Molecular characterization of Salmonella spp. and Listeria monocytogenes strains from biofilms in cattle and poultry slaughterhouses located in the federal District and State of Goiás, Brazil

Abstract

Listeria monocytogenes and Salmonella spp. are considered important foodborne pathogens that are commonly associated with foods of animal origin. The aim of this study was to perform molecular characterization of L. monocytogenes and Salmonella spp. isolated from biofilms of cattle and poultry slaughterhouses located in the Federal District and State of Goiás, Brazil. Fourteen L. monocytogenes isolates and one Salmonella sp. were detected in poultry slaughterhouses. No isolates were detected in cattle slaughterhouses. All L. monocytogenes isolates belonged to lineage II, and 11 different pulsotypes were detected. Pulsed-field gel electrophoresis analysis revealed the dissemination of two strains within one plant, in addition to the regional dissemination of one of them. The Salmonella isolate was identified via whole genome sequencing as Salmonella enterica serovar Minnesota ST548. In the sequence analysis, no premature stop codons were detected in the inlA gene of Listeria. All isolates demonstrated the ability to adhere to Caco-2 cells, while 50% were capable of invading them. Antimicrobial resistance was detected in 57.1% of the L. monocytogenes isolates, and resistance to sulfonamide was the most common feature. The tetC, ermB, and tetM genes were detected, and four isolates were classified as multidrug-resistant. Salmonella sp. was resistant to nine antimicrobials and was classified as multidrug-resistant. Resistance genes qnrB19, blaCMY-2, aac(6')-Iaa, sul2, and tetA, and a mutation in the parC gene were detected. The majority (78.5%) of the L. monocytogenes isolates were capable of forming biofilms after incubation at 37°C for 24 h, and 64.3% were capable of forming biofilms after incubation at 12°C for 168 h. There was no statistical difference in the biofilm-forming capacity under the different evaluated conditions. Salmonella sp. was capable of forming biofilms at both tested temperatures. Biofilm characterization was confirmed by collecting the samples consistently, at the same sampling points, and by assessing biofilm formation in vitro. These results highlight the potential risk of cross-contamination in poultry slaughterhouses and the importance of surveillance and pathogen control maintenance programs within the meat production industry.

Fig 1. Dendrogram and PFGE patterns of 14 isolates of Listeria monocytogenes restricted with AscI.

The data were analyzed using the BioNumerics software. Discrimination of pulsotypes, clusters, origins of isolates (Federal District, DF; or Goiás, GO), establishment of origin (A, B or C), and isolate identification are presented.

Fig 2

Sample collection points of the 11 L. monocytogenes pulsotypes detected in poultry slaughterhouses A, B and C located in Federal District and Goiás. The numbers in parenthesis represent the number of isolates belonging to each pulsotype, totaling 14 isolates.

Fig 3. Results of invasion and cell adhesion tests using Caco-2 cells for 14 Listeria monocytogenes isolates.

The values shown represent the average of the results, and the bars represent the standard deviation.

Fig 4. Results of the in vitro biofilm formation capacity test performed using polystyrene microplates (Djordjevic et al., 2002) and 12 Listeria monocytogenes isolates exhibiting biofilm formation capacity at 37°C and/or 12°C.

The bars represent the average value of the optical density of each test for each isolate (ODi), all performed in triplicate, subtracted from the average of the optical density of the negative control for each repetition (ODn).