Clonal spread and horizontal transfer mediate dissemination of phenicol-oxazolidinone-tetracycline resistance gene poxtA in enterococci isolates from a swine farm in China.

The emergence of the phenicol-oxazolidinone-tetracycline resistance gene poxtA becomes a significant challenge for public health, since it confers a decreased susceptibility not only to the last resort drug linezolid, but also to florfenicol and doxycycline widely used in veterinary medicine. To determine the dissemination mechanism of poxtA in enterococci isolates from different healthy pigs in the swine farm, a total of 178 florfenicol-resistant enterococci isolates were collected from 400 fresh faecal swabs in a swine farm in China. The poxtA gene was detected in 11 (6.18 %) enterococci isolates, including 8 E. faecium, 2 E. hirae and 1 E. casseliflavus isolates. Whole genome sequencing indicated that the eight poxtA-harbouring E. faecium strains belonged to four different sequence types, including ST156 and three new STs, ST1818, ST1819 and ST1820. Five out of the 11 poxtA-positive enterococci isolates also harboured optrA gene. Moreover, E. casseliflavus strain DY31 co-harboured poxtA, optrA and cfr. Seven different poxtA-harbouring plasmids were obtained through Nanopore combined with Illumina sequencing. The poxtA-harbouring plasmids exhibited high genetic variation, six out of which belonged to rep2 plasmid of Inc18 family. The poxtA gene was flanked by IS1216E in the left and/or right ends.The optrA and cfr genes were located on different plasmids, respectively, but those genes could be co-transferred with poxtA gene into the recipient E. faecalis strain by electrotransformation. Our study highlights that both clonal spread and horizontal transfer mediated by Inc18 plasmid and IS1216E promote the dissemination of poxtA in enterococci isolates from different healthy pigs in the swine farm.

Vagococcus sp. a porcine pathogen: molecular and phenotypic characterization of strains isolated from diseased pigs in Brazil.

INTRODUCTION: Vagococcus spp. is known for its importance as a systemic and zoonotic bacterial pathogen even though it is not often reported in pigs. This is related to the pathogen misidentification due to the lack of usage of more discriminatory diagnostic techniques. Here we present the first report of Vagococcus lutrae in swine and the characterization of Vagococcus fluvialis and Vagococcus lutrae isolated from diseased animals. METHODOLOGY: Between 2012 and 2017, 11 strains with morphological characteristics similar to Streptococcus spp. were isolated from pigs presenting different clinical signs. Bacterial identification was performed by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry and confirmed by 16S rRNA sequencing and biochemical profile. Strains were further genotyped by single-enzyme amplified fragment length polymorphism (SE-AFLP). Broth microdilution was used to determine the minimal inhibitory concentration of the antimicrobials of veterinary interest. RESULTS: Ten strains were identified as V. fluvialis and one was identified as V. lutrae. The SE-AFLP analysis enabled the species differentiation with specific clustering of all V. fluvialis separately from the V. lutrae strain. Most strains presented growth in the maximum antibiotic concentration values tested for eight of the 10 analyzed antimicrobial classes. CONCLUSIONS: The observed resistance pattern can represent a problem for veterinary and producers in the treatment of diseases associated Vagococcus spp. in swine production. Vagococcus species may also be a risk for pig industry workers. The data described here will be of great value in further understanding the behavior of this pathogen in animal production.

Transcriptional regulator SpxA1a controls the resistance of the honey bee pathogen Melissococcus plutonius to the antimicrobial activity of royal jelly.

