Detection of point mutations in the gyrA gene of Helicobacter pylori isolates in Slovenia.

BACKGROUND/AIMS: The prevalence of antibiotic-resistant H. pylori clinical isolates is a growing concern. So far, fluoroquinolones have not been used to treat H. pylori on a large scale, but recent studies have reported a high rate of quinolones-resistance in H. pylori too. The aim of our study was to asses the mechanism of resistance to ciprofloxacin in H. pylori clinical isolates from patients living in Slovenia. METHODOLOGY: Out of 397 H. pylori clinical isolates, obtained in the period 1997 to 2004, 33 (8.3%) ciprofloxacin-resistant H. pylori isolates were recognized. DNA sequences of the gyrA gene were determined and translated into amino acid sequences. RESULTS: Based on the results of this analysis, various point mutations in the ciprofloxacin-resistant clinical isolates were revealed. The most common mutations in H. pylori gyrA gene were found at codons corresponding to Asp91 (57.6%) and Asn87 (36.4%). Sequence analysis revealed amino acid substitutions also at codons Ala97 to Val, Ala129 to Thr and a double substitution at Asn87 to Lys and Val107 to Ile. CONCLUSIONS: These results suggest that H. pylori resistance to ciprofloxacin is already present in the Slovenian population and that it seems to be mediated through amino acid substitutions in the gyrA gene. Furthermore, the results obtained from the study also demonstrate no significant association between the type of gyrA mutation and the ciprofloxacin MIC level.

Virulence potential of Escherichia coli isolates from skin and soft tissue infections.

Escherichia coli strains frequently are isolated from skin and soft tissue infections (SSTI); however, their virulence potential has not yet been extensively studied. In the present study, we characterized 102 E. coli SSTI strains isolated mostly from surgical and traumatic wounds, foot ulcers, and decubitus. The strains were obtained from the Institute of Microbiology and Immunology, University of Ljubljana, Slovenia. Phylogenetic backgrounds, virulence factors (VFs), and antibiotic resistance profiles were determined. Correlations between VFs and phylogenetic groups were established and analyzed with regard to patient factors. Further, the associations of the three most prevalent antibiotic resistance patterns with virulence potential were analyzed. Our results showed that the majority of the studied strains (64%) [corrected] belonged to the B2 phylogenetic group. The most prevalent VF was ompT (80%), while toxin genes cnf1 and hlyA were found with prevalences of 32 and 30%, respectively. None of the investigated bacterial characteristics were significantly associated with patient gender, age, type of infection, or immunodeficiency. The most prevalent antibiotic resistance pattern was resistance to ampicillin (46%), followed by resistance to tetracycline (25%) and fluoroquinolones (21%). Strains resistant to ciprofloxacin exhibited a significantly reduced prevalence of cnf1 (P < 0.05) and usp (P < 0.01). Our study revealed that E. coli isolates from SSTIs exhibit a remarkable virulence potential that is comparable to that of E. coli isolates from urinary tract infections and bacteremia.

Prevalence and nosocomial spread of methicillin-resistant Staphylococcus aureus in a long-term-care facility in Slovenia.

OBJECTIVES: To determine the prevalence and incidence of methicillin-resistant Staphylococcus aureus (MRSA) colonization among residents and healthcare workers (HCWs) of a long-term-care facility (LTCF), to assess possible routes of nosocomial spread, and to determine genetic relatedness of the isolates. SETTING: A 351-bed community LTCF for the elderly. DESIGN AND PARTICIPANTS: Study investigators made two visits, approximately 3 months apart, to the facility. Samples for cultures were obtained from 107 residents during the first visit, 91 residents during the second visit, and 38 HCWs. RESULTS: The prevalence of MRSA colonization among residents was 9.3% during the first visit and 8.8% during the second visit. During the first visit, two HCWs were colonized. During the second visit, no HCWs were colonized. The colonization of HCWs suggested a potential role in the transmission of MRSA. Molecular typing showed that two of three roommates in one room had the same strain, whereas two in another room differed from one another. All isolates, except one, belonged to two related clonal groups. It seems that the clonal group to which most isolates belonged had the greatest potential for spreading among both residents and HCWs. CONCLUSIONS: Similar prevalence rates of MRSA colonization have been found in other European countries, but such studies have usually involved residents with better functional status than that of the participants in this study. Nosocomial spread of MRSA occurred in the facility examined, but not frequently. More attention should be focused on the hand hygiene of HCWs.

Risk factors for colonization with methicillin-resistant Staphylococcus aureus in a long-term-care facility in Slovenia.

