Biomedical subjects

Lucinda M C Hall

Publications and source records attributed to Lucinda M C Hall.

10 recordsLinked to original sources

PubMed2006

Mutators among CTX-M beta-lactamase-producing Escherichia coli and risk for the emergence of fosfomycin resistance.

OBJECTIVES: Fosfomycin is a possible oral treatment for lower urinary tract infections caused by Escherichia coli with CTX-M extended-spectrum beta-lactamases but is vulnerable to mutational resistance. Hypermutability among natural E. coli populations might facilitate the emergence of resistance to fosfomycin. We therefore examined the prevalence of mutators amongst urinary isolates of E. coli producing CTX-M beta-lactamases. METHODS: Urinary E. coli isolates with CTX-M beta-lactamases (n = 220) were screened for resistance to both rifampicin and fosfomycin, as well as a mutator phenotype, by rifampicin and fosfomycin disc assays. Mutation frequencies for 10 isolates, identified as mutators by the initial disc screen, were determined in triplicate on agar with rifampicin or fosfomycin at 4x MIC and with fosfomycin or nitrofurantoin at 256 mg/L. RESULTS: The disc screen identified 10 likely mutators and quantitative tests indicated that 9 of these had mutation frequencies of 8.0 x 10(-6)-1.5 x 10(-4) for fosfomycin and 0.1-2.3 x 10(-6) for rifampicin. These mutators were diverse in terms of PFGE type and 4 of the 10 were confirmed as strong mutators with rifampicin and fosfomycin. Only the strongest mutator isolate and hypermutable MutS(-) control strain consistently gave single-step mutants resistant to 256 mg/L fosfomycin. No nitrofurantoin-resistant mutants were selected from any isolate, although they could be selected from the hypermutable MutS(-) control strain. CONCLUSIONS: Mutator phenotypes were found among E. coli expressing CTX-M beta-lactamases and were independent of strain type. These had an increased propensity to fosfomycin resistance.

Anti-Bacterial Agents↗

PubMed2006

Effect of subinhibitory concentrations of antibiotics on mutation frequency in Streptococcus pneumoniae.

OBJECTIVES: To investigate the effect of subinhibitory concentrations of ciprofloxacin, streptomycin, trimethoprim, ampicillin and erythromycin on mutation frequency in Streptococcus pneumoniae. METHODS: Frequency of mutation to rifampicin resistance was determined in three clinical isolates grown with or without antibiotic treatment. dinB was analysed using PCR and DNA sequence determination. RESULTS: Subinhibitory levels of ciprofloxacin and streptomycin increased the frequency of mutation to rifampicin resistance between 2- and 5-fold for all three isolates, which is comparable to the increase seen in mismatch repair mutants of this species. These increases appeared not to be dependent on the function of the error-prone DNA polymerase encoded by dinB, since one of the isolates was a naturally occurring deletion mutant for this gene. Trimethoprim increased the mutation frequency for two isolates, but not the dinB mutant; ampicillin and erythromycin had no significant effect on mutation frequencies for any isolate. CONCLUSIONS: Exposure to quinolones and aminoglycosides at subinhibitory concentrations may result in increased mutability in pneumococci, as well as selecting for resistance per se.

Anti-Bacterial Agents↗

PubMed2006

Hypermutable bacteria isolated from humans--a critical analysis.

Hypermutable bacteria of several species have been described among isolates recovered from humans over the last decade. Interpretation of the literature in this area is complicated by diversity in the determination and definition of hypermutability, and this review outlines the different methods used. Inactivation of the mismatch repair gene mutS is often implicated in the mutator phenotype; the reported effect of mutS inactivation on mutation frequency varies widely between species, from under 10-fold to nearly 1,000-fold, but also varies among different reports on the same species. Particularly high proportions of mutators have been reported among Pseudomonas aeruginosa and other species in the cystic fibrosis lung, epidemic serogroup A Neisseria meningitidis, and Helicobacter pylori. Aspects of the biology of these infections that could be relevant to hypermutability are discussed, and some future directions that may increase our understanding of mutators among bacteria isolated from humans are considered.

Bacteria↗

PubMed2005

Resistance among Escherichia coli to sulphonamides and other antimicrobials now little used in man.

