Tc toxin activation requires unfolding and refolding of a ß-propeller.

Tc toxins secrete toxic enzymes into host cells using a unique syringe-like injection mechanism. They are composed of three subunits, TcA, TcB and TcC. TcA forms the translocation channel and the TcB-TcC heterodimer functions as a cocoon that shields the toxic enzyme. Binding of the cocoon to the channel triggers opening of the cocoon and translocation of the toxic enzyme into the channel. Here we show in atomic detail how the assembly of the three components activates the toxin. We find that part of the cocoon completely unfolds and refolds into an alternative conformation upon binding. The presence of the toxic enzyme inside the cocoon is essential for its subnanomolar binding affinity for the TcA subunit. The enzyme passes through a narrow negatively charged constriction site inside the cocoon, probably acting as an extruder that releases the unfolded protein with its C terminus first into the translocation channel.

Expanding the Ajudazol Cytotoxin Scaffold: Insights from Genome Mining, Biosynthetic Investigations, and Novel Derivatives.

Myxobacteria have proven to be a rich source of natural products, but their biosynthetic potential seems to be underexplored given the high number of biosynthetic gene clusters present in their genomes. In this study, a truncated ajudazol biosynthetic gene cluster in Cystobacter sp. SBCb004 was identified using mutagenesis and metabolomics analyses and a set of novel ajudazols (named ajudazols C-J, 3-10, respectively) were detected and subsequently isolated. Their structures were elucidated using comprehensive HR-MS and NMR spectroscopy. Unlike the known ajudazols A (1) and B (2), which utilize acetyl-CoA as the biosynthetic starter unit, these novel ajudazols were proposed to incorporate 3,3-dimethylacrylyl CoA as the starter. Ajudazols C-J (3-10, respectively) are characterized by varying degrees of hydroxylation, desaturation, and different glycosylation patterns. Two P450-dependent enzymes and one glycosyltransferase are shown to be responsible for the hydroxylation at C-8, the desaturation at C-15 and C-33, and the transfer of a d-ß-glucopyranose, respectively, based on mutagenesis results. One of the cytochrome P450-dependent enzymes and the glycosyltransferase were found to be encoded by genes located outside the biosynthetic gene cluster. Ajudazols C-H (3-8, respectively) exhibit cytotoxicity against various cancer cell lines.

Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial.

Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori-derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential.

Gene Organization, Expression, and Localization of Ribotoxin-Like Protein Ageritin in Fruiting Body and Mycelium of Agrocybe aegerita.

The edible mushroom Agrocybe aegerita produces a ribotoxin-like protein known as Ageritin. In this work, the gene encoding Ageritin was characterized by sequence analysis. It contains several typical features of fungal genes such as three short introns (60, 55 and 69 bp) located at the 5' region of the coding sequence and typical splice junctions. This sequence codes for a precursor of 156 amino acids (~17-kDa) containing an additional N-terminal peptide of 21 amino acid residues, absent in the purified toxin (135 amino acid residues; ~15-kDa). The presence of 17-kDa and 15-kDa forms was investigated by Western blot in specific parts of fruiting body and in mycelia of A. aegerita. Data show that the 15-kDa Ageritin is the only form retrieved in the fruiting body and the principal form in mycelium. The immunolocalization by confocal laser scanning microscopy and transmission electron microscopy proves that Ageritin has vacuolar localization in hyphae. Coupling these data with a bioinformatics approach, we suggest that the N-terminal peptide of Ageritin (not found in the purified toxin) is a new signal peptide in fungi involved in intracellular routing from endoplasmic reticulum to vacuole, necessary for self-defense of A. aegerita ribosomes from Ageritin toxicity.

Beyond diphtheria toxin: cytotoxic proteins of Corynebacterium ulcerans and Corynebacterium diphtheriae.

Diphtheria toxin is one of the best investigated bacterial toxins and the major virulence factor of toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans strains. However, also diphtheria toxin-free strains of these two species can cause severe infections in animals and humans, indicating the presence of additional virulence factors. In this study, we present a first characterization of two proteins with cytotoxic effect in corynebacteria. A putative ribosome-binding protein (AEG80717, CULC809_00177), first annotated in a genome sequencing project of C. ulcerans strain 809, was investigated in detail together with a homologous protein identified in C. diphtheriae strain HC04 (AEX80148, CDHC04_0155) in this study. The corresponding proteins show striking structural similarity to Shiga-like toxins. Interaction of wild-type, mutant and complementation as well as overexpression strains with invertebrate model systems and cell lines were investigated. Depending on the presence of the corresponding genes, detrimental effects were observed in vivo in two invertebrate model systems, Caenorhabditis elegans and Galleria mellonella, and on various animal and human epithelial and macrophage cell lines in vitro. Taken together, our results support the idea that pathogenicity of corynebacteria is a multifactorial process and that new virulence factors may influence the outcome of potentially fatal corynebacterial infections.

Evaluation of the anti-oxidant property and cytotoxic potential of the metabolites extracted from the bacterial isolates from mangrove Forest and saltern regions of South India.

