Detection of carbapenemase genes in Klebsiella pneumoniae isolates.

Introduction: Klebsiella pneumoniae is one of the leading causes of serious hospital-acquired infections worldwide among Enterobacteriaceae species. It is the most common producer of carbapenemases in many parts of the world. Objective: The aim of the study was to determine which enzymes were responsible for resistance to carbapenems in Klebsiella pneumoniae strains isolated at the Centre of Microbiology of Public Health Institute of Vojvodina. Methods: A total of 29 Klebsiella pneumoniae non-duplicated strains resistant to at least one carbapenem isolated from clinical samples of hospitalized patients between November 1st 2013 and April 30th 2014 were studied. The species identification and susceptibility were done using VITEK 2 (bioMérieux, Marcy-l'Étoile, France) system. Phenotypic conformation of carbapenemase production was done by double-disc synergy test. PCR technique was performed for detection of genes encoding production of carbapenemases (bls(KPC), bla(VIM), bla (NDM), bla(OXA-48)). Results: Isolates of Klebsiella pneumoniae resistant to at least one carbapenem showed positive on double- disc synergy test between meropenem and dipicolinic acid. All strains positive in phenotypic test contained bla(NDM) gene. In isolates resistant only to ertapenem, neither production of carbapenemases nor presence of genes encoding these enzymes were detected. Among these isolates, nine produced extended-spectrum ß-lactamase. Conclusion: The presence of NDM metallo-ß-lactamase was determined in all Klebsiella pneumoniae isolates resistant to at least one carbapenem.

Prevalence of quinolone resistance and mutations in the topoisomerase genes in Salmonella enterica serotype Enteritidis isolates from Serbia.

The prevalence of quinolone resistance was studied in Salmonella enterica serotype Enteritidis isolates collected during 2005-2010 in Southern Backa County, Serbia. A total of 878 clinical isolates were examined, among which 19 (2.2%) nalidixic acid (NAL)-resistant S. Enteritidis were detected by selection on agar plates containing 64 mg/L NAL. Antimicrobial susceptibility of the isolates was tested by the agar dilution method. According to Clinical and Laboratory Standards Institute (CLSI) breakpoints, ciprofloxacin (CIP) resistance was not present in the strains. Multiple drug resistance was rare, and resistance to NAL was most often present as a single resistance property. All but one NAL-resistant S. Enteritidis showed reduced susceptibility to CIP [i.e. minimum inhibitory concentration (MIC)≥0.125 mg/L]. This isolate of human origin had a CIP MIC of 0.064 mg/L and DNA sequencing revealed that it contained an Asp87Gly gyrA mutation. Most of the remaining isolates had MICs for NAL and CIP of 256 mg/L and 0.256 mg/L, respectively. Mutations in the Asp87 codon resulted in substitutions to Asn in most of the isolates, but Asp87Gly and Ser83Phe exchanges were also detected. No mutations were present in the gyrB, parC or parE genes. Although CIP resistance was absent, reduced susceptibility characterised by mutations in gyrA was apparent among S. Enteritidis isolates from Serbia.

[Frequency of vancomycin-resistant enterococci isolated from blood cultures from 2008 to 2010].

INTRODUCTION: Enterococci are important hospital-acquired pathogens. The most commonly isolated species of the genus, Enterococcus faecalis and Enterococcus faecium are the third to fourth-most prevalent nosocomial pathogens worldwide. The aim of this study was to determine the frequency of resistance to vancomycin and other antimicrobial agents of Enterococcus spp strains isolated from blood cultures of hospitalized patients. MATERIAL AND METHODS: During the three-year period, from 2008 to 2010, 132 strains of Enterococcus spp isolated from blood cultures of hospitalized patients were tested for their susceptibility to ampicillin, vancomycin, gentamycin (high-level resistance), erythromycin, chloramphenicol, teicoplanin, ciprofloxacin by disc diffusion method according to the Clinical and Laboratory Standards Institute recommendations. Susceptibility of vancomycin resistant E. faecium to the same antibiotics and to linezolid, quinopristin/dalfopristin and tigecyclin was determined using VITEK system. RESULTS AND DISCUSSION: Resistance to vancomycin was detected in 21 (15.9%) Enterococcus spp strains. The percentage of resistance to other antimicrobial agents varied from 23. l% for chloramphenicol to 81.3% for ciproflxacin. All vancomycin resistant enterococci were identified as E. faecium and belonged to phenotype VanA. The resistance to other antibiotics was very high, except for linezolid and quinopristin/dalfopristin (4.7%). The high-level aminoglycoside resistance was 87.6% for gentamycin and 95.2% for streptomycin. All isolates were resistant to ampicillin, teicoplanin and ciprofloxacin. CONCLUSION: The detected high frequency of multidrug-resistant isolates among vancomycin resistant enterococci is of great importance and suggests the need for further monitoring of susceptibility in order to take adequate measures to prevent and control spreading of resistant strains.

Resistance of Escherichia coli from healthy donors and from food--an indicator of antimicrobial resistance level in the population.

