Viral and Bacterial Zoonotic Agents in Dromedary Camels from Southern Tunisia: A Seroprevalence Study.

The rapid spread of SARS-CoV-2 clearly demonstrated the potential of zoonotic diseases to cause severe harm to public health. Having limited access to medical care combined with severe underreporting and a lack of active surveillance, Africa carries a high burden of neglected zoonotic diseases. Therefore, the epidemiological monitoring of pathogen circulation is essential. Recently, we found extensive Middle East respiratory syndrome coronavirus (MERS-CoV) prevalence in free-roaming dromedary camels from southern Tunisia. In this study, we aimed to investigate the seroprevalence, and thus the risk posed to public health, of two additional viral and two bacterial pathogens in Tunisian dromedaries: Rift Valley fever virus (RVFV), foot-and-mouth disease virus (FMDV), Coxiella burnetii and Brucella spp. via ELISA. With 73.6% seropositivity, most animals had previously been exposed to the causative agent of Q fever, C. burnetii. Additionally, 7.4% and 1.0% of the dromedaries had antibodies against Brucella and RVFV, respectively, while no evidence was found for the occurrence of FMDV. Our studies revealed considerable immunological evidence of various pathogens within Tunisian dromedary camels. Since these animals have intense contact with humans, they pose a high risk of transmitting serious zoonotic diseases during active infection. The identification of appropriate countermeasures is therefore highly desirable.

Prevalence of Middle East Respiratory Syndrome Coronavirus in Dromedary Camels, Tunisia.

Free-roaming camels, especially those crossing national borders, pose a high risk for spreading Middle East respiratory syndrome coronavirus (MERS-CoV). To prevent outbreaks, active surveillance is necessary. We found that a high percentage of dromedaries in Tunisia are MERS-CoV seropositive (80.4%) or actively infected (19.8%), indicating extensive MERS-CoV circulation in Northern Africa.

Isolation and characterization of lytic phage TUN1 specific for Klebsiella pneumoniae K64 clinical isolates from Tunisia.

BACKGROUND: Multidrug-resistant Klebsiella pneumoniae spp. (kp) are emerging agents of severe infections of the respiratory, urinary tract and wounds that can progress to fatal septicemia. The use of bacteriophages is currently being considered as an effective alternative or adjuvant to antibiotic therapy. RESULTS: In this study, we report capsule (K)-typing of 163 carbapenem-resistant Kp (CRKP) isolated 2014-2018 at the Military Hospital of Instruction of Tunis (MHT), Tunisia, by partial amplification and sequencing of the Kp wzi gene. The most prevalent K-type overall was K64 with 50.3% followed by K17 and K27 (22.7 and 11.0%, respectively). K64 Kp strains were most common and associated with increased case/fatality rates, especially at the intensive care unit (ICU). Using a K64 Kp strain we isolated and characterized a lytic Kp phage, vB_KpP_TUN1 (phage TUN1), from wastewater samples of the ICU at the MHT. TUN1 belongs to the Autographiviridae family and specifically digests K64 Kp capsules most probably via a depolymerase encoded by gp47. Furthermore, we successfully assembled phage TUN1 in a non-replicative host (E. coli) raising the possibility of in vitro assembly in the absence of live bacterial hosts. We propose that phage TUN1 is a promising candidate to be used as an adjuvant or an alternative to antibiotic therapy in CRKP infections, facilitating regulatory approval of phage therapy. CONCLUSIONS: K64, K17 and K27 are the most common wzi capsule types in this geographical location in Northern Africa. The lytic phage TUN1 efficiently lyses K64 Kp strains associated with increased case/fatality rates at body temperature. Together with its ability to be rescued in a non-replicative host these features enhance the utility of this phage as an antibacterial agent.

Two novel XRE-like transcriptional regulators control phenotypic heterogeneity in Photorhabdus luminescens cell populations.

