Synthesis and Evaluation of Selected Benzimidazole Derivatives as Potential Antimicrobial Agents.

A library of 53 benzimidazole derivatives, with substituents at positions 1, 2 and 5, were synthesized and screened against a series of reference strains of bacteria and fungi of medical relevance. The SAR analyses of the most promising results showed that the antimicrobial activity of the compounds depended on the substituents attached to the bicyclic heterocycle. In particular, some compounds displayed antibacterial activity against two methicillin-resistant Staphylococcus aureus (MRSA) strains with minimum inhibitory concentrations (MICs) comparable to the widely-used drug ciprofloxacin. The compounds have some common features; three possess 5-halo substituents; two are derivatives of (S)-2-ethanaminebenzimidazole; and the others are derivatives of one 2-(chloromethyl)-1H-benzo[d]imidazole and (1H-benzo[d]imidazol-2-yl)methanethiol. The results from the antifungal screening were also very interesting: 23 compounds exhibited potent fungicidal activity against the selected fungal strains. They displayed equivalent or greater potency in their MIC values than amphotericin B. The 5-halobenzimidazole derivatives could be considered promising broad-spectrum antimicrobial candidates that deserve further study for potential therapeutic applications.

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.

Probing bacterial uptake of glycosylated ciprofloxacin conjugates.

Mono- and disaccharide-functionalised conjugates of the fluoroquinolone antibiotic ciprofloxacin have been synthesised and used as chemical probes of the bacterial uptake of glycosylated ciprofloxacin. Their antimicrobial activities against a panel of clinically relevant bacteria were determined: the ability of these conjugates to inhibit their target DNA gyrase and to be transported into the bacteria was assessed by using in vivo and in vitro assays. The data suggest a lack of active uptake through sugar transporters and that although the addition of monosaccharides is compatible with the inhibition of DNA gyrase, the addition of a disaccharide results in a significant decrease in antimicrobial activity.

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.

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

BACKGROUND: Enteroaggregative 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. RESULTS: To 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. CONCLUSIONS: The 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.

Quantitative analysis of surfactant deposits on human skin by liquid chromatography/electrospray ionisation tandem mass spectrometry.

Surfactants are commonly used as cleansing agents and yet there are concerns that they may also have a role in skin irritation. The lack of suitable methods for the quantitative and qualitative analysis of surfactant deposition on skin has hindered the in-depth investigation of such effects. Here, we report the application of reversed-phase liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) assays for two surfactants commonly used in consumer products, namely sodium lauryl ether sulfate (SLES) and laurylamidopropyl betaine (LAPB), to a baseline study aiming to assess deposition levels on human skin. The linearity of the assays was established at 3-20 ng, with coefficient of variation below 5%. The detection limits were 100 pg for LAPB and 1 ng for SLES; quantitation limits were 500 pg for LAPB and 2.5 ng for SLES. The baseline study was conducted using a panel of 40 healthy volunteers. Skin extract samples were taken in triplicate from forearms, using ethanol. SLES was detected on most volunteers, with 75% of them having SLES deposits in the range of 100-600 ng/cm(2). LAPB was detected on the skin of all volunteers with 85% of them having deposit levels within the concentration range of 1-100 ng/cm(2). These results demonstrate the extent to which commonly used surfactants remain on the skin during the day. The analytical methods reported here can be applied to the investigation of surfactants in relation to general skin condition and to the development and optimisation of new consumer wash products.

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

BACKGROUND: The 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. RESULTS: We 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. CONCLUSIONS: A 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.

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.

Aerial dissemination of Clostridium difficile spores.

BACKGROUND: Clostridium 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. METHODS: In 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 Ringer's solution, alcohol shocked and plated out in triplicate onto Brazier's 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. RESULTS: On 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. CONCLUSION: This 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.

Commensal bacteria do translocate across the intestinal barrier in surgical patients.

BACKGROUND: The "gut origin of sepsis" hypothesis proposes that enteric bacteria may cause sepsis at distant extra-intestinal sites. Whilst there is much circumstantial evidence to support this hypothesis, there is no conclusive proof in humans. The nature of translocating bacteria remains unclear. The aim of this study was to establish the origin of Escherichia coli (E. coli) cultured from mesenteric lymph nodes (MLN) and determine if they belonged to any recognized pathotypes known to cause infections in humans. METHODS: MLN and faecal samples were obtained from 98 patients undergoing colonic resection. E. coli were isolated from 9/98 MLN samples. DNA fingerprints of MLN isolates were compared with faecal isolates from the same patient. MLN isolates were tested for adherence and invasion using HEp-2 epithelial cells, and screened for DNA markers indicative of different pathotypes of E. coli. MLN isolates were also tested for internalisation into Caco-2 cells. RESULTS: All the nine E. coli cultured from MLNs were found to have identical DNA fingerprints to at least one and often several E. coli isolates cultured from faecal samples of the same patient. 8/9 (89%) MLN isolates were weakly adherent and 2/9 (22.2%) were invasive. 8/9 (89%) tested negative for DNA markers. All the nine MLN strains were internalised by Caco-2 cells. CONCLUSION: This study confirms the gut origin of translocating bacteria. Most translocating E. coli do not belong to any recognised pathotype and are therefore normal commensal microflora. Our results suggest that bacterial translocation is more dependent upon the gut epithelium rather than the virulence properties of resident enteric bacteria.

Effects of probiotics on the gastrointestinal tract.

PURPOSE OF REVIEW: This review summarizes recent developments in our understanding of what gastrointestinal disorders probiotics can be of benefit for, focussing on conditions associated with infection or disruption of the normal gut flora. New insights into the effects administered strains can have in the gut, their safety, and potential for future development, will also be discussed. RECENT FINDINGS: Recent clinical studies have shown that probiotics can protect young children from diarrhoeal illness, including antibiotic-associated diarrhoea. They may also protect neonates from necrotizing enterocolitis. In adults, they can help prevent or reduce the severity of diarrhoeal illness, and ameliorate side-effects for those undergoing antibiotic therapy for infection. Researchers are looking at combining probiotics with prebiotics to enhance anti-inflammatory effects and restore colonization resistance of the commensal flora. New technologies are elucidating complex effects on gene expression in the gut, the probiotic, and bacterial pathogens. Recombinant strains capable of binding bacterial toxins are being developed as novel therapeutics against gastrointestinal infection. SUMMARY: Considering the clinical trial evidence of therapeutic benefit, probiotics are an underused treatment modality for prevention and amelioration of diarrhoeal illness. Better understanding of strain-specific effects, dosing regimens and any contraindications should help resolve this.

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 skin's 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.