Rapid assessment of the effect of ciprofloxacin on chromosomal DNA from Escherichia coli using an in situ DNA fragmentation assay

<p>Abstract</p> <p>Background</p> <p>Fluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs) by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP)-induced chromosomal DNA breakage in single-cell <it>Escherichia coli </it>by direct visualization of the DNA fragments that diffused from the nucleoid obtained after bacterial lysis in an agarose microgel on a slide.</p> <p>Results</p> <p>Exposing the <it>E. coli </it>strain TG1 to CIP starting at a minimum inhibitory concentration (MIC) of 0.012 μg/ml and at increasing doses for 40 min increased the DNA fragmentation progressively. DNA damage started to be detectable at the MIC dose. At a dose of 1 μg/ml of CIP, DNA damage was visualized clearly immediately after processing, and the DNA fragmentation increased progressively with the antibiotic incubation time. The level of DNA damage was much higher when the bacteria were taken from liquid LB broth than from solid LB agar. CIP treatment produced a progressively slower rate of DNA damage in bacteria in the stationary phase than in the exponentially growing phase. Removing the antibiotic after the 40 min incubation resulted in progressive DSB repair activity with time. The magnitude of DNA repair was inversely related to CIP dose and was noticeable after incubation with CIP at 0.1 μg/ml but scarce after 10 μg/ml. The repair activity was not strictly related to viability. Four <it>E. coli </it>strains with identified mechanisms of reduced sensitivity to CIP were assessed using this procedure and produced DNA fragmentation levels that were inversely related to MIC dose, except those with very high MIC dose.</p> <p>Conclusion</p> <p>This procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones.</p

A New and Efficient Enrichment Method for Metagenomic Sequencing of Monkeypox Virus

[Abstract] Background The methodology described in previous literature for Monkeypox virus (MPXV) sequencing shows low efficiency when using metagenomic approaches. The aim of the present study was to evaluate a new fine-tuned method for extraction and enrichment of genomic MPXV DNA using clinical samples and to compare it to a non-enrichment metagenomic approach. Results A new procedure that allows sample enrichment in MPXV DNA, avoiding wasting the sequencing capacity in human DNA, was designed. This procedure consisted of host DNA depletion using a saponin/NaCl combination treatment and DNase, together with high g-force centrifugations. After typical quality control, samples using the enrichment method contained around 96% of reads not classified as human DNA, while the non-enrichment protocol showed around 5-10%. When reads not belonging to Orthopoxvirus were removed, enriched samples kept about 50% of the original read counts, while non-enriched ones kept only 2-7%. Conclusions Results showed a very significant improvement in sequencing efficiency, increasing the number of reads belonging to MPXV, the depth of coverage and the trustworthiness of the consensus sequences. This, in turn, allows for more samples to be included in a single cartridge, reducing costs and time to diagnosis, which can be very important factors when dealing with a contagious disease.This work was supported by a grant from the SERGAS-Galician Healthcare Service (Program “Innova Saúde”) to GB, by CIBERINFEC and also by Instituto de Salud Carlos III (ISCIII) through the projects PI20/00413 to MP and PI21/00704 to G

A highly-safe live auxotrophic vaccine protecting against disease caused by non-typhoidal Salmonella Typhimurium in mice

