Gram-typing of mastitis bacteria in milk samples using flow cytometry.

Fast identification of pathogenic bacteria in milk samples from cows with clinical mastitis is central to proper treatment. In Denmark, time to bacterial diagnosis is typically 24 to 48 h when using traditional culturing methods. The PCR technique provides a faster and highly sensitive identification of bacterial pathogens, although shipment of samples to diagnostic laboratories delays treatment decisions. Due to the lack of fast on-site tests that can identify the causative pathogens, antibiotic treatments are often initiated before bacterial identification. The present study describes a flow cytometry-based method, which can detect and distinguish gram-negative and gram-positive bacteria in mastitis milk samples. The differentiation was based on bacterial fluorescence intensities upon labeling with biotin-conjugated wheat germ agglutinin and acridine orange. Initially 19 in-house bacterial cultures (4 gram-negative and 15 gram-positive strains) were analyzed, and biotin-conjugated wheat germ agglutinin and acridine orange florescence intensities were determined for gram-negative and gram-positive bacteria, respectively. Fluorescence cut-off values were established based on receiver operating characteristic curves for the 19 bacterial cultures. The method was then tested on 53 selected mastitis cases obtained from the department biobank (milk samples from 6 gram-negative and 47 gram-positive mastitis cases). Gram-negative bacteria in milk samples were detected with a sensitivity of 1 and a specificity of 0.74, when classification was based on the previously established cut-off values. However, when receiver operating characteristic curves were constructed for the 53 mastitis cases, results indicate that a sensitivity and specificity of 1 could be reached if cut-off values were reduced. This flow cytometry-based technique could potentially provide dairy farmers and attending veterinarians with on-site information on bacterial gram-type and prevent ineffective antimicrobial treatment in mastitis cases caused by gram-negative bacteria.

Brief report: biomarkers of aortic vascular prosthetic graft infection in a porcine model with Staphylococcus aureus.

Aortic vascular prosthetic graft infection (AVPGI) with Staphylococcus aureus is a feared post-operative complication. This study was conducted to evaluate the clinical signs and potential biomarkers of infection in a porcine AVPGI model. The biomarkers evaluated were: C-reactive protein (CRP), fibrinogen, white blood cells (WBC), major histocompatibility complex II (MHC II) density, lymphocyte CD4:CD8 ratio and tumour necrosis factor-alpha (TNF-α) in vitro responsiveness. Sixteen pigs were included in the study, and randomly assigned into four groups (n = 4): "SHAM" pigs had their infra-renal aorta exposed by laparotomy; "CLEAN" pigs had an aortic graft inserted; "LOW" and "HIGH" pigs had an aortic graft inserted and, subsequently, S. aureus were inoculated on the graft material (5 × 10(4) colony-forming units [CFU] and 1 × 10(6) CFU, respectively). Biomarkers were evaluated prior to surgery and on day 2, 5, 7, and 14 post-operatively in blood samples. Of all biomarkers evaluated, CRP was superior for diagnosing S. aureus AVPGI in pigs, with a sensitivity of 0.86 and a specificity of 0.75.