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Biomedical subjects

C A Ison

Publications and source records attributed to C A Ison.

At least 37 records · Page 2Linked to original sources
PubMed2001

Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in in vitro and in vivo systems.

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtC genes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously described H. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900-3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.

Animals↗
PubMed2000

A prospective social and molecular investigation of gonococcal transmission.

BACKGROUND: Gonorrhoea is a common infectious disease, poorly controlled despite effective treatments. Tracing chains of transmission is difficult, because sexual partners are commonly difficult or impossible to identify. We assess the use of gonococcal opa-typing in identifying transmission links not revealed through interview. METHODS: Epidemiological data and gonococcal isolates were collected prospectively from patients at two UK clinics in London and Sheffield. Social and epidemiological data were combined with molecular typing of gonococcal isolates by a new methodology based on the polymorphisms of the opa gene. FINDINGS: In London, interview data and opa-typing on samples from 215 cases showed a diverse population with few links. In Sheffield, interview data identified links between 51 (43%) of 120 cases, whereas opa-typing suggested a more connected population: 95 (79%) of cases had shared profiles. There was a highly significant correlation between the two distributions with epidemiological clusters appearing as a subset of the opa clusters. Two large opa clusters, of 18 and 43 cases, accounted for 50% of local cases of gonorrhoea. Discordance between epidemiological and opa-typing data was observed at highly connected points in the sexual network. INTERPRETATION: Opa-typing is a more powerful tool for epidemiological investigation of gonorrhoea transmission than earlier methods. Opa-typing can link infections that would otherwise remain unlinked, and may aid interventions to control endemic disease.

Adolescent↗
PubMed2000

A typing system for neisseria gonorrhoeae based on biotinylated oligonucleotide probes to PIB gene variable regions.

The porin proteins PIA and PIB of Neisseria gonorrhoeae are serotyping antigens for the serovar classification system and leading candidates for gonococcal vaccine development. Although serotyping has been a useful tool, this method can be insensitive to critical sequence changes in the por gene, including those in surface-exposed variable regions (VRs). A sensitive and specific typing system for N. gonorrhoeae has been developed that uses biotin-labeled oligonucleotide probes with chemiluminescence detection to type PIB gene VRs. The PIB VR types of geographically and temporally diverse gonococcal strains and sexual contact isolates were determined. por VR typing discriminated between most unrelated isolates and provided information about individual VR type that was not apparent from serovar designations. PIB VR typing avoids limited monoclonal antibody availability, interlaboratory variation, and the requirement for culture-based surveillance associated with gonococcal serotyping, and provides useful information about the molecular epidemiology of individual por gene VRs.

Amino Acid Sequence↗
PubMed2000

Rise in gonorrhoea in London, UK. London Gonococcal Working Group.

We report an alarming 34.9% overall increase in the number of gonococcal infections diagnosed during the same 3 month study period during 3 years. Between 1997 and 1998, an increase of 12.1% was detected, with a larger increase between 1998 and 1999, of 20.4%.

Adolescent↗
PubMed1999

Assessment of immune response to meningococcal disease: comparison of a whole-blood assay and the serum bactericidal assay.

A whole-blood assay (WBA), which assesses the complete bactericidal activity of blood, was compared with the serum bactericidal assay (SBA), which measures antibody and complement mediated cell lysis. Twenty children infected with serogroup B strains and 25 infected with serogroup C strains were studied 8-12 weeks after disease, and 29 healthy children were used as controls. The infecting strain (convalescent children only) and two reference strains, MC58 (B:15:P1.7, 16) and NCTC 8554 (C:NT:P1.5) were used. In children previously infected with a serogroup B strain, bactericidal activity was detected in 95% and 85% to their infecting strain by the WBA (>50% killing) and the SBA (s), respectively. Bactericidal activity to the reference serogroup B and C strain was detected by WBA in 70 and 75% of children, respectively, and the SBA in 45% and 20%. In contrast bactericidal activity was detected to both serogroup C strains in >80% of children previously infected with a serogroup C strain using either assay and in 48% (WBA) and 20% (SBA) to the reference serogroup B strain. Levels of bactericidal activity were detectable in fewer control children. Children convalescing from meningococcal disease develop an immune response to their infecting strain, detectable by both the WBA and SBA, which is independent of age. However, the WBA appears to be a more sensitive measure of bactericidal activity to heterologous strains than the SBA.

