Evaluation of Enterotoxins and Antimicrobial Resistance in Microorganisms Isolated from Raw Sheep Milk and Cheese: Ensuring the Microbiological Safety of These Products in Southern Brazil.

This study emphasizes the importance of monitoring the microbiological quality of animal products, such as raw sheep's milk and cheese, to ensure food safety. In Brazil, there is currently no legislation governing the quality of sheep's milk and its derivatives. Therefore, this study aimed to evaluate: (i) the hygienic-sanitary quality of raw sheep's milk and cheese produced in southern Brazil; (ii) the presence of enterotoxins and Staphylococcus spp. in these products; and (iii) the susceptibility of the isolated Staphylococcus spp. to antimicrobial drugs and the presence of resistance genes. A total of 35 samples of sheep's milk and cheese were examined. The microbiological quality and presence of enterotoxins were accessed using Petrifilm and VIDAS SET2 methods, respectively. Antimicrobial susceptibility tests were conducted using VITEK 2 equipment and the disc diffusion method. The presence of resistance genes tet(L), sul1, sul2, ermB, tetM, AAC(6)', tetW, and strA were evaluated through PCR. In total, 39 Staphylococcus spp. were obtained. The resistance genes tetM, ermB, strA, tetL, sul1, AAC(6)', and sul2 were detected in 82%, 59%, 36%, 28%, 23%, 3%, and 3% of isolates, respectively. The findings revealed that both raw sheep's milk and cheese contained Staphylococcus spp. that exhibited resistance to antimicrobial drugs and harbored resistance genes. These results underscore the immediate need for specific legislation in Brazil to regulate the production and sale of these products.

Evolution of the spontaneous sourdoughs microbiota prepared with organic or conventional whole wheat flours from South Brazil.

The purpose of this study was to compare the composition and stability of bacteria and fungi communities during the propagation of sourdoughs prepared with organic or conventional whole wheat (Triticum aestivum) flours from South Brazil. Sourdoughs were prepared and samples were collected during different fermentation times (0 to 216 h). Total DNA of sourdough samples were extracted and the 16S rRNA gene and Internal Transcribed Spacer region were sequenced by MiSeq-Illumina. A total of 43 and 56 OTUs were identified and defined as core taxa in the bacterial and fungal communities, respectively. The analysis revealed increases in the relative abundances of the lactic acid (Pediococcus pentosaceus, Weissella hellenica and Limosilactobacillus pontis) and acetic acid bacteria (Gluconobacter frateurii and Acetobacter tropicalis) during the sourdough propagation. The filaments fungi, Alternaria tenuissima, Fusarium culmorum, Fusarium petersiae and Microdochium seminicola remained more stable in organic than conventional during propagation cycles. After 216 h of fermentation, both sourdoughs were dominated by acid- and salt-tolerant yeast Issatchenkia orientalis (syn Pichia kudriavzevii, and Candida glycerinogenes). In conclusion, there were no significant differences in microbial communities among the sourdough samples. This study revealed that both flours contain autochthonous LAB, AAB, and yeasts with biotechnological applications in sourdough bread-making.

Antimicrobial resistance of enterococci isolated from food in South Brazil: Comparing pre- and post-RDC 20/2011.

Antimicrobial resistance has been attributed to the overuse of antibiotics. To control the use of antibiotics, Brazil adopted the RDC 20/2011. A comparison the antibiotic-resistance profile of bacterial has provided important insights into resistance evolution. Enterococci are ubiquitous bacteria recommended to be used as a sentinel organism, in national surveillance systems, for tracking antimicrobial resistance through the food chain. The present study aimed to evaluate the diversity and antimicrobial resistance of enterococci collected from food in South Brazil in 2017 (pos-RDC 20/11) for comparison with isolated in 2007 (pre-RDC 20/11). A total of 310 enterococci were isolated from vegetables and products of animal origin, identified by PCR and MALDI-TOF, tested for antimicrobial susceptibility and screened for resistance genes. Enterococcus casseliflavus was dominant in vegetables and E. faecalis in products of animal origin. Enterococcal isolates in 2017 were mostly sensitive to ampicillin, gentamicin, chloramphenicol, and vancomycin when compared to isolated collected in 2007. While resistance levels to most compounds remained relatively stable, multidrug resistance decreased by 24% during this period. Our results suggest that RDC 20/11 had a positive outcome in controlling the spread of antimicrobial resistance. This study provides baseline data to measure future changes in the prevalence of resistant enterococci.

