Enterotoxigenic Potential of Staphylococcus intermedius

Staphylococcal food poisoning (SFP) caused by enterotoxigenic staphylococci is one of the main food-borne diseases. In contrast to Staphylococcus aureus, a systematic screening for the enterotoxins has not yet been performed on the genomic level for the coagulase-positive species S. intermedius. Therefore, the enterotoxigenic potential of 281 different veterinary (canine, n = 247; equine, n = 23; feline, n = 9; other, n = 2) and 11 human isolates of S. intermedius was tested by using a multiplex PCR DNA-enzyme immunoassay system targeting the staphylococcal enterotoxin genes sea, seb, sec, sed, and see. Molecular results were compared by in vitro testing of enterotoxin production by two immunoassays. A total of 33 (11.3%) S. intermedius isolates, including 31 (12.6%) canine isolates, 1 equine isolate, and 1 human isolate, tested positive for the sec gene. In vitro production of the respective enterotoxins was detected in 30 (90.9%) of these isolates by using immunological tests. In contrast, none of 65 veterinary specimen-derived isolates additionally tested and comprising 13 (sub)species of coagulase-negative staphylococci were found to be enterotoxigenic. This study shows on both molecular and immunological levels that a substantial number of S. intermedius isolates harbor the potential for enterotoxin production. Since evidence for noninvasive zoonotic transmission of S. intermedius from animal hosts to humans has been documented, an enterotoxigenic role of this microorganism in SFP via contamination of food products may be assumed.     

Insertion elements in Staphylococcus intermedius

Staphylococcus intermedius cultures from dogs, pigeons, horses and mink were investigated for the prevalence of the insertion elements IS 256 and IS 257 in relation to their antibiotic resistance. Copies of IS 256 could not be detected in any of the Staph. intermedius isolates tested whereas single copies of IS 257 occurred in the isolates from dogs and horses. The mink strains did not harbour IS 257 elements, whereas Staph. intermedius isolates from pigeons carried multiple copies of IS 257 as predicted from the hybridization patterns obtained with a gene probe derived from the internal part of the IS 257 -encoded transposase gene. Independently of the origin of the Staph. intermedius isolates, all IS 257 copies were found to be located in the chromosomal DNA. The large number of chromosomal IS 257 copies in the pigeon strains might help to explain chromosomal multiresistance in many of those strains.     

Tetracycline resistance in Staphylococcus intermedius

Staphylococcus intermedius cultures commonly exhibited combined resistance to tetracycline and to minocycline. The respective tetracycline resistance genes could not be demonstrated to be plasmidborne as known from other staphylococcal species. They were found to be located in the chromosomal DNA. Southern blot hybridization with specific gene probes identified the tetracycline resistance genes of Staph. intermedius to belong to the class M of bacterial tet genes .     

Enterotoxigenic Staphylococcus aureus in bulk milk in Norway

AIMS: To investigate the presence of enterotoxigenic Staphylococcus aureus in bulk milk and in a selection of raw milk products. METHODS AND RESULTS: Samples of bovine (n = 220) and caprine (n = 213) bulk milk, and raw milk products (n = 82) were analysed for S. aureus. Isolates were tested for staphylococcal enterotoxin (SE) production (SEA-SED) by reversed passive latex agglutination and for SE genes (sea-see, seg-sej) by multiplex PCR. Staphylococcus aureus was detected in 165 (75%) bovine and 205 (96.2%) caprine bulk milk samples and in 31 (37.8%) raw milk product samples. Enterotoxin production was observed in 22.1% and 57.3% of S. aureus isolates from bovine and caprine bulk milk, respectively, while SE genes were detected in 52.5% of the bovine and 55.8% of the caprine bulk milk isolates. SEC and sec were most commonly detected. A greater diversity of SE genes were observed in bovine vs caprine isolates. CONCLUSIONS: Staphylococcus aureus seems highly prevalent in Norwegian bulk milk and isolates frequently produce SEs and contain SE genes. Enterotoxigenic S. aureus were also found in raw milk products. SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococcus aureus in Norwegian bovine and caprine bulk milk may constitute a risk with respect to staphylococcal food poisoning from raw milk products.     

