Subspecies differentiation and genotyping of Francisella tularensis strains isolated from clinical and environmental samples.

Molecular epidemiology is one of the most rapidly developing research area. In the light of past and modern technologies it has gained number of typing methods based on molecular biology techniques. In this report, the subspecies differentiation of Francisella tularensis and genotyping of strains isolated in Poland and other geographic locations were investigated using real-time PCR and multispacer typing (MST) methods respectively. In total, 49 strains of F. tularensis included 15 strains from Poland, for subspecies differentiation the real-time PCR method was applied. For molecular typing using MST method four intergenic spacers (IS) were sequenced and compared with those previously described and deposited in GenBank (NCBI). Phylogenetic testing was performed using with the UPGMA model using MEGA6 software. The real-time PCR enables to distinguish five strains belonged to type A and 44 to type B among deposited F. tularensis strains. The MST revealed previously described genotypes, as well as 23 new genotypes were detected. The use of real-time PCR and MST method are valuable in the analysis of F. tularensis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrate convenient molecular tools (real-time PCR and multispacer typing) for Francisella tularensis detection, differentiation and genotyping which can be applied for molecular epidemiological studies and providing useful information for scientific research and during natural tularaemia outbreaks.

Heterogeneity of methicillin-sensitive Staphylococcus pseudintermedius strains isolated from diseased dogs.

Thirty nine canine S. pseudintermedius strains were examined for antibiotic susceptibility and genetic polymorphisms. All strains were methicillin-sensitive S. pseudintermedius (MSSP). Resistance to penicillin was most prevalent (66.6%), followed by resistance to neomycin (56.4%), erythromycin (53.8%), clindamycin (48.7%), chloramphenicol (48.7%), and tetracycline (46.2%). Pulsed-field electrophoresis (PFGE) showed a high genetic polymorphism in the investigated strains.

Screening of Staphylococcus aureus nasal strains isolated from medical students for toxin genes.

Three hundred twenty-one students (156 students with no clinical exposure and 165 students with clinical exposure) were screened for nasal colonization by Staphylococcus aureus; 20.9% of students were S. aureus nasal carriers, and 40.3% of S. aureus isolates harbored toxin genes. The most prevalent genes were tst (15.0 %) and sec (13.4 %). Isolates with multiple genes were only found among clinical students (p = 0.045). Six of 11 PFGE clones were positive for toxin genes. Methicillin-resistant (MRSA) isolates were only detected in the clinical students (4.5 %). The exposure of students to the hospital environment neither radically increased S. aureus nasal carriage, nor the frequency of clinically important toxin gene presence, but it could have influenced the positive selection of toxigenic MRSA strains.

Genotypic screening of atypical Staphylococcus aureus strains isolated from clinical samples for presence of selected adhesin genes.

OBJECTIVE: The aim of this study was to screen Staphylococcus aureus negative for production of coagulase or clumping factor and for presence of selected adhesin genes. METHODS: Sixty coagulase-negative and 20 clumping factor-negative S. aureus strains were studied. Detection of methicillin resistance was performed using the agar screen technique with 6 mg/L of oxacillin and was confirmed by amplification mecA gene. The presence of bone binding protein (bbp), collagen binding protein (cna), fibronectin A binding protein (fnbA), fibronectin B binding protein (fnbB) and clumping factor A (clfA) genes was detected by multiplex PCR. RESULTS: Almost all (98%) of the strains were positive for clfA gene. There were fnbA and fnibB in 85%, cna in 54% and bbp in 5% of strains found. No correlation between presence of the particular genes and clinical samples was observed. The prevalence of fnbA, fnbB and cna was statistically higher in coagulase-negative than in clumping factor-negative strains (89, 89, 66 and 70, 70, 15%, respectively). Similarly, all of these genes were more often observed in MRSA than in MSSA atypical strains. The cna was detected only in coagulase-negative MRSA.

[Characteristics of clumping factor- and protein A-negative methicillin-resistant Staphylococcus aureus identified in Poland]. / Caractéristiques des souches de Staphylococcus aureus facteur agglutinant- et protéine- A-négatifs résistants à la méthicilline isolées en Pologne.

