Effects of pH on the Antibiotic Resistance of Bacteria Recovered from Diabetic Foot Ulcer Fluid An In Vitro Study.

BACKGROUND: This study investigated the resistance of bacteria isolated from diabetic foot ulcers (DFUs) to antibiotics frequently used in the management of the diabetic foot infections, at a range of pH values (pH 6.5, 7.5, and 8.5) known to exist in DFU wound fluid. This study aimed to determine whether changes (or atypical stasis) in wound fluid pH modulate the antibiotic resistance of DFU isolates, with potential implications in relation to the suppression/eradication of bacterial infections in DFUs. METHODS: Thirty bacterial isolates were recovered from DFU wound fluid, including Staphylococcus spp, Staphylococcus aureus, Escherichia coli, Streptococcus spp, Pseudomonas spp, and Pseudomonas aeruginosa. The resistances of these isolates to a panel of antibiotics currently used in the treatment of infected or potentially infected DFUs, ie, ciprofloxacin, amoxicillin-clavulanate, doxycycline, and piperacillin-tazobactam, at the previously mentioned pH values were determined by a modification of the Kirby-Bauer assay. RESULTS: The resistance of DFU isolates to clinically relevant antibiotics was significantly affected by the pH levels in DFU wound fluid. CONCLUSIONS: These findings highlight the importance of a more comprehensive understanding of the conditions in DFUs to inform clinical decision making in the selection and application of antibiotics in treating these difficult-to-heal wounds. The scale of the differences in the efficacies of antibiotics at the different pH values examined is likely to be sufficient to suggest reconsideration of the antibiotics of choice in the treatment of DFU infection.

Protein, cell and bacterial response to atmospheric pressure plasma grafted hyaluronic acid on poly(methylmethacrylate).

Hyaluronic acid (HA) has been immobilised on poly(methyl methacrylate) (PMMA) surfaces using a novel dielectric barrier discharge (DBD) plasma process for the purposes of repelling protein, cellular and bacterial adhesion in the context of improving the performance of ophthalmic devices. Grafting was achieved by the following steps: (1) treatment of the PMMA with a DBD plasma operating at atmospheric pressure, (2) amine functionalisation of the activated polymer surface by exposure to a 3-aminopropyltrimethoxysilane (APTMS) linker molecule and (3) reaction of HA with the surface bound amine. The mechanism and effectiveness of the grafting process was verified by surface analysis. XPS data indicates that the APTMS linker molecule binds to PMMA via the Si-O chemistry and has the required pendant amine moiety. The carboxylic acid moiety on HA then binds with this -NH2 group via standard carbodiimide chemistry. ToF-SIMS confirms the presence of a coherent HA layer the microstructure of which is verified by AFM. The plasma grafted HA coating surfaces showed a pronounced decrease in protein and cellular adhesion when tested with bovine serum albumin and human corneal epithelial cells, respectively. The ability of these coatings to resist bacterial adhesion was established using Staphylococcus aureus NTC8325. Interestingly, the coatings did not repel bacterial adhesion, indicating that the mechanism of adhesion of bacterial cells is different to that for the surface interactions of mammalian cells. It is proposed that this difference is a consequence of the specific HA conformation that occurs under the conditions employed here. Hence, it is apparent that the microstructure/architecture of the HA coatings is an important factor in fabricating surfaces intended to repel proteins, mammalian and bacterial cells.

The pH of wound fluid in diabetic foot ulcers -- the way forward in detecting clinical infection?

Infections within diabetic foot ulcers are often hard to detect and extremely difficult to treat. The normal signs and symptoms of infection including purulence, erythema, pain, tenderness, warmth and induration are frequently absent in such wounds necessitating exploration of other ways of rapidly and accurately detecting infection. This study considers diabetic wound fluid pH as a possible alternative means of monitoring infection status. CINAHL, Ovid SP and MEDLINE were searched for papers in English published between January 2004 to May 2014. Key search terms included wound fluid, exudate, wound, ulcer, diabetes, pH, healing, infection, bacteria. This paper considers the potential benefits of augmenting and supporting current clinical practice in the early determination of wound healing trajectory and infection status, by monitoring wound fluid pH. The evidence collected highlights the need for further research and suggests the potential of wound fluid analysis as a possible surrogate marker for detecting infection in diabetic foot ulcers.

