Antibiotic Susceptibility Patterns of ESβL Producers Isolated from the Mobile Phones.

Mobile phones have become an indispensable part of human lives for communication, education, and entertainment activities. This study aims to evaluate the diversity pattern of bacterial contaminants on mobiles and to check antibiotic resistance profiles in 105 samples. The study revealed a contamination of 51% in men and 49% in women, the highest in the 21- to 30-year age group, evidencing the extreme use of mobiles by teenagers. The study observed Gram-negative bacteria (63%) versus Gram-positive bacteria (37%). Overall, Gram-negative bacterial isolates showed the highest sensitivity to antibiotic nitrofurantoin (90%) and the lowest in ampicillin (35%). Gram positive has highest incidence of sensitivity towards tigecycline (100%) and lowest in cefoxitin (20%). ESβL producers were found to be 21.0% and highest being in Klebsiella oxytoca (35%) followed by Klebsiella pneumonia (31%). Staphylococcus pseudintermedius and Staphylococcus capitis have been identified on the mobile phones for the very first time. Interestingly, some soil microbes were also isolated and unfortunately found to have some antibiotic resistance like Raoultella ornithinolytica and Sphingomonas paucimobilis. The results revealed that mobiles were contaminated with multidrug-resistant (MDR) pathogens, and this study also showed that few of the saprophytic soil strains have antibiotic resistance, which can be an alarming situation that needs to be addressed.

1. Introduction

The mobile phone or smartphone have many attributes and characteristics that make it very attractive to both the young and the old. With the achievements in the field of technology, mobile phone, a portable electronic device for personal communication, has become an important part of one's life, and its benefits have made it user friendly. However, the disadvantage of overlooking the health hazard has been a concern because many users do not take care of personal hygiene while using these phones. The extensive usage of mobile devices, such as the touch screen, renders the highest possible transmission fomites of several pathogens across various age groups. Furthermore, different users handle them constantly exposing and acts as a good carrier to an array of microorganisms [1,2]. These mobile phones come in contact with many surfaces that are having germs during our daily activities. These germs gradually start to accumulate on our mobile phones over a period of time. It has been observed that with the constant handling of the mobile generates heat that acts a primary source for the growth of microbes that are normally found on the skin. Thus, sanitising the phone and hands play an important role in our health system. Disease causing bacteria are thus able to get transferred from person to person through the direct contact and fomites [3].

In the current situation, there has been an incessant use and handling of mobile phones, and these devices have also been exposed to a variety of different pathogenic bacteria [4]. These pathogens can survive on environmental surfaces, including mobiles and thus may perhaps act as a potential infectious source for humans. Mobiles tend to act as fomites for pathogenic microbes that are transmitted such as Staphylococcus aureus, Escherichia coli, and Pseudomonas spp [5]. It has been observed that these pathogens can be found to be multidrug resistant (MDR) [6]. These MDR microbes have now become a global issue that has led to an increase in the mortality and morbidity with an increased risk of treatment failure and health care costs [7].

Antibiotic resistance has an imperative issue that is related with both nosocomial and hospital infections and that has been drastically increasing. This presents an obstacle in treating the MDR pathogens that cause infections [8,9]. Many have reported various types of hospital-acquired infections and their role as pathogens causing severe infections [10-12]. However, to our knowledge, not much has been reported and very little has been documented on community-acquired pathogens on mobile phones. Hence, with respect to this, our aim is to survey the diversity of mobile phones, which can be varying with respect to the microbial diversity, occupation, habits, and the healthy lifestyle, to assess the antimicrobial resistance patterns of isolated microbes. In recent years, there is an increasing risk of bacterial contaminants, transmission, and antimicrobial resistance patterns due to mobile phones. This study showed that educating the society about the importance of hand hygiene and sanitising mobiles is necessary to reduce the bacterial contamination and limit the transmission of MDR strains.

2. Materials and Methods

2.1. Study Area, Size, and Data Collection

The study was conducted from January to April 2021, in the campus of Dayananda Sagar University, Bengaluru, India. During the study period, a total of 100 mobile samples were collected from the individual mobiles. Sociodemographic characteristics of the participants were taken into account with a self-administered questionnaire to collect the information such as age, sex, profession, use of mobile phones, and the habit of cleaning their mobile phones were considered.

2.2. Sample Collection

The samples were collected by using a sterile cotton swab that was dipped in the nutrient broth and rolled on the keys, screen, and back of the mobile phones.

