Biology subjects

Fagerlund, A.

Publications and source records attributed to Fagerlund, A..

5 recordsLinked to original sources

bioRxiv2023

Large-scale phenotypic and genomic characterization of Listeria monocytogenes susceptibility to quaternary ammonium compounds

Listeria monocytogenes is a significant concern for the food industry due to its ability to persist in the food processing environment. Decreased susceptibility to disinfectants is one of the factors that contribute to the persistence of L. monocytogenes. The objective of this study was to explore the diversity of L. monocytogenes susceptibility to quaternary ammonium compounds (QACs) using 1,671 L. monocytogenes isolates. This was used to determine the phenotype-genotype concordance and characterize genomes of the QAC sensitive and tolerant isolates for stress resistance, virulence and plasmid replicon genes. Distribution of QAC tolerance genes among 37,897 publicly available L. monocytogenes genomes were also examined. The minimum inhibitory concentration to QACs was determined by the broth microdilution method and non-sequenced isolates (n=1,244) were whole genome sequenced. Genotype-phenotype concordance was 99% for benzalkonium chloride, DDAC and a commercial QAC based sanitizer. Prevalence of QAC tolerance genes was 23% and 28% in our L. monocytogenes collection and in the global dataset, respectively. qacH was the most prevalent gene in our collection (61%), with 19% prevalence in the global dataset. Notably, bcrABC was most common (72%) globally, while 25% in our collection. Prevalence of emrC and emrE was comparable in both datasets, 7% and 2%, respectively. Replicon genes, indicative of plasmid harborage, were detected in 44% of the isolates and associated with the QAC tolerant phenotype. The presented analysis is based on the biggest L. monocytogenes collection in diversity and quantity for characterization of the L. monocytogenes QAC tolerance at both phenotypic and genomic levels. IMPORTANCEContamination of Listeria monocytogenes within the food processing environment is of concern to the food industry due to challenges in eradicating the pathogen once it becomes persistent in the environment. Genetic markers associated with increased tolerance to disinfectants have been identified, which alongside factors favor the persistence of L. monocytogenes in the production environment. By employing a comprehensive large-scale phenotypic testing and genomic analysis our study significantly enhances the understanding of the prevalence of quaternary ammonium compound (QAC) tolerant L. monocytogenes and the genetic determinants associated with the increased tolerance. Furthermore, we report on the prevalence of QAC tolerance genes among 37,897 publicly available L. monocytogenes sequences and their distribution within clonal complexes, isolation sources and geographical locations. As the propagation of QAC tolerance showed not be evenly distributed globally this highlights that understanding the development of L. monocytogenes disinfectant tolerance can be monitored using publicly available WGS data.

microbiology↗

bioRxiv2022

Pervasive Listeria monocytogenes are common in Norwegian food chains and associated with increased prevalence of stress survival and resistance determinants

To investigate the diversity, distribution, persistence, and prevalence of stress survival and resistance genes of Listeria monocytogenes clones dominating in food processing environments in Norway, genome sequences from 769 L. monocytogenes isolates from food industry environments, foods, and raw materials (of which 512 were sequenced in the present study) were subjected to wgMLST, SNP, and comparative genomic analyses. The dataset comprised isolates from nine meat and six salmon processing facilities in Norway collected over a period of three decades. The most prevalent clonal complex (CC) was CC121, found in ten factories, followed by CC7, CC8, and CC9, found in seven factories each. Overall, 72% of the isolates were classified as persistent, showing 20 or fewer wgMLST allelic differences towards an isolate found in the same factory in a different calendar year. Moreover, over half of the isolates (56%) showed this level of genetic similarity towards an isolate collected from a different food processing facility. These were designated as pervasive strains, defined as clusters with the same level of genetic similarity as persistent strains but isolated from different factories. The prevalence of genetic determinants associated with increased survival in food processing environments, including heavy metal and biocide resistance determinants, stress response genes and inlA truncation mutations, showed a highly significant increase among pervasive isolates, but not among persistent isolates. Furthermore, these genes were significantly more prevalent among the isolates from food processing environments compared to in isolates from natural and rural environments (n=218) and clinical isolates (n=111) from Norway. ImportanceListeria monocytogenes can persist in food processing environments for months to decades and spread through the food system by e.g., contaminated raw materials. Knowledge about the distribution and diversity of L. monocytogenes is of importance in outbreak investigations and essential to effectively track and control this pathogen in the food system. The current study presents a comprehensive overview of the prevalence of persistent clones and of the diversity of L. monocytogenes in Norwegian food processing facilities. The results demonstrate extensive spread of highly similar strains throughout the Norwegian food system, in that 56% of the 769 collected isolates from food processing factories belonged to clusters of L. monocytogenes identified in more than one facility. These strains were associated with an overall increase in the prevalence of plasmids and determinants of heavy metal and biocide resistance as well as other genetic elements associated with stress survival mechanisms and persistence.

