Use of an engineered honey to eradicate preformed biofilms of important wound pathogens: an in vitro study.

OBJECTIVE: We previously reported on the ability of SurgihoneyRO (SHRO), an engineered honey, to prevent biofilm formation in vitro, but data were lacking regarding the activity against preformed biofilms. This study aims to assess whether SHRO has any antibacterial activity against mature, preformed biofilms and whether there is any evidence to support the observed clinical effectiveness when SHRO has been used anecdotally on acute and chronic wounds where biofilm is most likely present. METHOD: We tested the in vitro antibacterial activity of SHRO against the mature biofilms of 16 clinically relevant wound pathogens, in terms of impacts on biofilm seeding and biofilm biomass. The honey was serially double diluted from 1:3 down to 1:6144, and the lowest dilution achieving a statistically significant reduction in biomass of ≥50%, compared with untreated controls, was recorded. RESULTS: All 16 bacterial isolates were susceptible to SHRO, with reduced biofilm seeding observed for all, and percentage reductions ranging from 58% (ACI_C59) to 94.3% (MDR_B) for the strongest concentration of honey (1:3). Furthermore at this concentration, biofilm seeding of the test biofilm was reduced by 80-94.3% (when compared with the positive control) for 12/16 isolates. We additionally demonstrated that SHRO has antibiofilm impacts, with the 24 hour exposure resulting in disruption of the biofilm, reduced seeding and reduced biomass. CONCLUSION: SHRO is effective at reducing seeding of preformed biofilms of clinically important wound pathogens in vitro, and also has antibiofilm activity. This supports the anecdotal clinical data for antibiofilm efficacy, and supports the use of SHRO as a promising topical wound care agent.

In vitro activity of an engineered honey, medical-grade honeys, and antimicrobial wound dressings against biofilm-producing clinical bacterial isolates.

OBJECTIVE: Honey is recognised to be a good topical wound care agent owing to a broad-spectrum of antimicrobial activity combined with healing properties. Surgihoney RO (SH1) is a product based on honey that is engineered to produce enhanced reactive oxygen species (ROS) and has been reported to be highly antimicrobial. The objective was to investigate the ability of the engineered honey and its comparators to prevent biofilm formation in vitro. METHOD: We tested the ability of three medical-grade honeys SH1, Activon manuka honey (MH) and Medihoney manuka honey (Med), alongside five antimicrobial dressings (AMDs) to prevent the formation of biofilms by 16 isolates. Honeys were serially double diluted from 1:3 down to 1:6144 and the lowest dilution achieving a statistically significant reduction in biomass of at least 50%, compared with untreated controls, was recorded. RESULTS: Although all the honeys were antibacterial and were able to prevent the formation of biofilms, SH1 was the most potent, with efficacy at lower dilutions than the medical honeys for five isolates, and equivalent dilutions for a further six. Additionally, SH1 was superior in antibacterial potency to three commercially available AMDs that contain honey. CONCLUSION: SH1 is effective at preventing bioflms from forming and is superior to medical honeys and AMDs in in vitro tests. DECLARATION OF INTEREST: Surgihoney RO was provided free of charge for testing by Matoke Holdings, UK and the hospital pharmacy provided the other honeys and dressings. This paper presents independent research funded by the National Institute for Health Research (NIHR). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

High-level and novel mechanisms of carbapenem resistance in Gram-negative bacteria from tertiary hospitals in Nigeria.

To determine the occurrence and molecular basis of carbapenem resistance in Gram-negative bacteria from tertiary hospitals in Nigeria, 182 non-duplicate Gram-negative bacterial isolates were investigated for antimicrobial susceptibility, presence of carbapenemases (tested phenotypically and genotypically), random amplified polymorphic DNA (RAPD) typing, plasmid sizing and replicon typing. Minimum inhibitory concentrations of carbapenems showed a high degree of resistance, with 67 isolates (36.8%) being resistant to all carbapenems, of which 40 (59.7%) produced enzymes able to hydrolyse imipenem. PCR and sequencing identified only 10 isolates (5.5%) carrying known carbapenemase genes, including bla(NDM), bla(VIM) and bla(GES). The majority of phenotypically carbapenem-resistant and carbapenemase-producing isolates did not carry a known carbapenemase gene. Transconjugant or transformant plasmid sizes were estimated to be 115 kb for bla(NDM)- and 93 kb for bla(VIM)-carrying plasmids. These plasmids were untypeable for replicon/incompatibility and transferred various other genes including plasmid-mediated quinolone resistance (PMQR) genes and bla(CTX-M-15). Typing showed that the isolates in this study were not clonally related. There is a high level of carbapenem resistance in Nigeria. As well as the globally relevant carbapenemases (bla(NDM), bla(VIM) and bla(GES)), there are other unknown gene(s) or variant(s) in circulation able to hydrolyse carbapenems and confer high-level resistance.

PCR slippage across the ML-2 microsatellite of the Cryptosporidium MIC1 locus enables development of a PCR assay capable of distinguishing the zoonotic Cryptosporidium parvum from other human infectious Cryptosporidium species.

