Agreement of methods to assess antimicrobial susceptibility using Escherichia coli isolates as target models.

Antimicrobial susceptibility tests (AST) conducted in vitro offer a range of methods to assess the antimicrobial resistance (AMR) of microorganisms. Escherichia coli, a widely distributed bacterium, is closely linked to the issue of AMR. In this way, the present study aimed to assess the agreement among different in vitro AST methods, including disk diffusion in agar, broth dilution, and agar dilution method. A total of 100 E. coli isolates were analyzed for their resistance levels against six antibiotics: amoxicillin, ceftiofur, ciprofloxacin, chloramphenicol, tetracycline, and sulfamethoxazole + trimethoprim, using the aforementioned AST methods. Standard breakpoint values were employed to classify isolates as resistant, intermediate, or susceptible, and comparisons among the AST methods were conducted by McNemar's test (P < .05). The obtained data demonstrated equivalence among the AST methods, highlighting the reliability of these standardized classical methodologies. This standardization aids in preventing the inappropriate use of antimicrobials and the dissemination of antimicrobial-resistant microorganisms.

Bacterial microbiota shifts in vacuum-packed beef during storage at different temperatures: Impacts on blown pack spoilage.

We aimed to evaluate the bacterial growth and diversity in vacuum-packed beef bags stored at different temperatures and to monitor blown-pack spoilage. We used culture-based methods and high-throughput sequencing to study the development of the main bacterial groups naturally present in beef stored at 4 and 15 °C for 28 days. The growth of sulfite-reducing clostridium (SRC) was impaired in beef bags stored at 4 °C; significant differences among SRC counts were observed in beef bags stored at 4 and 15 °C on days 14, 21, and 28 (P = 0.001). Blown pack was observed in most beef bags stored at 15 °C, from day 14 to day 28, but not in beef bags stored at 4 °C. A storage temperature of 4 °C was able to maintain a stable bacterial microbiota (most prevalent: Photobacterium, Hafnia-Obesumbacterium, and Lactococcus). Remarkable changes in microbial abundance occurred at 15 °C from day 14 to day 28, with a predominance of strict anaerobes (Bacteroides) and the presence of Clostridium spp. The relative frequencies of strict anaerobes and Clostridium were statistically higher in the beef bags stored at 15 °C (P < 0.001 and P = 0.004, respectively). The temperature influenced the microbial counts and relative abundance of spoilage bacteria, leading to blown pack spoilage.

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.

In Silico Detection of Integrons and Their Relationship with Resistance Phenotype of Salmonella Isolates from a Brazilian Pork Production Chain.

The pork production chain is an important reservoir of antimicrobial resistant bacteria. This study identified and characterized integrons in Salmonella isolates from a Brazilian pork production chain and associate them with their antibiotic resistance pattern. A total of 41 whole-genome sequencing data of nontyphoidal Salmonella were analyzed using PlasmidSPAdes and IntegronFinder software. Nine isolates (21.9%) had some integrons identified (complete and/or incomplete). Six complete class 1 integrons were found, with streptomycin resistance genes (aadA1, aadA2) alone or downstream of a trimethoprim resistance gene (dfrA1, dfrA12), and some also containing resistance genes for sulfonamides (sul1, sul3) and chloramphenicol (cmlA1). Class 2 integron was detected in only one isolate, containing dfrA1-sat2-aadA1 gene cassettes. Five isolates harbored CALINs-clusters attC but lacking integrases-with antimicrobial resistance genes typically found in integron structures. In all, integrons were observed among four serotypes: Derby, Bredeney, Panama, and monophasic var. Typhimurium I 4,[5],12:i:-. The association of integrons with antibiotic resistance phenotype showed that these elements were predominantly identified in multidrug resistance isolates, and six of the seven gentamicin-resistant isolates had integrons. So, surveillance of integrons in Salmonella should be performed to identify the potential for the spread of antimicrobial resistance genes among bacteria.

Staphylococcus aureus in Dairy Industry: Enterotoxin Production, Biofilm Formation, and Use of Lactic Acid Bacteria for Its Biocontrol.

