David Wareham qualified from the London Hospital Medical College in 1994 (MB BS). He trained in general medicine in East London and Essex gaining MRCP, DGM and DTM&H diplomas from the Royal College of Physicians before specialist training in Medical Microbiology (FRCPath). He was awarded a Clinical Training Fellowship to study aspects of Pseudomonas aeruginosa pathogenicity at Queen Mary University in 2002, gaining completing his PhD in 2006. He was appointed as Senior Clinical Lecturer in Microbiology at Barts and the London School of Medicine and Dentistry in 2005. He is an Honorary Consultant Microbiologist at Barts and The London, Newham University and Homerton NHS Trusts where he is responsible for aspects of intensive care microbiology.
Abstract: Background: Escherichia coli is the commonest cause of community and nosocomial urinary tract infection (UTI). Antibiotic treatment is usually empirical relying on susceptibility data from local surveillance studies. We therefore set out to determine levels of resistance to 8 commonly used antimicrobial agents amongst all urinary isolates obtained over a 12 month period.
Methods: Antimicrobial susceptibility to ampicillin, amoxicillin/clavulanate, cefalexin, ciprofloxacin, gentamicin, nitrofurantoin, trimethoprim and cefpodoxime was determined for 11,865 E. coli urinary isolates obtained from community and hospitalised patients in East London.
Results: Nitrofurantoin was the most active agent (94% susceptible), followed by gentamicin and cefpodoxime. High rates of resistance to ampicillin (55%) and trimethoprim (40%), often in combination were observed in both sets of isolates. Although isolates exhibiting resistance to multiple drug classes were rare, resistance to cefpodoxime, indicative of Extended spectrum β-lactamase production, was observed in 5.7% of community and 21.6% of nosocomial isolates.
Conclusion: With the exception of nitrofurantoin, resistance to agents commonly used as empirical oral treatments for UTI was extremely high. Levels of resistance to trimethoprim and ampicillin render them unsuitable for empirical use. Continued surveillance and investigation of other oral agents for treatment of UTI in the community is required.
Abstract: Acinetobacter spp. are increasingly reported as important causes of human infection. Many isolates exhibit multi-drug resistance, raising concerns over our ability to treat serious infections with these organisms. The impact of infection on clinical outcome as well as the importance of multi-drug resistance is poorly defined. A descriptive retrospective observational study was undertaken of all episodes of Acinetobacter bacteremia occurring in a UK tertiary care centre from 1998-2006. Demographics of infected patients, characteristics and antimicrobial susceptibility of infecting strains were recorded and the impact of antimicrobial therapy on all causes of 30-day mortality assessed. Three hundred ninety-nine episodes of Acinetobacter bacteremia were identified, with A. baumannii being the most frequently isolated species. Most episodes occurred in critical care and were associated with multidrug resistance, with carbapenem resistance rising from 0% in 1998 to 55% in 2006. Although bacteremia due to carbapenem-resistant Acinetobacter and a requirement for critical care were associated with a higher mortality, mortality was not reduced by the administration of appropriate empirical antimicrobial therapy. A prospective study is required to identify both the most effective intervention and those most likely to benefit from treatment.
Abstract: This study investigated the mechanisms of multidrug resistance (MDR) in an isolate of Bacteroides fragilis (WI1) from a patient with anaerobic sepsis. The MDR of WI1 affected susceptibility to beta-lactams, clindamycin, fluoroquinolones, metronidazole and tetracycline. In addition to its 5.31-Mb chromosome, WI1 possessed two low-copy-number plasmids, pHagl (5.6 kb) and pHag2 (9.9 kb), that were absent from B. fragilis NCTC 9343. Restriction digestion with EcoRV, HindIII and SstI, combined with DNA sequencing, revealed that pHAG2 contained a tet(Q) gene at base position 3689 that resided on the conjugative transposon CTn341. Genes cfiA (encoding a metallo-beta-lactamase) and erm(F) (encoding a macrolide-lincosamide-streptogramin B resistance determinant) were also found in WI1, but were absent from B. fragilis NCTC 9343. Nitrocefin hydrolysis revealed that WI1 had high beta-lactamase activity. Sequencing of the gyrA quinolone resistance-determining region revealed a mutation causing a Ser82 --> Phe substitution, and comparative quantitative real-time RT-PCR revealed that the cfiA, erm(F) and tet(Q) genes were all expressed in WI1. In addition, the resistance-nodulation-division efflux pump genes bmeB9 and bmeB15 were significantly over-expressed (12.30 +/- 0.42-fold and 3541.1 +/- 95.4-fold, respectively), and the efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone and reserpine significantly increased the susceptibility of the isolate to several unrelated antibiotics (p <0.005). These data suggested that WI1 was highly multidrug-resistant because of the additive effects of chromosome- and plasmid-encoded resistance determinants.
