The microbiome of diabetic foot ulcers: a comparison of swab and tissue biopsy wound sampling techniques using 16S rRNA gene sequencing.

BACKGROUND: Health-care professionals need to collect wound samples to identify potential pathogens that contribute to wound infection. Obtaining appropriate samples from diabetic foot ulcers (DFUs) where there is a suspicion of infection is of high importance. Paired swabs and tissue biopsies were collected from DFUs and both sampling techniques were compared using 16S rRNA gene sequencing. RESULTS: Mean bacterial abundance determined using quantitative polymerase chain reaction (qPCR) was significantly lower in tissue biopsies (p = 0.03). The mean number of reads across all samples was significantly higher in wound swabs [Formula: see text] = 32,014) compared to tissue ([Formula: see text] = 15,256, p = 0.001). Tissue biopsies exhibited greater overall diversity of bacteria relative to swabs (Shannon's H diversity p = 0.009). However, based on a presence/absence analysis of all paired samples, the frequency of occurrence of bacteria from genera of known and potential pathogens was generally higher in wound swabs than tissue biopsies. Multivariate analysis identified significantly different bacterial communities in swabs compared to tissue (p = 0.001). There was minimal correlation between paired wound swabs and tissue biopsies in the number and types of microorganisms. RELATE analysis revealed low concordance between paired DFU swab and tissue biopsy samples (Rho = 0.043, p = 0.34). CONCLUSIONS: Using 16S rRNA gene sequencing this study identifies the potential for using less invasive swabs to recover high relative abundances of known and potential pathogen genera from DFUs when compared to the gold standard collection method of tissue biopsy.

Biofilm contamination of high-touched surfaces in intensive care units: epidemiology and potential impacts.

The aim of this study was to determine the epidemiology (location, microbial load, microbiome, presence/absence of biofilm and pathogens, including ESKAPE-Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species, and antimicrobial susceptibility profiles) of the bacterial contamination on intensive care units (ICUs) surfaces. Fifty-seven high-touched surfaces were collected from adult, paediatric and neonatal ICUs from two large public Brazilian hospitals from central and north regions. Samples (c. 4 cm2 ) were subjected to culture (qualitative), qPCR targeting 16s rRNA gene (microbial load-bacteria per cm2 ), 16s rRNA amplicon sequencing (microbiome analysis) and scanning electron (SEM) or confocal laser scanning microscopy (CLSM) (biofilm presence). Multidrug resistant organisms (MROs) were detected using specific chromogenic agar. The average bacterial load was 1·32 × 104 bacteria per cm2 , container for newborn feeding bottles, stretcher mattress, humidicrib mattress filling and computer keyboards presented the higher bioburden. However, only 45·6% (26/57) were culture-positive, including 4/26 with MROs. ESKAPE organisms were detected in 51·8% of the samples subjected to next-generation sequencing. Viability staining and CLSM demonstrated live bacteria on 76·7% of culture-negative samples. Biofilm was present on all surfaces subjected to microscopy (n = 56), demonstrating that current cleaning practices are suboptimal and reinforcing that MROs are incorporated into hospital surfaces biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of healthcare facilities surfaces has been shown to play a major role in transmission of pathogens. The findings of this study show that dry surface biofilms are widespread and can incorporate pathogens and multidrug-resistant organisms (MROs). Biofilms on highly touched surfaces pose a risk to patients, as dry surface biofilms persist for long period and micro-organisms within biofilm have been shown to be transmitted. This study also provides a better understanding of microbial populations in hospital environments, reinforcing that pathogens and MROs are found incorporated into biofilm, which impacts the difficulty in cleaning/disinfection.

Understanding the microbiome of diabetic foot osteomyelitis: insights from molecular and microscopic approaches.

