Occurrence, genetic diversity and resistance profiles of Salmonella enterica from Brazilian sausages collected at production facilities.

This study aimed to investigate the occurrence and the genetic diversity of Salmonella enterica subsp. enterica in sausages from Southern Brazil, evaluate virulence genes and determine the phenotypic and genotypic basis of antimicrobial and sanitizer resistance. Salmonella was detected in sausage samples with an overall prevalence of 5.5%. The prevalent serovars were S. Infantis and S. Rissen. Pulsed-field gel electrophoresis (PFGE) analysis yielded nine distinct PFGE profiles, and some of them were recurrently recovered in the same establishment on different dates. Among tested isolates, 28.5% showed resistance to at least one antimicrobial agent and a multidrug-resistance (MDR) profile was observed in 21.4%. Resistance occurred most frequently to ampicillin, sulfonamide, trimethoprim/sulfamethoxazole, and trimethoprim. Regarding the genotypic antimicrobial resistance profile, S. Schwarzengrund carried tet(B), strA, strB, and sul2 genes. Benzalkonium chloride and chlorhexidine were more effective than peracetic acid and sodium hypochlorite, showing lower minimum inhibitory concentration values. Six Salmonella serovars were found, demonstrating a potential risk of salmonellosis associated with consuming this food. Salmonella carrying virulence genes, MDR profile, and tolerance to sanitizers is a public health concern and a challenge for the food industry, suggesting that new strategies should be developed to control this pathogen. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05809-w.

Listeria monocytogenes from food and food industry environments with reduced susceptibility to benzalkonium chloride, sodium hypochlorite, and peracetic acid.

The growing number of Listeria monocytogenes strains displaying increased tolerance to sanitizers widely applied in the food industry is becoming a problem. The aims of this study were to evaluate the susceptibility of L. monocytogenes isolates from food and food industry environments to sanitizers (benzalkonium chloride, sodium hypochlorite, peracetic acid, and chlorhexidine) and heavy metals (cadmium chloride), as well as to investigate the presence of the main genes related to efflux pumps. All 82 isolates showed reduced susceptibility to benzalkonium chloride (MIC from 16 to 128 µg mL-1), sodium hypochlorite (MIC of ≥ 2048 µg mL-1), and peracetic acid (MIC from 512 to ≥ 2048 µg mL-1), while 22 isolates showed reduced susceptibility to cadmium chloride (MIC > 70 µg mL-1). Susceptibility to chlorhexidine was found (MIC from 2 to 16 µg mL-1). PCR-based analysis revealed that mdrl and lde genes were harbored by 14.6% (12/82) and 40.2% (33/82) of the isolates, respectively. This study demonstrates the presence of L. monocytogenes from food and food industry environments with reduced susceptibility to sanitizers commonly used in food processing environments, highlighting the importance of continuous monitoring of the tolerance profile of this microorganism to sanitizers, as well as the need for strict control of sanitation conditions in food industries.

Genetic Profiles and Invasion Ability of Listeria monocytogenes Isolated from Bovine Carcasses in Southern Brazil.

ABSTRACT: The goals of this study were to evaluate the persistence and the virulence potential of Listeria monocytogenes isolated from beef carcasses obtained in processing facilities in the southern region of Rio Grande do Sul, Brazil, based on pulsed-field gel electrophoresis (PFGE), invasion ability in human colorectal carcinoma cells (HCT-116), internalin A (InlA) expression by Western blot, and identification of mutation points in inlA. PFGE profiles demonstrated that L. monocytogenes isolates were grouped based on their previously identified lineages and serogroups (lineage I: serogroup IIb, n = 2, and serogroup IVb, n = 5; lineage II: serogroup IIc, n = 5). Isolates with indistinguishable genetic profiles through this method were obtained from different slaughterhouses and sampling steps, with as much as a 3-year interval. Seven isolates showed high invasion ability (2.4 to 7.4%; lineage I, n = 6, and lineage II, n = 1) in HCT and expressed InlA. Five isolates showed low cell invasion ability (0.6 to 1.4%; lineage I, n = 1, and lineage II, n = 4) and did not express InlA, and two of them (lineage II, serogroup IIc) presented mutations in inlA that led to premature stop codon type 19 at position 326 (GAA → TAA). The results demonstrated that most L. monocytogenes isolates from lineage I expressed InlA and were the most invasive in HCT, indicating their high virulence potential, whereas most isolates from lineage II showed attenuated invasion because of nonexpression of InlA or the presence of premature stop codon type 19 in inlA. The obtained results demonstrated that L. monocytogenes with indistinguishable PFGE profiles can persist or be reintroduced in beef processing facilities in the studied region and that differences in their virulence potential are based on their lineages and serogroups.

Genetic diversity, biofilm and virulence characteristics of Listeria monocytogenes in salmon sushi.

