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

Authors: Pavan Kumar Pindi; P. Raghuveer Yadav; A. Shiva Shanker
Journal: BioMed Research International
 (2013)

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## Abstract

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 toAcinetobacter 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.


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## Body

## 1. 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 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,andKlebsiellain 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, Klebsiellasp., 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, Klebsiellasp., 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 asPseudomonas 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.


## 2. Materials and Methods

### 2.1. 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, 100μL 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.


### 2.2.16S 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].


### 2.3. 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).


## 2.1. 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, 100μL 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.


## 2.2.16S 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].


## 2.3. 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).


## 3. Results and Discussion

### 3.1. 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.


### 3.2. Identification of Isolated Strains from Drinking Water

A total of 55 bacterial strains were recovered from the 13 drinking water sources (Table1). 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,andLysinibacillus,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 Gammaproteobacteriawere 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 30006T  (98.5%).Table 1
Bacterial abundance from the drinking water samples collected from government hospitals, Mahabubnagar.
Serial number
Sample number
Place of sample collection
Number of isolates
Isolated strains
1
AL
Alampur
3
AL1, AL3, AL4
2
AP
Achampet
3
AP1, AP2, AP4
3
D
Shadnagar
4
D12, D22, D24, D25
4
GD
Gadwal
4
GD2, GD4–D6
5
JD
Jadcherla
4
JD1–JD4
6
JU
Jurala
4
JU1–JU4
7
KL
Kalwakurthy
5
KL1–KL5
8
MB
Mahabubnagar-1
4
MB1–MB4
9
MK
Makthal
3
MK2–MK4
10
NG
Nagar Kurnool
2
NG1, NG2
11
NR
Narayanpet
3
NR2–NR4
12
R
Mahabubnagar-2
12
R1, R2, R5–R8, R21–R23, R26, R30, R31
13
WN
Wanaparthy
4
WN1, WN3–WN5Table 2
Identification of the 55 bacterial strains isolated from the drinking water samples collected from different government hospitals, Mahabubnagar, based on BLAST analysis of the 16S rRNA gene sequences.
Sl no.
Strain no.
Nearest phylogenetic neighbor
16S rRNA gene sequence similarity %
Gram-negative bacterial strains
Proteobacteria
Alphaproteobacteria
1
D24
Methylobacterium chloromethanicumCM4T CP001298
99.6
2
AL1
Methylobacterium zatmaniiDSM 5688T AB175647
99.7
Betaproteobacteria
3
AP1
Cupriavidus pauculusLMG 3413T AF085226
100.0
4
AL3
Delftia lacustrisDSM 21246T EU888308
100.0
Gammaproteobacteria
