SNP-based high-resolution typing of Chlamydia psittaci from humans and wild birds in Sweden: circulation of the Mat116 genotype reveals the transmission mode to humans.

The incidence of Chlamydia psittaci respiratory tract infections in humans has increased in Sweden in recent years. This study aimed to identify the transmission route by genotyping C. psittaci from infected humans and birds. 42 human C. psittaci samples and 5 samples from C. psittaci-infected birds were collected. Genotyping was performed using ompA sequencing, Multi-locus sequence typing, and/or SNP-based high-resolution melting-PCR. Epidemiological data was also collected, and a phylogenetic analysis was conducted. Analysis of ompA provided limited resolution, while the SNP-based PCR analysis successfully detected the Mat116 genotype in 3/5 passerine birds and in 26/29 human cases, indicating a high prevalence of this genotype in the human population. These cases were associated with contact with wild birds, mainly through bird feeding during winter or other outdoor exposure. Human cases caused by other genotypes (psittacine and pigeon) were less common and were linked to exposure to caged birds or pigeons. The SNP-genotype Mat116 is rare, but predominated in this study. The use of SNP-based PCR provided a better understanding of the C. psittaci transmission from birds to humans compared to ompA analysis. In Sweden, human psittacosis appears mainly to be transmitted from garden birds during bird feeding in the winter season.

Prevalence of SARS-CoV-2 Omicron Sublineages and Spike Protein Mutations Conferring Resistance against Monoclonal Antibodies in a Swedish Cohort during 2022-2023.

Monoclonal antibodies (mAbs) are an important treatment option for COVID-19 caused by SARS-CoV-2, especially in immunosuppressed patients. However, this treatment option can become ineffective due to mutations in the SARS-CoV-2 genome, mainly in the receptor binding domain (RBD) of the spike (S) protein. In the present study, 7950 SARS-CoV-2 positive samples from the Uppsala and Örebro regions of central Sweden, collected between March 2022 and May 2023, were whole-genome sequenced using amplicon-based sequencing methods on Oxford Nanopore GridION, Illumina MiSeq, Illumina HiSeq, or MGI DNBSEQ-G400 instruments. Pango lineages were determined and all single nucleotide polymorphism (SNP) mutations that occurred in these samples were identified. We found that the dominant sublineages changed over time, and mutations conferring resistance to currently available mAbs became common. Notable ones are R346T and K444T mutations in the RBD that confer significant resistance against tixagevimab and cilgavimab mAbs. Further, mutations conferring a high-fold resistance to bebtelovimab, such as the K444T and V445P mutations, were also observed in the samples. This study highlights that resistance mutations have over time rendered currently available mAbs ineffective against SARS-CoV-2 in most patients. Therefore, there is a need for continued surveillance of resistance mutations and the development of new mAbs that target more conserved regions of the RBD.

SARS-CoV-2 variants of concern and spike protein mutational dynamics in a Swedish cohort during 2021, studied by Nanopore sequencing.

BACKGROUND: Since the beginning of the COVID-19 pandemic, new variants of significance to public health have emerged. Consequently, early detection of new mutations and variants through whole-genome sequencing remains crucial to assist health officials in employing appropriate public health measures. METHODS: We utilized the ARTIC Network SARS-CoV-2 tiled amplicon approach and Nanopore sequencing to sequence 4,674 COVID-19 positive patient samples from Uppsala County, Sweden, between week 15 and 52 in 2021. Using this data, we mapped the circulating variants of concern (VOC) in the county over time and analysed the Spike (S) protein mutational dynamics in the Delta variant throughout 2021. RESULTS: The distribution of the SARS-CoV-2 VOC matched the national VOC distribution in Sweden, in 2021. In the S protein of the Delta variant, we detected mutations attributable to variants under monitoring and variants of interest (e.g., E484Q, Q613H, Q677H, A222V and Y145H) and future VOC (e.g., T95I and Y144 deletion, which are signature mutations in the Omicron variant). We also frequently detected some less well-described S protein mutations in our Delta sequences, that might play a role in shaping future emerging variants. These include A262S, Q675K, I850L, Q1201H, V1228L and M1237I. Lastly, we observed that some of the Delta variant's signature mutations were underrepresented in our study due to artifacts of the used bioinformatics tools, approach and sequencing method. We therefore discuss some pitfalls and considerations when sequencing SARS-CoV-2 genomes. CONCLUSION: Our results suggest that genomic surveillance in a small, representative cohort can be used to make predictions about the circulating variants nationally. Moreover, we show that detection of transient mutations in currently circulating variants can give valuable clues to signature mutations of future VOC. Here we suggest six such mutations, that we detected frequently in the Delta variant during 2021. Lastly, we report multiple systematic errors that occurred when following the ARTIC Network SARS-CoV-2 tiled amplicon approach using the V3 primers and Nanopore sequencing, which led to the masking of some of the important signature mutations in the Delta sequences.

