Identification of Escherichia coli isolated from flies (Insecta: Diptera) that inhabit the environment of dairy farms harboring extraintestinal virulence markers.

AIMS: We investigate extraintestinal pathogenic genes (ExPEC) related to virulence of Escherichia coli in flies from the dairy environment. METHODS AND RESULTS: We collected 217 flies from nine dairy farms, which were submitted to microbiological culture. Fifty-one E. coli were identified using mass spectrometry. Eleven dipteran families were identified, with a predominance of Muscidae, and a minor frequency of Tachinidae, Drosophilidae, Sphaeroceridae, Ulidiidae, Syrphidae, Chloropidae, Calliphoridae, Sarcophagidae, and Piophilidae. A panel of 16 virulence-encoding genes related to ExPEC infections were investigated, which revealed predominance of serum resistance (traT, 31/51 = 60.8%; ompT, 29/51 = 56.9%), iron uptake (irp2, 17/51 = 33.3%, iucD 11/51 = 21.6%), and adhesins (papC, 6/51 = 11.8%; papA, 5/51 = 9.8%). CONCLUSIONS: Our findings reveal Dipterans from milking environment carrying ExPEC virulence-encoding genes also identified in clinical bovine E. coli-induced infections.

MALDI-TOF bacterial subtyping for rapid detection of biomarkers in Staphylococcus aureus from subclinical bovine mastitis.

AIMS: This study aimed to evaluate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bacterial subtyping for the rapid detection of biomarkers in Staphylococcus aureus from subclinical bovine mastitis. METHODS AND RESULTS: A total of 229 S. aureus isolates were obtained from milk samples collected from cows with subclinical mastitis using microbiological culture. Staphylococcus aureus isolates were also submitted to PCR analysis targeting the mecA and mecC genes, which are indicative of methicillin resistance. Confirmation of the species was achieved through MALDI-TOF MS analysis. To analyze antimicrobial resistance patterns, the MALDI BioTyper Compass Explorer and ClinProTools Bruker software were employed, and dendrograms were generated using Bionumerics software. CONCLUSIONS: MALDI-TOF MS successfully identified S. aureus at the species level, but no methicillin resistance was observed. Moreover, spectral typing displayed limited similarity when compared to pulsed-field gel electrophoresis (PFGE).

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.

Molecular characterization of persistent subclinical mastitis-causing Staphylococcus aureus from dairy farms.

The study aimed to evaluate the genetic diversity of Staphylococcus aureus causing subclinical mastitis (SM) isolated from dairy cows and to assess the effect of the infection status (transient vs. persistent) on the milk and component yield. A total of six dairy farms in São Paulo state were used for the selection of cows with SM caused by S. aureus. S. aureus strains (n = 56) obtained from three biweekly aseptic mammary quarter milk samplings (n = 1140 from 95 cows) were subjected to MALDI-TOF MS analysis for species confirmation and further PFGE analysis. Intramammary infections (IMI) caused by S. aureus were categorized as transient (T: when only one out of 3 milk samplings had positive isolation of any pulsotype) or persistent (P: when two (P2) or three (P3) milk samplings had positive isolation of identical pulsotype over the consecutive episodes of SM. The SmaI macrorestriction fragment profiles of 56 S. aureus isolates showed a dominant S. aureus clonal pattern (PFGE type A; n = 50; 89.3%) within and among the herds. The SM-causing S. aureus represented a reduction of quarter milk yield of 26.2% in transient and 54.8% in persistent cases as well as a reduction of total solid yield of 38.1% and 49.4%, respectively, when compared with the healthy control quarters. Overall, the greater chance of S. aureus to be persistent is when a dominant clonal pattern is present in the herd which consequently may be associated with the cause of accentuated milk loss.

Characterization of mammary pathogenic Escherichia coli reveals the diversity of Escherichia coli isolates associated with bovine clinical mastitis in Brazil.