5
JD2
Acinetobacter calcoaceticusDSM 30006T X81661
98.5
6
KL1
Acinetobacter calcoaceticusDSM 30006T X81661
98.5
7
KL5
Acinetobacter calcoaceticusDSM 30006T X81661
98.5
8
MB1
Acinetobacter calcoaceticusDSM 30006T X81661
98.4
9
MK4
Acinetobacter calcoaceticusDSM 30006T X81661
98.5
10
NR4
Acinetobacter calcoaceticusDSM 30006T X81661
98.4
11
GD5
Acinetobacter johnsoniiDSM 6963T X81663
99.9
12
NG1
Acinetobacter johnsoniiDSM 6963T X81663
99.5
13
AP2
Acinetobacter juniiLMG 998T AM410704
100.0
14
AP4
Acinetobacter juniiLMG 998T AM410704
100.0
15
GD2
Acinetobacter juniiLMG 998T AM410704
100.0
16
JD4
Acinetobacter juniiLMG 998T AM410704
100.0
17
KL3
Acinetobacter juniiLMG 998T AM410704
100.0
18
KL4
Acinetobacter juniiLMG 998T AM410704
100.0
19
MB3
Acinetobacter juniiLMG 998T AM410704
100.0
20
R1
Acinetobacter juniiLMG 998T AM410704
99.9
21
WN1
Acinetobacter juniiLMG 998T AM410704
99.9
22
WN3
Acinetobacter juniiLMG 998T AM410704
100.0
23
WN4
Acinetobacter juniiLMG 998T AM410704
100.0
24
WN5
Acinetobacter juniiLMG 998T AM410704
99.9
25
MB4
Acinetobacter ursingiiLUH 3792T AJ275038
99.6
26
JD1
Aeromonas aquariorumMDC47T EU085557
100.0
27
R31
Aeromonas aquariorumMDC47T EU085557
100.0
28
R30
Aeromonas aquariorumMDC47T EU085557
99.9
29
GD4
Aeromonas hydrophilasubsp.hydrophilaATCC 7966T CP000462
100.0
30
JD3
Aeromonas ichthiosmiaDSM 6393T X71120
99.8
31
MK2
Aeromonas ichthiosmiaDSM 6393T X71120
99.8
32
R26
Aeromonas punctatasubsp.punctataNCIMB 13016T X60408
99.2
33
NG2
Citrobacter freundiiDSM 30039T AJ233408
98.3
34
R21
Pantoea anthophilaLMG 2558T EF688010
99.8
35
R22
Pantoea anthophilaLMG 2558T EF688010
99.9
36
NR2
Pseudomonas aeruginosaLMG 1242T Z76651
99.9
37
R6
Pseudomonas alcaligenesLMG 1224T Z76653
100.0
38
R7
Pseudomonas alcaligenesLMG 1224T Z76653
99.8
39
R23
Pseudomonas alcaligenesLMG 1224T Z76653
99.8
40
D22
Pseudomonas otitidisMCC10330T AY953147
99.8
41
GD6
Pseudomonas otitidisMCC10330T AY953147
99.8
42
MB2
Pseudomonas otitidisMCC10330T AY953147
99.6
43
MK3
Pseudomonas otitidisMCC10330T AY953147
99.7
Gram-positive bacterial strains
Actinobacteria
44
R8
Kocuria rhizophilaDSM 11926T Y16264
100.0
Firmicutes
Bacilli
45
D12
Bacillus anthracisATCC 14578T AB190217
100.0
46
KL2
Bacillus aryabhattaiB8W22T EF114313
98.7
47
NR3
Bacillus aryabhattaiB8W22T EF114313
99.7
48
R5
Bacillus cereusATCC 14579T AE016877
100.0
49
JU1
Bacillus infantisSMC 4352-1T AY904032
99.5
50
JU3
Bacillus licheniformisATCC 14580T CP000002
99.9
51
JU4
Bacillus niabensis4T19T AY998119
98.9
52
AL4
Bacillus stratosphericus41KF2aT AJ831841
99.9
53
R2
Bacillus stratosphericus41KF2aT AJ831841
100.0
54
D25
Exiguobacterium indicum HHS31
T AJ846291
99.9
55
JU2
Lysinibacillus fusiformisNBRC 15717T AB271743
99.8
The accession numbers of the 55 strains are GU566304 and GU566358.Figure 1
The affiliation of the strains to the nearest phylogenetic neighbor and the percentage of 16S rRNA gene sequence similarities.Figure 2
The affiliation of the strains to the nearest phylogenetic neighbor and the percentage of 16S rRNA gene sequence similarities.


