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.

Motility in Periweissella Species: Genomic and Phenotypic Characterization and Update on Motility in Lactobacillaceae.

The genus Weissella and the recently described genus Periweissella, to which some previously named Weissella species have been reclassified as a result of a taxogenomic assessment, includes lactic acid bacteria species with high biotechnological and probiotic potential. Only one species, namely, Periweissella (P.) beninensis, whose type strain has been shown to possess probiotic features, has so far been described to be motile. However, the availability of numerous genome sequences of Weissella and Periweissella species prompted the possibility to screen for the presence of the genetic determinants encoding motility in Weissella and Periweissellas spp. other than P. beninensis. Herein, we performed a comprehensive genomic analysis to identify motility-related proteins in all Weissella and Periweissella species described so far, and extended the analysis to the recently sequenced Lactobacillaceae spp. Furthermore, we performed motility assays and transmission electron microscopy (TEM) on Periweissella type strains to confirm the genomic prediction. The homology-based analysis revealed genes coding for motility proteins only in the type strains of P. beninensis, P. fabalis, P. fabaria and P. ghanensis genomes. However, only the P. beninensis type strain was positive in the motility assay and displayed run-and-tumble behavior. Many peritrichous and long flagella on bacterial cells were visualized via TEM, as well. As for the Lactobacillaceae, in addition to the species previously described to harbor motility proteins, the genetic determinants of motility were also found in the genomes of the type strains of Lactobacillus rogosae and Ligilactobacillus salitolerans. This study, which is one of the first to analyze the genomes of Weissella, Periweissella and the recently sequenced Lactobacillaceae spp. for the presence of genes coding for motility proteins and which assesses the associated motility phenotypes, provides novel results that expand knowledge on these genera and are useful in the further characterization of lactic acid bacteria.

Identification and Characterization of Antibiotic-Resistant, Gram-Negative Bacteria Isolated from Korean Fresh Produce and Agricultural Environment.

The consumption of fresh produce and fruits has increased over the last few years as a result of increasing consumer awareness of healthy lifestyles. Several studies have shown that fresh produces and fruits could be potential sources of human pathogens and antibiotic-resistant bacteria. In this study, 248 strains were isolated from lettuce and surrounding soil samples, and 202 single isolates selected by the random amplified polymorphic DNA (RAPD) fingerprinting method were further characterized. From 202 strains, 184 (91.2%) could be identified based on 16S rRNA gene sequencing, while 18 isolates (8.9%) could not be unequivocally identified. A total of 133 (69.3%) and 105 (54.7%) strains showed a resistance phenotype to ampicillin and cefoxitin, respectively, while resistance to gentamicin, tobramycin, ciprofloxacin, and tetracycline occurred only at low incidences. A closer investigation of selected strains by whole genome sequencing showed that seven of the fifteen sequenced strains did not possess any genes related to acquired antibiotic resistance. In addition, only one strain possessed potentially transferable antibiotic resistance genes together with plasmid-related sequences. Therefore, this study indicates that there is a low possibility of transferring antibiotic resistance by potential pathogenic enterobacteria via fresh produce in Korea. However, with regards to public health and consumer safety, fresh produce should nevertheless be continuously monitored to detect the occurrence of foodborne pathogens and to hinder the transfer of antibiotic resistance genes potentially present in these bacteria.

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%.

Multidrug-resistant extended spectrum ß-lactamase (ESBL)-producing Escherichia coli from farm produce and agricultural environments in Edo State, Nigeria.

