Occurrence of Multidrug Resistant Pseudomonas aeruginosa Isolated from Waste Water Samples of Dhaka, Bangladesh

Pseudomonas aeruginosa is a prevalent gram-negative pathogenic bacterium ubiquitous in natural environment. Aquatic environment of wastewater serves as reservoirs of this bacteria and their wide resistance phenomenon to a number of antibiotics is frequently increasing. This study was conducted to determine the prevalence of P. aeruginosa in 10 industrial waste water and 10 tannery waste water samples of whole Dhaka and 65% (13/20) water samples were found positive for P. aeruginosa which was confirmed by both biochemical test & Biolog TM Microbial Identification System. Kirby Bauer disc diffusion method was applied for antimicrobial susceptibility testing and isolates showed resistance to most of the commercial antibiotics except neomycin, gentamycin, streptomycin, ciprofloxacin and nalidixic acid, hence confirmed the multidrug resistance (MDR) of P. aeruginosa in wastewater which is one of the life-threatening public health issues all over the world causing ineffectiveness of several antibiotics. So, it is recommended to make sure surface water or food samples not to be contaminated by this antibiotic resistant P. aeruginosa that might be transferred to animal and human. In these circumstances, not only the hygiene practice is the first and foremost prerequisite but also management practices with effective wastewater disposal system can also be a part of awareness. Additionally, appropriate and logical use of antibiotics must be applied to reduce the emergence of multidrug pathogens to environment.


INTRODUCTION
Pseudomonas species are considered as a leading organism found from soil, water, food and plants.It is an aerobic, gram-negative and motile rod bacterium belonging to the family Pseudomonadaceae.Widespread occurrence of this bacterium relating to water, is an increasing concern of public health now-a-days.Transmission of Pseudomonas sp. to human body initiates foodborne illness by taking food & water contaminated with this bacterium.Some species of Pseudomonas are medically significant because they are considered as opportunistic pathogens for humans and animals while others are very important in the agricultural sector. 1 Data covering 1986-2003 from the National Nosocomial Infections Surveillance system showed P. aeruginosa is the most common cause of pneumonia and urinary tract infection. 2he occurrence of P. aeruginosa in sewage effluent sample is very common.Although number of this bacteria is variable in fresh water bodies, its level is minimum in drinking water for good management and hygiene practices.P. aeruginosa can metabolize a wide variety of compounds, and proliferate in waters with low concentrations of dissolved compounds, showing its ability to adapt in environmental challenges with minimal nutritional requirements. 3This organism's ability to form biofilms, results their protection against adverse environmental conditions and also shows tolerance and resistance to antibiotics, which are secondary metabolites produced by microorganisms and chemically synthesized/semi-synthesized molecules inhibiting proliferation of others. 4Usage of antibiotics is increasing promptly and studies say that it will reach up to 200%. 5 Besides, it has been found that, very low percentages get completely metabolized by humans and animals whereas 20-90% get excreted through urine and feces reaching and contaminating the environment. 6As a result, most Pseudomonas aeruginosa infections are difficult to treat due to high levels of antibiotic resistance.The use of antibiotic as growth promoters and feed enhancer in farming and livestock management are responsible for spreading the antibiotic resistance genes in the environment. 7Besides, Antibiotic resistance is an accommodative genetic feature present in few bacterial subpopulations and due to this, they get survival capacity rather killing under therapeutic doses. 8Because antibiotics at low concentration create selective pressure on bacterial colonies which emphasize their resistance profile. 9,10Moreover, bacteria can explore multidrug resistance (MDR) creating a challenge to give treatments in hospitals. 11Not only that, several virulence factors are spread out due to multidrug resistance (MDR) in health care centers and others community settings impacting economic and social aspects, less productivity and high poverty rate. 12onsidering the concerning fact relating to multidrug resistance of P. aeruginosa, the present study was designed to isolate and identify environmental P. aeruginosa from 2 types of waste water samples and show their antibiotic resistance pattern.Besides, pH, temperature, DO (Dissolve Oxygen), TDS (Total Dissolve Solids), TVC (Total Viable Count), TC (Total Coliform) of the collected water samples were also documented as well.

