Phenotypic and Genotypic Characteristics of Non-Hemolytic L. monocytogenes Isolated from Food and Processing Environments

Abstract

Increasingly, Listeria monocytogenes (LM) with atypical phenotypic and genotypic characteristics are being isolated from food, causing problems with their classification and testing. From 2495 soil, food, and swab samples from the food industry, 262 LM isolates were found. A total of 30 isolates were isolated, mainly from soil and plant food, and were classified as atypical LM (aLM) because they lacked the ability to move (30/11.4%) and perform hemolysis (25/9.5%). The isolation environment affected aLM incidence, cell size, sugar fermentation capacity, antibiotic sensitivity, and the number of virulence genes. Therefore, despite several characteristics differentiating all aLMs/non-hemolytic isolates from reference LMs, the remaining phenotypic characteristics were specific to each aLM isolate (like a fingerprint). The aLM/non-hemolytic isolates, particularly those from the soil and meat industries, showed more variability in their sugar fermentation capacity and were less sensitive to antibiotics than LMs. As many as 11 (36.7%) aLM isolates had resistance to four different antibiotics or simultaneously to two antibiotics. The aLM isolates possessed 3-7 of the 12 virulence genes: prfA and hly in all aLMs, while iap was not present. Only five (16.7%) isolates were classified into serogroups 1/2c-3c or 4a-4c. The aLM/non-hemolytic isolates differed by many traits from L. immobilis and atypical L. innocua. The reference method of reviving and isolating LM required optimization of aLM. Statistical analyses of clustering, correlation, and PCA showed similarities and differences between LM and aLM/non-hemolytic isolates due to individual phenotypic traits and genes. Correlations were found between biochemical traits, antibiotic resistance, and virulence genes. The increase in the incidence of atypical non-hemolytic LM may pose a risk to humans, as they may not be detected by ISO methods and have greater antibiotic resistance than LM. aLM from LM can be distinguished based on lack of hemolysis, motility, growth at 4 °C, ability to ferment D-arabitol, and lack of six specific genes.

Keywords: antibiotic resistance; atypical Listeria monocytogenes; phenotyping; serotyping; sugar fermentation; virulence genes.

Figure 1

Dendrogram of reference LMs and atypical LMs, grouped by (A) selected morphological and biochemical characteristics, (B) ability to ferment sugars, (C) antibiotic resistance, and (D) presence of virulence genes. Groups of isolates highlighted in different colours on the dendrograms are indicated by the letters A–F.

Figure 2

PCA correlation between biochemical attributes evaluated for LM reference strains and aLM isolates.

Figure 3

PCA correlation between LM references and aLM isolates based on the analyzed phenotypic traits and genes. The groups LM references are indicated by A–C letters and groups aLM isolates by D–F letters.