Yael Danin-Poleg

Vibrio cholerae Strain Typing and Phylogeny Study Based on Simple Sequence Repeats

ABSTRACT Vibrio cholerae is the etiological agent of cholera. Its natural reservoir is the aquatic environment. To date, practical typing of V. cholerae is mainly serological and requires about 200 antisera. Simple sequence repeats (SSR), also termed VNTR (for variable number of tandem repeats), provide a source of high genomic polymorphism used in bacterial typing. Here we describe an SSR-based typing method that combines the variation in highly mutable SSR loci, with that of shorter, relatively more stable mononucleotide repeat (MNR) loci, for accurate and rapid typing of V. cholerae. In silico screening of the V. cholerae genome revealed thousands of perfect SSR tracts with an average frequency of one SSR every 152 bp. A panel of 32 V. cholerae strains, representing both clinical and environmental isolates, was tested for polymorphism in SSR loci. Two strategies were applied to identify SSR variation: polymorphism of SSR tracts longer than 12 bp (L-SSR) assessed by capillary fragment-size analysis and MNR polymorphism assessed by sequencing. The nine L-SSR loci tested were all polymorphic, displaying 2 to 13 alleles per locus. Sequence analysis of eight MNR-containing loci (MNR-multilocus sequence typing [MLST]) provided information on both variations in the MNR tract itself, and single nucleotide polymorphism (SNP) in their flanking sequences. Phylogenetic analysis of the combined SSR data showed a clear discrimination between the clinical strains belonging to O1 and O139 serogroups, and the environmental isolates. Furthermore, discrimination between 27 strains of the 32 strains was achieved. SSR-based typing methods combining L-SSR and MNR-MLST were found to be efficient for V. cholerae typing.

Predominant Effect of Host Genetics on Levels of Lactobacillus johnsonii Bacteria in the Mouse Gut

ABSTRACT The gut microbiota is strongly associated with the well-being of the host. Its composition is affected by environmental factors, such as food and maternal inoculation, while the relative impact of the hosts genetics have been recently uncovered. Here, we studied the effect of the host genetic background on the composition of intestinal bacteria in a murine model, focusing on lactic acid bacteria (LAB) as an important group that includes many probiotic strains. Based on 16S rRNA gene genotyping, variation was observed in fecal LAB populations of BALB/c and C57BL/6J mouse lines. Lactobacillus johnsonii, a potentially probiotic bacterium, appeared at significantly higher levels in C57BL/6J versus BALB/c mouse feces. In the BALB/c gut, the L. johnsonii level decreased rapidly after oral administration, suggesting that some selective force does not allow its persistence at higher levels. The genetic inheritance of L. johnsonii levels was further tested in reciprocal crosses between the two mouse lines. The resultant F1 offspring presented similar L. johnsonii levels, confirming that mouse genetics plays a major role in determining these levels compared to the smaller maternal effect. Our findings suggest that mouse genetics has a major effect on the composition of the LAB population in general and on the persistence of L. johnsonii in the gut in particular. Concentrating on a narrow spectrum of culturable LAB enables the isolation and characterization of such potentially probiotic bacterial strains, which might be specifically oriented to the genetic background of the host as part of a personalized-medicine approach.

ICEVchInd5 is prevalent in epidemic Vibrio cholerae O1 El Tor strains isolated in India

Integrative conjugative elements (ICEs) of the SXT/R391 family are self-transmissible mobile elements mainly involved in antibiotic resistance spread among γ-Proteobacteria, including Vibrio cholerae. We demonstrated that the recently described ICEVchInd5 is prevailing in V. cholerae O1 clinical strains isolated in Wardha province (Maharashtra, India) from 1994 to 2005. Genetic characterization by ribotyping and multiple-locus SSR analysis proved the same clonal origin for V. cholerae O1 isolates in Wardha province over an 11-year period and was used to assess the correlation between strain and ICE content among ours and different Indian reference strains. In silico analysis showed the existence of at least 3 sibling ICEs of ICEVchInd5 in V. cholerae O1 El Tor reference strains, isolated in the Indian subcontinent after 1992.

