Some Cultural Characteristics of Staphylococci Isolated from Milk

Observations were made on the value of certain selective media for isolation and identification of staphylococci from milk obtained from individual quarters and farm bulk tanks Aerobic fermentation of mannitol salt was unreliable for identification of coagulase positive strains. Coagulase positive strains tended to clump when grown in tryptose serum broth. Tellurite glycine agar plates were not entirely reliable for the selection of coagulase positive strains. Fibrinogen-tellurite-glycine plates were very useful for the isolation of staphylococci from milk in the presence of other microorganisms and for the simultaneous identification of coagulase positive strains. Plate coagulase tests utilizing fibrinogen tellurite glycine agar corresponded almost perfectly with conventional tube tests. Fibrinolytic strains were not uncommon and were found also among beta hemolysin producing coagulase positive strains.     

Comparison of methods for routine separation of coagulase-negative staphylococci from micrococci isolated from sheep

A total of 176 Gram-positive, catalase positive cocci strains, isolated from sheep were studied by different routine tests for the differentiation of staphylococci and micrococci, comparing their validity and usefulness. By glucose fermentation and growth in the anaerobic portion of thioglycolate 85 and 73.6% respectively of coagulase negative staphylococci were misclassified as Micrococcus spp. Susceptibility to lysostaphin was an adequate test for the differentiation of the strains. Atypical results in the production of acid from glycerol/erythromycin were obtained in 11.8% of the coagulase negative strains and 16.7% of micrococci. The combined use of the selective media furazolidone agar and Schleifer and Krämer medium resulted in a fast and useful separation of ovine staphylococci and micrococci. The bacteriolytic activity misclassified 32.2% of the coagulase negative strains.     

Evaluation of latex agglutination tests for establishing coagulase status of staphylococci from milk.

A total number of 640 staphylococci isolated from cows' milk were tested by latex agglutination and coagulase tests. About 50% of coagulase positive and 5% of coagulase negative staphylococci were positive to the latex agglutination tests. Latex agglutination tests were found to be not satisfactory for determining the coagulase status of staphylococci isolated from cows' milk.     

Distribution of bacteria and seasonal and regional effects in a new database for mastitis pathogens in Finland

Abstract

A database containing pathogen-specific information on mastitis has been established in Finland. The data consist of the results from routine milk samples collected from clinical and subclinical mastitis and submitted to laboratories all over the country. In the database, bacteriological information has been recorded on an individual cow basis. A total of 77,051 pathogen records submitted to the database from 1 January 2004 to 1 January 2006 were retrieved for this preliminary report of pathogen distribution. Staphylococcus aureus (18.26% and 17.73%) and coagulase negative staphylococci (CNS) (17.57% and 23.51%) were the most frequently isolated pathogens from the milk samples. Seasonal and regional differences in pathogen distribution were also found. The pathogen information is primarily used for targeting the antimicrobial treatment of the cow, but also for designing specific mastitis control strategies for the herds. Pathogen data can also be useful for national mastitis control programmes and breeding programmes of dairy cattle for the selection of more resistant animals.     

Vesicular Lesions Produced in Guinea Pigs by a Staphylococcus aureus Strain

A bacterium isolated from vesicular lesions on foot pads of guinea pigs reproduced lesions similar to those seen in experimental infections of guinea pigs with foot-and-mouth disease virus. (FMDV). These bacterial lesions were produced with an inactivated FMDV suspension. Identification as Staphylococcus aureus was determined by growth characteristics on nutrient and blood agar, Gram staining, fermentation of mannitol and coagulase positive reactions. In addition, the organism was sensitive to concentrations of penicillin and streptomycin commonly used in laboratory diluents.     

Characterization of staphylococci associated with clinical and subclinical bovine mastitis

