Comparative evaluation of direct plating and most probable number for enumeration of low levels of Listeria monocytogenes in naturally contaminated ice cream products

AbstractA precise and accurate method for enumeration of low level of Listeria monocytogenes in foods is critical to a variety of studies. In this study, paired comparison of most probable number (MPN) and direct plating enumeration of L. monocytogenes was conducted on a total of 1730 outbreak-associated ice cream samples that were naturally contaminated with low level of L. monocytogenes. MPN was performed on all 1730 samples. Direct plating was performed on all samples using the RAPID'L.mono (RLM) agar (1600 samples) and agar Listeria Ottaviani and Agosti (ALOA; 130 samples). Probabilistic analysis with Bayesian inference model was used to compare paired direct plating and MPN estimates of L. monocytogenes in ice cream samples because assumptions implicit in ordinary least squares (OLS) linear regression analyses were not met for such a comparison. The probabilistic analysis revealed good agreement between the MPN and direct plating estimates, and this agreement showed that the MPN schemes and direct plating schemes using ALOA or RLM evaluated in the present study were suitable for enumerating low levels of L. monocytogenes in these ice cream samples. The statistical analysis further revealed that OLS linear regression analyses of direct plating and MPN data did introduce bias that incorrectly characterized systematic differences between estimates from the two methods

Genomic Analysis of Closely Related Astroviruses▿

To understand astrovirus biology, it is essential to understand factors associated with its evolution. The current study reports the genomic sequences of nine novel turkey astrovirus (TAstV) type 2-like clinical isolates. This represents, to our knowledge, the largest genomic-length data set available for any one astrovirus type. The comparison of these TAstV sequences suggests that the TAstV species contains multiple subtypes and that recombination events have occurred across the astrovirus genome. In addition, the analysis of the capsid gene demonstrated evidence for both site-specific positive selection and purifying selection

Vibrio caribbeanicus sp. nov., Isolated from the Marine Sponge Scleritoderma cyanea

A Gram-negative, oxidase-positive, catalase-negative, facultatively anaerobic, motile, curved rod-shaped bacterium, strain N384T, was isolated from a marine sponge (Scleritoderma cyanea; phylum Porifera) collected from a depth of 795 feet (242 m) off the west coast of Curaçao. On the basis of 16S rRNA gene sequencing, strain N384Twas shown to belong to the genus Vibrio, most closely related to Vibrio brasiliensis LMG 20546T (98.8 % similarity), Vibrio nigripulchritudo ATCC 27043T (98.5 %), Vibrio tubiashii ATCC 19109T (98.6 %) and V. sinaloensis DSM 21326T (98.2 %). The DNA G+C content of strain N384Twas 41.6 mol%. An analysis of concatenated sequences of five genes (gyrB,rpoA, pyrH, mreB and ftsZ; 4068 bp) demonstrated a clear separation between strain N384T and its closest neighbours and clustered strain N384T into the ‘Orientalis’ clade of vibrios. Phenotypically, the novel species belonged to the arginine dihydrolase-positive, lysine decarboxylase- and ornithine decarboxylase-negative (A+/L−/O−) cluster. The novel species was also differentiated on the basis of fatty acid composition, specifically that the proportions of iso-C13 : 0, iso-C15 : 0, C15 : 0, iso-C16 : 0, C16 : 0, iso-C17 : 0, C17 : 1ω8c and C17 : 0 were significantly different from those found in V. brasiliensisand V. sinaloensis. The results of DNA–DNA hybridization, average nucleotide identity and physiological and biochemical tests further allowed differentiation of this strain from other described species of the genus Vibrio. Collectively, these findings confirm that strain N384T represents a novel Vibrio species, for which the name Vibrio caribbeanicus sp. nov. is proposed, with the type strain N384T ( = ATCC BAA-2122T = DSM 23640T). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene, topA,ftsZ, gapA, gyrB, mreB, pyrH, recA and rpoA gene sequences derived in this study are HM771333–HM771388 and HQ890462–HQ890466. The accession numbers for the sequences of strain N384T are GU223601 (16S rRNA), HM771337 (topA), HM771358 (ftsZ), HM771363 (gapA), HM771368 (gyrB), HM771373 (mreB), HM771378 (pyrH), HM771383 (recA), HM771388 (rpoA) and AEIU00000000 (whole-genome shotgun sequence). The accession numbers for the whole-genome shotgun sequences of V. brasiliensis LMG 20546T, V. sinaloensis DSM 21326T, V. splendidus ATCC 33789, V. ichthyoenteri ATCC 700023T, V. scophthalmi LMG 19158T, V. orientalis ATCC 33934T, V. tubiashii ATCC 19109T and V. nigripulchritudo ATCC 27043T are AEVS00000000, AEVT00000000, AFWG00000000, AFWF00000000, AFWE00000000, AFWH00000000, AFWI00000000 and AFWJ00000000

