Microbial pathogens from goat mastitis and phage-typing of Staphylococcus aureus isolates

Examination of milk from goats yielded 41 strains from 40 clinically affected halves; 15 were Staphylococcus aureus, 6 Staph. epidermis, 1 Streptococcus agalactiae, 2 Strept. dysgalactiae, 5 Strept. uberis, 2 Corynebacterium pyogenes, 3 Escherichia coli, 3 Pasteurella spp. and 4 Mycoplasma spp. One half had dual infection of Staph. aureus and Strept. dysgalactiae. Twenty two of the 297 milk samples from apparently normal halves also harboured pathogens comprising of 9 Staph. aureus, 1 Strept. agalactiae, 2 E. coli, 2 Pasteurella spp., 2 Candida albicans and 6 Mycoplasma spp. Most of the bacterial isolates were sensitive to many broad spectrum antibiotics. Twenty of the 24 Staph. aureus isolates were phase typable by a set of 23 human Staphylococcal International Phages suggesting the utility of these phages for the typing of goat strains. The isolates were grouped into 15 phage-types, many of which have been reported from human infections in Iraq. This indicates the possibility of association of human strains of Staph. aureus in caprine mastitis. No definite correlation could be noted between antibiogram and phage types of Staph. aureus strains.     

Phage typing coagulase-positive staphylococci from rooks and gulls

Phage typing was performed on 86 strains of Staphylococcus aureus and 25 strains of Staphylococcus intermedius from rooks and gulls with human, bovine, chicken and canine phages. Eighty per cent of the S aureus strains and 64 per cent of the S intermedius strains were typable. The S aureus biotype D strains of rook origin were specifically lysed at routine test dilution (RTD) by chicken phages from groups I or I + IV, by human phages belonging to groups I and M, and partly by canine phage 58. The other rook and gull S aureus strains did not show characteristic phage patterns. The S intermedius strains isolated from both species of birds could be typed only with canine phages and this correlated with their classification into biotypes. All the biotype 1 strains tested but only two of 12 biotype 2 strains were lysed with canine phages at RTD.     

Characteristics of Staphylococcus aureus isolated from lesions of horses.

Seventy-six Staphylococcus aureus strains isolated from various lesions of horses were characterized. All of the 76 strains were identified as biotypes B (38.2%) and C (61.8%). Of 55 strains tested, 42 (76.4%) were differentiated into 7 coagulase types. Coagulase types V and VII were predominant in the metritis strains. Coagulase type II was found most frequently in the strains from phlegmon, dermatitis, sinusitis, empyema sinus, and nasal catarrh. Forty-two (55.3%) of the 76 strains were differentiated into 24 phage patterns. Twenty (58.8%) of 34 typable strains from metritis were lysed by the human group I phage 52, and group II phages 3A, 3C, 55 and 71. Forty-five (59.2%) of the 76 strains were resistant to 1 or more of 6 antibiotics. Strains resistant to penicillin G, irrespective of source, were most frequent (95.6%). Forty (93.0%) of 43 strains resistant to penicillin G alone or in combination with other antibiotics produced beta-lactamase. Only 8 (10.5%) of the 76 strains produced enterotoxins A (n = 2), B (n = 1) or C (n = 5), and they all were isolated from metritis. Only 1 strain isolated from phlegmon and 2 from metritis produced exfoliative toxin (ET) and toxic shock syndrome toxin-1 (TSST-1), respectively. The latter 2 strains also produced enterotoxin C. The results of the present study showed the first evidence of the presence of both ET- and TSST-1-producing S. aureus isolated from horses.     

Prevalence and characterization of Staphylococcus aureus and enterotoxigenic Staphylococcus aureus in retail raw chicken meat throughout Japan

A total of 444 samples of raw chicken meat (thighs, breasts, wings, livers, gizzards, hearts and ovaries) that retailed at 145 different supermarkets in 47 prefectures in Japan were examined for contamination with Staphylococcus aureus in association with its enterotoxigenicity. S. aureus was isolated from 292 (65.8%) of the samples, and from 131 of the 145 supermarkets. There was no significant difference in the detection rate of S. aureus according to the type of meat examined. About 80% of 714 isolates belonged to the poultry (57.1%) and human biotypes (22.1%). Seventy-eight (21.7%) of 360 isolates were enterotoxigenic and isolated from 78 samples in 53 supermarkets in 31 prefectures. Staphylococcal enterotoxins (SEs) produced were SEB (50 isolates), SEA (14), SEC (8), SED (2), SEA+SEB (2), and SEA+SEC (2). Most of the enterotoxigenic isolates belonged to the human and poultry biotypes, coagulase type VII, VIII or IV, and were lysed by phages of group III. Identical SE types, biotypes, coagulase types and pulsed-field gel electrophoresis (PFGE) patterns were shown in isolates from different types of meat at the same supermarket and from samples taken from different supermarkets in the same prefectures or in isolates from samples obtained from several different prefectures. Among the 50 SEB-producing isolates, 27 yielded three similar PFGE patterns that differed by only a few fragments, suggesting that they were closely related genetically. The three patterns were found in isolates of samples that retailed at 17 supermarkets in 11 prefectures, indicating that they may be disseminated among raw chicken meat in Japan.     

