This study investigated an outbreak of neonatal sepsis in a neonatal intensive care unit attributed to coagulase-negative staphylococci (CoNS). The aims were to assess the association between intravenous catheters and CoNS sepsis, identify persistent CoNS strains, and determine antibiotic resistance patterns. Random amplified polymorphic DNA (RAPD) analysis identified two main clusters of CoNS isolates, with some strains showing similarity over 88%, suggesting persistence. Staphylococcus capitis was the most prevalent pathogen, infecting 80% of patients and showing multi-drug resistance. The results support that CoNS are a significant cause of neonatal intensive care unit sepsis, can infect via intravenous catheters, and persistent strains may cir

1. An Investigation into an Outbreak of Neonatal Sepsis in a Neonatal Intensive Care Unit, Presumptively
Attributed Due to Coagulase-Negative Staphylococci.
Rebecca L. Coxhill
School of Science and Technology, Nottingham Trent University, UK.
INTRODUCTION
Coagulase-Negative Staphylococci (CoNS) are the most significant pathogens causing
late-onset sepsis within the neonatal intensive care unit (NICU) [1]. The aim of this
study was to assess the association between the use of intravenous catheters [2] and
sepsis 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. These
strains often possess multiple antibiotic resistances and this study also aims to
identify potential patterns in resistance.
CONCLUSION
References
1. 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 Infect
52(4):282-289.
3. Vermont CL, et al., 1998. Persistence of clones of coagulase-negative staphylococci among premature neonates in neonatal intensive care
units: 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 Neonatal
Intensive 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: clinical
significance and antimicrobial susceptibility. J Microbiol Immunol Infec 32(1):26-32
6. Raimundo O, et al., 2002. Molecular epidemiology of coagulase-negative staphylococcal bacteraemia in a newborn intensive care unit. J Hosp
Infect 51(1):33-42.
RAPD was found to be an appropriate method for the genetic fingerprinting and
differentiation between strains of CoNS [6], though it cannot differentiate between
strains with different plasmid mediated antibiotic resistances. The results from this
study supports the theory in current literature that CoNS are a significant pathogens
causing sepsis within the NICU, that they can infect the neonate through the means
of 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 A
showed resistances to Cefoxitin, gentamicin and Fusidic acid, whereas pattern B
showed resistances to Cefoxitin, gentamicin and erythromycin (Table 1). Using a PCR
based method, the mecA gene was found in all samples, supporting the results from
the BSAC method, whereas the ermA gene could not be found in the erythromycin
resistant 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 Staphylococcus
capitis and the remaining 20% of isolates could be identified as Staphylococcus
haemolyticus.
RANDOM AMPLIFIED POLYMORPHIC DNA PCR
An arbitrary primer (OPA5; 5’ ACGCAGGCAC 3’) was used to type the isolates using
RAPD PCR [1] and BioNumerics 7.1 software was used to analyse the images of the
gel (Fig. 2) through cluster analysis (Fig. 1). This revealed the presence of 2 main
clusters (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 finally
the remaining isolate was S. capitis, with a similarity of 65% to all of the other
isolates.
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 the
NICU, especially amongst neonates with very low birth-weights (<1.5kg) [4, 5]. Using
RAPD, 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 has
acquired extra mutations after the colonisation of the neonates, due to a difference
in selective pressures. Also, isolates from neonate 3 presented a different antibiotic
susceptibility pattern to the isolates from neonates 4 and 5, suggesting the
dissemination of plasmids carrying resistance genes to erythromycin. Neonates 2-5
could be confirmed as presenting catheter-related sepsis due to a similarity of 93-
97% between the corresponding isolates, however, neonate 1 could not be
confirmed 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 Safe
DNA 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, isolate
15-2092; 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