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Isolation and characterization of antibiotic resistance bacteria in hospital

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Isolation and Characterization of Antibiotic Resistance Bacteria in Hospital Effluents

1Asad Ud-Daula, 1Abdur Rakib, 1Md. Hafizur Rahman, 2Md. Sabir Hossain,

2Md. Ibrahim Hossain, 3Md. Fuad Hossain and 2Md. Ibrahim Khalil 1Department of Applied Nutrition and Food Technology,

Islamic University, Kushtia-Jhenidah, Bangladesh. 2Department of Biochemistry and Molecular Biology,

Jahangirnagar University, Savar, Dhaka-1342, Bangladesh. 3Department of Microbiology, Jahangirnagar University,

Savar, Dhaka-1342, Bangladesh. E-mail: fuadhossain777@gmail.com, drmikhalil@yahoo.com

Abstract:-Antibiotic resistance has become a major clinical and public health problem within the lifetime of the peoples. The current worldwide increase in resistant bacteria and simultaneously, the downward trend in the development of new antibiotics have serious implications. This study isolated and characterized different antibiotic resistance bacteria present in the hospital effluents of Kushtia district in Bangladesh using selective growth medium in presence of amoxicillin, tetracycline and penicillin at different concentration. Salmonella spp, Escherichia coli and Shigella were found to be resistant to these antibiotics. It also finds that amoxicillin is more resistant to the microorganisms as compared to other two antibiotics. This study also showed that the gram-negative acquired resistant to antibiotics rather than gram-positive bacteria in the hospital effluents. Therefore a high level of antibiotic resistance bacteria is present in the hospital effluents which can be a big health hazard to the human beings. Key words: antibiotics, resistant bacteria, hospital effluents, municipal waste water

INTRODUCTION Antimicrobial resistance is a major public health crisis, eroding the discovery of antimicrobials and their application to clinical medicine. Antibiotics are used extensively to prevent or to treat microbial infections in human and animal being. Antibiotics are partially metabolized by patients and are then discharged into the hospital sewage system or directly into municipal waste water if used at home. Along with excreta, they pass through the sewage system and end up in the environment, mainly in the water compartment. According to the Centers for Disease Control and Prevention estimates, about 22000 tons of antibiotics are produced annually in the United States alone, 50% of it is dispensed to humans [1]. Residues of up to 10 different drugs have been found in sewage water at ≈ 6 ppb concentrations. Another research reported 30 to 60 drugs can be measured in a typical water sample [2]. A number of analysis reported that the simple of pit latrine, ventilated pit latrine, and pour-flush latrine, and the more advanced septic tank with soakaway or the aquaprivy contains many antibiotics while 25% contains three or more antibiotics such as sulfamethoxazole, trimethoprim, ciprofloxacin, ofloxacin, lincomycin, and penicillin G [3,4,5]. Among a wide variety of pharmaceutical compounds, antibiotics assume special significance due to: i) their extensive use (> 50,000,000 lbs produced annually in the US) in human therapy and veterinary medicine[6], ii) contribution from numerous sources (sewage treatment plants (STPs), confined animal feeding operations,