OBJECTIVE: To evaluate risk factors associated with methicillin-resistant Staphylococcus aureus (MRSA) colonization in a long-term-care facility (LTCF) for the elderly in Slovenia. SETTING: A 351-bed community LTCF for the elderly. DESIGN AND PARTICIPANTS: This was a case-control study. MRSA carriage was identified in 102 of 127 residents of the facility's nursing unit. Two swabs were taken: one from the anterior nares and one from the largest skin lesion. If no skin lesions were present, the axillae and the groin area were swabbed. Data were collected regarding gender, age, length of stay in the facility, underlying conditions, functional status, presence of wounds or pressure sores, presence of catheters, antibiotic treatments, and hospital admissions. RESULTS: We detected MRSA in 12 participants. Risk factors independently and significantly associated with MRSA colonization on the multivariate analysis were antibiotic treatments within 1 month before the investigation (odds ratio, 5.087; 95% confidence interval, 1.02 to 25.48; P = .048) and multiple hospital admissions in the 3 months before the investigation (odds ratio, 6.277; 95% confidence interval, 1.31 to 30.05; P = .022). CONCLUSIONS: This is the first assessment of risk factors for colonization with MRSA in an LTCF in Slovenia. MRSA poses a problem in this LTCE Our observations may be valuable in implementing active surveillance cultures in infection control programs in Slovenian LTCFs.

Possibility of application of new pneumococcal conjugate vaccines in children in Slovenia.

The emergence of pneumococcal strains resistant to penicillin caused a lot of problems in the therapy of invasive diseases, and added new dimensions to the role of immunisation. In addition to the currently available 23-valent pneumococcal polysaccharide vaccine (PPV) and a new 7-valent conjugate vaccine (PCV) (Prevnar, Wyeth Lederle), two new conjugate vaccines-a 9- and a 11-valent-are being developed. So far, the choice of most appropriate vaccines has depended on the established prevalence of serotypes causing invasive diseases and their antibiotic resistance in the Slovene children population. Between 1993 and 2001, 263 invasive pneumococcal strains isolated from children with invasive diseases were typed. During the period 1998-2001, the same 161 invasive strains were tested for their antibiotic sensitivity. Streptococcus pneumoniae was identified as the major cause of invasive bacterial diseases in the Slovene children population, especially in children under 4 years of age. Distribution by age groups showed the highest incidence in children aged 0-1 years. The predominant serotypes in all age groups were serotypes 14, 1, 19F, 23F, 6B, 18C and 6A. The distribution of penicillin-intermediate and penicillin-resistant strains showed the predominance of serotypes 23F, 14 and 19F. As concerns infection with S. pneumoniae serotypes, we have proved that children aged less than 5 years are more likely to be infected with penicillin-nonsusceptible or intermediate susceptible strains than older children. The 7-valent conjugate vaccine covers 74% of invasive strains in toddlers, but is less effective in older children. We can conclude that the 9-valent vaccine formulation is optimal for our country, but further cost-effectiveness analysis must be done for recommendation of wide use. At that moment it is reasonable to use the 7-valent conjugate vaccine for children with chronic cardiovascular, pulmonary, urinary and liver diseases, with asplenia, neoplasmia, diabetes, meningomyelocoele, before or after bone marrow transplantation and in cases of immunodeficiency.

Are there regional variations in the diagnosis, surveillance, and control of methicillin-resistant Staphylococcus aureus?

OBJECTIVE: To assess the way healthcare facilities (HCFs) diagnose, survey, and control methicillin-resistant Staphylococcus aureus (MRSA). DESIGN: Questionnaire. SETTING: Ninety HCFs in 30 countries. RESULTS: Evaluation of susceptibility testing methods showed that 8 laboratories (9%) used oxacillin disks with antimicrobial content different from the one recommended, 12 (13%) did not determine MRSA susceptibility to vancomycin, and 4 (4.5%) reported instances of isolation of vancomycin-resistant S. aureus but neither confirmed this resistance nor alerted public health authorities. A MRSA control program was reported by 55 (61.1%) of the HCFs. The following isolation precautions were routinely used: hospitalization in a private room (34.4%), wearing of gloves (62.2%), wearing of gowns (44.4%), hand washing by healthcare workers (53.3%), use of an isolation sign on the patient's door (43%), or all four. When the characteristics of HCFs with low incidence rates (< 0.4 per 1,000 patient-days) were compared with those of HCFs with high incidence rates (> or = 0.4 per 1,000 patient-days), having a higher mean number of beds per infection control nurse was the only factor significantly associated with HCFs with high incidence rates (834 vs 318 beds; P = .02). CONCLUSION: Our results emphasize the urgent need to strengthen the microbiologic and epidemiologic capacities of HCFs worldwide to prevent MRSA transmission and to prepare them to address the possible emergence of vancomycin-resistant S. aureus.

Immune response in chronic periapical parodonititis.

