Molecular Detection and Characterization of the Staphylococcus epidermidis and Staphylococcus haemolyticus Isolated from Hospitalized Patients and Healthcare Workers in Iran

Background The present study is aimed at surveying the antibiotics resistance profile, biofilm formation ability, staphylococcal cassette chromosome mec (SCCmec) types, and molecular epidemiology of Staphylococcus epidermidis and Staphylococcus haemolyticus isolated from hospitalized patients and healthcare workers in four teaching hospitals in Iran. Methods In total, 43 Staphylococcus epidermidis and 12 Staphylococcus haemolyticus were isolated from hospitalized patients, and 19 Staphylococcus epidermidis and 7 Staphylococcus haemolyticus isolated from healthcare workers were included in the present study. The antimicrobial resistance profile of isolates was determined using the disk diffusion method. Moreover, the resistance of isolates to methicillin was identified using the cefoxitin disk diffusion test. The microtiter-plate test was used for quantifying biofilm formation. Moreover, the frequency of icaA and icaD genes was determined using PCR assay. The molecular epidemiology of methicillin-resistant isolates was determined using SCCmec typing and pulsed-field gel electrophoresis methods. Results Among all coagulase-negative staphylococci isolates, the highest resistance rate (81.5%) was seen for cefoxitin and cotrimoxazole. All of the isolates were susceptible to linezolid. Out of the 66 mecA-positive isolates, the most common SCCmec type was the type I (n = 23; 34.8%) followed by type IV (n = 13; 19.7%). Using pulsed-field gel electrophoresis (PFGE) assay, 27 PFGE types including 14 common types and 13 singletons were obtained among 51 methicillin-resistant S. epidermidis (MRSE) isolates. Moreover, among 12 methicillin-resistant S. haemolyticus (MRSH) isolates, 8 PFGE types were detected, of which 5 PFGE types were singletons. Conclusion The high rate of resistance to antibiotics as well as the possibility of cross-infection shows the importance of a pattern shift in the management and controlling programs of coagulase-negative staphylococci, especially in healthcare centers. Clinical trial registration. The present study is not a clinical trial study. Thus, a registration number is not required.


Introduction
In recent years, coagulase-negative staphylococci (CoNS) as part of the skin and mucous membranes microbiota of humans had a profound influence on human life and health [1]. Among the CoNS, Staphylococcus epidermidis (S. epidermidis) and Staphylococcus haemolyticus (S. haemolyticus) are the most common species and can cause several infections, especially in patients with debilitating underlying diseases. These bacteria become resistant to a variety of antibiotics, especially beta-lactam drugs [2,3]. It is well known that the ability of biofilm formation makes bacteria to resistant to antimicrobial agents and to the host immune systems. Therefore, biofilm formation capacity is one of the most significant virulence factors that could lead to CoNS pathogenicity [4]. The emergence and increase of methicillin-resistant CoNS isolates are known as a public health concern [5]. In most cases, the mecA gene is responsible for the emergence of methicillin resistant (MR) isolates. It is revealed that MR-CoNS are often resistant to non-β-lactam antibiotics such as amikacin, ciprofloxacin, and clindamycin [6]. mecA gene carried by mobile genetic elements referred to as a staphylococcal cassette chromosome mec (SCCmec) [7]. So far, eleven types of SCCmec (types I to XI) have been described. However, only eight types (types I to VIII) have been established. All types I to XI have been described in S. aureus. To date, eight types of SCCmec (types I to VIII) were identified in CoNS isolates [8,9]. Notably, it has been reported that CoNS isolates can be transmitted between hospitalized patients (HPs) and healthcare workers (HCWs) [10]. Several studies revealed that the colonized HPs and HCWs are the main source of CoNS isolates [11,12]. However, considering that CoNS are considered a group of opportunistic pathogens, sufficient attention is not paid to the management and control of these bacteria [10,13,14]. Pulsed-field gel electrophoresis (PFGE) is an approved molecular typing method that can help in identifying the genetic relatedness between the staphylococcal isolates and their fingerprinting [15] In the present study, we used PFGE method to identify the genetic relatedness between the S. epidermidis and S. haemolyticus isolated from HPs and HCWs. Moreover, we surveyed the antibiotic resistance profile and biofilm formation capacity and determined the predominant SCCmec types of S. epidermidis and S. haemolyticus isolated from HPs and HCWs in four teaching hospitals in Iran.