OBJECTIVES: We investigated whether sulphonamide resistance in Escherichia coli remained prevalent in 2004, 9 years since the formal introduction of a UK prescribing restriction on co-trimoxazole. Resistance to other agents no longer in common use was also examined. METHODS: Consecutive urinary E. coli isolates were obtained at the diagnostic microbiology laboratory of the Royal London Hospital from January to March 2004. The presence of the sulphonamide resistance genes, sul1, sul2 and sul3, and the class I integrase gene, int1, were determined by PCR. RESULTS: Of the 391 E. coli isolates recovered in 2004, 45.5% were sulphonamide-resistant compared with 46.0% in 1999 and 39.7% in 1991. The sul2 gene remained the most prevalent sulphonamide resistance determinant, present in 81% of resistant isolates in 2004 compared with 79% and 67% in 1999 and 1991, respectively; 28% of resistant isolates carried both sul1 and sul2 genes; sul3 was not found. Resistance to streptomycin also remained common, whereas resistance to chloramphenicol and kanamycin had decreased since 1999. CONCLUSION: Sulphonamide resistance in E. coli persists undiminished despite the prolonged withdrawal of this antibiotic in the UK; resistance to streptomycin also seems stable whilst that to chloramphenicol and kanamycin is declining.

Anti-Bacterial Agents↗

PubMed2005

Sequence diversity and antigenic variation at the rag locus of Porphyromonas gingivalis.

The rag locus of Porphyromonas gingivalis W50 encodes RagA, a predicted tonB-dependent receptor protein, and RagB, a lipoprotein that constitutes an immunodominant outer membrane antigen. The low G+C content of the locus, an association with mobility elements, and an apparent restricted distribution in the species suggested that the locus had arisen by horizontal gene transfer. In the present study, we have demonstrated that there are four divergent alleles of the rag locus. The original rag allele found in W50 was renamed rag-1, while three novel alleles, rag-2 to rag-4, were found in isolates lacking rag-1. The three novel alleles encoded variants of RagA with 63 to 71% amino acid identity to RagA1 and each other and variants of RagB with 43 to 56% amino acid identity. The RagA/B proteins have homology to numerous Bacteroides proteins, including SusC/D, implicated in polysaccharide uptake. Monoclonal and polyclonal antibodies raised against RagB1 of P. gingivalis W50 did not cross-react with proteins from isolates carrying different alleles. In a laboratory collection of 168 isolates, 26% carried rag-1, 36% carried rag-2, 25% carried rag-3, and 14% carried rag-4 (including the type strain, ATCC 33277). Restriction profiles of the locus in different isolates demonstrated polymorphism within each allele, some of which is accounted for by the presence or absence of insertion sequence elements. By reference to a previously published study on virulence in a mouse model (M. L. Laine and A. J. van Winkelhoff, Oral Microbiol. Immunol. 13:322-325, 1998), isolates that caused serious disease in mice were significantly more likely to carry rag-1 than other rag alleles.

Alleles↗

PubMed2004

Enhancement of host fitness by the sul2-coding plasmid p9123 in the absence of selective pressure.

OBJECTIVES: Despite a 97% reduction in clinical sulphonamide usage, the prevalence of sulphonamide resistance among Escherichia coli has remained constant in the UK. Genetic linkage of sulphonamide resistance to other resistances is thought important for this maintenance, but the finding also implies that sulphonamide resistance exerts little fitness cost. To test this hypothesis, we examined the fitness impact of four naturally occurring sul2-coding plasmids upon their hosts. METHODS: The fitness impact of the plasmids upon E. coli was determined by pairwise growth competition in a minimal medium. The DNA sequence of plasmid p9123 was obtained by primer walking and PCR. RESULTS: Three of the four sul2-coding plasmids studied imposed fitness costs on their hosts. The fourth plasmid, a 6.2 kb resistance element carrying sul2, strA and strB designated p9123, conferred a 4% fitness advantage upon its original clinical host and also on E. coli K12 JM109. The complete sequence of p9123 revealed eight open reading frames, including five of unknown function. There was no obvious gene to which the fitness advantage might be attributed. CONCLUSIONS: The novel finding that p9123 can improve host fitness may explain why this plasmid and its close relatives are so widespread among enteric bacteria. In addition to other factors such as co-selection of sulphonamide resistance by other agents, the fitness advantage conferred by plasmids such as p9123 may have contributed to the maintenance of sulphonamide resistance in the UK in the absence of clinical selection pressure. These data indicate that once antibiotic resistance has been established on mobile genetic elements, it may be difficult to eliminate.

Anti-Infective Agents↗

PubMed2004

Klebsiella pneumoniae isolate from South Africa with multiple TEM, SHV and AmpC beta-lactamases.

