Antimicrobial resistance, virulence genes and biofilm formation in Enterococcus species isolated from milk of sheep and goat with subclinical mastitis

This study is designed to discuss the antimicrobial resistance, virulence determinants and biofilm formation capacity of Enterococcus spp. isolated from milk of sheep and goat with subclinical mastitis in Qena, Egypt. The obtained isolates were identified by the VITEK2 system and 16S rDNA sequencing as E. faecalis, E. faecium, E. casseliflavus and E. hirae. Overall, E. faecalis and E. faecium were the dominant species recovered from mastitic milk samples. The antimicrobial susceptibility test evidenced multidrug resistance of the isolates against the following antimicrobials: oxacillin (89.2.%), followed by vancomycin (75.7%) and linezolid (70.3%). Also, most of these isolates (73%) could form biofilms. For example, 18.9% of Enterococcus strains formed strong biofilm, whereas 32.4% of isolates formed moderate biofilm and 21.6% of isolates formed weak biofilm. The most prevalent resistance genes found in our isolates were blaZ (54%), vanA (40%), ermB (51.4%), tetM (13.5%) and optrA (10.8%). Moreover, asa1 (37.8%), cylA (42.3%), gelE (78.4%), esp (32.4%), EF3314(48.6%) and ace (75.5%) were the most common virulence genes. A significant correlation was found between biofilm formation, multidrug resistance and virulence genes of the isolates. This study highlights several aspects of virulence and harmfulness of Enterococcus strains isolated from subclinical mastitic milk, which necessitates continuous inspection and monitoring of dairy animals.

Prevalence of Mastitis Pathogens and Antimicrobial Susceptibility of Isolates From Cattle and Buffaloes in Northwest of Pakistan

Mastitis is the most prevalent disease of dairy animals, imparting huge economic losses to the dairy industry. There is always a dire need to monitor the prevalence of mastitis, its bacteriology, and evaluation of antimicrobial susceptibilities for mastitis control and prevention. Therefore, the objectives of this study were to investigate: (i) the prevalence of mastitis in cattle and buffaloes; (ii) identification of bacteria associated with mastitis; (iii) antimicrobial susceptibility of bacterial isolates. Milk samples (n = 1,566) from cattle (n = 1,096) and buffaloes (n = 470) were processed for detection of mastitis using the California mastitis test in the year 2018–19. A total of 633 mastitic milk samples were further processed for bacteriology and antimicrobial susceptibility testing by the disc diffusion method. Overall, the prevalence of clinical and subclinical mastitis was 17 and 57% in both species. Clinical mastitis was higher in cattle (20%) compared to buffaloes (11%), whereas subclinical was higher in buffaloes (66%) than cattle (53%). Besides, month-wise prevalence was higher in hot and humid months in both species. Staphylococci spp. (34%) were the most predominant bacterial isolates from mastitic milk, followed by Escherichia coli (19.4%), Streptococci spp. (9%), and Klebsiella spp. (8%). Most of the bacteria were susceptible to gentamicin (92%) and enrofloxacin (88%), when a panel of 16 different antimicrobials was tested. Nevertheless, most of the isolates were resistant to sulphamethoxazole (99%), lincomycin (98%), oxytetracycline (89%), ampicillin (86%), and doxycycline (85%). This study concludes a high prevalence of mastitis caused by Staphylococcal spp. in cattle and buffaloes belonging to the northwest of Pakistan, and gentamicin and enrofloxacin might be appropriate antimicrobial agents in the treatment of bovine mastitis.

