Antibiotic Susceptibility Profile of Staphylococcus Aureus and Micrococcus Luteus Isolated from Tap Water of Hayatabad Medical Complex and Cantonment General Hospital Peshawar
Masroor Munawar1, Muhammad Kamil Khan2, Kainatt Naeem3, Mishal Hameed4, Ihteshamul Haq5, Muhammad Shahab4, Shahzaib Khan4, Muhammad Latif4, Junaid Ahmad2*,Hazrat
Bilal6,Waseem Sajjad2,Muhammad Qasim Ail7, Muhammad Rehan Arif3
1 Department of Microbiology Sarhad University of Science and Information Technology
2Department of Microbiology Hazara University Mansehra, KPK, Pakistan.
3Institute of Food and Nutritional Sciences, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi
4Department of Microbiology, Faculty of Biological Science, Abbottabad University of Science and Technology, Abbottabad, Pakistan
5Department of Biotechnology and Genetic Engineering Hazara University Mansehra Kp Pakistan
6Institute of Health Sciences, Anhui Univeristy Hefei China.
7Institute of Food Science and Nutrition, University of Sargodha Corresponding Author: Junaid Ahmad2*
Email:[email protected]
Co-Corresponding Author: Ihteshamul Haq5*
Email:[email protected]
Abstract
The development of antibiotic resistance in pathogenic bacteria has been a problem. Hospitals are where the probability of developing multidrug-resistant (MDR) bacteria is much higher. We investigated the presence of MDR bacteria in tap water collected from Hayatabad Medical Complex and cantonment board hospital, Peshawar. Bacterial strains were isolated from tap water, and their antibiotic sensitivity profile was investigated. Six commonly used antibiotics; Co-trimoxazole, gentamycin, clindamycin, augmentin, erythromycin, and ciprofloxacin. Two bacterial strains were isolated from the tap water and identified as Staphylococcus aureus and Micrococcus luteus. Both the bacterial strains were found resistant to clindamycin and augmentin antibiotics. Micrococcus luteus also showed resistance to clindamycin while sensitive to the rest of the antibiotics used during the experiment. The antibiotics gentamycin, erythromycin, and ciprofloxacin were highly effective against the isolated bacterial strains. Further investigation is suggested to find how these bacteria reach hospitals' sources and storage tanks. It is also indicated that tap water from other hospitals should be investigated for MDR bacteria.
Keywords: Multidrug resistance, bacteria, tap water, Hospital
Introduction
Staphylococcus aureus and Micrococcus luteus are gram-positive and nosocomial pathogenic bacteria that cause skin infections, meningitis, arthritis, bacteremia, pneumonia, food poisoning, and endocarditis by facilitating tissue attachment, invasion, and evading from host immune response. A methicillin-resistant S. aureus (MRSA) may increase the treatment cost, and high mortality and
morbidity occur. However, new antibiotic discovery may also treat MRSA if not misused (Gnanamani, Hariharan, & Paul-Satyaseela, 2017). The researcher also reported that 17 previous cases of infective endocarditis are due to Micrococcus species (Khan, Aung, & Chaudhuri, 2019).
Antibiotic is a group of drugs that either kill or stop bacterial infection. In recent years the patient was first treated with antibiotics, due to which the antibiotic was misused and overused, and therefore bacteria become resistant and threat to public health (Spellberg & Gilbert, 2014). The microbial infection management was 1st documented in China and Greece. It was first discovered by Alexander, due to which it saved many lives because it treated serious bacterial infections in world war2 (Sengupta, Chattopadhyay, & Grossart, 2013). But soon after, those bacteria became resistant, and the need for new beta-lactam antibiotic drugs was discovered. In response to those, MDR bacteria were reported in America and soon after that pharmaceutical industry introduced some other new drugs to solve that problem(Sengupta et al., 2013; Spellberg & Gilbert, 2014). Antibiotic can save many patients live because it treats chronic infection like diabetes, end-stage renal disease, or rheumatoid arthritis (Gould & Bal, 2013). Moreover, it is also used in complicated surgery like organ transplants, joint replacements, or cardiac surgery (Gould & Bal, 2013; Wright, 2014).
The epidemiological studies show that resistance may be occurring by mutation and increased in many countries due to overuse of antibiotics available over the counter without prescription(Michael, Dominey-Howes, & Labbate, 2014). In bacteria, these resistance genes can be inherited by vertical and horizontal gene transfer(Read & Woods, 2014). The study reported that 30%-60% of antibiotics are incorrectly prescribed and used for ICUs, which increases resistance in bacteria (Lushniak, 2014;
Luyt, Bréchot, Trouillet, & Chastre, 2014).In many countries, i.e. the U.S, 80% of antibiotics are used in livestock, which humans ingest to prevent infection that improves the animal's health and produce a higher quality of yields product (Golkar, Bagasra, & Pace, 2014). The antibiotic in these countries is sold for hygienic purposes in pesticides. It also affects environmental microbes and limits the development of immunity in children and adults (Golkar et al., 2014; Michael et al., 2014).
