The Impact of Low-Level Benzalkonium Chloride Exposure on Staphylococcus spp. Strains and Control by Photoinactivation.

Exposure of bacteria to low concentrations of biocides can facilitate horizontal gene transfer, which may lead to bacterial adaptive responses and resistance to antimicrobial agents. The emergence of antibacterial resistance not only poses a significant concern to the dairy industry but also adds to the complexity and cost of mastitis treatment. This study was aimed to evaluate how selective stress induced by benzalkonium chloride (BC) promotes antibiotic non-susceptibility in Staphylococcus spp. In addition, we investigated the efficacy of photodynamic inactivation (PDI) in both resistant and susceptible strains. The study determined the minimum inhibitory concentration (MIC) of BC using the broth microdilution method for different Staphylococcus strains. The experiments involved pairing strains carrying the qacA/qacC resistance genes with susceptible strains and exposing them to subinhibitory concentrations of BC for 72 h. The recovered isolates were tested for MIC BC and subjected to disc diffusion tests to assess changes in susceptibility patterns. The results demonstrated that subinhibitory concentrations of BC could select strains with reduced susceptibility and antibiotic resistance, particularly in the presence of S. pasteuri. The results of PDI mediated by toluidine blue (100 µM) followed by 60 min irradiation (total light dose of 2.5 J/cm2) were highly effective, showing complete inactivation for some bacterial strains and a reduction of up to 5 logs in others.

Genetic and Antimicrobial Resistance Profiles of Mammary Pathogenic E. coli (MPEC) Isolates from Bovine Clinical Mastitis.

Mammary pathogenic E. coli (MPEC) is one of the main pathogens of environmental origin responsible for causing clinical mastitis worldwide. Even though E. coli are strongly associated with transient or persistent mastitis and the economic impacts of this disease, the virulence factors involved in the pathogenesis of MPEC remain unknown. Our aim was to characterize 110 MPEC isolates obtained from the milk of cows with clinical mastitis, regarding the virulence factor-encoding genes present, adherence patterns on HeLa cells, and antimicrobial resistance profile. The MPEC isolates were classified mainly in phylogroups A (50.9%) and B1 (38.2%). None of the isolates harbored genes used for diarrheagenic E. coli classification, but 26 (23.6%) and 4 (3.6%) isolates produced the aggregative or diffuse adherence pattern, respectively. Among the 22 genes investigated, encoding virulence factors associated with extraintestinal pathogenic E. coli pathogenesis, fimH (93.6%) was the most frequent, followed by traT (77.3%) and ompT (68.2%). Pulsed-field gel electrophoresis analysis revealed six pulse-types with isolates obtained over time, thus indicating persistent intramammary infections. The genes encoding beta-lactamases detected were as follows: blaTEM (35/31.8%); blaCTX-M-2/blaCTX-M-8 (2/1.8%); blaCTX-M-15 and blaCMY-2 (1/0.9%); five isolates were classified as extended spectrum beta-lactamase (ESBL) producers. As far as we know, papA, shf, ireA, sat and blaCTX-M-8 were detected for the first time in MPEC. In summary, the genetic profile of the MPEC studied was highly heterogeneous, making it impossible to establish a common genetic profile useful for molecular MPEC classification. Moreover, the detection of ESBL-producing isolates is a serious public health concern.

Rumen and lower gut microbiomes relationship with feed efficiency and production traits throughout the lactation of Holstein dairy cows.

Fermentation of dietary nutrients in ruminants' gastrointestinal (GI) tract is an essential mechanism utilized to meet daily energy requirements. Especially in lactating dairy cows, the GI microbiome plays a pivotal role in the breakdown of indigestible plant polysaccharides and supply most AAs, fatty acids, and gluconeogenic precursors for milk synthesis. Although the contribution of the rumen microbiome to production efficiency in dairy cows has been widely researched over the years, variations throughout the lactation and the lower gut microbiome contribution to these traits remain poorly characterized. Therefore, we investigated throughout lactation the relationship between the rumen and lower gut microbiomes with production efficiency traits in Holstein cows. We found that the microbiome from both locations has temporal stability throughout lactation, yet factors such as feed intake levels played a significant role in shaping microbiome diversity. The composition of the rumen microbiome was dependent on feed intake. In contrast, the lower gut microbiome was less dependent on feed intake and associated with a potentially enhanced ability to digest dietary nutrients. Therefore, milk production traits may be more correlated with microorganisms present in the lower gut than previously expected. The current study's findings advance our understanding of the temporal relationship of the rumen and lower gut microbiomes by enabling a broader overview of the gut microbiome and production efficiency towards more sustainable livestock production.