Identification of Staphylococcus intermedius Group by MALDI-TOF MS

The Staphylococcus intermedius Group includes S. intermedius, S. pseudintermedius and S. delphini, coagulase-positive bacteria commonly isolated from animals. The identification of organisms belonging to this group is presently carried out using molecular methods. This study assessed the suitability of MALDI-TOF MS for their identification. 69 strains of different biological and geographic origins, identified by partial hsp60 gene sequencing as S. intermedius (n = 15), S. pseudintermedius (n = 32) and S. delphini (n = 22), were analyzed by MALDI-TOF MS. The estimated sensitivity, specificity and efficiency were calculated. In addition we computed the agreement between the outcome of MALDI-TOF MS identification and partial hsp60 gene sequencing. The sensitivity of MALDI-TOF MS was higher for S. intermedius [0.95 (95% CI: 0.68-0.99)], than for S. pseudintermedius [0.78 (95% CI: 0.60-0.90)] and S. delphini [0.64 (95% CI: 0.41-0.83)], whereas the specificity was 1 for S. intermedius and S. delphini and 0.97 (95% CI: 0.86-0.99) for S. pseudintermedius. The Cohen's kappa coefficient indicated almost perfect agreement between MALDI-TOF MS and hsp60 gene sequencing for the identification of S. intermedius [0.96 (95% CI: 0.87-1.04)], and substantial agreement for S. delphini and S. pseudintermedius [0.70 (95% CI: 0.52-0.89) and 0.76 (95% CI: 0.616-0.92), respectively]. The overall efficiency of the proteomic identification ranged between 0.88 (95% CI: 0.78-0.95) for S. pseudintermedius and S. delphini and 0.99 (95% CI: 0.92-0.99) for S. intermedius. MALDI-TOF MS is thus a valuable and reliable tool for the rapid and accurate identification of bacteria belonging to the S. intermedius Group.     

In vitro indices of tissue adherence in Staphylococcus intermedius

In vitro indices of adherence showed that strains of Staphylococcus intermedius from lesions of canine pyoderma differed from strains isolated from normal carrier sites in that a significantly greater proportion of pyoderma strains adhered to extracellular matrix proteins whilst fewer adhered to polystyrene. Slime production and a hydrophobicity index did not differ between the groups. This suggests that exposure of extracellular matrix proteins due to underlying disease may result in the selection of a narrower spectrum of strains from amongst those at carrier sites.     

Cloning and sequence analysis of a plasmid-encoded chloramphenicol acetyltransferase gene from Staphylococcus intermedius.

The chloramphenicol acetyltransferase gene (cat) of a 3.9 kb chloramphenicol resistance (CmR) plasmid from Staphylococcus intermedius, designated pSCS1, was cloned into an Escherichia coli plasmid vector. Sequence analysis revealed a high degree of base similarity with the cat gene of the S. aureus CmR plasmid pC221 but there were several differences in the regulatory region. A lesser degree of similarity was observed between the cat gene of the S. intermedius plasmid and the cat gene of the S. aureus plasmid pC194.     

Clinical prevalence and antimicrobial susceptibility of Staphylococcus aureus and Staph. intermedius in dogs

AIMS: This study was undertaken to investigate whether the antibiotic resistance of Staphylococcus aureus and Staph. intermedius varies with the site of isolation, sex or age of dogs. METHODS AND RESULTS: A total of 867 isolates of Staph. aureus and 1339 isolates of Staph. intermedius were obtained from nose, eye, ear, reproductive extremity, urine, abscess, skin and throat isolates. Staphylococcus intermedius isolates were isolated most frequently and adult and male dogs were more common compared with juveniles and/or female dogs. Antimicrobial resistance was commonly found for penicillin G, lincomycin, tetracycline and trimethoprim-sulphamethoxazole in both Staphylococcus species. Surprisingly, we detected significant resistance to cloxacillin in male (67.1%) and female (69.4%) Staph. aureus isolates, irrespective of the anatomical site of isolation. The resistance or susceptibility of isolates of Staph. aureus from reproductive extremities and isolates of Staph. intermedius from ear, eye and abscess sites was associated with the age of the animal. CONCLUSIONS: Antimicrobial susceptibilities in Staph. aureus and Staph. intermedius often differed with regard to the site of isolation, sex and age of the animal. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing antimicrobial resistance in staphylococci in veterinary medicine complicates the empirical selection of antimicrobial agents. These complications reveal a continuously evolving, complicated multifactoral process of the site of isolation, sex and age of the animal.     

