Inactivation of the gene encoding the cationic antimicrobial peptide resistance factor MprF increases biofilm formation but reduces invasiveness of Listeria monocytogenes.

AIMS: To understand the genetics involved in surface attachment and biofilm formation of Listeria monocytogenes. METHODS AND RESULTS: An in vitro screen of a Himar1 transposon library of L. monocytogenes strain 15G01 identified three transposants that produced significantly different biofilm levels when compared to the wild-type strain; two mutants exhibited enhanced biofilm formation and one produced less biofilm biomass than the wild-type. The mutant 15G01 mprF::Himar1, which had a transposon insertion in the mprF gene, was selected for further analysis. The mutant produced a more densely populated biofilm on solid surfaces such as stainless steel and polystyrene, as determined using scanning electron and light microscopy. The 15G01 mprF::Himar1 mutant remained viable in biofilms, but showed an increase in sensitivity to the cationic antimicrobial gallidermin. The mutant also displayed reduced invasiveness in CaCo-2 intestinal cells, suggesting virulence properties are compromised by the inactivation of mprF. CONCLUSIONS: Biofilm formation and gallidermin resistance of L. monocytogenes is influenced by mprF, but this trait is associated with a compromise in invasiveness. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of pathogenic microorganisms in the food processing environment can cause a significant problem, especially when these microorganisms are established as biofilms. This study shows that the inactivation of the mprF gene results in enhanced biofilm formation and abiotic surface attachment of L. monocytogenes.

Tetrazolium reduction allows assessment of biofilm formation by Campylobacter jejuni in a food matrix model.

AIMS: To develop a staining method for specific detection of metabolically active (viable) cells in biofilms of the foodborne pathogen Campylobacter jejuni. METHODS AND RESULTS: Conversion of 2,3,5 triphenyltetrazolium chloride (TTC) to insoluble, red 1,3,5-triphenylformazan (TPF) was dependent on metabolic activity of Camp. jejuni. When used with chicken juice, TTC staining allowed quantification of Camp. jejuni biofilm levels, whereas the commonly used dye, crystal violet, gave high levels of nonspecific staining of food matrix components (chicken juice). The assay was optimized to allow for monitoring of biofilm levels and adapted to monitor levels of Camp. jejuni in broth media. CONCLUSIONS: Staining with TTC allows for the quantification of metabolically active Camp. jejuni and thus allows for quantification of viable cells in biofilms and food matrices. The TTC staining method can be adapted to quantify bacterial cell concentration in a food matrix model, where the accepted method of A600 measurement is not suitable due to interference by components of the food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: 2,3,5 Triphenyltetrazolium chloride (TTC) staining is a low-cost technique suitable for use in biofilm analysis, allowing rapid and simple imaging of metabolically active cells and increasing the methods available for biofilm assessment and quantification.

A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is a post-infectious disease in which the human peripheral nervous system is affected after infection by specific pathogenic bacteria, including Campylobacter jejuni. GBS is suggested to be provoked by molecular mimicry between sialylated lipooligosaccharide (LOS) structures on the cell envelope of these bacteria and ganglioside epitopes on the human peripheral nerves, resulting in autoimmune-driven nerve destruction. Earlier, the C. jejuni sialyltransferase (Cst-II) was found to be linked to GBS and demonstrated to be involved in the biosynthesis of the ganglioside-like LOS structures. Apart from a role in pathogenicity, we report here that Cst-II-generated ganglioside-like LOS structures confer efficient bacteriophage resistance in C. jejuni. By bioinformatic analysis, it is revealed that the presence of sialyltransferases in C. jejuni and other potential GBS-related pathogens correlated significantly with the apparent degeneration of an alternative anti-virus system: type II Clusters of Regularly Interspaced Short Palindromic Repeat and associated genes (CRISPR-Cas). Molecular analysis of the C. jejuni CRISPR-Cas system confirmed the bioinformatic investigation. CRISPR degeneration and mutations in the cas genes cas2, cas1 and csn1 were found to correlate with Cst-II sialyltransferase presence (p < 0.0001). Remarkably, type II CRISPR-Cas systems are mainly found in mammalian pathogens. To study the potential involvement of this system in pathogenicity, we inactivated the type II CRISPR-Cas marker gene csn1, which effectively reduced virulence in primarily cst-II-positive C. jejuni isolates. Our findings indicate a novel link between viral defence, virulence and GBS in a pathogenic bacterium.

