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Research Article

Molecular Detection of Multidrug Resistance Pathogenic Bacteria from Protective Materials Used By Healthcare Workers (HCW); Bangladesh Scenario

Afroza Sultana, Otun Saha, Ashfaqur Rahman Siddiqui, Apurbha Saha, Md. Saddam Hussain and Tarequl Islam
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Background and Objective: The increasing trend of Hospital acquired infections (HAIs), especially the ones caused by the multidrug resistant organisms has become a major public health concern. So that the aim of this study was to detect the dissemination of multidrug-resistant pathogenic bacteria on hands, gloves and masks of healthcare workers in some hospitals and pathology laboratories located in Noakhali and Dhaka, Bangladesh. Materials and Methods: A total of 106 fully fresh samples were collected. The samples were then subjected to various phenotypic cultural, biochemicals, antibiotic sensitivity along with molecular Polymerase Chain Reaction (PCR) analysis according to the standard procedures. Results: Approximately, 138 (57.5%) representative bacterial isolates were recovered among which the most frequently identified bacterium was E. coli 72 (52.17%) followed by Staphylococcus aureus 42 (30.43%), Salmonella typhi 14 (10.14%) and Pseudomonas aeruginosa 10 (7.25%). Among them total 33.33, 100 and 40% of Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa were pathogenic scanned through hlg, invA and OprI genes presence respectively. In contrast E. coli was tested through Congo red binding test where 36.11% were found pathogenic. The general frequency of Multiple Drug Resistance (MDR) pathogens were 79.31, 51.72, 68.97, 24.13, 6.89, 6.89, 17.24, 82.87, 24.13, 65.52 and 100% against ampicillin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, methicillin, streptomycin, tetracycline, nalidixic acid, doxycycline and imipenem consecutively. Conclusion: Healthcare personnel convey multiple drug resistance pathogenic bacteria in their protective materials which are potential source of nosocomial infections. Appropriate infection prevention measures, such as good hygiene practices and training for the healthcare workers should be taken to minimize the risks that are associated with the high rate of cross-contamination.

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Afroza Sultana, Otun Saha, Ashfaqur Rahman Siddiqui, Apurbha Saha, Md. Saddam Hussain and Tarequl Islam, 2018. Molecular Detection of Multidrug Resistance Pathogenic Bacteria from Protective Materials Used By Healthcare Workers (HCW); Bangladesh Scenario. Journal of Applied Sciences, 18: 48-55.

DOI: 10.3923/jas.2018.48.55

Received: August 04, 2018; Accepted: August 16, 2018; Published: October 30, 2018

Copyright: © 2018. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.


Different types of microorganisms are hidden in the hospital environments among them some are pathogenic and play role in spreading Hospital-acquired infections (HAI)1. Pathogenic bacteria accountable for HAI include Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Enterococci, Acinetobacter spp. and Coagulase-negative staphylococci. Some of them survive long time in the hospital environment naturally or through the formation of biofilms like Pseudomonas aeruginosa and Staphylococcus aureus and acquired resistance against commonly used disinfectants2. The ability of the bacterial population to resist antibiotics and disinfectants help them to reach patients through different sources and cause nosocomial infections3.

In hospitals different types of precautions are taken by the healthcare workers to prevent the transmission of harmful bacteria to the patients. That is why; hand gloves and mouth masks are used. Improper implementation of hand washing practices of HCW is still a barrier to achieve the expected outcome of the taken precautions 4. Thus, contaminated hand gloves, masks and even mobile phones are playing important in spreading multidrug resistant pathogenic bacteria5. HAIs normally occur after two days of staying in the hospitals6. HAIs are of major concerns as it leads the mortality and morbidity of the hospitalized patients greatly but one-third of such infections can be prevented by precise actions 7. Hands as well as gloves of HCWs are responsible for the spread of pathogenic bacteria causing nosocomial infections. The presence of antimicrobial resistant bacteria on contaminated gloves and hands of HCWs in hospitals poses a threat to public health8.

