Antibodies to coagulase of Staphylococcus aureus crossreact to Efb and reveal different binding of shared fibrinogen binding repeats.

Staphylococcus aureus pathology is caused by a plethora of virulence factors able to combat multiple host defence mechanisms. Fibrinogen (Fg), a critical component in the host coagulation cascade, plays an important role in the pathogenesis of this bacterium, as it is the target of numerous staphylococcal virulence proteins. Amongst its secreted virulence factors, coagulase (Coa) and Extracellular fibrinogen-binding protein (Efb) share common Fg binding motives and have been described to form a Fg shield around staphylococcal cells, thereby allowing efficient bacterial spreading, phagocytosis escape and evasion of host immune system responses. Targeting these proteins with monoclonal antibodies thus represents a new therapeutic option against S. aureus. To this end, here we report the selection and characterization of fully human, sequence-defined, monoclonal antibodies selected against the C-terminal of coagulase. Given the functional homology between Coa and Efb, we also investigated if the generated antibodies bound the two virulence factors. Thirteen unique antibodies were isolated from naïve antibodies gene libraries by antibody phage display. As anticipated, most of the selected antibodies showed cross-recognition of these two proteins and among them, four were able to block the interaction between Coa/Efb and Fg. Furthermore, our monoclonal antibodies could interact with the two main Fg binding repeats present at the C-terminal of Coa and distinguish them, suggesting the presence of two functionally different Fg-binding epitopes.

Simulated Pediatric Blood Cultures to Assess the Inactivation of Clinically Relevant Antimicrobial Drug Concentrations in Resin-Containing Bottles.

The bacteremia level as well as the administration of antibiotics before blood collection may significantly affect the recovery of bacterial pathogens from pediatric blood cultures in BacT/Alert Virtuo or Bactec FX BC systems, which remain the common techniques to diagnose bacteremia in pediatric patients. We simulated pediatric blood cultures with low or intermediate bacteremia level to evaluate BacT/Alert PF Plus and Bactec Peds Plus blood culture bottles for resin-based inactivation of 16 antibiotic-bacterium combinations. Overall, 105/192 (54.7%) of BacT/Alert PF Plus bottles and 69/192 (36.0%) of Bactec Peds Plus bottles allowed organisms to grow when exposed to antibiotics. In particular, both BacT/Alert PF Plus and Bactec Peds Plus bottles proved to be effective with piperacillin/tazobactam and Pseudomonas aeruginosa or with oxacillin and methicillin-susceptible Staphylococcus aureus (100% growth), whereas no effectiveness was apparent with ceftriaxone and Escherichia coli, Streptococcus agalactiae, or Streptococcus pneumoniae or with cefepime and E. coli (0% growth). In some relevant instances (e.g., with vancomycin and methicillin-resistant S. aureus or Streptococcus pneumoniae), BacT/Alert PF Plus bottles were superior to Bactec Peds Plus bottles. Together, these findings underscore the potentiality of resin-containing bottles to enhance diagnosis of bacteremia in pediatric patients on antimicrobial therapy. This is particularly true with one of the evaluated BC systems and with simulated intermediate bacteremia level only.

Corrigendum: In vitro Evaluation of BACT/ALERT® VIRTUO®, BACT/ALERT 3D®, and BACTEC™ FX Automated Blood Culture Systems for Detection of Microbial Pathogens Using Simulated Human Blood Samples.

[This corrects the article DOI: 10.3389/fmicb.2019.00221.].

Efficient Inactivation of Clinically Relevant Antimicrobial Drug Concentrations by BacT/Alert or Bactec Resin-Containing Media in Simulated Adult Blood Cultures.

We assessed the antimicrobial-inactivation capability of BacT/Alert (FA Plus and FN Plus) or Bactec (Plus Aerobic/F and Plus Anaerobic/F) media for 40 antibiotic-bacterium combinations in simulated adult blood cultures. Aside from high recovery rates (93.2% and 88.4%, respectively), we showed that at the lowest but clinically relevant antibiotic concentrations, both BacT/Alert and Bactec media recovered all the organisms tested with drugs except for Escherichia coli, which was tested in the presence of meropenem. Delayed recoveries were mainly associated with vancomycin.

Simplified Testing Method for Direct Detection of Carbapenemase-Producing Organisms from Positive Blood Cultures Using the NG-Test Carba 5 Assay.

We directly tested 484 organisms from clinical (n = 310) and simulated (n = 174) positive blood cultures using the NG-Test Carba 5 assay for carbapenemase-producing Enterobacterales detection. The assay identified all but 4 of the KPC (170/171), OXA-48-like (22/22), VIM (19/21), and NDM (14/15) producers with no false positives. Among the clinical Klebsiella pneumoniae organisms tested, 122 of 123 KPC, 1 of 1 OXA-48-like, and 1 of 2 VIM producers were detected by the assay. Some VIM and NDM producers yielded scant but still-readable bands with the assay. No organisms produced the IMPs that the assay was designed to detect.

In vitro Evaluation of BACT/ALERT® VIRTUO®, BACT/ALERT 3D®, and BACTEC™ FX Automated Blood Culture Systems for Detection of Microbial Pathogens Using Simulated Human Blood Samples.

Blood culture (BC) is still the standard for diagnosing bloodstream infections (BSIs), especially those caused by bacteria and fungi. Infection-complicating sepsis or septic shock often occurs at BSI onset, making necessary to improve the diagnostic yield of positive BCs. Among the BC systems currently available, the BACT/ALERT® VIRTUO® (VIRTUO) system has been developed to shorten time to detection (TTD) of positive BCs. In this study, we assessed TTD for 330 clinically relevant species including 14 Gram-positive, 14 Gram-negative, and 5 yeast isolates in spiked human blood samples that were tested in parallel with VIRTUO BACT/ALERT® 3D (BTA3D) and BACTEC™ FX (BACTEC) systems. We inoculated 30 colony-forming unit (CFU) from each microbial suspension into BACT/ALERT® Plus or BACTEC™ Plus (aerobic/anaerobic or pediatric) BC bottles, and we used two different blood volumes to simulate, respectively, the BCs collected from adult and pediatric patients. Of 2,610 bottles tested, 2,600 (99.6%) signaled positive in the three systems. Only the BACTEC system did not detect Staphylococcus lugdunensis isolates in anaerobic bottles. Among adult simulated cultures, the median TTD was significantly shorter for aerobic/anaerobic bottles incubated in VIRTUO (11.6 h and 10.1 h) compared to bottles incubated in either BTA3D (13.3 and 12.3 h) or BACTEC (13.5 and 12.2 h) system. Among pediatric simulated cultures, the median TTD was significantly shorter for bottles incubated in VIRTUO (11.2 h) compared to bottles incubated in either the BTA3D (13.0 h) or BACTEC (12.5 h) system. Compared to BTA3D and/or BACTEC systems, VIRTUO allowed faster growth detection for most of the 33 microbial species tested. Notable examples were Salmonella spp. (7.4 h by VIRTUO vs. 10.1 h and 9.2 h by either BTA3D or BACTEC) and Streptococcus agalactiae (8.1 h by VIRTUO vs. 10.3 and 9.4 h by either BTA3D or BACTEC). The few notable exceptions included Stenotrophomonas maltophilia and some Candida species. Together, these findings confirm that VIRTUO has greater potential of improving the laboratory detection of bacteremia and fungemia than the progenitor BTA3D or the competitor BACTEC system.