Mammary pathogenic Escherichia coli (MPEC) is one of the most common pathogens associated with clinical mastitis. We analyzed isolates obtained from milk samples of cows with clinical mastitis, collected from 10 farms in Brazil, to verify molecular and phenotypic characteristics. A total of 192 (4.5%) mammary pathogenic E. coli isolates were obtained from 4,275 milk samples analyzed, but we tested 161. We assigned most of these isolates to E. coli phylogroups B1 (52.8%) and A (36.6%), although phylogroups B2, C, D, E, and unknown also occurred. All isolates were assessed for the presence of several genes encoding virulence factors, such as adhesins (sfaDE, papC, afaBC III, ecpA, fimH, papA, and iha), toxins (hlyA, cnf1, sat, vat, and cdt), siderophores (iroN, irp2, iucD, ireA, and sitA), an invasion protein (ibeA), and serum resistance proteins (traT, KpsMTII, and ompT), and isolates from phylogroups B1, B2, and E showed up to 8 genes. Two isolates harbored the locus of enterocyte effacement (escN+) and lack the bundle-forming pilus (bfpB-) operon, which corresponds to a molecular profile of a subgroup of diarrheagenic E. coli (aEPEC), thus being classified as hybrid MPEC/aEPEC isolates. These isolates displayed a localized adherence-like pattern of adherence in HeLa cells and were able to promote F-actin polymerization underneath adherent bacteria. Based on the pulsed-field gel electrophoresis analyses, considerable genetic variability was observed. A low index of antimicrobial resistance was observed and 2 extended-spectrum ß-lactamase-producing E. coli were identified, both harboring blaCTX-M15 gene, and were classified as ST10 and ST993 using multilocus sequence typing. A total of 148 (91.2%) isolates were weak biofilm producers or formed no biofilm. Because raw milk is still frequently consumed in Brazil, the occurrence of virulence factor-encoding genes from extraintestinal or diarrheagenic E. coli added to the presence of extended-spectrum ß-lactamase-producing isolates can turn this veterinary medicine problem into a public health concern.

Virulence-encoding genes related to extraintestinal pathogenic E. coli and multidrug resistant pattern of strains isolated from neonatal calves with different severity scores of umbilical infections.

Umbilical infections in calves comprise a major cause of neonatal mortality and have been related to a variety of microorganisms. E. coli is an opportunistic enteropathogen characterized by a diversity of virulence factors (VF). Nonetheless, the gene profiles that encode VF associated with umbilical infections in calves and their effect on the clinical severity remains unclear. In this scenario, microbial identification (with an emphasis on E. coli), was carried out among 150 neonatal calves (≤30 days of age) with umbilical infections, where the omphalopathies were clinically scored as mild, moderate, or severe. Also, a panel of 16 virulence-encoding genes related to extraintestinal pathogenic E. coli (ExPEC) were investigated, i.e., fimbriae/adhesins (sfa/focDEa, papA, papC, afaBC), toxins (hlyA, sat, cnf1, cdt), siderophores (iroN, irp2, iucD, ireA), invasins (ibeA), and serum resistance (ompT, traT, kpsMT II). Bacteria and yeasts isolates were identified using mass spectrometry. Bacteria, yeasts, and fungi were isolated in 94.7% (142/150) of neonatal calves sampled. E. coli was the agent most frequently isolated (59/150 = 39.3%), in pure culture (27/59 = 45.8%) and combined infections (32/59 = 54.2%), although a great variety (n = 83) of other species of microorganisms were identified. Clinical severity scores of 1, 2, and 3 were observed in 32.2% (19/59), 23.7% (14/59), and 44.1% (26/59) of E. coli infections, respectively. The ExPEC genes detected were related to serum resistance (traT, 42/59 = 72.2%; ompT, 35/59 = 59.3%, kpsMTII, 10/59 = 17%), invasins (ibeA, 11/59 = 18.6%), siderophores (iucD, 9/59 = 15.3%; iroN, 8/59 = 13.6%), and adhesins/fimbriae (papA, 8/59 = 13.6%; papC, 15/59 = 9.6%). The presence of each virulence gene was not associated with the case's clinical score. Among all isolates, 89.8% (53/59) showed in vitro resistance to sulfamethoxazole/trimethoprim and 59.3% to ampicillin (35/59), while 94.1% (55/59) revealed a multidrug resistant profile. Great complexity of bacteria, yeast, and fungi species was identified, reinforcing the umbilical infections of neonatal calves as a polymicrobial disorder. The high occurrence of E. coli (39.3%) highlights the role of this pathogen in the etiology of umbilical infections in calves. Furthermore, a panel of ExPEC genes was investigated for the first time among calves that were clinically scored for case severity. The high prevalence of traT and ompT indicates that these serum resistance-related genes could be used as biomarkers for further investigations of ExPEC isolates from umbilical infections. Our results contribute to the etiological investigation, clinical severity scoring, antimicrobial resistance pattern, and virulence-related to ExPEC genes involved in umbilical infections of neonatal calves.

