Molecular Screening of Carbapenem-Resistant K. pneumoniae (CRKP) Clinical Isolates for Concomitant Occurrence of Beta-Lactam Genes (CTX-M, TEM, and SHV) in the Kingdom of Bahrain.

The emergence of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae, including CRKP infections, has resulted in significant morbidity and mortality worldwide. We aimed to explore the presence of bla genes (CTX-M, TEM, and SHV) in CRKP isolates. A total of 24 CRKP isolates were randomly selected from the Salmaniya Medical Complex Microbiology Laboratory. These isolates, which were positive for carbapenemases, were further explored for CTX-M, TEM, and SHV genes using PCR. All the CTX-M PCR amplicons were sent for sequencing. To determine genetic relatedness, molecular typing by ERIC-PCR was performed. The bla gene testing demonstrated that a significant proportion of these isolates harbored SHV, CTX-M, and TEM genes (100%, 91.6%, and 45.8%), respectively. Bioinformatic analyses confirmed CTX-M-15 in these isolates. ERIC-PCR analysis showed three clusters demonstrating genetic relatedness. The study findings reveal the concomitant carriage of the SHV and CTX-M-15 and a comparatively lower carriage of TEM genes in CRKP isolates. Our findings highlight the significance of routinely reporting the presence of antibiotic resistance genes along with regular antibiotic sensitivity reports, as this will aid clinicians in prescribing appropriate antibiotics.

Prevalence of Carbapenemases in Carbapenem-Resistant Acinetobacter baumannii Isolates from the Kingdom of Bahrain.

BACKGROUND: Acinetobacter baumannii is regarded as a significant cause of death in hospitals. The WHO recently added carbapenem-resistant Acinetobacter baumannii (CRAB) to its global pathogen priority list. There is a dearth of information on CRAB from our region. METHODS: Fifty CRAB isolates were collected from four main hospitals in Bahrain for this study. Bacterial identification and antibiotic susceptibility tests were carried out using the BD PhoenixTM and VITEK-2 compact, respectively. Using conventional PCR, these isolates were further screened for carbapenem resistance markers (blaOXA-51, blaOXA-23, blaOXA-24, blaOXA-40, blaIMP, blaNDM, blaVIM, and blaKPC). RESULTS: All of the isolates were resistant to imipenem (100%), meropenem (98%), and cephalosporins (96-98%), followed by other commonly used antibiotics. All these isolates were least resistant to gentamicin (64%). The detection of resistance determinants showed that the majority harbored blaOXA-51 (100%) and blaIMP (94%), followed by blaOXA-23 (82%), blaOXA-24 (46%), blaOXA-40 (14%), blaNDM (6%), blaVIM (2%), and blaKPC (2%). CONCLUSION: The study isolates showed a high level of antibiotic resistance. Class D carbapenemases were more prevalent in our CRAB isolate collection. The resistance genes were found in various combinations. This study emphasizes the importance of strengthening surveillance and stringent infection control measures in clinical settings to prevent the emergence and further spread of such isolates.

Clinical carbapenem-resistant Klebsiella pneumoniae isolates simultaneously harboring bla NDM-1, bla OXA types and qnrS genes from the Kingdom of Bahrain: Resistance profile and genetic environment.

