Emergence of vancomycin-resistant enterococci from vancomycin-susceptible enterococci in hospitalized patients under antimicrobial therapy.

OBJECTIVES: Enterococci are opportunistic pathogens with plastic genomes that evolve, acquire, and transmit antimicrobial-resistant determinants such as vancomycin resistance clusters. While vancomycin-resistant enterococci (VRE) have emerged as successful nosocomial pathogens, the mechanism by which vancomycin-susceptible enterococci (VSE) transform to VRE in hospitalized patients remains understudied. METHODS: Genomes of Enterococcus faecium from two critically ill hospitalized patients subjected to multiple antibiotic therapies, including broad-spectrum antibiotics, were investigated. To identify mechanisms of resistance evolution, genomes of vancomycin-susceptible and -resistant isolates were compared. RESULTS: While VSE isolates were initially identified, VRE strains emerged post-vancomycin therapy. Comparative genomics revealed horizontal transmission of mobile genetic elements containing the Tn1549 transposon, which harbours the vanB-type vancomycin resistance gene cluster. This suggests that broad-spectrum antibiotic stress promoted the transfer of resistance-conferring elements, presumably from another gut inhabitant. CONCLUSION: This is one of the first studies investigating VSE and VRE isolates from the same patient. The mechanism of transmission and the within-patient evolution of vancomycin resistance via mobile genetic elements under antibiotic stress is illustrated. Our findings serve as a foundation for future studies building on this knowledge which can further elucidate the dynamics of antibiotic stress, resistance determinant transmission, and interactions within the gut microbiota.

The population structure of vancomycin-resistant and -susceptible Enterococcus faecium in a low-prevalence antimicrobial resistance setting is highly influenced by circulating global hospital-associated clones.

Between 2010 and 2015 the incidence of vancomycin-resistant Enterococcus faecium (VREfm) in Norway increased dramatically. Hence, we selected (1) a random subset of vancomycin-resistant enterococci (VRE) from the Norwegian Surveillance System for Communicable Diseases (2010-15; n=239) and (2) Norwegian vancomycin-susceptible E. faecium (VSEfm) bacteraemia isolates from the national surveillance system for antimicrobial resistance in microbes (2008 and 2014; n=261) for further analysis. Whole-genome sequences were collected for population structure, van gene cluster, mobile genetic element and virulome analysis, as well as antimicrobial susceptibility testing. Comparative genomic and phylogeographical analyses were performed with complete genomes of global E. faecium strains from the National Center for Biotechnology Information (NCBI) (1946-2022; n=272). All Norwegian VREfm and most of the VSEfm clustered with global hospital-associated sequence types (STs) in the phylogenetic subclade A1. The vanB2 subtype carried by chromosomal Tn1549 integrative conjugative elements was the dominant van type. The major Norwegian VREfm cluster types (CTs) were in accordance with concurrent European CTs. The dominant vanB-type VREfm CTs, ST192-CT3/26 and ST117-CT24, were mostly linked to a single hospital in Norway where the clones spread after independent chromosomal acquisition of Tn1549. The less prevalent vanA VRE were associated with more diverse CTs and vanA carrying Inc18 or RepA_N plasmids with toxin-antitoxin systems. Only 5 % of the Norwegian VRE were Enterococcus faecalis, all of which contained vanB. The Norwegian VREfm and VSEfm isolates harboured CT-specific virulence factor (VF) profiles supporting biofilm formation and colonization. The dominant VREfm CTs in general hosted more virulence determinants than VSEfm. The phylogenetic clade B VSEfm isolates (n=21), recently classified as Enterococcus lactis, harboured fewer VFs than E. faecium in general, and particularly subclade A1 isolates. In conclusion, the population structure of Norwegian E. faecium isolates mirrors the globally prevalent clones and particularly concurrent European VREfm/VSEfm CTs. Novel chromosomal acquisition of vanB2 on Tn1549 from the gut microbiota, however, formed a single major hospital VREfm outbreak. Dominant VREfm CTs contained more VFs than VSEfm.

The chemotherapeutic drug methotrexate selects for antibiotic resistance.

