Patel, Robin

ABSTRACT Cell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency plasmid transfer in vitro in Enterococcus faecalis . To examine the role of pheromone signaling in vivo , we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF− recipient) in a germfree mouse model 3 days before donor inoculation and determined transfer frequencies of the pheromone-inducible plasmid pCF10. Plasmid transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid transfer was significantly lower with the CF− recipient. While rescue of the CF− mating defect by coculture with CF+ recipients is easily accomplished in vitro , no extracellular complementation occurred in vivo . This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 transfer in vivo . In the absence of CF+ recipients, a low level of transfer to CF− recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum. IMPORTANCE Horizontal gene transfer is a major factor in the biology of Enterococcus faecalis , an important nosocomial pathogen. Previous studies showing efficient conjugative plasmid transfer in the gastrointestinal (GI) tracts of experimental animals did not examine how the enterococcal sex pheromone response impacts the efficiency of transfer. Our study demonstrates for the first time pheromone-enhanced, high-frequency plasmid transfer of E. faecalis plasmid pCF10 in a mouse model in the absence of antibiotic or bacteriocin selection. Pheromone production by recipients dramatically increased plasmid transfer in germfree mice colonized initially with recipients, followed by donors. The presence of a coresident community of common gut microbes did not significantly reduce in vivo plasmid transfer between enterococcal donors and recipients. In mice colonized with enterococcal recipients, we detected plasmid transfer in the intestinal tract within 5 h of addition of donors, before transconjugants could be cultured from feces. Surprisingly, pCF10 carriage provided a competitive fitness advantage unrelated to antibiotic resistance or bacteriocin production.

ABSTRACT Rapid and accurate laboratory tests are important for the timely diagnosis and treatment of central nervous system infections. The FilmArray meningitis/encephalitis (ME) panel (BioFire Diagnostics, Salt Lake City, UT) is an FDA-cleared, multiplex molecular panel that allows the detection of 14 pathogens (bacterial [ n = 6], viral [ n = 7], and fungal [ n = 1] pathogens) from cerebrospinal fluid (CSF). In this study, we evaluated the performance characteristics of the FilmArray ME panel using clinical, residual CSF samples ( n = 291) that tested positive by a routine method(s) (e.g., bacterial culture, individual real-time PCR assay) for a pathogen represented on the ME panel. Of note, a subset ( n = 76) of the CSF specimens was collected during the prevaccine era and had been characterized as positive for a bacterial pathogen. The FilmArray ME panel demonstrated an overall percent positive agreement (PPA) of 97.5% (78/80) for bacterial pathogens, 90.1% (145/161) for viruses, and 52% (26/50) for Cryptococcus neoformans/C. gattii . Despite the low overall agreement (52%) between the ME panel and antigen testing for detection of C. neoformans/C. gattii , the percent positive agreement of the FilmArray assay for C. neoformans/C. gattii was 92.3% (12/13) when the results were compared directly to the results of routine fungal smear or culture. The FilmArray ME panel offers a rapid (∼60-min), syndrome-based approach for the detection of select meningitis and encephalitis pathogens.

Bacteria growing in biofilms cause a wide range of human infections. Biofilm bacteria are resistant to antimicrobics at levels 500 to 5,000 times higher than those needed to kill non-biofilm bacteria. In vitro experiments have shown that electric current can enhance the activity of some antimicrobial agents against certain bacteria in biofilms; this has been termed the “bioelectric effect”. Direct electrical current has already been safely used in humans for fracture healing. Application of direct electric current with antimicrobial chemotherapy in humans could theoretically abrogate the need to remove the device in device-related infections, a procedure associated with substantial morbidity and cost. In this article, we review what has been described in the literature with regards to the bioelectric effect. (Int J Artif Organs 2008; 31: 786–95)

