Claudia Jäckel

Campylobacter jejuni Group III Phage CP81 Contains Many T4-Like Genes without Belonging to the T4-Type Phage Group: Implications for the Evolution of T4 Phages

ABSTRACT CP81 is a virulent Campylobacter group III phage whose linear genome comprises 132,454 bp. At the nucleotide level, CP81 differs from other phages. However, a number of its structural and replication/recombination proteins revealed a relationship to the group II Campylobacter phages CP220/CPt10 and to T4-type phages. Unlike the T4-related phages, the CP81 genome does not contain conserved replication and virion modules. Instead, the respective genes are scattered throughout the phage genome. Moreover, most genes for metabolic enzymes of CP220/CPt10 are lacking in CP81. On the other hand, the CP81 genome contains nine similar genes for homing endonucleases which may be involved in the attrition of the conserved gene order for the virion core genes of T4-type phages. The phage apparently possesses an unusual modification of C or G bases. Efficient cleavage of its DNA was only achieved with restriction enzymes recognizing pure A/T sites. Uncommonly, phenol extraction leads to a significant loss of CP81 DNA from the aqueous layer, a property not yet described for other phages belonging to the T4 superfamily.

Primary Isolation Strain Determines Both Phage Type and Receptors Recognised by Campylobacter jejuni Bacteriophages

In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS) for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN) of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb), host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220) as well as receptors (CPS or flagella) recognised by the isolated phages.

Reduction of Campylobacter jejuni in Broiler Chicken by Successive Application of Group II and Group III Phages

Background Bacteriophage treatment is a promising tool to reduce Campylobacter in chickens. Several studies have been published where group II or group III phages were successfully applied. However, these two groups of phages are different regarding their host ranges and host cell receptors. Therefore, a concerted activity of group II and group III phages might enhance the efficacy of a treatment and decrease the number of resistant bacteria. Results In this study we have compared the lytic properties of some group II and group III phages and analysed the suitability of various phages for a reduction of C. jejuni in broiler chickens. We show that group II and group III phages exhibit different kinetics of infection. Two group III and one group II phage were selected for animal experiments and administered in different combinations to three groups of chickens, each containing ten birds. While group III phage CP14 alone reduced Campylobacter counts by more than 1 log10 unit, the concomitant administration of a second group III phage (CP81) did not yield any reduction, probably due to the development of resistance induced by this phage. One group of chickens received phage CP14 and, 24 hours later, group II phage CP68. In this group of animals, Campylobacter counts were reduced by more than 3 log10 units. Conclusion The experiments illustrated that Campylobacter phage cocktails have to be carefully composed to achieve the best results.

The Complete Genome Sequence of Bacteriophage CP21 Reveals Modular Shuffling in Campylobacter Group II Phages

ABSTRACT Campylobacter group II phages described so far share a high degree of sequence similarity. We report the 182,833-bp genomic sequence of the closely related group II phage CP21 and show that it has a completely different genomic organization. As in other group II phages, the CP21 genome is composed of large modules separated by long DNA repeat regions which obviously trigger recombination and modular shuffling.

Acquisition of virulence factors in livestock-associated MRSA: Lysogenic conversion of CC398 strains by virulence gene-containing phages

Staphylococcus aureus MRSA strains belonging to the clonal complex 398 (CC398) are highly prevalent in livestock and companion animals but may also cause serious infections in humans. CC398 strains in livestock usually do not possess well-known virulence factors that can be frequently found in other MRSA sequence types (ST). Since many staphylococcal virulence genes are residing on the genomes of temperate phages, the question arises why livestock-associated (LA-) CC398 strains are only rarely infected by those phages. We isolated and characterized four temperate phages (P240, P282, P630 and P1105) containing genes of the immune evasion cluster (IEC) and/or for the Panton-Valentine leucocidin (PVL). Sequence analysis of the phage genomes showed that they are closely related to known phages and that the DNA region encoding lysis proteins, virulence factors and the integrase exhibits numerous DNA repeats which may facilitate genomic rearrangements. All phages lysed and lysogenized LA-CC398 strains. Integration of IEC phage P282 was detected at ten sites of the hosts’ chromosome. The prophages were stably inherited in LA-CC398 and enterotoxin A, staphylokinase and PVL toxin were produced. The data demonstrate that lysogenic conversion of LA-CC398 strains by virulence-associated phages may occur and that new pathotypes may emerge by this mechanism.

