Carbapenemase VCC-1-Producing Vibrio cholerae in Coastal Waters of Germany.

During antimicrobial drug resistance testing for Vibrio spp. from coastal waters of Germany, we identified 4 nontoxigenic, carbapenem-resistant V. cholerae isolates. We used whole-genome sequencing to identify the carbapenemase gene blaVCC-1. In addition, a molecular survey showed that more blaVCC-1-harboring isolates are present in coastal waters of Germany.

Use of whole-genus genome sequence data to develop a multilocus sequence typing tool that accurately identifies Yersinia isolates to the species and subspecies levels.

The genus Yersinia is a large and diverse bacterial genus consisting of human-pathogenic species, a fish-pathogenic species, and a large number of environmental species. Recently, the phylogenetic and population structure of the entire genus was elucidated through the genome sequence data of 241 strains encompassing every known species in the genus. Here we report the mining of this enormous data set to create a multilocus sequence typing-based scheme that can identify Yersinia strains to the species level to a level of resolution equal to that for whole-genome sequencing. Our assay is designed to be able to accurately subtype the important human-pathogenic species Yersinia enterocolitica to whole-genome resolution levels. We also report the validation of the scheme on 386 strains from reference laboratory collections across Europe. We propose that the scheme is an important molecular typing system to allow accurate and reproducible identification of Yersinia isolates to the species level, a process often inconsistent in nonspecialist laboratories. Additionally, our assay is the most phylogenetically informative typing scheme available for Y. enterocolitica.

Evaluation of molecular methods to discriminate the closely related species Vibrio fluvialis and Vibrio furnissii.

Vibrio furnissii and Vibrio fluvialis are two closely related species which are regarded as emerging human pathogens. Human infections have been mainly associated with consumption of seafood or drinking of contaminated water. V. furnissii strains can be distinguished from V. fluvialis by their ability to produce gas from fermentation of carbohydrates. In this study, we compare two phenotypic (biochemical testing and matrix-assisted laser desorption/ionisation time of flight mass spectrometry, MALDI-TOF MS) and three genotypic techniques (rpoB sequencing, conventional PCR and real-time PCR) for determination of the two species. The methods were evaluated on a collection of 42 V. furnissii and 32 V. fluvialis strains, which were isolated from marine environments and from animals intended for food production. Four of the applied methods allowed the unambiguous discrimination of the two species, while the biochemical testing was the least reliable technique, due to a high variation in the phenotype of gas production from carbohydrates. In view of the One Health concept reliable diagnostic techniques are a prerequisite for preventive public health measurements, as pathogens isolated from animals can cross species borders and methods for detection of sources, reservoirs and ways of transmission of pathogenic bacteria are indispensable for the prevention of infectious diseases in humans and animals.

Pathogenic vibrios in environmental, seafood and clinical sources in Germany.

Bacteria of the family Vibrionaceae naturally occur in marine and estuarine environments. Only few species of Vibrionaceae are associated with human cases of gastroenteritis, ear and wound infections, caused by ingestion of seafood or contact with Vibrio containing water. Increasing consumption of seafood (fish, fishery products and shellfish) poses a possible source of Vibrio infections in Germany. Additionally, there is a growing concern that abundances of pathogenic vibrios may increase in German coastal waters as a result of e.g. climate change resulting in probably rising surface water temperatures. According to the One Health concept the VibrioNet consortium started in 2010 to investigate the occurrence and relevance of non-cholera vibrios of human concern in Germany. Vibrios from environmental, seafood and clinical sources were analyzed with the aim to find connections between different reservoirs or sources and to identify potential ways of transmission of these pathogens to assess the risk of infections associated with them. Potentially pathogenic strains mostly belong to the species Vibrio parahaemolyticus, Vibrio vulnificus and non-O1/non-O139 Vibrio cholerae. Investigations on imported seafood and mussels from primary production areas confirmed the frequent occurrence of these species. Moreover, studies of German coastal waters and sediments showed the presence and seasonality of these marine bacteria. So far the incidence of clinical cases of vibriosis in Germany is low. Between 1994 and 2013 thirteen cases of Vibrio spp. associated wound infections and/or septicaemia have been reported. However, the high prevalence of vibrios in aquatic environments and aquatic organisms is of concern and demands continued control of food and surveillance for clinical infections with pathogenic vibrios.

PVv3, a new shuttle vector for gene expression in Vibrio vulnificus.