Attempts were made to identify 900 species of staphylococci or micrococci recovered from samples of bovine milk examined for mastitis pathogens. The presence and identity of haemolysins was recorded together with results of disc diffusion antibiotic sensitivity tests. The occurrence of clinical mastitis was also noted and somatic cell counts (SCC) were performed on milk samples which were normal in appearance. Eight hundred and thirty-one coagulase positive staphylococci were obtained, of which 810 were S. aureus and 21 were S. intermedius. Of 65 coagulase negative staphylococci the species of 19 could not be determined by the identification systems used. The remainder were identified as S. hyicus sub sp. hyicus (1), S. hyicus sub sp. chromogenes (19), S. haemolyticus (17), S. hominis (3), S. epidermidis (4), S. capitis (1) and either S. hominis or S. warneri (1). Four other isolates could not clearly be assigned to the genus Staphylococcus or Micrococcus and were designated irregular strains. No micrococci were identified. The presence of alpha, beta, or delta haemolysins occurring singly or in various combinations was identified in 98.3% of coagulase positive staphylococci and in 60% of coagulase negative staphylococci. Epsilon haemolysin was detected in 47.6% of the coagulase negative staphylococci and in 9.5% of S. intermedius. All staphylococci were sensitive to tetracycline (30 microg), novobiocin (1.6 microg), nafcillin (30 microg), methicillin (10 microg) and cephalothin (30 microg) and variable numbers of each species were sensitive to penicillin (2 iu) and streptomycin (10 microg). One non-identified species of coagulase negative staphylococcus was sensitive to erythromycin (0.4 microg) the remaining staphylococci were resistant. Each of the four irregular strains was sensitive to erythromycin and novobiocin. Clinical mastitis was associated with 30.6% of coagulase positive staphylococci, 15.3% of coagulase negative staphylococci, and two of the four irregular strains (50%). Subclinical mastitis as determined by SCC of 500 x 10(3) or greater was associated with 92.7% of coagulase positive and 37.5% of coagulase negative staphylococci.     

The prevalence of methicillin-resistant staphylococci in healthy horses in the Netherlands

Two hundred healthy horses housed at 23 different farms and one clinic and 42 persons in close contact with these horses were screened for the presence of methicillin resistant staphylococci. Samples were taken from the nose and the pastern of the horses and from the nose and throat of the humans and incubated in selective media. Isolates were identified by standard techniques and their susceptibilities were tested using an agar diffusion method. Methicillin-resistant strains were tested for the presence of the mecA gene by PCR. In 45 horses (22.5%) and 15 humans (35.7%) mecA positive staphylococci were found. All isolates were coagulase negative staphylococci, except for one methicillin-resistant Staphylococcus aureus isolated from a veterinarian. Staphylococcus sciuri was the predominant species found among the methicillin resistant staphylococci (MRS) in the horses, whereas S. epidermidis predominated in the humans. From the horses, often more than one species of MRS could be isolated, resulting in a total of 175 mecA positive equine isolates. The equine isolates were predominantly susceptible to most antimicrobials tested, whereas the human isolates showed more resistance. In conclusion, no methicillin-resistant Staphylococcus aureus was found in healthy horses in the Netherlands, but methicillin-resistant coagulase negative staphylococci were found frequently. Further studies are needed in order to investigate whether horses can be a reservoir for MRS or the mecA gene for humans.     

Panton–Valentine leukocidin and some exotoxins of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and antimicrobial susceptibility profiles of staphylococci isolated from milks of small ruminants

The aims of this study were to determine the existence of pvl gene, some toxin genes, and mecA gene in Staphylococcus aureus strains isolated from sheep milk and to examine antimicrobial resistance profiles in staphylococci from sheep and goats' milk. The milk samples were collected from 13 different small ruminant farms in Kirikkale province from February to August 2009. A total of 1,604 half-udder milk samples from 857 ewes and 66 half-udder milk samples from 33 goats were collected. Staphylococcus spp. were isolated and identified from the samples. Toxin genes and mecA gene among S. aureus strains were determined by PCR. Antimicrobial susceptibility of staphylococci was examined by the disk diffusion method on Mueller-Hinton agar, and interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines. The prevalence of subclinical intramammary infection in both ewes and goats was 5.2%. The most prevalent subclinical mastitis agents were coagulase-negative staphylococci and S. aureus with prevalences 2.8% (n:46) and 1.3% (n = 21), respectively. The prevalence of resistances in isolated Staphylococcus spp. to penicilin G, tetracycline, erythromycin, gentamicin, and enrofloxacin were found as 26.9% (18), 7.5% (5), 6.0% (4), 3.0% (2), and 1.5% (1), respectively. Only 3 of the 21 S. aureus ewe isolates (13.4%) were shown to harbor enterotoxin genes being either seh, sej or sec. However, fourteen (66.6%) of the 21 S. aureus isolates had pvl gene while none of the isolates harbored mecA gene. In conclusion, Staphylococci were shown to be the most prevalent bacteria isolated from subclinical mastitis of ewes and goats and these isolates were susceptible to most of the antibiotics. In addition, S. aureus strains isolated from ewes were harboring few staphylococcal enterotoxin genes. However, Panton–Valentine leukocidin produced by S. aureus could be an important virulence factor and contribute to subclinical mastitis pathogenicity.     

Hemolytic Behavior of Staphylococci Isolated from Cows' Milk

Patterns of hemolysis produced by staphylococci isolated from milk were investigated on media prepared with bovine, sheep, horse, and rabbit erythrocytes under a variety of cultural circumstances.

