Differences between Danish bovine and human Staphylococcus aureus isolates in possession of superantigens.

PCR-assays for the detection of staphylococcal enterotoxins A-E, and H, toxic shock toxin 1, and exfoliative toxins A and B were evaluated against phenotypic methods, and performed well. Four hundred and fourteen isolates of Staphylococcus aureus from Danish cases of bovine mastitis were screened for genes encoding these superantigens. One hundred isolates from Danish human carriers were also included in the study. In contrast to the frequent presence of genes encoding and in vitro expression of superantigens among the human carrier isolates, only one of 414 isolates from bovine mastitis carried the genes encoding enterotoxin C and toxic shock toxin-1. These results further support the hypothesis that the bovine and human S. aureus reservoirs constitute two separate sub-populations of the species S. aureus. The results also show that these superantigens are generally not present in Danish S. aureus isolates from bovine mastitis, and thus play no essential role in the pathogenesis of bovine S. aureus mastitis.

The dynamics of Staphylococcus aureus intramammary infection in nine Danish dairy herds.

The aim of the present study was to examine the diversity of Staphylococcus aureus isolates from bovine intramammary infections (IMI) in nine dairy herds, and compare these with isolates from other sites on the cows by phage- and ribotyping. Whether colonisation of milkers with S. aureus could be a source of infection for bovine IMI was investigated. In addition, 100 epidemiologically unrelated S. aureus isolates from asymptomatic human carriers were also phage- and ribotyped to compare the human and bovine reservoir of S. aureus in Denmark. A total of 625 S. aureus isolates from bovine IMI, bovine skin lesions, milking personnel, and non-farm-related human carriers were included in the study. Certain types predominated in one or several herds during the study period of one-and-a-half to two years, whereas the presence of other types was of a more sporadic nature. Within the individual herds, there was a close correspondence between ribo- and phage types of S. aureus isolated from bovine intramammary infections and skin lesions. Isolates from milking personnel, however, were not identical to any of the predominant intramammary strains. Furthermore, several of the isolates from milking personnel showed ribo- and phage patterns identical to S. aureus isolates from human carriers. The findings of the present study underline the importance of strict milking hygiene and improvement of current mastitis therapy. The results support the hypothesis that some S. aureus mastitis strains are more contagious, virulent or persistent than others. The human reservoir of S. aureus does not play a major role as a source of bovine intramammary infections.

Frequency of alpha- and beta-haemolysin in Staphylococcus aureus of bovine and human origin. A comparison between pheno- and genotype and variation in phenotypic expression.

The phenotypic expression of haemolysins and the presence of genes encoding alpha and beta-haemolysin were determined in 105 Staphylococcus aureus isolates from bovine mastitis, 100 isolates from the nostrils of healthy humans, and 60 isolates from septicaemia in humans. Furthermore, the possible change in expression of haemolysins after subcultivation in human and bovine blood and milk was studied in selected isolates. Alpha-haemolysin was expressed phenotypically in 39 (37%) of the bovine isolates, in 59 (59%) of the human carrier isolates, and in 40 (67%) of the isolates from septicaemia. Beta-haemolysin was expressed in 76 (72%) bovine, 11 (11%) carrier, and 8 (13%) septicaemia isolates. Significantly more bovine than human isolates expressed beta-haemolysin and significantly fewer expressed alpha-haemolysin. Genotypically, the gene encoding alpha-haemolysin was detected in all isolates. A significant difference in the prevalence of the gene encoding beta-haemolysin between the bovine (96%), human carrier (56%) and isolates from septicaemia (57%) was found. Of the bovine isolates, 75% of those carrying the gene encoding beta-haemolysin expressed beta-haemolysin phenotypically, whereas only 20% of the carrier isolates and 24% of the septicaemia isolates did so. No change in expression of haemolysins could be observed after subcultivation of bovine isolates in human blood and milk. After 5 to 10 subcultures in bovine blood and 1 to 4 in bovine milk, 9 of 10 human isolates originally phenotypically beta-haemolysin negative initiated the expression of beta-haemolysin. This study showed that a larger proportion of S. aureus of bovine origin carry the beta-haemolysin gene compared to isolates from humans. Furthermore, a larger number of the isolates of bovine origin carrying the beta-haemolysin gene express this gene phenotypically compared to isolates of human origin.

