A multiplex oligonucleotide ligation-PCR as a complementary tool for subtyping of Salmonella Typhimurium

Subtyping below the serovar level is essential for surveillance and outbreak detection and investigation of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant 1,4,[5],12:i:- (S. 1,4,[5],12:i:-), frequent causes of foodborne infections. In an attempt to overcome the intrinsic shortcomings of currently used subtyping techniques, a multiplex oligonucleotide ligation-PCR (MOL-PCR) assay was developed which combines different types of molecular markers in a high throughput microsphere suspension array. The 52 molecular markers include prophage genes, amplified fragment length polymorphism (AFLP) elements, Salmonella genomic island 1 (SGI1), allantoinase gene allB, MLVA locus STTR10, antibiotic resistance genes, single nucleotide polymorphisms (SNPs) and phase 2 flagellar gene fljB. The in vitro stability of these markers was confirmed in a serial passage experiment. The validation of the MOL-PCR assay for subtyping of S. Typhimurium and S. 1,4,[5],12:i:- on 519 isolates shows that the method is rapid, reproducible, flexible, accessible, easy to use and relatively inexpensive. Additionally, a 100 % typeability and a discriminatory power equivalent to that of phage typing were observed, and epidemiological concordance was assessed on isolates of 2 different outbreaks. Furthermore, a data analysis method is provided so that the MOL-PCR assay allows for objective, computerised data analysis and data interpretation of which the results can be easily exchanged between different laboratories in an international surveillance network

MLVA as a tool for public health surveillance of human Salmonella Typhimurium : prospective study in Belgium and evaluation of MLVA loci stability

Surveillance of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is generally considered to benefit from molecular techniques like multiple-locus variable-number of tandem repeats analysis (MLVA), which allow earlier detection and confinement of outbreaks. Here, a surveillance study, including phage typing, antimicrobial susceptibility testing and the in Europe most commonly used 5-loci MLVA on 1,420 S. Typhimurium isolates collected between 2010 and 2012 in Belgium, was used to evaluate the added value of MLVA for public health surveillance. Phage types DT193, DT195, DT120, DT104, DT12 and U302 dominate the Belgian S. Typhimurium population. A combined resistance to ampicillin, streptomycin, sulphonamides and tetracycline (ASSuT) with or without additional resistances was observed for 42.5% of the isolates. 414 different MLVA profiles were detected, of which 14 frequent profiles included 44.4% of the S. Typhimurium population. During a serial passage experiment on selected isolates to investigate the in vitro stability of the 5 MLVA loci, variations over time were observed for loci STTR6, STTR10, STTR5 and STTR9. This study demonstrates that MLVA improves public health surveillance of S. Typhimurium. However, the 5-loci MLVA should be complemented with other subtyping methods for investigation of possible outbreaks with frequent MLVA profiles. Also, variability in these MLVA loci should be taken into account when investigating extended outbreaks and studying dynamics over longer periods

Two successive cesarean deliveries through separate posterior and anterior hysterotomy due to asymptomatic uterine torsion. A case report

Uterine torsion during pregnancy is a rare obstetrical complication that can be life-threatening for both mother and child. Although torsion usually presents with acute, non-specific symptoms, it can also occur without any symptoms and pose no immediate health threat. Ultimately, the diagnosis of torsion is often made only during cesarean section.We present a case of a patient who underwent two successive cesarean sections through separate posterior and anterior hysterotomy due to asymptomatic uterine torsion in both cases. During the first cesarean section an incision was inadvertently made in the posterior segment of the uterus. At the second cesarean section the degree of rotation was very different and an anterior hysterotomy was performed. The patient made an uneventful recovery after both deliveries.If access to the lower anterior uterine segment is not safely available due to uterine torsion, a hysterotomy in the lower posterior uterine segment can be performed. The risk of rupture of a posterior hysterotomy scar in future pregnancies is unclear

Development of a molecular subtyping method for Salmonella Typhimurium combining different types of markers in a Luminex xTAG assay