Study Design and Bacterial
Isolates. The present crosssectional study was performed from May 2020 to March 2021 in 3 teaching hospitals in Tabriz (northwest Iran) and one teaching hospital in Shahroud (northeast Iran). In the first step, all participants including HPs and HCWs signed an informed consent form, and clinical samples including blood, urine, tracheal tube, and pleural fluid were collected from them. Swab samples were taken with cotton sterile swabs from the hands of 80 healthcare providers (including 60 nurses and 20 physicians). All samples were cultured on blood agar and mannitol salt agar (Merck, Germany) plates and incubated at 37°C for 24 hours. After overnight incubation, Gram staining was performed on all colonies and phenotypic confirmation of CoNS isolates was carried out with catalase, oxidase, DNase, and coagulase tests, and growth in nutrient broth containing 6% sodium chloride (NaCl). The molecular identification of the Staphylococcus genus was performed based on the previously published work by Nahaei et al. [16]. Moreover, to final confirmation of S. epidermidis and S. haemolyticus, polymerase chain reaction (PCR) assay targeted the 16 s-23 s ribosomal DNA; internal transcribed spacer (ITS) region was applied based on the previously published studies by Cunha et al. [17] and Couto et al. [18].

Assessment of Biofilm Formation.
A quantitative assessment of biofilm formation was carried out by the microtiter-plate test (MTP) as described by Mohsenzadeh et al. [21]. Briefly, 2-3 colonies of the tested isolates were cultured in 10 mL of Trypticase Soy Broth (TSB) (Merck, Germany) containing 1% glucose (Sigma) and were incubated for 24 h at 37°C (adjusted to 0.5 McFarland standards). After overnight incubation, the suspension was shaken and then diluted at 1 : 100 in TSB containing 1% glucose. In the next step, 200 μL of the diluted solution (bacterial suspension) was added to each well of a microplate. Microplates were covered and incubated at 37 C for 18 h and following the process of incubation, the bacterial suspension was removed and all wells were washed five times with sterile distilled water. Afterward, the plates were stained with 0.1% crystal violet (Merck, Germany). Finally, the plates were washed and dried, and optical density (OD) was read at 570 nm. If the OD of the isolates was ≥0.12, they were classified as biofilm producers. The cut-off value was selected to differentiate between isolates that generated considerable amounts of biofilm and those that did not, taking into account the OD values for each experiment's negative controls. Also, S. epidermidis ATCC 12228 and S. epidermidis RP62A were used as negative and positive controls, respectively. Cetyltrimethylammonium Bromide (CTAB) procedure as described by Nahaei et al. [16]. For final confirmation of methicillin-resistant S. epidermidis (MRSE) and methicillinresistant S. haemolyticus (MRSH), the PCR method targeted the mecA gene was used. PCR conditions, the volume of materials, and primer sequence were set based on a previously published study by de Allori et al. [22]. Moreover, PCR analysis for icaA and icaD genes was performed using specific primers. The volume of materials, PCR conditions, and primer sequences have been described previously by Arciola et al. [23]. The PCR products were electrophoresed on 1.5% agarose gels in TBE buffer (89 mM Tris base, 89 mM boronic acid, 2 mM Na2, EDTA, pH 8.25). The gel was stained using the DNA-safe stain (SinaClon Co., Iran) and was observed under ultraviolet light. A 100 bp DNA ladder was used as a molecular size indicator.

SCCmec Typing.
To determine SCCmec types I to V among mecA-positive isolates, SCCmec typing was carried out using the multiplex-PCR method. The primer sequences, PCR condition, and volume of material were set based on a study performed by Ghanbari et al. [24]. DNA was amplified with a thermocycler (Eppendorf, Mastercycler Gradient; Eppendorf, Hamburg, Germany).

Pulsed-Field Gel Electrophoresis (PFGE).
Typing MRSE and MRSH was performed using the PFGE method as previously described by Talebi et al. [3]. Briefly, the enzyme digestion of the plugs was carried out with the restriction enzyme SmaI (New England Biolabs). A Salmonella serotype Braenderup strain (H9812) was used as a molecular size marker (kindly provided by the Research Center of Health Reference Laboratories, Tehran, Iran). In addition, DNA separation was carried out by programming two states under the following conditions in a pulsed-field electrophoresis system (CHEF DR-II; Bio-Rad Laboratories, Hercules, CA, USA) at the temperature of 14°C; voltage 6 V/cm; switch angle, 120°C; switch ramp 1-30 second for 20 hours. Ethidium bromide (SinaClon, Iran) 0.5 mg/mL was used for staining gels. For PFGE pattern analysis, BioNumerics software version 7.5 (Applied Maths, St-Martens-Latem, Belgium) was applied.
The unweighted pair group method by using mathematical averaging (UPGMA), dice correlation coefficient with 1.5% optimization, and a 1.5% tolerance setting was used for the calculation of dendrograms. Isolates were considered genetically indistinguishable, closely related, possibly related, and different when there were 0, 1, 2, and >3 banding differences, respectively [25].

