Mechanism of uptake of the catecholic (7)-α-formamido cephalosporin BRL 41897A by klebsiella pneumoniae

The catecholic cephalosporin BRL 41897 A is resistant to β-lactamases and is taken up by bacteria via the iron transport system. The uptake of this antibiotic in E.coli uses the Fiu and Cir outer membrane proteins, whereas in P. aerugtnosa it enters via the pyochelin transport system. In this thesis mutants of K. pneumoniae resistant to BRL 41897A were isolated using TnphoA mutagenesis and used to study the mechanism of uptake of BRL 41897A by K. pneumoniae. The activity of BRL 41897A towards the parent strain (M10) was increased in iron depleted media, whereas no significant differences in the resistant (KSL) mutants were observed. Three mutants (KSL19, KSL38and KSL59) produced decreased amounts of certain iron-regulated outer membrane proteins. The uptake of 55Fe-BRL 41897A by M10 in iron-deficient medium was higher than in iron-rich medium. This result indicated the involvement of an iron transport system in the uptake of BRL 41897A by K. pneumoniae. Uptake by the KSL mutants in iron-deficient culture was higher than that by M10. This result, supported by analysis of outer membrane and periplasmic proteins of the KSL mutants, indicates that loss of one outer membrane protein can be compensated by over expression of other outer membrane and/or periplasmic proteins. However, the increased uptake of BRL 41897A by the KSL mutants did not reflect increased activity towards these strains, indicating that there are defects in the transport of BRL 41897A resulting in failure to reach the penicillin binding protein target sites in the cytoplasmic membrane. Southern blotting of chromosomal digests and sequencing in one mutant (KSL19) showed that only one copy of TnphoA was inserted into its chromosome. A putative TnphoA inserted gene in KSL19, designated kslA, carrying a signal sequence was identified. Transformation of a fragment containing the kslA gene into KSL19 cells restored the sensitivity to BRL 41897A to that of the parent strain. Data base peptide sequence searches revealed that the kslA gene in the KSL19 has some amino acid homology with the E. coli ExbD protein, which is involved in stabilisation of the TonB protein. 

Neonatal enteral feeding tubes as loci for colonisation by members of the Enterobacteriaceae

Background The objective of this study was to determine whether neonatal nasogastric enteral feeding tubes are colonised by the opportunistic pathogen Cronobacter spp. (Enterobacter sakazakii) and other Enterobacteriaceae, and whether their presence was influenced by the feeding regime. Methods One hundred and twenty-nine tubes were collected from two neonatal intensive care units (NICU). A questionnaire on feeding regime was completed with each sample. Enterobacteriaceae present in the tubes were identified using conventional and molecular methods, and their antibiograms determined. Results The neonates were fed breast milk (16%), fortified breast milk (28%), ready to feed formula (20%), reconstituted powdered infant formula (PIF, 6%), or a mixture of these (21%). Eight percent of tubes were received from neonates who were 'nil by mouth'. Organisms were isolated from 76% of enteral feeding tubes as a biofilm (up to 107 cfu/tube from neonates fed fortified breast milk and reconstituted PIF) and in the residual lumen liquid (up to 107 Enterobacteriaceae cfu/ml, average volume 250 µl). The most common isolates were Enterobacter cancerogenus (41%), Serratia marcescens (36%), E. hormaechei (33%), Escherichia coli (29%), Klebsiella pneumoniae (25%), Raoultella terrigena (10%), and S. liquefaciens (12%). Other organisms isolated included C. sakazakii (2%),Yersinia enterocolitica (1%),Citrobacter freundii (1%), E. vulneris (1%), Pseudomonas fluorescens (1%), and P. luteola (1%). The enteral feeding tubes were in place between < 6 h (22%) to > 48 h (13%). All the S. marcescens isolates from the enteral feeding tubes were resistant to amoxicillin and co-amoxiclav. Of additional importance was that a quarter of E. hormaechei isolates were resistant to the 3rd generation cephalosporins ceftazidime and cefotaxime. During the period of the study, K. pneumoniae and S. marcescens caused infections in the two NICUs. Conclusion This study shows that neonatal enteral feeding tubes, irrespective of feeding regime, act as loci for the bacterial attachment and multiplication of numerous opportunistic pathogens within the Enterobacteriaceae family. Subsequently, these organisms will enter the stomach as a bolus with each feed. Therefore, enteral feeding tubes are an important risk factor to consider with respect to neonatal infections.

Staphylococci

Staphylococci are among the leading causes of nosocomial infections. Increasing insusceptibility to β-lactams and the glycopeptides complicates treatment of these infections. This review examines the current status and future perspectives for the therapy of infections caused by Staphylococcus aureus and coagulase-negative staphylococci. © 2007 Elsevier B.V. All rights reserved.

