A novel metagenomic approach uncovers phage genes as markers for increased disinfectant tolerance in mixed Listeria monocytogenes communities.

Listeria monocytogenes is an important human pathogen with a high mortality rate. Consumption of contaminated ready-to-eat food is the main mode of transmission to humans. Disinfectant-tolerant L. monocytogenes have emerged, which are believed to have increased persistence potential. Elucidating the mechanisms of L. monocytogenes disinfectant tolerance has been the focus of previous studies using pure cultures. A limitation of such approach is the difficulty to identify strains with reduced susceptibility due to inter-strain variation and the need to screen large numbers of strains and genes. In this study, we applied a novel metagenomic approach to detect genes associated with disinfectant tolerance in mixed L. monocytogenes planktonic communities. Two communities, consisting of 71 and 80 isolates each, were treated with the food industry disinfectants benzalkonium chloride (BC, 1.75 mg/L) or peracetic acid (PAA, 38 mg/L). The communities were subjected to metagenomic sequencing and differences in individual gene abundances between biocide-free control communities and biocide-treated communities were determined. A significant increase in the abundance of Listeria phage-associated genes was observed in both communities after treatment, suggesting that prophage carriage could lead to an increased disinfectant tolerance in mixed L. monocytogenes planktonic communities. In contrast, a significant decrease in the abundance of a high-copy emrC-harbouring plasmid pLmN12-0935 was observed in both communities after treatment. In PAA-treated community, a putative ABC transporter previously found to be necessary for L. monocytogenes resistance to antimicrobial agents and virulence, was among the genes with the highest weight for differentiating treated from control samples. The undertaken metagenomic approach in this study can be applied to identify genes associated with increased tolerance to other antimicrobials in mixed bacterial communities.

Draft genome sequences of a historical collection of Listeria monocytogenes from humans and other sources, 1926-1964.

Listeria monocytogenes can persistently contaminate food processing environments and tolerate sanitizers. Most sequenced strains are from clinical and environmental sources in the contemporary era, with relatively few prior to extensive food processing and sanitizer use. We report the genome sequences of a diverse panel of 83 strains from 1926 to 1964.

Draft Genome Sequences of 158 Listeria monocytogenes Strains Isolated from Black Bears (Ursus americanus) in the United States.

Listeria monocytogenes is responsible for severe foodborne disease and major economic losses, but its potential reservoirs in natural ecosystems remain poorly understood. Here, we report the draft genome sequences of 158 L. monocytogenes strains isolated from black bears (Ursus americanus) in the southeastern United States between 2014 and 2017.

Draft Genome Sequences of Closely Related Listeria monocytogenes Lineage III Strains Isolated from a Food Processing Environment and a Case of Human Listeriosis.

Listeria monocytogenes lineage III is genetically highly diverse, and closely related lineage III strains from food facilities and human listeriosis have not been reported. Here, we report the genome sequences of three closely related lineage III strains from Hawaii, namely, one isolated from a human case and two isolated from a produce storage facility.

Investigation of a Listeria monocytogenes Chromosomal Immigration Control Region Reveals Diverse Restriction Modification Systems with Complete Sequence Type Conservation.

Listeria monocytogenes is a Gram-positive pathogen responsible for the severe foodborne disease listeriosis. A chromosomal hotspot between lmo0301 and lmo0305 has been noted to harbor diverse restriction modification (RM) systems. Here, we analyzed 872 L. monocytogenes genomes to better understand the prevalence and types of RM systems in this region, designated the immigration control region (ICR). Type I, II, III and IV RM systems were found in 86.1% of strains inside the ICR and in 22.5% of strains flanking the ICR. ICR content was completely conserved within the same multilocus sequence typing-based sequence type (ST), but the same RM system could be identified in diverse STs. The intra-ST conservation of ICR content suggests that this region may drive the emergence of new STs and promote clone stability. Sau3AI-like, LmoJ2 and LmoJ3 type II RM systems as well as type I EcoKI-like, and type IV AspBHI-like and mcrB-like systems accounted for all RM systems in the ICR. A Sau3AI-like type II RM system with specificity for GATC was harbored in the ICR of many STs, including all strains of the ancient, ubiquitous ST1. The extreme paucity of GATC recognition sites in lytic phages may reflect ancient adaptation of these phages to preempt resistance associated with the widely distributed Sau3AI-like systems. These findings indicate that the ICR has a high propensity for RM systems which are intraclonaly conserved and may impact bacteriophage susceptibility as well as ST emergence and stability.

Horizontal Gene Transfer and Loss of Serotype-Specific Genes in Listeria monocytogenes Can Lead to Incorrect Serotype Designations with a Commonly-Employed Molecular Serotyping Scheme.