Statistical Analysis.
All results were included in SPSS software version 20 (SPSS Inc., Chicago, IL, USA) and analyzed using the chi-square test. A P value of < 0.05 was considered statistically significant. , tracheal tube (n = 9), urine (n = 5), and pleural fluid (n = 5).

Number of Bacteria and
In total, the majority of bacteria were recovered from the intensive care unit (ICU) (n = 55; 67.9%).

Biofilm Formation.
Results obtained from the MTP assay revealed that 89.1% (n = 49/55) of CoNS isolated from HPs had OD ≥ 0:12, indicating that they developed biofilms ( Table 3). Out of 49 CoNS isolates with biofilm formation capacity, 24 (43.6%) and 25 (45.4%) were positive and negative for icaA/icaD genes, respectively. In contrast, out of 6 CoNS isolates without biofilm formation capacity, 2 (3.6%) and 4 (7.3%) were positive and negative to icaA and icaD, respectively. There was no significant association between biofilm formation and the existence of icaA/icaD genes (P > 0:05). Our finding showed that 80.7% (n = 21/26) of CoNS isolates recovered from HCWs have biofilm formation capacity. Moreover, 64.2% of MDR isolates were biofilm producers. The statistical analysis showed a significant relationship between MDR and biofilm formation ability (P < 0:05).

Detection of mecA Gene and SCCmec
Types. The distribution of SCCmec types is shown in Table 3. Overall, among 62 S. epidermidis and 19 S. haemolyticus, 52 (83.9%) and 14 (73.7%) isolates were positive for the mecA gene. The SCCmec typing method was performed on 66 mecA-positive CoNS isolates. Frequency of SCCmec types were as follows: SCCmec type I (n = 23, 34.8%) SCCmec type IV (n = 13, 19.7%), SCCmec type I + III (n = 9, 13.6%), SCCmec type II (n = 4, 6.1%), SCCmec type III (n = 2; 3%), and SCCmec type III + V (n = 2; 3%). 19.7% (n = 13) of isolates were not typeable.  MRSH isolates was surveyed using the PFGE method. In total, 2 MRSE and 1 MRSH produced band sizes below 36 kb and were excluded from the study [26]. Out of 51 MRSE isolates, 27 PFGE types including 14 common types (CT: A-N) and 13 singletons were obtained ( Figure 1). CT-L was detected in 5 isolates, of which 4 isolates were obtained from HPs and 1 isolate was obtained from HCWs. Results showed that isolates J18 (isolated from the HPs) and J19 (isolated from the HCWs) had completely similar patterns, however, the SCCmec types of these isolates were different. It was noteworthy that both isolates J18 and J19 had MDR phenotypes. The CT-E consisted of 3 isolates recovered from HCWs and complete genetic similarity was observed between the 2 isolates. As shown in Figure 2, among 12 MRSH isolates, 8 PFGE types were detected, of which 5 PFGE types were singletons. PFGE type C consisted of 2 HPs isolates and 1 HCW isolate.

