Virulence factors and antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis in Rio de Janeiro

The study was conducted to characterize pheno-genotypically the virulence factors and resistance pattern of Staphylococcus aureus isolates from milk samples of cows with subclinical mastitis. All hemolytic isolates presented beta-hemolysin, and 38% of the non-hemolytic isolates were able to express hemolysins in the presence of a beta-hemolytic strain. The amplification of the coa-gene displayed four different size polymorphisms with about 400 bp, 600 bp, 700 bp and 900 bp. The spaA gene that encodes the IgG-binding region of protein A revealed sizes of 700 bp and 900 bp. The amplification of region X from spaA yielded a single amplicon for each isolate with the prevalent amplicon size being of 180 bp. Amplification of sae gene yielded an amplicon size of 920 bp in 71% of the isolates. Antibiotic resistance pattern revealed that 42% S. aureus were susceptible to all antimicrobials tested. Seven different antibiotic patterns were observed. Our results indicated that 47% and 25% of S. aureus strains exhibited resistance to penicillin and oxacillin respectively. All oxacillin-resistant isolates were mecA-positive.

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Staphylococcus aureus and bovine mastitis: molecular typing of methicillinresistance and clinical description of infected quarters

Journal of the Hellenic Veterinary Medical Society ◽

10.12681/jhvms.20826 ◽

2019 ◽

Vol 70 (2) ◽

pp. 1511

Author(s):

Α. OREIBY ◽

H. KHALIFA ◽

A. EID ◽

A. AHMED ◽

T. SHIMAMOTO ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Methicillin Resistance ◽

Animal Health ◽

Bovine Mastitis ◽

Clinical Signs ◽

Subclinical Mastitis ◽

Clinical Severity ◽

Resistance Pattern ◽

Gram Positive ◽

Staphylococcus Spp

This study targeted Staphylococcus aureus (S. aureus)-mastitis of bovine as a possible source of livestock-associated methicillin-resistant Staphylococcus aureus (MRSA), to describe clinical signs of mastitis associated with MRSA and methicillin-sensitive Staphylococcus aureus (MSSA). The study area was the Gharbia and Kafrelsheikh governorates, in the central and northern regions of the Egyptian Delta. Clinical examination of animals was performed to detect clinical mastitis (CM) and clinically normal cases were tested by the California mastitis test (CMT) to identify subclinical mastitis (SCM). Accordingly, 38 mastitis cases (68 infected quarters) were detected. Milk samples were collected aseptically and were cultured on Baird Parker agar. Fifty nine Gram-positive cocci-shaped isolates were selected and preserved. In addition, 33 Staphylococcus spp. isolates originated from bovine mastitis at the same study area were obtained from Animal Health Research Institute (AHRI). The cocci-shaped Gram-positive bacteria and AHRI Staphylococcus spp. isolates were used for molecular identification of S. aureus and MRSA. Molecular screening had yielded 17 S. aureus isolates, from which five isolates (29.41%) were MRSA and 12 isolates (70.59%) were MSSA. The five MRSA isolates were mecA positive, but mecC negative. Multilocus sequence typing (MLST) of the five MRSA isolates indicated that all were sequence type 1 (ST1). S. aureus-associated cases showed different clinical forms of mastitis, including subclinical, acute, chronic, and gangrenous. However, subclinical mastitis was the only detected form associated with MRSA, which may represent a potential hidden risk for humans. Phenotypic antimicrobial resistance pattern of MRSA isolates showed resistance to all of the tested β-lactam antimicrobials, with marked orgaresistance to tetracycline and gentamycin. Based on our knowledge, this is the first report to identify MRSA ST1 in Egypt. Bovine mastitis could be a source for the dissemination of MRSA to humans and other animals. Additionally,while methicillin-resistance may have no effect on the clinical severity of mastitis, it does affect therapeutic success, particularly when β-lactam antimicrobials are used.

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The molecular epidemiology of Staphylococcus aureus of bovine mastitis origin

Veterinarski arhiv ◽

10.24099/vet.arhiv.0808 ◽

2021 ◽

Vol 91 (1) ◽

pp. 1-10

Author(s):

Tawheed Ahmad Shafi ◽

◽

Baljinder K. Bansal ◽

Shukriti Sharma ◽

Dhiraj K. Gupta ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Molecular Epidemiology ◽

Protein A ◽

Bovine Mastitis ◽

Subclinical Mastitis ◽

Staphylococcal Protein A ◽

Spa Typing ◽

New Novel ◽

Spa Types ◽

High Diversity

The present study was aimed to understand the molecular epidemiology of Staphylococcus aureus (54 isolates), isolated from 422 milk samples obtained from 108 subclinical mastitis affected cows (CMT positive ≥1+ in at least one quarter). The molecular epidemiology of Staphylococcus aureus was studied using coagulase (coa) gene polymorphism, 16S-23S ribosomal spascer (RS-PCR) polymorphism and Staphylococcal protein A (Spa) typing. Staphylococcus aureus produced 7 coagulase genotypes and 8 RS genotypes respectively. Coagulase genotype GTIII (730 bp) was the most prevalent (35 strains) followed by GTV (900 bp, 7 strains) and GTIV (800 bp, 4 strains), whereas RS genotypes GTA accounted for the highest number of strains (31 strains), followed by GTB (11strains), GTH (4 strains) and GTE (3 strains). Coagulase genotype CTIII (730 bp) showed the highest diversity, as isolates within it produced 5 RS genotypes, the majority of them belonging to the RS genotype GTA (29 out of 31 strains). Forty out of 54 Staphylococcus aureus samples isolated in this study were correctly typed by spa typing, and were assigned to 21 known spa types, and one new novel spa type t18462. The study revealed high diversity within Staphylococcus aureus strains, consisting of 7 coagulase genotypes, 8 RS genotypes and 22 spa types.