The molecular study shows that resistance properties of bacteria, i.e. normal flora, were first reported in farm areas to humans when they consumed meat products (Bartlett, Gilbert, & Spellberg, 2013).
Due to economic crises in the society of pharmaceutical companies, a few antibiotics have been discovered (Bartlett et al., 2013; Piddock, 2012). Several new antibiotics have been cancelled due to the health regulatory authority (Gould & Bal, 2013; Piddock, 2012). Most of the antibiotic resistance bacteria (ARB) that are accelerated by antibiotic pressure are transferred from the hospital by the interactions of patients and Health Care Worker. Thus, their specific character and behaviour should be influenced. Many researchers reported that ARB might increase or decrease depending on the antibiotic class (Almagor et al., 2018). The antibiotic treatment may transmit the ARB when the patient misuse antibiotic due to which normal flora microbes eradicated and then ARB colonization occur. Thus 80% of colonized ARB-related patients are not treated with antibiotics(Pamer, 2016).The main aims and objectives of that study were to evaluate the antibiotic susceptibility profile of isolated bacterial strains from tap water in Hayatabad Medical Complex and Cantonment General Hospital.
Material and methods Sample collection
150 Tap water samples were collected from different wards of Hayatabad Medical Complex (HMC) and Cantonment General Hospital (CGH) Peshawar in sterilized glass bottles. The bottles were labelled properly, transported within an hour to the laboratory, and kept at 4℃ for analysis.
Serial dilution
Serial dilutions of each sample were made separately. Six test tubes that contained 9ml sterilized distilled water were taken. 1ml of the sample was mixed in the first test tube, then transferred 1ml of the dilution to the second test tube, then transferred 1ml dilution of the second test tube to the third test tube, and so on.
Isolation of Staphylococcus aureus and Micrococcus luteus
Nutrient agar and Mannitol Salt Agar (MSA) media isolated S. aureus and Micrococcus luteus. Each medium was prepared in an Erlenmeyer flask, sterilized in an autoclave at 121 ℃ for 15 mins, and then poured into Petri plates. After solidification, each dish was inoculated with 100 µl from10-2, 10-
4, and 10-6 dilution. The spread plate method was used to spread the inoculums on each media plate using a sterilized glass spreader. The plates were then incubated for 24hours at 37℃ temperature for bacterial growth.
Pure Culture preparation
The next day colony was observed, and again Nutrient agar and MSA media were prepared and used to prepare pure culture. Each colony was then selected and streaked on nutrient agar and MSA plates.
The plates were incubated at 37℃ for 24 hours to get pure culture.
Morphological and biochemical identification of S. aureus and Micrococcus luteus
The isolated bacteria, i.e. S. aureus and Micrococcus luteus, were examined by gram's staining test to differentiate between gram-positive and gram-negative bacteria and their morphology. Further identification of S. aureus and Micrococcus luteus was made by performing a series of biochemical tests using the taxonomic scheme of Bergey's Manual of Determinative Bacteriology such as Coagulase, Catalase, TSI, Oxidase, Indole Mannitol, and Lactose fermentation test.
Antibiotic susceptibility determination
An antibiotic susceptibility test was performed using the Kirby-Bauer disk diffusion method. Muller Hinton Agar has been accepted for international recognition for antimicrobial susceptibility testing.
The different antibiotic discs at the final concentrations that are indicated were used, i.e.
erythromycin, clindamycin, ciprofloxacin, gentamycin, augmentin, and cotrimoxazole. All these antibiotics were obtained from a local pharmacy store. The pure culture was picked from each sample, and each colony was transferred into 3 ml of sterile distilled water to prepare a bacterial suspension. Aliquots of 100μl from each suspension were spread-plated on Mueller-Hinton agar plates. Antibiotic discs were applied onto the plates using sterile needles, and the plates were incubated at 37°C for 24 hours. After incubation, the antibiotic inhibition zone diameters were measured, and the result was noted according to the Clinical and Laboratory Standards Institute (CLSI).
Result
Isolation of bacteria from hospital tap water
A total of 180 isolates were obtained from tap water samples collected from different wards of Hayatabad medical complex and Cantonment General Hospital Peshawar. Gram staining was done to observe the morphological character of all isolates. The gram staining result shows that all the isolated bacteria were gram-positive, as given in Table 1 and Fig 1. Table 1 presents the percentage presence of isolated bacterial strains.
Table 1. Show gram staining and occurrence of bacterial culture in the water sample.
S. No Bacterial isolates Gram reaction No. of isolates Percentages
1 Staphylococcus aureus Positive 150 83.33%
2 Micrococcus luteus Positive 30 16.99%
Total 180 100%
Figure 1. Show gram staining of gram-positive bacteria (A) Staphylococcus aureus and (B) Micrococcus luteus.
Identification of bacterial species through the biochemical test
These isolates were identified as Staphylococcus aureus and Micrococcus luteus through biochemical tests (Catalase test, Oxidase test, Indole, Coagulase test, TSI, Mannitol test, and Lactose fermentation test), morphology, and gram staining, as shown in Table 2. Figures 3-6 show the results of various biochemical tests.
