Neonatal infection with Staphylococcus capitis NRCS-A in Iceland: A 12-year longitudinal, retrospective study of strains from patients, staff and the environment in a neonatal intensive care unit

Abstract

Background

The multidrug resistant NRCS-A clone of Staphylococcus capitis (SC) has spread globally in neonatal intensive care units (NICUs) where it causes neonatal sepsis and colonisation of infants, fomites and staff. Whole genome sequencing (WGS) of international isolates has suggested a putative origin of the clone in Norway, which is closely related to Iceland geopolitically. No data have been available about the NRCS-A clone in Iceland where a case of neonatal sepsis in 2014 suggested its presence for the first time. This study examines the epidemiology of the clone in the single Icelandic NICU over a 12-year period and its relationship to international isolates.

Methods

The study involved retrospective examination of the occurrence and clinical significance of SC-NRCS-A isolated from blood cultures of infants during 2009 – 2020, and prospective screening for SC among infants, staff and the environment. WGS was done on selected isolates to verify the presence of the SC-NRCS-A clone, examine phylogenetic relationship within the Icelandic isolates, and compare them with an international collection of SC-NRCS-A.

Results

SC-NRCS-A was found in blood cultures from 28 infants, of which nine with sepsis, and was a frequent coloniser of infants and diverse fomites. In staff SC-NRCS-A was detected in nares, throat and scalp. WGS of 93 isolates from blood and prospective screening specimens and comparison with international isolates revealed that the Icelandic SC-NRCS-A was distributed into two clusters, one related to Norwegian and the other to Irish SC-NRCS-A isolates. Both clusters contained isolates representing all sample sources.

Conclusion

The study demonstrated interpatient transmission, widespread dissemination and persistence of the SC-NRCS-A clone in the Icelandic NICU. The close phylogenetic relationship of Icelandic isolates with those from Norway and Ireland suggests potential import to Iceland by NICU staff or infants receiving medical care in these neighbouring countries at some point before 2009 for the Irish clone and before 2014 for the Norwegian clone.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13756-025-01623-5.

Introduction

Coagulase negative staphylococci (CoNS) have been reported in up to 80% of cases of late-onset neonatal sepsis in industrialized countries [1–3] and while S. epidermidis is typically the most common species [4, 5] S. capitis (SC) has gained increasing importance due to global spread of the multidrug resistant NRCS-A clone in neonatal intensive care units (NICUs) [6]. SC-NRCS-A strains are almost universally methicillin and aminoglycoside resistant and adapt rapidly to local antibiotic pressure. In addition to vancomycin heteroresistance, which is seen in most isolates, resistance to rifampicin and fusidic acid has been reported from NICUs and correlated with high use of these agents [6]. SC-NRCS-A contaminates surfaces and fomites in NICUs [7, 8], colonises infants [7, 9, 10], may be found on staff [7, 8] and causes invasive infections and deaths in the most premature neonates [9, 10].

According to an in silico study of SC genomes from around the world, the emergence of the NRCS-A clone in the late 1960 s coincided with the establishment of NICUs and its expansion in the 1980 s has likely been facilitated by the rising use of vancomycin in neonatal units [6]. The clone has now been identified in more than 20 countries around the globe [6, 11, 12]. While the origin of the clone and mechanisms of spread remain largely unknown, the genomic study suggested a putative source of its emergence in Norway [6].

Hitherto no data have been available about the presence and diffusion of SC strains in Iceland. However, in 2014 a preterm neonate developed persistent sepsis with a vancomycin heteroresistant SC isolate. This was subsequently identified as the first recognised case of SC-NRCS-A in Iceland (index case), suggesting local presence of the SC-NRCS-A clone in Landspítali’s NICU, the only such unit in Iceland. Further cases were identified which prompted a comprehensive epidemiological study in the NICU, including retrospective examination of the occurrence, clinical characteristics and outcome of SC-NRCS-A sepsis over a 12-year period and prospective screening for SC among infants, staff and the environment. Analysis of SC isolates via whole genome sequencing (WGS) was undertaken to assess transmission dynamics and possible geographical sources of the clone.

