Abstract: Previously we have shown that the open reading frame 2 protein (ORF2 protein), which is encoded at the 3 ' end of serine protease of Aeromonas sobria (ASP), functions as a chaperone protein in periplasm in the production of ASP. Both proteins, ASP and ORF2 protein, associate in periplasm and ORF2 protein helps ASP to take an active form. ASP which is dissociated from ORF2 protein emerges in milieu . In this study, we examined the effect of sodium chloride (NaCl) in medium on ASP production by A. sobria. The ASP activity of culture supernatant was extremely decreased when A. sobria was cultured in medium containing 3.0% NaCl (concentration almost equivalent to sea water salinity). Our analysis showed that the transcription of asp by A. sobria is not inhibited by NaCl in medium and that A. sobria synthesizes and releases ASP in milieu even under the condition of 3.0% NaCl. However, these ASPs in milieu formed complex as with ORF2 proteins. This indicates that the maturation pathway of ASP is disturbed in A. sobria cultured in medium containing 3.0% NaCl. It is likely that ASP does not associate with ORF2 protein in the correct form in periplasam when A. sobria is cultured in medium containing 3.0% NaCl, though both proteins, ASP and ORF2 protein, make complexes and emerge outside of the cell. This idea suggests that the chaperone system of ASP possesses the ability to sense NaCl in surroundings and regulates the production of active ASP.

Abstract: Aeromonas sobria causes septic shock, a condition associated with high mortality. To study the mechanism of septic shock by A. sobria infection, we examined the vascular leakage (VL) activity of A. sobria serine proteinase (ASP), a serine proteinase secreted by this pathogen. Proteolytically active ASP induced VL mainly in a bradykinin (BK) B(2) receptor-, and partially in a histamine-H(1) receptor-dependent manner in guinea pig skin. The ASP VL activity peaked at 10 min to 1.8-fold of the initial activity with an increased BK B(2) receptor dependency, and attenuated almost completely within 30 min. ASP produced VL activity from human plasma apparently through kallikrein/kinin system activation, suggesting that ASP can generate kinin in humans. Consistent with the finding that a major part of the ASP-induced VL was reduced by a potent kallikrein inhibitor, soybean trypsin inhibitor that does not affect ASP enzymatic activity, ASP activated prekallikrein but not factor XII to generate kallikrein in a dose- and incubation time-dependent manner. ASP produced more VL activity directly from human low m.w. kininogen than high m.w. kininogen when both were used at their normal plasma concentrations. Intra-arterial injection of ASP into guinea pigs lowered blood pressure specifically via the BK B(2) receptor. These data suggest that ASP induces VL through prekallikrein activation and direct kinin release from kininogens, which is a previously undescribed mechanism of A. sobria virulence and could be associated with the induction of septic shock by infection with this bacterium. ASP-specific inhibitors, and kinin receptor antagonists, might prove useful for the treatment or prevention of this fatal disease.

Abstract: Resistance of Helicobacter pylori to clarithromycin is mostly associated with point mutations, generally adenine-to-guanine transition at either position 2142 or 2143 or an adenine-to-cytosine transversion at position 2142 in domain V, the macrolide binding pocket, of 23S rRNA gene. Furthermore, many H. pylori isolates carry plasmid DNAs of extremely variable size but so far no drug resistance properties were mediated by H. pylori plasmids. Previously, we found a common plasmid (pRMOHp) in clarithromycin resistant (MIC, 32 to >256 microgram/ml) H. pylori isolates tested. In this study, five clarithromycin-resistant Helicobacter pylori isolates were analyzed for point mutations in the 23S rRNA gene. Sequence analysis of all of the resistant isolates revealed an A-to-G transition mutation at position 2143. However, transformation experiments showed that pRMOHp is not associated with clarithromycin resistance in H. pylori isolates tested. Thus, the mechanism of resistance to clarithromycin may be solely related to the point mutation in the 23S rRNA gene.

