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.

The first vanE-type vancomycin resistant Enterococcus faecalis isolates in Norway - phenotypic and molecular characteristics.

OBJECTIVES: We aimed to characterize the vanE cluster and its genetic support in the first Norwegian vanE-type isolates and assess genetic relatedness to other vanE isolates. METHODS: Two vanE-type vancomycin resistant Enterococcus faecalis (vanE-VREfs) isolates (E1 and E2) recovered from the same patient 30 months apart were examined for antimicrobial susceptibility, genome sequence, vancomycin resistance induction, vanE transferability, genome mutation rate, and phylogenetic relationship to E. faecalis closed genomes and two vanE-VREfs from North America. RESULTS: The ST34 E1 and E2 strains expressed low-level vancomycin resistance and susceptibility to teicoplanin. Their vanE gene clusters were part of a non-transferable Tn6202. The histidine kinase part of vanSE was expressed although a premature stop codon (E1) and insertion of a transposase (E2) truncated their vanSE gene. The vancomycin resistance phenotype in E1 was inducible while constitutive in E2. E1 showed a 125-fold higher mutation rate than E2. Variant calling showed 60 variants but nearly identical chromosomal gene content and synteny between the isolates. Their genomes also showed high similarity to another ST34 vanE-VREfs from Canada. CONCLUSION: In-depth genomic analyses of the first two vanE-VREfs found in Europe identified a single chromosomal insertion site of two variants of vanE-conferring Tn6202. Single nucleotide polymorphism (SNP) and core genome multilocus sequence type (cgMLST) analyses show the genomes are different. This can be explained by the high mutation rate of E1 and acquisition of different mobile genetic elements; thus, we believe the two isolates from the same patient are genetically related. Genome similarities also suggest relatedness between the Canadian and Norwegian vanE-VREfs.

Co-localization of clinically relevant antibiotic- and heavy metal resistance genes on plasmids in Klebsiella pneumoniae from marine bivalves.

Klebsiella pneumoniae is an opportunistic pathogen frequently associated with antibiotic resistance and present in a wide range of environments, including marine habitats. However, little is known about the development, persistence, and spread of antibiotic resistance in such environments. This study aimed to obtain the complete genome sequences of antibiotic-resistant K. pneumoniae isolated from marine bivalves in order to determine the genetic context of antibiotic- and heavy metal resistance genes in these isolates. Five antibiotic-resistant K. pneumoniae isolates, of which four also carried heavy metal resistance genes, were selected for complete genome sequencing using the Illumina MiSeq platform and the Oxford Nanopore Technologies GridION device. Conjugation experiments were conducted to examine the transfer potential of selected plasmids. The average length of the complete genomes was 5.48 Mbp with a mean chromosome size of 5.27 Mbp. Seven plasmids were detected in the antibiotic-resistant isolates. Three IncFIB, one IncFIB/IncFII, and one IncFIB/IncHIB plasmid, respectively, carried antibiotic resistance genes such as qnrS1, aph(6)-Id and aph(3')-Ia, aadA1, and aadA2. Four of these plasmids also carried genes encoding resistance to copper (pco), silver (sil), and arsenic (ars). One plasmid carrying tet(D) and blaSHV-1 as well as pco, sil, and ars genes was transferred to Escherichia coli by conjugation. We show the co-occurrence of antibiotic- and heavy metal resistance genes on a conjugative IncFIB plasmid from K. pneumoniae from marine bivalves. Our study highlights the importance of the marine environment and seafood as a possible dissemination route for antimicrobial resistance and provides insights into the potential for co-selection of antibiotic resistance genes by heavy metals.

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.

Population dynamics and characteristics of Klebsiella pneumoniae from healthy poultry in Norway.

