Draft genome sequences of three Carnobacterium maltaromaticum isolates from two low pH bogs in Denmark.

Carnobacterium maltaromaticum is a lactic acid bacterium that is widely distributed in the environment, including freshwater. Here, we report the draft genome sequences of three C. maltaromaticum isolates from low pH Danish bogs, using the Illumina MiSeq platform.

Veterinary bacteriology in Denmark from the 1880s to 2022.

This paper gives an account of the history of veterinary bacteriology including clinical veterinary bacteriology as well as the area of veterinary public health in Denmark from the 1880s to 2022. We describe key persons, including B. Bang, C.O. Jensen, K.A. Jensen and others who made important contributions to the development of these areas of microbiological expertise, and we discuss how challenges ranging from bovine tuberculosis to bacterial antimicrobial resistance have been met. Further, we describe progress in research on important bacterial pathogens both with regard to animal clinical aspects and zoonotic food-related aspects. Finally, we describe current issues in relation to One Health and research organization.

Distribution and ecology of the generalist lactic acid bacterium Carnobacterium maltaromaticum in different freshwater habitats: Metabolic and antagonistic abilities.

We explored the distribution, metabolic and antagonistic activities of Carnobacterium maltaromaticum, isolated from freshwater locations in Denmark during winter or early spring. This species was widely distributed in such habitats although it was relatively rare in low pH locations. Isolates possessed a diverse metabolism, potentially enabling functional capacities independent of habitat. The intraspecies competition showed a relatively high degree of mostly low-intensity interactions, which overall were not correlated with phylogeny or location. Only a few isolates exhibited broad-spectrum inhibition activity, targeting species from other genera and families, including one isolate that exhibited a broad inhibitory activity due to H2 O2 production. Bioinformatic analyses revealed that the frequency of bacteriocinogenic systems was low, and only one unmodified bacteriocin, piscicolin 126, correlated with phenotypic antagonistic activity. Furthermore, most potential bacteriocin gene complexes were not complete. Overall, this study showed C. maltaromaticum to be a generalist (nomadic) species with a constant presence in freshwater habitats, especially those with pH values >5. General metabolic properties did not suggest a strong degree of adaptation to the freshwater environment, and bacteriocin-mediated antagonistic activities appeared to play a minimal ecological role.

Bacterial species diversity of traditionally ripened sheep legs from the Faroe Islands (skerpikjøt).

Skerpikjøt is a traditionally ripened sheep leg product from the Faroe Islands, constituting a relatively underexplored microbial ecosystem. The objective of this study is to achieve a deeper understanding of the microbial composition of this artisanal product. Nine ripened hind legs, obtained from three different producers, were assessed regarding their bacterial communities and contents of biogenic amines, including both surface and core samples. Biogenic amine concentrations were generally low, although one sample had a somewhat elevated concentration of cadaverine. Bacterial diversity was investigated by culture-dependent and culture-independent techniques. Gram-positive catalase-positive cocci (GCC) constituted the most abundant group. Within this group, Staphylococcus equorum was the most prevailing species, followed by Kocuria sp., Mammaliicoccus vitulinus, and Staphylococcus saprophyticus. Lactic acid bacteria prevailed in only one sample and were mainly represented by Latilactobacillus curvatus. Enterobacterial communities were characterised by the prevalence of Serratia proteamaculans. Despite the majority of GCC, Clostridium putrefaciens was the most abundant bacterial species in some core samples. Taken together, the culture-dependent and culture-independent identification methods gave complementary results.

Scientists' Assessments of Research on Lactic Acid Bacterial Bacteriocins 1990-2010.