Sushi is a ready-to-eat (RTE) food prepared from raw or cooked fish that is widely consumed worldwide. Listeria monocytogenes is the foodborne pathogen most commonly associated with RTE and fish products. The aim of the present study was to evaluate the presence of L. monocytogenes in salmon sushi commercialized in Pelotas city, Brazil, and to evaluate the genetic diversity, biofilm-forming ability in stainless steel, and virulence characteristics of the isolates. Four sampling events were carried out in seven specialized sushi establishments totaling 28 sushi pools. Listeria monocytogenes was detected in six samples (21.4%) from two establishments (28.6%). All isolates belonged to serotype 4b and carried the prfA, plcA, plcB, hlyA, mpl, actA, inlA, inlC, inlJ, and iap genes. The inlB gene was not detected in two isolates. The PFGE analysis grouped the isolates into four pulsotypes. All isolates had the ability to form biofilm on stainless steel and the average of biofilm formation counts varied between 6.4 and 7.2 log CFU.cm-2. The isolates harbored the biofilm-related genes agrA, agrB, agrC, agrD, and prfA, with the exception of two isolates that did not harbor the agrD gene. The presence of L. monocytogenes in RTE sushi is a concern, demonstrating that sushi consumption may be a risk of human listeriosis. Furthermore, it was possible to identify the persistence of this pathogen for at least one month (pulsotypes III and IV), in two establishments (A and G), highlighting the need for improving the cleaning and sanitation procedures in establishments that commercialize RTE sushi.

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.

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.

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.

First report of the Staphylococcus aureus isolate from subclinical bovine mastitis in the South of Brazil harboring resistance gene dfrG and transposon family Tn916-1545.

The aim of this work was to identify at the molecular level the species of coagulase-positive staphylococci isolates from clinical and subclinical bovine mastitis samples in Southern Brazil, and to evaluate the antimicrobial resistance profile, as well as the presence of resistance genes. According to the PCR assay, all 31 isolates were classified as Staphylococcus aureus. The isolates were tested for resistance to penicillin, ampicillin, oxacillin, cefoxitin, cephalothin, ceftiofur, streptomycin, tobramycin, teicoplanin, erythromycin, clindamycin, enrofloxacin, sulfonamide, trimethoprim-sulfamethoxazole, trimethoprim, and tetracycline by the disk diffusion method. Most of the isolates were resistant to sulfonamide (20), followed by ampicillin and clindamycin (16). Twenty isolates were multidrug-resistant. PCR was used for the detection of several antimicrobial resistance genes (ereB, ermB, ermC, tetA, tetB, tetK, tetL, tetM, tetO, Tn916-1545, strA, strB, sul1, sul2, dfrA, dfrG, dfrK, blaZ, mecA, and mecC). The most prevalent antimicrobial resistance genes were tetK and tetL, ereB, followed by tetM, Tn916-1545 and blaZ, detected in 11, nine and four isolates, respectively. For all the tetM gene positive isolates, the presence of conjugative transposons of the Tn916-1545 family was detected. The presence of multidrug-resistant isolates, antimicrobial resistance genes and transposons suggests a potential risk of spreading multi-resistance genes to other bacteria.

Occurrence and phenotypic and molecular characterization of Listeriamonocytogenes and Salmonella spp. in slaughterhouses in southern Brazil.

This study addressed the occurrence of Listeriamonocytogenes and Salmonella spp. in bovine carcasses at two slaughterhouses in southern Brazil. Then, the antimicrobial susceptibility profile and the virulence potential of the isolates were evaluated. Two hundred carcasses were sampled at four steps of the slaughter process, with L. monocytogenes being isolated in 12 and Salmonella spp. in 17 carcasses. All L. monocytogenes isolates carried the hlyA, prfA, plcA, plcB, actA, iap, mpl, inlA, inlB, inlC, and inlJ genes, while Salmonella spp. carried invA and hilA. Among the L. monocytogenes isolates, all of them presented virulence determinants and one showed multi-drug resistance. In relationship to Salmonella spp. isolates, many serogroups frequently related to outbreaks of foodborne diseases were identified and four isolates showed resistance to more than one antimicrobial agent. This data highlights the importance of a rigid hygienic-sanitary control during the slaughter process to reduce the risk of cross-contamination and lower the consumer exposure to L. monocytogenes and Salmonella spp. infections.

Staphylococcus aureus isolated from handmade sweets: Biofilm formation, enterotoxigenicity and antimicrobial resistance.

Staphylococcus aureus is the second most important pathogen involved in foodborne outbreaks in Brazil. Because of their widespread distribution and biofilm forming ability, handmade sweets are easily contaminated with S. aureus. The aim of this study was to isolate and identify coagulase-positive staphylococci (CPS) from handmade sweets produced in Pelotas City/Brazil. The virulence potential was checked by evaluating the presence of the staphylococcal enterotoxin genes, icaA and icaD genes, the biofilm forming potential and antimicrobial resistance of the isolates. It was find just S. aureus among the CPS isolates. All the S. aureus isolates had biofilm forming ability on stainless steel and more than half of them on polystyrene surfaces. The majority of the isolates carried the icaA (66.6%) and icaD (58.4%) genes and some of them had the genes encoding enterotoxins A (33.4%) and B (16.6%). Furthermore, the majority of the isolates (83%) were resistant to at least one of the tested antimicrobials and multidrug resistance was observed in 8.4% of the isolates. The isolates had virulence potential, and half of them were enterotoxigenic. In addition, the ability of all the isolates to produce biofilms highlights the danger posed by these potentially virulent microorganisms persisting in food manufacturing environments.