An efficient electroporation procedure for Vibrio vulnificus was designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 10(6) transformants per µg DNA were achieved. The vector stably replicated in both V. vulnificus and Escherichia coli and was also successfully introduced into Vibrio parahaemolyticus and Vibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and the vvhBA hemolysin operon were inserted into the vector and functionally expressed in Vibrio and E. coli.

German coasts harbor non-O1/non-O139 Vibrio cholerae with clinical virulence gene profiles.

Non-O1/non-O139 Vibrio cholerae (NOVC) are ubiquitous in aquatic ecosystems. In rare cases, they can cause intestinal and extra-intestinal infections in human. This ability is associated with various virulence factors. The presence of NOVC in German North Sea and Baltic Sea was observed in previous studies. However, data on virulence characteristics are still scarce. Therefore, this work aimed to investigating the virulence potential of NOVC isolated in these two regions. In total, 31 NOVC strains were collected and subjected to whole genome sequencing. In silico analysis of the pathogenic potential was performed based on the detection of genes involved in colonization and virulence. Phenotypic assays, including biofilm formation, mobility and human serum resistance assays were applied for validation. Associated toxin genes (hlyA, rtxA, chxA and stn), pathogenicity islands (Vibrio pathogenicity island 2 (VPI-II) and Vibrio seventh pathogenicity island 2 (VSP-II)) and secretion systems (Type II, III and VI secretion system) were observed. A maximum likelihood analysis from shared core genes revealed a close relationship between clinical NOVCs published in NCBI and environmental strains from this study. NOVC strains are more mobile at 37 °C than at 25 °C, and 68% of the NOVC strains could form strong biofilms at both temperatures. All tested strains were able to lyse erythrocytes from both human and sheep blood. Additionally, one strain could survive up to 60% and seven strains up to 40% human serum at 37 °C. Overall, the genetic virulence profile as well as the phenotypic virulence characteristics of the investigated NOVC from the German North Sea and Baltic Sea suggest potential human pathogenicity.

Human and animal isolates of Yersinia enterocolitica show significant serotype-specific colonization and host-specific immune defense properties.

Yersinia enterocolitica is a human pathogen that is ubiquitous in livestock, especially pigs. The bacteria are able to colonize the intestinal tract of a variety of mammalian hosts, but the severity of induced gut-associated diseases (yersiniosis) differs significantly between hosts. To gain more information about the individual virulence determinants that contribute to colonization and induction of immune responses in different hosts, we analyzed and compared the interactions of different human- and animal-derived isolates of serotypes O:3, O:5,27, O:8, and O:9 with murine, porcine, and human intestinal cells and macrophages. The examined strains exhibited significant serotype-specific cell binding and entry characteristics, but adhesion and uptake into different host cells were not host specific and were independent of the source of the isolate. In contrast, survival and replication within macrophages and the induced proinflammatory response differed between murine, porcine, and human macrophages, suggesting a host-specific immune response. In fact, similar levels of the proinflammatory cytokine macrophage inflammatory protein 2 (MIP-2) were secreted by murine bone marrow-derived macrophages with all tested isolates, but the equivalent interleukin-8 (IL-8) response of porcine bone marrow-derived macrophages was strongly serotype specific and considerably lower in O:3 than in O:8 strains. In addition, all tested Y. enterocolitica strains caused a considerably higher level of secretion of the anti-inflammatory cytokine IL-10 by porcine than by murine macrophages. This could contribute to limiting the severity of the infection (in particular of serotype O:3 strains) in pigs, which are the primary reservoir of Y. enterocolitica strains pathogenic to humans.

Spread of a distinct Stx2-encoding phage prototype among Escherichia coli O104:H4 strains from outbreaks in Germany, Norway, and Georgia.

Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) O104:H4 caused one of the world's largest outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in Germany in 2011. These strains have evolved from enteroaggregative E. coli (EAEC) by the acquisition of the Stx2 genes and have been designated enteroaggregative hemorrhagic E. coli. Nucleotide sequencing has shown that the Stx2 gene is carried by prophages integrated into the chromosome of STEC O104:H4. We studied the properties of Stx2-encoding bacteriophages which are responsible for the emergence of this new type of E. coli pathogen. For this, we analyzed Stx bacteriophages from STEC O104:H4 strains from Germany (in 2001 and 2011), Norway (2006), and the Republic of Georgia (2009). Viable Stx2-encoding bacteriophages could be isolated from all STEC strains except for the Norwegian strain. The Stx2 phages formed lysogens on E. coli K-12 by integration into the wrbA locus, resulting in Stx2 production. The nucleotide sequence of the Stx2 phage P13374 of a German STEC O104:H4 outbreak was determined. From the bioinformatic analyses of the prophage sequence of 60,894 bp, 79 open reading frames were inferred. Interestingly, the Stx2 phages from the German 2001 and 2011 outbreak strains were found to be identical and closely related to the Stx2 phages from the Georgian 2009 isolates. Major proteins of the virion particles were analyzed by mass spectrometry. Stx2 production in STEC O104:H4 strains was inducible by mitomycin C and was compared to Stx2 production of E. coli K-12 lysogens.

Unique cell adhesion and invasion properties of Yersinia enterocolitica O:3, the most frequent cause of human Yersiniosis.

Many enteric pathogens are equipped with multiple cell adhesion factors which are important for host tissue colonization and virulence. Y. enterocolitica, a common food-borne pathogen with invasive properties, uses the surface proteins invasin and YadA for host cell binding and entry. In this study, we demonstrate unique cell adhesion and invasion properties of Y. enterocolitica serotype O:3 strains, the most frequent cause of human yersiniosis, and show that these differences are mainly attributable to variations affecting the function and expression of invasin in response to temperature. In contrast to other enteric Yersinia strains, invasin production in O:3 strains is constitutive and largely enhanced compared to other Y. enterocolitica serotypes, in which invA expression is temperature-regulated and significantly reduced at 37°C. Increase of invasin levels is caused by (i) an IS1667 insertion into the invA promoter region, which includes an additional promoter and RovA and H-NS binding sites, and (ii) a P98S substitution in the invA activator protein RovA rendering the regulator less susceptible to proteolysis. Both variations were shown to influence bacterial colonization in a murine infection model. Furthermore, we found that co-expression of YadA and down-regulation of the O-antigen at 37°C is required to allow efficient internalization by the InvA protein. We conclude that even small variations in the expression of virulence factors can provoke a major difference in the virulence properties of closely related pathogens which may confer better survival or a higher pathogenic potential in a certain host or host environment.

Shiga toxin-producing Escherichia coli strains from cattle as a source of the Stx2a bacteriophages present in enteroaggregative Escherichia coli O104:H4 strains.

Enteroaggregative, Shiga toxin-producing E. coli (EAEC-STEC) O104:H4 strains are emerging pathogens causing life threatening diseases in humans. EAEC-STEC O104:H4 strains isolated between 2001 and 2011 were found to harbor a distinct type of Shiga toxin 2a- (Stx2a) encoding prophage. This phage type shows only <65% genetic similarity to so far described viable Stx phages due to differences in the modules for DNA replication, metabolism, regulation and host specificity. Stx production in EAEC is rarely observed and the source of the Stx2a phage in the EAEC-STEC O104:H4 strains is not known. We identified two DNA segments derived from orf15 and the cI gene of the O104:H4 Stx2a phage P13374 that are characteristic for Stx2a prophages present in EAEC-STEC O104:H4 strains. By PCR, these sequences were detected in 14 (5.8%) of 241 Stx2-positive STEC from animals and food. Infectious Stx2a phages could be isolated from four bovine STEC strains. These were found highly similar to P13374 for orf15, cI and stx2a sequences, the chromosomal integration site (wrbA), for phage DNA restriction profiles, virion morphology and superinfection immunity. Stx2a phages of the four bovine STEC strains formed lysogens on the E. coli K-12 strain C600. Phage P13374 from an EAEC-STEC O104:H4 outbreak strain and one of the bovine STEC phages (P13803) lysogenized the Stx-negative EAEC O104:H4 strain CB14647 by integrating in the wrbA gene of CB14647 and converted it into a Stx2a producer. Our findings provide experimental evidence that EAEC-STEC O104:H4 strains have evolved by uptake of Stx2a phages from the bovine reservoir.

Genotypic diversity and virulence characteristics of clinical and environmental Vibrio vulnificus isolates from the Baltic Sea region.