Hemolytic patterns were sharper and easier to interpret on erythrocyte agar plates when incubated at 37°C in an atmosphere containing 30% CO_2. Alpha-hemolysin production was greatly enhanced in this environment and was not detected at times when cultures were grown in air only. This was also true of deltahemolysin but to a lesser extent.

Very satisfactory identification of alpha, beta- and delta-hemolysins was obtained by streaking BEA plates with the unknown strains perpendicularly to a strain producing beta-hemolysin and incubating in 30% CO_2. This procedure avoided use of erythrocytes of different species of origin and use of staphylococcal alpha-antitoxin.

Prior action of the beta-hemolysin on bovine cells was found to completely inhibit hemolysis from alpha-hemolysin but the reverse was not true. A strain inhibiting beta-hemolysis in air failed to exhibit its antihemolytic properties for 24 hours when incubated in 30% CO_2. Patterns of hemolysis within areas subject to multiple hemolysins were found to reflect the nature and relative strength of contributing hemolysins or antihemolysins and could be modified by the time sequence of exposure to certain hemolysins or antihemolysins.

     

Hatchery hygiene evaluation as measured by microbiological examination of samples of fluff

The microbial content of 1,180 samples of fluff from hatchers at 18 chick and 6 turkey hatcheries has been determined. The samples were taken (a) 12–15 hr before removal of birds from the hatchers, (b) just before or after theremoval of chicks orpoults, (c) after fumigation and (d) inreplicate from the same hatcher.

Fifty‐eight per cent of the samples of chick fluff contained one million or more bacteria but only 24 per cent contained more than 100,000 coli‐aerogenes bacteria. Moulds were not found in 68 per cent of the samples. Some samples of fluff were examined for coagulase positive staphylococci and fluorescent pseudomonads but these organisms were generally not detected. Bacillus cereus was recovered in very large numbers from a few samples. Bacterial and coli‐aerogenes counts from poult fluff were generally higher than those from chick fluff but the numbers of pseudomonads and coagulase positive staphylococci recovered were generally negligible.

In an intensive study of samples of fluff from one turkey hatchery, it was found that the level of contamination of fluff increased during hatching and the laying season and was accompanied by a decrease in poult quality. Sampling from any location in the hatcher appears to provide a representative indication of overall contamination at that time. Fumigation of fluff and debris generally caused a reduction in the level of contamination in the fluff.

A suggested assessment of hatchery hygiene based on the microbial examination of fluff is given and the advantages and disadvantages of this method are discussed.     

Feline T-Lymphotropic Virus (FTLV) (Feline Immunodeficiency Virus Infection) in cats in New Zealand

AIM: To identify and enumerate colony forming units (cfu) of mastitis pathogens in bulk tank milk (BTM) from pasture-fed New Zealand dairy cows in the Waikato region.

METHODS: BTM samples from seven seasonal-calving dairy herds in the Waikato region were collected monthly from August to December 2004 (cows calved during July-September). Milk samples were cultured on blood aesculin and MacConkey agar plates for 24 h, and the number of mastitis pathogens identified and counted.

RESULTS: Colonies identified in BTM included aesculinpositive streptococci, Staphylococcus aureus, coagulase-negative staphylococci (CNS), and coliforms; counts ranged from zero to >1,000 cfu/ml. Counts >1,000 cfu/ml for total aesculin-positive streptococci, CNS and coliforms were present in 48%, 51% and 11% of BTM samples, respectively. Counts of Staph. aureus ranged from zero to 1,000 cfu/ml, but first appeared in BTM samples only in October. Staphylococcus aureus was repeatedly isolated in BTM from 4/7 farms during the testing period.

CONCLUSIONS: Counts of mastitis pathogens in this study appeared high relative to interpretive criteria set by other workers, which may indicate a high prevalence of mastitis risk factors on these farms. Interpretation of results is difficult as aesculinpositive streptococci, CNS and coliforms can be isolated from the environment as well as from cows with clinical or subclinical mastitis. Furthermore, Staph. aureus is inconsistently excreted from infected bovine mammary glands. More extensive study of this method is required in New Zealand to attempt to further validate the interpretation of results of bacterial culture of BTM.

CLINICAL RELEVANCE: This method may be used to monitor challenge from mastitis pathogens over time as part of milk quality control programmes. The technique may be of use as a screening test to provide information to veterinarians, affording them the opportunity to have an input into mastitis control on dairy farms in New Zealand.     

The Effect of Added Serum and Glucose, and Some Inherent Factors, on Phagocytosis in vitro by Milk Leukocytes from Several Cows

On four occasions leukocytes were obtained from mammary secretions of four cows following irritation by sterile distilled water.