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Relationship between virulence factor genes in bovineStaphylococcus aureussubclinical mastitis isolates and binding to anti-adhesin antibodies

Journal of Dairy Research ◽

10.1017/s0022029909990598 ◽

2009 ◽

Vol 77 (2) ◽

pp. 159-167 ◽

Cited By ~ 6

Author(s):

Melania Scarpa ◽

Renata Piccinini ◽

Paola Brun ◽

Alessia Grillo ◽

Giorgio Palù ◽

...

Keyword(s):

Virulence Factors ◽

Binding Protein ◽

Protein A ◽

Bovine Mastitis ◽

Subclinical Mastitis ◽

Effective Vaccine ◽

Hyperimmune Serum ◽

Staph Aureus ◽

Fibrinogen Binding ◽

Multiple Strains

Staphylococcus aureusis the most common aetiologic agent of contagious bovine mastitis. It is characterized by a wide array of virulence factors. The differences among strains jeopardize the development of effective vaccines againstStaph. aureusmastitis. We tested the immunogenicity of a peptide subunit vaccine coding for three different adhesion factors, fibrinogen-binding protein (Efb), fibronectin-binding protein A (FnbpA) and clumping factor A (ClfA). Then we evaluated the influence of some virulence factors on the ability of specific anti-adhesin antibodies to react with sixteenStaph. aureusstrains isolated from bovine subclinical mastitis. Immunization with the recombinant adhesins stimulated a strong humoural (IgG and IgA) and mucosal IgA immune response in all animals tested. Hyperimmune serum recognized with diverse efficiency the sixteenStaph. aureusstrains and this circumstance correlated well with the level of expression of adhesins. Among the different virulence factors considered to classify strains,spagene polymorphisms showed the strongest influence on isolate reactions to hyperimmune serum. Our results indicate the importance of a disease- and environment-specific analysis of isolates. Thus, as opposed to other pathogens to obtain an effective vaccine we should characterize multiple strains and identify the prevalent virulence factors expressed.

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Comparative sequence analysis of spa gene of Staphylococcus aureus isolated from bovine mastitis: characterization of an unusual spa gene variant

Journal of Dairy Research ◽

10.1017/s002202990600183x ◽

2006 ◽

Vol 73 (3) ◽

pp. 322-327 ◽

Cited By ~ 1

Author(s):

Amr El-Sayed ◽

Jörg Alber ◽

Christoph Lämmler ◽

Amir Abdulmawjood ◽

Michael Zschöck ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Protein A ◽

Bovine Mastitis ◽

Clinical Mastitis ◽

Comparative Sequence Analysis ◽

Border Region ◽

Binding Domains ◽

Igg Binding ◽

Spa Gene

The protein A encoding gene spa of four Staphylococcus aureus strains isolated from bovine clinical mastitis was amplified by PCR and sequenced. The four strains were selected after an initial screening of spa gene of 41 strains isolated from mastitic cows and were subjected to detailed investigations. According to the sequencing results the spa gene of three strains (M1, M2, M3) appeared with gene segments encoding five (E, D, A, B and C) and four (E, A, B and C) IgG binding domains for two (M1, M3) and one (M2) strain, respectively and with gene segments encoding four, two and two repeats of the octapeptide Xr-repeats for the strains M1, M2 and M3, respectively. For the remaining Staph. aureus strain (M4) gene segments encoding IgG binding domains E, D and A and a new domain BC with a size of 219 bp could be observed. The BC domain appears, with a deletion of a 123 bp segment from the border region between both domains, as fused domain of both previously characterized domains. The Xr-region of this strain had 11 octapeptide repeats.

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Molecular characterization of virulence factors in Staphylococcus aureus isolated from bovine subclinical mastitis in central Ethiopia

Annals of Microbiology ◽

10.1186/s13213-021-01639-3 ◽

2021 ◽

Vol 71 (1) ◽

Author(s):

Desiye Tesfaye Tegegne ◽

Gezahegne Mamo ◽

Hika Waktole ◽

Yohannes Equar Messele

Keyword(s):