Methods (see also Additional file 1)

Setting

Landspítali University Hospital is a 650-bed tertiary care institution serving more than 60% of the 380,000 population of Iceland. About 75% of births in the country occur in Landspítali. The hospital’s NICU is the only level III NICU in Iceland and manages all neonates requering level III care and preterms born at less than 34 weeks gestation. The unit has 22 beds and manages about 400 hospitalised infants (40% of whom are premature neonates) and close to 700 outpatients every year [13]. Infants requiring cardiothoracic surgery have been sent abroad for treatment since the inception of the Icelandic NICU in 1976. They were initially treated in London (United Kingdom), from 2000 to 2010 in Boston (USA) and from 2011 in Skåne (Sweden). Five Icelandic infants, on average, are treated annually in Skåne with about one subsequently returning to the Icelandic NICU. Whilst institutions in other countries have not received Icelandic infants on a regular basis a few infants have been treated in Copenhagen (Denmark).

Specimens and patient data

The information system of the Department of Microbiology was searched for all positive blood cultures (BCs) and for CoNS and SC isolated from blood and other normally sterile tissues from patients in the NICU and other wards of the Children’s Hospital during the period of January 1 st 2009 through December 31 st 2020. All CoNS were identified by MALDI-TOF MS Microflex LT (Bruker Daltonics, Billerica, MA, USA) from 2016 to 2020 and SC isolates were preserved at − 80°C. CoNS were generally not identified to species level prior to 2016 but 90% of isolates cultured from neonates in the NICU and paediatric wards during 2009—2015 had been preserved at − 20 °C or − 80 °C and were retrieved from storage for identification by MALDI-TOF MS. Inclusion criteria for the study population were: (i) SC was isolated from the above-mentioned specimen types and (ii) the infant was hospitalised in the NICU or was no more that 44-weeks-old (using corrected gestational age) if hospitalised in a paediatric ward at the time of the first SC-positive BC.

Screening

Weekly SC screening of NICU patients was initiated in early 2016 using a modified screening protocol for methicillin-resistant S. aureus (MRSA), with SC screening added to routine MRSA screening for epidemiological purposes without reporting the results. Infants who were not already known to be infected or colonised with SC were screened (nares, throat and perineum) from April 2016 through December 2020, and screening of staff took place in September 2016 (nares, throat and scalp) and April 2019 (nares and throat). The NICU environment was screened in April, August and December 2016 and in April 2019 by swabbing the outer and inner surfaces of incubators, including the baby tray and space underneath the tray, medical devices, including stethoscopes and blood-pressure monitors, and various surfaces in clinical areas, staff areas and parent areas, such as switches, remote controls, taps, drawers and horizontal surfaces. SC screening of staff and environment was added opportunistically to MRSA screening samples taken as a response to detection and outbreaks of MRSA in the NICU. All screening specimens were taken with swabs (eSwab, Copan Italia, Brescia, Italy).

Demographics and definitions

Demographic and clinical data were obtained from patient records. Neonates were defined as extremely premature, very premature and premature when born before 28 weeks’, at 28 to less than 32 weeks’ and before 37 weeks’ gestation, respectively [14]. Birthweights were defined as extremely low, very low and low if they were lower than 1000 g, 1500 g and 2500 g, respectively [15].

Episodes of SC-positive BCs were considered to be associated with sepsis when all of the following criteria were fulfilled: (i) SC infection was suspected by caregivers; (ii) infants met the criteria of the European Medicines Agency consensus [16] within three days of the SC-positive BC [17]; (iii) concomitant infections or non-infectious causes that could explain manifestations were absent. Episodes that did not meet the definition for sepsis were considered to represent SC bacteraemia or BC contamination (distinction between the two was not possible). A new episode of SC-positive BC was defined as occurring more than 14 days from the previous one [18]. Persistent SC bacteraemia was defined as three or more positive BCs, at least 48 h apart, during a single septic episode [19]. Infants were followed for six months for outcome. Information about NICU patient days for calculation of incidence rates were obtained from official hospital statistics [20]. Infants and staff members who had SC-positive screening specimens were presumed to be colonised and transient carriers [8], respectively.

Microbiology methods

During the study period, BCs were performed with BacT/ALERT® (Biomérieux, Marcy l’Etoile, France) and incubated for 5 days.

