Multidrug-Resistant Enteric Bacteria in the Water Sources of Kalgo Metropolis, Nigeria

Abubakar Maidamma¹, Umar Usman Liman², Tirmizhi Munkaila³, Bashar Haruna Gulumbe^1* , Junaidu Abubakar² and Uzairu Aminu¹

¹ Department of Microbiology, Faculty of Science, Federal University Birnin-Kebbi, Birnin Kebbi, Nigeria

² Department of Biochemistry and Molecular Biology, Faculty of Science, Federal University Birnin Kebbi, Nigeria

³ Department of Microbiology, Faculty of Science, Bauchi State University Gadau, Bauchi, Nigeria

* Corresponding Author: [email protected]

ABSTRACT

The high incidence of diseases, such as diarrhoea, typhoid fever, cholera, and bacillary dysentery among the populace, especially in remote and developing areas, has been traced to the domestic use of contaminated water. This study aimed to isolate enteric bacteria in major drinking water sources situated within the Kalgo metropolis, Nigeria. Three water samples were aseptically collected in triplicates from three different areas of the city. After serial dilution, the samples were inoculated on nutrient agar and each sample's total viable bacterial count was taken. The enteric bacteria were isolated and identified using standard protocols. The isolates were subjected to antibiotic sensitivity test using the Kirby Bauer disk diffusion method. The inhibition zones were measured in millimeters(mm). Seven (7) different species of bacteria were isolated from the collected water samples.The isolated organisms included Shigella spp., Serratia spp., Klebsiella spp., Escherichia coli, Proteus spp., Yersinia spp., and Salmonella spp. Among these bacterial isolates, E. coli and Salmonella spp.were found to have the highest frequency of 29.63% and 25.93%, respectively. While, Serratia spp.had the lowest frequency of 3.9%. The sensitivity profile showed that E. coli, Salmonella spp.,Klebsiella spp., Shigella spp., and Serratia spp. were resistant to ampicillin. In contrast, Proteus spp. and Yersinia spp. showed high and intermediate susceptibility, respectively.The presence of multidrug-resistant enteric bacteria in water samples suggests the need to improve water quality through its assessment and treatment before use.

Keywords: enteric bacteria, multidrug-resistant, Kalgo metropolis, Nigeria, water contamination, water sources

1. INTRODUCTION

Water is considered a universal solvent.Itssafety, adequacy, and accessibility should be ensured at the household level. Accessto safe drinking water can result in tangible benefits to human health. Continued efforts should be made to ensuresafe drinking water’s accessibility to all [1]. The most widespread health risk of drinking water is contamination, directly or indirectly, by human or animal excreta containing pathogenic microorganisms. Domestic use of contaminated water may cause different infections [2]. An estimated 4 billion cases of diarrhoeaare caused by contaminated drinking water annually, representing 5.7% of the global disease burden [3]. A review of 28 studies carried out by the World Bank makes it evident that the incidence of certain waterborne,water-based, and water sanitation-associated diseases is related to the quality and quantity of water and sanitation available to the users [4]. Most Nigerians living in remote areas do not have access to safe and reliable sanitation facilities. Additionally, most households do not have an adequate understanding of hygienic practices regarding food, water, and personal hygiene [5].

In addition to difficulties in accessingsafer and hygienic drinking water, metropolitan water can be contaminated with pathogens at itssources, during distribution, transportation, anddue to poor handling in households or at work place [6]. Human and animal excreta are the most common sources of exposure to microbes in water. The most significant microbial risks are associated with ingesting water contaminated with human or animal feces.Such water isinfected with pathogenic bacteria, viruses, protozoa, and helminths [1, 7].

A short-term increase in pathogen concentration in drinking water may increase disease risks considerably and may trigger waterborne disease outbreaks [8]. Many microorganismsthat cause serious diseases, such as typhoid fever, cholera, and dysentery can be directly associated with drinking contaminated water. A lowcost and friendly method known as bacteria indicator can be indirectly implemented to detect the contamination status of drinking water. Amongst the bacteria indicators are coliform bacteria that live in human and animal intestines and also in external environments. The presence of these bacteriain water beyond a certain threshold signifies its dangers [9]. No water intended for human consumption should contain E. coli in a 100ml water sample [10]. The presence of 10 E. coli counts per 100 ml of the water sample is acceptable but needs regular sanitary checks if it is un-chlorinated [6].

