Molecular basis of resistance to macrolides, lincosamides and streptogramins in Staphylococcus hominis strains isolated from clinical specimens

Coagulase-negative staphylococci (CoNS) are the most frequently isolated bacteria from the blood and the predominant cause of nosocomial infections. Macrolides, lincosamides and streptogramin B (MLSB) antibiotics, especially erythromycin and clindamycin, are important therapeutic agents in the treatment of methicillin-resistant staphylococci infections. Among CoNS, Staphylococcus hominis represents the third most common organism. In spite of its clinical significance, very little is known about its mechanisms of resistance to antibiotics, especially MLSB. Fifty-five S. hominis isolates from the blood and the surgical wounds of hospitalized patients were studied. The erm(C) gene was predominant in erythromycin-resistant S. hominis isolates. The methylase genes, erm(A) and erm(B), were present in 15 and 25 % of clinical isolates, respectively. A combination of various erythromycin resistance methylase (erm) genes was detected in 15 % S. hominis isolates. The efflux gene msr(A) was detected in 18 % of isolates, alone in four isolates, and in different combinations in a further six. The lnu(A) gene, responsible for enzymatic inactivation of lincosamides was carried by 31 % of the isolates. No erythromycin resistance that could not be attributed to the genes erm(A), erm(B), erm(C) and msr(A) was detected. In S. hominis, 75 and 84 %, respectively, were erythromycin resistant and clindamycin susceptible. Among erythromycin-resistant S. hominis isolates, 68 % of these strains showed the inducible MLSB phenotype. Four isolates harbouring the msr(A) genes alone displayed the MSB phenotype. These studies indicated that resistance to MLSB in S. hominis is mostly based on the ribosomal target modification mechanism mediated by erm genes, mainly the erm(C), and enzymatic drug inactivation mediated by lnu(A).


Introduction
Coagulase-negative staphylococci (CoNS) are part of the normal bacterial flora of human skin, but they have been increasingly recognized as opportunistic pathogens capable of causing various types of infections (Piette and Verschraegen 2009). Among clinically significant strains of CoNS, Staphylococcus hominis is ranked the third in importance only after S. epidermidis and S. haemolyticus. The S. hominis is a genetically diverse species, and it is believed that recombination plays a significant role in generating this diversity (Mendoza-Olazarán et al. 2013;Zhang et al. 2013;Szczuka et al. 2014). These bacteria can be responsible for blood stream infections, endocarditis, peritonitis, bone and joint infections (Kloos and Bannerman 1999;Kaufman and Fairchild 2004;Chaves et al. 2005;Sorlozano et al. 2010;Bouchami et al. 2011). Similar to other staphylococci, the formation of biofilm on medical devices, or on host tissues, is thought to be the one of the major pathogenic factors of S. hominis (Kaufman and Fairchild 2004;Götz et al. 2006;Chokr et al. 2006;Rodhe et al. 2006;Fredheim et al. 2009;Szczuka et al. 2015). Relatively high prevalence of methicillin resistance complicated the treatment of staphylococcal infections (Casey et al. 2007). Macrolides, lincosamides and streptogramin B antibiotics are the preferred alternative to penicillins and cefalosporins in the treatment of staphylococci infection. Moreover, erythromycin and clindamycin are recommended as second-line drugs for patients with a β-lactam allergy (Leclercq 2002;Gherardi et al. 2009). MLS B antibiotics are structurally distinct but functionally similar because they inhibit protein synthesis by binding to the 50S subunit (23S rRNA) of the bacterial ribosome. In staphylococci, resistance to MLS B is generally based on three mechanisms: the ribosomal target modification mediated by erm genes, the active efflux of antibiotics mediated by msr(A) and enzymatic drug inactivation mediated by lnu(A) (Leclercq 2002). The lnu(A) gene encodes lincosamide O-nucleotidyltransferase, which only inactivates lincosamides. Erythromycin-resistance methylase (erm) genes encode proteins which methylate adenine residue A2058 in the peptidyltransferase region of 23S rRNA domain V, which is part of the large (50S) ribosomal subunit and prevents the binding of the antibiotic to the target site (Leclercq 2002;Novotna et al. 2005). This methylation results in cross-resistance to macrolide, lincosamide and streptogramin B antibiotics (MLS B phenotype), which can be expressed either constitutively (cMLS B ) or inducibly (iMLS B ). Coagulase-negative staphylococci, with an iMLS B resistance phenotype are resistant to 14-membered and 15-membered macrolides, whereas CoNS with a cMLS B resistance phenotype are resistant to all MLS B antimicrobials. The msr(A) gene is involved in the active efflux of antibiotics, causing resistance to 14-and 15-membered macrolides as well as to streptogramin, but not to lincosamides (MS B phenotype). This makes clindamycin, as a treatment choice, effective (Lina et al. 1999;Leclercq 2002;Vimberg et al. 2015).
The main purpose of this study was to assess the molecular basis of resistance to MLS B antibiotics in clinical isolates of S. hominis.

