Genome Sequence of the Fish Brain Bacterium Clostridium tarantellae.

Eubacterium tarantellae was originally cultivated from the brain of fish affected by twirling movements. Here, we present the draft genome sequence of E. tarantellae DSM 3997, which consists of 3,982,316 bp. Most protein-coding genes in this strain are similar to genes of Clostridium bacteria, supporting the renaming of E. tarantellae as Clostridium tarantellae.

Phenotypic and Genotypic Characterization of Escherichia coli Causing Urinary Tract Infections in Kidney-Transplanted Patients.

Urinary tract infection (UTI), frequently caused by uropathogenic Escherichia coli (UPEC), is the most common infection after kidney transplantation (KTx). Untreated, it can lead to urosepsis and impairment of the graft function. We questioned whether the UPEC isolated from KTx patients differed from the UPEC of non-KTx patients. Therefore, we determined the genome sequences of 182 UPEC isolates from KTx and control patients in a large German university clinic and pheno- and genotypically compared these two isolated groups. Resistance to the ß-lactams, trimethoprim or trimethoprim/sulfamethoxazole was significantly higher among UPEC from KTx than from control patients, whereas both the isolated groups were highly susceptible to fosfomycin. Accordingly, the gene content conferring resistance to ß-lactams or trimethoprim, but also to aminoglycosides, was significantly higher in KTx than in control UPEC isolates. E. coli isolates from KTx patients more frequently presented with uncommon UPEC phylogroups expressing higher numbers of plasmid replicons, but interestingly, less UPEC virulence-associated genes than the control group. We conclude that there is no defining subset of virulence traits for UPEC from KTx patients. The clinical history and immunocompromised status of KTx patients enables E. coli strains with low uropathogenic potential, but with increased antibiotic resistance to cause UTIs.

Identification of Novel Biomarkers for Priority Serotypes of Shiga Toxin-Producing Escherichia coli and the Development of Multiplex PCR for Their Detection.

It would be desirable to have an unambiguous scheme for the typing of Shiga toxin-producing Escherichia coli (STEC) isolates to subpopulations. Such a scheme should take the high genomic plasticity of E. coli into account and utilize the stratification of STEC into subgroups, based on serotype or phylogeny. Therefore, our goal was to identify specific marker combinations for improved classification of STEC subtypes. We developed and evaluated two bioinformatic pipelines for genomic marker identification from larger sets of bacterial genome sequences. Pipeline A performed all-against-all BLASTp analyses of gene products predicted in STEC genome test sets against a set of control genomes. Pipeline B identified STEC marker genes by comparing the STEC core proteome and the "pan proteome" of a non-STEC control group. Both pipelines defined an overlapping, but not identical set of discriminative markers for different STEC subgroups. Differential marker prediction resulted from differences in genome assembly, ORF finding and inclusion cut-offs in both workflows. Based on the output of the pipelines, we defined new specific markers for STEC serogroups and phylogenetic groups frequently associated with outbreaks and cases of foodborne illnesses. These included STEC serogroups O157, O26, O45, O103, O111, O121, and O145, Shiga toxin-positive enteroaggregative E. coli O104:H4, and HUS-associated sequence type (ST)306. We evaluated these STEC marker genes for their presence in whole genome sequence data sets. Based on the identified discriminative markers, we developed a multiplex PCR (mPCR) approach for detection and typing of the targeted STEC. The specificity of the mPCR primer pairs was verified using well-defined clinical STEC isolates as well as isolates from the ECOR, DEC, and HUSEC collections. The application of the STEC mPCR for food analysis was tested with inoculated milk. In summary, we evaluated two different strategies to screen large genome sequence data sets for discriminative markers and implemented novel marker genes found in this genome-wide approach into a DNA-based typing tool for STEC that can be used for the characterization of STEC from clinical and food samples.

Characterization of Asymptomatic Bacteriuria Escherichia coli Isolates in Search of Alternative Strains for Efficient Bacterial Interference against Uropathogens.

Asymptomatic bacterial colonization of the urinary bladder (asymptomatic bacteriuria, ABU) can prevent bladder colonization by uropathogens and thus symptomatic urinary tract infection (UTI). Deliberate bladder colonization with Escherichia coli ABU isolate 83972 has been shown to outcompete uropathogens and prevent symptomatic UTI by bacterial interference. Many ABU isolates evolved from uropathogenic ancestors and, although attenuated, may still be able to express virulence-associated factors. Our aim was to screen for efficient and safe candidate strains that could be used as alternatives to E. coli 83972 for preventive and therapeutic bladder colonization. To identify ABU E. coli strains with minimal virulence potential but maximal interference efficiency, we compared nine ABU isolates from diabetic patients regarding their virulence- and fitness-associated phenotypes in vitro, their virulence in a murine model of sepsis and their genome content. We identified strains in competitive growth experiments, which successfully interfere with colonization of ABU isolate 83972 or uropathogenic E. coli strain 536. Six isolates were able to outcompete E. coli 83972 and two of them also outcompeted UPEC 536 during growth in urine. Superior competitiveness was not simply a result of better growth abilities in urine, but seems also to involve expression of antagonistic factors. Competitiveness in urine did not correlate with the prevalence of determinants coding for adhesins, iron uptake, toxins, and antagonistic factors. Three ABU strains (isolates 61, 106, and 123) with superior competitiveness relative to ABU model strain 83972 display low in vivo virulence in a murine sepsis model, and susceptibility to antibiotics. They belong to different phylogroups and differ in the presence of ExPEC virulence- and fitness-associated genes. Importantly, they all lack marked cytotoxic activity and exhibit a high LD50 value in the sepsis model. These strains represent promising candidates for a more detailed assessment of relevant fitness traits in urine and their suitability for therapeutic bladder colonization.

FIM$

In vivo whole-body imaging of small animals plays an important role for biomedical studies. In particular, animals like the fruit fly Drosophila melanogaster or the nematode Caenorhabditis elegans are popular model organisms for preclinical research since they offer sophisticated genetic tool-kits. Recording these translucent animals with high contrast in a large arena is however not trivial. Furthermore, fluorescent proteins are widely used to mark cells in vivo and report their functions. This paper introduces a novel optical imaging technique called FIM2c enabling simultaneous detection of the animals posture and movement as well as fluorescent markers like green fluorescent protein (GFP). FIM2c utilizes frustrated total internal reflection of two distinct wavelengths and captures both, reflected and emitted light. The resultant two-color high-contrast images are superb compared to other imaging systems for larvae or worms. This multipurpose method enables a large variety of different experimental approaches. For example, FIM2c can be used to image GFP positive cells/tissues/animals and supports the integration of fluorescent tracers into multitarget tracking paradigms. Moreover, optogenetic tools can be applied in large-scale behavioral analysis to manipulate and study neuronal functions. To demonstrate the benefit of our system, we use FIM2c to resolve colliding larvae in a high-throughput approach, which was impossible given the existing tools. Finally, we present a comprehensive database including images and locomotion features of more than 1300 resolved collisions available for the community. In conclusion, FIM2c is a versatile tool for advanced imaging and locomotion analysis for a variety of different model organisms.