In-Flight Transmission of a SARS-CoV-2 Lineage B.1.617.2 Harbouring the Rare S:E484Q Immune Escape Mutation.

We describe a flight-associated infection scenario of seven individuals with a B.1.617.2 (Delta) lineage, harbouring an S:E484Q point mutation. In Sweden, at least 10% of all positive SARS-CoV-2 samples were sequenced in each county; the B.1.717.2 + S:E484Q combination was not detected in Sweden before and was imported within the scenario described in this report. The high transmission rate of the delta lineage combined with the S:E484Q mutation, associated with immune escape in other lineages, makes this specific genetic combination a possible threat to the global fight against the COVID-19 pandemic. Even within the Omicron wave, the B.1.617.2 + S:E484Q variant appeared in community samples in Sweden, as it seems that this combination has an evolutionary gain compared to other B.1.617.2 lineages. The here described genomic combination was not detectable with the common fasta file-based Pango-lineage analysis, hence increasing the probability of the true global prevalence to be higher.

Cold case: The disappearance of Egypt bee virus, a fourth distinct master strain of deformed wing virus linked to honeybee mortality in 1970's Egypt.

In 1977, a sample of diseased adult honeybees (Apis mellifera) from Egypt was found to contain large amounts of a previously unknown virus, Egypt bee virus, which was subsequently shown to be serologically related to deformed wing virus (DWV). By sequencing the original isolate, we demonstrate that Egypt bee virus is in fact a fourth unique, major variant of DWV (DWV-D): more closely related to DWV-C than to either DWV-A or DWV-B. DWV-A and DWV-B are the most common DWV variants worldwide due to their close relationship and transmission by Varroa destructor. However, we could not find any trace of DWV-D in several hundred RNA sequencing libraries from a worldwide selection of honeybee, varroa and bumblebee samples. This means that DWV-D has either become extinct, been replaced by other DWV variants better adapted to varroa-mediated transmission, or persists only in a narrow geographic or host range, isolated from common bee and beekeeping trade routes.

Genomic and Phenotypic Characterisation of Campylobacter jejuni Isolates From a Waterborne Outbreak.

Campylobacter infections are the leading cause of bacterial gastroenteritis. In Europe, over 246,000 cases are confirmed annually. Infections are often transmitted via contaminated food, such as poultry products, but water may be the source of infection as well. The aim of this study was to characterise a selection of Campylobacter jejuni human isolates, together with a water isolate, from a waterborne outbreak in Norway in 2019, including human isolates from early, mid-, and late epidemic. The isolates were characterised with whole-genome sequencing, analysing the expression of putative virulence genes and demonstrating the pathogenic potential in an in vitro adhesion model using HT-29 cells. All isolates belonged to the multilocus sequence type 1701 and ST45 clonal complex. In the genomic analysis, the water isolate clustered somewhat separately from the human isolates. There was some variation between the human isolates, but the water isolate seemed to display the greatest pathogenic potential, demonstrated by the highest levels of virulence gene expression, adhesion to epithelial cells and IL-8 induction. These results suggest that the water isolate of the study has potential to cause human infections, and that some bacterial changes due to host or environmental adaptation, may occur during a waterborne Campylobacter epidemic. This is, to the best of our knowledge, the first study on C. jejuni isolates from a waterborne outbreak, including both human isolates and a water isolate, characterised with genomic and phenotypic approaches.

Early Phylogenetic Diversification of SARS-CoV-2: Determination of Variants and the Effect on Epidemiology, Immunology, and Diagnostics.