BACKGROUND: The insect pathogenic bacterium Photorhabdus luminescens exists in two phenotypically different forms, designated as primary (1°) and secondary (2°) cells. Upon yet unknown environmental stimuli up to 50% of the 1° cells convert to 2° cells. Among others, one important difference between the phenotypic forms is that 2° cells are unable to live in symbiosis with their partner nematodes, and therefore are not able to re-associate with them. As 100% switching of 1° to 2° cells of the population would lead to a break-down of the bacteria's life cycle the switching process must be tightly controlled. However, the regulation mechanism of phenotypic switching is still puzzling. RESULTS: Here we describe two novel XRE family transcriptional regulators, XreR1 and XreR2, that play a major role in the phenotypic switching process of P. luminescens. Deletion of xreR1 in 1° or xreR2 in 2° cells as well as insertion of extra copies of xreR1 into 2° or xreR2 into 1° cells, respectively, induced the opposite phenotype in either 1° or 2° cells. Furthermore, both regulators specifically bind to different promoter regions putatively fulfilling a positive autoregulation. We found initial evidence that XreR1 and XreR2 constitute an epigenetic switch, whereby XreR1 represses xreR2 expression and XreR2 self-reinforces its own gene by binding to XreR1. CONCLUSION: Regulation of gene expression by the two novel XRE-type regulators XreR1 and XreR2 as well as their interplay represents a major regulatory process in phenotypic switching of P. luminescens. A fine-tuning balance between both regulators might therefore define the fate of single cells to convert from the 1° to the 2° phenotype.

Whole genome sequencing and phylogenetic classification of Tunisian SARS-CoV-2 strains from patients of the Military Hospital in Tunis.

In the present work, two complete genome sequences of SARS-CoV-2 were obtained from nasal swab samples of Tunisian SARS-CoV-2 PCR-positive patients using nanopore sequencing. The virus genomes of two of the patients examined, a Tunisian soldier returning from a mission in Morocco and a member of another Tunisian family, showed significant differences in analyses of the total genome and single nucleotide polymorphisms (SNPs). Phylogenetic relationships with known SARS-CoV-2 genomes in the African region, some European and Middle Eastern countries and initial epidemiological conclusions indicate that the introduction of SARS-CoV-2 into Tunisia from two independent sources was travel-related.

Promoter Activation in Δhfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct Bioactivity Testing.

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.

Phenotypic Heterogeneity of the Insect Pathogen Photorhabdus luminescens: Insights into the Fate of Secondary Cells.

Photorhabdus luminescens is a Gram-negative bacterium that lives in symbiosis with soil nematodes and is simultaneously highly pathogenic toward insects. The bacteria exist in two phenotypically different forms, designated primary (1°) and secondary (2°) cells. Yet unknown environmental stimuli as well as global stress conditions induce phenotypic switching of up to 50% of 1° cells to 2° cells. An important difference between the two phenotypic forms is that 2° cells are unable to live in symbiosis with nematodes and are therefore believed to remain in the soil after a successful infection cycle. In this work, we performed a transcriptomic analysis to highlight and better understand the role of 2° cells and their putative ability to adapt to living in soil. We could confirm that the major phenotypic differences between the two cell forms are mediated at the transcriptional level as the corresponding genes were downregulated in 2° cells. Furthermore, 2° cells seem to be adapted to another environment as we found several differentially expressed genes involved in the cells' metabolism, motility, and chemotaxis as well as stress resistance, which are either up- or downregulated in 2° cells. As 2° cells, in contrast to 1° cells, chemotactically responded to different attractants, including plant root exudates, there is evidence for the rhizosphere being an alternative environment for the 2° cells. Since P. luminescens is biotechnologically used as a bio-insecticide, investigation of a putative interaction of 2° cells with plants is also of great interest for agriculture.IMPORTANCE The biological function and the fate of P. luminescens 2° cells were unclear. Here, we performed comparative transcriptomics of P. luminescens 1° and 2° cultures and found several genes, not only those coding for known phenotypic differences of the two cell forms, that are up- or downregulated in 2° cells compared to levels in 1° cells. Our results suggest that when 1° cells convert to 2° cells, they drastically change their way of life. Thus, 2° cells could easily adapt to an alternative environment such as the rhizosphere and live freely, independent of a host, putatively utilizing plant-derived compounds as nutrient sources. Since 2° cells are not able to reassociate with the nematodes, an alternative lifestyle in the rhizosphere would be conceivable.

Regulation of Phenotypic Switching and Heterogeneity in Photorhabdus luminescens Cell Populations.