Short communication: Association between the accessory gene regulator (agr) group and the severity of bovine mastitis caused by Staphylococcus aureus.

Staphylococcus aureus can elicit mild to more severe degrees of mastitis in cattle, depending on the response of the host's immune system and the virulence factors of the specific isolate. Several virulence factors are controlled by a global regulatory system, designated accessory gene regulator (agr). Thus, the objective was to examine associations between different capsular and agr types and the severity of bovine mastitis caused by S. aureus. All isolates were obtained from bovine subclinical (n = 50), mild clinical (n = 73), and moderate clinical mastitis cases (n = 28). Isolates containing the agrI gene and lacking the agr locus (agr-) were more prevalent among subclinical than clinical mastitis cases, whereas isolates containing the agrII and agrIII genes were more prevalent among clinical mastitis cases. The capsular types 5 (cap5) and 8 (cap8) were found in 42 and 44%, respectively, of the isolates obtained from subclinical cases and in 38.6 and 58.4%, respectively, of those isolated from clinical mastitis cases. Capsular type was not associated with type of mastitis (subclinical, mild clinical, or moderate clinical). We found a strong association between agr type and type of mastitis, suggesting that knowledge of S. aureus genetic profiles could be an additional tool to control this disease.

First investigation of Staphylococcus argenteus in a Brazilian collections of S. aureus isolated from bovine mastitis.

BACKGROUND: Staphylococcus argenteus is a new specie positive coagulase staphylococci. We investigate the presence of S. argenteus in isolates previously classified as S. aureus, obtained from the milk of cows with mastitis in Brazil. RESULTS: Among 856 S. aureus tested in chocolate agar, tryptone soya agar and salt egg yolk agar, white or colorless colonies were observed in 185 (21.6%) isolates. Regarding the ctrOPQMN operon, 111 (60%) presented the complete cluster. Despite some missing genes in this cluster, the remaining strains (74) were confirmed as S. aureus using the nrps gene. CONCLUSIONS: As far as we know, this is the first review of S. aureus collection in Brazil and S. argenteus does not appear to be a significant problem in Brazilian herds.

Biofilm production under different atmospheres and growth media by Streptococcus agalactiae isolated from milk of cows with subclinical mastitis.

Different methods to analyze Streptococcus agalactiae biofilm formation have been investigated, but standardized protocols have not been developed. We compared S. agalactiae biofilm production among different atmospheres and growth media. Biofilm formation was studied in 32 isolates from bovine mastitis cases grown in Tryptone Soy Broth (TSB), Todd Hewitt Broth (THB), Luria Bertani Broth (LB) and Brain Heart Infusion (BHI), under two atmospheres, aerobic and 5% CO2. Regardless of the culture medium, growth under 5% CO2 resulted in a greater proportion of biofilm formation (65.63%), as compared with aerobic conditions (39.84%). Regardless of the atmosphere, the chances of biofilm formation were greater for isolates grown in TSB, as compared with THB [Odds ratio (OR) = 3.02], BHI (OR = 4.57), or LB (OR = 10.20). Thus, we suggest the use of 5% CO2 atmosphere and TSB in biofilm formation assays by Group-B streptococci (GBS) isolated from intramammary infections.

Comparative analysis of four commercial on-farm culture methods to identify bacteria associated with clinical mastitis in dairy cattle.

Several multiple-media culture systems have become commercially available for on-farm identification of mastitis-associated pathogens. However, the accuracy of these systems has not been thoroughly and independently validated against microbiological evaluations performed by referral laboratories. Therefore, the purpose of the present study was to evaluate the performance of commercially available culture plates (Accumast, Minnesota Easy System, SSGN and SSGNC Quad plates) to identify pathogens associated with clinical mastitis in dairy cows. Milk samples from the affected quarter with clinical mastitis were aerobically cultured with the on-farm culture systems and by two additional reference laboratories. Agreeing results from both standard laboratories were denoted as the reference standard (RS). Accuracy (Ac), sensitivity (Se), specificity (Sp), positive and negative predictive values (PPV and NPV, respectively) and Cohen's kappa coefficient (k) of on-farm plates were determined based on the RS culture of 211 milk samples. All four plate-systems correctly identified ≥ 84.9% of milk samples with no bacterial growth. Accumast had greater values for all overall predictive factors (Ac, Se, Sp, PPV and NPV) and a substantial agreement (k = 0.79) with RS. The inter-rater agreements of Minnesota, SSGN, and SSGNC with RS were moderate (0.45 ≤ k ≤ 0.55). The effectiveness to categorize bacterial colonies at the genus and species was numerically different amongst the commercial plates. Our findings suggest that Accumast was the most accurate on-farm culture system for identification of mastitis-associated pathogens of the four systems included in the analysis.

