Molecular typing of toxic shock syndrome toxin-1- and Enterotoxin A-producing methicillin-sensitive Staphylococcus aureus isolates from an outbreak in a neonatal intensive care unit

International Journal of Medical Microbiology 305 (2015) 790–798

Contents lists available at ScienceDirect

International Journal of Medical Microbiology journal homepage: www.elsevier.com/locate/ijmm

Molecular typing of toxic shock syndrome toxin-1- and Enterotoxin A-producing methicillin-sensitive Staphylococcus aureus isolates from an outbreak in a neonatal intensive care unit Franziska Layer a,∗,1 , Andrea Sanchini b,f,1,2 , Birgit Strommenger a , Christiane Cuny a , Ann-Christin Breier c , Hans Proquitté d,3 , Christoph Bührer d , Karl Schenkel b,e , Jörg Bätzing-Feigenbaum g , Benedikt Greutelaers b , Ulrich Nübel a,4 , Petra Gastmeier c , Tim Eckmanns b , Guido Werner a a National Reference Centre for Staphylococci and Enterococci, Division Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Burgstraße 37, 38855 Wernigerode, Germany b Division of Healthcare Associated Infections, Surveillance of Antibiotic Resistance and Consumption, Department for Infectious Disease Epidemiology, Robert Koch Institute, Berlin, Germany c Institute of Hygiene and Environmental Medicine, Charité University Medical Centre, Berlin, Germany d Department of Neonatology, Charité University Medical Centre, Berlin, Germany e Department of Infectious Disease Prevention and Control, Community Health Office City of Berlin Mitte, Berlin, Germany f European Public Health Microbiology Training Programme (EUPHEM), European Centre for Disease Prevention and Control (ECDC), Stockholm, Sweden g Department of Infectious Disease Epidemiology and Environmental Health Protection, State Office for Health and Social Affairs, Federal State of Berlin, Berlin, Germany

a r t i c l e

i n f o

Keywords: Neonatal intensive care unit Methicillin-susceptible Staphylococcus aureus Toxic shock syndrome toxin-1 Outbreak

a b s t r a c t Outbreaks of Staphylococcus aureus are common in neonatal intensive care units (NICUs). Usually they are documented for methicillin-resistant strains, while reports involving methicillin-susceptible S. aureus (MSSA) strains are rare. In this study we report the epidemiological and molecular investigation of an MSSA outbreak in a NICU among preterm neonates. Infection control measures and interventions were commissioned by the Local Public Health Authority and supported by the Robert Koch Institute. To support epidemiological investigations molecular typing was done by spa-typing and Multilocus sequence typing; the relatedness of collected isolates was further elucidated by DNA SmaI-macrorestriction, microarray analysis and bacterial whole genome sequencing. A total of 213 neonates, 123 healthcare workers and 205 neonate parents were analyzed in the period November 2011 to November 2012. The outbreak strain was characterized as a MSSA spa-type t021, able to produce toxic shock syndrome toxin-1 and Enterotoxin A. We identified seventeen neonates (of which two died from toxic shock syndrome), four healthcare workers and three parents putatively involved in the outbreak. Whole-genome sequencing permitted to exclude unrelated cases from the outbreak and to discuss the role of healthcare workers as a reservoir of S. aureus on the NICU. Genome comparisons also indicated the presence of the respective clone on the ward months before the first colonized/infected neonates were detected. © 2015 Elsevier GmbH. All rights reserved.

1. Introduction

∗ Corresponding author. E-mail address: [email protected] (F. Layer). 1 These authors contributed equally to the manuscript. 2 Present address: Division of Mycotic and Parasitic Agents and Mycobacteria (FG16), Robert Koch Institute, Berlin, Germany. 3 Present address: Department of Pediatrics, University Hospital Jena, Jena, Germany. 4 Present address: Leibniz Institute DSMZ, Braunschweig, Germany. http://dx.doi.org/10.1016/j.ijmm.2015.08.033 1438-4221/© 2015 Elsevier GmbH. All rights reserved.

Premature neonates are particularly susceptible to Staphylococcus aureus infections (Borghesi and Stronati, 2008; Carey and Long, 2010). In neonatal intensive care units (NICUs), surgical procedures, understaffing, the use of invasive devices and mechanical ventilations are supportive factors increasing the risk for neonates to acquire S. aureus colonization or infection (Adams-Chapman and Stoll, 2002; Andersen et al., 2002; Heinrich et al., 2011). Furthermore healthcare-workers and parents serve as possible reservoirs.

F. Layer et al. / International Journal of Medical Microbiology 305 (2015) 790–798

Previous long-term studies revealed a heterogeneous population structure of S. aureus clones circulating in respective NICUs (Carey et al., 2010; Conceicao et al., 2012; Fujimura et al., 2004). Outbreaks of S. aureus in NICUs are common (Schwab et al., 2014) and usually those reported are caused by methicillin-resistant S. aureus strains (MRSA), including wellknown hospital- (HA-MRSA) and community-associated MRSA (CA-MRSA) (Koser et al., 2012; Nubel et al., 2013; Ramsing et al., 2013; Sanchini et al., 2013). In addition to classical HA-MRSA outbreaks also MRSA strains exhibiting special virulence factors like the Panton–Valentine–Leukocidin (PVL) (Ali et al., 2012; Sax et al., 2006) or toxic shock syndrome-toxin were reported among outbreaks in intensive care units (Kikuchi et al., 2003; Nakano et al., 2002). Reports about clusters of infections and colonizations involving methicillin-sensitive S. aureus (MSSA) are rarely reported, and if so, mainly describing exfoliative toxin producing strains causing bullous impetigo in neonates or PVL producing strains causing deep and recurrent skin and soft tissue infections (Bourigault et al., 2014; Gasch et al., 2012; Jursa-Kulesza et al., 2009; Kurlenda et al., 2009). Different molecular typing methods were developed during the last decades to trace the dissemination of S. aureus strains, ranging from DNA macrorestriction typing to sequence based typing (Nubel et al., 2011; Strommenger et al., 2006; Tenover et al., 1995) and the detection of genome wide single nucleotide polymorphisms (SNPs) by the use of whole-genome sequencing (Harris et al., 2013; Koser et al., 2012; SenGupta et al., 2014). In this study we report the epidemiological and molecular investigation of an MSSA outbreak in a NICU among preterm neonates caused by toxic shock syndrome-toxin-1 (TSST-1) – and Enterotoxin A (EntA)-producing MSSA.