Adolescent↗
PubMed1999

Neutrophil response to Neisseria meningitidis: inhibition of adhesion molecule expression and phagocytosis by recombinant bactericidal/permeability-increasing protein (rBPI21).

Polymorphonuclear neutrophil (PMNL) activation enhances microbial clearance but also contributes to the vascular damage and multiorgan failure associated with severe meningococcal sepsis. By use of a whole blood model of meningococcal bacteremia, loss of PMNL L-selectin and up-regulation of CD11b was observed in response to Neisseria meningitidis serogroups B and C, which is followed by opsonophagocytosis. PMNL priming with either Escherichia coli lipopolysaccharide (LPS) or FMLP prior to meningococcal challenge resulted in enhancement of both PMNL L-selectin shedding (1.5- to 4-fold) and phagocytosis (2- to 3-fold). Blockade of meningococcal LPS lipid A with recombinant bactericidal/permeability-increasing protein (rBPI21) resulted in partial inhibition of the PMNL activation and phagocytosis response to N. meningitidis. The effect of rBPI21 was reversed by excess E. coli LPS or FMLP. It is proposed that PMNL priming by N. meningitidis results in an exaggerated activation and phagocytosis response to the organism.

Adult↗
PubMed1999

Identification of the znuA-encoded periplasmic zinc transport protein of Haemophilus ducreyi.

The znuA gene of Haemophilus ducreyi encodes a 32-kDa (mature) protein that has homology to both the ZnuA protein of Escherichia coli and the Pzp1 protein of H. influenzae; both of these latter proteins are members of a growing family of prokaryotic zinc transporters. Inactivation of the H. ducreyi 35000 znuA gene by insertional mutagenesis resulted in a mutant that grew more slowly than the wild-type parent strain in vitro unless ZnCl(2) was provided at a final concentration of 100 microM. Other cations tested did not restore growth of this H. ducreyi mutant to wild-type levels. The H. ducreyi ZnuA protein was localized to the periplasm, where it is believed to function as the binding component of a zinc transport system. Complementation of the znuA mutation with the wild-type H. ducreyi znuA gene provided in trans restored the ability of this H. ducreyi mutant to grow normally in the absence of exogenously added ZnCl2. The wild-type H. ducreyi znuA gene was also able to complement a H. influenzae pzp1 mutation. The H. ducreyi znuA isogenic mutant exhibited significantly decreased virulence (P = 0.0001) when tested in the temperature-dependent rabbit model for experimental chancroid. This decreased virulence was not observed when the znuA mutant was complemented with the wild-type H. ducreyi znuA gene provided in trans.

Amino Acid Sequence↗
PubMed1999

Evaluation of the discriminatory power of typing methods for Neisseria gonorrhoeae.

A panel of 18 strains of Neisseria gonorrhoeae, known to be temporally and geographically diverse, was used to evaluate a number of typing systems, including conventional auxotyping and serotyping and the molecular methods of arbitrarily primed PCR (AP-PCR), amplified ribosomal-DNA restriction analysis (ARDRA), opa typing, and pulsed-field gel electrophoresis (PFGE). The discriminatory power of the different typing methods were determined with a collection of 87 clinical isolates from commercial sex workers in Indonesia, and Simpson's index of diversity was calculated. Of the two traditional techniques, auxotyping and serotyping, the latter gives the highest discriminatory index (DI) (DI, 0.846). The combination of auxotyping and serotyping yields a high DI (DI, 0. 928). D11344- and D8635-primed PCR showed low DIs of 0.608 and 0.622, respectively, but a combination of the two primers had a DI of 0. 849. The combination of serotyping with D11344-primed or D8635-primed PCR resulted in DIs of 0.936 and 0.937, respectively. ARDRA revealed a low DI of 0.743 alone but a DI of 0.955 in combination with serotyping. PFGE using the restriction enzyme BglII and opa typing produced the highest discrimination (DIs, 0.997 and 0. 996, respectively) for isolates of N. gonorrhoeae.

Bacterial Typing Techniques↗
PubMed1999

Susceptibility of gonococci isolated in London to therapeutic antibiotics: establishment of a London surveillance programme. London Gonococcal Working Group.

