Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk

Barreiro, Juliana Regina; Gonçalves, Juliano Leonel; Grenfell, Rafaella C.; Leite, Renata F.; Juliano, Luiz; Santos, Marcos Veiga dos

doi:10.1016/j.bjm.2018.04.012

Cited by 13 publications

(6 citation statements)

References 13 publications

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“…half (31) of these 60 samples. Statistical analysis with McNemar and Cohen’s kappa tests confirm the absence of any significant difference with a fair agreement between the qPCR and the MALDI‐TOF results, similarly to other recently published studies, although these were based on milk samples instead of isolates (Barreiro et al, ; Wilson et al, ). The reasons for the differences in the qualitative and quantitative results can be several, some dealing with the sampling method, others with the multiplex qPCR assays and still others with the bacterial growth or the MALDI‐TOF MS identification.…”

Section: Discussionsupporting

confidence: 85%

Comparison of quantitative PCR and MALDI‐TOF mass spectrometry assays for identification of bacteria in milk samples from cows with subclinical mastitis

et al. 2019

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Aims: The objective of this study was to compare qualitatively and quantitatively the results of identification of the bacteria present in milk samples from cows with subclinical mastitis using multiplex qPCR assay and matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS â ) after bacteriological growth. Methods and Results: A total of 182 samples were aseptically collected from 119 cows with high somatic cell counts (>2Á10 5 SCC per ml) on 11 farms in Belgium in 2014. The mutiplex qPCR assay was carried out on 350 µl of milk with the PathoProof â Complete-16kit. Ten microlitre of milk was streaked on Columbia blood agar and three selective agar plates. Growing colonies were identified by MALDI-TOF MS. Of the 182 samples, 90 gave positive results with either or both tests for one or two bacterial species/genera. Total qualitative agreement of the bacteria identified was observed in 41 mono-or bi-bacterial samples (46%) and partial agreement in 19 bi-bacterial samples at both or either tests (21%). The results of both tests on those mono-and bibacterial samples were not significantly different (McNemar test; P = 0Á395) with a fair agreement (Cohen's kappa test; k = 0Á375; P = 0Á055). Moreover, quantitative correlation between the qPCR intensity and the numbers of growing colonies was observed in half of the 60 samples with qualitative matching results. Conclusions: Both methods give identical qualitative and quantitative results with approximately a half and a quarter of the mono-and bi-bacterial samples respectively. Several reasons can explain the differences. The multiplex qPCR assay only targets the most important mammary gland pathogens and can detect DNA of bacteria both alive and dead. Conversely, bacteria only grow when alive and the MALDI-TOF MS databases do not include all bovine milkassociated bacterial species yet. Significance and Impact of the Study: This study further highlights the limitations and complementarity of the genetic and phenotypic tests for the identification of bacteria present in milk samples.

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Section: Discussionsupporting

confidence: 85%

Comparison of quantitative PCR and MALDI‐TOF mass spectrometry assays for identification of bacteria in milk samples from cows with subclinical mastitis

et al. 2019

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“…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.…”

