Kevin G. Kerr

Microbiological and Clinical Aspects of Infection Associated with Stenotrophomonas maltophilia

SUMMARY The gram-negative bacterium Stenotrophomonas maltophilia is increasingly recognized as an important cause of nosocomial infection. Infection occurs principally, but not exclusively, in debilitated and immunosuppressed individuals. Management of S. maltophilia-associated infection is problematic because many strains of the bacterium manifest resistance to multiple antibiotics. These difficulties are compounded by methodological problems in in vitro susceptibility testing for which there are, as yet, no formal guidelines. Despite its acknowledged importance as a nosocomial pathogen, little is known of the epidemiology of S. maltophilia, and although it is considered an environmental bacterium, its sources and reservoirs are often not readily apparent. Molecular typing systems may contribute to our knowledge of the epidemiology of S. maltophilia infection, thus allowing the development of strategies to interrupt the transmission of the bacterium in the hospital setting. Even less is known of pathogenic mechanisms and putative virulence factors involved in the natural history of S. maltophilia infection and this, coupled with difficulties in distinguishing colonization from true infection, has fostered the view that the bacterium is essentially nonpathogenic. This article aims to review the current taxonomic status of S. maltophilia, and it discusses the laboratory identification of the bacterium. The epidemiology of the organism is considered with particular reference to nosocomial outbreaks, several of which have been investigated by molecular typing techniques. Risk factors for acquisition of the bacterium are also reviewed, and the ever-expanding spectrum of clinical syndromes associated with S. maltophilia is surveyed. Antimicrobial resistance mechanisms, pitfalls in in vitro susceptibility testing, and therapy of S. maltophilia infections are also discussed.

Pseudomonas aeruginosa: a formidable and ever-present adversary

Pseudomonas aeruginosa is a versatile pathogen associated with a broad spectrum of infections in humans. In healthcare settings the bacterium is an important cause of infection in vulnerable individuals including those with burns or neutropenia or receiving intensive care. In these groups morbidity and mortality attributable to P. aeruginosa infection can be high. Management of infections is difficult as P. aeruginosa is inherently resistant to many antimicrobials. Furthermore, treatment is being rendered increasingly problematic due to the emergence and spread of resistance to the few agents that remain as therapeutic options. A notable recent development is the acquisition of carbapenemases by some strains of P. aeruginosa. Given these challenges, it would seem reasonable to identify strategies that would prevent acquisition of the bacterium by hospitalised patients. Environmental reservoirs of P. aeruginosa are readily identifiable, and there are numerous reports of outbreaks that have been attributed to an environmental source; however, the role of such sources in sporadic pseudomonal infection is less well understood. Nevertheless there is emerging evidence from prospective studies to suggest that environmental sources, especially water, may have significance in the epidemiology of sporadic P. aeruginosa infections in hospital settings, including intensive care units. A better understanding of the role of environmental reservoirs in pseudomonal infection will permit the development of new strategies and refinement of existing approaches to interrupt transmission from these sources to patients.

Aerial Dissemination of Clostridium difficile spores

BackgroundClostridium difficile-associated diarrhoea (CDAD) is a frequently occurring healthcare-associated infection, which is responsible for significant morbidity and mortality amongst elderly patients in healthcare facilities. Environmental contamination is known to play an important contributory role in the spread of CDAD and it is suspected that contamination might be occurring as a result of aerial dissemination of C. difficile spores. However previous studies have failed to isolate C. difficile from air in hospitals. In an attempt to clarify this issue we undertook a short controlled pilot study in an elderly care ward with the aim of culturing C. difficile from the air.MethodsIn a survey undertaken during February (two days) 2006 and March (two days) 2007, air samples were collected using a portable cyclone sampler and surface samples collected using contact plates in a UK hospital. Sampling took place in a six bedded elderly care bay (Study) during February 2006 and in March 2007 both the study bay and a four bedded orthopaedic bay (Control). Particulate material from the air was collected in Ringers solution, alcohol shocked and plated out in triplicate onto Braziers CCEY agar without egg yolk, but supplemented with 5 mg/L of lysozyme. After incubation, the identity of isolates was confirmed by standard techniques. Ribotyping and REP-PCR fingerprinting were used to further characterise isolates.ResultsOn both days in February 2006, C. difficile was cultured from the air with 23 samples yielding the bacterium (mean counts 53 – 426 cfu/m3 of air). One representative isolate from each of these was characterized further. Of the 23 isolates, 22 were ribotype 001 and were indistinguishable on REP-PCR typing. C. difficile was not cultured from the air or surfaces of either hospital bay during the two days in March 2007.ConclusionThis pilot study produced clear evidence of sporadic aerial dissemination of spores of a clone of C. difficile, a finding which may help to explain why CDAD is so persistent within hospitals and difficult to eradicate. Although preliminary, the findings reinforce concerns that current C. difficile control measures may be inadequate and suggest that improved ward ventilation may help to reduce the spread of CDAD in healthcare facilities.