Microbiological and physicochemical characteristics of buffalo milk used for dairy products in southern Brazil.

In Brazil, the buffalo milk market has been growing. However, identity and quality standards have not been established for this raw material, nor have proper distinctions between buffalo milk and bovine milk been defined. Currently, the State of Rio Grande do Sul (RS) has only three producers that supply raw material for officially marketed derivatives. The aim of this study was to determine the identity and quality standards of raw buffalo milk in this region. Samples were obtained biweekly from three farm cooling tanks between June 2017 and August 2018, to reach a total of 69 samples. The averages for the results of the physicochemical parameters fat, protein, lactose, total solids, SNF (solids-not-fat), calcium, density, FP, acidity and SCC were 5.5 g/100 g, 4.06 g/100 g, 5.07 g/100 g, 15.5 g/100 g, 9.96 g/100 g, 0.161 g/100 g, 1.034 g/ml, -0.527°C, 16°D and 95 × 103 cells/ml, respectively. With reference to the microbiological parameters, the mean of the Standard Plate Count (SPC) and thermotolerant coliforms were 9,0 × 104 CFU/ml and 1.6 × 102 MPN/ml, respectively. Regarding coagulase-positive staphylococci, 36 samples tested positive (52% of total). Neither Salmonella spp. nor Listeria monocytogenes, nor antibiotic or antiparasitic residues were detected in any sample. In conclusion, the buffalo milk used as raw material for dairy products in southern Brazil demonstrated satisfactory physicochemical and microbiological characteristics, in accordance with recent scientific literature.

Evaluation antibacterial and antibiofilm activity of the antimicrobial peptide P34 against Staphylococcus aureus and Enterococcus faecalis

ABSTRACT The adhesion ability of bacteria to abiotic surfaces has important implications in food industries, because these organisms can survive for long periods through the biofilm formation. They can be transferred from one place to another in the industry causing contamination of the food processing environment. In this study, the antibacterial and antibiofilm activities of the antimicrobial peptide P34, characterized as a bacteriocin-like substance (BLS P34) were tested against planktonic and sessile cells of Staphylococcus aureus and Enterococcus faecalis isolated from foods. The BLS P34 showed inhibitory effect against all planktonic cells of E. faecalis. The inhibition of biofilm formation and the eradication of pre-formed biofilm were evaluated with the crystal violet assay and with the reduction of 3-bromide [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium. The BLS P34 promoted a reduction of percentage of adhered microbial cells on the surface, not being able to perform the complete elimination of biofilm formation. The metabolic activity of S. aureus biofilms decreased considerably between 41-95%. However, E. faecalis cells showed up metabolically stimulated. The BLS P34 has the potential antibiofilm for the species S. aureus. Studies suggest more detailed approaches to a better understanding of the interactions between the antimicrobial and bacterial cells within the biofilm structure.

Evaluation antibacterial and antibiofilm activity of the antimicrobial peptide P34 against Staphylococcus aureus and Enterococcus faecalis.

The adhesion ability of bacteria to abiotic surfaces has important implications in food industries, because these organisms can survive for long periods through the biofilm formation. They can be transferred from one place to another in the industry causing contamination of the food processing environment. In this study, the antibacterial and antibiofilm activities of the antimicrobial peptide P34, characterized as a bacteriocin-like substance (BLS P34) were tested against planktonic and sessile cells of Staphylococcus aureus and Enterococcus faecalis isolated from foods. The BLS P34 showed inhibitory effect against all planktonic cells of E. faecalis. The inhibition of biofilm formation and the eradication of pre-formed biofilm were evaluated with the crystal violet assay and with the reduction of 3-bromide [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium. The BLS P34 promoted a reduction of percentage of adhered microbial cells on the surface, not being able to perform the complete elimination of biofilm formation. The metabolic activity of S. aureus biofilms decreased considerably between 41-95%. However, E. faecalis cells showed up metabolically stimulated. The BLS P34 has the potential antibiofilm for the species S. aureus. Studies suggest more detailed approaches to a better understanding of the interactions between the antimicrobial and bacterial cells within the biofilm structure.

