Isolation and characterisation of bacteria associated with flying insects in hospitals, with particualr emphasis on Clostridum difficile

Clostridium difficile is a bacterial healthcare-associated infection, which houseflies Musca domestica may transfer due to their synanthropic nature. The aims of this thesis were to determine the ability of M. domestica to transfer C. difficile mechanically and to collect and identify flying insects in UK hospitals and classify any associated bacteria. M. domestica exposed to independent suspensions of vegetative cells and spores of C. difficile were able to mechanically transfer the bacteria on to agar for up to 4 hours following exposure. C. difficile could be recovered from fly excreta for 96hrs and was isolated from the M. domestica alimentary canal. Also confirmed was the carriage of C. difficile by M. domestica larvae, although it was not retained in the pupae or in the adults that subsequently developed. Flying insects were collected from ultra-violet light flytraps in hospitals. Flies (order Diptera) were the most commonly identified. Chironomidae were the most common flies, Calliphora vicina were the most common synanthropic fly and ‘drain flies’ were surprisingly numerous and represent an emerging problem in hospitals. External washings and macerates of flying insects were prepared and inoculated onto a variety of agars and following incubation bacterial colonies identified by biochemical tests. A variety of flying insects, including synanthropic flies (e.g. M. domestica and C. vicina) collected from UK hospitals harboured pathogenic bacteria of different species. Enterobacteriaceae were the group of bacteria most commonly isolated, followed by Bacillus spp, Staphylococci, Clostridia, Streptococci and Micrococcus spp. This study highlights the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals. Also illustrated is the potential for flying insects to contribute to environmental persistence and spread of other pathogenic bacteria in hospitals and therefore the need to implement pest control as part of infection control strategies.

The housefly Musca domestica as a mechanical vector of Clostridium difficile

Background - Clostridium difficile is a bacterial healthcare-associated infection that may be transferred by houseflies (Musca domestica) due to their close ecological association with humans and cosmopolitan nature. Aim - To determine the ability of M. domestica to transfer C. difficile both mechanically and following ingestion. Methods - M. domestica were exposed to independent suspensions of vegetative cells and spores of C. difficile, then sampled on to selective agar plates immediately postexposure and at 1-h intervals to assess the mechanical transfer of C. difficile. Fly excreta was cultured and alimentary canals were dissected to determine internalization of cells and spores. Findings - M. domestica exposed to vegetative cell suspensions and spore suspensions of C. difficile were able to transfer the bacteria mechanically for up to 4 h upon subsequent contact with surfaces. The greatest numbers of colony-forming units (CFUs) per fly were transferred immediately following exposure (mean CFUs 123.8 +/− 66.9 for vegetative cell suspension and 288.2 +/− 83.2 for spore suspension). After 1 h, this had reduced (21.2 +/− 11.4 for vegetative cell suspension and 19.9 +/− 9 for spores). Mean C. difficile CFUs isolated from the M. domestica alimentary canal was 35 +/− 6.5, and mean C. difficile CFUs per faecal spot was 1.04 +/− 0.58. C. difficile could be recovered from fly excreta for up to 96 h. Conclusion - This study describes the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals, highlighting flies as realistic vectors of this micro-organism in clinical areas.

Studies on enhanced surface disinfection and skin antisepsis using chlorhexidine and eucalyptus oil

Healthcare associated infections may arise from many sources, including patient?s own skin flora and the clinical environment, and inflict a significant burden within the health service. Adequate and effective skin antisepsis and surface disinfection are therefore essential factors in infection control. Current EPIC guidelines recommend 2 % chlorhexidine (CHG) in 70 % isopropyl alcohol (IPA) for skin antisepsis however poor penetration has been reported. Eucalyptus oil (EO) is a known permeation enhancer, producing synergistic antimicrobial activity when combined with CHG. In this current study, the antimicrobial efficacy of EO and its main constituent 1,8-cineole were assessed against a panel of clinically relevant microorganisms, alone and in combination with CHG. The superior antimicrobial efficacy of EO compared with 1,8-cineole, and synergistic effects with CHG against planktonic and biofilm cultures, confirmed its suitability for use in subsequent studies within this thesis. Impregnation of EO, CHG and IPA onto prototype hard surface disinfectant wipes demonstrated significantly improved efficacy compared with CHG/IPA wipes, with clear reductions in the time required to eliminate biofilms. Optimisation of the EO/CHG/IPA formulation resulted in the development of Euclean® wipes, with simulated-use and time kill studies confirming their ability to remove microbial surface contamination, prevent cross contamination and eliminate biofilms within 10 minutes. The employment of isothermal calorimetry provided additional information on the type and rate of antimicrobial activity possessed by Euclean® wipes. A clinical audit of the Euclean® wipes at Birmingham Children?s Hospital, Birmingham, U.K. revealed divided staff opinion, with the highest cited advantage and disadvantage concerning the odour. Finally, skin penetration and cell toxicity studies of EO/CHG biopatches and Euclean® solution developed during this study, revealed no permeation into human skin following biopatch application, and no significant toxicity. These current studies enhance the knowledge regarding EO and its potential applications.