Background: Salmonella enterica serovar Typhimurium (S. Typhimurium) has become an important intestinal pathogen worldwide and is responsible for lethal invasive infections in populations at risk. There is at present an unmet need for preventive vaccines. Methods: IRTA GN-3728 genome was sequenced by Illumina and D-glutamate and D-glutamate/D-alanine knockout-auxotrophs were constructed. They were characterized using electron microscopy, growth/viability curves, reversion analysis, and motility/agglutination assays. Their potential as vaccine candidates were explored using two BALB/c mouse models for Salmonella infections: a systemic and an intestinal inflammation. Clinical signs/body weight and survival were monitored, mucosal lactoferrin and specific/cross-reactive IgA/IgG were quantified by enzyme-linked-immunosorbent assays and bacterial shedding/burden in fecal/tissues were evaluated. Results: The D-glutamate auxotroph, IRTA DmurI, is highly attenuated, immunogenic and fully protective against systemic infection. The IRTA DmurI Dalr DdadX double auxotroph, constructed to reinforce vaccine safety, showed a higher level of attenuation and was 100% effective against systemic disease. In the intestinal model, it proved to be safe, yielding a lowdegree of mucosal inflammation, short-term shedding and undetectable invasiveness in the long-term, while eliciting cross-reactive fecal IgA/serum IgG against clinically relevant multidrug-resistant (MDR) S. Typhimurium strains. It also conferred protection against homologous oral challenge, and protected mice from local and extra-intestinal dissemination caused by one MDR strain responsible for an international outbreak of highly severe human infections. Additionally, oral vaccination promoted extended survival after lethal heterologous infection. Conclusion: This study yielded a very safe S. Typhimurium vaccine candidate that could be further refined for mucosal application against disease in humans.This study was supported by a grant from SERGAS (The Galician Healthcare Service) (Programs “InnovaSaude” and “InnovaMicrolab”), the Spanish Network for Research in Infectious Diseases (RD16/0016/0006), CIBERINFEC, project PI18/00501, funded by Instituto de Salud Carlos III and cofunded by European Union (ERDF, “A way to make Europe”), and PI21/00704, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, awarded to GB, and by CZ Vaccines. VFV is funded by a predoctoral fellowship from Xunta de Galicia (IN606A-2019/012).S

N-acetylcysteine selectively antagonizes the activity of imipenem in Pseudomonas aeruginosa by an OprD-mediated mechanism

The modulating effect of N-acetylcysteine (NAC) on the activity of different antibiotics has been studied in Pseudomonas aeruginosa. Our results demonstrate that, in contrast to previous reports, only the activity of imipenem is clearly affected by NAC. MIC and checkerboard determinations indicate that the NAC-based modulation of imipenem activity is dependent mainly on OprD. SDS-PAGE of outer membrane proteins (OMPs) after NAC treatments demonstrates that NAC does not modify the expression of OprD, suggesting that NAC competitively inhibits the uptake of imipenem through OprD. Similar effects on imipenem activity were obtained with P. aeruginosa clinical isolates. Our results indicate that imipenem-susceptible P. aeruginosa strains become resistant upon simultaneous treatment with NAC and imipenem. Moreover, the generality of the observed effects of NAC on antibiotic activity was assessed with two additional bacterial species, Escherichia coli and Acinetobacter baumannii. Caution should be taken during treatments, as the activity of imipenem may be modified by physiologically attainable concentrations of NAC, particularly during intravenous and nebulized regimes.Ministerio de Ciencia e Innovación y Instituto de Salud Carlos III y European Development Regional Fund (ERDF) y Spanish Network for Research in Infectious Diseases REIPI RD12/0015 FIS PI13/0006

A rapid and simple method for constructing stable mutants of Acinetobacter baumannii

<p>Abstract</p> <p>Background</p> <p><it>Acinetobacter baumannii </it>is a multidrug-resistant bacterium responsible for nosocomial infections in hospitals worldwide. Study of mutant phenotypes is fundamental for understanding gene function. The methodologies developed to inactivate <it>A. baumannii </it>genes are complicated and time-consuming; sometimes result in unstable mutants, and do not enable construction of double (or more) gene knockout mutant strains of <it>A. baumannii</it>.</p> <p>Results</p> <p>We describe here a rapid and simple method of obtaining <it>A. baumannii </it>mutants by gene replacement via double crossover recombination, by use of a PCR product that carries an antibiotic resistance cassette flanked by regions homologous to the target locus. To demonstrate the reproducibility of the approach, we produced mutants of three different chromosomal genes (<it>omp33</it>, <it>oxyR</it>, and <it>soxR</it>) by this method. In addition, we disrupted one of these genes (<it>omp33</it>) by integration of a plasmid into the chromosome by single crossover recombination, the most widely used method of obtaining <it>A. baumannii </it>mutants. Comparison of the different techniques revealed absolute stability when the gene was replaced by a double recombination event, whereas up to 40% of the population reverted to wild-type when the plasmid was disrupting the target gene after 10 passages in broth without selective pressure. Moreover, we demonstrate that the combination of both gene disruption and gene replacement techniques is an easy and useful procedure for obtaining double gene knockout mutants in <it>A. baumannii</it>.</p> <p>Conclusions</p> <p>This study provides a rapid and simple method of obtaining stable mutants of <it>A. baumannii </it>free of foreign plasmidic DNA, which does not require cloning steps, and enables construction of multiple gene knockout mutants.</p