Klebsiella pneumoniae is an important opportunistic pathogen widely studied in relation to human infection and colonization. However, there is a lack of knowledge regarding other niches that K. pneumoniae may inhabit. K. pneumoniae isolated from healthy broiler and turkey flocks in Norway in 2018 have previously been described with regard to population structure, sequence types (STs), and the presence of virulence- and antimicrobial resistance (AMR) genes. In the present study we aimed to evaluate the dynamics of the K. pneumoniae population in poultry over time, with regards to AMR and virulence, and with a special focus on persistence of STs. A total of 391 flocks sampled in 2020 were included in the present study, of which 271 were from broiler flocks and 120 from turkey flocks. Similar to findings from 2018, the occurrence of K. pneumoniae was significantly higher based on culturing in turkey flocks (62.5%) compared to broiler flocks (24.0%). Major STs in 2020 included ST5827 (n = 7), ST37 (n = 7), ST370 (n = 7), ST17 (n = 5), and ST4710 (n = 5). Several STs persisted over time in both host species, including ST35, ST37, ST590, and ST17. This persistence may be due to local re-circulation or reintroduction from parent flocks. Of these five major STs, only ST590 carried AMR genes, indicating that the persistence was not associated with the presence of AMR genes. An ST4710 strain with a hypervirulence-encoding plasmid (p4710; iro5, iuc5) was recovered from turkeys in 2018. The same strain was present in turkeys in 2020, but the plasmid had lost the salmochelin locus. This loss may be attributed to reductive evolution due to the presence of several siderophores within the same isolates. In this study we also characterized a clinical ST4710 isolate from a turkey with airsacculitis. The isolate was closely related to two intestinal ST4710 isolates from healthy turkeys in 2018. These three isolates were sampled within the same location and time frame in 2018, and all carried the full p4710 virulence plasmid. These findings highlight the transmission- and infectious potential of ST4710 in turkeys.

Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17.

Klebsiella pneumoniae sequence type (ST) 17 is a global problem clone that causes multidrug-resistant (MDR) hospital infections worldwide. In 2008-2009, an outbreak of MDR ST17 occurred at a neonatal intensive care unit (NICU) in Stavanger, Norway. Fifty-seven children were colonized. We observed intestinal persistence of ST17 in all of the children for up to two years after hospital discharge. Here, we investigated the within-host evolution of ST17 in 45 of those children during long-term colonization and compared the outbreak with 254 global strains. Ninety-two outbreak-related isolates were whole-genome sequenced. They had capsule locus KL25, O locus O5 and carried yersiniabactin. During within-host colonization ST17 remained stable with few single nucleotide polymorphisms, no acquisition of antimicrobial resistance (AMR) or virulence determinants, and persistent carriage of a bla CTX-M-15-encoding IncFII(K) IncFIB(K) plasmid (pKp2177_1). The global collection included ST17 from 1993 to 2020 from 34 countries, that were from human infection (41.3%), colonization (39.3%) and respiratory specimens (7.3%), from animals (9.3%), and from the environment (2.7%). We estimate that ST17 emerged mid-to-late 19th century (1859, 95 % HPD 1763-1939) and diversified through recombinations of the K and O loci to form several sublineages, with various AMR genes, virulence loci and plasmids. There was limited evidence of persistence of AMR genes in any of these lineages. A globally disseminated sublineage with KL25/O5 accounted for 52.7 % of the genomes. It included a monophyletic subclade that emerged in the mid-1980s, which comprised the Stavanger NICU outbreak and 10 genomes from three other countries, which all carried pKp2177_1. The plasmid was also observed in a KL155/OL101 subclade from the 2000s. Three clonal expansions of ST17 were identified; all were healthcare-associated and carried either yersiniabactin and/or pKp2177_1. To conclude, ST17 is globally disseminated and associated with opportunistic hospital-acquired infections. It contributes to the burden of global MDR infections, but many diverse lineages persist without acquired AMR. We hypothesize that non-human sources and human colonization may play a crucial role for severe infections in vulnerable patients, such as preterm neonates.

Long-read sequencing for reliably calling the mompS allele in Legionella pneumophila sequence-based typing.