Abstract: Antibiotic resistance in Helicobacter pylori is a growing public health problem. Many H. pylori isolates carry plasmid DNAs of extremely variable size but so far no drug resistance properties were mediated by H. pylori plasmids. In this study, we investigate the primary antibiotic resistant rates of H. pylori to four different antibiotics. The frequency of plasmids as well as the relation between antibiotic resistance, genotypes, and plasmid carriage was also investigated. Primary clarithromycin, metronidazole, amoxicillin, and tetracycline susceptibility was tested against 149 H. pylori strains by E-test. Established procedures were employed for the isolation of plasmid DNAs from resistant isolates (n=25). We divided all resistant isolates in to three genotypes (I, II, and III) according to the ureB gene using PCR-RFLP. Overall primary resistance to clarithromycin and metronidazole was 14.7% and 5.3%, respectively. Dual clarithromycin and metronidazole resistance was present in 3.3% of the isolates. All strains were sensitive to amoxicillin and tetracycline. Of the 25 strains examined a common plasmid pRMOHp was found to be present in five (20%) of the resistant isolates. Interestingly all these five strains were resistant to clarithromycin only and belonged to the same genotype II. However, the unique pRMOHp was absent in clarithromycin-sensitive (n=54, tested) strains. In our geographical area, clarithromycin resistance is an emerging problem. Although, resistance of H. pylori to clarithromycin is mostly due to the point mutations in the 23S rRNA, but our data suggests a possibility that, there may be more than one mechanism of clarithromycin resistance in these isolates. The data of this study provide us with a basis for further characterization of the unique pRMOHp.

Abstract: Antimicrobial susceptibility of 120 Helicobacter pylori isolates to metronidazole, tetracycline, clarithromycin, and amoxicillin was determined, and 77.5, 15, 10, and 6.6% of the isolates, respectively, were resistant. Only rdxA inactivation and both rdxA and frxA inactivation were responsible for metronidazole resistance in 66% (8 of 12) and 33% (4 of 12) of the isolates, respectively.

Abstract: Twelve clarithromycin-resistant (MIC, > or = 1 microg/ml) Helicobacter pylori isolates were analyzed for point mutations in the 23S rRNA gene. Sequence analysis of all of the resistant isolates revealed a T-to-C transition mutation at position 2182. Transformation experiments confirmed that a single T-to-C transition mutation at position 2182 is associated with clarithromycin resistance.

Abstract: PCR surveillance of the rstR genes of CTX phages in Vibrio cholerae O1 and O139 showed no relationship between the incidence of disease and changes in the rstR but showed variations in their presence in O1 and O139 strains and the occurrence of multiple types in a few strains.

Abstract: Sixty-nine bacteriologically confirmed Streptococcus pneumonie strains were tested for antimicrobial susceptibility between 1998 and 1999. Of the 15 (21.7%) penicillin-resistant isolates, 2 (13.3%) showed complete resistance (MIC ≥ 2.0 microgram/ml) and 13 (86.7%) showed intermediate resistance (MIC, 0.12 to 1.0 microgram/ml). The overall percentages of all isolates resistant to ampicillin, chloramphenicol, erythromycin and co-trimoxazole were 5.8%, 7.2%, 10.1% and 84.1% respectively and 8.7% strains were multiple resistant. None of the strains were resistant to ceftriaxone. The study highlights that antimicrobial resistance has clearly emerged as a very serious problem with S. pneumoniae strains in Bangladesh. These findings suggest the need for routine screening of all pneumococcal isolates for penicillin resistance and re-evaluating indications for ampicillin and chloramphenicol use for the treatment of invasive pneumococcal disease.

Abstract: Shigella dysenteriae type 1 is the causative agent of the most severe form of bacillary dysentery, which occurs as epidemics in many developing countries. We isolated a bacteriophage from surface water samples from Bangladesh that specifically lyses strains of S. dysenteriae type 1. This phage, designated SF-9, belongs to the Podoviridae family and has a 41-kb double-stranded DNA genome. Further screening of water samples for the prevalence of the phage revealed 9 of 71 (12.6%) water samples which were positive for the phage. These water samples were also positive in PCR assays for one or more S. dysenteriae type 1-specific genes, including ipaBCD and stx1, and live S. dysenteriae type 1 was isolated from three phage-positive samples. The results of this study suggest that phage SF-9 may have epidemiological applications in tracing the presence of S. dysenteriae type 1 in environmental waters.

Abstract: Bacillary dysentery caused by Shigella species is a public health problem in developing countries including Bangladesh. Although, shigellae-contaminated food and drinks are often the source of the epidemic's spread, the possible presence of the pathogen and transmission of it through environmental waters have not been adequately examined. We analyzed surface waters collected in Dhaka, Bangladesh, for the presence of shigellae by a combination of PCR assays followed by concentration and culturing of PCR-positive samples. Analysis of 128 water samples by PCR assays for Shigella-specific virulence genes including ipaBCD, ipaH, and stx1 identified 14 (10.9%) samples which were positive for one or more of these virulence genes. Concentration of the PCR-positive samples by filtration followed by culturing identified live Shigella species in 11 of the 14 PCR-positive samples. Analysis of rRNA gene restriction patterns (ribotype) showed that the environmental isolates shared ribotypes with a collection of clinical isolates, but in contrast to the clinical isolates, 10 of the 11 environmental isolates were either negative or carried deletions in the plasmid-encoded invasion-associated genes ipaB, ipaC, and ipaD. However, all environmental Shigella isolates were positive for the chromosomal multicopy invasion-associated gene ipaH and all Shigella dysenteriae type 1 isolates were positive for the stx1 gene in addition to ipaH. This study demonstrated the presence of Shigella in the aquatic environment and dispersion of different virulence genes among these isolates which appear to constitute an environmental reservoir of Shigella-specific virulence genes. Since critical virulence genes in Shigella are carried by plasmids or mobile genetic elements, the environmental gene pool may contribute to an optimum combination of genes, causing the emergence of virulent Shigella strains which is facilitated in particular by close contact of the population with surface waters in Bangladesh.

