Monocytogenes Serotype 4b Strains Research Articles

Transcriptomic Response of L. monocytogenes to Co-Culture with S. cerevisiae

The aim of the present study was to assess the transcriptomic response of L. monocytogenes during co-culture with three S. cerevisiae strains. For this purpose, BHI broth was inoculated with 7 log CFU·mL−1 L. monocytogenes serotype 4b strain LQC 15257, isolated from a strawberry sample and 4 log CFU·mL−1 S. cerevisiae strains Y32, Y34 and Y37, isolated from spontaneous olive fermentation. Sampling took place after 24 and 48 h incubation at 5 and 20 °C. RNA was extracted, stabilized and the transcription of virulence associated genes prfA, sigB, hly, plcA, plcB, inlA, inlB, inlC and inlJ, was assessed by RT-qPCR. Co-culture with the yeast strains mostly affected the transcription of sigB and inlJ, the upregulation of which during growth at 5 °C for 24 h, reached 10.13 and 9.76 log2(fold change), respectively. Similarly, the effect that incubation time had on the relative transcription of the genes under study was dependent on the co-cultivating yeast strain. On the other hand, the effect of the yeast strain was less pronounced when the relative transcription of the genes under study was assessed between 20 °C and 5 °C. In that case, incubation temperature seemed to have an important effect since, in the 79.2% of the samples analyzed, upregulation was evident, irrespective of yeast strain presence. These results highlight the complex trophic relationships that take place during co-existence between L. monocytogenes and S. cerevisiae.

Characterization of a Novel Group of Listeria Phages That Target Serotype 4b Listeria monocytogenes

Listeria monocytogenes serotype 4b strains are the most prevalent clinical isolates and are widely found in food processing environments. Bacteriophages are natural viral predators of bacteria and are a promising biocontrol agent for L. monocytogenes. The aims of this study were to characterize phages that specifically infect serotype 4b strains and to assess their ability to inhibit the growth of serotype 4b strains. Out of 120 wild Listeria phages, nine phages were selected based on their strong lytic activity against the model serotype 4b strain F2365. These nine phages can be divided into two groups based on their morphological characteristics and host range. Comparison to previously characterized phage genomes revealed one of these groups qualifies to be defined as a novel species. Phages LP-020, LP-027, and LP-094 were selected as representatives of these two groups of phages for further characterization through one-step growth curve and inhibition of serotype 4b L. monocytogenes experiments. Listeria phages that target serotype 4b showed an inhibitory effect on the growth of F2365 and other serotype 4 strains and may be useful for biocontrol of L.monocytogenes in food processing environments.

A Comprehensive Evaluation of the Genetic Relatedness of Listeria monocytogenes Serotype 4b Variant Strains.

Recently, we have identified a link between four listeriosis incidents/outbreaks to a variant of Listeria monocytogenes (Lm) serotype 4b strains, 4bV. Although 4bV strains have been reported from clinical specimens as well as from foods, listeriosis outbreaks occurring in 2014–2016 were the first reported outbreaks involving 4bV in the USA. Since traditional typing methods do not detect members of this group, we undertook a systematic and retrospective analysis of all Lm in the NCBI WGS Sequence Read Archive database to investigate the burden of 4bV strains among all listeriosis cases. This analysis identified the presence of isolates causing sporadic cases as well as those associated with the aforementioned outbreaks, as determined by WGS and traditional epidemiology. In total, approximately 350 Lm 4bV strains were identified from multiple parts of the USA as well as from Australia and Chile, dating back to 2001. The genomic relatedness of these strains was compared using the CFSAN SNP Pipeline and multi-virulence-locus sequence typing (MVLST). Using the CFSAN Pipeline tool, the 4bV strains were found to group into seven clusters that were separate from 4b strains. All seven clades appeared to contain isolates from both clinical and non-clinical sources. Conversely, the MVLST analysis revealed that practically all of the strains belonged to a single clade, suggesting that 4bV strains from disparate geographic regions and sources are under varied selective pressure, restricting diversity across these six virulence loci while allowing more variability across the genome as a whole. Further evaluation of these 4bV strains identified genes potentially acquired from a lineage II source external to the lmo0733–lmo0739 region, as well as highly conserved SNPs unique to the 4bV strains when compared to those from other lineages. Taken together, these data suggest that 4bV strains have undergone adaptive responses to selective pressures that may enhance survival in the environment while maintaining the pathogenic potential of serotype 4b strains.

Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools.

The Gram-positive bacterium Listeria monocytogenes causes a significant percentage of the fatalities among foodborne illnesses in humans. Surface proteins specifically expressed in a wide range of L. monocytogenes serotypes under selective enrichment culture conditions could serve as potential biomarkers for detection and isolation of this pathogen via antibody-based methods. Our study aimed to identify such biomarkers. Interrogation of the L. monocytogenes serotype 4b strain F2365 genome identified 130 putative or known surface proteins. The homologues of four surface proteins, LMOf2365_0578, LMOf2365_0581, LMOf2365_0639, and LMOf2365_2117, were assessed as biomarkers due to the presence of conserved regions among strains of L. monocytogenes which are variable among other Listeria species. Rabbit polyclonal antibodies against the four recombinant proteins revealed the expression of only LMOf2365_0639 on the surface of serotype 4b strain LI0521 cells despite PCR detection of mRNA transcripts for all four proteins in the organism. Three of 35 monoclonal antibodies (MAbs) to LMOf2365_0639, MAbs M3643, M3644, and M3651, specifically recognized 42 (91.3%) of 46 L. monocytogenes lineage I and II isolates grown in nonselective brain heart infusion medium. While M3644 and M3651 reacted with 14 to 15 (82.4 to 88.2%) of 17 L. monocytogenes lineage I and II isolates, M3643 reacted with 22 (91.7%) of 24 lineage I, II, and III isolates grown in selective enrichment media (UVM1, modified Fraser, Palcam, and UVM2 media). The three MAbs exhibited only weak reactivities (the optical densities at 414 nm were close to the cutoff value) to some other Listeria species grown in selective enrichment media. Collectively, the data indicate the potential of LMOf2365_0639 as a surface biomarker of L. monocytogenes, with the aid of specific MAbs, for pathogen detection, identification, and isolation in clinical, environmental, and food samples. L. monocytogenes is traditionally divided into at least 12 serotypes. Currently, there are no monoclonal antibodies (MAbs) available that are capable of binding to the surface of L. monocytogenes strains representing all 12 serotypes. Such antibodies would be useful and are needed for the development of methods to detect and isolate L. monocytogenes from food samples. In our study, we aimed to identify surface proteins that possess regions of well-conserved amino acid sequences among various serotypes and then to employ them as antigen targets (biomarkers) for the development of MAbs. Through bioinformatics and protein expression analysis, we identified one of the four putative surface protein candidates, LMOf2365_0639, encoded by the genome of the L. monocytogenes serotype 4b strain F2365, as a useful surface biomarker. Extensive assessment of 35 MAbs raised against LMOf2365_0639 in our study revealed three MAbs (M3643, M3644, and M3651) that recognized a wide range of L. monocytogenes isolates.

Draft Genome Sequences of Listeria monocytogenes Serotype 4b Strains 944 and 2993 and Serotype 1/2c Strains 198 and 2932.

Listeria monocytogenes is a foodborne pathogen and the causative agent of listeriosis among humans and animals. The draft genome sequences of L. monocytogenes serotype 4b strains 944 and 2993 and serotype 1/2c strains 198 and 2932 are reported here.

Virulence Gene-Associated Mutant Bacterial Colonies Generate Differentiating Two-Dimensional Laser Scatter Fingerprints.

In this study, we investigated whether a laser scatterometer designated BARDOT (bacterial rapid detection using optical scattering technology) could be used to directly screen colonies of Listeria monocytogenes, a model pathogen, with mutations in several known virulence genes, including the genes encoding Listeria adhesion protein (LAP; lap mutant), internalin A (ΔinlA strain), and an accessory secretory protein (ΔsecA2 strain). Here we show that the scatter patterns of lap mutant, ΔinlA, and ΔsecA2 colonies were markedly different from that of the wild type (WT), with >95% positive predictive values (PPVs), whereas for the complemented mutant strains, scatter patterns were restored to that of the WT. The scatter image library successfully distinguished the lap mutant and ΔinlA mutant strains from the WT in mixed-culture experiments, including a coinfection study using the Caco-2 cell line. Among the biophysical parameters examined, the colony height and optical density did not reveal any discernible differences between the mutant and WT strains. We also found that differential LAP expression in L. monocytogenes serotype 4b strains also affected the scatter patterns of the colonies. The results from this study suggest that BARDOT can be used to screen and enumerate mutant strains separately from the WT based on differential colony scatter patterns. In studies of microbial pathogenesis, virulence-encoding genes are routinely disrupted by deletion or insertion to create mutant strains. Screening of mutant strains is an arduous process involving plating on selective growth media, replica plating, colony hybridization, DNA isolation, and PCR or immunoassays. We applied a noninvasive laser scatterometer to differentiate mutant bacterial colonies from WT colonies based on forward optical scatter patterns. This study demonstrates that BARDOT can be used as a novel, label-free, real-time tool to aid researchers in screening virulence gene-associated mutant colonies during microbial pathogenesis, coinfection, and genetic manipulation studies.

