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Journal of Clinical Microbiology, July 2007, p. 2301-2304, Vol. 45, No. 7
0095-1137/07/$08.00+0     doi:10.1128/JCM.00019-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Long Persistence of Methicillin-Susceptible Strains of Staphylococcus aureus Causing Sepsis in a Neonatal Intensive Care Unit{triangledown}

Carmen Gomez-Gonzalez,1 Concepción Alba,2 Joaquín R. Otero,1 Francisca Sanz,1 and Fernando Chaves1*

Servicio de Microbiología,1 Servicio de Neonatología, Hospital Universitario Doce de Octubre, Avenida de Córdoba sn, 28041 Madrid, Spain2

Received 4 January 2007/ Returned for modification 24 April 2007/ Accepted 12 May 2007


   ABSTRACT
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Molecular epidemiology of Staphylococcus aureus strains causing bacteremia in neonates during 2002 to 2005 revealed seven clones, with four MSSA clones responsible for 80% of the cases. Some clones persisted or reappeared throughout the study. Three bacteremic clones were found colonizing health care workers (HCWs), particularly clone C, which was harbored by at least 15% of HCWs.


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In spite of a decreasing incidence of Staphylococcus aureus infections in neonates in industrialized countries, this microorganism is still an important cause of bacteremia in neonatal intensive care unit (NICU) patients and has been associated with high mortality and morbidity (5, 9). In recent reports (14, 15), S. aureus was found to be responsible for 7.8% of cases of late-onset sepsis (LOS) and 3% of cases of early-onset sepsis (EOS) in very-low-birth-weight (<1,500 g) neonatal infants. Furthermore, methicillin-resistant Staphylococcus aureus (MRSA), either nosocomially or community acquired, has emerged as a challenging pathogen in NICUs (7, 13).

After gathering several cases of methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia in our NICU in 2005, we decided to investigate the epidemiology and clonality of S. aureus recovered from blood samples in neonates during a 4-year period (2002 to 2005). To date, the majority of attention has been focused on the characterization of nosocomial isolates of MRSA in the NICU setting, and there are few studies of molecular epidemiology of MSSA in the NICU (6). In order to gain insights into the dynamics of transmission of S. aureus in NICUs, we also conducted a molecular surveillance study in health care workers (HCWs) to determine their possible role as a reservoir of infection.

Our NICU is a 44-bed referral unit, 14 intensive care beds in a single large space and 30 intermediate intensive care beds in three separate rooms. During the period from 2002 to 2005, 4,369 patients were admitted to the NICU. Neonates with S. aureus bacteremia were included in the study. Medical charts were reviewed, and demographic, clinical, and microbiological data were collected. Blood samples were inoculated and processed in an automated blood culture system (BacT/ALERT [bioMérieux, Durham, NC] or BACTEC 9240 [BD Diagnostics, Sparks, MD]). Identification and susceptibility testing were performed by using conventional methods and the Wider system (Soria Melguizo, Madrid, Spain) (3). All isolates of S. aureus underwent confirmatory PCR analysis for the mecA gene (12). Molecular characterization of isolates was performed using pulsed-field gel electrophoresis (PFGE) following DNA extraction and digestion with SmaI according to previously described methods (4). Computer-assisted analysis of PFGE was performed with Bionumerics software (Applied Maths, Kortrijk, Belgium). A similarity coefficient of 80% was selected to define PFGE types (11). MRSA isolates underwent further staphylococcal cassette chromosome mec characterization by PCR (12). All isolates were tested for the presence of the Panton-Valentine leukocidin (PVL) genes by PCR (8, 10).

A point-prevalence active surveillance to detect nasal carriers of S. aureus, was performed on a voluntary basis in HCWs attending the NICU in June 2005. Samples were inoculated onto phenol red-mannitol-salt agar plates that were incubated at 37°C for 48 h. Isolation and identification of S. aureus were based upon standard microbiological procedures (1). Molecular characterization of S. aureus isolates was performed by PFGE, and the macrorestriction patterns were compared to those of neonates with bacteremia.

During the 4-year study period, 15 episodes of S. aureus bacteremia were identified in 14 different patients. Fourteen were MSSA, and only one was MRSA. Two cases were identified in 2002, four in 2003, four in 2004 (1 MRSA), and five in 2005. The demographic and clinical characteristics of the patients are summarized in Table 1. Eleven neonates (78.5%) were premature infants (gestational age of <37 weeks), and 9 (64.2%) had very low birth weights. All patients were hospitalized from birth. Twelve of 15 episodes (80%) were considered LOS (median age at presentation of 14.5 days; range, 6 to 61 days), and 3 (20%) were EOS. Most cases of LOS (75%) were catheter-related infections. The source of bacteremia in EOS was known in two cases: one episode was considered vertical transmission from the mother, and the other was catheter related.


