Identification of Opportunistic Pathogenic Bacteria in Drinking Water Samples of Different Rural Health Centers and Their Clinical Impacts on Humans

International drinking water quality monitoring programs have been established in order to prevent or to reduce the risk of contracting water-related infections. A survey was performed on groundwater-derived drinking water from 13 different hospitals in the Mahabubnagar District. A total of 55 bacterial strains were isolated which belonged to both gram-positive and gram-negative bacteria. All the taxa were identified based on the 16S rRNA gene sequence analysis based on which they are phylogenetically close to 27 different taxa. Many of the strains are closely related to their phylogenetic neighbors and exhibit from 98.4 to 100% sequence similarity at the 16S rRNA gene sequence level. The most common group was similar to Acinetobacter junii (21.8%) and Acinetobacter calcoaceticus (10.9%) which were shared by 7 and 5 water samples, respectively. Out of 55 isolates, only 3 isolates belonged to coliform group which are Citrobacter freundii and Pantoea anthophila. More than half (52.7%, 29 strains) of the phylogenetic neighbors which belonged to 12 groups were reported to be pathogenic and isolated from clinical specimens. Out of 27 representative taxa are affiliated have eight representative genera in drinking water except for those affiliated with the genera Exiguobacterium, Delftia, Kocuria, and Lysinibacillus.


Introduction
Human population growth wields several and various pressures on the quality and the quantity of drinking and fresh water resources and on the access to them. Safe drinking water remains inaccessible to several millions people in the globe. Safe drinking water, thus, reduces the load of waterborne diseases. Contamination of drinking water due to natural and manmade contaminants is frequently reported in developing countries where mainstream of the inhabitants survive in countryside and uptown areas with meager hygiene and waste clearance practices. In developing countries, poor water quality is the most important risk of child mortalities which are mainly through infectious diarrhea. In India, about 10% of the countryside and city populations do not have access to usual safe drinking water and several others are threatened [1].
Potable or drinking water is defined as having a satisfactory quality in terms of its physical, chemical, and bacteriological parameters so that it can be securely used for drinking and cooking. The most common and widespread health risks associated with drinking water in developing countries are of biological origin. The WHO estimates that about 1100 million people globally drink unsafe water, and the greater part of diarrheal disease in the globe (88%) is attributable to insecure water, sanitation, and hygiene [2]. Ten major waterborne diseases are responsible for over 28 billion disease episodes annually in developing countries. The quality of drinking water might be ascertained by its microbiological examination. The maximum threat from 2 BioMed Research International microbes (coliform) in water is related to consumption of drinking water, that is, contaminated with human or animal excreta. Coliform bacteria include members of the family Enterobacteriaceae, for example, Escherichia coli, Enterobacter aerogenes, Salmonella, and Klebsiella in water that are accountable for a variety of diseases like cholera, typhoid, dysenteries, bacillary dysentery, and so forth in human and livestock [3].
Recently, Suthar et al. [4,5] reported a wide range of pathogenic bacteria in potable water samples from some rural habitations of Northern Rajasthan, India. A total of ten bacterial species, E. coli, Pseudomonas aeruginosa, E. aerogenes, Klebsiella sp., Proteus vulgaris, Alcaligenes faecalis, Bacillus cereus, Staphylococcus aureus, Streptococcus lactis, and Micrococcus luteus, were isolated and identified from the potable water samples from this region [4] M. luteus, S. lactis, Klebsiella sp., and E. coli were the dominant microflora (recorded from 73.1% of the villages/towns) in the water samples. The presence of coliforms shows the danger of fecal pollution and a consequent hazard of contracting a disease through pathogenic organisms. Nonetheless, the disease-causing organisms (pathogens) mostly transmitted via drinking water are predominantly of fecal origin.
The results of the recent study on the detection of potentially pathogenic bacteria in the drinking water distribution system of a hospital in Hungary by Felföldi et al. [5] had shown the presence of Legionella, Pseudomonas aeruginosa, and also several other opportunistic pathogenic bacteria, such as Escherichia albertii, Acinetobacter lwoffii and Corynebacterium tuberculostearicum, emphasizing that drinking water systems, particularly those with stagnant water sections, could be the source of nosocomial infections.
Hospitals drinking (potable) water systems are the most important and controllable as well as the most overlooked source of nosocomial pathogens. Conventional culture-based microbiological water quality monitoring techniques take a long time (several days), and usually a small volume of water is sampled (typically 100 mL), which gives rise to inadequate detection limits with regard to drinking water safety. Furthermore, the presence of some important waterborne pathogens (such as Pseudomonas aeruginosa or legionellae) shows no correlation with conventional indicator organism counts. Water-related pathogens can also find niches in water systems (i.e., an association with biofilms or free-living amoebae), rendering their observation with conventional techniques more difficult. Molecular techniques provide new and rapid facilities for the detection of pathogens involved in nosocomial infections. Five representative end-points (taps) in the drinking water system of a hospital (Budapest, Hungary) were sampled in October 2005.

