Purification and Characterization of a Rabbit Serum Factor That Kills Listeria Species and Other Foodborne Bacterial Pathogens.

In an in-vitro assay, rabbit serum, but not human serum, killed Listeria monocytogenes, a foodborne pathogen. The aim of our study was to purify and partially characterize this killing factor. Listericidin was purified from rabbit serum by a single-step ion-exchange chromatography with DEAE-Sephadex A-50 and its antimicrobial activity was assessed by a microdilution method. Listericidin is a protein with a molecular weight of 9 kDa and an isoelectric point of 8.1. It kills L. monocytogenes at 4°C, 25°C, and 37°C, and its activity is resistant to heat (boiling) and acidic conditions (pH <2). Listericidin's activity is inhibited by sodium chloride and various growth media, is sensitive to proteolytic enzymes and is enhanced by calcium chloride, and is neutralized by monoclonal antibodies to human complement C3a. However, the listericidin reacts weakly with these antibodies in an ELISA. The first 33 N-terminal residues of listericidin (SVQLTEKRMDKVGQYTNKELRKXXEDGMRDNPM) have homology to various complement C3a components. Listericidin also kills other Listeria spp., Vibrio spp., Salmonella spp., Escherichia spp., Cronobacter spp., and Bacillus spp. The listericidin peptide purified in a single-step chromatography is pH and heat stable, and has a broad antimicrobial spectrum against major foodborne pathogens in addition to L. monocytogenes.

Analysis of the cellulose synthase operon genes, bcsA, bcsB, and bcsC in Cronobacter species: Prevalence among species and their roles in biofilm formation and cell-cell aggregation.

Cronobacter species are emerging food-borne pathogens that cause severe sepsis, meningitis, and necrotizing entercolitis in neonates and infants. Bacterial pathogens such as Escherichia coli and Salmonella species produce extracellular cellulose which has been shown to be involved in rugosity, biofilm formation, and host colonization. In this study the distribution and prevalence of cellulose synthase operon genes (bcsABZC) were determined by polymerase chain reaction (PCR) analysis in 231 Cronobacter strains isolated from clinical, food, environmental, and unknown sources. Furthermore, bcsA and bcsB isogenic mutants were constructed in Cronobacter sakazakii BAA894 to determine their roles. In calcofluor binding assays bcsA and bcsB mutants did not produce cellulose, and their colonial morphotypes were different to that of the parent strain. Biofilm formation and bacterial cell-cell aggregation were significantly reduced in bcsA and bcsB mutants compared to the parental strain. bcsA or bcsAB PCR-negative strains of C. sakazakii did not bind calcofluor, and produced less biofilm and cell-cell aggregation compared to strains possessing bcsAB genes. These data indicated that Cronobacter bcsABZC were present in all clinical isolates and most of food and environmental isolates. bcsA and bcsB genes of Cronobacter were necessary to produce cellulose, and were involved in biofilm formation and cell-cell aggregation.

Pan-genome analysis of the emerging foodborne pathogen Cronobacter spp. suggests a species-level bidirectional divergence driven by niche adaptation.

BACKGROUND: Members of the genus Cronobacter are causes of rare but severe illness in neonates and preterm infants following the ingestion of contaminated infant formula. Seven species have been described and two of the species genomes were subsequently published. In this study, we performed comparative genomics on eight strains of Cronobacter, including six that we sequenced (representing six of the seven species) and two previously published, closed genomes. RESULTS: We identified and characterized the features associated with the core and pan genome of the genus Cronobacter in an attempt to understand the evolution of these bacteria and the genetic content of each species. We identified 84 genomic regions that are present in two or more Cronobacter genomes, along with 45 unique genomic regions. Many potentially horizontally transferred genes, such as lysogenic prophages, were also identified. Most notable among these were several type six secretion system gene clusters, transposons that carried tellurium, copper and/or silver resistance genes, and a novel integrative conjugative element. CONCLUSIONS: Cronobacter have diverged into two clusters, one consisting of C. dublinensis and C. muytjensii (Cdub-Cmuy) and the other comprised of C. sakazakii, C. malonaticus, C. universalis, and C. turicensis, (Csak-Cmal-Cuni-Ctur) from the most recent common ancestral species. While several genetic determinants for plant-association and human virulence could be found in the core genome of Cronobacter, the four Cdub-Cmuy clade genomes contained several accessory genomic regions important for survival in a plant-associated environmental niche, while the Csak-Cmal-Cuni-Ctur clade genomes harbored numerous virulence-related genetic traits.

