Evaluation of the bacterial contamination of face masks worn by personnel in a center of COVID 19 hospitalized patients: A cross-sectional study

Abstract

Background

During the Coronavirus Pandemic, the use of masks has increased significantly. The lack of control on hygiene protocols and the need to use PPE properly increases the spread of bacterial infection. The purpose of this study was to investigate the degree of contamination and frequency of bacterial species isolated from surgical and N95 masks used by hospital personnel.

Methods

A total number of 175 masks were collected from staff working in Sina hospital (Hamadan province, Iran) during the first six months of 2022. The bacterial contamination of masks were evaluated and identified using biochemical kits. Antimicrobial susceptibility testing of the isolates were done using Kirby-Bauer methods and MIC were assessed for each isolate against different disinfectants (Sodium hypochlorite 5%, Hydrogen Peroxide 3%, Ethanol 70% and Deconex).

Results

Of 175 masks, 471 bacterial isolates were detected including 9 species. The most prevalent strain were Coagulase negative Staphylococcus (28%) followed by Acinetobacter (20.8%) and Pseudomonas (13.8%), while, Klebsiealla and Enterococcus were the least frequent species with the rate of 3.8% and 1.2%, respectively. The results of MIC methods indicated that all 471 strains were resistant to ehtanol70% and sensitive to hydrogen peroxide 3%. Furthermore, the mean average of Deconex inhibitory effect is lower than Sodium hypochlorite 5%.

Conclusions

According to the results of this study, there was a high prevalence of CoNS, Acinetobacter and Pseudomonas in hospital with a high resistance pattern against antibiotics especially Ampicillin and disinfectants.

1. Introduction

In 1934, Wells was the first to hypothesize the transmission of pathogens by droplets containing infectious microbes in the air through the respiratory system [1]. Later, many pathogenic agents that could be transmitted in this way were identified. When a sick person coughs or sneezes, droplets containing infectious particles are released into the air [2,3]. Despite all these research findings, the global community did not take the aerial transmission of diseases seriously until the acute respiratory syndrome occurred in 2003 and the human swine flu in 2009 [4,5].

After epidemies, many studies have been conducted to investigate ways to control and reduce infections caused by airborne pathogens. Respiratory infections can be transmitted by droplets of different sizes (from less than 5 microns to more than 10 microns). The smaller the droplets, the longer they remain in the air, and they can be transmitted to others at distances of more than one meter [5,6].

In the first three months of coronavirus pandemic, the World Health Organization announced that the corona virus is transmitted by large respiratory droplets and people can transmit this disease to each other in a distance of less than one meter. Recent research also shows that the transmission of this disease through the air is as important as acute respiratory syndrome [7]. Face masks were generally used in the surgical sections of hospitals to prevent post-surgery infections transmitted from mouth and nasal cavity of physician and staff. Today, due to the Corona pandemic, governments around the world have made it mandatory to use face masks to prevent disease transmission [8].

Application, duration of usage, and the removal of face masks are of high importance. These instructions may vary in different places, due to the fact that there are mixed bacteria in the air and surfaces. Plus, people who use masks can unintentionally transfer environmental microbes, including bacteria on surfaces, as well as the bacterial and viral species present in the mouth and nasal cavity, which can be transferred to the masks [9]. As a consequence, face masks can serve as bacterial carriers. Since the use of masks has increased during the corona pandemic, masks are left in the trash or on publicenvironments after use. Due to the fact that hospital environments are regularly cleaned with various disinfectants, bacteria may develop that are resistant to these disinfectants. The transfer of pathogenic bacteria to masks and their wide-spread in the environment can be a great potential risk for the public health [10].

Coronavirus as a respiratory disease is transmitted from one person to another through respiratory droplets. One of the best ways to prevent the transmission of this disease is to use face masks. Due to the contamination of the ambient air and various hospital surfaces with bacteria and the high resistance of hospital bacterial strains to various antibiotics and disinfectants, these bacteria can be transferred to masks by people and through the air [11].

The important point is that after using the mask, the masks are not eliminated in principle and are left in the trash cans or in public environments. Masks that contain dangerous hospital bacteria can seriously threaten the public health. To address this issue, the purpose of this study was to investigate the bacterial contamination of masks used by people working in various hospital sections (physicians, nurses, paramedics, service personnel, etc.). We also aimed to isolate the bacteria grown on the masks to investigate their resistance pattern to antibiotics and disinfectants.

