Comparison of four test procedures to identify Staphylococcus aureus isolated from bovine intramammary infections

A comparison was made between conventional tube coagulase, macrocupsular coagulase, latex agglutination, and miniaturized biochemical test systems for identification of Staphylococcus aureus of bovine origin. A total of 303 gram-positive, catalase-positive cocci of bovine origin were tested. Agreement between each pair of 4-hour tube coagulase, macrocupsular coagulase, latex agglutination, and miniaturized biochemical test results within isolates was greater than 95.0. Seventeen (5.6%) isolates were test negative for 4-hour tube coagulase, but test positive for 24-hour tube coagulase. Thirteen (76.5%) of these isolates were identified as S hyicus, 3 were S aureus, and 1 was not identified.     

Minimum inhibitory concentrations of 20 antimicrobial agents against Staphylococcus aureus isolated from bovine intramammary infections in Japan

Minimum inhibitory concentrations (MICs) of 20 antimicrobial agents were determined against 51 isolates of Staphylococcus aureus from bovine intramammary infections. Fourteen (27.4%) isolates were resistant to benzylpenicillin, but none of the isolates was resistant to cloxacillin, nafcillin, or cephems. Among aminoglycosides, gentamicin was the most active, with an MIC50 of 0.2 microg/ml, followed by kanamycin, with an MIC50 of 0.78 microg/ml. Five isolates (9.8%) were resistant to dihydrostreptomycin, three isolates (5.9%) to kanamycin and two isolates (3.9%) to gentamicin. Resistance to erythromycin was observed in two isolates (3.9%). Tylosin was less active than erythromycin, with MIC50s of 1.56 microg/ml versus 0.39 microg/ml, but none of the isolates was resistant to this antibiotic. Oxytetracycline MICs were situated in the range of 0.39-1.56 microg/ml for 48 susceptible isolates. Although 19 (37.3%) isolates were resistant to one or more antimicrobial agents, a single resistance pattern was most frequent: benzylpenicillin (12 isolates), dihydrostreptomycin (two isolates) and kanamycin (one isolate). There were no isolates resistant to antimicrobial agents such as methicillin, lincomycin, clindamycin, chloramphenicol, florfenicol and virginiamycin, which have not been approved for use in cattle husbandry in Japan.     

Herd-level risk factors for Staphylococcus aureus and Streptococcus agalactiae intramammary infections

Herd-level risk factors for high herd prevalence of Staphylococcus aureus and Streptococcus agalactiae intramammary infection were studied using data from Holstein herds participating in the New York State Quality Milk Promotion Services Program during the period 1978–1983. Using information from initial surveys and bacteriologic sampling of all herds, a set of possible risk factors for each organism was examined. Comparisons between herds with high and zero prevalence of each infection were made. Failure to teat dip and use of common cloths or sponges for udder preparation increased the odds of high prevalence for both organisms. Selective or no dry cow therapy, having tie stall or stanchion housing and using dry massage or no udder preparation increased the risk for Str. agalactiae, but not for S. aureus infection. Milking machine function played a role in the risk of both infections.     

Genetic diversity and antimicrobial susceptibility profiles among mastitis-causing Staphylococcus aureus isolated from bovine milk samples

OBJECTIVE: To determine whether particular antimicrobial susceptibility profiles of bovine mastitis-causing Staphylococcus aureus isolates were associated with specific S aureus genotypes. SAMPLE POPULATION:357 S aureus isolates recovered from milk samples submitted for diagnostic bacteriologic testing from 24 dairy herds. PROCEDURES: Antimicrobial susceptibility of S aureus isolates was assessed by determining minimum inhibitory concentrations (MICs) to 14 antimicrobial agents. After digestion of S aureus genomic DNA by SmaI, electrophoretic patterns were obtained via pulsed-field gel electrophoresis (PFGE) and used to classify isolates into types. Gels were analyzed, and data were used to prepare dendrograms. RESULTS: 308 of 357 (86%) S aureus isolates were susceptible to all antimicrobials evaluated. Forty-nine S aureus isolates were resistant to 1 or more antimicrobials; of these isolates, 37 were resistant only to penicillin, 9 were resistant to penicillin and erythromycin, 2 were resistant to tetracycline, and 1 was resistant to erythromycin. Isolates were assigned to 7 PFGE types. An association was found between PFGE type and antimicrobial susceptibility profile. Organisms with resistance to at least one of the tested antimicrobial agents were identified in only 4 of the 7 types of S aureus. CONCLUSIONS AND CLINICAL RELEVANCE: Antimicrobial resistance was uncommon among the mastitis-causing S aureus isolates identified in the milk samples. A limited number of genotypes were associated with mastitis in these herds. Antimicrobial resistance phenotypes were associated with particular S aureus PFGE types; this association may have implications for future treatment and control of S aureus-associated mastitis in cattle.     

