Large Multicountry Outbreak of Invasive Listeriosis by a Listeria monocytogenes ST394 Clone Linked to Smoked Rainbow Trout, 2020 to 2021.

Whole-genome sequencing (WGS) has revolutionized surveillance of infectious diseases. Disease outbreaks can now be detected with high precision, and correct attribution of infection sources has been improved. Listeriosis, caused by the bacterium Listeria monocytogenes, is a foodborne disease with a high case fatality rate and a large proportion of outbreak-related cases. Timely recognition of listeriosis outbreaks and precise allocation of food sources are important to prevent further infections and to promote public health. We report the WGS-based identification of a large multinational listeriosis outbreak with 55 cases that affected Germany, Austria, Denmark, and Switzerland during 2020 and 2021. Clinical isolates formed a highly clonal cluster (called Ny9) based on core genome multilocus sequence typing (cgMLST). Routine and ad hoc investigations of food samples identified L. monocytogenes isolates from smoked rainbow trout filets from a Danish producer grouping with the Ny9 cluster. Patient interviews confirmed consumption of rainbow trout as the most likely infection source. The Ny9 cluster was caused by a MLST sequence type (ST) ST394 clone belonging to molecular serogroup IIa, forming a distinct clade within molecular serogroup IIa strains. Analysis of the Ny9 genome revealed clpY, dgcB, and recQ inactivating mutations, but phenotypic characterization of several virulence-associated traits of a representative Ny9 isolate showed that the outbreak strain had the same pathogenic potential as other serogroup IIa strains. Our report demonstrates that international food trade can cause multicountry outbreaks that necessitate cross-border outbreak collaboration. It also corroborates the relevance of ready-to-eat smoked fish products as causes for listeriosis. IMPORTANCE Listeriosis is a severe infectious disease in humans and characterized by an exceptionally high case fatality rate. The disease is transmitted through consumption of food contaminated by the bacterium Listeria monocytogenes. Outbreaks of listeriosis often occur but can be recognized and stopped through implementation of whole-genome sequencing-based pathogen surveillance systems. We here describe the detection and management of a large listeriosis outbreak in Germany and three neighboring countries. This outbreak was caused by rainbow trout filet, which was contaminated by a L. monocytogenes clone belonging to sequence type ST394. This work further expands our knowledge on the genetic diversity and transmission routes of an important foodborne pathogen.

Genetic Characterization of Listeria from Food of Non-Animal Origin Products and from Producing and Processing Companies in Bavaria, Germany.

Reported cases of listeriosis from food of non-animal origin (FNAO) are increasing. In order to assess the risk of exposure to Listeria monocytogenes from FNAO, the genetic characterization of the pathogen in FNAO products and in primary production and processing plants needs to be investigated. For this, 123 samples of fresh and frozen soft fruit and 407 samples of 39 plants in Bavaria, Germany that produce and process FNAO were investigated for Listeria contamination. As a result, 64 Listeria spp. isolates were detected using ISO 11290-1:2017. Environmental swabs and water and food samples were investigated. L. seeligeri (36/64, 56.25%) was the most frequently identified species, followed by L. monocytogenes (8/64, 12.50%), L. innocua (8/64, 12.50%), L. ivanovii (6/64, 9.38%), L. newyorkensis (5/64, 7.81%), and L. grayi (1/64, 1.56%). Those isolates were subsequently sequenced by whole-genome sequencing and subjected to pangenome analysis to retrieve data on the genotype, serotype, antimicrobial resistance (AMR), and virulence markers. Eight out of sixty-four Listeria spp. isolates were identified as L. monocytogenes. The serogroup analysis detected that 62.5% of the L. monocytogenes isolates belonged to serogroup IIa (1/2a and 3a) and 37.5% to serogroup IVb (4b, 4d, and 4e). Furthermore, the MLST (multilocus sequence typing) analysis of the eight detected L. monocytogenes isolates identified seven different sequence types (STs) and clonal complexes (CCs), i.e., ST1/CC1, ST2/CC2, ST6/CC6, ST7/CC7, ST21/CC21, ST504/CC475, and ST1413/CC739. The core genome MLST analysis also showed high allelic differences and suggests plant-specific isolates. Regarding the AMR, we detected phenotypic resistance against benzylpenicillin, fosfomycin, and moxifloxacin in all eight L. monocytogenes isolates. Moreover, virulence factors, such as prfA, hly, plcA, plcB, hpt, actA, inlA, inlB, and mpl, were identified in pathogenic and nonpathogenic Listeria species. The significance of L. monocytogenes in FNAO is growing and should receive increasing levels of attention.