Royal jelly (RJ), a brood food of honey bees, has strong antimicrobial activity. Melissococcus plutonius, the causative agent of European foulbrood of honey bees, exhibits resistance to this antimicrobial activity and infects larvae orally. Among three genetically distinct groups (CC3, CC12 and CC13) of M. plutonius, CC3 strains exhibit the strongest RJ resistance. In this study, to identify genes involved in RJ resistance, we generated an RJ-susceptible derivative from a highly RJ-resistant CC3 strain by UV mutagenesis. Genome sequence analysis of the derivative revealed the presence of a frameshift mutation in the putative regulator gene spxA1a. The deletion of spxA1a from a CC3 strain resulted in increased susceptibility to RJ and its antimicrobial component 10-hydroxy-2-decenoic acid. Moreover, the mutant became susceptible to low-pH and oxidative stress, which may be encountered in brood foods. Differentially expressed gene analysis using wild-type and spxA1a mutants revealed that 45 protein-coding genes were commonly upregulated in spxA1a-positive strains. Many upregulated genes were located in a prophage region, and some highly upregulated genes were annotated as universal/general stress proteins, oxidoreductase/reductase, chaperons and superoxide dismutase. These results suggest that SpxA1a is a key regulator to control the tolerance status of M. plutonius against stress in honey bee colonies.

Microbicidal effects of slightly acidic hypochlorous acid water and weakly acidified chlorous acid water on foulbrood pathogens.

Paenibacillus larvae and Melissococcus plutonius are bacterial pathogens of honey bee brood. As decontamination of beekeeping equipment, including combs, is essential to control these pathogens, we evaluated the disinfecting effects of slightly acidic hypochlorous acid water (SAHAW) and weakly acidified chlorous acid water (WACAW) on the pathogens. Both disinfectants exhibited strong disinfecting effects in suspension tests under no organic matter conditions and reduced both pathogens by >5 log10 CFU/ml. Although the microbicidal activity of SAHAW with an available chlorine concentration (ACC) of 10-30 ppm was decreased by organic matter, it reduced viable P. larvae spores in combs more efficiently than H2O when the comb was not as dirty. However, its efficacy on combs decreased at 4°C and when overused or highly contaminated combs were tested. WACAW with an ACC of ≥600 ppm had a higher disinfecting capacity than SAHAW, and efficiently removed P. larvae spores from combs even under organic matter-rich and low-temperature conditions. However, even by WACAW, the amount of viable spores in combs was not markedly reduced depending on contamination levels and P. larvae genotypes. These results suggest the usefulness of both disinfectants for decontaminating beekeeping equipment depending on the situations expected.

Conjugative Delivery of CRISPR-Cas9 for the Selective Depletion of Antibiotic-Resistant Enterococci.

The innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) that are recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiont Enterococcus faecalis is associated with HAIs, and some strains are MDR. Therefore, novel strategies to control E. faecalis populations are needed. We previously characterized an E. faecalis type II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here, we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers to E. faecalis for the selective removal of antibiotic resistance genes. Using in vitro competition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistant E. faecalis by several orders of magnitude. Finally, we show that E. faecalis donor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinants in vivo Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.

Impact of species and antibiotic therapy of enterococcal peritonitis on 30-day mortality in critical care-an analysis of the OUTCOMEREA database.

INTRODUCTION: Enterococcus species are associated with an increased morbidity in intraabdominal infections (IAI). However, their impact on mortality remains uncertain. Moreover, the influence on outcome of the appropriate or inappropriate status of initial antimicrobial therapy (IAT) is subjected to debate, except in septic shock. The aim of our study was to evaluate whether an IAT that did not cover Enterococcus spp. was associated with 30-day mortality in ICU patients presenting with IAI growing with Enterococcus spp. MATERIAL AND METHODS: Retrospective analysis of French database OutcomeRea from 1997 to 2016. We included all patients with IAI with a peritoneal sample growing with Enterococcus. Primary endpoint was 30-day mortality. RESULTS: Of the 1017 patients with IAI, 76 (8%) patients were included. Thirty-day mortality in patients with inadequate IAT against Enterococcus was higher (7/18 (39%) vs 10/58 (17%), p = 0.05); however, the incidence of postoperative complications was similar. Presence of Enterococcus spp. other than E. faecalis alone was associated with a significantly higher mortality, even greater when IAT was inadequate. Main risk factors for having an Enterococcus other than E. faecalis alone were as follows: SAPS score on day 0, ICU-acquired IAI, and antimicrobial therapy within 3 months prior to IAI especially with third-generation cephalosporins. Univariate analysis found a higher hazard ratio of death with an Enterococcus other than E. faecalis alone that had an inadequate IAT (HR = 4.4 [1.3-15.3], p = 0.019) versus an adequate IAT (HR = 3.1 [1.0-10.0], p = 0.053). However, after adjusting for confounders (i.e., SAPS II and septic shock at IAI diagnosis, ICU-acquired peritonitis, and adequacy of IAT for other germs), the impact of the adequacy of IAT was no longer significant in multivariate analysis. Septic shock at diagnosis and ICU-acquired IAI were prognostic factors. CONCLUSION: An IAT which does not cover Enterococcus is associated with an increased 30-day mortality in ICU patients presenting with an IAI growing with Enterococcus, especially when it is not an E. faecalis alone. It seems reasonable to use an IAT active against Enterococcus in severe postoperative ICU-acquired IAI, especially when a third-generation cephalosporin has been used within 3 months.

