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Infection with carbapenem-resistant Enterobacteriaceae (CRE) has become an important challenge in health care settings and a growing concern worldwide. Since infection is preceded by colonization, an understanding of the latter may reduce CRE infections. We aimed to characterize the gut microbiota in CRE carriers, assuming that microbiota alterations precede CRE colonization. We evaluated the gut microbiota using 16S rRNA gene sequencing extracted of fecal samples collected from hospitalized CRE carriers and two control groups, hospitalized noncarriers and healthy adults. The microbiota diversity and composition in CRE-colonized patients differed from those of the control group participants. These CRE carriers displayed lower phylogenetic diversity and dysbiotic microbiota, enriched with members of the family Enterobacteriaceae Concurrent with the enrichment in Enterobacteriaceae , a depletion of anaerobic commensals was observed. Additionally, changes in several predicted metabolic pathways were observed for the CRE carriers. Concomitantly, we found higher prevalence of bacteremia in the CRE carriers. Several clinical factors that might induce changes in the microbiota were examined and found to be insignificant between the groups. The compositional and functional changes in the microbiota of CRE-colonized patients are associated with increased risk for systemic infection. Our study results provide justification for attempts to restore the dysbiotic microbiota with probiotics or fecal transplantation. IMPORTANCE The gut microbiota plays important roles in the host's normal function and health, including protection against colonization by pathogenic bacteria. Alterations in the gut microbial profile can potentially serve as an early diagnostic tool, as well as a therapeutic strategy against colonization by and carriage of harmful bacteria, including antibiotic-resistant pathogens. Here, we show that the microbiota of hospitalized patients demonstrated specific taxa which differed between carriers of carbapenem-resistant Enterobacteriaceae (CRE) and noncarriers. The difference in the microbiota also dictates alterations in microbiome-specific metabolic capabilities, in association with increased prevalence of systemic infection. Reintroducing specific strains and/or correction of dysbiosis with probiotics or fecal transplantation may potentially lead to colonization by bacterial taxa responsible for protection against or depletion of antibiotic-resistant pathogens., (Copyright © 2020 Korach-Rechtman et al.)

The gut microbiota is a complex ecosystem, affected by both environmental factors and host genetics. Here, we aim at uncovering the bacterial taxa whose gut persistence is controlled by host genetic variation. We used a murine model based on inbred lines BALB/c and C57BL/6J and their F 1 reciprocal hybrids (♀C57BL/6J × ♂BALB/c; ♀BALB/c × ♂C57BL/6J). To guarantee genetic similarity of F 1 offspring, including the sex chromosomes, we used only female mice. Based on 16S rRNA gene sequencing, we found that the genetically different inbred lines present different microbiota, whereas their genetically identical F 1 reciprocal hybrids presented similar microbiota. Moreover, the F 1 microbial composition differed from that of both parental lines. Twelve taxa were shown to have genetically controlled gut persistence, while none were found to show maternal effects. Nine of these taxa were dominantly inherited by the C57BL/6J line. Cohousing of the parental inbred lines resulted in a temporary and minor shift in microbiota composition, which returned back to the former microbial composition following separation, indicating that each line tends to maintain a unique bacterial signature reflecting the line. Taken together, our findings indicate that mouse genetics has an effect on the microbial composition in the gut, which is greater than maternal effect and continuous exposure to different microbiota of the alternative line. Uncovering the bacterial taxa associated with host genetics and understanding their role in the gut ecosystem could lead to the development of genetically oriented probiotic products, as part of the personalized medicine approach. IMPORTANCE The gut microbiota play important roles for their host. The link between host genetics and their microbial composition has received increasing interest. Using a unique reciprocal cross model, generating genetically similar F 1 hybrids with different maternal inoculation, we demonstrate the inheritance of gut persistence of 12 bacterial taxa. No taxa identified as maternally transmitted. Moreover, cohabitation of two genetically different inbred lines did not dramatically affect the microbiota composition. Taken together, our results demonstrate the importance of the genetic effect over maternal inoculation or effect of exposure to unlike exogenous microbiota. These findings may lead to the development of personalized probiotic products, specifically designed according to the genetic makeup., (Copyright © 2019 American Society for Microbiology.)