Colonization of rabbits with Staphylococcus aureus in flocks with and without chronic staphylococcosis.

Rabbits of 19 rabbitries were examined for the presence of Staphylococcus aureus in nine different body sites. Seven rabbitries experienced epidemically spreading signs of staphylococcosis while the other 12 rabbitries did not. S. aureus was isolated in all seven flocks that suffered from chronic problems of staphylococcosis and in 11 of the 12 clinically healthy flocks. The mean percentage of infected animals in these two groups was 90 and 43.3%, respectively. S. aureus was isolated from all body sites examined, but the ear and the perineum were often more intensely colonized. The number of animals colonized with S. aureus and the mean number of positive body sites in S. aureus positive rabbits were significantly higher in rabbitries with chronic staphylococcosis. This indicates that colonization capacity of S. aureus plays a role in epidemically spreading disease in rabbits. S. aureus isolates belonged to five different biotypes and 23 different phage types. Several different types simultaneously circulated in contaminated rabbitries and even simultaneously infected individual rabbits. Strains that belonged to the biotype-phage type combination mixed CV-C, 3A/3C/55/71 only occurred in rabbitries chronically dealing with signs of staphylococcosis. This may indicate a relationship between phenotypic strain properties and virulence of S. aureus.     

The effect of auto-vaccination therapy on the phenotypic variation of one clonal type of Staphylococcus aureus isolated from cows with mastitis

The aim of this study was to demonstrate the effect of auto-vaccine therapy on selected properties of Staphylococcus aureus strains, isolated from milk of cows with subclinical mastitis. The experiment was based on auto-vaccines which were prepared from S. aureus strains isolated from 16 cows. S. aureus strains isolated from cows on the 7th, 21st and 35th day following auto-vaccination, were analyzed phenotypically and genotypically. The isolated strains represented 17 biotypes all belonging to one clonal type. Increases of new biotypes of S. aureus were detected on the 35th day of therapy. Among 48 re-isolated strains, 18.75% (9/48) revealed single and 12.50% (6/48) multiple phenotypical changes. The present study demonstrated that during auto-vaccine therapy, S. aureus strains can change phenotypically, pointing out the necessity for using precise diagnostic methods, that would make possible a better assessment of the used therapy.     

Comparison of Staphylococcus aureus recovered from personnel in a poultry hatchery and in broiler parent farms with those isolated from skeletal disease in broilers.

Personnel from one broiler hatchery, and workers on 18 separate broiler parent farms which supply the hatchery, were tested for hand and nasal carriage of Staphylococcus aureus. In both locations, nasal carriage of S. aureus was more common than hand carriage. A total of 63 S. aureus strains were characterised by biotyping, protein A analysis and pulsed field gel electrophoresis (PFGE) typing. Of these, 36 were recovered from broiler hatchery personnel, 14 from broiler parent farm personnel and 13 from cases of skeletal disease in commercial broilers. Biotyping and protein A analysis indicated that none of the strains recovered from hatchery personnel were of the poultry biotype, but that two strains recovered from the hands of two broiler parent farm personnel could be grouped together with 12/13 of strains recovered from skeletal disease in broilers, as poultry biotypes. PFGE-typing could not distinguish 9/13 strains recovered from skeletal disease in broilers and one of the strains from the broiler parent farm personnel from isolate 24 (I. 24), which is the predominant S. aureus strain type associated with clinical disease in N. Ireland broiler flocks. The present study found no evidence of nasal carriage of S. aureus strains of poultry biotype by humans. The finding of hand carriage by broiler parent farm personnel, suggests that handling by personnel may contribute to the dissemination of I. 24 or other S. aureus strains associated with skeletal disease in broilers.     