2.3. Bacterial Isolation and Identification

Bacteria were isolated by inoculating swabs on MacConkey's agar, blood agar, and Mannitol salt agar (Hi-media, Mumbai, India). The inoculated plates were incubated at 37°C for 24 to 48 hours and observed for growth of the isolates. The primary bacterial identification was based on the morphological, cultural characteristics, and a series of biochemical tests. The test included Gram reaction, Indole test, MR and citrate test, Urease test, Mannitol motility, Triple sugar iron agar, Catalase, and Coagulase test [13].

2.4. Antimicrobial Susceptibility Test of Isolated Samples

The isolates were subjected for AST according to Clinical Laboratory Standards Institute [14] using Kirby–Bauer disk diffusion test on Muller–Hinton agar (MHA) (Hi-media, Mumbai, India). The isolates were inoculated in nutrient broth for 30 min and adjusted to the 0.5 McFarland standard. A sterile cotton swab was dipped in the bacterial culture and inoculated on the MHA plates, and the antibiotic disc was placed and incubated at 37 C for 24 hours. Results were interpreted by measuring zone of inhibition comparing with the standards.

Escherichia coli ATCC 25922 and S. aureus ATCC25923 were used as quality control strains. Following antibiotics were procured and used for the AST, which included amikacin (30 mcg), ampicillin (10 mcg), piperacillin/tazobactam (100/10 mcg), cefuroxime (30 mcg), cefuroxime Axetil (30 mcg), ceftriaxone (30 mcg), cefoperazone/sulbactam (75/30 mcg), cefepime (30 mcg), imipenem (10 mcg), meropenem (10 mcg), gentamicin (10 mcg), nalidixic acid (30 mcg), ciprofloxacin (5 mcg), tigecycline (15 mcg), nitrofurantoin (300 mcg), colistin (10 mcg), trimethoprim/sulfamethoxazole (23.75/1.25 mcg), cefoxitin (300 mcg), benzylpenicillin (2 mcg), oxacillin (1 mcg), levofloxacin (10 mcg), inducible clindamycin (2 mcg), erythromycin (15 mcg), clindamycin (2 mcg), linezolid (30 mcg), daptomycin (10 mcg), teicoplanin (30 mcg), vancomycin (30 mcg), tetracycline (30 mcg), and rifampicin (5 mcg).

2.5. Phenotypic ESβL Detection

ESβL producers were detected by performing Double Disc Synergy Test (DDST) as per the guidelines of CLSI (CLSI, 2018). The test isolates suspension for each of the pure bacterial isolates were prepared according to 0.5 McFarland constant that were swabbed on MHA plates. After 15 min, antibiotic discs containing amoxycillin (100 mcg) and cefotaxime (30 mcg) with amoxycillin/clavulanic acid (100/10 mcg) and cefotaxime/chlorophyllin (30/10 mcg) were placed at a distance of 20 mm apart. The plates were incubated at 37°C for 24 hrs. The results were interpreted by measuring the diameter of the inhibition zone. According to CLSI, an increase in <5 mm in the zone diameter around the Clavulanic acid combination discs versus the same discs alone confirmed the presence of ESβL producers.

2.6. Data Analysis

Data were entered and analysed using GraphPad Prism software and summarised frequencies and percentages has been presented in the tables and graphs.

3. Results

3.1. General Characterisation of the Study Population

A total of 100 mobile samples were collected from the various participants in the study. The distribution on the basis of gender were 51% males and 49% were females (Figure 1(a)). It has been observed that most of the youth use a mobile phone between the age of 15 to 25, and our study showed a higher usage of the same, with 46% in the age range of 21 to 30 group. Professionally, the population were group of students working in the laboratory as researchers, which also shows that the younger generation have more usage to the mobiles (Figure 1(b)), followed by 11 to 20 years, where the mean age is 18 and above of the students that showed an incidence of 30.47%.

Figure 1

(a). Gender-wise distribution of samples collected from mobile phones. (b). Age-wise distribution of mobile phones (p < 0.01 vs. Older age group, i.e., above 30 years of age).

3.2. Bacterial Isolation Rate and Efficacy of 70% Alcohol

The study revealed that of the 100 mobiles that were used, 96 mobiles showed a contamination with bacteria. Four of the mobiles had been decontaminated with alcohol, and therefore, the incidence rate was found to be 96%. Five mobile phones had shown to have multiple contaminants of the bacteria. A total of 105 bacterial species were identified from the contaminant mobile phones. Of which Gram-negative bacteria had a highest incidence rate with 62.85% (66 isolates) while, Gram-positive were 37.14% (39 isolates) as in Figure 2(a).