Antimicrobial resistance (AMR) is a major public health concern, especially the extended-spectrum ß-lactamase-producing (ESBL) Escherichia coli bacteria are emerging as a global human health hazard. This study characterized extended-spectrum ß-lactamase Escherichia coli (ESBL-E. coli) isolates from farm sources and open markets in Edo State, Nigeria. A total of 254 samples were obtained in Edo State and included representatives from agricultural farms (soil, manure, irrigation water) and vegetables from open markets, which included ready-to-eat (RTE) salads and vegetables which could potentially be consumed uncooked. Samples were culturally tested for the ESBL phenotype using ESBL selective media, and isolates were further identified and characterized via polymerase chain reaction (PCR) for ß-lactamase and other antibiotic resistance determinants. ESBL E. coli strains isolated from agricultural farms included 68% (17/25) from the soil, 84% (21/25) from manure and 28% (7/25) from irrigation water and 24.4% (19/78) from vegetables. ESBL E. coli were also isolated from RTE salads at 20% (12/60) and vegetables obtained from vendors and open markets at 36.6% (15/41). A total of 64 E. coli isolates were identified using PCR. Upon further characterization, 85.9% (55/64) of the isolates were resistant to ≥ 3 and ≤ 7 antimicrobial classes, which allows for characterizing these as being multidrug-resistant. The MDR isolates from this study harboured ≥1 and ≤5 AMR determinants. The MDR isolates also harboured ≥1 and ≤3 beta-lactamase genes. Findings from this study showed that fresh vegetables and salads could be contaminated with ESBL-E. coli, particularly fresh produce from farms that use untreated water for irrigation. Appropriate measures, including improving irrigation water quality and agricultural practices, need to be implemented, and global regulatory guiding principles are crucial to ensure public health and consumer safety.

Probiotic Potential and Safety Assessment of Type Strains of Weissella and Periweissella Species.

Although numerous strains belonging to the Weissella genus have been described in the last decades for their probiotic and biotechnological potential, others are known to be opportunistic pathogens of humans and animals. Here, we investigated the probiotic potential of two Weissella and four Periweissella type strains belonging to the species Weissella diestrammenae, Weissella uvarum, Periweissella beninensis, Periweissella fabalis, Periweissella fabaria, and Periweissella ghanensis by genomic and phenotypic analyses, and performed a safety assessment of these strains. Based on the results of the survival to simulated gastrointestinal transit, autoaggregation and hydrophobicity characteristics, as well as adhesion to Caco-2 cells, we showed that the P. beninensis, P. fabalis, P. fabaria, P. ghanensis, and W. uvarum type strains exhibited a high probiotic potential. The safety assessment, based on the genomic analysis, performed by searching for virulence and antibiotic resistance genes, as well as on the phenotypic evaluation, by testing hemolytic activity and antibiotic susceptibility, allowed us to identify the P. beninensis type strain as a safe potential probiotic microorganism. IMPORTANCE A comprehensive analysis of safety and functional features of six Weissella and Periweissella type strains was performed. Our data demonstrated the probiotic potential of these species, indicating the P. beninensis type strain as the best candidate based on its potential probiotic features and the safety assessment. The presence of different antimicrobial resistance profiles in the analyzed strains highlighted the need to establish cutoff values to perform a standardized safety evaluation of these species, which, in our opinion, should be mandatory on a strain-specific basis.

Serratia silvae sp. nov., Isolated from Forest Soil.

The Gram-negative, oxidase-negative, catalase-positive, rod-shaped strain Arafor3T was isolated from forest soil (France). Comparative 16S rRNA gene analysis and phylogenetic analysis based on (1) multilocus sequence analysis (MLSA) with four housekeeping genes (atpD, gyrB, infB and rpoB) and (2) genomes indicated that strain Arafor3T shared 98.83% 16S rRNA gene sequence similarity with the type strain of Serratia fonticola DSM 4576T and was closely related to this same strain in the MLSA and in the phylogenomic tree reconstruction. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) comparisons of strain Arafor3T with its nearest neighbor S. fonticola DSM 4576T showed 93.5% identity and 55.7% sequence similarity, respectively, and were lower than the 96% and 70% species-level cut-off values relating to these analyses (Logan et al. in Int J Syst Evol Microbiol 59:2114-21, 2009, https://doi.org/10.1099/ijs.0.013649-0 ). The strain differed from S. fonticola in that it was urease and arginine dihydrolase negative. The major fatty acids of strain Arafor3T are C16:0, C16:1 ω7c/C16:1 ω6c, C14:0, C14:0 3-OH/16:1 isoI, and C18:1 ω7c. The major respiratory quinone is Q8. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, and 6 unknown lipids. The mol G + C% content of the genomic DNA of strain Arafor3T was 53.49%. Hence, Arafor3T represents a novel species within the genus Serratia, for which the name Serratia silvae sp. nov. is proposed. The type strain is Arafor3T (=LMG 32338T = CIP 111939T).