Abstract: The type III secretion system (TTSS) of Pseudomonas aeruginosa enables delivery of a number of toxins involved in the disruption of eukaryotic epithelial surfaces. Whilst the ability to secrete ExoS facilitates invasion and internalization, the secretion of ExoU mediates acute cytotoxicity. In order to determine any association with the ability to secrete these toxins with the nature and severity of human infection, the TTSS genotypes and phenotypes of 163 clinical isolates were determined by multiplex PCR and Western blotting. An exoS+/exoU- genotype was associated with chronic infection in patients with cystic fibrosis whilst an exoS-/exoU+ genotype was associated with strains isolated from blood. Secretion of the ExoU protein was more commonly seen in isolates obtained from blood, suggesting this ability may be important in the development of acute invasive infection. Detection of TTSS toxins in clinical material may be useful in targeting antimicrobial therapy or identifying individuals infected with aggressive strains of P. aeruginosa.
Abstract: This report describes an unusual case of endocarditis caused by Capnocytophaga canimorsus as a result of dog bite. The diagnosis could be established only by molecular techniques after amplification of bacterial DNA from the infected cardiac valve. The epidemiology and management of Capnocytophaga infections is discussed, as well as the role of prophylactic antibiotics in preventing these infections after dog bites.
Abstract: Acinetobacter baumannii is increasingly recognized as an important multidrug-resistant nosocomial pathogen. Recent work has highlighted enhanced growth and heightened virulence in the presence of ethyl alcohols. As alcohol-based hand rubs (ABHRs) are extensively used in health care settings, the authors set out to determine whether the hand rubs could also influence the growth of prevalent multidrug-resistant strains circulating in UK hospitals. A significant increase in growth was observed when minimal media were supplemented with concentrations of 1 % and lower of four commercially available hand rubs. In addition, growth in ABHR-supplemented media resulted in secretion of proteins into the culture supernatant. One of these was identified as OmpA, which is recognized as having emulsifying activity, which could potentially confer enhanced pathogenicity to A. baumannii.
Abstract: OBJECTIVES: Clinical experience with linezolid in the treatment of infective endocarditis either alone or in combination with other agents is limited. We describe our experience in the treatment of two patients with IE due to multi-resistant Gram-positive bacteria. METHODS: One patient with MRSE and one with VRE endocarditis were treated with regimens containing linezolid. The killing kinetics of linezolid in combination with gentamicin or vancomycin against isolates of Staphylococcus epidermidis and Enterococcus faecalis were analysed in vitro. RESULTS: Clinical response and eradication of bacteraemia was achieved with linezolid therapy in both patients. Time-kill curve studies showed that linezolid was bacteriostatic against the MRSE and VRE isolates used. Combination with gentamicin or vancomycin did not produce synergy or antagonism but at best only marginal additive effect. CONCLUSIONS: Although bacteriostatic, linezolid provides an important therapeutic option in IE due to multi-resistant Gram-positive pathogens. It challenges the conventional wisdom that bactericidal synergy is required for the effective treatment of most cases of IE due to Gram-positive organisms.
Abstract: BACKGROUND: Acinetobacter baumannii has emerged as a major nosocomial pathogen worldwide. Many of the circulating strains exhibit multi-drug resistance remaining consistently susceptible only to polymyxins. In-vitro studies have reported that polymyxins combined with carbapenems, rifampicin or azithromycin are synergistic against these strains despite in-vitro resistance to these agents alone. The use of antimicrobial combinations have therefore been advocated for the treatment of severe A. baumannii infection in man. In order to determine whether such combinations are synergistic against the prevalent clones of multi-drug resistant A. baumannii causing infection in the UK, we performed synergy testing against representative isolates using two rapid Etest methods. METHODS: The activity of polymyxin in combination with imipenem, azithromycin or rifampicin was assessed against five strains of multi-drug resistant A. baumannii encoding OXA-23 carbapenemases. Synergy studies were performed by Etest-agar dilution and a combined Etest strip method. Synergy was defined as a FICI of < or = 0.5. RESULTS: All strains were resistant to beta-lactams, carbapenems, quinolones and aminoglycosides but susceptible to polymyxins. Marked synergy was not seen with polymyxin in combination with imipenem, rifampicin or azithromycin against any of the strains. Borderline synergy (FICI = 0.5) was seen against one strain belonging to OXA-23 clonal group 2, using the Etest-agar dilution method only. CONCLUSION: In-vitro synergy with polymxyin in combination with imipenem, rifampicin or azithromycin is highly strain and method dependent. As reliable synergy could not be demonstrated against the prevalent UK multi-drug resistant strains, use of such combinations should not be used for empirical treatment of these infections in the UK. The optimal treatment for serious multi-drug A. baumannii infection and the role of combination therapy should be addressed in a prospective clinical trial.
