Complete genome sequence of the novel virulent phage PMBT24 infecting Enterocloster bolteae from the human gut.

PMBT24, the first reported virulent bacteriophage infecting the anaerobic human gut bacterium Enterocloster bolteae strain MBT-21, was isolated from a municipal sewage sample and its genome was sequenced and analysed. Transmission electron microscopy revealed a phage with an icosahedral head and a long, non-contractile tail. The circularly permutated, 99,962-bp dsDNA genome of the pac-type phage has a mol% G + C content of 32.1 and comprises 173 putative ORFs. Using amino acid sequence-based phylogeny, phage PMBT24 showed similarity to other, hitherto non-published phage genomes in the databases. Our data suggested phage PMBT24 to present the type phage of a novel genus (proposed name Kielvirus) and novel family of phages (proposed name Kielviridae).

Molecular characterization and virulence profile of Klebsiella pneumoniae and Klebsiella oxytoca isolated from ill cats and dogs in Portugal.

Klebsiella spp. are important pathogens of humans and companion animals such as cats and dogs, capable of causing severe life-threatening diseases. The aim of this study was to characterize the molecular and phenotypic properties of Klebsiella pneumoniae and Klebsiella oxytoca isolated from ill companion animals by whole genome sequencing, followed by in vitro assessment of biofilm formation and in vivo pathogenicity using the Galleria mellonella model. Two LPS O-types were identified for all the K. pneumoniae isolates tested (O3B and O1/O2v2) and only one for K. oxytoca isolates (OL104), and the most common STs found were ST11 and ST266. Furthermore, a high diversity of K-locus types was found for K. pneumoniae (KL102; KL105; KL31, and KL13). Within K. pneumoniae, one specific O/K/ST-types combination (i.e., KL105-ST11-O1/O2v2) showed results that were of concern, as it exhibited a high inflammatory response at 12 h post-infection in G. mellonella with 80% of the larvae dead at 72 h post-infection. This virulence potential, on the other hand, did not appear to be directly related to the biofilm-forming capacity. Also, virulence and resistance scores obtained for this set of strains did surpass score 1. The present study demonstrated that Klebsiella spp. isolated from companion animals belonging to STs that can cause human infections and present virulence on an invertebrate model. Thus, this study underscores the role of dogs and cats as reservoirs of resistant Klebsiella spp. that could potentially be transmitted to humans.

Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens.

For successful elucidation of a food-borne infection chain, the availability of high-quality sequencing data from suspected microbial contaminants is a prerequisite. Commonly, those investigations are a joint effort undertaken by different laboratories and institutes. To analyze the extent of variability introduced by differing wet-lab procedures on the quality of the sequence data we conducted an interlaboratory study, involving four bacterial pathogens, which account for the majority of food-related bacterial infections: Campylobacter spp., Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The participants, ranging from German federal research institutes, federal state laboratories to universities and companies, were asked to follow their routine in-house protocols for short-read sequencing of 10 cultures and one isolated bacterial DNA per species. Sequence and assembly quality were then analyzed centrally. Variations within isolate samples were detected with SNP and cgMLST calling. Overall, we found that the quality of Illumina raw sequence data was high with little overall variability, with one exception, attributed to a specific library preparation kit. The variability of Ion Torrent data was higher, independent of the investigated species. For cgMLST and SNP analysis results, we found that technological sequencing artefacts could be reduced by the use of filters, and that SNP analysis was more suited than cgMLST to compare data of different contributors. Regarding the four species, a minority of Campylobacter isolate data showed the in comparison highest divergence with regard to sequence type and cgMLST analysis. We additionally compared the assembler SPAdes and SKESA for their performance on the Illumina data sets of the different species and library preparation methods and found overall similar assembly quality metrics and cgMLST statistics.

Characterisation of the Novel Filamentous Phage PMBT54 Infecting the Milk Spoilage Bacteria Pseudomonas carnis and Pseudomonas lactis.