2. Materials and methods

2.1. Mask collection

The masks were collected from Sina Hospital (the centeral location for hospitalization of Corona patients in the city of Hamadan, Iran) in the first six months of year 2022. Two types of masks were studied in this research: N95 and surgical masks. The masks were given to the staff working in different departments of the hospital daily, and then were collected from the people after using them, Based on the duration of face mask usages, participants were divided into four main groups including; less than 2 hours, between 3-4 hours, between 5-6 hours and between 7-8 hours. In general, 175 masks were collected. Each mask was placed in a container (sterile zippered plastic) and transferred in a cool box to the microbiology laboratory of Hamadan University of Medical Sciences [12].

2.2. Bacterial detection

In order to isolate the bacteria, first, the inner and outer parts of the masks were separated according to health protocols by Bio-Safety Cabinet (BSC) class2. In the next step, each part was immersed in a sterile physiological serum separately for 20 minutes. Then a 1 mL of supernatant was added to 9 mL of Tryptic Soy Broth (TSB) culture medium and incubated at 37°C for 4 hours to enhance bacterial growth. The resulting suspension (physiological serum and bacteria in TSB) was cultured on Eosin Methylene Blue (EMB) and blood agar and placed at 37°C overnight. After this period, in case of bacterial growth and colony formation, a suspension was prepared from each colony separately and the strain of each bacterium was identified by the Analytical Profile Index kit (API, Biomeriueux-USA). In this study, API 20E and 20NE kits were utilized for biochemical identification of gram negative bacteria and also phenotypic methods were used to identify Gram-positive strains [13]. Considering that the cost of DNA extraction and PCR methods was too high and on the other hand in this study only frequency was considered and genetic work such as checking virulence genes etc. was not considered, therefore we only used phenotypic tests for diagnosis. It should be noted that the use of API diagnostic kits and the usual phenotypic methods are scientifically standard and accepted methods for isolating bacteria.

2.3. Antimicrobial susceptibility testing

The antibiogram process was investigated by the Kirby-Bauer disc diffusion method. For each isolated sample, a suspension with a turbidity equivalent to 0.5 McFarland standards (density of a bacterial suspension with a 1.5 × 10⁸ Cfu/mL) was prepared and transferred to Mueller Hinton agar medium, and then antibiotic disks of Ciprofloxacin (5 μg), Gentamicin (10 μg), Erythromycin (15 μg), Imipenem (10 μg), Ceftazidim (30 μg), Amikacin (30 μg), Ampicillin (10 μg), Trimethoprim-Sulfamethoxazole (1.25-23.75μg) were placed on the plate and incubated at 37°C. After 24 hours, the growth inhibition diameter was measured. According to the Clinical & Laboratory Standards Institute (CLSI) guidelines [14], microorganism susceptibility pattern for each antibiotic is classified as sensitive, intermediate and resistant. The quality control strain used was ATCC E. faecalis 29212.

2.4. Assessing minimum inhibitory concentration

Broth Micro-dilution method was used in 96-well ELISA plates to quantitatively check the effectiveness of the desired disinfectants including Sodium hypochlorite (house-hold bleach) 5%, Hydrogen peroxide 3%, Ethanol 70% and Deconex solutions [15,16]. In the first step, serial dilutions of each of the disinfectants were prepared according to the following instruction: Wells 1 to 9 were considered as test wells with initial concentration of 50% of each disinfectant, following by 25%, 12.5%, 6.25%, 3.125%, 1.562%, 0.781%, 0.390% and 0.195% from the highest to the lowest concentration, respectively. Then, 100 microliter of each prepared dilution were added into wells. Well 10 was used as positive control (culture medium + bacteria), well 11 was used as negative control 1 (culture medium containing disinfectant) and well 12 was used as negative control 2 (only culture medium). Then 100 microliters of 1/20 dilution (preparation of microbial suspension equal to 0.5 McFarland) bacterial suspension was added to each well and the plate was placed in the incubator at a 37°C. After that, from each dilution of disinfectant, the lowest concentration that inhibited the growth of bacteria was considered as MIC [14].

