Prevalence of Methicillin-Resistant Staphylococcus aureus in Shrines

Methicillin-resistant Staphylococcus aureus (MRSA) infection in human beings and animals is concerning; it stands out as one of the leading agents causing nosocomial and community infections. Also, marginally increasing drug resistance in MRSA has limited therapeutic options. This study focuses on estimating the prevalence of MRSA in shrines, a place where human and animal interaction is frequent, sharing antibiotic-resistant bacteria, antibiotic-resistant genes, and diseases. A total of 120 environmental swabs were collected from targeted areas during the study period, March 2018 to May 2018. Staphylococcus aureus was identified by growth on mannitol salt agar (MSA), and MRSA by growth on mannitol salt agar containing 4 μg Oxacillin, Gram staining, and conventional biochemical test. Isolates of S. aureus were characterized by antibiotic susceptibility testing using the disc diffusion method. MRSA and methicillin-sensitive S. aureus (MSSA) proportion were 19% and 81%, respectively; a high rate of MRSA was observed in isolates from Thapathali (28.6%). MSSA isolates showed a high rate of resistance to erythromycin (64.7%). MRSA isolates were resistant to gentamicin (50%), cotrimoxazole (25%), erythromycin (50%), and ciprofloxacin (25%). The isolates were susceptible to linezolid (100%), clindamycin (100%), ciprofloxacin (75%), erythromycin (50%), tetracycline (100%), and cotrimoxazole (75%). Intermediate resistance was also found in gentamicin (50%). Of the 11 MSSA isolates that were erythromycin resistant and clindamycin sensitive, 6 (54.5%) showed the inducible clindamycin resistance (ICR) pattern and 2 MRSA isolates that were erythromycin resistant and clindamycin sensitive showed ICR pattern. Fifteen MSSA isolates were β-lactamase positive, whereas only two MRSA isolates showed β-lactamase production. There exists a minimal research work on infectious diseases that are shared between primates and animals. This study suggests the pervasiveness of MRSA/MSSA in the shrines, which may be a primary place for pathogen exchange between humans and primates.


Introduction
Staphylococcus aureus (S. aureus) has been continuously evolving and developing resistance to antibiotics since the medical use of penicillin began in 1942 [1]. Penicillin works by inhibiting penicillin-binding protein (PBP), which is crucial in cell wall synthesis of bacteria. e inhibition of PBP means the bacteria die from osmosis [2]. Bacteria soon began producing penicillinase enzyme, a specific type of ßlactamase, which hydrolyzes the antibiotic and makes it ineffective.
is production led to the introduction of semisynthetic penicillinase-resistant penicillin called methicillin. e working mechanism of methicillin is similar to that of penicillin with the difference of extra methoxy group that produces an enzyme that reduces the affinity for staphylococcal ß-lactamase [3]. Shortly, however, Staphylococcus aureus started exhibiting resistance to methicillin treatment.
ese resistant strains emerged in the United Kingdom [4] and became known as methicillin-resistant S. aureus (MRSA).
MRSA has become a prime nosocomial pathogen for patients in hospitals and nursing homes during the past ten years [5][6][7][8]. Moreover, community-acquired MRSA (CA-MRSA) infections are on the rise; the transmission of MRSA from the community beyond acute care hospital environments is receiving more attention [9][10][11]. Evidence suggests that contact with contaminated environmental surfaces is a significant transmission factor [12][13][14][15][16]. Outbreaks of CA-MRSA have occurred among individuals sharing close contact with others in schools, prisons, and locker rooms.
Still, other possible environmental reservoirs of MRSA have yet to be comprehensively explored [17,18]. Similarly, little is known about S. aureus or MRSA colonization and infection in nonhuman primates, which are essential research models for human disease [19][20][21][22].
Shrines can be considered a central location for the association of primates and people.
ere occurs a close contact between them as humans, both intentionally and unintentionally, feed the resident primates. Such close contact forms the basis for the transmission of infections, including MRSA. It is known that captured primates can acquire tuberculosis and MRSA from their human caregivers, but it is unclear in what conditions wild free-ranging primates both provide and receive pathogenic bacteria from human interaction [23]. Most of the studies done till today are confined in the hospital settings because of which other areas like CA-MRSA are in shadow. is may have been because of the immediate threat that hospital-acquired MRSA (HA-MRSA) brings to the population. Hence, this study can bring light to the condition of MRSA in shrines, which can be considered a perfect setting for the transfer of MRSA between humans and primates.

