Sanjib effective antibiotics whereas cefoxitin 42(37.5%) was

SanjibAdhikari1, Sujan Khadka1,2, Pabitra Shrestha1,and Sanjeep Sapkota11Department ofMicrobiology, Birendra Multiple Campus, Tribhuvan University, Chitwan, Nepal2Department ofLife Sciences, Biochemistry and Molecular Biology, Central China NormalUniversity, Wuhan, Hubei, P.R. China*Corresponding Author: Sujan Khadka,E-mail: [email protected] AbstractMobile phones widely used in ourday-to-day lives are potential reservoir for a number of bacteria including Staphylococcus aureus. Thestudy was carried out from February to May 2017 with a major objective toisolate methicillin resistant S.

aureus (MRSA) from mobile phones used by the staffs andstudents of Birendra Multiple Campus. Two hundred swabbed samples of mobilephones were collected from which 112 (56%) S.aureus were isolated by standard microbiological technique.

Gentamicin 101(90.2%)was the most effective antibiotics whereas cefoxitin 42(37.5%) was the leasteffective antibiotics. MRSA30(26.8%), VISA 58(51.

8%) and MDR- S.aureus 24(21.4%)were detected. Significantassociations were noted between the rate of occurrence of MRSA and MDRS. aureus with various attributessuch as the gender of the user, the way of handling the mobile phones and alsowith the length of use of mobile phones (P-value <0.01). Our findingsindicate the mobile phones can carry potentially threatening species of S.

aureus whichcan cause severe health hazards to humans. Awareness about regulardisinfection of mobile phones, hand hygiene, restricting the use of mobilephonesin contaminating areas and proper place for storing mobile phones canbe suggested. Keywords: Mobile phones,MRSA,VISA                                                                                                   IntroductionTheusers of mobile phones are increasing day by day with 4.61 billion users in2017 and is anticipated to become 5.07 billion in the year 2019 https://www.statista.com/statistics/274774/forecast-of-mobile-phone-users-worldwide/. Staphylococcus aureus is the commensalflora of several animals and humans 1.

The fact that mobile phone could createa key health hazard has been revealed by several researches. Because of thedual effect of constant handling and the heat generated by the phones, mobilephones provide a major breeding ground for all sorts of microorganisms that arenormally found on the human skin 2.The surface area of an adult human skin isabout 2m2 harboring about 1012 bacterial cells/person 3.The mobiles phone comes into contact withcontaminated human body parts with hands to hands, and hands to other partslike mouth, nose and ears, during the phone call 4.

Mobile phones may harbordifferent pathogenic bacteria because they are commonly handled regardless ofthe sanitation of hands and hardly disinfected 5. The use of suchmobile phone serves as a potential vehicle for the spread of nosocomialpathogens including multidrug-resistant pathogens such as MRSA 6.In recent years, community-acquired MRSA(CA-MRSA) strains, the rapidly becoming dominant pathogens in the community,have emerged 3.Vancomycin is the antibiotic for the treatmentof MRSA cases but several reports have shown that MRSA intermediate andresistant patterns against vancomycin and treatment of MRSA cases withvancomycin is extremely problematic 7,8. Presence of suchpathogenic bacteria like MRSA, VISA and MDR S.aureus in mobile phones can indicate the immediate medical attention toabate this issue. Althoughthe contamination of mobile phones of health workers has been studied, little informationregarding the contamination of personal mobile phones of people in thecommunity exists.

Bacterial flora on cell phones may vary in composition, numberand antibiotic sensitivity from person to person. This is probably the firststudy in Nepal that attempts to study the bacterial florapresenton the mobile phones incommunity samples and their antimicrobial susceptibility patterns.Inthis study, we have identified MRSA, VISA and MDR Staphylococcus aureus from mobile phones used bythe students and thestaffs and also have drawn a significant association between various attributesof users and mobile phones with the isolation rate of S. aureus. Materialsand methodsThis cross-sectional study was carriedout at the Microbiology laboratory of Birendra Multiple Campus,Bharatpur, Chitwan, Nepal from February to May, 2017.

 Sample Size: Atotal of 200 mobile phone swabs were collected amongst the staffs and studentsof Birendra Mutiple Campus.Culture of Specimen: Screens and keypads of mobile phones wereswabbed with the sterile cotton swabs soaked in normal saline and immersed in peptonewater and left for incubation at 37°Cfor 24 hours. On the following day, a loopful of the growth was streaked on MSA and again incubated for 24 hours at 37°C. Yellow colonies on MSA showing violet color with grapes like clusterson Gram’s staining were sub-cultured on NAand BA 9.Further, hemolysis was observed on blood agar for the identification of S. aureus.

