Antimicrobial Resistance Profile of Bacteria Isolated from Boreholes and Hand Dug Wells Water in Ngaoundere Municipality of Adamawa Region in Cameroon Bacteria, Microbial Resistance Index

Uncontrolled uses of antibiotics have led to rapid evolution of antibiot-ic-resistance bacteria and antibiotic resistance gene transfer, especially in a pool of aquatic system where resistance, intermediate and susceptible bacteria to some antibiotics strive together. Consequently, there is a transfer of resistance genes. In this study, bacteria of the Enterobacteriaceae family and some gram positive bacteria isolated from some boreholes and hand dug wells water of public use were tested on 19 antibiotics of different classes. This was achieved through a disk diffusion technique to determine the antimicrobial resistance profile of the said bacteria, microbial resistance index of the drugs used (and their ability to produce Beta-lactamase). These isolates were shown to demonstrate a very high resistance to the drugs used in the area. The resistance was highest in Escherichia coli 1 (73.68%) and lowest in Streptococcus pneumoniae (47.82%). These isolates also indicated very high levels of mul-ti-drug resistance. The minimum resistance index was 0.47, indicating that bacteria isolates were of fecal origin. It is evident from the present study that multiple antibiotic-resistant bacteria can thrive in water as an environmental re-servoir, and can therefore provide a route to multidrug-resistant pathogens to enter human and animal population.


Introduction
The global community of water, sanitation, hygiene researchers, practitioners and policy makers have to date inadequately addressed the challenge of the quality in relation to access to water and sanitation in development and humanitarian emergency contexts. Freshwater comprises 3% of the total water on earth, but only a small percentage (0.01%) of this freshwater is available for human use [1]. The burden of water-related diseases curtails efforts to improve public health in the developing world. Diarrhea most often related to unsafe drinking water, poor sanitation and inadequate hygiene is one of the leading causes of death among children under the age of five. The main risk for public health in water systems is that resistant genes are transferred from environmental bacteria to human pathogens. The potential of drinking water to transport microbial pathogens to a greater number of people, causing subsequent illness, is well documented in several countries at all levels of economic development [2]. The aquatic ecosystem plays a major role in the contamination and spread of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARG). Water acts as the most important mode for bacterial propagation and distribution of antibiotic resistance between man and environment. For the evolution of antibacterial resistance, urban water system, animal husbandry operations and pharmaceutical industry effluents are considered as the major hotspots [3]. The controlled process and engineered water cycle, which is operated in a local environment otherwise known as "urban cycle" is performed in two stages: firstly, the abstraction of surface water and groundwater for consumption; secondly, the treatment and disinfection of sewage before it's discharged into the environment. This serves as a path for the transfer of antibiotic resistance from the environment to human and back to the environment, as a large number of resistant bacteria enter the sewage through faeces, which is further carried into water bodies. Resistant bacteria and other pathogens in wastewater remain in close contact with sewage sludge bacteria and other microorganisms during biological treatment at wastewater treatment units, where the horizontal transfer resistant genes takes place [4]. Therefore, wastewater treatment plant becomes a hub of gene transfer and evolution of ARB. Resistant bacteria can also reach the ground water in an agricultural field due to manure spreading and animal grazing. New forms of resistance can be acquired by the bacterial strains in the environments. Pathogens that last longer and are dispersed in the environment cause greater hazard, than those that can be transmitted from one person to another. The spread of these resistant bacteria in the environment poses a serious threat to public health [5].
Hence, there is a growing concern regarding the occurrence of antibiotic resistance bacteria and antibiotic resistance genes in aquatic environments [6]. Direct or indirect contact with water (for drinking, or recreational use) contaminated by ARB could harm and infect the human population with antibiotic resistant pathogens, and/or ARG carried by bacteria may be transferred from microorganisms into humans, as a consequence of horizontal gene transfer [7]. Such Advances in Microbiology events would undermine our ability to prevent and control disease, and thus expose a great threat to public health. Little is known about the fate of ARG in drinking water systems, and it was recently proposed that ARG are emerging contaminants [8]. It is estimated that about half of the patients occupying African hospital beds suffer from water-borne illnesses due to lack of access to clean water and sanitation [9]. This holds true in Cameroon and in Vina division in particular where most of the population live below poverty level and cannot afford to have good antibiotics in pharmacies since most of the drugs sold in their vicinity are resistance to many infections. As such they turn to traditional medicine. As such it was necessary to carry out this exercise to determine the anti-microbial resistance profile of bacteria isolated from potable water source since it acts as a source transmission.