2. Materials and methods 2.1. Description of outbreak settings The hospital, with more than 3000 beds and more than 12,000 employees, is structured in four campuses in Berlin and offers delivery services and level-III neonatology wards at two locations about 5 km apart. Of 4700 births annually, 1300 took place in the “Mitte” campus, where the outbreak occurred. The neonatal unit of the Mitte campus is built of two units: the intensive care unit (A) and the intermediate intensive care unit (B). The A unit consists of three rooms, for a total of nine beds (3/3/3). The B unit consists of six rooms for a total of 11 beds (4/3/1/1/1, of which 6 are also intensive-care beds) including an extra room for 1 mother and 1 neonate. The sickest and smallest infants are treated in the unit A. When neonate conditions improve, the infant can be transferred to the B unit, transferred to another hospital closer to the parents’ residence or discharged. Regular screening (pharyngeal and rectal) is performed once a week on both units for all risk neonates (weight < 1500 g, ventilated, with central lines). Depending on organisms found, barrier care or even cohorting is initiated. In case of presumed infections, blood cultures and (if clinically indicated) a lumbar puncture is conducted. Moreover, swabs from trachea (if ventilated), the pharynx and a rectal swab are taken. In May 2012 three cases of serious infections caused by MSSA, with two neonates who died from toxic shock syndrome (TSS) in the NICU, prompted an outbreak investigation including a detailed characterization of the S. aureus strains. On May 30th 2012 the Local Public Health Authority of Berlin Mitte was informed regarding the outbreak and the investigation was supported by the Robert Koch Institute henceforward.

791

2.2. Case definitions and case finding Infected cases were defined as: a patient in A or B unit with signs of local or systemic infection, with an isolate of S. aureus characterized by spa type t021, positive for the tst gene and for the in vitro production of TSST-1. Colonized cases were defined as: a patient in A or B ward with an isolate of S. aureus characterized by spa type t021, positive for the tst gene and for the in vitro production of TSST-1. After the outbreak was recognized, medical registers were reviewed to identify further cases retrospectively. In order to assess the risk of an environmental reservoir, 140 environmental samples considered as likely sources of infections (swabs from incubators, medical equipment/devices, working surfaces, changing tables, scales, body care products, ultrasonic devices etc.) were screened for TSST-1-producing S. aureus. 2.3. Laboratory sampling and characterization of bacterial isolates All S. aureus strains isolated were sent to the German National Reference Centre for Staphylococci and Enterococci at the Robert Koch Institute (Wernigerode, Germany) for further characterization (n = 155; Supplementary Table S1). The isolates were cultured on sheep blood agar and S. aureus species were confirmed by the presence of the clumping factor and by the tube coagulase test using human plasma. Antimicrobial susceptibility testing was determined by using the broth microdilution method according to EUCAST guidelines and breakpoints for interpretation of the results. Strains were grown in tryptic soy broth at 37 ◦ C overnight and DNA was extracted using a DNeasy tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions with the modification that lysostaphin (100 ␮g/ml; Sigma, Munich, Germany) was added to the cell-lysis step. Specific PCRs were applied to detect the presence of genes encoding the toxic shock syndrome-toxin-1 (tst) (using oligonucleotides tst f (5 -CCCTTAAAGTATKGGCCAAAG-3 ) and tst r (5 -GTGGATCCGTCATTCATTGTT-3 ); cycling conditions: 1 × 95 ◦ C for 120 s, followed by 30 cycles of 95 ◦ C for 30 s, 50 ◦ C for 30 s, 72 ◦ C for 2 min, 72 ◦ C for 7 min) and Staphylococcal Enterotoxin A (sea) (Johnson et al., 1991). Production TSST-1 and Staphylococcal Enterotoxin A (EntA), B (EntB), C (EntC) and D (EntD) was determined by commercial toxin detection kits based on latex agglutination (Oxoid, Hampshire, UK). 2.4. Molecular typing of strains For spa-typing the amplification and sequencing of the polymorphic X-region of the protein A gene (spa) was performed according to (Harmsen et al., 2003; Strommenger et al., 2008). Spa-types were grouped into spa clonal complexes (spa-CC) using the BURP (Based Upon Repeat Pattern) algorithm of the Ridom Staphtype Software (version 2.2.1, Ridom GmbH, Würzburg, Germany). Multilocus sequence typing (MLST) on S. aureus was done as described elsewhere (Enright et al., 2000). The HARMONY protocol was used for obtaining Pulsed-field gel electrophoresis (PFGE) patterns of SmaI-digested genomic DNA (Cookson et al., 2007). Cluster analysis was performed with the BioNumerics software package (Applied Maths, Sint-Martens-Latem, Belgium), using the Dice coefficient, and visualized as a dendrogram by UPGMA with 1% tolerance and 1% optimization settings. For DNA microarray-based typing the AlereTM identibac S. aureus Genotyping Kit (AlereTM , Jena, Germany) was applied (Monecke et al., 2008). 2.5. Whole genome sequencing and analysis A total of 1 ng genomic DNA was used for library generation by the Nextera XT DNA Sample Prep Kit according to the

792

F. Layer et al. / International Journal of Medical Microbiology 305 (2015) 790–798

manufacturer’s recommendations (Illumina, San Diego, California, USA). Briefly, DNA was subjected to the Nextera Tagmentation Reaction, followed by Limited-Cycle PCR, thereby generating linker sequences. Generated libraries were normalized by utilizing magnetic beads as provided. Sequencing was carried out by means of illumina technology on a MiSeq instrument. Amplification was performed in paired-end modus using a v2 chemistry-based cartridge 500 on a 14 tile flow cell (500-Cycle Reagent Kit, illumina), providing >30-fold average coverage. Single-nucleotide polymorphisms (SNPs) were identified by mapping paired-end sequencing reads against the genome sequence of MRSA252. The genome sequence of MRSA 252 is representative for MRSA of the clonal complex ST30. The fully annotated genome is available from the EMBL/GenBank databases with accession number BX571856 (Holden et al., 2004). An alignment of SNPs in the non-repetitive core genome was used to reconstruct the isolates’ phylogeny by applying PhyML 3.0.1 (Guindon et al., 2010) and to calculate evolutionary rates and divergence times with the BEAST (Bayesian Evolutionary Analysis Sampling Trees) software (http://beast.bio.ed.ac.uk/) (Drummond and Rambaut, 2007). Results were virtually independent from clock models (strict, relaxed) and tree priors (constant population size, exponential growth, Bayesian skyline). Sequence reads were deposited in the European Nucleotide Archive (http://www.ebi.ac.uk/ena/) under the following accession number: PRJEB9867.