Sequence-based typing (SBT) of Legionella pneumophila is a valuable tool in epidemiological studies and outbreak investigations of Legionnaires' disease. In the L. pneumophila SBT scheme, mompS2 is one of seven genes that determine the sequence type (ST). The Legionella genome typically contains two copies of mompS (mompS1 and mompS2). When they are non-identical it can be challenging to determine the mompS2 allele, and subsequently the ST, from Illumina short-reads. In our collection of 233 L. pneumophila genomes, there were 62 STs, 18 of which carried non-identical mompS copies. Using short-reads, the mompS2 allele was misassembled or untypeable in several STs. Genomes belonging to ST154 and ST574, which carried mompS1 allele 7 and mompS2 allele 15, were assigned an incorrect mompS2 allele and/or mompS gene copy number when short-read assembled. For other isolates, mainly those carrying non-identical mompS copies, short-read assemblers occasionally failed to resolve the structure of the mompS-region, also resulting in untypeability from the short-read data. In this study, we wanted to understand the challenges we observed with calling the mompS2 allele from short-reads, assess if other short-read methods were able to resolve the mompS-region, and investigate the possibility of using long-reads to obtain the mompS alleles, and thereby perform L. pneumophila SBT from long-reads only. We found that the choice of short-read assembler had a major impact on resolving the mompS-region and thus SBT from short-reads, but no method consistently solved the mompS2 allele. By using Oxford Nanopore Technology (ONT) sequencing together with Trycycler and Medaka for long-read assembly and polishing we were able to resolve the mompS copies and correctly identify the mompS2 allele, in accordance with Sanger sequencing/EQA results for all tested isolates (n=35). The remaining six genes of the SBT profile could also be determined from the ONT-only reads. The STs called from ONT-only assemblies were also consistent with hybrid-assemblies of Illumina and ONT reads. We therefore propose ONT sequencing as an alternative method to perform L. pneumophila SBT to overcome the mompS challenge observed with short-reads. To facilitate this, we have developed ONTmompS (https://github.com/marithetland/ONTmompS), an in silico approach to determine L. pneumophila ST from long-read or hybrid assemblies.

Highly conserved composite transposon harbouring aerobactin iuc3 in Klebsiella pneumoniae from pigs.

Klebsiella pneumoniae is an important opportunistic pathogen associated with severe invasive disease in humans. Hypervirulent K. pneumoniae, which are K. pneumoniae with several acquired virulence determinants such as the siderophore aerobactin and others, are more prominent in countries in South and South-East Asia compared to European countries. This Klebsiella pathotype is capable of causing liver abscesses in immunocompetent persons in the community. K. pneumoniae has not been extensively studied in non-human niches. In the present study, K. pneumoniae isolated from caecal samples (n=299) from healthy fattening pigs in Norway were characterized with regard to population structure and virulence determinants. These data were compared to data from a previous study on K. pneumoniae from healthy pigs in Thailand. Lastly, an in-depth plasmid study on K. pneumoniae with aerobactin was performed. Culturing and whole-genome sequencing was applied to detect, confirm and characterize K. pneumoniae isolates. Phylogenetic analysis described the evolutionary relationship and diversity of the isolates, while virulence determinants and sequence types were detected with Kleborate. Long-read sequencing was applied to obtain the complete sequence of virulence plasmids harbouring aerobactin. A total of 48.8 % of the investigated Norwegian pig caecal samples (n=299) were positive for K. pneumoniae. Acquired virulence determinants were detected in 72.6 % of the isolates, the most prominent being aerobactin (69.2 %), all of which were iuc3. In contrast, only 4.6 % of the isolates from Thailand harboured aerobactin. The aerobactin operon was located on potentially conjugative IncFIBK/FIIK plasmids of varying sizes in isolates from both countries. A putative, highly conserved composite transposon with a mean length of 16.2 kb flanked by truncated IS3-family IS407-group insertion sequences was detected on these plasmids, harbouring the aerobactin operon as well as several genes that may confer increased fitness in mammalian hosts. This putative composite transposon was also detected in plasmids harboured by K. pneumoniae from several countries and sources, such as human clinical samples. The high occurrence of K. pneumoniae harbouring aerobactin in Norwegian pigs, taken together with international data, suggest that pigs are a reservoir for K. pneumoniae with iuc3. Truncation of the flanking ISKpn78-element suggest that the putative composite transposon has been permanently integrated into the plasmid, and that it is no longer mobilizable.

