Anna Rosander

Removal of Antibiotic Resistance Gene-Carrying Plasmids from Lactobacillus reuteri ATCC 55730 and Characterization of the Resulting Daughter Strain, L. reuteri DSM 17938

ABSTRACT The spread of antibiotic resistance in pathogens is primarily a consequence of the indiscriminate use of antibiotics, but there is concern that food-borne lactic acid bacteria may act as reservoirs of antibiotic resistance genes when distributed in large doses to the gastrointestinal tract. Lactobacillus reuteri ATCC 55730 is a commercially available probiotic strain which has been found to harbor potentially transferable resistance genes. The aims of this study were to define the location and nature of β-lactam, tetracycline, and lincosamide resistance determinants and, if they were found to be acquired, attempt to remove them from the strain by methods that do not genetically modify the organism before subsequently testing whether the probiotic characteristics were retained. No known β-lactam resistance genes was found, but penicillin-binding proteins from ATCC 55730, two additional resistant strains, and three sensitive strains of L. reuteri were sequenced and comparatively analyzed. The β-lactam resistance in ATCC 55730 is probably caused by a number of alterations in the corresponding genes and can be regarded as not transferable. The strain was found to harbor two plasmids carrying tet(W) tetracycline and lnu(A) lincosamide resistance genes, respectively. A new daughter strain, L. reuteri DSM 17938, was derived from ATCC 55730 by removal of the two plasmids, and it was shown to have lost the resistances associated with them. Direct comparison of the parent and daughter strains for a series of in vitro properties and in a human clinical trial confirmed the retained probiotic properties of the daughter strain.

Shotgun Phage Display - Selection for Bacterial Receptins or other Exported Proteins

Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins.

Phage display as a novel screening method to identify extracellular proteins.

Extracellular proteins are involved in many diverse and essential cell functions and in pathogenic bacteria, and they may also serve as virulence factors. Therefore, there is a need for methods that identify the genes encoding this group of proteins in a bacterial genome. Here, we present such a method based on the phage display technology. A novel gene III-based phagemid vector, pG3DSS, was constructed that lacks the signal sequence which normally orientates the encoded fusion protein to the Escherichia coli cell membrane, where it is assembled into the phage particle. When randomly fragmented DNA is inserted into this vector, only phagemids containing an insert encoding a signal sequence will give rise to phage particles displaying a fusion protein. These phages also display an E-tag epitope in fusion with protein III, which enables isolation of phages displaying a fusion protein, using antibodies against the epitope. From a library constructed from Staphylococcus aureus chromosomal DNA, genes encoding secreted as well as transmembrane proteins were isolated, including adhesins, enzymes and transport proteins.

Identification of extracytoplasmic proteins in Bradyrhizobium japonicum using phage display

A novel gene bank of Bradyrhizobium japonicum USDA110spc4 was constructed using pG3DSS, a phagemid vector designed for detecting genes encoding secreted proteins. In this phagemid, the phage protein III lacks its indigenous signal peptide required for protein secretion, thus recombinant fusion proteins are displayed on the phage surface only if a functional signal peptide is provided by an inserted DNA fragment. In addition, the N-terminal half of protein III has been replaced by a short linker region (the E-tag) that is recognized by a monoclonal antibody, which enables isolation of phages displaying a fusion protein. The expression library described here, therefore, provides a powerful means to affinity select for B. japonicum genes encoding extracytoplasmic proteins. In total, 182 DNA sequences were analyzed, among which 132 different putative extracytoplasmic proteins could be identified. The function of most proteins could be predicted and support an extracytoplasmic localization. In addition, genes encoding novel extracytoplasmic proteins were found. In particular, a novel family of small proteins has been identified that is characterized by a conserved pattern of four cysteine residues.