Klebsiella pneumoniae 2207, from Durban, was resistant to cefoxitin and beta-lactamase inhibitor combinations as well as oxyimino-aminothiazolyl cephalosporins. Beta-lactamases with isoelectric points of 5.4, 5.6, 7.6, 8.2 and 8.4 were found. DNA hybridisation identified two BamHI and three HindIII fragments carrying blaTEM, and two SalI fragments carrying blaSHV. At least two genes encoded TEM-1 enzyme; one blaSHV copy encoded SHV-5 but the other determined SHV-23, a novel SHV-5 variant with conservative amino-acid substitutions far from the catalytic site. The pI 8.4 activity was an AmpC-type enzyme. Determinants of the pI 5.6 and 7.6 activities were not identified.

Bacterial Proteins↗

PubMed2003

Variable susceptibility to piperacillin/tazobactam amongst Klebsiella spp. with extended-spectrum beta-lactamases.

MICs of piperacillin/tazobactam are conventionally determined by varying the concentration of piperacillin in the presence of a fixed 4 mg/L tazobactam. When tested in this way, the MIC distribution for Klebsiella isolates with extended-spectrum beta-lactamases (ESBLs) is strongly bimodal, such that many producers are inhibited at 16 + 4 mg/L whilst others require MICs of > or =512 + 4 mg/L. When, however, piperacillin/tazobactam was tested as a fixed 8:1 ratio, the MIC distribution became unimodal. If clavulanate 4 mg/L was combined with piperacillin, a unimodal MIC distribution was seen for ESBL-producing Klebsiella spp. but a bimodal distribution arose if the clavulanate concentration was reduced to 0.25 mg/L. These data for alternative combinations suggested that the bimodal MIC distribution seen for piperacillin + tazobactam 4 mg/L was a titration effect, not a reflection of some ESBLs being resistant to tazobactam. Even within single strains, as defined by serotype and DNA fingerprints, there was considerable variation in susceptibility to piperacillin + tazobactam 4 mg/L, with some representatives highly susceptible and others highly resistant. Some of the more resistant representatives produced more of their ESBL, or had a greater number of beta-lactamase types, but these associations were not universal. Elevated resistance to piperacillin + tazobactam was not associated with porin change in any ESBL producer examined, but has been found by others.

Bacterial Outer Membrane Proteins↗

PubMed2003

Evaluation of semiautomated multiplex PCR assay for determination of Streptococcus pneumoniae serotypes and serogroups.

A semiautomated method for the determination of five serotypes and three serogroups in Streptococcus pneumoniae was developed. Primers specific for serotypes 1, 3, 14, 19F, and 23F and serogroups 6, 19, and 23 were combined in three multiplex PCRs. Products were separated by capillary electrophoresis with a 7-min run time, and a serotype or serogroup was assigned on the basis of fragment size. The method was used to test 93 clinical isolates, and all isolates of the serotypes concerned were correctly detected. The strategy would allow the detection of multiple serotypes in a single sample. Detection of additional serotypes could be included as capsule locus sequences become available.

Automation↗

PubMed2002

Sulfonamide resistance in Haemophilus influenzae mediated by acquisition of sul2 or a short insertion in chromosomal folP.

Determinants of sulfonamide resistance were investigated in clinical isolates of Haemophilus influenzae from the United Kingdom and Kenya. The mechanism of sulfonamide resistance in H. influenzae has not previously been reported. Eight isolates requiring at least 1,024 microg of sulfamethoxazole per ml for inhibition carried the sul2 gene, a common mediator of acquired sulfonamide resistance in enteric bacteria. In other isolates with similarly high levels of resistance, the chromosomal gene encoding dihydropteroate synthase, folP, was found to carry an insertion of 15 bp together with other missense mutations relative to folP of H. influenzae strain Rd RM118 (MIC, 8 microg/ml); the folP sequence was identical in all seven such isolates investigated, although they represented three different strains by restriction pattern analysis. The 15-bp insertion was absent in isolates inhibited by sulfamethoxazole at 2 to 64 microg/ml (although these exhibited considerable divergence in folP sequence) and in highly resistant isolates carrying sul2. Transformation with a 599-bp fragment of folP containing the insertion but no other differences conferred high-level resistance on a recipient strain, confirming the role of the insertion. Other amino acid substitutions in dihydropteroate synthase may modulate the level of sulfonamide inhibition in susceptible isolates and those with more moderate levels of resistance. The two mechanisms of resistance, mediated by sul2 and modified folP, were detected in isolates from both the United Kingdom and Kenya.

Anti-Bacterial Agents↗