ABSTRACT We describe results from a multicenter study evaluating the Accelerate Pheno system, a first of its kind diagnostic system that rapidly identifies common bloodstream pathogens from positive blood cultures within 90 min and determines bacterial phenotypic antimicrobial susceptibility testing (AST) results within ∼7 h. A combination of fresh clinical and seeded blood cultures were tested, and results from the Accelerate Pheno system were compared to Vitek 2 results for identification (ID) and broth microdilution or disk diffusion for AST. The Accelerate Pheno system accurately identified 14 common bacterial pathogens and two Candida spp. with sensitivities ranging from 94.6 to 100%. Of fresh positive blood cultures, 89% received a monomicrobial call with a positive predictive value of 97.3%. Six common Gram-positive cocci were evaluated for ID. Five were tested against eight antibiotics, two resistance phenotypes (methicillin-resistant Staphylococcus aureus and Staphylococcus spp. [MRSA/MRS]), and inducible clindamycin resistance (MLSb). From the 4,142 AST results, the overall essential agreement (EA) and categorical agreement (CA) were 97.6% and 97.9%, respectively. Overall very major error (VME), major error (ME), and minor error (mE) rates were 1.0%, 0.7%, and 1.3%, respectively. Eight species of Gram-negative rods were evaluated against 15 antibiotics. From the 6,331 AST results, overall EA and CA were 95.4% and 94.3%, respectively. Overall VME, ME, and mE rates were 0.5%, 0.9%, and 4.8%, respectively. The Accelerate Pheno system has the unique ability to identify and provide phenotypic MIC and categorical AST results in a few hours directly from positive blood culture bottles and support accurate antimicrobial adjustment.

ABSTRACT The performance of a disk diffusion test using broth from positive blood cultures as inoculum (direct disk diffusion [dDD]) was evaluated for a collection of 20 challenge isolates of Enterobacteriaceae , Acinetobacter baumannii , and Pseudomonas aeruginosa . Isolates seeded into human blood were inoculated into Bactec Plus Aerobic/F, VersaTREK Redox 1, and BacT/Alert FA Plus bottles and incubated in the respective automated blood culture systems. Disk diffusion results were compared to reference disk diffusion results. Categorical agreement (CA) values for dDD, after removal of random errors due to natural MIC variation, were 87.8%, 88.4%, and 92.2% for the BacT/Alert, Bactec, and VersaTREK systems, respectively. No very major errors (VME) were observed, and major error (ME) rates were 3.0%, 2.3%, and 1.7%, respectively. Incubation of the dDD test samples for 6 h compared to incubation for 16 to 18 h resulted in 19.9% of tests having too light of growth to allow reading of zones of inhibition. Among the evaluable dDD tests, CA values were 58.9%, 76.6%, and 73.2% for the isolates seeded into the BacT/Alert, Bactec, and VersaTREK systems, respectively. VME rates for isolates seeded into these systems were 2.2%, 1.8%, and 3.0%, respectively, and ME rates were 25.4%, 6.1%, and 2.8%, respectively, at the 6-h reading. The best performance of dDD was found for blood cultures with bacterial concentrations in the range of 7.6 × 10 7 to 5.0 × 10 8 CFU/ml; CA values ranged from 94.7 to 96.2% for these concentrations after 18 h of incubation and from 76.9 to 84.1% after 6 h of incubation. These preliminary data demonstrate the potential accuracy of dDD testing by the clinical laboratory.

SUMMARY The recent development of commercial panel-based molecular diagnostics for the rapid detection of pathogens in positive blood culture bottles, respiratory specimens, stool, and cerebrospinal fluid has resulted in a paradigm shift in clinical microbiology and clinical practice. This review focuses on U.S. Food and Drug Administration (FDA)-approved/cleared multiplex molecular panels with more than five targets designed to assist in the diagnosis of bloodstream, respiratory tract, gastrointestinal, or central nervous system infections. While these panel-based assays have the clear advantages of a rapid turnaround time and the detection of a large number of microorganisms and promise to improve health care, they present certain challenges, including cost and the definition of ideal test utilization strategies (i.e., optimal ordering) and test interpretation.