A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis

BACKGROUND: Antibiotics which inhibit bacterial peptidoglycan biosynthesis are the most widely used in current clinical practice. Nevertheless, resistant strains increase dramatically, with serious economic impact and effects on public health, and are responsible for thousands of deaths each year. Critical clinical situations should benefit from a rapid procedure to evaluate the sensitivity or resistance to antibiotics that act at the cell wall. We have adapted a kit for rapid determination of bacterial DNA fragmentation, to assess cell wall integrity. RESULTS: Cells incubated with the antibiotic were embedded in an agarose microgel on a slide, incubated in an adapted lysis buffer, stained with a DNA fluorochrome, SYBR Gold and observed under fluorescence microscopy. The lysis affects the cells differentially, depending on the integrity of the wall. If the bacterium is susceptible to the antibiotic, the weakened cell wall is affected by the lysing solution so the nucleoid of DNA contained inside the bacterium is released and spread. Alternatively, if the bacterium is resistant to the antibiotic, it is practically unaffected by the lysis solution and does not liberate the nucleoid, retaining its normal morphological appearance. In an initial approach, the procedure accurately discriminates susceptible, intermediate and resistant strains of Escherichia coli to amoxicillin/clavulanic acid. When the bacteria came from an exponentially growing liquid culture, the effect on the cell wall of the beta-lactam was evident much earlier that when they came from an agar plate. A dose-response experiment with an E. coli strain susceptible to ampicillin demonstrated a weak effect before the MIC dose. The cell wall damage was not homogenous among the different cells, but the level of damage increased as dose increased with a predominant degree of effect for each dose. A microgranular-fibrilar extracellular background was evident in gram-negative susceptible strains after beta-lactam treatment. This material was digested by DNase I, hybridised with a specific whole genome probe, and so recognized as DNA fragments released by the bacteria. Finally, 46 clinical strains from eight gram-negative and four gram-positive species were evaluated blind for susceptibility or resistance to one of four different beta-lactams and vancomycin, confirming the applicability of the methodology. CONCLUSION: The technique to assess cell wall integrity appears to be a rapid and simple procedure to identify resistant and susceptible strains to antibiotics that interfere with peptidoglycan biosynthesis

Carbapenem Resistance in Acinetobacter nosocomialis and Acinetobacter junii Conferred by Acquisition of blaOXA-24/40 and Genetic Characterization of the Transmission Mechanism between Acinetobacter Genomic Species