Multiclonal human origin and global expansion of an endemic bacterial pathogen of livestock.

Most new pathogens of humans and animals arise via switching events from distinct host species. However, our understanding of the evolutionary and ecological drivers of successful host adaptation, expansion, and dissemination are limited. Staphylococcus aureus is a major bacterial pathogen of humans and a leading cause of mastitis in dairy cows worldwide. Here we trace the evolutionary history of bovine S. aureus using a global dataset of 10,254 S. aureus genomes including 1,896 bovine isolates from 32 countries in 6 continents. We identified 7 major contemporary endemic clones of S. aureus causing bovine mastitis around the world and traced them back to 4 independent host-jump events from humans that occurred up to 2,500 y ago. Individual clones emerged and underwent clonal expansion from the mid-19th to late 20th century coinciding with the commercialization and industrialization of dairy farming, and older lineages have become globally distributed via established cattle trade links. Importantly, we identified lineage-dependent differences in the frequency of host transmission events between humans and cows in both directions revealing high risk clones threatening veterinary and human health. Finally, pangenome network analysis revealed that some bovine S. aureus lineages contained distinct sets of bovine-associated genes, consistent with multiple trajectories to host adaptation via gene acquisition. Taken together, we have dissected the evolutionary history of a major endemic pathogen of livestock providing a comprehensive temporal, geographic, and gene-level perspective of its remarkable success.

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.

Characterization of Uropathogenic Escherichia coli Reveals Hybrid Isolates of Uropathogenic and Diarrheagenic (UPEC/DEC) E. coli.

(1) Background: Pathogenic Escherichia coli are divided into two groups: diarrheagenic (DEC) and extraintestinal pathogenic (ExPEC) E. coli. ExPEC causing urinary tract infections (UTIs) are termed uropathogenic E. coli (UPEC) and are the most common cause of UTIs worldwide. (2) Methods: Here, we characterized 112 UPEC in terms of phylogroup, serotype, the presence of virulence factor-encoding genes, and antimicrobial resistance. (3) Results: The majority of the isolates were assigned into the phylogroup B2 (41.07%), and the serogroups O6 (12.5%) and O25 (8.9%) were the most frequent. Five hybrid UPEC (4.5%), with markers from two DEC pathotypes, i.e., atypical enteropathogenic (aEPEC) and enteroaggregative (EAEC) E. coli, were identified, and designated UPEC/aEPEC (one isolate) and UPEC/EAEC (four isolates), respectively. Three UPEC/EAEC harbored genes from the pap operon, and the UPEC/aEPEC carried ibeA. The highest resistance rates were observed for ampicillin (46.4%) and trimethoprim/sulfamethoxazole (34.8%), while 99.1% of the isolates were susceptible to nitrofurantoin and/or fosfomycin. Moreover, 9.8% of the isolates were identified as Extended Spectrum ß-Lactamase producers, including one hybrid UPEC/EAEC. (4) Conclusion: Our data reinforce that hybrid UPEC/DEC are circulating in the city of Botucatu, Brazil, as uropathogens. However, how and whether these combinations of genes influence their pathogenicity is a question that remains to be elucidated.

