Methicillin-Resistant and Biofilm-Producing Staphylococcus aureus in Nasal Carriage among Health Care Workers and Medical Students

Antimicrobial resistance (AMR) is a global threat. It has been portrayed as a slow tsunami. Multidrug resistance and extensive drug resistance exacerbate the already-existing AMR problem. The aim of the study was to access the colonization of methicillin-resistant and biofilm-producing Staphylococcus aureus among healthcare workers (HCWs) and medical students (MSs). A cross-sectional study was designed. A total of 352 participants (176 were HCWs and 176 were MSs) were enrolled from different hospitals and medical colleges in Kathmandu, Nepal. Nasal cavity swab samples were collected and inoculated on Mannitol salt agar at standard in-vitro environmental conditions. Isolates were identified based on colony characteristics, staining properties, and biochemical tests. Identified isolates were tested for antibiotic susceptibility and biofilm production. Out of 352 participants, 65.3% were S. aureus carriers; among the carriers, 52.2% were HCWs and 47.8% were MSs. Of the total isolates, 47.4% isolates were methicillin-resistant S. aureus (MRSA) and 73.9% isolates were multidrug-resistant (MDR). Among MDR isolates, out of 109 MRSA isolates, 86.2% were MDR and out of 121 MSSA isolates, 62.8% were MDR where isolates were mainly resistant to erythromycin. In addition, 68.7% isolates were biofilm-forming; the results were similar in both MRSA and MSSA. Variables such as profession and educational level showed statistical significance (p < 0.05) with MRSA, MSSA, and biofilm producers. In conclusion, asymptomatic colonization of healthcare workers by drug-resistant S. aureus is increasing at alarming rates. This reflects the lack of proper hygiene practice as well as improper disinfection of workplace of study population.


Introduction
Staphylococcus aureus is a microbiota of human body; however, it can cause mild to fatal infections. Globally, around 30.0% and 50.0% of people carry S. aureus in their nasal cavities permanently [1,2]. In the early 1960s, methicillin-resistant S. aureus (MRSA) was frst reported in England [3], which is now ubiquitous all around the world. MRSA is a group of Gram-positive bacteria that genetically difer from other strains of S. aureus that cause an infection which does not respond to common antibiotics, including methicillin, amoxicillin, and penicillin [4]. Methicillin resistance is mediated by acquiring mecA gene on S. aureus chromosomal DNA which confers resistance to β-lactam antibiotics. Worldwide, the prevalence of MRSA varies from predispose to colonization of bacteria, even though majority of hosts remain asymptomatic carriers. Hospital environment, gowns, patients' care items, etc., contribute prominently to the pathogen transmission in healthcare setting. A study from England concluded that 58.0% healthcare workers (Healthcare worker: According to Te Public Health Service Act 2018, a healthcare worker is defned as anyone who registered in the concerned council as a healthcare worker under the prevailing law of Nepal) (HCWs) detected S. aureus carrier in nasal cavity [15]. In a hospital setting, HCWs and medical students (Medical student: a medical student is a person who is enrolled in an educational institution to pursue a medical education degree). MSs act as an asymptomatic carrier of the pathogen and transmit infection in community and hospital settings. Regular screening of methicillin-resistant and bioflmproducing S. aureus in HCWs and MSs could minimize the potential outbreak of the pathogen. Hitherto, in case of Nepal, limited studies have been conducted to determine the prevalence of methicillin-resistant and bioflm-producing S. aureus among HCWs and MSs. Tus, this study aimed to investigate the asymptomatic colonization of methicillinresistant and bioflm-producing S. aureus among HCWs and MSs in Kathmandu, Nepal.

Study Design, Study Area, and Sampling.
A crosssectional study was conducted between January, 2022, and June, 2022, in Kathmandu. Samples were recruited by a stratifed-random sampling technique. A total of 352 volunteer healthcare personnel (176 HCWs and 176 MSs) were enrolled. Te sample size was calculated using the formula n � Z2α/2pqN/e2 (N − 1) + Z2α/2pq, where α/2 � level of signifcance at 95% confdence interval (or 5% level of signifcance) and Zα/2 � 1.96. p � proportion/prevalence of S. aureus was 35.3% [16], thus p � 0.5, q � 0.5, N � total population size of the study area, and e � maximum allowance error which is assumed at 0.05. Nasal swab samples were collected from participants and a face-to-face interview was performed for sociodemographic characterization. Samples were processed and examined in the Department of Medical Laboratory Technology, Janamaitri Foundation Institute of Health Sciences, Lalitpur, Nepal.

