In-host evolution of Staphylococcus epidermidis in a pacemaker-associated endocarditis resulting in increased antibiotic tolerance

Treatment failure in biofilm-associated bacterial infections is an important healthcare issue. In vitro studies and mouse models suggest that bacteria enter a slow-growing/non-growing state that results in transient tolerance to antibiotics in the absence of a specific resistance mechanism. However, little clinical confirmation of antibiotic tolerant bacteria in patients exists. In this study we investigate a Staphylococcus epidermidis pacemaker-associated endocarditis, in a patient who developed a break-through bacteremia despite taking antibiotics to which the S. epidermidis isolate is fully susceptible in vitro. Characterization of the clinical S. epidermidis isolates reveals in-host evolution over the 16-week infection period, resulting in increased antibiotic tolerance of the entire population due to a prolonged lag time until growth resumption and a reduced growth rate. Furthermore, we observe adaptation towards an increased biofilm formation capacity and genetic diversification of the S. epidermidis isolates within the patient

A Selective Antibiotic for Lyme Disease

Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment

Prolonged bacterial lag time results in small colony variants that represent a sub-population of persisters

Persisters are a subpopulation of bacteria that are not killed by antibiotics even though they lack genetic resistance. Here we provide evidence that persisters can manifest as small colony variants (SCVs) in clinical infections. We analyze growth kinetics of Staphylococcus aureus sampled from in vivo conditions and in vitro stress conditions that mimic growth in host compartments. We report that SCVs arise as a result of a long lag time, and that this phenotype emerges de novo during the growth phase in various stress conditions including abscesses and acidic media. We further observe that long lag time correlates with antibiotic usage. These observations suggest that treatment strategies should be carefully tailored to address bacterial persisters in clinics.ISSN:2041-172

Engineering of bacteriophages Y2::dpoL1-C and Y2::luxAB for efficient control and rapid detection of the fire blight pathogen, Erwinia amylovora

Erwinia amylovora is the causative agent of fire blight, a devastating plant disease affecting members of the Rosaceae Alternatives to antibiotics for control of fire blight symptoms and outbreaks are highly desirable, due to increasing drug resistance and tight regulatory restrictions. Moreover, the available diagnostic methods either lack sensitivity, lack speed, or are unable to discriminate between live and dead bacteria. Owing to their extreme biological specificity, bacteriophages are promising alternatives for both aims. In this study, the virulent broad-host-range E. amylovora virus Y2 was engineered to enhance its killing activity and for use as a luciferase reporter phage, respectively. Toward these aims, a depolymerase gene of E. amylovora virus L1 (dpoL1-C) or a bacterial luxAB fusion was introduced into the genome of Y2 by homologous recombination. The genes were placed downstream of the major capsid protein orf68, under the control of the native promoter. The modifications did not affect viability of infectivity of the recombinant viruses. Phage Y2::dpoL1-C demonstrated synergistic activity between the depolymerase degrading the exopolysaccharide capsule and phage infection, which greatly enhanced bacterial killing. It also significantly reduced the ability of E. amylovora to colonize the surface of detached flowers. The reporter phage Y2::luxAB transduced bacterial luciferase into host cells and induced synthesis of large amounts of a LuxAB luciferase fusion. After the addition of aldehyde substrate, bioluminescence could be readily monitored, and this enabled rapid and specific detection of low numbers of viable bacteria, without enrichment, both in vitro and in plant material. IMPORTANCE: Fire blight, caused by Erwinia amylovora, is the major threat to global pome fruit production, with high economic losses every year. Bacteriophages represent promising alternatives to not only control the disease, but also for rapid diagnostics. To enhance biocontrol efficacy, we combined the desired properties of two phages, Y2 (broad host range) and L1 (depolymerase for capsule degradation) in a single recombinant phage. This phage showed enhanced biocontrol and could reduce E. amylovora on flowers. Phage Y2 was also genetically engineered into a luciferase reporter phage, which transduces bacterial bioluminescence into infected cells and allows detection of low numbers of viable target bacteria. The combination of speed, sensitivity, and specificity is superior to previously used diagnostic methods. In conclusion, genetic engineering could improve the properties of phage Y2 toward better killing efficacy and sensitive detection of E. amylovora cells