2.5. Statistical analysis

Analysis Of Variance(ANOVA) and T-tests were used to evaluate the relationship between the kinds of face masks and frequency of bacterial isolates and also the susceptibility patters of species to routine antibiotics and disinfectants. Data were analyzed statistically using Chi-Square test and difference was considered significant at P < 0.05 by SPSS software version 20 (Chicago, IL, USA).

3. Results

3.1. Sample collection

In this study, a total number of 175 mask from personnel of Sina hospital working in different wards were collected in the first six months of year 2022. Of these, 111 and 64 masks were obtained from women and men, respectively. After collection and transportation of samples into microbiology laboratory of Hamadan University of Medical Sciences, the masks were separated under BSC class 2 into inner and outer layer and a total of 350 sample were prepared. The highest frequency of age group incorporated in this study were between 31-40 years (70/175), in which men had higher frequency than women. This is while the 41-50 age group had women only. Fig. 1 depicts the frequency of staff included in this study with the data classified based on sex and age. As shown, the data were divided into four age groups included; 20-30 years, 31-40 years, 41-50 years and more than 51 years.

Fig. 1.

Frequency of gender and age of the studied subjects.

3.2. Bacterial detection

The results of API biochemical test indicated that an overall number of 9 bacterial species were isolated, 5 of which belonged to gram-negative bacteria including; E.coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella Spp and Enterobacter spp. 4 remained samples were gram-positive including; S. aureus, Coagulase-negative Staphylococcus, Enterococcus and β-hemolytic Streptococcus strains. Table 1 shows the frequency of isolated species from the inner and outer layers of N95 and surgical masks. A total of 471 cases of 9 bacterial species were detected from 350 mask samples (inner vs. outer layers). Surprisingly, the highest number of detected isolates belonged to the N95 face mask. Of the 175 mask, 246 number of strains were isolated from N95 whereas 225 isolates detected from surgical masks.

Table 1.

The frequency of bacteria species isolated from both kind of masks

Species Mask	Layer	E. coli	Acinetobacter	Pseudomonas	Coagulase-negative staphylococcus	Coagulase-positive staphylococcus	Klebsiella	Enterococcus	β-hemolytic Streptococcus	Enterobacter	Total
N95	Inner	17	34	12	31	3	9	3	6	3	246
	Outer	17	19	14	45	14	0	0	11	8
Surgical	Inner	6	17	25	37	14	6	0	3	11	225
	Outer	11	28	14	19	17	3	3	8	3
Total Frequency n (%)		51 (10.8)	98 (20.9)	65 (13.8)	132 (28)	48 (10.1)	18 (3.8)	6 (1.3)	28 (5.9)	25 (5.4)	471 (100)

Coagulase-negative staphylococcus (CoNS) was the most prevalent (28%) strain isolated from the inner and outer layers of both kinds of masks. The second and third ranks goes to Acinetobacter baumannii, (20.8%) and Pseudomonas aeruginosa (13.8%), respectively followed by, E.Coli (10.8%), S. aureus (10.1%), β-hemolytic streptococcus (5.9%), Enterobacter (5.4%). Klebsiella (3.8%) and Enterococcus (1.3%) had the lowest frequency among detected isolates. Out of 246 strains detected from the N95 masks, Acinetobacter and CoNS were more frequent in the inner and outer surface, respectively. Of the 225 strains isolated from the surgical masks, CoNS and Acinetobacter were more frequent rather than other species.

Additional data were collected to evaluate the relationship between frequencies of identified species with participants of different age groups which is depicted in Fig. 2. As mentioned, in almost all age groups, CoNS were observed as most frequent isolate. In contrast, Enterococcus, Enterobacter and Klebsiella had the lowest frequency in all different age groups.

Fig. 2.

The relationship between staff age groups and bacterial isolates frequency.

The data shows that.

• 1.In the age group of 20-30 years, Klebsiella were not been found and CoNS were more frequent than other isolates. Enterococcus had the lowest frequency.
• 2.In the age group of 31-40 years, all species except Enterococcus were found. The highest and lowest frequency belonged to CoNS and β-hemolytic Streptococcus, respectively.
• 3.In the age group 41-50 years, Acinetobacter had the highest frequency, whereas, Enterococcus had the lowest frequency.
• 4.In the age group upper than 50 years, isolates including CoNS and Acinetobacter were more frequent. In contrast, Klebsiella and E.coli hadn't been detected.

