Characterising the gut microbiome of stranded harbour seals (Phoca vitulina) in rehabilitation.

Animal rehabilitation centres provide a unique opportunity to study the microbiome of wild animals because subjects will be handled for their treatment and can therefore be sampled longitudinally. However, rehabilitation may have unintended consequences on the animals' microbiome because of a less varied and suboptimal diet, possible medical treatment and exposure to a different environment and human handlers. Our study describes the gut microbiome of two large seal cohorts, 50 pups (0-30 days old at arrival) and 23 weaners (more than 60 days old at arrival) of stranded harbour seals admitted for rehabilitation at the Sealcentre Pieterburen in the Netherlands, and the effect of rehabilitation on it. Faecal samples were collected from all seals at arrival, two times during rehabilitation and before release. Only seals that did not receive antimicrobial treatment were included in the study. The average time in rehabilitation was 95 days for the pups and 63 days for the weaners. We observed that during rehabilitation, there was an increase in the relative abundance of some of the Campylobacterota spp and Actinobacteriota spp. The alpha diversity of the pups' microbiome increased significantly during their rehabilitation (p-value <0.05), while there were no significant changes in alpha diversity over time for weaners. We hypothesize that aging is the main reason for the observed changes in the pups' microbiome. At release, the sex of a seal pup was significantly associated with the microbiome's alpha (i.e., Shannon diversity was higher for male pups, p-value <0.001) and beta diversity (p-value 0.001). For weaners, variation in the microbiome composition (beta diversity) at release was partly explained by sex and age of the seal (p-values 0.002 and 0.003 respectively). We mainly observed variables known to change the gut microbiome composition (e.g., age and sex) and conclude that rehabilitation in itself had only minor effects on the gut microbiome of seal pups and seal weaners.

Characterization of the flanking region of the Shiga toxin operon in Stx2a bacteriophages reveals a diversity of the NanS-p sialate O-acetylesterase gene.

Shiga toxin-producing E. coli (STEC) are diarrheagenic strains that can cause bloody diarrhea and hemolytic-uremic syndrome. Their main virulence factor, the Shiga toxin (Stx), is encoded by phages integrated into the bacterial chromosome. Stx phages are widely diverse and carry many genes with limited or unknown function. As the toxin subtype Stx2a is associated with highly pathogenic strains, this study was mainly focused on the characterization of the stx flanking region of Stx2a phages. Of particular interest was a sialate O-acetylesterase (NanS-p), which has been described previously to be encoded downstream stx in some phage genomes and may confer a growth advantage for STEC. Complete DNA sequences of Stx2a phages and prophages were retrieved from the GenBank database, and the genomic regions from anti-terminator Q to holin S genes were bioinformatically analyzed. Predicted NanSp sequences from phages encoding other Stx subtypes were also studied. Additionally, expression of nanS-p was quantified by qPCR in strains selected from our laboratory collection. The analysis of Stx2a phage genomes showed that all carried the Q, stx2a, nanS-p and S genes, but with allele diversity and other sequence differences. In particular, sequence differences were detected in each of the three domains of NanS-p esterases encoded by Stx2a phages and other Stx phages; however, nanS-p was not identified in the Stx2e, Stx2f and Stx2g phages analyzed. The expression of nanS-p increased in most stx2a-positive strains under phage inducing conditions, as was previously shown for stx2a. As the present work showed diversity at the Q-S region among Stx phages, and particularly in the encoded NanS-p enzyme, future studies will be necessary to evaluate if NanS-p variants differ in their activity and to assess the impact of the absence of nanS-p in certain Stx phages.

Widespread Detection of Yersiniabactin Gene Cluster and Its Encoding Integrative Conjugative Elements (ICEKp) among Nonoutbreak OXA-48-Producing Klebsiella pneumoniae Clinical Isolates from Spain and the Netherlands.