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(CAFOs)), iii) their ability to alter microbial community structure facilitating the development of antibiotic-resistant human pathogens[7], and iv) the potential to serve as indicators for the presence of resistant pathogens. Unused therapeutic drugs, antibiotics and disinfectants are present in the effluent of hospitals [8]. Ciprofloxacin, for example, was found in concentrations of between 0.7 and 124.5mg/L in hospital effluent and was assumed to be the main source of genotoxic effects measured with the umuC test in hospital effluent [9]. Ampicillin was found in concentrations of between 20 and 80 mg/L in the effluent of a large German hospital [10]. Quinolones and other pharmaceuticals have been detected in the effluents of hospitals up to l g/lit [9,11,12]. The occurrence of ß-lactams (including penicillins, cephalosporins carbapenems, monobactams, ß-lactamase inhibitors), has not been covered frequently, despite the fact that ß-lactams account for by far the highest proportion of consumption [13,14]. In one study ß-lactams were detected in hospital effluent [14]. Therefore, the continuous exposure of antibiotics to microorganisms especially bacteria become resistant to one or more antibiotics, they do not respond to therapy. Some bacteria are naturally resistant, whilst others become resistant following selection after prolonged antibiotic use. In addition unconscious misuse of these valuable therapeutic drugs to the sewage also produces resistance bacteria which cause threat to the human. These resistant bacteria can be transmitted from different predator and food-producing animals such as cattle, pigs and poultry, and the environment to humans, via the food chain. Thus, the emergence and dissemination of antibiotic resistant bacteria in hospitals and environments is an emerging challenge to humankind. The route of dissemination of resistant bacteria from patients to the environment was reported recently [15]. There are a handful of studies indicating that the wastewater treatment process is one of the routes disseminating antibiotic resistant bacteria into the environment [16,17]. Other literature claimed that the multi-drug resistant bacteria spread from hospital effluents to the municipal sewage system [18] and The presence of high numbers of resistant bacteria was due to the selection pressure of antibiotics released from the hospital into the sewage discharge system [19,20]. In the present thesis antimicrobial resistance bacteria have been isolated from hospital effluents of Kushtia district. The commonly prescribed antibiotics in Bangladesh such as amoxacillin, tetracycline and penicillin were applied in this study to isolate resistance bacteria in hospital effluents. The aim of the study is to isolation and characterization of antibiotic resistance bacteria of hospital effluents using selective growth medium. MATERIAL AND METHODS Instruments and Reagents The common laboratory instruments were used for this study is Laminar air flow (Heal Force, Model-H F safe7605, China), Autoclave (HIRAYAMA, Japan) and Incubator (Memmrt, Model-400, Japan). In addition the culture media such as nutrient broth medium (NB), nutrient agar media (NA) and selective media such as Salmonella Shigella Agar (SS-Agar), MacConkey agar, Muller Hinton agar and XDL agarwere used for this experiment and all media were purchased from Hi-Media, India. All media were prepared according to supplier guideline. Sample description Hospital effluents were collected in 500 ml bottle from Kusthia general hospital. The collection was usually made in early hours of the morning and transported to the laboratory within 2 hours for analysis. It is a mixture of water and dissolved or suspended solids and biological characteristics of human and animal origin. It also contains organisms such as bacteria, fungi, algae, protozoa, and viruses [21]. These organisms have potential concern due to the presence of disease causing pathogenic bacteria.