3.3. Antimicrobial susceptibility testing

To evaluate the antibacterial susceptibility behavior of each of the 9 detected isolates, eight different antibiotic disks including Imipenem, Ciprofloxacin, Gentamycin, Erythromycin, Ceftazidim, Trimethoprim-Sulfamethoxazol, Amikacin and Ampicillin disks were applied using disk-diffusion method, whose results are shown in Table 2. It should be noted, since the latest CLSI guidelines recognize the β-hemolytic Streptococcus isolate as highly susceptible to almost all antibiotics, this study did not test antimicrobial susceptibility for this strain and the whole 443 strains were evaluated for antimicrobial testing. The results show that the antibacterial susceptibility fo each strain as follows:

• 1.Acinetobacter had the highest resistance to Ceftazidim (85.7%) and Ampicillin (85.7%) and had the lowest resistant against Trimethoprim-Sulfamethoxazole (11.2%).
• 2.For E.coli, the highest and lowest resistance belonged to Ciprofloxacin (66.6%) and Ampicillin (9.08%), respectively. In-contrast, 78.4% of the E.coli strains were susceptible to Gentamycin and Ampicillin antibiotics whereas the lowest sensitivity belonged to Ciprofloxacin (21.5%).
• 3.Pseudomonas; the results supports that the highest resistance and sensitivity patterns of the strain belonged to Erythromycin (738%) and Gentamycin (61.5%), respectively. In-contrast, Pseudomonas depicted the lowest resistance and sensitivity to Gentamycin and Erythromycin, respectively.
• 4.Coagulase-negative Staphylococcus was most resistant and less sensitive to Ampicillin, while it showed the highest and lowest sensitivity and resistance to Ciprofloxacin, respectively.
• 5.75% of Staphylococcus aureus isolates were resistant to Erythromycin while they showed very good susceptibility pattern to Ciprofloxacin (81.2%).
• 6.Klebsiellashowed good sensitivity to almost all antibiotics except for Erythromycin and Gentamycin and showed a high resistance to Erythromycin (66.6%).
• 7.All Enterococcus isolates were sensitive to Ciprofloxacin and showed the highest resistance to Amikacin (83.3%).
• 8.Enterobacter showed the highest and lowest resistance to Trimethoprim-Sulfamethoxazole and Imipenem, respectively. The strains was very sensitive to Amikacin.

Table 2.

Antibacterial susceptibility test result

Overall, the highest susceptibility were obtained against Imipenem (61.4%) and highest resistance pattern were observed for Ampicillin (64.5%). More information about antibiotic susceptibility results are provided in Table 2.

3.4. Minimum inhibitory concentration (MIC) results

As mentioned earlier, antimicrobial susceptibility testing hasn't been applied for β-hemolytic streptococci following CLSI guidelines. All 443 samples were evaluated with the MIC method for each disinfectant. According to our data, all strains were resistant to Ethanol 70%, meaning that all 9 wells, from the highest to lowest concentrations, turbidity of growth observed in which Ethanol could not inhibit the growth of species. In contrast, the strains were highly susceptible to Hydrogen peroxide 3%. We observed no turbidity of growth in all wells. Hydrogen peroxide 3% had inhibitory effect on the growth of the species. The mean concentration rate of the 5% sodium hypochlorite and Deconex for each isolate is provided in Fig. 3. As demonstrated, CoNS had the highest sensitivity to 5% Sodium hypochlorite with the mean concentration of 3.97% and Enterococcus had the highest resistant rate with the rate of 9.37% meaning that 5% sodium hypochlorite can inhibit the growth of Enterobacter at high concentrations. While Enterobacter and Pseudomonas showed the highest sensitivity and resistance to Deconex with the rate of 3.12% and 9.50%, respectively.

Fig. 3.

The MIC results.

