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Biofilm-Forming Potential of Staphylococcus aureus Isolated from Clinical Mastitis Cases in New Zealand.

ABSTRACT: Biofilm formation is of growing concern in human and animal health. However, it is still unclear how biofilms are related to mastitis infections in dairy cattle. In this study, a comparison between two tests for biofilm formation and the association between biofilm and the presence of genes associated with biofilm formation were investigated for 92 Staphylococcus aureus isolates from clinical mastitis cases. Congo red agar (CRA) and microtitre test assay (MTA) in vitro phenotypic tests were used to evaluate biofilm formation. The presence of icaA, icaD, and bap genes associated with biofilm formation was confirmed using the polymerase chain reaction. Results show that most of the S. aureus isolates, though not possessing one of the biofilm-forming genes, were able to produce biofilms. MTA was more frequently positive in identifying biofilm-forming isolates than CRA.

SUBMITTER: Notcovich S

PROVIDER: S-EPMC5876566 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Biofilm-Forming Potential of Staphylococcus aureus Isolated from Clinical Mastitis Cases in New Zealand.

Notcovich Shirli S DeNicolo Gina G Flint Steve H SH Williamson Norman B NB Gedye Kristene K Grinberg Alex A Lopez-Villalobos Nicolas N

Veterinary sciences 20180119 1

Biofilm formation is of growing concern in human and animal health. However, it is still unclear how biofilms are related to mastitis infections in dairy cattle. In this study, a comparison between two tests for biofilm formation and the association between biofilm and the presence of genes associated with biofilm formation were investigated for 92 <i>Staphylococcus aureus</i> isolates from clinical mastitis cases. Congo red agar (CRA) and microtitre test assay (MTA) in vitro phenotypic tests we ...[more]

PMID: 29351199

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Project description:BackgroundBiofilm formation by Staphylococcus aureus is an important virulence attribute because of its potential to induce persistent antibiotic resistance, retard phagocytosis and either attenuate or promote inflammation, depending upon the disease syndrome, in vivo. This study was undertaken to evaluate the potential significance of strength of biofilm formation by clinical bovine mastitis-associated S. aureus in mammary tissue damage by using a mouse mastitis model.MethodsTwo S. aureus strains of the same capsular phenotype with different biofilm forming strengths were used to non-invasively infect mammary glands of lactating mice. Biofilm forming potential of these strains were determined by tissue culture plate method, ica typing and virulence gene profile per detection by PCR. Delivery of the infectious dose of S. aureus was directly through the teat lactiferous duct without invasive scraping of the teat surface. Both bacteriological and histological methods were used for analysis of mammary gland pathology of mice post-infection.ResultsHistopathological analysis of the infected mammary glands revealed that mice inoculated with the strong biofilm forming S. aureus strain produced marked acute mastitic lesions, showing profuse infiltration predominantly with neutrophils, with evidence of necrosis in the affected mammary glands. In contrast, the damage was significantly less severe in mammary glands of mice infected with the weak biofilm-forming S. aureus strain. Although both IL-1? and TNF-? inflammatory biomarkers were produced in infected mice, level of TNF-? produced was significantly higher (p<0.05) in mice inoculated with strong biofilm forming S. aureus than the weak biofilm forming strain.ConclusionThis finding suggests an important role of TNF-? in mammary gland pathology post-infection with strong biofilm-forming S. aureus in the acute mouse mastitis model, and offers an opportunity for the development of novel strategies for reduction of mammary tissue damage, with or without use of antimicrobials and/or anti-inflammatory compounds for the treatment of bovine mastitis.