OBJECTIVES: Rigorous visual evidence on whether or not biofilms are involved in diabetic foot osteomyelitis (DFO) is lacking. We employed a suite of molecular and microscopic approaches to investigate the microbiome, and phenotypic state of microorganisms involved in DFO. METHODS: In 20 consecutive subjects with suspected DFO, we collected intraoperative bone specimens. To explore the microbial diversity present in infected bone we performed next generation DNA sequencing. We used scanning electron microscopy (SEM) and peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) with confocal microscopy to visualize and confirm the presence of biofilms. RESULTS: In 19 of 20 (95%) studied patients presenting with DFO, it was associated with an infected diabetic foot ulcer. By DNA sequencing of infected bone, Corynebacterium sp. was the most commonly identified microorganism, followed by Finegoldia sp., Staphylococcus sp., Streptococcus sp., Porphyromonas sp., and Anaerococcus sp. Six of 20 bone samples (30%) contained only one or two pathogens, while the remaining 14 (70%) had polymicrobial communities. Using a combination of SEM and PNA-FISH, we identified microbial aggregates in biofilms in 16 (80%) bone specimens and found that they were typically coccoid or rod-shaped aggregates. CONCLUSIONS: The presence of biofilms in DFO may explain why non-surgical treatment of DFO, relying on systemic antibiotic therapy, may not resolve some chronic infections caused by biofilm-producing strains.

Transfer of dry surface biofilm in the healthcare environment: the role of healthcare workers' hands as vehicles.

BACKGROUND: Dry surface biofilms (DSBs) persist for extended periods in hospital, and may play a significant role in transmission of healthcare-associated infections. AIM: To determine whether DSBs may be transferred from hospital surfaces to healthcare workers' hands. METHOD: Twelve-day Staphylococcus aureus DSB was grown on polycarbonate and glass coupons in a CDC Biofilm Reactor®. A total of 1.8 × 106 and 8.8 × 105 bacteria grew on the polycarbonate and glass coupons respectively. Transmission was tested by lifting the coupon with forefinger and thumb of ungloved hands to a height of 30 cm, then touching horse blood agar (HBA) plates 19 sequential times. Transferred bacterial number was determined by colony-forming units. The effect of DSB wetting on biofilm transfer was tested with 5% neutral detergent treatment for 5 s. FINDINGS: Between 5.5 and 6.6% of the DSB bacteria were transferred to hands with one touch and ∼20% were then transferred to HBA with one touch, giving an overall transfer rate of 1.26% and 1.04% for polycarbonate and glass coupons, respectively. Detergent treatment had little effect on bacterial removal from coupons, but, for biofilm grown on polycarbonate, significantly increased transferral to HBA (P < 0.001) to 5.2%. Large numbers of bacteria were transferred by bare hands to multiple fomites. One-third of polycarbonate coupons transferred >1000 colonies during the first five sequential touches. Sufficient bacteria to cause infection were transmitted up to 19 times following one touch of the DSB. CONCLUSION: DSB bacteria are transferred by hands from one fomite to multiple fomites, suggesting that DSB may serve as a persistent environmental source of pathogens.

Evaluation of short exposure times of antimicrobial wound solutions against microbial biofilms: from in vitro to in vivo.