The antimicrobial activity of bacteriocins from lactic acid bacteria has constituted a very active research field within the last 35 years. Here, we report the results of a questionnaire survey with assessments of progress within this field during the two decades of the 1990s and the 2000s by 48 scientists active at that time. The scientists had research positions at the time ranging from the levels of Master's and Ph.D. students to principal investigators in 19 Asian, European, Oceanian and North American countries. This time period was evaluated by the respondents to have resulted in valuable progress regarding the basic science of bacteriocins, whereas this was not achieved to the same degree with regard to their applications. For the most important area of application, food biopreservation, there were some success stories, but overall the objectives had not been entirely met due to a number of issues, such as limited target spectrum, target resistance, poor yield as well as economic and regulatory challenges. Other applications of bacteriocins such as enhancers of the effects of probiotics or serving as antimicrobials in human clinical or veterinary microbiology, were not evaluated as having been implemented successfully to any large extent at the time. However, developments in genomic and chemical methodologies illustrate, together with an interest in combining bacteriocins with other antimicrobials, the current progress of the field regarding potential applications in human clinical microbiology and food biopreservation. In conclusion, this study illuminates parameters of importance not only for R&D of bacteriocins, but also for the broader field of antimicrobial research.

Novel cadaverine non-invasive biosensor technology for the prediction of shelf life of modified atmosphere packed pork cutlets.

Food waste in perishable products calls for the development of cost-efficient and real-time freshness and shelf life assessment tools. The current study evaluated a newly developed cadaverine biosensor for its ability to assess the sensory freshness stage and microbial quality of modified atmosphere packed (MAP) pork cutlets under a realistic supply chain scenario. The experiment compared the cadaverine levels measured by the biosensor to liquid chromatography - tandem mass spectrometry (LC-MS/MS) cadaverine concentrations, and associated these to the shelf life estimation and freshness states determined by sensory and microbial evaluations during an 18-day storage period (5 °C). Results underlined the potential of cadaverine as a freshness biomarker as well as the applicability of the biosensor as a shelf life prediction tool. This is supported by the correlations obtained between sensory odour freshness evaluation and total viable counts with biosensor cadaverine levels for which the r obtained were 0.97 (<0.001) and 0.95 (<0.001), respectively.

Identification of Potential Citrate Metabolism Pathways in Carnobacterium maltaromaticum.

In the present study, we describe the identification of potential citrate metabolism pathways for the lactic acid bacterium (LAB) Carnobacterium maltaromaticum. A phenotypic assay indicated that four of six C. maltaromaticum strains showed weak (Cm 6-1 and ATCC 35586) or even delayed (Cm 3-1 and Cm 5-1) citrate utilization activity. The remaining two strains, Cm 4-1 and Cm 1-2 gave negative results. Additional analysis showed no or very limited utilization of citrate in media containing 1% glucose and 22 or 30 mM citrate and inoculated with Cm 6-1 or ATCC 35586. Two potential pathways of citrate metabolism were identified by bioinformatics analyses in C. maltaromaticum including either oxaloacetate (pathway 1) or tricarboxylic compounds such as isocitrate and α-ketoglutarate (pathway 2) as intermediates. Genes encoding pathway 1 were present in two out of six strains while pathway 2 included genes present in all six strains. The two potential citrate metabolism pathways in C. maltaromaticum may potentially affect the sensory profiles of milk and soft cheeses subjected to growth with this species.

Characterization of antimicrobial-resistant Staphylococcus aureus from retail foods in Beijing, China.