Sample collection
A total of 20 water samples (first 10 from different industry effluent sites and next 10 from different tannery sites at Savar in Hemayetpur, Dhaka) were aseptically collected for detecting microbiological and physicochemical properties.Prior to collection, bottles were washed, rinsed thoroughly several times with distilled water and then autoclaved.For the accuracy of the results, the physicochemical parameters were measured immediately from the collected samples and the samples were labeled and transported to the laboratory for microbiological analysis.

Physico-chemical analysis
Temperature is the most responsible physico-chemical factor to determine the quality of water and is measured by thermometer. 13pH measures acidity and alkalinity of a solution.pH has a major influence on bacterial population growth, whereas pH value of 7 is the neutral one. 13he DO, TDS and pH of the samples were estimated at the point of collection using portable DO meter, TDS meter and pH meter respectively.

Microbiological analysis
Collected water samples were analyzed according to the standard method of APHA 1995 (American Public Health Association).TVC was performed by serial dilution method, followed by pour plating in plate count agar (PCA) media.The plates were incubated at 37°C for 24 hours.All the experiments were performed at 37°C for 24 hours in duplicate manner.To monitor water quality, the enumeration of total coliforms (TC) was done by following Most Probable Number (MPN).Presence of P. aeruginosa was detected by inoculating 10 ml of sample into 100 ml Tryptic soy broth (TSB). 14After proper mixing, broth was subjected to incubation for 48 hours at 35-37°C.After that, a loopful growth was streaked on cetrimide agar (CEA) plate from TSB and again incubated for 48 hours at 35-37°C.30 distinct colonies exhibited fluorescent green pigment were primarily selected as P. aeruginosa.

Biochemical confirmation of selected P. aeruginosa
Selected P. aeruginosa colonies were purified for 3 months continuously in Plate Count Agar (PCA) medium and 16 biochemical tests were performed for their possible identification according to Bergey's Manual for Systematic Bacteriology, Vol. 2. 15 All tests were done in duplicate and the incubation period was 37°C for 24 hours.

Biolog confirmation of P. aeruginosa
Preliminary selected P. aeruginosa were also confirmed by Biolog TM Microbial Identification System (Biolog Inc., USA) using GEN III Micro Plate TM .24 hours bacterial cultures were swabbed by inoculators and taken in inoculation fluid supplied by the company.The solution was poured into reservoir and 100 µl was transferred into each GEN III MicroPlate TM plate well by multichannel pipette tips followed by incubation of the plates at 37°C for 24 hours.After this period, plates were placed in Biolog TM Microbial Identification System machine and read by the system software OmniLog® which gave a specific bacterial name.

Antibiotic susceptibility pattern of P. aeruginosa
The antibiotic susceptibility testing was performed on Muller Hinton agar using Kirby Bauer disc diffusion method 16 against the following commercial antibiotics-ampicillin (AMP),