Bacterial strains
Fifty-five isolates of S. hominis were collected from the blood and surgical wound swabs of hospitalized patients. True bacteremia was diagnosed in 36 the of patients. The isolates were identified by using the VITEK 2 system (bioMérieux, France). Although the tuf sequencing gives perfect results in the identification of this species, the VITEK 2 offers very good results as well. Because the S. hominis is a genetically diverse species, we confirmed the identification of all tested S. hominis isolates by using the API STAPH. In this study, we included only those isolates whose identification was beyond any doubt. The isolates were stored at −70°C, in 50 % glycerol broth (BHI), until commencement of the study.

Characterization of resistance mechanisms
Phenotypic characterization of macrolides and lincosamides resistance was determined by the double-disc test, with erythromycin (15 μg) and clindamycin (2 μg) discs applied 20 mm apart. A 10-μl inoculum of a 0.5 McFarland suspension was spotted on Mueller-Hinton agar with antibiotic disc. After 18 h incubation at 35°C, blunting of the clindamycin zone of inhibition proximal to the erythromycin disc indicated the inducible type (D-shaped zone) of MLS B resistance, whereas resistance to both erythromycin and clindamycin indicated the constitutive type. Lack of a D-shaped zone in erythromycin-resistant and clindamycin-susceptible isolates was interpreted as the MS B efflux phenotype (Leclercq 2002;Aktas et al. 2007). The results were interpreted according to EUCAST recommendations. Isolates were also screened with a 30-μg cefoxitin disc and studied for the presence of mecA genes to test methicillin resistance (Geha et al. 1994). The bacterial genomic DNA was isolated from clinical isolates using the Genomic DNA Plus kit (A&A Biotechnology, Poland). For the detection of macrolide resistance genes (erm(A), erm(B), erm(C), msr(A), lun(A)) and mecA genes, PCR assays were performed as described by Lina et al. (1999), Le Bouter et al. (2011) and Geha et al. (1994). The STATISTICA software (10.00 StatSoft, Tulsa, OK, USA) was used for statistic analysis. Association between methicillin resistance and resistance to MLS B antibiotics was evaluated by using chi-square (χ 2 ) test. A P value of <0.05 was considered significant.

Results
The most prevalent resistance determinant was erm(C) which was detected in 25 of the isolates (45 %), followed by lnu(A), erm(B) and erm(A) detected in 17 (31 %), 14 (25 %) and 8 (15 %) isolates, respectively. The msr(A) gene was detected alone, in 4 isolates and in 6 isolates, in combination with other genes. As Table 1 shows, 14 distinct resistance genotypes could be observed in the S. hominis strains. Fourteen isolates were negative for all screened genes.
All isolates harbouring the erm(B) or erm(C) genes alone or in combination with other genes exhibited resistance to erythromycin. The erm(A) was never found alone and all erm(A)-positive isolates were resistant to erythromycin. Fourteen isolates, which were negative for all five resistance genes, displayed susceptibility to erythromycin and clindamycin. No isolates, resistant to clindamycin only, were found. Twenty eight erm-positive isolates were resistant to erythromycin but remained susceptible to clindamycin and exhibited the inducible MLS B phenotype. The remaining nine erm-positive isolates showed resistance to erythromycin and clindamycin, displaying the constitutive MLS B phenotype. It should be emphasized that the cMLSB phenotype was detected only in strains harbouring simultaneously erm and lnu(A). The four isolates, harbouring the mrsA gene alone, represented the MS B phenotype. Methicillin-resistant S. hominis isolates were significantly more often resistant to macrolides and lincosamides (93 % to erythromycin, 77 % to clindamycin) than methicillinsusceptible isolates (50 and 22 %, respectively; p<0.001).

