NewBioWorld A Journal of Alumni Association of Biotechnology (2024) 6(1):01-06
RESEARCH
ARTICLE
Isolation and characterization of multiple Drug resistant
bacteria isolated from poultry waste water
Sadhana
Jaiswal1, Deepali Rajwade2*, Preeti Mehta1, Chandrika Sarkar1
1Department
of Microbiology, Govt. Nagarjuna P.G. College of Science, Raipur, Chhattisgarh,
India.
2Department of Biotechnology, Govt. Nagarjuna P.G.
College of Science, Raipur, Chhattisgarh,
India.
Author’s
Email- skaushaljaiswal123@gmail.com,
ddrajwade@gmail.com, mehta.prity09@gail.com
*Corresponding Author Email- ddrajwade@gmail.com
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ARTICLE INFORMATION
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ABSTRACT
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Article history:
Received
12 April 2024
Received in revised form
15 June 2024
Accepted
Keywords:
MDR;
Poultry waste;
Antimicrobials;
Waste
water.
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India is one of the biggest producers of poultry all
over the world. Huge quantities of poultry meat consumption lead to
generation of large amount of poultry wastes. This waste may consist of many
disease-causing microorganisms and thus improper disposal of it may pose
severe environmental & Health risks. Irrational application of
antibiotics in poultry & other veterinary set ups may result in abundance
of microorganisms resistant to antibiotics is a serious problem for humanity.
This research work aims at the isolation &characterization of multiple
drug resistant bacteria from poultry sites. Water samples were taken from 10
different poultry sites of Raipur city, Chhattisgarh. The preliminary
identification of bacterial Isolates was done on the basis of Morphological
and Biochemical Characterization using a online software tool. The presence
of Multidrug resistant bacteria was screened using 12 common antibiotics. The
isolate showing multiple drug resistance was subjected to molecular
characterization using 16S RNA sequencing.
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Introduction
DOI: 10.52228/NBW-JAAB.2024-6-1-1
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Development of antimicrobial resistance is a one of the major threat to
mankind all over the world. Its risks are mostly related to the inability to
treat patients infected with antibiotic-resistant bacteria appropriately and a
greater possibility of these resistant infections spreading furthermore (Roca et al.
2015). Antibiotic abuse, such as their overuse in therapeutic treatments and as
growth boosters in animal production systems, is linked to the prevalence of
this resistance (Zwe et al.
2018).There is now enough data to conclude that the overuse of antibiotics and
animal resistance to antibiotics are interrelated, and that this contributes to
the overall burden of antibiotic-resistant infections (Hugo et al.
1998). The industrial sectors that produce chicken, meat and egg products are
currently regarded as some of the significant and rapidly expanding agri-food
sectors (Mottet et al.
2017; Berkhout 2020).
Since all production processes need hygiene and quality control, industrial
procedures are usually associated with the consumption of significant volumes
of freshwater (Kuyeli 2007).
Large volumes of wastewater that is extremely polluted are produced as a
consequence (Dlangamandla et al.
2016). Poultry waste has been found to contain a high load of pathogenic
organisms such as Pseudomonas aeruginosa, Salmonella, Shigella, E.
coli, Vibrio cholerae and Brucecella, along with their organic and
inorganic load (Shannon et al.
2007). Non-pathogenic bacteria include strains of the Streptococcus group,
total and faecal coliforms, of which Aeromonas sp. and Clostridium sp.
are the two main indicators (Kosamu et al.
2011). Most countries raise poultry with an extensive range of antimicrobials (FAO 2017).
Many of these antimicrobials are thought to be crucial for human treatment (Boamah et al.
2016). The indiscriminate uses of these vital antibiotics in animal husbandry
are probably going to hasten the development of antibiotic resistance in
pathogens and commensal organisms. This could lead to therapeutic failures,
financial losses, and the creation of a gene pool that could be passed on to
humans. Specifically, one major issue facing medical practitioners nowadays is the
emergence of multi-drug resistance in Gram-negative bacteria (Exner et al. 2017).
