High-Level Mupirocin Resistance in Staphylococcus spp. among Health Care Workers in a Tertiary Care Hospital.

BACKGROUND: Nasal carriage of Staphylococcus aureus is very common among health care workers, and treatment with mupirocin is one of the choicest antibiotics available. But with the rampant usage of mupirocin like other antibiotics, the emergence of mupirocin resistance is also on rise. This resistance is both low level as well as high level among the isolated strains. AIM: To screen for the high-level mupirocin resistance among the isolated Staphylococcus strains by Kirby Bauer disk diffusion method. MATERIALS AND METHODS: A total of 200 clinical isolates were tested for high level mupirocin resistance by disk diffusion method using Himedia disks. RESULTS: Among the 200 nasal swabs, 26 (13%) showed growth of S. aureus, whereas 174 (87%) showed the growth of coagulase negative staphylococcus (CONS) spp. Mupirocin resistance was observed only among CONS spp, which was 15% for low-level mupirocin and 8% for high-level mupirocin resistance. No mupirocin resistance was observed among the Staphylococcus spp. CONCLUSION: The identification of Mupirocin resistance will guide us to utilize the antibiotic in a judicious way to treat the nasal carriage effectively.

Bacteriological profile and perception on hand hygiene in school-going Children.

BACKGROUND: Handwashing is the most important daily activity to keep microbial infections at a distance. Schoolchildren tend to acquire most of the infections by not following the protocol of frequent handwashing which leads to frequent illnesses and absenteeism from school on a regular basis. MATERIALS AND METHODS: A cross-sectional study was conducted by means of collecting hand swabs from 133 schoolchildren to estimate the extent of germs present. Furthermore, student's perception on hand hygiene was assessed by means of questionnaire. RESULTS: Among the schoolchildren, majority (68.4%) of them felt washing hands is important. Almost 56.4% of students washed their hands before eating lunch, but only 64.7% of them used soaps for cleaning their hands. Furthermore, hand swabs of 133 schoolchildren showed the growth of potential pathogens such as Staphylococcus aureus, Escherichia coli, Klebsiella spp., and Enterococcus faecalis. CONCLUSIONS: Hands of schoolchildren were found to be contaminated and measures to inculcate the habit of frequent handwashing with soap are essential.

Comparative Study on the Vaginal Flora and Incidence of Asymptomatic Vaginosis among Healthy Women and in Women with Infertility Problems of Reproductive Age.

INTRODUCTION: The normal vaginal flora is highly complex, dominated by lactobacilli of doderlein that plays a vital role in maintaining the women's health and inhibits other pathogenic microorganisms. Fluctuation in local environment or exposure to any exogenous and endogenous sources changes the vaginal flora over a period of time. Disruption of the vaginal ecosystem changes the microflora of the healthy vagina, altering the pH and predisposing to lower reproductive tract infections. The change in the microflora of the female genital tract by pathogenic organisms may ascend from vagina to upper genital tract and may cause infertility. Although several studies demonstrate a higher prevalence of bacterial vaginosis in infertile population. The role of vaginal microbiome in infertility is not clear and need to be explored further. AIM: To compare the vaginal flora and analyse the incidence of asymptomatic vaginosis among healthy women and in women with infertility problems. MATERIALS AND METHODS: A cross-sectional study was conducted over a period of six months at Sri Lakshmi Narayana Medical College and Hospital Puducherry, India. A total of 200 high vaginal swabs were collected from Group 1 which included 84 healthy women with regular menstrual cycles without any gynaecological disorder and from Group 2, 116 women with infertility problems attending fertility clinic within the age group of 18 to 45 years. All swabs were subjected to routine aerobic, anaerobic and fungal culture. Saline wet mount was performed for the detection of clue cells and Trichomonas vaginalis, 10% KOH was performed for demonstration of budding yeast cells and pseudo hyphae, Gram's staining to determine the presence of yeast cells, leucocytes and bacterial morphotypes. The smear was also graded using Nugent scoring system. RESULTS: The vaginal flora of Group 1 was dominated by Lactobacillus (40, 27.8 %) followed by Micrococcus (22, 15.3 %), Enterococcus (16, 11.1%), Coagulase negative Staphylococcus spp. (12, 8.3%). Whereas in Group 2, the most dominant flora was Candida spp. (30, 26.5 %), Enterococcus (26, 23%) followed by Gram negative bacilli such as E. coli (16, 14.1 %). The percentage of Lactobacillus in Group 2 women with infertility problems was relatively low (4, 3.5%). Asymptomatic vaginosis was present in 32 (27.6 %) of Group 2 women compared to Group 1 women were only 6 (7.1%) had asymptomatic vaginosis. CONCLUSION: Women with infertility problems showed higher prevalence of asymptomatic vaginosis and abundance of Bacterial Vaginosis (BV) associated bacteria compared to healthy women. Hence, this study recommends the screening of vaginal flora as a routine for all women, especially in women undergoing infertility treatment and also suggests the importance of vaginal culture and sensitivity in routine practice.