In this study, we determined the presence of virulence factors in nonoutbreak, high-risk clones and other isolates belonging to less common sequence types associated with the spread of OXA-48-producing Klebsiella pneumoniae clinical isolates from The Netherlands (n = 61) and Spain (n = 53). Most isolates shared a chromosomally encoded core of virulence factors, including the enterobactin gene cluster, fimbrial fim and mrk gene clusters, and urea metabolism genes (ureAD). We observed a high diversity of K-Locus and K/O loci combinations, KL17 and KL24 (both 16%), and the O1/O2v1 locus (51%) being the most prevalent in our study. The most prevalent accessory virulence factor was the yersiniabactin gene cluster (66.7%). We found seven yersiniabactin lineages-ybt 9, ybt 10, ybt 13, ybt 14, ybt 16, ybt 17, and ybt 27-which were chromosomally embedded in seven integrative conjugative elements (ICEKp): ICEKp3, ICEKp4, ICEKp2, ICEKp5, ICEKp12, ICEKp10, and ICEKp22, respectively. Multidrug-resistant lineages-ST11, ST101, and ST405-were associated with ybt 10/ICEKp4, ybt 9/ICEKp3, and ybt 27/ICEKp22, respectively. The fimbrial adhesin kpi operon (kpiABCDEFG) was predominant among ST14, ST15, and ST405 isolates, as well as the ferric uptake system kfuABC, which was also predominant among ST101 isolates. No convergence of hypervirulence and resistance was observed in this collection of OXA-48-producing K. pneumoniae clinical isolates. Nevertheless, two isolates, ST133 and ST792, were positive for the genotoxin colibactin gene cluster (ICEKp10). In this study, the integrative conjugative element, ICEKp, was the major vehicle for yersiniabactin and colibactin gene clusters spreading. IMPORTANCE Convergence of multidrug resistance and hypervirulence in Klebsiella pneumoniae isolates has been reported mostly related to sporadic cases or small outbreaks. Nevertheless, little is known about the real prevalence of carbapenem-resistant hypervirulent K. pneumoniae since these two phenomena are often separately studied. In this study, we gathered information on the virulent content of nonoutbreak, high-risk clones (i.e., ST11, ST15, and ST405) and other less common STs associated with the spread of OXA-48-producing K. pneumoniae clinical isolates. The study of virulence content in nonoutbreak isolates can help us to expand information on the genomic landscape of virulence factors in K. pneumoniae population by identifying virulence markers and their mechanisms of spread. Surveillance should focus not only on antimicrobial resistance but also on virulence characteristics to avoid the spread of multidrug and (hyper)virulent K. pneumoniae that may cause untreatable and more severe infections.

Longitudinal study of the short- and long-term effects of hospitalisation and oral trimethoprim-sulfadiazine administration on the equine faecal microbiome and resistome.

BACKGROUND: Hospitalisation and antimicrobial treatment are common in horses and significantly impact the intestinal microbiota. Antimicrobial treatment might also increase levels of resistant bacteria in faeces, which could spread to other ecological compartments, such as the environment, other animals and humans. In this study, we aimed to characterise the short- and long-term effects of transportation, hospitalisation and trimethoprim-sulfadiazine (TMS) administration on the faecal microbiota and resistome of healthy equids. METHODS: In a longitudinal experimental study design, in which the ponies served as their own control, faecal samples were collected from six healthy Welsh ponies at the farm (D0-D13-1), immediately following transportation to the hospital (D13-2), during 7 days of hospitalisation without treatment (D14-D21), during 5 days of oral TMS treatment (D22-D26) and after discharge from the hospital up to 6 months later (D27-D211). After DNA extraction, 16S rRNA gene sequencing was performed on all samples. For resistome analysis, shotgun metagenomic sequencing was performed on selected samples. RESULTS: Hospitalisation without antimicrobial treatment did not significantly affect microbiota composition. Oral TMS treatment reduced alpha-diversity significantly. Kiritimatiellaeota, Fibrobacteres and Verrucomicrobia significantly decreased in relative abundance, whereas Firmicutes increased. The faecal microbiota composition gradually recovered after discontinuation of TMS treatment and discharge from the hospital and, after 2 weeks, was more similar to pre-treatment composition than to composition during TMS treatment. Six months later, however, microbiota composition still differed significantly from that at the start of the study and Spirochaetes and Verrucomicrobia were less abundant. TMS administration led to a significant (up to 32-fold) and rapid increase in the relative abundance of resistance genes sul2, tetQ, ant6-1a, and aph(3")-lb. lnuC significantly decreased directly after treatment. Resistance genes sul2 (15-fold) and tetQ (six-fold) remained significantly increased 6 months later. CONCLUSIONS: Oral treatment with TMS has a rapid and long-lasting effect on faecal microbiota composition and resistome, making the equine hindgut a reservoir and potential source of resistant bacteria posing a risk to animal and human health through transmission. These findings support the judicious use of antimicrobials to minimise long-term faecal presence, excretion and the spread of antimicrobial resistance in the environment. Video Abstract.