The Performances of Three Commercially Available Assays for the Detection of SARS-CoV-2 Antibodies at Different Time Points Following SARS-CoV-2 Infection.

The aim of this study was to evaluate the performances of three commercially available antibody assays for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies at different time points following SARS-CoV-2 infection. Sera from 536 cases, including 207 SARS-CoV-2 PCR positive, were tested for SARS-CoV-2 antibodies with the Wantai receptor binding domain (RBD) total antibody assay, Liaison S1/S2 IgG assay and Alinity i nucleocapsid IgG assay and compared to a two-step reference ELISA (SARS-CoV-2 RBD IgG and SARS-CoV-2 spike IgG). Diagnostic sensitivity, specificity, predictive values and Cohen's kappa were calculated for the commercial assays. The assay's sensitivities varied greatly, from 68.7% to 95.3%, but the specificities remained high (96.9-99.1%). The three tests showed good performances in sera sampled 31 to 60 days after PCR positivity compared to the reference ELISA. The total antibody test performed better than the IgG tests the first 30 days and the nucleocapsid IgG test showed reduced sensitivity two months or more after PCR positivity. Hence, the test performances at different time points should be taken into consideration in clinical practice and epidemiological studies. Spike or RBD IgG tests are preferable in sera sampled more than two months following SARS-CoV-2 infection.

Insights into the genetic diversity, antibiotic resistance and pathogenic potential of Klebsiella pneumoniae from the Norwegian marine environment using whole-genome analysis.

Klebsiella pneumoniae (Kp) can cause hospital- and community acquired infections. Although, Kp is widespread in the environment, very little is known about the genetic diversity and pathogenicity of Kp from the marine environment. The aim of our study was to understand the genetic diversity, resistome and pathogenic potential of 87 Kp isolates from the Norwegian marine environment, using whole-genome sequencing. We identified 50 sequence types, including globally disseminated sequence types associated with multidrug resistance or hypervirulence. Ten isolates carried the yersiniabactin loci. Acquired antibiotic resistance genes were identified in six Kp isolates. Heavy metal resistance genes were widespread among the isolates, with 71% carrying genes encoding resistance to copper, silver, arsenic, nickel and/or mercury. Co-occurrence of antibiotic resistance genes and heavy metal resistance genes was seen in five Kp isolates. Phylogenetic analysis revealed a close genetic relationship between Kp 2016-1200 ST25 isolated from blue mussels (Mytilus edulis) and a clinical isolate reported in Germany. To the best of our knowledge, this study provides the first comprehensive account of genetic diversity among Kp from the marine environment. Our study reveals high diversity of Kp in the Norwegian marine environment and seafood, including globally disseminated pathogenic sequence types carrying clinically relevant antibiotic resistance genes and virulence factors, as well as several heavy metal resistance genes.

Legionella pneumophila in Municipal Shower Systems in Stavanger, Norway; A Longitudinal Surveillance Study Using Whole Genome Sequencing in Risk Management.

Following an incidence of Legionnaires disease (LD) in 2007, where a municipal shower system was the likely source of infection, Stavanger municipality initiated a surveillance program for Legionella as part of establishing internal risk evaluation and prevention routines. More than 250 shower systems were examined for cultivatable Legionella pneumophila. The prevalence and diversity of serogroups (sg) and sequence types (STs) of L. pneumophila were mapped using available typing techniques over a period of more than 10 years (2010-2021). The surveillance showed an overall reduction in the L. pneumophila colonisation rate in municipal systems from 11 to 4.5% following prevention measures during the period, with the highest colonisation rate in complex systems (e.g., larger nursing homes and sports complexes). Further, an approximately even distribution between sg1 and 2-14 was seen. Whole genome sequencing (WGS) revealed that only a limited number of STs were detected, and they were consistent at specific locations over time. This study showed that environmental surveillance data in combination with available typing techniques and WGS can give the municipality a better tool for risk management and an overview of ST distributions that can be a valuable asset in future source investigations.

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.

Exploring Klebsiella pneumoniae in Healthy Poultry Reveals High Genetic Diversity, Good Biofilm-Forming Abilities and Higher Prevalence in Turkeys Than Broilers.

