DETECTION OF GENETIC DETERMINANTS OF ANTIBIOTIC RESISTANCE AND VIRULENCE FACTORS OF Mannheimia ISOLATES FROM BRONCHOPNEUMONIA CASES OF SHEEP AND GOAT, AND EXPLORATION OF ANTIMICROBIAL PROPERTIES OF PHYTOCHEMICALS
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Date
2024-02
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SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA
Abstract
The respiratory infections of small ruminants are a burning problem for the
communities who depend on sheep and goat rearing as major income source in India.
The Mannheimia species is one of the major pathogens implicated in pneumonia of
small ruminants and responsible for huge economic loses to livestock sector worldwide.
The Mannheimia is a Gram negative, non-motile, non-spore forming, oxidase positive, catalase positive coccobacillary/bipolar organism. In the present study a total of 92
nasal swab samples were collected form clinical cases of pneumonic sheep and goats,
out of which the Mannheimia was isolated from 30 samples (25 from sheep and 5 from
goats). Morphologically, all the isolates characterized as bipolar & cocco-bacillary in
Gram’s staining. Culturally, the Mannheimia isolates developed small pin-point pink
coloured colonies on MacConkey agar, and small greyish haemolytic colonies on Blood
agar. The phenotypically confirmed Mannheimia isolates were further genotyped by
using genus specific primers targeting the gene coding for 16S rRNA. The genotypic
primers specifically reacted and typed the isolates as belongs to the genera Mannheimia
with an amplified product size of 304 bp. Overall the prevalence of Mannheimia was
found to be 32.6%. The phylogenetic analysis revealed that the Mannheimia isolates
from Andhra Pradesh formed a separate clade with close relation to M. hemolytica,
Canada and M. caviae, Denmark strains.
The biofilm forming ability of the Mannheimia isolates was evaluated by three
methods such as Standard Tube method (ST), Congo red agar method (CRA) and
Microtiter plate assay (MTP). Among, MTP assay was considered as best method which
detected and quantified the biofilm production by 29 (96.6%) isolates. The antibiogram
pattern of Mannheimia isolates revealed that high resistance to Ampicillin (80%)
followed by Gentamicin (50%), Co-trimoxazole (43%), Tetracycline (40%),
Amoxicillin-Clavulanic acid (40%), Ceftriaxone (36.6%) and Enrofloxacin (26.6%). On
analyzing the multiple antibiotic resistance index (MAR), none of the samples revealed
a MAR index of less than 0.2, which indicates a potential threat of rising multi-drug
resistant (MDR) bacteria. The genes coding for antibiotic resistance of streptomycin and
extended spectrum β-lactamases (ESBL) were identified using gene specific primers in
multiplex PCR with a prevalence rate of 70% for strA gene, followed by 16% for
blaOXA, 13.3% for blaTEM and 10% for blaSHV genes. The genes coding for virulence
factor leukotoxin (Lkt) was found in only two isolates. The antimicrobial properties of
selected phytochemicals were evaluated which can be tested as alternative approach to
combat the threat of rising MDR strains of Mannheimia. The Eugenol, an extract of
Syzygium aeromaticum, was found to effective with MIC ranging between 32-2052
µg/ml against the Mannheimia isolates whereas the MIC of Cinnamic acid was found to
be ranging between 1.25 to 2.50 mg/ml.
In conclusion, our study provides the detailed information on prevalence of
Mannheimia in clinical cases of Pneumonia of sheep and Goats, its phylogenetic
relations, pathogenic potential, danger of rising MDR strains and phytochemicals,
Eugenol and Cinnamic acid, as alternative therapies to combat the MDR strains.