Risk of death in Klebsiella pneumoniae bloodstream infections is associated with specific phylogenetic lineages.

BACKGROUND: Klebsiella pneumoniae species complex (KpSC) bloodstream infections (BSIs) are associated with considerable morbidity and mortality, particularly in elderly and multimorbid patients. Multidrug-resistant (MDR) strains have been associated with poorer outcome. However, the clinical impact of KpSC phylogenetic lineages on BSI outcome is unclear. METHODS: In an 18-month nationwide Norwegian prospective study of KpSC BSI episodes in adults, we used whole-genome sequencing to describe the molecular epidemiology of KpSC, and multivariable Cox regression analysis including clinical data to determine adjusted hazard ratios (aHR) for death associated with specific genomic lineages. FINDINGS: We included 1078 BSI episodes and 1082 bacterial isolates from 1055 patients. The overall 30-day case-fatality rate (CFR) was 12.5%. Median patient age was 73.4, 61.7% of patients were male. Median Charlson comorbidity score was 3. Klebsiella pneumoniae sensu stricto (Kp) (79.3%, n = 858/1082) and K. variicola (15.7%, n = 170/1082) were the dominating phylogroups. Global MDR-associated Kp clonal groups (CGs) were prevalent (25.0%, n = 270/1082) but 78.9% (n = 213/270) were not MDR, and 53.7% (n = 145/270) were community acquired. The major findings were increased risk for death within 30 days in monomicrobial BSIs caused by K. variicola (CFR 16.9%, n = 21; aHR 1.86, CI 1.10-3.17, p = 0.02), and global MDR-associated Kp CGs (CFR 17.0%, n = 36; aHR 1.52, CI 0.98-2.38, p = 0.06) compared to Kp CGs not associated with MDR (CFR 10.1%, n = 46). CONCLUSION: Bacterial traits, beyond antimicrobial resistance, have a major impact on the clinical outcome of KpSC BSIs. The global spread of MDR-associated Kp CGs is driven by other mechanisms than antibiotic selection alone. Further insights into virulence determinants, and their association with phylogenetic lineages are needed to better understand the epidemiology of KpSC infection and clinical outcome.

Modulation of multidrug-resistant clone success in Escherichia coli populations: a longitudinal, multi-country, genomic and antibiotic usage cohort study.

BACKGROUND: The effect of antibiotic usage on the success of multidrug-resistant (MDR) clones in a population remains unclear. With this genomics-based molecular epidemiology study, we aimed to investigate the contribution of antibiotic use to Escherichia coli clone success, relative to intra-strain competition for colonisation and infection. METHODS: We sequenced all the available E coli bloodstream infection isolates provided by the British Society for Antimicrobial Chemotherapy (BSAC) from 2012 to 2017 (n=718) and combined these with published data from the UK (2001-11; n=1090) and Norway (2002-17; n=3254). Defined daily dose (DDD) data from the European Centre for Disease Prevention and Control (retrieved on Sept 21, 2021) for major antibiotic classes (ß-lactam, tetracycline, macrolide, sulfonamide, quinolone, and non-penicillin ß-lactam) were used together with sequence typing, resistance profiling, regression analysis, and non-neutral Wright-Fisher simulation-based modelling to enable systematic comparison of resistance levels, clone success, and antibiotic usage between the UK and Norway. FINDINGS: Sequence type (ST)73, ST131, ST95, and ST69 accounted for 892 (49·3%) of 1808 isolates in the BSAC collection. In the UK, the proportion of ST69 increased between 2001-10 and 2011-17 (p=0·0004), whereas the proportions of ST73 and ST95 did not vary between periods. ST131 expanded quickly after its emergence in 2003 and its prevalence remained consistent throughout the study period (apart from a brief decrease in 2009-10). The extended-spectrum ß-lactamase (ESBL)-carrying, globally disseminated MDR clone ST131-C2 showed overall greater success in the UK (154 [56·8%] of 271 isolates in 2003-17) compared with Norway (51 [18·3%] of 278 isolates in 2002-17; p<0·0001). DDD data indicated higher total use of antimicrobials in the UK, driven mainly by the class of non-penicillin ß-lactams, which were used between 2·7-times and 5·1-times more in the UK per annum (ratio mean 3·7 [SD 0·8]). This difference was associated with the higher success of the MDR clone ST131-C2 (pseudo-R2 69·1%). A non-neutral Wright-Fisher model replicated the observed expansion of non-MDR and MDR sequence types under higher DDD regimes. INTERPRETATION: Our study indicates that resistance profiles of contemporaneously successful clones can vary substantially, warranting caution in the interpretation of correlations between aggregate measures of resistance and antibiotic usage. Our study further suggests that in countries with low-to-moderate use of antibiotics, such as the UK and Norway, the extent of non-penicillin ß-lactam use modulates rather than determines the success of widely disseminated MDR ESBL-carrying E coli clones. Detailed understanding of underlying causal drivers of success is important for improved control of resistant pathogens. FUNDING: Trond Mohn Foundation, Marie Sklodowska-Curie Actions, European Research Council, Royal Society, and Wellcome Trust.