microbiology↗

bioRxiv2021

High diversity in the regulatory region of Stx-converting bacteriophage genomes

Shiga toxin (Stx) is the major virulence factor of enterohemorrhagic Escherichia coli (EHEC), and the stx genes are carried by temperate bacteriophages (Stx phages). The switch between lysogenic and lytic life cycle of the phage, which is crucial for Stx production and for severity of the disease, is regulated by the CI repressor. CI maintain latency by preventing transcription of the replication proteins. Three EHEC phage replication units (Eru1-3) in addition to the classical lambdoid replication region have been described previously, and Stx phages carrying the Eru1 replication region were associated with highly virulent EHEC strains. In this study, we have classified the Eru replication region of 419 Stx phages. In addition to the lambdoid replication region and the three already described Erus, ten novel Erus (named Eru4 to Eru13) were detected. The lambdoid type, Eru1, Eru4 and Eru7 seem to be widely distributed in Western Europe. Notably, EHEC strains involved in severe outbreaks in England and Norway carry Stx phages with Eru1, Eru2, Eru5 and Eru7 replication regions. Phylogenetic analysis of CI repressors from Stx phages revealed eight major clades that largely separate according to Eru type. The classification of replication regions and CI proteins of Stx phages provides an important platform for further studies aimed to assess how characteristics of the replication region influence the regulation of phage life cycle and, consequently, the virulence potential of the host EHEC strain. IMPORTANCEEHEC is an emerging health challenge worldwide and outbreaks caused by this pathogen tend to be more frequent and severe. Increased knowledge on how characteristics of the replication region influence the virulence of E. coli may be used for more precise identification of high-risk EHEC strains.

microbiology↗

bioRxiv2021

WGS analysis of Listeria monocytogenes from rural, urban, and farm environments in Norway: Genetic diversity, persistence, and relation to clinical and food isolates

Listeria monocytogenes is a ubiquitous environmental bacterium associated with a wide variety of natural and man-made environments, such as soil, vegetation, livestock, food processing environments, and urban areas. It is also among the deadliest foodborne pathogens, and knowledge about its presence and diversity in potential sources is crucial to effectively track and control it in the food chain. Isolation of L. monocytogenes from various rural and urban environments showed higher prevalence in agricultural and urban developments than in forest or mountain areas, and that detection was positively associated with rainfall. Whole genome sequencing (WGS) was performed for the collected isolates and for L. monocytogenes from Norwegian dairy farms and slugs, in total 218 isolates. The data was compared with available datasets from clinical and food associated sources in Norway collected within the last decade. Multiple examples of clusters of isolates with 0-8 wgMLST allelic differences were collected over time in the same location, demonstrating persistence of L. monocytogenes in natural, urban and farm environments. Furthermore, several clusters with 6-20 wgMLST allelic differences containing isolates collected across different locations, times and habitats were identified, including nine clusters harbouring clinical isolates. The most ubiquitous clones found in soil and other natural and animal ecosystems (CC91, CC11, and CC37) were distinct from clones predominating among both clinical (CC7, CC121, CC1) and food (CC9, CC121, CC7, CC8) isolates. The analyses indicated that ST91 was more prevalent in Norway than other countries and revealed a high proportion of the hypovirulent ST121 among Norwegian clinical cases. ImportanceListeria monocytogenes is a deadly foodborne pathogen that is widespread in the environment. For effective management, both public health authorities and food producers need reliable tools for source tracking, surveillance, and risk assessment. For this, whole genome sequencing (WGS) is regarded as the present and future gold standard. In the current study, we use WGS to show that L. monocytogenes can persist for months and years in natural, urban and dairy farm environments. Notably, clusters of almost identical isolates, with genetic distances within the thresholds often suggested for defining an outbreak cluster, can be collected from geographically and temporally unrelated sources. The work highlights the need for a greater knowledge of the genetic relationships between clinical isolates and isolates of L. monocytogenes from a wide range of environments, including natural, urban, agricultural, livestock, food production, and food processing environments, in order to correctly interpret and use results from WGS analyses.

microbiology↗

bioRxiv2020

MogR is a ubiquitous transcriptional repressor affecting motility, biofilm formation and virulence in the Bacillus cereus group

Flagellar motility is considered an important virulence factor in different pathogenic bacteria. In Listeria monocytogenes the transcriptional repressor MogR regulates motility in a temperature-dependent manner, directly repressing flagellar- and chemotaxis genes. The only other bacteria known to carry a mogR homolog are members of the Bacillus cereus group, which includes motile species such as B. cereus and Bacillus thuringiensis as well as the non-motile species Bacillus anthracis, Bacillus mycoides and Bacillus pseudomycoides. Furthermore, the main motility locus in B. cereus group bacteria, carrying the genes for flagellar synthesis, appears to be more closely related to L. monocytogenes than to Bacillus subtilis, which belongs to a separate phylogenetic group of Bacilli and does not carry a mogR ortholog. Here, we show that in B. thuringiensis, MogR overexpression results in non-motile cells devoid of flagella. Global gene expression profiling showed that 110 genes were differentially regulated by MogR overexpression, including flagellar motility genes, but also genes associated with virulence, stress response and biofilm lifestyle. Accordingly, phenotypic assays showed that MogR also affects cytotoxicity and biofilm formation in B. thuringiensis. Overexpression of a MogR variant mutated in two amino acids within the putative DNA binding domain restored phenotypes to those of an empty vector control. In accordance, introduction of these mutations resulted in complete loss in MogR binding to its candidate flagellar locus target site in vitro. In contrast to L. monocytogenes, MogR appears to be regulated in a growth-phase dependent and temperature-independent manner in B. thuringiensis 407. Interestingly, mogR was found to be conserved also in non-motile B. cereus group species such as B. mycoides and B. pseudomycoides, which both carry major gene deletions in the flagellar motility locus and where in B. pseudomycoides mogR is the only gene retained. Furthermore, mogR is expressed in non-motile B. anthracis. Altogether this provides indications of an expanded set of functions for MogR in B. cereus group species, beyond motility regulation. In conclusion, MogR constitutes a novel B. thuringiensis pleiotropic transcriptional regulator, acting as a repressor of motility genes, and affecting the expression of a variety of additional genes involved in biofilm formation and virulence.

microbiology↗