Prevalence of Waterborne bla NDM-1 Gene Producing Carbapenem-resistant Klebsiella pneumoniae from Al-Hillah River Water, Babylon Province, iraq

the current work suggested the occurrence of blaNDM-1 gene among Klebsiella pneumoniae recovered from surface waters of the Al-Hillah River. Between January and April 2015, water samples (101) were taken from seven different area of the Al-Hillah River, Babylon province, iraq. K.pneumoniae was reported in percentage of 35 (34.6%). the antibiotics susceptibility profile of K.pneumoniae was determined with disk diffusion assay. the most common resistance was detected for penicillins agents (ampicillin and cloxacillin) with 20(57.14%) and 17(48.57%) resistance rate, respectively. two isolates of K.pneumoniae were carbapenem- resistant. Phenotypic screening of metallo β-lactamase detection was carried out using imipenem–eDtA double disk synergy test for carbapenem resistant isolates, 2(100%) isolates with positive result. Conventional Polymerase Chain Reaction (PCR) test was used for detection NDM-1 beta-lactamase, 1 (50%) K.pneumoniae isolate harboring this gene.


iNtRODuCtiON
Resistant to antibiotics among bacteria has been recognized a universal risk to public and animal health globally. Aquatic system is vital habitats for pathogenic bacteria and a main route for circulation and transmission of antibiotic resistance genes in nature originating from different sources such as runoff from agricultural areas, hospital waste and domestic discharge. [1][2][3] The greater risk for human and environmental health is the migration of antibiotic resistance genes from surrounding environment to human and animal bacterial pathogens. [4][5][6] New Delhi metallo-beta-lactamase (NDM) is a recently described plasmid-borne carbapenemase gene belong to molecular class B beta-lactamase which readily transferred between bacteria and can lead to extreme drug-resistant phenotypes. [7][8][9] First outbreak due to K. pneumoniae ST11producing NDM-1gene during the COVID-19 Pandemic has been particularly detected in human cases of infection in a Portuguese Hospital Centre. 10 Other report achieved by Ebomah and Okoh 11 identified K. pneumoniae harboring bla NDM-1 and bla KPC from various environmental niches like farm soil in the eastern Cape Province, South Africa. Multi-drug resistant Enterobacteriaceae carrying NDM-1gene have been recognized worldwide, with many cases linked to international travel and tourism. [12][13][14] Our study aimed to provide insights on the potential the occurrence of Klebsiella pneumoniae obtained from waters of Al-Hillah river, detect resistant profiles of all isolates, determine carbapenems resistance profiles, as well as to detect bla NDM-1 gene by available phenotypic test and by conventional Polymerase Chain Reaction (PCR) assay among bacteria which resist carbapenem antibiotics.

Sampling
This study was employed from the beginning of January to the end of April 2015, surface water samples (101) were taken from seven selected sampling sites of the Al-Hillah River. It's the main river in Babylon province, Iraq which can be used for agricultural process and as a drinking waters for animals. The sites of this study located near by each of the following region: Ancient Babylon city, Al-Wardia region, Nationality office, Bab Al-Hussein region, Al-Attba street, Al-Farisi region and Al-Aifar region. Samples were placed on sterile glass bottles, then transported to the laboratory unit (college of sciences for women, Babylon university) by ice box for immediate processing and analysis.

Processing of Samples and Microbiological Analysis
Water samples were concentrated by filtration onto a sterile filter membrane (0.22 μm) (Millipore, Difco, USA). From each dilution (ten-fold), 0.1 ml was spread on plate count agar, then incubated at 37°C for 24-48 hrs under aerobic conditions. 15,16 Following incubation, bacterial colonies were sub-cultured onto different selective and enrichment media. Bacterial identification was carried out using standard biochemical and microbiological tests as described previously. [17][18][19]

Antimicrobial Assay
The identified K. pneumoniae isolates were assessed for antibiotics agents using disc diffusion test (Kirby-Bauer) on plates of Mueller-Hinton agar (Oxiod, England) 20