Filamentous bacteriophages are lysogenic and pseudo-lysogenic viruses that do not lyse their host but are often continuously secreted from the infected cell. They belong to the order Tubulavirales, which encompasses three families, with the Inoviridae being the largest. While the number of identified inoviral sequences has greatly increased in recent years due to metagenomic studies, morphological and physiological characterisation is still restricted to only a few members of the filamentous phages. Here, we describe the novel filamentous phage PMBT54, which infects the spoilage-relevant Pseudomonas species P. carnis and P. lactis. Its genome is 7320 bp in size, has a mol% GC content of 48.37, and codes for 13 open-reading frames, two of which are located on the (-) strand. The virion exhibits a typical filamentous morphology and is secreted from the host cell at various lengths. The phage was shown to promote biofilm formation in both host strains and, therefore, has potential implications for milk spoilage, as biofilms are a major concern in the dairy industry.

Does the high biodiversity of lactococcal bacteriophages allow predictions about their different UV-C susceptibilities?

Fermentation processes can only succeed if intact and active starter cultures are present. Bacteriophages, which can lyse bacteria and thus bring entire fermentation processes to a standstill, therefore pose a major threat. Cheese production, for example, is often affected. The by-product whey can be highly contaminated with bacteriophages (≤109 plaque-forming units/mL) and in this state, further utilization is a quality and processing risk. Therefore, an orthogonal process consisting of membrane filtration followed by UV-C irradiation could be applied to eliminate bacteriophages and to generate "phage-free" whey. In order to define suitable process parameters, 11 lactococcal bacteriophages belonging to different families and genera and differing in their morphology, genome size, heat resistance, and other attributes, were screened for their UV-C resistance in whey. P369 was found to be the most resistant and could thus be well-suited as a biomarker. Starting from a 4 log unit bacteriophage reduction by membrane filtration, another 5 log unit decrease should be realized when applying a UV-C dose of 5 J/cm2. A clear correlation of UV-C sensitivity to the chosen attributes studied such as bacteriophage morphology and genome size was difficult and ambiguous, presumably because other yet unidentified parameters are important. Mutation experiments were performed with the representative bacteriophage P008 by multiple cycles of UV-C irradiation and propagation. A few mutational events were found, but could not be linked to an artificially generated UV-C resistance, indicating that the process used would probably not lose its effectiveness over time.

Draft Genome Sequences of Three Lactic Acid Bacterial Strains Investigated for B Vitamin Biosynthesis.

The draft genome sequences of three lactic acid bacteria, namely, Limosilactobacillus reuteri 92071, Lactiplantibacillus plantarum 92117-i3, and Limosilactobacillus fermentum 92072, and the presence of genes involved in the biosynthesis of B vitamins were determined. Limosilactobacillus reuteri 92071 showed complete gene clusters for vitamin B12 biosynthesis, with a GC content of 38.52 mol%.

A next-generation sequencing approach for the detection of mixed species in canned tuna.

Tuna cans are relevant seafood products for which mixtures of different tuna species are not allowed according to European regulations. In order to support the prevention of food fraud and mislabelling, a next-generation sequencing methodology based on mitochondrial cytochrome b and control region markers has been tested. Analyses of defined mixtures of DNA, fresh tissue and canned tissue revealed a qualitative and, to some extent, semiquantitative identification of tuna species. While the choice of the bioinformatic pipeline had no influence in the results (p = 0.71), quantitative differences occurred depending on the treatment of the sample, marker, species, and mixture (p < 0.01). The results revealed that matrix-specific calibrators or normalization models should also be used in NGS. The method represents an important step towards a semiquantitative method for routine control of this analytically challenging food matrix. Tests of commercial samples uncovered mixed species in some cans, being not in compliance with EU regulations.

Fast Identification Method for Screening Bacteria from Faecal Samples Using Oxford Nanopore Technologies MinION Sequencing.

