Impact of the use of β-lactam antimicrobials on the emergence of Escherichia coli isolates resistant to cephalosporins under standard pig-rearing conditions

The aim of this study was to evaluate if the treatments with ceftiofur and amoxicillin are risk factors for the emergence of cephalosporin resistant (CR) E. coli in a pig farm during the rearing period. One hundred 7-day-old piglets were divided into two groups, a control (n = 50) group and a group parenterally treated with ceftiofur (n = 50). During the fattening period, both groups were subdivided in two. A second treatment with amoxicillin was administered in feed to two of the four groups, as follows: group 1 (untreated, n = 20), group 2 (treated with amoxicillin, n = 26), group 3 (treated with ceftiofur, n = 20), and group 4 (treated with ceftiofur and amoxicillin, n = 26). During treatment with ceftiofur, fecal samples were collected before treatment (day 0) and at days 2, 7, 14, 21, and 42 posttreatment, whereas with amoxicillin, the sampling was extended 73 days posttreatment. CR E. coli bacteria were selected on MacConkey agar with ceftriaxone (1 mg/liter). Pulsed-field gel electrophoresis (PFGE), MICs of 14 antimicrobials, the presence of cephalosporin resistance genes, and replicon typing of plasmids were analyzed. Both treatments generated an increase in the prevalence of CR E. coli, which was statistically significant in the treated groups. Resistance diminished after treatment. A total of 47 CR E. coli isolates were recovered during the study period; of these, 15 contained blaCTX-M-1, 10 contained blaCTX-M-14, 4 contained blaCTX-M-9, 2 contained blaCTX-M-15, and 5 contained blaSHV-12. The treatment with ceftiofur and amoxicillin was associated with the emergence of CR E. coli during the course of the treatment. However, by the time of finishing, CR E. coli bacteria were not recovered from the animals.

Garvicin A, a novel class IId bacteriocin from Lactococcus garvieae that inhibits septum formation in L. garvieae strains

Lactococcus garvieae 21881, isolated in a human clinical case, produces a novel class IId bacteriocin, garvicin A (GarA), which is specifically active against other L. garvieae strains, including fish- and bovine-pathogenic isolates. Purification from active supernatants, sequence analyses, and plasmid-curing experiments identified pGL5, one of the five plasmids found in L. garvieae [M. Aguado-Urda et al., PLoS One 7(6):e40119, 2012], as the coding plasmid for the structural gene of GarA (lgnA), its putative immunity protein (lgnI), and the ABC transporter and its accessory protein (lgnC and lgnD). Interestingly, pGL5-cured strains were still resistant to GarA. Other putative bacteriocins encoded by the remaining plasmids were not detected during purification, pointing to GarA as the main inhibitor secreted by L. garvieae 21881. Mode-of-action studies revealed a potent bactericidal activity of GarA. Moreover, transmission microscopy showed that GarA seems to act by inhibiting septum formation in L. garvieae cells. This potent and species-specific inhibition by GarA holds promise for applications in the prevention or treatment of infections caused by pathogenic strains of L. garvieae in both veterinary and clinical settings.

Fluoroquinolone efflux in Streptococcus suis is mediated by SatAB and not by SmrA

Streptococcus suis is an emerging zoonotic pathogen. With the lack of an effective vaccine, antibiotics remain the main tool to fight infections caused by this pathogen. We have previously observed a reserpine-sensitive fluoroquinolone (FQ) efflux phenotype in this species. Here, SatAB and SmrA, two pumps belonging to the ATP binding cassette (ABC) and the major facilitator superfamily (MFS), respectively, have been analyzed in the fluoroquinolone-resistant clinical isolate BB1013. Genes encoding these pumps were overexpressed either constitutively or in the presence of ciprofloxacin in this strain. These genes could not be cloned in plasmids in Escherichia coli despite strong expression repression. Finally, site-directed insertion of smrA and satAB in the amy locus of the Bacillus subtilis chromosome using ligated PCR amplicons allowed for the functional expression and study of both pumps. Results showed that SatAB is a narrow-spectrum fluoroquinolone exporter (norfloxacin and ciprofloxacin), susceptible to reserpine, whereas SmrA was not involved in fluoroquinolone resistance. Chromosomal integration in Bacillus is a novel method for studying efflux pumps from Gram-positive bacteria, which enabled us to demonstrate the possible role of SatAB, and not SmrA, in fluoroquinolone efflux in S. suis.

