Hospital Acquired Pathogenic Escherichia coli from Clinical and Hospital Water Samples of Quetta Balochistan

A study was conducted to determine the prevalence and drug resistance of Escherichia coli present in urinary tract infected patients and hospital drinking water. A total of eighty urine samples from clinically suspected patients and thirty tap water samples from hospital vicinity were collected and analyzed for the presence of E. coli. The isolates were preliminary identified based on morphological characteristics, biochemical test and further confirmed by polymerase chain reaction (PCR) using uidA primer. Isolates were subjected to antibiogram studies and analyzed for the presence of drug resistance (ESBL blaCTX-M-15, tetA, and TMP-SMX dfrA1) and pathogenicity associated pyelonephritis-associated pili (PAP) and Heat-labile (LT) toxin genes. Urine samples 19/80 (23.75%) and water samples 8/30 (26.7%) were found contaminated with E. coli. It was found that 12/19 (63%) bacterial isolates were extended spectrum beta-lactamase (ESBL) producers in clinical and 6/8 (75%) in water isolates whereas tetracycline resistance in clinical and water isolates was 11/19 (58%) and 6/8 (75%), respectively. The trimethoprim resistance gene was confirmed in 12/19 (63%) in clinical and 2/8 (25%) in water isolates. All the clinical and water isolates were found carrying pili PAP gene. It was concluded that the presence of drug resistant and pathogenic E. coli in clinical and water samples is extremely alarming for public health due to cross contamination and bacterial transfer from clinical samples to water and vice versa.


Introduction
Antibiotics are essential tool against pathogenic bacterial infections but the rapid emergence of antibiotic resistant pathogens is currently creating an alarming situation [1]. Drug resistance is a global public health concern, associated with more than 700,000 deaths annually [2]. is growing threat necessitates the action to prevent the emergence of new resistant bacterial strains and the spread of existing ones in the environment from humans and animals [3]. Antibiotic resistant bacteria have been found in wastewater, surface water, and drinking water. Water is regarded to be the most important medium for the transmission of antibiotic resistance. is is especially true in low-income nations, where lack of sanitation and hygiene practices aggravates the problem [4]. Hospital sanitation water is associated with the presence of antibiotic residues and resistant microorganisms which are delivered into the aquatic ecosystem [5]. Each year, hospitals in Pakistan are roughly generating 25,000 tons of wastes with antibiotic residues ranging from 1.4 g/L to 236.6 g/L, which is thrown into surface water untreated [6]. Due to lack of appropriate management, drinking water is regularly mixed with sewage water and contaminated hospital effluent that poses a serious threat to the human population. In such conditions, the presence of antibiotic resistant bacteria increases the possibility of horizontal genetic transmission between microbes, leading to the emergence of multidrug resistant bacteria [7,8]. E. coli can easily acquire drug resistance genes and is responsible for a variety of human diseases, including traveler's and infantile diarrhea, urinary tract infections, septicemia, hemolytic uremic syndrome, newborn meningitis, and pneumonia [9,10]. E. coli is a commensal, opportunistic pathogen that is found in animals' gastrointestinal tracts as well as in the environment, such as in water and soil [11]. In this work, E. coli bacteria isolated from clinical and water sources were evaluated for drug resistance pattern and virulence potential. e aim of this study was to determine the prevalence of pathogenic and drug resistant hospital acquired E. coli from clinical and hospital water samples. e findings of this study will be useful globally in antibiotic treatment and monitoring the resistance patterns over time in developing and underdeveloped regions.

Methodology
2.1. Sampling. Urine samples (n � 80) were collected aseptically in sterile sampling containers from the patients having clinical signs such as pelvic pain, burning sensation during urination, high fever, and blood in urine. All the samples were collected using convenience random sampling technique from Bolan Medical Complex Hospital and Provincial Sandeman Hospital Quetta, city of Pakistan from April to December 2018, following all the safety procedures and protocols. Informed consent was obtained from all the participants.
Drinking tap water samples (n � 30) were collected from hospital vicinity aseptically using sterilized glass bottles. All the samples (clinical and water) were transported to Center for Advanced Studies in Vaccinology and Bacteriology, University of Balochistan, and during transportation, temperature (0-4°C) was maintained. e samples were immediately (within 6 h of collection) processed for the isolation of target pathogen E. coli.

