Effects of a randomized controlled trial of a brief, student-nurse led, parent-based sexual health intervention on parental protective factors and HPV vaccination uptake.

BACKGROUND: Parents play a pivotal role in adolescent sexual health and Human Papillomavirus (HPV) vaccination. Nurses are on the frontlines of healthcare and play a critical role in promoting HPV vaccination and parent-child sexual health communication. We enhanced the Families Talking Together (FTT) parent-based sexual health curriculum to include adolescent vaccinations herein, FTT + HPV, and trained student nurses to provide a strong HPV vaccination and parent-child sexual health communication endorsement. METHODS: Using a randomized attention-controlled trial design, we examined the efficacy of FTT + HPV among 519 parents and their 11-14 year old youth recruited from medically underserved communities between 2015 and 2018. Participants were recruited from 22 after-school programs (e.g., Boys and Girls Clubs) and 19 charter schools. For parents, we examined protective factors including parent-child sexual health communication and parental involvement. For youth, we examined sexual health knowledge, parent-child sexual health communication, and parent-child connectedness. To assess HPV vaccination initiation and completion, we searched IMMTRAC immunization registry records for 85% of youth and used parental report for youth without registry records. Group differences were calculated using the estimated mean difference at one- and six months post-intervention with significance set at the p < 0.05 level. RESULTS: Baseline rates of HPV vaccination were low at 55.7%. No significant difference between the groups was seen in vaccination initiation or completion rates by one-month post-intervention. However, by six-months post intervention, there was a significant difference between the groups with 70.3% of the intervention group initiating the HPV vaccination series vs. 60.6% for the control group (p = 0.02). No difference between the groups was found for HPV series completion at six-months. There were significant differences in condom knowledge (p = 0.04), parent-child connectedness (p = 0.04), and communication frequency (p = 0.001) with greater improvement in the intervention vs. the control group. Rates of sexual activity remained low in both groups throughout the six-month follow-up period. CONCLUSION: A brief parent-based adolescent sexual health and HPV vaccination intervention delivered by student nurses can improve sexual health outcomes including protective parental factors, adolescent sexual health knowledge, and HPV vaccination initiation rates. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02600884 . Prospectively registered September 1, 2015.

Reduced pro-inflammatory responses to Staphylococcus aureus bloodstream infection and low prevalence of enterotoxin genes in isolates from patients on haemodialysis.

Patients with end-stage renal failure undergo regular haemodialysis (HD) and often develop episodes of Staphylococcus aureus bloodstream infection (BSI), which can re-occur. However, clinically, patients on HD, with S. aureus BSI, respond well to treatment, rarely developing overt signs of sepsis. We investigated the contributions of bacterial virulence and cytokine responses to the clinical course of S. aureus BSI in HD and non-HD patients. Seventy patients were recruited, including 27 (38.6 %) patients on HD. Isolates were spa-typed and virulence and antimicrobial resistance gene carriage was investigated using DNA microarray analysis. Four inflammatory cytokines, IL-6, RANTES, GROγ and leptin, were measured in patient plasma on the day of diagnosis and after 7 days. There was no significant difference in the prevalence of genotypes or antimicrobial resistance genes in S. aureus isolates from HD compared to non-HD patients. The enterotoxin gene cluster (containing staphylococcal enterotoxins seg, sei, sem, sen, seo and seu) was significantly less prevalent among BSI isolates from HD patients compared to non-HD patients. Comparing inflammatory cytokine response to S. aureus BSI in HD patients to non-HD patients, IL-6 and GROγ were significantly lower (p = 0.021 and p = 0.001, respectively) in HD patients compared to other patients on the day of diagnosis and RANTES levels were significantly lower (p = 0.025) in HD patients on day 7 following diagnosis. Lowered cytokine responses in HD patients and a reduced potential for super-antigen production by infecting isolates may partly explain the favourable clinical responses to episodes of S. aureus BSI in HD patients that we noted clinically.

