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This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology
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The current picture of human salmonellosis shows
Multilocus variable-number tandem repeat analysis (MLVA) has been increasingly used in Europe as a primary method for
In order to correctly allocate the available resources to prevent human foodborne diseases, it is important for risk managers to be able to accurately apportion sporadic cases of infection to specific animal hosts and to understand transmission routes of the pathogens (
Although different studies have demonstrated that the monophasic serovar emerged from
The aims of the present study were (i) to investigate the relationship between
Single isolates from 268 human sporadic cases and 325 veterinary isolates of
Distribution of isolates by serovar and source.
Serovar | Source | N∘ of isolates | N∘ of MLVA profiles | N∘ isolates with unshared MLVA profile (%)a |
---|---|---|---|---|
Human | 63 | 28 | 28 (44.4) | |
Pig | 73 | 44 | 47 (64.4) | |
Chicken | 20 | 15 | 17 (65) | |
Cattle | 18 | 15 | 11 (61.1) | |
Turkey | 8 | 6 | 3 (37.5) | |
Total | 182 | 93 | 106 (58.2) | |
Human | 205 | 53 | 38 (18.5) | |
Pig | 131 | 43 | 26 (19.8) | |
Chicken | 27 | 17 | 1 (3.7) | |
Cattle | 36 | 12 | 1 (2.8) | |
Turkey | 12 | 10 | 2 (16.7) | |
Total | 411 | 79 | 68 (16.5) |
Multilocus variable-number tandem repeat analysis was performed according to the protocol described by
A descriptive analysis of MLVA profile frequencies in the two serotypes and VNTR loci variability between human and non-human sources was conducted by using the R version 3.1.2 (
The diversity among the five VNTR (STTR9–STTR5–STTR6–STTR10pl–STTR3) was estimated according to the Simpson’s Diversity Index, which quantifies the variation of the number of repeats at each locus and assumes values ranging from 0.0 (indicative of complete absence of diversity) to 1.0 (indicative of complete diversity). To calculate the index, the online toll “DIversity and Confidence Extractor (V-DICE)” provided by the Health protection Agency’s Bioinformatics Unit (available at
The Asymmetric Island Model was applied as described originally by
The model was run considering
Moreover, in order to assess the sensitivity of the model to the sample size differences between sources, bootstrap samples of equal size were constructed for each source by sampling 100 times with replacement from the original sample. Also this new dataset, consisting of the original data for human isolates and the bootstrap samples for the source isolates, was used to run the model.
To quantify genetic differentiation between the different populations investigated (human and putative sources), analysis of molecular variance (AMOVA) was used. AMOVA explicitly extends the procedures and formats used in the traditional analysis of variance, in order to estimate the degree of genetic differentiation between-group and within-group at several hierarchical levels (
Although in the dataset contained more isolates of
Cluster analysis by UPGMA was performed to clarify the relationship between the two serovars (
The degree of polymorphism of MLVA profiles associated with the two serovars was quantified by calculating the diversity index (
Simpson’s index of diversity for the five VNTR of the MLVA scheme estimated for
Locus | Diversity index | Confidence interval∗ | K# | Max (pi)+ | Diversity index | Confidence interval∗ | K# | Max (pi)+ |
---|---|---|---|---|---|---|---|---|
STTR6 | 0.87 | 0.84–0.89 | 18 | 0.26 | 0.78 | 0.75–0.80 | 14 | 0.34 |
STTR5 | 0.82 | 0.79–0.86 | 17 | 0.34 | 0.72 | 0.70–0.75 | 11 | 0.40 |
STTR10 | 0.60 | 0.52–0.69 | 19 | 0.62 | 0.06 | 0.03–0.09 | 7 | 0.97 |
STTR3 | 0.51 | 0.44–0.58 | 10 | 0.66 | 0.13 | 0.09–0.18 | 8 | 0.93 |
STTR9 | 0.37 | 0.28–0.45 | 4 | 0.78 | 0.02 | 0.00–0.04 | 2 | 0.10 |
For
The unshared MLVA profiles, defined as profiles that were exclusively displayed by human isolates or by one specific source, comprised 58.2 and 16.5% of the total number of
List of MLVA profiles for
MLVA profile | N∘ of isolates |
|||||
---|---|---|---|---|---|---|
Human | Pig | Chicken | Cattle | Turkey | ||
3_12_10_0_211 | 2 | 4 | 2 | 3 | 0 | |
3_13_11_0_211 | 3 | 2 | 0 | 1 | 0 | |
3_12_11_0_211 | 7 | 3 | 0 | 0 | 0 | |
3_12_9_0_211 | 20 | 1 | 0 | 0 | 0 | |
3_15_11_0_311 | 1 | 3 | 0 | 0 | 0 | |
4_13_9_7_211 | 2 | 2 | 0 | 0 | 0 | |
3_12_10_0_211 | 21 | 16 | 4 | 7 | 2 | |
3_12_9_0_211 | 23 | 17 | 2 | 5 | 1 | |
3_12_7_0_211 | 1 | 3 | 1 | 2 | 1 | |
3_13_9_0_211 | 8 | 7 | 1 | 3 | 1 | |
3_11_8_0_211 | 8 | 1 | 2 | 2 | 0 | |
3_11_9_0_211 | 6 | 8 | 1 | 7 | 0 | |
3_12_11_0_211 | 8 | 8 | 2 | 1 | 0 | |
3_13_10_0_211 | 49 | 11 | 2 | 3 | 0 | |
3_12_8_0_211 | 5 | 8 | 4 | 1 | 0 | |
3_13_11_0_211 | 5 | 1 | 1 | 1 | 0 | |
3_12_12_0_211 | 3 | 2 | 1 | 0 | 1 | |
3_13_8_0_211 | 6 | 4 | 1 | 0 | 1 | |
3_10_10_0_211 | 3 | 3 | 1 | 0 | 0 | |
3_11_10_0_211 | 3 | 2 | 1 | 0 | 0 | |
3_11_11_0_211 | 1 | 2 | 0 | 0 | 1 | |
3_15_9_0_211 | 1 | 3 | 0 | 0 | 0 | |
3_11_12_0_211 | 3 | 0 | 0 | 0 | 1 | |
3_11_13_0_211 | 1 | 1 | 0 | 0 | 0 | |
3_13_7_0_211 | 2 | 2 | 0 | 0 | 0 | |
3_14_10_0_211 | 8 | 1 | 0 | 0 | 0 | |
3_14_11_0_211 | 2 | 2 | 0 | 0 | 0 |
With regard to
Cluster analysis based on similarities of MLVA profiles using MST for
The Asymmetric Island model attributed most human cases to the pig sources for both serovars (64.4%, 95% credibility interval [95% CI] 27.7–89.3% for
