Evaluation of Biofilm Formation and Prevalence of Multidrug-Resistant Strains of Staphylococcus epidermidis Isolated from Neonates with Sepsis in Southern Poland

Staphylococcus epidermidis strains play an important role in nosocomial infections, especially in the ones associated with biofilm formation on medical devices. The paper was aimed at analyzing the mechanisms of antibiotic resistance and confirming the biofilm-forming ability among S. epidermidis strains isolated from the blood of hospitalized newborns. Genetic analysis of resistance mechanism determinants included multiplex PCR detection of mecA, ermA, ermB, ermC, msrA, and mef genes. Biofilm analysis comprised phenotypic and genotypic methods including Christensen and Freeman methods and PCR detection of the icaADB gene complex. Among the tested S. epidermidis strains, 89% of the isolates were resistant to methicillin, 67%—to erythromycin, 53%—to clindamycin, 63%—to gentamicin, and 23%—to teicoplanin, while all the strains were susceptible to vancomycin and linezolid. The mecA gene was detected in 89% of the isolates, the ermC gene was the most common and present among 56% of the strains, while the msrA gene was observed in 11% isolates. Eighty-five percent of the strains were described as biofilm-positive by phenotypic methods and carried the icaADB gene cluster. Multidrug resistance and the biofilm-forming ability in most of the strains tested may contribute to antimicrobial therapy failure (p < 0.05)

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

<div><p>The aim of current study was to examine clonal structure and genetic profile of invasive <i>Staphylococcus aureus</i> isolates recovered from infants and children treated at the Jagiellonian University Children’s Hospital of Krakow, Poland. The 107 invasive <i>S</i>. <i>aureus</i> isolates, collected between February 2012 and August 2014, were analysed retrospectively. Antimicrobial susceptibility testing, <i>spa</i> 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 <i>S</i>. <i>aureus</i> 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, <i>spa</i>-CC 002), CC45 (<i>spa</i>-CC 015), CC7 or CC15 (t084, t091, <i>spa</i>-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, <i>spa</i> 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 <i>erm(A)</i>, <i>sea (N315)</i>, <i>egc-</i>cluster, <i>chp</i> was significantly higher in isolates obtained from children in this age group. The <i>spa</i> types t091 and t008 were underrepresented among patients aged 5 years or younger, whereas <i>spa</i> type t008, CC8 and presence of <i>splE</i> 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 (<i>spa</i> type, clonal complex or genetic content) was observed among the patients.</p></div

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

<p>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 [<i>sed/j/r</i>+]) and CC7-MRSA-IV. The <i>spa</i> types and/or <i>spa</i>-CCs are presented next to their respective CCs.</p

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

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

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

<p>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 <i>spa</i> 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.</p

Details of 15 paediatric patients from whom between 1 to 4 <i>S</i>. <i>aureus</i> isolates were collected at UCH during the study period.

<p>Details of 15 paediatric patients from whom between 1 to 4 <i>S</i>. <i>aureus</i> isolates were collected at UCH during the study period.</p

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

<p>The numbers in parentheses indicate the total number of isolates associated with each genotype (<i>spa</i> 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).</p

Identified statistically significant associations between age of patients and infection event classification/UCH departments/HA MRSA/CO MSSA/ MRSA/MLS_B/<i>spa</i> type/<i>spa-</i>CC/CC.

<p>Identified statistically significant associations between age of patients and infection event classification/UCH departments/HA MRSA/CO MSSA/ MRSA/MLS_B/<i>spa</i> type/<i>spa-</i>CC/CC.</p