Most bacterial identification methods require extensive culturing, strain purification and DNA extraction protocols. This leads to additional expenses and time lags when isolating specific bacteria from complex microbiological ecosystems. This study aimed to develop a fast and robust method for identification of lactobacilli, bifidobacteria and Bacteroides in human faecal samples. Bacteria from faecal samples were cultured anaerobically on selective media. Sonication-based DNA extraction was performed, followed by almost complete 16S rRNA gene polymerase chain reaction amplification and MinION sequencing with the Flongle adapter. Sequence analysis was performed using NanoCLUST, while RStudio was used for graphics. For 110 of the 125 colonies investigated, 100% of reads were attributed to a single species, while the remaining 15 colonies consisted of mixtures of up to three different species. The proposed bacterial identification method is advantageous for isolating particular bacteria for which there are no exclusively selective media, as it avoids lengthy colony purification and DNA purification methods, and yields a quick colony identification with high accuracy. Therefore, this method can be used for directly screening for pure cultures of target microorganisms and is suitable for the identification of bacteria in culturomics studies.

Morphological and Genetic Characterization of Eggerthella lenta Bacteriophage PMBT5.

Eggerthella lenta is a common member of the human gut microbiome. We here describe the isolation and characterization of a putative virulent bacteriophage having E. lenta as host. The double-layer agar method for isolating phages was adapted to anaerobic conditions for isolating bacteriophage PMBT5 from sewage on a strictly anaerobic E. lenta strain of intestinal origin. For this, anaerobically grown E. lenta cells were concentrated by centrifugation and used for a 24 h phage enrichment step. Subsequently, this suspension was added to anaerobically prepared top (soft) agar in Hungate tubes and further used in the double-layer agar method. Based on morphological characteristics observed by transmission electron microscopy, phage PMBT5 could be assigned to the Siphoviridae phage family. It showed an isometric head with a flexible, noncontractile tail and a distinct single 45 nm tail fiber under the baseplate. Genome sequencing and assembly resulted in one contig of 30,930 bp and a mol% GC content of 51.3, consisting of 44 predicted protein-encoding genes. Phage-related proteins could be largely identified based on their amino acid sequence, and a comparison with metagenomes in the human virome database showed that the phage genome exhibits similarity to two distantly related phages.

Characterization of antibiotic-resistant, coagulase-negative staphylococci from fresh produce and description of Staphylococcus shinii sp. nov. isolated from chives.

Coagulase-negative Staphylococcus (CoNS) species may possess antibiotic resistance genes and have been associated with nosocomial infections. In this study, 91 CoNS with decreased susceptibility to oxacillin were isolated from fresh produce using oxacillin containing agar plates. Their antibiotic resistances were determined phenotypically and all isolates were identified by rep-PCR, 16S rRNA and rpoB gene sequencing. Furthermore, the genomes of representative strains were sequenced in order to confirm species identification by phylogenomics. The majority (64 of 91) of the CoNS strains could be identified as Mammaliicoccus (M.) fleurettii, while 13 were identified as M. sciuri, 8 as M. vitulinus, 2 as Staphylococcus (S.) epidermidis and single strains each as S. warneri, S. xylosus, Staphylococcus spp. and S. casei. Most of the strains were generally susceptible to clinically-relevant antibiotics, but only few (< 7%) strains possessed multiple resistances. Both oxacillin and cefoxitin resistant isolates were considered to be presumptive methicillin-resistant CoNS. From whole genome sequencing data of 6 representative strains, the mecA gene, accessory genes and the SCC loci were compared, which revealed high variability between some of the strains. The major fatty acids of K22-5MT strain included anteiso-C15:0, iso-C15:0, iso-C17:0, anteiso-C17:0, C18:0, and C20:0. Average nucleotide identity and digital DNA-DNA hybridization values indicated that Staphylococcus strain K22-5MT was below the species delineation cutoff values for ANI (less than 91%) and DDH (less than 44.4%), with the most closely related species being the S. pseudoxylosus S04009T type strain. Thus, strain K22- 5MT (=DSM 112532T, =LMG 32324T) represents a novel species, for which the name Staphylococcus shinii sp. nov. is proposed.

Pseudomonas rustica sp. nov., isolated from bulk tank raw milk at a German dairy farm.