Coagulase and protein A polymorphisms do not contribute to persistence of nasal colonisation by Staphylococcus aureus.

The nasal carriage rate of Staphylococcus aureus was examined in a longitudinal study of 31 healthy Danish volunteers. Each person was classified as persistent (>8 positive cultures from 10 examinations), an intermittent carrier (50-80% positive cultures) or an ocassional carrier (positive cultures on 10-40% of ocassions only). One hundred and twenty strains from these persons were subjected to phage typing and random amplification of polymorphic DNA (RAPD) analysis. Phage and RAPD typing were in close agreement. RAPD confirmed the spread of a particular S. aureus clone (phage type 95) throughout Denmark. However, no common genotype or phenotype characteristics of S. aureus that could separate persistent from intermittent or incidental colonisers were identified. The immunoglobulin binding protein A and the prothrombin binding coagulase protein are both putative S. aureus virulence or defence factors. Analysis of polymorphisms in the variable repeat regions in the genes for these proteins showed no correlation between the number of repeat units and, consequently, the protein structure with the ability of strains to persist in the human nasal mucosa. The amount of protein A, detectable by its IgG binding activity, appeared not to be correlated to persistence of carriage. Thus protein A and coagulase gene polymorphisms do not seem to play a significant role in the propensity of S. aureus to colonise human nasal epithelium. Furthermore, based on the genetic heterogeneity encountered among the S. aureus strains it is suggested that within the current study population, no single clonal lineage of S. aureus has increased capability to colonise the human nasal epithelium.

Carriage of Staphylococcus aureus among 104 healthy persons during a 19-month period.

The present study was undertaken to investigate the frequency of the nasal carrier rate of Staphylococcus aureus. The investigation was performed on 104 healthy persons. The total number of swabs performed was 1498 and this resulted in isolation of 522 S. aureus strains. All strains have been identified, tested for antibiotic susceptibility, and phage-typed. The carrier-index (number of positive swabs/number of total swabs for each individual person) was compared with different sampling and culturing methods, phage type, age, and resistance to antibiotics. There was statistical difference in carrier rate according to sex (P < 0.05). Among the 104 persons 15 (14.4%) were persistent carriers, 17 (16.3%) intermittent carriers, 55 (52.9%) occasional carriers and 17 (16.3%) non-carriers. Among intermittent and occasional carriers the phage-type distribution was different from the S. aureus strains isolated from Danish hospitalized patients in 1992, while the persistent carriers had similar phage-type distribution.

No difference in enterotoxin production among Staphylococcus aureus strains isolated from blood compared with strains isolated from healthy carriers.

The production of enterotoxin A, B, C and D by 196 Staphylococcus aureus strains isolated from blood cultures and 95 strains from nasal carriers was investigated. Half of the bacteraemia strains were from patients who died with or because of their infection, the other half from patients who survived. The nasal strains were selected to match the bacteraemia strains regarding phage types. Overall, 30.6% of the bacteraemia strains and 40.0% of the nasal strains produced enterotoxins; enterotoxins B and C were the toxins produced most frequently in both groups. A similar incidence and pattern of enterotoxin production was found among the bacteraemia strains of S. aureus regardless of acquisition of the infection, the portal of entry, presence or absence of endocarditis and outcome of the infection. Thus, the concept that the enterotoxins play an important role in staphylococcal infections, apart from the diseases caused by the toxins per se such as food poisoning and toxic shock syndrome, cannot be substantiated by the results of the present study.