The genetic diversity of Vibrio vulnificus isolates from clinical and environmental sources originating from the Baltic Sea region was evaluated by multilocus sequence typing (MLST), and possible relationships between MLST clusters, potential genotypic and phenotypic traits associated with pathogenicity, and source of isolation were investigated. The studied traits included genotyping of polymorphic loci (16S rRNA, vcg, and pilF), presence/absence of potential virulence genes, including nanA, nab, and genes of pathogenicity regions, metabolic features, hemolytic activity, resistance to human serum, and cytotoxicity to human intestinal cells. MLST generated 35 (27 new) sequence types and divided the 53 isolates (including four reference strains) into two main clusters, with cluster I containing biotype 1 and 2 isolates of mainly environmental origin and cluster II containing biotype 1 isolates of mainly clinical origin. Cluster II isolates were further subdivided into two branches. Branch IIB included isolates from recent cases of wound infections that were acquired at the German Baltic Sea coastline between 2010 and 2011 and isolates from seawater samples of the same regions isolated between 1994 and 2010. Comparing the MLST data with the results of genotyping and phenotyping showed that strains of MLST cluster II possess a number of additional pathogenicity-associated traits compared to cluster I strains. Rapid microbiological methods such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry combined with typing of selected virulence-associated traits (e.g., serum resistance, mannitol fermentation, nanA, and pathogenicity region XII) could be used for risk assessment purposes regarding V. vulnificus strains isolated from the Baltic Sea region.

Genetic and Phenotypic Virulence Potential of Non-O1/Non-O139 Vibrio cholerae Isolated from German Retail Seafood.

Non-O1 and non-O139 Vibrio cholerae (NOVC) can cause gastrointestinal infections in humans. Contaminated food, especially seafood, is an important source of human infections. In this study, the virulence potential of 63 NOVC strains isolated from retail seafood were characterized at the genotypic and phenotypic levels. Although no strain encoded the cholera toxin (CTX) and the toxin-coregulated pilus (TCP), several virulence factors, including the HlyA hemolysin, the cholix toxin ChxA, the heat-stable enterotoxin Stn, and genes coding for the type 3 and type 6 secretion systems, were detected. All strains showed hemolytic activity against human and sheep erythrocytes: 90% (n = 57) formed a strong biofilm, 52% (n = 33) were highly motile at 37 °C, and only 8% (n = 5) and 14% (n = 9) could resist ≥60% and ≥40% human serum, respectively. Biofilm formation and toxin regulation genes were also detected. cgMLST analysis demonstrated that NOVC strains from seafood cluster with clinical NOVC strains. Antimicrobial susceptibility testing (AST) results in the identification of five strains that developed non-wildtype phenotypes (medium and resistant) against the substances of the classes of beta-lactams (including penicillin, carbapenem, and cephalosporin), polymyxins, and sulphonamides. The phenotypic resistance pattern could be partially attributed to the acquired resistance determinants identified via in silico analysis. Our results showed differences in the virulence potential of the analyzed NOVC isolated from retail seafood products, which may be considered for further pathogenicity evaluation and the risk assessment of NOVC isolates in future seafood monitoring.

Complete Genome Sequence of an Aeromonas rivuli Strain Isolated from Ready-to-Eat Food.

Aeromonads can be associated with diseases in animals and humans. Knowledge regarding Aeromonas rivuli, a species recently discovered in creek water in Germany, is still fragmentary. Here, we announce the complete genome sequence of Aeromonas rivuli strain 20-VB00005, which was recovered from ready-to-eat food.

Prevalence and Distribution of Potentially Human Pathogenic Vibrio spp. on German North and Baltic Sea Coasts.