The phylogenetic clustering of 95 SARS-CoV-2 sequences from the first 3 months of the pandemic reveals insights into the early evolution of the virus and gives first indications of how the variants are globally distributed. Variants might become a challenge in terms of diagnostics, immunology, and effectiveness of drugs. All available whole genome sequence data from the NCBI database (March 16, 2020) were phylogenetically analyzed, and gene prediction as well as analysis of selected variants were performed. Antigenic regions and the secondary protein structure were predicted for selected variants. While some clusters are presenting the same variant with 100% identical bases, other SARS-CoV-2 lineages show a beginning diversification and phylogenetic clustering due to base substitutions and deletions in the genomes. First molecular epidemiological investigations are possible with the results by adding metadata as travelling history to the presented data. The advantage of variants in source tracing can be a challenge in terms of virulence, immune response, and immunological memory. Variants of viruses often show differences in virulence or antigenicity. This must also be considered in decisions like herd immunity. Diagnostic methods might not work if the variations or deletions are in target regions for the detection of the pathogen. One base substitution was detected in a primer binding site.

Differences in virulence gene expression between human blood and stool Campylobacter coli clade 1 ST828CC isolates.

BACKGROUND: Campylobacter colonise the gastrointestinal tract of warm-blooded animals and are major enteropathogens in humans. C. coli is less common than C. jejuni and accounts for about 10% of the total number of Campylobacter infections although the two species seem to share many virulence determinants. Campylobacter bacteraemia is rare, estimated to occur in less than 1% of the infections, and the exact mechanisms regulating the progression of the infection from the gastrointestinal tract to the blood stream are unclear. Here, we looked at the contribution of C. coli to Campylobacter infections and further compared various virulence traits in C. coli clade 1 blood and stool isolates. RESULTS: We assessed the numbers of C. jejuni and C. coli among typed isolates in the PubMLST database and found that C. coli accounted for 25.9% of blood isolates, but only 8.9% of the stool isolates. Phylogenetic analysis of 128 C. coli clade 1 whole genome sequences deposited to NCBI revealed no specific clustering of the human blood, stool or animal isolates. Of the six C. coli isolates chosen for phenotypic analyses, stool isolates adhered significantly better to human HT-29 colon cancer cells than the blood isolates, while there was no difference in induced IL-8 levels between the isolates. Furthermore, the stool isolates had two- to fourfold higher RNA expression levels of the flpA, ciaB, iamA and cdt virulence genes than the blood isolates. Finally, we looked at the gene structure of the cdtA, B and C toxin genes and found numerous nucleotide additions and deletions disrupting the open reading frames. In contrast to 58% isolates of animal origin, only 38% and 32% of human blood and stool isolates, respectively, had all three cdt genes intact, a prerequisite to produce functional toxins. CONCLUSIONS: This study reveals interesting differences between C. coli clade 1 isolates of human and animal origin on one hand, and also between human blood and stool isolates, on the other. The results suggest that C. coli might downregulate and/or inactivate various virulence determinants as the isolates pass from the animal host to the human gastrointestinal tract and enter the human blood stream.

Staphylococcus argenteus and Staphylococcus schweitzeri are cytotoxic to human cells in vitro due to high expression of alpha-hemolysin Hla.

Staphylococcus argenteus and Staphylococcus schweitzeri are newly identified species of the S. aureus-related complex. S. argenteus, as occurring globally and showing significant prevalence and comparable infection and morbidity rates compared to S. aureus, is becoming clinically important. Whole genome sequencing has revealed the presence of several virulence genes but the molecular mechanisms of S. argenteus infection and virulence are largely unknown. Here, we studied the effect of a previously characterized clinical S. argenteus isolate on human cells in vitro. The clinical isolate, together with the S. argenteus type strain MSHR1132T and the S. schweitzeri type strain FSA084T, had a cytotoxic effect on the cells, which showed necrotic cell death after a few hours of treatment. The protein causing the cytotoxic effect was purified and identified by mass spectrometry as alpha-hemolysin, Hla, which is awell-known pore-forming toxin in S.aureus. The cytotoxic effect could be blocked with an antibody against Hla. S.argenteus showed 12-15 fold higher expression levels of hla at the RNA level and 4-6 fold higher expression levels at the protein level compared to S.aureus. The higher expression levels of hla were supported by higher RNA levels of the regulatory factors sarA and saeR. Also, the RNAIII component of the accessory gene regulator (agr) quorum sensing system was 8,000-10,000 fold higher in the S.argenteus isolates compared to S.aureus. This is the first study on the effect of S.argenteus on ahuman cell line and strengthens the idea of significant virulence of S.argenteus.