Objectives: Test the performance of topical antimicrobial wound solutions against microbial biofilms using in vitro, ex vivo and in vivo model systems at clinically relevant exposure times. Methods: Topical antimicrobial wound solutions were tested under three different conditions: (in vitro) 4% w/v Melaleuca oil, polyhexamethylene biguanide, chlorhexidine, povidone iodine and hypochlorous acid were tested at short duration exposure times for 15 min against 3 day mature biofilms of Staphylococcus aureus and Pseudomonas aeruginosa; (ex vivo) hypochlorous acid was tested in a porcine skin explant model with 12 cycles of 10 min exposure, over 24 h, against 3 day mature P. aeruginosa biofilms; and (in vivo) 4% w/v Melaleuca oil was applied for 15 min exposure, daily, for 7 days, in 10 patients with chronic non-healing diabetic foot ulcers complicated by biofilm. Results: In vitro assessment demonstrated variable efficacy in reducing biofilms ranging from 0.5 log10 reductions to full eradication. Repeated instillation of hypochlorous acid in a porcine model achieved <1 log10 reduction (0.77 log10, P = 0.1). Application of 4% w/v Melaleuca oil in vivo resulted in no change to the total microbial load of diabetic foot ulcers complicated by biofilm (median log10 microbial load pre-treatment = 4.9 log10 versus 4.8 log10, P = 0.43). Conclusions: Short durations of exposure to topical antimicrobial wound solutions commonly utilized by clinicians are ineffective against microbial biofilms, particularly when used in vivo. Wound solutions should not be used as a sole therapy and clinicians should consider multifaceted strategies that include sharp debridement as the gold standard.

Determination of bacterial species present in biofilm contaminating the channels of clinical endoscopes.

BACKGROUND: Outbreaks of endoscopy-related Carbapenem-resistant Enterobacteriaceae has highlighted failures in endoscope decontamination resulting in biofilm formation. Biofilms are tolerant to detergents and disinfectants. We evaluated decontaminated endoscope channels for residual bacterial contamination and biofilm presence. METHODS: 64 channels were collected from 12 gastroscopes and 11 colonoscopes. Aerobic bacteria were isolated from inside the endoscope tubing by scrapping, sonication, and aerobic plate culture. Total number of contaminating bacteria was determined by quantitative real-time PCR with 16s rRNA eubacterial universal primers. Microbial diversity was assessed using next generation DNA sequencing. Biofilm presence was visually confirmed by confocal laser scanning and scanning electron microscopy. RESULTS: 47% of channels were culture positive, with α-haemolytic Streptococci from gastroscopes and coliforms from colonoscopes the most frequently isolated species. Sphingomonas spp., Staphylococcus spp., Streptococcus spp., and Pseudomonas aeruginosa were also isolated. An average of 1.2 × 103 bacteria/cm contaminated air-water channels, 2.8 × 102 and 6.6 × 102 bacteria/cm contaminated gastroscope and colonoscope working channels, respectively. Biofilm was on all 39 channels examined and was principally composed of environmental bacteria, although all samples contained potential pathogens. CONCLUSION: Biofilm is present on many endoscope channels obtained from Australian hospitals. Any soil including biofilm can compromise disinfectant action.

Pattern of prescription of antibiotics among dental practitioners in Jeddah, KSA: A cross-sectional survey.

BACKGROUND: Inappropriate use of antibiotics by clinicians leads to antibiotic resistance, and is a serious global health concern. AIM: The aim of this study was to determine antibiotic prescription practices of dental practitioners and their adherence to professional guidelines while treating oral health problems among children. MATERIALS AND METHODS: All the dentists working in Jeddah were invited to participate and 126 dentists consented to be a part of this study. A two-part questionnaire was applied. First section explored the demographics and questions related to antibiotic use for certain dental clinical procedures, whereas the second section used five different clinical case scenarios to elicit the antibiotics prescription practices and adherence to professional guidelines by the dentists. RESULTS: Amoxicillin (73.8%) was the most commonly preferred antibiotic for most of the orofacial infections among the dentists. The overall adherence to the professional guidelines ranged from 9.5 to 45%. CONCLUSIONS: A lack of consistency in the antibiotic prescription pattern among dentists in Jeddah and overall low adherence to the professional guidelines was observed.

Next Generation DNA Sequencing of Tissues from Infected Diabetic Foot Ulcers.