Staphylococcus aureus is a well-known pathogen capable of producing enterotoxins during bacterial growth in contaminated food, and the ingestion of such preformed toxins is one of the major causes of food poisoning around the world. Nowadays 33 staphylococcal enterotoxins (SEs) and SE-like toxins have been described, but nearly 95% of confirmed foodborne outbreaks are attributed to classical enterotoxins SEA, SEB, SEC, SED, and SEE. The natural habitat of S. aureus includes the skin and mucous membranes of both humans and animals, allowing the contamination of milk, its derivatives, and the processing facilities. S. aureus is well known for the ability to form biofilms in food processing environments, which contributes to its persistence and cross-contamination in food. The biocontrol of S. aureus in foods by lactic acid bacteria (LAB) and their bacteriocins has been studied for many years. Recently, LAB and their metabolites have also been explored for controlling S. aureus biofilms. LAB are used in fermented foods since in ancient times and nowadays characterized strains (or their purified bacteriocin) can be intentionally added to prolong food shelf-life and to control the growth of potentially pathogenic bacteria. Regarding the use of these microorganism and their metabolites (such as organic acids and bacteriocins) to prevent biofilm development or for biofilm removal, it is possible to conclude that a complex network behind the antagonistic activity remains poorly understood at the molecular level. The use of approaches that allow the characterization of these interactions is necessary to enhance our understanding of the mechanisms that govern the inhibitory activity of LAB against S. aureus biofilms in food processing environments.

Pseudomonas carnis isolated from blue discolored fresh cheese and insights into the phylogeny.

Most Pseudomonas spp. are responsible for spoilage in refrigerated foods such as alteration in flavor, texture and appearance. Samples of Minas Frescal cheese with blue discoloration were analysed and contained a high Pseudomonas concentration (7.72 ± 0.36 log CFU/g). Out of the 26 Pseudomonas isolates that were analyzed in our study, 19 demonstrated the capability of producing a diffusible dark pigment. Thus, a pigment-producing isolate (C020) was selected by rep-PCR fingerprinting and subsequently subjected to whole-genome sequencing. The draft genome assembled comprises 42 contigs totaling 6,366,75 bp with an average G + C content of 59.97%, and the species prediction performed by TYGS server, based on the draft genome sequence, identified the C020 as Pseudomonas carnis. In order to investigate the phylogenetic relationships of this isolate with strains already identified of this species, we performed an analysis based on whole-genomic sequences. First, an analysis of all P. carnis genomes deposited in GenBank to date shows that 11% (4/37) are misidentified, and belong to the Pseudomonas paracarnis species. A comparative analysis based on phylogenomic analysis has showed that there is no evolutionary relationship between P. carnis strains carrying second copies of trp genes related to blue discoloration (trpABCDF). This finding reinforces the assertion that these genes are contained in a mobile genetic element. However, it is worth noting that all strains carrying these secondary gene copies have exclusively been isolated from food sources. This observation provides valuable insights into the potential origins and dispersion dynamics of this genetic trait within the species.

Multidrug Efflux System-mediated resistance in Staphylococcus aureus under a One Health approach.

The aim of the study was to track the spread of antimicrobial resistance among the different sectors of One Health through the detection of Multidrug-Efflux-System in multidrug-resistant Staphylococcus aureus isolates. Multidrug-resistant (MDR) and methicillin-resistant (MRSA) S. aureus isolates were selected: 25 of human, one of animal and eight of food origin. The efflux system genes norA, norB, norC, LmrS, tet38 and msrA were screened by PCR. The activity of the efflux systems was determined by the minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin in the presence and absence of CCCP and in the quantification of ethidium bromide efflux. Furthermore, biofilm formation was determined in the presence and absence of the CCCP. The molecular epidemiology of the isolates was traced with the aid of PFGE. The gene norC was the most prevalent, detected in all isolates and msrA was the least prevalent, detected in only two isolates from humans. There was no difference in the MICs of tetracycline and ciprofloxacin in the presence of CCCP, but 55.9% of isolates showed ethidium bromide efflux. The presence of CCCP decreased the biofilm formation. Regarding the molecular epidemiology, in three clusters was a mixture of the isolates from different origins. Therefore, S. aureus MDR with active multidrug efflux systems are circulating between One Health domains and it is necessary to consider strategies to decrease this circulation in order to prevent the dissemination of resistance mediated by MES.

Graduate Student Literature Review: Enterotoxigenic potential and antimicrobial resistance of staphylococci from Brazilian artisanal raw milk cheeses.

More than 30 types of artisanal cheeses are known in Brazil; however, microorganisms, such as Staphylococcus spp., can contaminate raw milk cheeses through different sources, from milking to processing. Staphylococcal food poisoning results from the consumption of food in which coagulase-positive staphylococci, mostly Staphylococcus aureus, have developed and produced enterotoxins. In addition, an emerging public health concern is the increasing antimicrobial resistance of some Staphylococcus strains. Furthermore, the ability of Staphylococcus spp. in sharing antibiotic resistance-related genes with other bacteria increases this problem. In light of these observations, this review aims to discuss the presence of, enterotoxins of, and antibiotic-resistant of Staphylococcus spp. in Brazilian artisanal cheese produced with raw milk.

