Methicillin resistance of staphylococci isolated from the skin of dogs with pyoderma

OBJECTIVE: To determine the methicillin-resistant profile of staphylococcal isolates from the skin of dogs with pyoderma. ANIMALS: 90 dogs with pyoderma. PROCEDURE: Staphylococci isolated from dogs with pyoderma were tested for susceptibility to methicillin by use of a standard disk diffusion test with oxacillin disks. The DNA extracted from the isolates was tested for the mecA gene that encodes the penicillin-binding protein 2a (PBP2a) by use of a polymerase chain reaction (PCR) assay. The expression of PBP2a was determined with a commercial latex agglutination assay. Species of staphylococcal isolates were identified by use of morphologic, biochemical, and enzymatic tests. RESULTS: Most of the isolated staphylococci were methicillin-susceptible, coagulase-positive Staphylococcus intermedius isolates. Whereas only 2 of 57 S. intermedius isolates were resistant to methicillin, approximately half of the isolates had the mecA gene and produced PBP2a. Staphylococcus schleiferi was the second most common isolate. Widespread resistance to methicillin was found among S. schleiferi isolates. More coagulase-negative S. schleiferi isolates were identified with mecA gene-mediated resistance to methicillin, compared with coagulase-positive S. schleiferi isolates. CONCLUSIONS AND CLINICAL RELEVANCE: The latex agglutination assay for the detection of PBP2a expression coupled with the PCR assay for the mecA gene may provide new information about emerging antimicrobial resistance among staphylococcal isolates.     

The antibiotic resistance of pathogenic staphylococci and streptococci isolated from dogs

The antibiotic resistance of strains of Staphylococcus aureus and beta–haemolytic streptococci of canine origin were assessed against eight drugs using the disc diffusion technique. Of 398 strains of Staph, aureus eight were resistant to neomycin, 15 to ampicillin, 23 to lincomycin, while of 87 streptococci, one was resistant to ampicillin, two to lincomycin and four to penicillin. In addition 4 of 340 Staph, aureus strains tested against cloxacillin were resistant.     

Antimicrobial susceptibility of coagulase-positive staphylococci isolated from Louisiana dogs

Disk diffusion susceptibility tests were done on 1,178 clinical strains of coagulase-positive staphylococci (CPS) isolated from dogs during a 7-year period. Relative decreases of 7% to 33% were found in the percentages of CPS sensitive to 8 antimicrobics. Relative percentages of CPS sensitive to 9 other antimicrobics were increased or decreased less than 5%. Sensitivity to the beta-lactam antibiotics showed the least relative change. Regression analysis demonstrated that the greatest change in percentage sensitivity of CPS occurred to gentamicin and cephalothin and the least change occurred to penicillin and ampicillin. Recent canine clinical isolates of CPS, specifically identified as Staphylococcus intermedius (n = 109), were uniformly sensitive to novobiocin, amikacin, tobramycin, spectinomycin, and amoxicillin-clavulanic acid. Twenty-two isolates were also sensitive to 17 other antimicrobics. Eighty-seven isolates were resistant to 1 or more antimicrobics tested. Resistance was most common to sulfonamides, penicillin G, ampicillin, tetracycline, and streptomycin. Differences in susceptibility results between S intermedius and unspecified CPS were not statistically significant.     

Identification and antimicrobial susceptibility of coagulase positive staphylococci isolated from healthy dogs and dogs suffering from otitis externa

A total of 67 strains of coagulase positive staphylococci isolated from healthy dogs and dogs suffering from otitis externa were studied. Twenty-two isolates were from healthy dogs (five from hound dogs and 17 from companion dogs) and 45 from dogs suffering otitis externa (14 from hound dogs and 31 from companion dogs). Presumptive identification was attempted using the following tests: production of acetoin, anaerobic utilization of mannitol, acid production from mannitol, presence of beta-galactosidase, and growth on P agar supplemented with different concentrations of acriflavine. Susceptibility of staphylococci to 16 antibiotics was determined. Most effective antibiotics were imipenem, amoxycillin/clavulanic acid, ciprofloxacin, tobramycin, gentamicin and marbofloxacin. Penicillin, ampicillin and polymyxin B showed the lowest activity. There were no significant differences in antimicrobial susceptibility among isolates from healthy dogs and dogs suffering from otitis externa.     