Whole-Genome Sequence Comparisons of Listeria monocytogenes Isolated from Meat and Fish Reveal High Inter- and Intra-Sample Diversity.

Interpretation of whole-genome sequencing (WGS) data for foodborne outbreak investigations is complex, as the genetic diversity within processing plants and transmission events need to be considered. In this study, we analyzed 92 food-associated Listeria monocytogenes isolates by WGS-based methods. We aimed to examine the genetic diversity within meat and fish production chains and to assess the applicability of suggested thresholds for clustering of potentially related isolates. Therefore, meat-associated isolates originating from the same samples or processing plants as well as fish-associated isolates were analyzed as distinct sets. In silico serogrouping, multilocus sequence typing (MLST), core genome MLST (cgMLST), and pangenome analysis were combined with screenings for prophages and genetic traits. Isolates of the same subtypes (cgMLST types (CTs) or MLST sequence types (STs)) were additionally compared by SNP calling. This revealed the occurrence of more than one CT within all three investigated plants and within two samples. Analysis of the fish set resulted in predominant assignment of isolates from pangasius catfish and salmon to ST2 and ST121, respectively, potentially indicating persistence within the respective production chains. The approach not only allowed the detection of distinct subtypes but also the determination of differences between closely related isolates, which need to be considered when interpreting WGS data for surveillance.

Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk.

Full-length SSU rRNA gene sequencing allows species-level identification of the microorganisms present in milk samples. Here, we used bulk-tank raw milk samples of two German dairies and detected, using this method, a great diversity of bacteria, archaea, and yeasts within the samples. Moreover, the species-level classification was improved in comparison to short amplicon sequencing. Therefore, we anticipate that this approach might be useful for the detection of possible mastitis-causing species, as well as for the control of spoilage-associated microorganisms. In a proof of concept, we showed that we were able to identify several putative mastitis-causing or mastitis-associated species such as Streptococcusuberis, Streptococcusagalactiae, Streptococcusdysgalactiae, Escherichiacoli and Staphylococcusaureus, as well as several Candida species. Overall, the presented full-length approach for the sequencing of SSU rRNA is easy to conduct, able to be standardized, and allows the screening of microorganisms in labs with Illumina sequencing machines.

Facklamia lactis sp. nov., isolated from raw milk.

Two strains of a Gram-staining-positive species were isolated from German bulk tank milk. On the basis of their 16S rRNA sequences they were affiliated to the genus Facklamia but could not be assigned to any species with a validly published name. Facklamia miroungae ATCC BAA-466T (97.3 % 16S rRNA sequence similarity), Facklamia languida CCUG 37842T (96.9 %), and Facklamia hominis CCUG 36813T (96.6 %) are the closest relatives. In the 16S rRNA phylogeny and in the core-genome phylogeny strains WS 5301T and WS 5302 form a well-supported, separate lineage. Pairwise average nucleotide identity calculated using MUMmer (ANIm) between WS 5301T and type strains of other Facklamia species is well below the species cut-off (95 %) and ranges from 83.4 to 87.7 %. The DNA G+C content of the type strain is 36.4 mol% and the assembly size of the genome is 2.2 Mb. Cells of WS 5301T are non-motile, non-endospore-forming, oxidase-negative, catalase-negative and facultatively anaerobic cocci. The fastidious species grows at 10-40 °C and with up to 7.0 % (w/v) NaCl in BHI supplemented with 5 g l-1 yeast extract. Major polar lipids are phosphatidylglycerol, diphosphatidylglycerol and two glycolipids. Predominant fatty acids are C16 : 1ω9c and C18 : 1ω9c. On the basis of their genomic, physiological and chemotaxonomic characteristics the strains examined in this study represent the same, hitherto unknown species. We propose the name Facklamia lactis sp. nov. for which WS 5301T (=DSM 111018T=LMG 31861T) is the type strain and WS 5302 (=DSM 111019=LMG 31862) is an additional strain of this novel species.

Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR.

The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers. KEY POINTS: • Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota. • Reduction of eukaryotic DNA by enzymatic digestion. • Shift of detected microbiome caused by high cycle numbers in library-PCR.

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.

Simultaneous quantification of the most common and proteolytic Pseudomonas species in raw milk by multiplex qPCR.

The heat-stable peptidase AprX, secreted by psychrotolerant Pseudomonas species in raw milk, is a major cause of destabilization and premature spoilage of ultra-high temperature (UHT) milk and milk products. To enable rapid detection and quantification of seven frequent and proteolytic Pseudomonas species (P. proteolytica, P. gessardii, P. lactis, P. fluorescens, P. protegens, P. lundensis, and P. fragi) in raw milk, we developed two triplex qPCR assays taking into account species-dependent differences in AprX activity. Besides five species-specific hydrolysis probes, targeting the aprX gene, a universal rpoB probe was included in the assay to determine the total Pseudomonas counts. For all six probes, linear regression lines between Cq value and target DNA concentration were obtained in singleplex as well as in multiplex approaches, yielding R2 values of > 0.975 and amplification efficiencies of 85-97%. Moreover, high specificity was determined using genomic DNA of 75 Pseudomonas strains, assigned to 57 species, and 40 other bacterial species as templates in the qPCR. Quantification of the target species and total Pseudomonas counts resulted in linear detection ranges of approx. 103-107 cfu/ml, which correspond well to common Pseudomonas counts in raw milk. Application of the assay using 60 raw milk samples from different dairies showed good agreement of total Pseudomonas counts calculated by qPCR with cell counts derived from cultivation. Furthermore, a remarkably high variability regarding the species composition was observed for each milk sample, whereby P. lundensis and P. proteolytica/P. gessardii were the predominant species detected. KEY POINTS: • Multiplex qPCR for quantification of seven proteolytic Pseudomonas species and total Pseudomonas counts in raw milk • High specificity and sensitivity via hydrolysis probes against aprX and rpoB • Rapid method to determine Pseudomonas contamination in raw milk and predict spoilage potential.

Pseudomonas cremoris sp. nov., a novel proteolytic species isolated from cream.

During a study investigating the microbiota of raw milk and its semi-finished products, strains WS 5106T and WS 5096 were isolated from cream and skimmed milk concentrate. They could be assigned to the genus Pseudomonas by their 16S rRNA sequences, but not to any validly named species. In this work, a polyphasic approach was used to characterize the novel strains and to investigate their taxonomic status. Examinations based on the topology of core genome phylogenomy as well as average nucleotide identity (ANIm) comparisons suggested a novel Pseudomonas species within the Pseudomonas fluorescens subgroup. With pairwise ANIm values of 90.1 and 89.8 %, WS 5106T was most closely related to Pseudomonas nabeulensis CECT 9765T and Pseudomonas kairouanensis CECT 9766T. The G+C content of strain WS 5106T was 60.1 mol%. Morphologic analyses revealed Gram-stain-negative, aerobic, catalase and oxidase positive, rod-shaped and motile cells. Proteolysis on skimmed milk agar as well as lipolysis on tributyrin agar occurred at both 28 and 6 °C. Tolerated growth conditions were temperatures between 4 and 34 °C, pH values between 6.0 and 8.0, and salt concentrations of up to 5 %. Fatty acid profiles showed a pattern typical for Pseudomonas, with C16 : 0 as the dominant component. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol and the dominating quinone was Q-9. Based on these results, it is proposed to classify the strains as a novel species, Pseudomonas cremoris sp. nov., with WS 5106T (=DSM 111143T=LMG 31863T) as type strain and WS 5096 (=DSM 111129=LMG 31864) as an additional strain.

