Survival of a serotype 4b strain and a serotype 1/2a strain of Listeria monocytogenes, isolated from a stone fruit outbreak investigation, on whole stone fruit at 4 °C.

In the summer of 2014, a multistate outbreak of listeriosis associated with contaminated stone fruit (peach and nectarine) was reported. A serotype 4b variant Listeria monocytogenes (Lm) strain of singleton Sequence Type (ST) 382 was isolated from clinical samples and stone fruit associated with the outbreak. A serotype 1/2b Lm strain of ST5, Clonal Complex 5 was isolated only from outbreak-associated stone fruit, not from clinical samples. Here we investigated the fate of the serotype 4b and 1/2b strains, at two inoculation levels (high level at 3.7 logCFU/fruit and low level at 2.7 logCFU/fruit), on the surfaces of white peach, yellow peach and yellow nectarine stored at 4 °C for 26 days. After rinsing the fruits, we determined the Lm levels in the rinsates and on the peels. We enumerated Lm using a direct plating method and compared two chromogenic agars. The Lm populations rapidly declined in the first 3 days and then declined more slowly until Day 19/21. The maximum decline was 1.6 logCFU/fruit on yellow peach inoculated with serotype 4b at high level. For fruits inoculated with high-level Lm, the lowest level of Lm (1.7 logCFU/fruit) was observed on for white peach inoculated with serotype 1/2b, and the highest level of Lm (2.6 logCFU/fruit) on Day 19/21 was observed on yellow peach inoculated with the serotype 1/2b strain. For fruits inoculated with low-level Lm, the lowest level of Lm (1.3 logCFU/fruit) was observed on yellow nectarine inoculated with either the serotype 4b or 1/2b strain, and the highest level of Lm (1.7 logCFU/fruit) on Day 19/21 was observed on yellow peach inoculated with ST382. The D-values ranged from 15 days to 28 days. Lm remained viable until the end of storage (Day 26), but the levels were not significantly different from those on Day 19/21. The types of stone fruit and Lm strain did not significantly affect the survival of Lm. These results demonstrate that contaminated stone fruit can carry a potential risk for causing listeriosis in susceptible populations. Comparison of direct plating results using two chromogenic agars showed that RAPID' L. mono and Agar Listeria Ottavani & Agosti performed equivalently for enumerating Lm on stone fruit. The fruit rinsing recovered 80% to 84% of Lm from fruit surfaces.

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

A 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.

Listeria monocytogenes in Stone Fruits Linked to a Multistate Outbreak: Enumeration of Cells and Whole-Genome Sequencing.

In 2014, the identification of stone fruits contaminated with Listeria monocytogenes led to the subsequent identification of a multistate outbreak. Simultaneous detection and enumeration of L. monocytogenes were performed on 105 fruits, each weighing 127 to 145 g, collected from 7 contaminated lots. The results showed that 53.3% of the fruits yielded L. monocytogenes (lower limit of detection, 5 CFU/fruit), and the levels ranged from 5 to 2,850 CFU/fruit, with a geometric mean of 11.3 CFU/fruit (0.1 CFU/g of fruit). Two serotypes, IVb-v1 and 1/2b, were identified by a combination of PCR- and antiserum-based serotyping among isolates from fruits and their packing environment; certain fruits contained a mixture of both serotypes. Single nucleotide polymorphism (SNP)-based whole-genome sequencing (WGS) analysis clustered isolates from two case-patients with the serotype IVb-v1 isolates and distinguished outbreak-associated isolates from pulsed-field gel electrophoresis (PFGE)-matched, but epidemiologically unrelated, clinical isolates. The outbreak-associated isolates differed by up to 42 SNPs. All but one serotype 1/2b isolate formed another WGS cluster and differed by up to 17 SNPs. Fully closed genomes of isolates from the stone fruits were used as references to maximize the resolution and to increase our confidence in prophage analysis. Putative prophages were conserved among isolates of each WGS cluster. All serotype IVb-v1 isolates belonged to singleton sequence type 382 (ST382); all but one serotype 1/2b isolate belonged to clonal complex 5. IMPORTANCE: WGS proved to be an excellent tool to assist in the epidemiologic investigation of listeriosis outbreaks. The comparison at the genome level contributed to our understanding of the genetic diversity and variations among isolates involved in an outbreak or isolates associated with food and environmental samples from one facility. Fully closed genomes increased our confidence in the identification and comparison of accessory genomes. The diversity among the outbreak-associated isolates and the inclusion of PFGE-matched, but epidemiologically unrelated, isolates demonstrate the high resolution of WGS. The prevalence and enumeration data could contribute to our further understanding of the risk associated with Listeria monocytogenes contamination, especially among high-risk populations.