The prevalence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) is currently increasing worldwide, prompting WHO to classify it as an urgent public health threat. CRKP is considered a difficult to treat organism owing to limited therapeutic options. In this study, a total of 24 CRKP clinical isolates were randomly collected from Salmaniya Medical Complex, Bahrain. Bacterial identification and antibiotic susceptibility testing were performed, on MALDI-TOF and VITEK-2 compact, respectively. The isolates were screened for carbapenem resistance markers (bla NDM, bla OXA-23, bla OXA-48 and bla OXA-51) and plasmid-mediated quinolone resistance genes (qnrA, qnrB, and qnrS) by monoplex PCR. On the other hand, only colistin-resistant isolates (n=12) were screened for MCR-1, MCR-2 and MCR-3 genes by monoplex PCR. Moreover, the Genetic environment of bla NDM, integrons analysis, and molecular characterization of plasmids was also performed. Antibiotic susceptibility revealed that all the isolates (100%) were resistant to ceftolozane/tazobactam, piperacillin/tazobactam, 96% resistant to ceftazidime, trimethoprim/sulfamethoxazole, 92% resistant to meropenem, gentamicin and cefepime, 88% resistant to ciprofloxacin, imipenem, and 37% resistant to amikacin. Ceftazidime/avibactam showed the least resistance (12%). 75% (n=12/16) were resistant to colistin and 44% (n=7/16) showed intermediate susceptibility to tigecycline. The detection of resistant determinants showed that the majority (95.8%) of CRKP harbored bla NDM-1, followed by bla OXA-48 (91.6%) bla OXA-51 (45.8%), and bla OXA-23 (41.6%). Sequencing of the bla NDM amplicons revealed the presence of bla NDM-1. Alarmingly, 100% of isolates showed the presence of qnrS. These predominant genes were distributed in various combinations wherein the majority were bla NDM-1 + bla OXA-51+ qnrS + bla OXA-48 (n =10, 41.7%), bla NDM-1 + bla OXA-23+ qnrS + bla OXA-48 (n=8, 33.3%), among others. In conclusion, the resistance rate to most antibiotics is very high in our region, including colistin and tigecycline, and the genetic environment of CRKP is complex with the carriage of multiple resistance markers. Resistance to ceftazidime/avibactam is uncommon and hence can be used as a valuable option for empirical therapy. Molecular data on resistance markers and the genetic environment of CRKP is lacking from this geographical region; this would be the first report addressing the subject matter. Surveillance and strict infection control strategies should be reinforced in clinical settings to curb the emergence and spread of such isolates.

Perception of online and face to face microbiology laboratory sessions among medical students and faculty at Arabian Gulf University: a mixed method study.

BACKGROUND: The COVID-19 pandemic has impacted all spheres of society including medical education and healthcare systems. In response to the pandemic, there has been a transition in medical education practice from traditional forms of teaching to online instruction delivery and virtual learning. Effective clinical microbiology education involves a combination of 'hands-on' practical learning and instructional delivery of scientific knowledge. Microbiology practical laboratories are critical learning environments offering 'hands-on' learning experiences that cannot be replicated through online learning. We conducted a mixed-methods study to understand the perception of online and face-to-face microbiology laboratory sessions among the medical students and microbiology faculty at Arabian Gulf University (AGU). METHODS: The study participants were third and fourth-year undergraduate medical students and faculty involved in delivering microbiology labs at AGU. The questionnaire consisted of questions ranging from perceived learning style to attitude towards online delivery of microbiology curriculum. After the questionnaire administration (google form), focus group discussion (FGD) was conducted for students and microbiology faculty separately. RESULTS: Among 168 students, 50.6% preferred face-to-face lab sessions as compared to 30.4% who preferred online labs, and 51.8% considered online labs to be an essential addition to face-to-face labs. Among the faculty, 85.7% preferred the face-to-face mode of teaching. All the faculty (100%) disagreed that all the microbiology labs teaching should be online. 57.2% considered online labs to be an essential addition to traditional face-to-face labs. Both faculty and students hold that a blended mode of instructional delivery is vital and indispensable for the transfer of skills and knowledge for microbiology students. CONCLUSION: The blended mode of delivering microbiology laboratory sessions in medical school is successful and well-received by both students and faculty. Students take the responsibility for furthering their own learning and understanding of concepts. Instructors have also noticed that blending learning strategies also successfully enhances the development of cognitive skills and problem-solving abilities in students. A review of the microbiology lab curriculum is necessary to identify content areas that can be delivered effectively through online, face-to-face lab sessions, or both, supported with appropriate tools and infrastructure.

Dysregulation of human beta-defensin-3 expression in the peripheral blood of patients with sepsis.

