Evaluation of chromogenic culture media for rapid identification of microorganisms isolated from cows with clinical and subclinical mastitis

Abstract

This study aimed to evaluate the diagnostic performance (specificity, Sp; sensitivity, Se; accuracy; positive predictive value; negative predictive value; and Cohen's kappa coefficient, κ, of agreement) of chromogenic culture media for rapid identification of microorganisms isolated from cows with clinical (CM) and subclinical mastitis (SCM). For this, 2 experiments were carried out: evaluation of (1) biplate, and (2) triplate of chromogenic culture media for rapid identification of mastitis-causing microorganisms. For the evaluation of diagnostic performance, identification of microorganisms by MALDI-TOF mass spectrometry was considered the standard methodology. In experiment 1, 476 milk samples collected from cows with CM and 660 from cows with SCM were evaluated by inoculation in 2 selective chromogenic culture media (CHROMagar) for gram-positive bacteria and another for gram-negative bacteria. In experiment 2, 476 milk samples from cows with CM and 500 from cows with SCM were evaluated by inoculation in triplate chromogenic culture media (Smartcolor2, Onfarm), selective for Streptococcus and Strep-like organisms, Staphylococcus, and gram-negative bacteria. In experiment 1 for the CM samples, the use of biplates with gram-positive and gram-negative culture media showed Se that ranged from 0.56 (0.32-0.81; Staphylococcus aureus) to 0.90 (0.80-0.99 Streptococcus uberis), Sp varied from 0.94 (0.92-0.96; Strep. uberis) to 1.00 (Prototheca spp. or yeast), and κ ranged from 0.47 (0.26-0.67; Staph. aureus) to 0.84 (0.78-0.9; Escherichia coli). The Se of biplates for SCM samples ranged from 0.50 (0.15-0.85; E. coli) to 0.94 (0.87-1.00; Staph. aureus), Sp varied from 0.95 (0.93-0.97; Strep. uberis) to 0.99 (0.98-1.00; Staph. aureus and Strep. Agalactiae or dysgalactiae), and κ ranged from 0.18 (0.00-0.40; Escherichia coli) to 0.88 (0.80-0.95; Staph. aureus). In experiment 2, the Se of the triplate chromogenic media in CM samples ranged from 0.09 (0.00-0.26; Serratia spp.) to 0.94 (0.85-1.00; Klebsiella spp. and Enterobacter spp.), Sp varied from 0.94 (0.92-0.96; Strep. agalactiae and Strep. dysgalactiae) to 1.00 (Serratia spp.) and κ ranged from 0.07 (0.00-0.24; Serratia spp.) to 0.85 (0.75-0.94; Klebsiella spp. and Enterobacter spp.). For SCM samples, the use of the triplate with the chromogenic culture media showed Se that varied from 0.25 (0.10-0.40; Lactococcus spp.) to 1.00 (Strep. Agalactiae or dysgalactiae), Sp ranged from 0.92 (0.90-0.94; Strep. Agalactiae and Strep. dysgalactiae) to 0.99 (0.98-1.00; Klebsiella spp. and Enterobacter spp.), and κ varied from 0.28 (0.00-0.72; E. coli) to 0.72 (0.60-0.82; Staph. aureus). Our results suggest that the diagnostic accuracy of the biplate and triplate of chromogenic culture media varies according to pathogen, and the results of chromogenic culture media may be useful for rapid decision-making on mastitis treatment protocols of the main mastitis-causing microorganisms, but their use for implementation of mastitis control measures will depend on each farm specific needs.

Diagnosing Intramammary Infection: Meta-Analysis and Mapping Review on Frequency and Udder Health Relevance of Microorganism Species Isolated from Bovine Milk Samples

“…For instance, during CM caused by E. coli, bacterial cells may be eliminated from the udder tissue, but the lipopolysaccharide endotoxin will still have a harming effect on the mammary gland and trigger clinical signs, thus leading to sampling when viable bacterial cells are not present anymore [ 47 ]. Furthermore, the protocol for bacteriological culture of the milk samples from CM cases could vary from one laboratory to another: the use of different culture media or the use of a supplementary enriched culture when no growth was observed after 24 h incubation of the CM samples may have impacted culture yield and the results interpretation [ 35 , 48 ].…”

Prevalence of bovine mastitis-related pathogens, identified by mass spectrometry in flies (Insecta, Diptera) captured in the milking environment

“…This discrepancy of results could be credited to the use of selective media in the present study, including a commercial three‐part chromogenic selective medium, developed for the microbial culture on farms of the pathogens related to bovine mastitis to facilitate decisions on treatment and control approaches (Granja et al . 2021). Here, the use of selective media probably favoured the isolation of enterobacteria from insects, as well as the classification of isolates by mass spectrometry, which has a high discriminative power on species‐level identification of microorganisms, including those related to bovine mastitis (Gonçalves et al .…”

Parasitocenoses in cattle and their circulation in small farms

“…Therefore, the underestimation of microorganisms capable of complex relationships between themselves and biofilm formation has led to the fact that today there are no guidelines that clearly regulate the actions of a veterinarian aimed at a clear diagnosis of diseases caused by associations of microorganisms. If a veterinarian does not consider at least one of the parasitocenosis members that is involved in the etiology of the disease, this will definitely lead to a decrease in the effectiveness of preventive and control measures [5,11,32].…”

“…In artificial biogeocenosis, conditionally pathogenic microflora that circulate on the farm can cause various associated diseases in farm animals. These are diseases such as mastitis, endometritis, vaginitis in adult animals and gastrointestinal, respiratory diseases in newborn young animals [1][2][3][4][5][6]. Due to the large number of parasitic forms in the external environment, the possibility of combining several pathogens in one organism increases.…”