Molecular Characterization of bla NDM-1 Gene
The modified method of Pospiech and Neuman (1995) 23 was used to extract DNA of pure K. pneumoniae isolates and kept at -20°C. Conventional PCR technique was used for amplification of bla NDM-1 gene with specific sets of primers (Bioneer, Korea) NDM-1/F (5-GGT TTG GCG ATC TGG TTTTC -3-) and NDM-1/R (5-CGGAATGGCTCATCACGATC -3-) (621bp). The final volume of 25 µl reaction mixture contained 5µl of DNA extract,12.5 µl of Go Taq Green Master Mix 2X (Promega, USA), 2.5 µl of each primer and 2.5 µl nuclease-free water. PCR conditions included: 1 min initial denaturation at 94°C, 30 cycles of 1 min denaturation at 94°C, 1 min annealing at 55°C and 2 min extension at 72°C followed by 10 min final extension step at 72°C 24,25 . Electrophoresis of the product was resolved on a 1.5% agarose gel stained with ethidium bromide, then the gel was observed using UV-Transilluminator for photodocumentation.

ReSultS AND DiSCuSSiON
Results identified 35/101(34.6%) isolates positive for K. pneumoniae in river waters (Table 1). Abd Al-Kareem et al. 26 proved the occurrence of 40 K. pneumoniae isolates from surface water of Tigris River in Baghdad city, Iraq. Our finding also consistent with the report detected by Ebomah and Okoh 11 who document 32 isolates as K. pneumoniae recovered from surface waters in the Eastern Cape Province, South Africa. Prevalence of K. pneumoniae in river water, Malaysia was (69%). 27 Another study in Hillah city recorded the occurrence of same bacteria in various clinical and environmental specimens. 28,29 The existence of K. pneumoniae in surface river water may be attributed to contamination of Al-Hillah River by different sources like discharge of hospitals mainly Babylon Teaching Hospital for Maternity and Pediatric, swimming of animals and discharge their wastes directly into river stream, runoff from agricultural areas, industrial effluents. Additionally, Hillah laboratories released their waste products into these waters which stimulate the proliferation and dissemination of pan-resistant strains and even evolve various mechanisms of resistance and pathogens.
Bacterial resistance to antimicrobial agents was ancient which can occur under selective pressure. However, due to the inappropriate prescription and massive use of antibiotics in medical therapy, agriculture and aquaculture, resistant bacteria have become a serious threat worldwide. 30,31 Susceptibility testing of bacterial isolates showed that most resistant agent was the penicillins antibiotics (ampicillin and cloxacillin) with 20(57.14%), 17(48.57%) resistance rate, respectively. Lihan et al. 32 recorded (31.6%) resistance rate for penicillin antibiotic by bacteria including K. pneumoniae in recreational river water of a community resort in Baram, Sarawak, Malysian, Barneo.
However, penems (meropenem and imipenem) antibiotics displayed lower rates of resistant with 2(5.71%) for each, ( The molecular screening of NDM-1 gene was performed using PCR technique on the two K. pneumoniae with resistant to carbapenem, the bla NDM-1 gene was detected in 1(50%) isolate only (Figure). Ahammad et al. 35 documented the presence of bla NDM-1 gene among coliform bacteria from Ganges river. Shah and Zahra 2 characterized 43 meropenem resistant bacteria from different water samples of which 3 K. pneumoniae isolates were harbored this gene in Islamabad, Pakistan.

CONCluSiON
This study report the finding of metallo beta lactamase of NDM-1 type containing K. pneumoniae in water samples from the Al-Hillah River. The presence of such highly resistant bacteria in water samples focus attention on the need to accelerate strategies to limit the emergence and spread of resistant organisms.

ACKNOWleDGMeNtS
I would like to acknowledge all people who provide assistance and cooperation to achieve this work.

FuNDiNG
None.

DAtA AvAilABility
All datasets generated or analyzed during this study are included in the manuscript.

etHiCS StAteMeNt
Not applicable.