Global ocean warming results in an increase of infectious diseases including an elevated emergence of Vibrio spp. in Northern Europe. The European Centre for Disease Prevention and Control reported annual periods of high to very high risks of infection with Vibrio spp. during summer months along the North Sea and Baltic Sea coasts. Based on those facts, the risk of Vibrio infections associated with recreational bathing in European coastal waters increases. To obtain an overview of the seasonal and spatial distribution of potentially human pathogenic Vibrio spp. at German coasts, this study monitored V. cholerae, V. parahaemolyticus, and V. vulnificus at seven recreational bathing areas from 2017 to 2018, including the heat wave event in summer 2018. The study shows that all three Vibrio species occurred in water and sediment samples at all sampling sites. Temperature was shown to be the main driving factor of Vibrio abundance, whereas Vibrio community composition was mainly modulated by salinity. A species-specific rapid increase was observed at water temperatures above 10°C, reaching the highest detection numbers during the heat wave event with abundances of 4.5 log10 CFU+1/100 ml of seawater and 6.5 log10 CFU+1/100 g of sediment. Due to salinity, the dominant Vibrio species found in North Sea samples was V. parahaemolyticus, whereas V. vulnificus was predominantly detected in Baltic Sea samples. Most detections of V. cholerae were associated with estuarine samples from both seas. Vibrio spp. concentrations in sediments were up to three log higher compared to water samples, indicating that sediments are an important habitat for Vibrio spp. to persist in the environment. Antibiotic resistances were found against beta-lactam antibiotics (ampicillin 31%, cefazolin 36%, and oxacillin and penicillin 100%) and trimethoprim-sulfamethoxazole (45%). Moreover, isolates harboring pathogenicity-associated genes such as trh for V. parahaemolyticus as well as vcg, cap/wcv, and the 16S rRNA-type B variant for V. vulnificus were detected. All sampled V. cholerae isolates were identified as non-toxigenic non-O1/non-O139 serotypes. To sum up, increasing water temperatures at German North Sea and Baltic Sea coasts provoke elevated Vibrio numbers and encourage human recreational water activities, resulting in increased exposure rates. Owing to a moderate Baltic Sea salinity, the risk of V. vulnificus infections is of particular concern.

Case Report: Vibrio fluvialis isolated from a wound infection after a piercing trauma in the Baltic Sea.

Vibrio spp. are Gram-negative bacteria found in marine ecosystems. Non-cholera Vibrio spp. can cause gastrointestinal infections and can also lead to wound infections through exposure to contaminated seawater. Vibrio infections are increasingly documented from the Baltic Sea due to extended warm weather periods. We describe the first isolation of Vibrio fluvialis from a wound infection acquired by an impalement injury in the shallow waters of the Baltic Sea. The severe infection required amputation of the third toe. Whole genome sequencing of the isolate was performed and revealed a genome consisting of two circular chromosomes with a size of 1.57 and 3.24 Mb.

Identification of genes essential for prey-independent growth of Bdellovibrio bacteriovorus HD100.

Bdellovibrio bacteriovorus HD100 is an obligate predatory bacterium that attacks and invades Gram-negative bacteria. The predator requires living bacteria to survive as growth and replication take place inside the bacterial prey. It is possible to isolate mutants that grow and replicate outside prey bacteria. Such mutants are designated host or prey independent, and their nutritional requirements vary. Some mutants are saprophytic and require prey extracts for extracellular growth, whereas other mutants grow axenically, which denotes the formation of colonies on complete medium in the absence of any prey components. The initial events leading to prey-independent growth are still under debate, and several genes may be involved. We selected new mutants by three different methods: spontaneous mutation, transposon mutagenesis, and targeted gene knockout. By all approaches we isolated mutants of the hit (host interaction) locus. As the relevance of this locus for the development of prey independence has been questioned, we performed whole-genome sequencing of five prey-independent mutants. Three mutants were saprophytic, and two mutants could grow axenically. Whole-genome analysis revealed that the mutation of a small open reading frame of the hit locus is sufficient for the conversion from predatory to saprophytic growth. Complementation experiments were performed by introduction of a plasmid carrying the wild-type hit gene into saprophytic mutants, and predatory growth could be restored. Whole-genome sequencing of two axenic mutants demonstrated that in addition to the hit mutation the colony formation on complete medium was shown to be influenced by the mutations of two genes involved in RNA processing. Complementation experiments with a wild-type gene encoding an RNA helicase, RhlB, abolished the ability to form colonies on complete medium, indicating that stability of RNA influences axenic growth.

Assessment of the mobilizable vector plasmids pSUP202 and pSUP404.2 as genetic tools for the predatory bacterium Bdellovibrio bacteriovorus.

Bdellovibrio and like organisms (BALOs) form the group of predatory bacteria which require Gram-negative bacteria as prey. Genetic studies with Bdellovibrio bacteriovorus can be performed with vectors which are introduced into the predator via conjugation. The usefulness of the two vectors pSUP202 and pSUP404.2 as genetic tools were assessed. Both vectors were transferable into B. bacteriovorus by conjugative matings with an Escherichia coli K12 strain as donor. The transfer frequency was higher for vector pSUP404.2 (approx. 10⁻¹-10⁻4) as for pSUP202 (approx. 10⁻5-10⁻6). Vector pSUP202 with a pMB1 origin is unstable in the predatory bacterium, whereas pSUP404.2 is stably maintained in the absence of selective antibiotics. pSUP404.2 harbors two plasmid replicons, the p15A ori and the RSF1010 replication region The copy number of pSUP404.2 was determined by quantitative PCR in B. bacteriovorus and averages seven copies per genome. pSUP404.2 harbors two resistance genes (chloramphenicol and kanamycin) which can be used for cloning either by selection for transconjugants or by insertional inactivation.

