The relative efficacy of multiple syringe tip disinfection techniques against virulent staphylococcus contamination.

BACKGROUND: A recent study confirmed significant contamination of syringe tips following routine anaesthesia practice of at least 6 h in duration. AIM: We assessed the relative efficacy of clinically relevant syringe tip disinfection techniques following contamination with the hyper transmissible and more pathogenic Staphylococcus aureus sequence type 5 (S. aureus ST5) strain characteristic associated with increased strength of biofilm formation and greater desiccation tolerance. METHODS: Syringe tips (N=40) contaminated with S. aureus ST5 were randomized to 70% isopropyl pads with 10 or 60 s of drying time, scrubbing alcohol disinfection caps with 10 or 60 s of dwell time, or to non-scrubbing alcohol disinfection caps with 60 s of dwell time. The primary outcome was residual 24-h colony forming units (cfu) >10. RESULTS: Scrubbing disinfection caps were more effective than alcohol pads (25% (12/48) <10 cfu for scrubbing caps (10- or 60-s dwell times) vs 0% (0/48) <10 cfu for alcohol pads (10 or 60 s of drying time), Holm-Sidak adjusted P=0.0016). Scrubbing disinfection caps were more effective than non-scrubbing alcohol disinfection caps (25% (12/48) <10 cfu for scrubbing alcohol caps (10- or 60-s dwell times) vs 2% (1/48) for non-scrubbing alcohol caps (60-s dwell time), adjusted P=0.0087). CONCLUSIONS: Scrubbing alcohol caps are more effective than alcohol pads or non-scrubbing disinfecting caps for microbial reduction of syringe tips contaminated with the more pathogenic S. aureus ST5.

Characterizing the molecular epidemiology of anaesthesia work area transmission of Staphylococcus aureus sequence type 5.

BACKGROUND: Staphylococcus aureus sequence type 5 (ST5) is an emerging global threat. AIM: To characterize the epidemiology of ST5 transmission in the anaesthesia work area. METHODS: The retrospective cohort study analysed transmitted, prophylactic antibiotic-resistant Staphylococcus aureus isolates involving anaesthesia work area reservoirs. Using whole-genome analysis, the epidemiology of ST5 transmission was characterized by reservoir(s) of origin, transmission location(s), portal of entry, and mode(s) of transmission. All patients were followed for at least 30 days for surgical site infection (SSI) development. FINDINGS: Forty-one percent (18/44; 95% confidence interval: 28-56%) of isolates were ST5. Provider hands were the reservoir of origin for 28% (5/18) of transmitted ST5 vs 4% (1/26) for other STs. Provider hands were the transmission location for 28% (5/18) of ST5 vs 7% (2/26) of other STs. Stopcock contamination occurred for 8% (1/13) of ST5 isolates vs 12% (3/25) of other STs. Sixty-three percent of transmission events occurring between cases on separate operative dates involved ST5. ST5 was more likely to harbour resistance traits (ST5 median (interquartile range) 3 (2-3) vs 2 (1-2) other STs; P < 0.001) and had greater resistance to cefazolin, piperacillin-tazobactam, and/or ciprofloxacin (ST5: 3 (2-3) vs 2 (1-3) other STs; P = 0.02). ST5 was associated with three of six SSIs. CONCLUSION: ST5 is prevalent among transmitted, prophylactic antibiotic-resistant isolates in the anaesthesia work area. Transmission involves provider hands and one patient to another on future date(s). ST5 is associated with a greater number of resistance traits and reduced in-vitro susceptibility vs other intraoperative meticillin-resistant S. aureus.

Transmission of Staphylococcus aureus in the anaesthesia work area has greater risk of association with development of surgical site infection when resistant to the prophylactic antibiotic administered for surgery.

