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.

Streptococcus pneumoniae serotype distribution in Vojvodina before the introduction of pneumococcal conjugate vaccines into the National Immunization Program.

Introduction: Streptococcus pneumoniae is the most common causative agent of bacterial pneumonia and meningitis. Mandatory childhood immunization against pneumococcal diseases is introduced in the new Law on Protection of Population against Communicable Diseases in Serbia. Objective: The objective of this study was to determine the prevalence of pneumococcal serotype distribution in Vojvodina region before routine use of pneumococcal conjugate vaccine in Serbia. Methods: A total of 105 isolates of Streptococcus pneumoniae were collected in the period from January 2009 to April 2016. Based on the results of serotyping in the National Reference Laboratory, we analyzed distribution of circulating serotypes and coverage of conjugate and 23-valent polysaccharide pneumococcal vaccines in different age groups. Results: Among 105 isolates, a total of 21 different serotypes of Streptococcus pneumoniae were determined. The most frequent serotypes were 3 (21.9%), 19F (20.0%), and 14 (10.5%). The serotype coverage of pneumococcal conjugate vaccines (PCV7, PCV10, and PCV13) was 48.6%, 54.3%, and 84.8%, respectively, while pneumococcal polysaccharide vaccine (PPV23) covered 89.5% of the total number of isolates in all age groups. Serotypes included in PCV7, PCV10, and PCV13 represented 72.0%, 76.0%, and 88.0% of the total number of isolates in children ≤5 years, respectively. Vaccine serotype coverage of PCV13 and PPV23 ranged from 87.1% to 90.3% in adults 50­64 years of age, and 77.8% to 85.2% in adults ≥65 years old. Conclusion: Serotype distribution of Streptococcus pneumoniae in the population fairly overlaps with the serotypes contained in pneumococcal vaccines, so that implementation of childhood immunization is justified. The study was done in the Province of Vojvodina but the findings may be applied to Serbia as a whole.

[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.

Chemical composition and antimicrobial activity of Satureja kitaibelii essential oil against pathogenic microbial strains.

Plant species Satureja kitaibelii Wierzb. ex Heuff. is used as a spice and as a natural preservative for food and herbal tea, owing to its characteristic scent and flavor as well as high antimicrobial activity. In the present study, the antimicrobial activity of isolated essential oil of S. kitaibelii was tested against a panel of 30 pathogenic microorganisms (foodborne microbes, selected multiresistant bacterial isolates from the patient wounds and dermatophyte isolates). Limonene (15.54%), p-cymene (9.99%), and borneol (8.91%) appeared as the main components in 44 identified compounds representing 98.44% of the oil. Essential oil of S. kitaibelii showed significant activity against a wide spectrum of foodborne microbes (MIC=0.18-25.5 microg mL(-1)) and multiresistant bacterial isolates (MIC=6.25-50.0 microg mL(-1)), as well as against dermatophyte strains (MIC=12.5-50.0 microg mL(-1)). These results demonstrate that S. kitaibelii essential oil could be used as a natural potential antimicrobial agent against pathogenic strains in the treatment of foodborne disease, wound and skin infections.

[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).

[Antimicrobial resistance of the bacteria isolated from blood cultures in 2007].

INTRODUCTION: Antimicrobial resistance become emerging global problem. Particularly dramatic increase in the number and type of bacteria resistant to numerous and structurally different antibiotics has taken place in the last two decades. MATERIALS AND METHODS: A total of 558 strains of different bacteria isolated from blood cultures during 2007 were tested to susceptibility to different antibiotics by disc diffusion method according to CLSI. All strains were isolated and identified in the laboratories of Microbiology Center at the Institute for Public Health in Novi Sad. A total of 7748 blood cultures were processed and microorganisms were isolated from 558 of them. Blood samples were taken from the patients hospitalized in the Institutes and Clinics of Novi Sad Clinical Center and Institute for Health Care of Children and Adolescents in Novi Sad RESULTS: The structure ofpathogens shows that the most frequent isolates are Gram-positive bacteria, above all coagulase- negative staphylococci with 285 isolates (51,1%) followed with Enterococcus spp (41 isolates - 6,8%). Percentage of resistance to Methicillin in staphylococci strains was 66,7%; all isolates was susceptible to Vancomycine. Enterococcus spp showed 13,2% resistance to Vancomycine. Among Gram-negative pathogens, we find that the most frequent is Klebsiella spp with 40 isolates (7,2%) and have high resistance to wide spectrum Cephalosporins - 82,5%. Acinetobacter spp participate with 41 isolates (7,3%) and 43,9% isolates are resistant to Imipenem Also, Pseudomonas aeruginosa (20 isolates) shows high resistance to Imipenem (40%) but lower resistance to wide spectrum Cephalosporines (27,8%). CONCLUSION: Having compared the resistance to antibiotics in 2002 and 2007 speak for the trend of growing antimicrobial resistance to the very antibiotics which are recommended as the empiric or initial therapy-wide spectrum Cephalosporins, Fluoroquinolons and Imipenem.

[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.