UVc-irradiation sublethal stress does not alter antibiotic susceptibility of staphylococci (MRSA, MSSA, and coagulase-negative staphylococci) to ß-lactam, macrolide, and fluoroquinolone antibiotic agents.

Skin tanning, either by exposure to natural sunlight or through use of UV sunbeds, has become a popular practice in the US, where it is estimated that approximately 1 million times per day someone in the US uses UV radiation for skin tanning, equating to 30 million Americans (circa 10% of the US population) who use a tanning bed. As well as exposing the host to periods of UV radiation, such practices also expose commensal skin bacteria, including Staphylococcus aureus, to such UV radiation. Previous work has indicated that environmental stresses on bacteria may lead to an upregulation of stress responses, in an attempt for the organism to combat the applied stress and remain viable. UV light may act as an environmental stress on bacteria, and so it was the aim of this study to examine the effect of UVc light on the antibiotic susceptibility of commensal skin bacteria, to determine if UV radiation would increase the antibiotic resistance of such skin flora and thus lead to a potential skin flora with increased antibiotic resistance. Previously, it has been shown that UVc light has a greater mutational effect on bacteria compared to lower-energy UV forms, including UVa and UVb light. Therefore, we decided to employ UVc light in our study to amplify the potential for mutational events occurring in skin staphylococci organisms (n=8) including methicillin-sensitive Staphylococcus aureus (n=2), methicillin-resistant Staphylococcus aureus (n=4), and coagulase-negative staphylococci (Staphylococcus haemolyticus) (n=2) were exposed to varying degrees of sublethal radiation via UVc light, and their minimum inhibitory concentration (MIC) susceptibility was determined by broth dilution assay against three classes of commonly used antibiotics, namely ß-lactams (penicillin), macrolides (erythromycin), and fluoroquinolones (ciprofloxacin). There was no significant difference between antibiotic susceptibility before UVc exposure and until maximum sublethal stress, prior to cell death due to fatal UVc exposure with the cells. These results indicate that UV environmental stress/exposure does not upregulate antibiotic resistance, and therefore these data indicate that UVc radiation does not lead to a more antibiotic-resistant population in the staphylococci organisms post-exposure.

Atmospheric pressure plasma induced grafting of poly(ethylene glycol) onto silicone elastomers for controlling biological response.

This study investigates the role that surface functionalisation of silicone elastomer (SE) by atmospheric pressure plasma induced graft immobilisation of poly(ethylene glycol) methyl ether methacrylate (PEGMA) plays in the attendant biological response. SE is used in modern ophthalmic medical devices and samples of the material were initially plasma treated using a dielectric barrier discharge reactor (DBD) to introduce reactive oxygen functionalities, prior to in situ grafting of two molecular weights of PEGMA (MW 1000 Da: PEGMA(1000), MW 2000 Da: PEGMA(2000)). The variously processed surfaces were characterised by water contact angle analysis, X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry and atomic force microscopy. Lens epithelial cells were then cultured on the PEGMA grafted SE surfaces. It was found that cells on the pristine surface were not well spread and had shrunken morphology. On the DBD pre-treated surfaces, the cells were well spread. On the PEGMA(1000) surface, the cells displayed evidence of shrinkage and were on the verge of detaching. Remarkably, on the PEGMA(2000) surface, no cell adhesion was detection. Bacterial adhesion to the surfaces was studied using Staphylococcus aureus NTC8325. There was no difference in the number of bacteria adhering to any of the surfaces studied.

Occurrence of fastidious Campylobacter spp. in fresh meat and poultry using an adapted cultural protocol.