Epidemiologic Study of Vibrio vulnificus Infections by Using Variable Number Tandem Repeats

A 3-year environmental and clinical Vibrio vulnificus survey using simple-sequence repeats typing shows that V. vulnificus biotype 3 constitutes ≈21% of the bacterium population in tested aquaculture ponds as opposed to ≈86% of clinical cases. Simple-sequence repeats proved to be a useful epidemiologic tool, providing information on the environmental source of the pathogen.

Genetic modification of iron metabolism in mice affects the gut microbiota

The composition of the gut microbiota is affected by environmental factors as well as host genetics. Iron is one of the important elements essential for bacterial growth, thus we hypothesized that changes in host iron homeostasis, may affect the luminal iron content of the gut and thereby the composition of intestinal bacteria. The iron regulatory protein 2 (Irp2) and one of the genes mutated in hereditary hemochromatosis Hfe , are both proteins involved in the regulation of systemic iron homeostasis. To test our hypothesis, fecal metal content and a selected spectrum of the fecal microbiota were analyzed from Hfe−/−, Irp2−/− and their wild type control mice. Elevated levels of iron as well as other minerals in feces of Irp2−/− mice compared to wild type and Hfe−/− mice were observed. Interestingly significant variation in the general fecal-bacterial population-patterns was observed between Irp2−/− and Hfe−/− mice. Furthermore the relative abundance of five species, mainly lactic acid bacteria, was significantly different among the mouse lines. Lactobacillus (L.) murinus and L.intestinalis were highly abundant in Irp2−/− mice, Enterococcus faecium species cluster and a species most similar to Olsenella were highly abundant in Hfe-/- mice and L. johnsonii was highly abundant in the wild type mice. These results suggest that deletion of iron metabolism genes in the mouse host affects the composition of its intestinal bacteria. Further studying the relationship between gut microbiota and genetic mutations affecting systemic iron metabolism in human should lead to clinical implications.

Vibrio vulnificus Typing Based on Simple Sequence Repeats: Insights into the Biotype 3 Group

ABSTRACT Vibrio vulnificus is an opportunistic, highly invasive human pathogen with worldwide distribution. V. vulnificus strains are commonly divided into three biochemical groups (biotypes), most members of which are pathogenic. Simple sequence repeats (SSR) provide a source of high-level genomic polymorphism used in bacterial typing. Here, we describe the use of variations in mutable SSR loci for accurate and rapid genotyping of V. vulnificus. An in silico screen of the genomes of two V. vulnificus strains revealed thousands of SSR tracts. Twelve SSR with core motifs longer than 5 bp in a panel of 32 characterized and 56 other V. vulnificus isolates, including both clinical and environmental isolates from all three biotypes, were tested for polymorphism. All tested SSR were polymorphic, and diversity indices ranged from 0.17 to 0.90, allowing a high degree of discrimination among isolates (27 of 32 characterized isolates). Genetic analysis of the SSR data resulted in the clear distinction of isolates that belong to the highly virulent biotype 3 group. Despite the clonal nature of this new group, SSR analysis demonstrated high-level discriminatory power within the biotype 3 group, as opposed to other molecular methods that failed to differentiate these isolates. Thus, SSR are suitable for rapid typing and classification of V. vulnificus strains by high-throughput capillary electrophoresis methods. SSR (≥5 bp) by their nature enable the identification of variations occurring on a small scale and, therefore, may provide new insights into the newly emerged biotype 3 group of V. vulnificus and may be used as an efficient tool in epidemiological studies.