Protracted transmission and persistence of ST80 vancomycin-resistant Enterococcus faecium clonal complex types CT2933, CT2932 and CT1916 in a large Irish hospital: a 39-month whole-genome sequencing study.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VREfm) are significant nosocomial pathogens. Sequence type (ST) 80 vanA-encoding VREfm predominate in Irish hospitals, but their transmission is poorly understood. AIMS: To investigate transmission and persistence of predominant complex type (CT) VREfm in two wards of an Irish hospital (H1) using whole-genome sequencing, and their intra- and inter-hospital dissemination. METHODS: Rectal screening (N = 330, September 2019 to December 2022) and environmental (N = 48, November 2022 to December 2022) E. faecium were investigated. Isolate relatedness was assessed by core-genome multi-locus sequence typing (cgMLST) and core-genome single nucleotide polymorphism (cgSNP) analysis. Likely transmission chains were identified using SeqTrack (https://graphsnp.fordelab.com/graphsnp) using cgSNP data and recovery location. Well-characterized E. faecium (N = 908) from seven Irish hospitals including H1 (June 2017 to July 2022) were also investigated. FINDINGS: Conventional MLST assigned isolates to nine STs (ST80, 82%). cgMLST identified three predominant ST80 CTs (CT2933, CT2932 and CT1916) (55% of isolates) of related isolates (≤20 allelic differences). cgSNP analysis differentiated these CTs into multiple distinct closely related genomic clusters (≤10 cgSNPs). Parisimonious network construction identified 55 likely inter- and intra-ward transmissions with epidemiological support between patients ≤30 days involving 73 isolates (≤10 cgSNPs) from seven genomic clusters. Numerous other likely transmissions over longer time periods without evident epidemiological links were identified, suggesting persistence and unidentified reservoirs contribute to dissemination. The three CTs predominated among E. faecium (N = 1286) in seven hospitals, highlighting inter-hospital spread without known epidemiological links. CONCLUSION: This study revealed the long-term intra- and inter-hospital dominance of three major CT ST80 VREfm lineages, widespread transmission and persistence, implicating unidentified reservoirs.

An emerging Panton-Valentine leukocidin-positive CC5-meticillin-resistant Staphylococcus aureus-IVc clone recovered from hospital and community settings over a 17-year period from 12 countries investigated by whole-genome sequencing.

BACKGROUND: A novel Panton-Valentine leukocidin (PVL)-positive meticillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC)5-MRSA-IVc ('Sri Lankan' clone) was recently described from Sri Lanka. Similar isolates caused a recent Irish hospital outbreak. AIM: To investigate the international dissemination and diversity of PVL-positive CC5-MRSA-IVc isolates from hospital and community settings using whole-genome sequencing (WGS). METHODS: Core-genome single nucleotide polymorphism (cgSNP) analysis, core-genome multi-locus sequence typing (cgMLST) and microarray-based detection of antimicrobial-resistance and virulence genes were used to investigate PVL-positive CC5-MRSA-IVc (N = 214 including 46 'Sri Lankan' clone) from hospital and community settings in 12 countries over 17 years. Comparators included 29 PVL-positive and 23 PVL-negative CC5/ST5-MRSA-I/II/IVa/IVc/IVg/V. RESULTS: Maximum-likelihood cgSNP analysis grouped 209/214 (97.7%) CC5-MRSA-IVc into Clade I; average of 110 cgSNPs between isolates. Clade III contained the five remaining CC5-MRSA-IVc; average of 92 cgSNPs between isolates. Clade II contained seven PVL-positive CC5-MRSA-IVa comparators, whereas the remaining 45 comparators formed an outlier group. Minimum-spanning cgMLST analysis revealed a comparably low average of 57 allelic differences between all CC5/ST5-MRSA-IVc. All 214 CC5/ST5-MRSA-IVc were identified as 'Sri Lankan' clone, predominantly spa type t002 (186/214) with low population diversity and harboured a similar range of virulence genes and variable antimicrobial-resistance genes. All 214 Sri Lankan clone isolates and Clade II comparators harboured a 9616-bp chromosomal PVL-encoding phage remnant, suggesting both arose from a PVL-positive meticillin-susceptible ancestor. Over half of Sri Lankan clone isolates were from infections (142/214), and where detailed metadata were available (168/214), most were community associated (85/168). CONCLUSIONS: Stable chromosomal retention of pvl may facilitate Sri-Lankan clone dissemination.

Immersion of debrided diabetic foot ulcer tissue in electrochemically generated pH neutral hypochlorous acid significantly reduces the microbial bioburden: whole-genome sequencing of Staphylococcus aureus, the most prevalent species recovered.

