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1.  Atypical Listeria innocua strains possess an intact LIPI-3 
BMC Microbiology  2014;14:58.
Background
Listeria monocytogenes is a food-borne pathogen which is the causative agent of listeriosis and can be divided into three evolutionary lineages I, II and III. While all strains possess the well established virulence factors associated with the Listeria pathogenicity island I (LIPI-1), lineage I strains also possess an additional pathogenicity island designated LIPI-3 which encodes listeriolysin S (LLS), a post-translationally modified cytolytic peptide. Up until now, this pathogenicity island has been identified exclusively in a subset of lineage I isolates of the pathogen Listeria monocytogenes.
Results
In total 64 L. innocua strains were screened for the presence of LIPI-3. Here we report the identification of an intact LIPI-3 in 11 isolates of L. innocua and the remnants of the cluster in several others. Significantly, we can reveal that placing the L. innocua lls genes under the control of a constitutive promoter results in a haemolytic phenotype, confirming that the cluster is capable of encoding a functional haemolysin.
Conclusions
Although the presence of the LIPI-3 gene cluster is confined to lineage I isolates of L. monocytogenes, a corresponding gene cluster or its remnants have been identified in many L. innocua strains.
doi:10.1186/1471-2180-14-58
PMCID: PMC3974016  PMID: 24606727
2.  The two peptide lantibiotic lacticin 3147 acts synergistically with polymyxin to inhibit Gram negative bacteria 
BMC Microbiology  2013;13:212.
Background
The emergence of bacterial drug resistance encourages the re-evaluation of the potential of existing antimicrobials. Lantibiotics are post-translationally modified, ribosomally synthesised antimicrobial peptides with a broad spectrum antimicrobial activity. Here, we focussed on expanding the potential of lacticin 3147, one of the most studied lantibiotics and one which possesses potent activity against a wide range of Gram positive species including many nosocomial pathogens. More specifically, our aim was to investigate if lacticin 3147 activity could be enhanced when combined with a range of different clinical antibiotics.
Results
Initial screening revealed that polymyxin B and polymyxin E (colistin) exhibited synergistic activity with lacticin 3147. Checkerboard assays were performed against a number of strains, including both Gram positive and Gram negative species. The resultant fractional inhibitory concentration (FIC) index values established that, while partial synergy was detected against Gram positive targets, synergy was obvious against Gram negative species, including Cronobacter and E. coli.
Conclusions
Combining lacticin 3147 with low levels of a polymyxin could provide a means of broadening target specificity of the lantibiotic, while also reducing polymyxin use due to the lower concentrations required as a result of synergy.
doi:10.1186/1471-2180-13-212
PMCID: PMC3849175  PMID: 24069959
Antimicrobial; Synergy; Lantibiotic; Bacteriocin; Lacticin 3147; Polymyxin
3.  In vivo activity of Nisin A and Nisin V against Listeria monocytogenes in mice 
BMC Microbiology  2013;13:23.
Background
Lantibiotics are post-translationally modified antimicrobial peptides, of which nisin A is the most extensively studied example. Bioengineering of nisin A has resulted in the generation of derivatives with increased in vitro potency against Gram-positive bacteria. Of these, nisin V (containing a Met21Val change) is noteworthy by virtue of exhibiting enhanced antimicrobial efficacy against a wide range of clinical and food-borne pathogens, including Listeria monocytogenes. However, this increased potency has not been tested in vivo.
Results
Here we address this issue by assessing the ability of nisin A and nisin V to control a bioluminescent strain of Listeria monocytogenes EGDe in a murine infection model.
More specifically, Balb/c mice were infected via the intraperitoneal route at a dose of 1 × 105 cfu/animal and subsequently treated intraperitoneally with either nisin V, nisin A or a PBS control. Bioimaging of the mice was carried out on day 3 of the trial. Animals were then sacrificed and levels of infection were quantified in the liver and spleen.
Conclusion
This analysis revealed that nisin V was more effective than Nisin A with respect to controlling infection and therefore merits further investigation with a view to potential chemotherapeutic applications.
doi:10.1186/1471-2180-13-23
PMCID: PMC3616995  PMID: 23374279
Antimicrobial; Lantibiotic; Bacteriocin; Peptide engineering; Mutagenesis; Nisin
4.  Directed evolution and targeted mutagenesis to murinize listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model 
BMC Microbiology  2010;10:318.
Background
Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells.
Results
We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26), multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlAm murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlAm*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlAm* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlAm strain.
Conclusions
We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced entry into human cells as previously observed. This murinized L. monocytogenes strain will provide a useful tool for the analysis of the gastrointestinal phase of listeriosis.
doi:10.1186/1471-2180-10-318
PMCID: PMC3016325  PMID: 21144051
5.  Enhancing bile tolerance improves survival and persistence of Bifidobacterium and Lactococcus in the murine gastrointestinal tract 
BMC Microbiology  2008;8:176.