3. Results 3.1. Description of the outbreak, infection control measures and interventions After two neonates succumbed with TSS enhanced control measures were initiated (at week 22 of 2012). All infants were screened twice a week through nasopharyngeal and rectal swabs and additionally tracheal swab, if ventilated. The care of neonates, which have been identified both as colonized and infected was modified as follows: by entering the room, surgical masks had to be worn; caring of the premature infants was only receivable by carrying an extra splash guard coat, additionally the wear of gloves was required in case of handling with secretions. Because three infants had developed serious infections with clinical deterioration and capillary leakage syndrome, further investigations such as spa typing and TSST-1/EntA toxins screening were also requested (week 22 of 2012). After having identified neonates with MSSA positive for both toxins (TSST-1 and EntA) all parents had to wear masks when visiting neonates. The medical staff members and the neonate parents were screened for MSSA by nasopharyngeal swab. All healthcare workers (HCWs) positive for an MSSA with TSST1 and EntA toxins were excluded from the working environment until they were tested negative. Decolonisation measures of HCWs and parents were commenced with antiseptic body washes and mupirocin nasal ointment. Follow-up screenings after decolonisation were performed to check for S. aureus eradication. All infected neonates received clindamycin and intravenous immunoglobulin. Infants below 1500 g received also intravenous immunoglobulin as prophylaxis. The National Reference Centre for Staphylococci received 155 MSSA strains, isolated from November 2011 (retrospective cases) to November 2012, for further characterisations and strain typing (see later; Supplementary Table S1). Seventeen neonates met the case definitions applied for infected or colonized cases. Furthermore four HCWs and three neonate relatives were colonized with a S. aureus spa-type t021, tst-positive. Clinical and demographic characteristics of neonates are summarized in Table 1. Neonate

couples 5–6, 8–9, 10–11 and 14–15 were twins. All neonates were born between November 15th 2011 and August 7th 2012 and were transferred immediately to the neonatal intensive care unit A. Two neonates died from S. aureus associated TSS after 18 and 20 days from birth, respectively. The remaining 15 neonates were either discharged from the A unit or transferred to the B unit before discharge. Length of stay on the ward for the surviving neonates ranged from 37 to 196 days of life (median 78 days). Positive testing for the outbreak strain in the neonates occurred between 4 and 91 days of life (median 14 days). All neonates, both colonized and infected, were born preterm between 24 and 29 weeks of gestation, their birth weight ranging from 610 to 1495 g (median 831 g). Twelve neonates were only colonized while five reported clinical infections (of which two died). The first case (colonized) and the last case (infected) were identified in week 48, 2011 and week 34, 2012, respectively (Fig. 1). 3.2. Characterization of bacterial isolates In total 213 neonates, 123 HCWs and 205 neonate parents were screened for MSSA. 56 neonates, 24 HCWs and 57 neonate relatives were tested MSSA-positive, resulting in 26.3%, 19.5% and 27.8% of MSSA colonization rates, respectively (overall MSSA colonization rate 25.3%). No MSSA were detected in environmental samples. A total of 155 MSSA isolates, including follow-up isolates taken after decolonization, were sent to the National Reference Centre for Staphylococci and Enterococci for further characterization (Supplementary Table S1). Thirty-four isolates were characterized as outbreak strains being spa type t021, positive for the tst- and sea-genes and accordingly positive for the production of TSST-1 and EntA. Relatedness of those isolates was further examined by SmaImacrorestriction using the first isolate of each individual (n = 24; follow-up isolates were excluded). The majority of isolates (n = 21) shared an identical macrorestriction pattern (Fig. 2, pattern “A”). A second cluster consisted of two strains, originating from a neonate and the corresponding parent. The third “group” comprised only one parental strain. DNA microarray-based typing assigned the 24 strains to CC30MSSA, agr III and capsule type 8 (Supplementary Table S2), but did not discriminate between the outbreak and non-outbreak isolates as defined by PFGE analysis. 3.3. Genome diversity, MSSA reservoirs and transmission routes Based on whole genome SNP data we were able to reconstruct the phylogenetic relationship of the isolates (Fig. 3A). Twenty-one MSSA isolates were related as they differed from each other by a maximum of 27 point mutations in the core genome (Fig. 3B). If we exclude the isolate from HCW A, the remaining 20 strains differ from each other by a maximum of 11 SNPs, indicating a close relationship between them. Bacterial genome sequencing therefore confirmed results of PFGE analysis and an epidemiological relationship between isolates. This cluster (Fig. 3B) comprised isolates from 16 neonates, four healthcare workers and one parent. The isolates of neonate 17 and his father formed a second clade, differing from each other by just one point mutation. SNP data also revealed that the isolate from parent 3 was unrelated, differing from the outbreak clade in approximately 1000 point mutations (therefore not shown in Figs. 3 and 4). Core genome sequences from neonates clustered in the outbreak clade did not show SNPs in four cases (Nos. 6, 9, 10, 16), further five strains differed from them just in 1–3 SNPs (Nos. 4, 5, 8, 11, 12, 13). Interestingly, twins always shared closely related but never identical isolates. Strains from neonate couples 5–6 and 8–9 differed in just one, couple 10–11 in two and couple 14–15 in eight SNPs. The

Table 1 Demographic and clinical data of the neonates colonized or infected with methicillin-susceptible Staphylococcus aureus (see case definitions) in the neonatal intensive care unit, November 2011–November 2012. Sex

Date of birth

Date of sample taken

Date tested MSSA positive

Age (days) tested MSSA positive

Type of sample

Birth weight (g)

Gestational age weeks (plus days)

Type of delivery

Case definition

Date of admission to A unit

Date of discharging

1 2 3

12-02017 12-02018 12-02019

F M M

15.11.11 09.12.11 25.01.12

na na na

28.11.11 27.12.11 09.02.12

13 18 15

610 942 470

24 (+2) 26 (+2) 27 (+1)

Caesarean Caesarean Caesarean

Colonized Colonized Colonized

15.11.11 09.12.11 25.01.12

29.05.12 02.03.12 06.05.12

4

12-02020

M

29.01.12

na

30.04.12

91

810

26 (+1)