The first fatal case of Vibrio vulnificus infection in Denmark.

Vibrio vulnificus can cause severe infections in humans and persons with preexisting liver disorders are especially at risk. In this paper we report what is to our knowledge the first fatal case of V. vulnificus infection in Denmark. The patient was a 68-year-old man with a history of chronic lymphatic leukemia and hepatic cirrhosis. Physicians should be aware of the clinical manifestations of this disease and should be especially attentive to patients at risk of acquiring the infection if there has been possible exposure to V. vulnificus by contact with seawater or contaminated material such as eels.

Evaluation of methods for the detection of nasal carriage of Staphylococcus aureus.

In the present study we investigate the optimal methodology for determination of the nasal carriage rate of Staphylococcus aureus. Tests were performed on 91 healthy laboratory staff. The reproducibility of different sampling, transportation, storage and culture methods was examined. We compared sterile dry cotton wool swabs with sterile dry cotton wool swabs impregnated with charcoal and 5% blood agar plates with mannitol salt agar plates after different incubation periods. Finally, we investigated the detection rate for S. aureus following direct plating compared to storage in Stuart's transport medium for 7 days. There were no differences in isolation rates from the right or left nostril using either cotton or charcoal swabs. Charcoal swabs gave an increased isolation rate as compared to cotton swabs, and incubation in broth enrichment medium containing 6.5% NaCl also increased the isolation rate. Storage in Stuart's transport medium for 7 days gave an increase in isolation rate as compared to direct plating on blood agar. With mannitol salt agar plates the increase in isolation rate when incubation was performed for from 2 to 4, 2 to 7, and 4 to 7 days was 5.9%, 16.7%, and 11.5%, respectively. For the detection of S. aureus nasal carriers we find the use of charcoal swabs and Stuart's transport medium combined with cultivation on mannitol salt agar for 7 days to be the optimal method.

Changing patterns among the subgroups of strains of Staphylococcus aureus of phage group II in Danish hospitals from 1961-91.

During the period 1961-91 a total of 567,635 strains of Staphylococcus aureus from hospitalized patients in Denmark have been characterized according to their antibiotic resistance, site of isolation and phage type. Strains of phage group II (typed by the phages 3A, 3C, 55 and 71) have been analysed further. The occurrence of group II strains was relatively constant (approximately 16%) from 1961 until 1983. Since then the frequency of group II strains increased; in 1991 they accounted for 22.7% of all S. aureus strains isolated. Strains of group II can, on the basis of their phage types, be divided in four subgroups: 3A, 71, 71+ and the 'rest of group II'. Furthermore, within these groups strains may differ from one another in respect to their sensitivity to phages. The increased isolation of group II strains during recent years was because of an increase in strains of subgroups 71+ and the 'rest of group II strains'. In 1991 these two subgroups accounted for 89.7% of all group II strains. Furthermore, an increasing number of group II strains, 71.4% in 1991, was typable only at RTD x 100. The increase in the number of group II strains was even throughout Denmark. All four subgroups of group II have, during the observation period, become more frequently resistant to penicillin and/or tetracycline. Strains typed at 100 x RTD of subgroup 71+ and the 'rest of group II' are more frequently antibiotic resistant than the rest of the group II strains. Strains of the increasing subgroups occurred most often in abscesses.

Non-typhoid Salmonella bacteraemia in Greater Copenhagen 1984 to 1988.