Screening methods

SC is generally salt-tolerant [21] and the SC-NRCS-A clone is resistant to oxacillin and gentamicin [22]; the species can thus be expected to grow in the presence of gentamicin on MRSA screening media. Swabs from patients, staff or environment were placed into heart infusion broth (Becton, Dickinson and Company, NJ, USA) with 6.5% NaCl and incubated at 37 °C for 18–24 h for pre-enrichment. The broth was subsequently subcultured on MRSA screening medium (oxacillin resistance screening agar base supplemented with oxacillin and polymyxin B, Oxoid, Cheshire, UK), to which a gentamicin disk (CN 10 µg, Oxoid, Cheshire, UK) was added, and incubated at 37 °C for 24 h. Colonies growing inside the gentamicin zone, or elsewhere on the medium in case of scarce growth, were identified by the use of MALDI-TOF MS Microflex LT and oxacillin and gentamicin resistance was confirmed by routine susceptibility testing [23].

Antibiotic susceptibility testing

Antibiotic susceptibility testing was performed on SC from blood and other sterile tissues. Isolates were tested against cefoxitin, erythromycin, clindamycin, tetracycline, gentamicin and sometimes vancomycin as part of routine service upon the first isolation. The following additional compounds were tested (for this study) on SC from blood isolated before 2017: ceftaroline, ciprofloxacin, daptomycin, fusidic acid, linezolid, mupirocin, rifampicin, trimethoprim-sulfamethoxazole, teicoplanin and tigecycline. The Kirby-Bauer disk diffusion method was used for all compounds (disks from Oxoid, Cheshire, UK) except ceftaroline, daptomycin, teicoplanin and vancomycin for which a gradient diffusion method (Etest, Biomérieux, Marcy l´Etoile, Lyon, France) was employed. Breakpoints from the Clinical and Laboratory Standards Institute (until March 2012) and the European Committee on Antimicrobial Susceptibility Testing [23, 24] were used for interpretation. Ceftaroline and mupirocin results were interpreted according to breakpoints for S. aureus.

PCR for NRCS-A associated genes

To facilitate selection for WGS, isolates from the period 2009—2016 were first screened for the presence of genes that are strongly associated with the NRCS-A clone [6, 25]. SC isolates cultured after 2016 were submitted directly to WGS. SC isolated from infants (invasive and colonising), the NICU environment and staff were thus submitted for PCR amplification of the chromosomal nsr gene and the ccrA/B and ccrC genes located on the composite SCCmec-SCCcad/ars/cop mobile element; PCR amplicons were checked by electrophoresis in agarose gel (Additional file 1).

WGS

All multidrug resistant SC isolates from blood and CSF (n = 43 from 28 infants) for which PCR results for nsr, ccrA/B and ccrC were positive or unknown were submitted to WGS (Additional file 1), as well as a sample of screening isolates from patients, staff and environment (Additional file 2, Table 1). Infant screening isolates (n = 24) were selected to obtain three to four per study year (about one per quarter) in addition to isolates from infants with SC-positive BCs. Environmental isolates (n = 15) were selected from each of the four screening periods aiming to represent diverse sampling sites. All isolates (n = 11) obtained from staff screening were selected. Furthermore, the WGS results of the Icelandic isolates were compared to those of 162 international isolates defined as the NRCS-A outbreak clade in a previous study by Wirth et al. [6]. Phylogenetic trees were built with the Icelandic isolates alone and with all isolates to examine the relatedness of isolates both inside the Icelandic NICU and at the global scale.

Infection control/cleaning methods

Routine cleaning of the NICU underwent changes throughout the study period, mostly in response to outbreaks involving conventional multidrug resistant bacteria in 2014—2015. After 2015 cleaning was carried out using soap and water, alcohol disinfection and Rely + On® Virkon® (LanXESS, Cologne, Germany) (further details in Additional file 1).

Results

Patient records

During the period of 2009–2020 there were 192 episodes of positive BCs in the neonatal unit (Additional file 1, image 1). SC (n = 29, 15.1% of all episodes and 29.3% of all CoNS episodes) was the second most common bacterial species isolated from BCs, after S. epidermidis (n = 51, 26.6% of all episodes). Other bacterial species of note were E. coli (n = 18, 9.4%), Streptococcus agalactiae (n = 13, 6.8%) and Staphylococcus aureus (n = 12, 6.3%).