Attribution estimates obtained by the Asymmetric Island model provided with the complete datasets for
Merged dataset | Bootstrap dataset | |||
---|---|---|---|---|
Pig | 64.4 (27.7–89.3) | 58.4 (1.8–96.2) | 67.0 (11.8–97.6) | 87.7 (69.1–98.4) |
Cattle | 21.5 (0.5–63.8) | 11.7 (0.3–40.8) | 20.6 (0.3–80.7) | 8.4 (0.3–25.4) |
Poultry | 14.1 (0.3–45.6) | 29.9 (0.5–91.8) | 12.4 (0.2–49.1) | 3.9 (0.1–14.3) |
For both serovars and for all sources, the attributions presented extremely large credibility intervals, leading to an excessive uncertainty, which hampered the robustness of the estimation model. As an attempt to improve the precision of the estimations, the sample size for each source was enlarged by merging the two original datasets into a unique dataset including all MLVA profiles associated with both
Since a possible bias of the source assignment could be the large difference in sample size among the putative sources investigated, the model was also run with a bootstrap dataset constructed sampling with replacement from each original source dataset and including 100 MLVA profiles per source. The bootstrap dataset provided the same ranking of sources as the merged dataset, but the relative importance of pigs as a source increased from 67.0% (95% CI 11.8–97.6) to 87.7% (95% CI 69.1–98.4), whereas for the other sources the attributions decreased. Moreover, the equal source size attribution led to a reduction of the uncertainty associate with the attribution estimates for all sources (
The AMOVA analysis was conducted on the merged dataset (including
Analysis of molecular variance (AMOVA), describing the population variation between human/non-human isolates, between isolates from different sources and within each isolate.
Covariance components |
||||
---|---|---|---|---|
Sigma | % | Φ-statistic ( |
||
Between human – non-human | 33.65 | 1.53 | ΦST | 0.0153 (0.164) |
Between different sources (human/pig/cattle/turkey/chicken) | 26.59 | 1.21 | ΦCT | 0.0123 (0.516) |
Within isolates | 2143.80 | 97.27 | ΦSC | 0.0273 |
Total variations | 2204.05 | 100.00 |
The analysis of the MLVA profiles of
This finding is in conformity with previous studies which compared the two serovars by using phenotypic methods (
In the present study, isolates were typed by using MLVA, which is classified as a highly discriminative subtyping method, since it targets highly unstable genetic markers (
Irrespective of the source of isolation, for
The presence of identical or closely related MLVA profiles among isolates from different animal sources and humans reinforced the evidence that food-producing animals have an active involvement in the dissemination of
The high discriminatory power of molecular subtyping methods makes source attribution difficult if sources are attributed simply based on the exact overlap of subtypes. Hence, population genetic models, taking into account the genetic relationship among isolates (based on analysis of mutations, recombination, and migrations), have been identified as valuable tools to further clarify the relevant host associations and to identify the key reservoirs when molecular subtyping data are available (
In the present study, the Asymmetric Island Model supplied with the MLVA profiles was used to infer sources of human infections. For both
These differences between countries in the relative contribution of different food sources to human salmonellosis can be explained by several factors, such as the differences in animal and food production systems, the food consumption and preparation habits, the epidemiology of the pathogen and the efficiency of surveillance programs in place in different regions (
Nevertheless, for both serovars and for all sources investigated the attributions provided by the Asymmetric Island Model presented large credibility intervals, leading to an excessive uncertainty, which hampered the robustness of the estimations. Unfortunately, merging the two original datasets into a unique dataset did not produce a substantial constriction of the credibility intervals.
Therefore, it seems relevant to enlarge the available datasets, so that even for the more rare sources at least 100 epidemiologically independent isolates can be available. Another important issue to take into account when putative sources of infection are inferred by using molecular data is the genetic differentiation between groups (sources), which must be higher than the within group heterogeneity in order to get robust estimations. In particular, in the case of a noteworthy heterogeneity within each source and a weak genetic differentiation among sources the degree of accuracy in the source assignments can be jeopardized (
Source attribution studies rely on subtyping methods which should have enough discriminatory power to identify links between human isolates and their putative sources, but they should not be too discriminatory, so that true epidemiological association between isolates might be missed. The current 5-loci MLVA scheme does not seem to fulfill this requirement, particularly for
Conceived and designed the experiments: LB, FB, JO, IL, AL, AR. Performed the experiments: FB, EC, ER, AL. Analyzed the data: LB, FB, EC, ER, AL, AR. Contributed strains: IL. Critical discussion about the results: LB, FB, EC, ER, JO, IL, AL, AR. Wrote the paper: LB, FB, AL, JO, AR. Final approval for the submitted document: LB, FB, EC, ER, JO, IL, AL, AR.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.