Shotgun-Metagenomics on Positive Blood Culture Bottles Inoculated With Prosthetic Joint Tissue: A Proof of Concept Study.

Clinical metagenomics is actively moving from research to clinical laboratories. It has the potential to change the microbial diagnosis of infectious diseases, especially when detection and identification of pathogens can be challenging, such as in prosthetic joint infection (PJI). The application of metagenomic sequencing to periprosthetic joint tissue (PJT) specimens is often challenged by low bacterial load in addition to high level of inhibitor and contaminant host DNA, limiting pathogen recovery. Shotgun-metagenomics (SMg) performed directly on positive blood culture bottles (BCBs) inoculated with PJT may be a convenient approach to overcome these obstacles. The aim was to test if it is possible to perform SMg on PJT inoculated into BCBs for pathogen identification in PJI diagnosis. Our study was conducted as a laboratory method development. For this purpose, spiked samples (positive controls), negative control and clinical tissue samples (positive BCBs) were included to get a comprehensive overview. We developed a method for preparation of bacterial DNA directly from PJT inoculated in BCBs. Samples were processed using MolYsis5 kit for removal of human DNA and DNA extracted with BiOstic kit. High DNA quantity/quality was obtained, and no inhibition was observed during the library preparation, allowing further sequencing process. DNA sequencing reads obtained from the BCBs, presented a low proportion of human reads (<1%) improving the sensitivity of bacterial detection. We detected a 19-fold increase in the number of reads mapping to human in a sample untreated with MolYsis5. Taxonomic classification of clinical samples identified a median of 96.08% (IQR, 93.85-97.07%; range 85.7-98.6%) bacterial reads. Shotgun-metagenomics results were consistent with the results from a conventional BCB culture method, validating our approach. Overall, we demonstrated a proof of concept that it is possible to perform SMg directly on BCBs inoculated with PJT, with potential of pathogen identification in PJI diagnosis. We consider this a first step in research efforts needed to face the challenges presented in PJI diagnoses.

Culturing periprosthetic tissue in BacT/Alert® Virtuo blood culture system leads to improved and faster detection of prosthetic joint infections.

BACKGROUND: Blood culture bottles (BCBs) provide a semiautomated method for culturing periprosthetic tissue specimens. A study evaluating BCBs for culturing clinical samples other than body fluids is needed before implementation into clinical practice. Our objective was to evaluate use of the BacT/Alert® Virtuo blood culture system for culturing periprosthetic tissue specimens. METHODS: The study was performed through the analysis of spiked (n = 36) and clinical (n = 158) periprosthetic tissue samples. Clinical samples were analyzed by the BCB method and the results were compared to the conventional microbiological culture-based method for time to detection and microorganisms identified. RESULTS: The BacT/Alert® Virtuo blood culture system detected relevant bacteria for prosthetic joint infection in both spiked and clinical samples. The BCB method was found to be as sensitive (79%) as the conventional method (76%) (p = 0.844) during the analyses of clinical samples. The BCB method yielded positive results much faster than the conventional method: 89% against 27% detection within 24 h, respectively. The median detection time was 11.1 h for the BCB method (12 h and 11 h for the aerobic and the anaerobic BCBs, correspondingly). CONCLUSION: We recommend using the BacT/Alert® Virtuo blood culture system for analyzing prosthetic joint tissue, since this detect efficiently and more rapidly a wider range of bacteria than the conventional microbiological method.

Coagulase-negative staphylococci in Southern Brazil: looking toward its high diversity.

