Genome-wide analysis in Escherichia coli unravels a high level of genetic homoplasy associated with cefotaxime resistance

Cefotaxime (CTX) is a third-generation cephalosporin (3GC) commonly used to treat infections caused by Escherichia coli. Two genetic mechanisms have been associated with 3GC resistance in E. coli. The first is the conjugative transfer of a plasmid harbouring antibiotic-resistance genes. The second is the introduction of mutations in the promoter region of the ampC β-lactamase gene that cause chromosome-encoded β-lactamase hyperproduction. A wide variety of promoter mutations related to AmpC hyperproduction have been described. However, their link to CTX resistance has not been reported. We recultured 172 cefoxitin-resistant E. coli isolates with known CTX minimum inhibitory concentrations and performed genome-wide analysis of homoplastic mutations associated with CTX resistance by comparing Illumina whole-genome sequencing data of all isolates to a PacBio sequenced reference chromosome. We mapped the mutations on the reference chromosome and determined their occurrence in the phylogeny, revealing extreme homoplasy at the -42 position of the ampC promoter. The 24 occurrences of a T at the -42 position rather than the wild-type C, resulted from 18 independent C>T mutations in five phylogroups. The -42 C>T mutation was only observed in E. coli lacking a plasmid-encoded ampC gene. The association of the -42 C>T mutation with CTX resistance was confirmed to be significant (false discovery rate T mutation of the ampC promotor as significantly associated with CTX resistance and underlines the role of recurrent mutations in the spread of antibiotic resistance

Aspergillus fumigatus - Aspergillus fumigatus differential expression upon itraconazole addition

The filamentous fungus Aspergillus fumigatus is an opportunistic pathogen which causes life-threatening diseases in immunocompromised patients. Resistance of A. fumigatus against azole class compounds continues to pose a threat to human health worldwide. How this fungus maintains fitness before any resistance causing mutations arise is not well understood. In this study, we use RNA-seq to demonstrate how A. fumigatus exerts its phenotypic plasticity when exposed to itraconazole, and which important cellular processes contribute to its adaptation to a new, stressful environment containing azole compounds. We conducted an in vitro assay in which we exposed 24h old mycelia of clinical A. fumigatus isolates grown at 37°C, to sublethal concentrations of itraconazole which might permit gradual adaptation of the fungus to the new, stressful environment. Mycelia were then harvested after 30, 60, 120 or 240 minutes respectively, and relative expression was compared to the mycelia harvested directly after 24h without the addition of itraconazol

Aspergillus fumigatus - Aspergillus fumigatus differential expression upon itraconazole addition

The filamentous fungus Aspergillus fumigatus is an opportunistic pathogen which causes life-threatening diseases in immunocompromised patients. Resistance of A. fumigatus against azole class compounds continues to pose a threat to human health worldwide. How this fungus maintains fitness before any resistance causing mutations arise is not well understood. In this study, we use RNA-seq to demonstrate how A. fumigatus exerts its phenotypic plasticity when exposed to itraconazole, and which important cellular processes contribute to its adaptation to a new, stressful environment containing azole compounds. We conducted an in vitro assay in which we exposed 24h old mycelia of clinical A. fumigatus isolates grown at 37°C, to sublethal concentrations of itraconazole which might permit gradual adaptation of the fungus to the new, stressful environment. Mycelia were then harvested after 30, 60, 120 or 240 minutes respectively, and relative expression was compared to the mycelia harvested directly after 24h without the addition of itraconazol

Aspergillus fumigatus - Aspergillus fumigatus differential expression upon itraconazole addition

The filamentous fungus Aspergillus fumigatus is an opportunistic pathogen which causes life-threatening diseases in immunocompromised patients. Resistance of A. fumigatus against azole class compounds continues to pose a threat to human health worldwide. How this fungus maintains fitness before any resistance causing mutations arise is not well understood. In this study, we use RNA-seq to demonstrate how A. fumigatus exerts its phenotypic plasticity when exposed to itraconazole, and which important cellular processes contribute to its adaptation to a new, stressful environment containing azole compounds. We conducted an in vitro assay in which we exposed 24h old mycelia of clinical A. fumigatus isolates grown at 37°C, to sublethal concentrations of itraconazole which might permit gradual adaptation of the fungus to the new, stressful environment. Mycelia were then harvested after 30, 60, 120 or 240 minutes respectively, and relative expression was compared to the mycelia harvested directly after 24h without the addition of itraconazol

Phenotypic plasticity and the evolution of azole resistance in Aspergillus fumigatus; an expression profile of clinical isolates upon exposure to itraconazole