Multidrug resistant (MDR) pathogenic bacteria for instance methicillin-resistant S. aureus (MRSA), broad spectrum beta-lactamase producing Enterobacteriaceae, ceftazidime-resistant P. aeruginosa, imipenem-resistant A. baumannii and vancomycin-resistant Enterococci are generally chance meeting in the healthcare facilitates9,10. As Bangladesh is a developing country, the rate is HAIs in Bangladesh is very high due to inadequate judgments, ignorance and proper consciousness11. Antimicrobial resistance pattern of HAIs related bacteria have changed a lot in the last few years and very few studies have been reported in this regards 12, 13. Moreover, the data related to it are not up to date in Bangladesh and thus are not reliable14. That is why; it examined the current condition of bacterial contamination of hands, hand gloves and mouth masks of HCWs and evaluated the pathogenicity and antimicrobial resistance pattern of isolated bacteria. Though the govt. is increasing the supervision system to control the nosocomial infections but the presence of multidrug resistance pathogenic bacteria and new emergence of pathogenic bacteria are increasing terribly. That is why, the study aimed to recover the dissemination of pathogenic bacteria on hands, gloves and masks of healthcare workers in some hospitals and pathology laboratories located in Noakhali and Dhaka and determination of their pathogenicity and antimicrobial resistance pattern.


Study design and initial processing: This study was accompanied within April 2017 and February 2018 at Noakhali (22.828973, 91.098944) and Dhaka (23.739944, 90.393575) regions in Bangladesh. A total 240 samples were taken from the healthcare worker of different hospitals and pathology centers. This sample includes gloves, surgical masks and direct Handler swab from HCWs. All the samples were transported to the laboratory using Nutrient agar (Oxoid, Basingstoke, UK) under refrigerated conditions and microbiological analyses were carried out immediately.

Isolation and identification of bacteria: For the primary isolation of bacterial population different selective and differential media for instance Salmonella-shigella (SS) agar, Mannitol salt agar (MSA), Cetrimide agar and Eosin Methylene Blue agar (EMB) media for Salmonella, Staphylococcus, Pseudomonas and E. coli were used respectively. Each suspected isolate was examined for Gram’s staining and followed by inoculation into aforementioned biochemical test such as oxidase test, indole test, urease test, citrate test, Triple Sugar Iron (TSI) test, catalase and MR-VP were performed according to the guideline of the Bergey’s Manual of Determinate Bacteriology15.

Preparation of template DNA: The DNA of each selected isolate was prepared using freshly cultured bacterial colonies on nutrient agar plates, suspended in 150 μL of sterile distilled water in a micro centrifuge tube, gently vortexed and boiled for 10 min in a water bath. After centrifugation at 10000 rpm for 5 min at room temperature (20°C), the supernatant was immediately used for PCR reactions13. The extracted DNA concentration was determined by using Nano Drop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE)16.

Screening of virulence genes to detect pathogenicity: Various individual pathogenic tests were performed for each isolated bacteria. For E. coli Congo red binding test was performed as it is associated with the pathogenicity of Escherichia coli17. invA, hlg and OprI can explain the viluencity in Salmonella, Staphylococci and Pseudomonas respectively (Table 1). Recommended primer set as per Rahn et al.18, Kumar et al.19 and Fazeli and Momtaz4 accordingly were utilized to amplify the specific gene in thermal cycler under specific PCR conditions for Salmonella, Staphylococci and Pseudomonas, respectively. The reaction was set as initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, extension at 72°C for 1 min and final extension was done at 72°C for 7 min. Subsequently, the amplified PCR product was visualized by agarose gel-electrophoresis (1.5% agarose gel).

Anti-biogram test: Disk diffusion method was used to evaluate the antimicrobial resistance pattern of the bacterial isolates 20. Fresh Bacterial inoculums having turbidity of a . 1-.5 nm using spectrophotometer. The antimicrobial susceptibility testing was performed using Mueller-hinton medium against ampicillin (10 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), erythromycin (15 μg), gentamicin (10 μg), methicillin (5 μg), streptomycin (10 μg), tetracycline (30 μg), nalidixic acid (30 μg), Doxycycline (30 μg), Imipenem (10 μg). The plates were incubated aerobically at 37°C for 18-24 h. The zones of inhibition were measured as stated by Clinical Laboratory Standards Institute (CLSI)) guidelines21.


The present study focused on isolation of multidrug resistant and pathogenic bacteria mainly E. coli, Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa in hands, gloves and masks of healthcare workers in some hospitals and pathology laboratories located in Noakhali and Dhaka, Bangladesh.

