High prevalence of plasmid-mediated quinolone resistance determinants in commensal members of the Enterobacteriaceae in Ho Chi Minh City, Vietnam

Antimicrobial-resistant pathogenic members of the Enterobacteriaceae are a well-defined global problem. We hypothesized that one of the main reservoirs of dissemination of antimicrobial resistance genes in Vietnam is non-pathogenic intestinal flora, and sought to isolate antimicrobial-resistant organisms from hospitalized patients and non-hospitalized healthy individuals in Ho Chi Minh City. The results identified substantial faecal carriage of gentamicin-, ceftazidime- and nalidixic acid-resistant members of the Enterobacteriaceae in both hospitalized patients and non-hospitalized healthy individuals. A high prevalence of quinolone resistance determinants was identified, particularly the qnrS gene, in both community- and hospital-associated strains. Furthermore, the results demonstrated that a combination of quinolone resistance determinants can confer resistance to nalidixic acid and ciprofloxacin, even in the apparent absence of additional chromosomal resistance mutations in wild-type strains and laboratory strains with transferred plasmids. These data suggest that intestinal commensal organisms are a significant reservoir for the dissemination of plasmid-mediated quinolone resistance in Ho Chi Minh City

Ceftazidime-avibactam resistance in Klebsiella pneumoniae sequence type 37: a decade of persistence and concealed evolution

The first reports of carbapenem-resistant Enterobacterales in our hospital date back to 2006. In that period, few ertapenem-resistant but meropenem-susceptible Klebsiella pneumoniae isolates belonging to sequence type (ST) 37 were retrieved from clinical samples. These strains produced the CTX-M-15 extended spectrum β-lactamase, OmpK35 was depleted due to a nonsense mutation, and a novel OmpK36 variant was identified. Yet, starting from 2010, Klebsiella pneumoniae carbapenemase (KPC)-producing ST512 isolates started prevailing and ST37 vanished from sight. Since 2018 the clinical use of the combination of ceftazidime-avibactam (CZA) has been introduced in clinical practice for the treatment of bacteria producing serine-β-lactamases, but KPC-producing, CZA-resistant K. pneumoniae are emerging. In 2021, four CZA-resistant ST37 isolates producing KPC variants were isolated from the same number of patients. blaKPC gene cloning in Escherichia coli was used to define the role of those KPC variants on CZA resistance, and whole genome sequencing was performed on these isolates and on three ST37 historical isolates from 2011. CZA resistance was due to mutations in the blaKPC genes carried on related pKpQIL-type plasmids, and three variants of the KPC enzyme have been identified in the four ST37 strains. The four ST37 isolates were closely related to each other and to the historical isolates, suggesting that ST37 survived without notice in our hospital for 10 years, waiting to re-emerge as a CZA-resistant K. pneumoniae clone. The ancestor of these contemporary isolates derives from ST37 wild-type porin strains, with no other mutations in chromosomal genes involved in conferring antibiotic resistance (parC, gyrA, ramR, mgrB, pmrB)

Energy-dependent accumulation of fluoroquinolones in quinolone- resistant Klebsiella pneumoniae strains

The intracellular accumulation of norfloxacin and perloxacin in Klebsiella pneumoniae was evaluated. The roles of lipopolysaccharide, capsule, and outer membrane proteins were not important for the intrabacterial accumulation of fluoroquinolones in isogenic strains with known outer membrane alterations. In fluoroquinolone-resistant clinical isolates also expressing GyrA alterations, an active efflux leading to decreased accumulation of the drugs enhanced their resistance to these agents.Comisión Interministerial de Ciencia y Tecnología PB96-019

The secondary resistome of multidrug-resistant Klebsiella pneumoniae.

Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of individual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of K. pneumoniae (ST258). We constructed a high-density transposon mutant library of >430,000 unique Tn5 insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Transposon Directed Insertion-site Sequencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential for growth during exposure to colistin (n = 35), imipenem (n = 1) or ciprofloxacin (n = 1) in addition to known resistance determinants, collectively termed the "secondary resistome". As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin resistance (dedA) restored colistin susceptibility by reducing the minimum inhibitory concentration from 8 to 0.5 μg/ml, 4-fold below the susceptibility breakpoint (S ≤ 2 μg/ml). This finding suggests that the secondary resistome is a potential target for developing antimicrobial "helper" drugs that restore the efficacy of existing antimicrobials

Analysis of drug resistance determinants in Klebsiella pneumoniae isolates from a tertiary-care hospital in Beijing, China.

published_or_final_versio

Axial distortion as a sensor of supercoil changes: a molecular model for the homeostatic regulation of DNA gyrase

Negative supercoiling stimulates transcription of many genes. In contrast, transcription of the genes coding for DNA gyrase is subject to a novel mechanism of autoregulation, wherein relaxation of the template DNA stimulates their transcription. Since DNA gyrase is the sole supercoiling activity in the eubacterial cell, relaxation-stimulated transcription (RST) could reflect an autoregulatory mechanism to maintain supercoil levels within the cell. Extensive deletion and mutational analyses of Escherichia coli gyrA promoter have shown that the -10 region is essential for RST; however, a molecular model has proved to be elusive. We find a strong bend centre immediately downstream of the -10 region in the gyrA promoter. On the basis of analysis of various mutants in the -10 region, we propose a model where axial distortion acts as a sensor of topological changes in DNA. Our model is consistent with earlier data with E. coli gyrA and gyrB promoters. We also extrapolate the model to explain the phenomenon of RST of gyr promoters in other organisms and contrast it with promoters induced by supercoiling

Antibiotikaresistenz und Pathogenität in den Gram-negativen Bakterien Pseudomonas aeruginosa und Klebsiella pneumoniae

