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Objectives: Third-generation cephalosporin-resistant Escherichia coli from community-acquired urinary tract infections are increasingly reported worldwide. We sought to determine and characterize the mechanisms of cefotaxime resistance employed by urinary E. coli obtained from primary care, over 12 months, in Bristol and surrounding counties in South-West England.Methods: Cefalexin-resistant E. coli isolates were identified from GP-referred urine samples using disc susceptibility testing. Cefotaxime resistance was determined by subsequent plating onto MIC breakpoint plates. β-Lactamase genes were detected by PCR. WGS was performed on 225 isolates and analyses were performed using the Center for Genomic Epidemiology platform. Patient information provided by the referring general practices was reviewed.Results: Cefalexin-resistant E. coli (n=900) isolates were obtained from urines from 146 general practices. Following deduplication by patient approximately 69% (576/836) of isolates were cefotaxime resistant. WGS of 225 isolates identified that the most common cefotaxime-resistance mechanism was blaCTX-M carriage (185/225), followed by plasmid-mediated AmpCs (pAmpCs) (17/225), AmpC hyperproduction (13/225), ESBL blaSHV variants (6/225) or a combination of both blaCTX-M and pAmpC (4/225). Forty-four STs were identified, with ST131 representing 101/225 isolates, within which clade C2 was dominant (54/101). Ciprofloxacin resistance was observed in 128/225 (56.9%) of sequenced isolates, predominantly associated with fluoroquinolone-resistant clones ST131 and ST1193.Conclusions: Most cefalexin-resistant E. coli isolates were cefotaxime resistant, predominantly caused by blaCTX-M carriage. The correlation between cefotaxime resistance and ciprofloxacin resistance was largely attributable to the high-risk pandemic clones ST131 and ST1193. Localized epidemiological data provide greater resolution than regional data and can be valuable for informing treatment choices in the primary care setting. [ABSTRACT FROM AUTHOR]

Background: In Klebsiella pneumoniae, loss-of-function mutations in the transcriptional repressors RamR and OqxR both have an impact on the production of efflux pumps and porins relevant to antimicrobial efflux/entry.Objectives: To define, in an otherwise isogenic background, the relative effects of OqxR and RamR loss-of-function mutations on envelope protein production, envelope permeability and antimicrobial susceptibility. We also investigated the clinical relevance of an OqxR loss-of-function mutation, particularly in the context of β-lactam susceptibility.Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. Antimicrobial susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and quantitative RT-PCR was used to measure transcript levels.Results: Loss of RamR or OqxR reduced envelope permeability in K. pneumoniae by 45%-55% relative to the WT. RamR loss activated AcrAB efflux pump production ∼5-fold and this reduced β-lactam susceptibility, conferring ertapenem non-susceptibility even in the absence of a carbapenemase. In contrast, OqxR loss specifically activated OqxAB efflux pump production >10 000-fold. This reduced fluoroquinolone susceptibility but had little impact on β-lactam susceptibility even in the presence of a β-lactamase.Conclusions: Whilst OqxR loss and RamR loss are both seen in K. pneumoniae clinical isolates, only RamR loss significantly stimulates AcrAB efflux pump production. This means that only RamR mutants have significantly reduced β-lactamase-mediated β-lactam susceptibility and therefore represent a greater clinical threat. [ABSTRACT FROM AUTHOR]