LRE-Finder, a Web tool for detection of the 23S rRNA mutations and the optrA, cfr, cfr(B) and poxtA genes encoding linezolid resistance in enterococci from whole-genome sequences.

OBJECTIVES: In enterococci, resistance to linezolid is often mediated by mutations in the V domain of the 23S rRNA gene (G2576T or G2505A). Furthermore, four genes [optrA, cfr, cfr(B) and poxtA] encode linezolid resistance in enterococci. We aimed to develop a Web tool for detection of the two mutations and the four genes encoding linezolid resistance in enterococci from whole-genome sequence data. METHODS: LRE-Finder (where LRE stands for linezolid-resistant enterococci) detected the fraction of Ts in position 2576 and the fraction of As in position 2505 of the 23S rRNA and the cfr, cfr(B), optrA and poxtA genes by aligning raw sequencing reads (fastq format) with k-mer alignment. For evaluation, fastq files from 21 LRE isolates were submitted to LRE-Finder. As negative controls, fastq files from 1473 non-LRE isolates were submitted to LRE-Finder. The MICs of linezolid were determined for the 21 LRE isolates. As LRE-negative controls, 26 VRE isolates were additionally selected for linezolid MIC determination. RESULTS: LRE-Finder was validated and showed 100% concordance with phenotypic susceptibility testing. A cut-off of 10% mutations in position 2576 and/or position 2505 was set in LRE-Finder for predicting a linezolid resistance phenotype. This cut-off allows for detection of a single mutated 23S allele in both Enterococcus faecalis and Enterococcus faecium, while ignoring low-level sequencing noise. CONCLUSIONS: A Web tool for detection of the 23S rRNA mutations (G2576T and G2505A) and the optrA, cfr, cfr(B) and poxtA genes from whole-genome sequences from enterococci is now available online.

Emergence of optrA-mediated linezolid resistance in enterococci from France, 2006-16.

OBJECTIVES: To describe the epidemiological trend of linezolid-resistant enterococci (LRE) collected in France from 2006 to 2016 and to extensively characterize LRE isolates. METHODS: The National Reference Center for Enterococci (NRC-Enc) received enterococcal isolates suspected to be VRE and/or LRE from all French hospitals between 2006 and 2016. LRE isolates were phenotypically characterized and their genomes were entirely sequenced by Miseq (Illumina). Transfer of linezolid resistance was attempted by filter mating experiments. RESULTS: Out of 3974 clinical isolates of enterococci received at the NRC-Enc over the period, 9 (0.2%) were LRE (MICs 8 to >32 mg/L), including 6 Enterococcus faecium and 3 Enterococcus faecalis. This overall prevalence significantly increased over the study period, reaching 0.8% in 2016. The five LRE isolated before 2016 were vanA-positive E. faecium whereas strains isolated in 2016 (one E. faecium and three E. faecalis) were susceptible to vancomycin. None of these isolates was part of an outbreak, while E. faecium strains were assigned to four different STs [17 (1), 80 (3), 412 (1) and 650 (1)] and all three E. faecalis belonged to ST480. Except for the strain isolated in 2010, all LRE were positive for optrA, which was located on plasmids (5/8) or in the chromosome (3/8). Plasmid transfer of optrA was successful in three cases. CONCLUSIONS: There has been a significant increase in the prevalence of LRE in France over time; this is due to the spread of optrA among E. faecium and E. faecalis human clinical isolates (VRE or not).