### 3.3. Biochemical Assay

Biochemical analysis of the genusBacillus 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 results for Indole, Methyl Red, Voges-Proskaeur, Oxidase, and Gelatinase. Genus Cupriavidus gave positive results for Citrate, Oxidase, Catalase, and Urease and negative results for Indole, Methyl red, Voges-Proskaeur, and Gelatinase. Delftia gave positive results for Citrate and Urease and negative ones for Indole, Methyl red, Voges-Proskaeur, Oxidase, Catalase, and Gelatinase. Genus Acinetobacter gave positive results for Citrate and Gelatinas and negative results for Indole, Methyl red, Voges-Proskaeur, Oxidase, Catalase, and Urease (A. ursingii gave positive results even for Citrate and Oxidase and Catalase). Genus Aeromonas (Ar. aquariorum gave positive results  for all except Catalase and Gelatinase, whereas Ar. hydrophila, Ar. Ichthiosmia, and Ar. punctata gave positive results for all tests except Methyl red). Genus Citrobacter gave positive results for Methyl red, Oxidase, Catalase, and Urease and negative results for Indole, VP, Citrate, and Gelatinase. Genus Pantoea gave positive results for VP, Citrate, and Catalase and negative results for Indole, Methyl red, Oxidase, Urease, and Gelatinase. Genus Pseudomonas gave positive results for Citrate, Oxidase, Catalase, and Gelatinase and negative ones for Indole, Methyl red, VP, and Urease. Kocuria rhizophila gave positive results for Oxidase and Catalase and negative ones for the remaining tests. Exiguobacterium indicum gave positive results for Methyl red, Citrate, and Catalase and negative ones for Indole,VP, Oxidase, Urease, and Gelatinase. Lysinibacillus fusiformis gave positive results for Oxidase and Gelatinase and negative results for the remaining tests (Table 3).Table 3
Biochemical tests for the strains obtained.
Sl no.
Strain no.
Nearest phylogenetic neighbor
Biochemical tests
Ind
MR
VP
Cit
Oxi
Cat
Ure
Gel
1
D24
Methylobacterium chloromethanicum CM4T  CP001298
−
−
−
+
−
+
+
−
2
AL1
Methylobacterium zatmanii DSM  5688TAB175647
−
−
−
+
−
−
+
−
3
AP1
Cupriavidus pauculus LMG   3413TAF085226
−
−
−
+
+
+
+
−
4
AL3
Delftia lacustrisDSM   21246TEU888308
−
−
−
+
−
−
+
−
5
JD2
Acinetobacter  calcoaceticusDSM  30006T X81661
−
−
−
+
−
−
+
+
6
KL1
Acinetobacter  calcoaceticusDSM  30006T X81661
−
−
−
+
−
−
−
+
7
KL5
Acinetobacter  calcoaceticusDSM  30006T X81661
−
−
−
+
−
−
−
+
8
MB1
Acinetobacter  calcoaceticusDSM  30006T X81661
−
−
−
+
−
−
−
+
9
MK4
Acinetobacter  calcoaceticusDSM  30006T X81661
−
−
−
+
−
−
−
+
10
NR4
Acinetobacter  calcoaceticusDSM  30006T X81661
−
−
−
+
−
−
−
+
11
GD5
Acinetobacter     johnsoniiDSM     6963T X81663
−
−
−
−
−
−
−
−
12
NG1
Acinetobacter     johnsoniiDSM     6963T X81663
−
−
−
−
−
−
−
−
13
AP2
Acinetobacter junii
LMG998T AM410704
−
−
−
−
−
−
−
−
14
AP4
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
15
GD2
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
16
JD4
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
17
KL3
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
18
KL4
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
19
MB3
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
20
R1
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
21
WN1
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
22
WN3
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
23
WN4
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
24
WN5
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
25
MB4
Acinetobacter      ursingiiLUH      3792T AJ275038
−
−
−
+
+
+
−
−
26
JD1
Aeromonas aquariorum MDC47T EU085557
+
+
+
+
+
−
+
−
27
R31
Aeromonas aquariorum MDC47T EU085557
+
+
+
+
+
−
+
−
28
R30
Aeromonas aquariorum MDC47TEU085557
+
+
+
+
+
−
+
−
29
GD4
Aeromonas  hydrophilasubsp.hydrophila ATCC 7966T  CP000462
+
−
+
+
+
+
+
+
30
JD3
Aeromonas     ichthiosmiaDSM     6393T X71120
+
−
+
+
+
+
+
+
31
MK2
Aeromonas     ichthiosmiaDSM     6393TX71120
+
−
+
+
+
+
+
+
32
R26
Aeromonas punctata subsp. punctateNCIMB 13016T  X60408
+
−
+
+
+
+
+
+
33
NG2
Citrobacter       freundiiDSM      30039TAJ233408
−
+
−
−
+
+
+
−
34
R21
Pantoea anthophila  LMG2558TEF688010
−
−
+
+
−
+
−
−
35
R22
Pantoea anthophila LMG2558T EF688010
−
−
+
+
−
+
−
−
36
NR2
Pseudomonas   aeruginosaLMG   1242T Z76651
−
−
−
+
+
+
−
+
37
R6
Pseudomonas   alcaligenesLMG   1224T Z76653
−
−
−
+
+
+
−
+
38
R7
Pseudomonas   alcaligenesLMG   1224T Z76653
−
−
−
+
+
+
−
+
39
R23
Pseudomonas   alcaligenesLMG   1224T Z76653
−
−
−
+
+
+
−
+
40
D22
Pseudomonas otitidis MCC10330TAY953147