Effect of slime on adherence of Staphylococcus aureus isolated from bovine and ovine mastitis

The interactions between slime, Staphylococcus aureus and ovine mammary gland epithelial cells (MGEC) were studied in vitro. Suspensions of radiolabelled bacteria incubated with slime significantly increased the ability of S. aureus strains to adhere to a filter. When suspensions of radiolabelled bacteria were incubated with MGEC treated with trypsin, the ability of slime to improve S. aureus adherence was also shown, indicating that it was not dependent on cell membrane proteins. The interaction of radiolabelled bacteria with slime prior to the adherence test with MGEC demonstrated that the adherence process requires the interaction between slime and bacteria. This interaction is inhibited by anti-slime antibodies. This study provides evidence that a specific interaction between bacteria coated with slime and MGEC could be a critical part of mammary gland infection.     

Population diversity of Staphylococcus aureus isolated from bovine mastitis in Brazilian dairy herds

Population diversity was evaluated in strains of Staphylococcus aureus isolated from bovine mastitis in Brazilian dairy herds by PCR-RFLP and sequencing of the 3'-terminal portion of the coagulase gene, and the susceptibility of strains to antimicrobials. The results showed great diversity in S. aureus population studied and the existence of predominant clones that account for most infections. No associations between the predominant types observed in the PCR-RFLP and the forms of presentation of the mastitis or to any of the different patterns of antimicrobial resistance were observed.     

Genetic diversity and antibiotic susceptibility of Staphylococcus aureus isolates from wild boars

We here report the occurrence of S. aureus in wild boars and characterize isolates genotypically and phenotypically in order to get knowledge about the occurrence of clonal lineages and genotypes in free-living wild animals. Forty-one S. aureus isolates obtained from 111 wild boars hunted in Lower Saxony, Germany, were investigated and compared to human and livestock isolates. The S. aureus belonged to multilocus sequence types ST1, ST7, ST30, ST133, ST425, ST804, ST890 and to the new ST3237, ST3238, ST3255 and ST3369. The livestock associated CC398-MRSA lineage, however, was not found. In addition to well-known spa types, the new types t14999, t15000, t15001 and t15002 were detected. Macrorestriction analysis revealed a variety of different SmaI fragment patterns. Most isolates were susceptible to all antimicrobials tested, including methicillin, and resistance was detected only to ampicillin, penicillin and erythromycin. PCR analysis confirmed the presence of staphylococcal enterotoxin genes (seh) in all t127-ST1 isolates. A high degree of genetic diversity was detected with many spa types and clonal lineages previously reported in humans and livestock animals.     

Intra-species diversity and epidemiology varies among coagulase-negative Staphylococcus species causing bovine intramammary infections

Although many studies report coagulase-negative staphylococci (CNS) as the predominant cause of subclinical bovine mastitis, their epidemiology is poorly understood. In the current study, the genetic diversity within four CNS species frequently associated with bovine intramammary infections, Staphylococcus haemolyticus, S. simulans, S. chromogenes, and S. epidermidis, was determined. For epidemiological purposes, CNS genotypes recovered from bovine milk collected on six Flemish dairy farms were compared with those from the farm environment, and their distribution within the farms was investigated. Genetic diversity was assessed by two molecular typing techniques, amplification fragment length polymorphism (AFLP) and random amplification of polymorphic DNA (RAPD) analysis. Subtyping revealed the highest genetic heterogeneity among S. haemolyticus isolates. A large variety of genotypes was found among environmental isolates, of which several could be linked with intramammary infection, indicating that the environment could act as a potential source for infection. For S. simulans, various genotypes were found in the environment, but a link with IMI was less obvious. For S. epidermidis and S. chromogenes, genetic heterogeneity was limited and the sporadic isolates from environment displayed largely the same genotypes as those from milk. The higher clonality of the S. epidermidis and S. chromogenes isolates from milk suggests that specific genotypes probably disseminate within herds and are more udder-adapted. Environmental sources and cow-to-cow transmission both seem to be involved in the epidemiology of CNS, although their relative importance might substantially vary between species.     