Identification and typing of Vagococcus salmoninarum using genomic and proteomic techniques.

This study reports on the characterization of Vagococcus salmoninarum using phenotypic, serological, antigenic, genetic and proteomic methods. All strains of V. salmoninarum were resistant to most of the antimicrobials tested, and only 10% of strains were sensitive to florfenicol. Serological analysis demonstrated a high antigenic homogeneity within the species. No cross-reaction was detected with other fish pathogenic species causing streptococcosis (Lactococcus garvieae, Streptococcus parauberis, Streptococcus iniae, Streptococcus agalactiae, Carnobacterium maltaromaticum) using serum against V. salmoninarum CECT 5810. Electrophoretic analysis of cell surface proteins and immunoblot supported the antigenic homogeneity within V. salmoninarum strains. Moreover, limited diversity was detected using genomic (RAPD, ERIC-PCR and REP-PCR) and MALDI-TOF-MS analyses. The phenotypic, genomic and proteomic methods tested allowed the rapid differentiation of V. salmoninarum from the other species causing streptococcosis. However, MALDI-TOF-MS is the most promising method for typing and characterization of V. salmoninarum.

Impact of the particulate matter from wastewater discharge on the abundance of antibiotic resistance genes and facultative pathogenic bacteria in downstream river sediments.

Wastewater treatment plants (WWTPs) are point sources for both, the release of antibiotic resistance genes (ARGs) and the discharge of antibiotics (ABs) into the environment. While it is well established that ARGs emission by WWTPs leads to an ARGs increase in receiving rivers, also the role of sub-inhibitory AB concentrations in this context is being discussed. However, the results obtained in this study suggest that, at environmental concentrations, ABs do not have an effect on resistance selection. Instead, we emphasize the significance of ARG transport and, in that respect, highlight the relevance of wastewater particles and associated microorganisms. We can show that ARGs (ermB, blaTEM,tetM, qnrS) as well as facultative pathogenic bacteria (FPB) (enterococci, Pseudomonas aeruginosa, Acinetobacter baumannii) inside the particulate fraction of WWTP effluent are very likely to remain in the riverbed of the receiving water due to sedimentation. Moreover, ARG and FPB abundances measured in the particulate fraction strongly correlated with the delta ARG and FPB abundances measured in the receiving river sediment (downstream compared to upstream) (R2 = 0.93, p < 0.05). Apparently, the sheer amount of settleable ARGs and FPB from WWTP effluent is sufficient, to increase abundances in the receiving riverbed by 0.5 to 2 log units.

Sexual Dimorphism in the Response to Broad-spectrum Antibiotics During T Cell-mediated Colitis.

BACKGROUND: Broad-spectrum antibiotics [Abx], including combination therapy with ciprofloxacin and metronidazole, are often prescribed during the treatment of inflammatory bowel disease [IBD] to alleviate symptoms, but with varying success. In this pilot study, we studied the effects of Abx on the course of experimental colitis, with a particular focus on sex as a determinant of the microbial and inflammatory responses. METHODS: The effects of Abx were tested on colonic inflammation and microbiome in male and female Rag-/- mice, using adoptive transfer of naïve T cells to induce colitis in a short-term [2-week] and long-term [9-week] study. RESULTS: We observed disparities between the sexes in both the response to adoptive T cell transfer and the effects of Abx. At baseline without Abx, female mice displayed a trend toward a more severe colitis than males. In both the short- and the long-term experiments, gut microbiota of some female mice exposed to Abx showed weak, delayed, or negligible shifts. Caecum weight was significantly lower in Abx-treated females. Abx exposure favoured a quick and persistent rise in Enterococcaceae exclusively in females. Males had higher relative abundance of Lactobacillaceae following Abx exposure relative to females. Abx-treated females trended toward higher colitis scores than Abx-treated males, and towards higher levels of IL-17A, NOS2, and IL-22. CONCLUSIONS: Although preliminary, our results suggest a differential response to both inflammation and Abx between male and female mice, The findings may be relevant to current practice and also as the basis for further studies on the differential gender effects during long-term antibiotic exposure in IBD.