Somatic cell counts and bacteria in milk from two nomadic herds in Abeokuta, Nigeria

Mastitis, the inflammation of the mammary gland, is a major endemic disease affecting dairy production worldwide. Costs of treatment and control of mastitis contributes to major losses to the dairy industry especially if the condition is not promptly and accurately diagnosed, thus necessitating the engagement of regular and reliable means of recognising intra-mammary infections. Somatic cell counting, which has been recognized as a major standard for mastitis diagnosis in milk, was utilized in this study as a direct measure of intramammary inflammation (IMI), along with bacterial culture and isolation to establish the presence of mammary infections in cows from two nomadic herds. Milk from all four quarters (composite samples) of each of 100 cows at various stages of lactation, were obtained and subjected to Levowitz-Weber staining of duplicate smears and direct microscopic counting of somatic cells. Culture and isolation of sterile milk swabs and subsequent identification by morphology, gram staining and biochemical tests were employed to assess the presence of mastitis-causing pathogens in the samples. Using ≤100,000 cells/ml as cut off for non-mastitic milk, sub-clinical mastitis (SM), was determined in 70 (70%) of the examined samples. Contagious pathogens, namely; Staphylococcus aureus, Staphylococcus species and others as well as environmental bacteria, E. coli and Enterococcus species amongst others, were isolated from the samples. This result circuitously indicates the level of losses to dairy production through poor milk quality and yield, costs of treatment and culling obtainable in the nomadic dairying venture. The need for routine evaluation of raw milk and other dairy products emanating from the itinerant pastoralists, using sensitive and reliable parameters to facilitate prompt diagnosis, targeted treatment and rapid control of cow to cow or herd to herd spread of IMI is highlighted in this study.

An Untargeted Metabolomics Investigation of Milk from Dairy Cows with Clinical Mastitis by 1H-NMR

Mastitis is one of the diseases with the highest incidence in dairy cows, causing huge economic losses to the dairy industry all over the world. The aim of the study was to characterize mastitic milk metabolome through untargeted nuclear magnetic resonance spectroscopy (1H-NMR). Taking advantage of the high reproducibility of 1H-NMR, we had the opportunity to provide quantitative information for all the metabolites identified. Fifty-four molecules were characterized, sorted mainly into the chemical groups, namely amino acids, peptides and analogues, carbohydrates and derivates, organic acids and derivates, nucleosides, nucleotides and analogues. Combined with serum metabolomic investigations, several pathways were addressed to explain the mechanisms of milk metabolome variation affected by clinical mastitis, such as tricarboxylic acid cycle (TCA cycle) and phenylalanine, tyrosine and tryptophan biosynthesis. These results provide a further understanding of milk metabolome altered by clinical mastitis, which can be used as a reference for the further milk metabolome investigations.

Genetic Characterization of CTX-M-2-Producing Klebsiella pneumoniae and Klebsiella oxytoca Associated With Bovine Mastitis in Japan

CTX-M-2-producing Klebsiella oxytoca (K. oxytoca) has not received much attention in animal husbandry compared with Klebsiella pneumoniae (K. pneumoniae), a major reservoir of extended-spectrum β-lactamase (ESBL) genes. Bacteriological examinations of 1,466 mastitic milk samples between October 2012 and December 2014 were conducted. Ninety-five K. pneumoniae isolates (total prevalence: 6.5%) and 81 K. oxytoca isolates (total prevalence: 5.5%) were obtained. Seventeen K. pneumoniae isolates obtained from 15 animals reared on 11 farms and 9 K. oxytoca isolates obtained from 9 animals reared on the same farm were phenotypically confirmed to be ESBL producers. All nine ESBL-producing K. oxytoca isolates were obtained from one farm between June and November 2013 and related to a significantly (p < 0.05) higher monthly prevalence of mild mastitis (in June, August, September, October, and November 2013). Pulsed-field gel electrophoresis (PFGE) patterns of ESBL-producing K. pneumoniae isolates were distinguished from each other by more than 6-band differences except for two isolates from two animals, whereas all nine K. oxytoca isolates showed an identical PFGE pattern. Transferability of the blaCTX−M−2 gene was found in 14 K. pneumoniae and 9 K. oxytoca isolates by conjugation analysis. Of these isolates, the blaCTX−M−2 gene was detected on plasmids belonging to the incompatibility (Inc) groups P and N derived from five K. pneumoniae and nine K. oxytoca isolates, respectively, although the plasmids from the remaining nine K. pneumoniae were untypeable. All the transconjugants exhibited elevated minimum inhibitory concentrations of ampicillin, cefotaxime, and ceftiofur compared with those in the wild-type, recipient strain. Restriction fragment length polymorphism analysis demonstrated that the IncN plasmids extracted from eight of nine transconjugants, which received resistance against β-lactams from K. oxytoca, showed an identical DraI digestion pattern. These results suggest that the CTX-M-2-producing K. oxytoca strain with the above-mentioned characteristics may have clonally spread within a farm, whereas the blaCTX−M−2 gene in K. pneumoniae possibly disseminated among the farms through different plasmids. Thus, monitoring of ESBL genes, including the blaCTX−M−2 gene, among causative agents of bacterial mastitis in cows can help to develop relevant treatments and control practices.