Identification and characterization of five new molecular serogroups of Cronobacter spp.

Cronobacter spp. (formerly Enterobacter sakazakii) is an emerging foodborne pathogen consisting of seven species including C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis (with three subspecies, dublinensis, lausannensis, and lactaridi), C. universalis, and C. condimenti. To date, 12 Cronobacter serogroups have been identified. In this study, MboII restriction fragment length polymorphism patterns and DNA sequences of O-antigen gene clusters were used to identify novel serogroups of Cronobacter spp. Sequence analysis of the O-antigen regions, located between galF and gnd, of strains with distinct restriction fragment length polymorphism patterns revealed five unique gene clusters. These new O-antigen gene clusters were species specific and were termed C. turicensis O3, C. muytjensii O2, C. dublinensis O1, C. dublinensis O2, and C. universalis O1. Polymerase chain reaction assays were developed using primers specific to O-antigen processing genes and used to screen a collection of Cronobacter strains to determine the frequency of these newly identified serotypes.

Induction of persistent colitis by a human commensal, enterotoxigenic Bacteroides fragilis, in wild-type C57BL/6 mice.

Enterotoxigenic Bacteroides fragilis (ETBF) causes diarrhea and is implicated in inflammatory bowel diseases and colorectal cancer. The only known ETBF virulence factor is the Bacteroides fragilis toxin (BFT), which induces E-cadherin cleavage, interleukin-8 secretion, and epithelial cell proliferation. A murine model for ETBF has not been characterized. Specific pathogen-free (SPF) C57BL/6J or germfree 129S6/SvEv mice were orally inoculated with wild-type ETBF (WT-ETBF) strains, a nontoxigenic WT strain of B. fragilis (WT-NTBF), WT-NTBF overexpressing bft (rETBF), or WT-NTBF overexpressing a biologically inactive mutated bft (rNTBF). In SPF and germfree mice, ETBF caused colitis but was lethal only in germfree mice. Colonic histopathology demonstrated mucosal thickening with inflammatory cell infiltration, crypt abscesses, and epithelial cell exfoliation, erosion, and ulceration. SPF mice colonized with rETBF mimicked WT-ETBF, whereas rNTBF caused no histopathology. Intestinal epithelial E-cadherin was rapidly cleaved in vivo in WT-ETBF-colonized mice and in vitro in intestinal tissues cultured with purified BFT. ETBF mice colonized for 16 months exhibited persistent colitis. BFT did not directly induce lymphocyte proliferation, dendritic cell stimulation, or Toll-like receptor activation. In conclusion, WT-ETBF induced acute then persistent colitis in SPF mice and rapidly lethal colitis in WT germfree mice. Our data support the hypothesis that chronic colonization with the human commensal ETBF can induce persistent, subclinical colitis in humans.

Identification and characterization of conjugative transposons CTn86 and CTn9343 in Bacteroides fragilis strains.

The related genetic elements flanking the Bacteroides fragilis pathogenicity island (PAI) in enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 and also present in pattern III nontoxigenic B. fragilis (NTBF) NCTC 9343 were defined as putative conjugative transposons (CTns), designated CTn86 and CTn9343, respectively (A. A. Franco, J. Bacteriol. 181:6623-6633, 2004). CTn86 and CTn9343 have the same basic structures except that their encoded transposases have low similarity and CTn9343 lacks the B. fragilis PAI and contains an extra 7-kb region not present in CTn86. In this study, using DNA hybridization and PCR analysis, we characterized the genetic element flanking the PAI in a collection of ETBF strains and the related genetic elements in a collection of NTBF pattern III strains. We found that in all 123 ETBF strains, the PAI is contained in a genetic element similar to CTn86. Of 73 pattern III strains, 26 (36%) present a genetic element similar to CTn9343, 38 (52%) present a genetic element similar to CTn9343 but lack the 7-kb region that is also absent in CTn86 (CTn9343-like element), and 9 (12%) present a genetic element similar to CTn86 but lacking the PAI (CTn86-like element). In addition to containing CTn86, ETBF strains can also contain CTn9343, CTn9343-like, or CTn86-like elements. CTn86, CTn9343, CTn86-like, and CTn9343-like elements were found exclusively in B. fragilis strains and predominantly in division I, cepA-positive strains.