We used next generation DNA sequencing to profile the microbiome of infected Diabetic Foot Ulcers (DFUs). The microbiota was correlated to clinical parameters and treatment outcomes to determine if directed antimicrobial therapy based on conventional microbiological cultures are relevant based on genomic analysis. Patients≥18years presenting with a new Diabetic Foot Infection (DFI) who had not received topical or oral antimicrobials in the two weeks prior to presentation, were eligible for enrolment. Tissue punch biopsies were obtained from infected DFUs for analysis. Demographics, clinical and laboratory data were collected and correlated against microbiota data. Thirty-nine patients with infected DFUs were recruited over twelve-months. Shorter duration DFUs (

Effect of cadexomer iodine on the microbial load and diversity of chronic non-healing diabetic foot ulcers complicated by biofilm in vivo.

Objectives: The performance of cadexomer iodine was determined against microbial populations from chronic non-healing diabetic foot ulcers (DFUs) complicated by biofilm in vivo , using molecular, microscopy and zymography methods. Methods: Chronic non-healing DFUs due to suspected biofilm involvement were eligible for enrolment. DNA sequencing and real-time quantitative PCR was used to determine the microbial load and diversity of tissue punch biopsies obtained pre- and post-treatment. Scanning electron microscopy and/or fluorescence in situ hybridization confirmed the presence or absence of biofilm. Zymography was used to determine levels of wound proteases. Results: Seventeen participants were recruited over a 6 month period. Scanning electron microscopy and or fluorescence in situ hybridization confirmed the presence of biofilm in all samples. Eleven participants exhibited log 10 reductions in microbial load after treatment (range 1-2 log 10 ) in comparison with six patients who experienced <1 log 10 reduction ( P = 0.04). Samples were tested for levels of wound proteases pre- and post-treatment. Reductions in the microbial load correlated to reductions in wound proteases pre- and post-treatment ( P = 0.03). Conclusions: To the best of our knowledge, this study represents the first in vivo evidence, employing a range of molecular and microscopy techniques, of the ability of cadexomer iodine to reduce the microbial load of chronic non-healing DFUs complicated by biofilm. Further analyses correlating log reductions to optimal duration of therapy and improvements in clinical parameters of wound healing in a larger cohort are required.

Self-management practices among type 2 diabetes patients attending primary health-care centres in Medina, Saudi Arabia.

The purpose of this study was to estimate the frequency of self-management activities among people who have type 2 diabetes in Saudi Arabia. The Arabic version of the Summary of Diabetes Self-care Activities questionnaire was used to identify self-management practices among 210 patients with type 2 diabetes mellitus. Only 15% of participants had a blood glucose level indicative of good glycaemic control (glycosylated haemoglobin ≤ 7 mmol/L). Most reported that they took their medication as prescribed, but many demonstrated low levels of compliance with other self-management practices (overall mean 3.7 days per week). Males and those with lower incomes were less likely to practise self-care activities. Most were given basic advice to undertake self-care activities, but only some were given more detailed information. There are opportunities to improve type 2 diabetes mellitus self-management practices in Saudi Arabia and increase the proportion of patients who achieve good glycaemic control.

Intensive care unit environmental surfaces are contaminated by multidrug-resistant bacteria in biofilms: combined results of conventional culture, pyrosequencing, scanning electron microscopy, and confocal laser microscopy.

BACKGROUND: Hospital-associated infections cause considerable morbidity and mortality, and are expensive to treat. Organisms causing these infections can be sourced from the inanimate environment around a patient. Could the difficulty in eradicating these organisms from the environment be because they reside in dry surface biofilms? AIM: The intensive care unit (ICU) of a tertiary referral hospital was decommissioned and the opportunity to destructively sample clinical surfaces was taken in order to investigate whether multidrug-resistant organisms (MDROs) had survived the decommissioning process and whether they were present in biofilms. METHODS: The ICU had two 'terminal cleans' with 500 ppm free chlorine solution; items from bedding, surrounds, and furnishings were then sampled with cutting implements. Sections were sonicated in tryptone soya broth and inoculated on to chromogenic plates to demonstrate MDROs, which were confirmed with the Vitek2 system. Genomic DNA was extracted directly from ICU samples, and subjected to polymerase chain reaction (PCR) for femA to detect Staphylococcus aureus and the microbiome by bacterial tag-encoded FLX amplicon pyrosequencing. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were performed on environmental samples. FINDINGS: Multidrug-resistant bacteria were cultured from 52% (23/44) of samples cultured. S. aureus PCR was positive in 50%. Biofilm was demonstrated in 93% (41/44) of samples by CLSM and/or SEM. Pyrosequencing demonstrated that the biofilms were polymicrobial and contained species that had multidrug-resistant strains. CONCLUSION: Dry surface biofilms containing MDROs are found on ICU surfaces despite terminal cleaning with chlorine solution. How these arise and how they might be removed requires further study.