Bacterial Isolation and Characterization.
Swab samples were inoculated onto Mannitol salt agar and Nutrient Agar incubated at 37°C for 24 hours. S. aureus colonies were identifed based on their Gram's staining properties, colony characteristics, and biochemical tests (catalase, coagulase, oxidase, and deoxyribonuclease tests).

Screening of Bioflm
Production. Te tissue culture plate method was employed for detection of bioflm-producing S. aureus and a quantitative test was performed as published elsewhere [14,18].

Statistical
Analysis. Data were entered and analyzed in Statistical Package for the Social Sciences (IBM Corp., USA) version 21. Chi-square test was performed for categorical data and a p value less than 0.05 was considered signifcant.

Sociodemographic Characteristics, Prevalence of S. aureus, and Association with MRSA and MSSA. An equal number of
HCWs and MSs (176 in each) were engaged, where females (82.1%) were higher than the male (17.9%) participants and mean age was 27.6 years. Te detailed demographic distributions and clinical traits are presented in Table 1. Out of 352 participants, 230 (65.3%) were positive for S. aureus carriage. In HCWs and MSs, S. aureus was isolated in 120 (52.2%) and 110 (47.8%) participants, respectively. Te rate of nasal carriage was higher in female (83.0%) than the male (16.9%) participants. Out of 230 bacterial isolates, 109 (47.4%) were found to be MRSA while MSSA were 121 (52.6%). Furthermore, 38 (34.9%) MRSA and 38 (31.4%) MSSA isolates exhibited strong bioflm-forming potency whereas 17 (15.6%) of MRSA and 20 (16.5%) of MSSA showed a moderate level of bioflm production. All bioflm producers MRSA and MSSA were highly resistant to erythromycin whilst strong bioflm-forming MRSA was also equally resistant to amoxicillin. However, there was no signifcant relation with the level of bioflm producers MRSA and MSSA with antibiotic resistance.

Multidrug Resistance Pattern.
Multidrug resistance (MDR) is defned as acquired nonsusceptibility to at least one agent in three or more antimicrobial categories [19]. A total of 230 S. aureus were isolated in which 170 (73.9%) isolates were MDR. Out of which, the maximum number of bacteria was resistant to two and three classes of antibiotics (n � 65 in both cases) while resistance to four drugs was seen in 24 isolates, resistance to fve was seen in 14 isolates, and resistance to six was seen in 2 isolates. Likewise, among 121 MSSA isolated, 76 (62.8%) were MDR where 26 showed resistance to three diferent classes of antibiotics, 6 showed resistance to four classes of antibiotics, and 4 showed resistance to fve drugs in varying patterns. Additionally, it was found that out of 109 MRSA, 94 (86.2%) were MDR of which 39 isolates displayed resistance to three classes of antibiotics, 18 displayed resistance to four classes of antibiotics, and 10 displayed resistance to fve classes drugs and 2 to six classes.

Antibiotic Resistance Pattern in MRSA and MSSA.
Most of the isolates were resistant to erythromycin in both MRSA (82.5%) and MSSA (68.6%). Meanwhile, secondhighest resistance was seen for amoxicillin in MRSA (73.4%) isolates and cotrimoxazole in MSSA (45.5%) as shown in Table 2. However, the correlation was statistically insignifcant (p value > 0.05).