−
−
−
+
+
+
−
+
41
GD6
Pseudomonas otitidis MCC10330T AY953147
−
−
−
+
+
+
−
+
42
MB2
Pseudomonas otitidis MCC10330T AY953147
−
−
−
+
+
+
−
+
43
MK3
Pseudomonas otitidis MCC10330T AY953147
−
−
−
+
+
+
−
+
44
R8
Kocuria rhizophilaDSM 11926T Y16264
−
−
−
−
+
+
−
−
45
D12
Bacillus       anthracisATCC 14578T AB190217
−
+
+
−
−
+
−
+
46
KL2
Bacillus aryabhattai B8W22T EF114313
−
+
+
−
−
+
−
+
47
NR3
Bacillus aryabhattai B8W22T EF114313
−
+
+
−
−
+
−
+
48
R5
Bacillus cereusATCC 14579T AE016877
−
+
+
−
−
+
−
+
49
JU1
Bacillus infantis SMC4352-1TAY904032
−
+
+
−
−
+
−
+
50
JU3
Bacillus licheniformis ATCC 14580 CP000002
−
+
+
−
−
+
−
+
51
JU4
Bacillus niabensis 4T19T AY998119
−
+
+
−
−
+
−
+
52
AL4
Bacillus stratosphericus 41KF2aTAJ831841
−
+
+
−
−
+
−
+
53
R2
Bacillus stratosphericus 41KF2aTAJ831841
−
+
+
−
−
+
−
+
54
D25
Exiguobacterium indicum HHS31
TAJ846291
−
+
−
+
−
+
−
−
55
JU2
Lysinibacillus fusiformis NBRC  15717TAB271743
−
−
−
−
+
−
−
+
Ind: Indole; MR: Methyl red; VP: Voges Proskeur; Cit: Citrate; Oxi: Oxidase; Cat: Catalase; Ure: Urease; and Gel: Gelatinase.Coliform is the name of a test adopted in 1914 by the Public Health Service for theEnterobacteriaceae 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 toBacillus cereus.In the present study, species that belonged to the generaAeromonas, 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], andPseudomonas[14] were reported from drinking water systems. Aeromonasspp. 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, Klebsiellasp., 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 includingAeromonas hydrophila [4], Bacillus cereus [4], Cupriavidus pauculus [15], andPseudomonas 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 aeruginosawhich 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).Table 4
Isolation of source of the type strains associate with disease.
Sl no.
Nearest phylogenetic neighbor
Isolation source
Associated human disease
Reference
1
Cupriavidus pauculus LMG 3413T AF085226
Isolated   from   a   variety   of   human clinical sources    including    blood,    wounds, sputum, urine, eye, throat, and peritoneal fluid. In addition strains have been isolated from pool water, ground water, and bottled mineral water
Sporadically cause human infections
Vandamme  et  al., 1999 [15]
2
Acinetobacter     johnsonii DSM 6963T X81663
Isolated from clinical specimens and from eviscerated chickens and may cause ropiness in milk. Isolated from duodenum
Vascular catheter-related bloodstream infection
Seifert et al., 1993 [16]
3
Acinetobacter juniiLMG 998T AM410704
Isolated from human clinical specimens like urine
Septicemia, community-acquired bacterial meningitis, peritoneal dialysis-related peritonitis, and infections associated with corneal perforation
Hung et al., 2009 [17]
4
Acinetobacter ursingii LUH 3792T  AJ275038
Isolated from blood of a hospitalized patient with endocarditis
Bacteremia
Loubinoux et al., 2003 [18]
5
Aeromonas hydrophilasubsp. hydrophila ATCC 7966T CP000462
Isolated from humans, animals, fish, and fresh water
Acute diarrheal disease
Ljungh et al., 1977 [19]
6
Citrobacter freundiiDSM 30039T AJ233408
Isolated from canal water
Opportunistic infections like neonatal meningitis
Badger et al., 1999 [20]
7
Pseudomonas  aeruginosa LMG 1242T  Z76651
Found in soil, water, skin flora, and most manmade environments throughout the world
Localized infection of eye, ear, skin, urinary, and respiratory. Bone, joint, gastrointestinal tract, central nervous system, and systemic infection with bacteremia. Secondary pneumonia. Endocarditis
8
Pseudomonas alcaligenesLMG 1224T  Z76653
Common soil and water inhabitant that has rarely been proven a human pathogen
Endocarditis
Valenstein et  al., 1983 [21]
9
Pseudomonas otitidis MCC 10330T  AY953147
Isolated from the ears of patients with acute otitis externa (inflammation of the ear)
Inflammation of the ear
Clark et al., 2006 [22]
10
Bacillus anthracisATCC 14578T AB190217
Isolated from the blood of sheep suffering from anthrax
Cutaneous anthrax, pulmonary anthrax, and gastrointestinal anthrax
11
Bacillus cereusATCC 14579T AE016877
Isolated from soil and food materials
Food poisoning
Todar, 2008 [23]
12
Bacillus infantisSMC 4352-1T AY904032
Isolated from a newborn child with sepsis
Sepsis
Ko et al., 2006 [24]
Out of 27 representative taxa are affiliated have eight representative genera in drinking water except for those affiliated with the generaExiguobacterium, Delftia, Kocuria, and Lysinibacillus.