The effects of three levels of bovine immune serum, three levels of glucose, and combinations of these on the ability of the leukocytes to ingest staphylococci in vitro were studied. Phagocytosis was estimated by examination of stained slides prepared after four hours contact at 37°C.

While 1% immune serum increased the percentage of milk leukocytes ingesting staphylococci, no improvement resulted from the addition of 3% or 5%. Addition of 20mg% glucose produced a larger increase than did 1% serum, and 40 and 60mg% gave further increases. Little advantage was obtained by combining serum and glucose at any of the levels studied.

Overall significant differences were found in the ability to ingest staphylococci by milk leukocytes from the four cows, and the previous observation that cells from some cows in vitro do not respond fully to added serum and glucose was confirmed. Neither of these phenomena was related to glycogen levels in blood or milk leukocytes as determined by the anthrone method, blood glucose levels, leukocyte viability, relative proportions of PMN's in circulating blood white cells, nor the amount of homologous blood serum in the mammary secretions. In two cows whose milk cells did not respond fully, very few blood PMN's were active.

     

Comparison of penicillin-G susceptibility testing methods of staphylococci isolated from bovine mastitis.

Penicillin-G susceptibility was analyzed on forty staphylococcal strains isolated from mastitic bovine quarters (29 coagulase positive and 11 coagulase negative) by the standard Kirby-Bauer agar diffusion method, the epsilon-Test, the Vetmic, turbidimetric MIC analysis in Iso-Sensitest Broth (ISB) and whey. Penicillinase production was tested. Parallel susceptibility tests were carried out in whole milk, whey and ISB using resazurin and triphenyltetrazolium as the indicators of bacterial activity. The traditional susceptibility testing methods (radial agar diffusion, MIC in broth culture) showed good agreement with each other and confirmed that the tests can be used interchangeably with the current breakpoint values (0.25 micrograms/ml and phi 26 mm). The tests carried out in whey showed good correlation with the traditional tests. However, the susceptibility testings in milk resulted in additional variation. Therefore, traditional susceptibility tests of Penicillin-G in artificial media are limited in estimation of bacterial susceptibility when they grow in whole milk. The relevance of this observation regarding mastitis therapy is discussed.     

Prevalence of coagulase-negative staphylococci in bovine mastitis in Zimbabwe

This study was carried out to determine the prevalence of coagulase-negative staphylococci in clinical and subclinical mastitis in commercial and small-scale farms in Zimbabwe. Thirty five quarter milk samples from clinical mastitis cases and 371 quarter milk samples from cows with subclinical mastitis were cultured for bacterial pathogens. The most frequent pathogens isolated in clinical mastitis were the enteric bacteria (31.4%), followed by coagulase negative staphylococci (22.9%) and then Staphylococcus aureus (17.1%), whereas in subclinical mastitis S. aureus (34.2%) and coagulase-negative staphylococci were (33.2%) the most common. Bacillus species were only isolated in milk samples from subclinical mastitis. Coagulase-negative staphylococci were observed in mixed infections with other bacteria in only 2.2 of the 406 milk samples from clinical and subclinical mastitis where they were isolated together with Bacillus species in 6 of the 9 mixed infection cases. About 95% of the milk samples from which 131 coagulase-negative staphylococci were isolated had correspondingly high somatic cell counts. The coagulase-negative staphylococci isolated most frequently were S. chromogenes (7.9%), S. epidermidis (7.4%) and S. hominis (5.9%). They were all associated with high somatic cell counts. All the coagulase-negative staphylococci isolates were susceptible to cloxacillin and erythromycin, and more than 90% of the isolates were susceptible to neomycin, penicillin and streptomycin. The highest resistance was to tetracycline (17.6%), followed by lincomycin (13.7%). About 8% of the isolates were resistant to both penicillin and streptomycin.     

Sensitivity to various antibiotics of coagulase-negative staphylococci isolated from samples of milk from Dutch dairy cattle

