Yijuan Xu

Culture-Dependent and -Independent Investigations of Microbial Diversity on Urinary Catheters

ABSTRACT Catheter-associated urinary tract infection is caused by bacteria, which ascend the catheter along its external or internal surface to the bladder and subsequently develop into biofilms on the catheter and uroepithelium. Antibiotic-treated bacteria and bacteria residing in biofilm can be difficult to culture. In this study we used culture-based and 16S rRNA gene-based culture-independent methods (fingerprinting, cloning, and pyrosequencing) to determine the microbial diversity of biofilms on 24 urinary catheters. Most of the patients were catheterized for <30 days and had undergone recent antibiotic treatment. In addition, the corresponding urine samples for 16 patients were cultured. We found that gene analyses of the catheters were consistent with cultures of the corresponding urine samples for the presence of bacteria but sometimes discordant for the identity of the species. Cultures of catheter tips detected bacteria more frequently than urine cultures and gene analyses; coagulase-negative staphylococci were, in particular, cultured much more often from catheter tips, indicating potential contamination of the catheter tips during sampling. The external and internal surfaces of 19 catheters were separately analyzed by molecular methods, and discordant results were found in six catheters, suggesting that bacterial colonization intra- and extraluminally may be different. Molecular analyses showed that most of the species identified in this study were known uropathogens, and infected catheters were generally colonized by one to two species, probably due to antibiotic usage and short-term catheterization. In conclusion, our data showed that culture-independent molecular methods did not detect bacteria from urinary catheters more frequently than culture-based methods.

In vivo gene expression in a Staphylococcus aureus prosthetic joint infection characterized by RNA sequencing and metabolomics: a pilot study.

BackgroundStaphylococcus aureus gene expression has been sparsely studied in deep-sited infections in humans. Here, we characterized the staphylococcal transcriptome in vivo and the joint fluid metabolome in a prosthetic joint infection with an acute presentation using deep RNA sequencing and nuclear magnetic resonance spectroscopy, respectively. We compared our findings with the genome, transcriptome and metabolome of the S. aureus joint fluid isolate grown in vitro.ResultFrom the transcriptome analysis we found increased expression of siderophore synthesis genes and multiple known virulence genes. The regulatory pattern of catabolic pathway genes indicated that the bacterial infection was sustained on amino acids, glycans and nucleosides. Upregulation of fermentation genes and the presence of ethanol in joint fluid indicated severe oxygen limitation in vivo.ConclusionThis single case study highlights the capacity of combined transcriptome and metabolome analyses for elucidating the pathogenesis of prosthetic infections of major clinical importance.

Microbiological diagnosis of device-related biofilm infections

Medical device‐related infections cause undue patient distress, increased morbidity and mortality and pose a huge financial burden on healthcare services. The pathogens are frequently distributed heterogeneously in biofilms, which can persist without being effectively cleared by host immune defenses and antibiotic therapy. At present, there is no ‘gold standard’ available to reveal the presence of device‐related biofilm infections. However, adequate sample collection and logistics, standardised diagnostic methods, and interpretation of results by experienced personnel are important steps in efficient diagnosis and treatment of these infections. The focus of this mini review is on prosthethic joint and cardiovascular implantable device infections, which exemplify permanent devices that are placed in a sterile body site. These device‐related infections represent some of the most challenging in terms of both diagnosis and treatment.

‘All in a box’ a concept for optimizing microbiological diagnostic sampling in prosthetic joint infections

BackgroundAccurate microbial diagnosis is crucial for effective management of prosthetic joint infections. Culturing of multiple intraoperative tissue samples has increased diagnostic accuracy, but new preparatory techniques and molecular methods hold promise of further improvement. The increased complexity of sampling is, however, a tough challenge for surgeons and assistants in the operation theatre, and therefore we devised and tested a new concept of pre-packed boxes with a complete assortment of swabs, vials and additional tools needed in the operating theatre for non-standard samples during a clinical study of prosthetic joint infections.FindingsThe protocol for the clinical study required triplicate samples of joint fluid, periprosthetic tissue, bone tissue, and swabs from the surface of the prosthesis. Separate boxes were prepared for percutaneous joint puncture and surgical revision; the latter included containers for prosthetic components or the entire prosthesis. During a 2-year project period 164 boxes were used by the surgeons, 98 of which contained a complete set of samples. In all, 1508 (89%) of 1685 scheduled samples were received.ConclusionWith this concept a high level of completeness of sample sets was achieved and thus secured a valid basis for evaluation of new diagnostics. Although enthusiasm for the project may have been a contributing factor, the extended project period suggests that the ‘All in a box’ concept is equally applicable in routine clinical settings with standardized but complex diagnostic sampling.

Differential Contributions of Specimen Types, Culturing, and 16S rRNA Sequencing in Diagnosis of Prosthetic Joint Infections.

ABSTRACT Prosthetic joint failure is mainly caused by infection, aseptic failure (AF), and mechanical problems. Infection detection has been improved with modified culture methods and molecular diagnostics. However, comparisons between modified and conventional microbiology methods are difficult due to variations in specimen sampling. In this prospective, multidisciplinary study of hip or knee prosthetic failures, we assessed the contributions of different specimen types, extended culture incubations, and 16S rRNA sequencing for diagnosing prosthetic joint infections (PJI). Project specimens included joint fluid (JF), bone biopsy specimens (BB), soft-tissue biopsy specimens (STB), and swabs (SW) from the prosthesis, collected in situ, and sonication fluid collected from prosthetic components (PC). Specimens were cultured for 6 (conventional) or 14 days, and 16S rRNA sequencing was performed at study completion. Of the 156 patients enrolled, 111 underwent 114 surgical revisions (cases) due to indications of either PJI (n = 43) or AF (n = 71). Conventional tissue biopsy cultures confirmed PJI in 28/43 (65%) cases and refuted AF in 3/71 (4%) cases; one case was not evaluable. Based on these results, minor diagnostic adjustments were made. Fourteen-day cultures of JF, STB, and PC specimens confirmed PJI in 39/42 (93%) cases, and 16S rRNA sequencing confirmed PJI in 33/42 (83%) cases. One PJI case was confirmed with 16S rRNA sequencing alone and five with cultures of project specimens alone. These findings indicated that JF, STB, and PC specimen cultures qualified as an optimal diagnostic set. The contribution of sequencing to diagnosis of PJI may depend on patient selection; this hypothesis requires further investigation.

Improved Diagnosis of Biofilm Infections Using Various Molecular Methods

Traditional culture-dependent methods and a number of culture-independent molecular methods including 16S rRNA gene polymerase chain reaction, construction of clone libraries, sequencing, phylogeny, fingerprinting, fluorescence in situ hybridization and quantitative PCR were used to describe the microbial composition of two types of biofilm-related infections, namely chronic venous leg ulcers and prosthetic joint infections. Multiple tissue biopsies were taken from each chronic wound, and different specimen types (joint fluid, tissue biopsy, bone biopsy and prosthesis scraping or sonication) were collected from prosthetic joint patients. The obtained results indicate that in these two types of infections the bacterial composition and yield may vary depending on the position and type of samples used for analysis. It emphasizes the need for multiple samplings in order to achieve better diagnosis and treatment of these biofilm-related infections. The most complete picture of microbial composition of biofilms is probably accomplished when several culture and culture-independent methods are used in parallel to characterize the pathogens.