Renal transplant recipients are predisposed to urinary tract infections caused by both common uropathogens and opportunistic bacteria resulting frequently in significant polymicrobial infections. In this study, a culture-independent 16S rRNA-based approach was established to identify unusual, fastidious, or anaerobic bacteria and to investigate bacterial diversity in urinary tract specimens. Similarly sized amplicons encompassing the V6 to V8 region of the 16S rRNA were analyzed with denaturing high-performance liquid chromatography (DHPLC) (WAVE System). Artificial mixtures of single amplicons from commonly encountered uropathogenic bacteria produced distinct peak profiles whose identities were confirmed by sequencing individually collected peak products. We evaluated the application of the method on 109 urinary tract specimens from renal transplant recipients; 100% correlation was found for culture-positive specimens, and DHPLC generated peak profiles. However, for culture-negative specimens, DHPLC facilitated the detection of novel peak profiles. DNA sequencing of these individual peaks was used to identify the bacteria involved. Thus, in PCR-positive but culture-negative samples the method allowed detection of previously known uropathogens such as Corynebacterium urealyticum and Gardnerella vaginalis, but also unusual agents including Anaerococcus lactolyticus, Bacteroides vulgatus, Dialister invisus, Fusobacterium nucleatum, Lactobacillus iners, Leptotrichia amnionii, Prevotella buccalis, Prevotella ruminicola, Rahnella aquatilis, and Streptococcus intermedius were detected as single pathogens or as constituents of polymicrobial infections. The method described is reproducible and rapidly and enables both DHPLC-based profiling and sequence-based investigation of microbial communities and polymicrobial infections. A detailed understanding of infections found in recipients of renal transplants will guide antibiotic therapy regimens and provide new perspectives for decreasing the risk of graft rejection.Currently, one of the major problems for successful kidney transplantation is the reaction of the immune system of the recipient against the donor organ, which in the case of unsuccessful immunosuppressive treatment can result in the loss of the transplant. In order to prevent or treat such rejection episodes and to maintain graft function the application of immunosuppressive agents is standard practice. The incidence of bacterial infection in renal transplant recipients is directly related to the net immunosuppressive effect achieved and the duration of time over which this therapy is administered. Bacterial urinary tract infections (UTIs) are frequently associated with the early onset of chronic rejection and may also lead to reduced transplant survival (15,18). Studies have shown that in 40 to 60% of transplant recipients the urinary tract is the source of septicemia and that in patients with urosepsis the recurrence rate was approximately 40% (1, 13).

An in vitro assay for adeno-aociated virus (AAV) DNA replication has been developed. The substrate is a plasmid containing the duplex form of AAV DNA in pBR322. The AAV insert is excised or rescued from the pas by extracts of uninfected cells. Replication was assayed by production of full-length excised AAV DNA resistant to Dpn I (ii) Only the excised AAV insert was replicated; pBR322 sequences were not. (iv) Replication was dependent on the presence of the AAV terminal repeat. (v) If the terminal 55 bases were deleted from both ends of the AAV insert, no rescue took place: replication occurred and both AAV and pBR322 sequences were replicated. We conclude that the AAV terminal repeat is essential for DNA replication but that under some conditions an initiation mechanism that does not involve hairpin priming may be used.The adeno-associated virus (AAV) genome has a palindromic inverted terminal repeat thought to serve as a "hairpin" primer to initiate DNA replication (1-3). In vivo AAV DNA replication appears to occur by a single-strand displacement mechanism leading to a duplex replicative intermediate covalently crosslinked at one end by the hairpin primer ( Fig. 1) (2, 3). To resolve the crosslink, the AAV-encoded rep 68/78 protein specifically nicks the hairpin at a point on the parental strand directly opposite the original 3' terminus (4). As a result of the nick, the 3'-terminal 125 bases are transferred from parental to progeny strand and are inverted in the process. The nick/transfer process leaves a 3'-OH at the end of the shortened parental strand, which then serves as a primer for synthesis to fill in the gapped region created by the transfer. In this reaction, the transferred hairpin sequence acts as the template.AAV DNA replication can be initiated from two very different substrates. In a permissive cell, in the presence of helper virus, the starting point for a productive infection is a small, linear, single-stranded DNA whose palindromic 3' terminus is free to form a hairpin and serve as a primer. Alternatively, in a latently infected cell the viral DNA is integrated as a duplex molecule into the host genome (5, 6). In the latter case, rescue or excision of the integrated genome is required for virion production. However, it is not known whether excision per se precedes the onset of free virion DNA synthesis or is a consequence of it. In vivo, the AAV rep gene is required in trans for rescue from the integrated state and for replication (7,8).A plasmid containing the duplex form of AAV DNA in pBR322 is fully infectious in adenovirus-infected HeLa cells (9,10 Plasmids. Plasmid pSM620 has been described (9). Plasmid pGM106 was created by insertion of the 4400-base-pair Bal I fragment of AAV into the Pst I site of pBR322 vector (14).Abbreviations: AAV, adeno-associated virus; SV40, simian virus 40;T antigen, large tumor antigen. 4673The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance w...

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Though numerous studies have shown that gene transfer occurs between distantly related bacterial genera under laboratory conditions, the frequency and breadth of horizontal transfer events in nature remain unknown. Previous evidence for natural intergeneric transfers came from studies of genes in human pathogens, bacteria that colonize the same host. We present evidence that natural transfer of a tetracycline resistance gene, tetQ, has occurred between bacterial genera that normally colonize different hosts. A DNA sequence comparative approach was taken to examine the extent of horizontal tetQ dissemination between species of Bacteroides, the predominant genus of the human colonic microflora, and between species of Bacteroides and of the distantly related genus PrevoteUa, a predominant genus of the microflora of the rumens and intestinal tracts of farm animals. Virtually identical tetQ sequences were found in a number of isolate pairs differing in taxonomy and geographic origin, indicating that extensive natural gene transmission has occurred. Among the exchange events indicated by the evidence was the very recent transfer of an allele of tetQ usually found in PrevoteUa spp. to a Bacteroides fragilis strain.

A novel denaturing high-performance liquid chromatography (DHPLC)-based technique allows rapid highresolution analysis of PCR products. We used this technique for unequivocal molecular identification of seven Candida species. We show the application of this PCR/DHPLC approach for direct detection and identification of yeast species from blood cultures and for detection of Candida colonization in the gastrointestinal tract of allogeneic transplant patients.