Cryptosporidium are ubiquitous and significant enteropathogens of all classes of vertebrates and a major cause of human morbidity and mortality worldwide. Of the 24 recognized species, the zoonotic Cryptosporidium parvum and the host-specific Cryptosporidium hominis cause the majority of cases of human cryptosporidiosis. Here, we report on structural and transcriptional variability between C. parvum and C. hominis at the MIC1 locus, which encodes a microneme localized thrombospondin-like domain containing protein previously demonstrated to be critical for host cell infection by C. parvum. We demonstrate, using reverse transcription quantitative PCR with the aid of genomic data from the EuPathDB site, that the transcribed product in C. hominis is both truncated and significantly down-regulated in the sporozoite. We hypothesize that CpMIC1 may be a genetic factor involved in facilitating the wider host range of C. parvum in comparison with the specific host range of C. hominis. Furthermore, we show that the presence of a microsatellite (ML-2) within the C. parvum MIC-1 locus enables the development of a PCR marker that can rapidly distinguish the zoonotic C. parvum from C. hominis and other significant human infectious Cryptosporidium species due to reproducible PCR slippage across the ML-2 microsatellite. Additionally, we demonstrate that this locus is tightly linked to the GP60 locus, a locus commonly used in the genetic characterization of C. parvum and C. hominis isolates. This marker should provide a robust and additional tool to aid in the rapid identification of C. parvum from other Cryptosporidium species.

An outbreak of Burkholderia multivorans beyond cystic fibrosis patients.

Strains of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens capable of causing serious infection in cystic fibrosis patients. Recently we identified a suspected outbreak of infection with Bcc strains at the University Hospital Olomouc. Seventy-four Bcc strains were isolated from 52 patients, most of whom (N = 48) did not suffer from cystic fibrosis. Most frequently (N = 46) Burkholderia multivorans was isolated and 24 (52.2%) of these strains were clonal. Fifteen of these strains were isolated from intensive care patients, five of whom died from hospital-acquired pneumonia. B. multivorans can cause serious outbreaks of infection beyond cystic fibrosis sufferers.

[Detection of Burkholderia cepacia complex strains in the University Hospital Olomouc]. / Záchyt kmenu Burkholderia cepacia komplex ve Fakultní nemocnici Olomouc.

BACKGROUND: The aim was to assess the epidemiology of Burkholderia cepacia complex strains isolated at the Department of Microbiology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, determine the most frequent strains and confirm or rule out potential clonal spread of the strains. MATERIAL AND METHODS: Over a period of eight months, all strains classified as Burkholderia cepacia complex were collected. Susceptibility to selected antimicrobial agents was determined and adequate molecular genetic methods were used to assess their genetic relationship. RESULTS: A total of 52 isolates were tested, with the most frequent (88.5 %) being genomovar II (Burkholderia multivorans). More than 46 % of them were genetically related; 58.3 % of them were detected in intensive care units. All isolates were highly resistant to antimicrobial agents. In four cases, deaths associated with Burkholderia multivorans infection were reported. CONCLUSION: It may be assumed that genetically related strains of Burkholderia multivorans spread from the hospital setting. As yet, the source of infection has not been determined and further investigations are needed.

Use of granulated sugar therapy in the management of sloughy or necrotic wounds: a pilot study.

OBJECTIVE: To determine the in vitro antimicrobial efficacy of three types of sugar and conduct a pilot clinical study with a view to developing a protocol for a randomised controlled trial (RCT). METHOD: In the in vitro studies three types of granulated sugar (Demerara, granulated beet sugar and granulated cane sugar) were tested to determine their minimum inhibitory concentrations (MICs) against 18 Gram-negative and Gram-positive bacteria in a micro-titre broth dilution assay; growth inhibition of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa in different concentrations of sugar (0.38-25%) was also tested over 12-hours in an agar diffusion assay. The pilot clinical study selected patients from a vascular surgical ward and a vascular outpatient department. All had acute or chronic exuding wounds, some of which were infected. White granulated sugar was applied to the wounds. The following parameters were assessed: surface area; wound characteristics including pain, malodour, appearance (slough/granulation); exudate level; pain level and bacterial load. Patients with diabetes had their blood sugar levels checked daily. All patients completed a short health questionnaire at the start and end of the study. Staff completed a satisfaction questionnaire at the end of the study. The study period was 21 days. RESULTS: In vitro tests demonstrated that sugar inhibits bacterial growth. All three types of sugars had MICs ranging from 6-25% in the bacterial strains tested. The diffusion tests showed that strains were able to grow well in low concentrations of sugar but were completely inhibited in higher concentrations. The two granulated sugars were found to be slightly more effective than Demerara sugar, so the latter was excluded from the clinical pilot study. Twenty-two patients (20 inpatients and two outpatients) with sloughy or necrotic wounds were recruited into the clinical study. Two patients had MRSA and two had Staphylococcus colonisation at baseline. Blood sugar levels remained stable in the seven patients with insulin-dependent diabetes mellitus. All wounds were clean/debrided in a mean of 11.13 days. Pain and malodour reduced markedly. Patient and staff surveys revealed overwhelming support for the sugar therapy. CONCLUSION: The pilot study achieved its aim of developing a protocol for a RCT. Preliminary data suggest that sugar is an effective wound cleansing and is safe to use in patients with insulin-dependent diabetes. In vitro studies demonstrate that sugar inhibits bacterial growth. CONFLICT OF INTEREST: None.