Here we present the description of a novel Pseudomonas species, designated Pseudomonas rustica sp. nov., which was isolated from raw milk samples obtained from Germany. Results of initial 16S rRNA gene sequence analysis assigned the strain into the genus Pseudomonas and showed Pseudomonas helmanticensis, Pseudomonas neuropathica and Pseudomonas atagonensis to be its closest relatives. Further studies including sequence analysis of the rpoB gene, multi-gene phylogenetic tree reconstruction, whole-genome sequence comparisons, cellular fatty acid analysis and chemotaxonomic characterization showed a clear separation from the known Pseudomonas species. Isolate MBT-4T was closely related to Pseudomonas helmanticensis, 'Pseudomonas crudilactis' and Pseudomonas neuropathica with average nucleotide identities based on blast values of 88.8, 88.8 and 88.6%, respectively. Therefore, the strain can be classified into the Pseudomonas koreensis subgroup of the Pseudomonas fluorescens group. The G+C content of strain MBT-4T was 58.9 mol%. The strain was catalase- and oxidase-positive, while the ß-galactosidase reaction was negative. Growth occurred between 4 and 30 °C and at pH values from pH 6.0 to 8.0. In conclusion, strain MBT-4T belongs to a novel species, for which the name Pseudomonas rustica sp. nov. is proposed. The type strain is MBT-4T (=DSM 112348T=LMG 32241T) and strain MBT-17 is also a representative of this species.

Novel Siphoviridae phage PMBT4 belonging to the group b Lactobacillus delbrueckii subsp. bulgaricus phages.

A novel Lactobacillus delbrueckii bacteriophage PMBT4 was isolated from the Nigerian fermented milk product nono. The phage possesses a long and thin, non-contractile tail and an isometric head, indicating that it belongs to the Siphoviridae family. A neck passage structure (`collar`), previously hypothesized to be encoded by two genes located in the Lactobacillus delbrueckii phage LL-K insertion sequence (KIS) element, as well as in two additional Lb. delbrueckii phages Ld17 and Ld25A, could also be observed on an estimated 1-5% of phage particles by transmission electron microscopy. However, neither mapping of high throughput sequencing data to KIS element genes from Lb. delbrueckii phages LL-K, Ld17 and Ld25A nor PCR amplification of the KIS element genes could corroborate the presence of these genes in the PMBT4 genome. The PMBT4 genome consists of 31,399 bp with a mol% GC content of 41.6 and exhibits high (95-96%) sequence homologies to Lb. delbrueckii phages c5, Ld3, Ld25A and Ld17, which assigned PMBT4 as a new member of this genus, i.e. the Cequinquevirus genus.

Characterization of the first Pseudomonas grimontii bacteriophage, PMBT3.

The complete genome sequence of the virulent bacteriophage PMBT3, isolated on the proteolytic Pseudomonas grimontii strain MBTL2-21, showed no significant similarity to other known phage genome sequences, making this phage the first reported to infect a strain of P. grimontii. Electron microscopy revealed PMBT3 to be a member of the family Siphoviridae, with notably long and flexible whiskers. The linear, double-stranded genome of 87,196 bp has a mol% G+C content of 60.4 and contains 116 predicted protein-encoding genes. A putative tellurite resistance (terB) gene, originally reported to occur in the genome of a bacterium, was detected in the genome of phage PMBT3.

First Molecular Characterization of Siphoviridae-Like Bacteriophages Infecting Staphylococcus hyicus in a Case of Exudative Epidermitis.

Exudative epidermitis (EE), also known as greasy pig disease, is one of the most frequent skin diseases affecting piglets. Zoonotic infections in human occur. EE is primarily caused by virulent strains of Staphylococcus (S.) hyicus. Generally, antibiotic treatment of this pathogen is prone to decreasing success, due to the incremental development of multiple resistances of bacteria against antibiotics. Once approved, bacteriophages might offer interesting alternatives for environmental sanitation or individualized treatment, subject to the absence of virulence and antimicrobial resistance genes. However, genetic characterization of bacteriophages for S. hyicus has, so far, been missing. Therefore, we investigated a piglet raising farm with a stock problem due to EE. We isolated eleven phages from the environment and wash water of piglets diagnosed with the causative agent of EE, i.e., S. hyicus. The phages were morphologically characterized by electron microscopy, where they appeared Siphoviridae-like. The genomes of two phages were sequenced on a MiSeq instrument (Illumina), resulting in the identification of a new virulent phage, PITT-1 (PMBT8), and a temperate phage, PITT-5 (PMBT9). Sequencing of three host bacteria (S. hyicus) from one single farm revealed the presence of two different strains with genes coding for two different exfoliative toxin genes, i.e., exhA (2 strains) and exhC (1 strain). The exhC-positive S. hyicus strain was only weakly lysed by most lytic phages. The occurrence of different virulent S. hyicus strains in the same outbreak limits the prospects for successful phage treatment and argues for the simultaneous use of multiple and different phages attacking the same host.