The source, cost and prevention of central venous catheter-related infection

Central venous catheters have become an integral part of patient management however they are associated with many complications including infection. Despite efforts being made to reduce the incidence of such infect ions the problem continues to increase and has resource implications for the Health Service. Studies relating to the source of microorganisms causing CVC-associated infection, the cost of such infections and the efficacy of an antimicrobial catheter have been undertaken. Thirty patients who required a CVC as part of their medical management and underwent cardiac surgery had the distal tips of their catheters sampled whilst in situ. Sampling took place within 1 h of catheter placement. Bacteria were isolated from 16% of the catheter distal tips sampled in situ. The guidewires used to insert the devices were also contaminated (50%). When CVC were inserted via a protective sheath, avoiding contact with the skin. the incidence of microbial contamination was reduced. These findings suggest that despite rigorous skin disinfection and strict aseptic technique, viable microorganisms are impacted onto the distal tip of CVC during the insertion procedure. Needleless intravascular access devices have been introduced in order to reduce the incidence of need1estick injury. However, it was unclear whether such connectors would act as a portal of entry for microorganisms to CVC. The efficacy of these devices was investigated. Within the controlled laboratory environment it was demonstrated that needleless devices, when challenged with microorganisms, did not allow the passage of microbes when flu id was injected. This therefore suggested that the devices should not increase the risk of catheter colonisation. When used in clinical practice however microbial contamination of the needleless connectors was 55 % in comparison to the routinely used luer connectors (23%). The cost of infections associated with CVC was determined. Twenty patients catheterised with a CVC designed for long term use who were admitted to hospital with a presumptive diagnosis of catheter-related infection were studied. The treatment given specifically for this infection was costed. The mean cost of such an infection was £ 1781.81. Throughout the UK this may amount to £1.565.906 per annum. The cost of infections associated with CVC designed for short term use was estimated to be between 5 and 7 million pounds per annum in the UK. In an attempt to reduce both the incidence and cost of catheter- related infection antimicrobial CVC have been developed. The efficacy of a novel polyurethane CVC impregnated on both the internal and external catheter surface with the quaternary ammonium compound benzalkonium chloride was investigated. Eighty eight patients received an antimicrobial catheter and 78 patients a conventional polyurethane CVC. The anti-microbial CVC resulted in a reduction in microbial colonisation of the external and internal polymer surfaces as compared to the control device. The observed reduction in microbial colonisation with the anti-microbial CVC may decrease the likelihood of subsequent infection offering a useful approach to the prevention of catheter-related infections.

Role of copper in reducing hospital environment contamination

The environment may act as a reservoir for pathogens that cause healthcare-associated infections (HCAIs). Approaches to reducing environmental microbial contamination in addition to cleaning are thus worthy of consideration. Copper is well recognised as having antimicrobial activity but this property has not been applied to the clinical setting. We explored its use in a novel cross-over study on an acute medical ward. A toilet seat, set of tap handles and a ward entrance door push plate each containing copper were sampled for the presence of micro-organisms and compared to equivalent standard, non-copper-containing items on the same ward. Items were sampled once weekly for 10 weeks at 07:00 and 17:00. After five weeks, the copper-containing and non-copper-containing items were interchanged. The total aerobic microbial counts per cm2 including the presence of ‘indicator micro-organisms’ were determined. Median numbers of microorganisms harboured by the copper-containing items were between 90% and 100% lower than their control equivalents at both 07:00 and 17:00. This reached statistical significance for each item with one exception. Based on the median total aerobic cfu counts from the study period, five out of ten control sample points and zero out of ten copper points failed proposed benchmark values of a total aerobic count of <5 cfu/cm2. All indicator micro-organisms were only isolated from control items with the exception of one item during one week. The use of copper-containing materials for surfaces in the hospital environment may therefore be a valuable adjunct for the prevention of HCAIs and requires further evaluation.