Reactive Oxygen Species-Inducible ECF σ Factors of Bradyrhizobium japonicum

Extracytoplasmic function (ECF) σ factors control the transcription of genes involved in different cellular functions, such as stress responses, metal homeostasis, virulence-related traits, and cell envelope structure. The genome of Bradyrhizobium japonicum, the nitrogen-fixing soybean endosymbiont, encodes 17 putative ECF σ factors belonging to nine different ECF σ factor families. The genes for two of them, ecfQ (bll1028) and ecfF (blr3038), are highly induced in response to the reactive oxygen species hydrogen peroxide (H2O2) and singlet oxygen (1O2). The ecfF gene is followed by the predicted anti-σ factor gene osrA (blr3039). Mutants lacking EcfQ, EcfF plus OsrA, OsrA alone, or both σ factors plus OsrA were phenotypically characterized. While the symbiotic properties of all mutants were indistinguishable from the wild type, they showed increased sensitivity to singlet oxygen under free-living conditions. Possible target genes of EcfQ and EcfF were determined by microarray analyses, and candidate genes were compared with the H2O2-responsive regulon. These experiments disclosed that the two σ factors control rather small and, for the most part, distinct sets of genes, with about half of the genes representing 13% of the members of H2O2-responsive regulon. To get more insight into transcriptional regulation of both σ factors, the 5′ ends of ecfQ and ecfF mRNA were determined. The presence of conserved sequence motifs in the promoter region of ecfQ and genes encoding EcfQ-like σ factors in related α-proteobacteria suggests regulation via a yet unknown transcription factor. By contrast, we have evidence that ecfF is autoregulated by transcription from an EcfF-dependent consensus promoter, and its product is negatively regulated via protein-protein interaction with OsrA. Conserved cysteine residues 129 and 179 of OsrA are required for normal function of OsrA. Cysteine 179 is essential for release of EcfF from an EcfF-OsrA complex upon H2O2 stress while cysteine 129 is possibly needed for EcfF-OsrA interaction.ISSN:1932-620

Engineered phages for efficient control and rapid detection of viable Erwinia amylovora cells

Vortra

Clonality and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus at the University Hospital Zurich, Switzerland between 2012 and 2014

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a global epidemic threat. The aim of this study was to determine which globally known MRSA lineages are currently present at our tertiary care hospital in Switzerland, a hospital with low MRSA prevalence. In light of the increasing prevalence of multi drug resistance including vancomycin resistance we also assessed antibiotic susceptibilities. METHODS: The 146 MRSA strains collected over two years (March 2012 until February 2014) at the University Hospital Zurich, Switzerland, were analyzed by PFGE analysis of SmaI digests in combination with spa-typing. In addition, representative isolates were analyzed by multi locus sequence typing (MLST). Susceptibilities to eight antibiotics were assessed using the Kirby-Bauer disc diffusion method. RESULTS: Isolates showed resistance to erythromycin (48%), ciprofloxacin (43%), clindamycin (31%), tetracycline (22%), and gentamicin (16%). All isolates were susceptible to vancomycin, 95% were susceptible to sulfamethoxazole/trimethoprim and rifampicin, respectively. PFGE analysis revealed 22 different patterns, with four major patterns that accounted for 53.4% of all MRSA isolates, and seven sporadic patterns. Spa typing revealed 50 different spa types with the predominant types being t008 (14%), t002 (10%), and t127 (9%). 82% of the MRSA isolates could be assigned to six clonal complexes (CCs) namely CC1 (10%), CC5 (23%), CC8 (18%), CC22 (17%), CC30 (11%), and CC45 (3%) based on spa-types, PFGE patterns, and MLST. Two isolates could not be typed by either PFGE analysis or spa-typing and three isolates had spa-types that have not yet been described. CONCLUSIONS: The combination of the two typing methods was more discriminatory as compared to the use of a single method. Several of the lineages that are predominant in Europe are present in our hospital. Resistances to antibiotics have decreased in comparison to a study conducted between 2004 and 2006