Rapid identification of respiratory bacterial pathogens from bronchoalveolar lavage fluid in cattle by MALDI-TOF MS

Abstract

Respiratory tract infections are a major health problem and indication for antimicrobial use in cattle and in humans. Currently, most antimicrobial treatments are initiated without microbiological results, holding the risk of inappropriate first intention treatment. The main reason for this empirical treatment is the long turnaround time between sampling and availability of identification and susceptibility results. Therefore the objective of the present study was to develop a rapid identification procedure for pathogenic respiratory bacteria in bronchoalveolar lavage fluid (BALf) samples from cattle by MALDI-TOF MS, omitting the cultivation step on agar plates to reduce the turnaround time between sampling and identification of pathogens. The effects of two different liquid growth media and various concentrations of bacitracin were determined to allow optimal growth of Pasteurellaceae and minimise contamination. The best procedure was validated on 100 clinical BALf samples from cattle with conventional bacterial culture as reference test. A correct identification was obtained in 73% of the samples, with 59.1% sensitivity (Se) (47.2-71.0%) and 100% specificity (Sp) (100-100%) after only 6 hours of incubation. For pure and dominant culture samples, the procedure was able to correctly identify 79.2% of the pathogens, with a sensitivity (Se) of 60.5% (45.0-76.1%) and specificity (Sp) of 100% (100-100%). In mixed culture samples, containing ≥2 clinically relevant pathogens, one pathogen could be correctly identified in 57% of the samples with 57.1% Se (38.8-75.5%) and 100% Sp (100-100%). In conclusion, MALDI-TOF MS is a promising tool for rapid pathogen identification in BALf. This new technique drastically reduces turnaround time and may be a valuable decision support tool to rationalize antimicrobial use.

Bovine Respiratory Disease Diagnosis

“…Relative to classic microbial culture, these culture-enriched direct MALDI-TOF MS techniques allow correct bacterial identification in 73% of the samples (sensitivity 5 59.1%; 95% confidence interval [CI], 47.2-71.0; specificity 5 100% [100-100]) within 6 hours. 33 The technique performed less well in polymicrobial samples and in samples with mixed infection. Also for M bovis, a culture-enriched direct MALDI-TOF MS technique was developed, which was 86.6% (95% BCI, 54-99) sensitive and 86.4% (95% BCI, 80-96) specific in a bayesian latent class model including PCR and microbial culture on solid agar.…”

“…More information can be derived from culture results if quantitative descriptions and at least the degree of contamination are described. A possible way to better describe culture results, previously used for research purposes, 10,33 is presented in Table 4. It is also important to realize that using selective media for Pasteurellaceae, as, for example, by adding bacitracin, ensures better growth and detection of these opportunistic pathogens, but information on the amount of pathogens and degree of contamination of the sample will be lost.…”

Rapid Identification of Mycoplasma bovis Strains from Bovine Bronchoalveolar Lavage Fluid with Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry after Enrichment Procedure

“…Nevertheless, prior isolation of M. bovis on specific solid medium is still necessary, and final identification can take up to 10 days (10,18,19). To reduce sample turnaround time, at present, there is great interest in the identification of bacteria by MALDI-TOF MS directly from the sample or after a short enrichment period in liquid broth as is already done for urine, blood, milk, and BALf specimens (20)(21)(22)(23). However, such a technique is currently not available for Mycoplasma spp., presumably because of difficulties such as their fastidious growth and overgrowth by other bacteria.…”

Storage time and temperature affect the isolation rate of Mannheimia haemolytica and Pasteurella multocida from bovine bronchoalveolar lavage samples

“…This study showed a longer duration of carriage in the nose and higher concentration for P. multocida compared to H. somni, however rates of M. haemolytica were too low for meaningful survival modelling. Retaining high concentrations of relevant bacteria in clinical samples can be an added value to diagnostics, both for clinical interpretation as for direct detection methods using for example matrix-assisted laser desorption/ionization-time-of-ight mass spectrometry (MALDI-TOF MS) [11]. A higher negative association between the presence of contaminants and the isolation rate of P. multocida was found compared to M. haemolytica.…”