Enteric bacteria are a large family of Gramnegative bacteria that include many harmless symbionts, as well as many of the more familiar pathogens, such as Salmonella, E. coli, Yersinia pestis, Klebsiella, and Shigella. Other diseasecausing bacteria in this family include Proteus, Enterobacter, Serratia, and Citrobacter. Enteric bacteria typically reside in the guts of animals and human beings [11]. Some are pathogenic, causing disease in certain animal species, while others can live in the gut without causing any health problems in a healthy individual. The enteric bacteria family causing water pollution composed of those in the order Enterobacteriales of the class Gamma proteobacteria in the phylum Proteobacteria [12].

Morphologically, members of the Enterobacteriaceae are rod-shaped and are typically 1–5 μm in length. They usually appear as medium-sized to large-sized grey colonies on blood agar, although some can express pigments (such as Serratia marcescens). Most members of Enterobacteriaceae have peritrichous, type I fimbriae involved in the adhesion of bacterial cells to their hosts. Some enterobacteria produce endotoxins.These reside in the cell wall and are released when the cell dies,then the cell wall disintegrates. Some members of the Enterobacteriaceae produce endotoxins that, when released into the bloodstream following cell lysis, cause a systemic inflammatory and vasodilatory response [13]. Hence, theproduction of endotoxins by bacteria can be among the significantreasons and integral mechanisms contributing to their multi-drug resistance. The strains of some species are harmless commensals, such as some strains of E. coli.Others are important human and animal pathogens and some strains are pathogenic to plants and insects. Their ubiquitous distribution indicates that it is inevitable that some members of the Enterobacteriaceaeenter the food chain [14].

Dumpsites are the most cost-effective and widely used method of waste disposal [15]. However, studies have linked dumpsites to groundwater contamination, necessitating the safety assessment of groundwater sources near dumpsites [16]. The presence of antibiotic residues in the environment is alarming because antibiotics might contribute to the appearance of resistant bacteria and could exert selective pressure [17]. The primarysource of antibiotics in aquatic environments is considered to be hospital sewage, followed by municipal, agricultural, and aquacultural wastewater, shown to be significant sources of antobiotic compounds and resistant bacteria [18]. Due to the presence of various contaminants in water sources and the high rate of multidrug-resistant enteric bacteria, it is crucial to regularly evaluate water quality in order to minimize the risk of waterborne diseases, improve overall hygiene for the society, and enhance the quality of life for both urban and rural communities, particularly in developing regions.

2. MATERIALS AND METHODS

2.1. Study Area

This study was conducted in Kalgo Metropolis,Kebbi State, Northwestern Nigeria. Kalgo is situated 10 km away from BirninKebbi, the capital of Kebbi State, located at latitude 12° 27' 57.8808" N and longitude 4° 11' 58.2864" E [19]. Itis one of the developing metropolises around Birnin Kebbi due to the establishment of Federal University Birnin Kebbi, where the number of students, staff, and businessmen is increasing daily. This increase in population and other anthropogenic activities pose a high risk to land and waterways.

2.2. Sample Collection and Handling

A total of 27 samples (threesamples in triplicate, each from different areas in Dutsin Dodo, TudunIllela, and Shiyar Dan Fili) were collected within the Kalgo metropolis fromOctober to November, 2022. Water sampleswith a volume of 100 ml were collected aseptically from a well, river, and borehole respectively using a sterile funnel and transferred into a cleancontainer. After the collection and transferof samples into the sterile container, it was tightly closed and transported to the laboratory. All samples collected were labeled with sample number, date of collection, and sample source. The sealed samples were then transferred to the laboratory for analysis.

2.3. Identification of Bacterial Isolates

One hundred (100) µl of serially diluted water samples were inoculated using the pour plate method on nutrient agar plates and incubated at 37°C for 24 h. Under the conditions described by Palanisamy et al. [17], the colonies for each sample were counted and expressed incolony-forming units per ml (cfu/ml). The colonies were then subcultured to obtain pure colonies. Bacterial isolates were identified using standard biochemical protocols [20].

2.4. Antimicrobial Susceptibility Testing

Susceptibility was determined by using the Kirby Bauer disk diffusion method as described by the National Committee for Clinical and Laboratory Standards. Bacteria colonies from nutrient agar were transferred into a tube containing 4-5ml of sterile normal saline. The suspension was visually adjusted to have a turbidity similar to 0.5 MacFarland standard. The inoculum was spread across the entire surface of the Muller Hinton agar plate using a sterile swab stick. The plate was rotated approximately 60^o between streaking to ensure even distribution. The inoculated plates were left to stand for at least 3 minutes but for no longer than 15 minutes before the disks were applied.The disc was gently pressed with sterile forceps to ensure contact with the medium. Commercial antibiotics disks (Abtex Biological Ltd, Liverpool. UK) were used. These includedtarivid (10μg), reflacine (10μg), ciproflox (10μg), gentamycin (10μg), streptomycin (30μg), ceporex (10μg), augumentin (30μg), aalidixicacid (30μg), septrin (30μg), and amplicin (30μg).