Staphylococcus aureus is an opportunistic pathogen leading to food poisoning as well as human infections. The present study examined the prevalence and characterization of antimicrobial-resistant S. aureus in sushi from 42 outlets and in pork products from eight outlets in Beijing, China. The total bacterial counts were between 3.0 and 8.9 log CFU/g (mean 5.5 ± 1.5 log CFU/g) in sushi products and 4.8 to 7.4 log CFU/g (mean 5.6 ± 0.8 log CFU/g) in pork products. The mean counts of coliforms were 2.7 and 2.9 log CFU/g in sushi and pork, respectively. Staphylococcus aureus was isolated from seven sushi outlets (13 isolates) and two pork outlets (2 isolates) with average counts below 2 log CFU/g in all cases. A total of 15 S. aureus isolates were further characterized. Six lineages of S. aureus were present, including ST398 (n = 5), ST25 (n = 4), ST15 (n = 2), ST59 (n = 2), ST8 (n = 1) and ST2631 (n = 1). Thirteen isolates contained the scn virulence marker, whereas four and eight isolates contained the virulence marker edinB and enterotoxin genes, respectively. Characterization of antimicrobial resistance profiles documented resistances to ampicillin (n = 15), penicillin (n = 14), ceftazidime (n = 6), erythromycin (n = 4), tetracycline (n = 3), clindamycin (n = 3), and gentamicin (n = 1). Three MRSA isolates were obtained, one from pork (ST398) and two from one sushi outlet (ST59). They were all resistant to at least three classes of antimicrobials and two of them contained the scn gene and enterotoxin genes. Twelve sushi isolates and one of the pork isolates contained the scn gene, indicating that they were of human origin. This emphasizes the potential importance of transmission through foods of antimicrobial-resistant S. aureus including MRSA. We also showed that S. aureus exhibited geographical variation with regards to ST profiles, antimicrobial-resistance and virulence genes when comparing isolates from sushi products sold in Beijing and Copenhagen, Denmark. Whereas food safety is not compromised by the presence of low amounts of S. aureus in sushi, this study shows that with regards to public health such foods may serve as vehicles for transmission of multidrug-resistant S. aureus and MRSA lineages.

Nisin Damages the Septal Membrane and Triggers DNA Condensation in Methicillin-Resistant Staphylococcus aureus.

Nisin is applied as a food preservative in processed foods and has the potential to be used synergistically with antibiotics for treatment of patients infected by antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus. The present study explores the antimicrobial effect of nisin on S. aureus viability and membrane integrity and, for the first time, used super-resolution microscopy to study morphological changes induced in S. aureus cells exposed to nisin. The exposure of S. aureus to nisin caused membrane depolarization and rapid killing. Super-resolution structured-illumination microscopy and transmission electron microscopy confirmed that nisin damages the cellular membrane and causes lysis of cells. Strikingly, condensation of chromosomal DNA was observed in all cells exposed to nisin, a phenotype not previously reported for this compound. Moreover, cells exposed to nisin were significantly smaller than non-exposed cells indicating the emergence of cell shrinkage. The strong association of DNA condensation with nisin exposure indicates that nisin interferes with chromosome replication or segregation in S. aureus.

The Diverse Search for Synthetic, Semisynthetic and Natural Product Antibiotics From the 1940s and Up to 1960 Exemplified by a Small Pharmaceutical Player.

The 1940s and 1950s witnessed a diverse search for not just natural product antibiotics but also for synthetic and semisynthetic compounds. This review revisits this epoch, using the research by a Danish pharmaceutical company, LEO Pharma, as an example. LEO adopted a strategy searching for synthetic antibiotics toward specific bacterial pathogens, in particular Mycobacterium tuberculosis, leading to the discovery of a new derivative of a known drug. Work on penicillin during and after WWII lead to the development of associated salts/esters and a search for new natural product antibiotics. This led initially to no new, marketable compounds, but concluded with the serendipitous discovery of fusidic acid, an antibiotic used to treat infections by Staphylococcus aureus, in 1960. The discovery process included contemporary approaches such as open innovation; targeting specific pathogens and/or specific organs in the patient; examining the effects of antimicrobial compounds on bacterial virulence as well as on antibiotic-resistant variants, and searching for antibiotic producers among microorganisms not previously well explored. These activities were promoted by the collaboration with a renowned Danish clinical microbiologist, K. A. Jensen, as well as company expertise in fermentation technologies, chemical synthesis and purification of bioactive compounds from organic materials.

Synthesis and Antimicrobial Properties of a Ciprofloxacin and PAMAM-dendrimer Conjugate.