## 3.1. 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.


## 3.2. Identification of Isolated Strains from Drinking Water

A total of 55 bacterial strains were recovered from the 13 drinking water sources (Table1). 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,andLysinibacillus,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 Gammaproteobacteriawere 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 30006T  (98.5%).Table 1
Bacterial abundance from the drinking water samples collected from government hospitals, Mahabubnagar.
Serial number
Sample number
Place of sample collection
Number of isolates
Isolated strains
1
AL
Alampur
3
AL1, AL3, AL4
2
AP
Achampet
3
AP1, AP2, AP4
3
D
Shadnagar
4
D12, D22, D24, D25
4
GD
Gadwal
4
GD2, GD4–D6
5
JD
Jadcherla
4
JD1–JD4
6
JU
Jurala
4
JU1–JU4
7
KL
Kalwakurthy
5
KL1–KL5
8
MB
Mahabubnagar-1
4
MB1–MB4
9
MK
Makthal
3
MK2–MK4
10
NG
Nagar Kurnool
2
NG1, NG2
11
NR
Narayanpet
3
NR2–NR4
12
R
Mahabubnagar-2
12
R1, R2, R5–R8, R21–R23, R26, R30, R31
13
WN
Wanaparthy
4
WN1, WN3–WN5Table 2
Identification of the 55 bacterial strains isolated from the drinking water samples collected from different government hospitals, Mahabubnagar, based on BLAST analysis of the 16S rRNA gene sequences.
Sl no.
Strain no.
Nearest phylogenetic neighbor
16S rRNA gene sequence similarity %
Gram-negative bacterial strains
Proteobacteria
Alphaproteobacteria
1
D24
Methylobacterium chloromethanicumCM4T CP001298
99.6
2
AL1
Methylobacterium zatmaniiDSM 5688T AB175647
99.7
Betaproteobacteria
3
AP1
Cupriavidus pauculusLMG 3413T AF085226
100.0
4
AL3
Delftia lacustrisDSM 21246T EU888308
100.0
Gammaproteobacteria
5
JD2
Acinetobacter calcoaceticusDSM 30006T X81661
98.5
6
KL1
Acinetobacter calcoaceticusDSM 30006T X81661
98.5
7
KL5
Acinetobacter calcoaceticusDSM 30006T X81661
98.5
8
MB1
Acinetobacter calcoaceticusDSM 30006T X81661
98.4
9
MK4
Acinetobacter calcoaceticusDSM 30006T X81661
98.5
10
NR4
Acinetobacter calcoaceticusDSM 30006T X81661
98.4
11
GD5
Acinetobacter johnsoniiDSM 6963T X81663
99.9
12
NG1
Acinetobacter johnsoniiDSM 6963T X81663
99.5
13
AP2
Acinetobacter juniiLMG 998T AM410704
100.0
14
AP4
Acinetobacter juniiLMG 998T AM410704
100.0
15
GD2
Acinetobacter juniiLMG 998T AM410704
100.0
16
JD4
Acinetobacter juniiLMG 998T AM410704
100.0
17
KL3
Acinetobacter juniiLMG 998T AM410704
100.0
18
KL4
Acinetobacter juniiLMG 998T AM410704
100.0
19
MB3
Acinetobacter juniiLMG 998T AM410704
100.0
20
R1
Acinetobacter juniiLMG 998T AM410704
99.9
21
WN1
Acinetobacter juniiLMG 998T AM410704
99.9
22
WN3
Acinetobacter juniiLMG 998T AM410704
100.0
23
WN4
Acinetobacter juniiLMG 998T AM410704
100.0
24
WN5
Acinetobacter juniiLMG 998T AM410704
99.9
25
MB4
Acinetobacter ursingiiLUH 3792T AJ275038
99.6
26
JD1
Aeromonas aquariorumMDC47T EU085557
100.0
27
R31
Aeromonas aquariorumMDC47T EU085557
100.0
28
R30
Aeromonas aquariorumMDC47T EU085557
99.9
29
GD4
Aeromonas hydrophilasubsp.hydrophilaATCC 7966T CP000462
100.0
30
JD3
Aeromonas ichthiosmiaDSM 6393T X71120
99.8
31
MK2
Aeromonas ichthiosmiaDSM 6393T X71120
99.8
32
R26
Aeromonas punctatasubsp.punctataNCIMB 13016T X60408
99.2
33
NG2
Citrobacter freundiiDSM 30039T AJ233408
98.3
34
R21
Pantoea anthophilaLMG 2558T EF688010
99.8
35
R22
Pantoea anthophilaLMG 2558T EF688010
99.9
36
NR2
Pseudomonas aeruginosaLMG 1242T Z76651
99.9
37
R6
Pseudomonas alcaligenesLMG 1224T Z76653
100.0
38
R7
Pseudomonas alcaligenesLMG 1224T Z76653
99.8
39
R23
Pseudomonas alcaligenesLMG 1224T Z76653
99.8
40
D22
Pseudomonas otitidisMCC10330T AY953147
99.8
41
GD6
Pseudomonas otitidisMCC10330T AY953147
99.8
42
MB2
Pseudomonas otitidisMCC10330T AY953147
99.6
43
MK3
Pseudomonas otitidisMCC10330T AY953147
99.7
Gram-positive bacterial strains
Actinobacteria
44
R8
Kocuria rhizophilaDSM 11926T Y16264
100.0
Firmicutes
Bacilli
45
D12
Bacillus anthracisATCC 14578T AB190217
100.0
46
KL2
Bacillus aryabhattaiB8W22T EF114313
98.7
47
NR3
Bacillus aryabhattaiB8W22T EF114313
99.7
48
R5
Bacillus cereusATCC 14579T AE016877
100.0
49
JU1
Bacillus infantisSMC 4352-1T AY904032
99.5
50
JU3
Bacillus licheniformisATCC 14580T CP000002
99.9
51
JU4
Bacillus niabensis4T19T AY998119
98.9
52
AL4
Bacillus stratosphericus41KF2aT AJ831841
99.9
53
R2
Bacillus stratosphericus41KF2aT AJ831841
100.0
54
D25
Exiguobacterium indicum HHS31
T AJ846291
99.9
55
JU2
Lysinibacillus fusiformisNBRC 15717T AB271743
99.8
The accession numbers of the 55 strains are GU566304 and GU566358.Figure 1
The affiliation of the strains to the nearest phylogenetic neighbor and the percentage of 16S rRNA gene sequence similarities.Figure 2
The affiliation of the strains to the nearest phylogenetic neighbor and the percentage of 16S rRNA gene sequence similarities.