BACKGROUND: The extent to which the transmission of prophylactic-antibiotic-resistant bacteria from the anaesthesia work area increases the risk of surgical site infection (SSI) is unknown. It was hypothesized that the risk of SSI would increase progressively from no transmission to transmission of prophylactic-antibiotic-resistant isolates. METHODS: This was a retrospective analysis of archival samples collected in two previously published studies with similar inclusion criteria and sample collection methodology (observational study 2009-2010 and randomized trial 2018-2019). Archival isolates were linked by barcode to all patient demographic and procedural information, including the prophylactic antibiotic administered, transmission and development of SSI. For this study, all archival isolates underwent prophylactic antibiotic susceptibility testing, and the ordered association of transmission of Staphylococcus aureus (no transmission, transmission of prophylactic-antibiotic-susceptible isolates and transmission of prophylactic-antibiotic-resistant isolates) with SSI was assessed. RESULTS: The risk of development of SSI was 2% (8/406) without S. aureus transmission, 11% (9/84) with transmission of S. aureus isolates that were susceptible to the prophylactic antibiotic used, and 18% (4/22) with transmission of prophylactic-antibiotic-resistant S. aureus isolates. The Cochrane-Armitage two-sided test for ordered association was P<0.0001. Treating these three groups as 0, 1 and 2, by exact logistic regression, the odds of SSI increased by 3.59 with each unit increase (95% confidence interval 1.92-6.64; P<0.0001). CONCLUSIONS: Transmission of S. aureus in the anaesthesia work area reliably increases the risk of SSI, especially when the isolates are resistant to the prophylactic antibiotic administered.

Desiccation tolerance is associated with Staphylococcus aureus hypertransmissibility, resistance and infection development in the operating room.

BACKGROUND: Desiccation tolerance increases Staphylococcus aureus survival and risk of transmission. A better understanding of factors driving intraoperative transmission of S. aureus pathogens may lead to innovative improvements in intraoperative infection control. AIMS: To determine whether desiccation tolerance is associated with intraoperative S. aureus transmission, and to examine typical transmission dynamics for desiccation-tolerant isolates in the operating room in order to provide the impetus for development of improved intraoperative infection control strategies. METHODS: S. aureus isolates (N=173) were collected from anaesthesia work area reservoirs in 274 operating room environments. Desiccation tolerance was assessed and the potential association with sequence type (ST) and clonal transmission was evaluated. Whole cell genome analysis and pulsed-field gel electrophoresis analysis were used to compare desiccation-tolerant isolates with causative organisms of infection. FINDINGS: S. aureus ST 5 isolates had greater desiccation tolerance than all other intraoperative STs [ST 5, N=34, median Day 2 colony-forming unit (cfu) survival 0.027% ± 0.029%; other STs, N=139, median Day 2 cfu survival 0.0091% ± 1.41%; corrected P=0.0001]. ST 5 was associated with increased risk of clonal transmission (relative risk 1.82, 95% confidence interval 1.23-2.71, P=0.003). ST 5 transmission was linked by whole cell genome analysis to postoperative infection. CONCLUSIONS: Increased desiccation tolerance is associated with intraoperative transmission of S. aureus ST 5 isolates that are linked to postoperative infection. Future work should determine whether attenuation of desiccation-tolerant, intraoperative ST 5 strains can impact the incidence of healthcare-associated infections.

The alpha-subunit of the epithelial sodium channel is an aldosterone-induced transcript in mammalian collecting ducts, and this transcriptional response is mediated via distinct cis-elements in the 5'-flanking region of the gene.

Aldosterone stimulates Na(+) reabsorption in the collecting ducts by increasing the activity of the epithelial sodium channel, ENaC. Systemic administration of aldosterone increases alpha ENaC mRNA expression in mammalian kidney, suggesting that the alpha ENaC gene is a target for aldosterone action in the distal nephron. To determine whether aldosterone increases alpha ENaC gene transcription, a portion of the alpha ENaC 5'- flanking region coupled to luciferase was transfected into MDCK-C7 cells, a collecting duct cell line with aldosterone-stimulated Na(+) transport. Both dexamethasone and aldosterone stimulated alpha ENaC-coupled reporter gene activity via the glucocorticoid receptor (GR), and this response correlated with the effect of these hormones on endogenous alpha ENaC expression. The aldosterone-stimulated alpha ENaC expression was blocked by actinomycin D, and aldosterone had no effect on alpha ENaC mRNA decay, confirming a transcriptional effect. In HT-29 cells, a GR/mineralocorticoid receptor (MR)-deficient colonic cell line with constitutive alpha ENaC expression, cotransfection with GR or MR restored aldosterone-stimulated alpha ENaC gene transcription, although aldosterone had a functional preference for MR. Analysis of deletion constructs confirmed that a single imperfect glucocorticoid response element (GRE) is necessary and sufficient to confer the aldosterone responsiveness to the alpha ENaC gene promoter in MDCK-C7 and HT-29 cells. These results confirm that alpha ENaC is an aldosterone-induced transcript in the collecting duct and delineates the molecular mechanism for this effect.