Chlorhexidine and the prevention of surgical site infection

Surgical site infections (SSI) are a prevalent health care-associated infection (HAl). Prior to the mid-19th century, surgical sites commonly developed postoperative wound complications. It was in the 1860's, after Joseph Lister introduced carbolic acid and the principles of antisepsis that postoperative wound infection significantly decreased. Today, patient preoperative skin preparation with an antiseptic agent prior to surgery is a standard of practice. Povidone-iodine and chlorhexidine gluconate are currently the most commonly used antimicrobial agents used to prep the patient's skin. In this current study, the epidemiology, diagnosis, surveillance and prevention of SSI with chlorhexidine were investigated. The antimicrobial activity of chlorhexidine was assessed. In in-vitro and in-vivo studies the antimicrobial efficacy of 2% (w/v) chlorhexidine gluconate (CHG) in 70% isopropyl alcohol (IPA) and 10% povidoneiodine (PVP-I) in the presence of 0.9% normal saline or blood were examined. The 2% CHG in 70% IPA solutions antimicrobial activity was not diminished in the presence of 0.9% normal saline or blood. In comparison, the traditional patient preoperative skin preparation, 10% PVP-I antimicrobial activity was not diminished in the presence of 0.9% normal saline, but was diminished in the presence of blood. In an in-vivo human volunteer study the potential for reduction of the antimicrobial efficacy of aqueous patient preoperative skin preparations compromised by mechanical removal of wet product from the application site (blot) was assessed. In this evaluation, 2% CHG and 10% povidone-iodine (PVP-I) were blotted from the patient's skin after application to the test site. The blotting, or mechanical removal, of the wet antiseptic from the application site did not produce a significant difference in product efficacy. In a clinical trial to compare 2% CHG in 70% IPA and PVP-! scrub and paint patient preoperative skin preparation for the prevention of SSI, there were 849 patients randomly assigned to the study groups (409 in the chlorhexidine-alcohol and 440 in the povidone-iodine group) in the intention-to-treat analysis. The overall surgical site infection was significantly lower in the 2% CHG in 70% IPA group than in the PVP-I group (9.5% versus 16.1 %, p=0.004; relative risk, 0.59 with 95% confidence interval of 0.41 to 0.85). Preoperative cleansing of the patient's skin with chlorhexidine-alcohol is superior to povidone-iodine in preventing surgical site infection after clean-contaminated surgery.

Gram-positive infections in patients with end-stage renal disease

Gram-positive microorganisms, specifically coagulase-negative staphylococci are the most common species recovered from clinical culture specimens of patients with end-stage renal disease. The propensity of coagulase-negative staphylococci (CNS) to cause infection in this patient group has been widely debated. However, it is still unclear how this usually avirulent commensal microorganism produces infection that contributes to high rates of morbidity and mortality in patients with end-stage renal disease. The aim of this thesis was to investigate the rate, geographical distribution, molecular and phenotypic mechanisms of Gram-positive microorganisms associated with infection in renal dialysis patients. In addition, it sought to assess the value of early serological diagnosis of dialysis catheter-associated infection and the effect of antimicrobial treatment regimens on the faecal carriage of enteric microorganisms. In this study, the incidence of haemodialysis catheter-associated infection was established with the Meditrend audit tool. This tool was used to assess the infection outcomes of catheter insertion and management procedures until the catheter was explanted. Introduction of a catheter management protocol decreased the incidence of catheter-related infection. Staphylococcal species recovered from episodes of haemodialysis catheter-associated infection and continuous ambulatory peritoneal dialysis (CAPD)-associated peritonitis were genotyped by determination of macrorestriction profiles with pulsed-field gel electrophoresis. This highlighted horizontal transfer of microorganisms between different patients and the environment. The phenotypic characteristics of these strains were also investigated to determine characteristics that could be used as markers for dialysis catheter-associated infection. The expression of elastase, lipase and esterase by CNS was significantly associated with infection. A rapid enzyme-linked immunosorbent assay incorporating a novel staphylococcal antigen (lipid S) was used to evaluate the early detection of anti-staphylococcal immunoglobulin gamma in patient sera. The comparison of culture positive and culture negative patients demonstrated a steady state of immune activation in both groups. However anti-lipid S serum antibody titres > 1000 were found to be a predictor of infection. The effect on faecal carriage of vancomycin resistant enterococci (VRE) and Clostridium difficile toxins in patients treated with CAPD when empiric cephalosporin therapy was substituted for piperacillin/tazobactam was investigated. The introduction of piperacillin/tazobactam demonstrated a decrease in the faecal carriage of VRE.