Discussion
Te nasal carriage rate of S. aureus among the participants was 65.3%, which is higher than that reported in studies from elsewhere in Nepal; 14.7%-15.7% [20,21]. Similarly, the prevalence of MRSA (47.4%) was higher than that in studies published from diferent settings of Nepal; 2.3%-41.3% [16,[22][23][24][25]. A meta-analysis study, between 2008 and 2020 in Nepal, noted that prevalence of MRSA ranged from 14.6% to 70.6% [26]. However, other studies from Nepal and Kenya have found a higher MRSA prevalence rate of 57.0% [11] and 53.4% [27] than ours. In this study, the high prevalence of MRSA could be because participants were healthcare personnel. Also, studies have found that Methicillin-resistantCoagulase Negative Staphylococci (MR-CoNS) acts a potential reservoir of mecA gene that facilitates MRSA colonization in human via horizontal crosspropagation of resistant genes when co-colonized resulting high prepondance of MRSA in nasal mucosa [28,29]. In addition, sufcient practices related to infection control and preventive measures, extensive and excessive antibiotic usage, and low socioeconomic status increase the chance of MRSA carriage. In this study, 73.9% isolates were MDR and mainly resistant to erythromycin followed by amoxicillin and cotrimoxazole. Out of 109 MRSA isolates, 94 (86.2%) were MDR. A similar study has noted that all the MRSA isolates were MDR [23]. Furthermore, bioflm producer S. aureus is two folds higher in number than the nonbioflm producers. However, bioflm producers were nearly equal in numbers in both MRSA and MSSA. On the contrary, a report from Poland has found that MRSA isolates have a higher ability to produce bioflms than MSSA [30]. Bioflms provide a protective barrier against the host defense, disinfectants, and antibiotics. It has been noted that incidence of methicillin resistance was nearly two folds higher in bioflm producers than the nonproducers [20]. Several other human pathogenic bacteria such as S. epidermidis, Enterococcus faecalis, Viridans streptococci, Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa produce bioflms [31]. Bioflm-producing bacteria are accountable for increased pathogenicity, long hospital stay, treatment failure with current antibiotic drugs, economic crisis [32], and may even contribute cancer development [33]. In our study, bioflm-producingS. aureus was mainly resistance to erythromycin and amoxicillin. Other studies noted that S. aureus isolates were mostly resistant to penicillin and erythromycin [34].
HCWs and MSs frequently encounter potentially lethal microorganism from patients and hospital environments. Terefore, they may transmit the disease to another person in hospital and community. A study revealed that the prevalence of MRSA is higher mainly in doctors and nurses [16]. In our study, there was a signifcant relation to the education level and profession with MRSA colonization. A similar study evidenced that the education level was signifcantly associated with the colonization of MRSA [35]. A study from Ethiopia reported that nasal carriage of MRSA was signifcantly associated with exposure time in hospital, sharing of clothing and sports equipment, and nose picking habit [36]. However, no signifcant relation to risk factors such as gender, age, occupation, experience, nasal irritation, and history of upper respiratory tract infection with MRSA colonization was recorded from Nepal [16].
Colonization of methicillin-resistant and bioflmproducing S. aureus could cause fatal infection in immunosuppressive clinical conditions. Asymptomatic colonization of the bacteria in HCWs and MSs could transmit the infection to hospitalized patients. S. aureus easily spreads through touching with contaminated hands. Periodic screening, decolonization, and adequate awareness among health professionals about the nasal carriages could minimize the risk of spread. Screening is also important in area where there is lack of sophisticated healthcare system, poor hygiene practice, and inadequate awareness.
Some constraints of this study are that the targeted population were all health professional which might have caused selection bias for high prevalence of MRSA. Also, this study has not examined co-colonization of MR-CoNS and MRSA as some studies have suggested that MR-CoNS are the repository for mecA gene and enhances MRSA colonization [28,29]. Furthermore, the identifcation of MRSA and bioflm detection were performed using conventional methods due to the lack of molecular laboratory.

Conclusion
Prevalence of methicillin-resistant, bioflm-producing, and multidrug-resistant S. aureus high among HCWs and MSs. Both the methicillin-resistant and bioflm producers are resistant to multiple antibiotics than nonmethicillinresistant and nonbioflm producers. Hygiene among healthcare professionals should be highly emphasized in infection control and prevention strategies. Te high prevalence of MRSA underscores the necessity for an effective surveillance system in Nepal.

Data Availability
Te data used to support the fnding of this study are available from the corresponding author upon request to authors.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
Bhuvan Saud designed the study. Gita Khatri, Bhuvan Saud, and Neetu Amatya carried out data collection, laboratory work, and data analysis. Bhuvan Saud and Neetu Amatya prepared the manuscript and carried out statistical analysis. Govinda Paudel and Vikram Shrestha made critical comment on the manuscript. All the authors fnalized and approved the manuscript.