Discussion
In general, CoNS isolates are part of the common human microbiota and are considered opportunistic pathogens which can cause several important infections, especially in patients who have medical indwelling equipment [27,28].
In recent years, CoNS have become resistant to the majority of commonly prescribed antibiotics [13,24]. In healthcare settings, it is presumed that these pathogens can be transmitted between patients and HCWs. Transmission of these opportunistic pathogens by HCWs could lead to the dissemination of infections in different wards of hospitals [14].
In the present study, we investigated the biofilm formation capacity of S. epidermidis and S. haemolyticus isolated from HPs and HCWs. Moreover, we surveyed the antibiotic resistance profile and genetic relationship between these CoNS isolates. Results of our study revealed that the resistance rate to several antibiotics including erythromycin, clindamycin, and trimethoprim-sulfamethoxazole was significantly higher among S. epidermidis and S. haemolyticus isolated from HPs. This may be a result of the indiscriminate use of antibiotics among HPs and the induction of selective pressure. However, there was no significant difference in resistance to gentamicin, ciprofloxacin, rifampicin, and cefoxitin among CoNS isolated from HPs and HCWs.
Our findings showed that 81.8% and 80.8% of CoNS isolated from HPs and HCWs were methicillin-resistant, respectively. Globally, several studies have surveyed the prevalence of resistance to methicillin among CoNS isolates. A study conducted in Iran reported that 74% of the S. epidermidis isolates were methicillin-resistant [15].   [30]. Resistances to antibiotics among CoNS isolated from HCWs show an adaptation of the microflora to a pressure induced by the antimicrobial agents' administration in hospital settings, where the colonization may occur.
In the present study, all CoNS isolates were susceptible to linezolid. This finding was in agreement with other studies conducted in Iran [31,32]. In contrast, a study performed in the USA revealed that resistance to linezolid among CoNS isolates ranged from 1% to 2% [33]. This result may be because linezolid is not prescribed for the treatment of CoNS infections in Iran.

Source
Type PFGE Ward Hospital SCCmec Bioflm icaA/D MDR  City   A1  J18  J19  J16  J23  J22  J20  J21  J15  J17  J14  J34  J11  J12  J8  J10  J9  J24  J53  J3  J4  J5  J6  J7  J1  J2  J63  J41  J42  J28  J43  J39  J40  J52  J61  J58  J59  J54  J55  J56  J57  J35  J37  J36  J32  J33  J38  J44  J45  J46  J47  J51   T  A1  T  A2  T  A3  T  ST1  T  B1  T  B2  T  C1  T  C2  T  D1  T  D2  T  E1  T  E2  T  E3  T  F1  T  F2  T  ST2  T  ST3  T  G1  T  G1  T  G2  T  H1  T  H2  T  I1  T  I2  T  I3  T  J1  T  J2  T  ST4  T  ST5  T  K1  T  K2  T  ST6  SH  ST7  SH  L1  T  L2  T  L3  T  L3  T  L4  T  ST8  SH  ST9  SH  ST10  SH  ST11  SH  M1  SH   HP  HCW  HP  HP  HP  HP  HP  HP  HP  HP  HP  HP  HP  HP  HCW  HCW  HP  HP  HCW  HCW  HCW  HP  HP  HCW  HCW  HCW  HP  HP  HP  HP  HCW  HP  HCW  HCW  HP  HP  HP  HP  HP  HCW  HCW  HP  HCW  HP  HP  M2   NICU  NICU  NICU  NICU  NICU  NICU  NICU  NICU  INF  NICU  NICU  NICU  INF  NICU  INF  ICU  DIA  ICU  ICU  ICU  ICU  ICU  INF  ICU  INF  ICU  ICU  INF  ICU  ICU  INF  ICU  INF  INF  ICU  ICU  ICU  ICU  DIA  ICU  INF  ICU  ICU  ICU  ICU   P  P  P  P  P  P  P  P  P H  H  H  H  H  H   NT  I  NT  NT  NT  I  I+III  I+III  I+III  I+III  I  II  I+III  I  NT  NT  III  I  I  I  IV  III+V  NT  I  II  I+III  NT  IV  III  III+V  I  I  IV  IV  NT  NT  I  NT  I  IV  IV  IV  IV  IV  I   YES  YES  YES  YES  YES  YES  YES  YES  NO