A study on knowledge, attitude and practice regarding voluntary blood donation among medical students in Puducherry, India.

Knowledge, attitude and practice studies have been used to understand the various factors that influence blood donation which is the basis for donor mobilization and retention strategies. Role of youngsters in voluntary blood donation is crucial to meet the demand of safe blood. The present study was aimed to assess the level of knowledge, attitude and practice regarding voluntary blood donation among the health care students. A validated and pre-tested questionnaire on knowledge, attitude and practice on blood donation were assessed among 371 medical students from Sri Lakshmi Narayana Institute of Medical Sciences and Research Institute, Puducherry, India. Result showed that knowledge on blood donation among respondents was 44.8% (1st year 36.7%, 2nd year 42.8% and 3rd year 54.9%). About 62.6% of non-donors (1st year 51%, 2nd year 61% and 3rd year 77%) showed positive attitude by expressing their willingness to donate blood while 22.8%.of the non-donors had negative attitude (1st year 33%, 2nd year 23% and 3rd year 13%). In practice 13.2% of students had donated blood (1st year 10%, 2nd year 13% and 3rd year 24%), in which 2.7% of male students alone donating blood on regular basis. Over all 3rd year student showed significantly higher knowledge compared with 1st years, in attitude and practice section 3rd year student's showed significantly higher positive attitude and practice than that of 1st and 2nd years. The present study reveals that there is a positive association among knowledge, attitude and practice on blood donation, which suggest that positive attitude and practice can be improved by inculcating knowledge on blood donation among college students to recruit and donate blood regularly, which will help to achieve 100% of blood donation on voluntary basis.

Morphometry examination of placenta in birth weight of full-term newborns in Puducherry, India.

irth weight is an important determinant of child survival, healthy growth and development. Low birth weight is a well-established risk factor for adverse long term health, particularly cardiovascular disease and metabolic syndrome. The ability of the fetus to grow and thrive in utero is presumed to be a function of the placenta. The present study was aimed to assess the morphometry examination of placenta in normal and low birth weight babies in the Union territory of Puducherry. Morphometry examination includes Placenta weight, number of cotyledons, maternal and fetal surface area and site of umbilical cord insertion were measured in normal and low birth weigh babies. Result showed among 200 subjects, mean birth weight of normal and low birth babies were 2806 and 2058 g, respectively. The prevalence rate of low birth babies (less than 2500 g) was 22%. The placental morphometry study namely placental weight, number of cotyledons, maternal and fetal surface area and insertion of umbilical cord at centre were significantly (p<0.001) reduced in the low birth weight babies when compared with normal birth weight babies. Study revealed that morphometry analysis of placenta significantly influences the birth weight of new born. In conclusion, study recommends the early measurements of placenta by non-invasive techniques like ultrasonography will be helpful in early prediction of low birth weight fetus in utero itself and for better management to avoid such low birth weight.

Evaluation of the growth inhibitory activities of Triphala against common bacterial isolates from HIV infected patients.

The isolation of microbial agents less susceptible to regular antibiotics and the rising trend in the recovery rates of resistant bacteria highlights the need for newer alternative principles. Triphala has been used in traditional medicine practice against certain diseases such as jaundice, fever, cough, eye diseases etc. In the present study phytochemical (phenolic, flavonoid and carotenoid) and antibacterial activities of aqueous and ethanol extracts of Triphala and its individual components (Terminalia chebula, Terminalia belerica and Emblica officinalis) were tested against certain bacterial isolates (Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei, S. flexneri, Staphylococcus aureus, Vibrio cholerae, Salmonella paratyphi-B, Escherichia coli, Enterococcus faecalis, Salmonella typhi) obtained from HIV infected patients using Kirby-Bauer's disk diffusion and minimum inhibitory concentration (MIC) methods. T. chebula was found to possess high phytochemical content followed by T. belerica and E. officinalis in both aqueous and ethanol extracts. Further, most of the bacterial isolates were inhibited by the ethanol and aqueous extracts of T. chebula followed by T. belerica and E. officinalis by both disk diffusion and MIC methods. The present study revealed that both individual and combined aqueous and ethanol extracts of Triphala have antibacterial activity against the bacterial isolates tested.