Quality of dialysis water and dialysate in haemodialysis centres: Highlight for occurrence of non-fermenting gram-negative bacilli.

AIMS: To evaluate the physicochemical and microbiological quality of dialysis water and dialysate samples from haemodialysis centres. METHODS AND RESULTS: Samples were fortnightly collected from three haemodialysis centres in Bauru City, Brazil, between July 2017 and June 2018, at the stages of post-reverse osmosis, reuse and dialysate. Analyses included determination of conductivity, fluoride, nitrate and sulphate; test for total coliform bacteria; count of heterotrophic bacteria; count and identification of non-fermenting gram-negative bacilli (NFGNB); drug susceptibility test; biofilm formation capacity; and genetic similarity among some isolated NFGNB. Of the analysed samples, only 4/72 (5.6%) had conductivity values ≥10 mS/cm, 4/216 (1.9%) presented total coliforms and 1/216 (0.5%) had heterotrophic bacteria count >100 CFU/ml. NFGNB were isolated from 99/216 (45.8%) samples, and the major identified micro-organisms included Herbaspirillum aquaticum/huttiense, Brevundimonas aurantiaca, Cupriavidus metallidurans, Pseudomonas aeruginosa and Ralstonia insidiosa. Isolates of P. aeruginosa and Burkholderia cepacia complex were sensitive to most antimicrobials and, together with isolates of Ralstonia insidiosa and Ralstonia pickettii, showed strong biofilm formation capacity. Some isolates expressed the same electrophoretic profile on pulsed-field gel electrophoresis, indicating the persistence of bacterial clones in the systems over time. CONCLUSIONS: NFGNB were observed in several dialysis water and dialysate samples from all investigated centres, which may represent a risk to the health of patients. SIGNIFICANCE AND IMPACT OF THE STUDY: Regular inclusion of actions for NFGNB control and monitoring in haemodialysis fluids are suggested for greater safety of the dialytic process.

Association between antimicrobial use and antimicrobial resistance of Streptococcus uberis causing clinical mastitis.

It is unknown whether overuse of antimicrobials against clinical mastitis (CM) from Streptococcus uberis is associated with increased antimicrobial resistance (AMR). Therefore, the aim of this study was to evaluate the association between antimicrobial use (AMU) and AMR in relation to the Strep. uberis causing CM in dairy herds. A total of 83 Strep. uberis isolates were selected from a collection created during a previous study evaluating the epidemiology of CM in dairy herds (n = 17) of southeastern Brazil. For each case of CM identified on farm, the following information was recorded: cow's identification number, affected mammary quarter, date of CM diagnosis, antimicrobial commercial names, number of administrations, and descriptions of protocol changes during the treatment. Streptococcus uberis isolates were confirmed by conventional culture, MALDI-TOF mass spectrometry, and quantitative multiplex PCR analyses. Thus, a total of 8 antimicrobials commonly used for CM treatment were evaluated for antimicrobial activity against Strep. uberis isolates. The minimum inhibitory levels of antimicrobials were determined at the lowest concentrations able to inhibit 50 and 90%, respectively, of Strep. uberis isolates. Data related to the antibiotics used for treatment of CM was used to calculate the frequency of administered antimicrobials as the number of defined daily doses (DDD). The highest frequencies of resistant Strep. uberis were observed for erythromycin (80.7% resistant, R), tetracycline (R = 59%), and penicillin G (R = 57.8%), whereas against ceftiofur only 10.8% of Strep. uberis isolates were resistant, and only 1.2% of the Strep. uberis isolates were resistant to enrofloxacin. Regarding the evaluation of resistance for antimicrobial classes, the highest frequency was observed for macrolides (R = 80.7%; 19.3% susceptible, S). Additionally, a frequency of 18.7% of Strep. uberis isolates were resistant to cephalosporins (S = 81.3%), respectively. Further, 94% of Strep. uberis isolates were multiresistant; all these isolates presented resistance to at least 3 different antimicrobial classes. The overall monthly average of antimicrobial treatment incidence (ATI) among the 17 herds enrolled in the study was 23.7 DDD per 1,000 lactating dairy cows [standard deviation (SD) = 13.9], ranging from 5.0 to 55.4 DDD per 1,000 cows in lactation-day. Cephalosporins and penicillins were the most commonly used antimicrobial classes among the evaluated herds (n = 16; 94.1%), followed by tetracyclines (n = 15 herds; 88.2%), fluoroquinolones (n = 14; 82.3%), and sulfonamides (n = 14; 82.3%). The tetracycline class had the highest ATI mean (5.0 DDD per 1,000 lactating cow-days, SD = 5.8), followed by fluoroquinolones (4.7 DDD per 1,000 lactating cow-days, SD = 6.0) and cephalosporins (3.8 DDD per 1,000 lactating cow-days, SD = 6.0). The overall use of antimicrobials was associated with the resistance of Strep. uberis to the antimicrobial tetracycline.