Study Site
The study was carried out in Ngaoundere urban council in Vina Division in the months of April and October 2017 and 2018. It covers a surface area of 17.196 km 2 and as from 2001 had a population of 247,420 [10] inhabitants. The capital is Ngaoundere, which had in 2005 a population of 152.700 inhabitants. It has one general hospital, integrated health centers in each council area and 3 private hospitals, with number pharmaceuticals shops to take care of the citizens. The city is divided into Ngaoundere I, II and III councils, which are rapidly growing. The main activities in the locality are cattle rearing and agriculture [10]. The monthly rainfall varies between 0 to 1500 mm [10]. The municipality is highly cosmopolite. With a good number of people from different parts of Cameroon and more especially from the far north who have escaped from the activities of Boko haram and a good number of refugees from Central African Republic. The Ngaoundere municipality has 97 officially known Figure 1 boreholes and a good number of wells not known all meant for public consumption.

Sample Collection
Sampling took place in the dry and rainy season during the months of April and October from fourteen (14) boreholes, (nine) 9 hand dug wells meant for public consumption were chosen based on population agglomeration. 500 mL sampling bottles were washed and rinsed with distilled water. Bottles were then sterilized under the laminar hood using UV light. For boreholes, water was pumped out for 3 -4 minutes to cool the metal pipe to eliminate the influence of water temperature with that of the metal pipe. Sampling bottles were rinsed with some of the boreholes water and then completely filled and covered. For wells, water was drawn using the rope and bucked found in the sampling points. Table 1 Sampling bottles were rinsed with well water, and then filled completely and covered. Water Samples were stored in a flask at −4˚C, and aseptically transported to the microbiology laboratory unit of the Institute of Agricultural Research for Development for analysis. Advances in Microbiology  Streptococcus spp., nutrient agar was used. All the media were prepared according to the manufacturers' instructions (Biolab).
Plates were incubated at 37˚C except for m-FC agar which were incubated at 45˚C for 18 -24 hours in a water bath. During the isolation of streptococcus, the inoculated nutrient agar plates with the filtrate were incubated for 72 hours at 37˚C. The colonies were enumerated, characterized, and recorded. The results were expressed as the number of fecal coliforms, total coliforms in 100 mL of water. Blue colonies from m-FC agar (presumptive coliforms), metallic-sheen colonies from m-Endo agar (presumptive total coliforms) as indicated by Eaton, Rice and Baird (2005) [12]. For sub-culturing, suspected bacteria were inoculated separately onto different bacteriological agar media under aseptic condition and incubated at 37˚C for 24 hours. Pure cultures were achieved as per procedures described by Cowan (1985) [13].

Assessment of Antimicrobial Susceptibility
Pure cultures of selected bacterial isolates were tested for susceptibility to different antimicrobial agents using in vitro disk diffusion (Kirby-Bauer) method as described by Quinn et al. (2002) [14]. Cultured broth was cross-checked with 0.5 McFarland standard before applying on Mueller Hinton agar and disk application. Nineteen different antimicrobial disks obtained from commercial sources (Becton Dickson and company and Liofilchem®, Bioanalyse) were selected for the testing and they included: Ceftazidime (CAZ) 30 mcg Amoxicillin (10 μg Table 2 plates were incubated at 37˚C for 18 -24 hours; and the ensuinginhibition zone diameters (IZDs) were measured to the nearest millimeter using a Mitutoyovernier caliper, this was repeated three times and the average readings of the zone of inhibition was calculated. The interpretation was done according to the guidelines of Clinical and Laboratory Standard Institute [15].

Detection of ESBL Producers
All 39 bacteria that showed reduced susceptibility to either ceftazidime were tested ESBL production using the standard double disk synergy tests. In this test,

Statistical Analysis
SPSS version 16 was used to calculate the percentages and Microsoft excel 2013 was used to draw the histograms.