Population structure and antimicrobial resistance among Klebsiella isolates sampled from human, animal, and environmental sources in Ghana: a cross-sectional genomic One Health study.

BACKGROUND: One Health approaches to address the increasing threat of antimicrobial resistance (AMR) are gaining attention. However, data on the distribution and movement of bacteria and their AMR-associated genes between clinical and non-clinical sources are scarce, especially from low-income and middle-income countries. We aimed to analyse Klebsiella isolates from various sources in Ghana and compare the prevalence of AMR with datasets from two other countries. METHODS: We conducted a cross-sectional genomic One Health study. Multiple clinical, environmental, and animal sources were sampled from 78 locations (eg, hospitals, residential areas, and farms) in and around Tamale, Ghana. Clinical samples were collected through routine screening and in cases of suspected infection between March 15 and Sept 15, 2019, and samples from the wider environment were collected during a dedicated sampling effort between the dates of Aug 19, 2018, and Sept 26, 2019. Sampling locations were approximately evenly distributed from the centre of the city and steadily outwards to capture both rural and urban locations. Samples with positive growth for Klebsiella were included. Isolates of Klebsiella were obtained from the samples using Simmons citrate agar medium and characterised by antimicrobial susceptibility testing and whole-genome sequencing. A comparative analysis with Klebsiella population surveys from Pavia, Italy, and Tromsø, Norway, was performed. AMR-associated and virulence genes were detected, and the population distribution of these genes was studied. FINDINGS: Of 957 samples collected around Tamale, Ghana, 620 were positive for Klebsiella spp. 573 Klebsiella isolates were successfully sequenced, of which 370 were Klebsiella pneumoniae. Only two hospital isolates were carbapenem-resistant. Extended-spectrum ß-lactamase (ESBL) genes were relatively common among the Ghanaian clinical isolates but rare in the environmental samples. Prevalence of ESBL genes in human-hospital disease samples was 64% (14 of 22 isolates) in Ghana and 44% (four of nine isolates) in Italy, and prevalence in human-hospital carriage samples was 7% (eight of 107) in Ghana and 13% (54 of 428) in Italy; the prevalence was higher in human-hospital disease samples than in human-hospital carriage samples in both countries, and prevalence across both samples in both countries was higher than in Norway. Ghanaian isolates showed evidence of high recombination rates (recombination events compared with point mutations [r/m] 9·455) and a considerable accessory gene overlap with isolates from Italy and Norway. INTERPRETATION: Although several AMR-associated gene classes were observed relatively frequently in non-clinical sources, ESBL, carbapenemase, and virulence genes were predominantly present only in hospital samples. These results suggest that interventions should be focused on clinical settings to have the greatest effect on the prevalence and dissemination of AMR-associated genes. FUNDING: European Research Council (742158), Academy of Finland EuroHPC grant, Trond Mohn Foundation (BATTALION grant), and Wellcome Trust.

Mge-cluster: a reference-free approach for typing bacterial plasmids.

Extrachromosomal elements of bacterial cells such as plasmids are notorious for their importance in evolution and adaptation to changing ecology. However, high-resolution population-wide analysis of plasmids has only become accessible recently with the advent of scalable long-read sequencing technology. Current typing methods for the classification of plasmids remain limited in their scope which motivated us to develop a computationally efficient approach to simultaneously recognize novel types and classify plasmids into previously identified groups. Here, we introduce mge-cluster that can easily handle thousands of input sequences which are compressed using a unitig representation in a de Bruijn graph. Our approach offers a faster runtime than existing algorithms, with moderate memory usage, and enables an intuitive visualization, classification and clustering scheme that users can explore interactively within a single framework. Mge-cluster platform for plasmid analysis can be easily distributed and replicated, enabling a consistent labelling of plasmids across past, present, and future sequence collections. We underscore the advantages of our approach by analysing a population-wide plasmid data set obtained from the opportunistic pathogen Escherichia coli, studying the prevalence of the colistin resistance gene mcr-1.1 within the plasmid population, and describing an instance of resistance plasmid transmission within a hospital environment.