Draft Genome Sequences of Five Staphylococcus saprophyticus Strains Isolated from African Fermented Nono in Nigeria.

Five Staphylococcus saprophyticus strains were isolated from the fermented milk product nono in Nigeria and were sequenced using an Illumina MiSeq platform. The genome sizes ranged from 2.53 to 2.60 Mbp, while the GC contents ranged from 32.99 to 33.07 mol%. The genomes possessed 2,505 to 2,687 protein-coding sequences.

Isolation and Characterization of Potential Starter Cultures from the Nigerian Fermented Milk Product nono.

Nono, an important traditional fermented dairy food produced from cow's milk in Nigeria, was studied for microbial diversity and for starter culture development for industrial production. On the basis of a polyphasic approach, including phenotypic and genotypic methods such as 16S rRNA gene sequencing, repetitive element PCR (rep-PCR) fingerprinting metagenomics, and whole genome sequencing, we identified Lactobacillus (Lb.) helveticus, Limosilactobacillus (L.) fermentum, Lb. delbrueckii, and Streptococcus (S.) thermophilus as predominant bacterial species involved with milk fermentation during traditional nono production in Nigeria, while the predominant yeast species in nono was identified as Saccharomyces cerevisiae. Using metagenomics, Shigella and potential pathogens such as enterobacteria were detected at low levels of abundance. Strains of the predominant lactic acid bacteria (LAB) were selected for starter cultures combination on the basis of their capacities for rapid growth in milk and reduction of pH below 4.5 and their gelling characteristic, which was demonstrated noticeably only by the S. thermophilus strains. Whole genome sequence analysis of selected bacterial strains showed the largest assembled genome size to be 2,169,635 bp in Lb. helveticus 314, while the smallest genome size was 1,785,639 bp in Lb. delbrueckii 328M. Genes encoding bacteriocins were not detected in all the strains, but all the LAB possessed genes potentially involved in diacetyl production and citrate metabolism. These bacteria isolated from nono can thus be used to improve the microbial safety quality of nono in Nigeria, in addition to improving technological parameters such as gelling viscosity, palatability, and product consistency.

Taxonomic Evaluation of the Heyndrickxia (Basonym Bacillus) sporothermodurans Group (H. sporothermodurans, H. vini, H. oleronia) Based on Whole Genome Sequences.

The genetic heterogeneity of Heyndrickxia sporothermodurans (formerly Bacillussporothermodurans) was evaluated using whole genome sequencing. The genomes of 29 previously identified Heyndrickxiasporothermodurans and two Heyndrickxia vini strains isolated from ultra-high-temperature (UHT)-treated milk were sequenced by short-read (Illumina) sequencing. After sequence analysis, the two H. vini strains could be reclassified as H. sporothermodurans. In addition, the genomes of the H.sporothermodurans type strain (DSM 10599T) and the closest phylogenetic neighbors Heyndrickxiaoleronia (DSM 9356T) and Heyndrickxia vini (JCM 19841T) were also sequenced using both long (MinION) and short-read (Illumina) sequencing. By hybrid sequence assembly, the genome of the H. sporothermodurans type strain was enlarged by 15% relative to the short-read assembly. This noticeable increase was probably due to numerous mobile elements in the genome that are presumptively related to spore heat tolerance. Phylogenetic studies based on 16S rDNA gene sequence, core genome, single-nucleotide polymorphisms and ANI/dDDH, showed that H. vini is highly related to H. sporothermodurans. When examining the genome sequences of all H.sporothermodurans strains from this study, together with 4 H. sporothermodurans genomes available in the GenBank database, the majority of the 36 strains examined occurred in a clonal lineage with less than 100 SNPs. These data substantiate previous reports on the existence and spread of a genetically highly homogenous and heat resistant spore clone, i.e., the HRS-clone.