BACKGROUND: Diabetic foot ulcer infections (DFUIs) are the leading cause of lower-limb amputations, mediated predominantly by Staphylococcus aureus. pH-neutral electrochemically generated hypochlorous acid (anolyte) is a non-toxic, microbiocidal agent with significant potential for wound disinfection. AIMS: To investigate both the effectiveness of anolyte for microbial bioburden reduction in debrided ulcer tissues and the population of resident S. aureus. METHODS: Fifty-one debrided tissues from 30 people with type II diabetes were aliquoted by wet weight and immersed in 1- or 10-mL volumes of anolyte (200 parts per million) or saline for 3 min. Microbial loads recovered were determined in colony forming units/g (cfu/g) of tissue following aerobic, anaerobic and staphylococcal-selective culture. Bacterial species were identified and 50 S. aureus isolates from 30 tissues underwent whole-genome sequencing (WGS). FINDINGS: The ulcers were predominantly superficial, lacking signs of infection (39/51, 76.5%). Of the 42/51 saline-treated tissues yielding ≥105 cfu/g, a microbial threshold reported to impede wound-healing, only 4/42 (9.5%) were clinically diagnosed DFUIs. Microbial loads from anolyte-treated tissues were significantly lower than saline-treated tissues using 1 mL (1065-fold, 2.0 log) and 10 mL (8216-fold, 2.1 log) immersion volumes (P<0.0005). S. aureus was the predominant species recovered (44/51, 86.3%) and 50 isolates underwent WGS. All were meticillin susceptible and comprised 12 sequence types (STs), predominantly ST1, ST5 and ST15. Whole-genome multi-locus sequence typing identified three clusters of closely related isolates from 10 patients indicating inter-patient transmission. CONCLUSIONS: Short immersions of debrided ulcer tissue in anolyte significantly reduced microbial bioburden: a potential novel DFUI treatment.

Transmission of endemic ST22-MRSA-IV on four acute hospital wards investigated using a combination of spa, dru and pulsed-field gel electrophoresis typing.

The transmission of meticillin-resistant Staphylococcus aureus (MRSA) between individual patients is difficult to track in institutions where MRSA is endemic. We investigated the transmission of MRSA where ST22-MRSA-IV is endemic on four wards using demographic data, patient and environmental screening, and molecular typing of isolates. A total of 939 patients were screened, 636 within 72 h of admission (on admission) and 303 >72 h after admission, and 1,252 environmental samples were obtained. Isolates were typed by spa, dru and pulsed-field gel electrophoresis (PFGE) typing. A composite dendrogram generated from the three sets of typing data was used to divide isolates into 'dendrogram groups' (DGs). Ten percent of patients (92/939) were MRSA-positive; 7 % (44/636) on admission and 16 % (48/303) >72 h after admission (p = 0.0007). MRSA was recovered from 5 % of environmental specimens (65/1,252). Most isolates from patients (97 %, 85/88) and the environment (97 %, 63/65) exhibited the ST22-MRSA-IV genotype. Four DGs (DG1, DG4, DG16 and DG17) accounted for 58 % of ST22-MRSA-IV isolates from patients. Epidemiological evidence suggested cross-transmission among 44/92 patients (48 %) but molecular typing confirmed probable cross-transmission in only 11 instances (13 %, 11/88), with the majority of cross-transmission (64 %; 7/11) occurring on one ward. In the setting of highly clonal endemic MRSA, the combination of local epidemiology, PFGE, spa and dru typing provided valuable insights into MRSA transmission.

Effective cleaning and decontamination of the internal air and water channels, heads and head-gears of multiple contra-angle dental handpieces using an enzymatic detergent and automated washer-disinfection in a dental hospital setting.