Haemophilus influenzae clinical isolates with plasmid pB1000 bearing blaROB-1: fitness cost and interspecies dissemination

Plasmid pB1000 is a mobilizable replicon bearing the bla(ROB-1) beta-lactamase gene that we have recently described in Haemophilus parasuis and Pasteurella multocida animal isolates. Here we report the presence of pB1000 and a derivative plasmid, pB1000', in four Haemophilus influenzae clinical isolates of human origin. Pulsed-field gel electrophoresis showed unrelated patterns in all strains, indicating that the existence of pB1000 in H. influenzae isolates is not the consequence of clonal dissemination. The replicon can be transferred both by transformation and by conjugation into H. influenzae, giving rise to recipients resistant to ampicillin and cefaclor (MICs, > or =64 microg/ml). Stability experiments showed that pB1000 is stable in H. influenzae without antimicrobial pressure for at least 60 generations. Competition experiments between isogenic H. influenzae strains with and without pB1000 revealed a competitive disadvantage of 9% per 10 generations for the transformant versus the recipient. The complete nucleotide sequences of nine pB1000 plasmids from human and animal isolates, as well as the epidemiological data, suggest that animal isolates belonging to the Pasteurellaceae act as an antimicrobial resistance reservoir for H. influenzae. Further, since P. multocida is the only member of this family that can colonize both humans and animals, we propose that P. multocida is the vehicle for the transport of pB1000 between animal- and human-adapted members of the Pasteurellaceae.

Novel blaROB-1-bearing plasmid conferring resistance to β-lactams in Haemophilus parasuis isolates from healthy weaning pigs

Haemophilus parasuis, the causative agent of Glässer's disease, is one of the early colonizers of the nasal mucosa of piglets. It is prevalent in swine herds, and lesions associated with disease are fibrinous polyserositis and bronchopneumonia. Antibiotics are commonly used in disease control, and resistance to several antibiotics has been described in H. parasuis. Prediction of H. parasuis virulence is currently limited by our scarce understanding of its pathogenicity. Some genes have been associated with H. parasuis virulence, such as lsgB and group 1 vtaA, while biofilm growth has been associated with nonvirulent strains. In this study, 86 H. parasuis nasal isolates from farms that had not had a case of disease for more than 10 years were obtained by sampling piglets at weaning. Isolates were studied by enterobacterial repetitive intergenic consensus PCR and determination of the presence of lsgB and group 1 vtaA, biofilm formation, inflammatory cell response, and resistance to antibiotics. As part of the diversity encountered, a novel 2,661-bp plasmid, named pJMA-1, bearing the blaROB-1 β-lactamase was detected in eight colonizing strains. pJMA-1 was shown to share a backbone with other small plasmids described in the Pasteurellaceae, to be 100% stable, and to have a lower biological cost than the previously described plasmid pB1000. pJMA-1 was also found in nine H. parasuis nasal strains from a separate collection, but it was not detected in isolates from the lesions of animals with Glässer's disease or in nontypeable Haemophilus influenzae isolates. Altogether, we show that commensal H. parasuis isolates represent a reservoir of β-lactam resistance genes which can be transferred to pathogens or other bacteria.

Improving safety and establishing episomal maintenance of Adeno-associated viral vectors

