Microplate-Test for the Rapid Determination of Bacteriophage-Susceptibility of Campylobacter Isolates—Development and Validation

Samuel Fischer,Günter Klein,Sophie Kittler,Gerhard Glünder,Markus M Heimesaat

doi:10.1371/journal.pone.0053899

Samuel Fischer, Günter Klein + Show 3 more

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https://doi.org/10.1371/journal.pone.0053899

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Abstract

A simple susceptibility test using 800 isolates of one Campylobacter strain with different degrees of susceptibility and four bacteriophages of the British phage typing scheme was developed and examined for its suitability. The test presented is economically cheaper and less time consuming than the conventional agar overlay plate assay and therefore enables the monitoring of changes in the susceptibility pattern during phage therapy under practical field conditions. The main objective of this study was to compare the simplified test with the conventional agar overlay plate assay. The conventional test describes for a population of Campylobacter: i. the rate of resistant isolates (0 plaques) and ii. the degree of susceptibility, also called relative efficiency of plating (EOP), for the remaining isolates. The simplified test divides the isolates into four susceptibility ranks, which are easily distinguishable to the naked eye. Ten Campylobacter isolates out of each rank were subjected to the conventional method for validation of the simplified test. Each resistance rank contained isolates showing certain degrees of susceptibility, reflecting decreasing susceptibility by an increase of the rank. Thus, the simplified test correlated well with the conventional method. Nevertheless, it can be suggested for a clear cut to summarise the first thee ranks as “high susceptible” and to mark out the fourth rank as reduced susceptible. Further test improvements will enable the monitoring of the degree of susceptibility and potentially also of resistance during phage therapy in the field. To ensure a long-lasting successful use of phage therapy, further studies on both the loss of susceptibility and the development of resistance of Campylobacter against phages combined with their impact on phage therapy will be necessary.

Highlights

Campylobacter as challenge Human campylobacteriosis is presently the most frequent foodborne zoonosis in Germany and many other industrial nations
A reduction of intestinal colonisation of broilers would lead to a considerable decline of human campylobacteriosis [8]
A risk assessment by the use of a mathematic model shows that a reduction of 2 lg of Campylobacter counts on broiler carcasses leads to a 30-fold decline in human campylobacteriosis caused by chicken meals [9]

Summary

Introduction

Campylobacter as challenge Human campylobacteriosis is presently the most frequent foodborne zoonosis in Germany and many other industrial nations. A reduction of intestinal colonisation of broilers would lead to a considerable decline of human campylobacteriosis [8]. A risk assessment by the use of a mathematic model shows that a reduction of 2 lg of Campylobacter counts on broiler carcasses leads to a 30-fold decline in human campylobacteriosis caused by chicken meals [9]. Phages as approach An investigation by Atterbury et al [10] showed that phages are able to reduce Campylobacter counts by up to 1.3 lg on the surface of experimentally contaminated broiler skins. The usage of phages for farm animals is of particular interest to reduce the administration of antibiotics. Antibiotic medication leads only to a temporary reduction, but not to an elimination of Campylobacter [16] and attracts long-lasting public criticism regarding increasing bacterial resistances

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