Natural

Colonized

29.01.12

16.05.12

5

12-01792

M

19.03.12

01.04.12

01.04.12

13

746

25 (+5)

Caesarean

Infected

19.03.12

16.07.12

6

12-01859

M

19.03.12

09.04.12

09.04.12

21

832

25 (+5)

Caesarean

Colonized

19.03.12

16.07.12

7

12-01793

F

23.04.12

07.05.12

07.05.12

14

940

26

Caesarean

Infected

23.04.12

11.05.12a

8 9 10 11

12-01794 12-02021 12-02022 12-01791

M M M M

30.04.12 30.04.12 07.05.12 07.05.12

07.05.12 na na 23.05.12

07.05.12 14.05.12 21.05.12 23.05.12

7 14 14 16

805 1100 995 526

28 (+2) 28 (+2) 29 (+2) 29 (+2)

Caesarean Caesarean Caesarean Caesarean

Colonized Colonized Colonized Infected

30.04.12 30.04.12 07.05.12 07.05.12

05.07.12 08.08.12 13.06.12 27.05.12a

12 13 14 15 16 17

12-02035 12-02039 12-02037 12-02078 12-02444 12-03128

F F F M F M

16.05.12 23.05.12 26.05.12 26.05.12 05.07.12 07.08.12

29.05.12 na 02.06.12 11.06.12 09.07.12 25.08.12

29.05.12 04.06.12 02.06.12 11.06.12 09.07.12 25.08.12

13 12 7 16 4 18

Throat swab Throat swab Tracheal secretion Tracheal secretion Tracheal secretion Tracheal secretion Tracheal secretion Throat swab Throat swab Throat swab Tracheal secretion Throat swab Throat swab Blood Nasal swab Nasal swab Tracheal secretion

830 540 1200 1495 1080 na

26 (+2) 27 (+3) 29 (+4) 29 (+4) 29 (+4) na

Caesarean Caesarean Caesarean Caesarean Caesarean na

Colonized Colonized Infected Colonized Colonized Infected

16.05.12 23.05.12 26.05.12 26.05.12 05.07.12 07.08.12

02.08.12 28.07.12 17.07.12 04.07.12 17.08.12 15.09.12

a

F. Layer et al. / International Journal of Medical Microbiology 305 (2015) 790–798

RKI-No.

Neonate No.

Died because of toxic shock syndrome; na: data are not available.

793

794

F. Layer et al. / International Journal of Medical Microbiology 305 (2015) 790–798

Fig. 1. Line list of the neonates colonized or infected with methicillin-susceptible Staphylococcus aureus (MSSA) defined by case definitions in the hospital neonatal intensive care unit, November 2011–November 2012 (TSS: toxic shock syndrome). The time-line represents birth, immediately transfer to the intensive care unit (A), transfer to the intermediate care unit (B) and discharge/death of the respective cases.

retrospectively identified isolates from the end of 2011 (Nos. 1 and 2) are placed on deeper branches in the ML-tree than those strains isolated more recently. These analyses suggest that the outbreak strain was already circulating on the ward in 2011. The integration of temporal data by performing BEAST analysis on the genome sequences of the closely related isolates (excluding neonate 17 and the father and parent 3) resulted in a maximum clade credibility tree shown in Fig. 4. Furthermore BEAST analysis revealed an evolutionary rate for the outbreak strain of

2.8 × 10−6 mutations per nucleotide per year (95% confidence intervals, 1.1 × 10−6 to 4.4 × 10−6 ). Whenever neonates were tested positive, HCWs colonized with the outbreak strain (as identified by PFGE) were also present. Therefore HCWs B, C and D served as possible sources. The nasal flora of HCW A is probably not the reservoir for the above described cluster; the respective isolate is related to the isolates from the neonates but differs by >27 point mutations. The divergence of the outbreak clade from the most recent common ancestor is dated back to approximately July 2009 (95% confidence intervals, 2007–2011; including HCW A) or March 2011 (95% confidence intervals, April 2010 to September 2011; excluding HCW A), respectively. The neonate corresponding to parent 1 was born in May 2012 and never tested MSSA positive, although it was screened weekly. Therefore its parent must have acquired the strains elsewhere; however environmental sampling on the ward did not reveal any MSSA strain. 4. Discussion

Fig. 2. Pulsed-field gel electrophoresis (PFGE) profiles of SmaI-digested genomic DNA of suspected outbreak isolates of methicillin-susceptible Staphylococcus aureus in the hospital neonatal intensive care unit, November 2011–November 2012 (No. 1–17, according to Table 1).

We report about an outbreak on a NICU characterized by infections and colonisations among preterm neonates related to a TSST-1- and EntA-producing MSSA strain (spa-type t021, ST30). The first colonized neonate case has been identified retrospectively in November 2011, 5 months before an outbreak was notified. Most of the cases (n = 10) were primarily identified between week 18 and 24 of the year 2012. After two premature infants died from TSS on account of the above mentioned MSSA strain the investigation of the outbreak was intensified. The control measures such as the enhanced hygienic measures and the preventive administration of gamma globulins may have prevented further TSS manifestations and contributed to terminate the outbreak. MSSA spa-type t021 and other MSSA belonging to the clonal complex ST30 are common among MSSA isolated from healthy carriers. A community-based study on S. aureus nasal colonization of adults in Northern Germany revealed that every fifths participant was colonized with S. aureus and ∼24% of these strains were grouped to clonal complex ST30 (Mehraj et al., 2014). Therefore spatyping was not the appropriate tool for distinguishing the outbreak strains from non-related strains in our setting, comprising not only isolates from neonates but also from parents and healthcareworkers. A similar situation can be found in hospital settings worldwide, where the high abundance of endemic HA-MRSA lineages compromises outbreak investigations based on spa-typing

F. Layer et al. / International Journal of Medical Microbiology 305 (2015) 790–798

795

Fig. 3. Maximum-likelihood (ML) phylogenetic trees based on the SNP-alignment of the core genomes of methicillin-susceptible Staphylococcus aureus from neonates (No. 1–17, according to Table 1), four healthcare workers (HCW A–D) and two parents (A). In B No. 17 and the father of No. 17 were excluded since these isolates were not part of the outbreak clade.