Bacterial Isolates Recovered from Freshwater in Boreholes and Wells in Ngaoundere Municipality
A total of 14 bore holes and 9 hand dug wells were sampled based on population agglomeration and the selection process was aided by the councilors of each municipality. Among the fourteen sampled bore holes Figure 2 five had fecal contaminates giving a percentage of 35.7%. This is contrary to the WHO policies which stipulate that any potable water in which fecal contamination is seen is completely out of use [17]. Nine hand dug wells of public use were equally se-  Bacteria isolated were placed in classes and prevalence per specie isolated in the dry and rainy season were placed together.
For convenient, those antibiotics that were intermediate were considered as resistance, hence Table 3

Identification of Multidrug Resistance (MDR) Strains
The antibiotic susceptibility analysis to detect multidrug-resistant (MDR) bacteria among enterobacteriaceae and some gram positive bacteria isolates was performed using 19 different antibiotics disks. The number of antibiotics to which each bacterium was resistant to in the disk diffusion test was noted for identification of multidrug resistant strains. Multidrug resistance (MDR) was understood as resistant to four or more antibiotics tested [19]. Multiple Antibiotic Resistance (MAR) Index Table 4 was calculated as a/b where: "a" represents the number of antibiotics to which the isolates were resistant to, while ''b'' represents the total number of antibiotics to which the isolate was exposed (MAR index for isolates = (a/b) [20]. Bacteria may exhibit intrinsic (primary) resistance to certain antimicrobial agents. Intrinsic resistance was based on either the lack or the inaccessibility of the antimicrobial target site among the bacteria in question. In other cases, intrinsically resistant bacteria are known to produce    inactivating enzymes, such as species-specific β-lactamases, contain multidrug transporters and/or exhibit permeability barriers [21].
Multi drug resistance was observed in all the isolates used in this study with the highest index found in E. coli 1.

Detection of ESBL in Isolates
See Figure 4 and Table 5.