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Preparation of stock solution of antibiotics The stock solution of tetraclylline (Square, Bangladesh), amoxicillin (ACME Lab. Ltd., Bangladesh) and penicillin (Opsonin Pharma, Bangladesh) were prepared respectively by dissolving 300 mg of them in nutrients broth medium in a 250 ml volumetric flask. The antibiotic was allowed to dissolve completely within 5 min. The volumetric flask was capped and stored at room temperature as recommended by the manufacturer. Isolation of antibiotic resistance of Bacteria Bacteria in the hospital effluents was grown in the liquid broth medium but here all types of bacteria and microbial community was presented in the culture. This culture was used as a starter for the subsequent steps of study. Three broad spectrum antibiotics namely; amoxicillin, penicillin and tetracycline were diluted to 12 ppm, 6 ppm, 3 ppm, 1.5 ppm, 0.75 ppm and 0.375 ppm in nutrient broth medium. 100 ml nutrient agar solution including precise dose of antibiotics was transferred into sterile 250 ml conical flask. Then 1 ml bacterial starter was inoculated into the conical flask and aseptically cultivated for 48 hours at 37oC. A crude culture of resistant bacteria was found. Then 20 µl aliquots of bacteria were inoculated on petridishes containing 10 ml growth media. The petridishes were placed in inverted position in the incubator for 24 hours at 37 0C for growth. The entire procedure was done in sterile condition. Preparation of bacterial sub-culture The bacterial sub–culture was prepared by streak plating technique to see whether they are alive or not. The petridishes containing 12 ppm concentration of tetracycline resistance bacteria was taken and opened. Bacterial inoculums was taken by streaking sterile loop and then transferred into a conical flask containing 100 ml sterile nutrient broth medium and subsequently cultivated at 37 0C for 24 hours. The entire procedure was done aseptically. The above procedure was also followed for petridish containing 12 ppm amoxicillin and penicillin resistance bacteria. Isolation of E. Coli MacConkey agar media was used for the E. coli conformation test. It’s slight reddish in color. MacConkey agar supports the growth of E. coli with inhibiting the growth of other bacteria. 10 ml MacConkey agar was transferred into sterile Petridishes. 20 µl aliquot of sub-culture of amoxycillin, penicillin and tetracycline resistance bacteria were transferred onto the petridishes respectively and spread them carefully. The petridishes were placed in inverted position in incubator for 24 hours at 37 0C. Isolation of Salmonella and Shigella species Salmonella Shigella agar (SS-agar) is a selective medium used to enumerate Salmonella and Shigella species. This media supports growth for Salmonella and Shigella species with inhibiting the growth of other bacteria. 10 ml ss-agar was transferred into sterile Petridishes. 20 µl aliquot of sub-culture of amoxycillin, penicillin and tetracycline resistance bacteria were transferred onto the petridishes respectively and spread them carefully. The petridishes were placed in inverted position in incubator for 24 hours at 370C. Isolation of salmonella species XDL agar media was used for the Salmonella conformation test. In spite of the presence of agar, extra agar was added to make the media more solid. XDL agar media was prepared in the same way as SS-agar media. XDL agar supports the growth of Salmonella with inhibiting the growth of other bacteria. 10 ml XDL agar media was transferred into sterile petridishes. 20 µl aliquot of sub-culture of amoxycillin,

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penicillin and tetracycline resistance bacteria were transferred onto the petridishes respectively and spread them carefully. The Petri dishes were placed in inverted position in incubator for 24 hours at 37 0C. Isolation of Neisseria and Moraxella species Müller-Hinton agar is microbiological growth medium that is commonly used for antibiotic susceptibility testing. It is also used to isolate and maintain Neisseria and Moraxella species as conformation test. 10 ml Hinton agar media was transferred into sterile petridishes. 20 µl aliquot of sub-culture of amoxycillin, penicillin and tetracycline resistance bacteria were transferred onto the petridishes respectively and spread them carefully. The Petri dishes were placed in inverted position in incubator for 24 hours at 37 0C. RESULTS Isolation of antibiotic resistant bacteria in the hospital effluents The bacteria grown in the liquid broth medium were aseptically transferred by a sterile loop into another broth medium containing three antibiotic tetracycline (TE), amoxicillin (AM) and penicillin (PE) respectively in a 250 ml conical flask at concentrations of 12 ppm, 6 ppm, 3 ppm, 1.5 ppm, 0.75 ppm and 0.375 ppm respectively. These bacteria in the presence of antibiotic were subjected to further growth at 370C in an incubator for 24 h. After the incubation time clearly showed that some bacteria were grown which indicates the resistant bacteria. Higher dose of antibiotics in nutrient agar medium showed few colonies of bacteria and lower dose of bacteria showed more colonies of bacteria. A comparatively strong resistance bacterium in response to 12 ppm antibiotics was used to characterization of bacteria. Higher dose (12 ppm) was chosen because this dose supposed to more potent to inhibit the growth of bacteria but bacteria still presence after treatment of this dose is considered to highly resistance to antibiotics. Characterization of the resistance bacteria This experimental bacterium was sub-cultured by streak plating technique. The antibiotic resistance bacteria in response to higher concentration of antibiotics (12 ppm) of AM, TE, and PE aseptically transferred onto petridishes containing selective growth media. When MacConkey's agar medium was used as a selective media to inhibit the growth of Gram-positive bacteria due to the presence of crystal violet and bile salts. Gram-negative bacteria grow well on MAC by utilizing lactose available in the medium. Lac+ (lactose fermenters) bacteria such as Escherichia coli, Enterobacter and Klebsiella will produce acid, which lowers the pH of the agar below 6.8 and results in the appearance of red/pink colonies. The bile salts precipitate in the immediate neighborhood of the colony, causing the medium surrounding the colony to become hazy. Non-lactose fermenting bacteria will be colorless. Bacteria produce red/pink colonies in presence of three antibiotic thus claim that Escherichia coli, Enterobacter and Klebsiella bacteria were present in the subculture as well hospital effluents which acquired resistant against these antibiotics as presented in Fig. 1.

