Control of Listeria monocytogenes in a biofilm by competitive-exclusion microorganisms

Abstract

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37 degrees C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10(3) CFU of L. monocytogenes/ml and 10(5) CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37 degrees C for 24 h, 15 degrees C for 14 days, 8 degrees C for 21 days, and 4 degrees C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37 degrees C, two at 15 and 8 degrees C, and three at 4 degrees C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4 degrees C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log(10) CFU of L. monocytogenes/cm(2)). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37 degrees C.

FIG. 1.

Inhibition at 37°C of L. monocytogenes (LM) by competitive-exclusion microorganisms (not including yeast isolates) in TSB-YE.

FIG. 2.

Inhibition at 15°C of L. monocytogenes (LM) by competitive-exclusion microorganisms (not including yeast isolates) in TSB-YE.

FIG. 3.

Inhibition at 8°C of L. monocytogenes (LM) by competitive-exclusion microorganisms (not including yeast isolates) in TSB-YE.

FIG. 4.

Inhibition at 4°C of L. monocytogenes (LM) by competitive-exclusion microorganisms (not including yeast isolates) in TSB-YE.

FIG. 5.

PCR amplification of nisin A (lanes 1 to 4) and nisin B (lanes 5 to 8) genes of competitive-exclusion microorganisms and genes of nisin A and B (ATCC 11454, control). Lanes: M, 100-bp DNA ladder; 1 and 5, ATCC 11454; 2 and 6, C-1-92; 3 and 7, C-1-152; 4 and 8, 143.