Effect of reduction of sodium content on the microbial ecology of Edam cheese samples.

Sodium intake is a major risk factor for non-communicable diseases. Consequently, reformulation of cheeses such as Edam to contain less sodium may contribute to lowering disease risk. However, sodium is essential for cheese manufacture, influencing starter culture bacteria activity and abundance during fermentation. This study aimed to assess the microbial diversity of reformulated Edam cheese samples with a reduced sodium content using culture-independent technique. The microbial diversity of samples produced using simple sodium reduction, as well as by substituting salt with a mineral salt compound containing potassium, were analysed in comparison to regular control Edam samples during manufacture and the subsequent 6-week ripening period using 16S rDNA metagenomics. In addition, a challenge test using Listeria (List.) innocua as a surrogate species for List. monocytogenes was performed. Reducing sodium content did not influence the microbiological composition of reformulated samples in comparison to that of regular samples. The starter culture bacteria dominated the microbial diversity and no increase in spoilage or potentially pathogenic bacterial growth was detected, including that of List. innocua. From a microbiological perspective, it can be concluded that lowering sodium content in Edam samples without affecting the microbial composition is achievable through simple sodium reduction and through implementation of a mineral salt replacement approach.

Polyphasic study of antibiotic-resistant enterobacteria isolated from fresh produce in Germany and description of Enterobacter vonholyi sp. nov. isolated from marjoram and Enterobacter dykesii sp. nov. isolated from mung bean sprout.

Forty-two antibiotic-resistant enterobacteria strains were isolated from fresh produce obtained from the northern German retail market. A polyphasic characterization based on both phenotypic and genotypic methods was used to identify predominant strains as Citrobacter (C.) gillenii, C. portucalensis, Enterobacter (En.) ludwigii, Escherichia (E.) coli and Klebsiella (K.) pneumoniae. 38.1% of the enterobacteria strains were resistant to tetracycline, while 23.8% and 9.5% of strains were resistant to streptomycin and chloramphenicol, respectively. A high percentage of Klebsiella (100%), Enterobacter (57.1%) and Citrobacter (42.9%) strains were also resistant to ampicillin, with some strains showing multiple resistances. For unequivocal species identification, the genomes of thirty strains were sequenced. Multilocus sequence analysis, average nucleotide identity and digital DNA-DNA hybridization showed that Enterobacter strains E1 and E13 were clearly clustered apart from Enterobacter species type strains below the species delineation cutoff values. Thus, strains E1T (=DSM 111347T, LMG 31875T) represents a novel species proposed as Enterobacter dykesii sp. nov., while strain E13T (=DSM 110788T, LMG 31764T) represent a novel species proposed as Enterobacter vonholyi sp. nov. Strains often possessed different serine ß-lactamase genes, tet(A) and tet(D) tetracycline resistance genes and other acquired antibiotic resistance genes. Typical plasmid replicon types were determined. This study thus accurately identified the enterobacteria from fresh produce as species belonging to the genera Citrobacter, Enterobacter, Escherichia and Klebsiella, but also showed that these can carry potentially transferable antibiotic resistance genes and may thus contribute to the spread of these via the food route.

Complete Genome Sequence of a Shiga Toxin-Producing Escherichia coli O26:H11 Strain (Sequence Type 21) and Two Draft Genome Sequences of Listeria monocytogenes Strains (Clonal Complex 1 [CC1] and CC59) Isolated from Fresh Produce in Germany.

The complete genome sequence of a Shiga toxin-producing Escherichia coli (STEC) O26:H11 strain, MBT-5 (sequence type 21 [ST21], stx 1a, stx 2a, eae, ehxA), and two draft genome sequences of Listeria monocytogenes strains MBT-6 and MBT-7 belonging to the virulent sequence types 1 (ST1, clonal complex 1 [CC1]) and 59 (ST59, CC59), respectively, were determined. The strains were isolated in 2015 from ready-to-eat mixed greens in Germany.