BACKGROUND: Dental handpieces (DHPs) are reusable invasive medical devices that must be cleaned, decontaminated, lubricated and steam sterilized after use. DHPs have a complex internal design including narrow channels, contamination of which can compromise sterilization. DHPs are not designed for routine disassembly, making cleaning/decontamination efficacy difficult to monitor. Washer-disinfection is the preferred method of decontaminating DHPs, but few studies have investigated its direct effectiveness at reducing microbial contamination internally. AIMS: To use contra-angle DHPs as a model system to investigate the effectiveness of washer-disinfection at reducing microbial contamination of internal components of multiple DHPs. METHODS: The air and water channels and heads of 10 disassembled contra-angle DHPs (BienAir, Biel/Bienne, Switzerland) were inoculated separately with 108 colony forming units (cfu) of Pseudomanas aeruginosa, Staphylococcus aureus, Enterococcus hirae or Candida albicans in the presence of 0.3% bovine serum albumin (BSA) (clean conditions), 3.0% BSA or 10% artificial test soil (dirty conditions). After reassembly, all 10 DHPs underwent washer-disinfection simultaneously in a Míele (Míele Ireland Ltd., Dublin, Ireland) PG8528 washer-disinfector and were tested for reductions in micro-organisms and protein. Additional experiments were undertaken with three lubricated DHPs inoculated with S. aureus and 10% test soil. All experiments were repeated in triplicate. FINDINGS: On average, an approximate 5 log or greater reduction in microbial cfu and a >93% reduction in protein from DHP heads and channels was consistently recorded following washer-disinfection for all DHPs under all conditions tested. CONCLUSIONS: The internal components of multiple DHPs can be effectively cleaned and decontaminated by washer-disinfection.

Meticillin-susceptible Staphylococcus aureus transmission among healthcare workers, patients and the environment in a large acute hospital under non-outbreak conditions investigated using whole-genome sequencing.

BACKGROUND: The role of meticillin-susceptible Staphylococcus aureus (MSSA) colonization of healthcare workers (HCWs), patients and the hospital environment in MSSA transmission events (TEs) is poorly understood. AIMS: The role of meticillin-resistant Staphylococcus aureus (MRSA) was investigated recently under non-outbreak conditions in a large hospital with a history of endemic MRSA over 2 years using whole-genome sequencing (WGS). Numerous potential MRSA TEs were identified. The present study investigated MSSA TEs from the same sources during the same 2-year hospital study. METHODS: HCW (N=326) and patient (N=388) volunteers on nine wards were tested for nasal and oral MSSA colonization over 2 years. Near-patient environment (N=1164), high-frequency touch sites (N=810) and air (N=445) samples were screened for MSSA. Representative MSSA and clinical isolates were sequenced and analysed by core genome multi-locus sequence typing. Closely related isolates (≤24 allelic differences) were segregated into related isolate groups (RIGs). Potential TEs involving MSSA in RIGs from HCWs, patients and patient infections were identified in combination with epidemiological data. FINDINGS: In total, 635 MSSA were recovered: clinical isolates (N=82), HCWs (N=170), patients (N=120), and environmental isolates (N=263). Twenty-four clonal complexes (CCs) were identified among 406/635 MSSA sequenced, of which 183/406 segregated into 59 RIGs. Numerous potential HCW-to-patient, HCW-to-HCW and patient-to-patient TEs were identified, predominantly among CC5-MSSA, CC30-MSSA and CC45-MSSA. HCW, patient, clinical and environmental isolates were identified in 33, 24, six and 32 RIGs, respectively, with 19/32 of these containing MSSA related to HCW and/or patient isolates. CONCLUSIONS: WGS detected numerous potential hospital MSSA TEs involving HCWs, patients and environmental contamination under non-outbreak conditions.

Meticillin-resistant Staphylococcus aureus transmission among healthcare workers, patients and the environment in a large acute hospital under non-outbreak conditions investigated using whole-genome sequencing.

BACKGROUND: The role of meticillin-resistant Staphylococcus aureus (MRSA) colonization of healthcare workers (HCWs), patients and the hospital environment in MRSA transmission in non-outbreak settings is poorly understood. AIMS: To investigate transmission events (TEs) involving HCWs, patients and the environment under non-outbreak conditions in a hospital with a history of endemic MRSA using whole-genome sequencing (WGS). METHODS: HCW (N = 326) and patient (N = 388) volunteers on nine wards were tested for nasal and oral MRSA colonization over two years. Near-patient environment (N = 1164), high-frequency touch sites (N = 810) and air (N = 445) samples were screened for MRSA. Representative MRSA and clinical isolates were analysed by WGS and core-genome multi-locus sequence typing (cgMLST). Closely related isolates (≤24 allelic differences) were segregated into related isolated groups (RIGs). FINDINGS: In total, 155 MRSA were recovered: clinical isolates (N = 41), HCWs (N = 22), patients (N = 37), environmental isolates (N = 55). Nine clonal complexes (CCs) were identified among 110/155 MRSA sequenced with 77/110 assigned to CC22. Seventy-nine MRSA segregated into 17 RIGs. Numerous potential TEs were associated with CC22-MRSA (RIGs 1-15), CC45-MRSA (RIG-16) and CC8-MRSA (RIG-17). RIG-1, (the largest RIG) contained 24 ST22-MRSA-IVh from six HCWs, six patients, four clinical and eight environmental samples recovered over 17 months involving 7/9 wards. TEs involving HCW-to-patient, HCW-to-HCW, patient-to-patient and environmental contamination by HCW/patient isolates were evident. HCW, patient, clinical and environmental isolates were identified in four, nine, seven and 13 RIGs, respectively, with 12/13 of these containing isolates closely related to HCW and/or patient isolates. CONCLUSIONS: WGS detected numerous potential hospital MRSA TEs involving HCWs, patients and the environment under non-outbreak conditions.