The Acquisition of the scr Gene Cluster Encoding Sucrose Metabolization Enzymes Enables Strains of Vibrio parahaemolyticus and Vibrio vulnificus to Utilize Sucrose as Carbon Source.

Most strains of Vibrio parahaemolyticus are unable to utilize sucrose as carbon source, though few exceptions exist. We investigated a sucrose-positive V. parahaemolyticus strain by whole-genome sequencing (WGS) and confirmed the presences of a genomic island containing sucrose utilization genes. A 4.7 kb DNA cluster consisting of three genes: scrA encoding a sucrose uptake protein, scrK encoding a fructokinase, and scrB coding for a sucrose-6-phosphate hydrolase, was PCR amplified and inserted into the Vibrio/Escherichia coli shuttle vector pVv3. Two recombinant plasmids, only differing in the orientation of the insert with respect to the pVv3-lacZα-fragment, conferred the E. coli K12 transformants the ability to utilize sucrose. The introduction of the two plasmids into sucrose-negative V. parahaemolyticus and V. vulnificus strains also results in a change of the sucrose utilization phenotype from negative to positive. By performing a multiplex PCR targeting scrA, scrK, and scrB, 43 scr-positive V. parahaemolyticus isolates from our collection of retail strains were detected and confirmed to be able to use sucrose as carbon source. Strains unable to utilize the disaccharide were negative by PCR for the scr genes. For in-depth characterization, 17 sucrose-positive V. parahaemolyticus were subjected to WGS. A genomic island with a nucleotide identity of >95% containing scrA, scrB, scrK and three additional coding sequences (CDS) were identified in all strains. The additional genes were predicted as a gene coding for a transcriptional regulator (scrR), a porin encoding gene and a CDS of unknown function. Sequence comparison indicated that the genomic island was located in the same region of the chromosome II in all analyzed V. parahaemolyticus strains. Structural comparison of the genomes with sequences of the sucrose utilizing species V. alginolyticus revealed the same genomic island, which indicates a possible distribution of this genetic structure by horizontal gene transfer. The comparison of all genome sequences based on SNP differences reveals that the presence of sucrose utilizing genes is found in genetically diverse V. parahaemolyticus strains and is not restricted to a subset of closely related strains.

Complete and Circularized Genome Assembly of a Human Isolate of Vibrio navarrensis Biotype pommerensis with MiSeq and MinION Sequence Data.

Vibrio navarrensis is a rare human pathogen. Strains of Vibrio navarrensis biotype pommerensis were isolated from seawater of the Baltic Sea. Recently, a strain of this biotype was recovered from a human patient. The isolate contains two circular chromosomes and a large plasmid with a size of 180 kb.

Entry exclusion and oriT of a conjugative system encoded by the cryptic plasmid p29930 of Yersinia enterocolitica.

The conjugative transfer system of Yersinia enterocolitica 29930 present on the cryptic plasmid p29930 comprises a mating pore formation system (Mpf) related to that of the IncX plasmid R6K and a DNA transfer and replication system (Dtr) with close relationship to the mob region of the mobilizable plasmid CloDF13. Two regions of the transfer system were selected for more detailed analyses of basic functions of conjugative transfer. The putative open reading frame orf22 located in the Mpf region confers the entry exclusion phenotype to possible recipient cells and inhibited conjugative transfer, when it was inserted into the coding region of the cat gene of pACYC184 in sense direction. Mobilization experiments with recombinant plasmids revealed that a 611bp fragment of the Dtr region containing two repeat sequences were required for a functional oriT by the conjugation system of Y. enterocolitica. While the conjugative transfer of cryptic plasmids harbouring the complete conjugation system had not been demonstrated previously, plasmid pBK17 containing the functional oriT was successfully mobilized from Y. enterocolitica strains into Escherichia coli, thus proving that the transfer system could contribute to the spread of these plasmids in nature.