Identification of Isolates: Those colonies from NAgiving positive Gram’s reaction were further tested for oxidase, catalase,coagulase, DNase and oxidative/fermentative tests forthe confirmation of S. aureus 9.AntibioticSusceptibility Test: Antibiotic Susceptibilitytests were performed by the disc diffusion method recommended by Clinical andLaboratory Standards Institute using Muller Hinton Agar 10. Firstly, MHAplates were swabbed by a bacterial suspension using sterile normal salinecomparable to 0.5 McFarland turbidity standards. Using sterile tweezers, antimicrobial discstetracycline (30 mcg), gentamicin (10 mcg), cloxacillin (5 mcg),amikacin (30 mcg), erythromycin (10 mcg), vancomycin (10 mcg), cefoxitin (30 mcg),cotrimoxazole (15 mcg),ciprofloxacin (5 mcg) and ceftriazone (30 mcg) were placed widely spacedaseptically on the surface of MHA plate. Tweezers were re-flamed afterapplication of each disc. The plates were then incubated at 37°C for 24 hours.

Following incubation, the diameter of inhibition zone weremeasured with a transparent ruler and expressed in millimeters (mm).  AST was performed for all the bacterialisolates. Multidrug resistance wasdefined as resistance to three or more of the antimicrobial agents belonging todifferent structural classes 11. For identification andstandardization of the Kirby-Bauer test, standard culture of S.

aureus ATCC25923 was used as a reference strain.Statistical analysis: Datawere tabulated and analyzed by using SPSS version 20. P-values less than 0.01 wereconsidered to have significant association.Ethical Considerations:This study was approved by the Department of Microbiology, Birendra MultipleCampus. A questionnaire was used to collect personal as well as behavioraldata. The questionnaire was arranged based on previous studies and according tothe authors own insights of probable factors that could contribute to thecontamination of mobile phones.

Informed consent was taken from the staffs andstudents of Birendra Multiple Campus prior to collecting samples and fillingthe structured questionnaire.  ResultsOut of  200 mobile phones swabbed, bacterial growth was foundon 112 (56%) samples. Of the 10 different antibiotics used, gentamicin101(90.2%) was found to be the most effective followed by amikacin 95(84.8%).

Incontrast, cefoxitin 42(37.5%) was the least active antibiotics. Thirty (26.8%) bacterialisolates were detected as methicillin resistant S. aureus (MRSA) whereas 58(51.

8%) bacterialisolates were found to be vancomycin intermediate S. aureus (VISA) and 24(21.4%) isolates were multiple drug resistant (MDR) (Table 1).Signification association was notedbetween the occurrence of bacterial isolates and various attributes. Mobilephones used in toilets and used for more than 2 years were found to be heavilycontaminated by S. aureus, MRSA andMDR isolates (P-value<0.01) whereas practice of disinfecting the mobilephones reduced their rates of isolation (P-value<0.

01).(Table 2).The number of MRSA isolates was quitehigher in the mobile phones carried in pockets than those carried inmobile-bags (P-value <0.01) (Table 3).A significant association was observed between the presence of MDR isolates andthe gender of the users. A large number of MDR S. aureus 16(66.

7%) was detected from the mobile phones used by females comparedto thoseused by males 8(33.3%) (P-value<0.01) (Table 4).DiscussionIn the present study, 56.

0% of themobile phones were found to harbor S.aureus. This finding is 16.

5% lower than the growth rate of S. aureus (MSSA) on mobile phones usedby non-health workers and almost 25.0% lower than S. aureus (Both MSSA and MRSA) on the mobile phones used by healthcare workers as reported by Chawla et al 12. Similaly, in our finding the rate of contamination of mobile phones by S.

aureus was found to be higher thanthe figures reached by studies on S.aureus contamination of mobile phones of health workers conducted inPalestine (27.0%) by Elmanama et al 13; in Turkey (52.

0%) by Ulger et al14; in Ethiopia (21.0%) by Gashaw et al 15 and in Nigeria (50.0%) by Ilusanya et al 16. Unlike the work byChawla et al 12 who reported none of the bacterial isolates from mobile phonesused by health-workers in India developed drug resistance, our findingsidentified 30(26.

8%) of the bacterial isolates were  methicillin resistant and 24(21.4%) weremultiple drug resistant.Elkholy et al reported 31.0% S.

aureus isolated from mobile phones weremethicillin resistant 4. A report by Heyba et al showed that MRSA was identified in 3(1.4 %) mobile phones amongwhich none was resistant to vancomycin 17.In our work, 26.8% of S. aureus were foundto be resistant against cefoxitin which is nearly similar to a study conductedby Kuhu Pal et al, who showed almost 21.0% of S. aureus isolated from mobile phones were resistant toCefoxitin 18.

None of the S. aureuswas noted to be resistant against vancomycin in Kuhu Pal’s work which isconsistent with our study. Similarly, Chawla et al also reported the presenceof 20.0% of MRSA on the mobilephones used by health care workers in teaching institution, Manipal, Karnataka,India. But no MRSA were detectedfrom non-health worker’s mobile phones in their study 12.

Similarly, inIndia, Bhat et al found 40.0% MRSAand 58.6% MSSA from mobile phones of medical personnel 19. In health caresettings, MRSA can cause terribleconsequences.

It can cause bloodstream infections, if not treated properly itcan also result sepsis and even deaths (https://www.cdc.gov/mrsa/healthcare/index.html). In another study, Kuhu Pal revealedthat conventional keypads phones (94.4%) were greatly contaminated thantouch-screen phones (67.

