Characterization of biofilm formation , antimicrobial resistance , and staphylococcal cassette chromosome mec analysis of methicillin resistant Staphylococcus hominis from blood cultures of children

Introduction: Methicillin resistant Staphylococcus hominis (MRSHo) has been recognized as an important human pathogen, particularly in immunocompromised patients. Methods: A total of 19 S. hominis isolates were collected from children at the Children’s Medical Centre, Tehran, Iran, from March 2012 to February 2013. MRSHo susceptibility against 13 antimicrobial and 3 antiseptic agents was determined using disk diffusion (DAD) and minimum inhibitory concentration (MIC), respectively. All isolates were subjected to polymerase chain reaction (PCR) assay for 15 distinct resistance genes, staphylococcal cassette chromosome mec (SCCmec), and arginine catabolic mobile elements (ACMEs). Biofilm production of the isolates was determined using a colorimetric microtiter plate assay. Results: Of the 19 isolates, 16 were resistant to oxacillin and harbored mecA. High resistance was also observed against trimethoprim/sulfamethoxazole (81.2%). All MRSHo isolates were susceptible to the three disinfectants tested (Septicidine-PC, Septi turbo, and Sayacept-HP). In total, 15 (78.9%) isolates produced biofilms. Three isolates had SCCmec types (V and VIII), 13 were untypable (UT), and 5 had ACME type II. Conclusions: The results indicate that MRSHo with high antibiotic resistance and unknown SCCmec might become a serious problem in the future for the treatment of patients such as children.


INTRODUCTION
Staphylococcus hominis, a member of coagulase-negative staphylococci (CoNS) found asymptomatically on the skin, arms, legs, and surfaces of the axilla, is an opportunistic pathogen capable of causing a wide variety of diseases including bacteremia, septicemia, and endocarditis especially in immunocompromised patients 1,2 .A growing concern is the emergence of methicillin resistant Staphylococcus hominis (MRSHo), which is the third most common pathogen among methicillin resistant CoNS (MRCoNS) in clinical isolates 3,4 .Resistance to methicillin is due to the acquisition of mecA, which encodes PBP2a -a transpeptidase with a low affinity for beta-lactam antibiotics such as penicillin.mecA is located on the staphylococcal cassette chromosome mec (SCCmec).Eleven main types of SCCmec differing in size and genetic composition have been assigned for staphylococcal isolates 5 .The capability of CoNS, including S. hominis, to produce disease depends on their ability to create a wide variety of virulence factors that contribute to colonization and disease incidence in hosts, such as biofilm production and the arginine catabolic mobile element (ACME) 6 .Arginine catabolic mobile elements includes two gene clusters, arc encoding a secondary arginine deiminase system and opp-3 encoding an ABC transporter 6 .In recent years, the widespread use of chemical disinfectants (Septicidine-PC, Sayacept-HP, and Septi turbo) in hospital settings played a critical role in the emergence of disinfectant-resistant microorganisms.Despite the incidence of methicillin resistance among staphylococci, there is currently little information available on the incidence and types of these resistances in many countries, such as Iran.The aim of this study was to investigate the antibiotic and antiseptic resistance patterns and prevalence of different SCCmec and ACME types among S. hominis isolates obtained from Iranian children.

Bacterial isolates
From March 2012 to February 2013, 256 CoNS strains were isolated from children at the Children's Medical Center, Tehran.Only one strain for each patient was selected.Isolates were identified as S. hominis, using conventional methods confirmed by the targeting of nuc 7,8 .In these isolates, the gene encoding methicillin resistance (mecA) was targeted by polymerase chain reaction (PCR) 9 .

Biofilm production
The capacity of isolates for biofilm production was determined using a colorimetric microtiter plate assay 15 .Briefly, MRSHo isolates were grown on Trypticase Soy Broth (TSB) overnight at 37°C (Merck, Darmstadt, Germany), and the cultures were then diluted 1:100 in TSB medium.Consequently, 150μL of this cell suspension was used to inoculate sterile flat-bottomed 96-well polystyrene microtiter plates.After 24 h incubation at 37°C, without shaking, the wells were gently washed three times with 200μL of phosphate buffered saline (PBS; Sigma-Aldrich, USA) and dried in an inverted position.For fixation of biofilms, 100μL of 99% methanol was added and after 15 min, supernatants were removed and the plate was air-dried.Next, 100μL of 1% crystal violet (CV; HiMedia, India) was added to all the wells.Excess CV was removed after 20 min by washing the plate under running tap water.Bound CV was released by adding 150μL of 33% acetic acid.The optical density (OD) of each well was measured at 590nm using a microtiter plate reader.All the tests were repeated three times.Uninoculated medium was used as a control to determine the background OD.The cut-off OD (ODc) was defined as three standard deviations above the mean OD of the negative control, and the final OD value of a tested strain was defined as the average OD of the strain reduced by the ODc value.The adherence ability of the tested strain was classified into four categories based on the OD 15 : non-adherent (OD < ODc), weakly adherent (ODc < OD < 2XODc), moderately adherent (2XODc < OD < 4XODc), and strongly adherent (4XODc < OD).The genes encoding intracellular adhesion (icaA and icaD) were targeted by PCR using the primers as previously described 9 .

