogrouping, macro-restriction digests and pulsed-field gel electrophoresis (PFGE), detection of virulence-related genes and antimicrobial-resistance phenotyping. Salmonella was recovered from barn floors from 3 pig farms (3/10), lairage floors (7/10), carcasses after bleeding (2/100) and final washing (1/100), palatine tonsils (45/100), mesenteric lymph nodes (43/100), utensils (3/120) and cuts (4/40). The most prevalent serogroup was O: 4 (82%) followed by O:3 (7.7%); O:9 (5.1%); O:8 (2.6%) and O:7 (2.6%). Recovered strains (n = 109) were classified into 24 different pulsotypes (XbaI restriction digest), which were arranged into five different clusters. Fourteen different virulence genotypes were observed based on 15 loci, and all isolates were positive for invA, sitC, pagC and tolC. There was a high prevalence of antimicrobial resistance against streptomycin (90.5%), tetracycline (88.1%), ampicillin (81.0%), chloramphenicol (71.4%), and ciprofloxacin (50.0%). No strain was resistant to ertapenem, meropenem or kanamycin. A majority (80.9%) of isolates were considered multidrug resistant (resistant to ≥3 antibiotic classes). This study provides valuable insight about the epidemiology of Salmonella in swine production, and despite the low presence of this pathogen in carcasses and meat cuts, the majority of isolates was multidrug resistant.

Listeria monocytogenes in Food-Processing Facilities, Food Contamination, and Human Listeriosis: The Brazilian Scenario.

Listeria monocytogenes is a foodborne pathogen that contaminates food-processing environments and persists within biofilms on equipment, utensils, floors, and drains, ultimately reaching final products by cross-contamination. This pathogen grows even under high salt conditions or refrigeration temperatures, remaining viable in various food products until the end of their shelf life. While the estimated incidence of listeriosis is lower than other enteric illnesses, infections caused by L. monocytogenes are more likely to lead to hospitalizations and fatalities. Despite the description of L. monocytogenes occurrence in Brazilian food-processing facilities and foods, there is a lack of consistent data regarding listeriosis cases and outbreaks directly associated with food consumption. Listeriosis requires rapid treatment with antibiotics and most drugs suitable for Gram-positive bacteria are effective against L. monocytogenes. Only a minority of clinical antibiotic-resistant L. monocytogenes strains have been described so far; whereas many strains recovered from food-processing facilities and foods exhibited resistance to antimicrobials not suitable against listeriosis. L. monocytogenes control in food industries is a challenge, demanding proper cleaning and application of sanitization procedures to eliminate this foodborne pathogen from the food-processing environment and ensure food safety. This review focuses on presenting the L. monocytogenes distribution in food-processing environment, food contamination, and control in the food industry, as well as the consequences of listeriosis to human health, providing a comparison of the current Brazilian situation with the international scenario.