Molecular detection of Staphylococcus aureus enterotoxin genes isolated from mastitic milk and humans in El-Behira, Egypt

Background and Aim: Milk is a chief source of many nutrients. However, we must also bear in mind that it is a potential source for many cases of food poisoning. This study was conducted to investigate the prevalence of cow mastitis and evaluate the presence of enterotoxins and antibiotic resistance patterns in Staphylococcus aureus isolated from milk and contact humans in El-Behira Province, Egypt. Materials and Methods: A total of 680 milk samples from 170 cows and 86 human samples consisting of 43 hand swabs and 43 nasal swabs were analyzed. The milk samples were subjected to the California mastitis test. Results: The general occurrence was 23.1% (157/680) where 48 quarters had clinical mastitis and 109 had subclinical mastitis. Subsequently, S. aureus was isolated in Baird-Parker agar where typical and atypical colonies were selected and submitted to coagulase and complementary tests. Out of 48 samples of mastitic milk studied, 16 (33.3%) showed contamination by S. aureus whereas 109 samples of subclinical mastitis showed contamination in only 18 (16.5%). On the opposite hand, of the 86 human samples, 33 revealed S. aureus contamination, corresponding to 38.37% of the samples. Furthermore, multiplex polymerase chain reaction targeting nuc and the staphylococcal enterotoxin-encoding genes sea, seb, sec, sed, and see were performed after culture, revealing that 88.2% (30/34) of milk samples and 93.9% (31/33) of human samples were variably positive to those genes. Conclusion: The use of nuc gene based PCR is an accurate and rapid method for S aureus isolates detection. A high prevalence of multiple drug-resistant isolates of S. aureus recovered from both human and milk represents further evidence for possible veterinary hazards as well as public hazards, especially to those that consume milk from this region.

Prevalence and distribution of multilocus sequence types of Staphylococcus aureus isolated from bulk tank milk and cows with mastitis in Pennsylvania