Materials.
e materials, equipment, and various reagents used in different stages of this study are listed in Appendix A.

Research Method.
e research method was quantitative, and primary data were collected from March 2018 to May 2018 from shrines inside Kathmandu valley. Samples were processed in the laboratory of Nepalese Farming Institute, Maitidevi, Kathmandu.

Study Variables.
e variables of the study were an occurrence of S. aureus, MRSA, different shrines, and antibiotic susceptibility profile.

Research Design.
e study was field-based and crosssectional.

Study Site and Its Justification.
e study was carried out in six shrines located at Kathmandu and Bhaktapur cities of Kathmandu valley.
No information is available about S. aureus/MRSA prevalence in shrines; hence, this study site has been selected for research.

Sample Size.
A total of 120 samples (environmental swabs) were collected from the shrine area.

Data Collection Techniques/Methods
(1) Specimen Collection and Transport. Using a sterile environmental swab (sponge swabs) moistened with buffered peptone broth, several surfaces around shrines frequently visited by humans and monkeys were gently swabbed. e collected swabs were kept in the vial, screw-capped, clearly labelled, and transported to the laboratory immediately to avoid contamination.
(2) Specimen Processing (a) Isolation of S. aureus e environment swabs were enriched in M-Staph broth and incubated for 48 hours in anaerobic conditions. e black precipitate obtained was directly inoculated in MSA agar for 24 hours. Mannitol fermenting colonies (yellow colonies) from MSA were subcultured on nutrient agar and incubated at 37°C for 24 hours. Golden yellow colonies on nutrient agar having round, convex, opaque, and smooth-glistening surface with a diameter of about 2-3 mm were indicative of S. aureus. Further phenotypic identification of the S. aureus was made by Gram staining, catalase test, oxidase test, coagulase test (slide and tube test), and oxidative/fermentative test ( Figure 1).
(b) Detection of MRSA e MRSA isolates were identified by growth on mannitol salt Agar (MSA) containing 4 μg/ml oxacillin (CLSI 2014). Four oxacillin-resistant S. aureus isolates were tested for the mecA gene-a molecular marker of methicillin resistance in S. aureus by modified Kirby-Bauer disc diffusion method using cefoxitin ( After incubation, the flattening of the clindamycin zone of inhibition adjacent to the erythromycin disc (referred to as a D-zone) was indicative of inducible clindamycin resistance ( Figure 3). (e) Detection of β-lactamase β-Lactamase production was detected by the penicillin disc diffusion zone-edge test recommended by CLSI (2014). e turbidity of the inoculum was made equivalent to a 0.5 McFarland standard. e lawn culture of the test inoculums was prepared by swabbing MHA with a sterile cotton swab dipped into inoculums. e penicillin (10 μg) disc was used for the detection of β-lactamase production.

(f ) Detection of DNase
DNase test was performed to determine the ability of an organism to produce the DNase enzyme, presumptively to differentiate Staphylococcus aureus that produces the enzyme deoxyribonuclease from other Staphylococci which do not produce deoxyribonuclease. e test organism was inoculated onto a small area of the DNase test agar plate, which was then incubated at 37°C for 24 hours. After incubation, the surface of agar was flooded with 1 N HCL solution ( Figure 4).

Occurrence of MRSA/MSSA in the Environmental
Samples. Out of 120 environmental samples collected from 6 different shrines located in the Kathmandu valley, a total of 21 S. aureus were isolated (17.5%); 4 isolates exhibited methicillin resistance (19%) (MRSA), and 17 isolates were methicillin susceptible (81%), as shown in Figure 5.

Distribution of MRSA among Different Sites.
Out of 21 S. aureus isolated, 4 were MRSA. e highest number of MRSA was isolated from apathali (2). One isolate was isolated from Pashupati and Nilbarahi. No MRSA was detected in Swayambhu, Bajrayogini, and Guheshwori (see Table 2).

Inducible Clindamycin Resistance in MSSA and MRSA.
Of the total 11 MSSA isolates that were erythromycin resistant and clindamycin sensitive, 6 (54.5%) showed the inducible clindamycin resistance pattern and 5 (45.5%) showed negatively inducible clindamycin resistance, whereas out of 2 MRSA that met the criteria, both were D-test positive (see Figure 6).