Staphylococcus aureus isolated from poultry in Australia. I. Phage typing and cultural characteristics

The phage typing and cultural characteristics of 574 strains of S. aureus of poultry origin in Australia were examined. With the avian phage set of Shimizu (1979) it was possible to type 74.2% of strains. A number of significant variations in the phage typing patterns of Australian strains compared to those reported from Japan and Europe were observed. A lower proportion of Australian strains were of avian phage group I and a higher proportion of group III. A high proportion of strains were of mixed lytic groups. No locally isolated phages were able to increase significantly the percentage of typeable strains, although four local phages appeared to be of greater value for phage typing poultry strains of S. aureus than some other phages of the avian phage set. The international (human) phage set was of limited value in typing Australian strains of poultry origin although four strains were identified which were indistinguishable from strains of human origin. Using cultural characteristics of the strains in conjunction with phage typing, the Australian strains of S. aureus were assigned to one of three major groups and nine subgroups. A list of typing phages considered to be valuable for use on Australian poultry strains of S. aureus is given.     

Isolation of Staphylococcus species from the tonsils of healthy cattle and phage patterns of isolates

Staphylococci were found in the tonsils of 121 (75.2%) of 161 cattle. There were 15 different species, 10 belonging to novobiocin-sensitive species. The most predominant species was S. simulans (79.3% of the 121 carriers), followed by S. aureus (20.7%), S. chromogenes (10.7%) and S. epidermidis (8.3%). The other 11 species were present in 0.8 to 5.8%. Twenty-six unidentifiable isolates were isolated from 26 (21.5%) carriers. Sixty-two (51.2%) of the 121 carriers yielded two to five Staphylococcus species together while only one species could be found in each of the other 59 (48.8%). Combinations of S. simulans and other species were most frequently encountered in 50 (41.3%) of the 121 carriers. Twenty-four (96.0%) out of 25 S. aureus isolates, 3 (42.9%) of 7 S. hyicus isolates and 45 (25.4%) of 177 coagulase-negative staphylococci (13 species and unidentifiable isolates) isolates were phage typable. Most of S. aureus isolates were lysed by bovine phages 119 (n = 16) or 116 (n = 5). Thirty-three (25.4%) of 45 coagulase-negative staphylococci typable isolates with Pulverer's phage set showed the phage pattern ph5/ph9/ph10/ph12/ph13/U4/U14/U16/++ +U20/U46. The tonsils of cattle thus appear to be a suitable environment for Staphylococcus species, particularly novobiocin-sensitive species.     

Food as a potential vector for antibiotic resistance. 1. Relevance of residues and selected foodborne infections and intoxicants

Food of animal origin has been considered as an important vector for the transfer of antibiotic resistances from animal to man. Such a transfer is possible by three ways: through antibiotic residues in food, through the transfer of resistant foodborne pathogens or through the ingestion of resistant parts of the original food microflora and resistance transfer to pathogenic microorganisms. A literature review and own investigations were performed in order to asses the potential impact of antibiotic resistance in food on the consumer health. In the first report Salmonella and Staph, aureus isolates were screened for their antibiotic resistance profiles. As a result it could be shown that residues in fresh meat or milk are quantitatively of minor interest. The resistance profile of Salmonella depended on the origin (pig or poultry), but only serovars could be identified which are generally not responsible for systemic infections. Staph. aureus isolates did not show any resistances relevant for human medicine. In these cases food can be considered as safe concerning its role as a vector for antibiotic resistances. However, a resistance monitoring seems to be necessary.     

Etiological agents of dairy cows' mastitis in western part of Poland

The purpose of the study was to determine the etiological mastitis agents in cows from herds located in the western part of Poland in years 2003-2005. Bacteriological examinations of 18,713 samples taken from California Mastitis Test (CMT) positive quarters were performed according to standard methods. Some Gram-negative bacilli and Gram-positive cocci were examined with the use of API tests (Analytical Profile Index). It was found that 32.7% of samples that were CMT positive were culture negative and 9.3% of samples were contaminated. Streptococcus species (15.7%), coagulase negative staphylococci (CNS) (14.6%), Staph. aureus (8.6%), Gram-negative bacilli (4.0%) and Corynebacterium species (3.8%) were most frequently isolated. Escherichia coli (52.3%) dominated among Gram-negative bacilli followed by Klebsiella pneumoniae (4.1%), Pseudomonas aeruginosa (3.6%), Enterobacter cloacae (3.6%), Serratia marcescens (3.1%), Pasteurella multocida (3.1%), Acinetobacter lwofii (3.1%), and 26 other bacteria species. Stahylococcus xylosus (31.5%), Staph. hyicus (12.2%), Staph. haemolyticus (12.2%), Staph. sciuri (11.6%), Staph. chromogenes (8.8%), Staph. epidermidis (8.3%) and Staph. simulans (6.1%) were the most frequent CNS. Streptococcus uberis (50%), Str. dysgalactiae (19.7%), Str. acidominimus (6.6%) and Enterococcus faecalis (5.3%) were mostly found among CAMP-negative streptococci. An increase in frequency of mastitis caused by non-agalactiae streptococci, Staph. aureus, A. pyogenes and yeast-like fungi and a decrease in that produced by Str. agalactiae in 2005 as compared to years 2003 or 2004 were observed.     

