Orsolya Dobay

Varying fitness cost associated with resistance to fluoroquinolones governs clonal dynamic of methicillin-resistant Staphylococcus aureus

The purpose of this study was to investigate the impact of fluoroquinolone resistance on the existence and dynamic of MRSA clones. Resistance to ciprofloxacin was induced in strains of community-acquired (CA) MRSA from various sequence types and the fitness cost suffered by mutant derivatives measured in a propagation assay. In addition, the fitness of fluoroquinolone resistant health care-associated (HA) MRSA isolates from major clones prevalent in Hungary were compared with each other and with those of the CA-MRSA derivatives. The genetic background of fluoroquinolone resistance and fitness cost in CA-MRSA was investigated. The fitness cost observed in the CA-MRSA derivatives proved diverse; the derivatives of the ST30-MRSA-IV strain suffered significantly greater fitness cost than those of the ST8-MRSA-IV and ST80-MRSA-IV isolates. Strains from the New York–Japan (ST5-MRSA-II), South German (ST228-MRSA-I) and EMRSA-15 (ST22-MRSA-IV) HA-MRSA clones proved more viable than CA-MRSA derivatives with similar MIC values to ciprofloxacin and HA-MRSA strains from the Hungarian/Brazilian clone (ST239-MRSA-III). Our strains from the New York–Japan, South-German and EMRSA-15 clones seem to have a competitive edge over the tested CA-MRSA isolates in the health care setting. The greater fitness observed in our New York–Japan and South-German strains could account for the replacement by them of the Hungarian/Brazilian clone in Hungary about ten years ago. Alterations in relevant genes were detected. The Ser80 → Phe mutation in the grlA gene may have seriously compromised viability. Surprisingly silent nucleotide substitutions in the grlB gene seemed to impact fitness in derivatives of the ST30-MRSA-IV isolate.

VANCOMYCIN RESISTANT ENTEROCOCCI (VRE) STILL PERSIST IN SLAUGHTERED POULTRY IN HUNGARY 8 YEARS AFTER THE BAN ON AVOPARCIN

In this report we examined the glycopeptide susceptibility of enterococci, isolated in 2005, from slaughtered animals, within the confines of Hungarian Antibiotic Resistance Monitoring System. We determined the presence of the van genes as well as their genetic relatedness in enterococci from poultry. Enterococcus sp. strains (n=175) were collected from intestinal samples of slaughtered poultry in 2005. The origin of the samples was registered at county level. After screening the strains with 30 mg vancomycin disc 19 (86%) intermediate resistant and 4 (3%) fully resistant strains were found. The distribution of minimum inhibitory concentration (MIC)-values among 23 enterococcus strains which were intermediate or resistant to vancomycin were 0.25 mg/L (4.4%), 2 mg/L (8.6%), 4 mg/L (8.6%), 8 mg/L (61%), 16 mg/L (8.6%) and 256 mg/L (8.6%). The MICs of teicoplanin were 0.25 mg/L (4.3%), 1 (8.6%), 4 mg/L (78.3%), 16 mg/L (4.3%) and 256 mg/L (4.3%). The two most vancomycin-resistant strains were vanA carriers (1 E. faecalis and 1 E. faeciuum). The farms that produced these strains can be reservoirs of VRE and the affected farms should change the technology of disinfection and breeding in order to prevent the emergence of high numbers of human VRE isolates in Hungary.

Distribution and genetic relatedness of vancomycin-resistant enterococci (VRE) isolated from healthy slaughtered chickens in Hungary from 2001 to 2004