Source of Samples.
Thirteen drinking water samples were collected from different government hospitals, Mahabubnagar District, Andhra Pradesh, India, on 22nd and 23rd of November, 2009. Fifty five strains were isolated from these 13 drinking water sources on nutrient agar medium at 37 ∘ C. For isolation of bacteria, 100 L of the water sample was plated on nutrient agar medium and incubated at 37 ∘ C for 2 days. Based on the different colony morphology from each sample, a total of 55 strains were selected and characterized in the present study.

16S rRNA Gene Sequencing and Phylogenetic Analysis.
For 16S rRNA gene sequencing, DNA was prepared using the Mo Bio microbial DNA isolation kit (Mo Bio Laboratories Inc., Solano Beach, CA, USA) and sequenced as described previously [6]. The resultant almost complete sequence of the 16S rRNA gene contained 1502 nucleotides. The 16S rRNA gene sequence of the isolate was subjected to BLAST sequence similarity search [7] and EzTaxon [8] to identify the nearest taxa. The entire related 16S rRNA gene sequences were downloaded from the database (http://www.ncbi.nlm.nih.gov/) aligned using the CLUSTAL X program [9] and the alignment corrected manually. Phylogenetic tree was constructed using tree-making algorithm and the Maximum likelihood (ML) using the PhyML program [10].

Biochemical
Tests. The biochemical tests were performed for all the strains obtained by using the HiMViCTM Biochemical Test Kit (HIMEDIA-KB001) and HiAssorted Biochemical Test Kit (HIMEDIA-KB002).

Characterization of the Bacterial Strains from Drinking
Water. All these isolates were from different drinking water sources, and all are mesophilic and could grow in the temperature range of 20 to 40 ∘ C with optimum growth temperature of 37 ∘ C. All the strains could grow without NaCl in the medium, and the tolerance to NaCl varied from 1 to 2%. All strains could grow either in the pH range of 6-8.

Identification of Isolated Strains from Drinking Water.
A total of 55 bacterial strains were recovered from the 13 drinking water sources (Table 1). Taxonomic analysis of all the 55 strains isolated from water samples indicated that 43 strains were Gram-negative and 12 Gram-positive. The nearest phylogenetic neighbor of all 55 isolated strains was identified following BLAST analysis of the 16S rRNA gene sequence. BLAST analysis indicated that twenty one strains belonged to the genus Acinetobacter, nine strains belonged to the genus Bacillus, eight strains belonged to the genus Pseudomonas, seven strains to the genus Aeromonas, two strains each belonged to the genera Methylobacterium and Pantoea, and one strain belonged to the genera Cupriavidus, Delftia, Exiguobacterium, Kocuria, and Lysinibacillus, respectively (Table 2). A total 27 different taxa were identified based on the BLAST analysis out of 55 strains. The most common group was similar to Acinetobacter junii (21.8%) and Acinetobacter calcoaceticus (10.9%) which were shared by 7 and 5 water samples, respectively. The phylogenetic trees constructed to determine their affiliations are shown in Figures 1 and 2. Out of 12 Gram-positive groups, 11 belonged to the phylum Firmicutes and 1 belonged to the phylum Actinobacteria (Table 2 and Figure 2). All 43 Gram-negative groups were belonged to the phylum Proteobacteria. Among the Proteobacteria, the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria were represented by 2, 2, and 39 groups, respectively (Table 2 and Figure 2). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the 21 Acinetobacter strains belonged to 4 groups, 9 Bacillus strains belonged to 7 groups, 8 Pseudomonas strains belonged  to 3 groups, 7 Aeromonas strains belonged to 5 groups, and 2 Methylobacterium strains formed two distinct groups (Figures 1 and 2). The affiliation of the strains to the nearest phylogenetic neighbor and the percentage of 16S rRNA gene sequence similarities are indicated in Table 2. Many of the strains are closely related to one another and exhibit 98.4 to 100% sequence similarity at the 16S rRNA gene sequence level like KL-1 and JD-2 which are closely related to one another (100%) and are affiliated to Acinetobacter calcoaceticus DSM 30006 T (98.5%).

