Surveillance for Colonization, Transmission, and Infection With Methicillin-Susceptible Staphylococcus aureus in a Neonatal Intensive Care Unit

Key Points Question What are the risk factors for the acquisition of methicillin-susceptible Staphylococcus aureus (MSSA) colonization and infections in a neonatal intensive care unit? Findings In this cohort study of 590 newborns, the acquisition of S aureus colonization and infection was monitored until hospital discharge. Colonization with MSSA was associated with lower birth weight, longer hospitalization, and higher odds of acquiring S aureus infection during hospitalization. Meaning These findings suggest that nasal colonization is a relevant risk factor for MSSA infection in a nonoutbreak neonatal intensive care unit setting.


Introduction
Preterm newborns, especially those with low to very low birth weight, are vulnerable to nosocomial infections, such as those caused by Staphylococcus aureus. Indeed, S aureus infections are among the leading causes of health care-associated infections in neonatal intensive care units (NICUs). [1][2][3] However, most studies focus on methicillin-resistant S aureus (MRSA), although infections with antibiotic-susceptible S aureus might be even more important in terms of morbidity and mortality, considering their reportedly higher prevalence when compared with MRSA infections. 2,4,5 Colonization with S aureus, in addition to extended hospital stay and low birth weight, is considered a significant risk factor for acquiring S aureus infections in NICUs. 6 Therefore, prevention of transmission and eradication of colonization is thought to be beneficial in reducing infections. [7][8][9][10] The colonization and transmission dynamics in newborns in a nonoutbreak setting are not yet fully understood, because most studies focus on transmissions in outbreak settings. 1,11 Commonly, colonization occurs in the postnatal phase and may be acquired through vertical transmission from mother to infant, or from health care workers (HCWs), parents, visitors, or the environment to infant. [12][13][14] In this study, we performed a retrospective analysis of all S aureus colonization and infection events during a 2-year period in the NICU of our tertiary hospital. Data from the weekly screenings for S aureus in all newborns admitted to the NICU were used to improve our understanding of the interplay between colonization and transmission. In addition, we assessed the performance of staphylococcal protein A (spa) typing using the polymorphic gene region encoding protein A as our standard typing method with whole-genome sequencing (WGS) to evaluate the latter's added benefit for routine surveillance of S aureus in a given setting.

Study Premise
In this cohort study, we enrolled all premature newborns admitted to the NICU of the Heidelberg University Hospital from January 1, 2018, to December 31, 2019. The NICU consists of 22 stationary beds that are assembled as two 4-bed rooms and six 2-bed rooms. The primary end point of this observational cohort study was S aureus infection during hospitalization with S aureus colonization as exposure. Secondary outcomes were transmission of S aureus in a nonoutbreak setting and the concordance of spa typing and WGS for routine surveillance and outbreak detection. Weekly screenings of newborns were conducted as part of the local infection control and screening policy for multidrug-resistant organisms as implemented by the Department of Medical Microbiology and Hygiene of Heidelberg University Hospital. The local ethics committee was consulted before the study begin and waived individual informed consent owing to deidentified data. This study adhered to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.

Routine Screening and Infection Control Measures
Screening for S aureus colonization was performed weekly in the postnatal period, together with screening for cephalosporin-and carbapenem-resistant gram-negative bacteria from the nasal (anterior nares) and rectal/perianal cavities using 2 separate swabs, 1 for each site.
Basic hygiene measures were applied for any patient contact, including patients colonized with methicillin-susceptible S aureus (MSSA). These measures included consistent hand disinfection in accordance with the 5 World Health Organization indications and the wearing of disposable gloves and protective gowns to avoid contamination of staff where direct contact with blood, secretions, excrement, mucous membranes, or nonintact skin is expected.
Furthermore, the following infection prevention and control measures were implemented: (1) disposable apron for nursing rounds and in case of contamination risk of the body front; (2) protective gown, if the child is carried; (3) regular and hygiene training and compliance observations (at least once a year), on-site visits (at least once a year), and quality meetings with the infection control team (at least 3 times a year); (4) 24-hour monitoring of automated hand disinfectant use at every bed site; (5) surgical face mask for the care of patients with MSSA or MRSA to avoid droplet contamination; (6) isolation room and contact precautions for patients with MRSA or cephalosporinand carbapenem-resistant gram-negative bacteria; and (7) decolonization of patients with MRSA using a 5-day scheme, including antiseptic nasal, oral, and skin treatment. No routine decolonization measures were implemented for MSSA.

