Genetic relatedness of methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolated from a dog and the dog owner.

In the present study four methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains isolated from a dog (n=3) and the anterior nares of the dog owner (n=1) were investigated by conventional and molecular methods. The species identity of the four S. pseudintermedius strains was confirmed by conventional methods, by PCR mediated amplification of S. intermedius/S. pseudintermedius specific segments of thermonuclease encoding gene nuc and by restriction fragment length polymorphism analysis of phosphoacetyltransferase encoding gene pta. Investigation of the four S. pseudintermedius for toxinogenic potential revealed that all four strains were positive for the exfoliative toxin encoding gene siet and the leukotoxin encoding genes lukS, lukF. The oxacillin and penicillin resistance of the four S. pseudintermedius strains could be determined by cultivation of the strains on oxacillin resistant screening agar base, ChromID MRSA Agar and Brilliance MRSA Agar and by multiplex PCR detecting the resistance genes mecA and blaZ. The genetic relatedness of the strains was studied by macrorestriction analysis of their chromosomal DNA using pulsed field gel electrophoresis (PFGE). According to PFGE all four S. pseudintermedius strains represent an identical bacterial clone indicating a cross transmission between the dog and the dog owner.

Synergistic and antagonistic hemolytic activities of bacteria of genus Arcanobacterium and CAMP-like hemolysis of Arcanobacterium phocae and Arcanobacterium haemolyticum with Psychrobacter phenylpyruvicus.

A total of 57 bacteria representing eight species of genus Arcanobacterium (A.) were investigated for hemolytic properties on blood agar containing sheep and rabbit blood and for CAMP-like reactions. An enhanced hemolysis on blood agar containing rabbit blood compared to sheep blood could be observed for A. haemolyticum, less pronounced for A. hippocoleae and A. pluranimalium. A synergistic hemolytic reaction with staphylococcal beta-hemolysin appeared to be constantly visible for A. hippocoleae, A. pluranimalium and A. pyogenes, with Streptococcus agalactiae for A. phocae and A. haemolyticum, with Rhodococcus equi for A. phocae, A. haemolyticum, A. pluranimalium and A. pyogenes and with A. haemolyticum for A. hippocoleae, A. pluranimalium and A. pyogenes, respectively. A reverse CAMP-reaction in the zone of staphylococcal beta-hemolysin could be observed for A.phocae and A.haemolyticum. In addition, a novel CAMP-like reaction could be noted between Psychrobacter phenylpyruvicus, identified by 16S rDNA sequencing, and A. phocae and A. haemolyticum. These synergistic or antagonistic hemolytic properties could possibly be used as additional criteria for identification of bacteria of genus Arcanobacterium.

Phenotypic and genotypic characterization of Staphylococcus aureus isolated from raw camel milk samples.

In the present study 320 milk samples collected from 160 apparently healthy camels of three different locations in Sudan were investigated for the presence of Staphylococcus aureus resulting in the isolation of this bacterial pathogen from 28 milk samples from 24 camels. Twenty-five S. aureus were identified phenotypically and by PCR mediated amplification of species-specific genes or gene segments. Investigation of the S. aureus for toxinogenic potential revealed that three S. aureus strains were positive for the enterotoxin encoding gene sec and the genes seg, sei, sem, sen and seo, representing the egc gene cluster. In addition all 25 S. aureus were positive for the superantigen-like encoding gene ssl7 (set1). Partial sequencing of gene sec of the three S. aureus strains yielded an almost complete sequence identity to the sequence of the sec variant sec2. However, all three sec2 genes of the present study showed a deletion of one base causing a frame shift and a corresponding earlier stop codon. According to the present results, the raw camel milk collected from three locations in Sudan seems to be, at least at this stage, of minor importance as vector causing staphylococcal food poisoning.

Molecular identification of Arcanobacterium bialowiezense and Arcanobacterium bonasi based on 16S-23S rRNA intergenic spacer region sequences.

In the present study, the 16S-23S rDNA intergenic spacer region (ISR) of Arcanobacterium (A.) bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483 and A. pyogenes DSM 20630 was amplified, sequenced and compared with the corresponding 16S rRNA gene sequences yielding comparable phylogenetic relationships. The ISR sequence of A. bialowiezense and A. bonasi allowed the design of species-specific oligonucleotide primers which could successfully be used for PCR-mediated identification of previously characterized A. bialowiezense and A. bonasi isolated from infections of the European bison. The presented molecular identification might help to improve a future diagnosis of both newly described bacterial pathogens.

Phenotypic and genotypic characteristics of methicillin/oxacillin-resistant Staphylococcus intermedius isolated from clinical specimens during routine veterinary microbiological examinations.