Fig. 4. Maximum clade credibility tree based on BEAST analysis of the genome sequences of methicillin-susceptible Staphylococcus aureus from neonates (No. 1–16, according to Table 1), healthcare workers (HCW A–D) and one parent. The time scale in days is indicated in the bottom of the graph. The tips of the tree are representing the isolation date of the respective strain. Purple bars indicate 95% Bayesian credibility intervals of bacterial divergence dates.

796

F. Layer et al. / International Journal of Medical Microbiology 305 (2015) 790–798

(Leopold et al., 2014; SenGupta et al., 2014; Strommenger et al., 2008). DNA macrorestriction typing of our spa-type t021 isolates revealed that 21 strains showed an identical PFGE-pattern, whereas three strains clustered into two separate clades. Genome sequencing showed later on that just 20 of the 21 isolates, showing an identical PFGE-pattern, were closely related, while one isolate (from HCW A) is related but differed in >27 SNPs. PFGE was the first widely applied and internationally standardized typing tool for S. aureus and even though it is no longer the gold standard it is still used in laboratories especially outside Europe to trace the spread of clonal lineages from continent to continent over extended periods of time (Cookson et al., 2007; McDougal et al., 2003; Nubel et al., 2011; Strommenger et al., 2006). But PFGE has a limited discriminatory power at a local scale, where individual strains dominate the population (Ghebremedhin et al., 2007; McDougal et al., 2003). DNA microarray-based typing determined all our suspected outbreak strains as MSSA belonging to clonal complex 30 and confirmed the presence of tst- and sea-genes in all cases, but differences between the strains allowing a discrimination were not found. This kind of analysis is useful for the assignment of isolates to distinct clonal lineages and to detect resistance-and virulence-associated markers (Monecke et al., 2008, 2011), but cannot be recommended for strain typing and outbreak detection and conformation. Genome sequencing of our isolates revealed that 16 isolates from neonates were closely related; their core genome was identical in four cases or differed just in a limited number of SNPs. Phylogenetic analysis also indicated that four healthcare-workers and one parent were colonized with the outbreak strain. Nosocomial transmission of S. aureus isolates to neonates via healthcare workers occur commonly in the NICUs (Geva et al., 2011; Laing et al., 2009; Mean et al., 2007; Raboud et al., 2005); the outbreak strain may also be re-introduced by a staff-carrier, as previously described (Harris et al., 2013). However, it may be also true that neonates may acquire the strain from their relatives, as previously reported, and then spread the strain to other neonates or the staff (Behari et al., 2004; Delaney et al., 2013; Huang et al., 2006; Kayiran et al., 2013; Mitsuda et al., 1996; Pinter et al., 2009). Concerning our t021 isolates molecular typing revealed just one pair of strains (neonate No. 17 and father of 17), where transmission from father to child, or vice versa, was conceivable. A WGS comparison of the outbreak isolates from the four pairs of twins revealed a common number of genome-wide SNPs, not different from isolates from unrelated neonates attending the ward at the same time. This is not surprising as the twins were nursed in separate beds. Furthermore none of the twin’s parents was colonized with MSSA spa t021. Considering the long duration of the outbreak and therefore the long persistence of this MSSA clone, it could be speculated that healthcare workers served as reservoirs of the outbreak strain. The core genomes analyzed here accumulated ∼6 mutations per year. This rate is concordant to evolutionary rates found for other S. aureus strain types (Harris et al., 2010; Nubel et al., 2010, 2013; Smyth et al., 2010). By using the divergence of the outbreak clade and the substitution rate we were able to date the most recent common ancestor back to July 2009 (including HCW A) or March 2011 (excluding HCW A), respectively, at least months before the first neonate tested positive in November 2011. Ideally, conclusions deduced from whole genome sequences are supported by epidemiological data, like in a study from Nubel et al. (2013). Harris et al. applied whole-genome sequencing to expand findings of the infection-control team and confirmed that MRSA carriage by a healthcare-worker allows an outbreak strain to persist during periods on an NICU (Harris et al., 2013). Spa-typing of S. aureus strains obtained from regular screening of neonates during the outbreak revealed that the neonates were also colonized with S. aureus belonging to clonal lineages other than the outbreak strain, some of them were even able to produce TSST-1

(Supplementary Table S1). Indicating the risk of acquiring toxinproducing strains we have to keep in mind, that there is a high prevalence of genes for pyrogenic toxin superantigens in the general S. aureus population colonizing humans (Becker et al., 2003). Studies describing the long-term epidemiology of S. aureus on NICUs are rare. Conceicao et al. demonstrated the presence of three major MSSA clones on a Portuguese NICU, causing infections in neonates and being present during the whole 30-month study period (Conceicao et al., 2012). Another study covered a 4-year period identifying persisting MSSA clones, which were responsible for more than one case of neonatal sepsis each year (GomezGonzalez et al., 2007). Surveys displaying shorter study periods also clearly indicated the polyclonal MSSA population structure on these wards (Mernelius et al., 2013; Silva Hde et al., 2009). Outbreaks on NICUs associated with TSST-producing strains are rarely reported. We identified only two papers from Japan characterizing TSST-positive MRSA strains causing neonatal toxic shock syndrome-like exanthematous disease in neonatal and perinatal wards (Kikuchi et al., 2003; Nakano et al., 2002). Our study highlighted that not only MRSA, but also MSSA strains can cause severe, life-threatening infections in neonates, especially if those strains are producers of particular virulence factors/toxins. Annual data of the National Reference Centre for Surveillance of Nosocomial Infections revealed that the real number of neonatal outbreaks within Germany is definitely higher than those outbreaks which are reported to the respective health authorities (Schwab et al., 2014). During the last years a lot of efforts were undertaken in Germany to establish and implement recommendations concerning screening on NICUs for multidrug-resistant bacteria and bacteria with special resistances (EpiBull Nr.42/2013 (including its amendment risk characterizing) und EpiBull Nr.2/2012). However, there is no consensus regarding the routine use of screening for susceptible strains such as MSSA in neonates. Although colonization of neonates with commensal bacteria cannot be avoided, an enhanced surveillance of MSSA infections in times of increased incidence could be useful to detect transmission ways and to prevent new infections (Conceicao et al., 2012; Gomez-Gonzalez et al., 2007). 5. Conclusions MSSA, exhibiting particular virulence factors, are facultative pathogens in NICUs. Parents and healthcare-workers may serve as possible reservoirs and transmitters. Therefore, the discrimination of closely related strains for analyzing an outbreak is essential. However, even by a combination of sophisticated, molecular typing techniques the resolution might be limited in outbreak situations involving highly prevalent S. aureus lineages. Whole-genome sequencing enabled us to exclude non-related cases from the outbreak scenario and to identify the most recent common ancestor of the outbreak clade. These analyses also indicated the presence of the respective clone on the ward at least months before detection of the first colonized/infected neonates. Whole-genome sequencing can provide new and more reliable insights into aspects of sourceidentification, re-introduction vs. ongoing transmission of identical strain types at a local level and thus has the potential to support infection-control measures in real-time. 5.1. Study limitations There are no screening data on health-care workers or mothers prior to May 2012, and no data on health-care workers room movements or health-care workers contact with neonates, which might have supported or disproved possible transmission route hypotheses.