Isolation and Identification.
e clinical samples were streak directly over the surface of sterile MacConkey agar (MCA) (Oxoid, UK) plates, while the water samples were first enriched in lactose broth for 6-16 h at 37°C, then transferred to MCA, and incubated at 37°C for 24 h [12].
Suspected lactose fermenting pink colonies from MCA were transferred to Eosin Methylene Blue (EMB) agar (Oxoid, UK) and incubated at 37°C for 16-24 h [13]. Colonies with green metallic sheen and typically resembling E. coli were further characterized. Gram's staining, Catalase, Oxidase, Sulfide, Indole Motility, Methyl Red, Voges Proskauer, Simmon citrate utilization, and urease tests were performed for the biochemical conformation of the E. coli isolates [12].

Molecular Identification.
e preliminary identified E. coli isolates were subjected to molecular identification using E. coli UidA primer that amplifies 147 bp gene size product [14]. Bacterial DNA was extracted by using the DNA Mini Kit (Qiagen), and polymerase chain reaction (PCR) was carried out by the use of specific oligonucleotide primers. PCR mixture (25 μl) was comprised of master mix (12.5 μl), molecular grade water (7.5 μl), 1 μl of primer (forward and reverse), and 3 μl of DNA template. Using a thermocycler (Bio-Rad, USA), PCR was carried out (Table 1). PCR products were analyzed by electrophoresis in 1.2% agarose gel, and visualization of bands was done in transilluminator (Dolphin View-Wealtec, USA) under UV light.

ESBL Conformation Test.
e double disc synergy test was done on MHA to determine extended spectrum betalactamase (ESBL) producing isolates. Ceftazidime and Cefotaxime discs were spaced 15 mm away from an amoxicillin-clavulanic acid (20/10 μg) disc in the plate's middle [17].

Molecular Detection of Drug Resistance Genes.
Genes responsible for drug resistances against selected antibiotics were determined using gene specific primers for the targeted genes including ESBL blaCTX-M-15 gene that amplifies 996 bp gene size product [13], tetracycline tetB gene that amplifies 634 bp gene size product [18], and TMP-SMX dfrA1 resistance gene that amplifies 367 bp gene size product [19].

Molecular Detection of Pathogenicity-Related Genes.
Two virulence genes PAP [20] and Heat-labile toxin [21] were targeted to determine the pathogenicity potential of the isolates by PCR. By using thermocycler, PCR mixture (25 μl) was used to amplify PAP gene (initial denaturing 3 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 58°C, 1 min at 72°C followed by final extension of 5 min at 72°C) and heat-labile toxin gene (initial denaturing 3 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 58°C, and 1 min at 72°C and final extension for 5 min at 72°C).

Statistical
Analysis. Statistical calculation of the data was done by using Microsoft Excel. Chi-square goodness-of-fit test was used for the determination of difference in resistance. p values < 0.05 were considered statistically significant.

Results and Discussion
A total 110 sample comprising 80 urine and 30 tap water samples were analyzed for the detection of E. coli. Out of total 110 samples, 25% (27/110) were found contaminated with E. coli, whereas 23.75% (19/80) urine sample was positive for the presence of the target pathogen, while 26.7% (8/30) water sample was confirmed with the presence of E. coli through conventional and molecular methods (PCR) targeting uidA gene (Figure 1).
Due to frequent and unnecessary use of antibiotics, E. coli is becoming drug resistant, making it a superbug and a huge public health risk. Antibiotic susceptibility of E. coli isolates must be determined for the proper and precise prescriptions. If proper management and precautions have not been taken for the prevention of drug resistance, the current death toll 700,000 per year can be increased to ten million mortality rate every year throughout the globe by year 2050 [22]. Drug resistance is a barrier to universal health care and achieving sustainable development goals in the areas of food security, clean water, sanitation, and health [23].
Contrary to our finding, Talukdar et al. [13] reported 63% prevalence of E. coli, in household water, which was relatively high compared to our findings. Similar to our study, Shimpoh et al. [24] used uidA primer to identify E. coli strains from clinical samples collected from Japanese patients and reported a high detection rate of 51%.