## 3.3. Biochemical Assay

Biochemical analysis of the genusBacillus 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 results for Indole, Methyl Red, Voges-Proskaeur, Oxidase, and Gelatinase. Genus Cupriavidus gave positive results for Citrate, Oxidase, Catalase, and Urease and negative results for Indole, Methyl red, Voges-Proskaeur, and Gelatinase. Delftia gave positive results for Citrate and Urease and negative ones for Indole, Methyl red, Voges-Proskaeur, Oxidase, Catalase, and Gelatinase. Genus Acinetobacter gave positive results for Citrate and Gelatinas and negative results for Indole, Methyl red, Voges-Proskaeur, Oxidase, Catalase, and Urease (A. ursingii gave positive results even for Citrate and Oxidase and Catalase). Genus Aeromonas (Ar. aquariorum gave positive results  for all except Catalase and Gelatinase, whereas Ar. hydrophila, Ar. Ichthiosmia, and Ar. punctata gave positive results for all tests except Methyl red). Genus Citrobacter gave positive results for Methyl red, Oxidase, Catalase, and Urease and negative results for Indole, VP, Citrate, and Gelatinase. Genus Pantoea gave positive results for VP, Citrate, and Catalase and negative results for Indole, Methyl red, Oxidase, Urease, and Gelatinase. Genus Pseudomonas gave positive results for Citrate, Oxidase, Catalase, and Gelatinase and negative ones for Indole, Methyl red, VP, and Urease. Kocuria rhizophila gave positive results for Oxidase and Catalase and negative ones for the remaining tests. Exiguobacterium indicum gave positive results for Methyl red, Citrate, and Catalase and negative ones for Indole,VP, Oxidase, Urease, and Gelatinase. Lysinibacillus fusiformis gave positive results for Oxidase and Gelatinase and negative results for the remaining tests (Table 3).Table 3
Biochemical tests for the strains obtained.
Sl no.
Strain no.
Nearest phylogenetic neighbor
Biochemical tests
Ind
MR
VP
Cit
Oxi
Cat
Ure
Gel
1
D24
Methylobacterium chloromethanicum CM4T  CP001298
−
−
−
+
−
+
+
−
2
AL1
Methylobacterium zatmanii DSM  5688TAB175647
−
−
−
+
−
−
+
−
3
AP1
Cupriavidus pauculus LMG   3413TAF085226
−
−
−
+
+
+
+
−
4
AL3
Delftia lacustrisDSM   21246TEU888308
−
−
−
+
−
−
+
−
5
JD2
Acinetobacter  calcoaceticusDSM  30006T X81661
−
−
−
+
−
−
+
+
6
KL1
Acinetobacter  calcoaceticusDSM  30006T X81661
−
−
−
+
−
−
−
+
7
KL5
Acinetobacter  calcoaceticusDSM  30006T X81661
−
−
−
+
−
−
−
+
8
MB1
Acinetobacter  calcoaceticusDSM  30006T X81661
−
−
−
+
−
−
−
+
9
MK4
Acinetobacter  calcoaceticusDSM  30006T X81661
−
−
−
+
−
−
−
+
10
NR4
Acinetobacter  calcoaceticusDSM  30006T X81661
−
−
−
+
−
−
−
+
11
GD5
Acinetobacter     johnsoniiDSM     6963T X81663
−
−
−
−
−
−
−
−
12
NG1
Acinetobacter     johnsoniiDSM     6963T X81663
−
−
−
−
−
−
−
−
13
AP2
Acinetobacter junii
LMG998T AM410704
−
−
−
−
−
−
−
−
14
AP4
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
15
GD2
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
16
JD4
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
17
KL3
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
18
KL4
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
19
MB3
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
20
R1
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
21
WN1
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