AIM: The aim of this study was to investigate the phenotypic and genotypic properties of clumping factor- and protein A- negative MRSA. METHODS: Two hundred ninety-four strains were studied. The production of protein A was determined by immunoblotting, clumping factor (CF) and coagulase by conventional tests with rabbit plasma. The presence of nuc gene and mecA gene was identified by PCR. The presence of clfA and spa genes was determined for CF- and protein A-negative strains and then phage typing was performed using two sets of phages and gene typing by RFLP of the spa gene. RESULTS: Sixteen (5.4%) CF- and protein A-negative MRSA were isolated. All of them produced coagulase and thermostable nuclease (except for one). nuc, mecA, spa and clfA genes were observed in all strains. The same RLFP pattern, but with different phage types and resistance profiles to antibiotics, was observed in all strains. CONCLUSIONS: CF- and protein A-negative methicillin resistant S. aureus isolated in Poland do not come from a single clone and the absence of CF and protein A is not caused by the deletion of clfA and spa genes.

[Predominance of multidrug resistant strains with reduced susceptibility to fusidic acid among methicillin-resistant Staphylococcus aureus strains (MRSA) isolated in the Gdánsk region]. / Dominacja wieloopornych szczepów o zmniejszonej wrazliwosci na kwas fusydowy wsród metycylinoopornych gronkowców zlocistych (MRSA) izolowanych w regionie gdanskim.

The aim of the present study was to follow the changes in the drug resistance among the methicillin-resistant Staphylococcus aureus strains (MRSA) isolated from clinical samples in various hospitals during 8 years, with particular consideration of Gdansk area. The study was carried out on 225 strains of MRSA from which 95 were isolated in the years 1990-1995 and 130 in the years 1997-1998. The drug susceptibility was determined by the disc-diffusion method. The sensitivity to fusidic acid was determined by both disc-diffusion method and minimal inhibitory concentration (MIC) using agar dilutions. The results obtained show that in 1997-1998 in the hospitals of Gdansk area have been appeared MRSA strains which have not occurred before. These strains were intermediately sensitive or resistant to fusidic acid and simultaneously resistant to doxycycline, gentamicin, erythromycin, clindamycin, ciprofloxacin and rifampin. They represented 66.2% of all MRSA strains isolated in 1997-1998 and were present in majority of the hospitals monitored. Only in this group of staphylococci the strains additionally resistant to mupirocin (6.2%) occurred. Among the MRSA strains with reduced susceptibility to fusidic acid 64.4% were intermediately sensitive (MIC 8-16 mg/L) and 15.4% were resistant to this drug (MIC > 16). In 1997-1998 the percentages of MRSA strains resistant to rifampin, clindamycin and ciprofloxacin increased significantly from 12.6% to 90.8%, from 42.1% to 92.3% and from 18.9% to 92.3% respectively. The percentage of the chloramphenicol resistant strains decreased from 14.7% to 0.8%. Like in 1990-1995, the MRSA strains resistant to vancomycin and teicoplanin were not found out in the same period of time.

[Changes of methicillin-resistant Staphylococcus aureus (MRSA) phagotypes in Gdansk in 1990-1998]. / Zmiany fagotypów gronkowców zlocistych, opornych na metycyline (MRSA) w regionie Gdanskim w latach 1990-1998.

The aim of the study was the assessment of changes in the occurrence of various MRSA phagotypes in hospitals in the Gdansk area in 1990-1998. The study was carried out on 175 MRSA strains: 45 strains isolated in 1990-1995 and 130 in 1997-1998. The studied staphylococci were obtained from various clinical materials from patients in 18 hospitals. Phagotyping was done with a set of 10 experimental phages from MRSA strains obtained from the Central Public Health Laboratory in London. Drug-resistance was determined by the disc-diffusion method and in case of strains with medium susceptibility to fusidic acid the minimal inhibitory concentration (MIC) was determined by serial dilutions on solid medium. The study showed among MRSA strains isolated in 1997-1998 a new, previously not known strains with phage pattern MR25/M5 predominated (57.7%). Its presence was found in various hospitals in that area. MRSA belonging to MR25/M5 phagotype were mostly resistant to doxycycline, gentamycin, erythromycin, clindamycin, ciprofloxacin, rifampicin and were resistant or only had medium susceptibility to fusidic acid. The MIC values for strains with medium susceptibility to fusidic acid (4-8 micrograms/ml) showed an evident decrease of the susceptibility to this antibiotic, formerly common in MRSA. At the same time, in 1997-1998 a considerable decrease was observed of the number of MRSA strains belonging to MR8/MR12/MR25/30/33/38/M5/622 phagotype (from 31.1% to 0.8%), and disappearance of strains with phagotypes MR25/56B/M3 and MR8/MR25/622, which in 1990-1995 accounted for 15.6% of the studied staphylococci.