Campylobacter coli Clade 3 Isolates Induce Rapid Cell Death In Vitro.

Campylobacter bacteria are major human enteropathogens. Campylobacter coli shows less genetic diversity than C. jejuni and clusters into three clades, of which clade 1 includes most human and farm animal isolates, while environmental C. coli isolates mainly belong to clades 2 and 3. Recently, we sequenced the whole genomes of eight C. coli clade 2 and 3 isolates cultivated from water, and here we studied their interaction with human HT-29 colon cancer cells compared to that of clinical clade 1 isolates. All C. coli clade 3 isolates already caused cell necrosis 1 to 2 h after inoculation, whereas none of the clade 1 and 2 isolates analyzed induced cell death. Isolates from clades 2 and 3 adhered to epithelial cells better than clade 1 isolates, but all isolates induced similar levels of interleukin-8 (IL-8). Alignment and phylogenetic analysis of the translated putative virulence genes cadF, flpA, iamA, ciaB, and ceuE revealed clade-specific protein sequence variations, with clade 1 and 2 sequences being more closely related and clade 3 sequences being further apart, in general. Moreover, when RNA levels were measured, clade 3 isolates showed significantly lower levels of expression of cadF, iamA, and ceuE than clade 2 isolates, while flpA expression levels were higher in clade 3 isolates. The cytolethal distending toxin genes were also expressed in clades 2 and 3, although there was no difference between clades. Our findings demonstrate differences between the effects of C. coli clade 1, 2, and 3 isolates on human cells and suggest that C. coli clade 3 might be more virulent than clade 2 due to the observed cytotoxicity.IMPORTANCECampylobacter coli is a common zoonotic cause of gastroenteritis in humans worldwide. The majority of infections are caused by C. coli clade 1 isolates, whereas infections due to clade 2 and 3 isolates are rare. Whether this depends on a low prevalence of clade 2 and 3 isolates in reservoirs important for human infections or their lower ability to cause human disease is unknown. Here, we studied the effects of C. coli clade 2 and 3 isolates on a human cell line. These isolates adhered to human cells to a higher degree than clinical clade 1 isolates. Furthermore, we could show that C. coli clade 3 isolates rapidly induced cell death, suggesting differences in the virulence of C. coli The exact mechanism of cell death remains to be revealed, but selected genes showed interesting clade-specific expression patterns.

Which methods are appropriate for the detection of Staphylococcus argenteus and is it worthwhile to distinguish S. argenteus from S. aureus?

PURPOSE: To further analyze a clinical isolate originally identified as methicillin-resistant Staphylococcus aureus (MRSA) using whole-genome sequencing and comparative genomics. MATERIALS AND METHODS: Classical diagnostic methods such as cultivation, biochemical tests, and PCR were supplemented with whole-genome sequencing and comparative genomics, to identify the isolate. RESULTS: The isolate was phenotypically similar to MRSA. However, the presence of the nuc gene could not be confirmed using PCR, while it was positive for the mecA gene. Whole-genome sequencing correctly identified the isolate as Staphylococcus argenteus. The isolate possessed several resistance genes, such as mecA, blaZ (ß-lactam antibiotics) and dfrG (trimethoprim). The nuc gene differed from that of MRSA. Six phylogenetic distinct clusters were identified by average nucleotide identity (ANI) analysis of all available S. argenteus whole-genome sequences. Our isolate, RK308, clustered with those isolated in Europe and Asia. CONCLUSION: Due to the invasive potential, the multi-drug resistance and the similarity to MRSA, S. argenteus should be included in the MRSA screening. Due to the divergent genome compared to MRSA, new PCR approaches have to be developed to avoid an unnoticed spreading of S. argenteus.

Survival of Campylobacter jejuni and Campylobacter coli water isolates in lake and well water.