Non-curliation of Escherichia coli O78:K80 isolates associated with IS1 insertion in csgB and reduced persistence in poultry infection

The elaboration of curli fimbriae by Escherichia coli is associated with the development of a lacy colony morphology when grown on colonisation factor antigen agar at 25 degrees C. Avian colisepticaemia E. coli isolates screened for curliation by this culture technique showed lacy and smooth colonial morphologies and the genetic basis of the non-curliated smooth colonial phenotype was analysed. Two smooth E. coli O78:K80 isolates possessed about 40 copies of the IS1 element within their respective genomes of which one copy insertionally inactivated the csgB gene, the nucleator gene for curli fibril formation. One of these two isolates also possessed a defective rpoS gene which is a known regulator of curli expression. In the day-old chick model, both smooth isolates were as invasive as a known virulent O78:K80 isolate as determined by extent of liver and spleen colonisation post oral inoculation but were less persistent in terms of caecal colonisation.

Infection risk associated with a closed luer access device

The potential for microbial contamination associated with a recently developed needleless closed luer access device (CLAD) (Q-Syte™ Becton Dickinson, Sandy, UT, USA) was evaluated in vitro. Compression seals of 50 multiply activated Q-Syte devices were inoculated with Staphylococcus epidermidis NCTC 9865 in 25% (v/v) human blood and then disinfected with 70% (v/v) isopropyl alcohol followed by flushing with 0.9% (w/v) sterile saline. Forty-eight of 50 (96%) saline flushes passed through devices that had been activated up to a maximum of 70 times remained sterile. A further 25 Q-Syte CLADs that had undergone multiple activations were challenged with prefilled 0.9% (w/v) sterile saline syringes, the external luer tips of which had been inoculated with S. epidermidis NCTC 9865 prior to accessing the devices. None of the devices that had been accessed up to 70 times allowed passage of micro-organisms, despite challenge micro-organisms being detected on both the syringe tip after activation and the compression seals before decontamination. These findings suggest that the Q-Syte CLAD may be activated up to 70 times with no increased risk of microbial contamination within the fluid pathway. The device may also offer protection from the external surface of syringe tips contaminated with micro-organisms. © 2005 Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

A randomized, prospective clinical trial to assess the potential infection risk associated with the PosiFlow® needleless connector

The microbial contamination rate of luers of central venous catheters (CVCs) with either PosiFlow® needleless connectors or standard caps attached was investigated. The efficacy of 70% (v/v) isopropyl alcohol, 0.5% (w/v) chlorhexidine in gluconate 70% (v/v) isopropyl alcohol and 10% (w/v) aqueous povidone-iodine to disinfect the intravenous connections was also assessed. Seventy-seven patients undergoing cardiac surgery who required a CVC as part of their clinical management were randomly allocated either needleless connectors or standard caps. Patients were also designated to receive chlorhexidine/alcohol, isopropyl alcohol or povidone-iodine for pre-CVC insertion skin preparation and disinfection of the connections. After 72 h in situ the microbial contamination rate of 580 luers, 306 with standard caps and 274 with needleless connectors attached, was determined. The microbial contamination rate of the external compression seals of 274 needleless connectors was also assessed to compare the efficacy of the three disinfectants. The internal surfaces of 55 out of 306 (18%) luers with standard caps were contaminated with micro-organisms, whilst only 18 out of 274 (6.6%) luers with needleless connectors were contaminated (P<0.0001). Of those needleless connectors disinfected with isopropyl alcohol, 69.2% were externally contaminated with micro-organisms compared with 30.8% disinfected with chlorhexidine/alcohol (P<0.0001) and 41.6% with povidone-iodine (P<0.0001). These results suggest that the use of needleless connectors may reduce the microbial contamination rate of CVC luers compared with the standard cap. Furthermore, disinfection of needleless connectors with either chlorhexidine/alcohol or povidone-iodine significantly reduced external microbial contamination. Both these strategies may reduce the risk of catheter-related infections acquired via the intraluminal route. © 2003 The Hospital Infection Society. Published by Elsevier Science Ltd. All rights reserved.