Klebsiella pneumoniae is a well-studied human pathogen for which antimicrobial resistant and hypervirulent clones have emerged globally. K. pneumoniae is also present in a variety of environmental niches, but currently there is a lack of knowledge on the occurrence and characteristics of K. pneumoniae from non-human sources. Certain environmental niches, e.g., animals, may be associated with high K. pneumoniae abundance, and these can constitute a reservoir for further transmission of strains and genetic elements. The aim of this study was to explore and characterize K. pneumoniae from healthy broilers and turkeys. A total of 511 cecal samples (broiler n = 356, turkey n = 155), included in the Norwegian monitoring program for antimicrobial resistance (AMR) in the veterinary sector (NORM-VET) in 2018, were screened for K. pneumoniae by culturing on SCAI agar. K. pneumoniae was detected in 207 (40.5%) samples. Among the broiler samples, 25.8% were positive for K. pneumoniae, in contrast to turkey with 74.2% positive samples (p < 0.01). Antibiotic susceptibility testing was performed, in addition to investigating biofilm production. Whole genome sequencing was performed on 203 K. pneumoniae isolates, and analysis was performed utilizing comparative genomics tools. The genomes grouped into 66 sequence types (STs), with ST35, ST4710 and ST37 being the most prevalent at 13.8%, 7.4%, and 5.4%, respectively. The overall AMR occurrence was low, with only 11.3% of the isolates showing both pheno- and genotypic resistance. Genes encoding aerobactin, salmochelin or yersiniabactin were detected in 47 (23.2%) genomes. Fifteen hypervirulent genomes belonging to ST4710 and isolated from turkey were identified. These all encoded the siderophore virulence loci iuc5 and iro5 on an IncF plasmid. Isolates from both poultry species displayed good biofilm-forming abilities with an average of OD595 0.69 and 0.64. To conclude, the occurrence of K. pneumoniae in turkey was significantly higher than in broiler, indicating that turkey might be an important zoonotic reservoir for K. pneumoniae compared to broilers. Furthermore, our results show a highly diverse K. pneumoniae population in poultry, low levels of antimicrobial resistance, good biofilm-forming abilities and a novel hypervirulent ST4710 clone circulating in the turkey population.

Rapid high-resolution detection of colistin resistance in Gram-negative bacteria using flow cytometry: a comparison with broth microdilution, a commercial screening test and WGS.

BACKGROUND: Even though both EUCAST and CLSI consider broth microdilution (BMD) as the reference method for antimicrobial susceptibility testing (AST) of colistin, the method exhibits potential flaws related to properties of the colistin molecule. OBJECTIVES: To develop a flow cytometry method (FCM) for colistin AST and to validate it against BMD, a commercial screening test and WGS. METHODS: Colistin-mediated loss of membrane integrity in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. was detected with the fluorescent probe YoPro-1 by FCM. An international collection of 65 resistant and 109 susceptible isolates were analysed and the colistin concentration required to reach the EC50 was compared with the BMD MIC and the presence of genotypic resistance markers. RESULTS: The overall FCM sensitivity and specificity for colistin resistance was 89% and 94%, with E. coli > K. pneumoniae > P. aeruginosa, whereas the performance for Acinetobacter spp. was poor. All tested E. coli were correctly categorized. Three K. pneumoniae isolates with genotypic findings consistent with colistin resistance were detected by FCM but not BMD. Compared with BMD, FCM delivered AST results with a 75% reduction of time. CONCLUSIONS: Here, we present a rapid FCM-based AST assay for qualitative and quantitative testing of colistin resistance in E. coli and K. pneumoniae. The assay revealed probable chromosomal colistin resistance in K. pneumoniae that was not detected by BMD. If confirmed, these results question the reliability of BMD for colistin testing.

Gastrointestinal carriage of Klebsiella pneumoniae in a general adult population: a cross-sectional study of risk factors and bacterial genomic diversity.