[Predominance of phage group II in Staphylococcus strains isolated from humans]. / Dominacja II grupy fagowej wsród szczepów Staphylococcus aureus izolowanych od ludzi.

2545 strains of Staphylococcus aureus isolated from 24 Polish laboratories in the years 1994-95 were investigated. Phage typing was performed according to the method of Blair and Williams using the present basic set of typing phages and additional phages 88, 89 and 187. The phages were employed in concentrations of RTD and 100 x RTD. The predominance of phage group II, reported elsewhere since 1980-ties, was found in the present study (23.6%). Strains of group III were second in frequency (16.6%), whereas strains of groups I and V, as well as type 95 occurred in small percentage (7.6%, 4% and 3.4% respectively). Strains of groups II and V have been rarely lysed by phages belonging to other groups. The use of additional phages resulted in typability of strains by 7.9%. Percentage of non-typable strains was high and amounted to 22.0%.

[Evaluation of the usefulness of new international experimental phages for typing methicillin resistant Staphylococcus aureus (MRSA)]. / Ocena przydatnosci nowego, miedzynarodowego zestawu eksperymentalnych fagów do typowania opornych na metycyline gronkowców zlocistych (MRSA).

The aim of the study was to determine the usefulness of the set of experimental phages obtained from the Central Public Health Laboratory in London for typing of MRSA strains in Poland. The study was performed on 150 MRSA strains isolated from various clinical materials in various regions of the country. The set of 10 experimental phages and the international basic set of 23 phages were used for typing. The results of the study showed that 76.8% of MRSA strains were typing with the experimental set of phages. The frequency of inhibition reactions was 19.9%. Only 3.3% of the strains were nontypable with the new phages while nearly half of the studied strains were nontypable with the basic set of phages. The studied strains were divided into 19 phagotypes. There was a high frequency of typable strains among MRSA typable and nontypable strains and those inhibited by the basic set of phages (71.4%-85.7%). These data indicate that the set of 10 experimental phages is useful for typing of MRSA strains isolated in Poland except for phage M3 which failed to react with almost all the strains and should be excluded from the proposed set.

[Sensitivity to disinfectants of methicillin resistant Staphylococcus aureus strains (MRSA) and gentamicin resistant strains]. / Wrazliwosc na srodki dezynfekcyjne metycylinoopornych gronkowców zlocistych (MRSA) i szczepów opornych na gentamycyne.

The aim of the study was to find out whether methicillin-resistant S. aureus strains (MRSA) are tolerant in a higher degree to disinfectants, and whether a correlation exists between lower sensitivity to these agents and resistance to gentamicin. The study was carried out on 30 strains of MRSA and 20 of MSSA isolated from various clinical materials in various regions of the country. Among the studied MRSA 24 strains were resistant and 6 were sensitive to gentamicin, and in MSSA 3 strains were resistant and 17 sensitive to this antibiotic. The sensitivity to four disinfectants: Manusan, Sterinol, Septyl R and Lysoformin Spezial was determined by measurement of MIC (minimal inhibitory concentration) in agar medium. Most MRSA in Poland showed decreased sensitivity to these disinfectants. Among gentamicin-sensitive and resistant MRSA strains the proportions of strains with higher tolerance of three disinfectants (Manusan, Sterinol and Lysoformin Spezial) were very similar. Reduced sensitivity to disinfectants was found in all gentamicin-resistant MSSA. These data indicate that S. aureus strains possess various mechanisms of tolerance of disinfectants. Nearly half the studied strains (46%) had decreased sensitivity to all three preparations (Manusan, Sterinol and Lysoformin Spezial) belonging to various chemical groups this seems to indicate that increased tolerance to these disinfectants is a non-specific feature of S. aureus strains.

Rapid assay for detection of methicillin-resistant Staphylococcus aureus using multiplex PCR.

The presence or absence of the mecA gene, the determinant of resistance to all beta-lactam antibiotics, was examined in clinical isolates of Staphylococcus aureus by multiplex polymerase chain reaction (MPCR). Two pairs of primers were used, which yielded two specific products; a 280-bp nuc- based PCR fragment (amplification product of the nuc gene encoding specific Staphylococcus aureus nuclease) and a 533-bp mecA-based PCR fragment (amplification product of the mecA gene). The MPCR system was designed to be incorporated into the work flow in clinical diagnostic laboratories as a routine analysis.

[Susceptibility of bacteria isolated from clinical material to ciprofloxacin]. / Wrazliwosc bakterii izolowanych z materialu klinicznego na cyprofloksacyne.

