Prevalence and molecular characterization of Extended Spectrum Beta-Lactamases (ESBLs) producing Escherichia Coli and Klebsiella Pneumoniae.

UNLABELLED: The aim of this study was to determine the prevalence of ESBL-producing Escherichia coli and Klebsiella pneumoniae as well as genes encoding ESBLs. MATERIAL AND METHODS: A total of 1207 non-repeat isolates of E. coli and K. pneumoniae were obtained from urine, tracheal aspirate, wound swab and blood from patients hospitalized at the University Clinics in Skopje. ESBL set and E-test were used for phenotypic detection of ESBL-production. Multiplex PCR was used to identify genes for different types of ESBLs in 100 ESBL positive strains (E. coli-52 and K. Pneumoniae-48), randomly selected. RESULTS: Out of 804 E. coli isolates and 403 K. pneumoniae isolates, 126 (15.7%) and 125 (31%) isolates were ESBL producers, respectively. The prevalence of ESBL-positive strains of E. coli in surgery clinics (42 out of total of 211-19.9%) and K. Pneumoniae (61 out of a total of 161-37.9%) was higher compared to those in the clinics of internal medicine (84 out of 593-14.2%) and (64 out of 242-26.4%), respectively. Only 87 of ESBL positive isolates could be typed for one or more genes. Among the isolates of E. coli and K. pneumoniae harbouring a single ESBL gene (39%), blaSHV, blaTEM and blaCTX-M were present in 19.5%, 16% and 3.4% strains, respectively. Two or more genes for ESBL were present in 61% of ESBL isolates; blaTEM+blaSHV being the most common combination. CONCLUSION: The majority of strains harboured two or more ESBL genes and the most common phenotypes were TEM, SHV and CTX-M. Identification of the genes is necessary for the surveillance of their transmission in hospitals.

Phenotypic and genetic relationship of Acinetobacter Baumannii isolates.

UNLABELLED: The interest in Acinetobacter continues to rise. One of the main reasons is the emergence of multi-resistant strains, which cause outbreaks of infection involving several patients in a ward, in the intensive care unit and in different areas of the hospital. Many outbreaks of its infection or colonization in surgical, neonatal and burn intensive care units have been reported, but the epidemiology of these infections remains unclear. AIM: To investigate the relationship among the isolates of Acinetobacter baumannii, comparing some of their phenotypic and genetic features. MATERIAL AND METHODS: A total of 20 Acinetobacter baumanni isolates were included in the study. 12 strains of Acinetobacter baumannii were obtained within a week in July 2010, from neonates hospitalized at the paediatric intensive care unit and on the neonatal ward. Three strains were isolated from neonates at the paediatric intensive care unit three months ago. All the Acinetobacter baumannii strains were isolated from tracheal aspirates obtained from neonates with infection of the lower respiratory tract. Five additional Acinetobacter baumannii strains were included in the study as controls. They were isolated from wound swabs taken from adult patients with wound infection, hospitalized at the University Traumatology Clinic. Susceptibility of the bacterial strains to 13 different antimicrobial agents was determined by the disk diffusion method (Kirby-Bauer). Additional testing of the susceptibility was performed by the VITEK 2 system. RAPD-PCR fingerprinting was carried out using the following primer (5' GAAACAGCTATGACCATG -3'). RESULTS: All A. baumannii isolates were multi-drug resistant. Antibiotic susceptibility-testing by the disk-diffusion method and automated VITEK 2 system showed 3 and 2 antimicrobial susceptibility patterns, respectively. RAPD-PCR assay of A. baumannii strains revealed two different RAPD-fingerprints. All the strains of A. baumannii isolated within a week in July 2010 from tracheal aspirates taken from neonates in the paediatric intensive care unit and neonates in the paediatric ward revealed the same RAPD-fingerprint, as well as 3 strains of A. baumannii isolated from tracheal aspirates taken from neonates in the paediatric intensive care unit three months ago. 5 strains of A. baumannii isolated from wound swabs of patients hospitalized at the Traumatology Clinic revealed a different RAPD-fingerprint. CONCLUSION: All the strains of A. baumannii isolated from neonates in the paediatric intensive care unit and paediatric ward were multi-drug resistant. Investigating the resistance patterns in multi-resistant isolates of Acinetobacter is a useful method which can predict the strain relationship. This method could be completed by at least one molecular method, such as the RAPD-PCR technique, which has shown itself to be a convenient and more reliable in interpreting the strain relationship of the A. baumannii isolates. Good infection control procedures, including phenotypic and molecular typing of A. baumannii isolates, are essential for preventing outbreaks of multi-drug resistant A. baumannii infections in our hospitals.

Risk factors for Toxoplasma infection in pregnant women in FYR of Macedonia.

