Isolation in Natural Host Cell Lines of Wolbachia Strains wPip from the Mosquito Culex pipiens and wPap from the Sand Fly Phlebotomus papatasi

Endosymbiotic intracellular bacteria of the genus Wolbachia are harboured by many species of invertebrates. They display a wide range of developmental, metabolic and nutritional interactions with their hosts and may impact the transmission of arboviruses and protozoan parasites. Wolbachia have occasionally been isolated during insect cell line generation. Here, we report the isolation of two strains of Wolbachia, wPip and wPap, during cell line generation from their respective hosts, the mosquito Culex pipiens and the sand fly Phlebotomus papatasi. wPip was pathogenic for both new C. pipiens cell lines, CPE/LULS50 and CLP/LULS56, requiring tetracycline treatment to rescue the lines. In contrast, wPap was tolerated by the P. papatasi cell line PPL/LULS49, although tetracycline treatment was applied to generate a Wolbachia-free subline. Both Wolbachia strains were infective for a panel of heterologous insect and tick cell lines, including two novel lines generated from the sand fly Lutzomyia longipalpis, LLE/LULS45 and LLL/LULS52. In all cases, wPip was more pathogenic for the host cells than wPap. These newly isolated Wolbachia strains, and the novel mosquito and sand fly cell lines reported here, will add to the resources available for research on host–endosymbiont relationships, as well as on C. pipiens, P. papatasi, L. longipalpis and the pathogens that they transmit.

The role of beta-hydroxybutyrate as inducer for adult cardiomyocyte proliferation during reprogramming in vivo

Background Regional loss of myocardium is the dominant cause of heart failure due to coronary artery disease. In mammals, the response to injury shows some similarities with border zone myocytes remodeling, but little proliferation and repair occur in adult mammalian hearts. Several studies have identified some cell cycle-related genes essential for dramatically inducing neonatal CM proliferation, but the same effects cannot be replicated in adult CMs. This implies that there is an underlying mechanism (an inducer) that is yet to be discovered, that should first be activated in order to subsequently increase adult CM proliferation efficiently in vivo. Purpose To determine the inducer for the purpose of efficiently increasing adult CM proliferation, and to examine its therapeutic potential for heart regeneration after injury. Methods Transgenic mice (αMHC-rtTA-OSKM), whose adult CMs can be specifically induced by Yamanaka's OSKM factors, were generated to systemically examine the early stage of iPSC reprogramming after tetracycline treatment. These induced reprogramming CMs were further isolated for gene ontology analysis by microarray to determine the specific inducer for the purpose of efficiently increasing CM proliferation. Finally, the inducer was applied to a mouse model of myocardial infarction (MI) to confirm its therapeutic potential for heart regeneration after injury. Results OSKM expressions of αMHC-rtTA-OSKM were confirmed to only increase in CMs after tetracycline treatment in vivo, and BrdU+ CM% was more than 2-times higher in isolated adult CMs from αMHC-rtTA-OSKM mice treated with tetracycline for 2 days compared to PBS-treated mice. More H3P+/cTnT+ population% was also confirmed in this CM-disorganized reprogramming hearts compared to normal hearts. Microarray data revealed that the morphological changes of the reprogrammed CMs were due to a metabolic switch shown in gene ontology analysis. Thus, we profiled the metabolic switch in normal and CM reprogramming hearts by perfusing them with 13C-labeled metabolic substrates through the Langendorff ex-vivo working heart perfusion system. Through 13C NMR spectroscopy we determined that TCA cycle metabolites were 2-times lower while β-hydroxybutyrate were 2-times higher in the reprogrammed hearts compared to that in control hearts. Next, we surprisingly found that β-hydroxybutyrate supplement in MI mice at post-MI day 3 improved the heart function but showed the opposite effects when supplement was administered at post-MI day 7. Thus, the timing of β-hydroxybutyrate supplement is important for increasing adult CM proliferation for effectiveness of heart regeneration after MI. Conclusion This is the first study to identify the metabolism shift to increase β-hydroxybutyrate as the inducer for the purpose of efficiently increasing adult CM proliferation, which supports heart function improvement when treating at short period immediately after MI. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): 2020 Academia Sinica Young Scholarship Travel Award

