A modified culture medium for improved isolation of marine vibrios.

Marcello Tagliavia,Monica Salamone,Angela Cuttitta,Paola Quatrini,Carmelo Bennici

doi:10.1002/mbo3.835

Abstract

Marine Vibrio members are of great interest for both ecological and biotechnological research, which often relies on their isolation. Whereas many efforts have been made for the detection of food‐borne pathogenic species, much less is known about the performances of standard culture media toward environmental vibrios. We show that the isolation/enumeration of marine vibrios using thiosulfate‐citrate‐bile salts‐sucrose agar (TCBS) as selective medium may be hampered by the variable adaptability of different taxa to the medium, which may result even in isolation failure and/or in substantial total count underestimation. We propose a modified TCBS as isolation medium, adjusted for marine vibrios requirements, which greatly improved their recovery in dilution plate counts, compared with the standard medium. The modified medium offers substantial advantages over TCBS, providing more accurate and likely estimations of the actual presence of vibrios. Modified TCBS allowed the recovery of otherwise undetected vibrios, some of which producing biotechnologically valuable enzymes, thus expanding the isolation power toward potentially new enzyme‐producers Vibrio taxa. Moreover, we report a newly designed Vibrio‐specific PCR primers pair, targeting a unique rpoD sequence, useful for rapid confirmation of isolates as Vibrio members and subsequent genetic analyses.

Highlights

Microbial enzymes have wide applications in several fields, rang‐ ing from food manufacturing/processing to biomedical‐pharma‐ ceutical applications
Data reported clearly indicate that some marine vibrios are unable to grow on selective culture media such as standard thiosulfate‐citrate‐bile salts‐ sucrose agar (TCBS) agar, and that they require adjusted medium composition for a suc‐ cessful isolation
All isolates, including the four characterized ones, showed different patterns of secrete proteolytic enzymes, enriched in molecular weights below 40 KDa (Figure 3). These results suggested their applicative potential as a source of low molecular weight‐secreted proteases, which fur‐ ther functional analyses showed to be largely serine proteases and metalloproteases highly active even in harsh conditions and low tem‐ peratures

Summary

| INTRODUCTION

Microbial enzymes have wide applications in several fields, rang‐ ing from food manufacturing/processing to biomedical‐pharma‐ ceutical applications. The extensive screening of environmental Vibrio isolates is still worthy as a promising strategy; its power relies on the maximum recovery of the Vibrio diversity originally present in the natural sample Besides their biotechnological potential, includ‐ ing biomedical applications (Salamone et al, 2015), the interest toward the genus Vibrio ( pathogenic species) in marine environments has increased in last years, due to their ecological role in natural ecosystems (Herbert, 1999; Zhang et al, 2015) and to their dynamics in aquatic environments in re‐ sponse to climate changes (Narracci, Acquaviva, & Cavallo, 2014; Vezzulli et al, 2012, 2016). TCBS has been widely employed for the isolation of pathogenic vibrios (V. cholerae, V. para‐ haemolyticus, V. vulnificus) from clinical specimens and food, as well as from the aquatic environment It is indicated as isolation/enumeration medium for all vibrios (except V. hollisae and V. metschnikovii), a bacterial group abundant mainly in estuarine and sea waters, as well as seafood (Madigan et al, 2014). A Vibrio‐specific primers pair, targeting a unique rpoD sequence, was designed in order to quickly further confirm isolates as Vibrio members

| MATERIALS AND METHODS

| Microbiological methods

Findings

| DISCUSSION