High levels of multidrug resistance in clinical isolates of Gram-negative pathogens from Nigeria.

In Nigeria, quinolones and ß-lactam antibiotics are widely used to treat bacterial infections. This study aimed to identify the prevalence of resistance to these drugs and to determine the mechanisms of resistance to these agents. In total, 134 non-duplicate, Gram-negative enteric isolates of 13 species from different hospitals were investigated for susceptibility to a panel of antibiotics, carriage of plasmid-mediated quinolone and ß-lactam resistance genes, production of extended-spectrum ß-lactamases (ESBLs), and mutations within topoisomerase genes. The level of resistance to all antibiotics tested was extremely high, with minimum inhibitory concentrations for 90% of the organisms (MIC(90) values) of ≥ 256 µg/mL for all drugs. Of the 134 isolates, 92 had mutations within the quinolone resistance-determining region (QRDR) of gyrA or within gyrA and parC. In addition, the plasmid-mediated quinolone resistance genes qnrA, qnrB, aac(6')-Ib-cr and qepA were identified. The qnrD allele, which has previously only been found in Salmonella isolates from China, was identified in two Proteus isolates and one Pseudomonas isolate. Of the 134 isolates, 23 (17.2%) carried aac(6')-Ib-cr, 11 (8.2%) carried a qnr variant and 5 (3.7%) were positive for qepA. Twenty-eight isolates (20.9%) produced ESBL variants, with a CTX-M variant being carried by 25 isolates (18.7%). In addition, six isolates (4.5%) carried ampC variants [ACT-1 (1 isolate), DHA-1 (4 isolates) and CMY-2 (1 isolate)]. This study demonstrates a very high level of multidrug resistance amongst Gram-negative enteric bacilli isolated from different sites from patients in Nigerian hospitals as well as the presence of a variety of plasmid-associated resistance genes, including some identified from Africa for the first time.

Psychoneuroimmunological outcomes and quality of life.

According to the American Psychological Association the field of psychoneuroimmunology comprises a rapidly expanding body of knowledge that strongly suggests a reciprocal interaction between mind (cognition) and body. This body of knowledge indicates that cognitive events have somatic consequences that impact directly on the immune system and disease processes, which in turn lead to maladaptive cognitive processes. Disease sufferers' thought processes and perceived quality of life may thus have a direct bearing on their somatic functioning and disease process, and interventions at this level may have a material impact on their prognosis.

Commonly used farm disinfectants can select for mutant Salmonella enterica serovar Typhimurium with decreased susceptibility to biocides and antibiotics without compromising virulence.

OBJECTIVES: To determine if one passage of Salmonella enterica serovar Typhimurium in the presence of farm disinfectants selected for mutants with decreased susceptibility to disinfectants and/or antibiotics. METHODS: Eight Salmonella Typhimurium strains including field isolates and laboratory mutants were exposed to either a tar oil phenol (PFD) disinfectant, an oxidizing compound disinfectant (OXC), an aldehyde based disinfectant (ABD) or a dairy sterilizer disinfectant (based on quaternary ammonium biocide) in agar. The susceptibility of mutants obtained after disinfectant exposure to antibiotics and disinfectants was determined as was the accumulation of norfloxacin. The proteome of SL1344 after exposure to PFD and OXC was analysed using two-dimensional liquid chromatography mass spectrometry. RESULTS: Strains with either acrB or tolC inactivated were more susceptible to most disinfectants than other strains. The majority (3/5) of mutants recovered after disinfectant exposure required statistically significantly longer exposure times to disinfectants than their parent strains to generate a 5 log kill. Small decreases in antibiotic susceptibility were observed but no mutants were multiply antibiotic-resistant (MAR). Notably exposure to ABD decreased susceptibility to ciprofloxacin in some strains. Mutants with increased disinfectant tolerance were able to survive and persist in chicks as well as in parent strains. Analysis of proteomes revealed significantly increased expression of the AcrAB-TolC efflux system after PFD exposure. CONCLUSIONS: Data presented demonstrate that efflux pumps are required for intrinsic resistance to some disinfectants and that exposure to disinfectants can induce expression of the AcrAB-TolC efflux system, but that single exposure was insufficient to select for MAR strains.

Absence of mutations in marRAB or soxRS in acrB-overexpressing fluoroquinolone-resistant clinical and veterinary isolates of Escherichia coli.

The amount of acrB, marA, and soxS mRNA was determined in 36 fluoroquinolone-resistant E. coli from humans and animals, 27 of which displayed a multiple-resistance phenotype. acrB mRNA was elevated in 11 of 36 strains. A mutation at codon 45 (Arg-->Cys) in acrR was found in 6 of these 11 strains. Ten of the 36 isolates appeared to overexpress soxS, and five appeared to overexpress marA. A number of mutations were found in the marR and soxR repressor genes, correlating with greater amounts of marA and soxS mRNA, respectively.