When Vibrio is not Vibrio: misidentified Aeromonas sp. associated with equine abortions and neonatal death, revealed by MALDI-TOF and 16S rRNA sequencing.

When identifying bacteria from equine abortions and neonatal deaths, some strains initially labeled as Vibrio species were actually Aeromonas species, as revealed by advanced testing methods like MALDI-TOF and 16S rRNA sequencing.

Background and Purpose of the Study

• The study aimed to investigate unexpected identifications of Vibrio species bacteria found in equine (horse) samples associated with abortions and neonatal deaths.
• Initial identifications using traditional biochemical testing (API 20NE) reported Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus, which was unusual as these are typically marine bacteria.
• Researchers suspected potential misidentification and therefore performed a re-evaluation using more advanced and specific bacterial identification techniques.

Methods Used for Re-evaluation

• A total of 27 bacterial strains were included, of which 25 were successfully cultured again for re-analysis.
• Identification methods used in re-evaluation included:
  • Biochemical testing (API 20NE and also API 20E, another biochemical panel)
  • MALDI-TOF mass spectrometry, a technique that identifies bacteria based on protein profiles
  • 16S rRNA gene sequencing, a molecular method that sequences a highly conserved bacterial gene
  • Vibrio-specific PCR assays, to detect DNA signatures unique to Vibrio species

Key Findings

• The original API 20NE tests confirmed the initial Vibrio identifications, indicating this biochemical test was not reliable in this case.
• All Vibrio-specific PCR tests were negative, supporting the suspicion that the strains were not Vibrio.
• MALDI-TOF and 16S rRNA sequencing analyses reclassified:
  • 24 out of 25 strains as Aeromonas species, a genus of bacteria normally found in freshwater and known to be opportunistic pathogens.
  • 1 strain as Acinetobacter species, another group of bacteria sometimes involved in infections.
• Use of API 20E biochemical panel could have helped avoid some misidentifications, correctly identifying more than half of the strains.

Significance of the Results

• Most of the misidentified strains came from equine fetuses and neonates in Normandy, France, and were related to infectious causes of death:
• The frequency of Aeromonas isolates increased significantly in recent years, with many strains isolated between 2014 and 2022, compared to very few or none in earlier periods (1996-2013).
• The findings suggest that Aeromonas species are emerging as important opportunistic pathogens implicated in equine abortions, stillbirths, and neonatal mortality.
• This has implications for diagnostic laboratories, which should consider using advanced identification methods when dealing with similar equine clinical samples to avoid misclassification and better guide treatment and epidemiological understanding.

Conclusions

• Traditional biochemical methods can lead to misidentification of bacteria, notably confusing Aeromonas species with Vibrio species in equine cases.
• Advanced methods like MALDI-TOF and 16S rRNA gene sequencing provide more accurate bacterial identification.
• Aeromonas species are emerging as significant pathogens in horses, particularly related to abortions and neonatal death.
• The study emphasizes the need to update diagnostic protocols and awareness among veterinarians and microbiologists dealing with equine infectious diseases.