High counts of thermophilic spore formers in dairy powders originate from persisting strains in processing lines.

During the last decades, thermophilic spore counts became a very important quality parameter for manufacturers with regard to powdered dairy products. Low-spore count powders are highly demanded but challenging to produce when high production volume and long process times are intended. In this study a detailed monitoring of microbial levels in three skim milk powder plants was conducted. Anoxybacillus flavithermus was found to be primarily responsible for increased spore levels with increasing spore numbers being detected after 6-8 h already during initial processing steps. Simultaneously, the species composition shifted from a diverse bulk tank milk microbiota where different Bacillus species represented around 90% of the thermophilic bacteria to a dominance of A. flavithermus in the end product. The analysis of A. flavithermus isolates from different powder batches with RAPD PCR revealed recurring patterns in each of the eight German manufacturers sampled over several months. The high relatedness of isolates exhibiting identical RAPD patterns was exemplified by cgMLST based on whole genome sequences. We assume that A. flavithermus strains persisted in production plants and were not eliminated by cleaning. It is concluded that such persisting strains recurrently recontaminated subsequent powder productions. The data highlight that a targeted optimization of cleaning and disinfection procedures is the most promising measure to effectively reduce thermophilic spore counts in German dairy powders.

Complementary Use of Cultivation and High-Throughput Amplicon Sequencing Reveals High Biodiversity Within Raw Milk Microbiota.

Raw milk microbiota are complex communities with a significant impact on the hygienic, sensory and technological quality of milk products. However, there is a lack of knowledge on factors determining their composition. In the present study, four bulk tank milk samples of two farms at two different time points were analyzed in detail for their microbiota using cultivation and 16S rRNA amplicon sequencing. Diversity in samples from the first time point was assessed via cultivation of 500 aerobic mesophilic bacterial isolates in each sample. A high biodiversity of 70 and 110 species per sample was determined, of which 25-28% corresponded to yet unknown taxa. The isolates were dominated by Gram-positive members of the genera Staphylococcus, Corynebacterium, Streptococcus, or Janibacter, whilst Chryseobacterium and Acinetobacter were most abundant among the Gram-negative taxa. At the second time point, samples of the same farms were analyzed via both cultivation (1,500 individual colonies each) and high-throughput 16S rRNA gene amplicon sequencing. The latter revealed a threefold higher biodiversity at the genus level, as anaerobic or fastidious species were also detected. However, cultivation identified genera not captured by sequencing, indicating that both approaches are complementary. Using amplicon sequencing, the relative abundance of a few genera was distorted, which seems to be an artifact of sample preparation. Therefore, attention needs to be paid to the library preparation procedure with special emphasis on cell lysis and PCR.

Fundicoccus ignavus gen. nov., sp. nov., a novel genus of the family Aerococcaceae isolated from bulk tank milk.

Three strains of a Gram-stain-positive, catalase-negative, facultative anaerobic, and coccoid species were isolated from German bulk tank milk. Phylogenetic analyses based on the 16S rRNA gene sequences indicated that the three strains (WS4937T, WS4759 and WS5303) constitute an independent phylogenetic lineage within the family Aerococcaceae with Facklamia hominis CCUG 36813T (93.7-94.1 %) and Eremococcus coleocola M1831/95/2T (93.5 %) as most closely related type species. The unclassified strains demonstrated variable growth with 6.5 % (w/v) NaCl and tolerated pH 6.5-9.5. Growth was observed from 12 to 39 °C. Their cell-wall peptidoglycan belongs to the A1α type (l-Lys-direct) consisting of alanine, glutamic acid and lysine. The predominant fatty acids were C16 : 1 ω9c, C16 : 0 and C18 : 1 ω9c and in the polar lipids profile three glycolipids, a phospholipid, phosphatidylglycerol, phosphoglycolipid and diphosphatidylglycerol were found. The G+C content of strain WS4937T was 37.4 mol% with a genome size of ~3.0 Mb. Based on phylogenetic, phylogenomic and biochemical characterizations, the isolates can be demarcated from all other genera of the family Aerococcaceae and, therefore, the novel genus Fundicoccus gen. nov. is proposed. The type species of the novel genus is Fundicoccus ignavus gen. nov., sp. nov. WS4937T (=DSM 109652T=LMG 31441T).