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Figure-1: E. coli resistant bacteria grown in the MacConkry agar showing bright, pink or red colonies. The petridish A, B and C indicates the tetracycline, amoxicillin and penicillin resistance bacteria respectively.

SS-agar (salmonella shigella agar) is the selective medium recommended for the isolation of Salmonella and Shigella species. Here lactose is the fermentable carbohydrate of bacteria. Brilliant green, bile and high concentrations of thiosulfate and citrate largely inhibit the gram-positive accompanying microbial flora. Thiosulfate in combination with iron acts as an indicator for hydrogen sulfide production which is indicated by blackening in the centres of the colonies. The presence of coliform bacteria is established by detecting degradation of lactose to acid with the pH indicator neutral red. Lactose-negative colonies are colorless. Lactose positive colonies are pink to red. Resistance bacteria in response to higher concentration (12ppm) of tetracycline antibiotics were collected and promptly inoculated onto SS-agar plate. After overnight incubation of bacteria produces blacken in centers of the colonies, colorless and pink or red colonies on the petridish indicates Salmonella and shigella. Similarly, amoxicillin and penicillin resistance Salmonella and Shigella bacteria were selected by producing blacken in centers of the colonies, colorless and pink or red colonies on the petridishes shown in the Fig-2.

Figure-2: Salmonella and shigella resistance to tetracycline grown in the SS-agar showing blaken in centers of the colonies and pink or red colonies. The petridish A, B and C indicates the tetracyclin, amoxicillin and

penicillin resistance bacteria respectively. XLD agar is a selective growth medium used in the isolation of Salmonella and Shigella species from clinical samples. It has a pH of approximately 7.4, leaving it with a bright pink or red appearance due to the indicator phenol red. Sugar fermentation lowers the pH and the phenol red indicator registers this by changing to yellow. Most gut bacteria, including Salmonella, can ferment the sugar xylose to produce acid;

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Shigella colonies cannot do this and therefore remain red. After exhausting the xylose supply Salmonella colonies will decarboxylate lysine, increasing the pH once again to alkaline and mimicking the red Shigella colonies. Salmonellae metabolize thiosulfate to produce hydrogen sulfide, which leads to the formation of colonies with black centers and allows them to be differentiated from the similarly colored Shigella colonies. Resistance bacteria in response to higher concentration of tetracycline antibiotics were incubated overnight; appearing colorless and pink or red colonies on the petridish indicates Salmonella and Shigella. Similarly amoxicillin and penicillin resistance Salmonella and shigella bacteria were selected by producing colourless and pink or red colonies on the petridishas shown in the Fig-3.

Figure-3: Salmonella and Shigella resistance to tetracycline grown in the XLD agar using showing red colonies, some with black centers. The petridish A, B and C indicates the tetracyclin, amoxicillin and