The role of water for transmission of Campylobacter jejuni and C. coli to humans might be underestimated, as factors important for bacterial viability in water are largely unknown. We have studied water survival of seven C. jejuni and eight C. coli isolates originally isolated from Swedish waters, together with selected reference strains, over eight days at 4 °C in the dark in untreated water collected from a local lake and a private well. To study seasonality, lake water samples were collected during spring and autumn. Samples for culturable bacterial counts were taken on days 2, 4, 6, and 8 and compared to the start inoculum. For C. jejuni, a significantly better survival was observed in autumn than in spring lake water. Furthermore, C. jejuni had a significantly better survival than C. coli in autumn lake and well water samples; the rate of culturability loss was almost double for C. coli in autumn lake water. These findings contribute to a better understanding on the seasonality of waterborne Campylobacter infections and the general predominance of C. jejuni.

Brucella abortus: determination of survival times and evaluation of methods for detection in several matrices.

BACKGROUND: Brucella abortus is a highly pathogenic zoonotic agent, tempting for the development of a rapid diagnostic method to enable adequate treatment and prevent further spread. Enrichment of the bacteria is often used as a first step in diagnostics to increase the bacterial number above the detection limit of the real-time PCR. The enrichment of Brucella spp. takes at least 3 days, which might be avoidable if sensitive PCR methods can be used. Since many matrices contain PCR inhibitors, the limit of detection (LOD) must be determined for each separate matrix. Another aim of this study was the determination of survival of Brucella abortus in the analyzed matrices. METHODS: The LOD for the detection of B. abortus in 14 matrices, relevant for human medicine, veterinary medicine and food and feed safety, was determined to evaluate the need of a pre-enrichment step prior to real-time PCR. The survival of B. abortus in the spiked matrices was tested by plate count in a 7-day interval for 132 days. RESULTS: The limit of detection for B. abortus in most matrices was in the range of 103-104 CFU/g for cultivation and 104-105 CFU/g for direct real-time PCR. The survival time of B. abortus was less than 21 days in apple purée and stomach content and 28 days in water while B. abortus remained viable at day 132 in milk, blood, spinach and minced meat. CONCLUSIONS: A direct PCR analysis without enrichment of bacteria saves at least 3 days. However, the limit of detection between direct PCR and plate count differs in a 10 fold range. We conclude that this lower sensitivity is acceptable in most cases especially if quick analysis are required.

Characterization of Swedish Campylobacter coli clade 2 and clade 3 water isolates.

Campylobacter jejuni and Campylobacter coli are important bacterial enteropathogens. Poultry is the best-known reservoir for Campylobacter infection but natural bodies of water have also been shown to be important pathways for transmission. Campylobacter can survive in cold water but most of the studies have focused on C. jejuni only. In this paper, we take a closer look at the biology and water survival strategies of C. coli. Eight C. coli isolates cultivated from raw (incoming) surface water at water plants in Sweden were characterized using whole-genome sequencing and phenotypical assays. Phylogenetic analysis assigned the Swedish water isolates to clades 2 and 3, known to include C. coli of environmental origin. In addition, 53 earlier published sequences of C. coli clade 2 and 3 from environmental waters were included for in silico analyses. Generally, clade 2 isolates had larger genomes, which included a functional tricarballylate utilization locus, while clade 3 isolates contained different genes involved in oxidative stress as well as putative virulence factors. The Swedish water isolates of clade 2 formed large, blurry bacterial colonies on agar, whereas clade 3 colonies were smaller. All Swedish isolates were motile, but clade 3 isolates formed larger motility zones on soft agar, and none of these isolates produced biofilm. Although water survival varied between the analyzed isolates, there were hardly any clade-specific significant differences. Our results highlight the diversity of C. coli in general, and show differences in metabolic capabilities and ways to handle oxidative stress between clade 2 and 3 water isolates.

Genomic and phenotypic characteristics of Swedish C. jejuni water isolates.