BioMed Research International
Biofilm formation capacity is known as an important feature used by CoNS isolates for persistence and survival in the host body and hospital settings [4]. Polysaccharide intercellular adhesin (PIA) is encoded by ica genes and is the main factor in biofilm formation among CoNS isolates [34]. Our findings revealed that 62.2% and 57.1% of CoNS isolated from HPs and HCWs were positive for icaA or icaD genes, respectively. In general, the high prevalence rates of icaA and icaD genes in CoNS isolated from HPs is a serious alarm and suggest that most CoNS isolates recovered from these patients are virulent. In the present study, results obtained from PCR assay revealed that the frequency of the icaA and icaD genes was more in MRSH compared to MRSE (78.8% versus 55.8%). In line with our study, several studies have reported that S. epidermidis and S. haemolyticus are potential biofilm producers [35,36]. Also, it was shown that biofilm formation was associated with the presence of icaA and icaD genes. In contrast, the results of a study showed that biofilm formation in S. haemolyticus is independent of the ica genes [37]. However, Pinheiro et al. have reported that S. haemolyticus isolates with biofilm-forming ability harbor ica genes [28]. Other genes such as aap, bap, and bhp may be involved in ica-independent biofilm formation [38].
In the present study, the SCCmec types I (34.8%) and IV (19.7%) had the highest frequency among all MRSE and MRSH isolates, respectively. Machado et al. from Brazil reported that SCCmec type I was the most prevalent SCCmec type among S. haemolyticus. In contrast, they showed that SCCmec type III was frequently detected among S. epidermidis isolates. [39]. In a study performed by Zong et al. in China, the SCCmec type III was the most prevalent SCCmec type among methicillin-resistant CoNS isolates [40]. Moreover, SCCmec type IV has been reported as the most frequent SCCmec type among CoNS isolates in Mexico [27] and France [41].
It is revealed that several factors such as differences in geographical locations and host species can be affected the distribution of various types of SCCmec in methicillinresistant CoNS isolates. The use of an appropriate method can be helpful in molecular epidemiology studies. SCCmec typing method is not an accurate and reliable procedure for the epidemiological study of CoNS isolates [4], therefore, we used from PFGE method to survey the genetic relatedness between the S. epidermidis and S. haemolyticus isolated from HPs and HCWs.
PFGE is the most appropriate method for CoNS epidemiology and surveillance analysis [25]. In the present study, PFGE showed high genetic diversity in the CoNS isolated from HPs. High genetic diversity among CoNS isolates revealed that the source of most isolates may be the patients themselves; however, the incidence of cross infections between the patients is also possible. The presence of CoNS isolates with similar PFGE types among HPs and HCWs suggested that HCWs can be a reservoir for transferring CoNS to HPs and vice versa.
In the present study, most of the methicillin-resistant CoNS isolates were isolated from ICU staff and patients. Some of these isolates showed identical patterns which confirmed person-to-person transmission within the ICU ward. However, the isolates from other wards showed a high heterogeneity, which may be due to the transfer of personnel and/or patients among different wards of the hospital. In general, CoNS isolates with similar PFGE types can be grouped into different SCCmec types [42,43]. The diversity of SCCmec among PFGE types may result from the high rate of SCCmec acquisition and frequent insertion/excision of SCCmec in the CoNS chromosome [44]. Transmission of CoNS isolates from hospital settings may increase the risk of colonization and disease in patients. As a result, HPs are at risk for secondary infections caused by CoNS such as bacteremia associated with vascular catheters, cellulitis, and skin ulcers.

Conclusion
In conclusion, the high prevalence rate of antibiotic resistance and biofilm production ability was observed in S. epidermidis and S. haemolyticus isolated from HPs and HCWs.  HP  A1  INF  H  II  NO  --SH  HP  A2  INF  H  II  YES  +  +  SH  HCW ST1  ICU  S  I  YES  +  +  T  HP  ST2  ICU  R  II  YES  --T  HP  ST3  INF  P  NT  YES  --T  HCW ST4 NICU  P  I  NO  +  +  T  HCW ST5  INF  P  I  YES  +  +  T  HP  B1  ICU  R  NT  YES  +  +  T  HP  B2  ICU  R  I  YES  +  +  T  HP  C1  ICU  S  I+III  YES  +  +  T  HP  C1  ICU  S  NT  YES  +  +  T  HCW  C2  ICU  S  I

Data Availability
All relevant data are within the manuscript.

Disclosure
This is a report of a database from the Ph.D. thesis of the first author registered at the Tabriz University of Medical Sciences

Conflicts of Interest
The authors confirm that they do not have any conflicts of interest.

Authors' Contributions
Mohammad Ali Noshak, Mohammad Ahangarzadeh Rezaee, and Alka Hasani managed the study concept and design. Mohammad Ali Noshak, Maryam Ebrahimi, and Taher Azimi were responsible for the acquisition of data and sampling. Mohammad Ali Noshak, Alka Hasani, and Alireza Dolatyar worked on the analysis and interpretation of data. Mohammad Ali Noshak and Mohammad Ahangarzadeh Rezaee were assigned on the original draft preparation. Alka Hasani, Mehdi Mirzaii, Mohammad Yousef Memar, and Taher Azimi contributed on the review and editing. Mohammad Ahangarzadeh Rezaee was assigned on the study supervision.