Figure 2

(a)-Distribution of Gram-positive and Gram-negative bacteria in the mobile phones (p < 0.01 vs. Gram-negative bacteria; Student's t-test). (b) (A) Occurrence of Gram-negative bacteria on mobile phones. (b) (B) Occurrence of Gram-positive bacterial load on mobile phone.

In the study, Gram-positive bacteria, coagulase-negative Staphylococci (CONS) were the most prevalent present with 15.38% followed by Staphylococcus citrus, Staphylococcus saprophyticus, Staphylococcus pseudintermedius, S. capitis, Staphylococcus hominis, Enterococcus faecalis, and the lowest was observed in Staphylococcus heamolyticus with 7.06% reviewed in Figure 2(b) (B). K. oxytoca (18.18%) is the most frequently observed contaminant on the mobiles in Gram-negative bacteria, followed by E. coli (13.6%) and least was observed with Pseudomonas aeruginosa (4.5%). The study also observed the other contaminants like Citrobacter fraundii, Citrobacter koserii, and Aeromonas hydrophilia species each accounting for 7.5%. The other organisms observed in the study were R. ornithinolytica (6%) and S. paucimobilis (6%) as in Figure 2(b) (A).

3.3. Antimicrobial Susceptibility of the Test Isolates

The bacterial isolates showed a variable antibacterial susceptibility pattern, Gram-positive bacteria showed a maximum resistance to cefoxitin (79.4%) followed by, clindamycin (69.2%), and erythromycin (66.6%) Table 1 (Figure 3). The lowest susceptibility was observed in vancomycin and tetracycline with 87.1%. It should be noted that none of the isolates was found to be resistant to tigecycline. Among the Gram-negative bacteria, ampicillin showed a higher resistance rate with 72.7% followed by cefuroxime with 54.5%. Lowest sensitivity was observed with the antibiotics trimethoprim/sulfamethoxazole and tigecycline with 99.4% and 93.9% respectively as in Table 2. Antibiotic trimethoprim/sulfamethoxazole had a higher sensitivity rate in Gram positive and Gram negative bacteria isolated from mobile phones.

Table 1

Antibiotic resistance in Gram-positive bacteria.

Antibiotics	Resistance	Percentage (%)	Sensitive	Percentage (%)
Tigecycline	0	0	39	100
Vancomycin	5	12.8	34	87.1
Tetracycline	5	12.8	34	87.1
Rifampicin	9	23	30	76.2
Trimethoprim/sulfamethoxazole	10	25.6	29	74.3
Teicoplanin	12	30.7	27	69.2
Nitrofurantoin	12	30.7	27	69.2
Ciprofloxacin	13	33.3	26	66.6
Levofloxacin	13	33.3	26	66.6
Gentamicin	14	35.8	25	64.1
Daptomycin	18	46.1	21	53.8
Linezolid	21	53.8	18	46.1
Oxacillin	23	58.9	6	41.0
Benzylpenicillin	23	58.9	16	41.0
Inducible clindamycin resistance	26	66.6	13	33.3
Erythromycin	26	66.6	13	33.3
Clindamycin	27	69.2	12	30.7
Cefoxitin	31	79.4	8	20.6

Figure 3

Distribution of ESβL producers in Gram-negative organism.

Table 2

Antibiotic resistance in Gram-negative bacteria.

Antibiotics	Resistance	Percentage (%)	Sensitive	Percentage (%)
Trimethoprim/sulfamethoxazole	3	4	63	99.4
Tigecycline	4	6	62	93.9
Ciprofloxacin	9	13.6	57	86.3
Amikacin	11	16.6	55	83.3
Imipenem	15	22.7	51	77.2
Meropenem	15	22.7	51	72.2
Nalidixic acid	15	22.7	51	77.2
Gentamicin	16	24.2	50	75.7
Piperacillin/tazobactam	18	27.7	48	72.7
Colistin	23	34.8	43	65.1
Cefoperazone/sulbactam	24	36.3	42	63.6
Ceftriaxone	25	37.8	41	62.1
Cefepime	27	40.9	39	59
Nitrofurantoin	27	40.9	39	40.9
Cefuroxime axetil	29	43.9	37	56
Cefuroxime	36	54.5	30	45.5
Ampicillin	48	72.7	18	27.7

The study revealed varying MIC results for the isolates that were isolated as interpreted in Table 3. It can be noted that the former antimicrobials like Gentamicin and Amikacin have a lowest value of MIC compared to the cephalosporins and other classes. It is alarming that some of the isolates also showed higher resistance values to carbapenems, oxacillin, and vancomycin.