RESULTS AND DISCUSSION
The initiative to create a resistance profile of waste water is a new undertaking in Bangladesh.The spread pattern of Multiple drug resistance (MDR) explores a threatening risk for human boosting up morbidity, mortality and cost. 12Untreated waste water may facilitate more spread of multi drug resistance (MDR).In the present study, the physico-chemical parameters of collected samples were measured and the results of the physico-chemical parameters of collected samples are shown in Table 1.
The pH level of samples ranged between 7.1-8.5 and the temperature was more or less 29°C.DO differed among the sampling areas, with a maximum of 4.85 ppm and a minimum of 3.17 ppm.In most of the cases, maximum dissolved oxygen concentrates vary with temperature.But for living organism, 4 mg/L (4ppm) of minimum DO should be in water otherwise living organism cannot survive. 17So, it can be said that our collected wastewater sites were not a good example for aquatic growth.
Figure 1 represents the total viable count (TVC) of waste water in Plate count agar media.Highest number of total bacterial count (5.2×10 4 ) was found in sample no 19 from tannery site.The total coliform no was also beyond the limit (>2400) that confirmed unsatisfactory water quality.P. aeruginosa was detected in 13 water samples showing fluorescent green pigment on CEA plate and among them, 30 colonies were subjected to biochemical test responses in different media.The overall same result for all selected colonies was presented in Table 2.
All strains gave positive result in citrate utilization, oxidase and catalase test.Besides, all the strains were motile and pigmented.On the other hand, these suspected colonies were found to be negative in indole test, methyl red test and VP test.Among the glucose, mannitol, sucrose and lactose, all the selected isolates were able to ferment glucose only.Finally, all the suspected colonies were biochemically confirmed as P. aeruginosa by comparing phenotypical data of pathogen with the published data of Bergey's Manual for Systematic Bacteriology, Vol. 2. 15 Moreover, the Biolog TM Microbial Identification System machine also clearly indicated the colonies as P. aeruginosa (Figure 2).
Being intrinsically resistant to several classes of antibiotics, P. aeruginosa limit the treatment choices.Unluckily now, problems with antimicrobial treatment are becoming complicated for their rising acquired or mutational resistance. 18ultiple reports of multidrug-resistant (MDR) P. aeruginosa have been found worldwide and this resistance is thought to be driven by several mechanisms including efflux mechanism, enzymatic deactivation, loss of outer membrane protein (porin) and target mutations. 19Similarly, in our study, antimicrobial susceptibility testing of P. aeruginosa showed resistance to most of the commercial antibiotics except neomycin, gentamycin, streptomycin, ciprofloxacin and nalidixic acid (Table 3).In a study of Frederick Bert 1997, 13.3% and 16.1% of P. aeruginosa were resistant to amikacin and ceftazidime respectively. 20Similarly, 66.7% resistance to ceftazidime was found in a study done by Shakir et al. 21This study was in agreement with Sulaiman and Abdulhasan who pointed on 66% resistance to ceftazidime. 22On the other hand, lower resistance against ceftazidime was found as 17.5% in Iraq. 23Another study of MM Loureiro et al., revealed high antimicrobial resistance percentage of this bacteria to ß-lactams, chloramphenicol, trimethoprim-sulfamethoxazole and tetracycline. 24Almost 91% sensitivity of P. aeruginosa against ciprofloxacin was reported by Dinesh Shubedi in 2017 whereas we found both resistant and susceptible phenotype among the isolates. 25A study done by Souli et al. shows the data from 23 countries on the European Antimicrobial Resistance Surveillance System (EARSS) on resistant rates of aminoglycosides, carbapenems, quinolones and ceftazidime antibiotics which were 0-51.9%;9-50.5%;7.2-51.9%and 4-48.5%,respectively.In this study, around 18% of the isolates were documented as multi drug resistant (MDR). 26ere are different kinds of wastewater systems, like municipal sewage systems, hospital wastewater systems which have higher risks of spreading MDR. 27In a study of Ghana, researchers found the presence of multidrug-resistant bacteria in hospital wastewater making up Escherichia coli (30.6%),Klebsiella pneumoniae (11.2%) and Pseudomonas mendocina 5.4%. 28In Saudi Arabia, Wang et al. conducted a study on a specific COVID-19 hospital wastewater and they assured antimicrobial resistance (AMR) due to the usage of antimicrobial agents during the pandemic. 29n China, various antibiotics were determined in frightening concentrations in hospital wastewater, containing high incidence of antibiotic resistance genes such as blaGES-1, qnrA, blaOXA-1, blaOXA-10 and blaTEM-1. 30

CONCLUSION
Wastewater creates great burning issues when the untreated or inadequately treated water mixes with our natural environment.As a result, number of diseases and health problems have been occurring every year.Wastewater is a potential source for irrigation purposes, but it can also be an issue of concern when a good number of P. aeruginosa are found in the water.If human or animals get exposed to P. aeruginosa from wastewater, severe infections can happen.Moreover, the chemical component discharged from industries can affect the oxygen demand, which ultimately affects the aquatic ecosystem also.Antibiotic resistant pathogen also creates emergence of antibiotic resistance which can spread through aquatic system to human body.So, it can be concluded that presence of multidrug resistant P. aeruginosa and their risk of transmission to human is an acute health issue.The most challenging part to prevent this bacterial disease outbreak is that, these bacteria are continuously changing their ways to fight against the antibiotics used to treat the infection.Our study provided information on environmental reservoirs with P. aeruginosa, recommending the importance of hygiene practice.Moreover, this work might be a good foundation for further research on multidrug resistance mechanism of P. aeruginosa and their pattern of transmission as well.

Table 2 .
Overall biochemical test results of selected isolates Lactose Negative Figure 1.Total bacterial count (cfu/ml) in PCA medium

Table 3 .
Antibiotic name, class, target and overall susceptibility test result of selected Pseudomonas aeruginosa