Discussion
A bacterium undergoes massive selection pressure due to the excessive use of antibiotics on it. The genetically adopted bacterium continues to reproduce and survive in the presence of antibiotic and transfers resistance to its following generations. The antibiotic slowly becomes ineffective towards these resistant bacteria [22]. There are specific targeted molecular structures in the bacterial cell which are attacked by the antibiotics. In case of spontaneous mutation, the antibiotic fails to locate these targets, hence the bacterium develops resistance.   and Piperacilline (Antipseudomonal penicillin + β-lactamase inhibitor). In contrast, Piperacilline was susceptible to all the bacteria species isolated he indicated also resistance to gentamycin and ofloxacin which to me were susceptible. Resistance of bacteria to antibiotics ranges from 100% in pefloxacine, ceftazidime, Amoxyllin, citizime Erythromycine to 14.2% in Amoxylineclauvulunic acid. E. coli 1 was also resistance to ciprolaxine (fluoroquinolones) and imepenem (carbapenems) as indicated by Carmen et al., (2016) [4]. It was also noticed that Samonella spp. were resistance to Doxicycliene (tetracycline), Ciproflolaxine (Floroquinones) and Netilimcin (Aminoglocosides). Ahmed (2011) [20] reported a 40% resistance of Salmonella spp. isolated from chickens to doxycline. Ciproflolaxine was 74% resistance to Salmonella spp. in this study. According to Hossain (2017) [26] 100% resistance was reported to ciproflolaxicine to bacteria isolated from urinary tract infections in India. Bulkholderia cepaceae of the family Pseudo monadaceae gave a resistance perctange of 47.6%. The Burkholderia cepacia organisms are opportunistic nosocomial pathogens capable of causing severe disease in immunocompromised individuals, especially those with cystic fibrosis [27]. Intrinsic resistance of Bulkholderia cepaceae has been reported in many other catergories of antibiotics except, ceftazidime and other extended-spectrum cephalosporins, But in this study we discovered that Bulkholderia was resistance to ceftazidme and cefepime all of the extended spectrum cepha- Klebsiella pneumoniae has become the most common pathogenic bacterium accountable for nosocomial infections due to its high virulence factor and general occurrence of resistance to most antibiotics [28]. Of the 19 antibiotics used in this study Klebsiella pneumoniae was resistant to 14 of them giving a resistance perctange of 73.68% as confirmed by Gajul et al., (2015) [28] Klebsiella pneumoniae showed very high resistance to different categorize of antibiotics such as Β-lactam; ceftazidime (CAZ); Penecillin G(P); Cephalosporines; cefepime (FEP); aminoglocosides; netilimcine (NET); fluoroquinones; cefepime, but it was 26.5% susceptible to other antobiotics like Amikacin, gentamycine (glycopeptides), Doxycycline (Tetracycline), Piperacillin and imepenem (β-lactam). High level of resistance observed within these gram-negative bacteria may be due to the non-respect of pharmaceutical norms and vendors of these pharmaceutical products who have not undergone any training of any sort equally the exposure of these product in fluctuating temperatures is a cause for concern for these products are degraded under high temperatures. The sources of supply to most of these fake pharmaceutical products are not often known in third world countries.
Streptococcus pneumoniae was seen to be 47.8% resistant to antibiotics tested, including Piperacillin, erythromycin, amoxicillin, Amoxylineclauvulunic acid ceftriaxone and Pefloxacine, and 52% susceptible the bacteria tested in this study. This is similar to the results obtained by Kandakai and Dido (2009) [5] who demonstrated that Streptococcus pneumoniae isolated from the pharynx was highly resistance to erythromycine and tetracycline, but susceptible to ciproflolaxcine. These results are also similar to those obtained by Emina et al., (2015) [29], when studying the antimicrobial susceptibility/resistance of S. pneumoniae isolated from the eyes, resistance was highest to erythromycine and lowest at ciproflolaxcine. Multi drug resistance was also noted in Streptococcus pneumoniae (tetracycline; aminoglycosides; netilimcin; Β-lactam; ceftazidime; Cephalosporine; cefepime). This may be due to the fact that people do not buy drugs from the right source and do not respect the dosage prescribed by the medical personnel.
Staphilococcus aureus was seen to have a high resistance index 0.68 of as seen in Figure 5. This is similar to the results obtained by Akanbi et al., (2017) [30] where they showed Staphilococcus aureus was resistance to a host of antibiotics like imipenem (96.76%), ciproflolaxcine (66.7%) and a host of others.
With an increase in the antibiotic load, the prevalence of acquiring resistance increases within a bacterial community. The MAR index is an excellent tool that  pneumoniae) to 0.73 (E. coli 1) Figure 5. According to Krumperman (1983) [31] and Tambekar (2011) [32], MAR index more than 0.4 is usually obtained from human fecal origin, while MAR indices less than 0.4 are from non-human fecal contamination. This confirms the fact that all the isolates were of fecal origin, considering the level of human activities and waste disposal in this community, it is obvious that all the bacteria isolated were of fecal origin. When antibiotics are seldom or never used, a MAR index value less than or equal to ≤0.2 is observed [33]. In this study, 100% of the bacterial isolates showed MAR value more than 0.4, which is a possible indication that the bacterial isolates have been exposed to several antibiotics. The high resistance seen may be due to the selective pressure exerted by the use of antibiotics in the management of bacterial infections in animals, humans, waste disposals, unhygienic nature of the surrounding of the boreholes and wells and poor sewage disposal systems.
It is unfortunate that enteropathogens like E. coli 1, Salmonella spp., Klebsiella pneumoniae, Streptococcus pneumoniae were resistant to most common antibiotics (erythromycine, Penicillin Amoxicillin) found in this zone (Table 3), but susceptible to antibiotics that are not assessable to common man such as Piperacillin, imepenem and levofloxacine except E. coli 1 and Bulkholderia cepaceae that were not susceptible to all the three mentioned antibiotics. This was confirmed by Shubra et al. (2014) [34]. Who demonstrated that bacterial isolates from water sources were found sensitive to imipenem and piperacillin/tazobactam, with maximum resistance found to ampicillin (57.5%). Wherever Enterobacteriaceae occur, ESBL resistance mechanism can be observed [36]. This resistance mechanism is currently the one whose dynamic spread causes the most difficulties. The water environment is conducive to transfer between species, where ESBL producing bacteria from various sources get in contact with a broad range of potential recipients. In this study the detection of ESBL was done using standard double disc diffusion technic and it was observed that there was synergy reaction between the Amoxicillin clavulunate and the ceftazidime, ceftriaxone and cefotaxime. This is contrary to the results obtained by Adelowo, (2018) [37] who detected very high presence of ESBL in hospital waste water and aquatic sources ( Figure 4 and Table 5).

Conclusion
The effectiveness of antibiotic treatment of diseases will decline due to the development of antibiotic resistance in the environment. It is evident from the present results that multiple antibiotic-resistant bacteria can thrive in water as an environmental reservoir, and can therefore provide a route to multidrug-resistant pathogens to enter human and animal population. The study of resistance in the environmental bacteria can help in guiding the development of strategies to counteract this resistance. From this study, it can also be said that there is a need to monitor antibiotic sensitivity at regular intervals. Treatment of water is essential not only to kill the pathogenic bacteria, but also to stop the transfer of antibiotic. Finally, legislation and education should be enforced to control the pharmaceutical products in our markets, the respect of dose prescribed by the medical officials and accessibility to these drugs both for humans and animals.