Infections caused by bacteria resistant to antibiotics are an increasing problem. Multivalent antibiotics could be a solution. In the present study, a covalent conjugate between Ciprofloxacin and a G0-PAMAM dendrimer has been synthesized and tested against clinically relevant Gram-positive and Gram-negative bacteria. The conjugate has antimicrobial activity and there is a positive dendritic effect compared to Ciprofloxacin itself.

Bacterial content and characterization of antibiotic resistant Staphylococcus aureus in Danish sushi products and association with food inspector rankings.

This study examined the prevalence and phenotypic and genotypic antibiotic resistance patterns of Staphylococcus aureus in sushi from 20 Danish outlets. Microbial quality of sushi products and food inspector ranking of outlets were assessed and results for thirteen of the outlets were compared with findings from a previous study in 2012. Inspector rankings were similar in the two studies. The mesophilic aerobic counts were slightly lower (p = 0.0296) in 2017 than in 2012 with average values of the 13 shops of 5.2 log CFU/g and 5.7 log CFU/g, respectively. In both studies E. coli was only found in the products from outlets that did not have consistently superior rankings. On the other hand prevalence and average counts of Staphylococcus spp. were slightly higher in 2017 (p = 0.0286) but no methicillin-resistant S. aureus (MRSA) were observed in the present study. Methicillin-sensitive S. aureus (MSSA) were, however, isolated from 18.7% of sushi products with an average count below 2 log CFU/g. Based on spa and multilocus sequence typing (MLST), isolates belonged to clonal complex CC7 (t2016), CC20 (t7836), CC45 (t065, t127, t362), CC88 (t1998) and CC398 (t164, t331, t1451). The Panton-Valentine leukocidin (PVL)-encoding gene lukF was detected only in isolates of the t065 spa-type whereas the scn gene from the ΦSa3 prophage was detected in 76.5% of the isolates, supporting that the majority of isolates were of likely human origin. Thirty-six isolates (70.6%) were resistant to at least one of the antibiotic compounds tested. Antibiotic resistance genes that confer resistance to ß-lactams (blaZ) and macrolides (ermC) were detected in 33.3% and 9.8% of isolates, respectively. The tet(K) gene that encode tetracycline resistance was only found in a t7836 strain. Overall, this study indicates that S. aureus in sushi products in Denmark do not represent a major food safety hazard due to, firstly, the low temperature and limited time of storage of product may prevent significant growth and production of toxic levels of enterotoxin of this species. Secondly, the S. aureus isolates obtained did not include MRSA variants and none of them encoded PVL that constitute one of the virulence factors in pathogenesis. Several MSSA isolates contained however genes encoding antibiotic resistance, which emphasize the potential role of foods as vehicles for transmission of such variants.

Antimicrobial Resistance and Virulence Gene Profiles of Methicillin-Resistant and -Susceptible Staphylococcus aureus From Food Products in Denmark.

Foods may potentially serve as vehicles for the transmission of antimicrobial-resistant variants of Staphylococcus aureus that are important in a human clinical context. Further, retail food products can be a cause of staphylococcal food poisoning. For these reasons and to account for source attribution and risk assessment, detailed information on the population structure, resistance, and virulence profiles of S. aureus originating from retail food products is necessary. In the current study, whole-genome sequences from 88 S. aureus isolates were subjected to bioinformatics analyses in relation to sequence types, antimicrobial resistance, and virulence profiles. The sequence types (ST) identified belonged to 13 clonal complexes (CC) with CC5 and CC398 being the most common. CC398 was identified as the dominant clone (n = 31). CC5 was identified as of avian origin, with the presence of φAVß prophage genes (n = 13). In total, 39.8% of the isolates contained multiple resistance genes, and methicillin-resistant Staphylococcus aureus (MRSA) isolates were found in CC8, CC9, and CC398. Genes conferring resistance to the antimicrobial classes of ß-lactams, tetracycline, and erythromycin were detected in this study, all of which are commonly used in Danish livestock production. The tst gene encoding the toxic shock syndrome toxin was for the first time identified in ST398 isolates, probably as a result of a single acquisition of a SaPI-like element. The sushi-CC398 isolates carrying the scn gene likely originated from a human reservoir, while the other isolates originated from livestock. Taken together, our results show that both human and animal reservoirs contribute to contamination in food products and that retail foods may serve as a vehicle of S. aureus between livestock and humans.