Antibiogram.
Twelve commercially available antibiotic discs were used to determine the antibiogram of the isolates. A majority of the isolates showed resistance to one or more antibiotics. Antimicrobial susceptibility test results revealed that few isolates showed susceptibility against chloramphenicol, amikacin, and imipenem while large number of E. coli isolates showed 100% (27/27) resistance to tetracycline, amoxicillin, amoxicillin-clavulanic acid, cefotaxime, and cefixime in clinical and water isolates ( Figure 2).
Out of 27 isolates, only 5 were susceptible to ceftazidime while other isolates showed resistance against various commercially available antibiotics. A research study conducted in Saudi Arabia reported that ESBL producing E. coli isolate demonstrated 100% resistance against amoxicillin/ clavulanic acid and showed higher resistance rate against ceftazidime 100% and amikacin 54% comparing with present results 80% and 50%, respectively [30]. e current study suggested that the commercially available antibiotics such as amikacin, ceftazidime, and imipenem are potent against ESBL E. coli isolates.
In comparison to our findings, a high prevalence rate (66%) of bla CTX-M-15 ESBL gene was found in E. coli isolate from water samples [31]. Comparing with present work, a research work reported lower prevalence rate of CTX-M-15 ESBL gene (43%) in E. coli clinical isolates [13]. Lyimo et al. [32] studied the prevalence of blacCTX-M-19  and blaCTX-M-15 in E. coli from water samples and reported lower prevalence comparing with the current study. In a study conducted in Pakistan, blaCTX-M gene was found in 29% E. coli isolates from water sample. BlaSHV and blaTEM were shown to impart resistance to several antibiotics in their investigation, and they were the most   widespread in E. coli, which contained the highest number of resistance genes, including blaCTXM, blaOXA, and blaNDM. [33].
Tetracycline (tetA, tetB, and tetC) and trimethoprimsulfamethoxazole resistance genes were investigated by Ibekwe et al. [34] in E. coli. e results of their study revealed that 10 E. coli isolates were positive for tetB gene and 10 isolates were also positive for dfrA1 gene. Comparing with the present study, a research study conducted in Iran, reported that tetB (53.63%) and dfrA1 genes (36.84%) were present in PCR-confirmed E. coli isolates [25]. Another study documented that tet B and TMP-SMX dfrA1 gene were highly prevalent in E. coli from clinical samples [35]. e virulence gene PAP was found in all (100%) the confirmed E. coli isolates ( Figure 6) while Heat-labile toxin gene was found absent in all isolates. In a similar study, it was reported that E. coli isolates tested for virulence gene PAP and Heat-labile toxin (LT) revealed 18% presence of PAP gene [36] and 12.7% LT gene detection in the isolates [37]. In another study conducted in South Africa by Nontongana et al. [26], 2% PAP gene and 47% LT gene presence were observed in the E. coli isolates. In comparison to the current study, literature variations in the prevalence of E. coli in clinical samples and antibacterial resistance might be due to different geographical regions, difference in the level of antibiotic exposure, and variation in E. coli strains. A detailed study by including larger sample size and collection of samples from different sampling sites around the region may provide more comprehensive findings.

Conclusion
It was concluded in this study that E. coli bacteria isolated form clinical and hospital vicinity water were resistant (100%) to Tetracycline, trimethoprim-sulfamethoxazole, Cefizime, and amoxicillin. Furthermore, drug resistance (ESBL blaCTX-M-15, tetA, and TMP-SMX dfrA1) and pathogenicity associated PAP genes were also present in the bacterial isolates, which presented a serious health concern. Precautions are required to reduce the incidence of multidrug resistant E. coli strains among humans. In order to achieve this, strict quality control procedures should also be used to ensure that water and wastewater are properly treated.

Data Availability
Major part of the data is presented in the manuscript, and the remaining data will be made available on reasonable request.

Ethical Approval
e study was conducted after the ethical approval from the Institutional Bioethics Committee, whereas the samples were collected after the patients' informed consent.

Conflicts of Interest
e authors declare that there are no conflicts of interest.