The primary mechanisms via which resistance is mediated in Gram-negative
bacteria include amp C β-lactamases, carbapenemases, and extended spectrum
β-lactamases (ESBL) (Schill et al.
2017). Therapeutic problems will arise from infections with these
multi-drug-resistant organisms (MDROs); the pipeline of antibiotics is closing,
and no new antimicrobial medicines are expected in the near future to treat
infections caused by MDROs (Bettiol
and Harbarth 2015).
Material
and Method
Sample
collection
To assess the microbiological quality of poultry
waste water 10 sampling sites were selected from different areas of Raipur city, Chhattisgarh.
Waste water samples from the poultry sites were collected in
sterile plastic bottles and brought to the Microbiology laboratory in mini
insulated ice box. To
prevent further growth of microorganisms in the collected sample, the sample were processed within 1
hours of their collection.
Physico-chemical
and microbiological analysis of sample
Temperature of the
water samples was measured
at the collection site using digital thermometer & pH was measured with the help of
Systronic 335 pH meter in the
laboratory. MPN test was performed by following the standard
method.
Isolation and characterization of bacterial isolates
An aliquot
of poultry wastewater sample was spread over the surface of
sterilized and solidified EMB agar media, incubated for 24 to 48 hours at 37±
1º C. The bacterial colonies appeared were further purified by sub-culturing.
Morphological characters
like colony colour, form, elevation, margin and the colony
density
were noted.
The shape and arrangement of the cells was observed byGram staining for each isolate. Various biochemical test like IMViC test, Catalase Test, Oxidase Test, Urease Test and Triple
Sugar Iron Agar test were carried out by following the standard methods.
Antibiotic susceptibility
test
The
isolates characterized biochemically were further subjected to antibiotic
susceptibility test in Muller Hinton agar medium by disk diffusion method .It is a
simple, practical, cost effective and well-standardized method. Commonly
prescribed antibiotics for gastrointestinal infection were used as test
antibiotics. Zone of inhibition was measured after an incubation
period of
24-48 hours.
Results
and Discussion
Physiochemical
Characterization and microbiological analysis of samples
The temperature & pH of water samples ranged from 29-35 and 4.5 -6.5 respectively.
As indicated in the table 1, most
of the water samples show neutral to slightly alkaline pH, which is
favourable for the growth of
maximum number of the bacteria. The MPN
values obtained ranged from 2-9 /100 mL where site 6 showed maximum MPN value. This
indicates the possibility of faecal contamination in poultry waste water which
is hazardous for human consumption .Similar study by Bhumbla et al. (2020) at
Udaypur Rajasthan reported that MPN value exceeding 10 is unsatisfactory for
human use.
Colony
characterization and morphology
The primary identification of the isolated was done on the
basis of morphological &
biochemical characteristics. 10
bacterial isolates were selected.
Antibiotic
resistance test of the bacteria
Antibiotic Susceptibility test was performed using
the disk diffusion method in Mueller Hinton agar media. The isolates showed
sensitivity against commonly used broad spectrum antibiotics like Nalidixic, Streptomycin,
Tetracycline, Kanamycin, Gentamicin and Neomycin. Isolate 7 and 10 were found
to be sensitive to all the used antibiotics. Whereas isolate 6 showed resistance against multiple drugs such as Cefazolin,
Chloramphenicol ,Amoxyclav & Sulphafurazole.
Table 1. Physiochemical
Characterization of the samples.
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Site
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Samples
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pH
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Temp.
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MPN
Index/
100ml
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Site
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SAMPLES
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pH
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Temp.
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MPN
Index/
100ml
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1.
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Bhanpuri, Raipur [CG]
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4.5
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33.33
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2
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6.
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Kota, Raipur [CG]
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5.7
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34.44
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9
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2.