Identification of immunogenic proteins in Treponema phagedenis-like strain V1 from digital dermatitis lesions by phage display

Digital dermatitis (DD) is a contagious claw disease causing lameness in cattle, affecting both animal welfare and economics. In this study, shotgun phage display was used to identify immunogenic proteins in a strain (V1) of the Treponema phylotype closely related to Treponema phagedenis, indicated as a key agent in the pathogenesis of DD. A genomic phage library was constructed and selected against antibodies from a rabbit immunized with live strain V1 bacteria. A homolog to the immunogenic protein TmpA of Treponema pallidum subsp. pallidum was identified, as well as a putative phage tail tape measure protein (Ttm), and a putative proline-rich repeat lipoprotein (PrrA). The complete amino acid sequences of these proteins were predicted from a genomic sequence of strain V1 generated by 454 Sequencing™. The presence of these genes in ten Treponema spp. field isolates was investigated by PCR. The tmpA and ttm genes were detected in all T. phagedenis-like isolates while prrA was detected in four out of seven. None of the genes were detected in the three Treponema pedis isolates investigated. Recombinant proteins were produced and used in indirect ELISAs. For all three proteins, a majority of serum samples from cattle with DD (n=8) showed higher optical density values than samples from cattle without DD (n=7).

Genetic analysis of a Treponema phagedenis locus encoding antigenic lipoproteins with potential for antigenic variation.

Digital dermatitis (DD) is a painful and debilitating claw disease in cattle. Spirochetes of the genus Treponema are found in high numbers in the lesions and are likely to be involved in the pathogenesis. The occurrence of Treponema phagedenis in DD lesions, especially near the interface of healthy and diseased tissue, suggests that this species contributes to the development and/or progression of the lesions. In this study we characterized a genetic locus in T. phagedenis that contains coding regions for three antigenic proteins, PrrA, VpsA, and VpsB. Comparative analysis of homologous loci from fifteen strains suggests that prrA may be transposed into or out of this locus. Alterations in the copy number of TA repeats within the putative promoter region may regulate VpsA/B expression. The vpsA and prrA genes occur in allelic variants in different T. phagedenis isolates and may provide one explanation for the antigenic variation observed in T. phagedenis DD isolates.

The occurrence of Treponema spp. in gingival plaque from dogs with varying degree of periodontal disease

Periodontal disease is common in dogs and is initiated by gingival plaque composed of several hundred bacterial species. Some of these species have specifically been pointed out as potential periodontal pathogens, such as Treponema spp. Treponema spp. are difficult to culture and therefore the majority have been detected by culture-independent methods, such as PCR (Polymerase Chain Reaction). This leaves many Treponema spp. uncharacterized and unnamed. In this study, treponemes were investigated in gingival plaque from dogs with varying degree of periodontal disease with the aim to describe their occurrence and diversity in dogs. The methods used were culture, phase-contrast microscopy, PCR targeting the 16SrRNA-tRNAIle intergenic spacer region (ISR2), sequencing of the ISR2 and phylogenetic analysis. Treponema spp. were detected in samples from 10 out of 11 dogs and isolates were obtained from six dogs. Both healthy and periodontitis affected dogs were Treponema positive. Phylogenetic analysis, based on ISR2 sequences, revealed a large diversity of treponemes in the study population that were found to be distributed mainly in two groups, corresponding to the human oral treponeme phylogroups II (Treponema denticola) and IV (Treponema maltophilum) genetic groups. They were generally more distantly related to other treponemes in these groups. Treponemes from dogs with periodontitis and dogs with mild gingivitis without periodontitis did not differ in any obvious way. The results indicate that several phylotypes of oral treponemes are common in dogs regardless of periodontal status.

Detection of Treponema phagedenis–like antibodies in serum and bulk milk from cows with and without digital dermatitis:

Digital dermatitis (DD) in dairy cows is a widespread disease linked to infection with Treponema. The traditional diagnostic method is clinical inspection, which is subjective and laborious. We explored the performance of 4 different immunogenic proteins from Treponema phagedenis in a new antibody ELISA for analysis of serum or milk. Analysis of samples from 390 cows in 25 herds showed that the ELISA could distinguish the majority of cows with DD from healthy cows. By changing the cutoff and applying parallel or serial testing, high sensitivity or specificity could be achieved. The investigation indicated that aggregated test results can be useful in the assessment of a herd’s DD status. In addition, analysis of bulk tank milk samples showed good agreement with results from individual cows. The test system could be useful in research on the epidemiology and immunology of DD.