Dissemination of carbapenemase-producing Enterobacterales through wastewater and gulls at a wastewater treatment plant in Sweden.

Here we report the detection of carbapenemase-producing Enterobacterales (CPE) isolated from Swedish wastewater and gull faeces. CPE have not been detected in samples from animals in Sweden preceding this report. Sampling of wastewater treatment plant (WWTP) inlet and outlet, sedimentation basins, surface seawater from key aquatic bird habitats and freshly deposited gull faeces was done on six separate occasions during May to September 2021. Following broth enrichment, selective screening of putative CPE was performed on mSuperCarba™ (CHROMagar). Species identification was done with MALDI-TOF. Antimicrobial susceptibility testing was performed according to EUCAST. In total, seventeen CPE were verified by genome sequencing carrying blaGES-5, blaIMI-3, blaOXA-181 or blaOXA-244. The blaGES-5 was carried on IncP plasmids in four different species; Escherichia coli ST10 isolated from WWTP outlet, Raoultella ornithinolytica isolated from WWTP inlet, outlet and sedimentation basins as well as gull faeces collected at the WWTP and Klebsiella spp. isolates from WWTP inlet and outlet. The genetic environment surrounding blaGES-5 was similar in two Citrobacter freundii causing human infections. The blaIMI-3 was carried on IncFII(Yp) plasmids in four Enterobacter ludwigii, isolated from WWTP outlet and gull faeces collected at a recreational city park 2 km from the WWTP. The blaOXA-181 was located on a COLKP3 plasmid found in an E. coli, while blaOXA-244 was chromosomally located in an E. coli ST10, both isolated from WWTP inlet. Phylogenetic analysis of R. ornithinolytica and E. ludwigii isolates indicate that the gulls carried strains related to those identified in the WWTP samples. The results thus add to the increasing evidence of WWTPs as anthropogenic reservoirs for mobile genetic elements with antibiotic-resistance functionality. Such environments could profoundly impact the dissemination and spread of such genetic elements via for example aquatic birds, thereby warranting further study and surveillance.

Detection of an IMI-2 carbapenemase-producing Enterobacter asburiae at a Swedish feed mill.

Occurrence of multidrug resistant Enterobacteriaceae in livestock is of concern as they can spread to humans. A potential introduction route for these bacteria to livestock could be animal feed. We therefore wanted to identify if Escherichia spp., Enterobacter spp., Klebsiella spp., or Raoutella spp. with transferable resistance to extended spectrum cephalosporins, carbapenems or colistin could be detected in the environment at feed mills in Sweden. A second aim was to compare detected isolates to previous described isolates from humans and animals in Sweden to establish relatedness which could indicate a potential transmission between sectors and feed mills as a source for antibiotic resistant bacteria. However, no isolates with transferable resistance to extended-cephalosporins or colistin could be identified, but one isolate belonging to the Enterobacter cloacae complex was shown to be carbapenem-resistant and showing carbapenemase-activity. Based on sequencing by both short-read Illumina and long-read Oxford Nanopore MinIon technologies it was shown that this isolate was an E. asburiae carrying a bla IMI-2 gene on a 216 Kbp plasmid, designated pSB89A/IMI-2, and contained the plasmid replicons IncFII, IncFIB, and a third replicon showing highest similarity to the IncFII(Yp). In addition, the plasmid contained genes for various functions such as plasmid segregation and stability, plasmid transfer and arsenical transport, but no additional antibiotic resistance genes. This isolate and the pSB89A/IMI-2 was compared to three human clinical isolates positive for bla IMI-2 available from the Swedish antibiotic monitoring program Swedres. It was shown that one of the human isolates carried a plasmid similar with regards to gene content to the pSB89A/IMI-2 except for the plasmid transfer system, but that the order of genes was different. The pSB89A/IMI-2 did however share the same transfer system as the bla IMI-2 carrying plasmids from the other two human isolates. The pSB89A/IMI-2 was also compared to previously published plasmids carrying bla IMI-2, but no identical plasmids could be identified. However, most shared part of the plasmid transfer system and DNA replication genes, and the bla IMI-2 gene was located next the transcription regulator imiR. The IS3-family insertion element downstream of imiR in the pSB89A was also related to the IS elements in other bla IMI-carrying plasmids.

