Clonal Structure and Characterization of Staphylococcus aureus Strains from Invasive Infections in Paediatric Patients from South Poland: Association between Age, spa Types, Clonal Complexes, and Genetic Markers

Abstract

The aim of current study was to examine clonal structure and genetic profile of invasive Staphylococcus aureus isolates recovered from infants and children treated at the Jagiellonian University Children's Hospital of Krakow, Poland. The 107 invasive S. aureus isolates, collected between February 2012 and August 2014, were analysed retrospectively. Antimicrobial susceptibility testing, spa typing and DNA microarray analysis were performed to determine clonal distribution, diversity and gene content in regard to patients characteristics. In total, 107 isolates were recovered from 88 patients with clinical symptoms of invasive bacterial infection. The final set of 92 non-duplicate samples included 38 MRSA isolates. Additionally, a set of 54 S. aureus isolates collected during epidemiological screening was genotyped and analysed. There were 72 healthcare-associated (HCA) and 20 community-onset (CO) infection events caused by 33 and 5 MRSA isolates, respectively. The majority of isolates were affiliated with the major European clonal complexes CC5 (t003, spa-CC 002), CC45 (spa-CC 015), CC7 or CC15 (t084, t091, spa-CC 084). Two epidemic clones (CC5-MRSA-II or CC45-MRSA-IV) dominated among MRSA isolates, while MSSA population contained 15 different CCs. The epidemiological screening isolates belonged to similar genetic lineages as those collected from invasive infection cases. The HCA infection events, spa types t003, t2642 or CC5 were significantly associated with infections occurring in neonates and children under 5 years of age. Moreover, carriage of several genetic markers, including erm(A), sea (N315), egc-cluster, chp was significantly higher in isolates obtained from children in this age group. The spa types t091 and t008 were underrepresented among patients aged 5 years or younger, whereas spa type t008, CC8 and presence of splE was associated with infection in children aged 10 years or older. The HCA-MRSA strains were most frequently found in children under 5 years, although the majority of invasive infections was associated with MSSA strains. Moreover, an association between age group of children from the study population and a specific strain genotype (spa type, clonal complex or genetic content) was observed among the patients.

Fig 1. Population structure of 161 S. aureus isolates, including 107 invasive and 54 epidemiological screening isolates, collected in UCH (Krakow, Poland) after BURP analysis with a cost of 4.

Clusters of linked spa types correspond to spa clonal complexes (spa-CCs). The spa types that were defined as founders of particular clusters are indicated in blue. In total, eight spa-CCs have been identified, with spa-CC 084, spa-CC 002, spa-CC 015 and spa-CC 037/021/012 accounting for 14 to 41 isolates, each. Eighteen spa types were regarded as singletons (n = 34).

Fig 2. A split network tree constructed from the hybridization results of all 107 invasive S. aureus isolates from paediatric patients and 54 epidemiological screening isolates collected in UCH (Krakow, Poland).

SplitsTree analysis determined 6 major (CC5, CC45, CC7, CC30, CC15, CC8) and 4 minor (CC1, CC10, CC22, CC395) clusters. There were 8 sporadic CCs represented by single/two isolates (CC9, CC25, CC101, CC97, CC779, CC12, CC188, CC398). Identified MRSA lineages included CC45-MRSA-IV (Berlin EMRSA), ST5/ST225-MRSA-II (Rhine-Hesse EMRSA/New York-Japan Clone), CC5-MRSA-IV (Paediatric clone [sed/j/r+]) and CC7-MRSA-IV. The spa types and/or spa-CCs are presented next to their respective CCs.

Fig 3. Distribution of 92 non-duplicate invasive S. aureus isolates assigned to particular spa type and microarray CCs genotypes collected over study duration.

The numbers in parentheses indicate the total number of isolates linked with each genotype. The three dominant genotypes (t003-CC5, t015-CC45, t091-CC7) were present in UCH (Krakow, Poland) for almost entire study period, although no trend could have been observed. Remaining spa type-CCs (n = 35) included 2 isolates each: t267-CC97, t564-CC5, t283-CC5, t360-CC15, t002-CC5; or 1 isolate each: t056-CC101, t209-CC9, t010-CC5, t088-CC5, t005-CC22, t012-CC30, t340-CC45, t116-CC45, t1911-CC8, t122-CC30, t2229-CC8, t124-CC45, t037-CC30, t026-CC45, t306-CC5, t3850-CC15, t349-CC25. MRSA (n = 38) included following major genotypes: t003-CC5 (n = 13), t015-CC45 (n = 12), t2642-CC45 (n = 5), and 6 minor: t026-CC45, t091-CC7, t116-CC45, t283-CC5, t340-CC45, t564-CC5 with 1-2 representative isolates each.

Fig 4. Distribution of 92 invasive S. aureus isolates in respect to spa types and microarray CCs genotypes collected from all examined departments of UCH (Krakow, Poland) during the study period between February 2012 and August 2014.

The numbers in parentheses indicate the total number of isolates associated with each genotype (spa type-microarray CC). MRSA (n = 38) included following genotypes: t003-CC5 (n = 13), t015-CC45 (n = 12), t2642-CC45 (n = 5), t283-CC5 (n = 2), t564-CC5 (n = 2), t026-CC45 (n = 1), t091-CC7 (n = 1), t116-CC45 (n = 1) and t340-CC45 (n = 1).