Utilization of Fourier-Transform Infrared Spectroscopy to Distinguish Salmonella Typhimurium from Other Salmonella Serotypes in Veterinary Isolates

Macy Rasmussen Author

08/07/2020 Added

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This project explored FT-IR as a diagnostic tool to identify Salmonella Typhimurium in veterinary isolates.

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[00:00:01.991]Hello and welcome to my presentation.
[00:00:04.697]My project was on the utilization
[00:00:06.941]of Fourirer-Transform infrared spectroscopy
[00:00:09.691]to distinguish Salmonella Typhimurium
[00:00:11.479]from other Salmonella serotypes
[00:00:13.339]in veterinary isolates.
[00:00:16.521]Salmonella enterica subspecies enterica
[00:00:19.241]is a leading cause of food borne illness
[00:00:21.351]in humans,
[00:00:22.281]and it affects a wide range of hosts,
[00:00:23.971]including food animals
[00:00:25.281]like cattle, swine, poultry, and others.
[00:00:29.736]There are many subtypes
[00:00:30.896]of this species of Salmonella,
[00:00:32.546]which are referred to as serotypes.
[00:00:36.025]These serotypes belong to larger
[00:00:37.945]groups called serogroups.
[00:00:40.243]Serogroups are distinguished
[00:00:41.933]based on O antigens
[00:00:43.273]found on the outer membrane
[00:00:44.633]of the bacterial cell.
[00:00:46.385]The serotypes within these groups
[00:00:48.455]are distinguished based on H antigens
[00:00:50.524]found on the flagella of the cell.
[00:00:52.792]Salmonella Typhimurium in serogroup B
[00:00:55.530]and Salmonella Enteritidis in serogroup D
[00:00:58.610]are the two serotypes
[00:00:59.960]most often identified
[00:01:01.240]in human Salmonellosis.
[00:01:03.320]In most countries,
[00:01:04.711]Salmonella Enteritidis is the
[00:01:06.311]more common serotype.
[00:01:08.140]Because of this,
[00:01:09.360]rapid diagnostic testing
[00:01:10.750]for Salmonella Enteritidis
[00:01:12.250]is more easily available
[00:01:13.780]than for Typhimurium.
[00:01:16.101]However, it is still important
[00:01:17.801]to rapidly diagnose Salmonella Typhimurium
[00:01:20.435]because it is a major pathogen.
[00:01:22.955]The goal of this research was
[00:01:24.555]to explore FT-IR as a
[00:01:25.995]diagnostic tool to distinguish
[00:01:27.690]Salmonella Typhimurium from other
[00:01:29.600]serogroup B Salmonella isolates
[00:01:31.430]by using hierarchal cluster agreement
[00:01:33.740]and principle component analysis
[00:01:35.663]to generate dendrograms
[00:01:36.973]and scatter plots of
[00:01:38.013]spectral data from the isolates.
[00:01:42.425]Around 70 Salmonella isolates
[00:01:44.478]from veterinary samples were used
[00:01:46.488]from different species including
[00:01:48.098]avian, bovine, porcine, and other species.
[00:01:51.335]All isolates were collected from
[00:01:53.045]diagnostic samples sent to the
[00:01:54.715]Nebraska Veterinary Diagnostic Center.
[00:01:57.817]The time of isolation ranged from
[00:01:59.637]1981 to 2020, so there was a wide range of
[00:02:02.673]time in which the isolates were collected.
[00:02:06.113]The isolates included
[00:02:07.583]22 Salmonella Typhimurium isolates,
[00:02:10.273]16 serogroup B non-Typhimurium isolates,
[00:02:14.273]11 serogroup C isolates,
[00:02:16.653]13 serogroup D isolates,
[00:02:19.318]5 serogroup E isolates,
[00:02:21.788]and 3 serogroup O isolates.
[00:02:24.360]The isolates were frozen in
[00:02:25.838]BHI and glycerol stock,
[00:02:27.998]plated on blood agar,
[00:02:29.408]and sub-cultured to another
[00:02:30.841]blood agar plate.
[00:02:32.148]The cultures were analyzed using
[00:02:33.698]MALDI-TOF mass spectrometry to ensure
[00:02:36.288]the culture was pure Salmonella.
[00:02:39.083]The cultures were plated on a TSA plate
[00:02:41.258]and incubated for 24 hours.
[00:02:43.668]After incubation, the samples were
[00:02:45.648]prepared by homogenizing a small amount
[00:02:47.988]of the culture in DI water,
[00:02:49.818]placing the suspensions on ice,
[00:02:51.528]and plating them on the IR Biotyper plate.
[00:02:55.039]Two manufacturer-given standards
[00:02:57.019]were used with each run
[00:02:58.624]to ensure the quality of the spectra.
[00:03:01.688]When selecting data,
[00:03:03.008]only spectra that fit within parameters
[00:03:04.998]for proper absorbance and noise
[00:03:06.688]were used in analysis.
[00:03:08.704]Dendrograms and scatter plots
[00:03:10.134]of several different groups of isolates