Rapid Whole-Genome Sequencing for Surveillance of Salmonella enterica Serovar Enteritidis

For Salmonella enterica serovar Enteritidis, 85% of isolates can be classified into 5 pulsed-field gel electrophoresis (PFGE) types. However, PFGE has limited discriminatory power for outbreak detection. Although whole-genome sequencing has been found to improve discrimination of outbreak clusters, whether this procedure can be used in real-time in a public health laboratory is not known. Therefore, we conducted a retrospective and prospective analysis. The retrospective study investigated isolates from 1 confirmed outbreak. Additional cases could be attributed to the outbreak strain on the basis of whole-genome data. The prospective study included 58 isolates obtained in 2012, including isolates from 1 epidemiologically defined outbreak. Whole-genome sequencing identified additional isolates that could be attributed to the outbreak, but which differed from the outbreak-associated PFGE type. Additional putative outbreak clusters were detected in the retrospective and prospective analyses. This study demonstrates the practicality of implementing this approach for outbreak surveillance in a state public health laboratory

Assessing the genome level diversity of <i>Listeria monocytogenes</i> from contaminated ice cream and environmental samples linked to a listeriosis outbreak in the United States

<div><p>A listeriosis outbreak in the United States implicated contaminated ice cream produced by one company, which operated 3 facilities. We performed single nucleotide polymorphism (SNP)-based whole genome sequencing (WGS) analysis on <i>Listeria monocytogenes</i> from food, environmental and clinical sources, identifying two clusters and a single branch, belonging to PCR serogroup IIb and genetic lineage I. WGS Cluster I, representing one outbreak strain, contained 82 food and environmental isolates from Facility I and 4 clinical isolates. These isolates differed by up to 29 SNPs, exhibited 9 pulsed-field gel electrophoresis (PFGE) profiles and multilocus sequence typing (MLST) sequence type (ST) 5 of clonal complex 5 (CC5). WGS Cluster II contained 51 food and environmental isolates from Facility II, 4 food isolates from Facility I and 5 clinical isolates. Among them the isolates from Facility II and clinical isolates formed a clade and represented another outbreak strain. Isolates in this clade differed by up to 29 SNPs, exhibited 3 PFGE profiles and ST5. The only isolate collected from Facility III belonged to singleton ST489, which was in a single branch separate from Clusters I and II, and was not associated with the outbreak. WGS analyses clustered together outbreak-associated isolates exhibiting multiple PFGE profiles, while differentiating them from epidemiologically unrelated isolates that exhibited outbreak PFGE profiles. The complete genome of a Cluster I isolate allowed the identification and analyses of putative prophages, revealing that Cluster I isolates differed by the gain or loss of three putative prophages, causing the banding pattern differences among all 3 <i>Asc</i>I-PFGE profiles observed in Cluster I isolates. WGS data suggested that certain ice cream varieties and/or production lines might have contamination sources unique to them. The SNP-based analysis was able to distinguish CC5 as a group from non-CC5 isolates and differentiate among CC5 isolates from different outbreaks/incidents.</p></div

<i>Asc</i>I-PFGE banding pattern changes due to the gain/loss of prophages 2 and 3.

<p>The <i>Asc</i>I-PFGE profile and corresponding brief two-enzyme PFGE profiles are listed on the right of the gel images. The corresponding prophage gain/loss profiles are listed to the right. + indicates the gain of a prophage and–indicates the loss of a prophage. Isolates exhibiting GX6A16.0061 contained both prophages 2 and 3. The loss of prophage 3 resulted in the change of a ~275 Kbp fragment in the gel pattern of GX6A16.0061 to ~237 Kbp in the gel patterns of GX6A16.0026 and GX6A16.0020, and this ~237 Kbp fragment and a ~240 Kbp fragment formed a duplet. The loss of prophage 2 resulted in the change of a ~392 Kbp fragment in the gel patterns of GX6A16.0061 and GX6A16.0026 to ~352 Kbp in the gel pattern of GX6A16.0020. The loss of both prophages 2 and 3 resulted in the pattern change from GX6A16.0061 to GX6A16.0020.</p

Putative prophages of the complete genome CFSAN029793 identified by a combination of PHAST [32], PHASTER [33] and prophage insertion site examination.

<p>Putative prophages of the complete genome CFSAN029793 identified by a combination of PHAST [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171389#pone.0171389.ref032" target="_blank">32</a>], PHASTER [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171389#pone.0171389.ref033" target="_blank">33</a>] and prophage insertion site examination.</p