Multiple distinct outbreaks of Panton-Valentine leucocidin-positive community-associated meticillin-resistant Staphylococcus aureus in Ireland investigated by whole-genome sequencing.

BACKGROUND: Panton-Valentine leucocidin (PVL)-positive community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) is increasingly associated with infection outbreaks. AIM: To investigate multiple suspected PVL-positive CA-MRSA outbreaks using whole-genome sequencing (WGS). METHODS: Forty-six suspected outbreak-associated isolates from 36 individuals at three separate Irish hospitals (H1-H3) and from separate incidents involving separate families associated with H2 were investigated by whole-genome multi-locus sequence typing (wgMLST). FINDINGS: Two clusters (CH1 and CH2) consisting of 8/10 and 6/6 PVL-positive t008 ST8-MRSA-IVa isolates from H1 and H2, respectively, were identified. Within each cluster, neighbouring isolates were separated by ≤5 allelic differences; however, ≥73 allelic differences were identified between the clusters, indicating two independent outbreaks. Isolates from the H3 maternity unit formed two clusters (CH3-SCI and CH3-SCII) composed of four PVL-negative t4667 ST5-MRSA-V and 14 PVL-positive t002 ST5-MRSA-IVc isolates, respectively. Within clusters, neighbouring isolates were separated by ≤24 allelic differences, whereas both clusters were separated by 1822 allelic differences, indicating two distinct H3 outbreaks. Eight PVL-positive t127 ST1-MRSA-V+fus and three PVL-negative t267 ST97-MRSA-V+fus isolates from two distinct H2-associated families FC1 (N = 4) and FC2 (N = 7) formed three separate clusters (FC1 (t127), FC2 (t127) and FC2 (t267)). Neighbouring isolates within clusters were closely related and exhibited ≤7 allelic differences. Intrafamilial transmission was apparent, but the detection of ≥48 allelic differences between clusters indicated no interfamilial transmission. CONCLUSION: The frequent importation of PVL-positive CA-MRSA into healthcare settings, transmission and association with outbreaks is a serious ongoing concern. WGS is a highly discriminatory, informative method for deciphering such outbreaks conclusively.

Characterisation of MRSA from Malta and the description of a Maltese epidemic MRSA strain.

Malta has one of the highest prevalence rates of methicillin-resistant Staphylococcus aureus (MRSA) in Europe. However, only limited typing data are currently available. In order to address this situation, 45 MRSA isolates from the Mater Dei Hospital in Msida, Malta, were characterised using DNA microarrays. The most common strain was ST22-MRSA-IV (UK-EMRSA-15, 30 isolates). Sporadic strains included ST36-MRSA-II (UK-EMRSA-16, two isolates), PVL-positive ST80-MRSA-IV (European Clone, one isolate), ST228-MRSA-I (Italian Clone/South German Epidemic Strain, one isolate) and ST239-MRSA-III (Vienna/Hungarian/Brazilian Epidemic Strain, one isolate). Ten MRSA isolates belonged to a clonal complex (CC) 5/ST149, spa type t002 strain. This strain harboured an SCCmec IV element (mecA, delta mecR, ugpQ, dcs, ccrA2 and ccrB2), as well as novel alleles of ccrA/B and the fusidic acid resistance element Q6GD50 (previously described in the sequenced strain MSSA476, BX571857.1:SAS0043). It also carried the gene for enterotoxin A (sea) and the egc enterotoxin locus, as well as (in nine out of ten isolates) genes encoding the toxic shock syndrome toxin (tst1) and enterotoxins C and L (sec, sel). While the presence of the other MRSA strains suggests foreign importation due to travel between Malta and other European countries, the CC5/t002 strain appears, so far, to be restricted to Malta.