Prevalence of foodborne and zoonotic viral pathogens in raw cow milk samples.

Foodborne and zoonotic viral pathogens are responsible for substantial morbidity and mortality worldwide. These viruses can be transmitted through foods such as dairy products to humans and cause several acute and chronic diseases. This study aimed to investigate the prevalence and profile of different foodborne and zoonotic viruses in raw cow milk samples. We collected 492 raw cow milk samples from local dairy markets in Qazvin, Iran. Then we evaluated the presence of hepatitis A virus, noroviruses, rotavirus, astrovirus, bovine leukaemia virus (BLV) and tick-borne encephalitis virus (TBEV) in samples using conventional and nested reverse transcription-polymerase chain reaction methods. We found that 34.95, 7.72, 25.81, 14.63, 66.86, 12.80 and 21.34% of raw milk samples were contaminated with norovirus GI, norovirus GII, hepatitis A virus, rotavirus, astrovirus, BLV and TBEV viruses, respectively. Interestingly, the samples collected from the city's south area revealed a higher prevalence of foodborne and zoonotic viruses. Astrovirus and its combination with norovirus GI were the most prevalent virus profiles. Also, the highest correlations were observed among the presence of rotavirus and hepatitis A viruses (0.36) and TBEV and norovirus GII (0.31). Considering the prevalence rate and virus profiles of different foodborne and zoonotic viruses in raw milk samples, hygiene practices and the pasteurization process are strongly suggested to be conducted throughout the cow milk production chain and in dairy industries to prevent infections with these pathogens.

Host DNA depletion can increase the sensitivity of Mycobacterium spp. detection through shotgun metagenomics in sputum.

Identification and phenotypic drug-susceptibility testing for mycobacteria are time-consuming and challenging but essential for managing mycobacterial infections. Next-generation sequencing (NGS) technologies can increase diagnostic speed and quality, but standardization is still lacking for many aspects (e.g., unbiased extraction, host depletion, bioinformatic analysis). Targeted PCR approaches directly on sample material are limited by the number of targets that can be included. Unbiased shotgun metagenomics on direct material is hampered by the massive amount of host DNA, which should be removed to improve the microbial detection sensitivity. For this reason, we developed a method for NGS-based diagnosis of mycobacteria directly from patient material. As a model, we used the non-tuberculous mycobacterium (NTM) Mycobacterium abscessus. We first compared the efficiency of three different DNA extraction kits for isolating DNA (quality and concentration). The two most efficient kits were then used in a follow-up study using artificial sputum. Finally, one extraction kit was selected and further evaluated for DNA isolation from a patients' sputum mixture spiked with M. abscessus at three concentrations (final concentrations 108, 107, 106 CFU/ml). The spiked sputum samples were processed with and without saponin treatment (ST) in combination with DNAse treatment prior to bacterial DNA extraction to evaluate the recovery of bacteria and depletion of host DNA by PCR and Illumina sequencing. While Ct values of the qPCR targeting mycobacterial ITS DNA remained rather stable, Ct values in the qPCR targeting the human ß-actin gene increased by five Ct values in ST samples. In subsequent Illumina sequencing, a decrease of 89% of reads mapped to the human genome was observed in ST samples. The percentage of reads mapped to M. abscessus (108 CFU/ml) increased by 89%, and the sequencing depth increased two times when undergoing ST. In conclusion, the sensitivity of M. abscessus detection in artificial sputum was increased using a saponin pre-treatment step. The saponin followed by the DNase I treatment approach could be efficiently applied to detect and characterize mycobacterial infections, including tuberculosis, directly from sputum.