During recent years the prevalence of coagulase-negative staphylococci in milk samples from Dutch dairy cows has increased. In 1999 16.2% of the bacteria isolated from milk collected from cows with subclinical mastitis were coagulase-negative staphylococci. In 2004 this proportion was 42.2%. The proportion of coagulase-negative staphylococci of the bacteria isolated from milk samples from cows with clinical mastitis was 7.3% in 1999 and 14.1% in 2004. In this study, the susceptibility of 108 coagulase-negative staphylococci to oxacillin, cefquinome, streptomycin, neomycin, penicillin, and the combination of nafcillin, penicillin, and streptomycin was tested. The isolates were cultured from milk collected from cows with mastitis and typed using the Api-Staph system. Eight species were identified. Staphylococcus chromogenes was the predominant species (41.7%), followed by Staphylococcus xylosus (15.7%) and Staphylococcus simulans (10.2%). With the agar dilution method all strains proved to be sensitive to cefquinome and 90% to oxacillin. Three isolates (2.8%) were mecA-positive. Despite the agar dilution results, these three isolates should be considered resistant to all beta-lactam antibiotics (penicillins, penicillins combined with a beta-lactamase inhibitor and all generations of cephalosporins). In the agar diffusion test, all isolates proved to be sensitive to the combination of nafcillin-penicillin-streptomycin, 99% were sensitive to neomycin and 1% intermediate sensitive, and 95% were sensitive to streptomycin, 4% resistant, and 1% intermediate sensitive. The coagulase-negative staphylococci were highly resistant to penicillin (37.4%), although the level of resistance varied between species, from 0% for Staphylococcus simulans to 100% for Staphylococcus saprophyticus. Because coagulase-negative staphylococci are resistant to several antibiotics, sensitivity testing is important for targeted treatment of mastitis.     

Production of staphylococcal enterotoxins and TSST-1 by coagulase negative staphylococci isolated from ruminant mastitis.

The production of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 by 40 coagulase negative staphylococci isolated from sheep, goat and cow mastitis was studied. Both ELISA double sandwich and Western blot were used to detect the production of these toxins. Only two strains of S. xylosus were enterotoxigenic, producing SEC. TSST-1 was seen to be produced by 5 strains of S. xylosus, 1 S. sciuri and 2 S. epidermidis. Results obtained by ELISA and by Western blot agreed in all cases except in one strain of S. epidermidis which was only positive using ELISA.     

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.     

Risk factors and impacts of clinical and subclinical mastitis in commercial meat-producing sheep flocks in Quebec, Canada

We conducted a prospective observational study on clinical and subclinical mastitis in 30 commercial meat-producing sheep flocks from 2 regions of the province of Quebec, Canada. A total of 2792 ewes selected in late gestation were followed from lambing to weaning of lambs. The incidence of clinical mastitis for the total lactation period (average of 58 days) ranged among flocks from 0 to 6.6%, with a median of 1.2%. The most frequently isolated bacteria from the cases of clinical mastitis, in pure or mixed culture, were Mannheimia haemolytica (26%), Staphylococcus aureus (23%), and coagulase-negative staphylococci (17%). Incidence of clinical mastitis was higher in ewes that gave birth to 3 or more lambs and from the Estrie region, and was associated with an increase in ewe mortality, an increase in lamb mortality at the litter level, and a decrease in lamb's weaning weight for lambs born in multiple litter size or from ewes ≥4 years old.Among 354 selected ewes with clinically normal udder at the end of lactation, 28.8% had potentially pathogenic bacteria isolated from milk. The most prevalent bacteria were S. aureus (9.3%) and coagulase-negative staphylococci (9.3%). The risk of having a positive culture in at least one half was different between the two regions. Prevalence of ewes (n = 261) with California Mastitis Test (CMT) positive result in at least one half was 24.1 and 14.9% using a cut-off of ≥1+ and ≥2+, respectively. Prevalence of culture-positive udder halves was 11.7% for CMT-negative compared with 53.6% for CMT 3+ halves. CMT status was positively associated with the isolation of coagulase-negative staphylococci, M. haemolytica, S. aureus, and various Streptococcus species, but not with other isolated bacteria. Additionally, prevalence of CMT-positive halves was higher in ewes from the Estrie region, aged of ≥4 years versus 1 year, having clinical mastitis previously detected in the lactation and/or with low body condition score. Lamb weaning weight was associated with CMT status of ewes, while weaning weight was not associated with milk culture results. More research is needed to understand the dynamic of milk SCC and IMI in ewes from meat-producing flocks, its economical impact and best ways to control it.     

Some Effects of the Source of Bovine Milk Leucocytes and Strain of Staphylococcus on their Interaction in Vitro

A method is described for the quantitative study in vitro of the bovine milk leucocyte: staphylococci interaction which is capable of demonstrating differences between groups of leucocytes, and, to some extent, stains of Staph. aureus.

By means of this method it has been possible to show statistically, that significant differences can occur, between quarters of the same udder, in the numbers of leucocytes competent to ingest staphylococci, and also in the numbers of staphylococci ingested.

Between 4 strains of Staph. aureus significant differences were noted in their ability to multiply in the presence of milk leucocytes; in the production of leucocidal factors; and in the reduction of Resazurin in whole, normal milk.