Intraregional hospital outbreak of OXA-244-producing Escherichia coli ST38 in Norway, 2020.

Infections with OXA-244-carbapenemase-producing Escherichia coli with sequence type (ST)38 have recently increased in Europe. Due to its low-level activity against carbapenems, OXA-244 can be difficult to detect. Previous assessments have not revealed a clear source and route of transmission for OXA-244-producing E. coli, but there are indications of non-healthcare related sources and community spread. Here we report a hospital-associated outbreak of OXA-244-producing E. coli ST38 involving three hospitals in Western Norway in 2020. The outbreak occurred over a 5-month period and included 12 cases identified through clinical (n = 6) and screening (n = 6) samples. The transmission chain was unclear; cases were identified in several wards and there was no clear overlap of patient stay. However, all patients had been admitted to the same tertiary hospital in the region, where screening revealed an outbreak in one ward (one clinical case and five screening cases). Outbreak control measures were instigated including contact tracing, isolation, and screening; no further cases were identified in 2021. This outbreak adds another dimension to the spread of OXA-244-producing E. coli ST38, illustrating this clone's ability to establish itself in the healthcare setting. Awareness of challenges concerning OXA-244-producing E. coli diagnostic is important to prevent further spread.

Nationwide, population-based observational study of the molecular epidemiology and temporal trend of carbapenemase-producing Enterobacterales in Norway, 2015 to 2021.

IntroductionNational and regional carbapenemase-producing Enterobacterales (CPE) surveillance is essential to understand the burden of antimicrobial resistance, elucidate outbreaks, and develop infection-control or antimicrobial-treatment recommendations.AimThis study aimed to describe CPE and their epidemiology in Norway from 2015 to 2021.MethodsA nationwide, population-based observational study of all verified clinical and carriage CPE isolates submitted to the national reference laboratory was conducted. Isolates were characterised by antimicrobial susceptibility testing, whole genome sequencing (WGS) and basic metadata. Annual CPE incidences were also estimated.ResultsA total of 389 CPE isolates were identified from 332 patients of 63 years median age (range: 0-98). These corresponded to 341 cases, 184 (54%) being male. Between 2015 and 2021, the annual incidence of CPE cases increased from 0.6 to 1.1 per 100,000 person-years. For CPE-isolates with available data on colonisation/infection, 58% (226/389) were associated with colonisation and 38% (149/389) with clinical infections. WGS revealed a predominance of OXA-48-like (51%; 198/389) and NDM (34%; 134/389) carbapenemases in a diversified population of Escherichia coli and Klebsiella pneumoniae, including high-risk clones also detected globally. Most CPE isolates were travel-related (63%; 245/389). Although local outbreaks and healthcare-associated transmission occurred, no interregional spread was detected. Nevertheless, 18% (70/389) of isolates not directly related to import points towards potentially unidentified transmission routes. A decline in travel-associated cases was observed during the COVID-19 pandemic.ConclusionsThe close-to-doubling of CPE case incidence between 2015 and 2021 was associated with foreign travel and genomic diversity. To limit further transmission and outbreaks, continued screening and monitoring is essential.

Community carriage of ESBL-producing Escherichia coli and Klebsiella pneumoniae: a cross-sectional study of risk factors and comparative genomics of carriage and clinical isolates.