Antibiotic Resistance and Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Infant Formula Milk.

BACKGROUND: Cronobacter sakazakii is a new emerging foodborne bacterial pathogen associated with severe lethal diseases such as meningitis, necrotizing enterocolitis, and septicemia in infants and neonates. Powdered infant formula milk (PIFM) has been recognized as one of the main transmission vehicles and contaminated sources of this pathogen. This study aimed to investigate the prevalence rate, genotypic and phenotypic antibiotic resistance profile, and clonal relatedness of C. sakazakii strains isolated from 364 PIFM samples collected from Tehran city, Iran. METHODS: Culture-based methods, Kirby-Bauer disk diffusion antibiotic resistance testing, conventional Polymerase Chain Reaction (PCR), and Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) assays were used in this study to detect and characterize the C. sakazakii isolates. RESULTS: We isolated 25 C. sakazakii strains from PIFM samples (6.86%). The isolates were highly resistant to amoxicillin-clavulanic acid, amoxicillin, ampicillin, cefoxitin, cefepime, erythromycin, ceftriaxone, ciprofloxacin, and chloramphenicol and susceptible to gentamicin, tetracycline, norfloxacin, and azithromycin antibiotics. The blaCTX-M-1 gene was detected in 96% of the isolates. The isolates were categorized into eight distinct clonal types using the ERIC-PCR method, showing a high genetic diversity among the isolates. However, there was a significant correlation between the genotypic and phenotypic antibiotic resistance properties of the isolates. CONCLUSIONS: Novel microbial surveillance systems for detecting multi-drug-resistant C. sakazakii are required to control the contamination of this foodborne pathogen in infant foods.

Exploring a prolonged enterovirus C104 infection in a severely ill patient using nanopore sequencing.

Chronic enterovirus infections can cause significant morbidity, particularly in immunocompromised patients. This study describes a fatal case associated with a chronic untypeable enterovirus infection in an immunocompromised patient admitted to a Dutch university hospital over nine months. We aimed to identify the enterovirus genotype responsible for the infection and to determine potential evolutionary changes. Long-read sequencing was performed using viral targeted sequence capture on four respiratory and one faecal sample. Phylogenetic analysis was performed using a maximum likelihood method, along with a root-to-tip regression and time-scaled phylogenetic analysis to estimate evolutionary changes between sample dates. Intra-host variant detection, using a Fixed Ploidy algorithm, and selection pressure, using a Fixed Effect Likelihood and a Mixed Effects Model of Evolution, were also used to explore the patient samples. Near-complete genomes of enterovirus C104 (EV-C104) were recovered in all respiratory samples but not in the faecal sample. The recovered genomes clustered with a recently reported EV-C104 from Belgium in August 2018. Phylodynamic analysis including ten available EV-C104 genomes, along with the patient sequences, estimated the most recent common ancestor to occur in the middle of 2005 with an overall estimated evolution rate of 2.97 × 10-3 substitutions per year. Although positive selection pressure was identified in the EV-C104 reference sequences, the genomes recovered from the patient samples alone showed an overall negative selection pressure in multiple codon sites along the genome. A chronic infection resulting in respiratory failure from a relatively rare enterovirus was observed in a transplant recipient. We observed an increase in single-nucleotide variations between sample dates from a rapidly declining patient, suggesting mutations are weakly deleterious and have not been purged during selection. This is further supported by the persistence of EV-C104 in the patient, despite the clearance of other viral infections. Next-generation sequencing with viral enrichment could be used to detect and characterise challenging samples when conventional workflows are insufficient.

MALDI-TOF MS Using a Custom-Made Database, Biomarker Assignment, or Mathematical Classifiers Does Not Differentiate Shigella spp. and Escherichia coli.