Whole-genome sequencing identifies highly related Pseudomonas aeruginosa strains in multiple washbasin U-bends at several locations in one hospital: evidence for trafficking of potential pathogens via wastewater pipes.

BACKGROUND: Hand washbasin U-bends have increasingly been associated with nosocomial outbreaks by Gram-negative bacteria, including Pseudomonas aeruginosa which is virtually ubiquitous in U-bends. Wastewater networks servicing U-bends are potential highways for trafficking pathogenic bacteria. AIM: To use P. aeruginosa to investigate trafficking of bacteria between hospital washbasin U-bends. METHODS: Twenty-five washbasin U-bends in five locations in Dublin Dental University Hospital (DDUH) were investigated for trafficking of P. aeruginosa: 10 in Clinic 2 (C2), 10 in the Accident & Emergency Department (A&E) and five in three other locations. In addition, washbasin tap samples (N=80) and mains and tap water samples (N=72) were cultured for P. aeruginosa. Selected P. aeruginosa isolates recovered over 29 months underwent whole-genome sequencing, and relatedness was interpreted using whole-genome multi-locus sequence typing and pairwise single nucleotide polymorphism (SNP) analysis. FINDINGS: P. aeruginosa was recovered from all U-bends but not from taps or water. Eighty-three U-bend isolates yielded 10 sequence types (STs), with ST560 and ST179 from A&E, C2 and two other locations predominating (70%). ST560 was also recovered from a common downstream pipe. Isolates within ST560 and ST179 were highly related regardless of source. ST560 was divided into Cluster I (N=25) and Cluster II (N=2) with average allelic differences and SNPs of three and zero, and two and five, respectively. The 31 ST179 isolates exhibited an average allelic difference and SNPs of three and 12, respectively. CONCLUSION: Highly related P. aeruginosa strains were identified in multiple U-bends in several DDUH locations, indicating trafficking via the wastewater network.

The oral cavity revealed as a significant reservoir of Staphylococcus aureus in an acute hospital by extensive patient, healthcare worker and environmental sampling.

BACKGROUND: Methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream infection rates have risen steadily in recent years, with a marked decline in the corresponding rates due to methicillin-resistant S. aureus (MRSA). Screening for MSSA carriage is not routinely undertaken and MRSA screening is not universal, so the extent of S. aureus colonisation pressure in nosocomial settings is unknown. METHODS: We conducted a prospective, observational study of patients and healthcare workers (HCWs) across nine inpatient wards in a tertiary referral hospital over a two-year period. Participants were screened for MSSA and MRSA using nasal swabs and oral rinses. Environmental surfaces and air were also tested for S. aureus using contact plates and active air sampling. FINDINGS: We enrolled 388 patients and 326 HCWs; and took 758 contact plate samples from surfaces and 428 air samples. MSSA was recovered from 24% of patients, 31.3% of HCWs, 16% of air samples and 7.9% of surface samples. MRSA was recovered from 6.4% of patients, 3.7% of HCWs, 2.5% of air samples and 2.2% of surface samples. Inclusion of the oral cavity in addition to the anterior nares in the sampling regimen identified 30 patients and 36 HCWs who exhibited exclusive oral colonisation. CONCLUSIONS: The oral cavity comprises a significant nosocomial reservoir for S. aureus that is currently under-appreciated. Oral screening should be considered both in terms of the colonisation pressure in a healthcare facility, and on an individual patient level, especially in patients where decolonisation attempts have repeatedly failed and those undergoing high risk procedures.

Hospital outbreak of linezolid-resistant and vancomycin-resistant ST80 Enterococcus faecium harbouring an optrA-encoding conjugative plasmid investigated by whole-genome sequencing.