This study used an adapted cultural protocol for the recovery of fastidious species of Campylobacter, to gain a more accurate understanding of the diversity of Campylobacter populations in fresh meats. Chicken (n=185), pork (n=179) and beef (n=186) were collected from supermarkets and butchers throughout the Republic of Ireland. Samples were enriched in Campylobacter enrichment broth for 24h under an atmosphere of 2.5% O(2), 7% H(2), 10% CO(2), and 80.5% N(2). The enriched samples were then filtered onto non-selective Anaerobe Basal Agar supplemented with lysed horse blood using mixed ester filter membranes. Isolates were identified by both genus and species-specific PCR assays and biochemical testing. The incidence of campylobacters on beef (36%) was significantly higher than on pork (22%) or chicken (16%), and far exceeds previously reported prevalence levels. The method was successful in recovering 7 species of Campylobacter, including the fastidious spp. C. concisus and C. mucosalis, from chicken meat, and 10 species, including C. concisus, C. curvus, C. mucosalis, C. sputorum, and C. upsaliensis, from minced beef. The isolation of C. concisus and C. upsaliensis from meat in this study is of particular significance, due to their emerging clinical relevance. The results of this study confirm that the diversity of Campylobacter species on fresh meats is greater than previously reported and highlights the bias of cultural methods towards the recovery of C. jejuni.

The effect of heat shrink treatment and storage temperature on the time of onset of "blown pack" spoilage.

This study determined the effects of (a) the short "heat shrink" treatment frequently applied to vacuum packed meats within normal commercial production, and (b) chill holding storage temperature, on the subsequent time to onset (TTO) of "blown pack" spoilage (BPS). Beef or lamb steaks were inoculated with 10³ CFU/cm² of spore suspensions of five gas producing clostridia, vacuum packed (VP) and treated as follows: no heat, 50°C/15 s, 70°C/10 s or 90°C/3 s. Samples were stored at -1.5, 1 or 4°C and examined daily to determine TTO of BPS. For each strain, pack treatment and storage temperature had significant (P<0.05 and P<0.001 respectively) effects on TTO of BPS, i.e. 90°C/3 s<70°C/10 s<50°C/15 s≤"no heat", and 4°C<1°C<-1.5°C. The study suggested that the meat industry could reduce the risks of BPS by avoiding higher temperature (90°C/3 s or 70°C/10 s) heat shrinking, and by storing VP meats at lower temperatures (e.g. -1.5°C).

Screening of clinical, food, water and animal isolates of Escherichia coli for the presence of blaCTX-M extended spectrum beta-lactamase (ESBL) antibiotic resistance gene loci.

A small study was carried out in order to examine the molecular presence of bla CTX-M gene phylogenetic groups in E. coli (n=263) isolated from food (n=54), water (n=7), animal sources (n=69), using consensus bla CTX-M primers and PCR, in addition to human faecal isolates (n=69) and VTEC O157:H7 (n=64). None of the clinically significant faecal VTEC O157:H7 isolates were shown to carry blaCTX-M type phylogenetic groups, nor were such phylogenetic groups observed in any of the food, water and animal isolates. One community faecal isolate (1/69; 1.4%), dating from 1997, carried this phylogenetic group. As recent work has indicated that a significant proportion of such phylogenetic groups are carried in community isolates of E. coli with little or no hospital contact, it is important that surveillance is increased to identify potential source(s) and reservoirs of such resistance in the community. Further prospective surveillance is thus required to help elucidate the origins of such phylogenetic group in the community. The significance of this study is that the ESBL-producing E. coli associated with local hospital outbreaks is not commonly found in local food, water or animal sources. In addition, given that ESBL-producing E. coli is now a significant organism, both in hospitals and nursing homes in Northern Ireland, this report demonstrates that such organisms were present in the community, as early as 1997.

Transcriptome analysis of alkali shock and alkali adaptation in Listeria monocytogenes 10403S.

Alkali stress is an important means of inactivating undesirable pathogens in a wide range of situations. Unfortunately, Listeria monocytogenes can launch an alkaline tolerance response, significantly increasing persistence of the pathogen in such environments. This study compared transcriptome patterns of alkali and non-alkali-stressed L. monocytogenes 10403S cells, to elucidate the mechanisms by which Listeria adapts and/or grows during short- or long-term alkali stress. Transcription profiles associated with alkali shock (AS) were obtained by DNA microarray analysis of midexponential cells suspended in pH 9 media for 15, 30, or 60 min. Transcription profiles associated with alkali adaptation (AA) were obtained similarly from cells grown to midexponential phase at pH 9. Comparison of AS and AA transcription profiles with control cell profiles identified a high number of differentially regulated open-reading frames in all tested conditions. Rapid (15 min) changes in expression included upregulation of genes encoding for multiple metabolic pathways (including those associated with Na+/H+ antiporters), ATP-binding cassette transporters of functional compatible solutes, motility, and virulence-associated genes as well as the σ(B) controlled stress resistance network. Slower (30 min and more) responses to AS and adaptation during growth in alkaline conditions (AA) involved a different pattern of changes in mRNA concentrations, and genes involved in proton export.

