Alison F. Kelly

Survival of Campylobacter jejuni during stationary phase: evidence for the absence of a phenotypic stationary-phase response.

ABSTRACT When Campylobacter jejuni NCTC 11351 was grown microaerobically in rich medium at 39°C, entry into stationary phase was followed by a rapid decline in viable numbers to leave a residual population of 1% of the maximum number or less. Loss of viability was preceded by sublethal injury, which was seen as a loss of the ability to grow on media containing 0.1% sodium deoxycholate or 1% sodium chloride. Resistance of cells to mild heat stress (50°C) or aeration was greatest in exponential phase and declined during early stationary phase. These results show that C. jejuni does not mount the normal phenotypic stationary-phase response which results in enhanced stress resistance. This conclusion is consistent with the absence of rpoS homologues in the recently reported genome sequence of this species and their probable absence from strain NCTC 11351. During prolonged incubation of C. jejuniNCTC 11351 in stationary phase, an unusual pattern of decreasing and increasing heat resistance was observed that coincided with fluctuations in the viable count. During stationary phase ofCampylobacter coli UA585, nonmotile variants and those with impaired ability to form coccoid cells were isolated at high frequency. Taken together, these observations suggest that stationary-phase cultures of campylobacters are dynamic populations and that this may be a strategy to promote survival in at least some strains. Investigation of two spontaneously arising variants (NM3 and SC4) of C. coli UA585 showed that a reduced ability to form coccoid cells did not affect survival under nongrowth conditions.

Antimicrobial activities of pomegranate rind extracts: enhancement by addition of metal salts and vitamin C

BackgroundPunica granatum L. or pomegranates, have been reported to have antimicrobial activity against a range of Gram positive and negative bacteria. Pomegranate formulations containing ferrous salts have enhanced although short-term, antibacteriophage activities which are rapidly diminished owing to instability of the ferrous combination. The aim of this study was to determine the antimicrobial activities of combinations of pomegranate rind extracts (PRE) with a range of metals salts with the added stabiliser vitamin C.MethodsPRE solutions, prepared by blending rind sections with distilled water prior to sterilisation by autoclaving or filtration, were screened with a disc diffusion assay using penicillin G as a control. Suspension assays were used to determine the antimicrobial activities of PRE alone and in combination with salts of the following metals; Fe (II), Cu (II), Mn (II) or Zn (II), and vitamin C, against a panel of microbes following exposure for 30 mins. The test organisms included Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis.ResultsThe screening assay demonstrated that PRE exhibited activity against the Gram positive organisms at 24 h with no observable effect on any of the Gram negative bacteria. However, after 12 h, zones of inhibition were only observed for Ps. aeruginosa. In contrast, using the suspension assay, addition of Cu (II) salts to PRE solutions extended the activities resulting in no detectable growth being observed for the PRE/Cu (II) combination against E. coli, Ps. aeruginosa and P. mirabilis. Minimal antimicrobial activity was observed following incubation with Fe (II), Mn (II) or Zn (II) salts alone or in combination with PRE against any of the organisms in the test panel. The addition of vitamin C markedly enhanced the activities of both PRE/Fe (II) and PRE/Cu (II) combinations against S. aureus.ConclusionThis is the first report demonstrating the enhanced efficacy of PRE/metal salt combinations in the presence of the stabilising agent vitamin C, to which all isolates were sensitive with the exception of B. subtilis. This study has validated the exploration of PRE along with additives such as metal salts and vitamin C as novel antimicrobial combinations.

The antimicrobial properties of copper surfaces against a range of important nosocomial pathogens

Hospital acquired infections (HAI) are a major problem worldwide and controlling the spread of these infections within a hospital is a constant challenge. Recent studies have highlighted the antimicrobial properties of copper and its alloys against a range of different bacteria. The objective of this study was to evaluate the antimicrobial properties of copper compared to stainless steel against a range of clinically important pathogens. These pathogens consisted of five isolates of each of the following organisms; meticillin resistantStaphylococcus aureus (MRSA),Pseudomonas aeruginosa, Escherichia coli, vancomycin-resistant Enterococci (VRE) and Panton-Valentine Leukocidin positive community acquired-MSSA (PVL positive CA-MSSA). MRSA,P. aeruginosa, E. coli, and CA-MSSA isolates were not detectable after a median time of 60 minutes. No detectable levels for all VRE isolates were determined after a median time of 40 minutes. However, for all isolates tested the stainless steel had no effect on the survival of the bacteria and levels remained similar to the time zero count. The results of this study demonstrate that copper has a strong antimicrobial effect against a range of clinically important pathogens compared to stainless steel and potentially could be employed to aid the control HAI.