Effect of nutritional interventions with quercetin, oat hulls, ß-glucans, lysozyme and fish oil on performance and health status related parameters of broilers chickens.

1. An experiment was conducted to evaluate the effects of technical feed ingredients between 14 and 28 d of age on performance and health status of broilers (d 14-35) fed diets with a high inclusion rate of rapeseed meal as a nutritional challenge. It was hypothesized that the feed ingredients would improve health status related parameters. 2. A total of 1008 one-day-old male Ross 308 chicks were distributed over 36 floor pens and allocated to one of six iso-caloric (AMEN 13 MJ/kg) growing diets (d 15-28): a control and five test diets supplemented with quercetin (400 mg/kg), oat hulls (50 g/kg), ß-glucan (100 mg/kg), lysozyme (40 mg/kg) or fish oil ω-3 fatty acids (40 g/kg), with six replicate pens per treatment. 3. Dietary inclusion of oat hulls and lysozyme resulted in a reduction in broiler performance during the first week after providing the experimental diets. 4. No effect of interventions on the microbiota diversity in the jejunum and ileum was observed. Ileal microbiota composition of birds fed oat hulls differed from the other groups, as shown by a higher abundance of the genus Enterococcus, mainly at the expense of the genus Lactobacillus. 5. In the jejunum, villus height and crypt depth of lysozyme-fed birds at d 28 were decreased compared to the control group. Higher total surface area of villi occupied by goblet cells and total villi surface area in jejunum (d 21 and 28) were observed in chickens fed oat hulls compared to other groups. 6. Genes related to the growth-factor-activity pathway were more highly expressed in birds fed ß-glucan compared to the control group, while the genes related to anion-transmembrane-transporter-activity pathway in the quercetin- and oat hull-fed birds were less expressed. The genes differently expressed between dietary interventions did not seem to be directly involved in immune related processes. 7. It was concluded that the tested nutritional interventions in the current experiment only marginally effected health status related parameters.

A frameshift mutation in the rRNA large subunit methyltransferase gene rlmA II determines the susceptibility of a honey bee pathogen Melissococcus plutonius to mirosamicin.

American foulbrood (AFB) and European foulbrood (EFB) caused by Paenibacillus larvae and Melissococcus plutonius, respectively, are major bacterial infections of honey bees. Although macrolides (mirosamicin [MRM] and tylosin) have been used to prevent AFB in Japan, macrolide-resistant P. larvae have yet to be found. In this study, we revealed that both MRM-resistant and -susceptible strains exist in Japanese M. plutonius and that a methyltransferase gene (rlmA II ) was disrupted exclusively in MRM-susceptible strains due to a single-nucleotide insertion. The M. plutonius RlmAII modified G748 of 23S rRNA, and the deletion of rlmA II resulted in increased susceptibility to MRM and the loss of modification at G748, suggesting that methylation at G748 by RlmAII confers MRM resistance in M. plutonius. The single-nucleotide mutation in MRM-susceptible strains was easily repaired by spontaneous deletion of the inserted nucleotide; however, intact rlmA II was only found in Japanese M. plutonius and not in a Paraguayan strain tested or any of the whole-genome-sequenced European strains. MRM has been used in apiculture only in Japan. Although M. plutonius is not the target of this drug, the use of MRM as a prophylactic drug for AFB may have influenced the antibiotic susceptibility of the causative agent of EFB.

