Simultaneous quantification of enterotoxins tilimycin and tilivalline in biological matrices using HPLC high resolution ESMS2 based on isotopically 15N-labeled internal standards

Glabonjat, Ronald A.; Kitsera, Maksym; Unterhauser, Katrin; Lembacher-Fadum, Christian; Högenauer, Christoph; Raber, Georg; Breinbauer, Rolf; Zechner, Ellen L.

doi:10.1016/j.talanta.2020.121677

Cited by 9 publications

(11 citation statements)

References 45 publications

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“…In this colonization model without a nonsteroidal anti-inflammatory drug (NSAID), mice are infected but do not develop colitis, which allows mice to survive for reliable measurements of fecal indole and tilimycin concentrations ( 35 ). The fecal metabolites of colonized mice were measured daily for 1 week postinfection using MS as described previously ( 43 ). Indole remained under the limit of quantitation throughout the experiment for animals colonized with the Δ tnaA mutant ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Bacterial Indole as a Multifunctional Regulator of Klebsiella oxytoca Complex Enterotoxicity

Ledala

Malik

Rezaul

et al. 2022

mBio

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Section: Resultsmentioning

confidence: 99%

“…Quantification of bacteria in inocula and in feces of colonized animals (CFU per gram of stool) was performed by plating of serial dilutions on tryptic soy (CASO) agar (40 μg/mL of kanamycin). Fecal metabolites were extracted and quantified as previously described ( 43 ).…”

Section: Methodsmentioning

confidence: 99%

Bacterial Indole as a Multifunctional Regulator of Klebsiella oxytoca Complex Enterotoxicity

Ledala

Malik

Rezaul

et al. 2022

mBio

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“…K. oxytoca is a pathobiont of the intestinal microbiota that can produce TV cytotoxin ( Beaugerie et al, 2003 ; Högenauer et al, 2006 ; Zollner-Schwetz et al, 2015 ; Glabonjat et al, 2021 ). After penicillin treatment, alteration of the enteric microbiota occurs, and overgrowth of K. oxytoca is originated in the colon causing a severe dysbiosis ( Schneditz et al, 2014 ; Hajishengallis and Lamont, 2016 ; Dornisch et al, 2017 ; von Tesmar et al, 2018 ; Alexander et al, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

cAMP Receptor Protein Positively Regulates the Expression of Genes Involved in the Biosynthesis of Klebsiella oxytoca Tilivalline Cytotoxin

et al. 2021

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Klebsiella oxytoca is a resident of the human gut. However, certain K. oxytoca toxigenic strains exist that secrete the nonribosomal peptide tilivalline (TV) cytotoxin. TV is a pyrrolobenzodiazepine that causes antibiotic-associated hemorrhagic colitis (AAHC). The biosynthesis of TV is driven by enzymes encoded by the aroX and NRPS operons. In this study, we determined the effect of environmental signals such as carbon sources, osmolarity, and divalent cations on the transcription of both TV biosynthetic operons. Gene expression was enhanced when bacteria were cultivated in tryptone lactose broth. Glucose, high osmolarity, and depletion of calcium and magnesium diminished gene expression, whereas glycerol increased transcription of both TV biosynthetic operons. The cAMP receptor protein (CRP) is a major transcriptional regulator in bacteria that plays a key role in metabolic regulation. To investigate the role of CRP on the cytotoxicity of K. oxytoca, we compared levels of expression of TV biosynthetic operons and synthesis of TV in wild-type strain MIT 09-7231 and a Δcrp isogenic mutant. In summary, we found that CRP directly activates the transcription of the aroX and NRPS operons and that the absence of CRP reduced cytotoxicity of K. oxytoca on HeLa cells, due to a significant reduction in TV production. This study highlights the importance of the CRP protein in the regulation of virulence genes in enteric bacteria and broadens our knowledge on the regulatory mechanisms of the TV cytotoxin.

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“…Direct confirmation of toxin production by the K oxytoca isolate is considered necessary for the diagnosis of AAHC (1). Currently, K oxytoca toxin production is confirmed by culture based MTT assay from in vitro cultures of strains, or directly by mass spectrometry-based metabolomic analyses (14,29,30), methods that are currently not available in routine diagnostic laboratories. The capacity to synthesize toxins in culture provides important information about the potential virulence of the isolate, but does not necessarily indicate whether toxins have been produced in substantial amounts in patients.…”

Section: Discussionmentioning

confidence: 99%

“…Tilimycin and tilivalline extraction from stool samples was performed as described previously (14). Briefly, samples were thawed at room temperature before chemical extraction.…”

Section: Toxin Detection In Stool Samplesmentioning

confidence: 99%

Toxin‐Producing Klebsiella oxytoca in Healthy Infants

Greimel¹,

Stampfer²,

Leitner

et al. 2021

J. pediatr. gastroenterol. nutr.

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Objectives: Klebsiella oxytoca is a gastrointestinal pathobiont with the potential to produce the toxins tilivalline and tilimycin, which cause antibiotic-associated hemorrhagic colitis. Overgrowth of toxigenic K oxytoca has recently been implicated in necrotizing enterocolitis. K oxytoca colonizes 2-9% of healthy adults, however, there is no systematic data on colonization in healthy children. We investigated K oxytoca colonization and its toxigenic properties in healthy infants. Methods: We sampled stool of healthy infants and determined K oxytoca colonization using stool culture and PCR ( pehX). Toxin in stool was measured with HPLC/high-resolution mass spectrometry. K oxytoca isolates were typed using multi-locus sequence typing (MLST) and Koxytoca toxin PCR (npsA/B). Cytotoxin production of isolates was analyzed by MTT assay. Results: K oxytoca was detected in 30 of 61 infants (49%) using stool culture and in 45 of 61 (73%) using PCR ( pehX). Toxin marker PCR (npsA/B) was positive in 66% of stool samples positive for K oxytoca PCR. Stool toxin levels were too low for quantitation but traces of tilivalline were detected. Contrarily, 49% of K oxytoca isolates demonstrated toxicity in the MTT assay. MLST revealed 36 distinct sequence types affiliated with all known K oxytoca sequence type clusters (A, B1 and B2). Conclusions: More than 70% of healthy infants were colonized with K oxytoca. Toxin quantities in stool of colonized healthy infants were below detection level, yet half of the isolates produced toxin in vitro demonstrating their pathobiont potential. The high occurrence of toxigenic K oxytoca in healthy infants has to be considered for future disease association studies.

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Simultaneous quantification of enterotoxins tilimycin and tilivalline in biological matrices using HPLC high resolution ESMS2 based on isotopically 15N-labeled internal standards

Cited by 9 publications

References 45 publications

Bacterial Indole as a Multifunctional Regulator of Klebsiella oxytoca Complex Enterotoxicity

Bacterial Indole as a Multifunctional Regulator of Klebsiella oxytoca Complex Enterotoxicity

cAMP Receptor Protein Positively Regulates the Expression of Genes Involved in the Biosynthesis of Klebsiella oxytoca Tilivalline Cytotoxin

Toxin‐Producing Klebsiella oxytoca in Healthy Infants

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