22
WN3
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
23
WN4
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
24
WN5
Acinetobacter junii LMG998T AM410704
−
−
−
−
−
−
−
−
25
MB4
Acinetobacter      ursingiiLUH      3792T AJ275038
−
−
−
+
+
+
−
−
26
JD1
Aeromonas aquariorum MDC47T EU085557
+
+
+
+
+
−
+
−
27
R31
Aeromonas aquariorum MDC47T EU085557
+
+
+
+
+
−
+
−
28
R30
Aeromonas aquariorum MDC47TEU085557
+
+
+
+
+
−
+
−
29
GD4
Aeromonas  hydrophilasubsp.hydrophila ATCC 7966T  CP000462
+
−
+
+
+
+
+
+
30
JD3
Aeromonas     ichthiosmiaDSM     6393T X71120
+
−
+
+
+
+
+
+
31
MK2
Aeromonas     ichthiosmiaDSM     6393TX71120
+
−
+
+
+
+
+
+
32
R26
Aeromonas punctata subsp. punctateNCIMB 13016T  X60408
+
−
+
+
+
+
+
+
33
NG2
Citrobacter       freundiiDSM      30039TAJ233408
−
+
−
−
+
+
+
−
34
R21
Pantoea anthophila  LMG2558TEF688010
−
−
+
+
−
+
−
−
35
R22
Pantoea anthophila LMG2558T EF688010
−
−
+
+
−
+
−
−
36
NR2
Pseudomonas   aeruginosaLMG   1242T Z76651
−
−
−
+
+
+
−
+
37
R6
Pseudomonas   alcaligenesLMG   1224T Z76653
−
−
−
+
+
+
−
+
38
R7
Pseudomonas   alcaligenesLMG   1224T Z76653
−
−
−
+
+
+
−
+
39
R23
Pseudomonas   alcaligenesLMG   1224T Z76653
−
−
−
+
+
+
−
+
40
D22
Pseudomonas otitidis MCC10330TAY953147
−
−
−
+
+
+
−
+
41
GD6
Pseudomonas otitidis MCC10330T AY953147
−
−
−
+
+
+
−
+
42
MB2
Pseudomonas otitidis MCC10330T AY953147
−
−
−
+
+
+
−
+
43
MK3
Pseudomonas otitidis MCC10330T AY953147
−
−
−
+
+
+
−
+
44
R8
Kocuria rhizophilaDSM 11926T Y16264
−
−
−
−
+
+
−
−
45
D12
Bacillus       anthracisATCC 14578T AB190217
−
+
+
−
−
+
−
+
46
KL2
Bacillus aryabhattai B8W22T EF114313
−
+
+
−
−
+
−
+
47
NR3
Bacillus aryabhattai B8W22T EF114313
−
+
+
−
−
+
−
+
48
R5
Bacillus cereusATCC 14579T AE016877
−
+
+
−
−
+
−
+
49
JU1
Bacillus infantis SMC4352-1TAY904032
−
+
+
−
−
+
−
+
50
JU3
Bacillus licheniformis ATCC 14580 CP000002
−
+
+
−
−
+
−
+
51
JU4
Bacillus niabensis 4T19T AY998119
−
+
+
−
−
+
−
+
52
AL4
Bacillus stratosphericus 41KF2aTAJ831841
−
+
+
−
−
+
−
+
53
R2
Bacillus stratosphericus 41KF2aTAJ831841
−
+
+
−
−
+
−
+
54
D25
Exiguobacterium indicum HHS31
TAJ846291
−
+
−
+
−
+
−
−
55
JU2
Lysinibacillus fusiformis NBRC  15717TAB271743
−
−
−
−
+
−
−
+
Ind: Indole; MR: Methyl red; VP: Voges Proskeur; Cit: Citrate; Oxi: Oxidase; Cat: Catalase; Ure: Urease; and Gel: Gelatinase.Coliform is the name of a test adopted in 1914 by the Public Health Service for theEnterobacteriaceae 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 toBacillus cereus.In the present study, species that belonged to the generaAeromonas, 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], andPseudomonas[14] were reported from drinking water systems. Aeromonasspp. 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, Klebsiellasp., 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 includingAeromonas hydrophila [4], Bacillus cereus [4], Cupriavidus pauculus [15], andPseudomonas 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 aeruginosawhich 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).Table 4
Isolation of source of the type strains associate with disease.
Sl no.
Nearest phylogenetic neighbor
Isolation source
Associated human disease
Reference
1
Cupriavidus pauculus LMG 3413T AF085226