Molecular identification of airborne bacteria associated with aerial spraying of bovine slurry waste employing 16S rRNA gene PCR and gene sequencing techniques.

Polymerase chain reaction amplification of the universal 16S ribosomal RNA (rRNA) gene was performed on a collection of 38 bacterial isolates, originating from air sampled immediately adjacent to the agricultural spreading of bovine slurry. A total of 16 bacterial genera were identified including both Gram-positive and Gram-negative genera. Gram-positive organisms accounted for 34/38 (89.5%) of total bacterial numbers consisting of 12 genera and included Staphylococcus (most common genus isolated), Arthrobacter (2nd most common genus isolated), Brachybacterium, Exiguobacterium, Lactococcus, Microbacterium and Sporosarcina (next most common genera isolated) and finally, Bacillus, Brevibacterium, Frigoribacterium, Mycoplana and Pseudoclavibacter. Gram-negative organisms accounted for only 4/38 (10.5%) bacterial isolates and included the following genera, Brevundimonas, Lysobacter, Psychrobacter and Rhizobium. No gastrointestinal pathogens were detected. Although this study demonstrated a high diversity of the microorganisms present, only a few have been shown to be opportunistically pathogenic to humans and none of these organisms described have been described previously as having an inhalational route of infection and therefore we do not believe that the species of organisms identified pose a significant health and safety threat for immunocompetant individuals.

Epidemiological study of E. coli O157:H7 isolated in Northern Ireland using pulsed-field gel electrophoresis (PFGE).

In Northern Ireland over the last 7 years, there is a mean of 41.9 laboratory reports per annum of human gastrointestinal infection (range 19-54) caused by Escherichia coli O157:H7. In the preceding years 1992-1996, reports were 5.4 per annum, whereas in 1997-2000, reports increased from 30 to 54 per annum. This high level has continued on an annual basis to date. The aim of this study was therefore to retrospectively examine this period of exponential increase in reports to help ascertain the genetic relatedness of strains employing pulsed-field gel electrophoresis (PFGE), as no data on the molecular epidemiology of E. coli O157:H7 in Northern Ireland has yet been published. Clinical isolates (n=84) were PFGE typed employing XbaI digestion and resulting band profiles demonstrated the presence of 13, 9 and 16 clonal types, for 1997, 1998 and 1999, respectively. In 1998, five clonal types remained from 1997 with the introduction of 4 new clonal types, whereas in 1999, 10 new clonal types were observed, accounting for over half (58%) of the E. coli O157 isolates for that year. These data suggest that, unlike gastrointestinal infections due to thermophilic campylobacters, there was considerable genetic evolution ofPFGE clonal types of E. coli O157, through the displacement and emergence of genotypes. Further studies are now required to find the environmental reservoirs of these common clonal types of clinical E. coli O157:H7 in Northern Ireland to help define sources and routes of transmission of this infection locally.

Comparison of the effect of ciprofloxacin and Tazocin on the incidence of meticillin-resistant Staphylococcus aureus (MRSA) in an Intensive Care Unit.

Meticillin-resistant Staphylococcus aureus (MRSA) is a very significant agent of recalcitrant healthcare-associated infections. A major risk of acquiring such infections is thought to be modulated by the use of particular antimicrobial therapies. The aim of this research was to evaluate prospectively the impact of using either ciprofloxacin or Tazocin (piperacillin+tazobactam) on the incidence of MRSA in an Intensive Care Unit (ICU). The 1-year (2 x 6 months) cross-over study was carried out in a medium-sized (426 beds) teaching hospital. During the first 6-month period, ciprofloxacin was used as the first-line broad-spectrum antibiotic therapy of choice. During the second 6-month period, Tazocin was used as first-line therapy. The incidence of hospital-acquired MRSA (i.e. colonised and/or infected) and rates of compliance of the ICU healthcare workers to optimal hand hygiene practices were recorded throughout the study. The study observed no statistically significant differences (P = 0.1) between MRSA incidence rates in the ICU during the ciprofloxacin (4.4/1000 bed-days) or Tazocin (11.4/1000 bed-days) arms of the study. Interestingly, observing healthcare workers' hand hygiene practices throughout the entire study showed that healthcare workers adhered to these practices 59.2% of the time during the ciprofloxacin arm and 66.0% during the Tazocin arm. The low incidence rates within the unit demonstrated the importance of infection control in limiting the spread of MRSA despite the extensive use of antibiotics in a high-risk setting.