Abstract: A strain of Listeria monocytogenes recovered from blood and cerebrospinal fluid had no detectable catalase activity, a characteristic used for primary identification. The sporadic occurrence of pathogenic catalase-negative strains highlights the need for a reconsideration of diagnostic criteria and questions the role of catalase in the pathogenesis of listeria infection.
Abstract: The Pseudomonas aeruginosa type III secretion system (TTSS), enabling direct injection of toxins into host cells, has been shown to be crucial to virulence in several models of P. aeruginosa pathogenesis. Using the strain PA14 and its isogenic mutant, PA14exsA, we investigated the role of the TTSS during infection of the nematode Caenorhabditis elegans. Although C. elegans N2 was killed by PA14 in an infection like process over 48 to 72 h the same effect was observed following infection with PA14exsA, implying that a functional TTSS was not essential for virulence. This was despite the TTSS being actively expressed during C. elegans infection as demonstrated by the use of green fluorescent reporter constructs and RT-PCR. However, compared to the wild type PA14, PA14exsA did display a reduced rate of killing of C. elegans strain AU1 which harbours a mutation in the sek-1 gene encoding a MAP kinase involved in nematode innate immunity. A fuller understanding of the mechanism of resistance to type III attack in C. elegans may lead to the identification and development of novel therapeutic targets affording protection to TTSS products in man.
Abstract: We describe the first reported case of anaerobic sepsis due to Bacteroides fragilis with simultaneous resistance to metronidazole, β-lactams, β-lactam/β-lactamase inhibitors, carbapenems, macrolides, and tetracyclines. Microbiological cure and clinical improvement was achieved with linezolid therapy, an agent that may be useful for the treatment of multidrug-resistant anaerobic infections.
Abstract: Aims: The diagnosis of deep seated bacterial infections, such as intra-abdominal abscesses, endocarditis, and osteomyelitis, can be difficult and delayed, thereby compromising effective treatment. This study assessed the efficacy of a new radioimaging agent, Tc-99m ciprofloxacin (Infecton), in accurately detecting sites of bacterial infection.
Methods: Eight hundred and seventy nine patients with suspected bacterial infection underwent Infecton imaging and microbiological evaluation. The sensitivity and specificity of Infecton in detecting sites of bacterial infection were determined with respect to Centres of Disease Control, World Health Organisation, and Dukes’s criteria.
Results: Five hundred and seventy four positive and 295 negative images were produced. These included 528 true positives, 46 false positives, 205 true negatives and 90 false negatives, giving an overall sensitivity of 85.4% and a specificity of 81.7% for detecting infective foci. Sensitivity was higher (87.6%) in microbiologically confirmed infections.
Conclusions: Infecton is a sensitive technique, which aids in the earlier detection and treatment of a wide variety of deep seated bacterial infections. The ability to localise infective foci accurately is also important for surgical intervention, such as drainage of abscesses. In addition, serial imaging with Infecton might be useful in monitoring clinical response and optimising the duration of antimicrobial treatment.
Abstract: Background:Plasmid-mediated quinolone resistance (PMQR) has been increasingly reported over the last ten years with three mechanisms described: Qnr proteins which protect target enzymes from quinolone inhibition, AAC(6’)-Ib-cr able to N-acetylate ciprofloxacin, and the QepA efflux pump which extrudes hydrophilic fluoroquinolones. PMQR has been associated with resistance to multiple antibiotic classes. QnrS has been found on plasmids with the metallo-β-lactamase blaVIM-1 in Italy, and qepA has been shown to be linked with the aminoglycoside ribosome methyltransferase gene rmtB in Japan and Belgium.The objectives of this study were to describe PMQR in clinical isolates in East London for the first time.