Isolated   from   a   variety   of   human clinical sources    including    blood,    wounds, sputum, urine, eye, throat, and peritoneal fluid. In addition strains have been isolated from pool water, ground water, and bottled mineral water
Sporadically cause human infections
Vandamme  et  al., 1999 [15]
2
Acinetobacter     johnsonii DSM 6963T X81663
Isolated from clinical specimens and from eviscerated chickens and may cause ropiness in milk. Isolated from duodenum
Vascular catheter-related bloodstream infection
Seifert et al., 1993 [16]
3
Acinetobacter juniiLMG 998T AM410704
Isolated from human clinical specimens like urine
Septicemia, community-acquired bacterial meningitis, peritoneal dialysis-related peritonitis, and infections associated with corneal perforation
Hung et al., 2009 [17]
4
Acinetobacter ursingii LUH 3792T  AJ275038
Isolated from blood of a hospitalized patient with endocarditis
Bacteremia
Loubinoux et al., 2003 [18]
5
Aeromonas hydrophilasubsp. hydrophila ATCC 7966T CP000462
Isolated from humans, animals, fish, and fresh water
Acute diarrheal disease
Ljungh et al., 1977 [19]
6
Citrobacter freundiiDSM 30039T AJ233408
Isolated from canal water
Opportunistic infections like neonatal meningitis
Badger et al., 1999 [20]
7
Pseudomonas  aeruginosa LMG 1242T  Z76651
Found in soil, water, skin flora, and most manmade environments throughout the world
Localized infection of eye, ear, skin, urinary, and respiratory. Bone, joint, gastrointestinal tract, central nervous system, and systemic infection with bacteremia. Secondary pneumonia. Endocarditis
8
Pseudomonas alcaligenesLMG 1224T  Z76653
Common soil and water inhabitant that has rarely been proven a human pathogen
Endocarditis
Valenstein et  al., 1983 [21]
9
Pseudomonas otitidis MCC 10330T  AY953147
Isolated from the ears of patients with acute otitis externa (inflammation of the ear)
Inflammation of the ear
Clark et al., 2006 [22]
10
Bacillus anthracisATCC 14578T AB190217
Isolated from the blood of sheep suffering from anthrax
Cutaneous anthrax, pulmonary anthrax, and gastrointestinal anthrax
11
Bacillus cereusATCC 14579T AE016877
Isolated from soil and food materials
Food poisoning
Todar, 2008 [23]
12
Bacillus infantisSMC 4352-1T AY904032
Isolated from a newborn child with sepsis
Sepsis
Ko et al., 2006 [24]
Out of 27 representative taxa are affiliated have eight representative genera in drinking water except for those affiliated with the generaExiguobacterium, Delftia, Kocuria, and Lysinibacillus.


## 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 in the hospitals suggested risks of secondary infection to the patients, staff, and also patient relatives. These observations were just a drinking water quality assessment, and no data on human health scenario in this region were recorded. Further detailed studies on the health issues of the patients and other people using this contaminated drinking water are still required to trace the impact of this water consumption on people in those hospitals. The lack of awareness about good sanitation and personal hygienic practices, among governmental rural and also urban hospitals is an important factor for poor drinking water quality in these sources.



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*Source: 348250-2013-06-03.xml*