BACKGROUND: Understanding drivers of antibiotic resistance evolution is fundamental for designing optimal treatment strategies and interventions to reduce the spread of antibiotic resistance. Various cytotoxic drugs used in cancer chemotherapy have antibacterial properties, but how bacterial populations are affected by these selective pressures is unknown. Here we test the hypothesis that the widely used cytotoxic drug methotrexate affects the evolution and selection of antibiotic resistance. METHODS: First, we determined methotrexate susceptibility (IC90) and selective abilities in a collection of Escherichia coli and Klebsiella pneumoniae strains with and without pre-existing trimethoprim resistance determinants. We constructed fluorescently labelled pairs of E. coli MG1655 differing only in trimethoprim resistance determinants and determined the minimum selective concentrations of methotrexate using flow-cytometry. We further used an experimental evolution approach to investigate the effects of methotrexate on de novo trimethoprim resistance evolution. FINDINGS: We show that methotrexate can select for acquired trimethoprim resistance determinants located on the chromosome or a plasmid. Additionally, methotrexate co-selects for genetically linked resistance determinants when present together with trimethoprim resistance on a multi-drug resistance plasmid. These selective effects occur at concentrations 40- to >320-fold below the methotrexate minimal inhibitory concentration. INTERPRETATION: Our results strongly suggest a selective role of methotrexate for virtually any antibiotic resistance determinant when present together with trimethoprim resistance on a multi-drug resistance plasmid. The presented results may have significant implications for patient groups strongly depending on effective antibiotic treatment. FUNDING: PJJ was supported by UiT The Arctic University of Norway and the Northern Norway Regional Health Authority (SFP1292-16/HNF1586-21) and JPI-EC-AMR (Project 271,176/H10). DIA was supported by the Swedish Research Council (grant 2017-01,527). The publication charges for this article have been funded by a grant from the publication fund of UiT The Arctic University of Norway.

Piggybacking on Niche Adaptation Improves the Maintenance of Multidrug-Resistance Plasmids.

The persistence of plasmids in bacterial populations represents a puzzling evolutionary problem with serious clinical implications due to their role in the ongoing antibiotic resistance crisis. Recently, major advancements have been made toward resolving this "plasmid paradox" but mainly in a nonclinical context. Here, we propose an additional explanation for the maintenance of multidrug-resistance plasmids in clinical Escherichia coli strains. After coevolving two multidrug-resistance plasmids encoding resistance to last resort carbapenems with an extraintestinal pathogenic E. coli strain, we observed that chromosomal media adaptive mutations in the global regulatory systems CCR (carbon catabolite repression) and ArcAB (aerobic respiration control) pleiotropically improved the maintenance of both plasmids. Mechanistically, a net downregulation of plasmid gene expression reduced the fitness cost. Our results suggest that global chromosomal transcriptional rewiring during bacterial niche adaptation may facilitate plasmid maintenance.

Growth phase-specific evolutionary benefits of natural transformation in Acinetobacter baylyi.

Natural transformation in bacteria facilitates the uptake and genomic integration of exogenous DNA. This allows horizontal exchange of adaptive traits not easily achieved by point mutations, and has a major role in the acquisition of adaptive traits exemplified by antibiotic resistance determinants and vaccination escape. Mechanisms of DNA uptake and genomic integration are well described for several naturally transformable bacterial species; however, the selective forces responsible for its evolution and maintenance are still controversial. In this study we evolved transformation-proficient and -deficient Acinetobacter baylyi for 175 days in serial transfer cultures where stress was included. We found that natural transformation-proficient populations adapted better to active growth and early stationary phase. This advantage was offset by the reduced performance in the late stationary/death phase. We demonstrate fitness trade-offs between adaptation to active growth and survival in stationary/death phase caused by antagonistic pleiotropy. The presented data suggest that the widely held assumption that recombination speeds up adaptation by rapid accumulation of multiple adaptive mutations in the same genetic background is not sufficient to fully account for the maintenance of natural transformation in bacteria.

Real-time polymerase chain reaction for detection of low-intensity Schistosoma japonicum infections in China.

More sensitive methods for diagnosing infection with Schistosoma japonicum are needed as control becomes more effective. We compared a real-time polymerase chain reaction (PCR) for stool samples with conventional diagnostic methods in a study of 1,727 persons from Anhui Province, China. Seroprevalence determined by using an indirect hemagglutination assay (IHA) was much higher (26.1%) than the prevalence in stool-based tests, which were 5.3%, 3.2%, and 3.0% for PCR, hatching test, and Kato-Katz thick smear, respectively. A large proportion of the positive stool samples were only positive in one or two tests. The PCR showed better agreement with IHA than the other two stool-based tests. A commonly used diagnostic algorithm with initial screening for antibodies and subsequent testing with the Kato-Katz thick smear of the seropositive results would have resulted in treatment of 22 people compared with 50 people if the PCR replaced the Kato-Katz thick smear. As prevalence and intensity decrease, the benefit of increased sensitivity using the PCR must be weighed against additional costs.