The global prevalence of infections caused by extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) is increasing, and for Escherichia coli, observations indicate that this is partly driven by community-onset cases. The ESBL-E population structure in the community is scarcely described, and data on risk factors for carriage are conflicting. Here, we report the prevalence and population structure of fecal ESBL-producing E. coli and Klebsiella pneumoniae (ESBL-Ec/Kp) in a general adult population, examine risk factors, and compare carriage isolates with contemporary clinical isolates. Fecal samples obtained from 4,999 participants (54% women) ≥40 years in the seventh survey of the population-based Tromsø Study, Norway (2015, 2016), were screened for ESBL-Ec/Kp. In addition, we included 118 ESBL-Ec clinical isolates from the Norwegian surveillance program in 2014. All isolates were whole-genome sequenced. Risk factors associated with carriage were analyzed using multivariable logistic regression. ESBL-Ec gastrointestinal carriage prevalence was 3.3% [95% confidence interval (CI) 2.8%-3.9%, no sex difference] and 0.08% (0.02%-0.20%) for ESBL-Kp. For ESBL-Ec, travel to Asia was the only independent risk factor (adjusted odds ratio 3.46, 95% CI 2.18-5.49). E. coli ST131 was most prevalent in both collections. However, the ST131 proportion was significantly lower in carriage (24%) versus clinical isolates (58%, P < 0.001). Carriage isolates were genetically more diverse with a higher proportion of phylogroup A (26%) than clinical isolates (5%, P < 0.001), indicating that ESBL gene acquisition occurs in a variety of E. coli lineages colonizing the gut. STs commonly related to extraintestinal infections were more frequent in clinical isolates also carrying a higher prevalence of antimicrobial resistance, which could indicate clone-associated pathogenicity.IMPORTANCEESBL-Ec and ESBL-Kp are major pathogens in the global burden of antimicrobial resistance. However, there is a gap in knowledge concerning the bacterial population structure of human ESBL-Ec/Kp carriage isolates in the community. We have examined ESBL-Ec/Kp isolates from a population-based study and compared these to contemporary clinical isolates. The large genetic diversity of carriage isolates indicates frequent ESBL gene acquisition, while those causing invasive infections are more clone dependent and associated with a higher prevalence of antibiotic resistance. The knowledge of factors associated with ESBL carriage helps to identify patients at risk to combat the spread of resistant bacteria within the healthcare system. Particularly, previous travel to Asia stands out as a major risk factor for carriage and should be considered in selecting empirical antibiotic treatment in critically ill patients.

Evolutionary and functional history of the Escherichia coli K1 capsule.

Escherichia coli is a leading cause of invasive bacterial infections in humans. Capsule polysaccharide has an important role in bacterial pathogenesis, and the K1 capsule has been firmly established as one of the most potent capsule types in E. coli through its association with severe infections. However, little is known about its distribution, evolution and functions across the E. coli phylogeny, which is fundamental to elucidating its role in the expansion of successful lineages. Using systematic surveys of invasive E. coli isolates, we show that the K1-cps locus is present in a quarter of bloodstream infection isolates and has emerged in at least four different extraintestinal pathogenic E. coli (ExPEC) phylogroups independently in the last 500 years. Phenotypic assessment demonstrates that K1 capsule synthesis enhances E. coli survival in human serum independent of genetic background, and that therapeutic targeting of the K1 capsule re-sensitizes E. coli from distinct genetic backgrounds to human serum. Our study highlights that assessing the evolutionary and functional properties of bacterial virulence factors at population levels is important to better monitor and predict the emergence of virulent clones, and to also inform therapies and preventive medicine to effectively control bacterial infections whilst significantly lowering antibiotic usage.

Rapid cross-border emergence of NDM-5-producing Escherichia coli in the European Union/European Economic Area, 2012 to June 2022.

Whole genome sequencing data of 874 Escherichia coli isolates carrying bla NDM-5 from 13 European Union/European Economic Area countries between 2012 and June 2022 showed the predominance of sequence types ST167, ST405, ST410, ST361 and ST648, and an increasing frequency of detection. Nearly a third (30.6%) of these isolates were associated with infections and more than half (58.2%) were predicted to be multidrug-resistant. Further spread of E. coli carrying bla NDM-5 would leave limited treatment options for serious E. coli infections.

The COVID-19 Pandemic Sparked Off a Large-Scale Outbreak of Carbapenem-Resistant Acinetobacter baumannii from the Endemic Strains at an Italian Hospital.