Methicillin/oxacillin resistance of 10 S. intermedius strains was investigated by conventional and molecular methods. The strains tested had been isolated in Germany during routine veterinary microbiological examinations of specimens from a small animal clinic between May and September 2005. Epidemiological relationships of the strains were studied by macrorestriction analysis of their chromosomal DNA using pulsed field gel electrophoresis (PFGE). Species identity of the 10 S. intermedius strains was confirmed by conventional methods and by PCR mediated amplification of S. intermedius specific segments of thermonuclease encoding gene nuc. As controls, four methicillin/oxacillin resistant S. intermedius (MRSI) strains obtained from specimens sent by four veterinarians and three selected methicillin/oxacillin sensitive S. intermedius (MSSI), also obtained from the small animal clinic, were tested. The 10 strains, representing approximately 6% of all S. intermedius isolated from the clinic throughout the time period mentioned above, and the four MRSI obtained from veterinarians, were methicillin/oxacillin and penicillin resistant using disk diffusion tests and could be cultivated on oxacillin resistant screening agar base (ORSAB). Both resistances could be confirmed by multiplex PCR detecting the resistance genes mecA and blaZ. The three MSSI were methicillin/oxacillin sensitive in all tests. Epidemiological investigation by macrorestriction analysis of the chromosomal DNA of the strains by pulsed field gel electrophoresis revealed that all 10 MRSI strains obtained from the clinic and the four MRSI strains obtained from veterinarians, in contrast to the three MSSI strains, represent identical or closely related bacterial clones possibly indicating a cross-infection of the animals in the clinic and the distribution of a single MRSI clone in the pet population.

Prevalence of genes encoding exfoliative toxins among Staphylococcus hyicus isolated in Russia and Germany.

In the present study, previously characterized Staphylococcus hyicus isolated in Russia (n=23) and Germany (n=17) were investigated for the prevalence of the exfoliative toxin encoding genes exhA, exhB, exhC and exhD by multiplex PCR resulting in the detection of exhD positive strains among the S. hyicus isolated from pigs with exudative epidermitis in Russia and the detection of exhC and exhD for one and two strains isolated from exudative epidermitis in Germany respectively. The toxin gene negative strains were generally isolated from apparently healthy pigs, from other animals and from specimens where the relation between the isolation of S. hyicus and the clinical symptoms remained unclear. Partial sequencing of the toxin genes of selected exhC and exhD positive strains and comparing the sequencing results with sequences of exhC and exhD reference strains revealed an almost complete identity. The results of the present study were in agreement with the findings of Andresen and Ahrens (J. Appl. Microbiol., 96, 2004, 1265) and Andresen (J. Vet. Rec., 157, 2005, 376) that the presented multiplex PCR could be used to investigate S. hyicus for toxinogenic potential and that there is an association between the presence of toxin genes in S. hyicus strains from exudative epidermitis. However, comparable with the S. hyicus strains isolated in Germany which were investigated previously by Andresen (J. Vet. Rec., 157, 2005, 376), exhD seems to predominate in S. hyicus strains from Russia.

Dissemination of the gene encoding exfoliative toxin of Staphylococcus intermedius among strains isolated from dogs during routine microbiological diagnostics.

Phenotypic properties and species-specific PCR tests based on the nuc gene of Staphylococcus intermedius and S. aureus, and a conserved region of 16S rDNA were used to identify 45 S. intermedius and four S. aureus isolated from samples of dogs during routine diagnostics. Four S. pseudintermedius strains used for control purposes reacted positively with the S. intermedius nuc PCR showing the close relationship between both species. Investigating the 45 S. intermedius and four S. pseudintermedius strains for the prevalence of the exfoliative toxin SIET encoding gene yielded the presence of the gene for 21 of the S. intermedius and two of the S. pseudintermedius strains. Partial sequencing of the toxin gene of a single S. intermedius strain and comparing this sequence with that obtained from GenBank revealed an almost complete identity. The presence of the exfoliative toxin gene could mainly be found among S. intermedius isolated from skin and wound infections and from otitis externa possibly indicating a role of this toxin for the clinical symptoms.

Identification of Staphylococcus hyicus by polymerase chain reaction mediated amplification of species specific sequences of superoxide dismutase A encoding gene sodA.

A species specific PCR test, based on manganese-dependent superoxide dismutase A encoding gene sodA, was developed for the identification of Staphylococcus hyicus, an important bacterial pathogen in pigs. The designed primers allowed a rapid and reliable identification of phenotypically characterized S. hyicus, isolated in Russia, Germany and Denmark. No cross reactivities could be observed investigating staphylococcal reference strains representing 18 different species and subspecies. The use of the described primers might improve a future diagnosis of this bacterial pathogen.