Laboratory Methods
Routine screening samples were processed in the microbiological diagnostics laboratory of the university hospital using the total laboratory automation system (Kiestra; Becton and Dickinson) as published elsewhere, with minor modifications. 15,16 Briefly, swabs were inoculated onto a biplate chromogenic medium for S aureus and MRSA detection (CHROMagar Staph aureus/MRSAII; BD Diagnostics) and Columbia blood agar with 5% sheep blood as a growth control for sampling validity.
Species identification was performed via matrix-assisted laser desorption ionization (MALDI-TOF; Bruker) using a score of at least 2.0 as a cutoff. Antibiotic susceptibility testing was not performed routinely for S aureus isolates from screening samples. The S aureus isolates were cryopreserved at −70°C for molecular typing.

Molecular Typing
Crude bacterial DNA was extracted using a rapid extraction method with lysostaphin (Genaxxon Bioscience) and proteinase K (Bio&Sell) as described previously. 17 Conventional routine spa typing was performed as described elsewhere 18 ; spa gene sequences were analyzed using Ridom StaphType software, version 2.2.1 (Ridom GmbH). Data on global frequency were obtained through the Ridom SpaServer website. 19

WGS and Data Analysis
Genomic DNA for WGS was extracted from overnight bacterial culture using a blood-typing kit (DNeasy Blood and Tissue Minikit; Qiagen GmbH) according to the manufacturer's protocol, with the addition of an initial lysis step with lysostaphin. Genomic DNA was sequenced (MiSeq system; Illumina, Inc; 2 × 250 base pairs [bp] paired-end) as described previously. 16 Raw sequences were trimmed for quality using Sickle, version 1.33 (parameters, q > 30; l > 45). 20 The cleaned sequences were then assembled using SPAdes, version 3.13.0. 21 Assembled contigs were curated for length (>500 bp) and coverage (>×10). Annotation was performed using Prokka, version 1.14.1 (based on Genetic Code

Statistical Analysis
Appropriate measures of the location and spread of the distribution of sociodemographic and clinical variables were tabulated by S aureus colonization or infection status. We used univariable and multivariable logistic regression to estimate the change in the odds of S aureus colonization and infection in the presence of putative risk factors of these outcomes, together with their 95% CIs, and tested against the null hypothesis (H 0 ) with an odds ratio [OR] of 1.00 using an α of .05. Test of the collinearity for correlations of the variables was performed using the (variance inflation factor) command following a regression model with all relevant variables. The mean of the variance inflation factor was 1.41 (range, 1.09-1.89), indicating no collinearity between variables. All statistics were performed in STATA, version 13 (StataCorp LLC).

Results
A total of 590 newborns treated in the NICU during the 2-year study period were included in this study, of whom 276 (46.8%) were female and 314 (53.2%) were male; 477 (80.9%) were preterm newborns; 449 (76.1%) were singletons; and 220 (37.3%) had a birthweight of less than 1500 g. Of 586 newborns with delivery mode data, 419 (71.5%) were delivered via cesarean section and 167 (28.5%) via natural vaginal delivery. Patient demographics are summarized in Table 1

Molecular Characteristics
The most prevalent (Ն10 isolates per cluster) MLST of the 123 sequenced S aureus isolates were ST45, ST30, ST15, and ST121. The most prevalent spa types, with more than 5 isolates each in the study population, were t084, t170, t2642, and t571. An overview of all spa types is summarized in eTable 2 in the Supplement. Panton-Valentine leukocidin was detected in 2 methicillin-susceptible isolates (ST152 t084 and ST34 t1758). One MRSA isolate belonged to ST225 t003 with SCCmec type II.

Transmission Clusters
Altogether, 123 colonization isolates were characterized by spa and WGS. Using spa types to identify potential transmissions, we obtained 27 potential events (Ն2 patients with identical spa types) involving 95 patients (eTable 2 in the Supplement). More than one-third (10 of 27 [37.0%]) of the In comparison, using a minimum spanning tree based on the hqSNVs to depict the phylogenetic relatedness among all colonization isolates (n = 123), we obtained 23 potential transmission clusters, of which only 6 (26.1%) involved 4 or more patients in the respective transmission groups (Figure 2). for the t002 spa transmission cluster ( Figure 2B), SNV analysis did not indicate a close genetic relationship (48-334 SNVs between isolates). Indeed, spa type t002, with more than 6.5% global frequency, belongs to the more common spa types; therefore, the accumulation of spa type t002 is not a reliable indicator for transmission events. Most WGS transmission clusters displayed plausible temporal-spatial overlap. However, for 2 clusters, t089 and t3375 ( Figure 2B), there was a gap of  spa Type several months between detection of new cases in the transmission cluster, which may be an indication of an external reservoir.