OBJECTIVES: To establish the in vitro susceptibility of gonococci isolated in the London area to antibiotics in current therapeutic use and to establish a sentinel surveillance system for monitoring trends in antibiotic resistant gonorrhoea in London. METHODS: Isolates of Neisseria gonorrhoeae from consecutive patients attending genitourinary medicine clinics at 10 hospitals in the London area were collected over a 3 month period, May to July 1997. The susceptibility to penicillin, ciprofloxacin, tetracycline, and spectinomycin was determined for each isolate. Isolates exhibiting either plasmid or chromosomally mediated resistance were additionally tested for susceptibility to agents used as alternative treatments including azithromycin, ceftriazone, and ofloxacin. The resistant isolates were also tested for plasmid profiles (penicillinase producing N gonorrhoeae, PPNG), type of tetM determinant (tetracycline resistant N gonorrhoeae, TRNG), and presence of gyrA and parC mutations (quinolone resistant N gonorrhoeae, QRNG). RESULTS: A total of 1133 isolates were collected which represents > 95% of the total gonococci isolated in the 3 months. Plasmid mediated resistance was exhibited by 48 (4.2%) isolates; six (0.5%) were PPNG, 15 (1.3%) were PP/TRNG, and 27 (2.4%) were TRNG. The majority of PPNG (18 of 20 tested) carried the 3.2 MDa penicillinase plasmid whereas the two types of tetM determinant were more evenly distributed. High level resistance to ciprofloxacin was detected in four (0.4%) isolates and double mutations were found in the quinolone resistance determining region (QRDR) of the gyrA gene in three QRNG with MICs of 16 mg/l and a single mutation in one isolate with a MIC of 1 mg/l to ciprofloxacin. No parC mutations were found. Of the remaining 1081 isolates, 86 (8.0%) were chromosomally mediated resistant N gonorrhoeae (CMRNG). CONCLUSIONS: A unique collection of gonococcal isolates has been established which can be used as a baseline for surveillance of susceptibility to antibiotics and for epidemiological purposes.

Adolescent↗
PubMed1999

A comparison of the use of Papanicolaou-stained cervical cytological smears with Gram-stained vaginal smears for the diagnosis of bacterial vaginosis in early pregnancy.

Our objective is to compare the efficacy of using Papanicolaou (PAP)-stained cervical cytology smears with a standardized method of interpreting Gram-stained vaginal smears for the diagnosis of bacterial vaginosis (BV) in pregnancy. High vaginal smears were Gram-stained and examined by a single observer to characterize 3 grades of vaginal flora and diagnose BV. Cervical smears were PAP-stained and examined for characteristic patterns of vaginal flora including evidence of BV by either a number of cytotechnicians or a single cytopathologist. The results of the 2 methods were compared. Seven hundred and forty-seven women attending an antenatal clinic in a district general hospital who consented to have a smear of vaginal secretions and cervical cytology in early pregnancy. The main outcome measure is the diagnosis of BV by different methods in a pregnant population. Compared with the Gram-stain method for the diagnosis of BV, there was good agreement between PAP-stain interpretation by a single observer but the agreement was not as good with PAP-stain interpretation by multiple cytotechnicians. When the grades were consolidated to normal (grade I) and abnormal flora (grades II and III), compared to Gram-stained smears, PAP cytology undertaken by several cytotechnicians had a sensitivity of 80.7% and a specificity of 90.7%. The sensitivity and specificity increased to 87% and 97%, respectively, when the PAP-stained smears were read by a single cytopathologist. Using kappa scores, only those readings made by a single cytopathologist were reliable. The setting in a cytopathology laboratory comprises multiple cytotechnicians, so that PAP-stain analysis of vaginal smears for the diagnosis of BV is likely to provide results which are less reliable than those obtained by Gram staining. The latter should be the first choice and every effort should be made to set up this service.

Cervix Uteri↗
PubMed1998

Naturally occurring isolates of Neisseria gonorrhoeae, which display anomalous serovar properties, express PIA/PIB hybrid porins, deletions in PIB or novel PIA molecules.