In vitro and in vivo activities of AM-112, a novel oxapenem

AM-112[1′R,5R,6R)-3-(4-amino-1,1-dimethyl-butyl)-6-(1′- hydroxyethyl)oxapenem-3-carboxylatel is a novel oxapenem compound which possesses potent β-lactamase-inhibitory properties. Fifty-percent inhibitory concentrations (IC50s) of AM-112 for class A enzymes were between 0.16 and 2.24 μM for three enzymes, compared to IC50s of 0.008 to 0.12 μM for clavulanic acid. Against class C and class D enzymes, however, the activity of AM-112 was between 1,000- and 100,000-fold greater than that of clavulanic acid. AM-112 had affinity for the penicillin-binding proteins (PBPs) of Escherichia coli DC0, with PBP2 being inhibited by the lowest concentration of AM-112 tested, 0.1 μg/ml. Ceftazidime was combined with AM-112 at 1:1 and 2:1 ratios in MIC determination studies against a panel of β-lactamase-producing organisms. These studies demonstrated that AM-112 was effective at protecting ceftazidime against extended-spectrum β-lactamase-producing strains and derepressed class C enzyme producers, reducing ceftazidime MICs by 16- and 2,048-fold. Similar results were obtained when AM-112 was combined with ceftriaxone, cefoperazone, or cefepime in a 1:2 ratio. Protection of ceftazidime with AM-112 was maintained against Enterobacter cloacae P99 and Klebsiella pneumoniae SHV-5 in a murine intraperitoneal sepsis model. The 50% effective dose of ceftazidime against E. cloacae P99 and K. pneumoniae SHV-5 was reduced from >100 and 160 mg/kg of body weight to 2 and 33.6 mg/kg, respectively, when it was combined with AM-112 at a 1:1 ratio. AM-112 demonstrates potential as a new β-lactamase inhibitor.

Phenotypic and genotypic characterisation of Clostrum Difficile

Clostridium difficile is at present one of the most common nosocomial infections in the developed world. Hypervirulent strains (PCR ribotype 027) of C. difficile which produce enhanced levels of toxins have also been associated with other characteristics such as a greater rate of sporulation and resistance to fluoroquinolones. Infection due to C. difficile PCR ribotype 027 has also been associated with greater rates of morbidity and mortality. The aim of this thesis was to investigate both the phenotypic and genotypic characteristics of two populations of toxigenic clinical isolates of C. difficile which were recovered from two separate hospital trusts within the UK. Phenotypic characterisation of the isolates was undertaken using analytical profile indexes (APIs), minimum inhibitory concentrations(MICs) and S-layer protein typing. In addition to this, isolates were also investigated for the production of a range of extracellular enzymes as potential virulence factors. Genotypic characterisation was performed using a random amplification of polymorphic DNA(RAPD) PCR protocol which was fully optimised in this study, and the gold standard method, PCR ribotyping. The discriminatory power of both methods was compared and the similarity between the different isolates also analysed. Associations between the phenotypic and genotypic characteristics and the recovery location of the isolate were then investigated. Extracellular enzyme production and API testing revealed little variation between the isolates; with S-layer typing demonstrating low discrimination. Minimum inhibitory concentrations did not identify any resistance towards either vancomycin or metronidazole; there were however significant differences in the distribution of antibiogram profiles of isolates recovered from the two different trusts. The RAPD PCR protocol was successfully optimised and alongside PCR ribotyping, effectively typed all of the clinical isolates and also identified differences in the number of types defined between the two locations. Both PCR ribotyping and RAPD demonstrated similar discriminatory power; however, the two genotyping methods did not generate amplicons that mapped directly onto each other and therefore clearly characterised isolates based on different genomic markers. The RAPD protocol also identified different subtypes within PCR ribotypes, therefore demonstrating that all isolates defined as a particular PCR ribotype were not the same strain. No associations could be demonstrated between the phenotypic and genotypic characteristics observed; however, the location from which an isolate was recovered did appear to influence antibiotic resistance and genotypic characteristics. The phenotypic and genotypic characteristics observed amongst the C. difficile isolates in this study, may provide a basis for the identification of further targets which may be potentially incorporated into future methods for the characterisation of C. difficile isolates.

Sensitivity of staphylococcus epidermidis to chlorhexidine and associated resistance properties

Staphylococcus epidermidis are common Gram-positive bacteria and are responsible for a number of life-threatening nosocomial infections. Treatment of S. epidermidis infection is problematic because the organism is usually resistant to many antibiotics. The high degree of resistance of this organism to a range of antibiotics and disinfectants is widely known. The aims of this thesis were to investigate and evaluate the susceptibility of isolates of S. epidermidis from various infections to chlorhexidine (CHX) and to other disinfectants such as benzalkonium chloride (BKC), triclosan (TLN) and povidone-iodine (PI). In addition, the mechanisms of resistance of S. epidermidis to chlorhexidine (the original isolates and strains adapted to chlorhexidine by serial passage) were examined and co-resistance to clinically relevant antibiotics investigated. In 3 of the 11 S. epidermidis strains passaged in increasing concentrations of chlorhexidine, resistance to the disinfectant arose (16-fold). These strains were examined further, each showing stable chlorhexidine resistance. Co-resistance to other disinfectants such as BKC, TLN and PI and changes in cell surface hydrophobicity were observed. Increases in resistance were accompanied by an increase in the proportion of neutral lipids and phospholipids in the cell membrane. This increase was most marked in diphosphatidylglycerol. These observations suggest that some strains of S. epidermidis can become resistant to chlorhexidine and related disinfectants/antiseptics by continual exposure. The mechanisms of resistance appear to be related to changes in membrane lipid compositions.

