Graciela Volz Lopes

Food isolate Listeria monocytogenes harboring tetM gene plasmid-mediated exchangeable to Enterococcus faecalis on the surface of processed cheese

The genetic basis of tetracycline resistance in a food isolate Listeria monocytogenes (Lm16) was evaluated. Resistance to tetracycline was associated with the presence of the tetM gene in plasmid DNA. The sequence of tetM showed 100% of similarity with the Enterococcus faecalis sequences found in the EMBL database, suggesting that Lm16 received this gene from E. faecalis. Various size bands were detected in the DNA plasmid analysis, the largest being approximately 54.38 kb. Transferability of the tetM gene was achieved in vitro by agar matings between Lm16 and E. faecalis JH2-2, proving the potential for the spread of tetM by horizontal gene transfer. Furthermore, the conjugation experiments were performed on the surface of processed cheese, confirming the transferability in a food matrix. PCR assays were used to confirm the identity of E. faecalis and to detect the tetM gene in transconjugant bacteria. Additionally, the minimal inhibitory concentration for tetracycline and rifampicin and plasmid profiling were performed. This is the first report of a food isolate L. monocytogenes carrying the tetM gene in plasmid DNA, and it highlights the potential risk of spreading antimicrobial resistance genes between different bacteria.

Foods introduced into Brazil through the border with Argentina and Uruguay: Pathogen detection and evaluation of hygienic-sanitary quality

This study aimed to evaluate the presence of pathogens in, and the hygienic-sanitary quality of, commercialized foods of animal origin at the international border region of Rio Grande do Sul, Brazil. In total, 270 samples of raw and processed foods of animal origin were collected in Paso de los Libres, Argentina (n = 65 raw meat, n = 47 dairy products, n = 28 processed meat) and Rivera, Uruguay (n = 60 raw meat, n = 31 dairy products, n = 29 processed meat), or were seized by the Brazilian International Agricultural Surveillance System (Brazil-Argentina border) (n = 9 raw meat, n = 1 bush meat). The samples were subjected to the enumeration of aerobic mesophilic bacteria, enterobacteria, and coagulase-positive staphylococci, and were tested for Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7. The virulence genes for Salmonella spp. (hilA, invA, spvC, pefA, and sefA), L. monocytogenes (prs, inlA, inlC, and inlJ) and E. coli O157:H7 (uspA, eae, rfbO157, fliCH7, stx1, stx2, and hlyA) were investigated using PCR assays. Raw products showed higher counts of aerobic mesophiles and enterobacteria compared to processed products (P < 0.05). There were no significant differences in aerobic mesophile or in enterobacterial counts between identical products according to origin (Argentina vs. Uruguay, P > 0.05). Escherichia coli O157:H7 was not detected in any of the samples tested. Salmonella spp. was detected in six (8%) raw products from Argentina. Listeria monocytogenes was isolated from five (6.66%) raw products originating in Argentina and 20 (16.66%) raw products from Uruguay. All 52 E. coli isolates carried the uspA gene, but only one carried the eae gene. The rfbO157, fliCH7, stx1, stx2, and hlyA genes were not detected. All Salmonella spp. isolates carried hilA and invA genes, but spvC, pefA, and sefA were not found. All L. monocytogenes isolates carried the prs gene; however, inlA, inlC, and inlJ genes were found in 20% of the isolates from Argentina and 95% of those from Uruguay. To our knowledge, this is the first microbiological study into the hygienic-sanitary quality of animal products in Brazils land border region. Salmonella spp. and L. monocytogenes were detected in products of animal origin, constituting a public health concern and emphasizing the need for an active surveillance system to reduce the risk of foodborne pathogen introduction into Brazil.

Genetic diversity of Campylobacter jejuni and Campylobacter coli isolated from poultry meat products sold on the retail market in Southern Brazil

ABSTRACT Campylobacter is regarded as the most common bacterial cause of gastroenteritis throughout the world and most cases of human campylobacteriosis can be traced back to the consumption of poultry meat. In Brazil, few studies evaluated the genetic relatedness among Campylobacter isolates. The aim of this research was to evaluate the genetic diversity of Campylobacter spp. isolated from poultry meat products sold on the retail market in Southern Brazil. The presumptive identification of Campylobacter was performed using traditional microbiological analysis, followed by molecular confirmation by PCR. The genetic diversity of isolates was analyzed by pulsed‐field gel electrophoresis (PFGE). Campylobacter spp. was isolated from 91.7% (33/36) of the samples, totaling 48 isolates. Campylobacter jejuni was the most prevalent species isolated (90.8%). PFGE data revealed 26 pulsotypes and 18 PFGE patterns composed of only 1 isolate. Campylobacter isolates exhibited high genetic diversity; however, some clones were recurrent in the poultry meat products sold on the retail market. As the south region of Brazil is an important producer and exporter of chicken meat, our results highlight the need to control this pathogen in the food chain in this area of the world to reduce the risks of exposing consumers to campylobacteriosis.