Effect of methanol intoxication on specific immune functions of albino rats.

Our previous studies revealed that methanol intoxication significantly altered the non-specific immune functions in albino rats. The present investigation focuses on the effect of methanol on certain specific immune functions of cell mediated immunity such as footpad thickness, leukocyte migration inhibition test (LMI) and antibody levels. In addition, serum interleukins (IL-2, IL-4, TNF-alpha and IFN-gamma), and splenic lymphocyte subsets were measured after an immune challenge. The specific immune function tests were carried out in three different groups of albino rats, which include control, 15 and 30 days methanol intoxication. Our study reports that animal body weight, organ weight ratio, lymphoid cell counts, footpad thickness, antibody titer, IL-2, TNF-alpha, IFN-gamma, Pan T cell, CD4, macrophages, MHC class II molecule expression, and B cell counts were significantly decreased compared to control animals nevertheless, LMI, IL-4, and DNA single strand breakage were increased significantly. Plasma corticosterone level was significantly increased in the 15 days group whereas the 30 days methanol intoxication group showed considerable decrease in corticosterone level compared with control animals. Therefore, our investigation concluded that repeated exposure of methanol profoundly suppressed the cell mediated and humoral immune functions in albino rats.

Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa biofilms are intrinsically resistant to antimicrobial chemotherapies. At present, very little is known about the physiological changes that occur during the transition from the planktonic to biofilm mode of growth. The resistance of P. aeruginosa biofilms to numerous antimicrobial agents that are substrates subject to active efflux from planktonic cells suggests that efflux pumps may substantially contribute to the innate resistance of biofilms. In this study, we investigated the expression of genes associated with two multidrug resistance (MDR) efflux pumps, MexAB-OprM and MexCD-OprJ, throughout the course of biofilm development. Using fusions to gfp, we were able to analyze spatial and temporal expression of mexA and mexC in the developing biofilm. Remarkably, expression of mexAB-oprM and mexCD-oprJ was not upregulated but rather decreased over time in the developing biofilm. Northern blot analysis confirmed that these pumps were not hyperexpressed in the biofilm. Furthermore, spatial differences in mexAB-oprM and mexCD-oprJ expression were observed, with maximal activity occurring at the biofilm substratum. Using a series of MDR mutants, we assessed the contribution of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps to P. aeruginosa biofilm resistance. These analyses led to the surprising discovery that the four characterized efflux pumps do not play a role in the antibiotic-resistant phenotype of P. aeruginosa biofilms.

Multidrug efflux in Pseudomonas aeruginosa: components, mechanisms and clinical significance.

Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an intrinsic resistance to multiple antimicrobial agents and the ability to develop high-level (acquired) multidrug resistance during antibiotic therapy. Much of this resistance is promoted by highly homologous three-component efflux systems of broad substrate specificity, of which four have been identified to date. These include MexA-Mexs-OprM and MexX-MexY-OprM, which are expressed constitutively in wild type cells and, thus, provide for intrinsic multidrug resistance, and MexC-MexD-OprJ and MexE-MexF-OprN, whose expression so far has only been seen in acquired multidrug resistant mutant strains. Additional homologues of these efflux systems are identifiable in the recently released genome sequence, though their roles, if any, in antimicrobial efflux are unknown. These tripartite pumps are composed of an integral cytoplasmic membrane drug-proton antiporter of the resistance-nodulation-cell division (RND) family of exporters, a channel-forming outer membrane efflux protein (or outer membrane factor [OMF]) and a periplasmic membrane fusion protein (MFP) that links the other two. In addition to a number of antimicrobials of clinical significance, these pumps also export dyes, detergents, disinfectants, organic solvents and acylated homoserine lactones involved in quorum-sensing. While the natural functional of these pumps remains undefined, the fact that they contribute to antimicrobial resistance in P. aeruginosa makes them reasonable targets for therapeutic intervention.

Assessing the activity of bacterial multidrug efflux pumps.