A novel suicide plasmid for efficient gene mutation in Listeria monocytogenes

Although several plasmids have been used in Listeria monocytogenes for generating mutants by allelic exchange, construction of L. monocytogenes mutants has been inefficient due to lack of effective selection markers for first and second recombination events. To address this problem, we have developed a new suicide plasmid, pHoss1, by using the pMAD plasmid backbone and anhydrotetracycline selection marker (secY antisense RNA) driven by an inducible Pxyl/tetO promoter. Expression of the secY antisense RNA eliminates merodiploids and selects for the loss of plasmid via a second allelic exchange, which enriches the number of mutants with deleted genes. To assess the effectiveness of pHoss1 for the generation of stable in-frame deletion mutations, we deleted the ispG and ispH genes of L. monocytogenes serotype 4b strain F2365. Results showed that identification of the second allelic exchange mutants was very efficient with 80–100% of the colonies yielding desired deletion mutants. L. monocytogenes' intestinal cell attachment was not altered when ispG and ispH genes were deleted. We expect that this new plasmid will be very useful for construction of marker-free deletion mutants in L. monocytogenes and in other Gram-positive bacteria, including Staphylococcus aureus and Bacillus cereus.

Genetic classification of Listeria monocytogenes serotype 4b strains, including epidemic clones, isolated from retail meat in the Tokyo metropolitan area.

A food-borne pathogen, Listeria monocytogenes serotype 4b, has been frequently isolated from patients with listeriosis, and numerous outbreaks of listeriosis are associated with this serotype. In the present study, we performed subtyping of L. monocytogenes serotype 4b strains on the basis of genetic analyses. Thirty-four isolates of serotype 4b were classified into 8 genotypes, namely genotypes 12, 15, 16, 17, 18, 23, 24, and 25, on the basis of the sequence for the partial iap gene. Genetic analyses revealed that genotype 16 and genotypes 24 and 25 belong to epidemic clone I (ECI) and ECII, respectively, which have been frequently associated with listeriosis outbreaks in the United States and Europe. The genotype isolated most frequently from retail meats in the Tokyo metropolitan area was genotype 12 (52%), followed by genotype 16 (29%), which belongs to ECI. We suggest that ECI is a common subtype of L. monocytogenes in retail meat in the area under investigation. On the other hand, ECII isolates were confirmed to be present in retail meat in Japan but were rare.

SecA2 is not required for secretion of the surface autolysin IspC in Listeria monocytogenes serotype 4b

Listeria monocytogenes is one of several Gram-positive bacteria known to contain an auxiliary ATPase (SecA2) involved in the Sec secretion of a subset of proteins important to bacterial pathogenesis, including autolysins. It is not known if IspC, a novel surface-associated autolysin essential for full virulence of L. monocytogenes serotype 4b, is SecA2-dependent for secretion. By creating a secA2 gene deletion (ΔsecA2) mutant from the wild type (WT) L. monocytogenes serotype 4b strain, in combination with the proteomic analysis of surface proteins and those secreted into the medium from both the mutant and the WT, we confirmed previous findings that two autolysins (p60 and NamA) are SecA2-dependent for secretion. However, this approach did not identify IspC as one of the surface proteins affected by the SecA2 deletion. Further experiments with immunofluorescence microscopy and Western blotting indicated that IspC was well displayed on the surface of both the ΔsecA2 mutant and WT cells, while p60 was not, clearly indicating that the secretion of IspC is not attributed to the SecA2 pathway. This finding sets IspC apart from other autolysins involved in virulence, such as p60 and NamA, in that SecA2 is not required for IspC secretion.