Presence of Classical Enterotoxin Genes, agr Typing, Antimicrobial Resistance, and Genetic Diversity of Staphylococcus aureus from Milk of Cows with Mastitis in Southern Brazil

Staphylococcus aureus is a common causative agent of bovine mastitis in dairy cows and commonly associated with foodborne disease outbreaks. The aim of this study was to evaluate the presence of enterotoxin genes, agr typing, antimicrobial resistance, and genetic diversity of S. aureus isolated from milk of cows with mastitis in dairy farms from southern Brazil. Results showed that 7 (22.6%) of 31 S. aureus isolates were positive for enterotoxin genes. Specifically, the genes encoding for enterotoxins A ( n = 4), C ( n = 2), and B ( n = 1) were detected. Isolates belonging to the agr group III (10 of 31, 32.2%) and agr group I (7 of 31, 22.5%) were the most common. To our knowledge, this is the first report of both agr I and III in the same S. aureus isolate from milk of cows with bovine mastitis. The antimicrobial resistance test showed that 54% of the isolates were multiresistant to antimicrobial agents. The macrorestriction analysis produced 16 different major SmaI pulsed-field gel electrophoresis patterns, with up to two subpatterns. Moreover, the presence of some S. aureus clones in a distinct area was observed. Although this study characterized a limited number of S. aureus isolates, the presence of classical enterotoxin genes and resistance to multiple antimicrobial agents reinforces the importance of this microorganism to animal and human health. In addition, similar genetic profiles have been identified in distinct geographic areas, suggesting clonal dissemination of S. aureus in dairy herds from southern Brazil.

Tolerance to benzalkonium chloride and antimicrobial activity of Butia odorata Barb. Rodr. extract in Salmonella spp. isolates from food and food environments

Salmonellosis, caused by the consumption of contaminated foods, is a major health problem worldwide. The aims of this study were to assess the susceptibility of Salmonella spp. isolates to benzalkonium chloride (BC) disinfectant and the antimicrobial activity of Butia odorata Barb. Rodr. extract against the same isolates from food and food environments. Moreover, phenotypic and genotypic resistance profiles, the presence of virulence genes and biofilm forming ability were determined. The minimum inhibitory concentration (MIC) of B. odorata extract against Salmonella spp. ranged from 10 to >19 mg.mL-1. Resistance to ampicillin, streptomycin, nalidixic acid, sulfonamide, trimethoprim/sulfamethoxazole, trimethoprim, tetracycline, and chloramphenicol was observed. In addition, multidrug resistance was observed in seven isolates (26.92%). The MIC of BC ranged from 32 to 64 mg.L-1, higher concentrations in comparison with wild-type MICs, and therefore were considered tolerant. Several resistance genes were detected, of which the most common were aadA, qacEΔ1, blaTEM, int1, sul1, and tetA. All isolates carried at least one virulence gene and produced biofilms on stainless steel surfaces at 10 and 22 °C. On the other hand, the B. odorata extract showed activity against Salmonella spp., and it has the potential to be used as a natural antimicrobial to control this important foodborne pathogen, despite its virulence potential and antimicrobial resistance profile.

Prevalence and expression of staphylococcal enterotoxin genes in Staphylococcus aureus isolated from food poisoning outbreaks

Staphylococcus aureus is an important pathogen of foodborne origin. The pathogen produces a variety of toxins that include the staphylococcal enterotoxins (SE). The present study aimed to evaluate the prevalence and expression of 5 SE genes (sea, seb, sec, sed, and see) in S. aureus isolated from outbreaks occurred in the state of Rio Grande do Sul, Brazil. All isolates, with the exception of 2, presented the same or higher transcriptional expression than the reference strains for at least 1 of these genes. The presence of SE genes combined with high levels of transcriptional expression suggests that 1 or more SEs were involved with the staphylococcal food poisoning outbreak analyzed in the present study.