Acid-shock of Campylobacter jejuni induces flagellar gene expression and host cell invasion.

The bacterial pathogen Campylobacter jejuni is the leading cause of foodborne gastroenteritis in the developed world, with the organism being transmitted by ingestion of contaminated and undercooked poultry. Exposure to acid is an inevitable stressor for C. jejuni during gastric passage, yet the effect of low pH on C. jejuni virulence is still poorly understood. Here, we investigate the effect of acid-shock on C. jejuni viability, gene expression and host-cell invasion. C. jejuni strain NCTC 11168 survived acid exposure at pH 3.5 and above for up to 30 min without a drop in viability, and this exposure induced the expression of flagellar genes transcribed from σ(54)-dependent promoters. Furthermore, acid-shock resulted in increased C. jejuni invasion of m-ICcl2 mouse small intestine crypt cells grown on transwells, but not when the cells were grown on flat-bottomed wells. This suggests that C. jejuni might be invading intestinal epithelial cells at the basolateral side, possibly after paracellular passage. We hypothesize that acid-shock prior to intestinal entry may serve as a signal that primes C. jejuni to express its virulence gene repertoire including flagellar motility genes, but this requires further study in the context of an appropriate colonization or disease model.

Campylobacter bacteremia: a rare and under-reported event?

Bacteria belonging to the species Campylobacter are the most common cause of bacterial diarrhoea in humans. The clinical phenotype associated with Campylobacter infections ranges from asymptomatic conditions to severe colitis and bacteremia. In susceptible patients, Campylobacter infections are associated with significant morbidity and mortality, with both host factors and bacterial factors being involved in the pathogenesis of bacteremia. In the host, age, gender and immune-compromising conditions may predispose for Campylobacter infections, whilst the most important bacterial determinants mentioned in the literature are cytotoxin production and flagellar motility. The role of sialylated lipo-oligosaccharide (LOS) and serum resistance in bacteremia is inconclusive at this time, and the clinical significance of Campylobacter bacteremia is not yet fully understood. More emphasis on the detection of Campylobacter species from blood cultures in susceptible patients at risk for Campylobacter infections will increase our understanding of the pathogenesis and the relevance of Campylobacter bacteremia.

PerR controls peroxide- and iron-responsive expression of oxidative stress defense genes in Helicobacter hepaticus.

Chronic intestinal and hepatic colonization with the microaerophilic murine pathogen Helicobacter hepaticus can lead to a range of inflammatory diseases of the lower digestive tract. Colonization is associated with an active cellular immune response and production of oxygen radicals. During colonization, H. hepaticus needs to cope with and respond to oxidative stress, and here we report on the role of the H. hepaticus PerR-regulator (HH0942) in the expression of the peroxidase-encoding katA (HH0043) and ahpC (HH1564) genes. Transcription of katA and ahpC was induced by hydrogen peroxide, and by iron restriction of growth media. This iron- and hydrogen peroxide-responsive regulation of katA and ahpC was mediated at the transcriptional level, from promoters directly upstream of the genes. Inactivation of the perR gene resulted in constitutive, iron-independent high-level expression of the katA and ahpC transcripts and corresponding proteins. Finally, inactivation of the katA gene resulted in increased sensitivity of H. hepaticus to hydrogen peroxide and reduced aerotolerance. In H. hepaticus, iron metabolism and oxidative stress defense are intimately connected via the PerR regulatory protein. This regulatory pattern resembles that observed in the enteric pathogen Campylobacter jejuni, but contrasts with the pattern observed in the closely related human gastric pathogen Helicobacter pylori.

Genomics of thermophilic campylobacter species.