Resistance to antibiotics in target bacterial populations has long complicated antibacterial chemotherapy. First described in the early 1980s (1, 2), efflux mechanism of resistance, whereby the antibiotic is actively (i.e., in an energy-dependent fashion) pumped from the bacterial cell, are being described with increasing frequency in recent years. Initial examples of bacterial antibiotic efflux systems were agent-specific, providing for export of and resistance to single agents (e.g., tetracyclines, chloramphenicol, macrolides) (3). More recently, bacterial drug efflux systems of broad substrate specificity have been described (4, 5). These systems are able to accommodate a wide variety of structurally unrelated antibiotics, contributing to intrinsic and acquired multiple antibiotic (multidrug) resistance.

Influence of mutations in the mexR repressor gene on expression of the MexA-MexB-oprM multidrug efflux system of Pseudomonas aeruginosa.

Several nalB-type multidrug-resistant mutants of Pseudomonas aeruginosa overexpressed MexAB-OprM and carried mutations in the local regulatory gene, mexR. Others, dubbed nalC types, carried mutations elsewhere and overexpressed MexAB-OprM less extensively than the nalB strains. Available evidence showed that MexR acted solely as repressor. Disruption of the mexR gene at various places suggested that the 5' end of mexR may be a part of the mexAB-oprM promoter.

Contribution of the MexAB-OprM multidrug efflux system to the beta-lactam resistance of penicillin-binding protein and beta-lactamase-derepressed mutants of Pseudomonas aeruginosa.

Deletion of the mexAB-oprM multidrug efflux operon substantially compromised the beta-lactam resistance of beta-lactamase-derepressed mutants of Pseudomonas aeruginosa, although it had only a modest impact on resistance of a penicillin-binding protein mutant. This highlights the multifactorial nature of beta-lactam resistance in this organism. Moreover, the contribution of efflux to the net resistance seen in some beta-lactam-resistant mutants suggests that inhibition of MexAB-OprM-mediated drug efflux might be an effective approach to overcoming beta-lactam resistance attributed to efflux as well as to other mechanisms of beta-lactam resistance.

Influence of the MexAB-OprM multidrug efflux system on quorum sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa nalB mutants which hyperexpress the MexAB-OprM multidrug efflux system produce reduced levels of several extracellular virulence factors known to be regulated by quorum sensing. Such mutants also produce less acylated homoserine lactone autoinducer PAI-1, consistent with an observed reduction in lasI expression. These data suggest that PAI-1 is a substrate for MexAB-OprM, and its resulting exclusion from cells hyperexpressing MexAB-OprM limits PAI-1-dependent activation of lasI and the virulence genes.

Influence of the TonB energy-coupling protein on efflux-mediated multidrug resistance in Pseudomonas aeruginosa.

TonB couples the energized state of the cytoplasmic membrane to the operation of outer membrane receptors responsible for Fe(III) siderophore uptake across the outer membrane of gram-negative bacteria. A tonB mutant of Pseudomonas aeruginosa deficient in iron siderophore uptake was shown in the present study to be hypersusceptible to a wide variety of antibiotics, reminiscent of the phenotype of mutants defective in the mexAB-oprM antibiotic efflux operon. This was not related to influences of a tonB mutation on the iron status of the cell, and indeed, intrinsic antibiotic susceptibility and mexAB-oprM expression were unaffected by iron levels in the growth medium. The presence of tonB on a multicopy plasmid increased the level of resistance of a MexAB-OprM+ strain but not that of a MexAB-OprM- strain to a variety of antimicrobial agents. mexAB-oprM expression was not, however, altered in a tonB deletion mutant, indicating that any influence of TonB on MexAB-OprM-mediated multidrug resistance was at the level of pump activity. Consistent with this, drug accumulation assays revealed that the tonB deletion mutant exhibited decreased levels of drug efflux. Still, the multidrug resistance of a nalB strain was not wholly abrogated by a tonB mutation, indicating that it is likely not an essential component of the efflux apparatus. Similarly, elimination of tonB from an nfxB strain only partially compromised MexCD-OprJ-mediated multidrug resistance. Intriguingly, the drug susceptibility of a mexAB-oprM deletion strain was increased following deletion of tonB, suggesting that TonB may also influence antibiotic resistance mediated by determinants other than MexAB-OprM (and MexCD-OprJ). Thus, TonB plays an important role in both intrinsic and acquired antibiotic resistance in P. aeruginosa.

Contribution of outer membrane efflux protein OprM to antibiotic resistance in Pseudomonas aeruginosa independent of MexAB.