Mastitis pathogens present in bulk tank milk from seven dairy herds in the Waikato region, 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 aesculin-positive 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 aesculin-positive 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.     

Fomites and reservoirs of Staphylococcus aureus causing intramammary infections as determined by phage typing: the effect of milking time hygiene practices

We studied the association of milkers' hands and milking unit liners as fomites, and teat skin as a reservoir, with S. aureus intramammary infections (IMI). Samples were collected from 40 commercial herds and S. aureus isolates were phage typed. Only 10 of 257 isolates were not typable. Of the milk samples, 8.4% had typable S. aureus; 4.5% and 9.2% of the skin and liner swabbings had typable S. aureus. Twenty-three different phage types were identified, 1 type was found in nearly 75% of the herds. Herds in which milking unit backflush was used were less likely to have the same phage type on the liners and the teat skin, and on the liners and milk samples, than herds that did not backflush. A greater percentage of liners had S. aureus of the same type as those causing S. aureus IMI, than skin swabbing solutions with the same type as that associated with IMI. Herds which did not use post-milking teat asepsis (teat dip) did not have a greater percentage of S. aureus isolates on the teat skin, nor were the S. aureus test skin isolates more likely to be of the same type as those causing intramammary infection. Results would suggest that the liner appears to be a significant fomite, that backflushing reduces its significance, and that teat skin is a less significant reservoir for S. aureus intramammary infection.     

The effects of various treatments on the microbial flora of whole poultry carcasses with particular reference to Staphylococcus aureus contamination.

Experiments are described in which the effects of dipping defrosted eviscerated poultry carcasses, for various periods, in acetic, lactic, succinic and citric acids, hypochlorite solution containing 200 ppm free Cl2 and in water at temperatures from 70 degrees C to 100 degrees C on the microbial flora were determined. 2. Hot (70 degrees C) water dipping for 2 min appeared to be the treatment most likely to succeed in reducing Staph. aureus, giving reductions of staphylococci of from 63 to greater than 99%, without completely eliminating other organisms. 3. A storage experiment showed this method to be relatively safe, provided the carcasses were refrigerated to less than 3 degrees C as soon as possible after treatment and held at this temperature until adequately cooked.     

A study of skin diseases in dogs and cats. III. III. Microflora of the skin of dogs with chronic eczema

The microflora of the skin was studied in 10 dogs with chronic eczema without clinical signs of secondary infection (Table I). The skin surface was swabbed at 7 different sites, making a total of 70 swabs, 25 of which were taken from visibly inflamed areas and 45 from apparently unaffected skin (Table II). Staph. aureus, Staph. epidermidis, micrococci, alpha-hemolytic streptococci, and Acinetobacter spp. were found consistently. Ten different Gram-negative bacteria, 3 different Gram-positive bacteria, and 2 yeasts were found to occur sporadically (Table III). Compared to a group of 10 healthy dogs a more prolific growth of aerobic microorganisms, a greater number of sites carrying Staph. aureus, and a higher recovery of Gram-negative transients were found in dogs with eczema (Table IV--VII). Within the group of dogs with eczema the growth of Staph. aureus was significantly heavier from eczematous skin areas than from clinically normal skin (Table VIII). In dogs with non-infective dermatitides the colonization of the skin by potentially pathogenic microorganisms may have to be considered in the clinical handling of these diseases.     