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confidence: 99%

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

et al. 2020

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Mycoplasma bovis is a leading cause of pneumonia in modern calf rearing. Fast identification is essential to ensure appropriate antimicrobial therapy. Therefore, the objective of this study was to develop a protocol to identify M. bovis from bronchoalveolar lavage fluid (BALf) with matrix-assisted laser desorption ionization–time of flight mass spectrometry MALDI-TOF MS and to determine the diagnostic accuracy in comparison with other techniques. BALf was obtained from 104 cattle, and the presence of M. bovis was determined in the following three ways: (i) rapid identification of M. bovis with MALDI-TOF MS (RIMM) (BALf was enriched and after 24, 48, and 72 h of incubation and was analyzed using MALDI-TOF MS), (ii) triplex real-time PCR for M. bovis, Mycoplasma bovirhinis, and Mycoplasma dispar, and (iii) 10-day incubation on selective-indicative agar. The diagnostic accuracy of the three tests was determined with Bayesian latent class modeling (BLCM). After 24 h of enrichment, M. bovis was identified with MALDI-TOF MS in 3 out of 104 BALf samples. After 48 and 72 h of enrichment, 32/104 and 38/100 samples, respectively, were M. bovis positive. Lipase-positive Mycoplasma-like colonies were seen in 28 of 104 samples. Real-time PCR resulted in 28/104 positive and 12/104 doubtful results for M. bovis. The BLCM showed a sensitivity (Se) and specificity (Sp) of 86.6% (95% credible interval [CI], 69.4% to 97.6%) and 86.4% (CI, 76.1 to 93.8) for RIMM. For real-time PCR, Se was 94.8% (CI, 89.9 to 97.9) and Sp was 88.9% (CI, 78.0 to 97.4). For selective-indicative agar, Se and Sp were 70.5% (CI, 52.1 to 87.1) and 93.9% (CI, 85.9 to 98.4), respectively. These results suggest that rapid identification of M. bovis with MALDI-TOF MS after an enrichment procedure is a promising test for routine diagnostics in veterinary laboratories.

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“…The isolated bacteria were identified by MALDI-TOF MS as described previously [ 27 , 28 , 29 ]. The direct colony method, which involves the application of bacterial colonies from growth plates directly onto the steel plate before testing by MALDI-TOF MS, was used.…”

Section: Methodsmentioning

confidence: 99%

Antimicrobial and Methicillin Resistance Pattern of Potential Mastitis-Inducing Staphylococcus aureus and Coagulase-Negative Staphylococci Isolates from the Mammary Secretion of Dairy Goats

Nelli

Voidarou

Venardou

et al. 2022

Biology

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Staphylococcus spp. is an important mastitis-inducing zoonotic pathogen in goats and is associated with antimicrobial resistance (AMR). The objectives of this study were to determine the prevalence and composition of staphylococci in individual mammary secretion (MS) samples of clinically healthy goats and to evaluate the phenotypic AMR pattern and the presence of methicillin resistance in the Staphylococcus spp. strains. Staphylococcus spp. isolates (n = 101) from the MS samples (n = 220) were identified to species level using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The antimicrobial susceptibility testing included a disk diffusion assay and the determination of the minimum inhibitory concentrations (MIC) of resistant strains (n = 46). Presumptive methicillin-resistant strains (n = 9) were assessed for the presence of mecA, mecC and SCCmec/orfx genes. Staphylococcus spp. isolates were recovered from 45.9% of the MS samples, of which, 72.3% was identified as coagulase-negative staphylococci (CoNS), with the remaining being Staphylococcus aureus. CoNS and S. aureus were most commonly resistant to ampicillin (56.2% and 57.1%, respectively), penicillin (26.0% and 39.3%, respectively), amoxicillin (26 % and 25 %, respectively) and cephalexin (12.3% and 25%, respectively) in the disk diffusion method. CoNS exhibited a broader AMR pattern and a higher percentage of resistant strains than S. aureus in the disk diffusion and MIC methods. Of the nine oxacillin- and cefoxitin-resistant strains, three S. aureus and five CoNS strains carried the mecA gene and, thus, were identified as methicillin-resistant. The mecC gene was not found in any of the studied strains. The presence of AMR and methicillin resistance in caprine S. aureus and CoNS poses a concern for animal and public health.

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Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk

Cited by 13 publications

References 13 publications

Comparison of quantitative PCR and MALDI‐TOF mass spectrometry assays for identification of bacteria in milk samples from cows with subclinical mastitis

Comparison of quantitative PCR and MALDI‐TOF mass spectrometry assays for identification of bacteria in milk samples from cows with subclinical mastitis

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

Antimicrobial and Methicillin Resistance Pattern of Potential Mastitis-Inducing Staphylococcus aureus and Coagulase-Negative Staphylococci Isolates from the Mammary Secretion of Dairy Goats

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