A Pseudomonas aeruginosa strain carrying an insertion of an omega Hg interposon in the mexB gene (mexB::omega Hg; strain K879) produced markedly reduced but still detectable levels of OprM, the product of the third gene of the mexAB-oprM multidrug efflux operon. By using a lacZ transcriptional fusion vector, promoter activity likely responsible for OprM expression in the mexB::omega Hg mutant was identified upstream of oprM. Introduction of the oprM gene, but not the mexAB genes, into a P. aeruginosa multidrug-susceptible delta mexAB-oprM mutant increased resistance to quinolones, cephalosporins, erythromycin, and tetracycline. A delta mexAB-oprM strain carrying the oprM gene accumulated markedly less antibiotic than the deletion strain without oprM. Antibiotic accumulation by the MexAB- OprM+ strain was markedly enhanced upon treatment of cells with the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that MexAB-independent OprM function likely involves an efflux process. Moreover, pretreatment of cells with CCCP prior to the accumulation assay abrogated any differences in accumulation levels between the MexAB- OprM+ and MexAB- OprM- strains, indicating that reduced drug accumulation by the OprM+ strain (in the absence of CCCP) cannot be due to OprM-mediated reduction in outer membrane permeability. It appears, therefore, the OprM can be expressed and function in a drug efflux capacity independent of MexAB.

Beta-lactamase inhibitors are substrates for the multidrug efflux pumps of Pseudomonas aeruginosa.

The MexAB-OprM multidrug efflux system exports a number of antimicrobial compounds, including beta-lactams. In an attempt to define more fully the range of antimicrobial compounds exported by this system, and, in particular, to determine whether beta-lactamase inhibitors were also accommodated by the MexAB-OprM pump, the influence of pump status (its presence or absence) on the intrinsic antibacterial activities of these compounds and on their abilities to enhance beta-lactam susceptibility in intact cells was assessed. MIC determinations clearly demonstrated that all three compounds tested, clavulanate, cloxacillin, and BRL42715, were accommodated by the pump. Moreover, by using beta-lactams which were readily hydrolyzed by the Pseudomonas aeruginosa class C chromosomal beta-lactamase, it was demonstrated that elimination of the mexAB-oprM-encoded efflux system greatly enhanced the abilities of cloxacillin and BRL42715 (but not clavulanate) to increase beta-lactam susceptibility. With beta-lactams which were poorly hydrolyzed, however, the inhibitors failed to enhance beta-lactam susceptibility in MexAB-OprM+ strains, although BRL42715 did enhance beta-lactam susceptibility in MexAB-OprM- strains, suggesting that even with poorly hydrolyzed beta-lactams this inhibitor was effective when it was not subjected to efflux. MexEF-OprN-overexpressing strains, but not MexCD-OprJ-overexpressing strains, also facilitated resistance to beta-lactamase inhibitors, indicating that these compounds are also substrates for the MexEF-OprN pump. These data indicate that an ability to inactivate MexAB-OprM (and like efflux systems in other bacteria) will markedly enhance the efficacies of beta-lactam-beta-lactamase inhibitor combinations in treating bacterial infections.

Expression of Pseudomonas aeruginosa multidrug efflux pumps MexA-MexB-OprM and MexC-MexD-OprJ in a multidrug-sensitive Escherichia coli strain.

The mexCD-oprJ and mexAB-oprM operons encode components of two distinct multidrug efflux pumps in Pseudomonas aeruginosa. To assess the contribution of individual components to antibiotic resistance and substrate specificity, these operons and their component genes were cloned and expressed in Escherichia coli. Western immunoblotting confirmed expression of the P. aeruginosa efflux pump components in E. coli strains expressing and deficient in the endogenous multidrug efflux system (AcrAB), although only the delta acrAB strain, KZM120, demonstrated increased resistance to antibiotics in the presence of the P. aeruginosa efflux genes. E. coli KZM120 expressing MexAB-OprM showed increased resistance to quinolones, chloramphenicol, erythromycin, azithromycin, sodium dodecyl sulfate (SDS), crystal violet, novobiocin, and, significantly, several beta-lactams, which is reminiscent of the operation of this pump in P. aeruginosa. This confirmed previous suggestions that MexAB-OprM provides a direct contribution to beta-lactam resistance via the efflux of this group of antibiotics. An increase in antibiotic resistance, however, was not observed when MexAB or OprM alone was expressed in KZM120. Thus, despite the fact that beta-lactams act within the periplasm, OprM alone is insufficient to provide resistance to these agents. E. coli KZM120 expressing MexCD-OprJ also showed increased resistance to quinolones, chloramphenicol, macrolides, SDS, and crystal violet, though not to most beta-lactams or novobiocin, again somewhat reminiscent of the antibiotic resistance profile of MexCD-OprJ-expressing strains of P. aeruginosa. Surprisingly, E. coli KZM120 expressing MexCD alone also showed an increase in resistance to these agents, while an OprJ-expressing KZM120 failed to demonstrate any increase in antibiotic resistance. MexCD-mediated resistance, however, was absent in a tolC mutant of KZM120, indicating that MexCD functions in KZM120 in conjunction with TolC, the previously identified outer membrane component of the AcrAB-TolC efflux system. These data confirm that a tripartite efflux pump is necessary for the efflux of all substrate antibiotics and that the P. aeruginosa multidrug efflux pumps are functional and retain their substrate specificity in E. coli.

