Shana de Mattos de Oliveira Coelho

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

Coelho S.M.O., Reinoso E., Pereira I.A., Soares L.C., Demo M., Bogni C & Souza M.M.S. 2009. Virulence factors and antimicrobial resistance in Staphylo- coccus aureus isolated from bovine mastitis in Rio de Janeiro. Pesquisa Veterinaria Brasileira 29(5):369-374. Departamento de Microbiologia e Imunologia Veterinaria, Instituto de Veterinaria, Universidade Federal Rural do Rio de Janeiro, Seropedica, RJ 23890-000, Brazil. E-mail: miliane@ufrrj.br 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.

Short communication: Profile of virulence factors of Staphylococcus aureus isolated from subclinical bovine mastitis in the state of Rio de Janeiro, Brazil

Staphylococcus aureus produces exoproteins that contribute to its ability to colonize the mammary gland such as hemolysins, coagulase, slime, and protein A. This study characterized phenotypically and genotypically these virulence factors in 50 Staph. aureus isolates. These isolates were obtained from milk samples from subclinical mastitis cases identified in 15 dairy cattle farms located in the state of Rio de Janeiro, Brazil. All of the confirmed Staph. aureus samples were PCR positive for the coa gene, which displayed 3 different size polymorphisms. The amplification of the spaA X region yielded a single amplicon for each isolate with the prevalent amplicon sized 315 bp. The Staph. aureus isolates were 24 and 16% positive for the hla and hlb genes, respectively, and 22 and 20% positive for the icaA and icaD genes, respectively. Amplification of the agr gene RNAIII was positive in 74% of the strains. Twenty-seven different profiles were identified among the samples, indicating a great diversity of Staph. aureus involved in the etiology of mastitis cases in the analyzed region. These findings are valuable to the comprehension of the distribution of the profiles of Staph. aureus strains isolated from subclinical mastitis cases in the state of Rio de Janeiro.

Mapeamento do perfil de resistência e detecção do gene mecA em Staphylococcus aureus e Staphylococcus intermedius oxacilina-resistentes isolados de espécies humanas e animais

Antimicrobial resistant Staphylococcus species represent an important cosmopolitan problem, and its spreading control is a significative challenge. Resistance pattern of Staphylococcus aureus and Staphylococcus intermedius species isolated from animals and humans clinical samples to different antibiotics was evaluated through disk diffusion method, where ampicillin and penicillin presented the highest level of resistance. The evaluation of the resistance to oxacillin, due to the heterogeneity of the response of the studied genus was carried out through the following tests: modified agar diffusion, agar screen and microdilution, and further correlation with the detection of mecA gene in samples that showed resistance in at least one of the susceptibility tests used. The correlation between the results obtained from phenotypic methods and the detection of resistance gene, considered as a reference method, was used in order to validate its sensitivity. Eighty clinical staphylococcal isolates (29 human and 51 animal isolates) were evaluated, 28 were oxacillin-resistant, mecA gene being detected in 12 samples. Susceptibility assessment tests to oxacillin presented above 50% of specificity, disk diffusion and agar screen being the most sensitive one, while modified disk diffusion presented the lowest sensibility rate. Ampicillin and penicillin presented the highest level of resistance.

Antimicrobial resistance and detection of mecA and blaZ genes in coagulase-negative Staphylococcus isolated from bovine mastitis

The present study evaluated the pheno- and genotypical antimicrobial resistance profile of coagulase-negative Staphylococcus (CNS) species isolated from dairy cows milk, specially concerning to oxacillin. Of 100 CNS isolates, the S. xylosus was the prevalent species, followed by S. cohnii, S. hominis, S. capitis and S. haemolyticus. Only 6% were phenotypically susceptible to the antimicrobial agents tested in disk diffusion assay. Penicillin and ampicillin resistance rates were significantly higher than others antimicrobials. Four isolates were positive to mecA gene (4%), all represented by the S. xylosus species. The blaZ gene was detected in 16% of the isolates (16/100). It was noticed that all mecA + were also positive to this gene and the presence of both genes was correlated to phenotypic beta-lactamic resistance. We conclude that CNS species from bovine milk presented significantly distinct antimicrobial resistance profiles, evaluated by phenotypic and genotypic tests, which has implications for treatment and management decisions.

