Environmental Persistence of Staphylococcus capitis NRCS-A in Neonatal Intensive Care Units: Role of Biofilm Formation, Desiccation, and Disinfectant Tolerance

Abstract

The clone Staphylococcus capitis NRCS-A is responsible for late-onset sepsis in neonatal intensive care units (NICUs) worldwide. Over time, this clone has evolved into three subgroups that are increasingly adapted to the NICU environment. This study aimed to decipher the mechanisms involved in NRCS-A persistence in NICUs. Twenty-six S. capitis strains belonging to each of the three NRCS-A clone subgroups and two other non-NRCS-A groups from neonates (alpha clone) or from adult patients ("other strains") were compared based on growth kinetics and ability to form biofilm as well as tolerance to desiccation and to different disinfectants. S. capitis biofilm formation was enhanced in rich medium and decreased under conditions of nutrient stress for all strains. However, under conditions of nutrient stress, NRCS-A strains presented an enhanced ability to adhere and form a thin biofilm containing more viable and culturable bacteria (mean 5.7 log₁₀ CFU) than the strains from alpha clone (mean, 1.1 log₁₀ CFU) and the "other strains" (mean, 4.2 log₁₀ CFU) (P < 0.0001). The biofilm is composed of bacterial aggregates with a matrix mainly composed of polysaccharides. The NRCS-A clone also showed better persistence after a 48-h desiccation. However, disinfectant tolerance was not enhanced in the NRCS-A clone in comparison with that of strains from adult patients. In conclusion, the ability to form biofilm under nutrient stress and to survive desiccation are two major advantages for clone NRCS-A that could explain its ability to persist and settle in the specific environment of NICU settings. IMPORTANCE Neonatal intensive care units (NICUs) host extremely fragile newborns, including preterm neonates. These patients are very susceptible to nosocomial infections, with coagulase-negative staphylococci being the species most frequently involved. In particular, a Staphylococcus capitis clone named NRCS-A has emerged worldwide specifically in NICUs and is responsible for severe nosocomial sepsis in preterm neonates. This clone is specifically adapted to the NICU environment and is able to colonize and maintain on NICU surfaces. The present work explored the mechanisms involved in the persistence of the NRCS-A clone in the NICU environment despite strict hygiene measures. The ability to produce biofilm under nutritional stress and to resist desiccation appear to be the two main advantages of NRCS-A in comparison with other strains. These findings are pivotal to provide clues for subsequent development of targeted methods to combat NRCS-A and to stop its dissemination.

Keywords: Staphylococcus capitis NRCS-A; biofilm; disinfection; environmental persistence; neonatal intensive care units.

FIG 1

Doubling times of S. capitis strains from the different groups in standard medium. The doubling time of each strain from the five S. capitis groups was calculated from growth kinetics in TSB medium. Results are shown as the median doubling time for each group with 95% confidence intervals. Statistical analyses were carried out using the Mann-Whitney nonparametric test with an α of 0.05.

FIG 2

Evaluation of biofilm formation among the five S. capitis groups under different growth conditions. (A) Total biomass (bacteria and biofilm matrix) evaluation by crystal violet staining. (B) Bacterial density in biofilms obtained by enumeration. The TSB medium allowed biofilm production under standard in vitro conditions, whereas TSB plus 1% glucose, TSB plus 4% NaCl, and RPMI medium represented a resource-rich environment, a hyperosmotic condition, and a nutritive stress condition, respectively. Results are shown as the mean OD₅₉₀ or number of CFU/biofilm for each group ± standard deviation. Statistical analyses were carried out using the Mann-Whitney nonparametric test with an α of 0.05.

FIG 3

S. capitis biofilm structure and matrix composition under nutrient stress. After biofilm formation under nutrient stress (RPMI medium), the different components of the matrix were marked and biofilms were observed using a Zeiss LSM 880 confocal microscope with a ×63 magnification. Blue, total DNA marked by Hoechst 33342; green, eDNA marked by TOTO-1 iodide; white, polysaccharides including PIA marked by WGA Alexa Fluor 647; red, proteins marked by Sypro ruby. Image acquisition was done individually for the four different matrix components (four different channels), and composite images were constructed using ImageJ software by superposition of the four channels. For strains AV74, AW14, and BD18, the images showing the general appearance of the biofilm did not allow for observation of the aggregates that were visible in other images. Since several images were taken for each strain, images of aggregates formed by these strains were added and framed in white.

FIG 4

Desiccation tolerance among the different groups of S. capitis. The percentage of persistence was obtained by comparison of the bacterial inocula at T₀ and after the desiccation stress (T₂₄ or T₄₈). Results are shown as the percentage of persistence of each group with 95% confidence intervals. Statistical analyses were carried out using the Mann-Whitney nonparametric test with an α of 0.05.

FIG 5

Effect of disinfectants on S. capitis at usual concentrations and time of contact. Results are shown as the mean log₁₀(N/N₀) ± standard deviation, with N equal to the remaining inoculum after treatment with disinfectant and N₀ equal to the remaining inoculum after treatment with water. Statistical analyses were carried out using the Mann-Whitney nonparametric test with an α of 0.05. CDG, chlorhexidine digluconate; SP, Surfanios Premium; BAC, benzalkonium chloride.