Characterization of Aerococcus viridans isolated from milk samples from cows with mastitis and manure samples

Abstract

Thirty-eight Aerococcus viridans isolates were obtained from milk from 478 cows with clinical mastitis in a farm during the periods between November 2011 and February 2012, and between December 2012 and March 2013. Additional isolates were obtained from processed manure (a mixture of composted manure, straw and hydrated lime) and bedding materials. The processed manure was later used to cover the floor of the stalls in barns as bedding materials. The temperatures recorded in the composted and processed manure were not as high as those generally observed during satisfactory composting. To reveal the association of A. viridans in manure-related products with intramammary infection in cows, isolates were characterized by their DNA fragment patterns as determined by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. Isolates obtained from milk, processed manure and bedding materials had identical DNA fragment patterns. Antimicrobial susceptibilities were determined for 29 isolates from milk, processed manure and bedding materials. Of these, 26 (89.7%) were resistant to clindamycin, whereas virtually all the isolates were susceptible to 12 other antimicrobials including cefalosporins that have been used to treat bovine mastitis in Japan. In vitro, three A. viridans isolates from milk and an isolate from processed manure survived for 3 hr in Good's buffer (pH 9) at high temperature (50°C). The results suggest that the processed manure and bedding materials in this farm were possible sources of A. viridans that caused infection in the cows with mastitis.

Fig. 1.

Numbers of milk samples of cows from which the following bacteria were isolated: A. viridans, E. coli, Streptococcus spp. other than S. agalactiae, coagulase-negative staphylococci, Pseudomonas aeruginosa and others. Horizontal lines under the months represent winter season in Japan.

Fig. 2.

SmaI-digested PFGE patterns of genome DNAs of representative A. viridans isolates. Lanes: 1 to 14, isolates from milk samples (MK) of 14 cows in farm A between November 2011 and February 2012; 15 and 16, MK of 2 cows in farm B, 17 to 22, MK of 6 cows in farm A between November 2012 and March 2013; 23 to 25, isolates from bedding materials (BM) sampled in January 2013 in farm A; 26 to 29, isolates from processed manure (PM) sampled in January 2013 in farm A. Lane M, lambda ladder. The lowercase letter below each of the lane numbers refers to the PFGE pattern (see RESULTS). The base sizes of the markers are indicated on the right of the panel.

Fig. 3.

Changes in viable cell counts of A. viridans and E. coli in (A) PBS (pH 7) and (B) the Good’s buffer (pH 9) at 50°C after 0 (solid column), 3 (grey), 6 (grid) and 24 (open) hr. A. viridans isolates #6, #9, #19 and #23 correspond to those with the same numbers in Fig. 2. E. coli isolates A and B were from milk samples of cows with mastitis. 700406 and 25922 represent the ATCC strains of A. viridans and E. coli, respectively. Dashed lines indicate the detection limit (3.0 × 10² cfu/ml). Significant differences between initial viable cell numbers and those post inoculation are indicated by asterisks.