Análise fenotípica e genotípica da virulência de Staphylococcus spp. e de sua dispersão clonal como contribuição ao estudo da mastite bovina

Mastitis is an inflammation of one or more mammary glands caused mainly by bacteria, among which the genus Staphylococcus plays an important role. Bacteria belonging to this genus are known to express virulence factors which allow their persistence and spread in the host. This study aimed to evaluate the phenotypic and genotypic aspects of virulence factors in Staphylococci spp. isolates from bovine mastitis clinical cases. A total of 272 milk samples from 8 farms in the South-Fluminense region of Rio de Janeiro were analyzed. The samples underwent conventional bacterial identification, yielding 250 Staphylococci spp. isolates. These were tested for the phenotypic detection of slime production by the microplate and Congo Red Agar methods. The hemolysins production, hemolytic synergism, caseinase and DNase production were also evaluated. The isolates were then assayed through the Polymerase Chain Reaction method to detect genes associated with virulence factors such as: capsule (cap5, cap8), fibronectin (fnbA, fnbB), slime (icaA, icaD) and hemolysins (hla e hlb). Regarding the number of isolates assessed, 58% (145/250) were identified as coagulase-negative Staphylococcus spp. and 42% (105/250) as coagulase-positive Staphylococcus spp. The latter comprised 36.2% (38/105) of isolates identified as S. aureus, 11.4% (12/105) as S. intermedius and 3.8% (4/105) belonging to the SIG group. The hemolisin production was not significant, whereas only 6,4% (16/250) produced alfa hemolysis, 4,8% (12/250) produced beta hemolysis and 1,6% (4/250) was able to produce both. Caseinase production was observed in 66.4% (166/250) and slime production assayed through the microplate method was positive in 76,8% (192/250). DNAse was detected in coagulase-negative Staphylococcus spp. (38/145) and in S. aureus (14/38). Low association between genetic detection of icaA (38/250) and icaD (54/250) and slime phenotypic expression (192/250) suggest that others genetic markers can be involved in this expression. Regarding gene amplification, the isolates did not show significant correlation between the genetic detection of icaA (38/250) and icaD (54/250) and slime production (192/250), indicating that other genetic markers may be involved in this trait expression. The frequency of the occurrence of the others studied genes was of 4% (10/250) for cap5 and cap8, 32,8% (82/250) for fnbA, 4,4% (11/250) for fnbB, 19,2% (48/250) for hla and 18% (45/250) for hlb. The major circulating strain profile on the farms encompassed slime and caseinase producer strains. The spaA gene was found in all of the S. aureus isolates, presenting varying amplicons sizes, with 300bp being the prevalent size. The amplification of the coa gene showed nine polymorphic variants, with 600bp being the prevalent amplicon. The agr gene was also detected in every S. aureus isolate, with an amplicon of 200bp. It was noticed that the presence or absence of the virulence genes assayed in this study were not correlated with the 6 distinct electrophoretic profiles obtained by PFGE.

Impairments of mecA gene detection in bovine Staphylococcus spp.

Staphylococcus aureus antimicrobial resistance, especially to beta-lactams, favors treatment failures and its persistence in herd environment. This work aimed to develop a more specific primer for mecA gene detection based on the comparison of the conserved regions from distinct host origins and also investigated the presence of homologue mecALGA251 in bovine strains. A total of 43 Staphylococcus spp. were included in this study, comprising 38 bovine S. aureus, two human and three equine coagulase-negative staphylococci (CNS). Phenotypical methicillin-resistance detection was performed through oxacillin agar-screening and cefoxitin disk-diffusion test. None isolate tested positive for mecALGA251 gene. For mecA gene PCR, new primers were designed based on the sequences of human S. aureus (HE681097) and bovine S. sciuri (AY820253) mecA. The new primers based on the S. aureus mecA sequence amplified fragments of human and equine CNS and the ones based on S. sciuri mecA sequence only yielded fragments for S. aureus bovine strains. Multiples alignments of mecA gene sequences from bovine, human and equine revealed punctual but significant differences in bovine strains that can lead to the mecA gene detection impairment. The observed divergences of mecA gene sequences are not a matter of animal or human origin, it is a specificity of bovine samples.