The ventilation of multiple-bed hospital wards: review and analysis.

BACKGROUND Although the merits of ventilating operating theatres and isolation rooms are well known, the clinical benefits derived from ventilating hospital wards and patient rooms are unclear. This is because relatively little research work has been done in the ventilation of these areas compared with that done in operating theatres and isolation rooms. Consequently, there is a paucity of good quality data from which to make important decisions regarding hospital infrastructure. This review evaluates the role of general ward ventilation to assess whether or not it affects the transmission of infection. METHODS A critical review was undertaken of guidelines in the United Kingdom and United States governing the design of ventilation systems for hospital wards and other multibed rooms. In addition, an analytical computational fluid dynamics (CFD) study was performed to evaluate the effectiveness of various ventilation strategies in removing airborne pathogens from ward spaces. RESULTS The CFD simulation showed the bioaerosol concentration in the study room to be substantially lower (2467 cfu/m(3)) when air was supplied and extracted through the ceiling compared with other simulated ventilations strategies, which achieved bioaerosol concentrations of 12487 and 10601 cfu/m(3), respectively. CONCLUSIONS There is a growing body of evidence that the aerial dispersion of some nosocomial pathogens can seed widespread environmental contamination, and that this may be contributing to the spread infection in hospital wards. Acinetobacter spp in particular appear to conform to this model, with numerous outbreaks attributed to aerial dissemination. This suggests that the clinical role of general ward ventilation may have been underestimated and that through improved ward ventilation, it may be possible to reduce environmental contamination and thus reduce nosocomial infection rates.

Bactericidal action of positive and negative ions in air

BackgroundIn recent years there has been renewed interest in the use of air ionisers to control of the spread of airborne infection. One characteristic of air ions which has been widely reported is their apparent biocidal action. However, whilst the body of evidence suggests a biocidal effect in the presence of air ions the physical and biological mechanisms involved remain unclear. In particular, it is not clear which of several possible mechanisms of electrical origin (i.e. the action of the ions, the production of ozone, or the action of the electric field) are responsible for cell death. A study was therefore undertaken to clarify this issue and to determine the physical mechanisms associated with microbial cell death.ResultsIn the study seven bacterial species (Staphylococcus aureus, Mycobacterium parafortuitum, Pseudomonas aeruginosa, Acinetobacter baumanii, Burkholderia cenocepacia, Bacillus subtilis and Serratia marcescens) were exposed to both positive and negative ions in the presence of air. In order to distinguish between effects arising from: (i) the action of the air ions; (ii) the action of the electric field, and (iii) the action of ozone, two interventions were made. The first intervention involved placing a thin mica sheet between the ionisation source and the bacteria, directly over the agar plates. This intervention, while leaving the electric field unaltered, prevented the air ions from reaching the microbial samples. In addition, the mica plate prevented ozone produced from reaching the bacteria. The second intervention involved placing an earthed wire mesh directly above the agar plates. This prevented both the electric field and the air ions from impacting on the bacteria, while allowing any ozone present to reach the agar plate. With the exception of Mycobacterium parafortuitum, the principal cause of cell death amongst the bacteria studied was exposure to ozone, with electroporation playing a secondary role. However in the case of Mycobacterium parafortuitum, electroporation resulting from exposure to the electric field appears to have been the principal cause of cell inactivation.ConclusionThe results of the study suggest that the bactericidal action attributed to negative air ions by previous researchers may have been overestimated.