F. Layer et al. / International Journal of Medical Microbiology 305 (2015) 790–798

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2015.08. 033. References Adams-Chapman, I., Stoll, B.J., 2002. Prevention of nosocomial infections in the neonatal intensive care unit. Curr. Opin. Pediatr. 14, 157–164. Ali, H., Nash, J.Q., Kearns, A.M., Pichon, B., Vasu, V., Nixon, Z., Burgess, A., Weston, D., Sedgwick, J., Ashford, G., Muhlschlegel, F.A., 2012. Outbreak of a South West Pacific clone Panton-Valentine leucocidin-positive meticillin-resistant Staphylococcus aureus infection in a UK neonatal intensive care unit. J. Hosp. Infect. 80, 293–298. Andersen, B.M., Lindemann, R., Bergh, K., Nesheim, B.I., Syversen, G., Solheim, N., Laugerud, F., 2002. Spread of methicillin-resistant Staphylococcus aureus in a neonatal intensive unit associated with understaffing, overcrowding and mixing of patients. J. Hosp. Infect. 50, 18–24. Becker, K., Friedrich, A.W., Lubritz, G., Weilert, M., Peters, G., Von Eiff, C., 2003. Prevalence of genes encoding pyrogenic toxin superantigens and exfoliative toxins among strains of Staphylococcus aureus isolated from blood and nasal specimens. J. Clin. Microbiol. 41, 1434–1439. Behari, P., Englund, J., Alcasid, G., Garcia-Houchins, S., Weber, S.G., 2004. Transmission of methicillin-resistant Staphylococcus aureus to preterm infants through breast milk. Infect. Control. Hosp. Epidemiol. 25, 778–780. Borghesi, A., Stronati, M., 2008. Strategies for the prevention of hospital-acquired infections in the neonatal intensive care unit. J. Hosp. Infect. 68, 293–300. Bourigault, C., Corvec, S., Brulet, V., Robert, P.Y., Mounoury, O., Goubin, C., Boutoille, D., Hubert, B., Bes, M., Tristan, A., Etienne, J., Lepelletier, D., 2014. Outbreak of skin infections due to Panton-Valentine Leukocidin-positive methicillin-susceptible Staphylococcus aureus in a French prison in 2010–2011. PLoS Curr., 6. Carey, A.J., Duchon, J., Della-Latta, P., Saiman, L., 2010. The epidemiology of methicillin-susceptible and methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit, 2000–2007. J. Perinatol. 30, 135–139. Carey, A.J., Long, S.S., 2010. Staphylococcus aureus: a continuously evolving and formidable pathogen in the neonatal intensive care unit. Clin. Perinatol. 37, 535–546. Conceicao, T., Aires de Sousa, M., Miragaia, M., Paulino, E., Barroso, R., Brito, M.J., Sardinha, T., Sancho, L., Carreiro, H., de Sousa, G., Machado Mdo, C., de Lencastre, H., 2012. Staphylococcus aureus reservoirs and transmission routes in a Portuguese Neonatal Intensive Care Unit: a 30-month surveillance study. Microb. Drug Resist. 18, 116–124. Cookson, B.D., Robinson, D.A., Monk, A.B., Murchan, S., Deplano, A., de Ryck, R., Struelens, M.J., Scheel, C., Fussing, V., Salmenlinna, S., Vuopio-Varkila, J., Cuny, C., Witte, W., Tassios, P.T., Legakis, N.J., van Leeuwen, W., van Belkum, A., Vindel, A., Garaizar, J., Haeggman, S., Olsson-Liljequist, B., Ransjo, U., Muller-Premru, M., Hryniewicz, W., Rossney, A., O’Connell, B., Short, B.D., Thomas, J., O’Hanlon, S., Enright, M.C., 2007. Evaluation of molecular typing methods in characterizing a European collection of epidemic methicillin-resistant Staphylococcus aureus strains: the HARMONY collection. J. Clin. Microbiol. 45, 1830–1837. Delaney, H.M., Wang, E., Melish, M., 2013. Comprehensive strategy including prophylactic mupirocin to reduce Staphylococcus aureus colonization and infection in high-risk neonates. J. Perinatol. 33, 313–318. Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214. Enright, M.C., Day, N.P., Davies, C.E., Peacock, S.J., Spratt, B.G., 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38, 1008–1015. Fujimura, S., Kato, S., Hashimoto, M., Takeda, H., Maki, F., Watanabe, A., 2004. Survey of methicillin-resistant Staphylococcus aureus from neonates and the environment in the NICU. J. Infect. Chemother. 10, 131–132. Gasch, O., Hornero, A., Dominguez, M.A., Fernandez, A., Suarez, C., Gomez, S., Camoez, M., Linares, J., Ariza, J., Pujol, M., 2012. Methicillin-susceptible Staphylococcus aureus clone related to the early pandemic phage type 80/81 causing an outbreak among residents of three occupational centres in Barcelona, Spain. Clin. Microbiol. Infect. 18, 662–667. Geva, A., Wright, S.B., Baldini, L.M., Smallcomb, J.A., Safran, C., Gray, J.E., 2011. Spread of methicillin-resistant Staphylococcus aureus in a large tertiary NICU: network analysis. Pediatrics 128, e1173–e1180. Ghebremedhin, B., Konig, W., Witte, W., Hardy, K.J., Hawkey, P.M., Konig, B., 2007. Subtyping of ST22-MRSA-IV (Barnim epidemic MRSA strain) at a university clinic in Germany from 2002 to 2005. J. Med. Microbiol. 56, 365–375. Gomez-Gonzalez, C., Alba, C., Otero, J.R., Sanz, F., Chaves, F., 2007. Long persistence of methicillin-susceptible strains of Staphylococcus aureus causing sepsis in a neonatal intensive care unit. J. Clin. Microbiol. 45, 2301–2304. Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O., 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321. Harmsen, D., Claus, H., Witte, W., Rothganger, J., Claus, H., Turnwald, D., Vogel, U., 2003. Typing of methicillin-resistant Staphylococcus aureus in a university