Novel LDPE/halloysite nanotube films with sustained carvacrol release for broad-spectrum antimicrobial activity

The emergence of antibiotic resistance of pathogenic bacteria has led to renewed interest in exploring the potential of plant-derived antimicrobials e.g., essential oils (EOs), as an alternative strategy to reduce microbial contamination. However, the volatile nature of EOs presents a major challenge in their incorporation into polymers by conventional high-temperature processing techniques. Herein, we employ halloysite nanotubes (HNTs) as efficient nano-carriers for carvacrol (a model EO). This pre-compounding encapsulation step imparts enhanced thermal stability to the carvacrol, allowing for its subsequent melt compounding with low-density polyethylene (LDPE). The resulting polymer nanocomposites exhibit outstanding antimicrobial properties with a broad spectrum of inhibitory activity against Escherichia coli, Listeria innocua in biofilms, and Alternaria alternata. Their antimicrobial effectiveness is also successfully demonstrated in complex model food systems (soft cheese and bread). This superior activity, compared to other studied carvacrol containing films, is induced by the significantly higher carvacrol content in the film as well as its slower out-diffusion from the hybrid system. Thus, these new active polymer nanocomposites presents an immense potential in controlling microbial contamination and biofilm related adverse effects, rendering them as excellent candidate materials for a wide range of applications.

Indication for Co-evolution of Lactobacillus johnsonii with its hosts

BackgroundThe intestinal microbiota, composed of complex bacterial populations, is host-specific and affected by environmental factors as well as host genetics. One important bacterial group is the lactic acid bacteria (LAB), which include many health-promoting strains. Here, we studied the genetic variation within a potentially probiotic LAB species, Lactobacillus johnsonii, isolated from various hosts.ResultsA wide survey of 104 fecal samples was carried out for the isolation of L. johnsonii. As part of the isolation procedure, terminal restriction fragment length polymorphism (tRFLP) was performed to identify L. johnsonii within a selected narrow spectrum of fecal LAB. The tRFLP results showed host specificity of two bacterial species, the Enterococcus faecium species cluster and Lactobacillus intestinalis, to different host taxonomic groups while the appearance of L. johnsonii and E. faecalis was not correlated with any taxonomic group. The survey ultimately resulted in the isolation of L. johnsonii from few host species. The genetic variation among the 47 L. johnsonii strains isolated from the various hosts was analyzed based on variation at simple sequence repeats (SSR) loci and multi-locus sequence typing (MLST) of conserved hypothetical genes. The genetic relationships among the strains inferred by each of the methods were similar, revealing three different clusters of L. johnsonii strains, each cluster consisting of strains from a different host, i.e. chickens, humans or mice.ConclusionsOur typing results support phylogenetic separation of L. johnsonii strains isolated from different animal hosts, suggesting specificity of L. johnsonii strains to their hosts. Taken together with the tRFLP results, that indicated the association of specific LAB species with the host taxonomy, our study supports co-evolution of the host and its intestinal lactic acid bacteria.

Genetic diversity of the human pathogen Vibrio vulnificus: a new phylogroup.

The biotype 3 group of the human pathogen Vibrio vulnificus emerged in Israel probably as a result of genome hybridization of two bacterial populations. We performed a genomic and phylogenetic study of V. vulnificus strains isolated from the environmental niche from which this group emerged - fish aquaculture in Israel. The genetic relationships and evolutionary aspects of 188 environmental and clinical isolates of the bacterium were studied by genomic typing. Genetic relations were determined based on variation at 12 variable number tandem repeat (VNTR, also termed SSR) loci. Analysis revealed a new cluster, in addition to the main groups of biotype 1& 2 and biotype 3. Similar grouping results were obtained with three different statistical approaches. Isolates forming this new cluster presented unclear biochemical profile nevertheless were not identified as biotype 1 or biotype 3. Further examination of representative strains by multilocus sequence typing (MLST) of 10 housekeeping genes and 5 conserved hypothetical genes supported the identification of this as yet undiscovered phylogroup (phenotypically diverse), termed clade A herein. This new clonal subgroup includes environmental as well as clinical isolates. The results highlight the fish aquaculture environment, and possibly man-made ecological niches as a whole, as a source for the emergence of new pathogenic strains.

Genome Sequence of the Pathogenic Bacterium Vibrio vulnificus Biotype 3

ABSTRACT We report the first genome sequence of the pathogenic Vibrio vulnificus biotype 3. This draft genome sequence of the environmental strain VVyb1(BT3), isolated in Israel, provides a representation of this newly emerged clonal group, which reveals higher similarity to the clinical strains of biotype 1 than to the environmental ones.