Characterization of trh2 Harbouring Vibrio parahaemolyticus Strains Isolated in Germany

Background Vibrio parahaemolyticus is a recognized human enteropathogen. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) as well as the type III secretion system 2 (T3SS2) are considered as major virulence factors. As tdh positive strains are not detected in coastal waters of Germany, we focused on the characterization of trh positive strains, which were isolated from mussels, seawater and patients in Germany. Results Ten trh harbouring V. parahaemolyticus strains from Germany were compared to twenty-one trh positive strains from other countries. The complete trh sequences revealed clustering into three different types: trh1 and trh2 genes and a pseudogene Ψtrh. All German isolates possessed alleles of the trh2 gene. MLST analysis indicated a close relationship to Norwegian isolates suggesting that these strains belong to the autochthonous microflora of Northern Europe seawaters. Strains carrying the pseudogene Ψtrh were negative for T3SS2β effector vopC. Transcription of trh and vopC genes was analyzed under different growth conditions. Trh2 gene expression was not altered by bile while trh1 genes were inducible. VopC could be induced by urea in trh2 bearing strains. Most trh1 carrying strains were hemolytic against sheep erythrocytes while all trh2 positive strains did not show any hemolytic activity. TRH variants were synthesized in a prokaryotic cell-free system and their hemolytic activity was analyzed. TRH1 was active against sheep erythrocytes while TRH2 variants were not active at all. Conclusion Our study reveals a high diversity among trh positive V. parahaemolyticus strains. The function of TRH2 hemolysins and the role of the pseudogene Ψtrh as pathogenicity factors are questionable. To assess the pathogenic potential of V. parahaemolyticus strains a differentiation of trh variants and the detection of T3SS2β components like vopC would improve the V. parahaemolyticus diagnostics and could lead to a refinement of the risk assessment in food analyses and clinical diagnostics.

Prevalence, Host Range, and Comparative Genomic Analysis of Temperate Ochrobactrum Phages

Ochrobactrum and Brucella are closely related bacteria that populate different habitats and differ in their pathogenic properties. Only little is known about mobile genetic elements in these genera which might be important for survival and virulence. Previous studies on Brucella lysogeny indicated that active phages are rare in this genus. To gain insight into the presence and nature of prophages in Ochrobactrum, temperate phages were isolated from various species and characterized in detail. In silico analyses disclosed numerous prophages in published Ochrobactrum genomes. Induction experiments showed that Ochrobactrum prophages can be induced by various stress factors and that some strains released phage particles even under non-induced conditions. Sixty percent of lysates prepared from 125 strains revealed lytic activity. The host range and DNA similarities of 19 phages belonging to the families Myoviridae, Siphoviridae, or Podoviridae were determined suggesting that they are highly diverse. Some phages showed relationship to the temperate Brucella inopinata phage BiPB01. The genomic sequences of the myovirus POA1180 (41,655 bp) and podovirus POI1126 (60,065 bp) were analyzed. Phage POA1180 is very similar to a prophage recently identified in a Brucella strain isolated from an exotic frog. The POA1180 genome contains genes which may confer resistance to chromate and the ability to take up sulfate. Phage POI1126 is related to podoviruses of Sinorhizobium meliloti (PCB5), Erwinia pyrifoliae (Pep14), and Burkholderia cenocepacia (BcepIL02) and almost identical to an unnamed plasmid of the Ochrobactrum intermedium strain LMG 3301. Further experiments revealed that the POI1126 prophage indeed replicates as an extrachromosomal element. The data demonstrate for the first time that active prophages are common in Ochrobactrum and suggest that atypical brucellae also may be a reservoir for temperate phages.

