Impact of daily octenidine skin washing versus nonwashing on antiseptic tolerance of coagulase-negative staphylococci in two neonatal intensive care units with different skin cleansing practices

Background: There is wide variation in practices regarding routine bathing/washing of babies in neonatal intensive care units (NICUs). Evidence is lacking as to the benefit of routine antiseptic washes for reducing infection. We aimed to compare the antiseptic tolerance of Coagulase Negative Staphylococci (CoNS) within two UK NICUs with very different approaches to skin washing. Methods: We compared antiseptic susceptibility of CoNS isolated from skin swabs of neonates admitted to the Norfolk and Norwich University Hospital (NNUH) NICU in December 2017–March 2018 with those isolated in the Bradford Royal Infirmary (BRI) NICU in January–March 2020. The NNUH does not practise routine whole-body washing whereas BRI practises daily whole-body washing from post-menstrual age 27 weeks using Octenisan wash lotion (0.3% octenidine; 1 minute contact time before washing off with sterile water). A total of 78 CoNS isolates from BRI and 863 from the NNUH were tested for susceptibility against the antiseptics octenidine (OCT) and chlorhexidine (CHX). Results: Isolates from the BRI with practice of routine washing did not show increased antiseptic tolerance to OCT or CHX. Isolates from the NNUH which does not practise routine whole-body washing and rarely uses octenidine, were comparatively less susceptible to both CHX and OCT antiseptics. Conclusions: Daily whole-body skin washing with OCT does not appear to select for CoNS isolates that are antiseptic tolerant towards OCT and CHX. There remains considerable uncertainty about the impact of different antiseptic regimes on neonatal skin microbiota, the benefit of routine washing, and the development of antiseptic tolerance in the NICU

Chlorhexidine gluconate usage is associated with antiseptic tolerance in staphylococci from the neonatal intensive care unit

Background: Intravascular catheters are essential for care in Neonatal Intensive Care Units (NICUs) but predispose infants to catheter-associated infections including late-onset sepsis, commonly caused by CoNS. Antiseptics are applied to prevent infection with chlorhexidine (CHG) and octenidine (OCT) the most common agents used. Objectives: To investigate the association between antiseptic use and bacterial susceptibility. Methods: CoNS isolates were collected from two NICUs with differing antiseptic regimens: Norwich, UK (using CHG) and Lubeck, Germany (using OCT). CoNS were isolated from different body sites of babies upon admission, and weekly thereafter. Antiseptic susceptibility testing was performed, and a selection underwent genome sequencing. Results: A total of 1274 isolates were collected. UK isolates (n = 863) were significantly less susceptible than German isolates (n = 411) to both CHG (mean MIC: 20.1 mg/L versus 8.9 mg/L) and OCT (mean MIC: 2.3 mg/L versus 1.6 mg/L). UK isolates taken on admission were more susceptible to CHG than subsequent isolates. No cross-resistance between the agents was seen. Genome sequencing of 122 CoNS showed the most common species to be Staphylococcus epidermidis and Staphylococcus haemolyticus and phylogenetic analysis suggested antiseptic tolerance evolved multiple times in independent lineages. There was no evidence of dominant antiseptic tolerant clones and carriage of genes previously implicated in antimicrobial susceptibility (qac, smr, norA/B), did not correlate with CHG or OCT susceptibility. Conclusions: Long-term CHG use may select for CHG and OCT tolerance in CoNS. This highlights the different potential for separate antiseptic regimens to select for resistance development. This could be an important factor in developing future infection control policies

Resolving the contributions of the membrane-bound and periplasmic nitrate reductase systems to nitric oxide and nitrous oxide production in Salmonella enterica serovar Typhimurium

The production of cytotoxic nitric oxide (NO) and conversion into the neuropharmacological agent and potent greenhouse gas nitrous oxide (N2O) is linked with anoxic nitrate catabolism by Salmonella enterica serovar Typhimurium. Salmonella can synthesize two types of nitrate reductase: a membrane-bound form (Nar) and a periplasmic form (Nap). Nitrate catabolism was studied under nitrate-rich and nitrate-limited conditions in chemostat cultures following transition from oxic to anoxic conditions. Intracellular NO production was reported qualitatively by assessing transcription of the NO-regulated genes encoding flavohaemoglobin (Hmp), flavorubredoxin (NorV) and hybrid cluster protein (Hcp). A more quantitative analysis of the extent of NO formation was gained by measuring production of N2O, the end-product of anoxic NO-detoxification. Under nitrate-rich conditions, the nar, nap, hmp, norV and hcp genes were all induced following transition from the oxic to anoxic state, and 20% of nitrate consumed in steady-state was released as N2O when nitrite had accumulated to millimolar levels. The kinetics of nitrate consumption, nitrite accumulation and N2O production were similar to those of wild-type in nitrate-sufficient cultures of a nap mutant. In contrast, in a narG mutant, the steady-state rate of N2O production was ~30-fold lower than that of the wild-type. Under nitrate-limited conditions, nap, but not nar, was up-regulated following transition from oxic to anoxic metabolism and very little N2O production was observed. Thus a combination of nitrate-sufficiency, nitrite accumulation and an active Nar-type nitrate reductase leads to NO and thence N2O production, and this can account for up to 20% of the nitrate catabolized

Characterisation of neonatal Staphylococcus capitis NRCS-A isolates compared with non NRCS-A Staphylococcus capitis from neonates and adults

