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. 2022 Jul 20;10(7):1472.
doi: 10.3390/microorganisms10071472.

Discrimination and Characterization of Escherichia coli Originating from Clinical Cases of Femoral Head Necrosis in Broilers by MALDI-TOF Mass Spectrometry Confirms Great Heterogeneity of Isolates

Marina Nees  1 , Michael Hess  1 , Claudia Hess  1
Affiliations

Discrimination and Characterization of Escherichia coli Originating from Clinical Cases of Femoral Head Necrosis in Broilers by MALDI-TOF Mass Spectrometry Confirms Great Heterogeneity of Isolates

Marina Nees et al. Microorganisms. .

Abstract

Escherichia coli, a major pathogen in poultry production, is involved in femoral head necrosis (FHN) in broiler birds. So far, the characterization and relationship of isolates in context with this disease are mainly based on phenotypic and genotypic characteristics. Previously, an involvement of diverse E. coli isolates was reported. MALDI-TOF MS has been successfully applied investigating the clonality of different bacteria. Therefore, its application to characterize a well-defined selection of E. coli isolates beyond the species level was tested. The isolates were derived from clinical cases of FHN as well as from healthy birds. Reproducibility studies to perform a standardized protocol were done, and LB agar as well as the usage of fresh bacterial cultures proved most appropriate. No distinct clustering in context with the origin of isolates, association with lesions, serotype, or PFGE profile was found. Most of the isolates belonging to phylogroup B2 revealed a characteristic peak shift at 9716 m/z and could be attributed to the same MALDI-TOF MS cluster. The present study confirmed the previously found pheno- and genotypic heterogeneity of E. coli involved in FHN on the proteomic level. The study also highlights the need for standardized protocols when using MALDI-TOF MS for bacterial typing, especially beyond species level.

Keywords: E. coli; proteomics; standardization.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Reproducibility of spectra on different solid agars shown for E. coli isolate 16/01363-1 KnoLi 2 (LB: Luria Broth agar; COS: Columbia agar with sheep blood; COLI: Coliformen agar; MCK: MacConkey agar). Differences in intensity and quantity of peaks are shown within the range of 4000–6000 m/z.
Figure 2
Figure 2
Influence of sub-cultivation (fresh culture vs. sub-cultivation step 3) on spectra quality with a decrease of peak intensity. Shown for E. coli isolate 16/1523-3 KnoRe 2 (n = 20 spectra). (A) Within the range of 4000–6000 m/z; (B) Within the range of 6000–8000 m/z; (C) Within the range of 8000–10000 m/z.
Figure 2
Figure 2
Influence of sub-cultivation (fresh culture vs. sub-cultivation step 3) on spectra quality with a decrease of peak intensity. Shown for E. coli isolate 16/1523-3 KnoRe 2 (n = 20 spectra). (A) Within the range of 4000–6000 m/z; (B) Within the range of 6000–8000 m/z; (C) Within the range of 8000–10000 m/z.
Figure 2
Figure 2
Influence of sub-cultivation (fresh culture vs. sub-cultivation step 3) on spectra quality with a decrease of peak intensity. Shown for E. coli isolate 16/1523-3 KnoRe 2 (n = 20 spectra). (A) Within the range of 4000–6000 m/z; (B) Within the range of 6000–8000 m/z; (C) Within the range of 8000–10000 m/z.
Figure 3
Figure 3
Additional peak (6535 m/z) detected in all E. coli strains when stored at 4 °C (24 h: green lines, 48 h: orange lines, 72 h: violet lines) compared to the fresh culture (black).
Figure 4
Figure 4
Score oriented MSP dendrogram based on 107 E. coli isolates revealing four distinct clusters (distance level 500).
Figure 5
Figure 5
Clustering of 50 randomly selected E. coli isolates from eight farms based on their farm origin (A), the presence of femoral head necrosis (B), their serotype (C), their PFGE profile (D), and their phylogroup (E).
Figure 5
Figure 5
Clustering of 50 randomly selected E. coli isolates from eight farms based on their farm origin (A), the presence of femoral head necrosis (B), their serotype (C), their PFGE profile (D), and their phylogroup (E).
Figure 5
Figure 5
Clustering of 50 randomly selected E. coli isolates from eight farms based on their farm origin (A), the presence of femoral head necrosis (B), their serotype (C), their PFGE profile (D), and their phylogroup (E).
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