Discussion
Diversified MRSA epidemiology is considered a significant health concern in clinical and community settings. Studies conducted to date have reported a high prevalence of MRSA colonization in hospital settings than community environments; however, no clear distinction criteria are present for the detection of MRSA origin. Furthermore, relatively little is known about the dynamics of S. aureus or MRSA colonization in nonhuman primates, which are essential research models for human disease [19,21,22,24].
Shrines, for the most part, are rife with primates. As people place offerings on a votive altar, they come in contact with the surface previously touched by primates or the primates in the hope of finding food, and roam in the vicinity making each susceptible to the transmission of infection. We characterized MRSA in environmental samples from such areas to predict the distribution of MRSA. is study has generated data to evaluate the condition of MRSA in shrines. A total of 120 environmental samples were collected from 6 shrine areas of Kathmandu.     [25]; a slightly high MRSA rate was observed in our study. e variance in the rate of MRSA in these studies might have been due to the difference in sample numbers and the area from which samples were obtained.
A relatively high number of MRSA was isolated from apathali (n � 2; 28.6%), whereas 3 shrines showed no occurrence of MRSA. is could be due to the close contact primates in apathali shared with the residents. e MRSA carriage depends on frequent contact with personnel's inanimate objects in the environment.
ere is limited research on whether primates receive pathogenic bacteria from human interaction or whether human beings can become the host for organisms transferred from these primates at shrines and parks. is inadequacy hampers our ability to compare the rates of transfer of bacteria.
MRSA showed high resistance to erythromycin and gentamicin (50%). Resistance towards ciprofloxacin and cotrimoxazole was moderate (25%). Correspondingly low resistance pattern of gentamicin (27.7%) was reported in CA-MRSA isolated from shrine areas in a study conducted by Al-Mohana et al., but the same study showed a high resistance pattern of erythromycin (72.7%) and ciprofloxacin (45.4%) [26]. Drugs that are highly preferred may result in resistance and are mediated by the acquisition of the genes that confer resistance to such antibiotics. A similar suggestion was given by Jaimes et al. that the development of antimicrobial resistance is nearly always as a repeated therapeutic or indiscriminate use of them [27]. e intermediate resistance was found against gentamicin (50%). Aminoglycoside antibiotics such as kanamycin, gentamicin, and streptomycin were effective against staphylococcal infections until strains evolved mechanisms to inhibit the aminoglycosides action-inhibition of the initiation complex formation and misreading of the mRNA. e MRSA strains were susceptible to linezolid (100%), erythromycin (50%), ciprofloxacin (75%), clindamycin (100%), cotrimoxazole (75%), and tetracycline (100%). Routine antimicrobial sensitivity of MRSA done by Mahmood et al. showed 28.7% to ciprofloxacin, 37.5% to gentamicin, 35% to clindamycin, 27.5% to erythromycin, 18% to fusidic acid, 8% to penicillin, 87% to moxifloxacin, 0% to oxacillin, 100% to vancomycin, teicoplanin, linezolid, and tigecycline [28]. Relatively high sensitivity towards erythromycin, clindamycin, and ciprofloxacin was seen in our study.
e full susceptibility of MRSA towards linezolid, clindamycin, and tetracycline indicates the narrow use of them in the MRSA chemotherapy. However, several reports show MRSA resistant to some of the listed drugs. In a study carried out by Belbase et al., few strains were resistant to tetracycline and clindamycin [29]. MRSA can be eradicated with a prescribed dose of linezolid [30], although treatment protocols differ, and serum levels of antibiotics generally vary from person to person and may affect the outcomes [31].
Macrolide-Lincosamide-Streptogramin B (MLS B ) has been used to treat skin and soft tissue infection caused by S. aureus, more often clindamycin, because of its good   International Journal of Microbiology pharmacokinetic properties leading to the evolution of resistance in many Staphylococci [32]. In the study, out of 11 erythromycin-resistant and clindamycin-sensitive MSSA isolates, 6 (54.5%) showed an inducible clindamycin resistance pattern. Two MRSA that met the criteria were D-test positive. Clindamycin preferably does MRSA infection treatment, but because of the high rate of inducible clindamycin resistance in MRSA and MSSA strains, there exists a high chance of failure showing this type of resistance. Such tests minimize the risk of failure of clindamycin therapy [29]. Two (50%) of the total 4 MRSA isolates and fifteen (88.2%) MSSA isolates out of 17 were β-lactamase producers. β-lactamase test was done to predict the outcome of susceptibility tests with β-lactam antimicrobials. e β-lactamase produced hydrolyzes the β-lactam ring, hence allowing it to achieve resistance to β-lactam drugs [33].
Our study indicated the prevalence of MRSA in the shrines. However, this study was focused on limited areas of Kathmandu valley only, rendering it unsuitable for making an assumption about the direction of transmission of MRSA between primates and humans and estimating accurate MRSA and MSSA persistence in the environment (Tables 5-12).

