Nisin resistance of Streptococcus bovis

Abstract

The growth of Streptococcus bovis JB1 was initially inhibited by nisin (1 microM), and nisin caused a more than 3-log decrease in viability. However, some of the cells survived, and these nisin-resistant cells grew as rapidly as untreated ones. To see if the nisin resistance was merely a selection, nisin-sensitive cells were obtained from agar plates lacking nisin. Results indicated that virtually any nisin-sensitive cell could become nisin-resistant if the ratio of nisin to cells was not too high and the incubation period was long enough. Isolates obtained from the rumen were initially nisin sensitive, but they also developed nisin resistance. Nisin-resistant cultures remained nisin resistant even if nisin was not present, but competition studies indicated that nisin-sensitive cells could eventually displace the resistant ones if nisin was not present. Nisin-sensitive, glucose-energized cells lost virtually all of their intracellular potassium if 1 microM nisin was added, but resistant cells retained potassium even after addition of 10 microM nisin. Nisin-resistant cells were less hydrophobic and more lysozyme-resistant than nisin-sensitive cells. Because the nisin-resistant cells bound less cytochrome c, it appeared that nisin was being excluded by a net positive (i.e., less negative) charge. Nisin-resistant cells had more lipoteichoic acid than nisin-sensitive cells, and deesterified lipoteichoic acids from nisin-resistant cells migrated more slowly through a polyacrylamide gel than those from nisin-sensitive cells. These results indicated that lipoteichoic acids could be modified to increase the resistance of S. bovis to nisin. S. bovis JB1 cultures were still sensitive to monensin, tetracycline, vancomycin, and bacitracin, but ampicillin resistance was 1,000-fold greater.

FIG. 1

The effect of nisin (1 μM) on the optical density (a) and viability (b) of S. bovis JB1 (●). The growth of untreated cultures (○) and those that had been previously treated with 1 μM nisin (▴) is also shown. The bars (b) show standard deviations (n = 3).

FIG. 2

The effect of nisin concentration on the lag time of S. bovis JB1. Inocula had been transferred repeatedly with 0 (○), 1 (●), or 10 (■) μM nisin prior to measurements of optical density.

FIG. 3

The effect of lysozyme (4 mg/ml added at the arrow) on the growth (optical density) of nisin-sensitive S. bovis JB1 cultures (○) and nisin-resistant cultures (●). The nisin-resistant cultures had been transferred repeatedly with 1 μM nisin.

FIG. 4

The effect of nisin on the intracellular potassium of nisin-sensitive S. bovis JB1 cells (○) and cells that had been treated with 1 μM nisin (●). The cells were washed and incubated in a buffer containing 20 mM glucose but lacking nitrogen. The experiments were repeated three times, and the coefficients of variation were always less than 10%.

FIG. 5

The potassium content of nisin-sensitive glucose-energized cells (1 optical density unit) that had been suspended in a medium with or without 1 μM nisin (light bars). The dark bars show the potassium content of nisin-sensitive cells that were suspended in the cell-free supernatants of nisin-sensitive and nisin-resistant cells. The nisin-sensitive and -resistant cells (2.0 optical density or 320 μg of protein/ml) were added to nisin-containing medium and were harvested by centrifugation prior to the addition of nisin-sensitive cells. The resistant cells had been transferred with 1 μM nisin. The bars show standard deviations (n = 3).

FIG. 6

The number of S. bovis cells that were naturally resistant to nisin (shaded bars). Cultures were serially diluted (10-fold increments) into medium containing 5 μM nisin (39°C, 48 h), and the dilution tubes were scored for growth. Isolates 1 to 4 were obtained from a cow fed hay. The resistant JB1 cells had been transferred several times with 1 μM nisin. The open bar shows a control without nisin. The error bars show standard deviations (n = 2).

FIG. 7

The number of S. bovis cells that were naturally resistant to antibiotics. Nisin-sensitive (a) or nisin-resistant (b) cultures were serially diluted (10-fold increments) into medium containing penicillin (50 μg/ml), ampicillin (50 μg/ml), vancomycin (10 μg/ml), tetracycline (10 μg/ml), bacitracin (10 μg/ml), or monensin (1 μM). The dilution tubes were scored for growth after a 48-h incubation at 39°C. The resistant JB1 cells had been transferred several times with 1 μM nisin. The error bars show standard deviations (n = 3).

FIG. 8

Polyacrylamide gels of dialyzed lipoteichoic acids from S. bovis. Lanes A and B show extracts that were derived from nisin-sensitive and nisin-resistant cells. Lanes C and D show the same extracts, but in this case the amount of anthrone-reactive material was normalized, and the extracts were treated with sodium hydroxide to remove lipid. The gels were stained with alcian blue and silver nitrate. The gels were run for approximately 2 h, and the migration distances are shown.