Background
The majority of commensal gastrointestinal bacteria used as probiotics are highly adapted to the specialised environment of the large bowel. However, unlike pathogenic bacteria; they are often inadequately equipped to endure the physicochemical stresses of gastrointestinal (GI) delivery in the host. Herein we outline a patho-biotechnology strategy to improve gastric delivery and host adaptation of a probiotic strain Bifidobacterium breve UCC2003 and the generally regarded as safe (GRAS) organism Lactococcus lactis NZ9000.
Results
In vitro bile tolerance of both strains was significantly enhanced (P < 0.001), following heterologous expression of the Listeria monocytogenes bile resistance mechanism BilE. Strains harbouring bilE were also recovered at significantly higher levels (P < 0.001), than control strains from the faeces and intestines of mice (n = 5), following oral inoculation. Furthermore, a B. breve strain expressing bilE demonstrated increased efficacy relative to the wild-type strain in reducing oral L. monocytogenes infection in mice.
Conclusion
Collectively the data indicates that bile tolerance can be enhanced in Bifidobacterium and Lactococcus species through rational genetic manipulation and that this can significantly improve delivery to and colonisation of the GI tract.
doi:10.1186/1471-2180-8-176
PMCID: PMC2577680  PMID: 18844989
6.  Development of a luciferase-based reporter system to monitor Bifidobacterium breve UCC2003 persistence in mice 
BMC Microbiology  2008;8:161.
Background
Probiotics such as bifidobacteria have been shown to maintain a healthy intestinal microbial balance and help protect against infections. However, despite these benefits, bifidobacteria still remain poorly understood at the biochemical, physiological and especially the genetic level. Herein we describe, for the first time, the development of a non-invasive luciferase-based reporter system for real-time tracking of Bifidobacterium species in vivo.
Results
The reporter vector pLuxMC1 is based on the recently described theta-type plasmid pBC1 from B. catenatulatum [1] and the luxABCDE operon from pPL2lux [2]. Derivatives of pLuxMC1, harbouring a bifidobacterial promoter (pLuxMC2) as well as a synthetically derived promoter (pLuxMC3) [3] placed upstream of luxABCDE, were constructed and found to stably replicate in B. breve UCC2003. The subsequent analysis of these strains allowed us to assess the functionality of pLuxMC1 both in vitro and in vivo.
Conclusion
Our results demonstrate the potential of pLuxMC1 as a real-time, non-invasive reporter system for Bifidobacterium. It has also allowed us, for the first time, to track the colonisation potential and persistence of this probiotic species in real time. An interesting and significant outcome of the study is the identification of the caecum as a niche environment for B. breve UCC2003 within the mouse gastrointestinal tract (GI) tract.
doi:10.1186/1471-2180-8-161
PMCID: PMC2564955  PMID: 18816375
7.  Development of multiple strain competitive index assays for Listeria monocytogenes using pIMC; a new site-specific integrative vector 
BMC Microbiology  2008;8:96.
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.
doi:10.1186/1471-2180-8-96
PMCID: PMC2440758  PMID: 18554399
8.  Relatedness between the two-component lantibiotics lacticin 3147 and staphylococcin C55 based on structure, genetics and biological activity 
BMC Microbiology  2007;7:24.
Background
Two component lantibiotics, such as the plasmid-encoded lacticin 3147 produced by Lactococcus lactis DPC3147 and staphylococcin C55 produced by Staphylococcus aureus C55, represent an emerging subgroup of bacteriocins. These two bacteriocins are particularly closely related, exhibiting 86% (LtnA1 and C55α) and 55% (LtnA2 and C55β) identity in their component peptides. The aim of this study was to investigate, for the first time for any two component bacteriocins, the significance of the relatedness between these two systems.
Results
So close is this relatedness that the hybrid peptide pairs LtnA1:C55β and C55α:LtnA2 were found to have activities in the single nanomolar range, comparing well with the native pairings. To determine whether this flexibility extended to the associated post-translational modification/processing machinery, the staphylococcin C55 structural genes were directly substituted for their lacticin 3147 counterparts in the ltn operon on the large conjugative lactococcal plasmid pMRC01. It was established that the lacticin LtnA1 post-translational and processing machinery could produce functionally active C55α, but not C55β. In order to investigate in closer detail the significance of the differences between LtnA1 and C55α, three residues in LtnA1 were replaced with the equivalent residues in C55α. Surprisingly, one such mutant LtnA1-Leu21Ala was not produced. This may be significant given the positioning of this residue in a putative lipid II binding loop.
Conclusion
It is apparent, despite sharing striking similarities in terms of structure and activity, that these two complex bacteriocins display some highly dedicated features particular to either system.
doi:10.1186/1471-2180-7-24
PMCID: PMC1858699  PMID: 17407564

Results 1-8 (8)