Escherichia coli, being an important part of normal intestinal flora, is a frequent carrier of antimicrobial drug resistance markers and food is the most important vector of antimicrobial resistance genes between humans and animals. The aim of this study was to confirm the presence and frequency of resistance markers in Escherichia coli from intestinal flora and from food as an indicator of antimicrobial resistance level in the population. The experiment included 100 fecal Escherichia coli isolates from healthy donors, 50 isolated in 2007 and 50 in 2010, and 50 from food samples. The resistance markers were found in all groups of isolates. The resistance to ampicillin and cotrimoxazole was most commonly found. The finding of multi-drug-resistant strains and resistance to ciprofloxacin is important. The frequency of resistance markers was similar in food and feces. The results of this study show the need to introduce systematic monitoring of antimicrobial resistance of these bacteria.

[Carbapenems resistance of Acinetobacter spp strains isolated from wound swabs during 2009-2010].

INTRODUCTION: Acinetobacter spp has become an important cause of nosocomial infections due to its great ability to survive and spread in a hospital setting and to develop resistance to many antibiotics. The aim of this study was to examine the resistance to carbapenems and other commonly used antibiotics in strains of Acinetobacter isolated from wound swabs. MATERIAL AND METHODS: In the Laboratories of Microbiology Center at the Institute for Public Health in Novi Sad wound swabs were taken from the patients hospitalized at the Institutes and Departments of the Clinical Center of Vojvodina in Novi Sad. A total of 271 Acinetobacter spp strains were tested to susceptibility on carbapenems and other most commonly used antibiotics by disk diffusion method according to the recommendation of the Clinical and Laboratory Standards Institute. RESULTS AND DISCUSSION: Acinetobacter spp (271 isolates) developed resistance to imipenem and meropenem (67.4% and 64.4%). The resistance to both cephalosporins III and IV generation and ciprofloxacin was 100%. The resistance to ampicillin-sulbactam was 71.4%. CONCLUSION: Our results show high resistance to carbapenems in Acinetobacter spp strains isolated from wound swabs. These facts suggest the need for continuous monitoring of susceptibility in order to take adequate measures to prevent and control spreading of resistant strains.

[Sensitivity of bacteria to antimicrobial drugs and interpretation of results].

INTRODUCTION: The discovery of antimicrobial drugs was a turning point in the permanent conflict between the mankind and microorganisms. However, due to the wide use and misuse of antibiotics in therapy and prophylaxis of infections the mankind is threatened by an alarming rise in the resistance of bacteria to drugs. Will this phenomenon turn us back to the pre-antibiotic era? DISCUSSION: The increasing resistance of bacteria has become a global public health problem: bacteria are showing a remarkable capacity to develop different mechanisms and avoid drug effect. Mechanisms of resistance are numerous and various: production of beta-lactamases (Ambler class A): TEM-I, TEM-2 and SHV-1 and mutants of classical enzymes with extended spectrum (ESBL) (e.g. in Klebsiella spp.) which results in the resistance to the 3rd generation cephalosporines and new metallo-beta-lactamases among Pseudomonas and Acinetobacter (resistance to carbapenems). The alteration of the target enzymes (PBP) leads to the Staphylococci resistance to methicillin and the responsible gene is mecA gene). The alteration of DNA gyrase due to the mutations of gyrA, gyrB, parC genes (accumulation of multiple mutations) results in the development of resistance to fluoroquinolones); and the active efflux system - "pumping out" of the drug from the bacterial cell leads to the resistance of a wide spectrum of different antibiotics. In order to choose the most efficient drug for therapy, it is necessary to investigate susceptibility of bacteria to antimicrobial agents. For that purpose, a disc-diffusion method according to CLSl standard procedure is performed. For invasive strains it is often necessary to determine minimal inhibitory concentrations (MIC) of antimicrobials. The methods that are in use are agar-dilution methods, E-test and automated MIC determination by VITEK 2 system. CONCLUSION: By molecular-biological methods it is possible to identify the mechanisms of resistance and detect the specific genes behind it (mecA gene). The targeted therapy prevents compromising of antibiotics valuable in treatment of severe infections (carbapenems).

[Frequency of methicilin-resistant Staphylococcus aureus strains in clinical spacimens obtained from hospitalized patients in 2007].

INTRODUCTION: Staphylococcus aureus is a major cause of both hospital- and community-acquired infections worldwide. Since the introduction of methicillin into clinical use, methicillin-resistant S. aureus strains have emerged with increasing frequency throughout the world. The aim of the study was to determine frequency of resistance to methicillin and other most commonly used antibiotics of S aureus strains isolated from hospitalized patients. MATERIAL AND METHODS: During the period 1.1.-31. 12. 2007, 226 strains of S. aureus isolated from hospitalized patients were tested for their susceptibility to penicillin, methicillin, erythromycin, clindamycin, gentamycin, sulfamethoxazolle-trimethoprim, fusidic acid and vancomycin using disc diffusion technique. RESULTS: Resistance to methicillin was detected in 20 (7.5%) S. aureus strains. All of them were susceptible to vancomycin. The resistance to other antimicrobial agents varied from 5% for sulfamethoxazolle-trimethoprim to 75% to gentamycin. Among methicillin susceptible strains, high resistance (84.1%) was found to penicillin only The resistance to other antimicrobial agents was low, ranging from 0 for fusidic acid and vancomycin to 12.2% for gentamycin. CONCLUSION: Our results show low rate of methicillin-resistant S. aureus strains compared to other authors "findings, but also the presence of multidrug-resistant isolates and isolates susceptible to vancomycin only These facts suggest the need for further monitoring of susceptibility in order to take adequate measures to prevent and control spreading of resistant strains.