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.

First Genome-Based Characterisation and Staphylococcal Enterotoxin Production Ability of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus Strains Isolated from Ready-to-Eat Foods in Algiers (Algeria).

Staphylococcus aureus is a pathogenic microorganism of humans and animals, able to cause foodborne intoxication due to the production of staphylococcal enterotoxins (SEs) and to resist antibiotic treatment as in the case of methicillin-resistant S. aureus (MRSA). In this study, we performed a genomic characterisation of 12 genetically diverse S. aureus strains isolated from ready-to-eat foods in Algiers (Algeria). Moreover, their ability to produce some classical and new staphylococcal enterotoxins (SEs) was investigated. The 12 S. aureus strains resulted to belong to nine known sequence types (STs) and to the novel ST7199 and ST7200. Furthermore, S. aureus SA46 was assigned to the European clone MRSA-ST80-SCCmec-IV. The 12 strains showed a wide endowment of se and sel (staphylococcal enterotoxin-like toxin) genes (sea, seb, sed, seg, seh, sei, selj, sek, sem, sen, seo, seq, ser, selu2, selw, selx, sey, sel30; ψent1-ψent2), including variants and pseudogenes, and harboured the enterotoxin gene cluster (egc) types 1 and 5. Additionally, they produced various amounts of SEA (64.54-345.02 ng/mL), SEB (2871.28-14739.17 ng/mL), SED (322.70-398.94 ng/mL), SEH (not detectable-239.48 ng/mL), and SER (36,720.10-63,176.06 ng/mL) depending on their genotypes. The genetic determinants related to their phenotypic resistance to ß-lactams (blaZ, mecA), ofloxacin (gyrA-S84L), erythromycin (ermB), lincomycin (lmrS), kanamycin (aph(3')-III, ant(6)-I), and tetracyclin (tet(L), tet(38)) were also detected. A plethora of virulence-related genes, including major virulence genes such as the tst gene, determinant for the toxic shock syndrome toxin-1, and the lukF-PV and lukS-PV genes, encoding the panton-valentine leukocidin (PVL), were present in the S. aureus strains, highlighting their pathogenic potential. Furthermore, a phylogenomic reconstruction including worldwide foodborne S. aureus showed a clear clustering based on ST and geographical origin rather than the source of isolation.

Novel Insights Into the Phylogeny and Biotechnological Potential of Weissella Species.

In this study, the genomes of the Weissella (W.) beninensis, W. diestrammenae, W. fabalis, W. fabaria, W. ghanensis, and W. uvarum type strains were sequenced and analyzed. Moreover, the ability of these strains to metabolize 95 carbohydrates was investigated, and the genetic determinants of such capability were searched within the sequenced genomes. 16S rRNA gene and genome-based-phylogeny of all the Weissella species described to date allowed a reassessment of the Weissella genus species groups. As a result, six distinct species groups within the genus, namely, W. beninensis, W. kandleri, W. confusa, W. halotolerans, W. oryzae, and W. paramesenteroides species groups, could be described. Phenotypic analyses provided further knowledge about the ability of the W. beninensis, W. ghanensis, W. fabaria, W. fabalis, W. uvarum, and W. diestrammenae type strains to metabolize certain carbohydrates and confirmed the interspecific diversity of the analyzed strains. Moreover, in many cases, the carbohydrate metabolism pathway and phylogenomic species group clustering overlapped. The novel insights provided in our study significantly improved the knowledge about the Weissella genus and allowed us to identify features that define the role of the analyzed type strains in fermentative processes and their biotechnological potential.

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.

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.

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.

Draft Genome Sequence of Bacillus subtilis subsp. subtilis MD 32, Isolated from the Korean Fermented Food Kimchi.