The ddn Trp20Stop Mutation and Its Association with Lineage 4.5 and Resistance to Delamanid and Pretomanid in Mycobacterium tuberculosis.

High-confidence resistance mutations for new and repurposed anti-TB drugs, such as delamanid (DLM) and pretomanid (Pa), are rare and more data are needed in order to correctly interpret the results generated by genotypic drug susceptibility testing. In this study performed on clinical Mycobacterium tuberculosis complex isolates, we report that in the Swedish strain collection the ddn mutation Trp20Stop is found exclusively among DLM and Pa resistant (Pa MIC >16 mg/L) isolates assigned to lineage 4.5.

Cross-Sectional Variations in Structure and Function of Coral Reef Microbiome With Local Anthropogenic Impacts on the Kenyan Coast of the Indian Ocean.

Coral reefs face an increased number of environmental threats from anthropomorphic climate change and pollution from agriculture, industries and sewage. Because environmental changes lead to their compositional and functional shifts, coral reef microbial communities can serve as indicators of ecosystem impacts through development of rapid and inexpensive molecular monitoring tools. Little is known about coral reef microbial communities of the Western Indian Ocean (WIO). We compared taxonomic and functional diversity of microbial communities inhabiting near-coral seawater and sediments from Kenyan reefs exposed to varying impacts of human activities. Over 19,000 species (bacterial, viral and archaeal combined) and 4,500 clusters of orthologous groups of proteins (COGs) were annotated. The coral reefs showed variations in the relative abundances of ecologically significant taxa, especially copiotrophic bacteria and coliphages, corresponding to the magnitude of the neighboring human impacts in the respective sites. Furthermore, the near-coral seawater and sediment metagenomes had an overrepresentation of COGs for functions related to adaptation to diverse environments. Malindi and Mombasa marine parks, the coral reef sites closest to densely populated settlements were significantly enriched with genes for functions suggestive of mitigation of environment perturbations including the capacity to reduce intracellular levels of environmental contaminants and repair of DNA damage. Our study is the first metagenomic assessment of WIO coral reef microbial diversity which provides a much-needed baseline for the region, and points to a potential area for future research toward establishing indicators of environmental perturbations.

Revisiting colistin susceptibility testing: will adding calcium to Mueller-Hinton agar improve the detection of colistin resistance?

OBJECTIVE: The aim was to investigate whether adding calcium to Mueller-Hinton agar for gradient MIC or disc diffusion tests could improve separation between colistin-susceptible and -resistant populations of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. and if this method could provide a reliable screening test for colistin resistance in routine laboratories. METHODS: An isolate collection of 57 E. coli, K. pneumoniae, P. aeruginosa and Acinetobacter spp. was tested. Ca2+ in concentrations from 2.5 to 40 mM was added to the Mueller-Hinton agar plates used for gradient MIC and disc diffusion tests. Broth microdilution (ISO 20776-1) MIC determination was used as reference. Escherichia coli and K. pneumoniae were investigated for colistin resistance genes. RESULTS: Results were similar for gradient tests and disc diffusion for all species. Correlation between phenotypic expression of resistance and resistance genes was not absolute. Addition of Ca2+ to Mueller-Hinton agar improved separation between colistin-susceptible and -resistant isolates for E. coli. For K. pneumoniae, separation was improved for isolates with mcr genes, but not for isolates harbouring other colistin resistance mechanisms. To further increase the concentrations of Ca2+ did not improve the separation between susceptible and resistant isolates of E. coli and K. pneumoniae. For P. aeruginosa and Acinetobacter species, addition of Ca2+ did not improve separation between susceptible and resistant populations. DISCUSSION: The results from this study show that addition of Ca2+ to the Mueller-Hinton agar does not sufficiently improve detection of colistin resistance by gradient MIC or disc diffusion tests for use in a routine laboratory.

