MLSB GENOTYPE IS PREDOMINANT MOLECULAR GENETIC MECHANISM AMONG ERYTHROMYCIN-RESISTANT STREPTOCOCCUS PNEUMONIAE FOR THE PERIOD : 2006-2008 .

A. Alexandrova1, L. Setchanova1, M. Sredkova2, I. Haydoushka3, B. Markova4, U. Proevska4, K. Bojkova5, I. Mitov1 1Department of Microbiology, Medical University, Sofia 2Department of Microbiology, Medical University, Pleven 3Department of Microbiology, Medical University, Plovdiv 4Alexander Hospital, Sofia 5Department of Microbiology, Medical University, Varna Journal of IMAB Annual Proceeding (Scientific Papers) 2009, book 1


INTRODUCTION
Streptococcus pneumoniae is an important pathogen in many community-acquired respiratory infections such as bacterial sinusitis, otitis media, community acquired pneumonia and in more invasive infections like meningitis and bacteremia.
The pneumococcus is becoming increasingly resistant to a variety of antibiotics (7,8).Nowadays multiply resistant pneumococci that are resistant to penicillin, erythromycin, clindamycin, tetracycline, chloramphenicol and trimetoprimsulfametoxazol have been reported very often (2,3).The present study examined the in-vitro sus-ceptibility to macrolides of S. pneumoniae isolates from four main University hospitals in Bulgaria.The main aims were to deter-mine the frequency of resistance to macrolides in our country, and to investigate the mecha-nisms of macrolide resistance.Resistance seems to be associated mainly with efflux mechanisms, but nowadays target site modification by the erm(B) gene is more common.
Pneumococcal macrolide resistance can be caused by erm(B) (erythromycin ribosome methylase)-encoded methylation of a residue in the 23S rRNA (1).Phenotypically, this results in the MLSB resistance phenotype, with resistance to all macrolides plus lincosamides and streptogramin B antibiotics.An alternative resistance mechanism involves mef (E) (macrolide efflux), resulting in M-type resistance to 14-and 15-membered macrolides, but not to 16-membered macrolides, lincosamides or streptogramin B antibiotics.The simultaneous presence of mef(E) and erm(B) has been reported also.
Initial identification of isolates was done by test for optochin susceptibility as a primary identifica-tion method and if it was necessary, by another test such as bile solubility.

Susceptibility testing
All macrolide resistant strains S.pneumoniae were tested both by Agar Dilution method and MIC testing for penicillin G, erythromycin and clindamycin on Mueller-Hinton agar (Oxoid, England) containing 5% sheep blood.Incubation (5% CO2) was for 24 h at 37°C.

Identification of resistance phenotypes
Resistance phenotypes were determined by triple disk test with erythromycin, clindamycin and rokitamycin (4) for all macrolide-resistance strains S.pneumoniae which have erythromycin susceptibility d" 20 mm.All isolates (0.5x McFarland standard) were tested on Mueller-Hinton agar (Oxoid, England) containing 5% sheep blood.
Serotyping.The pneumococci were serotyped by slide agglutination with factor-specific antisera (Statens Serum Institute, Copenhagen, Denmark) and observed by phase contrast microscopy (Binocular, Carl Zeiss).Pneumococcal diagnostic antisera are intended for identification and typing of pneumococci by means of the capsular reaction test.A positive reaction is the result of an in situ immunoprecipitation, leading to a change in the refractive index.In addition the bacteria agglutinate.

Molecular identification methods
The presence of erythromycin resistance genes erm(B) and mef(E) were identified by conventional PCR according to the method of Sutcliffe et al. ( 6) Following growth overnight on blood agar in 5 % CO2, single colonies were grown in 5 ml of Todd Hewitt Broth with Yeast extract (BBL,Becton,Dickinson and Company,) for 6 h, and this was followed by extraction of DNA.All erythromycin-resistant isolates were analysed further by PCR.
PCR buffers and DNA polymerase were supplied by Abgene,UK and all DNA primers were purchased from Alpha DNA,Canada.
The PCR mix was with a magnesium concentration of 2 mM being preferred for the erm (B) primer set and 4 mM for the mef (E) primer set.Electrophoresis on 1% agarose gels in 40 mM Tris acetate-2 mM EDTA buffer was used to distinguish PCR products.The size of each PCR product was estimated using standard molecular weight markers (Super DNA Ladder -Low100-bp, Abgene,UK).

RESULTS
Macrolide resist-ance was found in 150 strains (36.3%) out of all 413 tested S. pneumoniae.The invasive isolates of all ERSP from blood, cerebrospinal fluids, ear fluids and excudates were 25 (16.67%).
We observed 87.34% penicillin resistance (Pen-R) and intermediate penicillin resistance (Pen-I) among erythromycinresistant pneumococcus.Of these Pen-R and -I isolates 71.0 % were with MLSB type.Resistant to clindamycin were more than a half (62%) or 93 strains of all ERSP isolates.
The most prevalence of macrolide resistant pneumococci we observed in children under 5 years old -120 patients (80.0%).In this age group 32.5% showed M phenotype and 67.5% of erythromycin-resistant S.pneumoniae were with MLSB phenotype.The patients over 65 years old with infections, caused by ERSP were 17 (11.33%)and these between 18-65 years old were 13 (8.67%).

Macrolide resistance genotypes
Two different molecular genetic mechanisms of macrolide resistance have been described among pneumococci: targed alteration in a result of methylase expression and erythromycin active cell efflux, encoded by erm (Â) and mef (Å) genes.
PCR analysis of the 150 macrolide-resistant pneumococcal isolates showed that 64.7% isolates harboured only the erm (B) gene and 29.3%only the mef (Å) gene.Nine (6.0%) isolates carried both erm (B) and mef (Å) gene.Erm (B)-positive isolates generally had higher MICs for erythromycin and clindamycin, while mef (E)-positive isolates had lower erythromycin MICs and remained susceptible to clindamycin.(Tabl.According to specimen type, erythromycin resistance was found in 22.97% of isolates from sputum or the lower (Â) gene, and serotype 14 was predominant among strains harboring mef (E) gene.

CONCLUSION
Analysis of the genetic basis of macrolide resistance revealed two main genotypes, namely erm (B) and mef (E).
The study shows that resistance was asso-ciated predominantly with MLSB cross-resistance to macrolides and lincosamides respectively with erm (B) and rare mef (E)positive isolates.The efflux mechanism of macrolide resistance, which were predominant before in our country (5), now give place to widely spread MLSB phenotype and genotype reffering with target modification.This indicate that erm (B) remains the major macrolide-resistance mechanism among pneumococci in Bulgaria.
The dissemination of macrolide resistance encoded by erm (Â) gene were 30% in 2000, increased to 43% in 2005 and 70% in 2008.
Serotypes 6Â, 19F and 19A were the most prevalent among ERSP with erm (Â) gene, and serotype 14 was predominant among strains harboring mef (E) gene.
Ìacrolides are widespread used antibiotics in therapy of pneumococcus infections in our country and ERSP increased with accelerated rates: from 20% in 2000, to 25% in 2005 (5), till 36% in the present research.Controlling the spread of resistant pneumococci through appropriate and judicious prescribing to reduce selective pressure is necessary.Efforts should be made to prevent pneumococcal infections in highrisk patients through vaccination.