Antibiotic resistance and enterotoxin genes in Staphylococcus sp. isolates from polluted water in Southern Brazil.

The aim of this study was to evaluate the species distribution, antibiotic-resistance profile and presence of enterotoxin (SE) genes in staphylococci isolated from the Dilúvio stream in South Brazil. Eighty-eight staphylococci were identified, 93.18% were identified as coagulase-negative (CNS) and 6.82% coagulase-positive (CPS). Fourteen Staphylococcus species were detected and the most frequently were Staphylococcus cohnii (30.48%) and S. haemolyticus (21.95%). Resistance to erythromycin was verified in 37.50% of the strains, followed by 27.27% to penicillin, 12.50% to clindamycin, 6.81% to trimethoprim-sulfamethoxazole, 5.68% to chloramphenicol and 2.27% to norfloxacin. None of the investigated strains showed gentamicin and ciprofloxacin resistance. The strains were tested for the presence of sea, seb, sec, sed and see genes by PCR and only CNS strains (43.18%) showed positive results to one or more SE genes. The scientific importance of our results is due to the lack of data about these topics in polluted waters in Brazil. In conclusion, polluted waters from the Dilúvio stream may constitute a reservoir for disseminating antibiotic-resistance and enterotoxin into the community. In addition, the detection of staphylococci in the polluted waters of the Dilúvio stream indicated a situation of environmental contamination and poor sanitation conditions.

Virulência e formação de biofilme microbiano por Enterococcus faecalis isolados de swabs cloacais de frangos de corte infectados com Eimeria spp / Virulence and biofilm formation by Enterococcus faecalis isolates from cloacal swabs of broilers infected with Eimeria spp

A dinâmica da microbiota no trato gastrointestinal (TG) de animais pode ser afetada por patógenos, tais como Eimeria spp. Os enterococos são bactérias saprófitas que colonizam o TG de mamíferos e aves. A influência sobre a microbiota intestinal está relacionada com a capacidade de adaptação das bactérias em se aderir às células hospedeiras e de colonizar as células das mucosas. O objetivo deste estudo foi analisar a frequência de genes de virulência ace, agg e operon do bopABCD em Enterococcus faecalis isolados de swabs cloacais de frangos de corte desafiados com Eimeria spp e alimentados com dietas padrões suplementadas ou não com anticoccidiano (monesina) e também avaliar a capacidade dessas cepas em formar biofilmes sob condições in vitro. Um total de 70 E. faecalis foram selecionadas e o gene agg foi mais freqüente em cepas isoladas de frangos de corte alimentados com anticoccidiano (92,3%) quando comparado ao grupo que não recebeu anticoccidiano (70,5%). Por outro lado, os genes ace e do operon bopABCD não demostraram nenhuma diferença significativa entre os dois grupos de frangos (P>0,005). Os E. faecalis isolados de frangos de corte alimentados com anticoccidiano demostraram uma maior frequência de fortes aderentes quando crescendo em meio suplementado com glicose (92,3-88,5%) e urina (77%), quando comparado com enterococos isolados de frangos que não receberam anticoccidiano. Observou-se que E. faecalis isolados de frangos tratados com anticoccidiano mostraram uma maior frequêencia dos genes dos fatores de virulência e de perfil de fortes formadores de biofilme, o que indica uma melhor adaptação dos isolados em ambiente intestinal saudável.