Staphylococcus Aureus ◽

Dairy Cows ◽

Virulence Factors ◽

Virulence Genes ◽

Animal Health ◽

Subclinical Mastitis ◽

Biochemical Tests ◽

Milk Samples ◽

Lactating Dairy Cows ◽

Genomic Study

Abstract Purpose Staphylococcus aureus (S. aureus) is the most important pathogen involved in bovine mastitis in dairy production. S. aureus produces a spectrum of extracellular protein toxins and virulence factors which are thought to contribute to the pathogenicity of the organism. The aim of this work was to isolate and molecular characterize S. aureus associated with bovine subclinical mastitis in the central part of Ethiopia. Methods A total of 265 lactating dairy cows from various dairy farms in four different geographical locations were screened by the California mastitis test (CMT) for bovine subclinical mastitis. One hundred thirty CMT-positive milk samples were collected and transported to the laboratory. Different biochemical tests and polymerase chain reaction (PCR) were used for the identification of S. aureus isolates. Finally, PCR was performed for molecular detection of virulence genes. Results From a total of 265 lactating dairy cows screened, 49% (n = 130) were positive for bovine subclinical mastitis. One hundred thirty mastitic milk samples were subjected to bacterial culturing, and one hundred (76%) S. aureus isolates were identified based on phenotypic characters. Sixty-eight confirmed S. aureus isolates were obtained using PCR. The confirmed S. aureus isolates were tested for six virulence genes (tsst-1, hlb, eta, sea, clfA, and icaD) using PCR. Of the six virulence genes screened from all the isolates, only two (clfA and eta) were detected in the isolates. Out of 68 isolates, 25% and 22% were possessed the eta and clfA genes, respectively. Conclusion The presence of Staphylococcus aureus having virulence genes (eta and clfA) revealed that mastitis is a major concern nowadays affecting animal health, milk quality, and yield. Further genomic study of these isolates will provide broad new insights on virulence.

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Expression of icaA and icaD genes in biofilm formation in Staphylococcus aureus isolates from bovine subclinical mastitis

Pesquisa Veterinária Brasileira ◽

10.1590/1678-5150-pvb-6645 ◽

2021 ◽

Vol 41 ◽

Author(s):

Viviane F. Marques ◽

Huarrisson A. Santos ◽

Thomas H. Santos ◽

Dayanne A. Melo ◽

Shana M.O. Coelho ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Phenotypic Expression ◽

Bovine Mastitis ◽

Subclinical Mastitis ◽

Transcriptional Analysis ◽

Important Species ◽

High Prevalence ◽

Staphylococcus Spp

ABSTRACT: Staphylococcus spp. plays a significant role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most important species due to the high prevalence and the difficulty of in vivo treatment that is related to the expression of virulence factors and biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolated from bovine mastitis and to evaluate the expression and regulation of genes involved in its production. MALDI-TOF and phenogenotypic identification assays were performed to characterize the isolates. The phenotypic biofilm production and the presence of icaA and icaD and bap genes were evaluated. The Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Furtherly, Real-time PCR (qPCR) was performed at chosen times to quantify the expression of icaA, icaD and hld genes in three selected isolates. All 20 strains were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II showed a prevalence of 70%. Transcriptional analysis revealed increased expression of ica genes at eight hours of growth. These results confirm that polysaccharides production mediated by the icaADBC operon genes is an essential mechanism to the biofilm formation and contributes to the early stages of bacterial growth.

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Within-Host Adaptation of Staphylococcus aureus in a Bovine Mastitis Infection Is Associated with Increased Cytotoxicity

International Journal of Molecular Sciences ◽

10.3390/ijms22168840 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8840

Author(s):

Katharina Mayer ◽

Martin Kucklick ◽

Helene Marbach ◽

Monika Ehling-Schulz ◽

Susanne Engelmann ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Bovine Mastitis ◽

Adaptive Strategy ◽

Subclinical Mastitis ◽

Growth Conditions ◽

Typical Feature ◽

Host Adaptation ◽

Bovine Mammary Epithelial Cells ◽

Strain Pair

Within-host adaptation is a typical feature of chronic, persistent Staphylococcus aureus infections. Research projects addressing adaptive changes due to bacterial in-host evolution increase our understanding of the pathogen’s strategies to survive and persist for a long time in various hosts such as human and bovine. In this study, we investigated the adaptive processes of S. aureus during chronic, persistent bovine mastitis using a previously isolated isogenic strain pair from a dairy cow with chronic, subclinical mastitis, in which the last variant (host-adapted, Sigma factor SigB-deficient) quickly replaced the initial, dominant variant. The strain pair was cultivated under specific in vitro infection-relevant growth-limiting conditions (iron-depleted RPMI under oxygen limitation). We used a combinatory approach of surfaceomics, molecular spectroscopic fingerprinting and in vitro phenotypic assays. Cellular cytotoxicity assays using red blood cells and bovine mammary epithelial cells (MAC-T) revealed changes towards a more cytotoxic phenotype in the host-adapted isolate with an increased alpha-hemolysin (α-toxin) secretion, suggesting an improved capacity to penetrate and disseminate the udder tissue. Our results foster the hypothesis that within-host evolved SigB-deficiency favours extracellular persistence in S. aureus infections. Here, we provide new insights into one possible adaptive strategy employed by S. aureus during chronic, bovine mastitis, and we emphasise the need to analyse genotype–phenotype associations under different infection-relevant growth conditions.

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