Staphylococcal cassette chromosome mec and arginine catabolic mobile element typing
The primers used to detect SCCmec types and their variants, the conditions of PCR, and the size of the amplified products were each used as described by Ito et al. 16 The ACME typing PCR was performed with template deoxyribonucleic acid (DNA) from each isolate to determine if they harbored parts of ACME genes I to III 17 .
Of the 16 oxacillin-resistant MRSHo isolates, only 3 were typed by SCCmec typing: 2 were type VIII and 1 was type V (Table 2).Analysis of the mec and ccr complexes among the 13 untypable (UT) isolates revealed that four had mec complex A + ccrC (UT1); two, mec complex A + ccrAB1 and ccrC (UT2); one, mec complex B + ccrC (UT3); one, mec complex A and c1 + ccrAB1 (UT4); one, mec complex A and no ccr (UT5); one, only ccrAB1 and ccrC (UT6); three, neither ccr nor mec (UT7).Some of the isolates within the same UT group had different junk yard motifs.In ACME typing, only arcC was positive in 5 (31.2%) isolates, which were considered ACME type II.All isolates were negative for the opp-3 gene.

DISCUSSION
In this study, the methicillin resistance rate was slightly higher than that reported in other studies conducted in the USA (80-82%), Mexico (81%), and Tunis (75%) 2-4,18 , and the MDR rate was higher than that reported in the Tunis study (94%) 2 .The different MRSHo rates in different countries may reflect differences in the doses and dosing schedules used and in the spread of particular clones.In the current study, the reduced vancomycin susceptibility observed in some clinical isolates could preclude full resistance, which is in contrast to the findings of Palazzo et al. 19 , who observed 83.3% of isolates with resistance to vancomycin.In this study, we also observed high levels of resistance to gatifloxacin and tigecycline.Unfortunately, fluoroquinolones, especially ciprofloxacin and gatifloxacin, are widely used for the treatment of various bacterial infections in Iran, which may contribute to the emergence of resistant strains 20 .Moreover, several studies that used MIC methods revealed that 100% of CoNS were susceptible to tigecycline [21][22][23] .Given that we used disk diffusion to determine the tigecycline susceptibility of our MRSHo isolates, MIC-based methods might indicate that all of our isolates were susceptible.
In the current study, some strains with or without resistance genes (smr and qacA/B) had the same MIC, which may be owing to the presence of other resistance genes.In a Norway study that worked on cattle and goats, smr was detected in one of two S. hominis isolates, which is higher than our results in humans 24 .With regards to biofilm production, our results were similar to those obtained in certain studies conducted in Argentina and Mexico 25,26 ; however, they contrasted with that of a different Mexico study 4 , which reported high biofilm production in most strains Therefore, the low and moderate biofilm production may be related to factors other than the presence of ica.Given that different ACME types help to maintain S. epidermidis in the environment, the presence of ACME type II in some of these strains may improve their ability to remain and colonize.The SCCmec types observed in our study have previously been found in the USA (type VIII), China (type VIII and V), and Tunis (type VIII); nevertheless, none of these studies reported additional segments with these types 2,3,27 .The two strains in our study with SCCmes type VIII (strain 1 and 12) exhibited approximately the same antibiotic resistance (MDR) and biofilm production (weak), but strain 12 had qacA/B and an extra ccrC gene; these two strains were identified with an interval of 10 months.A combination of mec complex class A with ccr type 5 and mec complex class A with ccr type 1 has been reported before 2,4,27 .We also found isolates harboring ccrC, which is similar to that in the USA study 3 .A limitation of our study is that Multiplex SCCmec: Staphylococcal cassette chromosome mec; ACME: arginine catabolic mobile element; MRSHo: methicillin resistant Staphylococcus hominis ; untypable (UT) 1: mec complex A + ccrC; UT2: mec complex A + ccrAB1 and ccrC; UT3: mec complex B + ccrC; UT4: mec complex A and c1 + ccrAB1; UT5: mec complex A -ccr; UT6: only ccrAB1 and ccrC; UT7: neither ccr nor mec genes.PCR could not able to detect new SCCmec elements due to wide variation in ccr genes.Therefore, sequence-based methods are recommended.
In conclusion, the horizontal transfer of genes between species leads to increases in antibiotic-and disinfectant-resistant genes and the emergence of new types of SCCmec, all of which can influence the clinical features of MRSA in the future.In addition, MRSHo with high antibiotic resistance and unknown SCCmec type might become a serious problem for the treatment of patients with immune deficiencies, such as children.

TABLE1
Antibiotic and disinfectant resistance and biofilm production of MRSHo.

TABLE 2
SCCmec and ACME types of MRSHo isolates.