Shigella spp. and E. coli are closely related and cannot be distinguished using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) with commercially available databases. Here, three alternative approaches using MALDI-TOF MS to identify and distinguish Shigella spp., E. coli, and its pathotype EIEC were explored and evaluated using spectra of 456 Shigella spp., 42 E. coli, and 61 EIEC isolates. Identification with a custom-made database resulted in >94% Shigella identified at the genus level and >91% S. sonnei and S. flexneri at the species level, but the distinction of S. dysenteriae, S. boydii, and E. coli was poor. With biomarker assignment, 98% S. sonnei isolates were correctly identified, although specificity was low. Discriminating markers for S. dysenteriae, S. boydii, and E. coli were not assigned at all. Classification models using machine learning correctly identified Shigella in 96% of isolates, but most E. coli isolates were also assigned to Shigella. None of the proposed alternative approaches were suitable for clinical diagnostics for identifying Shigella spp., E. coli, and EIEC, reflecting their relatedness and taxonomical classification. We suggest the use of MALDI-TOF MS for the identification of the Shigella spp./E. coli complex, but other tests should be used for distinction.

Application of shotgun metagenomics sequencing and targeted sequence capture to detect circulating porcine viruses in the Dutch-German border region.

Porcine viruses have been emerging in recent decades, threatening animal and human health, as well as economic stability for pig farmers worldwide. Next-generation sequencing (NGS) can detect and characterize known and unknown viruses but has limited sensitivity when an unbiased approach, such as shotgun metagenomics sequencing, is used. To increase the sensitivity of NGS for the detection of viruses, we applied and evaluated a broad viral targeted sequence capture (TSC) panel and compared it to an unbiased shotgun metagenomic approach. A cohort of 36 pooled porcine nasal swab and blood serum samples collected from both sides of the Dutch-German border region were evaluated. Overall, we detected 46 different viral species using TSC, compared to 40 viral species with a shotgun metagenomics approach. Furthermore, we performed phylogenetic analysis on recovered influenza A virus (FLUAV) genomes from Germany and revealed a close similarity to a zoonotic influenza strain previously detected in the Netherlands. Although TSC introduced coverage bias within the detected viruses, it improved sensitivity, genome sequence depth and contig length. In-depth characterization of the swine virome, coupled with developing new enrichment techniques, can play a crucial role in the surveillance of circulating porcine viruses and emerging zoonotic pathogens.

Future potential of metagenomics in microbiology laboratories.

Rapid and sensitive diagnostic strategies are necessary for patient care and public health. Most of the current conventional microbiological assays detect only a restricted panel of pathogens at a time or require a microbe to be successfully cultured from a sample. Clinical metagenomics next-generation sequencing (mNGS) has the potential to unbiasedly detect all pathogens in a sample, increasing the sensitivity for detection and enabling the discovery of unknown infectious agents. High expectations have been built around mNGS; however, this technique is far from widely available. This review highlights the advances and currently available options in terms of costs, turnaround time, sensitivity, specificity, validation, and reproducibility of mNGS as a diagnostic tool in clinical microbiology laboratories. The need for a novel diagnostic tool to increase the sensitivity of microbial diagnostics is clear. mNGS has the potential to revolutionize clinical microbiology. However, its role as a diagnostic tool has yet to be widely established, which is crucial for successfully implementing the technique. A clear definition of diagnostic algorithms that include mNGS is vital to show clinical utility. Similarly to real-time PCR, mNGS will one day become a vital tool in any testing algorithm.

Long-read sequencing-based in silico phage typing of vancomycin-resistant Enterococcus faecium.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are successful nosocomial pathogens able to cause hospital outbreaks. In the Netherlands, core-genome MLST (cgMLST) based on short-read sequencing is often used for molecular typing. Long-read sequencing is more rapid and provides useful information about the genome's structural composition but lacks the precision required for SNP-based typing and cgMLST. Here we compared prophages among 50 complete E. faecium genomes belonging to different lineages to explore whether a phage signature would be usable for typing and identifying an outbreak caused by VRE. As a proof of principle, we investigated if long-read sequencing data would allow for identifying phage signatures and thereby outbreak-related isolates. RESULTS: Analysis of complete genome sequences of publicly available isolates showed variation in phage content among different lineages defined by MLST. We identified phage present in multiple STs as well as phages uniquely detected within a single lineage. Next, in silico phage typing was applied to twelve MinION sequenced isolates belonging to two different genetic backgrounds, namely ST117/CT24 and ST80/CT16. Genomic comparisons of the long-read-based assemblies allowed us to correctly identify isolates of the same complex type based on global genome architecture and specific phage signature similarity. CONCLUSIONS: For rapid identification of related VRE isolates, phage content analysis in long-read sequencing data is possible. This allows software development for real-time typing analysis of long-read sequencing data, which will generate results within several hours. Future studies are required to assess the discriminatory power of this method in the investigation of ongoing outbreaks over a longer time period.