The microbiota dynamics in the gastrointestinal tract (GT) of animals can be disrupted by pathogens, such as Eimeria spp. Enterococci are saprophytic bacteria that colonize the GT of mammals and birds. The influence on the intestinal microbiota is related to the adaptive capacity of bacteria to adhere to host cells and colonize the mucosal cells. The aim of this study was to analyze the frequency of virulence genes ace, agg and bopABCD operon in Enterococcus faecalis isolated from cloacal swabs of broilers challenged with Eimeria spp. and fed with a standard diet supplemented or not with anticoccidial (monensin), and, also to evaluate for the ability of these strains to form biofilms under in vitro conditions. A total of 70 E. faecalis were selected and the agg gene was more frequent in strains isolated from the broilers treated with anticoccidial (92.3%) when compared to the group that not received anticoccidial (70.5%). On the other hand, the ace and bopABCD operon genes showed no significant difference between the two groups of broilers (P>0.005). The E. faecalis isolated from the broilers treated with anticoccidial showed a higher frequency of strong biofilm formation when growing in medium supplemented with glucose (92.3-88.5%) and urine (77%) when compared with enterococci isolated from broilers that not received anticoccidial. It was observed that E. faecalis isolated from broilers treated with anticoccidial showed a higher frequency of virulence factors genes and stronger biofilms formation, indicating better adaptation of the isolates in healthy intestinal environment.

Differential expression of cysteine desulfurases in soybean.

BACKGROUND: Iron-sulfur [Fe-S] clusters are prosthetic groups required to sustain fundamental life processes including electron transfer, metabolic reactions, sensing, signaling, gene regulation and stabilization of protein structures. In plants, the biogenesis of Fe-S protein is compartmentalized and adapted to specific needs of the cell. Many environmental factors affect plant development and limit productivity and geographical distribution. The impact of these limiting factors is particularly relevant for major crops, such as soybean, which has worldwide economic importance. RESULTS: Here we analyze the transcriptional profile of the soybean cysteine desulfurases NFS1, NFS2 and ISD11 genes, involved in the biogenesis of [Fe-S] clusters, by quantitative RT-PCR. NFS1, ISD11 and NFS2 encoding two mitochondrial and one plastid located proteins, respectively, are duplicated and showed distinct transcript levels considering tissue and stress response. NFS1 and ISD11 are highly expressed in roots, whereas NFS2 showed no differential expression in tissues. Cold-treated plants showed a decrease in NFS2 and ISD11 transcript levels in roots, and an increased expression of NFS1 and ISD11 genes in leaves. Plants treated with salicylic acid exhibited increased NFS1 transcript levels in roots but lower levels in leaves. In silico analysis of promoter regions indicated the presence of different cis-elements in cysteine desulfurase genes, in good agreement with differential expression of each locus. Our data also showed that increasing of transcript levels of mitochondrial genes, NFS1/ISD11, are associated with higher activities of aldehyde oxidase and xanthine dehydrogenase, two cytosolic Fe-S proteins. CONCLUSIONS: Our results suggest a relationship between gene expression pattern, biochemical effects, and transcription factor binding sites in promoter regions of cysteine desulfurase genes. Moreover, data show proportionality between NFS1 and ISD11 genes expression.

Diversity of mutations in the atpC gene coding for the c Subunit of F0F1 ATPase in clinical isolates of optochin-resistant Streptococcus pneumoniae from Brazil.

We report the characteristics of four optochin-resistant (Opt(r)) Streptococcus pneumoniae isolates from Brazil. All four Opt(r) isolates presented mutations in the nucleotide sequence coding for the c subunit of F(0)F(1) ATPase. Two isolates showed mutations in codons 23 (leading to the deduced amino acid substitution isoleucine instead of alanine) and 49 (serine instead of alanine, a novel type of mutation detected at this position), respectively. Two additional novel mutations, both located in codon 45, were detected in the other two isolates, corresponding to leucine or valine (instead of phenylalanine). The data indicate that three previously unrecognized alterations were detected in the atpC gene of S. pneumoniae and that Opt resistance among Brazilian pneumococcal isolates is not related to a specific pneumococcal serotype, antimicrobial-resistance profile, or clonal group.