Biochemical Assay. Biochemical analysis of the genus
Bacillus gave positive results for Indole, Citrate, Oxidase, and Urease and positive results for Methyl red Voges-Proskaeur, Catalase, and Gelatinase tests. Genus Methylobacterium gave positive results for Citrate, Catalase, and Urease and negative   (Table 3). Coliform is the name of a test adopted in 1914 by the Public Health Service for the Enterobacteriaceae family. It is the commonly used bacterial indicator of sanitary quality of foods and water. Coliform bacteria live in soil or vegetation and in the gastrointestinal tract of animals. Coliform bacteria enter water supplies from the direct disposal of waste into streams or lakes, or from runoff, from wooded areas, pastures, feedlots, septic tanks, and sewage plants into streams or groundwater. Coliform bacteria are not a single type of bacteria, but a group of bacteria that includes many strains, such as E. coli. They are ubiquitous in nature, and many types are harmless. Coliform bacteria belong to the genera Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella, Pantoea, and Serratia. Out of 55 isolates only 3 isolates belonged to coliform group which are NG2 which is phylogenetically close to Citrobacter freundii and R21 and R22 which are phylogenetically close to Pantoea anthophila. This shows that the water samples NG and R collected from Nagarkurnool and Mahabubnagar-2 might be contaminated with fecal pollution and a consequent hazard of contracting a disease through pathogenic organisms. Nonetheless, the disease-causing organisms (pathogens) mostly transmitted via drinking water are predominantly of fecal origin. A total of 2 and 12 strains were isolated from the samples NG and R which include 3 coliform bacteria, and out of 14 strains, 7 were likely to be pathogenic which include NG1 which is phylogenetically close to Acinetobacter johnsonii, R1 which is phylogenetically close to Acinetobacter junii, NG2 which is phylogenetically close to Citrobacter freundii, R6, 7, and 23 which are phylogenetically close to Pseudomonas alcaligenes, and R5 which is phylogenetically close to Bacillus cereus.
In the present study, species that belonged to the genera Aeromonas, Methylobacterium, Bacillus, and Pseudomonas were isolated which is supported by the different studies     where several novel species which belong to the genera Aeromonas [11], Methylobacterium [12], Bacillus [13], and Pseudomonas [14] were reported from drinking water systems. Aeromonas spp. are ubiquitous bacteria found in diverse aquatic environments worldwide such as bottled water, chlorinated water, well water, and heavily polluted waters. Aeromonas hydrophila survives easily in waters polluted by feces and seems resistant to various disinfectants, insecticides, and chemicals. Recent studies on drinking water in rural areas in Northern Rajasthan, India, [4] and in drinking water distribution system of a hospital in Hungary, [5] showed the presence of several bacteria in the drinking water which included Acinetobacter lwoffii, Alcaligenes faecalis, Bacillus cereus, Corynebacterium tuberculostearicum, Enterobacter aerogenes, Escherichia coli, Escherichia albertii, Klebsiella sp., Micrococcus luteus, Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus lactis. In the present study, species belonging to the genus Acinetobacter, Bacillus cereus, and Pseudomonas aeruginosa which are opportunistic pathogens were isolated from 9 drinking water samples such as AP, GD, JD, KL, MB, NG, NR, R, and WN out of 13.
Of the 27 groups, only few (GD4, R5, AP1, and NR2) of the phylogenetic neighbors had been isolated earlier from the drinking water sources including Aeromonas hydrophila [4], Bacillus cereus [4], Cupriavidus pauculus [15], and Pseudomonas aeruginosa [4,5]. More than half (52.7%, 29 strains) of the phylogenetic neighbors which belonged to 12 groups were reported to be pathogenic and were isolated from clinical specimens, and other 15 groups were reported to be isolated from several distinct habitats. Most of the drinking water sources studied for bacterial diversity include bacteria which are phylogenetically close to pathogenic bacteria except one sample AL from which 3 strains AL1, AL3, and AL4 were isolated which belong to the phylogenetic neighbors Methylobacterium zatmanii, Delftia lacustris, and Bacillus stratosphericus, respectively. The isolates which are likely to be pathogenic include strain AP1 which is phylogenetically close to Cupriavidus pauculus which causes human infections sporadically and were isolated earlier from a variety of human clinical sources and also from drinking water sources [15] (Table 4); strains GD5 and NG1 which are phylogenetically close to Acinetobacter johnsonii which causes vascular catheter-related bloodstream infection were isolated from clinical specimens and milk [16] (Table 4); strains AP2, AP4, GD2, JD4, KL3, KL4, MB3, R1, WN1, WN3, WN4, and WN5 which are phylogenetically close to Acinetobacter junii which causes septicemia, meningitis, peritonitis, and so forth were isolated from human clinical specimens like urine [17] (Table 4); strain MB4 which is phylogenetically close to Acinetobacter ursingii which causes bacteremia was isolated from blood [18] (Table 4); strain GD4 which is phylogenetically close to Aeromonas hydrophila subsp. Hydrophila which causes acute diarrheal disease was isolated from humans, animals, fish, and fresh water [11,19] (Table 4); strain NG2 which is phylogenetically close to Citrobacter freundii which causes opportunistic infections was isolated from canal water [20] (Table 4); strain NR2 which is phylogenetically close to Pseudomonas aeruginosa which causes several infections in immunocompromised individuals was isolated from wide variety habitats including clinical samples and also from drinking water samples [4,5] (Table 4); strains R6, R7, and R23 which are phylogenetically close to Pseudomonas alcaligenes which causes endocarditis occasionally were isolated from soil, water, and also from clinical samples [21] (Table 4); strains D22, GD6, MB2, and MK3 which are phylogenetically close to Pseudomonas otitidis which causes inflammation of the ear were isolated from the ear [22] (Table 4); strain D12 which is phylogenetically close to Bacillus anthracis which causes anthrax disease was isolated from sheep blood (Table 4); strain R5 which is phylogenetically close to Bacillus cereus which causes food poisoning was isolated from the soil and also food materials [23] (Table 4); and strain JU1 which is phylogenetically close to Bacillus infantis which causes sepsis was isolated from clinical specimens [24] (Table 4).

Conclusion
This study is an overview of drinking water quality in the rural and urban parts of Mahabubnagar District, India. Recent investigations on bacterial contaminations of drinking water