Field-Adapted Full Genome Sequencing of Peste-Des-Petits-Ruminants Virus Using Nanopore Sequencing.

Peste-des-petits-ruminants virus (PPRV) is currently the focus of a control and eradication program. Full genome sequencing has the opportunity to become a powerful tool in the eradication program by improving molecular epidemiology and the study of viral evolution. PPRV is prevalent in many resource-constrained areas, with long distances to laboratory facilities, which can lack the correct equipment for high-throughput sequencing. Here we present a protocol for near full or full genome sequencing of PPRV. The use of a portable miniPCR and MinION brings the laboratory to the field and in addition makes the production of a full genome possible within 24 h of sampling. The protocol has been successfully used on virus isolates from cell cultures and field isolates from tissue samples of naturally infected goats.

Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole.

BACKGROUND: Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways. METHODS: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10-9 M, 10-11 M, 10-13 M), pyrantel citrate (10-6 M, 10-8 M, 10-10 M), thiabendazole (10-5 M, 10-7 M, 10-9 M) or without anthelmintics (control) at 37 °C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system. RESULTS: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj < 0.05 and log2 fold change ≥ 1 or ≤ - 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunit lgc-37, which was upregulated only in worms exposed to 10-9 M of ivermectin. CONCLUSIONS: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described in P. univalens and future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance.

Identification of Transmission Routes of Campylobacter and On-Farm Measures to Reduce Campylobacter in Chicken.

An in-depth analysis was performed on Swedish broiler producers that had delivered chickens with Campylobacter to slaughter over several years, in order to identify possible transmission routes and formulate effective measures to prevent chickens being colonized with Campylobacter. Between 2017 and 2019, 626 samples were collected at farm level and Campylobacter was isolated from 133 (21.2%). All C. jejuni and C. coli isolated from these samples were whole-genome sequenced, together with isolates from the corresponding cecum samples at slaughter (n = 256). Core genome multi-locus sequence typing (cgMLST) analysis, using schemes consisting of 1140 and 529 genes for C. jejuni and C. coli, respectively, revealed that nearby cattle, contaminated drinking water, water ponds, transport crates, and parent flocks were potential reservoirs of Campylobacter. A novel feature compared with previous studies is that measures were implemented and tested during the work. These contributed to a nationwide decrease in Campylobacter-positive flocks from 15.4% in 2016 to 4.6% in 2019, which is the lowest ever rate in Sweden. To conclude, there are different sources and routes of Campylobacter transmission to chickens from different broiler producers, and individual measures must be taken by each producer to prevent Campylobacter colonization of chickens.

Simulating Illumina metagenomic data with InSilicoSeq.

Motivation: The accurate in silico simulation of metagenomic datasets is of great importance for benchmarking bioinformatics tools as well as for experimental design. Users are dependant on large-scale simulation to not only design experiments and new projects but also for accurate estimation of computational needs within a project. Unfortunately, most current read simulators are either not suited for metagenomics, out of date or relatively poorly documented. In this article, we describe InSilicoSeq, a software package to simulate metagenomic Illumina sequencing data. InsilicoSeq has a simple command-line interface and extensive documentation. Results: InSilicoSeq is implemented in Python and capable of simulating realistic Illumina (meta) genomic data in a parallel fashion with sensible default parameters. Availability and implementation: Source code and documentation are available under the MIT license at https://github.com/HadrienG/InSilicoSeq and https://insilicoseq.readthedocs.io/. Supplementary information: Supplementary data are available at Bioinformatics online.

Resistance to pyrantel embonate and efficacy of fenbendazole in Parascaris univalens on Swedish stud farms.