The presence of the vanA gene was determined in enterococci from healthy poultry, originating from the Hungarian resistance monitoring system between 2001 and 2004. Enterococci (n = 562) were collected from intestinal samples of slaughtered broiler chickens. The presence of van genes was detected by polymerase chain reaction (PCR). The vancomycin-resistant enterococcus (VRE) strains carried only the vanA gene. Genus- and species-level identification of the vanA gene carrier strains was carried out by PCR using specific primers. In 2001, 25 out of the 289 isolated strains (8.6%) were vanA carriers (1 Enterococcus mundtii, 13 E. durans and 11 E.faecium). In 2002 (n = 87), 20 (23%) strains were vanA positive (11 E. durans and 9 E. faecium). In 2003 and 2004, none of the strains (n = 95 and 91, respectively) were positive for the most common van genes. In 2003, there was only one strain for which higher minimum inhibitory concentrations (MIC) of vancomycin (4 mg/L) and teicoplanin (8 mg/L) were found. In 2004 there were three strains for which the MIC of vancomycin was 8 mg/L, and 2 strains and 1 strain with teicoplanin MICs of 4 mg/L and 8 mg/L, respectively. The potential similarity of these strains was studied by pulsed-field gel electrophoresis (PFGE). The VRE strains were not closely related to one another. The annual data of vancomycin resistance indicate an association between the recovery of vancomycin-resistant enterococci and the use of avoparcin in animal feeds. This study indicates that with the reduced use of antibiotics in food animals, it is possible to decrease the rate of resistant bacteria. Although the use of avoparcin had been banned in 1998, the VRE strains disappeared only five years later.

Genotypic and phenotypic characterisation of invasive Streptococcus pneumoniae isolates from Hungary, and coverage of the conjugate vaccines

Background and aims The 7-valent conjugate pneumococcal vaccine (Prevenar) was introduced as a recommended (but not yet obligatory) vaccine in Hungary in April 2009 and there was a sharp increase in the number of children vaccinated. Hence there is an urgent need for in-depth epidemiological data on invasive pneumococci before vaccination becomes widespread. Such a study has never been done before in Hungary. Methods 144 pneumococcal isolates, obtained from invasive infections or pneumonia, were collected from eight Hungarian diagnostic laboratories between 2000 and 2008. After confirmation of species identity, their susceptibilities to nine antibiotics were determined by Etest and agar dilution method. The serotypes and pulsed-field gel electrophoresis genotypes of the strains were also determined. Results In this cohort, most of the isolates were from patients at the extreme of life. Only 1.4% of the strains were resistant to penicillin, but nearly 40% were resistant to erythromycin (mainly due to erm(B) gene). Higher incidences of resistance were found in the very young and very old. The most prevalent serotypes in the cohort in descending order were 14, 6A, 6, 6B, 23F, 3, 19F and 11A. Conclusions Results showed a similar but not identical profile to previously examined strains causing pulmonary infections in Hungary. The serotypes could be correlated to patient groups. Furthermore, there were examples of serotype switching in strains showing identical genotype but different serotype. The study also shows a good coverage by the conjugate vaccines over the invasive pneumococcal strains in Hungary based on the detected serotypes.

Taguchi optimisation of a multiplex pneumococcal serotyping PCR and description of 11 novel serotyping primers

Recently, a PCR-derived method for serotyping Streptococcus pneumoniae has been devised to substitute the conventional antiserum phenotypic method. The method initially used a multiplex PCR reaction, dividing the isolates into 6 different groups based on the detected PCR gel pattern. In order to optimise and refine this crucial step, the Taguchi technique was employed, which can evaluate the individual effect of six parameters (in this case: primers, MgCl2, nucleotide mix, polymerase and buffer), with only 18 experiments; varying the parameter levels in an orthogonal matrix which suppresses the interactions between them. With this method, clear and sharp bands were observed in 5 experiments out of the 18, while the PCR did not work reliably in the remaining cases. In addition, the PCR-based technique could be rendered more economic by the 10-fold lowering of the quantities of two primers. The modified reaction yielded identical results to those obtained with the original method. Furthermore, we have designed serotype-specific primers for 11 new serotypes. The most important ones are those that can distinguish the very closely related, but equally important serotypes 6A and 6B.

Serological evidence of adenovirus infection in cats.