Table 3

MIC values for respective antibiotics obtained for the isolates from mobiles.

Antibiotics	Interpretative criteria (mcg) as per CLSI guidelines/MIC values of the study isolates obtained
	S	I	R
Amikacin	16/46 isolates	32/12 isolates	64/8 isolates
Ampicillin	8/12 isolates	16/5 isolates	32/49 isolates
Cefepime	2 (>1 mcg)/24 isolates	4–8/11 isolates	16/31 isolates
Imipenem	2 (0.25 mcg)/46 isolates	4/20 isolates	8/None
Meropenem	1 mcg (0.25 mcg)/46 isolates	2/None	4/20 isolates
Benzylpenicillin	0.12/23 isolates	—	0.25/16 isolates
Gentamicin	4/16 isolates	8/11 isolates	18/5 isolates
	1 mcg/51 isolates
Vancomycin	2 (1 mcg)/21 isolates	4–8/5 isolates	16/5 isolates
Oxacillin	2 (1 mcg)/12 isolates	—	4/24 isolates

3.4. Extended Spectrum Beta Lactamases (ESβL)

This study also aimed at the phenotypic detection of Gram-negative bacteria resistant to cephalosporins. The expression of ESβL production by mobile contaminants was found to be highest in K. oxytoca (5.28%) followed by Klebsiella pneumoniae (4.62%) and the least in Erewina spp and Enterobacter spp (2.64%). The ESβL expressed by the bacteria has further increased resistance development to antibiotics such as ceftazidime, gentamycin, cefepime, and ceftriaxone. However, of the four ESβL producers other seven turned out to be non-ESβL.

3.5. Multiple Antimicrobial Resistances

With the increase in the antimicrobials, there has been a drastic increase in the MDR strains ranging with the resistance among two drugs upto 18 antibiotics. Here, maximum of 4.5% to 16 antimicrobials (most seen in K. pneumoniae) and 12.8% for 14 drugs (mostly in CONS) in Gram-negative and Gram-positive bacteria, respectively tabulated in Tables 4 and 5.

Table 4

MDR strains in Gram negative bacteria.

Organism	Number of antimicrobials	No: of isolates	Percentage (%)
S. paucimobilis	3	4	6.0
Enterobacter aerogenes	3	4	6.0
Aeromonas hydrophilia	3	5	7.5
Escherichia. coli	7	4	6.0
K. oxytoca	7	4	6.0
Klebsiella spp	8	4	6.0
K. oxytoca	12	4	18.1
Klebsiella pneumonia	15	4	6.0
Erewina spp	15	4	6.0
Klebsiella pneumonia	16	3	4.5
Total (n = 66)

Table 5

MDR strains in Gram-positive bacteria.

Organism	Number of antimicrobials	No: of isolates	Percentage (%)
S. pseudintermedius	7	5	12.8
Staphylococcus heamolyticus	8	3	7.6
Staphylococcus hominis	8	5	12.8
Staphylococcus citrus	9	4	10.2
S. capitis	10	5	12.8
Staphylococcus saprophyticus	10	4	10.2
CONS	14	5	12.8
Total (n = 39)

4. Discussion

This study demonstrated the incidence rate of microbes in 96% mobiles similar to the earlier study [15] where they found a contamination rate of 98.3% among healthcare workers. Parallel to this study, many such studies have repeatedly reported the spread of various diseases and infections due to the improper sanitation of touch-screen phones [16]. To complement this, Kotris et al. [17] showed a higher risk of contamination with a rate of 84%. In addition, several studies have briefed about the mobile contamination across the globe which includes Turkey (94.5% and 90.98%), India (95%), and other parts of the world [10,11,18,19]. On the contrary, lower values have been reported from Saudi Arabia (43.6%), India (40.62%), Iran (32%), Turkey (61.3%), and Nigeria (62%) [5,20-22]. The students and medical staff contained a high percentage of bacterial contaminants on their mobiles, wherein dental students had an incidence of 98% [23]. In our study, the bacterial contaminants with a higher percentage were in youth between the age of 18 and 25 years, corresponding to a total of 76%. The results vary due to the fact that many participants in our study did not clean their mobile phones, and there was an increased risk of contamination rates. We also observed high contamination load, this might be due to the overuse of mobiles at work place, rest rooms, and even at the dining with lack of proper hand washing practice. This evidences the extensive addiction of mobile phones in all classes of individuals irrespective of their profession, location of use, age, gender, and financial status.