Role of sigma B factor in the alkaline tolerance response of Listeria monocytogenes 10403S and cross-protection against subsequent ethanol and osmotic stress.

Many of the considerable abilities of Listeria monocytogenes to persist and grow in a wide range of adverse environmental conditions are thought to be at least partly under the control of the alternative sigma factor (sigmaB), encoded by the sigB gene. However, little is known about the role of this master regulon in the impressive ability of Listeria to persist and grow under conditions of alkaline pH. In this study, Northern blot analysis of parent Listeria mRNA revealed that alkali adaptation (pH 9.5 for 1 h) significantly increased the expression of sigB-derived mRNA. The study included a comparison of the relative survival of mid-exponential populations of adapted and nonadapted parent type (sigmaB expressing) and mutant (not sigmaB expressing, deltasigB) Listeria strains during subsequent alkaline (pH 12.0), osmotic (25% NaCl, wt/vol), or ethanol (16.5%) stress. Alkali-adapted parent strains were more resistant to pH 12.0 than were adapted deltasigB type strains, but both alkali-adapted parent and deltasigB strains were more resistant to pH 12.0 than were nonadapted strains. Alkali-adapted parent strains were more resistant to osmotic stress than were adapted deltasigB type strains. No significant differences in viability were observed between alkali-adapted parent and deltasigB strains after ethanol stress, suggesting that cross-protection against osmotic stress is mediated by sigmaB whereas cross-protection against ethanol is sigmaB independent. Overall, alkali-induced cross-protection against osmotic and ethanol challenges may have serious implications for food safety and human health because such stress conditions are routinely used as part of food preservation and surface cleaning processes.

Genomic and proteomic analysis of the Alkali-Tolerance Response (AlTR) in Listeria monocytogenes 10403S.

BACKGROUND: Information regarding the Alkali-Tolerance Response (AlTR) in Listeria monocytogenes is very limited. Treatment of alkali-adapted cells with the protein synthesis inhibitor chloramphenicol has revealed that the AlTR is at least partially protein-dependent. In order to gain a more comprehensive perspective on the physiology and regulation of the AlTR, we compared differential gene expression and protein content of cells adapted at pH 9.5 and un-adapted cells (pH 7.0) using complementary DNA (cDNA) microarray and two-dimensional (2D) gel electrophoresis, (combined with mass spectrometry) respectively. RESULTS: In this study, L. monocytogenes was shown to exhibit a significant AlTR following a 1-h exposure to mild alkali (pH 9.5), which is capable of protecting cells from subsequent lethal alkali stress (pH 12.0). Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. The observed variability between results of cDNA arrays and 2D gel electrophoresis may be accounted for by posttranslational modifications. Interestingly, several alkali induced genes/proteins can provide a cross protective overlap to other types of stresses. CONCLUSION: Alkali pH provides therefore L. monocytogenes with nonspecific multiple-stress resistance that may be vital for survival in the human gastrointestinal tract as well as within food processing systems where alkali conditions prevail. This study showed strong evidence that the AlTR in L. monocytogenes functions as to minimize excess alkalisation and energy expenditures while mobilizing available carbon sources.

Recovery and transfer of Salmonella typhimurium from four different domestic food contact surfaces.

