Culture-independent identification of pathogenic bacteria and polymicrobial infections in the genitourinary tract of renal transplant recipients

Abstract

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.

FIG. 1.

Flow chart for DHPLC/WAVE System-based analysis of the 16S rRNA from isolated bacteria and clinical samples. (A) Schematic representation of the compiled 16S rRNA from the 5′ to the 3′ end. Hypervariable regions are depicted in white boxes, and highly conserved regions in black (; this study). Primers 0933F and 1407R encompass the V6 to V8 region (474 bp). Amplicon A harbors a 40-bp GC clamp at the 5′ end and amplicon B a 10-bp GC clamp. Amplicon C is devoid of a GC clamp. F, forward; R, reverse; V, variable. (B) SYBR-Gold-stained agarose gel electrophoresis of amplicon C from the 16S rRNA of selected uropathogens. Lane 1, E. faecalis; lane 2, P. vulgaris; lane 3, E. coli; lane 4, K. pneumoniae; lane 5, P. aeruginosa; lane 6, S. saprophyticus.

FIG. 2.

Distinct peak profiles of selected uropathogens. Amplicon C (0933F/1407R) was analyzed with the analytical gradient mixed species at 62.0°C. Ten microliters of each PCR-product was used for injection. The retention times are the time points when the DNA fragments eluted from the column of the WAVE System and are indicated in minutes. The amount of DNA eluted is indicated as the absorbance in millivolts. The experiments were done in triplicate. The major and the smaller peaks are indicated by m and s, respectively. The duration of one analysis was 16 min.

FIG.3.

Analysis of artificially mixed PCR products of selected uropathogens. Several equimolar mixtures of amplicon C (0933F/1407R) from selected uropathogens were analyzed with the analytical gradient mixed species at 62.0°C. Twenty microliters of each mixture was used for injection. The retention times are the time points when the DNA fragments are eluted from the column of the WAVE System and are indicated in minutes. The amount of DNA eluted is indicated as the absorbance (in millivolts). The experiments were done in triplicate. (A) mixture of the species E. faecalis and E. faecium; (B) E. faecalis and E. coli; (C) E. coli and P. aeruginosa; (D) mixture of the three species S. saprophyticus, K. pneumoniae, and S. agalactiae; (E) mixture of the four species S. saprophyticus, E. faecalis, K. pneumoniae, and E. coli. Panel F: mixture of the five species S. aureus, E. faecalis, K. pneumoniae, E. coli, and P. aeruginosa. The duration of one analysis was 16 min.

FIG. 4.

Culture-independent analysis of urinary tract specimen and comparison with the culture on agar plates. Total DNA of urinary tract specimens was extracted, amplicon C (0933F/1407R) was generated and analyzed with the analytical gradient mixed species at 62.0°C. Twenty microliters of each PCR product was used for injection. The retention times are the time points when the DNA fragments are eluted from the column of the WAVE System and are indicated in minutes. The amount of DNA eluted is indicated as the absorbance (in millivolts). The .experiments were done in triplicate. UR659: E. faecalis and E. coli were detected by both culture and PCR. UR540: E. faecalis and K. pneumoniae were detected by both culture and PCR. UR542: G. vaginalis was detected only by PCR. Coinfection with C. albicans was detected on Sabouraud agar. UR881: R. aquatilis was detected only by PCR. Coinfection with C. albicans was detected on Sabouraud agar. UR543: L. iners and G. vaginalis were detected only by PCR. UR539: A. lactolyticus, P. buccalis, P. ruminicola, and G. vaginalis were detected only by PCR. The duration of one analysis was 16 min.