Background The prevalence of azole resistance in clinical and environmental Aspergillus fumigatus isolates is rising over the past decades, but the molecular basis of the development of antifungal drug resistance is not well understood. This study focuses on the role of phenotypic plasticity in the evolution of azole resistance in A. fumigatus. When A. fumigatus is challenged with a new stressful environment, phenotypic plasticity may allow A. fumigatus to adjust their physiology to still enable growth and reproduction, therefore allowing the establishment of genetic adaptations through natural selection on the available variation in the mutational and recombinational gene pool. To investigate these short-term physiological adaptations, we conducted time series transcriptome analyses on three clinical A. fumigatus isolates, during incubation with itraconazole. Results After analysis of expression patterns, we identified 3955, 3430, 1207, and 1101 differentially expressed genes (DEGs), after 30, 60, 120 and 240 min of incubation with itraconazole, respectively. We explored the general functions in these gene groups and we identified 186 genes that were differentially expressed during the whole time series. Additionally, we investigated expression patterns of potential novel drug-efflux transporters, genes involved in ergosterol and phospholipid biosynthesis, and the known MAPK proteins of A. fumigatus. Conclusions Our data suggests that A. fumigatus adjusts its transcriptome quickly within 60 min of exposure to itraconazole. Further investigation of these short-term adaptive phenotypic plasticity mechanisms might enable us to understand how the direct response of A. fumigatus to itraconazole promotes survival of the fungus in the patient, before any “hard-wired” genetic mutations arise

Phenotypic plasticity and the evolution of azole resistance in Aspergillus fumigatus; An expression profile of clinical isolates upon exposure to itraconazole

Background: The prevalence of azole resistance in clinical and environmental Aspergillus fumigatus isolates is rising over the past decades, but the molecular basis of the development of antifungal drug resistance is not well understood. This study focuses on the role of phenotypic plasticity in the evolution of azole resistance in A. fumigatus. When A. fumigatus is challenged with a new stressful environment, phenotypic plasticity may allow A. fumigatus to adjust their physiology to still enable growth and reproduction, therefore allowing the establishment of genetic adaptations through natural selection on the available variation in the mutational and recombinational gene pool. To investigate these short-term physiological adaptations, we conducted time series transcriptome analyses on three clinical A. fumigatus isolates, during incubation with itraconazole. Results: After analysis of expression patterns, we identified 3955, 3430, 1207, and 1101 differentially expressed genes (DEGs), after 30, 60, 120 and 240 min of incubation with itraconazole, respectively. We explored the general functions in these gene groups and we identified 186 genes that were differentially expressed during the whole time series. Additionally, we investigated expression patterns of potential novel drug-efflux transporters, genes involved in ergosterol and phospholipid biosynthesis, and the known MAPK proteins of A. fumigatus. Conclusions: Our data suggests that A. fumigatus adjusts its transcriptome quickly within 60 min of exposure to itraconazole. Further investigation of these short-term adaptive phenotypic plasticity mechanisms might enable us to understand how the direct response of A. fumigatus to itraconazole promotes survival of the fungus in the patient, before any "hard-wired" genetic mutations arise.</p

Development of an algorithm to discriminate between plasmid- and chromosomal-mediated AmpC β-lactamase production in Escherichia coli by elaborate phenotypic and genotypic characterization

Objectives: AmpC-β-lactamase production is an under-recognized antibiotic resistance mechanism that renders Gram-negative bacteria resistant to common β-lactam antibiotics, similar to the well-known ESBLs. For infection control purposes, it is important to be able to discriminate between plasmid-mediated AmpC (pAmpC) production and chromosomal-mediated AmpC (cAmpC) hyperproduction in Gram-negative bacteria as pAmpC requires isolation precautions to minimize the risk of horizontal gene transmission. Detecting pAmpC in Escherichia coli is challenging, as both pAmpC production and cAmpC hyperproduction may lead to third-generation cephalosporin resistance. Methods: We tested a collection of E. coli strains suspected to produce AmpC. Elaborate susceptibility testing for third-generation cephalosporins, WGS and machine learning were used to develop an algorithm to determine ampC genotypes in E. coli. WGS was applied to detect pampC genes, cAmpC hyperproducers and STs. Results: In total, 172 E. coli strains (n=75 ST) were divided into a training set and two validation sets. Ninety strains were pampC positive, the predominant gene being blaCMY-2 (86.7%), followed by blaDHA-1 (7.8%), and 59 strains were cAmpC hyperproducers. The algorithm used a cefotaxime MIC value above 6 mg/L to identify pampC-positive E. coli and an MIC value of 0.5 mg/L to discriminate between cAmpC-hyperproducing and non-cAmpC-hyperproducing E. coli strains. Accuracy was 0.88 (95% CI=0.79-0.94) on the training set, 0.79 (95% CI=0.64-0.89) on validation set 1 and 0.85 (95% CI=0.71-0.94) on validation set 2. Conclusions: This approach resulted in a pragmatic algorithm for differentiating ampC genotypes in E. coli based on phenotypic susceptibility testing

