Biology subjects

Bonten, M.

Publications and source records attributed to Bonten, M..

6 recordsLinked to original sources

bioRxiv2020

In vitro evolution of colistin resistance in the Klebsiella pneumoniae complex follows multiple evolutionary trajectories with variable effects on fitness and virulence characteristics.

The increasing prevalence of multidrug-resistant Gram-negative opportunistic pathogens, including Klebsiella pneumoniae, has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic lipopeptide antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPS). Colistin resistance in K. pneumoniae is mediated through loss of these phosphate groups, or modification with cationic groups (e.g. 4-amino-4-deoxy-L-arabinose (L-Ara4N), or phosphoethanolamine), but also hydroxylation of acyl-groups of lipid A. Here, we study the in vitro evolutionary trajectories towards colistin resistance in clinical K. pneumoniae complex strains (three K. pneumoniae sensu stricto strains and one K. variicola subsp. variicola strain) and their impact on fitness and virulence characteristics. Through population sequencing during the in vitro evolution experiment, we found that resistance develops through a combination of single nucleotide polymorphisms (SNPs), insertion and deletions (indels), and the integration of insertion sequence (IS) elements, affecting genes associated with LPS biosynthesis and modification, and capsule structures. The development of colistin resistance decreased the maximum growth rate of one K. pneumoniae sensu stricto strain, but not in the other three K. pneumoniae sensu lato strains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and L-Ara4N addition. Colistin-resistant K. pneumoniae sensu stricto strains exhibited cross-resistance to LL-37, in contrast to the K. variicola subsp. variicola strain that did not change in susceptibility to LL-37. Virulence, as determined in a Caenorhabditis elegans survival assay, was higher in two colistin-resistant strains. Our study suggests that nosocomial K. pneumoniae complex strains can rapidly develop colistin resistance de novo through diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the lifespan of this last-resort antibiotic for the treatment of infections with multidrug-resistant Klebsiella. Author summaryBacteria that frequently cause infections in hospitalised patients are becoming increasingly resistant to antibiotics. Colistin is a positively charged antibiotic that is used for the treatment of infections with multidrug-resistant Gram-negative bacteria. Colistin acts by specifically interacting with the negatively charged LPS molecule in the outer membrane of Gram-negative bacteria. Colistin resistance is mostly mediated through modification of LPS to reduce its negative charge. Here, we use a laboratory evolution experiment to show that strains belonging to the Klebsiella pneumoniae complex, a common cause of multidrug-resistant hospital-acquired infections, can rapidly accumulate mutations that reduce the negative charge of LPS without an appreciable loss of fitness. Colistin resistance can lead to cross-resistance to an antimicrobial peptide of the human innate immune system, but can increase susceptibility to serum, and virulence in a nematode model. These findings show that extensively resistant K. pneumoniae complex strains may rapidly develop resistance to the last-resort antibiotic colistin via different evolutionary trajectories, while retaining their ability to cause infections.

microbiology↗

bioRxiv2020

Microevolution of acquired colistin resistance in Enterobacteriaceae from ICU patients receiving selective decontamination of the digestive tract.

Colistin is an antibiotic that targets the lipopolysaccharides present in the membranes of Gram-negative bacteria. It is used as last-resort drug to treat infections with multidrug-resistant strains. Colistin is also used in selective decontamination of the digestive tract (SDD), a prophylactic therapy used in patients hospitalised in intensive care units (ICUs) to selectively eradicate opportunistic pathogens in the oropharyngeal and gut microbiota. In this study, we aimed to unravel the mechanisms of acquired colistin resistance in Gram-negative opportunistic pathogens obtained from SDD-treated patients. Routine surveillance of 428 SDD-treated patients resulted in thirteen strains with acquired colistin resistance (Escherichia coli n=9; Klebsiella aerogenes, n=3; Enterobacter asburiae, n=1) from five patients. Genome sequence analysis showed that these isolates represented multiple distinct colistin-resistant clones, but that within the same patients, colistin-resistant strains were clonally related. We identified previously described mechanisms that lead to colistin resistance, i.e. a G53 substitution in the response regulator PmrA/BasR, and the acquisition of the mobile colistin resistance gene mcr-1.1, but we also observed novel variants of basR with an 18-bp deletion, and a G19E substitution in the sensor histidine kinase BasS. We experimentally confirmed these variants to contribute to reduced colistin susceptibility. In a single patient, we observed that colistin resistance in a single E. coli clone evolved through two unique variants in basRS. We show that prophylactic use of colistin during SDD can select for colistin resistance in species that are not intrinsically colistin-resistant. This highlights the importance of continued surveillance for the emergence of colistin resistance in patients treated with SDD.

microbiology↗

bioRxiv2019

Mechanisms of colistin resistance in Escherichia strains isolated from bloodstream infections

Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one E. albertii strain. These were the only colistin-resistant strains out of 1140 bloodstream Escherichia isolates collected in a tertiary hospital over a ten-year period (2006 - 2015). Core genome phylogenetic analysis showed that each patient was colonised by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr-genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen. ImportanceMultidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole genome sequence analysis and experimental validation to characterise the mechanisms through which E. coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.

microbiology↗

bioRxiv2019

Confounding by indication of the safety of de-escalation in community-acquired pneumonia: a simulation study embedded in a prospective cohort.