The aim of the study was to identify risk factors for Toxoplasma gondii infection in pregnant women in FYR of Macedonia. Retrospective analysis of serological and epidemiological data in a series of 235 pregnant women from Macedonia, tested for Toxoplasma infection between January 2004 and December 2005, showed an overall prevalence of infection of 20.4%. Exposure to transmission factors significantly increased the risk of infection (RR = 1.989, 95 % CI = 1.041-3.800, p = 0.037). The single infection transmission factor that was a predictor of infection in the whole series was exposure to soil (RR = 1.946, 95% CI = 1.026-3.692, p = 0.041). Based on prevalence and the established risk factors for Toxoplasma infection in Macedonia, the health education programme as a sustainable measure for the prevention of congenital toxoplasmosis should focus on educating women of generative age to avoid contact with soil (farming, gardening), and/to adhere to strict hygienic practices afterwards.

Isolation, identification and antimicrobial susceptibility of brucella blood culture isolates.

UNLABELLED: Isolation of slowly growing and fastidious Brucella spp strains from clinical specimens is difficult, because of varying factors, including species specificities, stadium of disease, and previous antibiotic treatment of the patients. The use of automated blood culture systems has overcome some cultivation problems. The automated identification system such as VITEK 2 compact allows more precise identification, as well. AIM: To present our own experience in the isolation of Brucella species from blood cultures, by the Bact/Alert automated system, identification by the VITEK 2 compact system and antimicrobial susceptibility of isolated strains. MATERIAL AND METHODS: Patients from various regions of Macedonia hospitalized in the University Infectious Diseases and Febrile Condition Clinic in Skopje. FAN blood culture bottles (aerobic and anaerobic) of the Bact/Alert system were used, inoculated with 5-10 ml of blood, incubated under continuous agitation and monitored for up to 5 days or until they became positive (in our cases for 2-3 days). Confirmations of all isolates were made by the VITEK 2 automated system on GN cards. RESULTS: During a period of three years, 113 blood cultures from patients with diagnosis of brucellosis hospitalized at the above-mentioned clinic were examined. A total of 16 blood cultures from different patients were positive (14.2%), showing Gram negative bacilli, oxidase positive small colonies on Columbia agar media. The isolates were identified as four biochemically different types of B. mellitensis, mainly within 8 hours. Susceptibility testing by the disk diffusion method on Muller Hinton agar showed sensitivity of all strains to cephalosporin, tetracycline, aminoglycoside and quinolone antibiotic groups. CONCLUSION: With the BacT/Alert system Brucella spp. were isolated in 14.2% of suspected cases of brucellosis. Isolation was done within 2-3 days. Only B. melitensis from the Brucella genus could be identified by the VITEK 2 system and some biochemical differences could be detected. The VITEK 2 system is not able to determine the susceptibility of B. melitensis. The Disk-diffusion method used in this study showed sensitivity to all tested antibiotics, although not recommended by CLSI for the Brucella genus.

Phenotypes and genes of resistance of pneumococci to penicillin isolated from children.

UNLABELLED: (Full text is available at http://www.manu.edu.mk/prilozi). In recent decades, the increase of Streptococcus pneumoniae strains resistant to beta-lactams, to other classes of antimicrobial drugs and especially to penicillin (penicillin-resistant pneumococcus - PRP) has further complicated the treatment of pneumococcal infection. Penicillin resistance in pneumococci is due to the development of altered penicillin-binding proteins (PBPs) in the bacterial cell wall. PBPs are known as six different variants (PBP1a, 1b, 2x, 2a, 2b and 3). AIM: to compare the presence and types of genes responsible for penicillin resistance in Streptococcus pneumoniae isolates with the minimal inhibitory concentrations (MIC) of penicillin as well as their correlation within the period of childhood. MATERIAL AND METHODS: A total of 45 pneumococci obtained from nasal swabs and tracheal aspirates of children treated at the University Paediatric Clinic in Skopje were examined. According to age, the children were grouped as 1-3, 4-6 and 7-10 years. the oxacillin test (1microg) was used as a rapid screening test for the detection of PRP. MIC of penicillin were determined using the agar dilution method and interpreted according to NCCLS as resistant (if MIC are > 2 microg/ml), intermediate resistant (between 0,12-1.0 microg/ml) and susceptible (< 0,06 microg/ml). The genes pbp2b and pbp 2x, which are the genes mainly responsible for the onset of PRP, were detected using polymerase chain reaction (PCR). RESULTS: the oxacillin test showed that 38 pneumococci were resistant and 7 susceptible to penicillin. MIC of penicillin showed that 7 strains were resistant, 33 strains were intermediate resistant (12, 18, and 3 with MIC of 0.5 microg/ml, 0.25 microg/ml and 0.12 microg/ml, respectively) and 5 susceptible. According to MIC, of the total 40 resistant/intermediate resistant pneumococci, in 22 genes pbp2b and/or pbp2x, were confirmed (3 resistant strains with both genes; 7 intermediate resistant and 3 resistant strains with pbp2x genes; whereas 8 intermediate resistance and 1 susceptible strain with pbp2b). In a total of 11 strains (10 intermediate resistant and one resistant according to MIC), pbp2b and/or pbp2x genes were not detected, and their resistance is probably due to some other mechanisms or other genes that code PBP. The largest number of the examined pneumococci (32) were isolated from children aged 1-3 years and in 18 of them either pbp2b or pbp2x genes were detected. CONCLUSION: the oxacillin test is not suitable for discriminating the intermediate resistant and resistant pneumococci, while it is relevant for the detection of susceptible strains. Penicillin resistance of pneumococci that were causes of infection in children was on a lower level (15.5% resistant strains with MIC 1double dagger2 mg/ml and 73.3% intermediate resistant strains with MIC 0.12double dagger1 microg/ml). Pbp2b and/or pbp2x genes were detected in 22 of the examined strains and all of them except one were intermediate resistant or resistant. The Pbp2b gene is mostly present in the intermediate resistant strains and because it was detected in one susceptible strain, this gene is responsible for a low level of resistance. The pbp2x gene was detected in all the resistant strains and that is why we could conclude that it was coding the high level of resistance. Streptococcus pneumoniae was predominantly isolated from the age group 1-3 years where the PRP were not significant (Chi square; p > 0.05). Key words: Streptococcus pneumoniae, Penicillin resistance, Minimal Inhibitory Concentration (MIC), Genes of Resistance.