Beneficial features of pediococcus: from starter cultures and inhibitory activities to probiotic benefits.

Pediococci are lactic acid bacteria (LAB) which have been used for centuries in the production of traditional fermented foods. There fermentative abilities were explored by the modern food processing industry in use of pediococci as starter cultures, enabling the production of fermented foods with distinct characteristics. Furthermore, some pediococci strains can produce bacteriocins and other antimicrobial metabolites (AMM), such as pediocins, which are increasingly being explored as bio-preservatives in various food matrices. Due to their versatility and inhibitory spectrum, pediococci bacteriocins and AMM are being extensively researched not only in the food industry, but also in veterinary and human medicine. Some of the pediococci were evaluated as potential probiotics with different beneficial areas of application associated with human and other animals' health. The main taxonomic characteristics of pediococci species are presented here, as well as and their potential roles and applications as starter cultures, as bio-preservatives and as probiotic candidates.

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.

Why knowledge is the best way to reduce the risks associated with raw milk and raw milk products.

In an age of flexible conditions about mandatory milk pasteurisation, this opinion-based research reflection supports the view that the knowledge and the awareness of milk-borne infections are key requirements to decrease the risks associated with raw milk. Providing an analysis of the current potential risks related to consumption of raw milk and raw milk products, we discuss the main reasons to continue to be vigilant about milk-borne pathogens and the current scenario in relation to the formal and clandestine sale of raw milk. Finally, we select some highly effective strategies to reduce the risks associated with raw milk in food services. Regardless of whether a country regulation allows or prohibits the trade of raw milk and its products, this is not the time to be negligent.

Biofilm growth by Listeria monocytogenes on stainless steel and expression of biofilm-related genes under stressing conditions.

This research was carried out to investigate the differences in adhesion and growth during biofilm formation of L. monocytogenes from different sources and clonal complexes. Biofilm by L. monocytogenes (isolates CLIST 441 and 7: both lineage I, serotype 1/2b, CC3; isolates 19 and 508: both lineage II, serotype 1/2c, CC9) was grown on stainless steel coupons under different stressing conditions (NaCl, curing salts and quaternary ammonium compounds-QAC), to determine the expression of different genes involved in biofilm formation and stress response. CLIST 441, which carries a premature stop codon (PMSC) in agrC, formed high-density biofilms in the presence of QAC (7.5% w/v) or curing salts (10% w/v). Reverse Transcriptase-qPCR results revealed that L. monocytogenes isolates presented differences in transcriptional profile of genes related to biofilm formation and adaptation to environmental conditions. Our results demonstrated how L. monocytogenes can survive, multiply and form biofilm under adverse conditions related to food processing environments. Differences in transcriptional expression were observed, highlighting the role of regulatory gene networks for particular serotypes under different stress responses.

The ability of Lactococcus lactis subsp. lactis bv. diacetylactis strains in producing nisin.

Lactococcus lactis subsp. lactis bv. diacetylactis is a relevant microorganism for the dairy industry because of its role in the production of aromatic compounds. Despite this technological property, the identification of bacteriocinogenic potential of obtained strains can offer the additional positive aspect of biosafety. A panel of 15 L. lactis subsp. lactis bv. diacetylactis strains was characterised for the presence and expression of bacteriocin related genes, and further investigated regarding the nisin operon. Eight strains were positive only for nisA, and one strain (SBR4) presented a full nisin operon, with sequencing that was shown to be similar to nisin Z. Only SBR4 presented inhibitory activity against 16 microbial target strains. The growth curves of selected targets strains confirmed the inhibitory activity of SBR4 and consequently the nisin production. This research has demonstrated the inhibitory potential of L. lactis subsp. lactis bv. diacetylactis strain, SBR4, due to its ability to produce nisin Z. This biopreservative potential, associated to previously characterised technological properties, allow the indication of this strain as a promising candidate to be used by the dairy industry as a starter or adjunct culture.

Comparison of Meat Juice Serology and Bacteriology for Surveillance of Salmonella in the Brazilian Pork Production Chain.