Antibacterial activity of low amperage and low voltage electric current (DC)

Infection is a major clinical problem associated with the use of intravenous catheters.The efficacy of a direct electric current (10µA, 9V) via electrode-conducting carbon impregnated catheters to prevent colonisation of catheters by micro-organisms was investigated. The range of organisms susceptible to 10µA was determined by a zone of inhibition test. The catheters acting as the anode and the cathode were inserted into a nutrient agar plate inoculated with a lawn of bacteria. There was no zone of inhibition observed around the anode. Organisms susceptible to 10µA at the cathode were Staphylococcus aureus (2 strains), Staphylococcus epidermidis (5 strains), Escherichia coli and Klebsiella pneumoniae (2 strains each), and one strain of the following micro-organisms: Staphylococcus hominis, Proteus mirabilis, Pseudomonas aeruginosa and Candida albicans. The zones ranged from 6 to 16 mm in diameter according to the organisms under test. The zone size was proportional to the amperage (10 - 100 µA) and the number of organisms on the plate. Ten µA did not prevent adhesion of staphylococci to the cathode nor did it affect their growth in nutrient broth. However, it was bactericidal to adherent bacteria on the cathodal catheter and significantly reduced the number of bacteria on the catheter after 4 to 24 h application of electricity. The antimicrobial activity of low amperage electric current under anaerobic conditions and in the absence of chloride ions against bacteria attached to the surface of a current carrying electrode was also investigated.The mechanisms of the bactericidal activity associated with the cathode were investigated with S. epidermidis and S. aureus. The inhibition zone was greatly reduced in the presence of catalase. There was no zone around the cathode when the test was carried out under anaerobic conditions. Hydrogen peroxide was produced at the cathode surface under aerobic conditions, but not in the absence of oxygen. A salt-bridge apparatus was used to demonstrate further that hydrogen peroxide was produced at the cathode, and chlorine at the anode. The antimicrobial activity of low amperage electric current under anaerobic conditions and in the absence of chloride ions against bacteria attached to the surface of a current carrying electrode was also investigated. Antibacterial activity was reduced under anaerobic conditions, which is compatible with the role of hydrogen peroxide as a primary bactericidal agent of electricity associated with the cathode. A reduction in chloride ions did not significantly reduce the antibacterial activity suggesting chlorine plays only a minor role in the bactericidal activity against organisms attached to anodal electrode surfaces. The bactericidal activity of electric current associated with the cathode and H202 was greatly reduced in the presence of 50 μM to 0.5 mM magnesium ions in the test menstrum. Ten μA applied via the catheters did not prevent the initial biofilm growth by the adherent bacteria but reduced the number of bacteria in the biofilm by 2 log order aiter 24 h. The results suggested that 10 μA may prevent the colonisation of catheters by both the extra~ and intra-luminal routes. The localised production of hydrogen peroxide and chlorine and the intrinsic activity due to electric current may offer a useful method for the eradication of bacteria from catheter surfaces.

RAPD for the typing of coagulase-negative staphylococci implicated in catheter-related bloodstream infection

Objectives: A rapid random amplification of polymorphic DNA (RAPD) technique was developed to distinguish between strains of coagulase-negative staphylococci (CoNS) involved in central venous catheter (CVC)-related bloodstream infection. Its performance was compared with that of pulsed-field gel electrophoresis (PFGE). Methods: Patients at the University Hospital Birmingham NHS Foundation Trust, U.K. who underwent stem cell transplantation and were diagnosed with CVC-related bloodstream infection due to CoNS whilst on the bone marrow transplant unit were studied. Isolates of CoNS were genotyped by PFGE and RAPD, the latter employing a single primer and a simple DNA extraction method. Results: Both RAPD and PFGE were highly discriminatory (Simpson's diversity index, 0.96 and 0.99, respectively). Within the 49 isolates obtained from blood cultures of 33 patients, 20 distinct strains were identified by PFGE and 25 by RAPD. Of the 25 strains identified by RAPD, nine clusters of CoNS contained isolates from multiple patients, suggesting limited nosocomial spread. However, there was no significant association between time of inpatient stay and infection due to any particular strain. Conclusion: The RAPD technique presented allows CoNS strains to be genotyped with high discrimination within 4 h, facilitating real-time epidemiological investigations. In this study, no single strain of CoNS was associated with a significant number of CVC-related bloodstream infections. © 2005 Published by Elsevier Ltd on behalf of the British Infection Society.