The thermophilic Campylobacter species C. jejuni and C. coli are important human pathogens, which are major causes of bacterial gastroenteritis. The recent progress in genomics techniques has allowed for a rapid increase in our knowledge of the molecular biology of Campylobacter species, but needs to be matched by concurrent increases in our understanding of the unique biology of these organisms. Campylobacter species display significant levels of genomic variation via natural transformation, phase variation, plasmid transfer and infection with bacteriophages, and this poses a continuous challenge for studies on pathogenesis, physiology, epidemiology and evolution of Campylobacter. In this chapter we will review the current state of the art of the genomics of thermophilic Campylobacter species, and opportunities where genomics can further contribute to our understanding of the biology of these successful human pathogens.

A pro-inflammatory genotype predisposes to Barrett's esophagus.

INTRODUCTION: Severity of mucosal inflammation is shown to be associated with Barrett's esophagus (BE) development in animals. It has therefore been postulated that a strong pro-inflammatory host response predisposes to BE. AIM: To determine the impact of cytokine gene polymorphisms on the development of BE. METHODS: The multiplex SNaPshot method was used to determine interleukin (IL)-12B (A+1188C), IL-10 (C-592A, C-819T, A-1082G), IL-8 (A-251T), IL-6 (G-174C) and IL-2 (G-330T) gene polymorphisms in 255 patients with BE and 247 patients with reflux esophagitis (RE). RESULTS: The presence of the IL-12B C-allele, which is associated with increased IL-12p70 expression, was more frequently observed in BE than in RE patients [odds ratio (OR) 1.8; 95% confidence interval (CI) 1.2-2.7; P = 0.007). The risk of BE was increased in patients in whom the IL-12B C-allele coincided with a hiatal hernia (OR 2.9; 95% CI 1.32-6.58; P = 0.008). The IL-10(-1082) GG genotype, which is associated with higher IL-10 levels, was also associated with a decreased risk of BE when it was associated with the IL-12B C-allele, indicating IL-10-dependent down-regulation of IL-12p70 expression. A combination of the IL-12B AA genotype and the IL-10 AA or AG genotypes was associated with RE (OR 1.4; 95% CI 1.05-1.85; P = 0.011). CONCLUSION: A genetic profile predisposing to a strong pro-inflammatory host response, mediated by IL-12p70 and partially dependent on IL-10, is associated with BE. This risk further increases when this genotype coincides with a hiatal hernia, suggesting that exposure to gastroesophageal reflux in the presence of a pro-inflammatory genetic background is a driving force in the development of BE.

The homeodomain protein CDX2 is an early marker of Barrett's oesophagus.

BACKGROUND: In Barrett's oesophagus (BO), squamous epithelium is replaced by specialised intestinal epithelium (SIE). Transcription factors associated with intestinal differentiation, such as CDX2, may be involved in BO development. AIM: To investigate CDX2 expression in BO, squamous epithelium, and oesophageal adenocarcinoma (ADC). METHODS: CDX2 expression was assessed in 245 samples-167 biopsies of the columnar lined segment and 38 squamous epithelial biopsies of 39 patients with histologically confirmed BO (10 with ADC). Forty biopsies from 20 patients with reflux oesophagitis (RO) without BO were also evaluated. CDX2 protein was investigated immunohistochemically in 138 biopsies from 16 patients with BO, four with ADC, and 20 with RO. Cdx2 and Muc2 mRNA were detected semiquantitatively using 88 BO biopsies and squamous epithelium from 19 BO patients, and when present from ADC. RESULTS: SIE was present in 53/79 biopsies from the columnar lined segment; CDX2 protein was seen in all epithelial cells, but not in biopsies containing only gastric metaplastic epithelium (26/79), or in squamous epithelium (0/40) of patients with RO. Cdx2 mRNA was detected in all biopsies with goblet cell specific Muc2 transcription-indicative of SIE. Low Cdx2 mRNA expression was seen in 6/19 squamous epithelium samples taken 5 cm above the squamocolumnar junction of BO patients. CONCLUSION: CDX2 protein/mRNA is strongly associated with oesophageal SIE. Cdx2 mRNA was present in the normal appearing squamous epithelium of one third of BO patients, and may precede morphological changes seen in BO. Therefore, pathways that induce Cdx2 transcription in squamous epithelial cells may be important in BO development.