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Khamtari, Raipur [CG]
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6.3
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32.22
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2
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7.
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Daganiya Market, Raipur [CG]
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5.5
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33.66
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4
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3.
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Khamtari, Raipur [CG]
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6.5
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35.5
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6
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8.
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Hirapur, Raipur [CG]
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5.8
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35.5
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4
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4.
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Khamtari, Raipur [CG]
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5.5
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30.5
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7
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9.
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Changurabhata, Raipur [CG}
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6.0
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30.22
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2
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5.
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Kota, Raipur [CG]
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4.8
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29.44
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7
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10.
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Sunder Nagar, Raipur [CG}
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4.7
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32.22
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2
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Table 2.
Colony Characterization and Morphology of Bacteria
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Isolate no.
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Color
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Form
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Elevation
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Margin
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Density
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Gram P/N
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Shape
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Isolate no.
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Color
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Form
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Elevation
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Margin
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Density
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Gram P/N
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Shape
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1
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Blue
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Circular
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Raised
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Entire
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Opaque
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N
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Rod
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11
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Blue
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Irregular
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Flat
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Undulate
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Opaque
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N
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Rod
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2
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Pink
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Punctiform
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Raised
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Undulate
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Opaque
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N
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Rod
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12
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Pink
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Irregular
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Pulvinate
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Entire
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Opaque
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N
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Rod
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3
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Purple
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Punctiform
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Flat
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Undulate
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Opaque
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N
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Rod
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13
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Green
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Punctiform
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Flat
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Undulate
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Opaque
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N
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Rod
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4
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Green
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Punctiform
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Flat
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undulate
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Opaque
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N
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Rod
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14
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Purple
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Punctiform
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Flat
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Entire
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Opaque
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N
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Rod
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5
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Pink
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Irregular
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Pulvinate
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Entire
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Opaque
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N
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Rod
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15
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Pink
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Irregular
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Raised
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Undulate
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Opaque
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N
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Rod
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6
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Blue
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Circular
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Raised
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Entire
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Opaque
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N
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Rod
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16
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Green
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Punctiform
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Flat
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Undulate
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Opaque
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N
|
Rod
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7
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Purple
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Punctiform
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Raised
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circular
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Opaque
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N
|
Rod
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17
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Purple
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Punctiform
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Raised
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Circular
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Opaque
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N
|
Rod
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8
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Pink
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Irregular
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Pulvinate
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Undulate
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Opaque
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N
|
Rod
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18
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Pink
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Punctiform
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Raised
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Undulate
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Opaque
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N
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Rod
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9
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Green
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Circular
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Flat
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Undulate
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Opaque
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N
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Rod
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19
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Blue
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Circular
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Raised
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Entire
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Opaque
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N
|
Rod
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10
|
Purple
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Irregular
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Raised
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Undulate
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Opaque
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N
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Rod
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20
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Pink
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Irregular
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pulvinate
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Entire
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Opaque
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N
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Rod
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Table 3. Biochemical
Characterization of bacterial isolates
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Sample
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IMVIC
TEST
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TSIA
TEST
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Urease
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Catalase
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Oxidase
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Indole Test
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MR test
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VP Test
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Citrate test
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Colour of
Slant/ Butt
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Lactose fermentation
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Fructose
Fermentation
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Dextrose
Fermentation
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Gas Production
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H2S production
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1.
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-
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-
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-
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+
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Y/B
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+
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+
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-
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+
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+
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-
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+
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+
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2.
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-
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+
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-
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+
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Y/B
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-
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-
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+
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+
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+
|
-
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+
|
-
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3.
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-
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+
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-
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+
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Y/Y
|
-
|
-
|
+
|
+
|
-
|
-
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+
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-
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4.
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-
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-
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+
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+
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Y/Y
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+
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+
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+
|
-
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-
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-
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+
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-
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5.