The C-terminal region of Bacteroides fragilis toxin is essential to its biological activity.

To evaluate the role of the C-terminal region in Bacteroides fragilis toxin (BFT) activity, processing, and secretion, sequential C-terminal truncation and point mutations were created by site-directed mutagenesis. Determination of BFT activity on HT29/C1 cells, cleavage of E-cadherin, and the capacity to induce interleukin-8 secretion by wild-type BFT and C-terminal deletion mutants showed that deletion of only 2 amino acid residues at the C terminus significantly reduced BFT biological activity and deletion of eight or more amino acid residues obliterated BFT biologic activity. Western blot and reverse transcription-PCR analyses indicated that BFT mutants lacking seven or fewer amino acid residues in the C-terminal region are processed and expressed similar to wild-type BFT. However, BFT mutants lacking eight or more amino acids at the C terminus are expressed similar to wild-type BFT but are unstable. We concluded that the C terminus of BFT is not tolerant of modest amino acid deletions, suggesting that it is biologically important for BFT activity.

Mutation of the zinc-binding metalloprotease motif affects Bacteroides fragilis toxin activity but does not affect propeptide processing.

To evaluate the role of the zinc-binding metalloprotease in Bacteroides fragilis toxin (BFT) processing and activity, the zinc-binding consensus sequences (H348, E349, H352, G355, H358, and M366) were mutated by site-directed-mutagenesis. Our results indicated that single point mutations in the zinc-binding metalloprotease motif do not affect BFT processing but do reduce or eliminate BFT biologic activity in vitro.

The Bacteroides fragilis pathogenicity island is contained in a putative novel conjugative transposon.

The genetic element flanking the Bacteroides fragilis pathogenicity island (BfPAI) in enterotoxigenic B. fragilis (ETBF) strain 86-5443-2-2 and a related genetic element in NCTC 9343 were characterized. The results suggested that these genetic elements are members of a new family of conjugative transposons (CTns) not described previously. These putative CTns, designated CTn86 and CTn9343 for ETBF 86-5443-2-2 and NCTC 9343, respectively, differ from previously described Bacteroides species CTns in a number of ways. These new transposons do not carry tetQ, and the excision from the chromosome to form a circular intermediate is not regulated by tetracycline; they are predicted to differ in their mechanism of transposition; and their sequences have very limited similarity with CTnDOT or other described CTns. CTn9343 is 64,229 bp in length, contains 61 potential open reading frames, and both ends contain IS21 transposases. Colony blot hybridization, PCR, and sequence analysis indicated that CTn86 has the same structure as CTn9343 except that CTn86 lacks a approximately 7-kb region containing truncated integrase (int2) and rteA genes and it contains the BfPAI integrated between the mob region and the bfmC gene. If these putative CTns were to be demonstrated to be transmissible, this would suggest that the bft gene can be transferred from ETBF to nontoxigenic B. fragilis strains by a mechanism similar to that for the spread of antibiotic resistance genes.

Modulation of bft expression by the Bacteroides fragilis pathogenicity island and its flanking region.

To establish a recombinant system for high-level expression of biologically active Bacteroides fragilis toxin (BFT), we studied the expression of bft in non-toxigenic B. fragilis (NTBF) strains. The bft gene and the B. fragilis pathogenicity island (BfPAI) were cloned into NTBF strains with two distinct genetic patterns: (i) pattern II, strains lacking the BfPAI and its flanking region; and (ii) pattern III, strains lacking the BfPAI but containing its flanking region. Analysis of BFT activity of these recombinant strains on HT29/C1 cells showed that both the BfPAI and its flanking regions are important to optimal BFT activity. Reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that the BfPAI and its flanking regions modulate bft expression. Further experiments demonstrated that the approximately 700 bp region upstream of bft is the BfPAI region critical for optimal bft expression. We conclude that both the region flanking the BfPAI and approximately 700 bp region upstream of bft are crucial to maximal BFT production by ETBF strains.