Development of an algorithm to discriminate between plasmid- and chromosomal-mediated AmpC β-lactamase production in Escherichia coli by elaborate phenotypic and genotypic characterization

Objectives: AmpC-β-lactamase production is an under-recognized antibiotic resistance mechanism that renders Gram-negative bacteria resistant to common β-lactam antibiotics, similar to the well-known ESBLs. For infection control purposes, it is important to be able to discriminate between plasmid-mediated AmpC (pAmpC) production and chromosomal-mediated AmpC (cAmpC) hyperproduction in Gram-negative bacteria as pAmpC requires isolation precautions to minimize the risk of horizontal gene transmission. Detecting pAmpC in Escherichia coli is challenging, as both pAmpC production and cAmpC hyperproduction may lead to third-generation cephalosporin resistance. Methods: We tested a collection of E. coli strains suspected to produce AmpC. Elaborate susceptibility testing for third-generation cephalosporins, WGS and machine learning were used to develop an algorithm to determine ampC genotypes in E. coli. WGS was applied to detect pampC genes, cAmpC hyperproducers and STs. Results: In total, 172 E. coli strains (n=75 ST) were divided into a training set and two validation sets. Ninety strains were pampC positive, the predominant gene being blaCMY-2 (86.7%), followed by blaDHA-1 (7.8%), and 59 strains were cAmpC hyperproducers. The algorithm used a cefotaxime MIC value above 6 mg/L to identify pampC-positive E. coli and an MIC value of 0.5 mg/L to discriminate between cAmpC-hyperproducing and non-cAmpC-hyperproducing E. coli strains. Accuracy was 0.88 (95% CI=0.79-0.94) on the training set, 0.79 (95% CI=0.64-0.89) on validation set 1 and 0.85 (95% CI=0.71-0.94) on validation set 2. Conclusions: This approach resulted in a pragmatic algorithm for differentiating ampC genotypes in E. coli based on phenotypic susceptibility testing

Neisseria meningitidis Serogroup Z Meningitis in a Child with Complement C8 Deficiency and Potential Cross Protection of the MenB-4C Vaccine

Complement deficient patients are susceptible to rare meningococcal serogroups. A 6-year-old girl presented with serogroup Z meningitis. This led to identification of a C8 deficiency. The MenB-4C vaccine induced cross-reactive antibodies to serogroup Z and increased in vitro opsonophagocytic killing and may thus protect complement deficient patients

The potential role of drug transporters and amikacin modifying enzymes in M. avium

ABSTRACT: Objectives: Mycobacterium avium (M. avium) complex bacteria cause opportunistic infections in humans. Treatment yields cure rates of 60% and consists of a macrolide, a rifamycin, and ethambutol, and in severe cases, amikacin. Mechanisms of antibiotic tolerance remain mostly unknown. Therefore, we studied the contribution of efflux and amikacin modification to antibiotic susceptibility. Methods: We characterised M. avium ABC transporters and studied their expression together with other transporters following exposure to clarithromycin, amikacin, ethambutol, and rifampicin. We determined the effect of combining the efflux pump inhibitors berberine, verapamil and CCCP (carbonyl cyanide m-chlorophenyl hydrazone), to study the role of efflux on susceptibility. Finally, we studied the modification of amikacin by M. avium using metabolomic analysis. Results: Clustering shows conservation between M. avium and M. tuberculosis and transporters from most bacterial subfamilies (2–6, 7a/b, 10–12) were found. The largest number of transporter encoding genes was up-regulated after clarithromycin exposure, and the least following amikacin exposure. Only berberine increased the susceptibility to clarithromycin. Finally, because of the limited effect of amikacin on transporter expression, we studied amikacin modification and showed that M. avium, in contrast to M. abscessus, is not able to modify amikacin. Conclusion: We show that M. avium carries ABC transporters from all major families important for antibiotic efflux, including homologues shown to have affinity for drugs included in treatment. Efflux inhibition in M. avium can increase susceptibility, but this effect is efflux pump inhibitor– and antibiotic-specific. Finally, the lack of amikacin modifying activity in M. avium is important for its activity