This study assessed an enzyme-linked immunosorbent assay (ELISA) based assay to detect Salmonella in swine as a potential tool to predict the presence of Salmonella in swine carcasses. The following samples were collected from 10 swine batches: blood (n = 100); environment (barn floor, n = 10, and lairage floor, n = 10); meat juice (n = 100, obtained after defrosting of diaphragm); tonsils (n = 100); mesenteric lymph nodes (MLNs) (n = 100); and carcasses after bleeding (n = 100), after singeing (n = 100), after evisceration (n = 100), and after final rinsing (n = 100). Blood and meat juice were subjected to ELISA to detect antibodies against Salmonella, and other samples were subjected to Salmonella detection by ISO 6579. Salmonella was detected in 3 samples from barn floors, 7 lairage floors, 45 tonsils, 43 MLNs and in 3 carcasses. Based on ELISA, Salmonella positive samples were: 86 and 46 blood serum (20% and 40% cut-offs) and 68 and 46 meat juice (20% and 40% cut-offs). Optical density readings from blood serum and meat juice presented a high and significant correlation (r = 0.93, p < 0.001), and a substantial agreement for Salmonella detection (K = 0.69, ELISA 40% cut-off). The agreement between ELISA and microbiological analysis for Salmonella detection in pig carcasses were absent or poor, with the exception of results obtained by ELISA 40% cut-off from blood serum and meat juice with MLNs (K = 0.49 and 0.50, respectively) and tonsils (K = 0.29 and 0.30, respectively). Based on the obtained results, meat juice can be considered an alternative to blood serum as a matrix for ELISA for preliminary detection of Salmonella, allowing the identification of potential sources of contamination during slaughtering.

Lactobacillus curvatus UFV-NPAC1 and other lactic acid bacteria isolated from calabresa, a fermented meat product, present high bacteriocinogenic activity against Listeria monocytogenes.

BACKGROUND: Bacteriocins produced by lactic acid bacteria (LAB) can be considered as viable alternatives for food safety and quality, once these peptides present antimicrobial activity against foodborne pathogens and spoilage bacteria. Fermented foods, such as artisanal sausages and cured meats, are relevant sources of LAB strains capable of producing novel bacteriocins, with particular interest by the food industry. RESULTS: Three LAB strains (firstly named as Lactobacillus curvatus 12, L. curvatus 36 and Weissella viridescens 23) were obtained from calabresa by presenting promising bacteriocinogenic activity, distinct genetic profiles (rep-PCR, RAPD, bacteriocin-related genes) and wide inhibitory spectrum. Among these strains, L. curvatus 12 presented higher bacteriocin production, reaching 25,000 AU/mL after incubation at 25, 30 and 37 °C and 6, 9 and 12 h. Partially purified bacteriocins from L. curvatus 12 kept their inhibitory activity after elution with isopropanol at 60% (v/v). Bacteriocins produced by this strain were purified by HPLC and sequenced, resulting in four peptides with 3102.79, 2631.40, 1967.06 and 2588.31 Da, without homology to known bacteriocins. CONCLUSIONS: LAB isolates obtained from calabresa presented high inhibitory activity. Among these isolates, bacteriocins produced by L. curvatus 12, now named as L. curvatus UFV-NPAC1, presented the highest inhibitory performance and the purification procedures revealed four peptides with sequences not described for bacteriocins to date.

Distribution, adhesion, virulence and antibiotic resistance of persistent Listeria monocytogenes in a pig slaughterhouse in Brazil.

Listeria monocytogenes is a relevant pathogen usually associated with meat and ready-to-eat products. This study aimed to assess the distribution, adhesion, virulence and antibiotic resistance of L. monocytogenes in a pork production chain. Environment, carcass and food samples (n = 894) were obtained from different steps of a pork production chain over a 6-month period (10 samplings), including from farms and the slaughterhouse (reception, slaughtering, processing, storage and end products). L. monocytogenes was detected in samples from the reception (lairage floor, 1/10), slaughtering (drains, 2/20) and cutting room stages (conveyor belts in the final packing stage - 11/20, knife - 1/40, and cutting boards - 1/20). Positive results for conveyor belts were recorded in seven consecutive samplings. L. monocytogenes isolates (n = 87) were characterized as belonging to serogroup IVb and presented positive PCR results for inlA, inlB, inlC, inlJ, hlyA, plcA, actA and iap. Isolates were selected according to the original samples (n = 31) and subjected to Pulsed Field Gel Electrophoresis (PFGE), demonstrating their high clonal identity (98.4-100%). According to PFGE results and their original samples, isolates were selected (n = 16) and subjected to phenotypic assay to assess their adhesion potential and tested for resistance against 15 antibiotics; all tested isolates presented weak adhesion potential and were resistant to ampicillin. The present study demonstrated the persistence of L. monocytogenes in the pork processing facility, indicating the potential risk for cross-contamination with a potential virulent and resistant clone.

Isolation of Enterococcus faecium, characterization of its antimicrobial metabolites and viability in probiotic Minas Frescal cheese.

