Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Acinetobacter baumannii and Interlaboratory Validation of an Optimized Multiple-Locus VNTR Analysis Typing Scheme

Abstract

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial outbreaks, mostly occurring in intensive care units. Due to the multiplicity of infection sources, reliable molecular fingerprinting techniques are needed to establish epidemiological correlations among A. baumannii isolates. Multiple-locus variable-number tandem-repeat analysis (MLVA) has proven to be a fast, reliable, and costeffective typing method for several bacterial species. In this study, an MLVA assay compatible with simple PCR-and agarose gel-based electrophoresis steps as well as with high-throughput automated methods was developed for A. baumannii typing. Preliminarily, 10 potential polymorphic variable-number tandem repeats (VNTRs) were identified upon bioinformatic screening of six annotated genome sequences of A. baumannii. A collection of 7 reference strains plus 18 well-characterized isolates, including unique types and representatives of the three international A. baumannii lineages, was then evaluated in a two-center study aimed at validating the MLVA assay and comparing it with other genotyping assays, namely, macrorestriction analysis with pulsed-field gel electrophoresis (PFGE) and PCR-based sequence group (SG) profiling. The results showed that MLVA can discriminate between isolates with identical PFGE types and SG profiles. A panel of eight VNTR markers was selected, all showing the ability to be amplified and good amounts of polymorphism in the majority of strains. Independently generated MLVA profiles, composed of an ordered string of allele numbers corresponding to the number of repeats at each VNTR locus, were concordant between centers. Typeability, reproducibility, stability, discriminatory power, and epidemiological concordance were excellent. A database containing information and MLVA profiles for several A. baumannii strains is available from http://mlva.u-psud.fr/.The Gram-negative bacterium Acinetobacter baumannii has emerged worldwide as a major nosocomial pathogen and a serious threat to patients in intensive care units (ICUs) (18,27). Hallmarks of A. baumannii infection are resistance to a broad range of antimicrobial agents, the tendency for epidemic spread, and long-term persistence in the hospital setting (4,8,27).Most A. baumannii clinical strains from multiple hospital outbreaks throughout the world have been referred to a few epidemic lineages. Two of these, called international clonal lineages I and II, were first identified in northwestern Europe in the early 1980s and then worldwide (8, 17), while a third clone, called international clonal lineage III, was later detected in France, Netherlands, Italy, and Spain, probably persisting in European hospitals since the 1990s (17, 34).Several typing methods have been developed to trace A. baumannii epidemiology from the local to the global scale. Among them, macrorestriction analysis with pulsed-field gel electrophoresis (PFGE) (23) and multilocus sequence typing (MLST) (2, 7) are currently considered the methods of choice for epidemi...

Significant spread of extensively drug-resistant Acinetobacter baumannii genotypes of clonal complex 92 among intensive care unit patients in a university hospital in southern Iran

“…Such information is also essential to enable infection control professionals to develop and balance control strategies aimed at reducing the spread and disease burden of multidrug-resistant (MDR) A. baumannii in the hospital setting. Several valuable techniques have been utilized and have contributed to our understanding of the genotypic characterization of A. baumannii isolates in the healthcare setting: PFGE [9,12,13], multilocus sequence typing (MLST) [12,14,15] and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) [13,16].…”

Comparison of Multiple-Locus Variable-Number Tandem-Repeat Analysis with Pulsed-Field Gel Electrophoresis Typing of Acinetobacter baumannii in China

“…The 223-bp flanking length of Abaum_0826 was mentioned by Pourcel et al in Table S2 of their article (10).…”

“…Taking into account the intrinsic variability of VNTRs, Pourcel et al proposed criteria for the assignment of strains to MLVA complexes: (i) identical L-repeat VNTR profiles and (ii) a maximum of three differences of no more than two repeats at S-repeat VNTR loci (10). It is obviously not suitable to determine the clonal relationships of some isolates used in this study, e.g., HN049 and HN051, which shared the same PFGE profile but differed at Abaum_0826 as null and 16, respectively (Fig.…”

“…1). Abaum_0826 is entirely located within coding regions of the pilus assembly protein (FilE) (10). To determine whether the absence of Abaum_0826 amplification was due to mismatches in the target region of the primers or due to complete deletion of this locus, two primers located in the conserved region of filE, FilE-F (CAGTTAAATCCTTCTGTTC AGC) and FilE-R (AACATCCACGGCAGAAGA), were designed.…”

“…MLVA typing using the MLVA-8 scheme was performed as described previously (10), except that the primers were fluorescently labeled and PCR products were sized on an automated DNA sequencer to accurately determine the number of tandem repeats. The polymorphism index of individual loci or combined VNTR loci was calculated using the Hunter-Gaston diversity index (HGDI) (18).…”