Comparative Genomic Analysis Reveals Ecological Differentiation in the Genus Carnobacterium.

Lactic acid bacteria (LAB) differ in their ability to colonize food and animal-associated habitats: while some species are specialized and colonize a limited number of habitats, other are generalist and are able to colonize multiple animal-linked habitats. In the current study, Carnobacterium was used as a model genus to elucidate the genetic basis of these colonization differences. Analyses of 16S rRNA gene meta-barcoding data showed that C. maltaromaticum followed by C. divergens are the most prevalent species in foods derived from animals (meat, fish, dairy products), and in the gut. According to phylogenetic analyses, these two animal-adapted species belong to one of two deeply branched lineages. The second lineage contains species isolated from habitats where contact with animal is rare. Genome analyses revealed that members of the animal-adapted lineage harbor a larger secretome than members of the other lineage. The predicted cell-surface proteome is highly diversified in C. maltaromaticum and C. divergens with genes involved in adaptation to the animal milieu such as those encoding biopolymer hydrolytic enzymes, a heme uptake system, and biopolymer-binding adhesins. These species also exhibit genes for gut adaptation and respiration. In contrast, Carnobacterium species belonging to the second lineage encode a poorly diversified cell-surface proteome, lack genes for gut adaptation and are unable to respire. These results shed light on the important genomics traits required for adaptation to animal-linked habitats in generalist Carnobacterium.

Bacterial viruses enable their host to acquire antibiotic resistance genes from neighbouring cells.

Prophages are quiescent viruses located in the chromosomes of bacteria. In the human pathogen, Staphylococcus aureus, prophages are omnipresent and are believed to be responsible for the spread of some antibiotic resistance genes. Here we demonstrate that release of phages from a subpopulation of S. aureus cells enables the intact, prophage-containing population to acquire beneficial genes from competing, phage-susceptible strains present in the same environment. Phage infection kills competitor cells and bits of their DNA are occasionally captured in viral transducing particles. Return of such particles to the prophage-containing population can drive the transfer of genes encoding potentially useful traits such as antibiotic resistance. This process, which can be viewed as 'auto-transduction', allows S. aureus to efficiently acquire antibiotic resistance both in vitro and in an in vivo virulence model (wax moth larvae) and enables it to proliferate under strong antibiotic selection pressure. Our results may help to explain the rapid exchange of antibiotic resistance genes observed in S. aureus.

Aeromonas salmonicida subsp. salmonicida strains isolated from Chinese freshwater fish contain a novel genomic island and possible regional-specific mobile genetic elements profiles.

Two strains of Aeromonas salmonicida, YK and BG, were isolated from largemouth bronze gudgeon and northern whitefish in China, and identified as A. salmonicida subsp. salmonicida based on phylogenetic analysis of vapA and 16S rRNA gene sequences. YK and BG originated from freshwater fish, one of which belonged to the cyprinid family, and the strains showed a difference in virulence. Subsequently, we performed whole genome sequencing of the strains, and comparison of their genomic sequences to the genome of the A449 reference strain revealed various genomic rearrangements, including a new variant of the genomic island AsaGEI in BG, designated as AsaGEI2c This is the first report on a GEI of A. salmonicida strain from China. Furthermore, both YK and BG strains contained a Tn7 transposon inserted at the same position in the chromosome. Finally, IS-dependent rearrangements on pAsa5 are deemed likely to have occurred, with omission of the resD gene in both strains as well as omission of genes related to the IncF conjugal transfer system in the YK isolate. This study demonstrates that A. salmonicida subsp. salmonicida can infect non-salmonids (cyprinids) in addition to salmonids, and that AsaGEI2c might be useful as a geographical indicator of Chinese A. salmonicida subsp. salmonicida isolates.