The dramatic increase of infections caused by multidrug-resistant Gram-negative bacteria is an emerging global problem and possibly one of the greatest challenges of modern medicine. Bacterial pathogens devise various mechanisms to withstand the activity of a wide range of antimicrobial compounds and there is an alarming increase of multi- or even pandrug-resistant isolates. The aims of this thesis were i) to elucidate the molecular mechanisms of fluoroquinolone resistance in the opportunistic pathogen Pseudomonas aeruginosa and ii) to describe the transcriptomic landscape of Klebsiella pneumoniae to correlate gene transcription with the clinical relevant phenotypes of biofilm formation, virulence and antibiotic resistance. In this context, we evaluated the quantitative contributions of quinolone target alteration and efflux pump expression to fluoroquinolone resistance in Pseudomonas aeruginosa by applying a combination of directed resequencing methods, quantitative real-time PCRs and whole-transcriptome sequencing (RNA-Seq) on a broad and cross-sectional panel of 172 clinical isolates. This comprehensive data showed the role of distinct mutations in the quinolone resistance-determining regions of gyrA and parC. The combination with further mutations (e.g. in gyrB and parE) or enhanced efflux exhibited additive effects Furthermore, we exploited the power of deep transcriptome profiling by RNA-seq to shed light on the transcriptomic landscape of 37 clinical K. pneumoniae isolates of diverse phylogenetic origin. We identified a large set of 3346 genes which were expressed in all isolates. While these core-transcriptome profiles were largely homogenous among isolates of the same sequence type, they varied substantially between groups of different sequence types. This detailed information on differentially expressed genes was linked with the clinically relevant phenotypes of biofilm formation, bacterial virulence and antibiotic resistance. This allowed the identification of a low biofilm-specific gene expression profile within the group of ST258 isolates, the dominant clonal lineage associated with KPC-carbapenemase spread, as a sequence type-specific trait. Moreover, the analysis revealed that antimicrobial resistance in this panel of clinical isolates can be explained by the occurrence of only a few dominant resistance determinants.Der dramatische Anstieg von Infektionen durch multiresistente, gramnegative Bakterien ist ein weltweites Problem, welches möglicherweise eine der größten Herausforderungen moderner Medizin darstellt. Bakterielle Krankheitserreger besitzen verschiedenste Mechanismen, um der Aktivität einer Vielzahl antimikrobieller Verbindungen zu widerstehen und zeigen eine alarmierende Zunahme von multi- oder sogar pan-resistenten Isolaten. Die Ziele der vorliegenden Arbeit waren i) die molekularen Mechanismen der Fluorchinolonresistenz im opportunistischen Erreger Pseudomonas aeruginosa zu erklären und ii) die generelle Genexpression von Klebsiella pneumoniae zu beschreiben und mit den klinisch relevanten Phänotypen der Biofilmbildung, Virulenz und Antibiotikaresistenz zu korrelieren. In diesem Zusammenhang untersuchten wir den quantitativen Einfluss von Mutationen und Veränderung der Expression von Effluxpumpen auf die Fluorchinolonresistenz in Pseudomonas aeruginosa durch eine Kombination von Resequenzierung, quantitativer realtime-PCR und Transkriptom-Sequenzierung (RNA-Seq) anhand einer Sammlung von 172 klinischen Isolaten. Diese umfassenden Daten zeigten die dominierende Rolle bestimmter Mutationen in gyrA und parC, während die Kombination mit weiteren Mutationen (zum Beispiel in gyrB und parE) oder verstärkter Efflux zwar eine additive Wirkung hatte, aber höchstwahrscheinlich nicht zum hohen Resistenzniveau in der Klinik beiträgt. Darüber hinaus nutzen wir die Möglichkeiten hoch-auflösenden Transkriptom-Profilings mittels RNA-Seq um die generelle Gentranskription 37 klinischer K. pneumoniae Isolate unterschiedlichster Herkunft aufzuklären und identifizierten eine große Anzahl von 3346 Genen, die in allen Isolaten exprimiert wurden. Während dieses Kern-Transkriptom weitgehend homogen zwischen Isolaten des gleichen Sequenztypen war, variierte es deutlich zwischen Gruppen unterschiedlicher Sequenztypen. Diese detaillierten Informationen über differentiell exprimierte Gene wurde mit den klinisch relevanten Phänotypen der Biofilmbildung, bakterieller Virulenz und Antibiotikaresistenz verknüpft. Dieses erlaubte die Identifizierung eines Biofilm-spezifischen Genexpressionsprofil in der Gruppe der ST258-Isolate, welche hauptverantwortlich für die Verbreitung der KPC-Carbapenemase sind, als ein Sequenztyp-spezifisches Merkmal. Außerdem ergab die Analyse, dass die Antibiotikaresistenz durch das Auftreten nur weniger, dominanter Resistenzdeterminanten erläutert werden kann

Trade-offs between antibacterial resistance and fitness cost in the production of metallo-b-lactamases by enteric bacteria manifest as sporadic emergence of carbapenem resistance in a clinical setting

Meropenem is a clinically important antibacterial reserved for treatment of multiresistant infections. In meropenem-resistant bacteria of the family Enterobacterales, NDM-1 is considerably more common than IMP-1, despite both metallo-β-lactamases (MBLs) hydrolyzing meropenem with almost identical kinetics. We show that bla(NDM-1) consistently confers meropenem resistance in wild-type Enterobacterales, but bla(IMP-1) does not. The reason is higher bla(NDM-1) expression because of its stronger promoter. However, the cost of meropenem resistance is reduced fitness of bla(NDM-1)-positive Enterobacterales. In parallel, from a clinical case, we identified multiple Enterobacter spp. isolates carrying a plasmid-encoded bla(NDM-1) having a modified promoter region. This modification lowered MBL production to a level associated with zero fitness cost, but, consequently, the isolates were not meropenem resistant. However, we identified a Klebsiella pneumoniae isolate from this same clinical case carrying the same bla(NDM-1) plasmid. This isolate was meropenem resistant despite low-level NDM-1 production because of a ramR mutation reducing envelope permeability. Overall, therefore, we show how the resistance/fitness trade-off for MBL carriage can be resolved. The result is sporadic emergence of meropenem resistance in a clinical setting