The Listeria monocytogenes serotype 4b autolysin IspC has N-acetylglucosaminidase activity

IspC is a novel peptidoglycan (PG) hydrolase that is conserved in Listeria monocytogenes serotype 4b strains and is involved in virulence. The aim of this study was to establish the hydrolytic bond specificity of IspC. Purified L. monocytogenes peptidoglycan was digested by recombinant IspC and the resulting muropeptides were separated by reverse phase high-performance liquid chromatography. The structure of each muropeptide was determined using matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry in combination with MALDI-post-source decay mass spectrometry. The structure of muropeptides resulting from IspC-mediated hydrolysis indicated that IspC has N-acetylglucosaminidase activity. These muropeptides also had a high proportion of N-acetylated glucosamine residues. To determine whether IspC is more effective at hydrolysing N-acetylated peptidoglycan than de-N-acetylated peptidoglycan, a peptidoglycan deacetylase (PgdA) in-frame deletion mutant was created. This mutant was shown to have fully N-acetylated peptidoglycan and was more susceptible to hydrolysis by IspC when compared with the partially de-N-acetylated wild-type peptidoglycan. This indicates that IspC is more efficient when hydrolysing a fully N-acetylated peptidoglycan substrate. The finding that IspC acts as an N-acetylglucosaminidase is consistent with its categorization, based on amino acid sequence, as a member of the GH73 family. As with other members of this family, de-N-acetylation seems to be an important mechanism for regulating the activity of IspC.

Listeriosis Outbreak in Dairy Cattle Caused by an UnusualListeria MonocytogenesSerotype 4b Strain

A listeriosis outbreak, in dairy cattle, with a high case mortality and acute death after onset of symptoms was investigated using gross pathology and bacteriologic approaches, including molecular characterization of a clinical Listeria monocytogenes isolate. In a herd of 315 animals, 9 animals showed clinical symptoms consistent with listeriosis, including 3 animals that died within 2-4 days after acute onset of clinical signs, 4 animals that were euthanized, and 2 that survived. Initial EcoRI ribotyping and serotyping indicated that this outbreak was caused by an unusual L. monocytogenes serotype 4b strain, which was classified into lineage III. Further characterization of this isolate by DNA sequencing-based subtyping methods indicated that the strain responsible for this outbreak represented a unique genotype as supported by its classification into a new sigB allelic type, which has not been identified previously among >290 isolates, and by compelling phylogenetic evidence. While lineage III isolates are generally rare, they seem to be more common among L. monocytogenes isolates from animals with clinical signs of listeriosis. This is the first report of a particularly severe clinical course of disease associated with infection by a lineage III strain. The high prevalence of Listeria spp., including L. monocytogenes, in the farm environments may favor emergence and evolution of novel, and possibly more virulent, L. monocytogenes strains. Continued monitoring of animal listeriosis cases and outbreaks may not only improve animal health but also aid in the early discovery of newly emerging L. monocytogenes strains.

Characterization of Virulence Properties of Listeria monocytogenes Serotype 4b Strains of Different Origins

In this study, the virulence heterogeneity of Listeria monocytogenes serotype 4b strains of different origins was analysed on different levels. On one hand, the survival of L. monocytogenes strains in synthetic gastric fluid was studied. On the other hand, the pathogenic potential of strains with different inlB expression levels was analysed in an A/J mouse model for gastrointestinal listeriosis. Differences in survival capacity in gastric fluid and in in vivo virulence potential were observed between the tested strains. No clear correlation between the origin and the obtained data could be made. However, these results confirm the existence of heterogeneity in virulence potential of L. monocytogenes serotype 4b strains.