Summary. The widespread presence of adenoviruses in various species makes it probable that infection and the carrier state also exist in cats. On the basis of these considerations, investigations were carried out to find antibodies against adenovirus in sera from different cat populations kept under different conditions. For the antibody detection, purified adenovirus was used in an indirect ELISA. To produce positive serum, SPF cats were immunized with a purified hexon preparation. Altogether 632 field sera of different origin were tested. Among field samples, adenovirus seropositivity varied between 10–26%.

A marked shift in the serotypes of pneumococci isolated from healthy children in Szeged, Hungary, over a 6-year period.

Streptococcus pneumoniae is an important pathogen with significant morbidity and mortality rates worldwide, especially among children <5 years. Healthy carriers are the most important sources of pneumococcal infections, and the nasopharyngeal colonisation is the most prevalent among children attending communities such as day-care centres (DCCs). The conjugate pneumococcal vaccines (PCVs) were shown to have an impact on the colonisation, and so play an important role in inhibiting infections. In this study we compared the nasal carriage of healthy children attending DCCs in Szeged, Hungary in 2003/2004, when nobody was vaccinated, and in 2010, when already 1/5 of the children received PCV-7. Significant differences were observed in the serotype distribution, representing a marked shift from the previously widespread vaccine-types (mostly 6A or 14) to others (11A and 23F). The new serotypes showed higher antibiotic susceptibility. The bacterium exchange between children was clear from the pulsed-field gel electrophoresis (PFGE) patterns, and the circulation of certain international clones plays also a role in these dynamic changes.

Phenotypic and genetic characterisation of methicillin-resistant Staphylococcus aureus strains isolated from the university hospitals of Debrecen

The purpose of this study was to characterise methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in 2005 at the university hospitals of Debrecen, Hungary. Three hundred and thirty-nine MRSA strains were isolated from 102 patients at 18 different clinics. Their sensitivity to oxacillin and ten other antibiotics was determined. For genotypic analysis, phage typing and pulsed-field gel electrophoresis (PFGE) was performed. The rate of MRSA strains increased to 7.2% in 2005, especially at the clinics of surgery, pulmonology and paediatrics. No vancomycin- or teicoplanin-resistant strains were found. The resistance to erythromycin, clindamycin and ciprofloxacin was nearly 100% and multi-resistance was very frequent. Fifty-eight percent of the isolates belonged to mixed phage types and 8% was non-typable. One PFGE clone contained 58.2% of all strains and two further major clones were found at a separately located clinical block, indicating intra-hospital spread. We can conclude that MRSA exhibits an increasing nosocomial problem also in Hungary.

The First Steps Towards Fluoroquinolone Resistance in Hungarian Pneumococci

Abstract The incidence of fluoroquinolone resistance among Hungarian routine laboratory Streptococcus pneumoniae isolates, collected in 2000-2002, in common with other European countries, was very low; only 5/304 strains (1.64%) were resistant to ciprofloxacin (MIC = 4 μg/ml), and the other fluoroquinolones showed full efficacy. However, we could identify the Lys-137-Asp amino acid change, caused by a point mutation in the QRDR of the parC gene, in five strains. Additionally, we observed a definite shift in the minimum inhibitory concentrations (MICs) of all fluoroquinolones towards higher values throughout the study period. These two findings, coupled with the increasing consumption figures of fluoroquinolones, suggest that pneumococcal resistance looks poised to develop in Hungary.

Detection of Staphylococcus aureus nasal carriage in healthy young adults from a Hungarian University

Asymptomatic carriage of Staphylococcus aureus in healthy individuals has a high prevalence, especially in children and young adults. Nasal colonisation is a well-known risk factor for subsequent severe infection, or can be the source of transmission of this bacterium to other susceptible persons. In this study, we have surveyed the nasal carriage rate of students of the Semmelweis University, by screening 300 volunteers. We have determined the antibiotic sensitivity of the isolates by Etest, and their genetic relatedness by pulsed-fieled gel electrophoresis. The nasal carriage rate of S. aureus was found to be 29.3%, and that of MRSA only 0.67% (2/300). The isolates were generally sensitive to antibiotics, except for macrolides. We could observe a noticeably great genetic diversity, even among strains deriving from students of the same university group.