Disrupting the ArcA regulatory network increases tetracycline susceptibility of TetREscherichia coli

ABSTRACTThere is an urgent need for strategies to discover secondary drugs to prevent or disrupt antimicrobial resistance (AMR), which is causing >700,000 deaths annually. Here, we demonstrate that tetracycline resistant (TetR) Escherichia coli undergoes global transcriptional and metabolic remodeling, including down-regulation of tricarboxylic acid cycle and disruption of redox homeostasis, to support consumption of the proton motive force for tetracycline efflux. Targeted knockout of ArcA, identified by network analysis as a master regulator among 25 transcription factors of this new compensatory physiological state, significantly increased the susceptibility of TetRE. coli to tetracycline treatment. A drug, sertraline, which generated a similar metabolome profile as the arcA knockout strain also synergistically re-sensitized TetRE. coli to tetracycline. The potentiating effect of sertraline was eliminated upon knocking out arcA, demonstrating that the mechanism of synergy was through action of sertraline on the tetracycline-induced ArcA network in the TetR strain. Our findings demonstrate that targeting mechanistic drivers of compensatory physiological states could be a generalizable strategy to re-sensitize AMR pathogens to lost antibiotics.

Isolation of Tetracycline-Resistant Chlamydia suis from a Pig Herd Affected by Reproductive Disorders and Conjunctivitis

Due to various challenges in diagnosing chlamydiosis in pigs, antibiotic treatment is usually performed before any molecular or antibiotic susceptibility testing. This could increase the occurrence of tetracycline-resistant Chlamydia (C.) suis isolates in the affected pig population and potentiate the reoccurrence of clinical signs. Here, we present a case of an Austrian pig farm, where tetracycline resistant and sensitive C. suis isolates were isolated from four finishers with conjunctivitis. On herd-level, 10% of the finishers suffered from severe conjunctivitis and sows showed a high percentage of irregular return to estrus. Subsequent treatment of whole-herd using oxytetracycline led to a significant reduction of clinical signs. Retrospective antibiotic susceptibility testing revealed tetracycline resistance and decreased susceptibility to doxycycline in half of the ocular C. suis isolates, and all isolates were able to partially recover following a single-dose tetracycline treatment in vitro. These findings were later confirmed in vivo, when all former clinical signs recurred three months later. This case report raises awareness of tetracycline resistance in C. suis and emphasizes the importance of preventative selection of tetracycline resistant C. suis isolates.

Long-lasting effects of antibiotics on bacterial communities of adult flies

ABSTRACT Insect symbionts benefit their host and their study requires large spectrum antibiotic use like tetracycline to weaken or suppress symbiotic communities. While antibiotics have a negative impact on insect fitness, little is known about antibiotic effects on insect microbial communities and how long they last. We characterized the bacterial communities of adult cabbage root fly Delia radicum in a Wolbachia-free population and evaluated the effect of tetracycline treatment on these communities over several generations. Three D. radicum generations were used: the first- and second-generation flies either ingested tetracycline or not, while the third-generation flies were untreated but differed with their parents and/or grandparents that had or had not been treated. Fly bacterial communities were sequenced using a 16S rRNA gene. Tetracycline decreased fly bacterial diversity and induced modifications in both bacterial abundance and relative frequencies, still visible on untreated offspring whose parents and/or grandparents had been treated, therefore demonstrating long-lasting transgenerational effects on animal microbiomes after antibiotic treatment. Flies with an antibiotic history shared bacterial genera, potentially tetracycline resistant and heritable. Next, the transmission should be investigated by comparing several insect development stages and plant compartments to assess vertical and horizontal transmissions of D. radicum bacterial communities.