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.

Environmental persistence and virulence of Salmonella spp. Isolated from a poultry slaughterhouse.

Salmonella spp. is responsible for severe foodborne disease, and is one of the main agents involved in foodborne outbreaks worldwide. Contamination occurs mainly as a result of poultry and egg consumption since they can carry some serotypes pathogenic to humans. The aim of the study was to evaluate the persistence and pathogenic potential of Salmonella spp. (n = 40) isolated from poultry slaughterhouse mats, using adhesion and invasion assays, antimicrobial susceptibility by disc diffusion, and biofilm production as phenotypic tests and genotypic analyses. Polystyrene mats presented 3.2 times greater chance of isolating Salmonella than canvas mats. Besides, we observed resistance to tetracycline (17.5%), ampicillin (10%), cefotaxime (7.5%), trimethoprim-sulfamethoxazole (5%), and chloramphenicol (2.5%). All strains possessed the invA, sipB, sipD, ssaR, sifA, sitC, iroN, tolC, flgK, fljB, and flgL genes. The genes sopB and sipA were both present in 92.5% of the isolates, while sopD and spvB were observed in 90% and 32.5% of strains, respectively. All strains adhered to and invaded HeLa cells. Regarding biofilm production, 31 (77.5%) strains were able to produce biofilm on polystyrene microplates. Using PFGE, we detected the persistence of clones in the environment for up to 18 fromthe 20 weeks. The ability of these strains to produce a biofilm and thus persist in the environment and disperse through contact surfaces in the processing plant favors the contamination of food, aggravated by the pathogenic potential of these isolates demonstrated by their adhesion capacity, invasion and resistance to various antibiotic agents.

Short communication: Investigation of extra-intestinal pathogenic Escherichia coli virulence genes, bacterial motility, and multidrug resistance pattern of strains isolated from dairy cows with different severity scores of clinical mastitis.

