1. Environmental stress exacerbate the emergence of Multidrug
Resistant Bacteria – Global health hazard
Hemlata and ArchanaTiwari*
School of Sciences, Noida International University, Gautam Budh Nagar, India
Abstract: Environmental pollution triggers myriad of wildlife and public health hazards. Wastewater treatment as well as hospital effluents are “hot spots” for the transmission of resistant bacteria. Multidrug resistant bacteria (MDR) have been found widely in aquatic environments majorly in rivers where they originate from anthropogenic sources; such as hospital, municipal, and aquaculture effluents. Both anthropogenic and naturally occurring MDR in aquatic ecosystem triggers human health as infected by MDR through drinking water, aquatic edibles and direct contact with water bodies. Aquatic environment has direct intimate contact with human life due to continuity of the water body that may transport antibiotic resistance to every corner of the world as compared with soil. Waste water effluents nurture resistance genes and genetic elements that promote their genetic exchange between bacteria. Commensals or pathogens are important sources of resistance genes that cause human infections and disease. This leads to infections causing higher morbidity and mortality and imposing huge costs on our society. The selective pressure of above discussed factors emerges the new category of multidrug resistant bacteria named Extended Spectrum of β-lactamases (ESBLs) common ones include TEM, SHV, OXA, and recently evolved CTX-M of third generation cephalosporins for the treatment of severe infections. In this study water samples was collected from tertiary phase effluent spot Yamuna action sewage treatment plant, Haryana; that harbors a variety of MDR later on enter in ecosystem via food chain or food web and causing health hazard. We focused on prevalence of MDR genes in environment. 110 non-duplicate isolates screened, 70 were found to be positive for ESBL production through clinical and laboratory standards institute (CLSI) agency of USA. Environmental Selective pressure of different kinds of wastes accelerates the emergence of multi drug resistance bacteria. Innovative advanced water treatments approaches need to be investigated that are focused on the quality treatment and effectively measure.
Introduction: Ecological nutrient cycling is the pathway of exchange of organic and inorganic materials and environment pollution through STP’s harbor the growth of Multidrug resistant bacteria. The selective pressure and exposure to heavy metals, pesticides, and herbicides as non-biodegradable naturally selects organisms with the genes of resistance necessary for their survival. These metals are used in various industries from which effluents are consequently discharged into the environment and accelerates this notorious phenomenon
Methodology:
Isolation and Screening of bacterial isolates: Water sample was collected from tertiary phase effluent spot of Yamuna action sewage treatment plant, Palwal, Haryana. Sample was serially diluted and spread on LA plates for selection of morphologically non-duplicate colonies and streaked to get pure culture.
ESBL Test: Bacterial isolates were screened for ESBL production by preliminary test and Phenotypic Disc Confirmatory Test (PDCT) according to CLSI guidelines.
Antibiotic Sensitivity test: Resistant isolates were tested for susceptibility towards wide range of antibiotics using Kirby-Bauer method and their MIC was determined.
16S rDNA profiling: 16S rRNA genes were amplified using gene specific primers and sequenced.
Amplification of blaCTX genes: ESBL genes blaCTX were amplified from genomic DNA using gene specific primers and sequenced.
Results: A total of thirty colonies growing on Luria agar plates were used for screening of ESBL production.
Screenings of isolates for ESBLs production:
Preliminary Test: Twelve isolates were found
positive in preliminary test for ESBL.
Phenotypic Disc Confirmatory Test (PDCT): A total of twelve isolates were confirmed for ESBL production in
PDCT showing a ≥ 5 mm increase
in zone diameter for antibiotic CAZ
and CTX + clavulanic acid than
antibiotic alone.
Antibiotic Sensitivity test: Phenotypicaly ESBL positive E.coli isolates were subjected to susceptibility tests
towards eight different antibio-
tics for the determination of
Zone of Inhibition (ZOI).
16S rRNA gene based characterization of bacterial isolates:
PCR successfully amplified approximately 650bp fragment corresponding to partial 16S rRNA gene. The amplified product was sequenced.
PCR amplification of blaCTX gene: ̴ 860 bp fragment corresponding to
complete blaCTX gene was successfully amplified from genomic DNA of eleven ESBL positive isolates.
Fig 1: Bacterial colonies growing on LA media. M
̴ 650 bp
Fig 5: PCR based amplification of 16S rRNA gene (~650 bp).
Lane M: 100 bp DNA ladder
Lane 1-12: Amplified 16S rRNA gene in different isolates.
Table 1: Zone of inhibition of CAZ, CTX and CTR.
Isolates
Ceftazidime
Cefotaxime
Ceftriaxone
AT1 21 28
AT2 20
AT3 23
AT6 22 30
AT7 19 25 24
AT8 18
AT9 12
AT13 34
AT14 27 33
AT15 26 31
AT16
AT18 EC KP 09 16
16S rRNA Gene
Oligo Sequence
Forward Seq.
GGCGGACGGGTGAGTAATG
Reverse Seq.
ATCCTGTTTGCTCCCCACG
Fig 2: Plates showing zone of inhibition
for antibiotics CAZ, CTX and CTR.
blaCTX Gene
Oligo Sequence
Forward Seq.
SCVATGTGCAGYACCAGTAA
Reverse Seq.
GCTGCCGGTYTTATCVCC
C M
̴860 bp
Table 2: ZOI (mm) of antibiotic and antibiotic + clavulanic acid
Bacterial
Isolates
Ceftazidime
Ceftazidime + Clavulanate
Cefotaxime
Cefotaxime + Clavulanate
AT1 30 31 26
AT2 20 21 24 22
AT3 33 35
AT6 38
AT7 11 28 29 32
AT8 25
AT9 13
AT13 39 36
AT14 34
AT15
AT16 27
AT18
35s EC KP 09 16 23
Fig 3: Plates showing a ≥ 5 mm
increase in zone of inhibition
Fig 6: PCR based amplification of blaCTXgene (~860 bp).
Lane M: 100 bp DNA ladder .
Lane 1-11: Amplified blaCTX-M gene product.
Lane +C: Positive control
Fig 7: Phylogenetic Tree of identified isolates with different variants of blaCTX-M.
Table 3: ZOI (mm) of different antibiotics against bacterial isolates. AK
AMP
CIP CX
CXM
IPM OF
TOB
Isolates
ZOI
S/I/R
AT1 17 S 12 R 06 18 22 I
AT2 15 16 20 28
AT3 11 13 10
AT6 19
AT7 21 24 27
AT8 25
AT9 37 30 41 35
AT13
AT14
AT15 14 23
AT16 08
AT18 26 EC 31 KP
Conclusion:
Bacterial isolates collected from STP were found to harbour ESBL genes which are of common occurrence among clinically important isolates.
Out of twelve ESBL+ isolates, eleven were found positive for blaCTX-M genes representing a high prevalence of ESBL genes among environmental isolates.
Isolates resistant to β-Lactams were also tolerant to other classes of antibiotics presenting a multidrug resistant phenotype.
Study of resistance pattern in environmental isolates would help to prevent development and spread of resistance genes.
Fig 4: Zone of inhibition of different antibiotics against bacterial isolate. 
References
Hemlata, Arif Tasleem Jan and Archana Tiwari (2016). CDM. 17,
Davies J and Davies D (2010). Microbiol. Mol Biol Rev. 74:
WHO (2014) Global Tuberculosis Report.
Berdy J (2012). J Antibiot. (Tokyo). 65: 385–395.
Positive control : Klebsiella pneumoniae (ATCC ); Negative control: E.coli (ATCC 25922)
Acknowledgement :