Campylobacter jejuni is the most common cause of bacterial gastroenteritis. Major reservoirs are warm-blooded animals, poultry in particular, but Campylobacter can also be transmitted via water. In this paper, we have taken a closer look at the biology and potential virulence of C. jejuni water isolates. Seven C. jejuni isolates from incoming surface water at water plants in Sweden were characterized with whole genome sequencing and phenotypical testing. Multi locus sequence typing analysis revealed that these isolates belonged to groups known to include both common (ST48CC) and uncommon (ST1275CC, ST683, ST793 and ST8853) human pathogens. Further genomic characterization revealed that these isolates had potential for arsenic resistance (due to presence of arsB gene in all isolates), an anaerobic dimethyl sulfoxide oxidoreductase (in three isolates) and lacked the MarR-type transcriptional regulator gene rrpB (in all but one isolate) earlier shown to be involved in better survival under oxidative and aerobic stress. As putative virulence factors were concerned, there were differences between the water isolates in the presence of genes coding for cytolethal distending toxin (cdtABC), Type VI secretion system and sialylated LOS, as well as in biofilm formation. However, all isolates were motile and could adhere to and invade the human HT-29 colon cancer cell line in vitro and induce IL-8 secretion suggesting potential to infect humans. This is, to the best of our knowledge, the first study where C. jejuni water isolates have been characterized using whole genome sequencing and phenotypical assays. We found differences and shared traits among the isolates but also potential to infect humans.

Time to review the gold standard for genotyping vancomycin-resistant enterococci in epidemiology: Comparing whole-genome sequencing with PFGE and MLST in three suspected outbreaks in Sweden during 2013-2015.

Vancomycin-resistant enterococci (VRE) are a challenge to the health-care system regarding transmission rate and treatment of infections. VRE outbreaks have to be controlled from the first cases which means that appropriate and sensitive genotyping methods are needed. The aim of this study was to investigate the applicability of whole genome sequencing based analysis compared to Pulsed-Field Gel Electrophoresis (PFGE) and Multi-Locus Sequence Typing (MLST) in epidemiological investigations as well as the development of a user friendly method for daily laboratory use. Out of 14,000 VRE - screening samples, a total of 60 isolates positive for either vanA or vanB gene were isolated of which 38 were from patients with epidemiological links from three suspected outbreaks at Uppsala University Hospital. The isolates were genotypically characterised with PFGE, MLST, and WGS based core genome Average Nucleotide Identity analysis (cgANI). PFGE was compared to WGS and MLST regarding reliability, resolution, and applicability capacity. The PFGE analysis of the 38 isolates confirmed the epidemiological investigation that three outbreaks had occurred but gave an unclear picture for the largest cluster. The WGS analysis could clearly distinguish six ANI clusters for those 38 isolates. As result of the comparison of the investigated methods, we recommend WGS-ANI analysis for epidemiological issues with VRE. The recommended threshold for Enterococcus faecium VRE outbreak strain delineation with core genome based ANI is 98.5%. All referred sequences of this study are available from the NCBI BioProject number PRJNA301929.

A novel real-time PCR assay for specific detection of Brucella melitensis.

BACKGROUND: Brucellosis is a zoonosis that occurs worldwide. The disease has been completely eradicated in livestock in Sweden in 1994, and all cases of confirmed human brucellosis are imported into Sweden from other countries. However, due to an increase in the number of refugees and asylum seekers from the middle-east to Sweden, there is a need to improve the current diagnostic methodology for Brucella melitensis. Whilst culture of Brucella species can be used as a diagnostic tool, real-time PCR approaches provide a much faster result. The aim of this study was to set up a species-specific real-time PCR for the detection of all biovars of Brucella melitensis, which could be used routinely in diagnostic laboratories. METHODS: A Brucella melitensis real-time PCR assay was designed using all available genomes in the public database of Brucella (N = 96) including all complete genomes of Brucella melitensis (N = 17). The assay was validated with a collection of 37 Brucella species reference strains, 120 Brucella melitensis human clinical isolates, and 45 clinically relevant non-Brucella melitensis strains. RESULTS: In this study we developed a single real-time PCR for the specific detection of all biovars of Brucella melitensis. CONCLUSIONS: This new real-time PCR method shows a high specificity (100%) and a high sensitivity (1.25 GE/µl) and has been implemented in the laboratories of four governmental authorities across Sweden.

Massilia norwichensis sp. nov., isolated from an air sample.