Antibiotic resistant Klebsiella pneumoniae is a leading public health threat and gastrointestinal carriage is an established risk factor for subsequent infections during hospitalization. Our study contributes new knowledge of risk factors for gastrointestinal carriage and the genomic population structure of K. pneumoniae colonizing humans in a representative sample of a general population in a community setting. Altogether, 2,975 participants (54% women) >40 y in the population-based Tromsø Study: Tromsø7, Norway (2015-2016) were included. Fecal samples were screened for K. pneumoniae, which were characterized using whole-genome sequencing. Risk factors for carriage were analyzed using multivariable logistic regression on data from questionnaires and the Norwegian Prescription Database. Prevalence of K. pneumoniae gastrointestinal carriage was 16.3% (95% CI 15.0-17.7, no gender difference). Risk factors associated with carriage included age ≥60 y, travel to Greece or Asia past 12 months (adjusted odds ratio 1.49, 95% CI 1.11-2.00), Crohn's disease/ulcerative colitis (2.26, 1.20-4.27), use of proton pump inhibitors (1.62, 1.18-2.22) and non-steroidal anti-inflammatory drugs past 6 months (1.38, 1.04-1.84), and antibiotic use the last month (1.73, 1.05-2.86). Prevalence was higher among those having used combinations of drug classes and decreased over time with respect to preceding antibiotic use. The K. pneumoniae population was diverse with 300 sequence types among 484 isolates distributed across four phylogroups. Only 5.2% of isolates harbored acquired resistance and 11.6% had virulence factors. Identification of risk factors for gastrointestinal carriage allows for identification of individuals that may have higher risk of extraintestinal infection during hospitalization. The findings that specific diseases and drugs used were associated with carriage show an impact of these possibly through modulating the human gut microbiota promoting colonization. The diverse population structure of carriage isolates reflects the ecologically adaptive capacity of the bacterium and challenges for vaccine prospects and the identification of reservoirs as a potential source for human colonization.

Use of probiotics to reduce infections and death and prevent colonization with extended-spectrum beta-lactamase (ESBL)-producing bacteria among newborn infants in Tanzania (ProRIDE Trial): study protocol for a randomized controlled clinical trial.

BACKGROUND: Extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) has emerged as an urgent global health threat and is by the World Health Organization ranked as priority 1 among pathogens in need of new treatment. Studies have shown high mortality in Tanzanian children with ESBL-E infections. Gut colonization of ESBL-E, which is a potential risk factor of ESBL-E infections, is reported to be very high among children in Tanzania. Probiotics may potentially reduce gut colonization of multidrug-resistant bacteria. However, there is limited data on whether probiotics may reduce ESBL-E carriage in infants. The ProRIDE Trial aims to evaluate whether the use of probiotics can reduce morbidity and mortality among infants in Haydom, Tanzania, and whether this effect is associated with a reduction in ESBL-E colonization and/or infections. METHODS/DESIGN: This large randomized double-blinded placebo-controlled trial aims to recruit 2000 newborn infants at Haydom Lutheran Hospital and the surrounding area in the period of November 2020 to November 2021. Participants will be enrolled from days 0 to 3 after birth and randomized to receive probiotics or placebo for 4 weeks. Participants will be followed-up for 6 months, during which three visits will be made to collect clinical and demographic information, as well as rectal swabs and fecal samples which will be subjected to laboratory analysis. The primary composite outcome is the prevalence of death and/or hospitalization at 6 months of age. DISCUSSION: As the use of probiotics may give a more favorable gut composition, and thereby improve health and reduce morbidity and mortality, the results may have implications for future therapy guidelines in Africa and internationally. TRIAL REGISTRATION: ClinicalTrials.gov NCT04172012. Registered on November 21, 2019.

Emergence and dissemination of antimicrobial resistance in Escherichia coli causing bloodstream infections in Norway in 2002-17: a nationwide, longitudinal, microbial population genomic study.