[00:03:11.934]were generated using mainly PCA.
[00:03:14.244]However, linear discriminant analysis
[00:03:16.938]was used when PCA did not
[00:03:18.638]show clear distinction.
[00:03:22.593]The first set of isolates analyzed
[00:03:24.677]was all 70 isolates.
[00:03:26.662]A dendrogram was generated using PCA.
[00:03:29.698]The cutoff was at 1.971,
[00:03:32.329]which was the optimal value.
[00:03:34.358]Three major clusters were generated
[00:03:36.208]in this analysis.
[00:03:37.628]Cluster 1 had a purity labeled “bad.”
[00:03:40.516]It was made up of 58.8% group C isolates
[00:03:43.616]and contained group D, B, and O isolates.
[00:03:48.193]Cluster 2 had a purity of “medium,”
[00:03:50.314]and it was made up of 72.2%
[00:03:52.504]group D isolates and contained
[00:03:54.524]all the group E isolates that were ran.
[00:03:57.674]Cluster 3 was also of “medium” purity,
[00:04:00.124]and had 97.7% group B isolates,
[00:04:03.114]with one group C isolate.
[00:04:06.902]Figure one shows the three
[00:04:08.152]clusters that were generated.
[00:04:11.740]The third cluster contained 36 of the 38
[00:04:14.600]group B isolates ran,
[00:04:16.090]and only had one non-group B
[00:04:18.030]isolate within it.
[00:04:21.993]The second analysis contained all
[00:04:23.633]38 group B isolates.
[00:04:25.409]The cutoff value was set to 0.359.
[00:04:30.893]Figure 2 shows that there was inconsistent
[00:04:33.119]grouping of Salmonella Typhimurium
[00:04:35.139]isolates, which are labeled on the side.
[00:04:37.765]The other serotypes did not exhibit
[00:04:39.769]consistent clustering either.
[00:04:44.401]Because PCA did not demonstrate good
[00:04:46.833]grouping of the Typhimurium isolates,
[00:04:48.904]LDA was used to analyze the same
[00:04:50.753]38 group B isolates.
[00:04:52.968]LDA looks for differences between
[00:04:54.803]two or more groups of data
[00:04:56.573]that are identified before analysis.
[00:04:59.213]The two groups identified were
[00:05:00.953]Typhimurium and non-Typhimurium.
[00:05:04.405]21 of the 22 Typhimurium isolates were
[00:05:07.168]located in the triangle labeled
[00:05:08.940]“Typhimurium”.
[00:05:10.567]15 of the 16 non-Typhimurium isolates
[00:05:13.499]were located in the
[00:05:14.469]“non-Typhimurium” triangle.
[00:05:16.594]Two outliers to the right of the triangles
[00:05:18.703]slightly skewed the groupings,
[00:05:20.163]but a majority of the isolates
[00:05:21.983]showed grouping.
[00:05:25.675]To compare the results of group B analysis
[00:05:28.045]using LDA and PCA,
[00:05:29.613]a scatter plot of the group B isolates
[00:05:32.081]made with PCA is shown in figure 4.
[00:05:36.313]Barring the outliers,
[00:05:37.726]there is heavy overlap among the
[00:05:39.247]different serotypes, meaning there was not
[00:05:41.463]great distinction made in the
[00:05:42.853]different types.
[00:05:47.378]The dendrogram and scatter plot of
[00:05:49.054]spectra from all isolates made using
[00:05:50.765]PCA showed promising discrimination
[00:05:52.945]between the group B isolates
[00:05:54.995]and the other serogroups
[00:05:56.655]because 94.7% of the group B isolates
[00:05:59.385]were located in one cluster.
[00:06:01.494]However, the PCA analysis of only
[00:06:04.204]group B isolates did not
[00:06:05.564]show discrimination,
[00:06:06.844]and the Salmonella Typhimurium isolates
[00:06:08.716]showed inconsistent scattering throughout
[00:06:10.671]the other serotypes.
[00:06:12.896]LDA provided better separation of the
[00:06:14.964]Typhimurium isolates from the
[00:06:16.608]non-Typhimuriums.
[00:06:18.715]Although the PCA results did not show
[00:06:20.765]discrimination at the serotype level,
[00:06:23.211]analysis using LDA did.
[00:06:25.660]Further study could look into
[00:06:27.040]what variables were used to distinguish
[00:06:28.937]between the two groups in the analysis
[00:06:30.987]and focus on those portions of spectra.
[00:06:34.407]The results of this study demonstrated
[00:06:36.317]that FT-IR and Bruker Biotyper software
[00:06:38.957]have the potential to be a tool for
[00:06:40.837]identification of Salmonella
[00:06:42.492]at the serogroup level.
[00:06:44.718]Thank you for your time.

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Utilization of Fourier-Transform Infrared Spectroscopy to Distinguish Salmonella Typhimurium from Other Salmonella Serotypes in Veterinary Isolates

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