Molecular typing of Staphylococcus aureus isolated from cows, goats and sheep with intramammary infections on the basis of gene polymorphisms and toxins genes

We investigated 116 Staphylococcus aureus isolates from cows, goats and sheep with intramammary infections (IMI) in Italy to provide information about the spread of enterotoxigenic strains and to compare strains isolated from different ruminant species. The isolates were typed by restriction fragment length polymorphism (RFLP) analysis of the coagulase (coa) gene, by analysis of polymorphisms of the X region of protein A (spa) gene and by detection of genes encoding enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, sej and sel). Seven different coa types and 12 different spa types were distinguished. On the basis of polymerase chain reaction-RFLP, 29 different coa subtypes were identified. Two different coa subtypes accounted for 49% and 67% of bovine and ovine isolates respectively. Only seven coa subtypes were observed in isolates from more than one host species and no coa subtype was present in isolates from all three ruminant species. Furthermore, 85 of the isolates (73%) harboured at least one enterotoxin gene (se) with a predominance of sea, sed and sej among isolates from bovine IMI, and sec and sel among isolates from caprine and ovine IMI. Comparing the S. aureus isolates on the basis of gene polymorphisms and presence of se genes, significant differences were found in distributions of genotypes among isolates from cows, goats and sheep.     

Comparison of phenotypic and genotypic detection of penicillin G resistance of Staphylococcus aureus isolated from bovine intramammary infection

Resistance of Staphylococcus aureus to penicillin G is common among isolates from bovine mastitis. We determined phenotypic resistance to penicillin G for 151 S. aureus isolates derived from dairy cows with intramammary infection by two methods. The methods were determination of minimum inhibitory concentration (MIC) by a standard agar dilution technique and direct testing of beta-lactamase production using a chromogenic cephalosporin, nitrocefin. The results from these tests were compared with the presence of the beta-lactamase (blaZ) gene in the isolates, which was detected by polymerase chain reaction (PCR). Testing beta-lactamase production with nitrocefin was more predictive for the presence of the blaZ gene than the agar dilution method and the results of the former agreed highly with the presence of the blaZ gene in the isolates. In contrast, the resistance breakpoint generally used in the agar dilution method may be too high for prediction of penicillin resistance in S. aureus isolates with borderline MICs. Using this method, 40% of the isolates possessing the blaZ gene were classified as susceptible; however, majority of these isolates produced beta-lactamase when tested with nitrocefin.     

Efficacy of vaccination and antimicrobial treatment to eliminate chronic intramammary Staphylococcus aureus infections in dairy cattle

OBJECTIVE: To determine whether a combination of vaccination and extended intramammary antimicrobial treatment would eliminate chronic intramammary Staphylococcus aureus infections in lactating dairy cows. DESIGN: Randomized controlled clinical trial. ANIMALS: 50 dairy cows with chronic mastitis caused by S aureus. PROCEDURE: Cows were identified and paired within herd on the basis of days in milk, lactation number, milk production, and numbers of quarters infected. Treated cows (n=20) received 3 doses of a polyvalent S aureus bacterin on days 1, 15, and 21 of the study along with intramammary administration of pirlimycin in all 4 quarters once daily for 5 treatments (days 16 to 20). Control cows (n=23) received no treatment. Follow-up samples for bacteriologic culture were collected for at least 3 months after treatment to determine treatment success rates. RESULTS: Significantly more S aureus infections were eliminated from treated cows (8/20 [40%]), compared with control cows (2/23 [9%]). The proportion of infected quarters that yielded negative results throughout the follow-up period was also significantly higher in treated cows (13/28 [46%]) than in control cows (2/41 [5%]). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that a combination of vaccination and antimicrobial treatment can be successful in eliminating some cases of chronic intramammary S aureus infections in dairy cattle. However, it is important to consider extended treatment protocols carefully because many cows are likely to remain infected with S aureus despite treatment and vaccination.     

Genetic analysis of exfoliative toxin A-producing Staphylococcus aureus isolated from mastitic cow's milk

Exfoliative toxin A (ETA), produced by Staphylococcus aureus, is the causative agent of staphylococcal scalded-skin syndrome (SSSS) in children. Recently, we reported that ETA was detected by reverse passive latex agglutination in three isolates of S. aureus from cow's milk, but that these ETA-positive isolates did not cause the so-called Nikolsky sign in neonatal mice. In this study, therefore, the eta gene encoding ETA and regulatory genes of these bovine isolates were analyzed by the polymerase chain reaction (PCR) and sequencing. The eta gene was amplified from three bovine isolates by PCR and their resulting nucleotide sequences found to correspond to the eta gene from the human isolate, except for three nucleotides in the upstream region of the eta open reading frame (ORF). An accessory gene regulator (agr), which is a global regulatory locus, was detected in these bovine isolates by PCR amplification. In addition, the ORF (J-4), located 120 bp upstream from the eta ORF of the human isolate, was also amplified from these bovine isolates, with their nucleotide sequences differing at 32 positions from the human isolate. Bovine and human ORF J-4 equally enhanced production of ETA in the recombinants of the eta gene, suggesting that the variation in bovine ORF J-4 may be not be the cause of the difference in amount of ETA produced by bovine and human isolates.     