OBJECTIVES: Sepsis is a serious medical condition caused by the body's systemic inflammatory response to infections. The antimicrobial peptides, human beta-defensins, play a key role in modulating host immune responses, and aberrant expression of human beta-defensins has been implicated in many infections and inflammatory diseases. However, little is known about the expression of human beta-defensin-3 in systemic infectious diseases. METHODS: We investigated the gene expression and protein level of human beta-defensin-3 in peripheral whole blood from 107 participants-67 patients with sepsis and 40 healthy controls-and evaluated the feasibility of human beta-defensin-3 as an indicator for sepsis. Total RNA was extracted from peripheral blood samples, and relative mRNA expression of human beta-defensin-3 was determined by reverse transcription-quantitative polymerase chain reaction. Plasma concentration of human beta-defensin-3 was measured by enzyme-linked immunosorbent assay. Pearson's correlation analysis was performed to assess the relationship between human beta-defensin-3 mRNA and protein levels. Receiver operating characteristic analysis was performed to evaluate the value of human beta-defensin-3 as a biomarker for sepsis. RESULTS: Human beta-defensin-3 mRNA expression was significantly downregulated in sepsis patients compared to controls (p = 0.001). The mean fold change of mRNA expression (±standard error) was 0.82 ± 0.63 in sepsis patients and 1.39 ± 1.09 in controls. Plasma concentration of human beta-defensin-3 (pg/mL) was significantly lower in sepsis patients compared to healthy controls (p = 0.039). The mean protein concentration (±standard error) was 539.6 ± 39.4 in sepsis patients and 715.5 ± 53 in controls. There was a significant correlation between human beta-defensin-3 mRNA expression and the corresponding protein level in sepsis patients (r = 0.358, p = 0.04), but not in healthy controls (r = 0.124, p = 0.51). For discriminating sepsis patients from healthy controls, the area under the receiver operating characteristic curve was 0.722 (95% confidence interval: 0.597-0.847, p = 0.002) for human beta-defensin-3 mRNA and 0.689 (95% confidence interval: 0.557-0.827, p = 0.009) for human beta-defensin-3 protein. CONCLUSION: This is the first study to show the downregulation of human beta-defensin-3 gene expression and protein level in sepsis, which may contribute to the complex immunological imbalance in sepsis. The significant correlation between human beta-defensin-3 mRNA expression and protein concentration suggests that mRNA expression could be used to predict protein level. Our study also showed a potential role of human beta-defensin-3 as a blood-based biomarker for sepsis. More studies on the clinical significance of human beta-defensin-3 in sepsis could further support a biomarker development.

Student Perception of Microbiology Laboratory Skills Learning Through a Problem-Based Learning Curriculum: Arabian Gulf University Experience.

INTRODUCTION: The curriculum at medical school at Arabian Gulf University is centered on small group learning and real-life problems provided to students and guiding students to learn actively. In microbiology, laboratory skills are taught in an innovative manner using mini cases and different lab sessions and are integrated with other basic sciences. This article describes the format and pattern of laboratory skills sessions conducted using PBL methods at Arabian Gulf University and discusses the perception of students towards PBL in laboratory skill learning and way forward for the same. METHODS: The study sample size was 110. The students' perception of the laboratory skills teaching methods was assessed through an exit survey at the end of each session. A semi-structured self-administered survey instrument was prepared, and the questions were arranged in two sessions and focused on identifying the relevance, timing, strengths, and weaknesses of the teaching method and recommendations to improve the same. RESULTS: We observed that more than 50% of the participants agreed or strongly agreed that the time given for PBL was adequate, topics discussed were relevant, presentations were clear, pre-session briefing and Case-Based Studies (team-based learning (TBL)) helped in their learning. The participants identified the demonstration of experiments and hands-on experience provided in the laboratory were most helpful. When enquired about the difficulty, among 48% of the participants, 80% observed that the slides used in the learning/teaching were lengthy. CONCLUSION: The use of PBL in a lab setting promotes active learning. In the heart of PBL, TBL is a powerful tool in the educational process offering the students deep comprehension and allowing them to gain practical and intellectual skills.

Characterization of cephalosporin-resistant clinical Enterobacteriaceae for CTX-M ESBLs in Bahrain.

OBJECTIVE: To detect the presence of specific CTX-M class of extended spectyum ß-lactamases (ESBLs) in a collection of cephalosporin-resistant Enterobacteriaceae isolates from Bahrain. METHODS: A subset of 80 cephalosporin-resistant Enterobacteriaceae collected from Salmaniya Medical Complex, Bahrain, were characterized further for the presence of specific genogroups of CTX-M ß-lactamases by multiplex- and monoplex- PCRs. The primers used for the multiplex and monoplex PCRs were of genogroups- 1, 2, 8, 9 and 25. Sequencing of the representative isolates was performed to find the circulating CTX-M-types. RESULTS: A total of 93.8% (75/80) isolates showed the amplicons corresponding to any of the genogroups (1, 2, 8, 9, 25) and the remaining 6.2% isolates turned out negative in multiplex PCR. Some of the isolates demonstrated multiple bands corresponding to the sizes of different genogroups. Further confirmation with respective monoplex PCR on these 75 isolates demonstrated that 93.3% (70/75) harbored CTX-M genogroup-1 and 6.7% (5/75) harbored genogroup-9. We did not find the presence of genogroups 2, 8, and 25 in these isolates by monoplex PCR. Sequencing results of genogroup-1 isolates demonstrated the presence of CTX-M-15-like ESBL, however, discrepant results were noticed in genogroup-9 isolates, sequencing showed them as CTX-M-55-like ESBL. CONCLUSIONS: This is the first report from Bahrain characterizing the CTX-M genogroups of ESBLs and reporting the emergence of blaCTX-M-55-like gene in this region.

