Assessing antibiotic tolerance of Staphylococcus aureus derived directly from patients by the Replica Plating Tolerance Isolation System - REPTIS

Antibiotic tolerant Staphylococcus aureus pose a great challenge to clinicians as well as to microbiological laboratories and are one reason for treatment failure. Antibiotic tolerant strains survive transient antibiotic exposure despite being fully susceptible in vitro . Thus, fast and reliable methods to detect tolerance in the routine microbiology laboratory are urgently required. We therefore evaluated the feasibility of the replica plating tolerance isolation system (REPTIS) to detect antibiotic tolerance in S. aureus isolates derived directly from patients suffering from different types of infections and investigated possible connections to clinical presentations and patient characteristics. One hundred twenty-five S. aureus isolates were included. Replica plating of the original resistance testing plate was used to assess regrowth in the zones of inhibition, indicating antibiotic tolerance. Bacterial regrowth was assessed after 24 and 48 hours of incubation and an overall regrowth score (ORS) was assigned. Regrowth scores were compared to the clinical presentation. Bacterial regrowth was high for most antibiotics targeting protein synthesis and relatively low for antibiotics targeting other cellular functions such as DNA-replication, transcription and cell wall synthesis, with the exception of rifampicin. Isolates with a blaZ penicillinase had lower regrowth in penicillin and ampicillin. Low ORSs were more prevalent among isolates recovered from patients with immunosuppression or methicillin-resistant S. aureus (MRSA) isolates. In conclusion, REPTIS is useful to detect antibiotic tolerance in clinical microbiological routine diagnostics. Further studies should evaluate the impact of rapid detection of antibiotic tolerance as a clinical decision-making tool for tailored antibiotic treatments.

Bacteria-Targeted Fluorescence Imaging of Extracted Osteosynthesis Devices for Rapid Visualization of Fracture-Related Infections

Purpose: Fracture-related infection (FRI) is a serious complication in orthopaedic trauma surgery worldwide. Especially the distinction of infection from sterile inflammation and the detection of low-grade infection are highly challenging. The objective of the present study was to explore the use of bacteria-targeted fluorescence imaging for enhanced detection of FRI on extracted osteosynthesis devices as a step-up towards real-time image-guided trauma surgery.Methods: Extracted osteosynthesis devices from 13 patients, who needed revision surgery after fracture treatment, were incubated with a near-infrared fluorescent tracer composed of the antibiotic vancomycin and the fluorophore IRDye800CW (i.e. vanco-800CW). Subsequently, the devices were imaged and vanco-800CW fluorescence signals were correlated to the results of microbiological culturing and to bacterial growth upon replica plating of the imaged devices on blood agar.Results: Importantly, compared to culturing, the bacteria-targeted fluorescence imaging of extracted osteosynthesis devices with vanco-800CW allows for a prompt diagnosis of FRI, reducing the time-to-result from days to less than 30 min. Moreover, bacteria-targeted imaging will provide surgeons with real-time visual information on the presence and extent of infection.Conclusion: Here we present the first clinical application of fluorescence imaging for detection of FRI. We conclude that imaging with vanco-800CW can provide early, accurate and real-time visual diagnostic information on FRI in the clinical setting, even in case of low-grade infections.

Replica Plating

This chapter describes how Esther Lederberg, in daring to re-purpose her compact makeup pad as a kind of ink stamp, developed the new Replica Plating Technique but later her husband Joshua seemed to claim sole credit for this discovery. The Lederbergs demonstrated the effectiveness of the new plating technique in their study of spontaneous bacterial mutations, using the technique to resolve a longstanding question of bacterial mutations: Did mutations occur spontaneously or was some other process of adaptation involved? While at first, Joshua defended Esther’s co-equal contribution to the invention of replica plating, in later publications, he seemed to claim sole credit, by ignoring her contributions. This is a classic example of the Matilda Effect, when a male colleague is given sole credit for an invention by unfairly ignoring the female colleague’s contribution. Some of the Lederbergs’ colleagues recognize Esther as the primary inventor of replica plating, since she had the reputation of an experimental genius; others assumed that the creative insight was Joshua’s.