Genetic Organization of the aprX-lipA2 Operon Affects the Proteolytic Potential of Pseudomonas Species in Milk.

Psychrotolerant Pseudomonas species are a main cause of proteolytic spoilage of ultra-high temperature (UHT) milk products due to the secretion of the heat-resistant metallopeptidase AprX, which is encoded by the first gene of the aprX-lipA2 operon. While the proteolytic property has been characterized for many different Pseudomonas isolates, the underlying aprX-lipA2 gene organization was only described for a few strains so far. In this study, the phylogenomic analysis of 185 Pseudomonas type strains revealed that the presence of aprX is strongly associated to a monophylum composed of 81 species, of which 83% carried the aprX locus. Furthermore, almost all type strains of known milk-relevant species were shown to be members of the three monophyletic groups P. fluorescens, P. gessardii, and P. fragi. In total, 22 different types of aprX-lipA2 genetic organizations were identified in the genus, whereby 31% of the species tested carried the type 1 operon structure consisting of eight genes (aprXIDEF prtAB lipA2). Other genetic structures differed from type 1 mainly in the presence and location of genes coding for two lipases (lipA1 and lipA2) and putative autotransporters (prtA and prtB). The peptidase activity of 129 strains, as determined on skim milk agar and in UHT-milk, correlated largely with different aprX-lipA2 gene compositions. Particularly, isolates harboring the type 1 operon were highly proteolytic, while strains with other operon types, especially ones lacking prtA and prtB, exhibited significantly lower peptidase activities. In conclusion, the phylogenomic position and the aprX-lipA2 gene organization specify the proteolytic potential of Pseudomonas isolates. In addition, however, an interplay of several environmental factors and intrinsic traits influences production and activity of AprX, leading to strain-specific proteolytic phenotypes.

Brevilactibacter flavus gen. nov., sp. nov., a novel bacterium of the family Propionibacteriaceae isolated from raw milk and dairy products and reclassification of Propioniciclava sinopodophylli as Brevilactibacter sinopodophylli comb. nov.

Eight facultatively anaerobic rod-shaped bacteria were isolated from raw milk and two other dairy products. Results of phylogenetic analyses based on 16S rRNA gene sequences showed that the isolates are placed in a distinct lineage within the family Propionibacteriaceae with Propioniciclava sinopodophylli and Propioniciclava tarda as the closest relatives (94.6 and 93.5 % similarity, respectively). The cell-wall peptidoglycan contained meso-diaminopimelic acid, alanine and glutamic acid and was of the A1γ type (meso-DAP-direct). The major cellular fatty acid was anteiso-C15 : 0 and the major polar lipids were diphosphatidylglycerol, phosphatidyglycerol and three unidentified glycolipids. The quinone system contained predominantly menaquinone MK-9(H4). The G+C content of the genomic DNA of strain VG341T was 67.7 mol%. The whole-cell sugar pattern contained ribose, rhamnose, arabinose and galactose. On the basis of phenotypic and genetic data, eight strains (VG341T, WS4684, WS4769, WS 4882, WS4883, WS4901, WS4902 and WS4904) are proposed to be classified as members of a novel species in a new genus of the family Propionibacteriaceae, for which the name Brevilactibacter flavus gen. nov., sp. nov. is proposed. The type strain is VG341T (=WS4900T=DSM 100885T=LMG 29089T) and seven additional strains are WS4684, WS4769, WS4882, WS4883, WS4901, WS4902 and WS4904. Furthermore, we propose the reclassification of P. sinopodophylli as Brevilactibacter sinopodophylli comb. nov.