Prevalence of virulence factors in Staphylococcus intermedius isolates from dogs and pigeons

Background

The genetic diversity of Anaplasma platys (Rickettsiales: Anaplasmataceae) strains is currently poorly defined. The present study was designed to characterize A. patys strains in dogs from Palermo, Sicily, Italy, using a combination of PCR and sequence analysis of the 16S rDNA, heat shock operon groESL and citrate synthase (gltA) genes.

Results

Blood was collected from 344 dogs (111 pet dogs, 122 pound dogs and 111 hunting dogs) during 2003–2005 in the Province of Palermo, Sicily, Italy. The prevalence of A. platys in dogs in Sicily, as demonstrated by PCR and sequence analysis of the 16S rDNA, groESL and gltA genes, was 4%. None of the samples were positive for A. marginale, A. centrale, A. ovis and A. phagocytophilum DNA. Three different gltA genotypes of A. platys were identified in dogs from Sicily. Two of the gltA sequences of Sicilian A. platys strains were different from sequences reported previously. However, one of the gltA, 16S rDNA and groESL sequences were identical to the sequence of A. platys strains from other regions of the world characterized previously.

Conclusion

At least three different strains of A. platys were identified in dogs from Sicily by PCR and sequence analyses of the 16S rDNA, groESL and gltA genes. The results reported herein suggested that genetic diversity of A. platys strains may be similar to A. ovis, but lower than the diversity reported for A. marginale and A. phagocytophilum. This lower genetic diversity may have resulted from restricted movement of infected hosts compared to A. marginale-infected cattle and/or the limited host range of A. ovis and A. platys as compared with A. phagocytophilum. These results expand our knowledge about A. platys and encourage further research for analysis of the genetic variation of A. platys strains worldwide.     

Staphylococcus intermedius produces a functional agr autoinducing peptide containing a cyclic lactone

The agr system is a global regulator of accessory functions in staphylococci, including genes encoding exoproteins involved in virulence. The agr locus contains a two-component signal transduction module that is activated by an autoinducing peptide (AIP) encoded within the agr locus and is conserved throughout the genus. The AIP has an unusual partially cyclic structure that is essential for function and that, in all but one case, involves an internal thiolactone bond between a conserved cysteine and the C-terminal carboxyl group. The exceptional case is a strain of Staphylococcus intermedius that has a serine in place of the conserved cysteine. We demonstrate here that the S. intermedius AIP is processed by the S. intermedius AgrB protein to generate a cyclic lactone, that it is an autoinducer as well as a cross-inhibitor, and that all of five other S. intermedius strains examined also produce serine-containing AIPs.     

Enterotoxigenic E. coli in humans.

E. coli strains isolated from persons having diarrhoeal disease were tested for the production of TS enterotoxin. The production of TSE was demonstrated in 2.5% in a series of 80 strains isolated from children under one year of age, having acute diarrhoea. TSE was produced by 8.4% of E. coli strains out of 59 strains isolated from patients over one year of age. Among these strains, an interesting E. coli strain was isolated from the patient T. J., which produced TSE for more than 15 months. The production of TLE was tested though not proved in all strains by experiment on an isolated intestinal loop of an adult rabbit. The test on suckling mice so far appears to be the most suitable test for the demonstration of TSE. The results were considered positive when the index (the ratio of the weight of the whole intestine to the weight of the rest of the body) was higher than 0.08 while indices up to 0.078 were considered negative. E. coli strains with indices of intermediate values and strains with temporary production of TSE, occurring particularly in very small children, deserve special attention. The height of the indices was not influenced by a 30-minute exposure at 60 degrees C, but a decrease in the values of the indices was observed after boiling for a period of 15 min. The occurrence of E. coli strains producing TSE is evidently small in humans in European countries but, without doubt, they are important in the aetiology of diarrhoeal diseases.     

Bacteroides intermedius binds fibrinogen.

