1 Fisheries Laboratory, Department of Zoology, University of Azad Jammu and Kashmir, King Abdullah

Overview

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Key numbers

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mean risk of illness (%) due to Bacillus species was recorded in children (56.67% to
70.0% compared to the adults (47.0% to 50.0%). All isolated spring water-associated
square kilometers. The weather in this area is highland and subtropical. Temperatures range from 42°C to −3°C, and the
at 37°C, the isolates were moved to a glass slide. Catalase activity was detected by dropwise addition of 3% H2O2; the
growth at 37°C on nutritional agar, 1% oxidase reagent was added to the isolates. The presence of the oxidase enzyme
In the KOH test, a loopful of bacterial growth on a microscope slide was combined with two drops of 3% KOH.
taining 100 mg/L tryptophan, a precursor of IAA. Each test tube was filled with Kovac’s reagent after incubation, and the
changed from bright yellow to orange-brown, indicating a successful cyanide manufacturing outcome (48,49).
by pink, whilst negative urease activity was indicated by orange (43). In lipolytic activity, bacterial isolates were cultivated
for 24 hours at 35 ± 2°C on nutritional agar plates supplemented with 1% Tween 80. After incubation, the formation of a
10 ⁻ ⁶/0.000001), Very low risk (10 ⁻ ⁶ – 10 ⁻ ⁵), generally acceptable (10 ⁻ ⁵ – 10 ⁻ ⁴), Low risk (> 10 ⁻ ⁴), may be acceptable with
justification, Exceeds WHO health targets → intervention required, and High risk (> 10 ⁻ ²/1%) – unacceptable.
°C and 60–80% humidity for 10–12 hours/day. At the end of the experiment, water samples were collected, and micro-
(Mean ± SD) using the software Excel 16. All water samples were analyzed in triplicate. One-way analysis of variance
Table 1). All SWABs were gram-positive rods and endospore-forming. Differential and selective cultural media showed that
indole acetic acid (Table 1). Similarly, all SWABs produced urease enzymes except SWAB-9, SWAB-10, SWAB-12,
SWAB-2 only produced ammonia, and SWAB-5 and SWAB-12 produced proteases (Table 1). It was observed that all
Table 1. Biochemical characterization of spring water-associated bacteria.
Table 2 indicates that all isolated SWABs (SWAB-1 to SWAB-19) were resistant against Amoxycillin (AMC-30), Aztreonam
The range of amplified PCR products was recorded as 932 bps- 1240 bps (Table III). Extracted DNA from some samples,
homology percentage was recorded for all spring water-associated bacterial isolates (Table 3).
Table 2. Antibiogram analysis of spring water-associated bacteria via agar disc diffusion method.
Table 3. BLAST Analysis of spring water-associated bacterial strains.
SWAB-5 (1240 bps) Bacillus albus 1400 99% 0 87.91 1460 OM232457.1
Bacillus wiedmannii 1740 99% 0 93.42 1426 OM510278.1
Table 3. (Continued)
SWAB-8 (1112 bps) Lysinibacillus fusiformis 2091 98% 0.0 99.48% 1444 JQ071512.1
Lysinibacillus sphaericus 2087 98% 0.0 99.39% 1465 MF000302.1
Lysinibacillus fusiformis 2089 98% 0.0 99.39% 1454 PP565089.1
Lysinibacillus sphaericus 2087 98% 0.0 99.48% 1514 KF228925.1
SWAB-9 (1152 bps) Lysinibacillus sphaericus 2087 98% 0.0 99.39% 1465 MF000302.1
Lysinibacillus fusiformis 2091 98% 0 99.48 1444 JQ071512.1
Lysinibacillus fusiformis 2089 98% 0 99.39 1454 PP565089.1
Lysinibacillus fusiformis 2087 98% 0 99.48 1482 GU125642.1
Lysinibacillus sp. 2087 98% 0 99.48 1493 OR902475.1
Lysinibacillus fusiformis 2087 98% 0 99.48 1444 MN826508.1
Lysinibacillus sp. VKK-5OL 2087 98% 0 99.48 1506 JX871464.1
Bacillus nitratireducens 1805 98% 0.0 94.58% 1427 MK418697.1
Bacillus wiedmannii 1740 99% 0 93.42 1426 OM510278.1
Bacillus nitratireducens 1748 99% 0.0 93.12% 1427 MK418697.1
SWAB-13 (1152 bps) Bacillus wiedmannii 1740 99% 0.0 93.42% 1426 OM510278.1
Table 3. (Continued)

Methods (brief)

cereus, Bacillus anthracis, Lysinibacillus fusiformis, Bacillus wiedmannii, uncultured
instantly. These spring water samples are used for drinking by the local population. Muzaffarabad, situated in Pakistan’s
garden soil and vermicompost were collected from the University of Azad Jammu and Kashmir (UAJ&K), Muzaffarabad,
2.2.1. Isolation of bacteria. A 100 ml of spring water was collected and placed at room temperature for bacterial
associated with spring water samples. Nutrient broth medium (NBM) was used for bacterial growth. Serial dilutions of 10 ⁻ ²
the sample (10 µl) was spread onto nutrient agar medium (NAM) and incubated overnight at 37°C. The next day, multiple
original sample was calculated using the formula: CFU = (Number of colonies × Dilution factor)/ Volume of inoculum. A
then autoclaved. After that, a wire loop was used to aseptically inoculate the bacterial sample. A few drops of phenol red
on bacterial isolates from spring water samples to evaluate their adaptive tolerance to heavy metal contamination, which
2.2.5. Molecular analysis. Genomic DNA was isolated from samples using the QIAGEN DNeasy Mini Kit, utilizing
5 min), and 35 cycles, and the expected product size was 1.4 kb to 1.6 Kb. After PCR analysis, 19 samples were sent
and macerated using a mortar and pestle. The crude aqueous extracts were collected, filtered using Whatman No. 1 filter
For the ex-situ phytoremediation of spring water, B. rapa and S. oleracea were used. Spring water samples such as
to high CFU/mL thresholds. Garden soil was collected from the University grounds, Muzaffarabad, and all physiochem-
°C and 60–80% humidity for 10–12 hours/day. At the end of the experiment, water samples were collected, and micro-
ically the Winkler procedure, and chemical oxygen demand (COD) of the sample spring waters was determined using
(Mean ± SD) using the software Excel 16. All water samples were analyzed in triplicate. One-way analysis of variance
The range of amplified PCR products was recorded as 932 bps- 1240 bps (Table III). Extracted DNA from some samples,

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The five most recent substantive edits to this page, classified major (evidence or structure moved), correction (a published value or statement was wrong and has been fixed), or minor (narrative rewritten without changing the underlying evidence). Each description is derived from what the edit did to this page; the linked commit is the authoritative record, routine regeneration passes are excluded, and the full version history lives in git. When DOI minting comes online (see schema docs), each entry below will also link to a version-pinned DataCite DOI.

Commit	Date	Change	Description
b01ec52c	2026-08-04	major	2 sections added
d49e450f	2026-08-03	major	5 sections added; narrative text revised