Discussion
Coagulase-negative staphylococci have been recognized as an important cause of nosocomial infections and are the most frequently isolated bacteria from blood (Krediet et al. 2004;Hira et al. 2007;Piette and Verschraegen 2009). These pathogens have developed an increased resistance to antimicrobial agents, especially to methicillin and other semisynthetic penicillins. Among CoNS, S. haemolyticus has the highest tendency to develop resistance to multiple antibiotics (Rodríguez-Aranda et al. 2009). S. hominis isolates display a lower virulence than S. haemolyticus and have been recognized, less frequently, as significant human pathogens. However, there are reports indicating that S. hominis can be responsible for nosocomial outbreaks (Chaves et al. 2005;d'Azevedo et al. 2008;Palazzo et al. 2008;Sorlozano et al. 2010;Ruiz de Gopegui et al. 2011;Roy et al. 2014). Nevertheless, there is limited information on their resistance to antibiotics, especially to macrolides, lincosamides and streptogramin B. As mentioned above, MLS B are used against staphylococcal infection in penicillin-allergic patients and in methicillin-resistant staphylococci (MRS)-infected patients. In particular, the use of clindamycin is regarded as a valid choice in the treatment of soft-tissue and bone infections (Lina et al. 1999;Leclercq 2002;Gherardi et al. 2009). The present data indicates that 16 % of S. hominis strains were resistant to clindamycin, whereas 75 % displayed resistance to erythromycin. In German studies, only 19 % of S. hominis strains were erythromycin resistant (Gatermann et al. 2007). Most of these strains displayed the constitutive MLS B phenotype, as opposed to our study, which demonstrated that the majority of S. hominis expressed the inducible MLS B phenotype. It should be emphasized, that coagulase-negative staphylococci, with an iMLS B resistance phenotype are resistant to 14-membered and 15-membered macrolides, but susceptible to lincosamides, streptogramin B and 16-membered macrolides. Although, iMLS B CoNS are in vitro resistant to erythromycin and in vitro sensitive to clindamycin, prescribing clindamycin may lead to treatment failure. In our studies, more than half of the S. hominis isolates were resistant to methicillin. Additionally, methicillin resistance was closely associated with resistance to erythromycin, which narrows the therapeutic options. It is well known that glycopeptides are the treatment of choice for infections caused by the multi-resistant staphylococci. However, due to the emergence of vancomycin-resistant staphylococci, a reduction in the use of this antibiotic has  been recommended. Recently, Won and Kim (2013) has reported the emergence of vancomycin-resistant S. hominis. Also, the emergence of resistance to relatively new antibiotics, such as linezolid and quinupristin/dalfopristin, has also been noted, in clinical S. hominis strains (Petinaki et al. 2005;Ruiz de Gopegui et al. 2011). This study indicated that the resistance to macrolides and lincosamides in S. hominis is mostly based on the ribosomal target modification mechanism mediated by erm genes; mainly the erm(C) and enzymatic drug inactivation, mediated by lnu(A). The erm(C) genes are predominant among coagulase-negative staphylococci from European countries, Canada and Korea (Martineau et al. 2000;Lim et al. 2002;Novotna et al. 2005;Gatermann et al. 2007;Gherardi et al. 2009). However, these data largely concerns the most frequently isolated coagulasenegative strains i.e. S. epidermidis and S. haemolyticus, whereas little is known about the distribution of MLS B resistance genes in other staphylococci species, including S. hominis. Recently, Le Bouter et al. (2011) characterized resistance to macrolides, lincosamides and streptogramin B in 72 S. saprophyticus strains isolated from urine specimens. They found that the distribution of MLS B resistance genes in S. saprophyticus is different from that generally reported for S. epidermidis and S. haemolyticus. The results of this study show that erm(A) and erm(B) genes were present more frequently in S. hominis than in other staphylococcal species as previously described (Martineau et al. 2000;Gatermann et al. 2007;Gherardi et al. 2009). For example, in a study conducted in Korea, erm(B) genes were present only in 3.3 % of isolates (Lim et al. 2002). The efflux of macrolides due to msr(A) is a mechanism found only in a minority of S. hominis. Previously obtained data indicates that msr(A) genes were present in 11-24 % of coagulase-negative staphylococci (Aktas et al. 2007;Bouchami et al. 2007;Gatermann et al. 2007). In contrast, in S. saprophyticus, the efflux mechanisms were the most common mechanisms of resistance to MLS B antibiotics (Le Bouter et al. 2011). We observed a high occurrence of the lnu(A) gene, which confer resistance to lincomycin, but clindamycin remains active (Leclercq 2002). Overall, this study suggested that S. hominis may constitute a reservoir for MLS B genes, in particular erm(C) and lnu(A), among coagulase-negative staphylococci. These resistance genes are often located on plasmids or transposons and may be transferable to more pathogenic staphylococcal species (Leclercq 2002).
Our results indicated that the uncommon pathogen, S. hominis had a high prevalence of erythromycin resistance and most of these strains display the inducible MLS B phenotype. Ribosomal modification and drug inactivation are the main mechanisms of MLS B resistance, in S. hominis strains.
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