Staphylococcus capitis is a frequent cause of late-onset sepsis in neonates admitted to Neonatal Intensive Care Units (NICU). One clone of S. capitis, NRCS-A has been isolated from NICUs globally although the reasons for the global success of this clone are not well understood. We analysed a collection of S. capitis colonising babies admitted to two NICUs, one in the UK and one in Germany as well as corresponding pathological clinical isolates. Genome analysis identified a population structure of three groups; non-NRCS-A isolates, NRCS-A isolates, and a group of ‘proto NRCS-A’ – isolates closely related to NRCS-A but not associated with neonatal infection. All bloodstream isolates belonged to the NRCS-A group and were indistinguishable from strains carried on the skin or in the gut. NRCS-A isolates showed increased tolerance to chlorhexidine and antibiotics relative to the other S. capitis as well as enhanced ability to grow at higher pH values. Analysis of the pangenome of 138 isolates identified characteristic nsr and tarJ genes in both the NRCS-A and proto groups. A CRISPR-cas system was only seen in NRCS-A isolates which also showed enrichment of genes for metal acquisition and transport. We found evidence for transmission of S. capitis NRCS-A within NICU, with related isolates shared between babies and multiple acquisitions by some babies. Our data show NRCS-A strains commonly colonise uninfected babies in NICU representing a potential reservoir for potential infection. This work provides more evidence that adaptation to survive in the gut and on skin facilitates spread of NRCS-A, and that metal acquisition and tolerance may be important to the biology of NRCS-A. Understanding how NRCS-A survives in NICUs can help develop infection control procedures against this clone

Impact of daily octenidine skin washing on antiseptic tolerance of coagulase-negative staphylococci in two neonatal intensive care units with different bathing practices

Background: Coagulase Negative Staphylococci (CoNS) are responsible for 80-90 % of catheter related sepsis in neonates which can cause life threatening and damaging effects. NICUs within the UK use various practices to decolonise neonates to prevent infection ranging from regular full body bathing to localised skin decontamination before insertion of indwelling devices. There is a disparity in bathing practices for infants admitted onto neonatal units, with some choosing to regularly bathe infants and others not, and some routinely washing with skin antiseptics, and others not. Aim: To compare the abundance of CoNS within two UK NICUs with different approaches to skin bathing and to test their tolerances to antiseptics. Methods: A collection of CoNS from two UK based NICUs with differing bathing routines for neonates were collated and tested for susceptibility to the antiseptics in use, octenidine and chlorhexidine. Findings: Regular bathing of neonates in octenidine did not decrease the abundance of organisms on neonatal skin. Isolates from the unit where octenidine was in frequent use did not show any increased antiseptic tolerance. Isolates from the unit where regular bathing was not routine practice were less susceptible to both antiseptics. Conclusion: Frequent whole-body skin washing with octenidine does not appear to result in a lasting reduction in numbers of organisms found on the skin but also does not appear to select for antiseptic tolerant CoNS

High-frequency phosphorus monitoring of the River Kennet, UK: are ecological problems due to intermittent sewage treatment works failures?

The River Kennet in southern England has exhibited excessive benthic algal growth and associated ecological problems, such as loss of macrophytes and invertebrates, since the 1980s. These ecological problems were attributed to regular peaks in phosphorus concentration, which were widely attributed to intermittent failures of the Marlborough sewage treatment works (STW). This study deployed highfrequency phosphorus auto-analysers to monitor the total reactive phosphorus (TRP) concentrations of Marlborough STW final effluent and the downstream River Kennet at hourly and 30 minute resolution respectively, between 2008 and 2009. This monitoring confirmed that the Marlborough STW was operating well within its 1000 mg l-�1 annual mean total phosphorus consent limit, with mean total P and soluble reactive P concentrations of 675 and 345 mg l-�1 respectively. There were two occasions where effluent TRP concentration exceeded 1000 mg l-�1, and only one of these resulted in a peak in TRP concentration of over 100 mg l-�1 in the River Kennet at Mildenhall. The other nine peaks of over 100 mg l-�1 in the River Kennet during the monitoring period were associated with storm events, indicating that diffuse-source inputs and remobilisation of stored within-channel phosphorus were the cause of the peaks in river concentration, rather than Marlborough STW. The value of high-frequency environmental monitoring and the problems associated with using nutrient auto-analysers in the field are discussed. Seasonal phosphorus consents for STWs could provide a useful and cost effective means to improve both water quality and river ecology in the upper River Kennet

Sticking together: Independent evolution of biofilm formation in different species of staphylococci has occurred multiple times via different pathways

Various species of staphylococci cause a wide range of infections, including implant-associated infections which are often difficult to treat due to the presence of biofilms. Whilst some proteins involved in biofilm formation are known, the differences in biofilm production between staphylococcal species remains understudied. Currently biofilm formation by Staphylococcus aureus is better understood than for other members of the genus as more research effort has focused on this species. We assembled a panel of 385 non-aureus Staphylococcus isolates of 19 species from prosthetic joint infection as well as other clinical sources and reference strains. We assessed the biofilm forming ability of all strains using a high-throughput crystal violet assay. This identified distinct biofilm formation categories and we then compared the prevalence of Pfam domains and identified those which distinguished the categories as well as using machine learning to identify amino acid 20-mers linked to biofilm formation. This identified some domains within proteins already positively linked to biofilm formation but we also identified important domains not previously linked to biofilm formation. RT-qPCR confirmed the expression of selected genes predicted to encode important domains within biofilms in Staphylococcus epidermidis. The prevalence and distribution of biofilm associated domains showed a link to phylogeny, suggesting different Staphylococcus species have independently evolved different mechanisms of biofilm production. This work has identified different routes to biofilm formation in diverse species of Staphylococcus as well as suggesting independent evolution of biofilm has occurred multiple times across the genus. Understanding the mechanisms of biofilm formation in any given species is likely to require detailed study of relevant strains and the ability to generalise across the genus may be limited