797

hospital setting by using novel software for spa repeat determination and database management. J. Clin. Microbiol. 41, 5442–5448. Harris, S.R., Cartwright, E.J., Torok, M.E., Holden, M.T., Brown, N.M., Ogilvy-Stuart, A.L., Ellington, M.J., Quail, M.A., Bentley, S.D., Parkhill, J., Peacock, S.J., 2013. Whole-genome sequencing for analysis of an outbreak of meticillin-resistant Staphylococcus aureus: a descriptive study. Lancet Infect. Dis. 13, 130–136. Harris, S.R., Feil, E.J., Holden, M.T., Quail, M.A., Nickerson, E.K., Chantratita, N., Gardete, S., Tavares, A., Day, N., Lindsay, J.A., Edgeworth, J.D., de Lencastre, H., Parkhill, J., Peacock, S.J., Bentley, S.D., 2010. Evolution of MRSA during hospital transmission and intercontinental spread. Science 327, 469–474. Heinrich, N., Mueller, A., Bartmann, P., Simon, A., Bierbaum, G., Engelhart, S., 2011. Successful management of an MRSA outbreak in a neonatal intensive care unit. Eur. J. Clin. Microbiol. Infect. Dis. 30, 909–913. Holden, M.T., Feil, E.J., Lindsay, J.A., Peacock, S.J., Day, N.P., Enright, M.C., Foster, T.J., Moore, C.E., Hurst, L., Atkin, R., Barron, A., Bason, N., Bentley, S.D., Chillingworth, C., Chillingworth, T., Churcher, C., Clark, L., Corton, C., Cronin, A., Doggett, J., Dowd, L., Feltwell, T., Hance, Z., Harris, B., Hauser, H., Holroyd, S., Jagels, K., James, K.D., Lennard, N., Line, A., Mayes, R., Moule, S., Mungall, K., Ormond, D., Quail, M.A., Rabbinowitsch, E., Rutherford, K., Sanders, M., Sharp, S., Simmonds, M., Stevens, K., Whitehead, S., Barrell, B.G., Spratt, B.G., Parkhill, J., 2004. Complete genomes of two clinical Staphylococcus aureus strains: evidence for the rapid evolution of virulence and drug resistance. Proc. Natl. Acad. Sci. U. S. A. 101, 9786–9791. Huang, Y.C., Chou, Y.H., Su, L.H., Lien, R.I., Lin, T.Y., 2006. Methicillin-resistant Staphylococcus aureus colonization and its association with infection among infants hospitalized in neonatal intensive care units. Pediatrics 118, 469–474. Johnson, W.M., Tyler, S.D., Ewan, E.P., Ashton, F.E., Pollard, D.R., Rozee, K.R., 1991. Detection of genes for enterotoxins, exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction. J. Clin. Microbiol. 29, 426–430. Jursa-Kulesza, J., Kordek, A., Kopron, K., Wojciuk, B., Giedrys-Kalemba, S., 2009. Molecular studies of an impetigo bullosa epidemic in full-term infants. Neonatology 96, 61–68. Kayiran, P.G., Can, F., Kayiran, S.M., Ergonul, O., Gurakan, B., 2013. Transmission of methicillin-sensitive Staphylococcus aureus to a preterm infant through breast milk. J. Matern. Fetal Neonat. Med. 27, 527–529. Kikuchi, K., Takahashi, N., Piao, C., Totsuka, K., Nishida, H., Uchiyama, T., 2003. Molecular epidemiology of methicillin-resistant Staphylococcus aureus strains causing neonatal toxic shock syndrome-like exanthematous disease in neonatal and perinatal wards. J. Clin. Microbiol. 41, 3001–3006. Koser, C.U., Holden, M.T., Ellington, M.J., Cartwright, E.J., Brown, N.M., Ogilvy-Stuart, A.L., Hsu, L.Y., Chewapreecha, C., Croucher, N.J., Harris, S.R., Sanders, M., Enright, M.C., Dougan, G., Bentley, S.D., Parkhill, J., Fraser, L.J., Betley, J.R., Schulz-Trieglaff, O.B., Smith, G.P., Peacock, S.J., 2012. Rapid whole-genome sequencing for investigation of a neonatal MRSA outbreak. N. Engl. J. Med. 366, 2267–2275. Kurlenda, J., Grinholc, M., Krzyszton-Russjan, J., Wisniewska, K., 2009. Epidemiological investigation of nosocomial outbreak of staphylococcal skin diseases in neonatal ward. Antonie Van Leeuwenhoek 95, 387–394. Laing, I.A., Gibb, A.P., McCallum, A., 2009. Controlling an outbreak of MRSA in the neonatal unit: a steep learning curve. Arch. Dis. Child. Fetal. Neonat. Ed. 94, F307–F310. Leopold, S.R., Goering, R.V., Witten, A., Harmsen, D., Mellmann, A., 2014. Bacterial whole-genome sequencing revisited: portable, scalable, and standardized analysis for typing and detection of virulence and antibiotic resistance genes. J. Clin. Microbiol. 52, 2365–2370. McDougal, L.K., Steward, C.D., Killgore, G.E., Chaitram, J.M., McAllister, S.K., Tenover, F.C., 2003. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J. Clin. Microbiol. 41, 5113–5120. Mean, M., Mallaret, M.R., Andrini, P., Recule, C., Debillon, T., Pavese, P., Croize, J., 2007. A neonatal specialist with recurrent methicillin-resistant Staphylococcus aureus (MRSA) carriage implicated in the transmission of MRSA to newborns. Infect. Control Hosp. Epidemiol. 28, 625–628. Mehraj, J., Akmatov, M.K., Strompl, J., Gatzemeier, A., Layer, F., Werner, G., Pieper, D.H., Medina, E., Witte, W., Pessler, F., Krause, G., 2014. Methicillin-sensitive and methicillin-resistant Staphylococcus aureus Nasal carriage in a random sample of non-hospitalized adult population in Northern Germany. PLOS ONE 9, e107937. Mernelius, S., Lofgren, S., Lindgren, P.E., Blomberg, M., Olhager, E., Gunnervik, C., Lenrick, R., Thrane, M.T., Isaksson, B., Matussek, A., 2013. The effect of improved compliance with hygiene guidelines on transmission of Staphylococcus aureus to newborn infants: the Swedish Hygiene Intervention and Transmission of S. aureus study. Am. J. Infect. Control. 41, 585–590. Mitsuda, T., Arai, K., Fujita, S., Yokota, S., 1996. Demonstration of mother-to-infant transmission of Staphylococcus aureus by pulsed-field gel electrophoresis. Eur. J. Pediatr. 155, 194–199. Monecke, S., Coombs, G., Shore, A.C., Coleman, D.C., Akpaka, P., Borg, M., Chow, H., Ip, M., Jatzwauk, L., Jonas, D., Kadlec, K., Kearns, A., Laurent, F., O’Brien, F.G., Pearson, J., Ruppelt, A., Schwarz, S., Scicluna, E., Slickers, P., Tan, H.L., Weber, S., Ehricht, R., 2011. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS ONE 6, e17936. Monecke, S., Slickers, P., Ehricht, R., 2008. Assignment of Staphylococcus aureus isolates to clonal complexes based on microarray analysis and pattern recognition. FEMS Immunol. Med. Microbiol. 53, 237–251.