Cattle, weather and water: mapping Escherichia coli O157:H7 infections in humans in England and Scotland

Entero-haemorrhagic Escherichia coli O157:H7 is a zoonotic pathogen, responsible for a relatively small number of food poisoning and illness outbreaks each year, when compared with other food-borne bacteria capable of causing infections in the population. Nevertheless, E. coli O157:H7 is a bacterial pathogen associated with severe human illnesses including bloody diarrhoea and haemolytic uremic syndrome occurring in both outbreak and sporadic settings. In England and Wales approximately 1% of all laboratory-confirmed cases of food poisoning are the result of E. coli O157:H7; however, in Scotland this figure increases to 3%. When the size of the population is taken into account and the rate of E. coli O157:H7 confirmed cases per 100,000 population is examined, the rate of E. coli 0157:H7 infections in Scotland is much greater than England and Wales. The routes of transmission have changed over time, with new routes of transmission such as farm visits emerging. The prevalence of E. coli O157:H7 has a seasonal dependency, with greater faecal shedding of the organism in the warmer months; this is directly mirrored in the increased reporting of E. coli O157:H7 infection among hospitalized patients. This review attempts to suggest why this phenomenon occurs, paying particular attention to weather, animal movement and private water supplies.

Anti-microbial activities of pomegranate rind extracts: enhancement by cupric sulphate against clinical isolates of S. aureus, MRSA and PVL positive CA-MSSA

BackgroundRecently, natural products have been evaluated as sources of antimicrobial agents with efficacies against a variety of micro-organisms.MethodsThis report describes the antimicrobial activities of pomegranate rind extract (PRE) singularly and in combination with cupric sulphate against methicillin-sensitive and -resistant Staphylococcus aureus (MSSA, MRSA respectively), and Panton-Valentine Leukocidin positive community acquired MSSA (PVL positive CA-MSSA).ResultsPRE alone showed limited efficacy against MRSA and MSSA strains. Exposure to copper (II) ions alone for 2 hours resulted in moderate activity of between 102 to 103 log10 cfu mL-1 reduction in growth. This was enhanced by the addition of PRE to 104 log10 cfu mL-1 reduction in growth being observed in 80% of the isolates. However, the PVL positive CA-MSSA strains were more sensitive to copper (II) ions which exhibited moderate activities of between 103 log10 cfu mL-1 reduction in growth for 60% of the isolates.ConclusionPRE, in combination with Cu(II) ions, was seen to exhibit moderate antimicrobial effects against clinical isolates of MSSA, MRSA and PVL positive CA-MSSA isolates. The results of this study indicate that further investigation into the active ingredients of natural products, their mode of action and potential synergism with other antimicrobial agents is warranted. This is the first report of the efficacy of pomegranate against clinical PVL positive CA-MSSA isolates.

The application of high resolution diffusion NMR to the analysis of manuka honey

The application of DOSY (Diffusion Ordered SpectroscopY) NMR as a technique for the virtual separation of key components of manuka honey and the implications for future discriminatory analysis of honey types is reported for the first time. The scope and the limitations of DOSY NMR are considered using the recently conceived DOSY Tool Box processing software and preliminary anti-bacterial data for the different honey types is reported.

Description of a “Phoenix” Phenomenon in the Growth of Campylobacter jejuni at Temperatures Close to the Minimum for Growth

ABSTRACT When Campylobacter jejuni cultures that had been grown in broth at 39°C were subcultured into fresh medium at 30°C, there was a transient period of growth followed by a decline in viable-cell numbers before growth resumed once more. We propose that this complex behavior is the net effect of the growth of inoculum cells followed by a loss of viability due to oxidative stress and the subsequent emergence of a spontaneously arising mutant population that takes over the culture.

