Biofilm Formation and Disinfectant Susceptibility of Persistent and Nonpersistent Listeria monocytogenes Isolates from Gorgonzola Cheese Processing Plants

Abstract

The aim of this study was to investigate whether the biofilm-forming ability and/or the disinfectant susceptibility accounted for the persistence of Listeria monocytogenes in Gorgonzola cheese processing plants. For this purpose, a set of 16 L. monocytogenes isolates collected in the 2004-2007 period was analyzed, including 11 persistent isolates collected in different years, within the collection period, and displaying identical or highly correlated pulsotypes. The evaluation of biofilm-forming ability was assessed using crystal violet (CV) staining and the enumeration of viable cells on stainless steel coupons (SCC). Absorbance values obtained with CV staining for persistent and non-persistent isolates were not significantly different (rm-ANOVA p>0.05) and the cell counts from non-persistent isolates showed to be higher than of persistent isolates (rm-ANOVA p<0.05). A simulation of disinfectant treatments was performed on spot inoculated coupons in clean and dirty conditions, according to EN 13697, and on biofilms on SSC, grown in nutrient-rich (dirty) and limiting (clean) conditions using acid acetic-hydrogen peroxide (P3) and acid citric-hydrogen peroxide (MS) commercial disinfectants. The treatment was considered effective when a 4 Log reduction in viable cell count was observed. The Log reductions of persistent and non-persistent isolates, obtained with both the assays in clean and dirty conditions, were compared and no significant differences were detected (rm-ANOVA p>0.05). A greater influence of organic matter on MS could explain why P3 was efficient in reducing to effective levels the majority of the isolates at the lowest concentration suggested by the manufacturer (0.2% [v/v]), while the same purpose required an higher concentration (1% [v/v]) of MS.In conclusion, our results demonstrate that the persistence of these isolates in Gorgonzola cheese processing plants was linked neither to the biofilm forming ability nor to their susceptibility to hydrogen peroxide-based disinfectants, therefore other factors should contribute to the persistent colonization of the dairies.

Listeria innocua and Listeria monocytogenes strains from dairy plants behave similarly in biofilm sanitizer testing

“…The evaluation of biofilm-forming ability was performed by quantification of biofilm biomass on microtiter plates (polystyrene), using both the crystal violet (CV) method (Borucki, Peppin, White, Loge, & Call, 2003) and a method of enumeration of viable cells on stainless steel coupons (SSC), as described by Costa et al (2016). Biofilms were grown in tryptic soy broth with 0.6% yeast extract (TSB-YE, Biokar Diagnostics, Beauvais, France) at 25 °C for 24 (CV) and 48 h (SSC).…”

“…The evaluation of the biofilm susceptibility to the disinfectants was performed on biofilms grown on SCC in nutrient-limiting (1/10 diluted TSB-YE) and in nutrient-rich (TSB-YE) medium at 25 °C for 48 h, as described by Costa et al (2016). Two commercial HP-based disinfectants, commonly employed at the dairies where the isolates were collected, were used: P3-oxonia active (ECOLAB S.r.l) containing acetic acid and peracetic acid (designated herein as P3), and Mida San 315 (Christeyns Food Hygiene S.r.l) containing citric acid (designated herein as MS).…”

“…The reasons for persistence are not known, but strong biofilm forming ability and disinfectant susceptibility do not seem to be prerequisites (Costa, Bertolotti, Brito, & Civera, 2016). The present study aimed to investigate whether L. innocua biofilms may be used as surrogates of the pathogenic L. monocytogenes biofilms.…”

Persistent Listeria monocytogenes Isolates from a Poultry-Processing Facility Form More Biofilm but Do Not Have a Greater Resistance to Disinfectants than Sporadic Strains

“…However, an association between resistance to disinfectants and the persistence of the pathogen in different food processing environments has been demonstrated only in a few cases [11,52,[75][76][77][78]. A clear link between persistence and increased disinfectant resistance was not recorded in some other studies [65,68,[79][80][81]. To the best of our knowledge, this is the first report where the greater susceptibility to biocides of the biofilms by L. monocytogenes was observed in the case of the group of persistent strains ( Table 2).…”

“…Some studies showed that persistent strains of L. monocytogenes formed more biofilm than non-persistent strains [5,13,49,52,54,64,65]. Others observed no difference between persistent strains as opposed to sporadic strains [44,[65][66][67][68]. Thus, the association between the ability of L. monocytogenes strains to form biofilms and their persistence in a food-processing plant environment is not clear.…”