Pseudomonas haemolytica sp. nov., isolated from raw milk and skimmed milk concentrate.

Two strains, WS 5063T and WS 5067, isolated from raw cow's milk and skimmed milk concentrate, could be affiliated as members of the same, hitherto unknown, Pseudomonas species by 16S rRNA and rpoD gene sequences. Multilocus sequence and average nucleotide identity (ANIm) analyses based on draft genome sequences confirmed the discovery of a novel Pseudomonas species. It was most closely related to Pseudomonas synxantha DSM 18928T with an ANIm of 91.4 %. The DNA G+C content of WS 5063T was 60.0 mol %. Phenotypic characterizations showed that the isolates are rod-shaped, motile, catalase- and oxidase-positive, and aerobic. Growth occurred at 4-34 °C and at pH values of pH 5.5-8.0. Both strains showed strong ß-haemolysis on blood agar. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The dominant quinone was Q-9 (90 %), but noticeable amounts of Q-8 (9 %) and traces of Q-7 were also detected. Fatty acid profiles were typical for Pseudomonas species and exhibited C16 : 0 as a major component. Based on these results, we conclude that both strains belong to a novel species, for which the name Pseudomonas haemolytica sp. nov. is proposed. The type strain is WS 5063T (=DSM 108987T=LMG 31232T) and an additional strain is WS 5067 (=DSM 108988=LMG 31233).

Pseudomonas saxonica sp. nov., isolated from raw milk and skimmed milk concentrate.

A polyphasic approach was used to investigate the taxonomic status of two bacterial strains, WS 5072T and WS 5092, isolated from skimmed milk concentrate and raw cow's milk. The 16S rRNA and rpoD gene sequences affiliated the strains to the same, hitherto unknown, Pseudomonas species. Further examinations of the draft genomes based on multilocus sequence analysis and average nucleotide identity confirmed the presence of a novel Pseudomonas species. It was most closely related to Pseudomonas fragi DSM 3456T with 86.3 % ANIm. The DNA G+C content of strain WS 5072T was 56.3 mol%. Cells were aerobic, Gram-negative, catalase and oxidase positive, rod-shaped and motile. Growth occurred at 4-34 °C, pH 5.5-8.0 and with salt concentrations of up to 7 %. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The dominating quinone was Q-9 with 94 %, with noticeable amounts of Q-8 (5 %) and traces of Q-7 and Q-10. Fatty acid profiles showed a composition common for Pseudomonas with the major component C16 : 0. Based on these results, the novel species Pseudomonas saxonica sp. nov. is proposed, with the type strain WS 5072T (=DSM 108989T=LMG 31234T) and the additional strain WS 5092 (=DSM 108990=LMG 31235).

Resistance of thermophilic spore formers isolated from milk and whey products towards cleaning-in-place conditions: Influence of pH, temperature and milk residues.

The occurrence of thermophilic spore formers in dairy powders is a major concern for producers worldwide. This study aims to investigate the resistance of thermophilic endospores towards cleaning solutions typically used for cleaning-in-place in dairy manufacturing plants. From eleven tested strains, all were able to survive an alkaline treatment (NaOH) at 65 °C for 10 min (0.5%), whereas at concentrations of 2% eight strains withstood the treatment. Acid solutions were more sporicidal. At 0.5% of HNO3, only three strains survived the treatment. Milk impurities reduced the inactivation effect of the NaOH solutions towards thermophilic spore formers. For two selected strains, a detailed kinetic inactivation in NaOH and HNO3 solutions at different temperatures was performed and non-log-linear inactivation curves were observed. This study highlights the risk of reusing cleaning solutions in dairies.

Proposal of Lysobacter pythonis sp. nov. isolated from royal pythons (Python regius).

