Molecular identification of the source of an uncommon tularaemia outbreak, Germany, autumn 2016

Abstract

Background In 2016, an uncommon outbreak of oropharyngeal tularaemia involving six human cases occurred in Germany, caused by drinking contaminated fresh must after a grape harvest. Aim We describe the details of laboratory investigations leading to identification of the outbreak strain, its characterisation by next generation sequencing (NGS) and the finding of the possible source of contamination. Methods We incubated wine samples in different media and on agar plates. NGS was performed on DNA isolated from young wine, sweet reserve and an outbreak case’s lymph node. A draft genome of the outbreak strain was generated. Vertebrate-specific PCRs using primers targeting the mitochondrial cytochrome b gene and product analyses by blast search were used to identify the putative source of must contamination. Results No bacterial isolate could be obtained. Analysis of the draft genome sequence obtained from the sweet reserve attributed this sequence to Francisella tularensis subsp. holarctica , belonging to the B.12/B.34 phylogenetic clade (erythromycin-resistant biovar II). In addition, the DNA sequence obtained from the case’s isolate supported our hypothesis that infection was caused by drinking contaminated must. The vertebrate-specific cytochrome b sequence derived from the young wine and the sweet reserve could be assigned to Apodemus sylvaticus (wood mouse), suggesting that a wood mouse infected with F. tularensis may have contaminated the must. Conclusion The discovered source of infection and the transmission scenario of F. tularensis in this outbreak have not been observed previously and suggest the need for additional hygienic precautionary measures when processing and consuming freshly pressed must.

Outbreak of Tularemia in a Group of Hunters in Germany in 2018—Kinetics of Antibody and Cytokine Responses

“…DNA extraction was performed from all throat swabs, one eye swab, and all EDTA and whole blood samples. In addition, DNA extraction was conducted out of bacterial colony material (A-1338) or lymph node material (A-1299/6) and bone marrow material (A-1299/7) from hare using the QIAGEN DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions as described recently [ 5 ]. DNA elution was performed in 100 µL of QIAGEN Elution Buffer (Qiagen).…”

“…Amplification was performed in an Applied Biosystems 7500 Real-Time PCR System (ThermoFisher Scientific, Langenselbold, Germany), each run including 40 cycles. The block PCR of the region of difference 1 (RD1-PCR) was used for the subspecies differentiation of F. tularensis as described recently [ 5 ]. The PCR was carried out using 15–100 ng of template DNA according to the protocol described by Broekhuijsen et al [ 8 ].…”

“…in combination with the extraction and amplification control targeting KoMa2 were performed with the following oligonucleotides and probes for mazG: mazG-F 5′-ggATCTgATCgTAgCgACggA-3′, mazG-R 5′-CgTCCAATgTCTCACTggAAAA-3′, Bru-mazG-TM-Multi: 5′-FAM-TgCCTTACATgggCgAACTCgAACgT-BHQ-1-3′ and for IS711: BruIS-F 5′-gCCATCAgATTgAATgCTTTTTTAAC-3′, BruIS-R 5′-AACCAgATCATAgCgCATgCg-3′; Bru-IS-TM-Multi 5′-Cy5-CgCTgCgATgCgAgAAAACATTgACC-BHQ-2-3′. The oligonucleotides and probes targeting KoMa2, the PCR assay, and conditions are described in Jacob et al [ 5 ].…”

“…Whole genome sequencing (WGS) from DNA of sample A-1338, one Francisella isolate from a hare belonging to the outbreak, was performed and analyzed regarding the biovar and genetic clade of the respective strain [ 9 ]. Library pool sequencing was performed in paired-end mode on a MiSeq Instrument (Illumina, San Diego, CA, USA); for the next generation sequencing of sample A-1338-1 Illumina sequencing in combination with Nextera XT library generation was used (Illumina), as recently described [ 5 ].…”

“…Hare hunters in endemic regions for tularemia are at special risk of the disease, typically due to underestimation or lack of awareness. In countries with a low prevalence of the disease, such as Germany [ 3 ], physicians might consider tularemia as a differential diagnosis quite late in the course of the disease, leading to a delay in specific diagnostics and treatment [ 4 , 5 , 6 ].…”