The por gene of Neisseria gonorrhoeae encodes the Protein I porin responsible for serovar specificity. In this study the por genes have been sequenced from clinical isolates which exhibited anomalous serovar reactivity. One group of 'intermediate' strains differed significantly from both Protein IA and IB strains, were more closely related to IA but appeared to represent a distinct class of Protein I. Another strain was closely related to Protein IB of serovar IB-6 but contained a deletion of six amino acids in surface exposed loop 6 which removed epitopes recognized by IB specific monoclonal antibodies. The third group of strains, which reacted with both IA and IB specific monoclonal antibodies, expressed hybrid Protein I molecules containing both IA and IB epitopes. These strains appeared to originate from a double crossover between Proteins IA and IB with the amino and carboxy terminal residues homologous to IB while the surface exposed loop 6 demonstrated close homology to IA. This is the first demonstration of naturally occurring gonococci expressing a hybrid Protein IA/IB.

Amino Acid Sequence↗
PubMed1998

Activation of complement by mannose-binding lectin on isogenic mutants of Neisseria meningitidis serogroup B.

Mannose-binding lectin (MBL) is a serum protein that has been demonstrated to activate the classical complement pathway and to function directly as an opsonin. Although MBL deficiency is associated with a common opsonic defect and a predisposition to infection, the role of the protein in bacterial infection remains unclear. We have investigated MBL binding to Neisseria meningitidis serogroup B1940 and three isogenic mutants, and the subsequent activation of the two major isoforms of C4 (C4A and C4B) by an associated serine protease, MASP. The mutants lacked expression of the capsular polysaccharide (siaD-), the lipo-oligosaccharide (LOS) outer core that prevented LOS sialylation (cpsD-), or both capsule and LOS outer core (cps-). Using flow cytometry, it was possible to detect strong MBL binding to the cps- and cpsD- mutants over a wide range of concentrations. In contrast, minimal or no MBL binding was detected on the parent organism, with binding to siaD- only at higher MBL concentrations. C4 was activated and bound by mutants that had previously bound MBL/MASP, but there was no significant difference in the amounts of C4A and C4B bound. When sialic acid residues were removed from the parent organism by neuraminidase treatment, the binding of both MBL and C4 increased significantly. Our results suggest that MBL may bind to and activate complement on these encapsulated organisms, and the major determinants of these effects are the LOS structure and sialylation.

Bacterial Proteins↗
PubMed1998

Genotyping of Neisseria gonorrhoeae.

Genotyping of Neisseria gonorrhoeae has proved highly discriminatory. It has been used to differentiate between isolates of the same serovar, to determine the genetic relatedness of antibiotic resistant isolates and to identify isolates from sexual contacts. This level of resolution has been possible because Neisseria gonorrhoeae exhibits a high degree of genetic variability. The validity of using any typing technique to study temporal or geographical differences of an organism known to be non-clonal should, however, be carefully considered.

Journal Article↗
PubMed1998

Gonococcal resistance to beta-lactams and tetracycline involves mutation in loop 3 of the porin encoded at the penB locus.

penB is a chromosomal mutation that confers resistance to beta-lactams and tetracyclines and reduced susceptibility to quinolones in Neisseria gonorrhoeae. It is linked to the porin gene (por) and requires the increased expression of an efflux pump due to mtr. Transformation of a susceptible gonococcus (strain H1) with chromosomal DNA from strain FA140 (penA mtr penB; porin serovar IB1) and conjugal transfer of a beta-lactamase-expressing plasmid was used to produce isogenic strains for determination of equilibrium periplasmic penicillin concentrations by the method of Zimmermann and Rosselet (W. Zimmermann and A. Rosselet, Antimicrob. Agents Chemother. 12:368-372, 1977). In transformants with the Mtr and PenB phenotypes, equilibrium concentrations of penicillin were reduced. DNA sequence analysis of por from isogenic penB and penB+ transformants revealed 14 sequence differences; nine of these differences resulted in amino acid changes. Three amino acid changes were found in the putative gonococcal equivalent of the pore-constricting loop 3 of Escherichia coli OmpF. Two of these changes (Gly-101-Ala-102-->Asp-Asp) result in an increased negative charge at this position in por loop 3. PCR products comprising the complete por gene from strain FA140 were transformed into strain H1-2 (penA mtr; porin serovar IB-3), with the resulting transformants having the antibiotic susceptibility phenotype associated with penB. penB-like mutations were found in loop 3 of clinical isolates of gonococci with chromosomally mediated resistance to penicillin. We conclude that penB is a mutation in loop 3 of por that reduces porin permeability to hydrophilic antibiotics and plays an important role in the development of chromosomally mediated resistance to penicillin and tetracycline in gonococci.