Cross-Contamination and Biofilm Formation by Salmonella enterica Serovar Enteritidis on Various Cutting Boards.

Cross-contamination is one of the main factors related to foodborne outbreaks. This study aimed to analyze the cross-contamination process of Salmonella enterica serovar Enteritidis from poultry to cucumbers, on various cutting board surfaces (plastic, wood, and glass) before and after washing and in the presence and absence of biofilm. Thus, 10 strains of Salmonella Enteritidis were used to test cross-contamination from poultry to the cutting boards and from thereon to cucumbers. Moreover, these strains were evaluated as to their capacity to form biofilm on hydrophobic (wood and plastic) and hydrophilic materials (glass). We recovered the 10 isolates from all unwashed boards and from all cucumbers that had contacted them. After washing, the recovery ranged from 10% to 100%, depending on the board material. In the presence of biofilm, the recovery of salmonellae was 100%, even after washing. Biofilm formation occurred more on wood (60%) and plastic (40%) than glass (10%) boards, demonstrating that bacteria adhered more to a hydrophobic material. It was concluded that the cutting boards represent a critical point in cross-contamination, particularly in the presence of biofilm. Salmonella Enteritidis was able to form a biofilm on these three types of cutting boards but glass showed the least formation.

Milk microbiome and bacterial load following dry cow therapy without antibiotics in dairy cows with healthy mammary gland.

Preventive infusion of antibiotics in the mammary gland of cows consumes 11 tons/year of medically relevant antimicrobials, yet, this practice might not be critical to prevent new infections in the healthy mammary gland of cows. Here, we used next-generation sequencing and quantitative real-time PCR to determine the impact of dry cow therapy without antibiotics on milk microbiome and bacterial load, respectively. Cows diagnosed as negative for mastitis at dry off were randomly allocated to receive antibiotic (intramammary ceftiofur hydrochloride) and teat sealant or just teat sealant. Firmicutes was the most abundant phylum, and Corynebacterium, Acinetobacter, and Staphylococcus, often involved in mastitis cases, were the most abundant genera across treatments and time. However, there were no effects of antimicrobial on milk microbiome and bacterial load. Bacterial load was greater at seven days postpartum than at dry off. Dry cow therapy based on teat sealant without antibiotics can be used with no detrimental impacts on milk microbiome and bacterial load in cows with a healthy mammary gland.

Presence of Immune Evasion Cluster and Molecular Typing of Methicillin-Susceptible Staphylococcus aureus Isolated from Food Handlers.

The hands and noses of food handlers colonized by Staphylococcus aureus are an important source of food contamination in restaurants and food processing. Several virulence factors can be carried by mobile elements in strains of S. aureus, including the immune evasion cluster (IEC). This gene cluster improves the capacity of S. aureus to evade the human immune response. Many studies have reported the transmission of strains between animals and humans, such as farm workers that have close contact with livestock. However, there are few studies on the transmission between food and food handlers. The aim of this study was to detect the IEC and the mecA gene in strains isolated from food handlers and to type these strains using the spa typing method. Thirty-five strains of S. aureus isolated from the noses and hands of food handlers in three different kitchens were analyzed for the presence of the mecA gene and IEC and by spa typing. All strains were negative for the mecA gene, and the presence of IEC was observed in 10 (28.6%) strains. Fifteen different spa types were observed, with the most frequent being t127 (42.85%) and t002 (11.42%). Strains from the two most prevalent spa types and a novel spa type were typed by multilocus sequence typing. spa types t127, t002, and t13335 were determined to be multilocus sequence types (ST) ST-30, ST-5, and ST-45, respectively. The food handlers may have been contaminated by these strains of S. aureus through food, which is suggested by the low frequency of IEC and by ST that are observed more commonly in animals.