First field-based evidence of association of Proteus mirabilis causing large scale mortality in Indian major carp farming

Diseases caused by opportunistic pathogens have been generally overlooked where a pathogen causes mild clinical signs with sporadic mortality at the farm level. Here, we identified an opportunistic Gram negative bacterium, Proteus mirabilis, from Indian major carp, Labeo rohita, exhibiting disease and mortality in the culture farm and subsequently confirmed for its virulence in experimental challenge study. The bacterium was identified as P. mirabilis based on phenotypical, biochemical characters and 16s rDNA PCR followed by virulence/species specific gene PCRs targeting urease, metalloprotease and haemolysin genes. Experimental challenge of healthy rohu juveniles with 107 cfu/fish reproduced the clinical signs and caused 100% mortality within 3 days of challenge. The affected fish, as well as the experimentally infected fish exhibited haemorrhages on the body along with red patches over the operculum and lower abdomen before dying. Histopathological analysis of the posterior kidney of infected rohu revealed significant renal damage showing massive glomerular congestion, focal periglomerular necrosis and periglomerular mononuclear aggregation. Skin tissues showed massive inflammatory reaction in epidermis, dermis, and myositis below the dermal layer. Further, the presence of amplicons for integrons I and II was also determined by concurrent analysis, thus implying significant horizontal stability and mobility in the gene capture system of this isolate along with enhanced resistance to antibiotics. This isolate was found to be multi-drug resistant showing resistance to 10 classes of antibiotic groups. Phylogenetic analysis based on 16s rRNA gene revealed its close association with other reported P. mirabilis from different locations with common ancestry and formed a distinct cluster along with other Proteus species. This study seems to be first record of sole involvement of MDR P. mirabilis causing mortality in fish farm, which was mostly considered as a favourable microbe in aquaculture systems.