The aim of this study was to isolate Enterococcus faecium from raw milk samples, to characterize its antimicrobial metabolites, and to evaluate its viability in a probiotic Minas Frescal cheese. For this, antagonist activity against Listeria monocytogenes, safety aspects and biochemical, genotypic, and probiotic characteristics of the isolates were evaluated. Minas Frescal cheese was manufactured with the isolate that showed the best characteristics in vitro, and its viability in the product was evaluated. It was observed that of the 478 lactic acid bacteria isolates, only isolate E297 presented antagonist activity, genes encoding for enterocin production and absence of virulence factors. Besides that, E297 presented probiotic characteristics in vitro, and maintained its viability (8.09 log CFU mL-1) for 14 days of cold storage, when it was added to cheese. Therefore, isolate E297 can be considered a promising microorganism for the manufacture of probiotic foods, especially Minas Frescal cheese.

Occurrence and characterization of Listeria monocytogenes from beef jerky processing line.

Beef jerky is a ready-to-eat product that does not require refrigeration at the point of sale. Here, we evaluated the occurrence of Listeria monocytogenes in the production process of beef jerky, the presence of virulence genes and the genomic relatedness of the isolates, to assess the safety of the final product. The raw material, surfaces with and without contact with the product and the final product were evaluated along the beef jerky processing line. The samples were evaluated by VIDAS immunoassay system, and the L. monocytogenes isolates were confirmed and evaluated for the presence of several virulence genes by PCR. Listeria monocytogenes was identified in six of the 84 samples (7.14%), and no genetic relationship was observed among isolates. Samples of raw material (2/7), food contact surface (1/56), and work surfaces without contact with food (3/14) presented contamination by L. monocytogenes. The final product was not contaminated, demonstrating that barriers to multiplication of pathogens used during the production process were effective for its control.

Beneficial properties of lactic acid bacteria naturally present in dairy production.

BACKGROUND: Consumers are increasingly demanding for natural and beneficial foods, in order to improve their health and well-being. Probiotics play an important role in such demand, and dairy foods are commonly used as vehicles for such bacteria, represented predominantly by lactic acid bacteria. Due to consumers demand, food industry is constantly looking for novel bacterial strains, leading to studies that aims the isolation and characterization of their beneficial features. This study aimed to characterize the naturally occurring lactic acid bacteria obtained from a dairy environment, in order to assess their potential use as probiotics. RESULTS: Preliminary screening and PCR analysis, based on 16S rRNA sequencing, were applied to select and identify 15 LAB strains from the genera Lactobacillus (n = 11), Pediococcus (n = 2) and Weissella (n = 2). All strains showed resistance to low pH and the evaluated bile salt concentrations in vitro. The API ZYM test characterized the enzymatic activity of the strains, and a high ß-galactosidase activity was observed in 13 strains. All strains presented resistance to simulated gastric (3 h) and intestinal (4 h) conditions in vitro, the ability to auto- and co-aggregate with indicator microorganisms and a high cell surface hydrophobicity. Most of the strains were positive for map and EFTu beneficial genes. All strains exhibited strong deconjugation of bile salts in vitro and all assimilated lactose. CONCLUSIONS: The phenotypes exhibited in vitro and the presence of beneficial genes revealed the beneficial potential of the studied strains, demanding further analyses in a food matrix and in vivo to allow the development of a functional product, with health-related properties.

Staphylococcus aureus undergoes major transcriptional reorganization during growth with Enterococcus faecalis in milk.

Previous studies have demonstrated the antagonistic potential of lactic acid bacteria (LAB) present in raw milk microbiota over Staphylococcus aureus, albeit the molecular mechanisms underlying this inhibitory effect are not fully understood. In this study, we compared the behavior of S. aureus ATCC 29213 alone and in the presence of a cheese-isolated LAB strain, Enterococcus faecalis 41FL1 in skimmed milk at 30 °C for 24 h using phenotypical and molecular approaches. Phenotypic analysis showed the absence of classical staphylococcal enterotoxins in co-culture with a 1.2-log decrease in S. aureus final population compared to single culture. Transcriptional activity of several exotoxins and global regulators, including agr, was negatively impacted in co-culture, contrasting with the accumulation of transcripts coding for surface proteins. After 24 h, the number of transcripts coding for several metabolite responsive elements, as well as enzymes involved in glycolysis and acetoin metabolism was increased in co-culture. The present study discusses the complexity of the transcriptomic mechanisms possibly leading to S. aureus attenuated virulence in the presence of E. faecalis and provides insights into this interspecies interaction in a simulated food context.