vacA genotypes in Helicobacter pylori strains isolated from patients with and without duodenal ulcer in Bahrain.

BACKGROUND: The vacuolating cytotoxin and the cytotoxinassociated protein, encoded by vacA and cagA, respectively, are important virulence determinants of Helicobacter pylori. OBJECTIVE: The aim of this study was to perform vacA genotyping and evaluate its association with cagA genotype and clinical outcome. METHODS: One hundred and twenty H. pylori strains were isolated from dyspeptic patients (29 with peptic ulcer, 91 with non-ulcer dyspepsia). Genotype was determined by PCR. RESULTS: Seventy-nine (66%) of 120 strains had the vacA signal sequence genotype s1 and 41 (34%) had the type s2. The vacA middle-region types m1 and m2 were detected in 56 (47%) and 64 (53%) strains, respectively. The combinations s1-m1 (n=56 [47%] and s2-m2 (41 [34%]) occurred more frequently than s1-m2 (23 [19.2%]; p=0.001). No strain with the combination s2-m1 was found. All patients with peptic ulcers harbored type s1 strains compared to 75 (82.4%) of 91 patients with non-ulcer dyspepsia (p=0.01). The vacA genotype s1 was associated with the presence of cagA (p <0.0001). The cagA gene was detectable in 38 (31.6%) of 120 isolates and present in all 29 patients with ulcer compared to nine of 91 with non-ulcer dyspepsia (p <0.001). CONCLUSION: Helicobacter pylori strains of vacA type s1 and the combination of s1-m1 were associated with peptic ulceration and the presence of cagA gene.

Campylobacter jejuni induces diverse kinetics and profiles of cytokine genes in INT-407 cells.

OBJECTIVE: To examine the kinetic ability of embryonic human epithelial INT-407 cells to express messenger ribonucleic acid mRNA for various cytokines and chemokines in response to Campylobacter jejuni C. jejuni stimulation. METHODS: In an experimental single-blind study, cultured embryonic human epithelial INT-407 cells were treated with different concentrations of viable C. jejuni, its sonicated, and filtered supernatant. A modified non-radioactive in situ hybridization using probe cocktails was used to measure mRNA levels for the pro-inflammatory cytokines interleukin IL-1beta, IL-6, interferon-gamma IFN-gamma, tumour necrosis factor TNF-alpha, transforming growth factor TGF-beta1, and IL-8, and the anti-inflammatory cytokines, IL-4 and IL-10. The study was carried out from September 2005 to March 2007 at the Department of Microbiology, Immunology, and Infectious Diseases, College of Medicine, Arabian Gulf University, Bahrain. RESULTS: Viable C. jejuni, sonicated bacteria and filtered supernatant induced high mRNA expression for the pro-inflammatory cytokines IL-1beta, IL-6, IFN-gamma, TNF-alpha, TGF-beta1, and IL-8, which peaked at the 12 hours post stimulation. Anti-inflammatory cytokines IL-4 and IL-10 mRNA expression were induced maximally at 3 hours post stimulation mainly by sonicated bacteria and filtrated supernatant, however, not with living bacteria. Untreated embryonic human epithelial INT-407 cells expressed low amount of mRNA for the various cytokines and chemokines at all time points. For each cytokine, 4 samples were used per time hour. CONCLUSION: This study demonstrated that embryonic human epithelial INT-407 cells in response to viable C. jejuni or its cytotoxins can alter cytokine and chemokine mRNA expression patterns and kinetics suggesting a potential role for theses mediators in the immunopathogenesis of the infection caused by this pathogen, which might be relevant for future immunotherapeutic interventions during severe bacterial infections.