Acinetobacter baumannii is a nosocomial pathogen that poses a serious threat due to the rise of incidence of multidrug-resistant (MDR) strains. During the COVID-19 pandemic, MDR A. baumannii clones have caused several outbreaks worldwide. Here, we describe a detailed investigation of an MDR A. baumannii outbreak that occurred at Policlinico San Matteo (Pavia, Italy). A total of 96 A. baumannii strains, isolated between January and July 2020 from 41 inpatients (both SARS-CoV-2 positive and negative) in different wards, were characterized by phenotypic and genomic analyses combining Illumina and Nanopore sequencing. Antibiotic susceptibility testing revealed that all isolates were resistant to carbapenems, and the sequence analysis attributed this to the carbapenemase gene blaOXA-23. Virulence factor screening unveiled that all strains carried determinants for biofilm formation, while plasmid analysis revealed the presence of two plasmids, one of which was ~100 kbp long and encoded a phage sequence. A core genome-based phylogeny was inferred to integrate outbreak strain genomes with background genomes from public databases and the local surveillance program. All strains belonged to the globally disseminated sequence type 2 (ST2) clone and were mainly divided into two clades. Isolates from the outbreak clustered with surveillance isolates from 2019, suggesting that the outbreak was caused by two strains that were already circulating in the hospital before the start of the pandemic. The intensive spread of A. baumannii in the hospital was enhanced by the extreme emergency situation of the first COVID-19 pandemic wave that resulted in reduced attention to infection prevention and control practices. IMPORTANCE The COVID-19 pandemic, especially during the first wave, posed a great challenge to the hospital management and generally promoted nosocomial pathogen dissemination. MDR A. baumannii can easily spread and persist for a long time on surfaces, causing outbreaks in health care settings. Infection prevention and control practices, epidemiological surveillance, and microbiological screening are fundamental in order to control such outbreaks. Here, we sequenced the genomes of 96 isolates from an outbreak of MDR A. baumannii strains using both short- and long-read technology in order to reconstruct the outbreak events in fine detail. The sequence data demonstrated that two endemic clones of MDR A. baumannii were the source of this large hospital outbreak during the first COVID-19 pandemic wave, confirming the effect of COVID-19 emergency disrupting the protection provided by the use of the standard prevention procedures.

Strong pathogen competition in neonatal gut colonisation.

Opportunistic bacterial pathogen species and their strains that colonise the human gut are generally understood to compete against both each other and the commensal species colonising this ecosystem. Currently we are lacking a population-wide quantification of strain-level colonisation dynamics and the relationship of colonisation potential to prevalence in disease, and how ecological factors might be modulating these. Here, using a combination of latest high-resolution metagenomics and strain-level genomic epidemiology methods we performed a characterisation of the competition and colonisation dynamics for a longitudinal cohort of neonatal gut microbiomes. We found strong inter- and intra-species competition dynamics in the gut colonisation process, but also a number of synergistic relationships among several species belonging to genus Klebsiella, which includes the prominent human pathogen Klebsiella pneumoniae. No evidence of preferential colonisation by hospital-adapted pathogen lineages in either vaginal or caesarean section birth groups was detected. Our analysis further enabled unbiased assessment of strain-level colonisation potential of extra-intestinal pathogenic Escherichia coli (ExPEC) in comparison with their propensity to cause bloodstream infections. Our study highlights the importance of systematic surveillance of bacterial gut pathogens, not only from disease but also from carriage state, to better inform therapies and preventive medicine in the future.

Detection of Klebsiella pneumoniae human gut carriage: a comparison of culture, qPCR, and whole metagenomic sequencing methods.

Klebsiella pneumoniae is an important opportunistic healthcare-associated pathogen and major contributor to the global spread of antimicrobial resistance. Gastrointestinal colonization with K. pneumoniae is a major predisposing risk factor for infection and forms an important hub for the dispersal of resistance. Current culture-based detection methods are time consuming, give limited intra-sample abundance and strain diversity information, and have uncertain sensitivity. Here we investigated the presence and abundance of K. pneumoniae at the species and strain level within fecal samples from 103 community-based adults by qPCR and whole metagenomic sequencing (WMS) compared to culture-based detection. qPCR demonstrated the highest sensitivity, detecting K. pneumoniae in 61.2% and 75.8% of direct-fecal and culture-enriched sweep samples, respectively, including 52/52 culture-positive samples. WMS displayed lower sensitivity, detecting K. pneumoniae in 71.2% of culture-positive fecal samples at a 0.01% abundance cutoff, and was inclined to false positives in proportion to the relative abundance of other Enterobacterales present. qPCR accurately quantified K. pneumoniae to 16 genome copies/reaction while WMS could estimate relative abundance to at least 0.01%. Quantification by both methods correlated strongly with each other (Spearman's rho = 0.91). WMS also supported accurate intra-sample K. pneumoniae sequence type (ST)-level diversity detection from fecal microbiomes to 0.1% relative abundance, agreeing with the culture-based detected ST in 16/19 samples. Our results show that qPCR and WMS are sensitive and reliable tools for detection, quantification, and strain analysis of K. pneumoniae from fecal samples with potential to support infection control and enhance insights in K. pneumoniae gastrointestinal ecology.