Antimicrobial susceptibility of Prototheca zopfii isolated from bovine intramammary infections

In vitro antimicrobial susceptibility tests were carried out on 48 strains of Prototheca zopfii, an achlorophyllous algae causing refractory mastitis in dairy cows; 27 antimicrobials were evaluated. All strains were susceptible to both myxin and nystatin. In addition, 22 strains were susceptible to amphotericin B, 21 to polymyxin B, and 18 to gentamicin. Only 1 strain was susceptible to kanamycin. All strains were resistant to ampicillin, bacitracin, carbenicillin, cephalothin, chloramphenicol, clotrimazole, cloxacillin, erythromycin, flucytosine, ketoconazole, lincomycin, miconazole, neomycin, nitrofurazone, novobiocin, oleandomycin, penicillin, rifampin, streptomycin, tetracycline, and vancomycin.     

Epidemiological study of non-contagious intramammary infections in nine commercial dairy herds following a Staphylococcus aureus control programme

Changes in prevalence in intramammary infection, by pathogen type, in herds applying a stringent contagious mastitis control programme was studied. Enrollment of 1651 lactating cows and collection of milk samples was made in this ancillary study to a cohort study of the dynamics of mastitis prevalence after adoption of a strict contagious mastitis control programme that targeted the elimination of mastitis caused by Staphylococcus aureus. Nine commercial dairies in Italy were used. Aseptic collection of milk samples from all lactating cows was performed at the time of enrollment, from all cows within 7-14 days of entering the lactating herd after the date of enrollment, and from all lactating cows at 2, 4, 7, 10 and 14 months after the date of enrollment. Prevalence of intramammary infection by pathogen type was determined from culture of milk samples. Application of the strict contagious mastitis programme did not lead to an increased risk of non-contagious mastitis. The risk of coliform, environmental streptococcal and coagulase-negative staphylococcal intramammary infections decreased after adoption of the programme. The data reported herein indicate that the overall risk for any intramammary infections decreases with adoption of a strict contagious mastitis programme, and that such a programme therefore does not necessarily lead to an increase in environmental mastitis.     

Epidemiologic study of intramammary infections with Staphylococcus aureus during a control program in nine commercial dairy herds

OBJECTIVE: To determine the epidemiologic pattern of intramammary infections (IMIs) with Staphylococcus aureus during implementation of a control program in 9 commercial dairy herds. DESIGN: Cohort study. ANIMALS: 1,651 lactating cows and 53,098 quarter milk samples. PROCEDURES: Nine herds located in different regions of Italy were enrolled. Control of S. aureus infections followed the general principles of contagious mastitis control and was based on precise diagnostic procedures and strict control and segregation of infected cows. All lactating cows in each herd were tested, and those free of S. aureus IMI were enrolled as the cohorts. Further additions to the cohort group were cows and heifers free of S. aureus IMI, as determined from aseptically collected milk samples taken approximately 7 and 14 days after calving. RESULTS: After the ninth month of the program, incidence decreased to < 2 new IMIs/100 cow-months in 7 of the herds. At the end of the study, 8 of 9 herds had an incidence of < or = 1 new IMI/100 cow-months. Heifers were most at risk of developing an IMI. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that control of S. aureus IMIs can be achieved by use of a control program based on segregation and use of hygienic and therapeutic protocols. Analysis of incidence and identification of risk factors in a herd could avoid the possible shortcomings of the program, maximizing the probability of success.     

Genetic diversity and methicillin resistance of Staphylococcus aureus originating from buffaloes with mastitis in Iran

The aims of the present study were to investigate the genetic diversity and methicillin resistance in S. aureus isolates recovered from mastitis-affected buffaloes. Five hundred seventy-eight milk samples were obtained from buffaloes with mastitis in three provinces, Iran. Ninety-one of the 578 tested samples contained S. aureus (15.74%), in two cases were methicillin resistant S. aureus (MRSA). Isolates were typed by spa typing, followed by MLST on some representative isolates and SCCmec typing for MRSA strains. The presence of genes encoding Panton–Valentine leukocidin (PVL) was also tested by PCR. Eight spa types were identified, with t3576 (n = 18), t7311 (n = 18) and t937 (n = 17) were the most common, followed by t304 (n = 11), t7308 (n = 9), t521 (n = 7), t267 (n = 6), and t527 (n = 5). MLST revealed four different sequence types (STs) including ST97 (related to t521 and t527 spa types), ST352 (related to t267), ST291 (related to t304 and t937) and ST522 (related to t7338, t7311 and t3576). Two MRSA were identified as t304-ST291-SCCmecIV and t7311-ST522-SCCmecIV. No PVL-positive S. aureus were found. A significant difference in geographical distribution of genotypes was observed, with some types being prevalent in all studied provinces (P < 0.001). The results demonstrated genetic diversity among the S. aureus strains involved in mastitis in buffaloes. This study also provides evidence of the presence of MRSA belonging to genotypes which have been earlier reported in human infections, emphasizing the need for their epidemiological monitoring.     