BackgroundObservational studies have demonstrated that de-escalation of antimicrobial therapy is independently associated with lower mortality. This most probably results from confounding by indication. Reaching clinical stability is associated with the decision to de-escalate and with survival. However, studies rarely adjust for this confounder. We quantified the potential confounding effect of clinical stability on the estimated impact of de-escalation on mortality in patients with community-acquired pneumonia.\n\nMethodsData were used from the Community-Acquired Pneumonia immunization Trial in Adults (CAPiTA). The primary outcome was 30-day mortality. We performed Cox proportional-hazards regression with de-escalation as time-dependent variable and adjusted for baseline characteristics using propensity scores. The potential impact of unmeasured confounding was quantified through simulating a variable representing clinical stability on day three, using data on prevalence and associations with mortality from the literature.\n\nResultsOf 1,536 included patients, 257 (16.7%) were de-escalated, 123 (8.0%) were escalated and in 1156 (75.3%) the antibiotic spectrum remained unchanged. The adjusted hazard ratio of de-escalation for 30-day mortality (compared to patients with unchanged coverage), without adjustment for clinical stability, was 0.36 (95%CI: 0.18-0.73). If 90% to 100% of de-escalated patients were clinically stable on day three, the fully adjusted hazard ratio would be 0.53 (95%CI: 0.26-1.08) to 0.90 (95%CI: 0.42-1.91), respectively. The simulated confounder was substantially stronger than any of the baseline confounders in our dataset.\n\nConclusionsWith plausible, literature-based assumptions, clinical stability is a very strong confounder for the effects of de-escalation. Quantification of effects of de-escalation on patient outcomes without proper adjustment for clinical stability results in strong negative bias. As a result, the safety of de-escalation remains to be determined.

epidemiology↗

bioRxiv2018

A pilot study of a novel molecular host response assay to diagnose infection in patients after high-risk gastro-intestinal surgery

AimSeptiCyte LAB measures the expression of four host-response RNAs in blood to distinguish sepsis from sterile inflammation. Sequential monitoring of this assay may have diagnostic utility in patients at high risk for postoperative infectious complications.\n\nMethodsIn this pilot study we studied esophagectomy patients who had developed a complication within 30 days following surgery as well as a random sample of 100 uncomplicated postoperative patients. PAXgene blood samples were collected postoperatively and whenever a complication occurred. SeptiCyte scores (ranging 0-10 with increasing likelihood of infection) were compared to post-hoc physician adjudication of infection likelihood using strict definitions.\n\nResultsAmong 370 esophagectomy patients, 120 (32%) subjects developed a complication requiring ICU (re)admission, 63 (53%) of whom could be analyzed. Immediate postoperative SeptiCyte LAB scores were highly variable, yet similar for patients having a complicated and uncomplicated postoperative course (median score of 2.4 (IQR 1.6-3.3) versus 2.2 (IQR 1.3-3), respectively). Scores increased as complications developed, but this rise was higher for 34 subjects having confirmed infection (median difference 4.7 (IQR 4.1-5.8)) then for 12 subjects with a non-infectious complication (2.1 (IQR 0.4-3.6); p<0.0001). When 17 cases with undetermined infectious status were excluded, addition of SeptiCyte LAB to CRP resulted in improved diagnostic discrimination (AUC 0.88 (95%CI 0.77-0.99)) compared to CRP alone (AUC 0.76 (95%CI 0.61-0.91); p=0.04).\n\nConclusionsSequential measurement of SeptiCyte LAB may have clinical utility in surgical patients at high risk of postoperative infection, but its diagnostic performance in this setting needs to be further evaluated.

epidemiology↗

bioRxiv2017

Validation Of A Novel Molecular Host Response Assay To Diagnose Infection In Hospitalized Patients Admitted To The ICU With Acute Respiratory Failure

PurposeThe discrimination between infectious and non-infectious causes of acute respiratory failure (ARF) in hospitalized patients admitted to the intensive care unit (ICU) is difficult. Using a novel diagnostic test measuring the expression of four RNA biomarkers in blood (SeptiCyte LAB) we aimed to distinguish between infection and inflammation in this setting.\n\nMethodsWe enrolled hospitalized patients with ARF requiring prompt intubation in the ICU from 2011 to 2013. We excluded patients having an established infection diagnosis or an evidently non-infectious reason for intubation. Blood samples were collected upon ICU admission. Test results were categorized into four probability bands (with higher bands indicating a higher probability of infection) and compared with the plausibility of infection as rated by post-hoc assessment using predefined definitions.\n\nResultsOf 467 included patients, 373 (80%) were treated for a suspected infection at admission. Plausibility of infection was classified as ruled-out, undetermined, or confirmed in 41 (11%), 135 (36%), and 197 (53%) of these, respectively. Overall, the pre-test probability of infection was 42%. Test results correlated with the plausibility of infection (Spearmans rho 0.332; p<0.001). After exclusion of undetermined cases, positive predictive values were 29%, 54%, and 76% for probability bands 2, 3, and 4, respectively, whereas the negative predictive value for band 1 was 76%. However, SeptiCyte LAB did not outperform CRP when comparing diagnostic discrimination (AUC 0.731; 95%CI 0.677-0.786 vs. 0.727; 95%CI 0.666-0.788).\n\nConclusionIn a setting of hospitalized patients admitted to the ICU with ARF, the diagnostic value of SeptiCyte LAB seems limited.

epidemiology↗