Intestinal Dysbiosis Secondary to Proton-Pump Inhibitor Use.

BACKGROUND: Gut dysbiosis associated with the use of proton-pump inhibitors (PPIs) has been found to lead to the occurrence of infectious and inflammatory adverse events. A longitudinal observational cohort study has demonstrated the heightened risk of death associated with PPI use. SUMMARY: We evaluated meta-analyses to determine the association between PPI use and infectious and inflammatory diseases. Meta-analyses showed that PPI use is a potential risk for the development of enteric infections caused by Clostridium difficile, as well as small intestinal bacterial overgrowth, spontaneous bacterial peritonitis, community-acquired pneumonia, hepatic encephalopathy, and adverse outcomes in inflammatory bowel disease. We also examined changes in the composition and function of the gut microbiota with the use of PPIs. PPI use significantly increased the presence of Streptococcaceae and Enterococcaceae, which are risk factors for C. difficile infection, and decreased that of Faecalibacterium, a commensal anti-inflammatory microorganism. Key Message: High-throughput, microbial 16S rRNA gene sequencing has allowed us to investigate the association between the gut microbiome and PPI use. Future prospective comparison studies are necessary to confirm this association, and to develop new strategies to prevent complications of PPI use.

High-level resistance of Melissococcus plutonius clonal complex 3 strains to antimicrobial activity of royal jelly.

Melissococcus plutonius is the causative agent of European foulbrood of honey bee larvae. Among its three genetically distinct groups (CC3, CC12 and CC13), CC3 strains have been suggested to be more virulent at the colony level. Honey bee larvae are fed royal or worker jellies by adult bees, and these jellies exhibit antimicrobial activity. Since M. plutonius orally infects larvae via brood food, we herein investigated the resistance of M. plutonius to the antimicrobial activity of royal jelly (RJ). The results obtained revealed that M. plutonius strains were more resistant to RJ and its component, 10-hydroxy-2-decenoic acid, than the other species tested. Moreover, among the M. plutonius strains examined, CC3 strains exhibited the strongest resistance to antimicrobial activity; they temporarily proliferated and survived for a long period in 50% RJ-containing broth. However, resistance was not observed when freshly cultured bacteria were used, it was only detected after a preculture on agar media for five or more days, suggesting that, under certain conditions, CC3 strains change their physiological state to that which is advantageous for survival in brood food. This high-level RJ resistance of CC3 strains may contribute to their virulence in the field.

Effect of Exogenous Proline on Metabolic Response of Tetragenococcus halophilus under Salt Stress.

This study investigated the effect of proline addition on the salt tolerance of Tetragenococcus halophilus. Salt stress led to the accumulation of intracellular proline in T. halophilus. When 0.5 g/l proline was added to hyperhaline medium, the biomass increased 34.6% (12% NaCl) and 27.7% (18% NaCl) compared with the control (without proline addition), respectively. A metabolomic approach was employed to reveal the cellular metabolic responses and protective mechanisms of proline upon salt stress. The results showed that both the cellular membrane fatty acid composition and metabolite profiling responded by increasing unsaturated and cyclopropane fatty acid proportions, as well as accumulating some specific intracellular metabolites (environmental stress protector). Higher contents of intermediates involved in glycolysis, the tricarboxylic acid cycle, and the pentose phosphate pathway were observed in the cells supplemented with proline. In addition, addition of proline resulted in increased concentrations of many organic osmolytes, including glutamate, alanine, citrulline, N-acetyl-tryptophan, and mannitol, which may be beneficial for osmotic homeostasis. Taken together, results in this study suggested that proline plays a protective role in improving the salt tolerance of T. halophilus by regulating the related metabolic pathways.

Safety assessment of Tetragenococcus halophilus isolates from doenjang, a Korean high-salt-fermented soybean paste.