Salivary microRNAs in oral cancer.

OBJECTIVE: This study investigated the use of three salivary microRNAs (miRNA-21, miRNA-184, and miRNA-145) as possible markers for malignant transformation in oral mucosal lesions. MATERIALS AND METHODS: Salivary whole unstimulated samples were collected from a study group of 100 subjects, consisting of 20 clinically healthy controls, 40 patients with oral potentially malignant disorders (PMDs) [20 with dysplastic lesions and 20 without dysplasia], 20 with biopsy-confirmed oral squamous cell carcinoma (OSCC), and 20 with recurrent aphthous stomatitis (RAS) as disease controls. Total RNA was isolated and purified from saliva samples using the microRNA Isolation Kit (Qiagen, UL). miRNA expression analysis was performed using qRT-PCR (Applied Biosystems). RESULTS: There was a highly significant increase in salivary miRNA-21 and miRNA-184 in OSCC and PMD (with and without dysplasia) when compared to healthy and disease controls (P < 0.001). Conversely, miRNA-145 levels showed a highly significant decrease in OSCC and PMD overall (P < 0.001). RAS cases showed no significant difference from normal controls in any measured miRNA (P > 0.05). The only microRNA to discriminate between OSCC and PMD with dysplasia was miRNA-184. When receiver operating characteristic curves were designed for the three miRNAs, cutoff points delineating the occurrence of malignant change were a fourfold increase in miRNA-21 with specificity 65% and sensitivity 65%, a 0.6 decrease in miRNA-145, with specificity 70% and sensitivity 60%, and a threefold increase of miRNA-184, with specificity 75% and sensitivity 80%. Calculating the area under the curve revealed that miRNA-184 was the only one among the studied miRNAs that provided good diagnostic value. CONCLUSION: Salivary determination of the miRNAs tested might furnish a noninvasive, rapid adjunctive aid for revealing malignant transformation in oral mucosal lesions, particularly miRNA-184.

Self-management practices among type 2 diabetes patients attending primary health-care centres in Medina, Saudi Arabia

The purpose of this study was to estimate the frequency of self-management activities among people who have type 2 diabetes in Saudi Arabia. The Arabic version of the Summary of Diabetes Self-care Activities questionnaire was used to identify self-management practices among 210 patients with type 2 diabetes mellitus. Only 15% of participants had a blood glucose level indicative of good glycaemic control [glycosylated haemoglobin </= 7 mmol/L]. Most reported that they took their medication as prescribed, but many demonstrated low levels of compliance with other self-management practices [overall mean 3.7 days per week]. Males and those with lower incomes were less likely to practise self-care activities. Most were given basic advice to undertake self-care activities, but only some were given more detailed information. There are opportunities to improve type 2 diabetes mellitus self-management practices in Saudi Arabia and increase the proportion of patients who achieve good glycaemic control