The median (interquartile range, IQR) gestational age and birth weight for the 29 SC-positive infants were 27.6 weeks (24.9—30) and 905 g (693—1363), respectively, and the median day of life at the first SC-positive BC was 9 days (IQR 8–17.5) (Additional file 2, Table 2). For comparison, the median (IQR) for gestational age and birth weight for 4603 infants admitted to the NICU during the study period were 38 weeks (34–40) and 3146 g (2252–3796), respectively. The 29 infants had a total of 34 SC-related BC episodes. While only nine infants (31%) were assessed as having one or more sepsis episodes, vancomycin was administered to 23 infants. Two infants died before 6 months of age from causes unrelated to SC (infant 2015–3 and infant 2017–1). Thirty-eight blood isolates from 23 of the 29 infants were tested in PCR assay and all but one (from infant 2014–4) were positive for the NRCS-A clone (Additional file 2, Table 1). The average incidence of infants with SC-positive BC during the study period was 0.6 per 100 NICU admissions and 0.5 per 1000 patient days, and the incidence of infants with SC-related sepsis was 0.2 per 100 NICU admissions and 0.1 per 1000 patient days (Additional file 2, Table 3).

All seven infants with SC in BCs during the screening period (April 2016—December 2020) were identified as colonised at some point during their NICU stay. The timing of positive screen tests ranged from 21 days before to 80 days after an SC-positive BC.

Screening specimens

SC colonisation was detected in 296 infants from April 2016 to December 2020. The average incidence of new positives each week was 18% (ranging from 0–100%) and the average prevalence of infants colonised with SC (new positives and known positives) during weekly screening was 47% (ranging from 0–100%) (Additional file 1, image 2). Comparing the screening sites where SC was first detected in new positive neonates, there was no difference between perineum and the combined nares and throat sites.

Staff screening for SC was carried out on three occasions. In an anonymous pilot screen of 15 staff members in June 2016 there was one positive member (6.7%). In September 2016 48 staff members were screened (nares, throat, scalp) and eight were found to have SC carriage (16.7%); five were positive in nasal and/or throat specimens (throat only = 3, nares and throat = 1, nares and scalp = 1), whilst three were positive only from scalp specimens, suggesting that this may be an important sample site to detect SC in staff. In April 2019 screening (nares and throat only) of 26 staff members revealed only two positive results (7.7%). This lower proportion of positivity might have been due to the omission of scalp specimens. Of the eight positive staff members from September 2016 only three were rescreened in 2019 with one being positive on both occasions.

Environmental screening was first carried out in April 2016 to assess SC contamination in the unit. This revealed heavy contamination with 52% (13/25) of sites testing positive. Repeat screening in August 2016, after increased emphasis was placed on cleaning procedures due to SC presence in the NICU, showed 30% (7/23) positivity rate. In April 2019, when screening was done immediately after additional extensive cleaning of the unit, contamination rate was still high, with SC identified from 44% (15/34) of tested sites; the positivity rate was 25% (5/25 sites) the following week after repeat cleaning. All types of areas were positive throughout the different screening periods, including infant bay areas (n = 12), staff work rooms, stations, toilets and medicine preparation room (n = 11), various medical equipment (n = 7, including ultrasound, scales, stethoscopes, trolleys with medicines and iv equipment), shared parent areas (n = 7, including the milk collection room in all screening periods, parent rooms, upholstered armchairs and toilet). Infant incubators were screened on two separate occasions after cleaning, with two out of six and one out of four incubators found to be contaminated when ready to be used for new infants.

Antibiotic susceptibility testing (AST)

Susceptibility rates for the 28 SC-NRCS-A isolates from BC were 0% for cefoxitin and gentamicin, 89% for erythromycin, 96% for clindamycin and 100% for tetracycline and vancomycin (vancomycin MICs ranged from 1 to 3 µg/mL). AST results for other agents, tested on the first blood isolate from 22 infants, revealed susceptibility rates of 100% for ceftaroline, linezolid, mupirocin, rifampicin and tigecycline, 95% for daptomycin, 91% for fusidic acid and teicoplanin, 82% for ciprofloxacin, and 77% for trimethoprim-sulfamethoxazole.

WGS of S. capitis

All 93 isolates from BCs, infant, environment and staff screening belonged to the outbreak lineage of the NRCS-A clone. When analysed alongside international isolates belonging to the NRCS-A “outbreak” lineage [6] the Icelandic collection was split into two clusters (Figs. 1 and 2). A smaller cluster (“Irish cluster”), closely related to Irish isolates, was represented throughout the entire study period, whereas a larger one (“Norwegian cluster”), closely related to Norwegian isolates, was present only from 2014 onwards. After early 2016 (when screening was implemented) the majority of infant (83%), environmental (80%) and staff (73%) screening isolates belonged to the Norwegian cluster, and the same was true for BC isolates (75%).