Adeno-associated viral (AAV) vectors are among the most widely used gene transfer systems in basic and pre-clinical research and have been employed in more than 160 clinical trials. AAV vectors are commonly produced in producer cell lines like HEK293 by co-transfection with a so-called vector plasmid and one (in this work) or two so-called helper plasmids. The vector plasmid contains the transgene cassette of interest (TEC) flanked by AAV’s inverted terminal repeats (ITRs) which serve as packaging signals, whereas the helper plasmid provides the required AAV and helper virus functions in trans. A pivotal aspect of AAV vectorology is the manufacturing of AAV vectors free from impurities arising during the production process. These impurities include AAV vector preparations that contain capsids containing prokaryotic sequences, e.g. antibiotic resistance genes originating from the producer plasmids. In the first part of the thesis we aimed at improving the safety of AAV vectors. As we found that encapsidated prokaryotic sequences (using the ampicillin resistance gene as indicator) cannot be re-moved by standard purification methods we investigated whether the producer plasmids could be replaced by Minicircles (MCs). MCs are circular DNA constructs which contain no functional or coding prokaryotic sequences; they only consist of the TEC and a short sequence required for production and purification. MC counterparts of a vector plasmid encoding for enhanced green fluorescent (eGFP) protein and a helper plasmid encoding for AAV serotype 2 (AAV2) and helper Adenovirus (Ad) genes were designed and produced by PlasmidFactory (Bielefeld, Germany). Using all four possible combinations of plasmid and MCs, single-stranded AAV2 vectors (ssAAV) and self-complementary AAV vectors (scAAV) were produced and characterized for vector quantity, quality and functionality. The analyses showed that plasmids can be replaced by MCs without decreasing the efficiency of vector production and vector quality. MC-derived scAAV vector preparations even exceeded plasmid-derived preparations, as they displayed up to 30-fold improved transduction efficiencies. Using MCs as tools, we found that the vector plasmid is the main source of encapsidated prokaryotic sequences. Remarkably, we found that plasmid-derived scAAV vector preparations contained a much higher relative amount of prokaryotic sequences (up to 26.1 %, relative to TEC) compared to ssAAV vector preparations (up to 2.9 %). By replacing both plasmids by MCs the amount of functional prokaryotic sequences could be decreased to below the limit of quantification. Additional analyses for DNA impurities other than prokaryotic sequences showed that scAAV vectors generally contained a higher amount of non-vector DNA (e.g. adenoviral sequences) than ssAAV vectors. For both, ssAAV and scAAV vector preparations, MC-derived vectors tended to contain lower amounts of foreign DNA. None of the vectors tested could be shown to induce immunogenicity. In summary we could demonstrate that the quality of AAV vector preparations could be significantly improved by replacing producer plasmids by MCs. Upon transduction of a target tissue, AAV vector genomes predominantly remain in an episomal state, as duplex DNA circles or concatemers. These episomal forms mediate long-term transgene expression in terminally differentiated cells, but are lost in proliferating cells due to cell division. Therefore, in the second part of the thesis, in cooperation with Claudia Hagedorn and Hans J. Lipps (University Witten/Herdecke) an AAV vector genome was equipped with an autonomous replication element (Scaffold/matrix attachment region (S/MAR)). AAV-S/MAR encoding for eGFP and a blasticidin resistance gene and a control vector with the same TEC but lacking the S/MAR element (AAV-ΔS/MAR) were produced and transduced into highly proliferative HeLa cells. Antibiotic pressure was employed to select for cells stably maintaining the vector genome. AAV-S/MAR transduced cells yielded a higher number of colonies than AAV-ΔS/MAR-transduced cells. Colonies derived from each vector transduction were picked and cultured further. They remained eGFP-positive (up to 70 days, maximum cultivation period) even in the absence of antibiotic selection pressure. Interestingly, the mitotic stability of both AAV-S/MAR and control vector AAV-ΔS/MAR was found to be a result of episomal maintenance of the vector genome. This finding indicates that, under specific conditions such as the mild selection pressure we employed, “common” AAV vectors persist episomally. Thus, the S/MAR element increases the establishment frequency of stable episomes, but is not a prerequisite.

Augmentation de l’expression de la chaine α1 de la laminine 111, un potentiel traitement pour la Dystrophie musculaire de Duchenne

La protéine hétérotrimérique laminine-111 permet le lien entre la matrice-extracellulaire et l’intégrine α7β1 du sarcolemme, remplaçant ainsi dans les muscles dystrophiques, des liens normalement assurés par le complexe de la dystrophine. L’injection de laminine-111 dans des souris mdx a permis, entre autre, l’augmentation de l’expression de l’intégrine α7β1, d’empêcher les bris du sarcolemme lors de la contraction musculaire, de restaurer un niveau normal de la créatine kinase sérique, ainsi que d’augmenter la résistance et la force dans les muscles déficients en dystrophine. Ces résultats suggèrent que l’augmentation de la laminine-111 est un potentiel traitement pour la DMD. Les chaines β1 et γ1 de la laminine sont déjà exprimées dans le muscle humain adulte, mais la chaine α1 de la laminine (Lamα1) est exprimée uniquement pendant le stade très précoce 16 cellules de l’embryogenèse. Nous avons donc développé une méthode alternative à l’injection répétée de Laminine-111 en induisant l’expression endogène du gène LAMA1, afin de reformer le complexe trimérique α1β1γ1, la laminine 111. Ceci a été réalisé avec une technologie récente, le système CRISPR/Cas9, dont la Cas9 a été désactivée (dCas9) puis couplée à un domaine d’activation de la transcription, le VP160 (dCas9-VP160). L’utilisation d’un ou plusieurs ARN guides (ARNg) a permis de cibler le promoteur du gène LAMA1. L’ARNm de Lamα1 (qRT-PCR) ainsi que la protéine (immunohistochimie et immunobuvardage) n’ont pas été détecté dans le contrôle négatif, des myoblastes murins (C2C12). Cependant, une expression significative a été observée dans ces myoblastes transfectés avec des plasmides codant pour dCas9-VP160 et un ARNg. L’analyse protéique in vivo, dans des muscles de souris électroporés avec le même plasmide, a démontré une forte augmentation de la chaine α1 de la laminine. Des augmentations plus importantes de l’ARNm de Lamα1 ont été observées en utilisant 2 ARNg, suggérant un effet synergique. L’augmentation de l’expression de Lamα1 par le système de CRISPR/Cas9 devrait être étudiée d’avantage afin de vérifier si cette stratégie pourrait s’avérer efficace dans des cas de myopathies.