Inner membrane efflux components are responsible for beta-lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa.

A major feature of the MexAB-OprM multidrug efflux pump which distinguishes it from the MexCD-OprJ and MexEF-OprN multidrug efflux systems in Pseudomonas aeruginosa is its ability to export a wide variety of beta-lactam antibiotics. Given the periplasmic location of their targets it is feasible that beta-lactams exit the cell via the outer membrane OprM without interaction with MexA and MexB, though the latter appear to be necessary for OprM function. To test this, chimeric MexAB-OprJ and MexCD-OprM efflux pumps were reconstituted in delta mexCD delta oprM and delta mexAB delta oprJ strains, respectively, and the influence of the exchange of outer membrane components on substrate (i.e., beta-lactam) specificity was assessed. Both chimeric pumps were active in antibiotic efflux, as evidenced by their contributions to resistance to a variety of antimicrobial agents, although there was no change in resistance profiles relative to the native pumps, indicating that OprM is not the determining factor for the beta-lactam specificity of MexAB-OprM. Thus, one or both of inner membrane-associated proteins MexA and MexB are responsible for drug recognition, including recognition of beta-lactams.

Porins of Haemophilus influenzae type b mutated in loop 3 and in loop 4.

Porin (341 amino acids; mass of 37,782 Da) in the outer membrane of Haemophilus influenzae type b (Hib) permits diffusion into the periplasm of small solutes up to a molecular mass of 1400 Da. Molecular modeling of Hib porin identified its structural similarities to OmpF of Escherichia coli and disclosed for Hib porin a shorter length of loop 3 and a longer length of loop 4. By site-directed mutagenesis of the porin gene ompP2, mutant porins were constructed to contain 6 or 12 amino acid deletions either in loop 3 or in surface-exposed loop 4. Wild type Hib porin and mutant porins were expressed in a nontypeable H. influenzae strain deleted for the ompP2 gene. The mutant porins were purified and reconstituted into planar bilayers, tested for channel formation and compared with wild type Hib porin. Mutant Haemophilus porin possessing a 6-amino acid deletion in loop 3 displayed a broad distribution of single channel conductance values, while deletion of 12 amino acids from the same loop destabilized the porin channel. By comparison, deletion of 6 or of 12 amino acids from loop 4 of Hib porin resulted in an increased single channel conductance (1.15 and 1.05 nanosiemens, respectively) compared with wild type Hib porin (0. 85 nanosiemens). The C3 epitope of the poliovirus VP1 capsid protein was inserted either into loop 3 or into loop 4 of Hib porin. By flow cytometry, the C3 epitope was detected as surface-exposed in strains expressing C3 insertion in loop 4; in strains expressing C3 insertion in loop 3, the epitope was inaccessible. We propose that loop 4 of Hib porin, although surface-accessible, is oriented toward the central axis of the pore and that deletions in this loop increase the single channel conductance by widening the pore entrance.

Purification and refolding of recombinant Haemophilus influenzae type b porin produced in Bacillus subtilis.

The major diffusion channel in the outer membrane of Haemophilus influenzae type b (Hib) is porin (341 amino acids; Mr 37 782). The Hib porin gene was cloned and overexpressed in Bacillus subtilis. Recombinant Hib porin (Bac porin), having aggregated into inclusion bodies, was purified under denaturing conditions and subsequently refolded. To compare Bac porin that is intrinsically devoid of lipooligosaccharides versus native Hib porin, the properties of Bac porin were assessed by the following four criteria: circular dichroism spectroscopy, channel formation in planar bilayers, resistance to trypsin digestion and formation of the conformational epitope recognized by an anti-Hib porin monoclonal antibody. We conclude that in the absence of lipooligosaccharides, Bac porin was refolded into a functional form which closely resembled the structure of Hib porin.