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+
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+
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-
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-
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R/B
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-
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-
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+
|
-
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+
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-
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+
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-
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6.
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-
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-
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-
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+
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R/Y
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-
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-
|
-
|
+
|
-
|
-
|
+
|
+
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7.
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-
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-
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-
|
+
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R/Y
|
+
|
+
|
+
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+
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-
|
-
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+
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+
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8.
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-
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-
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-
|
+
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Y/Y
|
-
|
-
|
+
|
-
|
-
|
+
|
+
|
+
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9.
|
+
|
-
|
+
|
+
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Y/B
|
+
|
+
|
+
|
-
|
+
|
-
|
+
|
+
|
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10.
|
-
|
-
|
-
|
+
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R/Y
|
-
|
-
|
-
|
+
|
-
|
-
|
+
|
+
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Multiple
antibiotic resistance [MAR] Index
Multiple antibiotic resistance (MAR) index is an
effective tool for bacterial source tracking. This method helps in
differentiating between human and nonhuman faecal sources (Scott et.al.
2002). MAR indices
of the present isolates, belonging to Coliform bacteria against the tested
antibiotics were calculate based on the formula as follows (Tambekar et al.
2005).
MAR index for isolates = a⁄b
Where,
a = Number of antibiotics to which
the isolate is resistant
b = Number of antibiotics tested.
The overall resistance of isolates to the
antibiotics is represented as MAR index. Whereas MAR index of the sampling
sites ranged from 0 to 0.33, maximum being for the
isolate 6.
Most of
the bacterial isolates
indicated high degree of antibiotic resistance to Ampicillin, and Amoxiclav
whereas less resistance to Cefazolin, Nalidixic, Tetracycline, Gentamicin and
Neomycin. Similar results were reported by Mane et al. 2014 where multidrug-resistant isolates
showed high resistance against Ampicillin and sensitivity towards Gentamicin and Amikacin. The presence of Multiple Drug Resistance
(MDR) bacteria in the environment with susceptible ones increases the chances
of antibiotic
resistance being transferred to the sensitive ones. It may contribute to the general
increase and dissemination of bacterial resistance and may be a source of
resistance genes for pathogenic bacteria (Pontes et al. 2009). In
previous reports MDR E. coli strains were isolated from water
that demonstrated high resistance for ampicillin &
various other antibiotics
(Sharma and Rai
2012). Sirisha et al. (2017) isolated many multiple drug resistant bacteria
from Kundu river water.
Probablistic identification of bacteria
On the basis of morphological and biochemical
characteristics the bacterial isolates were identified using an online tool
ABIS. The table 5 shows
the probable bacterial species.
Conclusion
The results observed in the present study indicates
the prevalence of MDR in the poultry waste water. These bacteria are capable of
causing various threatening diseases to the mankind and in addition can harm
the environment too. The present study has strongly
implicated that the Microbiological standards of Poultry waste water must be
developed to a large extent to validate the safety standards. So, from the view
of prevention, contamination should be controlled by applying various strict
preventive measures to secure the lives and environment. Several bacterial
species are the primary sourse of infections in poultry and other animal
husbandry. Most of these infections are linked to food borne outbreaks, live
animal contact, poor hygiene, and environmental exposure. With the emergence of
antimicrobial resistance, the pathogenicity and virulence of these organisms
have increased and treatment options are diminishing and also more expensive.
To reduce the emergence of antimicrobials, the use of tradition herbal products
should be encouraged, many researchers (Verma et al. 2023; Chandra et al. 2023;
Singh et al. 2021) reported the antimicrobial efficacy of various herbs and
spices against gram positive and gram negative bacteria. Prevention of irrational use
of antibiotics, incorporation of eco-friendly & environment sensitive
practices in poultry and other animal settlements need to be followed.
Conflict of interest Authors declare that they have no conflict of
interest.
Funding
information not applicable.
Ethical
approval not applicable.
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