Project description:This study aimed to evaluate virulence factors and genetic markers of antimicrobial resistance in 400 Staphylococcus aureus strains isolated from bovine mastitis in four Brazilian states, as well as to assess the association between these characteristics and field information. Virulence factors and drug resistance genes were identified by PCR screening. Biofilm-forming and hemolytic phenotype were detected using Congo red Tryptic Soy Broth and defibrinated sheep blood agar, respectively. Of all isolates, 83.5% were biofilm-forming and 98.5% strains exhibited biofilm gene icaAD, and a significant association between phenotype and genotype for biofilm was observed (P = 0.0005). Hemolysin genes were observed in 82.85% (hla+hlb+), 16.5% (hla+) and 0.75% (hlb+) isolates, whereas the hemolytic phenotype exhibited was complete and incomplete hemolysis in 64.25%, complete in 28.25%, incomplete in 4.75%, and negative in 2.75% of the strains. Virulence factors genes luk, seb, sec, sed, and tst were observed in 3.5%, 0.5%, 1%, 0.25%, and 0.74% isolates, respectively. The gene blaZ was detected in 82.03% of penicillin-resistant isolates, whereas tetK and aac(6')-Ie-aph(2')-Ia were observed in 33.87% and 45.15% of the tetracycline and aminoglycosides-resistant isolates, respectively. Fluoroquinolone resistance gene mepA was detected for the first time in S. aureus from bovine mastitis. Resistance genes tetM (3.22%), tetL (1.61%), ermA (14.29%), ermB (14.29%), ermC (33.3%), ermT (9.52%), ermY (4.76%), msrA (9.52%), and mphC (9.52%) were also detected among resistant isolates. No association between virulence factors or antimicrobial-resistant genes and year of isolation, geographic origin, or antimicrobial resistance profile was observed. Our results showed that S. aureus strains isolated from bovine mastitis in the four Brazilian states sampled are mainly biofilm-forming and hemolytic, whereas virulence genes associated with enterotoxins, luk and tst, were less frequently observed. Moreover, a wide variety of resistance genes that confer resistance to almost all classes of antimicrobial agents approved for use in animals and humans were found. Overall, the data point to a great pathogenic potential of S. aureus associated with bovine mastitis and to the non-negligible risks to public health of staphylococcal infections from animal origin.

Project description:Whole-genome sequencing was carried out on 30 Staphylococcus (S.) aureus isolates from dairy cows with subclinical mastitis from all five provinces of Rwanda. Twenty-five of the isolates produced enough sequence to be analyzed using core genome multilocus sequence typing (cg-MLST). The isolates group into three main clusters. The largest cluster contain isolates of sequence type (ST) 152 (n = 6) and the closely related ST1633 (n = 2). These sequence types have previously mainly been encountered in humans. The isolates of the second-largest cluster belong to ST5477 (n = 5),so far exclusively isolated from cows in Rwanda. The third cluster consists of isolates of ST97 (n = 4), which is a well-known bovine-adapted sequence type. These three clusters were all widespread over the country. Isolates of the usually human-adapted sequence types 1 (n = 2) and 5 (n= 1) were found and a single isolate of ST2430, previously found among humans in Africa. Finally, four isolates of novel sequence types were found: ST7108 (n = 2), ST7109 (n = 1), and ST7110 (n = 1). The blaZ penicillin resistance gene was found in 84% of the isolates and was in all cases corroborated by phenotypic resistance determination. Five (20%) of the isolates carried a tetracycline resistance gene, tet(K) or tetM, and three of these five also displayed phenotypic resistance while two isolates carried a tetM-gene but were yet tetracycline susceptible. Seven (28%) isolates carried the dfrG gene conferring resistance to trimethoprim. Four of these isolates indeed were resistant to trimethoprim while three isolates were sensitive. The str gene conferring resistance to aminoglycosides was found in three isolates; however, none of these displayed resistance to gentamycin. Our data revealed a high diversity of the sequence types of S. aureus isolates from cows with subclinical mastitis in Rwanda. Two major clusters of ST97 and ST5477 are likely to be bovine adapted and cause mastitis while the third cluster of ST152 usually have been found in humans and may signify a recent transmission of these types from human to cows, for example from hand milking. The high prevalence of this sequence type among dairy cows may pose zoonotic threat. The sequence types were widely distributed without any geographic correlation. Penicillin resistance, the most common type of resistance with a prevalence over 80%, but also tetracycline and trimethoprim resistance were displayed by several isolates.

Dataset Information

Biofilm-Forming Potential of Staphylococcus aureus Isolated from Clinical Mastitis Cases in New Zealand.

Publications

Biofilm-Forming Potential of Staphylococcus aureus Isolated from Clinical Mastitis Cases in New Zealand.

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