Listeria monocytogenes is a Gram-positive, facultative intracellular foodborne pathogen capable of causing severe, invasive illness (listeriosis). Three serotypes, 1/2a, 1/2b, and 4b, are leading contributors to human listeriosis, with 4b including the major hypervirulent clones. The multiplex PCR scheme developed by Doumith and collaborators employs primers targeting specific lineages (e.g., lineage II-specific lmo0737, lineage I-specific LMOf2365_2059) or serotypes (e.g., serotype 4b-specific LMOf2365_1900). The Doumith scheme (DS) is extensively employed for molecular serotyping of L. monocytogenes due to its high accuracy, relative ease, and affordability. However, for certain strains, the DS serotype designations are in conflict with those relying on antibody-based schemes or whole-genome sequence (WGS) analysis. In the current study, all 27 tested serotype 4b strains with sequence type 782 (ST782) within the hypervirulent clonal complex 2 (CC2) were designated 1/2b/3b using the DS. These strains lacked the serotype 4b-specific gene LMOf2365_1900, while retaining LMOf2365_2059, which, together with prs, yields the DS 1/2b/3b profile. Furthermore, 15 serotype 1/2a strains of four STs, mostly from water, were designated 1/2b/3b using the DS. These strains lacked the lmo0737 cassette but harbored genomic islands with LMOf2365_2059, thus yielding the DS 1/2b/3b profile. Lastly, we investigated a novel, dual 1/2a-1/2b profile obtained using the DS with 21 serotype 1/2a strains of four STs harboring both the lmo0737 cassette and genomic islands with LMOf2365_2059. The findings suggest that for certain strains and clones of L. monocytogenes the DS designations should be viewed with caution and complemented with alternative tools, e.g., traditional serotyping or WGS analysis. IMPORTANCE Listeria monocytogenes is a foodborne pathogen responsible for severe illness (listeriosis), especially in pregnant women and their fetuses, immunocompromised individuals, and the elderly. Three serotypes, 1/2a, 1/2b, and 4b, account for most human listeriosis, with certain serotype 4b clonal complexes (CCs) overrepresented in human disease. Serotyping remains extensively employed in Listeria epidemiologic investigations, and a multiplex PCR-based serotyping scheme is widely used. However, the PCR gene targets can be lost or gained via horizontal gene transfer, leading to novel PCR profiles without known serotype designations or to incorrect serotype assignments. Thus, an entire serotype 4b clone of the hypervirulent CC2 would be misidentified as serotype 1/2b, and several strains of serotype 1/2a would be identified as serotype 1/2b. Such challenges are especially common in novel clones from underexplored habitats, e.g., wildlife and surface water. The findings suggest caution in application of molecular serotyping, while highlighting Listeria's diversity and potential for horizontal gene transfer.

Draft Genome Assemblies of Two Campylobacter novaezeelandiae and Four Unclassified Thermophilic Campylobacter Isolates from Canadian Agricultural Surface Water.

This report presents the draft genome sequences of two Campylobacter novaezeelandiae and four unclassified Campylobacter isolates from Canadian agricultural surface water. Phylogenomic analysis revealed that the six isolates formed unique clades, closely related to the disease-causing species C. jejuni, C. coli, and C. hepaticus.

Chemical composition and antioxidant activity of ultrasound-assisted extract of the endemic plant Haberlea rhodopensis Friv.

The antioxidant capacity of extracts from leaves of Haberlea rhodopensis Friv. obtained by a highly efficient and simple method of the ultrasound-assisted extraction was investigated. The total polyphenolic content was determined spectrophotometrically using the Folin - Ciocalteu's phenol reagent and the polyphenols composition was analyzed by HPLC method. Antioxidant activity of the extract was evaluated as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2-azinobis-3 ethyl benxothiazoline-6-sulphonic acid (ABTS) cation decolorization activity assays and the ferric reducing/antioxidant power method (FRAP). The total phenolics of the extract were determined to be 148.71 ± 5.33 mg GAE on a dry weight basis. The HPLC analysis showed a large number of flavonoids and phenolic acids present in the ultrasound ethanol extract of H. rhodopensis. Results also revealed that the major flavonoids were luteolin (2730.18 µg/g DW), hesperidin (928.56 µg/g DW) and kaempferol (578.52 µg/g DW), whereas the most abundant phenolic acids identified in the extract were ferulic acid (630.48 µg/g DW) and sinapic acid (580.80 µg/g DW). DPPH(•) and ABTS(•+) values were 0.803 ± 0.007 and 0.769 ± 0.040 mmol TE/g DW, respectively and FRAP values were 1.362 ± 0.05 mmol TE/g DW.

Rapid identification of Campylobacter jejuni from poultry carcasses and slaughtering environment samples by real-time PCR.

The objective of this study was to develop a real-time PCR assay for rapid identification of Campylobacter jejuni and to apply the method in analyzing samples from poultry processing. A C. jejuni-specific primer set targeting a portion of the C. jejuni hippuricase gene was developed. The specificity of the newly designed primer pair was verified using 5 C. jejuni strains and 20 other bacterial strains. Sensitivity was determined to be as low as 1 genome copy per reaction. A total of 73 samples were collected at different sites along the processing line during 2 visits to a poultry slaughterhouse and were examined by direct plating onto modified charcoal cefoperazone deoxycholate agar or after enrichment in Bolton broth followed by plating on modified charcoal cefoperazone deoxycholate agar. The newly developed real-time PCR assay was used to identify the presumptive colonies as belonging to C. jejuni. A real-time PCR assay targeting 16S ribosomal RNA was also applied to determine Campylobacter spp. prevalence. Results from the real-time PCR analysis indicated considerable variability in Campylobacter contamination, with incidence rates of 72.7 and 27.6% for sampling days A and B, respectively. Campylobacter was isolated from 100% of prescalded and preeviscerated carcasses on sampling day A. In contrast, on sampling day B, the highest number of Campylobacter-positive carcasses was recovered after evisceration (60%). The chilling process significantly reduced (P < 0.05) Campylobacter population, but the percentage of positive samples on sampling day A increased to 80%. All samples collected from the processing environment, except scalding tank 3 and the prechiller and chiller tanks, were 100% positive on day A, whereas no campylobacters were isolated from machinery on sampling day B. Our results revealed the widespread of C. jejuni in poultry processing and proved that the newly developed real-time PCR assay is a simple, specific, and inexpensive method for rapid C. jejuni identification. The newly developed PCR method can be easily used in laboratories for reliable and unambiguous identification of C. jejuni in poultry samples.