Bacterial isolation and presumptive identification: From all samples, initially 138 isolates with characteristic colonies were detected positive based on the colony characteristic on SS Agar, MSA, Cetrimide agar and EMB Agar. For presumptive identification of E. coli, Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa. A panel of biochemical test (IMViC, TSI and Urease) was performed. Out of 240 samples, after completing all biochemical tests, 138 (57.5%) isolates were presumptively identified. Among all isolates, 42 (30.43%) were Staphylococcus, 10 (7.25%) Pseudomonas, 14 (10.14%), Salmonella followed by 72 (52.17%) Escherichia coli. Overall, a high prevalence of pathogenic bacteria was reported in Handler swab samples where the percentage was 49.28. Bacteria were isolated in all the abattoir types; the most prevalent bacteria were E. coli while Pseudomonas was the lowest. No Salmonella was detected in this study from face mask samples (Table 2).

Screening of virulence character to detect pathogenicity: Overall, pathogenic E. coli was detected in 26 (36.11%) congo red binding test. In contrast 14 (100%), 33.33% (14) and 40% (4) pathogenic invA, hlg, Oprl genes were detected in Salmonella typhi, S. aureus and Pseudomonas aeruginosa respectively in this study (Fig. 1).

Table 1: Primers used for the detection of Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa by PCR
Image for - Molecular Detection of Multidrug Resistance Pathogenic Bacteria from Protective Materials Used By Healthcare Workers (HCW); Bangladesh Scenario
Virulence genes (A) hlg (937 bp), (B) invA (284 bp) and (C) Oprl (249 bp) were used for Staphylococci, Salmonella and Pseudomonas, respectively

Table 2: Bacteria distribution in the different Samples of HCWs examined in this study
Image for - Molecular Detection of Multidrug Resistance Pathogenic Bacteria from Protective Materials Used By Healthcare Workers (HCW); Bangladesh Scenario

Image for - Molecular Detection of Multidrug Resistance Pathogenic Bacteria from Protective Materials Used By Healthcare Workers (HCW); Bangladesh Scenario
Fig. 1(a-c):
Amplification products of virulence genes: (a) hlg (937 bp) for Staphylococci, (Lane 1: 100 bp ladder as molecular size DNA marker. Lane 2: +ve control, Lanes 3: -ve control, Lane 4: 10 represent group (Staphylococci)), (b) invA (284 bp) for Salmonella, (Lane 1: 100 bp, Lane 2: +ve control, Lanes 3: -ve control, Lane 4: 10 represent group (Salmonella)) and (c) Oprl (249 bp) for Pseudomonas, (Lane 1: 100 bp, Lane 2: +ve control, Lanes 3: -ve control, Lane 4: 10 represent group (Pseudomonas))

Antimicrobial susceptibility testing: It conducted antibiogram profile for all pathogenic isolates. Out of the 14 isolated S. aureus; 14 (100%), 10 (71.43%), 8 (57.14%), 14 (100%), 4 (28.57%), 10 (71.43%), 10 (71.43%) and 14 (100%) isolates were resistant to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, methicillin, streptomycin, tetracycline, Imipenem. Similarly, out of the 6 isolated P. aeruginosa, 4 (66.67%), 4 (66.67%), 2 (33.33%), 2 (33.33%), 4 (66.67%), 4 (66.67%) isolates were resistant to ampicillin, chloramphenicol, ciprofloxacin, tetracycline, doxycycline, imipenem. In total 18 (69.23%), 18 (69.23%), 12 (46.14%), 22 (84.61%), 22 (84.61%) and 26 (100%) isolates of E. coli were resistant to ampicillin, chloramphenicol, ciprofloxacin, tetracycline, doxycycline and imipenem, respectively. Out of the 14 isolates of Salmonella typhi 10 (71.43%), 8 (57.14%), 6 (42.86%), 4 (28.57%), 14 (100%), 14 (100%), 12 (85.71%), 14 (100%) isolates were resistant to ampicillin, chloramphenicol, ciprofloxacin, gentamicin, tetracycline, nalidixic acid , doxycycline and imipenem (Fig. 2). Finally, we also estimated the total isolated bacteria among them 79.31, 51.72, 68.69, 24.13, 6.89, 6.89, 17.24, 82.72, 24.13, 65.52 and 100% isolates were resistant to Ampicillin, Chloramphenicol, Ciprofloxacin, Erythromycin, Gentamicin, Methicillin, Streptomycin, Tetracycline, Nalidixic acid, Doxycycline, Imipenem antibiotics, respectively (Fig. 3, Table 3).


Due to the lack of insufficient research on the degree of bacterial infections in the hospitals mainly on healthcare and pathology centers’ workers, yearly medical expenditures going up in the healthcare facilitates. Not only that, the healthcare facilitates are opening infection management system because of various unwanted infectious.