Table 2. Biochemical and morphological characterization of isolated strains.
S. No. Biochemical test Staphylococcus aureus Micrococcus luteus
1 Catalase Positive (+) Positive (+)
2 Coagulase Positive (+) Negative (-)
3 Mannitol Negative (-) Positive (+)
4 Lactose test Negative (-) Negative (-)
5 Oxidase Negative (-) Positive (+)
6 Indole Negative (-) Negative (-)
7 TSI A/A A/K
Key: A/A: (acidic slant/acidic butt), A/K: (acidic butt/alkaline slant), TSI: (Triple Sugar Iron).
Figure 2. Showing catalase test performed for bacterial isolates
Figure 3. Showing coagulase test performed for bacterial isolate
Figure 4. Showing mannitol test for bacterial isolates
Figure 5. Showing lactose test for bacterial isolates
Antibiotic susceptibility profile
Table 3 and fig. 7 and 8 show the antibiotic susceptibility patterns of each isolate. Staphylococcus aureus was highly susceptible to Gentamycin, Erythromycin, and Ciprofloxacin and resistant to Clindamycin and Augmentin antibiotics while showing intermediate sensitivity to Co-trimoxazole.
Moreover, Micrococcus luteus was highly sensitive to Gentamycin, Erythromycin, and Ciprofloxacin while resistant to Co-trimoxazole, Clindamycin and Augmentinantibiotic.
Table 3. Antibiotic susceptibility profiles of bacterial isolates S.No. Antibiotic Mean diameter of inhibition zone
Staphylococcus aureus Micrococcus luteus
1 Cotrimoxazole 18 mm 8 mm
2 Gentamycin 30mm 25mm
3 Clindamycin 5 mm 6 mm
4 Augmentin 1 mm 5mm
5 Erythromycin 28 mm 28mm
6 Ciprofloxacin 28 mm 28mm
7 Ceftriaxone 18 mm 15 mm
Note:Zoneofinhibition:0.13mm=resistance,14-17mm=intermediate,18mm and above =sensitivity.
Figure 6.Antibiotic susceptibility profile of Staphylococcus aureus
Figure 7. Antibiotic susceptibility profile of Micrococcus luteus
Discussions
The emergence of resistance in pathogenic bacteria against antibiotics has become a major public health concern. The extensive use of antibiotics in hospitals is considered one of the causes of spreading resistance in pathogenic bacteria (Xi et al., 2009). Several investigators have isolated antibiotic-resistant bacterial strains from solid and liquid waste hospitals (Adnan, Sultana, Islam, Nandi, & Hossain, 2013). There are chances that pathogenic bacteria could get entry into the hospital drinking water sources and storage tanks. We have investigated the presence of multidrug-resistant pathogenic bacterial strains in tap water collected from HMC and CGH in Peshawar. We have isolated two pathogenic bacterial strains, i.e. Staphylococcus aureus and Micrococcus luteus, which showed resistance to different antibiotics used during the experiments. Both the isolated species are pathogenic and have produced several infections in humans(Gnanamani et al., 2017). Previously, these bacteria have been isolated from hospital wastewater (treated and non-treated), but we have separated them from hospital tap water in the current study.
Kalaiselviet al. (2016) isolated Staphylococcus aureus strains from hospital-generated recycled water and checked their antibiotic resistance profile. They found that the isolated S. aureus was resistant to several antibiotics, including gentamycin, erythromycin, and ciprofloxacin (Kalaiselvi, Mangayarkarasi, Balakrishnan, & Chitraleka, 2016). Our results also show that the isolated Staphylococcus aureus was resistant to ampicillin and cotrimoxazole while susceptible to gentamycin, clindamycin, Ofloxacin and Ciprofloxacin. In another study, Thompson et al. (2013) isolated S. aureus from the hospital environment, resistant to ciprofloxacin and gentamycin. The bacterial Micrococcus luteus were found resistant to Augmentin, Co-trimoxazole and Clindamycin.
The implication of S. aureus in toxic shock syndrome generates immediate concern about the possibility that drinking water may be a source of this syndrome (Thompson, Gündoğdu, Stratton, &
Katouli, 2013).
The presence of antibiotic-resistant bacteria in tap water may be due to inadequate/ unhygienic sanitation systems. The prevalence of antibiotic-resistant bacteria poses a great challenge to clinicians, and the consumption of water containing these antibiotic microbes may prolong the treatment of water-borne diseases. This implies that treating water-borne diseases with these antibiotics may be inappropriate and require new and most expensive antibiotics. This study recommended that stringent measures be taken to prevent their occurrence.
Conclusion
Emergences of resistance in bacteria against antibiotics have compromised the effectiveness of
antibiotics used against these bacteria. From this study, it has been concluded that tap water was collected from Hayatabad medical complex and cantonment board hospital Peshawar contain MDR bacterial strains and is not safe for drinking and washing purposes.The bacterial strains S. aureus and M. luteusare pathogenic, and their presence in hospital tap water is a serious public health concern.
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