A MALDI-TOF MS database for fast identification of Vibrio spp. potentially pathogenic to marine mollusks.

In mollusk aquaculture, a large number of Vibrio species are considered major pathogens. Conventional methods based on DNA amplification and sequencing used to accurately identify Vibrio species are unsuitable for monitoring programs because they are time-consuming and expensive. The aim of this study was, therefore, to develop the MALDI-TOF MS method in order to establish a rapid identification technique for a large panel of Vibrio species. We created the EnviBase containing 120 main spectra projections (MSP) of the Vibrio species that are potentially responsible for mollusk diseases, comprising 25 species: V. aestuarianus, V. cortegadensis, V. tapetis and species belonging to the Coralliilyticus, Harveyi, Mediterranei, and Orientalis clades. Each MSP was constructed by the merger of raw spectra obtained from three different media and generated by three collaborating laboratories to increase the diversity of the conditions and thus obtain a good technique robustness. Perfect discrimination was obtained with all of the MSP created for the Vibrio species and even for very closely related species as V. europaeus and V. bivalvicida. The new EnviBase library was validated through a blind test on 100 Vibrio strains performed by our three collaborators who used the direct transfer and protein extraction methods. The majority of the Vibrio strains were successfully identified with the newly created EnviBase by the three laboratories for both protocol methods. This study documents the first development of a freely accessible database exclusively devoted to Vibrio found in marine environments, taking into account the high diversity of this genus. KEY POINTS: • Development of a MALDI-TOF MS database to quickly affiliate Vibrio species. • Increase of the reactivity when faced with Vibrio associated with mollusk diseases. • Validation of MALDI-TOF MS as routine diagnostic tool.

Contagious agalactia monitoring in caprine herds through regular bulk tank milk sampling.

Surveillance and control of Mycoplasma spp. responsible for contagious agalactia (CA) in caprine herds are important challenges in countries with a large small-ruminant dairy industry. In the absence of any clinical signs, being able to determine the potential circulation of mycoplasmas within a herd could help to prevent biosecurity issues during animal exchanges between farms and improve health management practices. The objective of this study was to determine whether regular sampling of bulk tank milk was suitable for such surveillance. Twenty farms were sampled once a month for 2 yr and CA-responsible mycoplasmas were detected by real-time PCR on DNA extracted from milk, using 3 different DNA extraction methods. The pattern of mycoplasma excretion in bulk tank milk was assessed over time and several herd characteristics were recorded together with any event occurring within the herds. In general, the results obtained with the different detection methods were comparable and mainly agreed with the culture results. Several patterns of excretion were observed but were not related to herd characteristics (size, breed, and so on). Recurrence of the same (sub)species and same pulsed-field gel electrophoresis subtype during the 2-yr period is indicative of the considerable persistence of mycoplasmas. This persistence was associated with intermittent excretion. In conclusion, bulk tank milk sampling could be valuable for controlling CA in caprine herds provided it is repeated several times, yet to be defined, per year and analyzed using an appropriate methodology and the right cut-off for interpretation.

Identification and subtyping of clinically relevant human and ruminant mycoplasmas by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) recently emerged as a technology for the identification of bacteria. In this study, we aimed to evaluate its applicability to human and ruminant mycoplasmal identification, which can be demanding and time-consuming when using phenotypic or molecular methods. In addition, MALDI-TOF MS was tested as a subtyping tool for certain species. A total of 29 main spectra (MSP) from 10 human and 13 ruminant mycoplasma (sub)species were included in a mycoplasma MSP database to complete the Bruker MALDI Biotyper database. After broth culture and protein extraction, MALDI-TOF MS was applied for the identification of 119 human and 143 ruminant clinical isolates that were previously identified by antigenic or molecular methods and for subcultures of 73 ruminant clinical specimens that potentially contained several mycoplasma species. MALDI-TOF MS resulted in accurate (sub)species-level identification with a score of ≥1.700 for 96% (251/262) of the isolates. The phylogenetically closest (sub)species were unequivocally distinguished. Although mixtures of the strains were reliably detected up to a certain cellular ratio, only the predominant species was identified from the cultures of polymicrobial clinical specimens. For typing purposes, MALDI-TOF MS proved to cluster Mycoplasma bovis and Mycoplasma agalactiae isolates by their year of isolation and genome profiles, respectively, and Mycoplasma pneumoniae isolates by their adhesin P1 type. In conclusion, MALDI-TOF MS is a rapid, reliable, and cost-effective method for the routine identification of high-density growing mycoplasmal species and shows promising prospects for its capacity for strain typing.

[Pediatric management of early-onset neonatal sepsis: guidelines adherence in Lille's perinatal care network]. / Suivi des recommandations de prise en charge pédiatrique de l'infection maternofoetale: enquête au sein du réseau de soins périnatals de la métropole lilloise.

UNLABELLED: Neonatal early-onset infection has been recognized as a significant cause of perinatal mortality and morbidity, in spite of recent improvement in perinatal care. In France, Anaes guidelines were published in 2002. OBJECTIVES: To evaluate paediatricians' knowledge about these official guidelines in Lille's perinatal network. MATERIALS AND METHODS: Every network's paediatrician, working in a hospital maternity or in a neonatology unit received a questionnaire, to evaluate his knowledge about early onset sepsis diagnosis, indication and type of per partum antibiotherapy, and immediate neonatal care, according to perinatal factors. RESULTS: Response rate was 55%. Thirty-five per cent of paediatricians underestimated gastric sample's diagnostic role and 41% prescribed a triple antibiotherapy. Duration of antibiotic treatment was too long in 56% of cases. Biologic tests were prescribed in excess in 71% of cases when a streptoccocal B mother's colonization was present. Per partum antibiotherapy was appropriated in only 29% of responses. CONCLUSION: This study underlines imperfect official guidelines' knowledge, leading to ecological, bacterial, and economical consequences.