A greater incidence of Gram-negative bacteria (62.8%) is observed in our study, compared to Gram-positive bacteria (37.14%). On the contrary, Karkee et al. [24] much higher rate of Gram-positive bacteria (79.81%) and Kokate et al. [25] reported (71.87%) observed versus Gram-negative (20.19%) in hospital settings. Several studies also reported similar findings among medical personnel and students with a higher incidence of Gram-positive pathogens (85% and 83.87%) and Gram-negative pathogens (15% and 16.13%) [5,26]. In correlation with our current findings with respect to the aetiology of microbes isolated from the mobile surface, the isolation rate was found to be almost similar to the others. Such identical studies have been reported by researchers Aroroa et al. [20Sadat-Ali et al 21] and Sadat-Ali et al. [20], in which the frequently isolated Gram-positive bacteria identified as CONS, as reported in our study. In addition, we also found several highly pathogenic and commensalistic microbes such as E. coli, Klebsiella spp, and Enterobacter spp, indicating that mobile phones can be a significant source of spread of community infections. However, a lot of work has been conducted and have reported these bacterial contaminants on mobile phones in health care workers [5,22]. Contrary, some reported the presence of Acinetobacter sps which were not found in this study [10,18,27]. However, we observed some of the soil microbes on the mobile surfaces with antibiotic resistance like R. ornithinolytica and S. paucimobilis.

In this study, the isolated bacteria had variable sensitivities to the antibiotics tested. It had different variability patterns compared to the other studies with higher rates [5,22,28]. A drastic resistance rate in these microbes noticed in this study might be due to the inappropriate repetition of using antibiotics against recommended in a safe way. A report by Gashaw et al. [15] showed a higher resistance to ceftriaxone and ciprofloxacin with 71.7% and 89.1%, in gram-positive bacteria, compared to our observation in our studies, which had a lower resistance rate. Their study also observed a higher rate for amoxicillin and trimethoprim-sulfamethoxazole. While, they denoted a higher sensitivity to ciprofloxacin to E. coli with 100%. However, Gram-negative isolates in this study had resistance to ampicillin [29] while ciprofloxacin and chloramphenicol had reduced rates. An increased rate of MDR strains was also observed compared to their reports conducted with 1.7% for six antimicrobials.

In our study, a remarkable increase in the incidence rate of Staphylococcus and Enterococcus species was found. Alongside, we could also observe S. pseudintermedius and S. capitis, these organisms are not been documented as contaminants amongst all studies done so far related in community on mobile phones. The study denoted a high resistance rate to cefoxitin 79.4% and 58.9% to penicillin group that includes benzylpenicillin and oxacillin. While, a recent study conducted by Campista-Leon et al. [30] showed gram-positive bacteria alone, indicating the presence of Staphylococcus (84.6%), Bacillus, and Enterococcus species (7.7%). In addition, this study also showed erythromycin resistant with 92.3%, ampicillin and penicillin (76.9%), dicloxacillin (61.5%), cephalothin (38.5%), and cefotaxime and ceftriaxone (7.7%). In our studies, we used extensive antimicrobials, and varying results were observed with the resistance rates. The rising antibiotic resistance designates that in the near future, treating the patients with infectious diseases will be difficult, if we do not take proper measures to prevent their rate of contamination and safety measures to prevent and contained for their transmission.

5. Conclusion

Mobile phones have become a necessary need of every individual, irrespective of their profession with new breeding grounds for the multidrug pathogens leading to infections. With high antibiotic resistance rate that has been observed in our study, it shows that there is a need for the decontamination of the mobile phones. Increasing antibiotic resistance patterns is a huge challenge in the treatment of pathogenic infections. Based on our study, it appears crucial to make people aware about the possibility of MDR risk being transferred by mobile phones in workplace. With regular cleaning and sanitising, the implementation of appropriate infection prevention guidelines will help reduce possible risks. The study also noted some important saprophytic soil microbes, such as R. ornithinolytica and S. paucimobilis, which are unfortunately found to have resistance to certain antibiotics. The presence of clinically significant MDR strains poses a potential risk to user health, further disseminating the antibiotic resistant mechanism in the community by these microbes, we recommend hand hygiene and disinfecting mobiles to prevent cross-infection by these microbes.