Antimicrobial pomegranate rind extracts: enhancement by Cu(II) and vitamin C combinations against clinical isolates of Pseudomonas aeruginosa

Abstract Recently, natural products have been further evaluated as sources of antimicrobial agents with efficacies against a variety of microorganisms. This study reports the antimicrobial activities of pomegranate rind extract (PRE) in combination with Fe(II) and Cu(II) salts against extended-spectrum multidrug-resistant Pseudomonas aeruginosa. Antimicrobial suspension assays were carried out using aqueous extract of pomegranate alone or in combination with metals salts against P. aeruginosa. The extract:metal salt combination was also enhanced with the addition of vitamin C. Marked activities were observed for the aqueous PRE/Cu(II) preparations, which were greatly enhanced by the addition of the reductant vitamin C. In contrast, the aqueous PRE/Fe(II) preparations were inactive, regardless of addition of vitamin C. The combination of PRE and Cu(II) salts and vitamin C showed the greatest activity against clinical isolates of P. aeruginosa. These results warrant further investigation of PRE as a potential source of new antimicrobial agents.

The withdrawal of antimicrobial treatment as a mechanism for defeating resistant microorganisms

Antimicrobial resistance is a major concern in health care and farming settings throughout the world. The level of antimicrobial resistance continues to increase and the requirement for a novel and possibly dramatic change in therapy choices is required. One possible mechanism for overcoming resistance is the actual removal of antimicrobial treatment from the therapeutic armoury. This review examines the potential for success of a policy advocating the reduction of antimicrobial use and additionally the withdrawal of such treatments. Evidence from agriculture suggests that the removal of certain drugs from animal husbandry can result in concomitant falls in certain drug resistances in human patients.

Enhancement of antimicrobial activities of whole and sub-fractionated white tea by addition of copper (II) sulphate and vitamin C against Staphylococcus aureus; a mechanistic approach

BackgroundEnhancement of antimicrobial plant products e.g. pomegranate extract by copper (II) sulphate is known. Such combinations have applications in various settings, including the identification of novel compositions to study, treat and control infection.MethodsA combination of white tea (WT) (made allowing 10 minutes infusion time at 100°C) was combined with 4.8 mM copper (II) sulphate and tested for antimicrobial effect on the viability of Staphylococcus aureus NCTC 06571. Comparisons were made with green (GT) and black (BT) teas. A WT sub-fraction (WTF < 1000 Da) was tested with copper (II) sulphate and 4.8 mM vitamin C. pH measurements of samples were taken for controls and to observe any changes due to tea/agent interaction. Catalase was used to investigate hydrogen peroxide release. UV-vis. was used to compare WT and WTF.ResultsA 30 minute incubation at room temperature of copper (II) sulphate alone and combined with WT reduced the viability of S. aureus NCTC 06571 by c.a 1 log10 cfu mL-1. GT and BT with copper (II) sulphate negated activity to buffer values. Combined with copper (II) sulphate, vitamin C, WTF and, vitamin C plus WTF all reduced the viability of S. aureus NCTC 06571 by c.a. 3.5 log10 cfu mL-1. Independent experiments showed the results were not due to pH effects. Adding WT or WTF to copper (II) sulphate resulted in increased acidity. Copper (II) sulphate alone and combined with WT required c.a 300 μg mL-1 (final concentration) catalase to restore S. aureus viability, WTF with copper (II) sulphate and added vitamin C required c.a 600 μg mL-1. WT and WTF UV-visible spectra were similar.ConclusionsWT showed no efficacy in the combinations tested. WTF was enhanced with copper (II) sulphate and further with vitamin C. WT and WTF increased acidity of copper (II) sulphate possibly via the formation of chemical complexes. The difference in WT/WTF absorbance possibly represented substances less concentrated or absent in WTF. Investigations to establish which WTF component/s and in what proportions additives are most effective against target organisms are warranted.