Objectives: In Klebsiella pneumoniae, overproduction of RamA results in reduced envelope permeability and reduced antimicrobial susceptibility but clinically relevant resistance is rarely observed. Here we have tested whether RamA overproduction can enhance acquired β-lactam resistance mechanisms in K. pneumoniae and have defined the envelope protein abundance changes upon RamA overproduction during growth in low and high osmolarity media.Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. β-Lactam susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and transcript levels were quantified using real-time RT-PCR.Results: RamA overproduction enhanced β-lactamase-mediated β-lactam resistance, in some cases dramatically, without altering β-lactamase production. It increased production of efflux pumps and decreased OmpK35 porin production, though micF overexpression showed that OmpK35 reduction has little impact on envelope permeability. A survey of K. pneumoniae bloodstream isolates revealed ramA hyperexpression in 3 of 4 carbapenemase producers, 1 of 21 CTX-M producers and 2 of 19 strains not carrying CTX-M or carbapenemases.Conclusions: Whilst RamA is not a key mediator of antibiotic resistance in K. pneumoniae on its own, it is potentially important for enhancing the spectrum of acquired β-lactamase-mediated β-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production. [ABSTRACT FROM AUTHOR]

Objectives: In Klebsiella pneumoniae, overproduction of RamA and RarA leads to increased MICs of various antibiotics; MarA and SoxS are predicted to perform a similar function. We have compared the relative effects of overproducing these four AraC-type regulators on envelope permeability (a combination of outer membrane permeability and efflux), efflux pump and porin production, and antibiotic susceptibility in K. pneumoniae.Methods: Regulators were overproduced using a pBAD expression vector. Antibiotic susceptibility was measured using disc testing. Envelope permeability was estimated using a fluorescent dye accumulation assay. Porin and efflux pump production was quantified using proteomics and validated using real-time quantitative RT-PCR.Results: Envelope permeability and antibiotic disc inhibition zone diameters both reduced during overproduction of RamA and to a lesser extent RarA or SoxS, but did not change following overproduction of MarA. These effects were associated with overproduction of the efflux pumps AcrAB (for RamA and SoxS) and OqxAB (for RamA and RarA) and the outer membrane protein TolC (for all regulators). Effects on porin production were strain specific.Conclusions: RamA is the most potent regulator of antibiotic permeability in K. pneumoniae, followed by RarA then SoxS, with MarA having very little effect. This observed relative potency correlates well with the frequency at which these regulators are reportedly overproduced in clinical isolates. [ABSTRACT FROM AUTHOR]

Objectives: To further understand the mechanisms used to regulate expression of the blr regulon of Aeromonas hydrophila T429125, including three unlinked beta-lactamase genes, ampH, cepH and imiH, and to examine the role of the 'cre/blr-tag' DNA sequence (TTCAC) in transcriptional control exerted by the two-component system, BlrAB.Methods: Genes linked to blrAB-ampH were cloned using standard methods; gene expression was measured by RT-PCR or beta-lactamase assays; transcription start sites were determined by reversed-transcript analysis; cepH promoter probe reporter constructs including cre/blr-tag deletions were generated by PCR; and BlrA was overexpressed in Escherichia coli using the pBAD plasmid.Results: The blrD gene, encoding a putative inner membrane protein, was found to be located downstream of blrAB-ampH. RT-PCR analysis showed that blrD is part of the A. hydrophila blr regulon, and transcript start-point determinations revealed that blr-regulon promoters (including that of blrD) are preceded by at least one cre/blr-tag. Targeted deletion of the 16 bp cepH cre/blr-tag dimer blocked BlrA-induced overproduction of cepH in E. coli.Conclusions: This is the first report of non-beta-lactamase genes being co-ordinately regulated with a normally co-resident beta-lactamase gene, and the first direct evidence for a role of the cre/blr-tag sequence in the regulation of transcription by BlrA. [ABSTRACT FROM AUTHOR]

Legionella pneumophila Philadelphia-1 (ATCC 33152) produces a serine active site beta-lactamase. The chromosomal gene that encodes this enzyme, loxA, has been cloned by PCR using information from the L. pneumophila Philadelphia-1 genome sequencing project. LoxA is a class 2d penicillinase, and its sequence puts it into the molecular class D beta-lactamase family, although phylogenetic analysis shows that LoxA forms a distinct branch in the OXA family along with the LoxA homologue, OXA-29, from Legionella gormanii ATCC 33297(T). Upstream of loxA on the L. pneumophila Philadelphia-1 chromosome is a two-gene locus similar to that found linked to the beta-lactamase genes of Gram-positive bacteria. The unit consists of loxI, encoding a homologue of the Gram-positive beta-lactamase expression regulator, and pbpX, encoding a putative penicillin-binding transpeptidase. Despite the presence of beta-lactamase regulator homologues, we could find no evidence of LoxA induction upon challenge of L. pneumophila Philadelphia-1 with beta-lactams. [ABSTRACT FROM AUTHOR]