We assessed the safety of 49 Tetragenococcus halophilus strains isolated from doenjang in Korea. Minimum inhibitory concentration assays showed that all strains can be considered as susceptible to ampicillin, erythromycin, penicillin G, tetracycline, and vancomycin, but resistant to ciprofloxacin based on the Enterococcus breakpoint values provided by the European Committee on Antimicrobial Susceptibility testing in 2015. Ciprofloxacin resistance was sufficiently high to consider the potential for acquisition of transmissible determinants. Two strains exhibiting potentially acquired resistance to chloramphenicol and gentamicin, and chloramphenicol alone, were identified. None of the strains exhibited α-hemolytic activity or biofilm formation; two strains exhibited weak ß-hemolytic activity. Doenjang isolates produced an average of 3338.6 ppm of tyramine in the laboratory, considerably higher than the levels produced by two reference strains. All of the test strains exhibited similar cadaverine, histamine, and putrescine production patterns. Most T. halophilus strains could grow at a NaCl concentration >18%, exhibited acid production at 15% NaCl, and expressed strain-specific protease and lipase activities. The potential acquisition of transmissible determinants for antibiotic resistance and tyramine production identified in this study necessitate the need for a thorough safety assessment of T. halophilus before it can be considered for use in food fermentation processes.

Food to some, poison to others - honeybee royal jelly and its growth inhibiting effect on European Foulbrood bacteria.

Honeybee colonies (Apis mellifera) serve as attractive hosts for a variety of pathogens providing optimal temperatures, humidity, and an abundance of food. Thus, honeybees have to deal with pathogens throughout their lives and, even as larvae they are affected by severe brood diseases like the European Foulbrood caused by Melissococcus plutonius. Accordingly, it is highly adaptive that larval food jelly contains antibiotic compounds. However, although food jelly is primarily consumed by bee larvae, studies investigating the antibiotic effects of this jelly have largely concentrated on bacterial human diseases. In this study, we show that royal jelly fed to queen larvae and added to the jelly of drone and worker larvae, inhibits not only the growth of European Foulbrood-associated bacteria but also its causative agent M. plutonius. This effect is shown to be caused by the main protein (major royal jelly protein 1) of royal jelly.

Lysophosphatidylcholine acts in the constitutive immune defence against American foulbrood in adult honeybees.

Honeybee (Apis mellifera) imagines are resistant to the Gram-positive bacterium Paenibacillus larvae (P. larvae), causative agent of American foulbrood (AFB), whereas honeybee larvae show susceptibility against this pathogen only during the first 48 h of their life. It is known that midgut homogenate of adult honeybees as well as a homogenate of aged larvae exhibit strong anti-P. larvae activity. A bioactivity-guided LC-HRMS analysis of midgut homogenate resulted in the identification of 1-oleoyl-sn-glycero-3-phosphocholine (LPC) pointing to a yet unknown immune defence in adult honeybees against P. larvae. Antimicrobial activity of LPC was also demonstrated against Melissococcus plutonius, causative agent of European Foulbrood. To demonstrate an AFB-preventive effect of LPC in larvae, artificially reared larvae were supplemented with LPC to evaluate its toxicity and to assess whether, after infection with P. larvae spores, LPC supplementation prevents AFB infection. 10 µg LPC per larva applied for 3 d significantly lowered mortality due to AFB in comparison to controls. A potential delivery route of LPC to the larvae in a colony via nurse bees was assessed through a tracking experiment using fluorescent-labelled LPC. This yet undescribed and non-proteinous defense of honeybees against P. larvae may offer new perspectives for a treatment of AFB without the utilization of classic antibiotics.

Facile Synthesis of Azaarene-Substituted Hydroxycoumarins Possessing High Biological Activities via Three-Component C(sp(3))-H Functionalization.

An unprecedented three-component C(sp(3))-H functionalization of 2-alkylazaarenes with aryl aldehydes and 4-hydroxycoumarins was realized, providing azaarene-substituted 3-benzyl-4-hydroxycoumarins in good to excellent yields. These new target compounds displayed broad-spectrum antibacterial activities, providing a new type of antibacterial skeleton.