Cyclic Lipodepsipeptides Produced by Pseudomonas spp. Naturally Present in Raw Milk Induce Inhibitory Effects on Microbiological Inhibitor Assays for Antibiotic Residue Screening

Abstract

Two Pseudomonas strains, identified as closely related to Pseudomonas tolaasii, were isolated from milk of a farm with frequent false-positive Delvotest results for screening putative antibiotic residues in raw milk executed as part of the regulatory quality programme. Growth at 5 to 7°C of these isolates in milk resulted in high lipolysis and the production of bacterial inhibitors. The two main bacterial inhibitors have a molecular weight of 1168.7 and 1140.7 Da respectively, are heat-tolerant and inhibit Geobacillus stearothermophilus var. calidolactis, the test strain of most of the commercially available microbiological inhibitor tests for screening of antibiotic residues in milk. Furthermore, these bacterial inhibitors show antimicrobial activity against Staphylococcus aureus, Bacillus cereus and B. subtilis and also interfere negatively with yoghurt production. Following their isolation and purification with RP-HPLC, the inhibitors were identified by NMR analysis as cyclic lipodepsipeptides of the viscosin group. Our findings bring to light a new challenge for quality control in the dairy industry. By prolonging the refrigerated storage of raw milk, the keeping quality of milk is influenced by growth and metabolic activities of psychrotrophic bacteria such as pseudomonads. Besides an increased risk of possible spoilage of long shelf-life milk, the production at low temperature of natural bacterial inhibitors may also result in false-positive results for antibiotic residue screening tests based on microbial inhibitor assays thus leading to undue production loss.

Impact of a Stereocentre Inversion in Cyclic Lipodepsipeptides from the Viscosin Group: A Comparative Study of the Viscosinamide and Pseudodesmin Conformation and Self‐Assembly

“…[12b, [15][16]18] For example, although several strains produce viscosin as the main CLP, in other strains massetolide A is the most abundant with viscosin appearing as a minor product. [12a] Gerard et al [12b] demonstrated that the NRPSs exhibit a certain degree of flexibility in selecting the free amino acids, thus providing an explanation for this variation.…”

Characterization of Milkisin, a Novel Lipopeptide With Antimicrobial Properties Produced By Pseudomonas sp. UCMA 17988 Isolated From Bovine Raw Milk

“…As far as we know, only two Pseudomonas strains isolated from dairy product were shown to produce cyclic lipopeptides. However, those two strains, closely related to P. tolaasii , were shown to produce isoforms belonging to another family (viscosin) ( Reybroeck et al, 2014 ).…”

Nitrogen gas flushing can be bactericidal: the temperature-dependent destiny of Bacillus weihenstephanensis KBAB4 under a pure N2 atmosphere

“…The bacterium Pseudomonas “ reactans ” was recently re-classified as P. fluorescens (Ghequire et al, 2013 ). The incidence of CLP producers in milk is not well known, but a recent report demonstrated the implication of CLP production in false positive results when screening for antibiotic residues in milk (Reybroek et al, 2014 ). Considering that tolaasin had an inhibitory effect on Bacillus subtilis and B. megaterium (Rainey et al, 1991 ) and that C1 is also a CLP producer, both representatives of two different types of CLPs producers, C1 and P. tolaasii LMG 2342 T , were selected in this study.…”