Molecular Characterisation of Vancomycin-Resistant Enterococcus faecium Isolates Belonging to the Lineage ST117/CT24 Causing Hospital Outbreaks.

Background: Vancomycin-resistant Enterococcus faecium (VREfm) is a successful nosocomial pathogen. The current molecular method recommended in the Netherlands for VREfm typing is based on core genome Multilocus sequence typing (cgMLST), however, the rapid emergence of specific VREfm lineages challenges distinguishing outbreak isolates solely based on their core genome. Here, we explored if a detailed molecular characterisation of mobile genetic elements (MGEs) and accessory genes could support and expand the current molecular typing of VREfm isolates sharing the same genetic background, enhancing the discriminatory power of the analysis. Materials/Methods: The genomes of 39 VREfm and three vancomycin-susceptible E. faecium (VSEfm) isolates belonging to ST117/CT24, as assessed by cgMLST, were retrospectively analysed. The isolates were collected from patients and environmental samples from 2011 to 2017, and their genomes were analysed using short-read sequencing. Pangenome analysis was performed on de novo assemblies, which were also screened for known predicted virulence factors, antimicrobial resistance genes, bacteriocins, and prophages. Two representative isolates were also sequenced using long-read sequencing, which allowed a detailed analysis of their plasmid content. Results: The cgMLST analysis showed that the isolates were closely related, with a minimal allelic difference of 10 between each cluster's closest related isolates. The vanB-carrying transposon Tn1549 was present in all VREfm isolates. However, in our data, we observed independent acquisitions of this transposon. The pangenome analysis revealed differences in the accessory genes related to prophages and bacteriocins content, whilst a similar profile was observed for known predicted virulence and resistance genes. Conclusion: In the case of closely related isolates sharing a similar genetic background, a detailed analysis of MGEs and the integration point of the vanB-carrying transposon allow to increase the discriminatory power compared to the use of cgMLST alone. Thus, enabling the identification of epidemiological links amongst hospitalised patients.

Virulence Factors of Enteric Pathogenic Escherichia coli: A Review.

Escherichia coli are remarkably versatile microorganisms and important members of the normal intestinal microbiota of humans and animals. This harmless commensal organism can acquire a mixture of comprehensive mobile genetic elements that contain genes encoding virulence factors, becoming an emerging human pathogen capable of causing a broad spectrum of intestinal and extraintestinal diseases. Nine definite enteric E. coli pathotypes have been well characterized, causing diseases ranging from various gastrointestinal disorders to urinary tract infections. These pathotypes employ many virulence factors and effectors subverting the functions of host cells to mediate their virulence and pathogenesis. This review summarizes new developments in our understanding of diverse virulence factors associated with encoding genes used by different pathotypes of enteric pathogenic E. coli to cause intestinal and extraintestinal diseases in humans.

Centre-specific bacterial pathogen typing affects infection-control decision making.

Whole-genome sequencing is becoming the de facto standard for bacterial outbreak surveillance and infection prevention. This is accompanied by a variety of bioinformatic tools and needs bioinformatics expertise for implementation. However, little is known about the concordance of reported outbreaks when using different bioinformatic workflows. In this multi-centre proficiency testing among 13 major Dutch healthcare-affiliated centres, bacterial whole-genome outbreak analysis was assessed. Centres who participated obtained two randomized bacterial datasets of Illumina sequences, a Klebsiella pneumoniae and a Vancomycin-resistant Enterococcus faecium, and were asked to apply their bioinformatic workflows. Centres reported back on antimicrobial resistance, multi-locus sequence typing (MLST), and outbreak clusters. The reported clusters were analysed using a method to compare landscapes of phylogenetic trees and calculating Kendall-Colijn distances. Furthermore, fasta files were analysed by state-of-the-art single nucleotide polymorphism (SNP) analysis to mitigate the differences introduced by each centre and determine standardized SNP cut-offs. Thirteen centres participated in this study. The reported outbreak clusters revealed discrepancies between centres, even when almost identical bioinformatic workflows were used. Due to stringent filtering, some centres failed to detect extended-spectrum beta-lactamase genes and MLST loci. Applying a standardized method to determine outbreak clusters on the reported de novo assemblies, did not result in uniformity of outbreak-cluster composition among centres.