Prevalence and Level of Listeria monocytogenes in Ice Cream Linked to a Listeriosis Outbreak in the United States.

A most-probable-number (MPN) method was used to enumerate Listeria monocytogenes in 2,320 commercial ice cream scoops manufactured on a production line that was implicated in a 2015 listeriosis outbreak in the United States. The analyzed samples were collected from seven lots produced in November 2014, December 2014, January 2015, and March 2015. L. monocytogenes was detected in 99% (2,307 of 2,320) of the tested samples (lower limit of detection, 0.03 MPN/g), 92% of which were contaminated at <20 MPN/g. The levels of L. monocytogenes in these samples had a geometric mean per lot of 0.15 to 7.1 MPN/g. The prevalence and enumeration data from an unprecedented large number of naturally contaminated ice cream products linked to a listeriosis outbreak provided a unique data set for further understanding the risk associated with L. monocytogenes contamination for highly susceptible populations.

Differentiation of Salmonella strains from the SARA, SARB and SARC reference collections by using three genes PCR-RFLP and the 2100 Agilent Bioanalyzer.

Rapid molecular typing methods are important tools in surveillance and outbreak investigations of human Salmonella infections. Here we described the development of a three-genes PCR-RFLP typing method for the differentiation of Salmonella species, subspecies and serovars using the Agilent 2100 Bioanalyzer. The fliC, gnd, and mutS genes were PCR-amplified in 160 Salmonella strains representing the two Salmonella species, six subspecies, and 41 different serovars of S. enterica subspecies enterica. PCR products were individually cut with two different restriction enzymes and the resulting 930 restriction patterns were collected using the Agilent 2100 Bioanalyzer followed by cluster analysis. Both species of Salmonella were differentiated by conventional PCR. All of S. bongori tested were gnd PCR negative due to a mismatch at the 3'-end in one the PCR primers. Salmonella subspecies were differentiated into third-teen homogeneous groups representing each of the six subspecies by cluster analysis of restriction patterns generated from the mutS gene cut with AciI. S. enterica subspecies enterica serovars were further differentiated by the combination of the three target genes and five out the six sets of restriction patterns with a discriminatory power of 0.9725 by cluster analysis. The combined RFLP results of five sets of restriction patterns allowed us to assign each of the 160 strains to one of 128 restriction types. During inoculation studies we were able to identify S. Saintpaul and Typhimurium from 24 h pre-enrichment samples using the described method. The use of fliC, gnd, and mutS PCR-RFLP with the Agilent 2100 Bioanalyzer can provide an accessible and automated alternative method for differentiation of Salmonella pathogens.

Detection of live Salmonella sp. cells in produce by a TaqMan-based quantitative reverse transcriptase real-time PCR targeting invA mRNA.

Salmonella enterica contamination in foods is a significant concern for public health. When DNA detection methods are used for analysis of foods, one of the major concerns is false-positive results from the detection of dead cells. To circumvent this crucial issue, a TaqMan quantitative real-time RT-PCR (qRT-PCR) assay with an RNA internal control was developed. invA RNA standards were used to determine the detection limit of this assay as well as to determine invA mRNA levels in mid-exponential-, late-exponential-, and stationary-phase cells. This assay has a detection limit of 40 copies of invA mRNA per reaction. The levels of invA mRNA in mid-exponential-, late-exponential-, and stationary-phase S. enterica cells was approximately 1 copy per 3 CFU, 1 copy per CFU, and 4 copies per 10(3) CFU, respectively. Spinach, tomatoes, jalapeno peppers, and serrano peppers were artificially contaminated with four different Salmonella serovars at levels of 10(5) and less than 10 CFU. These foods were analyzed with qRT-PCR and with the FDA's Bacteriological Analytical Manual Salmonella culture method (W. A. Andrews and T. S. Hammack, in G. J. Jackson et al., ed., Bacteriological analytical manual online, http://www.cfsan.fda.gov/ approximately ebam/bam-5.html, 2007). Comparable results were obtained by both methods. Only live Salmonella cells could be detected by this qRT-PCR assay, thus avoiding the dangers of false-positive results from nonviable cells. False negatives (inhibition of the PCR) were also ruled out through the use of an RNA internal control. This assay allows for the fast and accurate detection of viable Salmonella spp. in spinach, tomatoes, and in both jalapeno and serrano peppers.

Modeling the physiological state of the inoculum and CO2 atmosphere on the lag phase and growth rate of Listeria monocytogenes.

