Development of multiple strain competitive index assays for Listeria monocytogenes using pIMC; a new site-specific integrative vector

Abstract

Background: The foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays.

Results: We have constructed a new site-specific integrative vector, pIMC, based on pPL2, for the selection of L. monocytogenes from complex samples. The pIMC vector was further modified through the incorporation of IPTG inducible markers (antibiotic and phenotypic) to produce a suite of four vectors which allowed the discrimination of multiple strains from a single sample. We were able to perform murine infection studies with up to four EGDe isolates within a single mouse and showed that the tags did not impact upon growth rate or virulence. The system also allowed the identification of subtle differences in virulence between strains of L. monocytogenes commonly used in laboratory studies.

Conclusion: This study has developed a competitive index assay that can be broadly applied to all L. monocytogenes strains. Improved statistical robustness of the data was observed, resulting in fewer mice being required for virulence assays. The competitive index assays provide a powerful method to analyse the virulence or fitness of L. monocytogenes in complex biological samples.

Figure 1

Plasmid maps for pIMC and IPTG inducible marker expression in pIMC3. The plasmid (A) pIMC was created by SOE PCR (see Material and Methods) from oligonucleotides described in Table 1. The backbone of pIMC, derived from pPL2 [11] encodes the p15A low copy E. coli origin of replication and RP4 conjugative origin of transfer. No gram-positive origin of replication is present on the plasmid, therefore upon transformation, chromosomal integration into L. monocytogenes tRNA^ARG is directed by the Listeriophage PSA integrase. Antibiotic selection is supplied by the chloramphenicol acetytransferase (cat) fused to the highly expressed listerial promoter (Phelp) [13]. Restriction sites labelled on pIMC are unique and for sequencing purposes, T3 and T7 primer binding sites are present before the KpnI and after the SacI restriction sites, respectively. The plasmid sequence is accessible under the EMBL nucleotide accession number AM940001. A comparison of chloramphenicol selection (7.5 μg/ml) is shown in (B), with pIMC exhibiting uniform colony size compared to pPL2 transformed EGDe. (C) Antibiotic markers (kanamycin (aphA3), erythromycin (ermAM) and tetracycline (tetM)) and the beta-glucuronidase marker (gusA) were subcloned from pIMK3 into pIMCa as a SacI/PstI fragment (see Materials and Methods).

Figure 2

Growth kinetics within Balb/c mice after quadruple intravenous infection with EGDe::pIMC3 derivatives. (A) A tail vein injection with L. monocytogenes isolate EGDe was administered into 15 Balb/c mice with a total inoculum of 2 × 10⁴ CFU per 100 μl. The inoculum was composed of an equal ratio of EGDe wt (5 × 10³ CFU) tagged with the four different pIMC3 plasmids. On each subsequent day, 5 mice were sacrificed, with the spleens and livers enumerated on different BHI agars as described in the materials and methods. Bars correspond to EGDe; green (::pIMC3kan), red (::pIMC3ery), grey (::pIMC3tet) and blue (::pIMC3gus). Data are presented as mean CFU with standard deviation from five organs. The relative virulence ratio (RVR) (B) was calculated from the data presented in (A). Per mouse, the proportion of each strain comprised within the organ (liver or spleen) (output %) was divided by the proportion of the strain in the initial inoculum (input %) and presented as an individual data point (using the formula in additional file 2). The mean RVR was calculated from the average of 5 organs from each strain. A value of 1 indicates no change in the relative ratio. Symbols correspond to EGDe; green circle (::pIMC3kan), red diamond (::pIMC3ery), grey triangle (::pIMC3tet) and blue square (::pIMC3gus). (C) A tail vein infection with L. monocytogenes isolate EGDe was administered into 15 Balb/c mice with a total inoculum of 2 × 10⁴ CFU per 100 μl. The inoculum was composed of EGDe wt tagged with pIMCkan (0.75%), pIMCery (2.75%), pIMCtet (13.25%) and pIMCgus (83.25%) in a skewed ratio. The number on each bar gives the mean percentage for each strain recovered from the spleen or liver. Bars correspond to EGDe; green (::pIMC3kan), red (::pIMC3ery), grey (::pIMC3tet) and blue (::pIMC3gus).

Figure 3

Growth kinetics within Balb/c mice after IV infection with three L. monocytogenes strains. (A) A tail vein injection containing an equal ratio of three L. monocytogenes strains (EGDe, 10403S and F2365) was administered into 15 Balb/c mice with a total inoculum of 2 × 10⁴ CFU per 100 μl. Each of the strains was tagged with a different antibiotic marker. On each subsequent day, 5 mice were sacrificed, with the spleens and livers enumerated on different BHI agars as described in the materials and methods. Bars correspond to; green (EGDe::pIMC3kan), red (10403S::pIMC3ery) and grey (F2365::pIMC3tet). Data are presented as mean CFU per organ with standard deviation. Statistical analyses were conducted using the one sample T-test (see additional file 1) measuring the raw CFU differences between two strains to calculate the mean difference per organ over 5 mice. The relative virulence ratio (RVR) (B) was calculated from the data presented in (A) as described in figure legend 2. Symbols correspond to; green circle (EGDe::pIMC3kan), red diamond (10403S::pIMC3ery) and grey triangle (F2365::pIMC3tet). Statistical analyses were conducted using the one sample T-test measuring the RVR differences between two strains to calculate the mean difference of 5 organs (see additional file 2). The calculated P-values are presented, with values below 0.05 considered significant (P < 0.05 (*), P < 0.005 (**) and P < 0.0005(***)). (C) The competitive index score relative to EGDe (set as 1) was calculated by dividing the percentage of the strain (10403S or F2365) within an organ by the percentage value obtained for EGDe. The CI score per organ were plotted (with the same symbols as in (B)) and the mean CI written below. A CI score of 1 denotes no difference in the virulence compared to EGDe (for CI calaulation see additional figure 3). Underlined scores denote were statistical significance from (B) was observed.