In this study, we successfully applied the MLVA method to analyze uropathogenic E. coli strains isolated from clinical samples.
MLVA method does not require a high level of expertise and is capable to provide reasonable results about monitoring of outbreaks and clonal spread of bacterial isolates over a short period of time. It has been successfully applied to investigate the clonal relationship and epidemiology of clinical E. coli isolates [21, 25, 26]. In this study, the MLVA technique was able to detect 56 different genotypes among 70 E. coli strains isolated from patients with UTI in a major hospital in Tehran, Iran, indicating a high diversity of UPEC isolates genotypes.
Also, MLVA technique could classify the stains in 5 clonal complexes. In examining UPGMA dendrograms, uropathogenic E. coli isolates were classified according to the similarity in 4 clusters (clusters A to D). Each complex group represents an evolutionary relationship between the isolates. In other words, isolates belonging to each group have a genetic association with each other and are likely to have the same origin. In our study, Complex No. 1 was the largest clonal group. The strains associated with Complex No. 1 appeared to be the dominant pathogens of UPEC in the hospital under study.
In the present study, we used markers that can be easily separated by electrophoresis on the agarose gel. On the other hand, the evolution rate of different VNTRs differentiated the clonal relationships. Typically, loops that have larger replication sizes are slower than circuits that have smaller duplicate sizes. But loci with a slower evolutionary velocity are suitable for the investigation of clonal relationships between isolates that have evolved over a more extended period. Therefore, the type of VNTR locus is also in the analysis [27, 28].
The applied technique could classify E. coli isolates in 5 clonal complexes. Each complex group represents the evolutionary relationship between isolates. In other words, isolates belonging to each group have a genetic link with each other and are likely to have the same origin. In our study, Complex No. 1 was the largest clonal group. The strains in Complex No. 1 appeared to be the prevalent strains of UPEC disease, with about two-thirds of the population receiving all strains.
The ms32 has the highest number of alleles. The ms06 and ms23 loci have the smallest amount of alleles, and the ms06, ms09, ms21, and ms23 loci have null alleles. In the dendrogram analysis, The UPGMA of uropathogenic E. coli isolates is classified according to the similarity in 4 clusters (clusters A to D). We also found that to better evaluate VNTR loci for the study of epidemics or genotyping, more and more numbers of E. coli are needed. Besides, the use of a higher number of VNTR loci will be more beneficial for better differentiation of clonal groups.
Mellor et al. used the MLVA method to determine the genetic diversity of E. coli O157 producing Shiga toxin venom. There was some evidence that E. coli O157 strains collected from different regions may also show genetically diversity. To study the amount of this variation, Shiga toxin bacteriophage insertion sites (SBI), lineage-specific polymorphisms (LSPA-6), multilocus variable-number tandem repeat analysis (MLVA), and a tir 255T > A polymorphism were used to enquire 606 isolates from Australian and U.S. cattle and human. All analyses of collected data showed a significant connection between the source of origin and multilocus genotypes (P < 0.0001) and it confirms the evolution of enterohemorrhagic E. coli O157 in Australia and the United States .
MLVA was also used by Bustamante et al. for subtyping of 202 STEC isolates from different sources to get information about the genetic diversity of native STEC in Argentina. Two different MLVA protocols were used in their investigation, one for O157 and the other for a generic E. coli assay. As a result of their studies, MLVA was mentioned as a very sensitive tool to STEC subtyping and it showed the diversity in many serotypes .
Naseer et al. used the MLVA and multi-locus sequence typing (MLST) for subtyping of a total of 100 E. coli isolates from Spain, UK, Sweden, and Norway (n = 19, 20, 24 and 37 respectively) and the obtained data showed equivalence between the MLST profile and MLVA typing results .
In a study in Norway by Sekse et al, on 142 E. coli O26 isolated from 491 fecal, PFGE and MLVA was performed for investigation of the genetic relationship between the strains and 63 different PFGE profiles and 22 MLVA profiles were identified. Despite some differences in result between MLVA and PFGE, comparison by partition mapping showed good overall agreement between two methods. A few isolates of EPEC O26: H11 had a close relationship to STEC O26: H11 .
VNTRs regions are widely used for bacterial subtyping but it should not be overlooked that the hyper variability in VNTR loci can be problematic in trying to predict the relationships between isolates. It is also worth noting that knowing the rate of VNTR mutation and its effective factors can introduce the MLVA for legal epidemiological or microbiological research .
In a study in Japan on a total of 57 isolates from patients, 20 types were identified by using MLVA while they were classified into 23 PFGE types and the results were almost the same. In another study in the same city, a total of 24 isolates from different sources and one outbreak occurred in central parts of the city was investigated by MLVA an PFGE. The results showed a good correlation between these both methods. However, the MLVA typing proved to be a much more comfortable and more rapid method for the analysis of E. coli O157: H7 strain relatedness to identify transmission paths. Hence, MLVA method was appeared to be a convenient method for typical typing in many laboratories, including public health agencies and even in hospitals [33–36].