The Equine Faecal Microbiota of Healthy Horses and Ponies in The Netherlands: Impact of Host and Environmental Factors.

Several studies have described the faecal microbiota of horses and the factors that influence its composition, but the variation in results is substantial. This study aimed to investigate the microbiota composition in healthy equids in The Netherlands under standard housing and management conditions and to evaluate the effect of age, gender, horse type, diet, pasture access, the season of sampling and location on it. Spontaneously produced faecal samples were collected from the stall floor of 79 healthy horses and ponies at two farms. The validity of this sampling technique was evaluated in a small pilot study including five ponies showing that the microbiota composition of faecal samples collected up to 6 h after spontaneous defaecation was similar to that of the samples collected rectally. After DNA extraction, Illumina Miseq 16S rRNA sequencing was performed to determine microbiota composition. The effect of host and environmental factors on microbiota composition were determined using several techniques (NMDS, PERMANOVA, DESeq2). Bacteroidetes was the largest phylum found in the faecal microbiota (50.1%), followed by Firmicutes (28.4%). Alpha-diversity and richness decreased significantly with increasing age. Location, age, season, horse type and pasture access had a significant effect on beta-diversity. The current study provides important baseline information on variation in faecal microbiota in healthy horses and ponies under standard housing and management conditions. These results indicate that faecal microbiota composition is affected by several horse-related and environment-related factors, and these factors should be considered in future studies of the equine faecal microbiota.

First detection of porcine respirovirus 1 in Germany and the Netherlands.

Porcine respirovirus 1, also referred to as porcine parainfluenza virus 1 (PPIV-1), was first detected in deceased pigs from Hong Kong in 2013. It has since then been found in the USA, Chile and most recently in Hungary. Information on the pathogenicity and global spread is sparse. However, it has been speculated to play a role in the porcine respiratory disease complex. To investigate the porcine virome, we screened 53 pig samples from 26 farms within the Dutch-German border region using shotgun metagenomics sequencing (SMg). After detecting PPIV-1 in five farms through SMg, a real-time reverse transcriptase PCR (RT-qPCR) assay was designed, which not only confirmed the presence of the virus in 1 of the 5 farms but found an additional 6 positive farms. Phylogenetic analysis found the closest match to be the first detected PPIV-1 strain in Hong Kong. The Dutch-German region represents a significant area of pig farming within Europe and could provide important information on the characterization and circulation of porcine viruses, such as PPIV-1. With its recent detection in Hungary, these findings suggest widespread circulation of PPIV-1 in Central Europe, highlighting the need for further research on persistence, pathogenicity and transmission in Europe.

Effects of Clinical Wastewater on the Bacterial Community Structure from Sewage to the Environment.

This study pertains to measure differences in bacterial communities along the wastewater pathway, from sewage sources through the environment. Our main focus was on taxa which include pathogenic genera, and genera harboring antibiotic resistance (henceforth referred to as "target taxa"). Our objective was to measure the relative abundance of these taxa in clinical wastewaters compared to non-clinical wastewaters, and to investigate what changes can be detected along the wastewater pathway. The study entailed a monthly sampling campaign along a wastewater pathway, and taxa identification through 16S rRNA amplicon sequencing. Results indicated that clinical and non-clinical wastewaters differed in their overall bacterial composition, but that target taxa were not enriched in clinical wastewater. This suggests that treatment of clinical wastewater before release into the wastewater system would only remove a minor part of the potential total pathogen load in wastewater treatment plants. Additional findings were that the relative abundance of most target taxa was decreased after wastewater treatment, yet all investigated taxa were detected in 68% of the treated effluent samples-meaning that these bacteria are continuously released into the receiving surface water. Temporal variation was only observed for specific taxa in surface water, but not in wastewater samples.