BACKGROUND: Linezolid is an antibiotic used to treat infections caused by multi-drug-resistant Gram-positive bacteria. Linezolid resistance in enterococci has been reported with increasing frequency, with a recent rise in resistance encoded by optrA, poxtA or cfr. AIM: To investigate a hospital outbreak of linezolid- and vancomycin-resistant Enterococcus faecium (LVREfm) using whole-genome sequencing (WGS). METHODS: Thirty-nine VREfm from patient screening (19 isolates, 17 patients) and environmental sites (20 isolates) recovered in October 2019 were investigated. Isolates were screened using polymerase chain reaction for optrA, poxtA and cfr, and underwent Illumina MiSeq WGS. Isolate relatedness was assessed using E. faecium core genome multi-locus sequence typing (cgMLST). One LVREfm underwent MinION long-read WGS (Oxford Nanopore Technologies) and hybrid assembly with MiSeq short-read sequences to resolve an optrA-encoding plasmid. FINDINGS: Twenty isolates (51.3%) were LVREfm and optrA-positive, including the LVREfm from the index patient. A closely related cluster of 28 sequence type (ST) 80 isolates was identified by cgMLST, including all 20 LVREfm and eight linezolid-susceptible VREfm, with an average allelic difference of two (range 0-10), indicating an outbreak. Nineteen (95%) LVREfm harboured a 56,684-bp conjugative plasmid (pEfmO_03). The remaining LVREfm exhibited 44.1% sequence coverage to pEfmO_03. The presence of pEfmO_03 in LVREfm and the close relatedness of the outbreak cluster isolates indicated the spread of a single strain. The outbreak was terminated by enhanced infection prevention and control (IPC) and environmental cleaning measures, ceasing ward admissions and ward-dedicated staff. CONCLUSION: WGS was central in investigating an outbreak of ST80 LVREfm. The rapid implementation of enhanced IPC measures terminated the outbreak.

Biofilm problems in dental unit water systems and its practical control.

Dental chair units (DCUs) contain integrated systems that provide the instruments and services for a wide range of dental procedures. DCUs use water to cool and irrigate DCU-supplied instruments and tooth surfaces during dental treatment. Water is supplied to these instruments by a network of interconnected narrow-bore (2-3 mm) plastic tubes called dental unit waterlines (DUWLs). Many studies over the last 40 years demonstrated that DUWL output water is often contaminated with high densities of micro-organisms, predominantly Gram-negative aerobic heterotropic environmental bacteria, including Legionella and Pseudomonas species. Untreated DUWLs host biofilms that permit micro-organisms to multiply and disperse through the water network and which are aerosolized by DCU instrument use, thus exposing patients and staff to these micro-organisms, to fragments of biofilm and bacterial endotoxins. This review concentrates on how practical developments and innovations in specific areas can contribute to effective DUWL biofilm control. These include the use of effective DUWL treatment agents, improvements to DCU supply water quality, DCU design changes, development of automated DUWL treatment procedures that are effective at controlling biofilm in the long-term and require minimal human intervention, are safe for patients and staff, and which do not cause deterioration of DCU components following prolonged use.

Contribution of whole-genome sequencing to understanding of the epidemiology and control of meticillin-resistant Staphylococcus aureus.

In recent years, approaches to tracking the spread of meticillin-resistant Staphylococcus aureus (MRSA) as part of outbreak management have used conventional DNA-based methods, including pulsed-field gel electrophoresis and spa typing. However, when a predominant clone is present, these methods may be insufficiently discriminatory. A literature search was undertaken to highlight how whole-genome sequencing (WGS) has revolutionized the investigation of outbreaks of MRSA, including intrahospital spread and MRSA in the community, and to review its future potential. WGS provides enhanced isolate discrimination, as it permits the entire genomic DNA sequence of isolates to be determined and compared rapidly. Software packages used for the analysis of WGS data are becoming increasingly available. To date, WGS has been more sensitive in confirming outbreaks, often persisting for prolonged periods, previously undetected by conventional molecular typing. The evolving dynamic of spread from the community to hospitals, within and between hospitals, and from hospitals to the community is only becoming clear with WGS studies, and is more complex and convoluted than widely appreciated. Also, WGS can exclude cross-transmission, when isolates are different. The challenges now are to make WGS technology more amenable for routine use, and to develop an evidence-based consensus for sequence difference thresholds for isolates that are deemed to be part of the same outbreak, including protracted outbreaks. Using such data in a timely way will provide increased sensitivity in detecting cross-transmission events at an earlier stage, with the potential to prevent outbreaks, and have a positive impact on infection prevention and control.

CYP56 (Dit2p) in Candida albicans: characterization and investigation of its role in growth and antifungal drug susceptibility.