4. Discussion

This study endeavored to evaluate the prevalence of bacterial species isolated from the inner and outer layers of N95 and surgical mask used by personnel of Hamadan Sina hospital, IRAN. Currently, the increasing incidence of microbial resistance is competing with the Corona pandemic in parallel, while both are responsible for the high rates of mortality and morbidity. One of the factors that accounts for microbial resistances is the excessive use of disinfectants and underestimating hygienic protocols as placing contaminated masks on surface areas leading to direct transmission of bacterial community on the surfaces to the mask, as well as the transmission of infectious agents form personnel protective equipment to the surfaces. In the meantime, the horizontal gene transferring of the microbial resistance between bacterial species also takes place.

In this study, the load of bacterial contamination of N95 masks was expected to be much lower than surgical due to the higher thickness of the layers but the most bacterial contamination were isolated from N95 masks (246/471- 52.2%) compared to the surgical masks (225/471- 47.8%). This can have various causes, such as the inappropriate structure of the mask or the incorrect assertion of the manufacturing company, or lack of observing hygienic protocols related to the mask by hospital staff, including excessive use of the mask, or placing the mask on contaminated surfaces and reusing it.

Bacterial analysis detected 9 bacterial isolates, 5 gram-negative and 4 gram-positive strains. Overall, most predominant species from both surgical and N95 masks were CoNS and Acinetobacter which were reported to be 28% and 20.8%, respectively which is proposed that burden bacterial colonization of Sina hospital wards or surfaces is Coagulase negative Staphylococcus and Acinetobacter. In contrast, Enterococcus was found less (1.2%). It is important to clarify the reason if the high prevalence of Coagulase-negative staphylococci (CoNS; S. epidermidis, S. lugdunensis, S. haemolyticus) is related to hospital (nosocomial) infections or just it is a skin and hand contamination. If the reason is related to CoNS colonization due to hospital surfaces and medical devices, using disinfectant becomes important. However, the results of this study completely matches with article reviews that reported CoNS strains have the most frequency in clinical cares [17]. Another study also reported that CoNS and S. aureus are the predominant bacterial species connected to device-associated infections which is consistent with the results of this study that CoNS were more prevalent in the samples study [18]. On the other hand, CoNS are a part of normal flora of skin, and can be easily transported to the mask due to the direct contact of the mask with the face skin, as well as moving the mask by hand, both surfaces of the mask become infected with this bacterium [19]. Azimi et al., also reported CoNS were most frequent isolate detected from children's specimen hospitalized during 2013-2018 in Mofid hospital, Tehran, IRAN [20]. In a more detailed study and comparison at the endemic level, Sedighi et al., also evaluated the contamination level of personal equipment of the health care providers in a teaching hospital located in Hamadan province, Iran. Their results highly supports the fact that CoNS colonization (82.4%) in clinical cares is common [21]. Moreover, Acinetobacter was frequently detected from the mask samples in this study which can be related to the fact that the most common site for Acinetobacter infection is respiratory tract, which might be the nasopharynx of the staff working in a hospital. Besides, Acinetobacter is one of the most prevalent isolates tending to attach to surfaces like medical devices inhospitals and easily transported to the staff PPE such as masks and gloves [22].

Our results indicate that the highest resistance among the strains were related to Ampicillin (64.5%) rather than other antibiotics, in which Acinetobacter and CoNS showed the highest resistance rate to Ampicillin that were 85.7% and 80.3% respectively. This can be due to the intrinsic or acquired ability of the organism to hydrolyze the β-lactam ring and inactive the antibiotics. OXA-1 β-lactamase gene located in DNA is responsible for resistance to Ampicillin [23]. The high resistance rate to Ampicillin can be due to the availability and also uses as empirical therapy makes the drug accessible and gradually cause resistance toward a member of penicillin family as it observed in a retrospective study in a teaching hospital in Ghana [24].

Tavakoly et al. also conducted a retrospective study on clinical isolates of hospitalized patients in Rasht, IRAN, whose results are in agreement with the data of the current study. Both data demonstrated high efficacy of Imipenem in clinical isolates and also high resistant of the strains to Ampicillin [25]. Expectably, as most isolates could hydrolyze the β-lactam ring and become resistant to Ampicillin, Carbapenem family (e.g. Imipenem) could be an effective alternative therapy in cases of Penicillin family resistance as demonstrated in this study.