What is the context?Klebsiella pneumoniae is a major cause of healthcare-associated infections and a key contributor to the spread of resistance to last-line antimicrobials.Gastrointestinal colonization by K. pneumoniae is a risk factor for developing infection and can facilitate the spread of antimicrobial resistance.Culture-based detection may lack sensitivity and is ill-suited to detecting within-sample K. pneumoniae abundance and diversity.Developing molecular methods to improve K. pneumoniae abundance and strain diversity detection are essential in understanding human gut colonization and ecology.What is new? We compared culture-based detection of K. pneumoniae within human fecal samples to two molecular-based techniques: 1) qPCR, which amplifies K. pneumoniae species complex-specific DNA targets with high sensitivity, and 2) whole metagenomic sequencing (WMS), which sequences the entire DNA content of complex microbial communities.Our findings show:qPCR had the highest sensitivity, detecting K. pneumoniae in all (52/52) culture-positive samples and 11/51 and 23/47 culture-negative samples, using a direct-fecal and culture-sweep method, respectively. qPCR could accurately quantify K. pneumoniae to 16 genome copies/reaction.WMS had lower sensitivity, positive in 37/52 culture-positive samples, and demonstrated false positives arising from closely related bacterial species. Relative abundance estimates could be performed to 0.01%.WMS performed accurate strain-level detection of K. pneumoniae to 0.1% relative abundance and could detect within-sample strain diversity.What is the impact?qPCR and WMS are valid methods for the detection and characterization of colonizing K. pneumoniae with potential to enhance our understanding of the gastrointestinal ecology of this important pathogen.

Evolution of ß-lactamase-mediated cefiderocol resistance.

BACKGROUND: Cefiderocol is a novel siderophore ß-lactam with improved hydrolytic stability toward ß-lactamases, including carbapenemases, achieved by combining structural moieties of two clinically efficient cephalosporins, ceftazidime and cefepime. Consequently, cefiderocol represents a treatment alternative for infections caused by MDR Gram-negatives. OBJECTIVES: To study the role of cefiderocol on resistance development and on the evolution of ß-lactamases from all Ambler classes, including KPC-2, CTX-M-15, NDM-1, CMY-2 and OXA-48. METHODS: Directed evolution, using error-prone PCR followed by selective plating, was utilized to investigate how the production and the evolution of different ß-lactamases cause changes in cefiderocol susceptibility determined using microbroth dilution assays (MIC and IC50). RESULTS: We found that the expression of blaOXA-48 did not affect cefiderocol susceptibility. On the contrary, the expression of blaKPC-2, blaCMY-2, blaCTX-M-15 and blaNDM-1 substantially reduced cefiderocol susceptibility by 4-, 16-, 8- and 32-fold, respectively. Further, directed evolution on these enzymes showed that, with the acquisition of only 1-2 non-synonymous mutations, all ß-lactamases were evolvable to further cefiderocol resistance by 2- (NDM-1, CTX-M-15), 4- (CMY-2), 8- (OXA-48) and 16-fold (KPC-2). Cefiderocol resistance development was often associated with collateral susceptibility changes including increased resistance to ceftazidime and ceftazidime/avibactam as well as functional trade-offs against different ß-lactam drugs. CONCLUSIONS: The expression of contemporary ß-lactamase genes can potentially contribute to cefiderocol resistance development and the acquisition of mutations in these genes results in enzymes adapting to increasing cefiderocol concentrations. Resistance development caused clinically important cross-resistance, especially against ceftazidime and ceftazidime/avibactam.

Evolutionary Instability of Collateral Susceptibility Networks in Ciprofloxacin-Resistant Clinical Escherichia coli Strains.

Collateral sensitivity and resistance occur when resistance development toward one antimicrobial either potentiates or deteriorates the effect of others. Previous reports on collateral effects on susceptibility focus on newly acquired resistance determinants and propose that novel treatment guidelines informed by collateral networks may reduce the evolution, selection, and spread of antimicrobial resistance. In this study, we investigate the evolutionary stability of collateral networks in five ciprofloxacin-resistant, clinical Escherichia coli strains. After 300 generations of experimental evolution without antimicrobials, we show complete fitness restoration in four of five genetic backgrounds and demonstrate evolutionary instability in collateral networks of newly acquired resistance determinants. We show that compensatory mutations reducing efflux expression are the main drivers destabilizing initial collateral networks and identify rpoS as a putative target for compensatory evolution. Our results add another layer of complexity to future predictions and clinical application of collateral networks. IMPORTANCE Antimicrobial resistance occurs due to genetic alterations that affect different processes in bacteria. Thus, developing resistance toward one antimicrobial drug may also alter the response toward others (collateral effects). Understanding the mechanisms of such collateral effects may provide clinicians with a framework for informed antimicrobial treatment strategies, limiting the emergence of antimicrobial resistance. However, for clinical implementation, it is important that the collateral effects of resistance development are repeatable and temporarily stable. Here, we show that collateral effects caused by resistance development toward ciprofloxacin in clinical Escherichia coli strains are not temporarily stable because of compensatory mutations restoring the fitness burden of the initial resistance mutations. Consequently, this instability is complicating the general applicability and clinical implementation of collateral effects into treatment strategies.