The purpose of our study was to investigate the immune response in chronical periapical parodontitis (CPP) by using multidisciplinary approach. 30 CPP samples were obtained after surgical removal - apicoectomy. Each CPP sample was examined by histological, bacteriological and flow cytometrical (FC) analysis of lymphocytes infiltrating CPP samples. Ten percent of bacteriological samples were sterile, others had significant aerobic and anaerobic growth. We used patohistologic and microbiologic findings and compare them to the results of imunological analysis. By FC we found a significant increase in proportions of T lymphocytes expressing interleukin-2 receptors and ICAM-1 compared to peripheral blood lymphocytes. Proportions of T helper cells that produce interferon-gama (IFN-gamma) was higher in CPP samples predominantly colonized by anaerobic bacteria. There were no differences in IL-4 expression by T cells in both groups (anaerobic and streptoccoccal). Among anaerobic CPP samples differences in proportion of T cells that express IL-2 receptors expression was also found between samples colonised by P. acnes and Bacteroides sp. Oral streptococci cause relatively limited tissue destruction and induce Th2 type of immune response accompanied by non-cytotoxic inflammatory reaction. On the contrary, anaerobic bacteria induce Th1 type of immune response that cause more severe inflammatory reaction (type 4 of hypersensitivity that damage the tissue by the action of cytotoxic T cell activation.

Diminished Th1-type cytokine production in gastric mucosa T lymphocytes after H. pylori eradication in duodenal ulcer patients.

Helicobacter pylori infects an estimated 50% of the world population, however only a small proportion of individuals develop clinical symptoms of gastritis, peptic ulceration or gastric cancer. The variations in disease presentation may be due to differences in bacterial virulence and/or immune response to the pathogen. In the previous study we reported an increased expression of the IL-2 receptor in duodenal ulcer (DU) patients infected with H. pylori. This study examined intracellular lymphokine production in gastric mucosa infiltrating T lymphocytes in DU patients before and after H. pylori eradication. T lymphocytes were isolated from gastric mucosa biopsies by using mechanical and enzymatic tissue desegregation. Ficoll-purified lymphocytes were incubated with monoclonal antibodies and analysed by using 3-colour flow cytometry analysis for intracellular interferon gamma (IFNgamma) and interleukin 4 (IL-4) expression in order to define Th1 and Th2 cell population. We demonstrated a significant decrease in the proportion of Th1 cells infiltrating gastric mucosa 6 and 12 months after H. pylori eradication. Our results suggest the importance of the local immune response in the development of H. pylori related gastritis.

A mouse model of chronic bacterial lesions (a cotton trap) for studying oral bacteria - lymphocyte interactions.

We established a mouse model of chronic bacterial infection (cotton trap) to get a deeper insight into interactions between immune cells and bacterial strains, that are most commonly isolated from periapical processes. We have used flow cytometry to identify the presence of intracellular cytokines of activated T cells collected from cotton traps, previously infected with different strains of bacteria and implanted subcutaneously into the back of the mice. We provide an evidence that anaerobic bacteria (Bacteroides sp.) and nocardiae are more effective in inducing cytotoxic immunity and Th1 response compared to oral streptococci. Differences in immune response against anaerobic bacteria when compared to streptococci are probably dependent on some non-specific immune cell stimulation (e.g. by bacterial cell wall components), nevertheless the role of specific antigen-dependent immune mechanism can not be excluded.

Use of semi-quantitative and quantitative culture methods and typing for studying the epidemiology of central venous catheter-related infections in neonates on parenteral nutrition.

To study the epidemiology - especially the impact of contaminated stopcocks - on central venous catheter (CVC) infection and catheter-related sepsis (CRS), semi-quantitative (SQ) and quantitative (Q) culture methods and typing of coagulase-negative staphylococci (CNS) were employed in 49 neonates with clinical signs of sepsis while receiving parenteral nutrition in the paediatric intensive care unit. The patients were divided into two groups according to stopcock contamination: group A consisted of 18 patients (36%) with contaminated stopcocks and group B consisted of 31 patients (64%) with sterile stopcocks. Five specimens were obtained from each patient, in addition to that from the stopcock: a swab taken from the skin surrounding the catheter puncture site; the CVC tip; the intradermal segment (IDC); and samples of parenteral fluid and blood. A total of 294 specimens (392 sites) was cultured and micro-organisms were identified. All CNS isolated were typed by biotyping, antibiogram, plasmid analysis and pulsed-field gel electrophoresis (PFGE), and the discriminatory power of the typing methods was compared. The CVC tips were infected in 25 patients (51%); 15 (83%) in group A and 10 (32%) in group B. Sepsis was detected in 24 neonates (49%), 13 in group A and 11 in group B. This was catheter-related in 15 patients (63%), 12 in group A and 3 in group B. CNS were recovered from 13 (52%) of 25 infected CVCs, nine in group A and four in group B. Sixty-five CNS isolates were recovered from these patients and belonged to 14 biotypes, 22 antibiograms, 22 plasmid profiles and 26 PFGE types. Typing showed that in six of nine patients in group A, CNS of the same type were recovered from the catheter tip and the stopcock, in one patient the catheter tip and skin isolates were the same and in two others the catheter tip isolates were different from stopcock and skin isolates. In all four patients in group B, different CNS types were recovered from CVC tips and skin. Bacteraemia was caused by CNS in 14 patients (58%), six in group A and eight in group B. Typing confirmed that nine cases (six in group A and three in group B) were catheter-related but five were not. SQ and Q culture methods and typing, especially by PFGE, allowed the study to determine that bacteria from contaminated stopcocks were frequently the source of CVC infection and CRS.