An Investigation into an Outbreak of Neonatal Sepsis in a Neonatal Intensive Care Unit, Presumptively Attributed Due to Coagulase-Negative Staphylococci.

Rebecca L. CoxhillSchool of Science and Technology, Nottingham Trent University, UK.

INTRODUCTION

Coagulase-Negative Staphylococci (CoNS) are the most significant pathogens causinglate-onset sepsis within the neonatal intensive care unit (NICU) [1]. The aim of thisstudy was to assess the association between the use of intravenous catheters [2] andsepsis attributed to CoNS, as well as to determine the presence of persistent strains[3], which have both been incriminated in outbreaks of sepsis in the NICU. Thesestrains often possess multiple antibiotic resistances and this study also aims toidentify potential patterns in resistance.

CONCLUSION

References1. Marchant EA, et al, 2013. Neonatal Sepsis due to Coagulase-Negative Staphylococci. Clin Dev Immunol 2013(586076).2. Casey AL, et al., 2006. RAPD for the typing of coagulase-negative staphylococci implicated in catheter-related bloodstream infection. J Infect52(4):282-289.3. Vermont CL, et al., 1998. Persistence of clones of coagulase-negative staphylococci among premature neonates in neonatal intensive careunits: two-center study of bacterial genotyping and patient risk factors. J Clin Microbiol 36(9):2485-2490.4. Van Der Zwet WC, et al., 2002. Nosocomial Spread of a Staphylococcus capitis Strain with Heteroresistance to Vancomycin in a NeonatalIntensive Care Unit. J Clin Microbiol 40(7):2520- 2525.5. Wang SM, et al., 1999. Staphylococcus capitis bacteremia of very low birth weight premature infants at neonatal intensive care units: clinicalsignificance and antimicrobial susceptibility. J Microbiol Immunol Infec 32(1):26-326. Raimundo O, et al., 2002. Molecular epidemiology of coagulase-negative staphylococcal bacteraemia in a newborn intensive care unit. J HospInfect 51(1):33-42.

RAPD was found to be an appropriate method for the genetic fingerprinting anddifferentiation between strains of CoNS [6], though it cannot differentiate betweenstrains with different plasmid mediated antibiotic resistances. The results from thisstudy supports the theory in current literature that CoNS are a significant pathogenscausing sepsis within the NICU, that they can infect the neonate through the meansof an intravenous catheter and that persistent strains may be endemic.

METHODS AND RESULTS

ANTIBIOTIC SUSCEPTIBILITY TESTING

The BSAC method, two different susceptibility patterns were identified. Pattern Ashowed resistances to Cefoxitin, gentamicin and Fusidic acid, whereas pattern Bshowed resistances to Cefoxitin, gentamicin and erythromycin (Table 1). Using a PCRbased method, the mecA gene was found in all samples, supporting the results fromthe BSAC method, whereas the ermA gene could not be found in the erythromycinresistant strains.

API STAPH

Using the API Staph kit (bioMérieux, Marcy-l’Étoile, France) and the apiweb™identification software, 80% of the isolates could be identified as Staphylococcuscapitis and the remaining 20% of isolates could be identified as Staphylococcushaemolyticus.

RANDOM AMPLIFIED POLYMORPHIC DNA PCR

An arbitrary primer (OPA5; 5’ ACGCAGGCAC 3’) was used to type the isolates usingRAPD PCR [1] and BioNumerics 7.1 software was used to analyse the images of thegel (Fig. 2) through cluster analysis (Fig. 1). This revealed the presence of 2 mainclusters (1 and 2) and an unrelated strain (3). Cluster 1 consisted of two isolates of S.haemolyticus and an isolate identified to be S. capitis, with a similarity of >84%.Cluster 2 consisted of six isolates of S. capitis with a similarity of >88% and finallythe remaining isolate was S. capitis, with a similarity of 65% to all of the otherisolates.

FIGURE 1 UPGMA dendrogram cluster analysis of the RAPD profiles using

BioNumerics 7.1 software. Each isolate is denoted by its individual strain

identification number. The RAPD types are labelled A-F and are sorted into 2 major

clusters (1 & 2) and unrelated strains (3). The red line at 82% represents the

threshold for related strains, and the blue line at 93% represents the threshold for

identical strains.

RAPD type

A

A

B

C

C

D

D

E

E

F

% Similarity

Isolate

15-3033

15-3034

15-1001

15-6254

15-6255

15-4094

15-4095

15-2092

15-2093

15-1002

1

2

3

DISCUSSION

Multidrug-resistant S. capitis was the most prevalent pathogen in this study,infecting 80% of patients (Table 1) which has been an emerging pathogen in theNICU, especially amongst neonates with very low birth-weights (<1.5kg) [4, 5]. UsingRAPD, the strains isolated from neonates 3-5 were found to be very similar (>88%),but not identical, suggesting that there may be a persistent clone which hasacquired extra mutations after the colonisation of the neonates, due to a differencein selective pressures. Also, isolates from neonate 3 presented a different antibioticsusceptibility pattern to the isolates from neonates 4 and 5, suggesting thedissemination of plasmids carrying resistance genes to erythromycin. Neonates 2-5could be confirmed as presenting catheter-related sepsis due to a similarity of 93-97% between the corresponding isolates, however, neonate 1 could not beconfirmed as presenting catheter-related sepsis.

TABLE 1 The details of the outbreak displayed in chronological order of isolation.

a B, Blood culture sample; C, catheter culture sample.b P-Hem, Pulmonary haemorrhage; NEC, Necrotising enterocolitis.c A, Resistances to Cefoxitin, Gentamicin and Fusidic acid; B, Resistances to Cefoxitin,Gentamicin and Erythromycin.

FIGURE 2 Images of the RAPD profiles in 2% agarose gel containing 1% SYBR SafeDNA stain. M, 100bp molecular marker; 1, isolate 15-1001; 2, isolate 15-1002; 3,isolate 15-3033; 4, isolate 15-3034; 5, isolate 15-6254; 6, isolate 15-6255; 7, isolate15-2092; 8, isolate 15-2093; 9, isolate 15-4094; 10, isolate 15-4095.

bp

10037

200

600

1000

2000

M 1 2 3 4 5 6 7 8 9 10 M

1500

bp

800

400