The complete DNA sequence of Candida albicans DIT2, encoding cytochrome P450 family 56 (CYP56), was obtained, and heterologous expression was achieved in Escherichia coli, where CYP56 was targeted to the membrane fraction. In reconstituted assays with the purified enzyme, CYP56 was shown to catalyze the conversion of N-formyl tyrosine into N,N'-bisformyl dityrosine, a reaction that was dependent on cytochrome P450 reductase, NADPH, and oxygen, yielding a turnover of 21.6 min(-1) and a k(s) of 26 microM. The Hill number was calculated as 1.6, indicating that two molecules of the substrate could bind to the protein. Azole antifungals could bind to the heme of CYP56 as a sixth ligand with high affinity. Both chromosomal alleles of CYP56 were disrupted using the SAT1 flipper technique, and CYP56 was found to be nonessential for cell viability under the culture conditions investigated. Susceptibility to azole drugs that bind to cytochromes P450 was tested, and the mutant showed unaltered susceptibility. However, the mutant showed increased susceptibility to the echinocandin drug caspofungin, suggesting an alteration in 1,3-glucan synthase and/or cell wall structure mediated by the presence of dityrosine. Phenotypically, the wild-type and mutant strains were morphologically similar when cultured in rich yeast extract-peptone-dextrose medium. However in minimal medium, the cyp56Delta mutant strain exhibited hyphal growth, in contrast to the wild-type strain, which grew solely in the yeast form. Furthermore, CYP56 was essential for chlamydospore formation.

Minimizing microbial contamination risk simultaneously from multiple hospital washbasins by automated cleaning and disinfection of U-bends with electrochemically activated solutions.

BACKGROUND: Outbreaks of infection associated with microbial biofilm in hospital hand washbasin U-bends are being reported increasingly. In a previous study, the efficacy of a prototype automated U-bend decontamination method was demonstrated for a single non-hospital pattern washbasin. It used two electrochemically activated solutions (ECA) generated from brine: catholyte with detergent properties and anolyte with disinfectant properties. AIM: To develop and test a large-scale automated ECA treatment system to decontaminate 10 hospital pattern washbasin U-bends simultaneously in a busy hospital clinic. METHODS: A programmable system was developed whereby the washbasin drain outlets, U-bends and proximal wastewater pipework automatically underwent 10-min treatments with catholyte followed by anolyte, three times weekly, over five months. Six untreated washbasins served as controls. Quantitative bacterial counts from U-bends were determined on Columbia blood agar, Reasoner's 2A agar and Pseudomonas aeruginosa selective agar following treatment and 24 h later. FINDINGS: The average bacterial densities in colony-forming units/swab from treated U-bends showed a >3 log reduction compared with controls, and reductions were highly significant (P<0.0001) on all media. There was no significant increase in average bacterial counts from treated U-bends 24 h later on all media (P>0.1). P. aeruginosa was the most prevalent organism recovered throughout the study. Internal examination of untreated U-bends using electron microscopy showed dense biofilm extending to the washbasin drain outlet junction, whereas treated U-bends were free from biofilm. CONCLUSION: Simultaneous automated treatment of multiple hospital washbasin U-bends with ECA consistently minimizes microbial contamination and thus the associated risk of infection.

Nonadditive effects of leaf litter species diversity on breakdown dynamics in a detritus-based stream.

Since species loss is predicted to be nonrandom, it is important to understand the manner in which those species that we anticipate losing interact with other species to affect ecosystem function. We tested whether litter species diversity, measured as richness and composition, affects breakdown dynamics in a detritus-based stream. Using full-factorial analyses of single- and mixed-species leaf packs (15 possible combinations of four dominant litter species; red maple [Acer rubrum], tulip poplar [Liriodendron tulipifera], chestnut oak [Quercus prinus], and rhododendron [Rhododendron maximum]), we tested for single-species presence/absence (additive) or species interaction (nonadditive) effects on leaf pack breakdown rates, changes in litter chemistry, and microbial and macroinvertebrate biomass. Overall, we found significant nonadditive effects of litter species diversity on leaf pack breakdown rates, which were explained both by richness and composition. Leaf packs containing higher litter species richness had faster breakdown rates, and antagonistic effects of litter species composition were observed when any two or three of the four litter species were mixed. Less-consistent results were obtained with respect to changes in litter chemistry and microbial and macroinvertebrate biomass. Our results suggest that loss of litter species diversity will decrease species interactions involved in regulating ecosystem function. To that end, loss of species such as eastern hemlock (Tsuga canadensis) accompanied by predicted changes in riparian tree species composition in the southeastern United States could have nonadditive effects on litter breakdown at the landscape scale.