Identification of Enterococcus spp. by MALDI-TOF mass spectrometry isolated from clinical mastitis and bulk tank milk samples.

BACKGROUND: Throughout a three-year study period, 1,577 bovine clinical mastitis samples and 302 bulk tank samples were analyzed from ten Brazilian dairy herds. Enterococcus spp. was isolated and identified in 93 (5.9%) clinical mastitis samples. In addition, 258 Enterococcus spp. were isolated from the bulk tank samples of the same herds. The identification of Enterococcus spp. isolated from bulk tanks and milk samples of clinical mastitis were accomplished by phenotypic characteristics and confirmed by MALDI-TOF Mass Spectrometry (MS). Fisher test was performed to verify the difference between bulk tanks and mastitis samples. RESULTS: The following species were identified from clinical mastitis: E. saccharolyticus (62.4%), E. faecalis (19.4%), E. faecium (15.1%), E. hirae (1.1%), E. mundtii (1.1%), E. durans (1.1%). Furthermore, from 258 bulk tank milk samples, eight enterococci species were isolated: E. faecalis (67.8%), E. hirae (15.1%), E. faecium (4.6%), E. saccharolyticus (4.6%), E. mundtii (3.1%), E. caseliflavus ( 2.7%), E. durans (1.2%), E. galinarum (0.8%). CONCLUSIONS: The difference in species predominance in bulk tank samples (67.8% of E. faecalis) and clinical mastitis (62.4% of E. saccharolyticus) was unexpected and caught our attention. Although Enterococcus spp. are traditionally classified as an environmental mastitis agent, in the present study, E. saccharolyticus behaved as a contagious agent of mastitis, which consequently changed the control patterns to be implemented.

Prevalence of bovine mastitis-related pathogens, identified by mass spectrometry in flies (Insecta, Diptera) captured in the milking environment.

Diptera (Insecta) are able to transmit approximately 200 pathogenic microorganisms to humans and animals, causing more than 65 diseases, including bovine mastitis, which constitutes a major cause of economic losses in the dairy industry. In this scenario, 217 adult specimens of Diptera were collected from nine farms and identified to the family and/or species level. Of the 11 families recorded, Muscidae was the most prevalent (152 out of 217; 70%). All Diptera specimens were subjected to microbiological culture using conventional and selective media, and isolates were then identified at the species level by mass spectrometry. In total, 275 microorganisms were identified, with a predominance of pathogens related to environmental bovine mastitis (166/275 = 60·4%), that is, Enterococcus species (70/275 = 25·4%) and Escherichia coli (49/275 = 17·8%). Nontraditional agents related to bovine mastitis (called miscellaneous) were detected in 28% (77/275), as well as microorganisms with well-known zoonotic behaviour (e.g. Bacillus cereus). This is study contributes with knowledge of diversity of microorganisms carried by Diptera in the dairy environment, including pathogens associated with environmental and contagious bovine mastitis, and agents with human relevance. To our knowledge, a three-part chromogenic selective medium used to microbial culture of milk on farms was used for the first time to identification of pathogens in Diptera.

Species diversity and antimicrobial resistance patterns of Enterococcus spp. isolated from mastitis cases, milking machine and the environment of dairy cows.

The objectives were to study the diversity of Enterococcus spp. isolated from mastitis cases, milking equipment and the environment of dairy cows; and to determine in vitro resistance of isolates to antimicrobials that are relevant to human and animal health. Ten dairy farms were visited to collect samples from mastitis cases, faeces, bedding, aisles, water and milking equipment. Identification of Enterococcus at the species level and antimicrobial resistance testing was performed by MALDI-TOF and the disk-diffusion method, respectively. Of 365 isolates, Enterococcus hirae was the most prevalent, being more likely to be isolated from faeces than from milk (odds ratio (OR) = 39·2), liners (OR = 5·4) or bedding (OR = 2·2). Enterococcus saccharolyticus was the most prevalent in milk samples. The chances of isolating Enterococcus faecalis from milk were higher than from aisles (OR = 12·5), faeces (OR = 5·3), bedding (OR = 3·6) or liners (OR = 3·0). The odds of isolating Enterococcus faecium from faeces were higher than from liners (OR = 7·3), bedding (OR = 2·5) or aisles (OR = 2·4). Of 360 tested isolates, 1·9, 0·3 and 0·6% were resistant to penicillin, vancomycin and teicoplanin, respectively. Our results suggest that Enterococcus species can occupy specific ecological niches on dairy farms and pose a risk to public and animal health.