A retrospective survey of non-typhoid Salmonella bacteraemia in the period 1984 to 1988 was carried out by the five departments of clinical microbiology in Greater Copenhagen. A total of 168 patients were identified. A gradual increase was observed from 11 cases in 1984 to 58 cases in 1988. The corresponding incidence per 100,000 inhabitants in Copenhagen rose from 0.9 in 1984 to 5.0 in 1988. During the same period the total registered incidence of human Salmonella infections in Denmark increased from 17.6 to 67.4 per 100,000 inhabitants. The serotype most often isolated from bacteraemic patients was Salmonella dublin followed by Salmonella enteritidis and Salmonella typhimurium. Salmonella dublin demonstrated enhanced invasive and pathogenic properties. Predisposing factors were present in 56% of the patients; the most common was malignant disease. A fatal or complicated course of the bacteraemia was observed more frequently in patients with underlying diseases than in persons who had previously been healthy. A total of 17% of the patients died; one-fifth of these had a ruptured aortic aneurysm. It is concluded that the substantial increase in the number of cases and the often serious course taken by the infection demonstrate a need for increased efforts at prophylaxis.

Tuberculous meningitis. 23 cases from a 12-year period (1976-1987).

Twenty-three patients with tuberculous meningitis were reviewed to see whether clinical features or initial laboratory findings could discriminate between these patients and other patients with bacterial meningitis. Nineteen patients were Danes and four immigrants. Preexisting diseases were found in eight cases. Duration of symptoms could be related to neurological sequelae, but not to death. The initial clinical picture was indistinguishable from meningitis of other causes. Microscopy of the cerebrospinal fluid (CSF) was negative in all but two cases, where acid fast bacilli were found. CSF cytology and biochemistry could not discriminate from other causes bacteria of meningitis although CSF/blood glucose ratio in 56% was below 0.3. One of the most important pieces of information in establishing an early diagnosis in tuberculous meningitis is the anamnestic information, and therapy often has to be started without knowing the microbiological data.

[Bacteremia caused by zoonotic Salmonella types in greater Copenhagen in 1984-1988]. / Bakteriaemi forårsaget af zoonotiske Salmonella-typer i Storkøbenhavn 1984-1988.

The five departments of clinical microbiology in Greater Copenhagen have together carried out a retrospective review of bacteraemia caused by the zoonotic Salmonella serotypes in the period 1984-1988 in the municipalities of Copenhagen and Frederiksberg and in the County of Copenhagen. A gradual increase in frequency was observed from 11 cases in 1984 to 58 cases in 1988. The serotype most commonly isolated was Salmonella dublin followed by Salmonella enteritidis and Salmonella typhimurium. S. dublin was found to be more invasive and more virulent than the other serotypes. Predisposing factors were present in 56% of the patients; the commonest of these was malignant disease. Fatal or complicated course of the disease were observed more frequently in predisposed patients than in persons who had previously been healthy. A total of 17% of the patients died and one fourth of these had ruptured aortic aneurysm probably on account of Salmonella arteritis. 20% developed recurrence of bacteraemia while in the remaining patients the disease ran an uncomplicated course. It is concluded that the marked increase in the number of cases and the serious course taken by the infection demonstrate a definite need for increased prophylactic efforts in the food industry.

[Purulent meningitis in Denmark]. / Purulent meningitis i Danmark.

The epidemiology of meningococcal disease in Denmark has changed in recent years, with a rise in the number of cases in the 14-18 year age group. During the last decade 80 per cent of the meningococci belonged to Group B and 20 per cent to Group C. The treatment has followed the lines of the recommendations issued by the Rigshospitalet but some hospital departments have now chosen other preparations, as appears from the study presented in the article.

[Fatal septicemia caused by Salmonella dublin]. / Septikaemi forårsaget af Salmonella dublin med letalt forløb.

A case of Salmonella Dublin infection which ran a lethal course in a woman aged 49 years is described. Salmonella Dublin was first isolated in Denmark in recent years and appears to be associated with more serious clinical pictures than the other zoonotic Salmonella serotypes. The incidence of S. Dublin is increasing particularly in France and Belgium. It was first isolated from human cases in Denmark in recent years. In the clinical microbiological department in the County of Copenhagen, S. Dublin constitutes approximately 1% of the zoonotic Salmonella serotypes which are isolated from faeces while it is one of the commonest Salmonella serotypes isolated from blood.