# In House Rapid, Simple Multiple‐Locus Variable‐Number Tandem Repeat Analysis (MLVA): A Reliable Tool for Enterobacter hormaechei Genotyping

## TL;DR

A new MLVA method was developed to quickly and cheaply track Enterobacter hormaechei strains, useful for infection control but not a full replacement for whole-genome sequencing.

## Contribution

A novel MLVA protocol targeting eight VNTR loci for rapid and cost-effective genotyping of E. hormaechei.

## Key findings

- The MLVA protocol showed discriminatory power comparable to MLST for E. hormaechei genotyping.
- MLVA distinguished some strains within the same sequence type and identified unique profiles in potential outbreaks.
- The method is a useful first-line tool for outbreak detection but requires WGS for high-resolution analysis.

## Abstract

Enterobacter hormaechei, a prominent species within the Enterobacter cloacae complex, is a significant cause of nosocomial infections and is frequently associated with multidrug resistance. Rapid genomic comparison helps guide timely infection control measures. This study aimed to develop a simple and rapid multiple‐locus variable‐number tandem‐repeat analysis (MLVA) protocol for epidemiological surveillance of E. hormaechei. Eight variable‐number tandem‐repeat (VNTR) regions were selected for amplification using multiplex PCR, followed by gel electrophoresis. The method's discriminatory power was evaluated on 46 unrelated strains from 22 French hospitals. Then, suspected related strains from three potential outbreaks, including ESBL‐ and/or NDM‐producing isolates were compared. An independent collection of 22 VIM‐producing strains was also analyzed. Whole‐genome sequencing (WGS) was used as the gold standard. Among 46 unrelated E. hormaechei strains, representing the five subspecies, MLVA and MLST showed similar discriminatory power (36 MLVA profiles vs. 33 STs, Hunter and Gaston diversity indices 0.9833 vs. 0.9824, respectively). Isolates with different ST typically had distinct MLVA profiles, except for three instances where different STs shared similar profiles. In STs represented by multiple strains, MLVA sometimes distinguished strains sharing the same ST. Among the three potential outbreak, epidemic strains exhibited unique MLVA profiles, with genetic distances of 0–11 SNPs using WGS, while unrelated isolates had different MLVA profiles, indicating this technique's potential as an effective screening tool for clonal groups. Similar results were observed for VIM‐producing E. hormaechei, with consistent MLVA profiles within the same STs. The MLVA protocol developed is a rapid, cost‐effective method for E. hormaechei epidemiological investigations that can quickly rule out unrelated strains. However, highly discriminatory techniques like WGS remain necessary when profiles are similar.

We developed a new MLVA protocol targeting eight VNTR loci for the rapid and cost‐effective surveillance of Enterobacter hormaechei. Tested on diverse clinical isolates, it showed a discriminatory power comparable to MLST and was able to distinguish some strains within the same sequence type. This method offers a valuable first‐line tool for outbreak detection, although WGS remains necessary for high‐resolution analysis.