S aureus Colonization in Twins and Triplets
In 12 of the 48 sets of twins in our study cohort (25.0%), both newborns had S aureus colonization.

Discussion
In our study, most newborns admitted to the NICU were premature (delivery at gestational age of <37 weeks and birthweight of Յ1500 g). Premature newborns generally require longer NICU stays, 28 providing ample opportunities to acquire S aureus colonization during hospitalization. Overall, 22.9% of the newborns in our study population were colonized by S aureus, which is consistent with the expected general colonization proportion (20%-30%) in the population. Thus, these findings may represent a physiological process instead of a pathological phenomenon. 29-31 A significant percentage of these infants (23.7%) acquired colonization in the first week after delivery, and 71.9% had a positive in S aureus screening result within their first month of life. Nasal colonization appears to be the primary and most important reservoir in this population, because only 3.7% of the colonized infants were exclusively colonized in the perianal/rectal region. Our findings are consistent with published data on the colonization sites of infants in the NICU setting. 11,32 Overall, the acquisition of S aureus infection was a rare event. Only 1.0% and 1.7% of all NICU patients in our study acquired S aureus bloodstream infections or any type of S aureus infections, respectively. Our data imply that hospitalized infants with S aureus colonization at the NICU develop S aureus infections more frequently than those without colonization, independent of their length of stay. All our S aureus infections in carriers were endogenous; that is, colonization and infection strains were clonally identical. Indeed, colonization with S aureus, regardless of methicillin resistance, has frequently been described as one of the major risk factors for the acquisition of S aureus infections, both in adult and pediatric populations. 1,33,34 Length of stay in the NICU has been reported as one of the contributing factors for S aureus infections, 6 13 The fact that genetically closely related clones were detected in unrelated newborns indicates a different acquisition route.
The genetic and epidemiological link suggests that patient-to-patient transmission via hands of HCWs could contribute to the acquisition of colonization in newborns in the NICU. 2,37 In contrast to adult patients, these newborns are not mobile and are confined within their own incubators, so that direct newborn-to-newborn contact and transmission would have to be mediated by mobile vectors.
Indeed, the role of HCWs as vectors for transmission has been reported numerously. 11,37,38 Because mandatory screening of HCWs in Germany requires specific approval and compliance, which would only be granted for outbreak investigations, we cannot incorporate S aureus from HCWs in our study.
Nevertheless, molecular typing, along with epidemiological patient data and transmission patterns, suggests the involvement of HCWs as a transmission vector and warrants further scrutiny.
High discriminatory molecular typing such as WGS can be costly and may not be readily accessible. The spa typing method seems to be reliable and affordable for surveillance, especially when interpreted in conjunction with the global spa type frequency. This method will probably exaggerate the magnitude of an outbreak owing to the limited discriminatory power compared with WGS, 41 but most importantly, spa typing did not produce very major errors (ie, false-negative findings).

Limitations
Our study has some limitations. This was a single-center study, so generalization of the findings may be limited. The incidence rates of infections differ significantly between hospitals, and the hygiene standards and measures are not harmonized on a national level. Nevertheless, the large sample size allows us to investigate the transmission of S aureus in a nonoutbreak NICU setting. Although the sample size may be adequate to study transmissions within in the hospital setting, the sample size may be too small to accurately assess the incidence of infection events, and thus the incidence may be underestimated or overestimated. Numerous studies on the risk factors for S aureus infections in patients in the NICU were performed in outbreak settings and focused mainly on MRSA; therefore, the relevance and impact of the risk factors may have been exaggerated against an elevated incidence for colonization and infection owing to large outbreak clusters. Furthermore, our NICU has only shared rooms (2-bed or 4-bed), so the impact of private rooms or preemptive isolation cannot be assessed in our study setting.

Conclusion
This cohort study found that the incidence of S aureus infection was very low in a nonoutbreak NICU setting. Our data suggest that nasal colonization is a relevant risk factor for S aureus infection in neonates and that minor transmission events occur in a nonoutbreak setting. Furthermore, molecular characterization suggested the presence of transmission vectors or source in facilitating the acquisition and spread of S aureus colonization in premature newborns in a NICU setting, which warrants further investigation and validation.