INTRODUCTION: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens in nosocomial infections and may serve as a reservoir of mobile genetic elements such as the staphylococcal cassette chromosome mec (SCCmec) encoding methicillin resistance. Molecular characterization of SCCmec types combined with advanced molecular typing techniques may provide essential information for understanding the evolution and epidemiology of CoNS infections. We therefore aimed to investigate the SCCmec distribution, multidrug-resistance (MDR), and biofilm formation in CoNS blood culture isolates from a hospital in Southern Brazil. METHODS: We analyzed 136 CoNS blood culture isolates obtained during 2002-2004 from patients admitted to a tertiary care hospital in Brazil. SCCmec types I to V were determined using multiplex PCR. The clonal relationship of Staphylococcus epidermidis was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Molecular epidemiological data were interpreted along with data on biofilm formation, presence of the icaD gene, and MDR. RESULTS: The most prevalent species were S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis harboring mainly SCCmec types II, III, and V. Overall, the presence of multiple SCCmec was associated with non-MDR, except for S. epidermidis. S. epidermidis isolates showed a high prevalence of icaD, but had low phenotypic biofilm formation. PFGE and MLST revealed high genetic diversity in the S. epidermidis population. CONCLUSIONS: Our results suggest a major shift in SCCmec types within a short period and reveal a different behavior of S. epidermidis with regard to the association between the presence of multiple SCCmec types and MDR profile.

Coagulase-negative staphylococci in Southern Brazil: looking toward its high diversity

Abstract: INTRODUCTION: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens in nosocomial infections and may serve as a reservoir of mobile genetic elements such as the staphylococcal cassette chromosome mec (SCCmec) encoding methicillin resistance. Molecular characterization of SCCmec types combined with advanced molecular typing techniques may provide essential information for understanding the evolution and epidemiology of CoNS infections. We therefore aimed to investigate the SCCmec distribution, multidrug-resistance (MDR), and biofilm formation in CoNS blood culture isolates from a hospital in Southern Brazil. METHODS: We analyzed 136 CoNS blood culture isolates obtained during 2002-2004 from patients admitted to a tertiary care hospital in Brazil. SCCmec types I to V were determined using multiplex PCR. The clonal relationship of Staphylococcus epidermidis was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Molecular epidemiological data were interpreted along with data on biofilm formation, presence of the icaD gene, and MDR. RESULTS: The most prevalent species were S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis harboring mainly SCCmec types II, III, and V. Overall, the presence of multiple SCCmec was associated with non-MDR, except for S. epidermidis. S. epidermidis isolates showed a high prevalence of icaD, but had low phenotypic biofilm formation. PFGE and MLST revealed high genetic diversity in the S. epidermidis population. CONCLUSIONS: Our results suggest a major shift in SCCmec types within a short period and reveal a different behavior of S. epidermidis with regard to the association between the presence of multiple SCCmec types and MDR profile.

Core genome conservation of Staphylococcus haemolyticus limits sequence based population structure analysis.

The notoriously multi-resistant Staphylococcus haemolyticus is an emerging pathogen causing serious infections in immunocompromised patients. Defining the population structure is important to detect outbreaks and spread of antimicrobial resistant clones. Currently, the standard typing technique is pulsed-field gel electrophoresis (PFGE). In this study we describe novel molecular typing schemes for S. haemolyticus using multi locus sequence typing (MLST) and multi locus variable number of tandem repeats (VNTR) analysis. Seven housekeeping genes (MLST) and five VNTR loci (MLVF) were selected for the novel typing schemes. A panel of 45 human and veterinary S. haemolyticus isolates was investigated. The collection had diverse PFGE patterns (38 PFGE types) and was sampled over a 20 year-period from eight countries. MLST resolved 17 sequence types (Simpsons index of diversity [SID]=0.877) and MLVF resolved 14 repeat types (SID=0.831). We found a low sequence diversity. Phylogenetic analysis clustered the isolates in three (MLST) and one (MLVF) clonal complexes, respectively. Taken together, neither the MLST nor the MLVF scheme was suitable to resolve the population structure of this S. haemolyticus collection. Future MLVF and MLST schemes will benefit from addition of more variable core genome sequences identified by comparing different fully sequenced S. haemolyticus genomes.