Methicillin-resistant staphylococci isolated from animals

Staphylococci isolated from animals (n=311) were screened for methicillin resistance by oxacillin agar screening. Oxacillin-resistant strains were tested for the presence of the mecA gene by PCR. Isolates were identified by standard techniques and 16S rDNA analysis, and their antimicrobial susceptibilities were tested using an agar diffusion method. MecA-positive strains were further analyzed using pulsed-field gel electrophoresis (PFGE). From 11 multidrug-resistant staphylococci, 6 were mecA-positive: 2 methicillin-resistant Staphylococcus aureus (MRSA) and 4 Staphylococcus haemolyticus. Screening of 300 staphylococci (100 S. aureus, 100 S. intermedius and 100 coagulase-negative staphylococci (CNS)) randomly chosen from the strain collection of the Veterinary Microbiological Diagnostic Center yielded five oxacillin-resistant coagulase-negative staphylococci, four of which were mecA-positive. PFGE showed that all mecA-positive staphylococci isolated from animals had distinct patterns. However, one MRSA isolated from a flank fistula of a dog showed homology to a human epidemic MRSA cluster, suggesting that transfer of MRSA between humans and dogs might occur.     

In vitro antimicrobial resistance of staphylococci isolated from canine urinary tract infection

This study determined the diversity of species and antimicrobial resistance of staphylococci isolated from dogs with a presumptive diagnosis of urinary tract infection (UTI). Urine samples from 348 dogs with clinical signs of UTI, according to clinical examination and urinalysis, were processed for isolation of Staphylococcus. Colonies in pure culture were identified by biochemical reactions and tested for susceptibility to 15 antimicrobials. Seventy isolates of staphylococci were obtained (20.1%). Staphylococcus pseudintermedius was the most frequent species (32.8%), followed by S. epidermidis (18.6%), S. simulans (15.7%), S. schleiferi schleiferi (11.4%), S. aureus (11.4%), S. schleiferi coagulans (7.2%) and S. saprophyticus (2.9%). All the isolates were resistant to at least 1 drug and 77.1% were multiresistant. The study reports the alarming antimicrobial resistance of members of the Staphylococcus genus isolated from canine UTI and highlights the importance of coagulase-negative staphylococci in its etiology.     

Prevalence of antibiotic resistance genes in staphylococci isolated from ready-to-eat meat products

Prevalence of mecA, blaZ, tetO/K/M, ermA/B/C, aph, and vanA/B/C/D genes conferring resistance to oxacillin, penicillin, tetracycline, erythromycin, gentamicin, and vancomycin was investigated in 65 staphylococcal isolates belonging to twelve species obtained from ready-to-eat porcine, bovine, and chicken products. All coagulase negative staphylococci (CNS) and S. aureus isolates harbored at least one antibiotic resistance gene. None of the S. aureus possessed more than three genes, while 25% of the CNS isolates harbored at least four genes encoding resistance to clinically used antibiotics. In 15 CNS isolates the mecA gene was detected, while all S. aureus isolates were mecA-negative. We demonstrate that in ready-to-eat food the frequency of CNS harboring multiple antibiotic resistance genes is higher than that of multiple resistant S. aureus, meaning that food can be considered a reservoir of bacteria containing genes potentially contributing to the evolution of antibiotic resistance in staphylococci.     

Evaluation of methicillin resistance in Staphylococcus intermedius isolated from dogs

Methicillin and multi-drug resistance were investigated in 136 Staphylococcus intermedius strains of canine origin. The large majority of isolates were found to be mecA-negative by polymerase chain reaction, whereas only four strains were positive for the mecA gene. All mecA-positive strains were confirmed as methicillin-resistant by complementary tests, except for oxacillin disk diffusion, which yielded one false-negative result. A significantly higher resistance to fusidic acid, lincosamides, and cotrimoxazole was observed in methicillin-resistant S. intermedius (MRSI) compared with methicillin-susceptible strains. Although the prevalence of MRSI in dogs appeared to be low, methicillin resistance was confirmed to be associated with multi-drug resistance, suggesting the importance of antimicrobial susceptibility testing of canine S. intermedius strains.     