A Gram-negative, rod-shaped and motile bacterial isolate, designated strain NS9(T), isolated from air of the Sainsbury Centre for Visual Arts in Norwich, UK, was subjected to a polyphasic taxonomic study including phylogenetic analyses based on partial 16S rRNA, gyrB and lepA gene sequences and phenotypic characterization. The 16S rRNA gene sequence of NS9(T) identified Massilia haematophila CCUG 38318(T), M. niastensis 5516S-1(T) (both 97.7% similarity), M. aerilata 5516S-11(T) (97.4%) and M. tieshanensis TS3(T) (97.4%) as the next closest relatives. In partial gyrB and lepA sequences, NS9(T) shared the highest similarities with M. haematophila CCUG 38318(T) (94.5%) and M. aerilata 5516-11(T) (94.3%), respectively. These sequence data demonstrate the affiliation of NS9(T) to the genus Massilia. The detection of the predominant ubiquinone Q-8, a polar lipid profile consisting of the major compounds diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol and a polyamine pattern containing 2-hydroxyputrescine and putrescine were in agreement with the assignment of strain NS9(T) to the genus Massilia. Major fatty acids were summed feature 3 (C16:1ω7c and/or iso-C15 : 0 2-OH), C16:0, C18: 1ω7c and C10:0 3-OH. Dissimilarities in partial lepA and gyrB gene sequences as well as results from DNA-DNA hybridizations demonstrate that strain NS9(T) is a representative of an as-yet undescribed species of the genus Massilia that is also distinguished from its close relatives based on physiological and biochemical traits. Hence, we describe a novel species, for which we propose the name Massilia norwichensis sp. nov., with the type strain NS9(T) ( = CCUG 65457(T) =LMG 28164(T)).

How to Show the Real Microbial Biodiversity? A Comparison of Seven DNA Extraction Methods for Bacterial Population Analyses in Matrices Containing Highly Charged Natural Nanoparticles.

A DNA extraction that comprises the DNA of all available taxa in an ecosystem is an essential step in population analysis, especially for next generation sequencing applications. Many nanoparticles as well as naturally occurring clay minerals contain charged surfaces or edges that capture negatively charged DNA molecules after cell lysis within DNA extraction. Depending on the methodology of DNA extraction, this phenomenon causes a shift in detection of microbial taxa in ecosystems and a possible misinterpretation of microbial interactions. With the aim to describe microbial interactions and the bio-geo-chemical reactions during a clay alteration experiment, several methods for the detection of a high number of microbial taxa were examined in this study. Altogether, 13 different methods of commercially available DNA extraction kits provided by seven companies as well as the classical phenol-chloroform DNA extraction were compared. The amount and the quality of nucleic acid extracts were determined and compared to the amplifiable amount of DNA. The 16S rRNA gene fragments of several taxa were separated using denaturing gradient gel electrophoresis (DGGE) to determine the number of different species and sequenced to get the information about what kind of species the microbial population consists of. A total number of 13 species was detected in the system. Up to nine taxa could be detected with commercially available DNA extraction kits while phenol-chloroform extraction lead to three detected species. In this paper, we describe how to combine several DNA extraction methods for the investigation of microbial community structures in clay.

Brucellosis outbreak in a Swedish kennel in 2013: determination of genetic markers for source tracing.

Brucellosis is a highly infectious zoonotic disease but rare in Sweden. Nonetheless, an outbreak of canine brucellosis caused by an infected dog imported to Sweden was verified in 2013. In total 25 dogs were tested at least duplicated by the following approaches: real-time PCR for the detection of Brucella canis, a Brucella genus-specific real-time PCR, selective cultivation, and microscopic examination. The whole genome of B. canis strain SVA13 was analysed regarding genetic markers for epidemiological examination. The genome of an intact prophage of Roseobacter was detected in B. canis strain SVA13 with whole genome sequence prophage analysis (WGS-PA). It was shown that the prophage gene content in the American, African and European isolates differs remarkably from the Asian strains. The prophage sequences in Brucella may therefore serve of use as genetic markers in epidemiological investigations. Phage DNA fragments were also detected in clustered, regularly interspaced short palindromic repeats (CRISPR) in the genome of strain SVA13. In addition to the recommendations for genetic markers in Brucella outbreak tracing, our paper reports a validated two-step stand-alone real-time PCR for the detection of B. canis and its first successful use in an outbreak investigation.