The aim of the study was to evaluate susceptibility to ciprofloxacin of bacteria species isolated from different specimens of clinical materials. The investigated strains (830) were identified using conventional methods. The antibacterial activity of ciprofloxacin was studied by the method of drug dilution in Mueller-Hinton agar. Among aerobic bacteria, only 5 strains (0.6%) were resistant to ciprofloxacin in concentration 4 micrograms/ml; two belonged to E. coli species, two to S. aureus and one was a Streptococcus group B., Corynebacterium sp. MIC90 = 0.12 microgram/ml turned out to be most resistant, next, Gram-negative rods MIC90 = 0.5 microgram/ml, coagulase-negative and positive staphylococci and Neisseria sp. MIC90 = 1.0 microgram/ml. Enterococcus faecalis and anaerobes were the least susceptible to ciprofloxacin. Their MIC90 was 2 and 16 micrograms/ml respectively. Among 20 anaerobic strains, up to 10 were resistant to ciprofloxacin, mainly from Bacteroides and Clostridium genus.

[Occurrence of methicillin-resistant strains in Poland and their characteristics]. / Wystepowanie metycylinoopornych gronkowców w Polsce i ich charakterystyka.

The investigation was performed on 923 strains of S. aureus isolated from clinical material obtained from several regions of Poland. Resistance to methicillin was tested by a dilution method on a solid Mueller-Hinton medium supplemented with 2% of NaCl. Strains exhibiting MIC higher than 4 micrograms/ml were determined as resistant. Resistance to other antibiotics (P, Am, CB, CF, CH, MA, Ge, Bs, E, L, Dx) was tested by a disc method. Bacteriophage typing of S. aureus was performed by a method described by Blair and Williams in RTD and RTD x 100, using basic set of phages and additional phages (88, 89, 187). MRSA were present in various regions of the country with similar frequency (from 3.3% to 8.3%). In one center only the percentage was as high as 52.3%. High percentage of MRSA was noted in burned patients (59.9) and these strains were obtained at one center. Within the MRSA III phage group was dominating as well as non-typable strains and inhibited at 100 x RTD-35.4%. MRSA most frequently were typing with phages 88-40.9%, 89-35.0%, 85-24.0%, and they rarely belonged to the phage group II. Among MRSA strains higher percentage of antibiotic-resistance was noted, as compared with other strains. About 60% of MRSA strains were resistant to 6-8 antibiotics. The dominating resistance concerned penicillins, cephalosporins, aminoglycosides, tetracyclines and erythromycin.

[Susceptibility of methicillin-sensitive and methicillin-resistant staphylococci to ciprofloxacin]. / Wrazliwosc gronkowców metycylino-wrazliwych i metycylino-opornych na ciprofloksacyne.

Investigations were carried on 352 strains of Staphylococcus aureus and 105 strains of coagulase-negative staphylococci, isolated from various clinical materials and derived from some regions of the country. Methicillin-resistance was tested by an antibiotic dilution method in solid Mueller-Hinton medium with addition of 2% NaCl. Staphylococci with MIC higher than 4 micrograms/ml were considered as resistant. The same method for testing resistance to ciprofloxacin was used. Only one strain (S. aureus) was resistant to both ciprofloxacin and methicillin. All remaining strains of staphylococci were ciprofloxacin-susceptible. The MIC for all of them was not higher than 2 micrograms/ml, regardless of resistance to methicillin. Some slight differences in MIC50 and MIC90 values were found between MRSA and MSSA and they were, respectively, 1 microgram/ml and 0.25 microgram/ml, and 2 micrograms/ml and 1 microgram/ml. Mean MIC of ciprofloxacin for MRSA was 1.1 microgram/ml and for MSSA it amounted to 0.4 microgram/ml. Range of MIC was following: MRSA--0.12 microgram/ml--16 micrograms/ml and MSSA--0.12-2 micrograms/ml. Coagulase-negative staphylococci, both methicillin-resistant and methicillin-sensitive, exhibited same value for MIC50, MIC90 and MIC range and they were following: 0.5 microgram/ml, 1 microgram/ml and 0.12-1 micrograms/ml. Both groups differed slightly in mean MIC values which was 0.6 microgram/ml for methicillin-resistant strains and 0.47 microgram/ml for methicillin-sensitive staphylococci. It seems that within staphylococci isolated in Poland there is no correlation between resistance to methicillin and ciprofloxacin, which is frequently pointed out by other authors.