Phenotypic and genotypic characterisation of thymine auxotrophy in Escherichia coli isolated from a patient with recurrent bloodstream infection.

INTRODUCTION: Thymine auxotrophic in vitro mutants of Escherichia coli were first reported in the mid-20th century. Later, thymine-dependent clinical strains of E. coli as well as other Enterobacterales, Enterococcus faecalis and Staphylococcus aureus have been recognized as the cause of persistent and recurrent infections. OBJECTIVES: The aim of this study was to characterize the phenotype and investigate the molecular basis of thymine auxotrophy in ten E. coli isolates obtained at different time points from a patient with recurrent bloodstream infection (BSI) due to a chronic aortic graft infection treated with Trimethoprim/sulfamethoxazole (TMP-SMX). METHODS: Clinical data was obtained from hospital records. Growth characterization and antimicrobial susceptibility testing to TMP-SMX was performed on M9 agar and in MH broth with different thymine concentrations (0.5, 2, 5, 10 and 20 µg/mL), on Mueller-Hinton (MH) and blood agar. Whole genome sequencing (WGS) was performed on all E. coli isolates. RESULTS: E. coli were isolated from ten consecutive BSI episodes from a patient with chronic aortic graft infection. Six of these isolates were resistant to TMP-SMX when assayed on blood agar. Growth experiments with added thymine confirmed that these isolates were thymine-dependent (thy-), and revealed growth defects (slower growth rate and smaller colony size) in these isolates relative to thy+ isolates (n = 4). WGS indicated that all isolates were of the same clonal lineage of sequence type 7358. Genomic analysis revealed a G172C substitution in thyA in all TMP-SMX resistant isolates, while mutations affecting genes involved in the deoxyribose salvage pathway (deoB and deoC) were identified in eight isolates. CONCLUSION: This case highlights the risk of resistance development to TMP-SMX, especially for long-term treatment, and the possible pitfalls in detection of growth-deficient subpopulations from chronic infections, which could lead to treatment failure.

Intracellular Transposition and Capture of Mobile Genetic Elements following Intercellular Conjugation of Multidrug Resistance Conjugative Plasmids from Clinical Enterobacteriaceae Isolates.

Mobile genetic elements (MGEs) are often associated with antimicrobial resistance genes (ARGs). They are responsible for intracellular transposition between different replicons and intercellular conjugation and are therefore important agents of ARG dissemination. Detection and characterization of functional MGEs, especially in clinical isolates, would increase our understanding of the underlying pathways of transposition and recombination and allow us to determine interventional strategies to interrupt this process. Entrapment vectors can be used to capture active MGEs, as they contain a positive selection genetic system conferring a selectable phenotype upon the insertion of an MGE within certain regions of that system. Previously, we developed the pBACpAK entrapment vector that results in a tetracycline-resistant phenotype when MGEs translocate and disrupt the cI repressor gene. We have previously used pBACpAK to capture MGEs in clinical Escherichia coli isolates following transformation with pBACpAK. In this study, we aimed to extend the utilization of pBACpAK to other bacterial taxa. We utilized an MGE-free recipient E. coli strain containing pBACpAK to capture MGEs on conjugative, ARG-containing plasmids following conjugation from clinical Enterobacteriaceae donors. Following the conjugative transfer of multiple conjugative plasmids and screening for tetracycline resistance in these transconjugants, we captured several insertion sequence (IS) elements and novel transposons (Tn7350 and Tn7351) and detected the de novo formation of novel putative composite transposons where the pBACpAK-located tet(A) is flanked by ISKpn25 from the transferred conjugative plasmid, as well as the ISKpn14-mediated integration of an entire 119-kb, blaNDM-1-containing conjugative plasmid from Klebsiella pneumoniae. IMPORTANCE By analyzing transposition activity within our MGE-free recipient, we can gain insights into the interaction and evolution of multidrug resistance-conferring MGEs following conjugation, including the movement of multiple ISs, the formation of composite transposons, and cointegration and/or recombination between different replicons in the same cell. This combination of recipient and entrapment vector will allow fine-scale experimental studies of factors affecting intracellular transposition and MGE formation in and from ARG-encoding MGEs from multiple species of clinically relevant Enterobacteriaceae.