Trends in antibiotic sensitivity pattern and molecular detection of tet(O)-mediated tetracycline resistance in campylobacter jejuni isolates from human and poultry sources.

This study was conducted to determine the trends in Campylobacter antibiotic resistance occurring in our setting and to assess the differences in the isolates using patterns of plasmid profiles. One hundred Campylobacter jejuni strains of human and poultry origin isolated in 2002-2003 (phase A) and 2005-2006 (phase B) in the Kingdom of Bahrain were evaluated. Susceptibility to erythromycin, ciprofloxacin and tetracycline was determined, and plasmid extraction and polymerase chain reaction detection of the tet(O) gene was carried out. A single erythromycin-resistant isolate was identified, in sharp contrast to the high ciprofloxacin resistance which also showed an increment in phase B. Tetracycline resistance was higher in chicken (80.9%) compared to human (41.3%) isolates (P<0.01). Most isolates harbored two plasmids (23 kb and 35 kb) with significant correlation between tetracycline resistance and plasmid carriage in chicken isolates. The findings show continued effectiveness of erythromycin for campylobacteriosis but an increasing trend of high ciprofloxacin and tetracycline resistance. Tetracycline resistance is most likely due to the transfer of plasmids carrying the tet(O) gene between isolates.

Multiplex PCR for direct identification of Campylobacter spp. in human and chicken stools.

Differentiation between Campylobacter jejuni and Campylobacter coli is problematic in clinical specimens due to fastidious growth requirements and limited biochemical tests. This study describes a rapid, multiplex PCR protocol for the direct detection and differentiation of C. jejuni and C. coli in stools. An evaluation was carried out of this multiplex protocol based on the detection of cadF (genus specific), and hipO (C. jejuni) and asp (C. coli) genes, using stool from patients with Campylobacter enteritis and chicken. Protocol sensitivity was assessed and specificity determined using a panel of enteric bacteria, and evaluation of 30 diarrhoeic stool specimens culture negative for Campylobacter. Of the 114 specimens (54 human and 60 chicken) evaluated by the protocol, 70 (61.4 %) were identified as C. jejuni, 35 (30.7 %) as C. coli and 9 (7.9 %) as a mixed infection/colonization with both species. All mixed infections were identified as C. jejuni by culture. Among the stool specimens that were culture negative for Campylobacter, two (6.7 %) were C. jejuni positive by multiplex PCR. The protocol sensitivity limit was 0.015-0.016 ng C. jejuni and C. coli DNA mul(-1) in the specimen. There was no cross-reaction with the reference strains assessed. Comparison of hippurate test and multiplex PCR demonstrated 17 isolates with false-positive hippurate enzymic activity and 7 with false-negative activity. This rapid protocol (turnaround time 6 h) is highly sensitive and specific for direct evaluation of stool for these pathogens. It has significant application for routine clinical diagnostic and epidemiological purposes.

Clinical relevance of virulence genes in Campylobacter jejuni isolates in Bahrain.

There are no data describing the genetic make-up of Campylobacter strains (an important aetiological agent of diarrhoea) circulating in the Arabian Gulf region. Here, the molecular characterization of two virulence genes in Campylobacter jejuni from Bahrain and the relationship with clinical infection are reported. Molecular screening for cytolethal distending toxin (cdtB) and invasion-associated marker (iam) genes was carried out on C. jejuni stool isolates collected from January 2002 to January 2004 in Bahrain. The molecular characterization was correlated with the patients' socio-demographic and clinical parameters. Of the 96 C. jejuni strains tested, 50 (52 %) were cdtB+/iam+, 30 (31 %) were cdtB+/iam- and 16 (17 %) were cdtB-/iam-. Sixty-nine per cent (66/96) of patients were less than 3 years old, with significantly higher detection of cdtB+/iam+ and cdtB+/iam- strains (P < 0.001 and P < 0.01, respectively) in this age group. Seventy patients (73 %) were symptomatic. In the group that were less than 3 years old, 62 and 85 % of those with cdtB+/iam+ and cdtB+/iam- strains, respectively, were symptomatic compared with 100 % for those over 3 years of age. However, the presence of cdtB-/iam- strains still resulted in clinical infection in the children under 3 years but not in the older patients. This is the first report describing the molecular characterization of virulence genes in Campylobacter isolates from this region. The findings indicate that strains of different virulence genetic make-up are circulating in the population, with children under the age of 3 years being most vulnerable. Further work on the molecular characterization, gene expression and determination of the invasive phenotypes of C. jejuni strains circulating in different regions is needed.

