Prevalence and antimicrobial resistance profile of Listeria spp. isolated from raw fish

Abstract

Background: Listeria spp. contamination in food, particularly antimicrobial-resistant strains, poses an escalating concern for public health. This study investigated the prevalence and antimicrobial resistance profiles of Listeria spp. isolated from raw fish collected from diverse sources in Northern Poland.

Methods: A total of 750 raw fish samples were collected from standing freshwater tanks, flowing freshwater reservoirs, and a saltwater reservoir. Isolation was performed following the standard protocol, which describes the horizontal method for detecting Listeria spp., including Listeria monocytogenes. The antimicrobial resistance profiles of isolated strains were performed using the disk diffusion method. Antimicrobial resistance genes were identified using PCR, targeting 11 genes associated with resistance to β-lactams, macrolides, glycopeptides, and sulfonamides. Statistical analyses included Phi correlation coefficients, hierarchical clustering, and logistic regression to examine associations between phenotypic resistance patterns and antimicrobial resistance genes.

Results: Listeria spp. was isolated from 13.9% of samples (104 positive samples), comprising L. seeligeri (34.6%), L. welshimeri (28.8%), L. monocytogenes (23.1%), and L. innocua (13.5%). Phenotypic antimicrobial susceptibility testing revealed universal resistance to oxacillin (100%) across all Listeria spp. isolates. High resistance levels were also observed for cefotaxime (97.1%), cefoxitin (92.3%), rifampicin (92.3%), clindamycin (96.2%), and trimethoprim-sulfamethoxazole (91.3%). Alarmingly, 98.1% of all Listeria spp. isolates exhibited multidrug resistance (MDR), reaching 100% MDR among L. monocytogenes isolates. Specifically, L. monocytogenes isolates exhibited complete resistance to meropenem, cefoxitin, cefotaxime, rifampicin, and trimethoprim-sulfamethoxazole, and significant resistance to ciprofloxacin (91.7%), clindamycin (83.3%), tetracycline (75.0%), erythromycin (75.0%), benzylpenicillin (70.8%), and nitrofurantoin (70.8%). Molecular analysis identified blaTEM (100%), ampC (37.5%), and ereB (37.5%) as the most prevalent antimicrobial resistance genes in L. monocytogenes.

Conclusions: The exceptionally high prevalence of multidrug-resistant Listeria spp., particularly L. monocytogenes, in raw fish underscores a critical public health risk, suggesting the urgent need for ongoing surveillance and robust risk mitigation strategies in aquaculture and seafood processing. The elevated antimicrobial resistance levels may also indicate aquatic environmental contamination, warranting further investigation into the sources and broader ecological implications of antimicrobial resistance in these ecosystems.

Keywords: Listeria monocytogenes; Aquaculture; Multidrug resistance; Raw fish; Seafood safety.

Fig. 1

Distribution of Listeria spp. isolates by sample origin and species. Figure provides a overview of the distribution of Listeria spp. isolates obtained from raw fish samples across different aquatic environments and fish types. (A) A donut chart illustrates the distribution of the 750 raw fish samples collected, with the inner ring representing the aquatic environment: saltwater, flowing freshwater, standing freshwater, and the outer ring segments further detailing the types of fish sampled within each environment: crucian carp, herring, sturgeon, and trout. The sample size (n) for each environment and fish type is indicated within the chart segments, alongside representative images of each fish species. (B) A bar chart presents the total counts of each Listeria spp. isolated (L. innocua, L. monocytogenes, L. seeligeri, L. welshimeri) and a summary category for the total number Listeria spp., NAD- Not Assigned to Listeria spp. Detection (C) A stacked bar chart details the distribution of Listeria spp. across the four fish types: crucian carp, herring, sturgeon, and trout, displayed on the x-axis with representative images. The y-axis indicates the number of samples, with color-coded stacked bars representing the count of each Listeria spp. isolated from each fish type, as detailed in the legend. (D) Another stacked bar chart shows the distribution of Listeria spp. across the three aquatic environments: flowing freshwater, saltwater, and standing freshwater. The y-axis denotes the number of samples, with stacked, color-coded bars indicating the counts of each Listeria spp. isolated from each environment, as specified in the legend

Fig. 2

Phenotypic antimicrobial resistance distribution and correlation analyses of Listeria spp. isolates from raw fish. (A) A dendrogram on the left illustrates the hierarchical clustering of antimicrobials based on the resistance profiles observed across the Listeria spp. isolates. The clustering, performed using Euclidean distance and Ward’s linkage, groups antimicrobials with similar resistance patterns. (B) A horizontal stacked bar chart on the right displays the antibiotic resistance distribution for each of the 17 tested antimicrobials. Each bar is segmented and color-coded to show the proportion of isolates classified as Resistant (red), Intermediate (yellow), and Susceptible (green) according to EUCAST and CLSI breakpoints [16, 17]. The percentage values for each category are indicated within the bar segments. (C) A heatmap at the bottom left presents the Phi correlation matrix for antimicrobial resistance profiles. (D) A heatmap at the bottom right shows the Phi correlation matrix for antimicrobial resistance (antimicrobials listed on the y-axis) versus the presence of antimicrobial resistance genes (genes listed on the x-axis)

Fig. 3

Multidrug Resistance (MDR) stratification across Listeria spp. isolates from raw fish. Figure presents a stratified analysis of MDR among Listeria spp. isolates, categorized by species and overall MDR distribution, where multidrug resistance is defined as resistance to at least one antimicrobial drug in more than three antimicrobial classes. (A) Total MDR stratification across all Listeria spp. isolates. This panel displays a histogram overlaid with a Kernel Density Estimate (KDE) curve, providing an overview of the overall distribution of MDR levels in the entire dataset and summarizing MDR prevalence across all Listeria isolates. (B) MDR stratification for L. innocua isolates. (C) MDR stratification for L. monocytogenes isolates. (D) MDR stratification for L. seeligeri isolates. (E) MDR stratification for L. welshimeri isolates. In all subplots, the x-axis represents the number of antimicrobial groups to which isolates are resistant, and the y-axis indicates the percentage of samples at each resistance level. Each bar in the histograms is annotated with the percentage and count (n) of isolates at each resistance level. Different letters above the graphs indicate significantly different MDR distribution patterns among Listeria species (p < 0.05), as determined by post hoc chi-square analysis