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-resistantKlebsiella 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 Klebsiella 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

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.publishedVersio

Cytokine release from alveolar macrophages exposed to ambient particulate matter: Heterogeneity in relation to size, city and season

BACKGROUND: Several studies have demonstrated an association between exposure to ambient particulate matter (PM) and respiratory and cardiovascular diseases. Inflammation seems to play an important role in the observed health effects. However, the predominant particle component(s) that drives the inflammation is still not fully clarified. In this study representative coarse (2.5–10 μm) and fine (0.1–2.5 μm) particulate samples from a western, an eastern, a northern and a southern European city (Amsterdam, Lodz, Oslo and Rome) were collected during three seasons (spring, summer and winter). All fractions were investigated with respect to cytokine-inducing potential in primary macrophages isolated from rat lung. The results were related to the physical and chemical parameters of the samples in order to disclose possible connections between inflammatory potential and specific characteristics of the particles. RESULTS: Compared on a gram-by gram basis, both site-specific and seasonal variations in the PM-induced cytokine responses were demonstrated. The samples collected in the eastern (Lodz) and southern (Rome) cities appeared to be the most potent. Seasonal variation was most obvious with the samples from Lodz, with the highest responses induced by the spring and summer samples. The site-specific or seasonal variation in cytokine release could not be attributed to variations in any of the chemical parameters. Coarse fractions from all cities were more potent to induce the inflammatory cytokines interleukin-6 and tumour necrosis factor-α than the corresponding fine fractions. Higher levels of specific elements such as iron and copper, some polycyclic aromatic hydrocarbons (PAHs) and endotoxin/lipopolysaccaride seemed to be prevalent in the coarse fractions. However, variations in the content of these components did not reflect the variation in cytokine release induced by the different coarse fractions. Addition of polymyxin B did not affect the particle-induced cytokine release, indicating that the variations in potency among the coarse fractions are not explained by endootoxin. CONCLUSION: The inflammatory potential of ambient PM demonstrated heterogeneity in relation to city and season. The coarse particle fractions were consistently more potent than the respective fine fractions. Though a higher level of some elements, PAH and endotoxin was found in the coarse fractions, the presence of specific components was not sufficient to explain all variations in PM-induced cytokine release

Antibiotic sensitivity screening of Klebsiella spp. and Raoultella spp. isolated from marine bivalve molluscs reveal presence of CTX-M-producing K. pneumoniae

Klebsiella spp. are a major cause of both nosocomial and community acquired infections, with K. pneumoniae being responsible for most human infections. Although Klebsiella spp. are present in a variety of environments, their distribution in the sea and the associated antibiotic resistance is largely unknown. In order to examine prevalence of K. pneumoniae and related species in the marine environment, we sampled 476 batches of marine bivalve molluscs collected along the Norwegian coast. From these samples, K. pneumoniae (n = 78), K. oxytoca (n = 41), K. variicola (n = 33), K. aerogenes (n = 1), Raoultella ornithinolytica (n = 38) and R. planticola (n = 13) were isolated. The number of positive samples increased with higher levels of faecal contamination. We found low prevalence of acquired resistance in all isolates, with seven K. pneumoniae isolates showing resistance to more than one antibiotic class. The complete genome sequence of cefotaxime-resistant K. pneumoniae sensu stricto isolate 2016-1400 was obtained using Oxford Nanopore and Illumina MiSeq based sequencing. The 2016-1400 genome had two contigs, one chromosome of 5,088,943 bp and one plasmid of 191,744 bp and belonged to ST1035. The β-lactamase genes blaCTX-M-3 and blaTEM-1, as well as the heavy metal resistance genes pco, ars and sil were carried on a plasmid highly similar to one found in K. pneumoniae strain C17KP0055 from South-Korea recovered from a blood stream infection. The present study demonstrates that K. pneumoniae are prevalent in the coastal marine environment and that bivalve molluscs may act as a potential reservoir of extended spectrum β-lactamase (ESBL)-producing K. pneumoniae that may be transmitted through the food chain.publishedVersio

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.publishedVersio

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.publishedVersio

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.publishedVersio

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

Emergence and rapid global dissemination of CTX-M-15-associated Klebsiella pneumoniae strain ST307

Objectives: Recent reports indicate the emergence of a new carbapenemase-producing Klebsiella pneumoniae clone, ST307. We sought to better understand the global epidemiology and evolution of this clone and evaluate its association with antimicrobial resistance (AMR) genes. Methods: We collated information from the literature and public databases and performed a comparative analysis of 95 ST307 genomes (including 37 that were newly sequenced). Results: We show that ST307 emerged in the mid-1990s (nearly 20 years prior to its first report), is already globally distributed and is intimately associated with a conserved plasmid harbouring the blaCTX-M-15 ESBL gene and several other AMR determinants. Conclusions: Our findings support the need for enhanced surveillance of this widespread ESBL clone in which carbapenem resistance has occasionally emerged.publishedVersio

Comparison of organic and conventional food and food Production. Part V: Human health – pesticide residues

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