Clindamycin-resistance in methicillin-resistant Staphylococcus aureus isolated from animals

Clindamycin is widely used in veterinary medicine to treat a variety of bacterial infections. Resistance to clindamycin amongst staphylococci may be due to modification of the ribosomal target site. Resistance due to this mechanism is encoded by the erm genes, which may be either constitutively or inducibly expressed. Inducible expression of resistance is not usually demonstrable by routine laboratory susceptibility testing, however, a relatively simple double disc agar diffusion test (D-test) may be used to detect the presence of inducible resistance. Two hundred and eighty-five isolates of methicillin-resistant Staphylococcus aureus (MRSA) isolated from infections in animals were tested using the D-test for the presence of inducible clindamycin resistance. One hundred and seven (71%) of the strains tested demonstrated inducible resistance to clindamycin, which were not detected on initial susceptibility testing. It has been shown that there is potential for staphylococci to develop resistance to clindamycin whilst on treatment when inducible resistance mechanisms are present. Therefore, routine screening for inducible resistance may be prudent.     

Biofilm production by Staphylococcus aureus associated with intramammary infection

Biofilm production by 221 Staphylococcus aureus isolates from 45 dairy herds was evaluated. Isolates were from composite milk of 117 cows, from teat skin of 70 cows, and from 34 milking machine unit liners. Of S. aureus from milk samples, 41.4% were biofilm producers, as compared to 24.7 and 14.7% of the isolates collected from skin and liners. Pulsed field gel electrophoresis (PFGE) best categorized S. aureus biofilm producers as compared to phage typing and binary typing. PFGE types that were significantly associated with isolation from milk as opposed to teat skin or liners, had isolates that were more likely to produce biofilm than PFGE types that were isolated from milk, skin and liners at similar frequencies. By contrast, PFGE type A was significantly associated with isolation from teat skin and had few biofilm producers. PFGE type Q, which is exclusively a milk, isolate produced more biofilm as evidenced by absorbance values. Given S. aureus that are associated with milk are more likely to produce biofilm as compared to extramammary sources (teat skin and milking unit liners), suggests that biofilm production is a risk factor for infection.     

Effect of intramammary infusion of bacterial lipopolysaccharide on experimentally induced Staphylococcus aureus intramammary infection

Mastitis due to Staphylococcus aureus is a significant problem in the dairy industry and is refractory to antibiotic treatment and/or vaccine prevention. Relative to other mastitis-causing pathogens, S. aureus elicits a diminutive host inflammatory response during intramammary infection. To determine whether induction of a heightened inflammatory response could influence outcome of infection, the highly pro-inflammatory molecule bacterial lipopolysaccharide (LPS) was infused into udder quarters experimentally infected with S. aureus. Relative to S. aureus-infected udder quarters receiving saline, quarters infused with LPS demonstrated a heightened inflammatory response as demonstrated by the induction of TNF-alpha and higher milk somatic cell counts and albumin levels. Although there was no overall effect on bacterial clearance, a trend toward reduced bacterial numbers during the immediate pro-inflammatory response following LPS infusion was observed suggesting that this novel approach to treating S. aureus intramammary infection may warrant further investigation.     

Species of Staphylococcus isolated from animal infections

One hundred randomly selected clinical strains of staphylococci were identified by species using a commercial micromethod system. Eight species of staphylococci were identified. Staphylococcus intermedius was the most frequent (n = 74) species identified and accounted for 70/74 (94.6%) of the coagulase-positive strains and 70/79 (88.6%) of the total isolates from dogs. Other species identified, in order of their frequency, included S. epidermidis (8), S. aureus (7), S. simulans (4), S. sciuri (2), S. xylosus (2), S. hyicus (2) and S. saprophyticus (1). These results show that at least 8 different species of staphylococci can be recovered from animal infections and that coagulase-positive species such as S. intermedius may be more common than S. aureus. The relative significance of these other species in animal infections needs to be assessed.