In previous studies, the growth of L. monocytogenes has been modeled under different CO2 headspace concentrations; however, the inoculum cells were always in the stationary phase. In this study, the growth of L. monocytogenes under different CO2 concentrations as affected by the physiological state of the cells was investigated. Exponential-growth-phase, stationary-phase, dried, and starved cells were prepared and inoculated at 5 degrees C into brain heart infusion broths that had been preequilibrated under atmospheres of 0, 20, 40, 60, or 80% CO2 (the balance was N2). Lag-phase duration times (LDTs) and exponential growth rates were determined by enumerating cells at appropriate time intervals and by fitting the data to a three-phase linear function that has a lag period before the initiation of exponential growth. Longer LDTs were observed as the CO2 concentration increased, with no growth observed at 80% CO2. For example, the LDTs for exponential-phase, stationary-phase, starved, and dried cells were 2.21, 8.27, 9.17, and 9.67 days, respectively, under the 40% CO2 atmosphere. In general, exponential-growth-phase cells had the shortest LDT followed by starved cells and stationary-phase cells. Dried cells had the longest LDT. Exponential growth rates decreased as the CO2 concentrations increased. Once exponential growth was attained, no retained differences among the various initial physiological states of the cells for any of the atmospheres were observed in the exponential growth rates. The exponential growth rates under 0, 20, 40, 60, and 80% CO2 averaged 0.39, 0.37, 0.23, 0.23, and 0.0 log CFU/day, respectively. Dimensionless factors were calculated that describe the inhibitory action of CO2 on the LDTs and exponential growth rates for the various physiological states.

Quantitative contamination and transfer of Escherichia coli from foods by houseflies, Musca domestica L. (Diptera: Muscidae).

The housefly, Musca domestica L. (Diptera: Muscidae), is recognized as an important factor in the dissemination of various infectious diseases such as cholera, shigellosis, and salmonellosis. They can also serve as a cross-contamination vector for other foodborne pathogens. However, the potential for bacterial transfer by houseflies has been demonstrated in a qualitative rather than quantitative manner. In this study, the numbers of bacteria a housefly can carry on its body and transfer to a clean surface after exposure to a sugar-milk aqueous solution, steak, and potato salad contaminated with a fluorescent gene Escherichia coli (8 log10 CFU/ml) were determined. In the first series of experiments to quantify bacterial numbers on the flies, about 40-60 flies were transferred into a sterile cage, exposed to the food for 30 min, the flies immobilized and the attached E. coli on each fly enumerated. Detectable E. coli (>1.7 log10 CFU/fly) were found on 43% (29/67), 53% (23/43), and 62% (32/52) of the flies in the cages with sugar/milk, steak, and potato salad, respectively. For the positive flies, the geometric mean carriage (log10 CFU/fly) was 2.93+/-1.24 for sugar-milk, 3.77+/-1.28 for steak, and 2.25+/-0.64 for the potato salad. In the second series of experiments, the transfer of bacteria by individual flies from contaminated food to the inner surface of a sterile jar per each landing was determined. E. coli transferred from the sugar-milk was 3.5+/-0.7 log10 CFU/fly-landing, 3.9+/-0.7 for steak and 2.61+/-1.16 for the potato salad. From the initial contamination levels of bacteria and the number of transferred bacteria, it can be calculated that flies contaminate clean surfaces with approximately 0.1 mg of food per landing.

Thermal inactivation, growth, and survival studies of Listeria monocytogenes strains belonging to three distinct genotypic lineages.

Twenty-one Listeria monocytogenes strains belonging to three different genotypic lineages were evaluated for differences between lineages and between individual strains with respect to thermal inactivation, growth, and survival. Three sets of heat inactivation conditions (60 degrees C, pH 6.0, and 0.5 M lactate; 55 degrees C, pH 6.0, and 0.5 M lactate; and 50 degrees C, pH 4.0, and 0.5 M lactate) were used on strains grown in modified brain heart infusion (BHI) broth with and without glucose. Two sets of growth conditions (35 degrees C, pH 6.5, and 0.1 M lactate and 5 degrees C, pH 6.5, and 0.1 M lactate) were used with modified BHI broths to determine lag phases and exponential growth rates. Two sets of conditions (28 degrees C, pH 4.0, and 1 M lactate and 28 degrees C, pH 4.5, and 0.5 M lactate) were used with modified BHI broth to determine survival times (D-values). Thermal inactivation D-values were consistently lowest for lineage III, but differences were not significant for any set of conditions tested. Some significant differences were observed between lineages with respect to some of the growth and survival conditions tested. Extensive strain-to-strain variation was observed for all parameters tested. Average coefficients of variation for the thermal inactivation, growth, and survival studies were 0.31, 0.18, and 0.26, respectively. Strain-to-strain variations were approximately equal to the uncertainties associated with the analytical procedures. The results obtained indicate a diversity among strains encountered in food processing that must be accounted for in process calculations and risk assessments.