Survival and Growth of Burkholderia cepacia Within the Free-Living Amoeba Acanthamoeba polyphaga

Abstract The aim of this study was to investigate whether Burkholderia cepacia is capable of survival and growth within the free-living amoeba Acanthamoeba polyphaga using a differential immunofluorescence assay of bacterial-amoebal cocultures and viable counts of bacteria determined after amoebal lysis. The numbers of intra-amoebal bacteria and the numbers of infected amoebae increased over time; although, when heat-killed bacteria were used, no intracellular bacteria were observed. These findings should be taken into account in future studies of environmental reservoirs of Burkholderia cepacia.

Staphyloferrin A as siderophore-component in fluoroquinolone-based Trojan horse antibiotics

A series of fluoroquinolone conjugates was synthesised by linking the carboxylic acid functionality of the carboxylate-type siderophore staphyloferrin A and its derivatives to the piperazinyl nitrogen of ciprofloxacin and norfloxacin via amide bond formation. Four siderophore-drug conjugates were screened against a panel of bacteria associated with infection in humans. Whilst no activity was found against ciprofloxacin- or norfloxacin-resistant bacteria, one of the conjugates retained antibacterial activity against fluoroquinolone-susceptible strains although the structure of its lysine-based siderophore component differs from that of the natural siderophore staphyloferrin A. In contrast, three ornithine-based siderophore conjugates showed significantly reduced activity against strains that are susceptible to their respective parent fluoroquinolones, regardless of the type of fluoroquinolone attached or chirality at the ornithine Cα-atom. The loss of potency observed for the (R)- and (S)-ornithine-based ciprofloxacin conjugates correlates with their reduced inhibitory activity against the target enzyme DNA gyrase.

Typing of epidemiologically associated environmental and clinical strains of Listeria monocytogenes by random amplification of polymorphic DNA

One hundred and three strains of Listeria monocytogenes (Lm), Listeria seeligeri and Listeria innocua of clinical, food, and environmental origin were examined by generating randomly amplified polymorphic DNA (RAPD). Using one 10 bp and two 13 bp random primers, epidemiologically related strains, previously shown to be indistinguishable by phage typing, yielded identical RAPD profiles. Strains isolated from the hands of three workers in a retail food establishment showed the presence of a single predominant Lm isolate. RAPD analysis is a rapid, reproducible, and relatively inexpensive method for the differentiation of epidemiologically linked isolates of Listeria spp.

The Survival of Listeria monocytogenes on Fingertips and Factors Affecting Elimination of the Organism by Hand Washing and Disinfection

The survival of Listeria monocytogenes applied to the fingertips was investigated using both an impression plate and an elution method. When suspended in saline, L. monocytogenes NCTC 9863 survived for up to 60 min on fingertips, but survival times were greatly extended when the inoculum was suspended in milk. Survival was not apparently affected by skin lipids, the skins normal flora, or the fat content of the milk. Different serotypes displayed similar results for the percentage persistence over a 2-h period when suspended in milk except for an isolate of L. monocytogenes serotype 7 which had a greater percentage survival than other organisms tested. In contrast, Escherichia coli C600 failed to survive for one hour under the same conditions. Hand washing with either soap or a water-based chlorhexidine hand cleanser usually failed to decontaminate fingertips to which an inoculum of 104/CFU per fingertip suspended in milk was applied, but a solution of chlorhexidine gluconate in methanol was found to be effective.

A new selective differential medium for isolation ofStenotrophomonas maltophilia

A new selective differential medium for the isolation ofStenotrophomonas (formerlyXanthomonas) maltophilia was developed. The medium, VIA agar, contained vancomycin, imipenem, and amphotericin B as selective agents and incorporated a mannitol/bromothymol blue indicator system. Compared withXanthomonas maltophilia Selective Medium (XMSM), VIA agar was less inhibitory toStenotrophomonas maltophilia and was more selective than XMSM in preventing the growth of unwanted bacteria from contaminated specimens. Although vancomycin-resistant strains ofEnterococcus faecium may grow on VIA agar, these can be easily distinguished fromStenotrophomonas maltophilia because of mannitol fermentation.