Anna M. Snelling

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.

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.

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.

Synthesis of citrate-ciprofloxacin conjugates.

Two regioisomeric citrate-functionalized ciprofloxacin conjugates have been synthesized and their antimicrobial activities against a panel of clinically-relevant bacteria have been determined. Cellular uptake mechanisms were investigated using wild-type and ompF deletion strains of Escherichia coli K-12.

Comparative evaluation of the hygienic efficacy of an ultra-rapid hand dryer vs conventional warm air hand dryers

Aims:  To compare an ultra‐rapid hand dryer against warm air dryers, with regard to: (A) bacterial transfer after drying and (B) the impact on bacterial numbers of rubbing hands during dryer use.

Acinetobacter spp. and the Clinical Environment

Infections associated with Acinetobacter spp. are an increasing problem in hospitals around the world, with many infections being multiply resistant to antibiotics. Acinetobacter spp. are particularly hardy and can survive on dry surfaces for long periods of time. They have also been cultured from the air on hospital wards. While it is known that environmental factors can contribute to the spread of infections caused by Acinetobacter spp., the precise role of the clinical environment in this process remains unclear. This review paper investigates this issue and seeks to identify environmental factors which may influence the spread of infections associated with Acinetobacter spp.

The commonly-used DNA probe for diffusely-adherent Escherichia coli cross-reacts with a subset of enteroaggregative E. coli

BackgroundThe roles of diffusely-adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) in disease are not well understood, in part because of the limitations of diagnostic tests for each of these categories of diarrhoea-causing E. coli. A HEp-2 adherence assay is the Gold Standard for detecting both EAEC and DAEC but DNA probes with limited sensitivity are also employed.ResultsWe demonstrate that the daaC probe, conventionally used to detect DAEC, cross-reacts with a subset of strains belonging to the EAEC category. The cross hybridization is due to 84% identity, at the nucleotide level, between the daaC locus and the aggregative adherence fimbriae II cluster gene, aafC, present in some EAEC strains. Because aaf-positive EAEC show a better association with diarrhoea than other EAEC, this specific cross-hybridization may have contributed to an over-estimation of the association of daaC with disease in some studies. We have developed a discriminatory PCR-RFLP protocol to delineate EAEC strains detected by the daaC probe in molecular epidemiological studies.ConclusionsA PCR-RFLP protocol described herein can be used to identify aaf-positive EAEC and daaC-positive DAEC and to delineate these two types of diarrhoeagenic E. coli, which both react with the daaC probe. This should help to improve current understanding and future investigations of DAEC and EAEC epidemiology.

Probing linker design in citric acid-ciprofloxacin conjugates.

A series of structurally related citric acid-ciprofloxacin conjugates was synthesised to investigate the influence of the linker between citric acid and ciprofloxacin on antibacterial activities. Minimum inhibitory concentrations (MICs) were determined against a panel of reference strains and clinical isolates of bacteria associated with infection in humans and correlated with the DNA gyrase inhibitory activity. The observed trend was rationalised by computational modelling.

IS3 profiling identifies the enterohaemorrhagic Escherichia coli O-island 62 in a distinct enteroaggregative E. coli lineage

BackgroundEnteroaggregative Escherichia coli (EAEC) are important diarrhoeal pathogens that are defined by a HEp-2 adherence assay performed in specialist laboratories. Multilocus sequence typing (MLST) has revealed that aggregative adherence is convergent, providing an explanation for why not all EAEC hybridize with the plasmid-derived probe for this category, designated CVD432. Some EAEC lineages are globally disseminated or more closely associated with disease.ResultsTo identify genetic loci conserved within significant EAEC lineages, but absent from non-EAEC, IS3-based PCR profiles were generated for 22 well-characterised EAEC strains. Six bands that were conserved among, or missing from, specific EAEC lineages were cloned and sequenced. One band corresponded to the aggR gene, a plasmid-encoded regulator that has been used as a diagnostic target but predominantly detects EAEC bearing the plasmid already marked by CVD432. The sequence from a second band was homologous to an open-reading frame within the cryptic enterohaemorrhagic E. coli (EHEC) O157 genomic island, designated O-island 62. Screening of an additional 46 EAEC strains revealed that the EHEC O-island 62 was only present in those EAEC strains belonging to the ECOR phylogenetic group D, largely comprised of sequence type (ST) complexes 31, 38 and 394.ConclusionsThe EAEC 042 gene orf1600, which lies within the EAEC equivalent of O-island 62 island, can be used as a marker for EAEC strains belonging to the ECOR phylogenetic group D. The discovery of EHEC O-island 62 in EAEC validates the genetic profiling approach for identifying conserved loci among phylogenetically related strains.