Subtyping of Salmonella enterica serovar Typhimurium, a frequent cause of food-borne diseases, is critical for surveillance and identification, tracking and ultimately confinement of outbreaks. We present a proof of concept of a molecular subtyping method for S. Typhimurium which combines different types of markers in a multiplex assay with detection by the Luminex xTAG technology in a high-throughput format. Selected markers include, amongst others, markers based on AFLP fragments, prophage genomes, sequence repeats, antibiotic resistance genes and SNPs. The optimal multiplex design was evaluated and a ligation dependent amplification (LDA) assay was found to be the most efficient strategy to target the selected markers. The in vitro stability of the selected markers was verified. To determine the discriminatory ability, S. Typhimurium isolates of most common phage types in Belgium were subjected to the proposed subtyping method. The capability to identify outbreaks was tested with strains from two outbreaks in Belgium that occurred in 2008 and 2011. The resulting profiles were examined for correlation with phage types, MLVA and antimicrobial resistance profiles. The potential of the method based on the xTAG technology to provide a new subtyping scheme for S. Typhimurium will be discussed.status: publishe

Design of multiplex assays for molecular subtyping of pathogens with the Luminex xMAP technology

Multiplex assays are a powerful tool for molecular subtyping of pathogens, which is crucial for rapid diagnosis, surveillance and identification and containment of outbreaks. The Luminex xMAP technology allows detection of up to 500 different analytes per sample in a high-throughput format through a liquid bead suspension array. Different types of Luminex assays can be envisaged. Each assay has its advantages and limitations, some of which are related to the inherent design of the primers and probes used. Software that predicts melting temperatures, hybridization structures and specificity for different types of oligonucleotides, and thereby simulating the multiplex assays in silico can facilitate the design of multiplex assays by reducing time and cost of experimentation. We have compared two commercial software packages for the development and in silico simulation of a ligation dependent amplification (LDA) assay and a direct hybridization assay for the subtyping of a pathogen. The main findings, limitations and challenges will be discussed.status: publishe

Molecular subtyping of Salmonella Typhimurium combining different types of markers in a multiplex liquid bead suspension array

Subtyping of Salmonella enterica subsp. enterica serovar Typhimurium, a frequent cause of food-borne diseases, is critical for surveillance and identification, tracking and ultimately confinement of outbreaks. We present a preliminary proof of concept of a molecular subtyping method for S. Typhimurium which combines different types of markers in a multiplex assay with detection on a liquid bead suspension array in a high-throughput format. Selected markers include, amongst others, markers based on AFLP fragments, prophage genomes, sequence repeats, antibiotic resistance genes and SNPs. The optimal multiplex design was evaluated and a ligation dependent amplification (LDA) assay was found to be the most efficient strategy to target the selected markers. The in vitro stability of the selected markers was verified. To determine the discriminatory ability, S. Typhimurium isolates of most common phage types in Belgium were subjected to the proposed subtyping method. The capability to identify outbreaks was tested with strains from two outbreaks in Belgium that occurred in 2008 and 2011. The resulting profiles were examined for correlation with phage types, MLVA and antimicrobial resistance profiles. The potential of the new method to provide a new subtyping scheme for S. Typhimurium will be discussed.status: publishe

Optimized MOL-PCR for characterization of microbial pathogens

Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium

A multiplex oligonucleotide ligation-PCR as a complementary tool for subtyping of Salmonella Typhimurium

Subtyping below the serovar level is essential for surveillance and outbreak detection and investigation of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant 1,4,[5],12:i:- (S. 1,4,[5],12:i:-), frequent causes of foodborne infections. In an attempt to overcome the intrinsic shortcomings of currently used subtyping techniques, a multiplex oligonucleotide ligation-PCR (MOL-PCR) assay was developed which combines different types of molecular markers in a high-throughput microsphere suspension array. The 52 molecular markers include prophage genes, amplified fragment length polymorphism (AFLP) elements, Salmonella genomic island 1 (SGI1), allantoinase gene allB, MLVA locus STTR10, antibiotic resistance genes, single nucleotide polymorphisms (SNPs) and phase 2 flagellar gene fljB. The in vitro stability of these markers was confirmed in a serial passage experiment. The validation of the MOL-PCR assay for subtyping of S. Typhimurium and S. 1,4,[5],12:i:- on 519 isolates shows that the method is rapid, reproducible, flexible, accessible, easy to use and relatively inexpensive. Additionally, a 100 % typeability and a discriminatory power equivalent to that of phage typing were observed, and epidemiological concordance was assessed on isolates of 2 different outbreaks. Furthermore, a data analysis method is provided so that the MOL-PCR assay allows for objective, computerised data analysis and data interpretation of which the results can be easily exchanged between different laboratories in an international surveillance network.status: publishe

Evaluation of whole genome sequencing for routine subtyping of Salmonella Typhimurium for routine surveillance and outbreak investigation

status: publishe