Antimicrobial resistance studies in staphylococci and streptococci isolated from cows with mastitis in Argentina

Background Staphylococcus aureus and Streptococcus agalactiae are the main cause of clinical mastitis in dairy cattle in Argentina, whereas coagulase-negative staphylococci (CNS) and environmental streptococci are the main cause of subclinical mastitis. Bacteria isolated from infected animals show increasing antimicrobial resistance.ObjectivesThis study aims to determine the antimicrobial resistance of staphylococci and streptococci isolated from milk with mastitis, and to genotypically characterize the methicillin-resistant (MR) staphylococci.MethodsIsolation was performed on blood agar and identification was based on biochemical reactions. Antimicrobial susceptibility was according to the Clinical and Laboratory Standards Institute guidelines. The antimicrobial resistance genes, SCCmec type and spa type were detected by the polymerase chain reaction method.ResultsWe isolated a total of 185 staphylococci and 28 streptococci from 148 milk samples. Among the staphylococcal isolates, 154 were identified as CNS and 31 as S. aureus. Among the 154 CNS, 24.6% (n = 38) were resistant to penicillin, 14.9% (n = 23) to erythromycin, 17.5% (n = 27) to clindamycin, 6.5% (n = 10) to cefoxitin and oxacillin. Among the S. aureus isolates, 16.1% (n = 5) were resistant to penicillin, 3.2% (n = 1) to cefoxitin and oxacillin (MRSA). Six MR isolates (5 CNS and 1 MRSA) were positive to the mecA gene, and presented the SCCmec IVa. The MRSA strain presented the sequence type 83 and the spa type 002. Among the 28 streptococcal isolates, 14.3% (n = 4) were resistant to penicillin, 10.7% (n = 3) to erythromycin and 14.3% (n = 4) to clindamycin.ConclusionsThe present findings of this study indicate a development of antimicrobial resistance in main bacteria isolated from cows with mastitis in Argentina.     

Coagulase negative staphylococci isolated from cow milk

The species composition was determined in the set of 52 randomly selected strains of coagulase-negative staphylococci, which were isolated from the milk of dairy cows in 1989. Of this set of strains, the following species were identified: Staphylococcus hyicus subsp. chromogenes (26.9% strains of the set), S. hyicus subsp. hyicus (10.3%), S. xylosus (19.3%), S. saprophyticus (11.5%), S. warneri (9.6%), S. haemolyticus (9.6%), S. hominis (3.8%). Attention is drawn to the increasing occurrence and significance of coagulase-negative staphylococci from the point of view of mastitis in dairy cows.     

Biochemical characterization of staphylococci isolated from goats

Caprine strains of staphylococci show strong and early results in carbohydrate fermentation tests. Their ability to produce DNase and acetoin is weak but they possess a reasonably high degree of phosphatase activity. All the 82 isolates of Staphylococcus aureus isolated from clinically healthy and sick goats produced acid from D-(+)-glucose aerobically at 37 degrees C within 24 h, 98.8% fermented both D-mannitol and maltose, 98.7% fermented sucrose, and 81.7% fermented D-(+)-xylose with acid production, but none fermented L-sorbose. Of the ten coagulase-negative staphylococci characterized to be of animal origin, three produced DNase, but none fermented dulcitol and L-sorbose. All fermented D-(+)-glucose, maltose and D-mannitol, eight fermented D-(+)-xylose and D-sorbitol with acid production. Whilst other coagulase-positive and coagulase-negative staphylococci showed different antibiotic susceptibility patterns, the ten coagulase-negative staphylococci characterized to be of animal origin showed similar antibiotic susceptibility patterns.     

