In this study, MLST resolved 14 L. monocytogenes strains recovered from two factory surveys into five distinct groups. Furthermore, MLST improved on the discriminatory power of the rep – PCR typing scheme previously applied to this strain set, separating one rep – PCR group and removing another all together. Rep – PCR is an established, robust method for strain differentiation in L. monocytogenes, and has been used in a number of studies (Versalovic et al., 1991; Versalovic et al., 1994; Jersek et al., 1999 and others). That method measures rapidly evolving
genomic variation rather than the slowly evolving variation observed through MLST (Maiden et al., 1998). However, it is unlikely that this could explain the variation
37 observed between rep - PCR and MLST in this study. In typing the current strain set using rep – PCR, visual differentiation of agarose gel electrophoresis patterns was applied, rather than one of the computer assisted differentiation systems available (Rademaker et al., 2000). The increased resolution obtained by MLST, although anticipated to some extent due to its nucleotide basis, may, in part, reflect the subjective nature of visual differentiation of rep – PCR groups. On this premise, future works utilising rep – PCR as a strain differentiation method should, as recommended by Rademaker et al. (2000), employ a computer assisted differentiation system to limit observational discrepancies.
Of the five MLST‘s identified in the current work, three corresponded to previously described ST‘s, with each of these recovered from human infections on multiple occasions. These sequence types (ST – 3, 9 and 155) have numerous entries on the MLST database corresponding to serotypes 1/2a (ST – 155), 1/2b (ST – 3) and 1/2c (ST – 9). On this basis, it seems that these serotypes correspond to these
sequence types, and suggests that MLST may be an effective, indirect determinant of strain serotype. Notably, serotype 1/2a and 1/2b strains of L. monocytogenes are associated (along with serotype 4b) with the majority of human L. monocytogenes infections (Lukinmaa et al., 2003). Given that these strains were recovered from a food processing environment, and that ST – 3 was a persistent contaminant of these facilities based on the surveys conducted, these strains potentially constitute very high risk L. monocytogenes populations, and may represent a significant risk to public health.
Numerous studies have shown that it is possible to use molecular
methodologies to determine the route of L. monocytogenes contamination within factory environments (e.g. Alessandria et al., 2010). The relatedness of each of the MLST‘s identified in this study implied that they may have resided, and been dispersed throughout, the food processing facility for a prolonged period of time. Further to this, the distribution of MLST‘s throughout the factory, including multiple rooms and equipments, suggest that the strains are being actively disseminated throughout the factory. This confounded identification of a specific reservoir, and suggested a self perpetuating, rather than external, contamination source. Active dissemination by daily factory processes appears likely, evidenced by the presence of L. monocytogenes ST‘s on the wheels of forklifts, on moveable trolleys, and in water puddles located in walk ways. Given these and other locations where
38 highly probable that L. monocytogenes from this food facility will, or has been, transferred into the food supply chain. This is of serious concern and may be indicative of a systemic microbial control failure within the facility.
The current study compared the sequence types of the factory isolates against all previously known Australian ST‘s. To date, eighteen MLST‘s have been assigned to Australian L. monocytogenes isolates, encompassing 109 isolates. As already stated, three of the factory ST‘s corresponded to known Australian ST‘s, with the remaining two not previously described. Phylogenetic comparison showed that all of the ST‘s, including those recovered from the factory, clustered according to
L. monocytogenes strain serotype and lineage. This was presented as three main tree arms corresponding to lineage I, II and III strains, with branching within these arms correlated to serotypes characteristic of the lineage. This permitted inferential assignment of lineage and serotype to those factory L. monocytogenes strains corresponding to known ST‘s, and allowed prediction of likely lineages for the strains corresponding to novel ST‘s. However, given that there appears to be a relational bias corresponding to lineage and serotype, the phylogenetic relationship of the survey factory L. monocytogenes isolates must be discounted, as this may simply represent evolutionary descent for the species as a whole.
Lineage I strains are significantly more common from human sources, while lineage II strains are significantly more common from food sources, and lineage III isolates are rarely recovered from either food or humans (Gray et al., 2004).
Sequence types corresponding to two of these lineages, however, were recovered from the factory survey, including ST – 3 (lineage I), ST – 9 (lineage II) and ST – 155 (lineage II). These results highlight the presence of high risk L. monocytogenes strains within the survey factory.
The use of MLST as a means of identifying high risk sub – populations of microbes within complex environments is not new. The method is regularly
employed in epidemiological investigations / studies, as well as works investigating the prevalence and distribution of specific pathogen subtypes within environments, such a multi – drug resistant Escherichia coli (Simoes et al., 2010; Urwin and Maiden, 2003). At present, MLST of L. monocytogenes has largely been focused on isolates of clinical origin. Given that L. monocytogenes has an environmental reservoir, knowledge of the distribution of this organism in both natural and anthropogenic settings could substantially aid investigations on its movements,
39 microecology and infection foci, and help to guide the logic of prevention and
intervention strategies.
In the present study, MLST identified high risk L. monocytogenes strains associated with persistent contamination of a food processing factory. However, in some instances, sporadic and persistent contaminants belonged to the same sequence type (e.g. DS_81 and DS_25). Although MLST has been shown capable of
identifying high – risk L. monocytogenes populations in this study, knowledge of the factors underpinning the incongruent distribution of environmentally persistent strains is also of considerable importance. The acquisition of persistent
L. monocytogenes strains presents an opportunity to study environmental persistence in this species in greater detail. Further work using the environmentally persistent and sporadic strains from this study to investigate factors that may contribute to environmental persistence, such as biofilm production and physiological stress, is required. This could improve knowledge of the mechanisms affording environmental persistence in L. monocytogenes and help guide preventative strategies directed at this organism. This aim forms the basis of the following experimental chapters of this thesis.
40