MIC method showed all strains to be resistant to Ethanol 70%, meaning that the turbidity of bacterial growth were observed in each 9 well. Rozman et al. conducted a review study on the mechanisms of disinfectant resistance between clinical isolates. The data also supports that there was an increasing resistance among many disinfectants including Ethanol 70-95%. This can be due to the cross-resistance mechanism among microorganisms. Ethanol can effectively dissolve proteins and lipids of bacterial cell membrane, as well as alter membrane pH and enhance the efflux pumps. Horizontal gene transferring among organisms can cause a mutation in core genome of chromosomal nucleotides rpoB gene B position of RNA subunits, which makes a bacterial cell completely resistant to commercial disinfectant including ethanol. Therefore, increasing rate of resistance to ethanol during the last decades can be a result of overuse and misuse of disinfectants, making an opportunity for microorganism cells to have gene exchanges [26], so it is not recommended to use ethanol as the disinfectant for medical devices (25-CDC).

Our results also showed high susceptibility of all strains to Hydrogen peroxide (H2O2). This can be due to the release of free hydroxyl radicals which have detrimental effects on proteins and lipids of yeast and bacterial cell membrane and also disrupts the S-S ligands of bacterial DNA [26]. As reported by Center for Disease Control and prevention (CDC), hydrogen peroxide has a strong antimicrobial efficacy on a wide range of microorganisms including bacteria, fungi, and viruses and also spores. According to the our experiments, hydrogen peroxide have bactericidal and virucidal and also sporicidal activity in health-care settings if applied in 1 minutes and also Mycobactericidal and fungicidal action in 5 minutes [27].

Another finding of this study is the high susceptibility of CoNS to Sodium hypochlorite 5% (3.97%) while Enterococcus was barely inhibited by the effect of household bleach with the highest resistant rate (9.37%). This agrees with the reports by Lineback et al. indicating high susceptibility of Staphylococcus and Pseudomonas to hydrogen peroxide and sodium hypochlorite [28]. This can prove the effectiveness of chlorine compounds in clinical cares as they have broad spectrum of activity and also less toxicity (25-CDC).

Deconex is another biocide solution used in this study. According to the results, Enterobacter was the most sensitive strain (3.12%) against the inhibitory effect of Dexonex while Pseudomonas was the most resistant isolate against Deconex with the rate of 9.50%. The results demonstrated the mean average for the minimum inhibitory concentration of Deconex (6.98%) is lower than Sodium hypochlorite 5% (7.44%). This implies that it might have higher inhibitory effect when applied as disinfectant which is consistent with the reports by Amin et al. and Monirzadeh et al. [29]. Moreover, Pseudomonas is a frequent isolate in clinical setting which shows high degree of tolerance to many antibiotics and disinfectant due to the ability of bacterium to live in biofilms and also overexpression of genes boosting resistance to antibiotics as confirmed by Goodarzi et al. that Pseudomonas species can tolerate a range of biocide including Deconex [30].

5. Conclusion

It is inferable that applying PPE such as face masks without following the health protocols can be a source of contamination itself, which in turn leads to. Spread of many MDR pathogens such as CoNS, Acinetobacter and Pseudomonas in hospital environments. Additionally, our results do not advocate use of 70% Ethanol 70% as hospital disinfectant. This is because it is not sporicidal and cannot penetrate protein-rich materials. Moreover, according to CDC guidelines, 70% ethanol is not suggested for disinfecting medical instruments and surfaces since it evaporates soon and the remaining material which contains more water than ethanol, cannot have enough antimicrobial effect other than a mere use as hand sanitizer. Our results highly support using hydrogen peroxide 3-6% and Deconex in hospital cares as disinfectant applied on medical devices such as ventilators, soft contact lenses and patient's rooms.

Ethics statement

The study protocol was approved by the ethic committee of Hamadan University of medical sciences (Ethic code: IR.UMSHA.REC.1400.139).

Declaration of competing interest

Authors declare no conflict of interest.

CRediT authorship contribution statement

Milad Yousefimashouf: Methodology, Writing – original draft. Rasoul Yousefimashouf: Funding acquisition, Supervision. Mohammad Sina Alikhani: Methodology. Hamid Hashemi: Conceptualization. Pezhman Karami: Methodology. Zahra Rahimi: Methodology. Seyed Mostafa Hosseini: Conceptualization, Methodology, Project administration, Writing – review & editing.