Ochrobactrum anthropi is resistant to most cephalosporins and penicillins due, at least in part, to the inducible expression of a single β-lactamase. The β-lactamase gene has been cloned and sequenced. It encodes an AmpC-type class 1 serine active-site enzyme that hydrolyses mainly cephalosporins and is resistant to inhibition by clavulanic acid. Expression of the ampC gene is inducible via a typical AmpR regulator, which is encoded upstream of ampC. Inducible expression is retained following cloning of O. anthropi ampR–ampC into Escherichia coli, confirming that the signal for AmpR activation in O. anthropi is the same as that used in the Enterobacteriaceae. This is the first reported example of an AmpC β-lactamase outside of the γ-subdivision of the bacterial kingdom. Genomic searches of other non-γ-subdivision bacteria revealed a homologous ampR–ampC cluster in the plant symbiont, Sinorhizobium meliloti. [ABSTRACT FROM PUBLISHER]

The class 1 cephalosporinase (CepH) and class 2d oxacillinase (AmpH) from an Aeromonas hydrophila clinical isolate, strain T429125, have been cloned and sequenced. Both enzymes are typical of their equivalents in other species of Aeromonas. Both cloned β-lactamase genes were expressed at a low level in a standard laboratory Escherichia coli strain, but when cloned into a cre deletion E. coli mutant, they were expressed at significantly higher levels. Specific disruption of the creB gene resulted in similar increased levels of β-lactamase expression, so it was concluded that CreB represses the transcription of ampH and cepH in a cre+ E. coli strain. The expression of cepH was four times that of ampH in the δcre mutant because of an additional factor encoded on the cloned T429125 chromosomal fragment containing cepH. This factor was able to trans-activate expression of co-resident ampH in the δcre mutant such that expression of the two genes was approximately equal. The entire cepH-containing fragment was sequenced, but it contained no genes that were obviously related to any known class of DNA-binding protein. [ABSTRACT FROM PUBLISHER]

We have examined the production of β-lactamases by 11 clinical and nine environmental isolates of Plesiomonas shigelloides from Czechoslovakia, the Czech Republic and Cuba. Ten isolates (50%) expressed detectable amounts of a chromosomally encoded, non-inducible β-lactamase, though all isolates showed a broadly similar resistance profile: low-level resistance to ampicillin and higher-level resistance to carbenicillin. All strains were susceptible to cephalosporins and meropenem. Three clinical isolates expressed a β-lactamase similar to a class 2c carbenicillinase, with a pI of 5.2 and three expressed an enzyme similar to a class 2d oxacillinase, with a pI of 5.3. The environmental isolates produced a variety of penicillinases, indicating that there is a reservoir of heterogeneous β-lactamase genes in this species. [ABSTRACT FROM PUBLISHER]

It has been reported that Stenotrophomonas maltophilia L1 and L2 beta-lactamase expression is coordinated. We have isolated S. maltophilia mutants where (i) L1 is constitutively hyper-expressed and L2 is inducible; (ii) L2 is hyper-expressed and L1 is inducible; and (iii) L1 and L2 are constitutively hyper-expressed. The frequency of isolating type 1 and 2 mutants is c. 10(-7), indicating that promoter mutations are probably not involved and providing strong evidence that L1 and L2 expression is not, after all, coordinated. The frequency of isolating type 3 mutants is c. 10(-9), however, implying that there is a significant overlap between the regulatory mechanisms. [ABSTRACT FROM AUTHOR]