Enterotoxin production by strains of Staphylococcus aureus isolated from animals and main in Nigeria

Enterotoxin production by 194 strains of Straphylococcus aureaus isolated from animals and man was investigated. The organisms were also classified into biotypes, irrespective of sources. Human biotypes isolated from people had the highest proportion of enterotoxigenic strains (39%). The animal biotypes isolated from human beings were not enterotoxinenic, but 8.8% of the human biotypes isolated from animals yielded enterotoxin. About 18% of the animal biotypes isolated from animals were enterotoxigenic, while 25% of the untypable biotypes from all the sources investigated were enterotoxin producers. The predominant enterotoxins were A and B, with A accounting for 55%5% of the entrotoxins produced by human biotypes. Enterotoxins A, B and C were produced by some animal biotypes, and three canine biotypes produced enterotoxin combinations, BE, CE and ABE, respectively. From goat isolates, only four of the caprine biotypes were enterotoxigenic, including the only isolate producing enterotoxins B, C and E. The isolates obtained from horses and chickens were non-enterotoxigenic regardless of the biotypes. It is concluded that strains producing enterotoxins A and B may be responsible for outbreaks of staphylococcal food poisoning in Nigeria. The close association between man and his animals in this environment may account for the high prevalence (47%) of human biotypes in animals.     

PERINATAL LAMB MORTALITY IN WESTERN AUSTRALIA 4. Neonatal Infections

Infection was found in 14.5% of dead lambs classified as delayed or late post-parturient deaths or in 7.5% of the 4,417 lambs autopsied. Eighteen species of bacteria were isolated from these acquired neonatal infections. Many infections resulted from umbilical contamination at birth, some by ingestion, some were superimposed complications upon an already existing condition such as starvation, others occurred as post-marking sequellae. Neonatal infections were divided into hebdomadal (first week) and post-hebdomadal (7 to 28 days). The microorganisms isolated from hebdomadal deaths were Cl. septicum, C. pyogenes, Staph, aureus, Past. haemolytica, Past, multocida , and E. coli , from post-hebdomadal deaths Staph, aureus, F. necrophorum, C. pyogenes, Corynebacterium spp, E. coli, Cl. septicum, Cl. perfringens, Past, haemolytica. Streptococcus spp, pleomorphic gram-negative rod, A. lignieresii, Dermatophilus congolensis and miscellaneous species. It was estimated that acquired neonatal infections were responsible for one-thirteenth of the perinatal lamb deaths in the agricultural area of Western Australia or for approximately 1% of births.     

Prediction of penicillin resistance in Staphylococcus aureus isolates from dairy cows with mastitis, based on prior test results

AIM: To gauge how well prior laboratory test results predict in vitro penicillin resistance of Staphylococcus aureus isolates from dairy cows with mastitis. METHODS: Population-based data on the farm of origin (n=79), genotype based on pulsed-field gel electrophoresis (PFGE) results, and the penicillin-resistance status of Staph. aureus isolates (n=115) from milk samples collected from dairy cows with mastitis submitted to two diagnostic laboratories over a 6-month period were used. Data were mined stochastically using the all-possible-pairs method, binomial modelling and bootstrap simulation, to test whether prior test results enhance the accuracy of prediction of penicillin resistance on farms. RESULTS: Of all Staph. aureus isolates tested, 38% were penicillin resistant. A significant aggregation of penicillin-resistance status was evident within farms. The probability of random pairs of isolates from the same farm having the same penicillin-resistance status was 76%, compared with 53% for random pairings of samples across all farms. Thus, the resistance status of randomly selected isolates was 1.43 times more likely to correctly predict the status of other isolates from the same farm than the random population pairwise concordance probability (p=0.011). This effect was likely due to the clonal relationship of isolates within farms, as the predictive fraction attributable to prior test results was close to nil when the effect of within-farm clonal infections was withdrawn from the model. CONCLUSIONS: Knowledge of the penicillin-resistance status of a prior Staph. aureus isolate significantly enhanced the predictive capability of other isolates from the same farm. In the time and space frame of this study, clinicians using previous information from a farm would have more accurately predicted the penicillin-resistance status of an isolate than they would by chance alone on farms infected with clonal Staph. aureus isolates, but not on farms infected with highly genetically heterogeneous bacterial strains.     

The bacterial flora of the normal canine vagina compared with that of vaginal exudates

The aerobic bacterial flora (excluding Mycoplasma spp.) of the vagina of sixty-two clinically normal bitches was determined and the species most frequently isolated, in decreasing order, were: Escherichia colt , Streptococcus 'viridans', Staphylococcus aureus and Streptococcus cards .
From the vaginal exudates of seventy-two bitches with vaginitis, the order of frequency was: E. coli , Strep, canis and Staph. aureus .
The only consistent difference between the bacterial flora from the two sources was the relatively higher numbers of bacteria in the vaginal exudates.