3. RESULTS

3.1. Total Bacterial Load of the Isolates

The results for bacterial load from water samples ranged from 3.1x10^-5 to 2.6x10^-6 in Dutsin dodo, 2.0x10^-4 to 2.2x10^-5 in TudunIllela, and 1.0x10^-6 to 3.0x10^-6 in Shiyar Dan Fili,where Shiyar Dan fili (river water) was found to have the highest bacterial load. The high bacterial levels in water sources can be attributed to several factors, including uncovered water sources and exposure to dust, as well as the use of contaminated toilet articles to fetch water and the lack of proper sanitation of reservoirs. This is demonstrated in Table 1.

3.2. Identification of Bacterial Isolate

In this study, 27 (3 samples in each area in the triplicate) bacteria were isolated from 3 locations inthe Kalgo metropolis. The isolates identified were Shigella spp.,Serratia spp., Klebsiella spp., Escherichia coli, Proteus spp., Yersinia spp, and Salmonella spp. Out of the 27 bacteria genera isolated, E.coli and Salmonella spp.were found to have the highest occurence, while Serratia spp. occurred the least, as shown in (Table 2).

Table 1. Total Viable Plate Count of BacterialIsolates (cfu/ml)

Table 2. Identification of Bacterial Isolates from Water Samples

3.3. Frequency of Occurrence of Enteric Bacteria Isolated from Water per Area

The study revealed that in area 1 (Dutsin Dodo), the water sample analyzed showed a high frequency of occurrence of bacteria, with E. coli having the highest percentage at 33.33% (3 occurrences). In contrast,Klebsiella, Yersinia, andSerratiawere found to have the lowest frequency of 1(11.11%) each. In area 2 (TudunIllela),E. colihad the highest frequency of occurrence of 3 (33.33%), whileShigella and Yersiniawere found to have the lowest frequency of 1(11.11%) each. While, area 3 (Shiyar Dan Fili) showed the highest frequency of Salmonella with 3 (33.33%) and the lowest frequency of occurrence of Klebsiella and Yersinia with 1(11.11%) each, as shown in Table 3.

The total frequency of occurrence of isolates indicates that out of the seven (7) bacteria genera isolated, E. coli (29.63%) and Salmonella spp.(25.93%)showed the highest percentage of occurrence, respectively. In contrast,Serratia spp.showed the lowest percentageof occurrence (3.70%), as illustrated in the pie chart.

Table 3.Frequency of Occurrence of Enteric Bacteria Isolated from Water Sample per Area

Figure 1. Percentage of Occurance of Bacterial Isolates

Table 4. Zones of Inhibition in Millimeters(mm) Obtained using the Kirby Bauer Disk Diffusion Method

Key: S: susceptible. I: intermediate, R:  resistance, CN: gentamycin, SXT:  septrin, CEP: ceporex , PEF:

4. DISCUSSION

Many microorganisms have been reportedlyfound in contaminated water. Monitoring and detecting indicator organisms and disease-causing microorganisms is asignificantpart of sanitary microbiology. Bacteria found in intestinal tract generally do not survive in an aquatic environment. They remain under physiological stress and gradually lose their ability to form colonies in different sensitivity media. Bacterial transmission typically takes place through the fecal-oral pathway, which can occur directly or indirectly, such as when individuals come into contact with healthcare workers. A substantial contributing factor to the risk of contracting and getting infected by enteric bacteria is the practice of inadequate hygiene. The frequency of these enteric bacteria differs significantly from person to person, which is a multi-factor determinant.

The bacterial loads in water samples varied, with the highest load found in Shiyar Dan Fili (river water) ranging from 1.0x10^-6 to 3.0x10^-6. In comparison, Dutsin dodo had a range of 3.1x10^-5 to 2.6x10^-6, and Tudun Illela ranged from 2.0x10^-4 to 2.2x10^-5. The results revealed that all water samples collected from the river were highly contaminated. This finding coincides with the study conducted in Sagamu, Nigeria,which found that most of the river water (72%) was contaminated by E. coli, Salmonella spp., Proteus spp., and Klebsiella spp., respectively [21].