We report the genome sequence of the pathogenic Vibrio vulnificus biotype 1 clade B, which is suggested to have a common ancestor with biotype 3. This draft genome of the clinical strain V252, isolated in Israel, represents the clonal clade B group that contains both clinical and environmental strains., (Copyright © 2015 Efimov et al.)

Here, we report the genome sequence of Lactobacillus johnsonii, a member of the gut lactobacilli. This draft genome of L. johnsonii strain 16 isolated from C57BL/6J mice enables the identification of bacterial genes responsible for host-specific gut persistence., (Copyright © 2015 Buhnik-Rosenblau et al.)

Modern man-made environments, including urban, agricultural, and industrial environments, have complex ecological interactions among themselves and with the natural surroundings. Microbial source tracking (MST) offers advanced tools to resolve the host source of fecal contamination beyond indicator monitoring. This study was intended to assess karst spring susceptibilities to different fecal sources using MST quantitative PCR (qPCR) assays targeting human, bovine, and swine markers. It involved a dual-time monitoring frame: (i) monthly throughout the calendar year and (ii) daily during a rainfall event. Data integration was taken from both monthly and daily MST profile monitoring and improved identification of spring susceptibility to host fecal contamination; three springs located in close geographic proximity revealed different MST profiles. The Giach spring showed moderate fluctuations of MST marker quantities amid wet and dry samplings, while the Zuf spring had the highest rise of the GenBac3 marker during the wet event, which was mirrored in other markers as well. The revelation of human fecal contamination during the dry season not connected to incidents of raining leachates suggests a continuous and direct exposure to septic systems. Pigpens were identified in the watersheds of Zuf, Shefa, and Giach springs and on the border of the Gaaton spring watershed. Their impact was correlated with partial detection of the Pig-2-Bac marker in Gaaton spring, which was lower than detection levels in all three of the other springs. Ruminant and swine markers were detected intermittently, and their contamination potential during the wet samplings was exposed. These results emphasized the importance of sampling design to utilize the MST approach to delineate subtleties of fecal contamination in the environment., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We report the genome sequence of the environmental Vibrio vulnificus biotype 1_cladeA. This draft genome of the CladeA-yb158 strain, isolated in Israel, represents this newly emerged clonal group that contains both clinical and environmental strains., (Copyright © 2015 Danin-Poleg et al.)

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.

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 host's 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.

Most procedures for detecting pathogens in liquid media require an initial concentration step. However, poor recovery efficiencies of conventional methods, such as filtration, often lead to low sensitivity. Here, we describe a strategy for concentrating bacteria using their binding affinity for an oligoacyllysine (OAK), a novel peptide-mimetic antimicrobial compound. We show that the resin-linked OAK (ROAK) efficiently captures a variety of pathogens in different media, upon brief incubation with ROAK beads or after continuous flow through a ROAK-packed column. Using Escherichia coli expressing green fluorescent protein, we show that binding occurs rapidly during incubation and persists after filtration as visualized by confocal microscopy. The high binding affinity of bacteria was confirmed by surface plasmon resonance technology using an OAK-linked chip. ROAK-bound bacteria remained viable and were readily identifiable by real-time PCR after ethanol elution. A single ROAK bead is estimated to capture about 3,000 bacterial cells in culture medium, in contaminated saline or tap water. ROAK beads can be regenerated for multiple uses after brief ethanol treatment. Collectively, the data support the notion that OAK-based coating of polymeric surfaces might represent a useful means for medium filtration as well as for concentration of bacteria.

