Introduction
Coagulase-negative staphylococci (CoNS) ubiquitously colonize human skin and mucosal membranes, and due to this fact, they were for a long time considered harmless commensals [
1]. Nowadays, however, they are increasingly important etiologic agents of hospital-acquired infections (HAIs), including central line-associated bloodstream infections (CLABSIs) and surgical-site infections (SSIs). Among human CoNS,
Staphylococcus epidermidis represents the most frequently isolated species [
2]. The increasing prevalence of antibiotic-resistant CoNS from nosocomial infections have been reported in Europe for some time [
3‐
6], including especially worrisome methicillin-resistant
S. epidermidis (MRSE) [
7]. Since 2000, linezolid, a representative of the oxazolidynones has become an important addition in treatment for uncomplicated and complicated skin and skin structure infections and hospital- and community-acquired pneumonia caused by Gram-positive pathogens [
8]. Shortly after the introduction into hospital practice, the first case of linezolid-resistant
Staphylococcus aureus was reported in the USA in 2001 [
9]. Linezolid-resistant
S. epidermidis (LRSE) are increasingly observed in European countries, such as Portugal, Germany, Greece, Italy, Ireland, and France [
10‐
17]. Linezolid resistance determinants may be acquired by staphylococci due to mutations selected during prolonged linezolid therapy and by horizontal gene transfer [
18‐
21]. The G2576T mutation in the loop V of 23S rRNA is the principal determinant of the resistance; however, other mutations such as C2190T, T2502A, C2532T, and G2603T are observed as well [
11,
22‐
24]. Mutations in the genes of ribosomal proteins L3 (
rplC gene), L4 (
rplD gene), and L22 (
rplV gene) are also relatively frequently encountered among LRSE [
8]. As reviewed by Mendes et al. [
8], the alterations in L3 and L4 as a resistance mechanism appeared later in time and the complexity and number of such alterations in LRSE increased since 2014. The A157R modification in L3 was observed in the USA and Italy up to date [
8]. Transferable genes conferring linezolid resistance in staphylococci include the
cfr gene encoding ribosomal methyltransferase gene and the
optrA and
poxtA genes of ribosomal protection proteins [
25‐
28]. Among these, only
cfr was reported among LRSE so far [
29]. Next emerging problem is that linezolid and methicillin resistance are often combined, so it is also important to characterize the SCC
mec cassette elements in such isolates [
16,
30‐
31].
Here, we present a report on a possible LRSE spread in the University Children’s Hospital (UCH) in Krakow, Poland, between 2015 and 2017. The aim of this study was to genetically characterize the LRSE strains, determine their clonal relationships, linezolid resistance mechanisms and refer the results to patients’ characteristics.
Discussion
Linezolid is an effective treatment for multidrug-resistant Gram-positive bacteria and despite its broad use for almost 20 years, it still exhibits excellent activity against staphylococci. Linezolid resistance among
S. epidermidis remains uncommon worldwide but the increasing resistance in European countries such as Greece, Spain, Portugal, Italy, France, and Ireland has been reported [
10‐
17]. The LRSE outbreaks occur occasionally and are mainly associated with ICUs [
15]. Here, we describe the first emergence of LRSE and MRSE in Poland in a pediatric ICU. The emergence of LRSE strains is associated with an increased prior linezolid usage. In our hospital, linezolid was introduced into practice in 2005. The first LRSE strain was isolated from cerebrospinal fluid at 12.01.2014 from the Oncology and Hematology Department. Since 2015, the increased number of LRSE was isolated from invasive infections with a highest number of isolates in 2017.
Pediatric patients are at particular risk of bacterial infections due to their immature immune system, and this risk is especially evident in premature newborns and babies undergoing medical procedures, such as surgery, presence of catheters, and prolonged/extensive antimicrobial treatment. Restriction of linezolid usage was associated with disappearance of the resistant strains from the affected ICU.
In Poland, the nosocomial
S. epidermidis population is dominated by strains belonging to MLST clonal complex 2 (CC2) [
http://eburst.mlst.net]. These clones are multiresistant, seem to persist in hospital environment, and evolve quickly due to mutations, recombination events, and frequent transmission of mobile genetic elements [
46]. In the present study, all isolates belonged to ST2, a presumable ancestral type of CC2. In Germany, in 2015, Bender et al. described 12 (33%) LRSE belonging to ST2 [
11], O’Connor et al. described 9 isolates (100%) as ST2 [
13], and Barros et al. described one in a Portuguese hospital [
10]. In 2018, Dortet et al. described an outbreak caused by ST2, ST5, and ST22 of LRSE in France [
15]. In other European countries, five different STs were identified: (i) ST22 in Greece, Germany, and Spain [
11‐
12,
16,
31]; (ii) ST23 in Italy and Germany [
11,
14,
16]; (iii) ST83 in Italy [
14]; and (iv and v) ST5 and ST168 in Germany [
11,
16].
PFGE analyses are widely used for detection of the spread of a single clone at the local level [
47], and we also applied this technique in our study. The restriction patterns of
SmaI revealed two closely related PFGE types among isolates. Furthermore, all isolates shared the antimicrobial resistance phenotypes and determinants. Linezolid resistance was associated with acquisition of the A157R mutation in the ribosomal protein L3 and the presence of
cfr gene. Such L3 alterations were described previously to impact linezolid susceptibility [
48‐
49]. As shown in other studies, the
cfr gene can co-occur with other linezolid resistance mechanisms [
26,
50‐
51]. Importantly, the presence of a highly similar
cfr plasmids in different genetic backgrounds was confirmed [
17], and their acquisition via horizontal gene transfer in LRSE has been shown [
11,
14]. The evidence for the presence of endemic LRSE clones that circulate in hospital settings was also reported [
10,
12]. These strains differ from commensal
S. epidermidis isolates and become more successful in the hospital environment [
6]. All isolates carried also the
icaADBC locus, which is responsible for the production of polysaccharide intercellular adhesin (PIA), playing an important role in formation of biofilm by the bacterium [
52‐
53]. Since, the linezolid have been used to treat biofilm-associated
S. epidermidis infections, the circulation of LRSE with a biofilm-associated operon constitutes a real threat for patients [
54‐
55].
All studied isolates shared also the atypical composition of their SCC
mec elements. Such situation is indeed observed in MRCoNS also by others [
3,
56]. Chen et al. showed the multiple
ccr complexes composition in CoNS strains. The authors showed the
ccrAB3 and
ccrAB4 genes in
S. hominis and
S. capitis [
57]. Our study revealed not only the multiple
ccr but also a combination of SCC
mec cassette II and III elements in all our LRSE strains. The detection of new SCC
mec cassettes composition of all LRSE strains revealed the acquisition of antibiotic resistance determinants within hospital environment and revealed that CoNS strains are a reservoir of antibiotic resistance genes which can be easily spread to
S. aureus strains. To fully elucidate the structure of SCC
mec element in studied isolates, the whole-genome sequencing is indispensable.
In conclusion, we have reported the first emergence of LRSE in Poland. Recently, linezolid is used more frequently, especially in pediatric patients for treatment of severe infections. As the ST2 LRSE is identified not only in Poland but in many European countries, it is very important to start or continue the surveillance, infection control, and antimicrobial guidelines against linezolid-resistant staphylococcal strains.
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