The aims of this study were to determine the species of Parascaris present in foals in Sweden and to establish whether anthelmintic resistance to pyrantel and fenbendazole is present on Swedish stud farms. Ascarid eggs collected from different regions in Sweden were karyotyped and were all identified as Parascaris univalens, characterized by one chromosomal pair. Faecal egg count reduction tests were performed on a total of 142 foals on 9 farms between September 2016 and May 2017. Healthy foals with at least 150 eggs per gram faeces (EPG) were included in the study and treated with oral pastes of pyrantel embonate or fenbendazole according to manufacturer instructions. The efficacy of the drugs was calculated by a Bayesian model using the R package "eggCounts". In accordance with the American Association of Equine Practitioners, parasites were classified as resistant to pyrantel if the reduction in EPG was ≤ 85% and to fenbendazole if the observed efficacy was ≤ 90%. Four of eleven groups treated with pyrantel had an observed efficacy of ≤ 85%, and as many as 43% of the foals treated with pyrantel excreted eggs 10-16 days after treatment. In contrast, one of the six groups treated with fenbendazole had an observed efficacy of ≤ 90%, and only 6% of all foals were excreting eggs 10-16 days after treatment. Since resistance to ivermectin has earlier been shown to be widespread in Parascaris spp. in Sweden it is likely that multiresistant populations are present on Swedish stud farms. This is the first study showing the existence of pyrantel-resistant Parascaris spp. in Europe, and the first ever study where anthelmintic resistance has been shown in P. univalens.

Detection of novel syntrophic acetate-oxidizing bacteria from biogas processes by continuous acetate enrichment approaches.

To enrich syntrophic acetate-oxidizing bacteria (SAOB), duplicate chemostats were inoculated with sludge from syntrophic acetate oxidation (SAO)-dominated systems and continuously supplied with acetate (0.4 or 7.5 g l-1 ) at high-ammonia levels. The chemostats were operated under mesophilic (37°C) or thermophilic (52°C) temperature for about six hydraulic retention times (HRT 28 days) and were sampled over time. Irrespective of temperature, a methane content of 64-69% and effluent acetate level of 0.4-1.0 g l-1 were recorded in chemostats fed high acetate. Low methane production in the low-acetate chemostats indicated that the substrate supply was below the threshold for methanization of acetate via SAO. Novel representatives within the family Clostridiales and genus Syntrophaceticus (class Clostridia) were identified to represent putative SAOB candidates in mesophilic and thermophilic conditions respectively. Known SAOB persisted at low relative abundance in all chemostats. The hydrogenotrophic methanogens Methanoculleus bourgensis (mesophilic) and Methanothermobacter thermautotrophicus (thermophilic) dominated archaeal communities in the high-acetate chemostats. In line with the restricted methane production in the low-acetate chemostats, methanogens persisted at considerably lower abundance in these chemostats. These findings strongly indicate involvement in SAO and tolerance to high ammonia levels of the species identified here, and have implications for understanding community function in stressed anaerobic processes.

MetLab: An In Silico Experimental Design, Simulation and Analysis Tool for Viral Metagenomics Studies.

Metagenomics, the sequence characterization of all genomes within a sample, is widely used as a virus discovery tool as well as a tool to study viral diversity of animals. Metagenomics can be considered to have three main steps; sample collection and preparation, sequencing and finally bioinformatics. Bioinformatic analysis of metagenomic datasets is in itself a complex process, involving few standardized methodologies, thereby hampering comparison of metagenomics studies between research groups. In this publication the new bioinformatics framework MetLab is presented, aimed at providing scientists with an integrated tool for experimental design and analysis of viral metagenomes. MetLab provides support in designing the metagenomics experiment by estimating the sequencing depth needed for the complete coverage of a species. This is achieved by applying a methodology to calculate the probability of coverage using an adaptation of Stevens' theorem. It also provides scientists with several pipelines aimed at simplifying the analysis of viral metagenomes, including; quality control, assembly and taxonomic binning. We also implement a tool for simulating metagenomics datasets from several sequencing platforms. The overall aim is to provide virologists with an easy to use tool for designing, simulating and analyzing viral metagenomes. The results presented here include a benchmark towards other existing software, with emphasis on detection of viruses as well as speed of applications. This is packaged, as comprehensive software, readily available for Linux and OSX users at https://github.com/norling/metlab.