Virulence factors and antibiotic resistance in Enterococcus faecalis isolated from urine samples.

Haemolysin, enterococcal surface protein (Esp), aggregation substance and gelatinase are some markers that have been proposed as possible enterococci virulence factors. The aim of this study was to detect the presence of haemolysin, gelatinase and enterococcal surface protein in enterococci isolated from urine and to determine their susceptibility to antimicrobial agents. A total of 50 strains of Enterococcus faecalis isolated from urine samples was examined. UTI agar (Oxoid) was used for the isolation and identification of the strains as Enterococcus spp. The differentiation of the species was done by the Vitek automated system (GPI-card). Haemolysin production was detected phenotypically on Columbia CNA agar as a zone of beta haemolysis around the streak. Production of gelatinase was determined as a clear halo around the colonies on tripticase soy agar supplemented with 1.5% skim milk. Esp was proved by detection of the esp gene using PCR after DNA extraction. Antibiotic sensitivity to ampicillin, ceftriaxone, vancomycin, nitrofurantoin and ciprofloxacin was examined by the agar diffusion method. In 16 Enterococcus faecalis strains (32%) all the virulence factors were present. Two factors were found in 19 (38%) strains and only one in 11 strains. There were only 4 strains without any virulence factor. Esp was the most frequently determined factor (in 38 isolates). All the strains were susceptible to vancomycin and nitrofurantoin; 12 isolates were resistant to ampicillin, 17 to ceftriaxone and 14 to ciprofloxacin. No relationship was found between virulence factors and resistance to an antibiotic.

Methicillin-resistant Staphylococcus aureus: comparison of susceptibility test methods with mecA gene analysis for determining oxacillin (methicillin) resistance in our clinical isolates.

The aim of the study was to determine which of the following susceptibility test methods, using recommended or modified NCCLS, best detects oxacillin resistance: disk diffusion, agar screen, and broth dilution. PCR for mecA was used as "gold standard". We studied 120 Staphylococcus aureus isolates received from different patients hospitalized at the Clinical center in Skopje from May 2001 to November 2003. There were no two isolates from the same patient. Methicillin resistance in Staphylococcus aureus strains was performed according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS). PCR appears to be promising. Since variations among the methods exist and no acceptable guidelines are formulated, a combination of conventional methods, including either 3 microg of oxacillin/ml. in Mueller Hinton broth or one of the screen agar plates (6 microg/ml), alone or with PCR should be the method of choice for the detection of MRSA. (Tab. 4, Ref. 12.)

Investigation of antimicrobial activity of essential oils of several Macedonian Thymus L. species (Lamiaceae).

Antimicrobial activity of twenty specimens of essential oils of eleven Thymus species, naturally occurring in the Macedonian flora, was investigated by agar diffusion and broth dilution methods. Inhibition of growth and microbicidal action was examined on three Gram positive bacteria (Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae). In spite of wide variability in essential oil composition, ranging from traces of thymol to the amount of about 50% thymol in oils, all examined samples of Thymus essential oil possessed strong antibacterial activity. Zones of inhibition of growth (for 25% dilution of oils) was from 10-54 mm in diameters. MICs ranging from 0.012-0.1% while MMCs were from 0.025-0.4% for essential oils that contained large amounts of phenols and 0.2-1.6% for those which contained traces of phenols and large amounts of geraniol, linalool and (Z + E)-citral.