Fig. 1.

Phylogenetic tree of 162 international and 93 Icelandic SC-NRCS-A isolates. The international collection corresponds to 162 NRCS-A outbreak strains, from blood cultures, previously sequenced and reported in Wirth et al., 2020 [6]. The Icelandic isolates form two main clusters representing all specimen types included in the study. The smaller (“Irish”) cluster branches off the phylogenetic tree along with Irish strains, whereas the larger (“Norwegian”) cluster branches off alongside Norwegian strains. Two additional isolates cluster with Norwegian strains at a different position on the tree

Fig. 2.

Phylogenetic tree of 93 Icelandic SC-NRCS-A isolates. Both main clusters, a smaller Irish (green circle) and a larger Norwegian cluster (pink triangle), comprise isolates from all specimen types. The Irish cluster was already present in 2009 and throughout the study period, whereas the Norwegian cluster was first identified in 2014, and from 2016 almost all NRCS-A isolates detected belonged to the Norwegian cluster. Two additional isolates (pink square), related to Norwegian strains (see Fig. 1), were cultured from a single neonate in 2020. Labels A – D illustrate examples of closely related strains that were isolated months and years apart from all specimen types (see also Fig. 3). Isolate AD69 is the French prototype S. capitis NCRS-A strain CR01 [25], which is also included in the international collection shown in Fig. 1. Labels for coloured dots represent strains from staff skin („staff “), infant blood (“sepsis” and “contam/bacteremia”), infant screening (“carriage”) and the NICU environment (“environment”)

Genes associated with antimicrobial resistance included the β-lactam resistance genes mecA (in 100% of isolates), carried by the SCCmec type V element—included in the composite cassette SCCmec/SCC cad/ars/cop previously described in SC [25]—and blaZ, blaR, or blaI (98%), the aminoglycoside resistance genes aac(6')-Ie-aph(2'')-Ia and (AGly) aph-Stph (100% for both), and a multidrug efflux pump gene norA and its regulator mgrA (100%) which can confer resistance to fluoroquinolones and antiseptic compounds including those containing quaternary ammonium. Genome analysis confirmed that all isolates were found to have the nsr gene.

Analysis of eight serial blood isolates from infant 2014–2 (Additional file 1, clinical vignette) revealed that compared to the infant’s first isolate the seven following isolates (from the second septicaemic episode) harboured a total of 22 mutations. The majority of these were not found in subsequent isolates. One of the conserved mutations occurred in the intergenic region between ebh and rnhA.

Isolate comparison suggests that SC-NRCS-A strains remain minimally changed within the unit for months up to many years and that closely related strains are found in different sampling sources. Figure 3 shows examples of closely related strains that were cultured from blood and screening of neonates, the NICU environment and staff with intervals ranging from two months to more than 5 years.

Fig. 3.

Closely related SC-NRCS-A strains from different periods and sources. Panels A – D illustrate examples of closely related SC-NRCS-A strains that were isolated months to years apart from various sources in the NICU (see also Fig. 2). The dots represent specimen sources, i.e. blood from sepsis episode (dark blue) and bacteraemia/blood contamination (light blue), swabs from infant screening (green), staff screening (yellow), and the NICU environment (purple). The grey arrows show time intervals (d, days; m, months; y, years) between events. A: Blood culture isolates from neonates hospitalised almost 5 years apart. B: Blood culture isolates from five neonates in 2010 were closely related to an isolate found on a staff member in 2016. C: Blood culture isolates from neonates hospitalised a year apart were closely related to isolates found almost 4 years later in screening specimens from an infant and an echocardiogram probe. D: Blood culture isolates from two infants in 2015–2016, whose NICU stay was separated by 2 months, clustered closely with environmental (toilet) and staff member isolates sampled during and almost 4 months after the second infant’s admission, respectively

Six infants had both blood and screening isolates analysed by WGS, with paired isolates being identical on three occasions (5, 10 and 13 day interval) and closely related on two occasions (21 and 74 day interval). The screening isolate from the remaining infant was identical to a blood isolate obtained six days prior (both in the Norwegian cluster) but differed from a same day blood isolate (in the Irish cluster). As for staff members, three of the eight that were positive in 2016 were rescreened in 2019 with one (labeled NN and NN4 on Fig. 2) being positive on both occasions but with isolates from different clusters.