Bacillus subtilis subsp. subtilis MD 32 was isolated from kimchi. The strain was sequenced using an Illumina MiSeq platform, and the genome size was 4,238,856 bp with a GC content of 43.41 mol%. The genome encoded 4,396 proteins, with 45 tRNAs, 6 rRNAs, and 5 noncoding RNAs.

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.

Microbial quality and safety of milk and milk products in the 21st century.

Milk and milk products have been utilized by humans for many thousands of years. With the advent of metagenomic studies, our knowledge on the microbiota of milk and milk products, especially as affected by the environment, production, and storage parameters, has increased. Milk quality depends on chemical parameters (fat and protein content and absence of inhibitory substances), as well as microbial and somatic cells counts, and affects the price of milk. The effects of hygiene and effective cooling on the spoilage microbiota have shown that proteolytic and lipolytic bacteria such as Pseudomonas or Acinetobacter spp. predominate the spoilage bacterial populations. These bacteria can produce heat-stable proteases and lipases, which remain active after pasteurization and thus can spoil the milk during prolonged storage. Additionally, milk can become contaminated after pasteurization and therefore there is still a high demand on developing better cleaning and sanitation regimes and equipment, as well as test systems to (quantitatively) detect relevant pathogenic or spoilage microorganisms. Raw milk and raw milk cheese consumption is also increasing worldwide with the growing demand of minimally processed, sustainable, healthy, and local foods. In this context, emerging and re-emerging pathogens once again represent a major food safety challenge. As a result of global warming, it is conceivable that not only microbiological risks but also chemical risks relating to presence of mycotoxins or plant toxins in milk will increase. Herein, we provide an overview of the major microbial hazards occurring in the 21st century.

Complete Genome Sequence of Tetracycline-Resistant Serratia liquefaciens S1, Isolated from Mixed Greens, Obtained Using Illumina MiSeq and Oxford Nanopore MinION Sequencing.

Serratia liquefaciens strain S1 was isolated from mixed greens and sequenced with short-read Illumina MiSeq technology and long-read MinION technology. Hybrid sequence assembly generated a complete single circular chromosome and two extrachromosomal contigs, which together encoded 5,098 proteins, 85 tRNAs, and 22 rRNAs.

Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk.

In this study, 53 Staphylococcus (S.) aureus strains were typed by 16S-23S rDNA intergenic spacer region (ISR) typing and staphylococcal enterotoxin gene (SEg) typing for all the staphylococcal enterotoxin (se) and staphylococcal enterotoxin-like toxin (sel) genes known to date, revealing a higher discriminatory power than that of multi locus sequence typing. Six strains, one of each ISR- and SEg-type, were genome sequenced and the ability to produce some classical and new SEs when growing in milk was investigated. The manual analysis of the six genomes allowed us to confirm, correct and expand the results of common available genomic data pipelines such as VirulenceFinder. Moreover, it enabled us to (i) investigate the actual location of se and sel genes, even for genes such as selY, whose location (in the core genome) was so far unknown, (ii) find novel allelic variants of se and sel genes and pseudogenes, (iii) correctly annotate se and sel genes and pseudogenes, and (iv) discover a novel type of enterotoxin gene cluster (egc), i.e. the egc type 5 in strains 356P and 364P, while S. argenteus MSHR1132 harbored the egc type 6. Four of the six S. aureus strains produced sufficient amounts of SEA, SEC, SED and SEH in milk to cause staphylococcal food poisoning (SFP), with S. aureus 372 P being the highest producer of SED in milk found to date, producing as much as ca. 47,300 ng/mL and 49,200 ng/mL of SED, after 24 and 48 h of incubation in milk at 37 °C, respectively. S. aureus 372 P released a low amount of SER in milk, most likely because the seR gene was present as a pseudogene, putatively encoding only 51 amino acids. These findings confirm that not only the classical SEs, but also the new ones can represent a potential hazard for the consumers' health if produced in foods in sufficient amounts. Therefore, the detection of SEs in foods, especially if involved in SFP cases, should focus not only on classical, but also on all the new SEs and SEls known to date. Where reference methods are unavailable, the presence of the relevant genes, by using the conventional and real time PCR protocols we exhaustively provided herein, and their nucleotide sequences, should be investigated.