Escherichia coli is a major pathogen involved in the etiology of environmentally derived bovine mastitis and is characterized by a variety of virulence factors (VF). Mammary infections with E. coli have shown a wide range of clinical signs, causing changes in milk (score 1, or mild), abnormal appearance of milk and udder inflammation (score 2, or moderate), and abnormalities in milk, udder inflammation, and systemic signs of illness (score 3, or severe). Nevertheless, to date, the profile of the genes related to the virulence of the pathogen in mammary infections and the severity scores of cases have not been thoroughly elucidated. Therefore, a panel of 18 virulence-encoding genes associated with extra-enteric pathogenicity of E. coli (ExPEC) were investigated in addition to in vitro swimming and swarming motility profiles and antimicrobial susceptibility/resistance patterns among 114 E. coli strains isolated from cows with clinical mastitis and different severity scores. Of 114 clinical cases, 39.5, 54.4, and 6.1% were mild, moderate, and severe, respectively. The main genes related to VF harbored by isolates were adhesins (fimH 100%; ecpA 64.0%, fimA 31.6%), serum resistance (traT 81.6%; ompT 35.1%), siderophores (irp2 9.6%), and hemolysin (hlyA 7%). Among the isolates studied, 99.1% showed in vitro resistance to bacitracin and cloxacillin, and 98.2% to lincosamin. Of the total isolates, 98.2% were considered multidrug resistant based on the multiple antimicrobial resistance index. No significant difference was observed between mean swimming (13.8 mm) and swarming (13.5 mm) motility, as well as severity scores of clinical mastitis and the ExPEC genes studied. The isolation of strains resistant to various antimicrobials, even though tested only in vitro, highlights the importance of rational use of antimicrobials for mastitis treatment. The high prevalence of the genes related to serum resistance (traT and ompT) and adhesion (ecpA) of the pathogen, in addition to main associations between the genes fimH, ecpA, and traT among cows with severity scores of 1 (15%) and 2 (22.6%), indicates that the genes traT, ecpA, and ompT could be further studied as biomarkers of ExPEC for clinical intramammary infections. In addition, the ExPEC genes ompT (protectin), ibe10 (invasin), and ecpA (adhesin) were investigated for the first time among cows with mastitis, where scores of clinical severity were assessed. Results of this study contribute to the characterization of virulence mechanisms and antimicrobial resistance profile of ExPEC variants that affect dairy cows with different scores of clinical mastitis.

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.

Identification and characterization of atypical enteropathogenic and Shiga toxin-producing Escherichia coli isolated from ground beef and poultry breast purchased in Botucatu, Brazil.

Atypical enteropathogenic (serotypes O4:H16, O8:H25, O68:H2, O105:H7, and OR:H25) and Shigatoxigenic (ONT:H46) Escherichia coli were isolated from samples of ground beef and poultry breast purchased in Botucatu, Brazil. Phenotypic and molecular characterization indicated the potential of these isolates to adhere to host epithelial cells and cause damage.

Diversity of strategies used by atypical enteropathogenic Escherichia coli to induce attaching and effacing lesion in epithelial cells.

PURPOSE: This study aimed to characterize 82 atypical enteropathogenic Escherichia coli (aEPEC) isolates, obtained from patients with diarrhea in Brazil, regarding their adherence patterns on HeLa cells and attaching and effacing (AE) lesion pathways. METHODOLOGY: The adherence and fluorescence-actin staining (FAS) assays were performed using HeLa cells. AE lesion pathways were determined through the detection of tyrosine residue 474 (Y474) phosphorylation in the Tir protein, after its translocation to host cells, and by PCR assays for tir genotyping and detection of Tir-cytoskeleton coupling protein (tccP) genes. RESULTS: Regarding the adherence pattern, determined in the presence of d-mannose, 12 isolates (14.6 %) showed the localized adherence (LA)-like pattern, 3 (3.7  %) the aggregative adherence pattern and 4 (4.9  %) a hybrid LA/diffuse adherence pattern. In addition, 36 (43.9  %) isolates displayed an undefined adherence, and 26 (31.7  %) were non-adherent (NA), while one (1.2 %) caused cell detachment. Among the 26 NA aEPEC isolates, 11 showed a type 1 pilus-dependent adherence in assays performed without d-mannose, while 15 remained NA. Forty-eight (58.5 %) aEPEC were able to trigger F-actin accumulation underneath adherent bacteria (FAS-positive), which is an important feature of AE lesions. The majority (58.3 %) of these used the Tir-Nck pathway, while 39.6  % may use both Tir-Nck and Tir-TccP pathways to induce AE lesions. CONCLUSION: Our results reveal the diversity of strategies used by aEPEC isolates to interact with and damage epithelial host cells, thereby causing diarrheal diseases.

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.