penicillin resistance bacteria respectively. Muller-Hinton agar is a microbiological growth medium that is commonly used for antibiotic susceptibility testing. It is also used to isolate and maintain Neisseria and Moraxella species. But there were no growth bacteria in this media. Resistance bacteria in response to higher concentration of Tetracycline, amoxicillin and Penicillin antibiotics were collected and promptly inoculated onto Muller-Hinton agar plate. After overnight incubation, there were no growth bacteria colonies on the petridish. DISCUSSION Antibiotic resistance is a major public health threat, and the presence of resistant organisms in environmental waters is an emerging concern around the world. However, high incidence of antibiotics resistant bacteria in this study region appeared to be analogous to what was predicted by many previous studies. Indiscriminate use of antibiotics, lack of proper knowledge and negligence toward disease increase the occurrence of antibiotics resistant bacteria isolates in the hospital effluents. In Bangladesh there is clear evidence of abuse of antibiotics for which emergence of multi-drug resistant bacteria are continuously increasing[22]. In the present study, an attempt was made to isolation and characterization of antibiotic resistance bacteria of hospital effluents samples of the Kusthia general hospital using antibiotic sensitive tests. The subculture of the bacteria in selective media clearly demonstrates that the colifirm bacteria E. coli, Salmonella and Shigella are reasistant to tertracycline, amoxaciline and penicillin. It was also found that the amoxicillin and penicillin are the more resistance to these bacteria than tetracycline.

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Examination after 24 hours of incubation revealed considerable differences between the antibiotics and also differences in the degree of susceptibility to the antibiotic. There was also a high degree of resistance to amoxicillin. The same result was reported in previous study and indicated that the highest resistance rates were found in E. coli strains of a sewage treatment plant which treats not only municipal sewage but also sewage from a hospitals [23]. Accordingly, hospital waste effluent could increase the numbers of resistant bacteria in the recipient sewers by both mechanisms of introduction and selection for resistant bacteria [24]. The volume of antibiotics used in hospitals and private households released into effluent and municipal sewage indicates a selection pressure on bacteria [19]. Waste effluent from hospitals contains high numbers of resistant bacteria and antibiotic residues at concentrations able to inhibit the growth of susceptible bacteria [25,26]. From the study it was found that three antibiotics were resistant to bacteria. Such high incidence of multidrug resistance may presumably be due to indiscriminate use of antibiotics at the present time, which may eventually the drug sensitive microorganisms from antibiotic saturated environment [20]. The isolates were found to be resistant to three antibiotics including some potent and reliable drugs. Therefore, if the current practice of the use of antibiotics is uphold, emergence of resistant bacteria to these useful drugs will follow and soon a grave situation will arise when there will be no drug to treat life threaten infections caused by these resistant bacteria. Hospital effluents isolates were resistant to amoxicillin. These results suggest a high frequency of amoxicillin resistant among bacteria. On the other hand penicillin and tetracycline was also found to be resistant. The higher prevalence of resistance to antimicrobial agents in this environment could be due to widespread, indiscriminate use of antibiotics. The formulation and implementation of a national drug policy by governments are fundamental to ensure rational drug use. Proper use of drugs has to be promoted by providing objective information and training. One limitation of this study was failure to select all antimicrobial agents commonly used for resistance evaluation. Those antimicrobial agents not included into this study were either due to being not used for routine susceptibility tests or not available during the study period. In brief, this study showed that the hospital effluents had high antibiotic resistance isolates. The low efficacy of hospital effluents treatment may contribute to the dissemination of such multi-drug resistant bacteria from the hospital to the environment by draining those bacteria into the city sewage pool or directly into the water bodies such as lakes, rivers or oceans. The fate of sewage isolates should be investigated in the future in order to assess the impact of hospital effluents on the spread of antimicrobial resistance in the community. CONCLUSION Antibiotic resistant bacteria have been a source of ever-increasing therapeutic problem with profound health. The current worldwide increase in resistant bacteria and, simultaneously, the downward trend in the development of new antibiotics have serious implications. Antibiotic resistance bacteria were isolated from hospital effluents. The levels of resistance of bacteria to various antibiotics differed considerably. Hospital effluents samples were collected in Kushtia district. The antibiotics included for the study were amoxicillin, tetracycline and penicillin. The most frequently identified bacteria were Salmonella spp, Escherichia coli and shigella which found to be resistant or highly resistant to amoxicillin, tetracycline and penicillin in the hospital influent. From the present investigation we can conclude that the release of wastewater from the hospital under study was associated with an increase in the prevalence of antibiotic resistance. According to the results of this study, factors other than the indiscriminate use of antibiotics in human medicine may disrupt the microbial balance in favor of resistant bacteria.

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