Putative virulence factors of extra-intestinal Escherichia coli isolated from bovine mastitis with different clinical scores.

We investigated the genes kpsMTII, iucD, sfaDE, afaBC, papA and papC, (proposed to be involved in extra-intestinal pathogenic Escherichia coli-ExPEC), phylogroup classification and the in vitro swimming and swarming motility in 50 E. coli isolated from bovine mastitis with different clinical severity scores (mild, moderate and severe). The aforementioned genes were detected in 12 (n = 12/50; 24·0%) isolates. kpsMTII and iucD were the most frequent genes identified in six (n = 6/50; 12·0%) and four (n = 4/50; 8·0%) of the isolates, respectively. In only one (n = 1/50; 2·0%) isolate, more than one gene was simultaneously identified: iucD and kpsMTll were detected whereas sfaDE and afaBC were not detected. Mild, moderate and severe clinical signs were observed in 40·0% (n = 20/50), 28·0% (n = 14/50) and 32·0% (n = 16/50) of the cases. Commensal phylogroups B1 (n = 19/50; 38·0%) and A (n = 19/50; 38·0%) were prevalent; whereas pathogenic phylogroups B2 and D were observed in only 10·0% (n = 5/50). Swarming and swimming motilities were observed in 90·0% (n = 45/50) and 68·0% (n = 34/50) of the isolates, respectively; and there was a significant association (P = 0·0036) between swarming motility and severe clinical cases (score 3). To the best of our knowledge, this is the first study where clinical severity of bovine mastitis cases and the genes proposed to classify ExPEC were assessed in relation to swarming and swimming motility. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli is classified as extra-intestinal (ExPEC) when strains contain at least two of the genes kpsMTII, iucD, sfaDE, afaBC and papA and/or papC. We investigated in vitro motility and the presence of these genes in 50 E. coli isolated from bovine mastitis with different clinical scores (mild, moderate and severe). Clinical severity was not associated with the genes studied. Swarming motility was associated with severe cases (score 3) of clinical mastitis. Results of this study contribute to a better understanding of the factors that determine the severity of clinical mastitis.

Diarrhoeagenic Escherichia coli and Escherichia albertii in Brazil: pathotypes and serotypes over a 6-year period of surveillance.

Diarrhoeagenic Escherichia coli (DEC) is a leading cause of infectious diarrhoea worldwide. In recent years, Escherichia albertii has also been implicated as a cause of human enteric diseases. This study describes the occurrence of E. coli pathotypes and serotypes associated with enteric illness and haemolytic uremic syndrome (HUS) isolated in Brazil from 2011 to 2016. Pathotypes isolated included enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC). PCR of stool enrichments for DEC pathotypes was employed, and E. albertii was also sought. O:H serotyping was performed on all DEC isolates. A total of 683 DEC and 10 E. albertii strains were isolated from 5047 clinical samples. The frequencies of DEC pathotypes were 52.6% (359/683) for EPEC, 32.5% for EAEC, 6.3% for ETEC, 4.4% for EIEC and 4.2% for STEC. DEC strains occurred in patients from 3 months to 96 years old, but EPEC, EAEC and STEC were most prevalent among children. Both typical and atypical isolates of EPEC and EAEC were recovered and presented great serotype heterogeneity. HUS cases were only associated with STEC serotype O157:H7. Two E. albertii isolates belonged to serogroup O113 and one had the stx2f gene. The higher prevalence of atypical EPEC in relation to EAEC in community-acquired diarrhoea in Brazil suggests a shift in the trend of DEC pathotypes circulation as previously EAEC predominated. This is the first report of E. albertii isolation from active surveillance. These results highlight the need of continuing DEC and E. albertii surveillance, as a mean to detect changes in the pattern of pathotypes and serotypes circulation and provide useful information for intervention and control strategies.