Genes associated to lactose metabolism illustrate the high diversity of Carnobacterium maltaromaticum.

The dairy population of Carnobacterium maltaromaticum is characterized by a high diversity suggesting a high diversity of the genetic traits linked to the dairy process. As lactose is the main carbon source in milk, the genetics of lactose metabolism was investigated in this LAB. Comparative genomic analysis revealed that the species C. maltaromaticum exhibits genes related to the Leloir and the tagatose-6-phosphate (Tagatose-6P) pathways. More precisely, strains can bear genes related to one or both pathways and several strains apparently do not contain homologs related to these pathways. Analysis at the population scale revealed that the Tagatose-6P and the Leloir encoding genes are disseminated in multiple phylogenetic lineages of C. maltaromaticum: genes of the Tagatose-6P pathway are present in the lineages I, II and III, and genes of the Leloir pathway are present in the lineages I, III and IV. These data suggest that these genes evolved thanks to horizontal transfer, genetic duplication and translocation. We hypothesize that the lac and gal genes evolved in C. maltaromaticum according to a complex scenario that mirrors the high population diversity.

Predation and selection for antibiotic resistance in natural environments.

Genes encoding resistance to antibiotics appear, like the antibiotics themselves, to be ancient, originating long before the rise of the era of anthropogenic antibiotics. However, detailed understanding of the specific biological advantages of antibiotic resistance in natural environments is still lacking, thus limiting our efforts to prevent environmental influx of resistance genes. Here, we propose that antibiotic-resistant cells not only evade predation from antibiotic producers but also take advantage of nutrients released from cells that are killed by the antibiotic-producing bacteria. Thus, predation is potentially an important mechanism for driving antibiotic resistance during slow or stationary phase of growth when nutrients are deprived. This adds to explain the ancient nature and widespread occurrence of antibiotic resistance in natural environments unaffected by anthropogenic antibiotics. In particular, we suggest that nutrient-poor environments including indoor environments, for example, clean rooms and intensive care units may serve as a reservoir and source for antibiotic-producing as well as antibiotic-resistant bacteria.

Injuries can prolong lifespan in Drosophila melanogaster males.

Previous studies have shown that a range of different stresses can increase mean lifespan. Here we investigated the effect of injuries and bacterial inoculation on mean lifespan in lines selected for increased longevity and their controls. The three lines from each selection regime were subjected to one of five treatments ranging from control, over perforating the cuticle with a sterile needle, to inoculating with peptidoglycan or one of two strains of live bacteria. The flies were subjected to the infection stress at two ages and the experiment was conducted on both males and females of replicate lines of each selection regime. The individual lines and sexes differed in response to the treatment. However, overall the sterile injury of young males resulted in prolonged mean lifespan from both selection regimes, whereas inoculating had no additional effect to stabbing with a sterile needle. In middle-aged males only treatment with peptidoglycan had a significant hormetic effect and this was only in longevity-selected flies. In females only one of the tested contrasts was significant and here the effect of the treatment was to reduce average lifespan. As could be expected, the results showed a significant interaction between the effects of sex and infection on survival.

Effects of Listeria monocytogenes EGD-e and Salmonella enterica ser. Typhimurium LT2 chitinases on intracellular survival in Dictyostelium discoideum and mammalian cell lines.

Some bacterial pathogens produce chitinases as virulence factors during host infection. The molecular target of such enzymes in non-chitinous hosts remains uncertain. We studied the importance of Listeria monocytogenes EGD-e and Salmonella enterica ser. Typhimurium LT2 chitinases for intracellular survival in Dictyostelium discoideum, and for Salmonella, also infection of mammalian cell lines, and a mouse model. The Salmonella chitinase did not contribute significantly to infection of D. discoideum, mammalian cell lines or mice. However, survival in D. discoideum was clearly reduced for Listeria mutants deficient of ChiB (8-fold) or deficient of both ChiA and ChiB (22-fold). Our findings suggest that chitinases from the two species play different roles in virulence.