The presence of any coliform bacteria in drinking water is used as an indicator of fecal or other related sourcesof contamination. In the current study, seven (07)species of bacteria were isolated from the collected water samples. Out of the 27 bacteria genera isolated, E. coli (29.63%) and salmonella spp. (25.93%) showedthe highest percentage of occurence, while Serratiamercescens (3.70%) showedthe least occurrence. This finding is in agreement with the recent findings of Aliero et al. [22] regardingantibiotics resistance pattern of coliform bacteria isolated from slaughterhouse wastewater in the Jega local government.Jega is situated 10 km ahead of the Kalgo metropolis, where three Gramnegative bacterianamely E. coli, Pseudomonas aeruginosa, and Enterobacteraerogenes were identified.E. coli was the most frequently isolated in slaughterhouse wastewater 39 (45.8%).

This study further correlates with the bacteriological and physicochemical analyses of Aliero dam water by Gulumbe et al. [23]. The analyses detected Yersinia enterocolitic a and Staphylococcus aureuswith the highest percentage of occurrence of 20% each,Bacillus megaterium, Salmonella sp, and Escherichia coli each with 12.5% occurrence,and Klebsiella, Vibrio, and Shigella spp. with the lowest percentage of occurrence with 7.5% each. This correlation might result from the connection betweenthe waterways and rivers from Aleiro to Jega down to the Kalgo metropolis river, since Aleiro and Jega are geographically ahead of the Kalgo metropolis. The presence of these organisms can be linked to human activities, as human beings are reservoirs of various organisms. This also aligns with the findings of Ouseph [24]. In this research,the presence of enteric bacteria in healthy human carriers was investiagted. He reported that healthy human beings carry a significant number of enteric bacteria, such as Salmonella spp., Shigella spp., Citrobacter spp., Yersinia spp., E. coli, Klebsiella spp., Proteus and Enterobacter spp., with E. coli and Salmonella being the most frequently isolated bacteria.

Table 4shows that the resistance displayed by all the organisms to the various antibiotics is problematic, especially the resistance to three or more antibiotics, an indication of multi-drug resistance. The resultsshowed that ampicillin, which has 12.5% susceptible, 12.5% intermediate, and 75% resistant isolates, is the antibiotic with the highest number of resistance isolates. In comparison, ofloxacin which has 85.7% susceptible and 14.38% intermediate isolates is the antibiotic with the highest number of susceptible isolates. These results coincide with the results of the study conducted in Ghana, where ahigh percentage of resistance to ampicillin and tetracycline was observed [25]. Epidemiological evidence suggests that the increasing resistance prevalence of tetracycline, ampicillin, and many other antibiotics is directly linked to their usage [26, 27].

It was also observed that none of the isolates tested was susceptible to all the antimicrobial drugs used in this study. This is a cause for concern because many clinicians fall back on quinolones to treatGramnegative pathogens in the face of drug resistance [28, 29]. Ciprofloxacin resistance in Portugal was 25.8% and 24.3% in Italy, while in Germany and Netherlands, it was 15.2% and 6.8%, respectively [30]. Similarly, in a study conducted by Yusuf et al. [8] in Nigeria, up to 18% resistance to ciprofloxacin was reported. In another report, Thomas et al. [30] showed a contrasting situation with K. pneumonia in which all K. pneumoniawere susceptible to ciprofloxacin. The increasing resistance to several antimicrobial drugs has been ascribed to their inappropriate usage (including their overuse, misuse, suboptimal dosage, and non-compliance with the treatment duration), which leads to selection pressure. Sougakoff and Jarlier [31] reported that the abuse and misuse of antimicrobial agents for growth promotion and prevention of diseases has put a selective pressure that induces more resistance among bacteria.To reduce the risk of the contamination of water bodies and consumers, sanitary microbiologists should continue to monitor and detect indicators and disease-causing microorganisms [32].

4.1. Conclusion

The current study found that the bacterial load in drinking water collected from three sources in the Kalgo metropolis varied, with the highest load in Shiyar Dan Fili (river water). The most common indicator organisms for faecal contamination were Shigella spp., Serratia spp., Klebsiella spp., E.coli, Proteus spp., Yersinia spp., and Salmonellaspp.E. coli and Salmonella spp.had the highest percentage of occurrence among the 27 isolated bacteria genera. The results also showed that none of the isolates were susceptible to all the tested antibiotics, indicating a resistance profile that remains a cause for concern. This poses a threat to human health and calls for further investigation and measures to improve drinking water quality.

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