Abstract: Data on the seroprevalence of antibodies protective against the varicella-zoster virus are needed to develop strategies to prevent varicella infections in Bangladesh. Of 1209 patients evaluated at referral-level health facilities in Dhaka, 943 (78%) had no known history of chickenpox and were tested by latex agglutination for the presence of varicella-zoster antibody in serum. Forty-one per cent (386) of the 943 specimens tested were negative. Seropositivity was highest among neonates (83%), declined sharply to 19% in those aged 7-12 months, and thereafter rose steadily with age until a plateau of 85% was reached after the age of 16 years. This first report of varicella-zoster antibody seroprevalence in Bangladesh suggests that, as in other tropical areas, a significant proportion of children, adolescents and adults are susceptible. Children aged from 15 months to early adolescence might be the most important group to target with the vaccine currently available. However, to ensure successful immunisation, further, population-based seroprevalence data are needed, as are an assessment of the vaccine's acceptability and the accessibility of the target population. Incomplete coverage of young children could result in delayed acquisition, and, ultimately, in more severe disease.

Abstract: Streptococcus pneumoniae (the pneumococcus) is a human pathogen that causes life-threatening, invasive diseases such as pneumonia, bacteremia, and meningitis with high morbidity and mortality throughout the world; young children and the elderly are particularly susceptible. In developing countries, an estimated five million children under the age if five years die each year from pneumonia, with S. pneumioniae being the single most common causative agent. In Bangladesh it is a common cause of pneumonia and the second leading cause of meningitis, with high mortality. Diagnosis of pneumococcal infection is difficult. In this study, surface protein antigens from predominant serotypes of S. pneumoniae in Bangladesh were analyzed in an attempt to establish whether they could be used as a diagnostic marker. For this, rabbits were immunized with the sonicated whole cell extract of the serotype 7F, and the sera were reacted with the surface materials extracted from different serotypes predominant in Bangladesh. The rabbit polyclonal antibody detected only one antigenic band of 74-kDa molecular weight from each of the surface materials of different serotypes. All these data confirm each other and strongly suggests that the pneumococcal 74-kDa protein antigen is a immunogenic protein and have common epitopes in different predominant serotypes of S. pneumoniae. Thus, this unique 74-kDa pneumococcal surface protein could be used as a diagnostic as well as an epidemiological marker.

Abstract: Streptococcus pneumoniae (the pneumococcus) is a human pathogen that causes life threatening, invasive diseases such as pneumonia, bcteremia, and meningitis with high morbidity and mortality throughout the world; young children and elderly are particularly susceptible. In developing countries, as estimated five million children under the age of five years die each year from pneumonia, with S. pneumoniae being the single most common causative agent. In Bangladesh it is a common cause of pneumonia and the second leading cause of meningitis, with high mortality. In this study we have tried to demonstrate the protective efficacy of the unique 74 kDa pneumococcal surface antigen against pneumococcal infection in mice. For this rabbits were immunized with the sonicated whole cell extract of the serotype 7F, and the sera were reacted with the surface materials extracted from different serotypes predominant in Bangladesh. The rabbit polyclonal antibody detected only one antigenic band of 74 kDa molecular weight from surface materials of different serotypes. Sera from 40 pneumonia or meningitis patients also recognized the common 74 kDa antigenic component from the surface materials of 7F and 12F. Whereas, this antigenic band was absent, when normal and healthy human sera were tested. All these data confirm each other and strongly suggests that the pneumococcal 74 kDa protein antigen is an immunogenic protein and have common epitopes in different serotypes. In the mice challenge experiments, eleven of the twelve mice, which were immunized with the 74 kDa protein, survived the live cell challenge. Whereas, all the control mice died within a period of 17 days. These mice challenge experiment clearly demonstrate strong protective activity of the 74 kDa protein in the immunized mice. The data of this study provide us with a basis for further characterization of the unique 74 kDa pneumococcal surface protein as an agent for diagnosis and immunotherapy against S. pneumoniae.