Introduction

This chapter relates how one day in 1950, Esther Zimmer Lederberg cleverly re-purposed her compact makeup pad and invented replica plating. This whimsical experiment led to an elegant technique for duplicating many bacterial clones in one step, a clever invention that epitomized her experimental creativity. The chapter shows how the Lederbergs established the field of bacterial genetics years before the birth of molecular biology and together discovered bacterial sex (or horizontal gene transfer, HGT) the peculiar processes that enable bacteria to rapidly spread their genes, leading to antibiotic resistance and the evolution of new species. The stellar reputation of her brilliant husband and collaborator, however, diminished Esther Lederberg’s legacy. The systematic bias against giving due credit for achievements of women scientists whose work is misattributed to their scientific colleagues is known as the Matilda Effect. Esther Lederberg’s story is sadly similar to those of many exemplary women scientists.

A Hidden Legacy

This biography of Esther Zimmer Lederberg highlights the importance of her research work, which revealed the unique features of bacterial sex, essential for our understanding of molecular biology and evolution. A Hidden Legacy relates how, she and her husband Joshua Lederberg established the new field of bacterial genetics together, in the decade leading up to the discovery of the DNA double helix. Their impressive series of achievements include: the discovery of λ‎ bacteriophage and of the first plasmid, known as the F-factor; the demonstration that viruses carry bacterial genes between bacteria; and the elucidation of fundamental properties of bacterial sex. This successful collaboration earned Joshua the 1958 Nobel Prize, which he shared with two of Esther’s mentors, George Beadle and Edward Tatum. Esther Lederberg’s contributions, however, were overlooked by the Nobel committee, an example of institutional discrimination known as the Matilda Effect. Esther Lederberg should also have been recognized for inventing replica plating, an elegant technique that she originated by re-purposing her compact makeup pad as a kind of ink stamp for conveniently transferring bacterial colonies from one petri dish to another. Instead, the credit for the invention is given to her famous husband, or, at best, to Dr. and Mrs. Lederberg. Within a few years of winning the Nobel Prize, Joshua Lederberg divorced his wife, leaving Esther without a laboratory, cut off from research funding, and facing uncertain employment. In response, she created a new social circle made up of artists and musicians, including a new soulmate. She devoted herself to a close-knit musical ensemble, the Mid-Peninsula Recorder Orchestra, an avocation that flourished for over forty years, until the final days of her life.

Assessing antibiotic tolerance of Staphylococcus aureus derived directly from patients by the Replica Plating Tolerance Isolation System- REPTIS

Antibiotic tolerant Staphylococcus aureus pose a great challenge to clinicians as well as to microbiological laboratories and are one reason for treatment failure. Antibiotic tolerant strains survive transient antibiotic exposure despite being fully susceptible in vitro. Thus, fast and reliable methods to detect tolerance in the routine microbiology laboratory are urgently required. We therefore evaluated the feasibility of the replica plating tolerance isolation system (REPTIS) to detect antibiotic tolerance in S. aureus isolates derived directly from patients suffering from different types of infections and investigated possible connections to clinical presentations and patient characteristics. One hundred twenty-five S. aureus isolates were included. Replica plating of the original resistance testing plate was used to assess regrowth in the zones of inhibition, indicating antibiotic tolerance. Bacterial regrowth was assessed after 24 and 48 hours of incubation and an overall regrowth score (ORS) was assigned. Regrowth scores were compared to the clinical presentation. Bacterial regrowth was high for most antibiotics targeting protein synthesis and relatively low for antibiotics targeting other cellular functions such as DNA-replication, transcription and cell wall synthesis, with the exception of rifampicin. Isolates with a blaZ penicillinase had lower regrowth in penicillin and ampicillin. Low ORSs were more prevalent among isolates recovered from patients with immunosuppression or methicillin-resistant S. aureus (MRSA) isolates. In conclusion, REPTIS is useful to detect antibiotic tolerance in clinical microbiological routine diagnostics. Rapid detection of antibiotic tolerance offers a new diagnostic readout that might allow more tailored treatments in the future.