The bacterial strains 4284/11T and 812/17 isolated from the respiratory tract of two royal pythons in 2011 and 2017, respectively were subjected to taxonomic characterization. The 16S rRNA gene sequences of the two strains were identical and showed highest sequence similarities to Lysobacter tolerans UM1T (97.2%) and Luteimonas aestuarii DSM 19680T (96.7 %). The two strains were identical in the sequences of the 16S-23S rRNA internal transcribed spacer (ITS) and partial groEL gene sequences and almost identical in genomic fingerprints. In the ITS sequence Ly. tolerans DSM 28473T and in the groEL nucleotide sequence Luteimonas mephitis DSM 12574T showed the highest similarity. In silico DDH analyses using genome sequence based ANIb and gANI similarity coefficients demonstrated that strain 4284/11T represents a novel species and revealed Ly. tolerans UM1T as the next relative (ANIb = 76.2 %, gANI = 78.0 %). Based on the topology of a core gene phylogeny strain 4284/11T could be assigned to the genus Lysobacter. Chemotaxonomic characteristics including polyamine pattern, quinone system, polar lipid profile and fatty acid profile were in accordance with the characteristics of the genera Lysobacter and Luteimonas. Strains 4284/11T and 812/17 could be differentiated from the type strains of the most closely related species by several physiological tests. In conclusion we are here proposing the novel species Lysobacter pythonis sp. nov. The type strain is 4284/11T (= CCM 8829T = CCUG 72164T = LMG 30630T) and strain 812/17 (CCM 8830) is a second strain of this species.

Dynamic Proteome Alteration and Functional Modulation of Human Saliva Induced by Dietary Chemosensory Stimuli.

Saliva flow measurements and SDS-PAGE separation of human whole saliva freshly collected after oral stimulation with citric acid (sour), aspartame (sweet), iso-α-acids (bitter), mono sodium l-glutamate (umami), NaCl (salty), 6-gingerol (pungent), hydroxy-α-sanshool (tingling), and hydroxy-ß-sanshool (numbing), followed by tryptic digestion, nano-HPLC-MS/MS, and label-free protein quantitation demonstrated a stimulus- and time-dependent influence of the dietary chemosensates on salivation and the salivary proteome composition. Gene ontology enrichment analysis showed evidence for stimulus-induced alterations of the saliva proteome to boot an efficient molecular defense network of the oral cavity, e.g., 6-gingerol increased salivary lactoperoxidase activity, catalyzing the oxidation of thiocyanate to produce the antimicrobial and antifungal hypothiocyanate, from 0.37 ± 0.02 to 0.91 ± 0.05 mU/mL 45 s after stimulation. In comparison, oral citric acid stimulation induced an increase of myeloperoxidase activity, catalyzing the chloride oxidation to generate antimicrobial hypochloride in saliva, from 0.24 ± 0.04 to 0.70 ± 0.1 mU/mL as well as an increase of salivary levels of lysozyme, exhibiting antimicrobial activity on Gram-positive bacteria, from 6.0-10 to 100-150 µg/mL. Finally, microbial growth experiments clearly demonstrated for the first time that the increase of the salivary lysozyme abundance upon oral citric acid stimulation translates into an enhanced biological function, that is an almost complete growth inhibition of the two lysozyme-sensitive Gram-positive bacteria tested.

Genome sequence of the novel virulent bacteriophage PMBT14 with lytic activity against Pseudomonas fluorescens DSM 50090R.

Psychrotrophic gram-negative Pseudomonas spp. represent a serious problem in the dairy industry as they can cause spoilage of milk and dairy products. Bacteriophages have moved into focus as promising biocontrol agents for such food spoilage bacteria. The virulent Siphoviridae phage PMBT14 was isolated on a mutant variant of P. fluorescens DSM 50090 challenged with an unrelated virulent P. fluorescens DSM 50090 Podoviridae phage (i.e., mutant strain DSM 50090R). PMBT14 has a 47,820-bp dsDNA genome with 76 predicted open reading frames (ORFs). Its genome shows no significant sequence similarity to that of known phages, suggesting that PMBT14 represents a novel phage. Phage PMBT14 could be a promising biocontrol agent for P. fluorescens in milk or dairy foods.