Human papillomavirus infection among women attending health facilities in the Kingdom of Bahrain.

OBJECTIVE: To investigate the occurrence of human papillomavirus (HPV) infection and the associated risk factors in Bahrain's female population. METHODS: This study was carried out between March to December 2004, which includes cervical scrapings for Pap smear and HPV-DNA testing using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis, obtained from 100 women attending the Gynecology Clinic at Salmaniya Medical Center and Sheikh Sabah Health Center in the Kingdom of Bahrain. We distributed questionnaires that include the sociodemographic data as well as information on risk factors such as smoking, parity, and the contraceptive used. RESULTS: Eleven women (11%) with normal cytology were HPV-positive. The RFLP analysis detected HPV-types 16, 18, 45, 62 and 53. Positive women were significantly older (43.3 +/- 10.1 years) than negatives (36.5 +/- 9.9 years; p=0.04), however, there was no difference in age of first sexual contact (positive: 18.1 +/- 5.7 years versus negative: 20.6 +/- 4.4 years). Polygamy, smoking and hormonal contraception was not identified as risk factors, but positive women showed higher parity. CONCLUSION: In this study on HPV infection in Bahrain, the 11% positivity with high risk HPV types, in the presence of normal cytology suggests that in addition to the cervical cancer screening program, offer of HPV testing deserves consideration.

Effect of antibiotic sub inhibitory concentration on cytolethal distending toxin production by Campylobacter jejuni.

OBJECTIVES: To investigate the effect of pre-exposure antibiotics on cytolethal distending toxin (CDT) production and toxigenic effect of C. jejuni. METHODS: Sonicates and filtrates were prepared from known cdt+ and cdt- isolates of C. jejuni which had been pre-exposed to varying concentrations (MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC) of erythromycin and ciprofloxacin. The CDT toxigenic effect was examined using INT 407 and HeLa cells. RESULTS: A trend of increased toxigenic effect was observed with pre-exposure to antibiotics. This was more pronounced with erythromycin pre-exposure compared to ciprofloxacin. Although a trend of increasing toxigenic effect with decreasing antibiotic concentration was demonstrable, some differences were observed between isolates. In one isolate the increased toxigenic effect was statistically significant (P<0.05) at 1/4 MIC in INT 407 cells and at 1/8 MIC in HeLa cells. CONCLUSIONS: This study provides evidence of an association between CDT production by C. jejuni and pre-exposure to antibiotics. Pre-exposure to ciprofloxacin and erythromycin at concentrations below MICs could potentiate CDT activity. Further work is needed to elucidate the mechanism involved. We recommend that these antibiotics be used in the treatment of C. jejuni enteritis only when strongly indicated and with careful monitoring of patients.

Campylobacter-stimulated INT407 cells produce dissociated cytokine profiles.

OBJECTIVES: To study the action of factors produced by living Campylobacter jejuni (C. jejuni) against those present within sonicated and filtrated bacteria on induction of potential cytokines by the human intestinal cell line INT407. METHODS: We used immunohistochemical technique modified to detect intracellular production of cytokines protein and RT-PCR to read RNA messages for evaluation of de novo cytokine synthesis. RESULTS: The data herein display dissociation of cytokine profiles induced on by living C. jejuni. Exposure of INT407 cells to 10(6) live bacteria showed the highest numbers of cytokine producing cells of all examined cytokines. IFN-gamma was the highest induced cytokine followed by IL-10, TNF-alpha and lastly IL-4. Also, abrogation of induction of the proinflammatory cytokines IFN-gamma and TNF-alpha but not the antiinflammatory cytokines IL-4 and IL-10 by sonicated and filtrated bacteria was depicted. At the mRNA level, TNF-alpha signals were noted in accordance with its protein levels since increased TNF-alpha mRNA signals were registered only after stimulation with living bacteria. Very low or no induction of TNF-alpha was registered with non-stimulated cells. CONCLUSIONS: These results illustrate for the first time a role for factors from living bacteria in directing the immune response towards Th1 type. Characterization of such factors may be essential for future immunotherapeutic interventions during severe bacterial infections.