[Abstract] Carbapenem resistance is increasing among Gram-negative bacteria, including the genus Acinetobacter. This study aimed to characterize, for the first time, the development of carbapenem resistance in clinical isolates of Acinetobacter junii and Acinetobacter nosocomialis conferred by the acquisition of a plasmid-borne blaOXA-24/40 gene and also to characterize the dissemination of this gene between species of Acinetobacter. Carbapenem-resistant A. nosocomialis HUAV-AN66 and A. junii HUAV-AJ77 strains were isolated in the Arnau de Vilanova Hospital (Spain). The genomes were sequenced, and in silico analysis were performed to characterize the genetic environment and the OXA-24/40 transmission mechanism. Antibiotic MICs were determined, and horizontal transfer assays were conducted to evaluate interspecies transmission of OXA-24/40. Carbapenems MICs obtained were ≥64 mg/L for HUAV-AN66 and HUAV-AJ77. Genome analysis revealed the presence in both strains of a new plasmid, designated pHUAV/OXA-24/40, harboring the carbapenem-resistance gene blaOXA-24/40 and flanked by sequences XerC/XerD. pHUAV/OXA-24/40 was successfully transferred from A. nosocomialis and A. junii to a carbapenem-susceptible A. baumannii strain, thus conferring carbapenem resistance. A second plasmid (pHUAV/AMG-R) was identified in both clinical isolates for the successful horizontal transfer of pHUAV/OXA-24/40. blaOXA-24/40-carrying plasmids of the GR12 group and showing high identity with pHUAV/OXA-24/40 were identified in at least 8 Acinetobacter species. In conclusion the carbapenemase OXA-24/40 is described for the first time in A. nosocomialis and A. junii. In both isolates the blaOXA-24/40 gene was located in the GR12 pHUAV/OXA-24/40 plasmid. GR12 plasmids are implicated in the dissemination and spread of carbapenem resistance among Acinetobacter species. IMPORTANCE Acinetobacter baumannii is one of the most relevant pathogens in terms of antibiotic resistance. The main resistance mechanisms are the carbapenem-hydrolyzing class D β-lactamases (CHDLs), especially OXA-23 and OXA-24/40. In addition to A. baumannii, there are other species within the genus Acinetobacter, which in general exhibit much lower resistance rates. In this work we characterize for the first time two clinical isolates of Acinetobacter nosocomialis and Acinetobacter junii, isolated in the same hospital, carrying the carbapenemase OXA-24/40 and displaying high resistance rates to carbapenems. By means of bioinformatics analysis we have also been able to characterize the mechanism by which this carbapenemase is horizontally transferred interspecies of Acinetobacter spp. The dissemination of carbapenemase OXA-24/40 between non-baumannii Acinetobacter species is concerning since it prevents the use of most β-lactam antibiotics in the fight against these resistant isolates.This work was supported by Projects PI17/01482 and PI20/01212 awarded to A.B. and PI18/00501 to G.B., all within in the National Plan for Scientific Research, Development and Technological Innovation 2013–2016 and funded by the ISCIII - General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe.” The work was also supported by CIBERINF (CIBER de Enfermedades Infecciosas). The study was also funded by project IN607A 2020/05 (GAIN- Agencia Gallega de Innovación - Consellería de Economía, Emprego e Industria) awarded to G.B. and IN607D 2021/12 awarded to A.B. This work was also supported by Planes Nacionales de I+D+i2008 to 2011/2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectiosus Diseases (REIPI RD16/0016/006) cofinanced by European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth 2014–2020. J.A.-S.was financially supported by the Rio Hortega program (ISCIII, CM19/00219), J.C.V.-U. was financially supported by the pFIS program (ISCIII, PI17/01482), C.L.-M. was financially supported by IN606A-2019/029 Grant (Xunta de Galicia) and P.G.-S. was financially supported by IN607A 2020/05 Grant (Xunta de Galicia)Xunta de Galicia; IN607A 2020/05Xunta de Galicia; IN607D 2021/12Xunta de Galicia; IN606A-2019/02

Multidrug-Resistant Acinetobacter baumannii Harboring OXA-24 Carbapenemase, Spain

In February 2006, a patient colonized with a multidrug-resistant sequence type 56 Acinetobacter baumannii strain was admitted to a hospital in Madrid, Spain. This strain spread rapidly and caused a large outbreak in the hospital. Clinicians should be alert for this strain because its spread would have serious health consequences

Fast assessment of resistance to carbapenems and ciprofloxacin of clinical strains of Acinetobacter baumannii

Infections caused by multidrug-resistant Acinetobacter baumannii constitute a major life-threatening problem worldwide, and early adequate antibiotic therapy is decisive for success. For these reasons, rapid detection of antibiotic susceptibility in this pathogen is a clinical challenge. Two variants of the Micromax kit were evaluated for a rapid detection in situ of susceptibility or resistance to meropenem or ciprofloxacin, separately, in 322 clinical isolates. Release of the nucleoid is the criterion of susceptibility to the beta-lactams (carbapenems), whereas diffusion of DNA fragments emerging from the nucleoid characterizes the quinolone activity. All the susceptible and resistant strains were correctly categorized in 100 min according to the MIC results and CLSI criteria. Thus, our technology is a promising tool for rapid identification of carbapenem and quinolone resistance of A. baumannii strains in hospital settingsThis work has been supported by grants from the European Community, FP 7, ID: 278232 (MagicBullet), Xunta de Galicia 10CSA916020PR, and by REIPI, Spanish Network for Research in Infectious Diseases (Instituto de Salud Carlos III, RD06/0008/0025) and the Fondo de Investigaciones Sanitarias (PS09/00687) to G.B