Methods:We screened for mechanisms of PMQR in enterobacteriaciae obtained from noscomial and community acquired urinary tract and bloodstream infections. PCR with primers specific for qnrA,B and S, qepA, and aac(6’)-Ib genes was carried out on consecutive non duplicate isolates resistant to ciprofloxacin and / or gentamicin
Results:83 isolates were included, 96.4% of which were resistant to ciprofloxacin, 48.2% to gentamicin, 51.8% were resistant to cefpodoxime, 32.5% of which produced ESBLs. qnrS was present in 4 (4.8%) isolates, in association with and without ESBL production. None of the isolates with qnrS had the blaVIM-1 gene. qnrA or qnrB were not found in any isolates. qepA was present in 3 (3.6%) isolates, none of which carried the rmtB gene or produced ESBLs. The aac(6’)-Ib gene was found in 50 (60.2%) isolates.
Conclusions:We report quinolone resistant isolates carrying the qnrS and qepA genes for the first time in the UK. Unlike previous reports from other regions, there was no association between qnrS and blaVIM-1, or between qepA and rmtB. In contrast to previous UK surveys we found no qnrA-positive isolates. Analysis of the genetic backgrounds and plasmids encoding the qnrS and qepA genes is underway.
Abstract: Background: Antisense oligonucelotides (oligos) which bind complementary mRNA sequences are a well established means of modifying eukaryotic gene expression. Only a few compounds, usually targeting essential genes, have undergone investigation in bacteria. Inhibition of antibiotic resistance genes is an approach with the potential to restore susceptibility to long abandoned antibiotics and target only resistant bacteria. In order to identify effective and potent antisense sequences a novel bioinformatic algorithm, Sfold, able to predict accessible sites in the secondary structure of the target mRNA was used. Sequences were assessed in a cell free expression system for their ability to inhibit a number of generic β-lactamases in-vitro.
Methods: Genes encoding TEM-1, SHV-11, KPC-4 and VIM-2 were amplified by PCR from clinical isolates. Amplicons were cloned in a pEXP5-NT TOPO vector and expressed as N-terminal polyhistidine tagged proteins. Sfold software was used to identify sites in the secondary structure of TEM, SHV, KPC and VIM mRNA and the 5 highest scoring oligos selected as inhibitors. Genes were expressed in a cell free system for 3 hours +/- oligos alone and in combination. Efficiency of gene inhibition was determined by densitometric analysis following SDS-PAGE and immunoblotting for the histidine tag.
Results: Oligos at a final concentration of 5 μM resulted in widely varying degrees of inhibition (12- 85%). The most effective oligos resulting in 69% (TEM), 64% (SHV), 85% (KPC) and 45% (VIM) inhibition targeted the following nucleotide positions: TEM-1 271-290, SHV-11 458-477, KPC-4 312-331, and VIM-2 391-410. When all 5 oligos were used in combination >95% inhibition was observed for all genes.
Conclusion: An in-vitro assay was designed for the evaluation of candidate antisense sequences for use of inhibitors of antibiotic resistance genes. A number of potent oligos were identified which will be linked with peptide carrier molecules or packaged into phages to assess their role as novel inhibitors in-vivo.
Abstract: Pseudomonas aeruginosa is a major nosocomial and opportunistic pathogen involved in a wide range of localised and systemic human infections. Amongst the many virulence factors enabling colonisation and invasion of host tissues, P. aeruginosa possesses a type III secretion system (TTSS), enabling direct injection of toxins into eukaryotic cells. In this study the contribution of the TTSS to the overall virulence of P. aeruginosa was assessed. A P. aeruginosa isogenic mutant unable to perform type III secretion, was constructed by insertional inactivation into the global regulatory gene exsA in the strain PA14. Mutation of exsA abolished contact dependent cytotoxicty towards epithelial cell lines but did not lead to attenuated virulence in a Caenorhabditis elegans – P. aeruginosa pathogenicity model despite being actively expressed as demonstrated by the use of green fluorescent reporter constructs and RT-PCR. Delayed killing by the TTSS mutant was observed when a C. elegans mutant with impaired innate immunity was used, highlighting the importance of host defence in the pathogenesis of P. aeruginosa infection. In order to define the role of type III effectors in human infections, a collection of clinical P. aeruginosa isolates from blood and cystic fibrosis sputum were examined. The TTSS effector genotype and the ability to secrete specific TTSS toxins were found to correlate with the nature and severity of human disease. The detection of components of the TTSS in clinical material may therefore provide useful prognostic information and aid selection of patients for aggressive antimicrobial therapy or treatment with novel inhibitors of the TTSS.