Human amiloride-sensitive epithelial Na+ channel gamma subunit promoter: functional analysis and identification of a polypurine-polypyrimidine tract with the potential for triplex DNA formation.

The mRNA for the epithelial Na(+) channel gamma subunit (gammaENaC) is regulated developmentally in the lung, colon and distal nephron and in response to Na(+) deprivation and systemic corticosteroids in the distal colon. Because such regulation is likely to be at the level of gene transcription, we examined the function of the promoter and other 5' flanking elements of the human gammaENaC gene. The proximal 5' flanking region contains two GC boxes but does not contain a TATA box. A 450 bp human gammaENaC fragment (-459 to +40) directed the expression of luciferase in H441 cells and primer extension analysis in transfected cells confirmed the correct initiation of human gammaENaC-luciferase chimaeric transcripts. By deletional analysis, GC boxes at -21 and -52 were found to be critical for this promoter activity. To begin to identify transcription factors that bind to the core promoter, a double-stranded oligonucleotide that corresponded to this region was synthesized and tested in a gel mobility-shift assay. Incubation of this radiolabelled oligonucleotide with nuclear extracts from H441 and FRTL5 cells resulted in the formation of four specific and distinct DNA-protein complexes. On the basis of antibody 'supershift' assays, one of these factors corresponds to Sp1, whereas the other three correspond to Sp3. Further upstream, an approx. 300 nt (-1143 to -839) polypurine-polypyrimidine tract (PPy tract) containing internal mirror repeats was identified. When contained in a supercoiled plasmid, the approx. 1200 nt 5' flanking region was sensitive to S1 endonuclease, which was consistent with the formation of an intramolecular triplex DNA ('H-DNA') structure with an unpaired single strand. High-resolution mapping with S1 endonuclease and sequencing of S1-generated clones confirmed that all S1-sensitive sites were within the PPy tract. Finally, a negative regulatory element was identified between -1525 and -1296 that functioned in lung, colon and collecting duct cell lines.

Glucocorticoid induction of epithelial sodium channel expression in lung and renal epithelia occurs via trans-activation of a hormone response element in the 5'-flanking region of the human epithelial sodium channel alpha subunit gene.

In airway and renal epithelia, the glucocorticoid-mediated stimulation of amiloride-sensitive Na+ transport is associated with increased expression of the epithelial Na+ channel alpha subunit (alphaENaC). In H441 lung cells, 100 nM dexamethasone increases amiloride-sensitive short-circuit current (3.3 microA/cm2 to 7.5 microA/cm2), correlating with a 5-fold increase in alphaENaC mRNA expression that could be blocked by actinomycin D. To explore transcriptional regulation of alphaENaC, the human alphaENaC 5'-flanking region was cloned and tested in H441 cells. By deletion analysis, a approximately 150-base pair region 5' to the upstream promoter was identified that, when stimulated with 100 nM dexamethasone, increased luciferase expression 15-fold. This region, which contains two imperfect GREs, also functioned when coupled to a heterologous promoter. When individually tested, only the downstream GRE functioned in cis and bound GR in a gel mobility shift assay. In the M-1 collecting duct line Na+ transport, malphaENaC expression and luciferase expression from alphaENaC genomic fragments were also increased by 100 nM dexamethasone. In a colonic cell line, HT29, trans-activation via a heterologously expressed glucocorticoid receptor restored glucocorticoid-stimulated alphaENaC gene transcription. We conclude that glucocorticoids stimulate alphaENaC expression in kidney and lung via activation of a hormone response element in the 5'-flanking region of halphaENaC and this response, in part, is the likely basis for the up-regulation of Na+ transport in these sites.