BACKGROUND: The clonal diversity underpinning trends in multidrug resistant Escherichia coli causing bloodstream infections remains uncertain. We aimed to determine the contribution of individual clones to resistance over time, using large-scale genomics-based molecular epidemiology. METHODS: This was a longitudinal, E coli population, genomic, cohort study that sampled isolates from 22 512 E coli bloodstream infections included in the Norwegian surveillance programme on resistant microbes (NORM) from 2002 to 2017. 15 of 22 laboratories were able to share their isolates, and the first 22·5% of isolates from each year were requested. We used whole genome sequencing to infer the population structure (PopPUNK), and we investigated the clade composition of the dominant multidrug resistant clonal complex (CC)131 using genetic markers previously reported for sequence type (ST)131, effective population size (BEAST), and presence of determinants of antimicrobial resistance (ARIBA, PointFinder, and ResFinder databases) over time. We compared these features between the 2002-10 and 2011-17 time periods. We also compared our results with those of a longitudinal study from the UK done between 2001 and 2011. FINDINGS: Of the 3500 isolates requested from the participating laboratories, 3397 (97·1%) were received, of which 3254 (95·8%) were successfully sequenced and included in the analysis. A significant increase in the number of multidrug resistant CC131 isolates from 71 (5·6%) of 1277 in 2002-10 to 207 (10·5%) of 1977 in 2011-17 (p<0·0001), was the largest clonal expansion. CC131 was the most common clone in extended-spectrum ß-lactamase (ESBL)-positive isolates (75 [58·6%] of 128) and fluoroquinolone non-susceptible isolates (148 [39·2%] of 378). Within CC131, clade A increased in prevalence from 2002, whereas the global multidrug resistant clade C2 was not observed until 2007. Multiple de-novo acquisitions of both blaCTX-M ESBL-encoding genes in clades A and C1 and gain of phenotypic fluoroquinolone non-susceptibility across the clade A phylogeny were observed. We estimated that exponential increases in the effective population sizes of clades A, C1, and C2 occurred in the mid-2000s, and in clade B a decade earlier. The rate of increase in the estimated effective population size of clade A (Ne=3147) was nearly ten-times that of C2 (Ne=345), with clade A over-represented in Norwegian CC131 isolates (75 [27·0%] of 278) compared with the UK study (8 [5·4%] of 147 isolates). INTERPRETATION: The early and sustained establishment of predominantly antimicrobial susceptible CC131 clade A isolates, relative to multidrug resistant clade C2 isolates, suggests that resistance is not necessary for clonal success. However, even in the low antibiotic use setting of Norway, resistance to important antimicrobial classes has rapidly been selected for in CC131 clade A isolates. This study shows the importance of genomic surveillance in uncovering the complex ecology underlying multidrug resistance dissemination and competition, which have implications for the design of strategies and interventions to control the spread of high-risk multidrug resistant clones. FUNDING: Trond Mohn Foundation, European Research Council, Marie Sklodowska-Curie Actions, and the Wellcome Trust.

Microbial risk factors for treatment failure of pivmecillinam in community-acquired urinary tract infections caused by ESBL-producing Escherichia coli.

The aim of this study was to identify microbial risk factors for treatment failure of pivmecillinam in community-acquired urinary tract infections (ca-UTIs) caused by ESBL-producing Escherichia coli. Eighty-nine ESBL-producing E. coli isolated from women suffering from ca-UTIs were included. The susceptibilities to mecillinam were determined using MIC gradient strip. Whole genome sequencing was performed on a MiSeq platform, and genome assembly was performed using SPAdes v3.11.0. Neither mecillinam MICs nor ESBL genotypes were associated with treatment outcome of patients treated with pivmecillinam. Specific STs, however, showed significant differences in treatment outcome. Patients infected with ST131 were more likely to experience treatment failure compared to patients infected with non-ST131 (p 0.02) when adjusted for pivmecillinam dose, mecillinam MIC and severity of infection. Patients infected with ST69 were more often successfully treated compared to patients infected with non-ST69 (p 0.04). Patients infected with blaCTX-M-15 ST131 strains were more likely to experience treatment failure than those infected with non-blaCTX-M-15 ST131 strains (p 0.02). The results suggest that specific STs are associated with the clinical efficacy of pivmecillinam. Further studies with a larger number of strains, including a larger number of mecillinam resistant strains, are needed to confirm these results.