798

F. Layer et al. / International Journal of Medical Microbiology 305 (2015) 790–798

Nakano, M., Miyazawa, H., Kawano, Y., Kawagishi, M., Torii, K., Hasegawa, T., Iinuma, Y., Ohta, M., 2002. An outbreak of neonatal toxic shock syndrome-like exanthematous disease (NTED) caused by methicillin-resistant Staphylococcus aureus (MRSA) in a neonatal intensive care unit. Microbiol. Immunol. 46, 277–284. Nubel, U., Dordel, J., Kurt, K., Strommenger, B., Westh, H., Shukla, S.K., Zemlickova, H., Leblois, R., Wirth, T., Jombart, T., Balloux, F., Witte, W., 2010. A timescale for evolution, population expansion, and spatial spread of an emerging clone of methicillin-resistant Staphylococcus aureus. PLoS Pathog. 6, e1000855. Nubel, U., Nachtnebel, M., Falkenhorst, G., Benzler, J., Hecht, J., Kube, M., Brocker, F., Moelling, K., Buhrer, C., Gastmeier, P., Piening, B., Behnke, M., Dehnert, M., Layer, F., Witte, W., Eckmanns, T., 2013. MRSA transmission on a neonatal intensive care unit: epidemiological and genome-based phylogenetic analyses. PLoS ONE 8, e54898. Nubel, U., Strommenger, B., Layer, F., Witte, W., 2011. From types to trees: reconstructing the spatial spread of Staphylococcus aureus based on DNA variation. Int. J. Med. Microbiol. 301, 614–618. Pinter, D.M., Mandel, J., Hulten, K.G., Minkoff, H., Tosi, M.F., 2009. Maternal-infant perinatal transmission of methicillin-resistant and methicillin-sensitive Staphylococcus aureus. Am. J. Perinatol. 26, 145–151. Raboud, J., Saskin, R., Simor, A., Loeb, M., Green, K., Low, D.E., McGeer, A., 2005. Modeling transmission of methicillin-resistant Staphylococcus aureus among patients admitted to a hospital. Infect. Control Hosp. Epidemiol. 26, 607–615. Ramsing, B.G., Arpi, M., Andersen, E.A., Knabe, N., Mogensen, D., Buhl, D., Westh, H., Ostergaard, C., 2013. First outbreak with MRSA in a Danish neonatal intensive care unit: risk factors and control procedures. PLoS ONE 8, e66904. Sanchini, A., Spitoni, M.G., Monaco, M., Raglio, A., Grigis, A., Petro, W., Menchini, M., Pesenti, A., Goglio, A., Pantosti, A., 2013. Outbreak of skin and soft tissue infections in a hospital newborn nursery in Italy due to community-acquired methicillin-resistant Staphylococcus aureus USA300 clone. J. Hosp. Infect. 83, 36–40.

Sax, H., Posfay-Barbe, K., Harbarth, S., Francois, P., Touveneau, S., Pessoa-Silva, C.L., Schrenzel, J., Dharan, S., Gervaix, A., Pittet, D., 2006. Control of a cluster of community-associated, methicillin-resistant Staphylococcus aureus in neonatology. J. Hosp. Infect. 63, 93–100. Schwab, F., Geffers, C., Piening, B., Haller, S., Eckmanns, T., Gastmeier, P., 2014. How many outbreaks of nosocomial infections occur in German neonatal intensive care units annually? Infection 42, 73–78. SenGupta, D.J., Cummings, L.A., Hoogestraat, D.R., Butler-Wu, S.M., Shendure, J., Cookson, B.T., Salipante, S.J., 2014. Whole-genome sequencing for high-resolution investigation of methicillin-resistant Staphylococcus aureus epidemiology and genome plasticity. J. Clin. Microbiol. 52, 2787– 2796. Silva Hde, A., Pereira, E.M., Schuenck, R.P., Pinto, R.C., Abdallah, V.O., Santos, K.R., Gontijo-Filho, P.P., 2009. Molecular surveillance of methicillin-susceptible Staphylococcus aureus at a neonatal intensive care unit in Brazil. Am. J. Infect. Control 37, 574–579. Smyth, D.S., McDougal, L.K., Gran, F.W., Manoharan, A., Enright, M.C., Song, J.H., de Lencastre, H., Robinson, D.A., 2010. Population structure of a hybrid clonal group of methicillin-resistant Staphylococcus aureus, ST239-MRSA-III. PLoS ONE 5, e8582. Strommenger, B., Braulke, C., Heuck, D., Schmidt, C., Pasemann, B., Nubel, U., Witte, W., 2008. spa Typing of Staphylococcus aureus as a frontline tool in epidemiological typing. J. Clin. Microbiol. 46, 574–581. Strommenger, B., Kettlitz, C., Weniger, T., Harmsen, D., Friedrich, A.W., Witte, W., 2006. Assignment of Staphylococcus isolates to groups by spa typing, SmaI macrorestriction analysis, and multilocus sequence typing. J. Clin. Microbiol. 44, 2533–2540. Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H., Swaminathan, B., 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33, 2233–2239.