Image for - Molecular Detection of Multidrug Resistance Pathogenic Bacteria from Protective Materials Used By Healthcare Workers (HCW); Bangladesh Scenario
Fig. 2: Percentage of resistance of isolated microorganisms against specific antibiotics

Table 3: Antibiogram pattern of isolated pathogenic bacteria
Image for - Molecular Detection of Multidrug Resistance Pathogenic Bacteria from Protective Materials Used By Healthcare Workers (HCW); Bangladesh Scenario
NA: Not applicable, R: Resistance, I: Intermediate, S: Sensitive

For the dramatic increase of hospital infections recently various important accessories like clothes, hands and mobile phones of healthcare workers for instance doctors, specialists and nurses were inspected. The consequences revealed that healthcare workers’ mobile phones, wear uniforms healthcare workers’ gowns, gloves and shirt sleeves possible means of bacterial dissemination between patients and hospitals personnel22-26.

Image for - Molecular Detection of Multidrug Resistance Pathogenic Bacteria from Protective Materials Used By Healthcare Workers (HCW); Bangladesh Scenario
Fig. 3: Antibiotic resistance pattern of isolated microorganism against different antibiotics

So far in our country, there are not adequate researches on the level of bacterial infections especially targeting the healthcare workers’ accessory materials. That is why, it projected to recover pathogenic bacteria from hands, mask and hand swab samples of hospital and pathology workers.

As a result of isolation of pathogenic bacteria from hospitals and pathology centers workers’ hands, Staphylococcus aureus, Pseudomonas aeruginosa, E. coli and Salmonella typhi were isolated where 49.28% were from Handler swab samples, 31.88% were from gloves swab samples and 18.84% were from masks. The rate of isolation of bacterial pathogens in the handler swab samples was higher than the face mask samples. Similar trends was reported by Chaka et al.27, Hall et al.28 and Luksamijarulkul et al.29 where bacteria were isolated from hands, gloves and other accessories of healthcare workers and wherein 78, 81.1, 15±9 CFU mL1/piece and 61.53% of hand swabs, gloves, mask and female fingernails were contaminated respectively.

Multiple drug resistance was also usual in Gram-negative and Gram-positive bacterial isolates to commonly used antibiotics in these study areas. Staphylococcus aureus, Pseudomonas aeruginosa, E. coli and Salmonella typhi were 100% resistant to imipenem and also resistance to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, methicillin, streptomycin, tetracycline, nalidixic acid, doxycycline in various ranges which is consistence with the study of Rocha et al.1 who also recovered these four types of bacteria to be multidrug resistance in different levels. In the case of S. aureus, got only 2 MRSA phenotypically which is similar to Kim and Jeong30 who also found 2 strains of MRSA from 104 samples of dentists depending on the presence of mecA gene.

According to the discussion, it can be considered that the possible source of bacterial infection from hospital system could be originated from healthcare workers’ accessories. Moreover, environment as a source for the chance of infection could not be declined, as prevalence of drug resistance bacteria is a common scenario in Bangladesh in environmental effluents. The resistance pattern observed is also a huge challenge in treating infectious diseases with the commonly available drugs. Despite a relatively high level of knowledge about the potential role of examined samples as bacterial reservoirs, the habits of hygienic practices were very poor. This poor hygiene may help to disperse the MDR bacteria to the hospitalized patient as nosocomial infection which is a great threat now a day. So, regular cleaning and disposal by health care workers and implementation of appropriate infection prevention guideline will help in reducing the possible risks associated with those accessories of health care workers and reduce rate of nosocomial infection.


This study discovers the sources of MDR bacteria from the hospital environment that can be beneficial for the patient who has admitted into the hospital and getting various lives threading nosocomial infection. Due to nosocomial infection which they have got after hospital admission every year numerous people suffer and even died due. Hospital is multidimensional environments where lots of patients come with various types of disease. So there are lots of chance to spread out contagious diseases and pathogenic bacteria from one person to another in the hospital. But knowing the sources of spreading disease one can easily prevent this spreading lots of factors may play role in this whereas HCW is one of the major as they come close enough to the patient. So by maintaining proper’s hygiene about their protective material nosocomial infection rate can be reduced at such a significant rate. This study will help the researcher to uncover the critical areas contributes to the spreading of pathogenic bacteria that many researchers were not able to explore. Thus a new theory on reducing nosocomial infection as well as pathogenic MDR bacteria dispersal may be arrived at.


We would like to thank Noakhali Private Hospital, Noakhali.

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