Stability of plasmid pBR322 within Escherichia coli cells

nsertion of plasmids into the bacterial cells is of great significance especially in course of the transfer of drug resistance, virulence and other traits. Retention of plasmids within the host bacteria is therefore an important factor for bacterial homeostasis. Current study inferred the pBR322 plasmid stability within the Escherichia coli competent cells. The calcium chloride heat shock method was used for the transformation purpose. The plasmid retention phenomenon was assessed through the replica plating. The results positively showed the plasmid retention within E. coli.

A genome-wide screen for genes affecting spontaneous direct-repeat recombination in Saccharomyces cerevisiae

ABSTRACTHomologous recombination is an important mechanism for genome integrity maintenance, and several homologous recombination genes are mutated in various cancers and cancer-prone syndromes. However, since in some cases homologous recombination can lead to mutagenic outcomes, this pathway must be tightly regulated, and mitotic hyper-recombination is a hallmark of genomic instability. We performed two screens in Saccharomyces cerevisiae for genes that, when deleted, cause hyper-recombination between direct repeats. One was performed with the classical patch and replica-plating method. The other was performed with a high-throughput replica-pinning technique that was designed to detect low-frequency events. This approach allowed us to validate the high-throughput replica-pinning methodology independently of the replicative aging context in which it was developed. Furthermore, by combining the two approaches, we were able to identify and validate 35 genes whose deletion causes elevated spontaneous direct-repeat recombination. Among these are mismatch repair genes, the Sgs1-Top3-Rmi1 complex, the RNase H2 complex, genes involved in the oxidative stress response, and a number of other DNA replication, repair and recombination genes. Since several of our hits are evolutionary conserved, and repeated elements constitute a significant fraction of mammalian genomes, our work might be relevant for understanding genome integrity maintenance in humans.

Complete Killing of Agar Lawn Biofilms by Systematic Spacing of Antibiotic-Loaded Calcium Sulfate Beads

Background: Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are the major causative agents of acute and chronic infections. Antibiotic-loaded calcium sulfate beads (ALCSB) are used in the management of musculoskeletal infections such as periprosthetic joint infections (PJI). Methods: To determine whether the number and spatial distribution of ALCSB are important factors to totally eradicate biofilms, ALCSBs containing vancomycin and tobramycin were placed on 24 h agar lawn biofilms as a single bead in the center, or as 16 beads placed as four clusters of four, a ring around the edge and as a group in the center or 19 beads evenly across the plate. Bioluminescence was used to assess spatial metabolic activity in real time. Replica plating was used to assess viability. Results: For both strains antibiotics released from the beads completely killed biofilm bacteria in a zone immediately adjacent to each bead. However, for PA extended incubation revealed the emergence of resistant colony phenotypes between the zone of eradication and the background lawn. The rate of biofilm clearing was greater when the beads were distributed evenly over the plate. Conclusions: Both number and distribution pattern of ALCSB are important to ensure adequate coverage of antibiotics required to eradicate biofilms.

Selection and identification of xylose-fermenting yeast strains for ethanol production from lignocellulosic biomass

Ethanol production from lignocellulosic biomass is of economic interest due to the pressure to reduce fossil fuels consumption and land use for non-edible crops. Xylose is one of the main sugars obtained by hydrolysis of hemicellulose fraction of biomass, but industrial yeasts cannot ferment it. This work aimed to select, characterize and identify xylose-fermenting yeasts from Brazilian microorganisms collections with potential use in ethanol production. Xylose assimilation was tested by replica plating, and fermentation was tested with Durham tubes. Xylose-fermenting strains had their fermentative capacity quantified and compared to a reference strain (Scheffersomyces stipitis UFMG-IMH 43.2) and were identified by molecular techniques. Three strains isolated from plant exudates were able to ferment xylose and showed fermentative parameters similar to the reference strain. Two strains were identified as Candida parapsilosis and one was identified as Meyerozyma guilliermondii. The findings show the potential biotechnological use of these microorganisms.