Isolation of enterotoxigenic strains of staphylococci from dogs

The ability of 309 staphylococcal isolates from household dogs to produce enterotoxin, coagulase, thermonuclease and hemolysin was investigated. A total of 52 (16.8%) isolates from 45 out of 150 dogs examined were enterotoxigenic when tested for enterotoxin types A, B and C. Based on sites sampled, 33 (20.5%) out of 161 isolates from the anterior nares were enterotoxigenic while from dorsal skins 19 (12.8%) out of 148 isolates were enterotoxigenic. Staphylococcal enterotoxin C(SEC) was predominantly produced as 21 (6.8%) isolates elaborated it and also accounted for 40.4% of all enterotoxins produced by isolates. Staphylococcal enterotoxins A(SEA) and B(SEB) were produced by 10 (3.2%) and 16 (5.2%) strains, respectively. Mixed enterotoxin types AB, AC and BC were produced by 1,3 and 1 strains, respectively. With human plasma, 17.1% of coagulase-positive and 15.0% of coagulase-negative strains were enterotoxigenic. However, using canine plasma, 19.1% and 6.9% of the coagulase-positive and negative isolates, respectively, were enterotoxigenic. The incidence of enterotoxigenicity was 16.9% amongst thermonuclease-positive isolates and 16.3% for thermonuclease-negative strains.Alpha hemolysin was predominantly produced by 180 (60.2%) isolates and 19.9% of these were enterotoxigenic. Beta hemolysin was produced by 36 (11.7%) isolates with 13.9% enterotoxigenic, while 87 (28.2%) exhibited gamma hemolytic pattern amongst which 11.5% were enterotoxigenic.Based on data provided on coagulation of human and canine plasmas and hemolytic patterns, it is concluded that a large proportion of canine isolates from this environment are not of canine biotypes, but are most probably human biotypes.     

Antimicrobial resistance determinants among anaerobic bacteria isolated from footrot

Antibiotic resistance has been evaluated among 36 Gram negative and anaerobic bacilli (10 Bacteroides, 11 Prevotella, 7 Porphyromonas and 8 Fusobacterium strains) isolated from clinical cases of caprine and ovine footrot (necrotic pododermatitis). The initial analysis on this bacterial consortium evaluates the relationships existing among antimicrobial resistance determinants, phenotype expression and mobilization potential. The Bacteroides strains were generally resistant to penicillins, first-generation cephalosporins, tetracycline and erythromycin, and expressed low level of β-lactamase activity. The main determinants found among the Bacteroides strains were cepA and tetQ genes, conferring resistance to β-lactams and tetracycline, respectively. A general susceptibility to β-lactams was shown for most Prevotella, Porphyromonas and Fusobacterium strains, where none of the β-lactamase genes described in Bacteroides was detected. Resistance to tetracycline and/or erythromycin was found among the three bacterial groups. Although tetQ genes were detected for several Prevotella and Porphyromonas strains, a unique ermF positive was revealed among Prevotella strains. The expression of resistance markers was not related with the polymorphism of their coding sequences. However, the finding of sequence signatures for conjugative transposons in the vicinities of tetQ and ermF suggests a mobilization potential that might have contributed to the spread of antimicrobial resistance genes.     

Methicillin resistance genes and in vitro biofilm formation among Staphylococcus aureus isolates from bovine mastitis in India

IntroductionBiofilms, an assemblage of microbial cells irreversibly associated with a surface and enclosed in a matrix of polysaccharide material pose serious health challenges, resulting in high economic losses. The emergence of methicillin-resistant S. aureus (MRSA) infections and ability to form biofilms in dairy animals is of emerging concern for livestock and public health owing to their association with serious infections. The present study was undertaken to examine the presence of methicillin resistance genes among the biofilm forming Staphylococcus aureus strains isolated from cases of acute and subacute bovine mastitis. A total of 150 mastitic milk samples referred to Veterinary Clinical Complex, Shuhama (Aulesteng) SKUAST-K were screened in present study. The methicillin resistant Staphylococcus aureus isolates were also screened for in vitro biofilm forming ability.ResultsA total of 80 (53.33%) S. aureus isolates were recovered from cases of bovine mastitis of which 20 (25%) were methicillin (mecA) gene positive. Of the 20 mecA positive isolates, 20% were positive for SCCmec I, 35% for SCCmec IV and 45% for SCCmec V subtypes. In vitro antibiotic sensitivity testing of MRSA revealed complete resistance towards methicillin and other pencillin group of antibiotics.ConclusionA significant correlation was observed between in vitro biofilm formation and presence of methicillin resistance gene in S aureus isolates recovered from acute and subacute mastitis. The Staphylococcus aureus isolates positive for methicillin resistance gene (mecA) were either strong or moderate biofilm formers.