Vibrio cholerae has been shown to adhere to cornstarch granules. The present work explored the mechanisms involved in this adhesion and the possibility of its occurrence in vivo. The findings suggest that both specific and nonspecific interactions are involved in the adhesion. Nonspecific hydrophobic interactions may play a role, since both V. cholerae and cornstarch granules exhibited hydrophobic properties when they were tested using a xylene-water system. In addition, the presence of bile acids reduced the adhesion. The adhesion also involves some specific carbohydrate-binding moieties on the cell surface, as reflected by reduced adhesion following pretreatment of the bacteria with proteinase K and sodium m-periodate. Further investigations supported these observations and showed that media containing low-molecular-weight carbohydrates had a significant inhibitory effect. Binding cell lysate to starch granules and removing the adhered proteins using either glucose or bile acids led to identification (by liquid chromatography-tandem mass spectrometry analysis) of several candidate V. cholerae outer membrane-associated starch-binding proteins. Different sets of proteins were isolated by removal in a glucose solution or bile acids. When the upper gastrointestinal tract conditions were simulated in vitro, both bile salts and the amylolytic activity of the pancreatic juices were found to have an inhibitory effect on the adherence of V. cholerae to starch. However, during acute diarrhea, this inhibitory effect may be significantly reduced due to dilution, suggesting that adhesion does occur in vivo. Such adhesion may contribute to the beneficial effects observed following administration of granular starch-based oral rehydration solutions to cholera patients.

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.

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.

Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. Cholera can become epidemic and deadly without adequate medical care. Appropriate rehydration therapy can reduce the mortality rate from as much as 50% of the affected individuals to <1%. Thus, oral rehydration therapy (ORT) is an important measure in the treatment of this disease. To further reduce the symptoms associated with cholera, improvements in oral rehydration solution (ORS) by starch incorporation were suggested. Here, we report that V. cholerae adheres to starch granules incorporated in ORS. Adhesion of 98% of the cells was observed within 2 min when cornstarch granules were used. Other starches showed varied adhesion rates, indicating that starch source and composition play an important role in the interaction of V. cholerae and starch granules. Sugars metabolized by V. cholerae showed a repressive effect on the adhesion process. The possible mechanisms involved are discussed. Comparing V. cholerae adhesion with the adhesion of other pathogens suggests the involvement of starch degradation capabilities. This adhesion to granular starch can be used to improve ORT.

DNA-based methods are increasingly important for bacterial typing. The high number of polymorphic sites present among closely related bacterial genomes is the basis for the presented method. The method identifies multilocus genomic polymorphisms in intergenic regions termed AILP (amplified intergenic locus polymorphism). For each locus, a pair of unique PCR primers was designed to amplify an intergenic sequence from one open reading frame (ORF) to the adjacent ORF. Presence, absence, and size variation of the amplification products were identified and used as genetic markers for rapidly differentiating among strains. Polymorphism was evaluated using 18 AILP sites among 28 strains of Listeria monocytogenes and 6 strains of Listeria spp. and 30 AILP markers among 27 strains of Escherichia coli. Up to four alleles per locus were identified among Listeria strains, and up to six were identified among E. coli strains. In both species, more than half of the AILP sites revealed intraspecies polymorphism. The AILP data were applied to phylogenetic analysis among Listeria and E. coli strains. A clear distinction between L. monocytogenes and Listeria spp. was demonstrated. In addition, the method separated L. monocytogenes into the three known lineages and discriminated the most common virulent serotypic group, 4b. In E. coli, AILP analysis separated the known groups as well as the virulent O157:H7 isolates. These findings for both Listeria and E. coli are in agreement with other phylogenetic studies using molecular markers. The AILP method was found to be rapid, simple, reproducible, and a low-cost method for initial bacterial typing that could serve as a basis for epidemiological investigation.

Multilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains of Escherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensal E. coli strains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of the E. coli reference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria.

Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. The disease does not persist in a chronic state in humans or animals. The pathogen is naturally present as a free-living organism in the environment. Recently, it was suggested that egg masses of the nonbiting midge Chironomus sp. (Diptera) harbor and serve as a nutritive source for V. cholerae, thereby providing a natural reservoir for the organism. Here we report that V. cholerae O9, O1, and O139 supernatants lysed the gelatinous matrix of the chironomid egg mass and inhibited eggs from hatching. The extracellular factor responsible for the degradation of chironomid egg masses (egg mass degrading factor) was purified from V. cholerae O9 and O139 and was identified as the major secreted hemagglutinin/protease (HA/P) of V. cholerae. The substrate in the egg mass was characterized as a glycoprotein. These findings show that HA/P plays an important role in the interaction of V. cholerae and chironomid egg masses.