Conclusions
MRSA and MSSA carriage rates were 19% and 81%, respectively. All MSSA and MRSA isolates were susceptible to linezolid, tetracycline, and clindamycin; however, high resistance to erythromycin followed by ciprofloxacin in MSSA isolates, and erythromycin and gentamicin in MRSA isolates were observed in the study.
ough the results obtained are concerning and suggest an extensive application of antibiotics, it might not be best to use the results as a sole indicator of antibiotic overuse. Moreover, the use of molecular techniques such as polymerase chain reaction, real-time polymerase chain reaction, or nucleic acid sequence-based amplification in the detection of Staphylococcus aureus strains would have exhibited a better result. Further evaluation is essential to understand the extent of MRSA, including shrines from outside of Kathmandu valley.

Appendix
List of materials and preparation of culture media are as follows: (d) acetone alcohol (1 : 1) 50 ml of ethanol (95%) with 50 ml acetone was mixed in a brown bottle. e date was labelled, and the solution was stored at room temperature.
Biochemical test reagent is as follows: (1) Turbidity standard equivalent to McFarland 0.5 6 International Journal of Microbiology (i) 0.6 ml of 1% barium chloride dehydrate (BaCl 2 ·H 4 O 2 ) was mixed with 99.4 ml of 1% sulphuric acid. (ii) en, the mixture was mixed completely to form a turbid suspension. (iii) A small volume of the turbid solution was transferred into screw-capped tubes of the same size as used for preparing test and control inoculums. (iv) e tubes were then stored in the dark and vigorously agitated in a vortex mixture before use.
e standard, when stored in a well-sealed container in the dark at room temperature (20-28°C), may be kept for up to 6 months.    To 100 ml distilled water, 3 ml of hydrogen peroxide is mixed with 100 ml of distilled water to prepare catalase reagent.     (ii) Tube coagulase (free coagulase) test procedure (1) ree test tubes were taken and labelled as T (test), P (positive), and N (negative). (2) About 0.2 ml of fresh plasma was pipetted in each test tube. (3) About 0.8 ml of test broth culture was added to the tube labelled as "T," 0.8 ml of a standard culture of S. aureus was added to the one tube labelled as "P," and 0.8 ml of sterile broth was added to another tube labelled as "N." (4) After mixing gently, all the tubes were incubated at 37°C and the clot formation was examined at an interval of one hour for up to 4 hours, by tilting the tubes. (5) e negative tubes were kept at room temperature overnight and were reexamined. Oxidative and fermentative test (1) Two tubes of oxidative and fermentative test medium were prepared. (2) e colony of the test organism was transferred using straight wire by stabbing halfway to the bottom of the tubes. (3) One tube of each pair was covered with a 1-cm layer of sterile mineral oil or liquid paraffin. Another tube was left open without any covering. (4) Both tubes were incubated at 35 for 48 hours (slowgrowing bacteria may take 3 to 4 days before results can be observed).
Acid production was detected in the medium by the appearance of a yellow color. S. aureus was detected by acid production on both (open and covered) tubes. e acid produced changed the pH indicator, bromothymol blue, from green to yellow.

DNase test
(1) With the help of a sterile loop, colonies from 18 to 24 hours were picked and inoculated onto a small area of the DNase test agar plate. S. aureus ATCC was used as positive control and E. coli ATCC 25922 as a negative control. (2) e plate was incubated at 37°C for 18-24 hours.
(3) After incubation, the surface of agar was flooded with 1 N HCL solution, and excess acid was removed. (4) e clear zone around the colonies was observed within five minutes, which indicated the positive test. Zone diameter/MIC interpretive criteria for S. aureus in Table 13.

Data Availability
No data were used to support this research work. 8 International Journal of Microbiology

Conflicts of Interest
e authors declare that they have no conflicts of interest.