A total of 163 S. aureus isolates; 113 from mastitic milk (MM) and 50 from bulk tank milk (BTM) (2008, 2013–2015) submitted for bacteriologic analysis at the Penn State Animal Diagnostic Laboratory were examined for their phenotypic and genotypic characteristics. Multi-locus sequence typing (MLST) analysis identified 16 unique sequence types (STs) which belonged to eight clonal complexes (CCs). Majority of the isolates were variants of CC97 (68.7%) and CC151 (25.1%). CC97 comprised of seven STs, of which two were new STs (ST3273, ST3274), while CC151 comprised of three STs of which ST3272 was identified for the first time. Several farms had more than one ST type that were either members of the same clonal complex or unrelated STs. On one farm, six different STs of both categories were seen over the years within the farm. It was observed that ST352 and ST151 were the two main clonal populations in cattle not only in Pennsylvania but also globally. Most isolates were susceptible to all the antibiotics evaluated. 6.7% of isolates showed resistance to vancomycin and penicillin. Two isolates of ST398 showed multidrug resistance (>3 antibiotics) against clindamycin, erythromycin, tetracycline, and penicillin. It was noted that 59 of 163 (36.2%) isolates encoded for enterotoxigenic genes. Enterotoxin genes seg/sei accounted for ~85% of enterotoxin positive isolates. Toxic shock syndrome gene tsst-1 alone was positive in two isolates (ST352, ST 2187). 97.5% of CC151 isolates were enterotoxin seg/sei positive. Most isolates were positive for lukED (95%) and lukAB (96.3%) leukotoxin genes. Bovine specific bi-component leucocidin lukMF’ was present in 54% of isolates. A prominent observation of this study was the explicit association of lukMF’ with lineages ST151 and ST352. In conclusion, the findings of the study, suggest that small number of S. aureus STs types (ST352, ST2187, ST3028, and ST151) are associated with majority of cases of bovine mastitis in Pennsylvania dairy farms. It was observed that one ST of S. aureus predominated in the herd and this ST can coexist with several other ST types of S. aureus strains. When STs were interpreted along with virulence, leucocidin genes and antimicrobial resistance, ST-variants allowed better interpretation of the S. aureus molecular epidemiologic findings specifically for tracing recurrence or persistence of infections in cow over time, among cows in the herd, and between herds in Pennsylvania.

Alterations in Quality Parameters of Mastitic Milk

Quality milk production in modern dairy systems is facing many challenges. Salient in them is mastitis which is responsible for decline in milk production, altered milk composition and compromised udder health. The malaise consists of multiple bacterial etiologies which can be broadly classified into contagious pathogens and environmental pathogens S. aureus is being isolated invariably in all epidemiological studies, followed by E. coli. Pathogenic virulence in mastitis is often accounted due to microbial ability of producing wide array of virulence factors that enhances pathogenicity and sustainment potential in the epithelial linings of udder. Mastitis affects quality parameters of milk i.e. constitutional as well as mineral profile due to local damage and inflammatory mediators. It decreases the lactose secretion because of oxidative stress generated due to the formation of free radicals in the milk. In mastitic milk, IgG2 becomes the predominant antibody which is thought to be the main opsonin supporting neutrophil phagocytosis in the bovine mammary gland. Therefore, it plays a significant role in the battle against mastitis pathogens. Mastitis infected cow shows a notable elevated level of the sodium and chlorine and demoted level of calcium, potassium and inorganic phosphorus. In micro minerals, mastitis affects are pretty much same as in most macro minerals i.e. lower down their concentration in milk secretion. Consistent preventive strategy alongside strict surveillance and biosecurity is recommended for combating this challenge.

Phylogenetic genotyping, virulence genes and antimicrobial susceptibility of Escherichia coli isolates from cases of bovine mastitis

The study described in this research communication used phylogenetic genotyping to identify virulence genes and antimicrobial susceptibility in Escherichia coli recovered from cases of bovine mastitis. From 385 mastitic milk samples, 30 (7.8%) isolates were confirmed as E. coli. Most isolates (80%) belonged to phylo-group A. These 30 E. coli isolates were also screened for 11 different virulence genes. The majority of isolates (63%) harbored no virulence gene. Only 11 (37%) isolates tested positive for two virulence genes, either the iron uptake gene iucD in 3 (10%) isolates or the serum resistance gene traT in 2 (7%) isolates or both traT and iucD in 6 (20%) isolates. The E. coli isolates showed highest susceptibility to gentamicin, meropenem, and pipracillin. Most isolates were resistant to ampicillin, cefotaxime and streptomycin. This study suggests that mastitis causing E. coli might originate from commensal bacteria and that the presence of these virulence genes, common in extra-intestinal pathogenic E. coli (ExPEC) strains could be attributed to high genetic variability of mastitis-causing E. coli.