The binding of Bacteroides intermedius VPI 8944 to human fibrinogen has been characterized. The binding is time dependent, at least partially reversible, saturable, and specific. On an average, a maximum of 3,500 fibrinogen molecules bind per bacterial cell, with a dissociation constant of 1.7 X 10(-11) M. These bacteria also exhibit a fibrinogenolytic activity which can be partially inhibited by protease inhibitors. Bacteria release fibrinogenolytic activity into the surrounding medium without loss of binding activity, but more pronounced fibrinogen breakdown occurs when 125I-labeled fibrinogen is associated with the bacteria, suggesting that fibrinogen is degraded at the cell surface. Fibrinogen binding by B. intermedius might represent a mechanism of bacterial tissue adherence.     

Interspecies lysogenization in staphylococci: transfer of bacteriophage converting enterotoxin A from a clinical strain of Staphylococcus aureus to Staphylococcus intermedius

The ability of lysogenization was examined of 50 S. intermedius strains and of 77 strains belonging to 14 different species of coagulase-negative staphylococci using 8 enterotoxin A converting bacteriophages isolated from S. aureus. All the examined bacteriophages showed lytic activity against at least 1 of 11 susceptible strains of S. intermedius to them. Lytic activity towards coagulase-negative staphylococci was observed for 6 of 8 examined bacteriophages. Two bacteriophages were active against 1 of 9 examined S. capitis strains, one against 1 of 11 examined S. haemolyticus strains, four against 1 of 6 examined S. lugdunensis strains, three against 1 of 6 examined S. warneri strains and one against 1 of 5 examined S. xylosus strains. Lysogenization with bacteriophage f421-1 able to convert positively enterotoxin A and staphylokinase and negatively beta-haemolysin of one S. intermedius strain was successful. S. intermedius lysogenized with phi 421-1 was able to produce both enterotoxin A and staphylokinase and lost ability to produce beta-haemolysin. Our results showed a broad lytic spectrum and interspecies host range of some S. aureus bacteriophages and the ability of interspecies transfer of bacteriophages between S. aureus and S. intermedius.     

Acetobacter intermedius,sp. nov

Strains of a new species in the genus Acetobacter, for which we propose the name A. intermedius sp. nov., were isolated and characterized in pure culture from different sources (Kombucha beverage, cider vinegar, spirit vinegar) and different countries (Switzerland, Slovenia). The isolated strains grow in media with 3% acetic acid and 3% ethanol as does A. europaeus, do, however, not require acetic acid for growth. These characteristics phenotypically position A. intermedius between A. europaeus and A. xylinus, DNA-DNA hybridizations of A. intermedius-DNA with DNA of the type strains of Acetobacter europaeus, A. xylinus, A. aceti, A. hansenii, A. liquefaciens, A. methanolicus, A. pasteurianus, A. diazotrophicus, Gluconobacter oxydans and Escherichia coli HB 101 indicated less than 60% DNA similarity. The important features of the new species are described. Acetobacter intermedius strain TF2 (DSM11804) isolated from the liquid phase of a tea fungus beverage (Kombucha) is the type strain.     

Biotyping of Enterotoxigenic Staphylococcus aureus by Enterotoxin Gene Cluster (egc) Polymorphism and spa Typing Analyses

Thirty-five Staphylococcus aureus strains, including 10 reference strains and 25 strains recovered from clinical specimens and food samples, were analyzed by PCR REA (restriction endonucleases analysis) of the egc operon and spa typing. Nineteen spa types and seven different egc operons, including four putative new egc variants, were revealed. In 13 strains, allelic variants of sei and/or seg were found. By an analysis of their nucleotide sequence identities, a new homogeneous cluster of a sei variant, called the sei variant, was detected in six strains. In addition, the prototype sei was shown to be more polymorphic than assumed so far. Seven strains possessed the recently described seg variant, also exhibiting several nucleotide exchanges. spa typing was more effective than REA egc grouping as a typing technique. Since, in some cases, the REA typing method was able to discriminate strains showing the same spa type, it must be considered for PCR approaches involved in diagnostic procedures and may be useful for epidemiological studies. Hence, the polyphasic approach used in this study can be reliably and advantageously applied for typing egc-positive S. aureus strains.