Discussion

In this comprehensive 12-year national study on the epidemiology of SC in an Icelandic NICU there were 29 infants with SC-positive BC. WGS confirmed the SC-NRCS-A clone in 28 infants and nine of these were identified with sepsis using European Medicines Agency criteria.

SC was prevalent in the neonates, in all areas of the NICU environment and on staff, with closely related SC-NRCS-A isolates from different time points and sources. The study confirmed long term persistence of strain types in the unit and close relationship between strains from BCs and those found both in the environment and on staff. Phylogenetic analysis of the Icelandic isolates alongside an international collection [6] suggested that SC-NRCS-A was introduced into Iceland on two separate occasions, before or during 2009 (“Irish cluster”) and 2014 (“Norwegian cluster”). The Irish cluster was already present in 2009 and throughout the study period, whereas the Norwegian cluster was first identified in 2014, suggesting later introduction into the unit. From 2016 almost all NRCS-A isolates detected belonged to the Norwegian cluster, suggesting that this clone might be more successful, having to an extent replaced the Irish clone.

Incidence of S. capitis-related sepsis and significance of positive blood cultures

Few studies have described the incidence of SC-related bloodstream infections (BSI) in NICUs, which is expected to vary depending on inclusion criteria and endemicity of the SC-NRCS-A clone [2, 26]. Incidence rates were 0.2 per 1000 patient days in our study, 0–2.24 per 1000 patient days in 36 French NICUs [26] and 19.1 in a Taiwanese NICU [27]. Similarly, the prevalence rates of BSI among infants with SC-positive BCs vary widely. Thus, while 31% of infants in our study met sepsis criteria, other investigators have reported rates ranging from 26 to 91% [9, 28]. This is likely explained by different inclusion criteria, and the assessment being confounded by concomitant immaturity, multimorbidity and management side effects [29].

Previous studies have described persistent SC-related BSI during vancomycin therapy [30] and a higher number of persistently positive BCs in vancomycin-treated infants with SC versus other CoNS infections [2], in keeping with vancomycin heteroresistance of SC-NRCS-A. Persistent bacteraemia was seen in two infants in our study (2014–1, 2014–2) when vancomycin trough concentrations were consistently under 15–20 mg/L. In the more prolonged and severe index case (2014–2), the bacteraemia only resolved once vancomycin concentrations were consistently above 20 mg/L, whilst in the other case vancomycin concentrations around 15 mg/L were sufficient. Recent data indicate that high vancomycin trough levels are needed for SC-NRCS-A, and some have suggested 10–25 mg/L [31].

Infant, environmental and staff screening for S. capitis

SC was continuously present in the NICU during the five-year period when neonates were screened, with just under half the neonates becoming colonised. This prevalence appears higher than previously reported (29–36%) [7, 31, 32], which may partly be due to different, more sensitive screening methods in our study, as other studies either did not use selective screening methods [7, 32] and/or only used a single screening site [7, 31, 32]. The average yearly prevalence rates in our unit were relatively consistent, ranging from lowest in 2016 and 2020 (40%) to highest in 2017 (60%). However, the weekly prevalence rates varied considerably (0–100%) and continuous screening over a 5 year period showed that colonisation rates fluctuated in periods over time so that colonisation rates reported from shorter time periods [7, 31, 32] could be affected by such variation. There were a number of sustained periods when incidence rates of new positive infants were low, with rare weeks when there were no neonates colonised with SC in the unit (Additional file 1, image 2). Nevertheless, and most likely due to persistent environmental contamination and/or possibly staff carriage, SC always re-emerged in neonates, with up to five new positive infants in some weeks after or during a period of sustained low positivity. We were unable to find explanations for these high incidence weeks, although some of the periods of low incidence of new SC colonisation occurred following detection of MRSA and/or ESBL cases in the unit, which usually prompts enhanced cleaning and adherence to infection control procedures. However, our results, like those of other investigators [7, 8, 33] indicate that SC-NRCS-A persists in the NICU environment despite routine cleaning and infection control methods. Furthermore, periods of enhanced cleaning had limited short term effect and no real long term effect in reducing the burden of SC environmental contamination, with SC detected in multiple environmental screening specimens even directly after enhanced cleaning was carried out. Persistence of SC-NRCS-A in the NICU environment has been attributed to enhanced ability to form biofilms under nutrient stress and to survive desiccation [34]. Tolerance to some disinfectant molecules, other than those used in our NICU, has also been described [7, 34], and attempts to eradicate SC may thus require specific detergents and cleaning methods.