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  		TABLE 1. Demographic and clinical characteristics of neonates with Staphylococcus aureus bacteremia

 
Molecular characterization of isolates performed using PFGE showed seven clones (Table 1). Four clones were responsible for more than one case of sepsis during the 4-year study period, with some clones persisting for several years (Table 2). Clone C was the most frequent, affecting four infants; clone E affected three infants; clone B affected two infants causing three episodes of bacteremia, and clone D affected two neonates. Three other isolates presented unique PFGE patterns (A, E, and F). Clone E corresponded to a MRSA strain which presented certain genetic determinants, staphylococcal cassette chromosome mec type IV and PVL genes, characteristics of community-associated MRSA strains (17). We do not know how this neonate acquired this MRSA strain, but recent reports have illustrated the penetration of a community pathogen into the NICU, where nosocomial transmission may rapidly spread the microorganism (7, 13).


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  		TABLE 2. Temporal distribution of different S. aureus clones causing sepsis in neonates

 
The purpose of the present molecular epidemiological study of S. aureus strains isolated from blood was to gain insight into the clonal structure of S. aureus and to determine whether particular clones of these organisms were spreading within our NICU. To address this, we elected to study isolates of S. aureus that were recovered from blood because of their undoubted clinical significance and the potential utility of such an investigation in defining the epidemiology of invasive S. aureus infections. Although we recognize that these isolates represent the "tip of the iceberg" in terms of the total S. aureus population, we believe that molecular epidemiological studies of these pathogenic organisms can provide a clearer view of the overall clonal diversity within this species. Our study revealed four predominant MSSA clones responsible for 80% of cases of S. aureus bacteremia, including 11 cases (91.7%) with LOS and one case with EOS. Some of these MSSA clones persisted in our NICU between 2 or 3 years (clones B and C), and others (clones D and G) reappeared after 1 or 2 years of apparent eclipse.

In order to gain insight into the dynamics of transmission of S. aureus in our NICU, we performed a point-prevalence active surveillance in HCWs. Although it was performed in a limited time frame and not all HCWs were screened, we found 26.6% (16/60) of HCWs colonized with seven different MSSA clones, and four of the MSSA clones were also found in neonates (Fig. 1). No HCW was colonized with a MRSA. A predominant MSSA clone (clone C) was colonizing 15% of HCWs and caused 26.7% of cases of sepsis in three consecutive years. The long persistence of this MSSA clone could be explained because HCWs may have served as the reservoir to colonize neonates. Other studies have reported the implication of HCWs as the reservoir of either MSSA or MRSA outbreaks (2, 16). In our NICU, most cases of S. aureus sepsis were catheter-related infections. Probably, these neonates were first colonized with MSSA strains acquired from HCWs, and subsequent infections of vascular catheters in these immunocompromised patients caused bloodstream infections.


Figure 1
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  		FIG. 1. Dendrogram containing PFGE patterns of S. aureus strains isolated from blood (neonates) and nasal samples (HCW). Isolates were considered to be genetically related according to PFGE if gel patterns demonstrated ≥80% similarity upon analysis using the Dice coefficient and the unweighted-pair group method using average linkages.

 
Understanding the epidemiology and clonality of S. aureus infections has important implications for future efforts to control the emergence and spread not only of multidrug-resistant strains but also of multidrug-susceptible microorganisms. Given that these infections are potentially preventable, we believe that active surveillance of MSSA infection during periods of increased incidence would be useful in the detection of unsuspected transmission caused by breakdowns in infection control practices.


   ACKNOWLEDGMENTS
 
This work was supported by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III-FEDER, Spanish Network for the Research in Infectious Diseases (REIPI RD06/0008) and by Fundación Mutua Madrileña.


   FOOTNOTES
 
* Corresponding author. Mailing address: Servicio de Microbiología, Hospital Universitario Doce de Octubre, Avenida de Cordoba sn, Madrid 28041, Spain. Phone: (34) 91-3908239. Fax: (34) 91-5652765. E-mail: fchaves.hdoc{at}salud.madrid.org Back

{triangledown} Published ahead of print on 23 May 2007. Back


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Journal of Clinical Microbiology, July 2007, p. 2301-2304, Vol. 45, No. 7
0095-1137/07/$08.00+0     doi:10.1128/JCM.00019-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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