Biofilm formation by Staphylococcus haemolyticus.

Infections due to coagulase-negative staphylococci (CoNS) most frequently occur after the implantation of medical devices and are attributed to the biofilm-forming potential of CoNS. Staphylococcus haemolyticus is the second most frequently isolated CoNS from patients with hospital-acquired infections. There is only limited knowledge of the nature of S. haemolyticus biofilms. The aim of this study was to characterize S. haemolyticus biofilm formation. We analyzed the biofilm-forming capacities of 72 clinical S. haemolyticus isolates. A detachment assay with NaIO(4), proteinase K, or DNase was used to determine the main biofilm components. Biofilm-associated genes, including the ica operon, were analyzed by PCR, and the gene products were sequenced. Confocal laser scanning microscopy (CLSM) was used to elucidate the biofilm structure. Fifty-three isolates (74%) produced biofilms after growth in Trypticase soy broth (TSB) with glucose, but only 22 (31%) produced biofilms after growth in TSB with NaCl. It was necessary to dissolve the biofilm in ethanol-acetone to measure the optical density of the full biofilm mass. DNase, proteinase K, and NaIO(4) caused biofilm detachment for 100%, 98%, and 38% of the isolates, respectively. icaRADBC and polysaccharide intercellular adhesin (PIA) production were found in only two isolates. CLSM indicated that the biofilm structure of S. haemolyticus clearly differs from that of S. epidermidis. We conclude that biofilm formation is a common phenotype in clinical S. haemolyticus isolates. In contrast to S. epidermidis, proteins and extracellular DNA are of functional relevance for biofilm accumulation, whereas PIA plays only a minor role. The induction of biofilm formation and determination of the biofilm mass also needed to be optimized for S. haemolyticus.

Extended-spectrum beta-lactamase-type SHV-12-producing Enterobacteriaceae causing septicemia in Tanzanian children: vectors for horizontal transfer of antimicrobial resistance.

Septicemia caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae was associated with high mortality in Tanzanian children. Conjugation experiments on the SHV-12-producing Enterobacteriaceae isolates showed that ESBL-encoding genes were transferred on large plasmids together with genes encoding resistance to aminoglycosides, resistance to ceftazidime, gentamicin, doxycycline, trimethoprim-sulfamethoxazole, and chloramphenicol.

Coagulase-negative staphylococcal sepsis in neonates. Association between antibiotic resistance, biofilm formation and the host inflammatory response.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens causing late onset sepsis in neonates. They are often multiresistant to antibiotics, and the ability to form biofilm is considered their main virulence determinant. METHODS: During a 12-year period, we identified 150 neonates having 164 suspected septic episodes with growth of CoNS in blood culture. We examined the relationship between antibiotic resistance, phenotypic biofilm production and genetic determinants for biofilm formation in different CoNS species and their correlation with neonatal inflammatory response. RESULTS: Eighty-five episodes were classified as true sepsis, and 79 episodes of CoNS growth in blood culture were considered contaminations. Sixty-one percent of Staphylococcus epidermidis isolates produced biofilm compared with 26% of CoNS non-epidermidis (P < 0.001). We observed no difference in phenotypic biofilm production or genetic determinants for biofilm formation between invasive isolates and contaminants. C-reactive protein levels as a marker of inflammatory response were higher in CoNS sepsis caused by methicillin and aminoglycoside resistant versus susceptible isolates (P = 0.031). In contrast, there was a significant association between a lower C-reactive protein response and biofilm-positive isolates (P = 0.018). Antibiotic resistance was significantly correlated with biofilm production in S. epidermidis, but not in other CoNS species. CONCLUSIONS: CoNS sepsis with biofilm-forming strains was associated with a decreased host inflammatory response, potentially limiting the immune system to counteract the infection. The impact of antibiotic resistance and virulence determinants on clinical outcome of neonatal CoNS sepsis warrants additional clinical studies.