Domestic food contact surfaces can play an important role in the transmission of foodborne disease, yet debate continues as to which surface materials pose the greatest risk to consumer health in terms of cross-contamination during food preparation. Salmonella Typhimurium was inoculated onto stainless steel, Formica, polypropylene, or wooden surfaces (25 cm2) in the presence or absence of protein (tryptic soy broth supplemented with 5% horse serum) and held at room temperature. The pathogen was recovered from the test surfaces immediately after inoculation (T=0) and every hour for up to 6 h, by a conventional microbiological sampling technique and by direct transfer onto a model ready-to-eat food (cucumber slices). On all surfaces, pathogen numbers declined during the 6-h holding period, with the most rapid reductions occurring within the first hour. The presence of protein significantly increased (P < 0.05) the number of bacteria recovered from all surface types. However, regardless of application medium or holding time, the number of bacteria recovered from Formica (in all cases) and stainless steel (in most cases) was significantly higher than were the numbers on polypropylene or wood. Similarly, regardless of application medium or holding time, significantly higher bacterial numbers were transferred to the model food from Formica or stainless steel than from polypropylene or wooden surfaces. These differences were greater when the bacteria were applied in a protein-rich medium and the test surfaces held for 1 h or more. The results of this study emphasize that differences, both in recoverability and in the number of bacteria transferred to the model food rather than simply reflecting differences in pathogen survival, may also reflect differences in the ability of the test bacteria to remobilize from the different surface types. However, the results also demonstrate a fundamental problem when choosing food contact surfaces, i.e., that those characteristics that make a surface "easy to clean" may also render it more likely to release contaminating pathogens during common food preparation practices.

Morphological changes in Listeria monocytogenes subjected to sublethal alkaline stress.

Scanning electron microscopy (SEM) studies revealed that exposure to 4lethal alkaline stress induced statistically significant (P<0.05) changes in mean cell length, radius and volume in Listeria monocytogenes and a derived sigma(B) deficient mutant. Bacterial morphology was altered at pH values above 9.0, to include single filamentous or elongated chain forms. Such filamentation and chain formation was observed in the parent strain and in the sigma(B) deficient strain, and in buffered and non-buffered media. Giemsa staining revealed that the filaments were multi-nucleate, with nucleoids spaced along the length of the atypical cells. In buffered media, longer alkaline exposure was associated with increases in the frequency and length of filamentation. In non-buffered medium, longer exposure was associated with gradual decline in length and the frequency of observation of filaments. Transfer of alkaline treated cells to neutral conditions was associated with the formation of septa within filaments, cell division, and a rapid return to normal morphology, i.e. within 3 h. The observed effects, and their reversibility, may be important in increasing the alkaline tolerance of this pathogen during phagocytosis within the innate human immune system response, and in adaptation/survival in food environments treated with alkali detergents and/or sanitisers. Such atypical cells may be associated with increased survival of L. monocytogenes in adverse environments and may also contribute to qualitative and quantitative underestimation of this important pathogen in food processing environments, with potential implications in public health.

Role of branched-chain fatty acids in pH stress tolerance in Listeria monocytogenes.

In alkaline conditions, Listeria monocytogenes cells develop higher proportions of branched-chain fatty acids (FAs), including more anteiso forms. In acid conditions, the opposite occurs. Reduced growth of pH-sensitive mutants at adverse pH (5.0/9.0) was alleviated by the addition of 2-methylbutyrate (an anteiso-FA precursor), suggesting that anteiso-FAs are important in adaptation to adverse pH. The balance between anteiso- and iso-FAs may be more important than changes in the amounts and/or degrees of saturation of FAs in pH adaptation.

Habituation to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia) is associated with reduced susceptibility to antibiotics in human pathogens.

OBJECTIVES: To investigate the effect of sub-lethal challenge with tea tree oil (TTO) on the antibiotic susceptibility profiles of significant human pathogens and commensals. METHODS: The study compared the antibiotic susceptibility (Etest) patterns of Escherichia coli, Staphylococcus aureus/methicillin-resistant S. aureus (MRSA) and Salmonella spp. after broth culture for 72 h in the presence or absence of sub-lethal concentrations of TTO (0.25%, 0.25% and 0.1%). RESULTS: All habituated cultures (exposed to sub-lethal concentrations of TTO) displayed reduced susceptibility to a range of clinically relevant antibiotics compared with non-habituated (control) cultures. CONCLUSIONS: Although TTO may be an effective antimicrobial agent when appropriately used at bactericidal concentrations, its application at sub-lethal concentrations may contribute to the development of antibiotic resistance in human pathogens.