Phenotypic heterogeneity in bacterial cell populations allows genetically identical organisms to different behavior under similar environmental conditions. The Gram-negative bacterium Photorhabdus luminescens is an excellent organism to study phenotypic heterogeneity since their life cycle involves a symbiotic interaction with soil nematodes as well as a pathogenic association with insect larvae. Phenotypic heterogeneity is highly distinct in P. luminescens. The bacteria exist in two phenotypic forms that differ in various morphologic and phenotypic traits and are therefore distinguished as primary (1°) and secondary (2°) cells. The 1 cells are bioluminescent, pigmented, produce several secondary metabolites and exo-enzymes, and support nematode growth and development. The 2° cells lack all these 1°-specific phenotypes. The entomopathogenic nematodes carry 1° cells in their upper gut and release them into an insect's body after slipping inside. During insect infection, up to the half number of 1° cells undergo phenotypic switching and convert to 2° cells. Since the 2° cells are not able to live in nematode symbiosis any more, they cannot re-associate with their symbiosis partners after the infection and remain in the soil. Phenotypic switching in P. luminescens has to be tightly regulated since a high switching frequency would lead to a complete break-down of the nematode-bacteria life cycle. Here, we present the main regulatory mechanisms known to-date that are important for phenotypic switching in P. luminescens cell populations and discuss the biological reason as well as the fate of the 2° cells in the soil.

Phenotypic and genomic comparison of Photorhabdus luminescens subsp. laumondii TT01 and a widely used rifampicin-resistant Photorhabdus luminescens laboratory strain.

BACKGROUND: Photorhabdus luminescens is an enteric bacterium, which lives in mutualistic association with soil nematodes and is highly pathogenic for a broad spectrum of insects. A complete genome sequence for the type strain P. luminescens subsp. laumondii TT01, which was originally isolated in Trinidad and Tobago, has been described earlier. Subsequently, a rifampicin resistant P. luminescens strain has been generated with superior possibilities for experimental characterization. This strain, which is widely used in research, was described as a spontaneous rifampicin resistant mutant of TT01 and is known as TT01-RifR. RESULTS: Unexpectedly, upon phenotypic comparison between the rifampicin resistant strain and its presumed parent TT01, major differences were found with respect to bioluminescence, pigmentation, biofilm formation, haemolysis as well as growth. Therefore, we renamed the strain TT01-RifR to DJC. To unravel the genomic basis of the observed differences, we generated a complete genome sequence for strain DJC using the PacBio long read technology. As strain DJC was supposed to be a spontaneous mutant, only few sequence differences were expected. In order to distinguish these from potential sequencing errors in the published TT01 genome, we re-sequenced a derivative of strain TT01 in parallel, also using the PacBio technology. The two TT01 genomes differed at only 30 positions. In contrast, the genome of strain DJC varied extensively from TT01, showing 13,000 point mutations, 330 frameshifts, and 220 strain-specific regions with a total length of more than 300 kb in each of the compared genomes. CONCLUSIONS: According to the major phenotypic and genotypic differences, the rifampicin resistant P. luminescens strain, now named strain DJC, has to be considered as an independent isolate rather than a derivative of strain TT01. Strains TT01 and DJC both belong to P. luminescens subsp. laumondii.

Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro.

The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat/K M) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k cat/K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat/K M values >107 M-1 min-1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships.

Functional dissection of the Pax6 paired domain: Roles in neural tube patterning and peripheral nervous system development.

During development of the CNS, stem and progenitor cell proliferation, cell fate designation, and patterning decisions are tightly regulated by interdependent networks of key transcriptional regulators. In a genetic approach we analyzed divergent functionality of the PAI and RED sub-domains of the Pax6 Paired domain (PD) during progenitor zone formation, motor and interneuron development, and peripheral connectivity at distinct levels within the neural tube: within the hindbrain, mutation of the PAI sub-domain severely affected patterning of the p3 and pMN domains and establishment of the corresponding motor neurons. Exit point designation of hypoglossal axons was disturbed in embryos harboring either mutations in the PD sub-domains or containing a functional Pax6 Null allele. At brachial spinal levels, we propose a selective involvement of the PAI sub-domain during patterning of ventral p2 and pMN domains, critically disturbing generation of specific motor neuron subtypes and increasing V2 interneuron numbers. Our findings present a novel aspect of how Pax6 not only utilizes its modular structure to perform distinct functions via its paired and homeodomain. Individual sub-domains can exert distinct functions, generating a new level of complexity for transcriptional regulation by one single transcription factor not only in dorso-ventral, but also rostro-caudal neural tube patterning.