Laure Galofaro

Agence Nationale de Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail, Île-de-France, France

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Publications (5)9.79 Total impact

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    Full-text · Article · Nov 2010 · Journal of Antimicrobial Chemotherapy
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    ABSTRACT: Streptococcus uberis is an environmental pathogen commonly causing bovine mastitis, an infection that is generally treated with penicillin G. No field case of true penicillin-resistant S. uberis (MIC > 16 mg/liter) has been described yet, but isolates presenting decreased susceptibility (MIC of 0.25 to 0.5 mg/liter) to this drug are regularly reported to our laboratory. In this study, we demonstrated that S. uberis can readily develop penicillin resistance in laboratory-evolved mutants. The molecular mechanism of resistance (acquisition of mutations in penicillin-binding protein 1A [PBP1A], PBP2B, and PBP2X) was generally similar to that of all other penicillin-resistant streptococci described so far. In addition, it was also specific to S. uberis in that independent resistant mutants carried a unique set of seven consensus mutations, of which only one (Q554E in PBP2X) was commonly found in other streptococci. In parallel, independent isolates from bovine mastitis with different geographical origins (France, Holland, and Switzerland) and presenting a decreased susceptibility to penicillin were characterized. No mosaic PBPs were detected, but they all presented mutations identical to the one found in the laboratory-evolved mutants. This indicates that penicillin resistance development in S. uberis might follow a stringent pathway that would explain, in addition to the ecological niche of this pathogen, why naturally occurring resistances are still rare. In addition, this study shows that there is a reservoir of mutated PBPs in animals, which might be exchanged with other streptococci, such as Streptococcus agalactiae, that could potentially be transmitted to humans.
    Full-text · Article · Mar 2010 · Antimicrobial Agents and Chemotherapy
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    ABSTRACT: Objectif: S. uberis, du groupe pyogenes, est un pathogène majeur de mammites bovines. Ces infections sont souvent traitées par la pénicilline G, mais aucun phénotype de résistance n'a encore été décrit. Des souches de sensibilité diminuée ont été détectées chez les bovins et l'objectif de cette étude était d'en identifier les déterminants, en recherchant les mutations des PLPs (Protéines Liant la Pénicilline) chez ces souches de terrain et chez des mutants expérimentaux. Méthode: Les mutants ont été obtenus sous pression de sélection par cyclages à la pénicilline G. Les mutations ont été détectées chez les mutants et les souches de terrain par séquençage du domaine de transpeptidation des PLP1A, 2B et 2X identifiées in silico. Les PLPs ont été visualisées par marquage à la bocillin-FL et les modifications de la structure du peptidoglycane analysées par HPLC. Résultats: S. uberis possède 5 PLPs fortement homologues à celles des autres streptocoques (PLP1A, 1B, 2A de classe A; PLP 2B, 2X de classe B). En environ 30 cycles, les CMI ont progressé de 0.016 mg/L à 2 mg/L. En parallèle, des mutations se sont accumulées dans la PLP2X (dont la substitution Q554E caractéristique des streptocoques), 2B et 1A. Sept mutations ont systématiquement été conservées dans les mutants de laboratoire (CMI=2 mg/L). L'effet de ces modifications sur l'affinité des PLPs pour la pénicilline et sur la structure du peptidoglycane a été démontré. Ces mutations spécifiques ont aussi été détectées dans les isolats cliniques non cyclés (CMI de 0.125-0.25 mg/L). Par ailleurs, aucune PLP mosaïque n'a été identifiée. Conclusion: S. uberis peut résister à la pénicilline G par cyclages successifs, contrairement à S. pyogenes. La sensibilité diminuée est toujours associée à des mutations ponctuelles (dont 7 hautement conservées) dans les PBP2X, 2B et 1A, sans PLP mosaïque. S. uberis est donc apte à développer cette résistance in vitro comme dans l'environnement. A ce jour chez les bovins, la résistance à la pénicilline G chez S. uberis n'a pas le succès connu pour S. pneumoniae chez l'homme, dont la cavité buccale est probablement plus propice aux échanges génétiques que la mamelle, mais ces résultats soulignent désormais la nécessité d'un suivi renforcé de ce phénotype dans cette filière animale.
    No preview · Article · Dec 2009
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    Marisa Haenni · Jean-Yves Madec · Laure Galofaro
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    ABSTRACT: Staphylococcus spp are known to be responsible of about one third of bovine mastitis
    Preview · Article · Sep 2009
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    ABSTRACT: Background: S. uberis is a member of the streptococcal pyogenic group and a causative agent of bovine mastitis, which are frequently treated with penicillin. Despite the report of decreased susceptibility in certain environmental isolates, the ability of S. uberis to develop penicillin-resistance has not been documented yet. Thus, since multimodular PBPs are commonly considered as the major resistance determinants in penicillin-resistance, we looked for PBP mutations occurring during penicillin-resistance selection in the S. uberis reference strain ATCC 19436 and 9 susceptible environmental isolates. In parallel, we determined the PBP modifications accumulated in environmental bacteria naturally presenting a decreased susceptibility to penicillin. Methods: PBPs were identified by standard in silico analyses. Liquid cultures of S. uberis were cycled with increasing concentrations of penicillin and the increase in MIC was followed. Mutations were determined by sequencing the transpeptidase domains of PBP1A, PBP2B and PBP2X genes. Results: S. uberis carries 5 multimodular PBPs, which share homologies with other streptococcal PBPs and were named accordingly (class A PBP1A, 1B and 2A; class B PBP2B and 2X). By culture cycling, resistance increased stepwise from an MIC of about 0.016 μg/ml to 1-2 μg/ml in ca. 30 cycles. And the same trend was observed for each cycled isolate. PBP mutations were detected in PBP2X (including the characteristic streptococcal Q554E substitution), PBP2B and PBP1A, and accumulated concomitantly to the increase of resistance. Moreover, a high number of silent mutations was identified in these genes. Point mutations, but no mosaic-like PBPs, were also detected in the environmental isolates. Conclusion: In S. uberis, resistance development was readily obtained by culture cycling, contrarily to S. pyogenes. In the cycled strains as well as in the environmental isolates, decreased susceptibility was associated with point mutations in pbp genes, and no mosaic PBPs were detected in the natural strains. Consequently, while fully able to develop strongly decreased susceptibilities under laboratory conditions, S. uberis isolated from cattle mastitis might only present limited levels of penicillin-resistance probably because of the absence of a long-lasting bacterial community on the site of infection, contrarily to what is occurring in the buccal cavity for oral streptococci.
    No preview · Article · Jun 2009