Testing scalp in staff screening added three SC-NRCS-A positive members to the five found positive in nares or throat only, suggesting that detection in staff may be underestimated if scalp is not included. We also detected SC with an AST pattern strongly suggestive of SC-NRCS-A on a parent (found incidentally during MRSA screening and not included in results). Staff and parents could act as intermittant vectors, transmitting SC between neonates, dispersing SC throughout the unit and re-contaminating previously cleaned areas. This complicated ecological cycle may partly explain the persistence of SC-NRCS-A and is reflected in our findings of widespread, long-term environmental contamination affecting almost equally all area types in the NICU (clinical areas, staff rooms, parent areas, medical equipment).

Whole genome sequencing

Phylogenetic proximity of Icelandic isolates to Norwegian and Irish isolates

At least two potential routes could have contributed to transmission of strains from Norway to Iceland. First, many healthcare professionals in Iceland, including neonatologists, are trained or seek temporary work experience in Norway and Sweden. Second, infants requiring cardiothoracic surgery have been treated in Sweden since 2011 and some have been admitted back to the Icelandic NICU upon return. However, since no Swedish isolates were included in the international WGS analysis [6], it was not possible to determine whether Icelandic NRCS-A isolates were more likely to have originated from Sweden than Norway. The route and time of entry of the Irish clone remains more speculative as exchange of health care workers between Iceland and Ireland has traditionally been minimal, if any, and no infant has been transferred from Ireland to the Icelandic NICU in the past five decades.

Spatial and temporal relatedness of SC isolates

No significant genetic differences were observed between isolates based on their source, i.e. sepsis, bacteraemia/blood contamination and screening of infants, environment and staff, suggesting that sepsis-related SC strains are not more virulent than those isolated from other sources. This is consistent with previous findings [35, 36]. However, WGS analysis of the NRCS-A clone in a New Zealand NICU showed neonatal blood and skin screening isolates to be indistinguishable from environmental SC isolates [7], whilst being generally distinct from isolates carried by NICU staff. This contrasts with our findings of staff carrying isolates not only temporally related to neonatal and environmental isolates, but also closely related to neonatal BC isolates from up to six years before.

Antibiotic resistance phenotypes and genotypes

Resistance to fusidic acid was rare and all isolates were rifampicin susceptible. This contrasts with results for NICU-related SC isolates from New Zealand, where a high prevalence of a fusidic acid resistance and a fusB (far1)-containing plasmid [7] was associated with high fusidic use, and from a French NICU where common rifampicin resistance was in line with high rifampicin use [6]. Fusidic acid resistance and presence of fusB were also found in the majority of SC-NRCS-A isolates from English NICUs [37].

Limitations of the study

As the study period started in 2009, we could not ascertain the presence of the NRCS-clone in Iceland before this time. However, the fact that three and six infants had SC-positive BCs during 2009 and 2010, respectively, compared to none to four infants/year in the following years suggests that the clone may have already been established in the unit before 2009.

Conclusion

This nationwide study of a single Icelandic NICU demonstrated the SC-NRCS-A clone in neonatal sepsis, colonisation of infants and carriage by staff. Furthermore, persistent environmental contamination was shown for patient, staff and parental areas within the NICU despite repeated and extensive cleaning. Infection control measures sufficient to eradicate MRSA from the NICU had limited efficacy against SC. The close phylogenetic relationship of Icelandic isolates with those from Ireland and Norway suggested import to Iceland from these neighbouring countries at some point before 2009 and 2014 respectively, potentially with NICU staff and/or infants. This raises the question of whether staff and/or infants should be screened for SC-NRCS-A carriage before transfer from an affected NICU to a unit free of this multiresistant clone, although the question of how to perform decolonisation remains open.

Authors’ contributions

Design of study: IH, KOH, FL and MaBu. Data collection and analysis: IH, KOH, ATh, MéBo, JDS, AEJ and ThTh. Interpretation of data: IH, KOH, ATh, MéBo and MaBu. Writing of original manuscript: IH, KOH, MéBo and MaBu. Review and editing of manuscript: all authors. All authors have read and agreed to the published version of the manuscript.

Funding

Grant from Landspítali University Hospital, Agence Nationale de la Recherche, France, (project NeoSCap ANR 19-CE17-0004-01)

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The study was approved by the National Bioethics Committee (permission no. 16–155) (a waiver of consent was granted).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.