Development of a multilocus variable-number of tandem repeat typing method for Listeria monocytogenes serotype 4b strains

Listeria monocytogenes serotype 4b strains have been identified as the causative agent in many human listeriosis epidemics as well as in a considerable number of sporadic cases. Due to the genetic homogeneity of serotype 4b isolates, development of rapid subtyping methods with high discriminatory power for serotype 4b isolates is required to allow for improved outbreak detection and source tracking. In this study, multilocus variable-number tandem repeat analysis (MLVA) was developed and used to characterize 60 serotype 4b isolates from various sources. All isolates were also characterized by automated EcoRI ribotyping, single enzyme pulsed-field gel electrophoresis (PFGE) with ApaI, and a multilocus sequence typing (MLST) scheme targeting six virulence and virulence-associated genes. Discriminatory power of MLVA (as determined by Simpson Index of Discrimination) was higher than the discriminatory power of any of the other three methods. MLVA markers targeted were found to be stable and did not change when three isolates were passaged daily for 70 days. Cluster analyses of MLVA, PFGE and MLST consistently grouped the same isolates into three major clusters, each of which includes one of the three major L. monocytogenes epidemic clones (i.e., ECI, ECIa and ECII). We conclude that the MLVA method described here (i) provides for more discriminatory subtyping of L. monocytogenes serotype 4b strains than the other three methods, (ii) identifies three major groups within the serotype 4b, which are consistent with the groups identified by other subtyping methods, and (iii) is easy to interpret. Use of MLVA may thus be recommended for subtyping of serotype 4b isolates, including as a secondary more discriminatory subtyping method that could be used after initial isolate characterization by PFGE or ribotyping.

Pathogenicity of Food and Clinical Listeria monocytogenes Isolates in a Mouse Bioassay

Serotype distributions of Listeria monocytogenes in clinical samples and foods often differ. It is unknown whether such differences reflect a variation in the virulence of strains or are due to other factors that are not directly related to the strains' ability to cause illnesses. Fifty-two food and eight clinical isolates of L. monocytogenes were obtained from France, Japan, and the United States. Their pathogenicity in nonimmunocompromised female ICR mice was determined by intraperitoneal (i.p.) injection of the mice with test strains at 108 to 109 CFU per mouse. Five mice were injected with each Listeria strain and observed for 5 days. Listeria isolates that caused at least one death in 5 days were considered pathogenic. Isolates that caused no deaths in 5 days were considered nonpathogenic. All strains except Listeria innocua and one L. monocytogenes serotype 4b strain (RM3-1) isolated from bovine raw milk were pathogenic to nonimmunocompromised mice. Three food isolates of L. monocytogenes serotype 1/2c were weakly pathogenic to nonimmunocompromised mice, killing a maximum of 50% of mice at 108 CFU. Strains with no pathogenicity or reduced pathogenicity were further tested for their pathogenicity to immunocompromised mice. Each strain was inoculated i.p. into five mice at 103 to 1010 CFU per mouse. No deaths of immunocompromised mice inoculated with 108 CFU were observed, but 20 to 40% of the mice died when inoculated with 109 CFU of L. monocytogenes RM3-1. The three L. monocytogenes serotype 1/2c isolates were also weakly pathogenic to immunocompromised mice, with two of the three isolates killing ≤60% of mice at doses of ≤108 CFU. The hemolytic activity of the three weakly pathogenic serotype 1/2c isolates was similar to that of pathogenic strains. However, the nonpathogenic strain RM3-1 was not found to be hemolytic on horse blood agar. We have identified several L. monocytogenes strains with reduced virulence levels. Further characterization of such isolates may aid in understanding factors affecting the variation in virulence among strains.

Characterization by DNA restriction endonuclease analysis of Listeria monocytogenes strains related to the Swiss epidemic of listeriosis

Listeria monocytogenes strains responsible for outbreaks of listeriosis were studied by using serotyping and phage typing. An additional approach based on restriction endonuclease analysis (REA) of the chromosomal DNA was used to characterize L. monocytogenes strains collected from various sources during and after a Swiss outbreak of listeriosis (1983 to 1987). Among the 169 wild-type strains of Listeria spp. that were examined, 161 (95%) belonged to the species L. monocytogenes, of which 109 were of human origin. Ten different REA profiles were obtained from the 120 L. monocytogenes serotype 4b strains tested. All 57 serotype 4b strains that were identified as Swiss epidemic strains by phage typing clustered in two closely related REA profiles. In particular, 10 L. monocytogenes 4b strains isolated from the brand of soft cheese responsible for the outbreak and from its direct environment were indistinguishable from isolates from 40 patients by both phage typing and REA analysis. However, 5 of the 17 non-phage-typeable L. monocytogenes strains and 18 L. monocytogenes strains with a phage type different from those of the Swiss epidemic types showed the same profile. REA enabled the characterization of non-phage-typeable strains and, thus, seems a promising tool for L. monocytogenes typing, especially during epidemiological investigations.