Prevalence of Cronobacter spp. in tropical seafood from Mumbai, India: Comparative study of isolation media and PCR detection.

Abstract

Cronobacter species are opportunistic emerging pathogens associated with diverse foods of plant and animal origin. Considering the diversity of the Cronobacter group of bacteria and their co-existence with closely related Enterobacterales in the aquatic environment, their isolation from fish and shellfish is a challenge. This study aimed to investigate the incidence of Cronobacter in finfish, shellfish, and dried fish, and to compare nine combinations of enrichment broth-selective isolation media for efficient isolation of Cronobacter spp. Seventy-five seafood samples collected from five different retail markets were subjected to multiple selective-enrichment methods to isolate Cronobacter, which were presumptively identified by biochemical tests followed by confirmation with genus- and species-specific PCRs. Of 75 seafood samples analyzed, 24 (32%) were positive for Cronobacter spp. The highest incidence was in dried fish (21 samples, 47.72%), followed by 19 (43.18%) fresh finfish and four (9.09%) shellfish samples. Forty-four isolates from these samples were identified as Cronobacter spp. by PCR. Species-specific PCR further categorized these as C. sakazakii (25), C. malonaticus (16) and C. turicensis (1), while two isolates remained unidentified at species level. Enrichment in Cronobacter screening broth or Rappaport Vassiliadis medium, followed by isolation on the chromogenic Cronobacter sakazakii agar was found to be the most effective combination for the isolation of Cronobacter spp. from seafood. Dried fish is an important reservoir of C. sakazakii owing to its desiccation tolerance and absence of competing microbiota in dried fish. Although C. sakazakii is the only known pathogen among Cronobacter spp., improved and specific methods to identify diverse members of this genus are needed.