Rapid serotype-independent differential detection of biofilm-positive and biofilm-negative Salmonella using Fourier transform infrared biotyping

Abstract

Foodborne illnesses caused by Salmonella represent a global one health challenge, with biofilm-forming strains exhibiting enhanced public health risks due to their ability to persist due to resistance to antimicrobial agents, disinfectants, and environmental stresses. While food-safety and public health investigation primarily focus on Salmonella identification and source tracing, they often overlook the biofilm-forming capacity of isolates, limiting their predictive value for risks posed by biofilm producing Salmonella. This study assessed fourier transform infrared (FTIR) biotyping for rapid serotype-independent differentiatial detection of biofilm-positive (BFP) from biofilm-negative (BFN) Salmonella. A total of 270 Salmonella strains representing 12 common serotypes were classified using three conventional biofilm assays (congo red and coomassie brilliant blue agar test, calcofluor test, and tube test) into true BFP (n = 80), true BFN (n = 64), and uncertain (n = 59) biofilm producers. Biofilm production for each group was also assessed with a microtiter plate assay. FTIR biotyping was applied to a subset of 115 strains (61 BFP, 54 BFN). Using spectral windows of 1180-1050 cm^-1 and 1400-1200 cm^-1, FTIR biotyping accurately differentiated BFP from BFN strains with 93.4 % sensitivity, 83.3 % specificity, and 88.6 % overall accuracy. FTIR biotyping differentiated 59 strains with uncertain biofilm status into BFN (n = 45) and BFP (n = 14). FTIR biotyping provides a rapid, sensitive and specific method for detection of biofilm-forming Salmonella strains. Incorporating FTIR biotyping for biofilm detection in current Salmonella surveillance and source-tracing protocols can enhance food safety risk assessments and improve Salmonella outbreak prevention.

Keywords: Biofilm; FTIR spectroscopy; IR Biotyper; Salmonella.

Fig. 1

Distribution of biofilm morphotypes among 270 Salmonella strains representing 12 serotypes based on three conventional assays. Representative images showing six distinct CRCBB colony morphotypes after 96 h of incubation at 25 ± 1 °C. Microscopic images were captured at 4× magnification under a light microscope. The calcofluor and tube test results for strains within each of the CRCBB morphotypes are included for comparison.

Fig. 2

Distribution of true biofilm-positive (a), true biofilm-negative (b), and uncertain strains (c) among 270 Salmonella strains representing 12 serotypes. N indicates total number of strains positive by specific tests in Fig. 2(a) and (c), whereas negative by specific tests in Fig. 2(b). True biofilm-positive (BFP) strains (n = 80) were identified as consistently producing biofilms in all three biofilm assays. In contrast, true biofilm-negative (BFN) strains (n = 64) consistently tested negative in all three tests. The strains (n = 126) with inconsistent results were identified as strains with uncertain (U) biofilm status (c).

Fig. 3

The median OD₄₉₀ values for true biofilm-negative (n = 64), uncertain (n = 126), and true biofilm-positive (n = 80) groups of Salmonella strains. Error bars represent the standard error of the mean (SEM). The differences in median OD₄₉₀ among the three biofilm class groups (true BFN, true BFP, and Uncertains) were statistically significant (P < 0.01, Kruskal-Wallis test, and pairwise rank sum test with Bonferroni correction).

Fig. 4

Dendrogram displaying the hierarchical clustering analysis results for 345 spectra of 115 true BFN (n = 54) and true BFP (n = 61) Salmonella strains representing 12 different serotypes from three measurements, using the wavenumber-window of 1180–1050 and 1400–1200 cm⁻¹ and correlation and unweighted pair group method with arithmetic mean (UPGMA) for clustering. The automatically calculated cutoff value is −0.154 and is indicated as a vertical blue line. The columns at the end represent the strain ID (first column), biofilm production status (second column with grey representing BFN and red representing BFP), and serotype of the strain (different colors represent different serotypes). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Dendrogram showing hierarchical clustering analysis of challenge set 1 consisting of ten Salmonella strains with uncertain biofilm status along with a training set of 15 Salmonella strains each representing true biofilm-positive and true biofilm-negative groups using correlation and UPGMA. The columns at the right of the cluster represent the strain ID (first column), biofilm production status (second column with grey representing BFN, red representing BFP, and sky blue representing uncertain status), and serotype of the strain (third column with different colors representing different serotypes). Using a consistent cut-off of 0.15, four strains with U biofilm status clustered within the BFP and six strains with U biofilm status clustered within the BFN cluster. Using a similar approach, five additional challengesets of 9 or 10 strains each with U biofilm status were tested (see supplementary file 2). The cluster distribution of these additional strains from each challenge set (b) and the distribution of CRCBB morphotypes among the challenge strain sets (c) are shown for comparison. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)