Biofilm Formation by Multidrug-Resistant Serotypes of Salmonella Isolated from Fresh Products: Effects of Nutritional and Environmental Conditions

Abstract

Salmonella serotypes can develop biofilms in fresh food products. This study focused on determining the antimicrobial resistance profile and the effects of different growth media and environmental conditions on biofilm formation by multidrug-resistant serotypes of Salmonella. All 49.4% of the Salmonella strains (five serotypes) were multidrug resistant. Assessment of the ability to form biofilms using the crystal violet staining method revealed that 95.6% of the strains of Salmonella were strong biofilm producers in 96-well polystyrene microtiter plates. Overall, 59.3% of the Salmonella strains showed the rdar (red dry and rough colony) morphotype, 2.1% pdar (pink dry and rough colony), 27.4% bdar (brown dry and rough colony) and 10.9% saw (smooth and white colony), at two temperatures (22 and 35 °C). Mono-species biofilms of Salmonella serotypes showed a mean cell density of 8.78 log10 CFU/cm2 ± 0.053 in TSBS (1/20 diluted TSB (tryptic soy broth) + 1% strawberry residues) and 8.43 log10 CFU/cm2 ± 0.050 in TSBA (1/20 diluted TSB + 1% avocado residues) on polypropylene type B (PP) (p < 0.05). In addition, epifluorescence microscopy and scanning electron microscopy (SEM) enabled visualizing the bacteria and extracellular polymeric substances of biofilms on PP. Salmonella form biofilms depending on the serotype of the strains and the environmental conditions. Mono-species biofilms formed by Salmonella serotypes respond to nutrient limitation with the use of simplified culture media such as TSBA and TSBS.

Conditions of In Vitro Biofilm Formation by Serogroups of Listeria monocytogenes Isolated from Hass Avocados Sold at Markets in Mexico

“…In total, 18 L. monocytogenes isolates were evaluated in this study, which were previously obtained from Hass avocados sold at retail markets in Guadalajara, Mexico [12]. Stocks of L. monocytogenes were stored using the protocol described by Avila-Novoa et al [24]. Working cultures of L. monocytogenes were maintained in tryptic soy broth (TSB; Becton Dickinson Bioxon, Le Pont de Claix, France) with 0.6% yeast extract (TSBYE) (Sigma-Aldrich, St. Louis, MO, USA) and confirmed by PCR.…”

“…Bacterial DNA was extracted from 24 h/30 • C cultures in TBSYE using the protocol described by Avila-Novoa et al [24]. All L. monocytogenes strains were investigated for the detection of prfA, plcA, hly, mpl, actA, plcB, inlA, and inlB genes by PCR using the protocol by Montero et al [25].…”

“…Biofilms were developed on PP coupons (polypropylene type B (2 × 0.7 × 0.2 cm; Plásticos Tarkus, Jalisco, Mexico)), using the protocol described by Avila-Novoa et al [24] for 240 h at 30 • C in 10 mL of 4 different media: TSB, 1/10 diluted TSB, TSBA, and 1/10 diluted TSBA, and inoculated with 100 µL of the corresponding strain of L. monocytogenes (~10 8 CFU/mL). The quantification was carried out as described by Avila-Novoa et al [24]. L. monocytogenes ATCC 19111 was used as the positive control.…”

“…After incubation at 30 • C for 240 h, the PP coupons were removed and processed using the protocol described by Avila-Novoa et al [24]. Alternatively, SEM was performed using the methodology described by Borucki et al [22] and Fratesi et al [28].…”

“…Previously mature biofilms cultivated in TSB at 30 • C for 240 h in polystyrene microtiter plates were washed with 0.9% NaCl. The major components of L. monocytogenes biofilms were treated with (i) proteinase K (Promega, Madison, WI, USA) and (ii) 0.5 mg/mL DNase I (Roche, Mannheim, Germany) using the protocol described by Avila-Novoa et al [24].…”