Virulence Profiles of Vibrio vulnificus in German Coastal Waters, a Comparison of North Sea and Baltic Sea Isolates

Vibrio vulnificus is a halophilic bacterium of coastal environments known for sporadically causing severe foodborne or wound infections. Global warming is expected to lead to a rising occurrence of V. vulnificus and an increasing incidence of human infections in Northern Europe. So far, infections in Germany were exclusively documented for the Baltic Sea coast, while no cases from the North Sea region have been reported. Regional variations in the prevalence of infections may be influenced by differences in the pathogenicity of V. vulnificus populations in both areas. This study aimed to compare the distribution of virulence-associated traits and genotypes among 101 V. vulnificus isolates from the Baltic Sea and North Sea in order to assess their pathogenicity potential. Furthermore, genetic relationships were examined by multilocus sequence typing (MLST). A high diversity of MLST sequences (74 sequence types) and differences regarding the presence of six potential pathogenicity markers were observed in the V. vulnificus populations of both areas. Strains with genotypes and markers associated with pathogenicity are not restricted to a particular geographic region. This indicates that lack of reported cases in the North Sea region is not caused by the absence of potentially pathogenic strains.

A multiplex real-time PCR for the detection and differentiation of Campylobacter phages

Campylobacter jejuni and C. coli are important food-borne pathogens that are widespread in animal husbandry. To combat Campylobacter along the food chain, the application of lytic phages has been shown to be a promising tool. Campylobacter phages are currently classified into three groups, of which group II and group III phages are the most common. Members of each group are closely related, whereas the two groups share only little DNA similarity. Moreover, while group III phages are specific for C. jejuni, group II phages additionally infect C. coli. Phage cocktails intended to be used for applications should be composed of various phages that differ in their host range and growth kinetics. The isolation of phages is generally performed by plaque assays. This approach has the limitation that phages are merely identified by their lytic activity on certain indicator strains and that relatively high numbers of phages must be present in a tested sample. Therefore, a more sensitive molecular detection system would be beneficial, which allows a pre-screening of samples and the quick detection and discrimination of group II and group III phages. New phages can then be isolated by use of indicator strains that may be different from those typically applied. On the basis of available Campylobacter phage genome sequences, we developed a multiplex PCR system for group II and group III phages selecting the tail tube gene and the gene for the base plate wedge, respectively, as target. Phages of both groups could be identified with primers deduced from the putative tail fiber gene. Efficient release of phage DNA from capsids was achieved by an extended heat treatment or digestion of phage particles with proteinase K/SDS yielding a detection limit of 1 pfu/ml. Individual detection of group II phages, group III phages and of both groups was studied with artificially contaminated chicken skin. To recover phages that had strongly adhered to the skin, stomaching was the most efficient technique. The developed PCR protocol was employed to detect Campylobacter phages in food and environmental samples. In 50 out of 110 samples group II and/or group III phages were identified.

Genetic Diversity of Brucella Reference and Non-reference Phages and Its Impact on Brucella-Typing

Virulent phages have been used for many years to type Brucella isolates, but until recently knowledge about the genetic makeup of these phages remains limited. In this work the host specificity and genomic sequences of the original set (deposited in 1960) of VLA Brucella reference phages Tb, Fi, Wb, Bk2, R/C, and Iz were analyzed and compared with hitherto described brucellaphages. VLA phages turned out to be different from homonymous phages in other laboratories. The host range of the phages was defined by performing plaque assays with a wide selection of Brucella strains. Propagation of the phages on different strains did not alter host specificity. Sequencing of the phages TbV, FiV, WbV, and R/CV revealed nucleotide variations when compared to same-named phages previously described by other laboratories. The phages Bk2V and IzV were sequenced for the first time. While Bk2V exhibited the same deletions as WbV, IzV possesses the largest genome of all Brucella reference phages. The duplication of a 301 bp sequence in this phage and the large deletion in Bk2V, WbV, and R/CV may be a result of recombination caused by repetitive sequences located in this DNA region. To identify new phages as potential candidates for lysotyping, the host range and Single Nucleotide Polymorphisms (SNPs) of 22 non-reference Brucella phages were determined. The phages showed lysis patterns different from those of the reference phages and thus represent novel valuable candidates in the typing set.