Anti-Bacterial Agents↗
PubMed1998

Drift in susceptibility of Neisseria gonorrhoeae to ciprofloxacin and emergence of therapeutic failure.

Ciprofloxacin, 500 mg, was introduced as the first-line therapy for gonorrhea at St. Mary's Hospital, London, in 1989, when a surveillance program was initiated to detect the emergence of resistance. Isolates of Neisseria gonorrhoeae from consecutive patients attending the Jefferiss Wing, Genitourinary Medicine Clinic at St. Mary's Hospital, between 1989 and 1997 have been tested for susceptibility to ciprofloxacin by using an agar dilution breakpoint technique. Isolates considered potentially resistant (MIC, >0.12 microg/ml) were further characterized by determination of the MICs of ciprofloxacin, nalidixic acid, and penicillin, auxotyped and serotyped, and screened for mutations in the DNA gyrase gene, gyrA, and the topoisomerase IV gene, parC. A total of 4,875 isolates were tested. While the majority of isolates were highly susceptible (MIC, </=0.008 microg of ciprofloxacin/ml), there was a drift toward reduced susceptibility in N. gonorrhoeae isolated between 1993 and 1996 (P < 0.001). In 1997 this drift was reduced but remained above pre-1993 levels. Isolates from 18 patients were classed as potentially resistant (MIC, >0.12 microg/ml); all of these belonged to serogroup B, and NR/IB-1 was the most common auxotype/serovar class. The infections in 14 of the 18 patients were known to be acquired abroad, and 5 were known to result in therapeutic failure. The surveillance program has established that ciprofloxacin is still a highly effective antibiotic against N. gonorrhoeae in this population. However, it has identified a drift in susceptibility which may have resulted from increased usage of ciprofloxacin. High-level resistance has now emerged, although treatment failure is still uncommon.

Anti-Infective Agents↗
PubMed1998

GroEL heat shock protein of Haemophilus ducreyi: association with cell surface and capacity to bind to eukaryotic cells.

The Haemophilus ducreyi homolog of GroEL, a 58.5-kDa heat shock protein (Hsp), is a dominant protein produced not only in response to heat stress but also under in vitro growth conditions. Extracellular localization of the 58.5-kDa Hsp was investigated by whole-cell enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy and in supernatants of washed bacteria by immunoblotting with a Haemophilus ducreyi GroEL-specific mouse monoclonal antibody (BB11). To investigate binding of the Hsp to eukaryotic cells, the 58.5-kDa Hsp was purified by ion-exchange and size exclusion chromatography; incubated with HEp-2 cells, HeLa cells, and human fibroblasts; and then analyzed by immunoblotting. Direct involvement of the 58.5-kDa Hsp in the adherence of H. ducreyi to HEp-2 cells was investigated by using an inhibition assay. An epitope of the 58.5-kDa Hsp was detected by whole-cell ELISA on all of the strains tested, suggesting that it is associated with the cell surface. This was also supported by immunoelectron microscopy results. In supernatants of washed bacteria, the 58.5-kDa Hsp was detected by immunoblotting after 10 h of cultivation. The 58.5-kDa Hsp bound to the eukaryotic cells tested but exerted only limited (about 20%) inhibition of H. ducreyi adherence to HEp-2 cells. These results demonstrate that the 58.5-kDa Hsp of H. ducreyi is associated with the bacterial surface, binds to eukaryotic cells, and partially influences H. ducreyi adherence to HEp-2 cells, indicating possible involvement of the 58.5-kDa Hsp in the attachment of bacteria to host cells and to each other.

Animals↗
PubMed1998

The role of effective diagnosis for the control of gonorrhoea in high prevalence populations.

Gonorrhoea in the UK is now a highly focal problem that is localized to certain inner city areas where it presents a significant public health concern. However, the majority of men and a proportion of women infested with Neisseria gonorrhoeae do not experience symptoms of sufficient intensity to prompt them to seek medical advice and the current strategy of treating symptomatic individuals in specialist clinics with subsequent tracing of sexual partners is therefore failing to control the spread of infection in these areas. One method for addressing this problem would be the extension of effective diagnostic services into the clinical environments most likely to be accessed by these high-risk individuals. This paper reviews the scientific literature examining the various methodologies available for the identification of N. gonorrhoeae and assesses their suitability for the diagnosis of gonorrhoea in these alternative clinical settings.

DNA Probes↗