86%) 18. In contrast, in our study, 86.6% oftouch-screen phones and 13.4% of key-pad phones were found to be contaminated.

In our study, 37.0% users were foundto have never used any disinfectants their mobile phones. This figure is quitelesser than a research carried out by Sadat-Ali in Saudi Arabia who reportedthat 76.0% of the clinicians had never disinfected their mobile phones 20.Similarly, a work done at one of the hospitalsin Kuwait by Heyba et al pointedout that 66.5% of the participants had neverdisinfected their mobile phones 17. Microbialcontaminations are the risks related with the irregular cleaning of phones 21. Our study shows a significantassociation between the disinfection process and the rate of contamination by S.

aureus (P-value<0.01), MRSA (P-value <0.01) and MDR S.  aureus(P-value<0.

01).The rate of incidence of contaminationof mobile phones held by females in IUG (Islamic University of Gaza) (52.0%)was lower than that of male counterparts (79.0%) 13. Auhim’s findings in Iraqwere consistent with this, which showed that the rate of bacterialcontamination of personal mobile phones of males was 85.0%compared with 80.0% of females 22.

Similarly, inthe present study, we observed a higher rate of incidence of bacterialcontamination in the mobile phones carried by males (52.7%) than those carriedby the females (47.3%). We also noted a significantassociation between the isolation rate of MDR S. aureus and gender of the users (P-value <0.

01). Female users hadtheir mobile phones more contaminated with MDR S. aureus than the males. In a research work performed by SalhaH.M and Al-Zahrani in 2012, it was found that fingernails can also harbor MDR S.

aureus 23. Females havelonger nails which can directly transmit MDR S. aureus to the mobile phones while using.In a studypresented in a meeting of the Infectious Disease Society of America in SanFrancisco, researchers showed that artificial and natural nails longer than 3millimeters beyond the tip of the finger, or the length of a pencil tip, transportmore harmful bacteria and yeast under them as compared to the short nails (http://abcnews.go.com/Health/story?id=117161). Akinyemi et al concluded that S.

aureuswas the most encountered bacterial agent, probably becausethis type of bacteria proliferates in optimum temperatures, as phones are keptwarm in pockets, handbags and brief cases 24. Our study reveals that 83.3% MRSA were isolated fromthe mobile phones carried in the pants and shirts and only 5(16.7%) bacteriawere isolated from mobile phones carried in bags. A studyconducted by Kuhu Pal illustrated that a large number of users carry theirphones in clothes than in bags but in this study the rate of contamination wasfound to be higher in mobile phones stored in bags (95.4%) than thosecarried in clothes (84.6%) 19.

There was no significant association between the rate of isolation of MDR S. aureus and the storage of mobilephones (P-value>0.01) in our study.

Similarly, no significant associationwas noted in the study conducted by Kuhu Pal regarding the rate of isolation S. aureus and storage of mobile phones (P-value>0.1) 19.A study conducted by Zakaiet al revealed that 59.0% medical students used their mobile phones in thetoilets 25.This finding is comparatively higher than our study inwhich 40.2% of the respondents used their mobile phones in toilets.

. Mobilephones can act as fomites as they are contaminated by users from areas such astoilets, hospitals and kitchens, which are burdened with microorganisms 26. Further, Giannini et al reported thathospitals toilets are the source for MRSA 27.The MRSA are very difficult to kill andare significantly dangerous as stated by San Diego County Health and HumanServices Agency which also reported that MRSA is spread by person-to-personcontact and is frequently harbored in toilet bowls and dirty environment (https://healthyliving.

azcentral.com/bacteria-found-in-toilet-bowls12320100.html). Using mobile phones in such environments can be one of thereasons for presence of MRSA on the mobile phone surface. It is also revealed in our study that the mobilephones used in the toilets were tremendously contaminated with S aureus (P-value<0.

01) and also asignificant number of MRSA (P-value <0.01) and MDRS. aureus (P-value<0.01) were found in the mobile phones used intoilets.Furthermore,in our study, mobile phones used for more than 24 months were found to be highlycontaminated with MRSA, VISA and MDR S.aureus.

This may be due to the fact that older phones are more hospitableto S. aureus for proliferation.ConclusionOccurrenceof MRSA, VISA and MDR S.

aureus inthe mobile phones as observed in the present study can be a potential threat tohumans and medical fraternity as well. Therefore, increase in awareness todecontaminate the mobile phones by effective disinfectant would be useful. Properand judicious use of antibiotic should be recommended to prevent the emergenceof MDR bacteria. Using mobile phones in the contaminating areas like toiletsshould be discouraged. Good hygienic practices of users arenecessary in order to prevent cross-contamination.

 Author’scontribution: Sujan Khadka, SanjibAdhikariand Pabitra Shrestha conceived and designed thestudy, accomplished the laboratory works and analyzed the data. Sujan Khadka,Sanjib Adhikari and Santosh Khanal drafted the manuscript. Sanjib Adhikarimonitored the study. All the authors state that there is noconflict of interests. Acknowledgements: The authors would like to appreciate the studentsand staffs of Birendra Multiple campus for providing mobile phone samples and respondingenthusiastically to the questionnaire developed.