Suscetibilidade à azitromicina de agentes bacterianos isolados de processos infecciosos em diferentes sítios de animais de companhia

Avaliou-se o perfil de suscetibilidade bacteriana de diferentes sitios infecciosos frente aos antimicrobianos de eleicao e determinaram-se o perfil de atividade in vitro e a concentracao inibitoria minima (CIM) da azitromicina. Diferentes testes fenotipicos detectaram resistencia a azitromicina em 45% de Staphylococcus spp. e 65% dos bastonetes Gram-negativo. A CIM50 para S. aureus foi 4,0μg/mL para S. intermedius 1,0μg/mL, Staphylococcus spp. coagulase-negativo >512μg/mL e bastonetes Gram-negativo 256μg/mL. Investigou-se, tambem, uma possivel resistencia cruzada entre oxacilina e azitromicina por meio da deteccao do gene mecA em Staphylococcus spp. Foi possivel detectar resistencia a azitromicina em nove (15%) isolados de Staphylococcus spp. mecA positivo.

Caracterização fenotípica da resistência a antimicrobianos e detecção do gene mecA em Staphylococcus spp. coagulase-negativos isolados de amostras animais e humanas

Coagulase-negative staphylococci (SCN) make part of the normal microbiota skin and although they have been considered saprophytics for years, nowadays their clinical significance as an etiologic agent has increased. In this study, 72 SCN isolates obtained from external ear canals of dogs, bovine mastitis and human nosocomial infections were evaluated. Staphylococcus xylosus was the most prevalent microorganism in animal samples and S. cohnii subsp. cohnii in human samples. SCN isolates were evaluated in order to establish a phenotypical resistance pattern towards the most indicated antibiotics for staphyloccocal infections. A high level of resistance to penicillin and ampicillin was detected. The most efficient antibiotics evaluated were gentamicin, vancomicin and the association between ampicillin and sulbactam. To certify the heterogeneous resistance pattern, oxacillin resistance was phenotypically detected by a modified-disc-diffusion test, agar screen, broth micro-dilution and agar dilution. The presence of the mecA gene was detected in 5.6% of the SCN isolates by Polimerase Chain Reaction (PCR).

Biofilm production and beta-lactamic resistance in Brazilian Staphylococcus aureus isolates from bovine mastitis

Staphylococcus spp. play an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant species due to the production of virulence factors such as slime, which is required for biofilm formation. This study aimed to evaluate biofilm production and its possible relation to beta-lactamic resistance in 20 S. aureus isolates from bovine mastitic milk. The isolates were characterized by pheno-genotypic and MALDI TOF-MS assays and tested for genes such as icaA, icaD, bap, agr RNAIII, agr I, agr II, agr III, and agr IV, which are related to slime production and its regulation. Biofilm production in microplates was evaluated considering the intervals determined along the bacterial growth curve. In addition, to determine the most suitable time interval for biofilm analysis, scanning electron microscopy was performed. Furthermore, genes such as mecA and blaZ that are related to beta-lactamic resistance and oxacillin susceptibility were tested. All the studied isolates were biofilm producers and mostly presented icaA and icaD. The Agr type II genes were significantly prevalent. According to the SEM, gradual changes in the bacterial arrangement were observed during biofilm formation along the growth curve phases, and the peak was reached at the stationary phase. In this study, the penicillin resistance was related to the production of beta-lactamase, and the high minimal bactericidal concentration for cefoxitin was possibly associated with biofilm protection. Therefore, further studies are warranted to better understand biofilm formation, possibly contributing to our knowledge about bacterial resistance in vivo.

Resistência bacteriana e ação das bacteriocinas de Lactobacillus spp em Staphylococcus aureus isolados de mastite bovina

Staphylococcus aureus is the major pathogen causing intramammary infections in dairy cattle worldwide. Among the factors that contribute to its spread and infectious potential is the ability to overcome the mechanisms of antimicrobials activity. The present work investigated the antimicrobial resistance pattern and sensibility to bacteriocins produced by strains of Lactobacillus spp of 30 isolates of S. aureus from mastitis. From this, 29 are beta-lactamase producers. Eight isolates (26.6%) showed resistance to at least four antibiotics being considered multiresistent. All of them were mecA-positive. Otherwise, all isolates tested showed sensibility to at least one of the four bacteriocin producer strains. Due to the significant depletion of the efficacy of antimicrobials, pathogen growth inhibition by bacteriocins seems an alternative of biological control in infectious processes.