A nationwide genomic study of clinical Klebsiella pneumoniae in Norway 2001-15: introduction and spread of ESBLs facilitated by clonal groups CG15 and CG307.

OBJECTIVES: To use the nationwide Norwegian surveillance programme on resistant microbes in humans (NORM) to address longitudinal changes in the population structure of Klebsiella pneumoniae isolates from 2001-15, focusing on the emergence and dissemination of ESBL-producing K. pneumoniae in Norway. METHODS: Among blood (n = 6124) and urinary tract (n = 5496) surveillance isolates from 2001-15, we used Illumina technology to whole genome sequence 201 ESBL-producing isolates from blood (n = 130) and urine (n = 71), and 667 non-ESBL isolates from blood. Complete genomes for four isolates were resolved with Oxford Nanopore sequencing. RESULTS: In a highly diverse collection, Klebsiella variicola ssp. variicola caused 24.5% of Klebsiella pneumoniae species complex (KpSC) bacteraemias. ESBL production was limited to K. pneumoniae sensu stricto (98.5%). A diverse ESBL population of 57 clonal groups (CGs) were dominated by MDR CG307 (17%), CG15 (12%), CG70 (6%), CG258 (5%) and CG45 (5%) carrying blaCTX-M-15. Yersiniabactin was significantly more common in ESBL-positive (37.8%) compared with non-ESBL K. pneumoniae sensu stricto isolates (12.7%), indicating convergence of virulence and resistance determinants. Moreover, we found a significantly lower prevalence of yersiniabactin (3.0%, 37.8% and 17.3%), IncFIB (58.7%, 87.9% and 79.4%) and IncFII plasmid replicons (40.5%, 82.8% and 54.2%) in K. variicola ssp. variicola compared with ESBL- and non-ESBL K. pneumoniae sensu stricto isolates, respectively. CONCLUSIONS: The increase in Norwegian ESBL-producing KpSC during 2010-15 was driven by CG307 and CG15 carrying blaCTX-M-15. K. variicola ssp. variicola was a frequent cause of invasive KpSC infection, but rarely carried ESBLs.

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.

A high-throughput multiplexing and selection strategy to complete bacterial genomes.

BACKGROUND: Bacterial whole-genome sequencing based on short-read technologies often results in a draft assembly formed by contiguous sequences. The introduction of long-read sequencing technologies permits those contiguous sequences to be unambiguously bridged into complete genomes. However, the elevated costs associated with long-read sequencing frequently limit the number of bacterial isolates that can be long-read sequenced. Here we evaluated the recently released 96 barcoding kit from Oxford Nanopore Technologies (ONT) to generate complete genomes on a high-throughput basis. In addition, we propose an isolate selection strategy that optimizes a representative selection of isolates for long-read sequencing considering as input large-scale bacterial collections. RESULTS: Despite an uneven distribution of long reads per barcode, near-complete chromosomal sequences (assembly contiguity = 0.89) were generated for 96 Escherichia coli isolates with associated short-read sequencing data. The assembly contiguity of the plasmid replicons was even higher (0.98), which indicated the suitability of the multiplexing strategy for studies focused on resolving plasmid sequences. We benchmarked hybrid and ONT-only assemblies and showed that the combination of ONT sequencing data with short-read sequencing data is still highly desirable (i) to perform an unbiased selection of isolates for long-read sequencing, (ii) to achieve an optimal genome accuracy and completeness, and (iii) to include small plasmids underrepresented in the ONT library. CONCLUSIONS: The proposed long-read isolate selection ensures the completion of bacterial genomes that span the genome diversity inherent in large collections of bacterial isolates. We show the potential of using this multiplexing approach to close bacterial genomes on a high-throughput basis.

Bacterial genomic epidemiology with mixed samples.

Genomic epidemiology is a tool for tracing transmission of pathogens based on whole-genome sequencing. We introduce the mGEMS pipeline for genomic epidemiology with plate sweeps representing mixed samples of a target pathogen, opening the possibility to sequence all colonies on selective plates with a single DNA extraction and sequencing step. The pipeline includes the novel mGEMS read binner for probabilistic assignments of sequencing reads, and the scalable pseudoaligner Themisto. We demonstrate the effectiveness of our approach using closely related samples in a nosocomial setting, obtaining results that are comparable to those based on single-colony picks. Our results lend firm support to more widespread consideration of genomic epidemiology with mixed infection samples.