L'objectif de la presente etude etait d'estimer la frequence des activites d'auto-prise en charge chez des personnes atteintes de diabete de type 2 en Arabie saoudite.La version en langue arabe du questionnaire Summary of Diabetes Self-care Activities a ete utilisee pour identifier les pratiques d'auto-prise en charge chez 210 patients atteints de diabete sucre de type 2.Seuls 15 % des participants presentaient un taux glycemique revelateur d'un bon controle de leur glycemie [hemoglobine glycosylee </= 7 mmol/L]. La plupart indiquaient avoir pris leur traitement comme prescrit,mais ils etaient nombreux a presenter des taux d'observance faibles pour d'autres pratiques d'auto-prise en charge [moyenne d'observance globale hebdomadaire:3,7 jours sur sept]. Les hommes et les personnes ayant des revenus plus faibles etaient moins susceptibles de pratiquer des activites d'autosoins.Pour la majorite,ils avaient recu des conseils elementaires pour realiser des activites d'autosoins,mais seule une minorite d'entre eux avait eu des informations plus detaillees.Des opportunites existent pour ameliorer les pratiques d'auto-prise en charge du diabete de type 2 en Arabie saoudite et augmenter le pourcentage de patients qui parviennent a un bon controle du taux de glycemie

Randomised controlled trial of the efficacy of HybenX in the symptomatic treatment of recurrent aphthous stomatitis.

BACKGROUND: The treatment of recurrent aphthous stomatitis (RAS) is principally directed towards reducing the pain and duration of each episode of ulceration; however, there remain few agents for which there is definitive evidence of benefit. AIMS: The aims of the present study were to determine the efficacy of HybenX (Epien Medical Inc., Minneapolis, MN, USA), vs another device used for the treatment of RAS (Salicept; Carrington Laboratories Inc., lrving, TX, USA) to reduce the symptoms and duration of RAS and determine the safety of HybenX for this clinical application. MATERIAL AND METHODS: Sixty-three individuals (36 male, 27 female, group median age 25 years, range 17.8-57.9 years) were entered into a prospective randomised controlled trial of HybenX vs an occlusive covering device (Salicept oral patches; Carrington Laboratories). RESULTS: Painful symptoms over a 5-day posttreatment period were reduced by both agents although HybenX was statistically more effective at day 2 than Salicept, and there was a trend for HybenX to cause greater pain reduction than Salicept over this 5-day period. Both agents gave rise to few adverse side effects--a total of nine adverse events in eight patients were recorded. All were unlikely to be related to the treatment devices. HybenX was only applied on one occasion to the HybenX group, while individuals in the Salicept group were able to self medicate as required. The mean number of Salicept patches used per day per subject was three (s.d. 3.3) on day 1 posttreatment, 3.4 (s.d. 3.1) on day 2 and 2.7 (s.d. 1.9) on day 3. Thereafter, the number of applications fell to a mean of 0.8 on day 7. CONCLUSION: It is concluded that HybenX safely and effectively reduces the painful symptoms of RAS.

Onset and progression of clinical manifestations of orofacial granulomatosis.

BACKGROUND: There remain few studies describing in detail the early occurrence and long-term progression of clinical manifestations of orofacial granulomatosis (OFG) in a substantial number of patients. OBJECTIVES: The aim of this study was to determine the early and late clinical manifestations of a large case series of patients with OFG. PATIENTS/METHODS: Clinically relevant data of 49 patients with OFG who attended an Oral Medicine unit in the UK were examined retrospectively. The analyzed parameters included occurrence and typology of initial manifestations at onset and with respect to long-term follow-up. RESULTS: Five major patterns of disease onset were observed. Recurrent facial swelling with/without intra-oral manifestations was the single most common presentation at onset followed by intra-oral ulcers, and other intra-oral and neurological manifestations. The majority of patients later developed a spectrum of additional features. CONCLUSIONS: OFG results in multiple manifestations at different time points. The disease onset is characterized by manifestations other than facial swelling in about half of affected individuals. However, patients can develop cosmetically unacceptable lip/facial swelling at a later stage. Nearly all affected individuals ultimately develop lip/facial swelling while about half of all patients develop oral ulceration.