The objective was to evaluate the anticancer and antioxidant activities of the methanolic extracts of halophilic bacteria, isolated from soil samples of Marakkanam saltern and Pichavaram mangrove forest, India. Radical Scavenging activity, reducing power, and metal ion chelation ability was used to evaluate the antioxidant potential of the metabolic extracts, whereas cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The methanolic extract of Bacillus VITPS7 exhibited significant antioxidant property. Bacillus VITPS14 and Bacillus VITPS16 extracts were cytotoxic against HeLa cell lines but not to A549 cell lines. Colorimetric assays for the presence of specific metabolites including, total flavonoid and ß carotene content were performed. The presence of these specific classes of metabolites was confirmed by UV-Visible spectrophotometry, Nuclear Magnetic Resonance (NMR) spectroscopy and Gas Chromatography-Mass Spectrometry (GC-MS). Specific NMR signals revealed the presence of aromatic and unsaturated metabolites whereas GC-MS analysis indicated the presence of metabolites such as squalene and methyl hexadeconate. The present study thus reports for the first time the presence of squalene in Bacillus VITPS12 and Planococcus maritimus VITP21, in addition to other metabolites that contribute to the observed antioxidant or/and cytotoxicity, thus revealing the therapeutic potential of these selected halophilic bacterial isolates.

Two novel cyclic hexapeptides from the genetically engineered Actinosynnema pretiosum.

Two novel cyclic hexapeptides designated actinosynneptides A (1) and B (2), together with three tryptophan containing diketopiperazines, namely cyclo(L-Trp-L-Trp) (3), cyclo(L-Trp-N-MeL-Trp) (4), and cyclo(N-MeL-Trp-N-MeL-Trp) (5), were isolated from the culture of the genetically engineered strain HGF052::asm18 derived from Actinosynnema pretiosum ATCC31565. Their structures were elucidated on the basis of spectroscopic analyses and single-crystal X-ray diffractions. Compound 1 is the first example of 3-amino-6-hydroxy-2-piperidone-containing cyclic peptides, and 1 and 2 showed moderate cytotoxic activities against HeLa and PC3 cell lines.

Improved insecticidal activity of a recombinant baculovirus expressing spider venom cyto-insectotoxin.

Baculoviruses have a long history of safe use as specific, environmentally friendly insecticides that provide alternatives to chemical pesticides for controlling insect pests. However, their use has been limited by several factors, particularly their slow pathogenicity. In this study, we constructed a recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) and an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) that expressed an insect-specific cyto-insectotoxin (Cit1a) from the venom of the central Asian spider Lachesana tarabaevi. Cit1a is a comparatively long linear cytolytic molecule that contains a predicted α-helix structure composed of two short membrane-acting antimicrobial peptides (MAMPs) that are joined together in a "head-to-tail" shape. Cit1a fused to polyhedrin gene (polh) (polh-cit1a) was expressed in the nuclei as polyhedra in silkworm larvae, Bm5 and Sf9 cells. An early death of Bm5 and Sf9 cells by recombinant BmNPV/Polh-Cit1a and AcMNPV/Polh-Cit1a was observed compared with control viruses that lacked the toxin gene. The infected cells showed a loss of cytoplasm, membrane integrity, and structural changes, suggesting that recombinant baculovirus-infected cells were killed by the necrosis caused by Cit1a. In addition, the BmNPV/Polh-Cit1a showed a significant reduction in the median lethal time (LT50) against silkworm larvae compared with those of control BmNPV that lacked the cit1a gene.

[Secondary Metabolites from Marine Microorganisms. I. Secondary Metabolites from Marine Actinomycetes].

Review represents data on new active metabolites isolated from marine actinomycetes published in 2007 to 2014. Marine actinomycetes are an unlimited source of novel secondary metabolites with various biological activities. Among them there are antibiotics, anticancer compounds, inhibitors of biochemical processes.

Penicillium verruculosum SG: a source of polyketide and bioactive compounds with varying cytotoxic activities against normal and cancer lines.

A newly isolated fungus Penicillium verruculosum SG was evaluated for the production and characterization of bioactive colored secondary metabolites using solid-state fermentation along with their cytotoxic activities against normal and cancer cell lines. Logical fragmentation pattern following column chromatography, thin layer chromatography and liquid chromatography and mass spectrometry of crude culture filtrate of fungus revealed the presence of different polyketide pigments and other bioactive compounds. Cytotoxicity of the selected colored fractions of fungal filtrate containing different compounds revealed IC50 (µg/ml) values ranging from 5 to 100. It was significantly higher in case of orevactaene (5 + 0.44) and monascorubrine followed by pyripyropene (8 + 0.63) against cancer cell line KA3IT. Overall, these compounds considerably showed less toxicity toward normal cell lines NIH3T3, HSCT6, HEK293 and MDCK. XRD of a yellow crystalline compound (224.21 m/z) confirmed its 3-dimensional structure as phenazine 1 carboxylic acid (C13H8N2O2) (broad spectrum antibiotic), and it is first time reported in fungi.

Bacteriocin production, antibiotic susceptibility and prevalence of haemolytic and gelatinase activity in faecal lactic acid bacteria isolated from healthy Ethiopian infants.

The objective of this study was to characterise lactic acid bacteria (LAB) isolated from faecal samples of healthy Ethiopian infants, with emphasis on bacteriocin production and antibiotic susceptibility. One hundred fifty LAB were obtained from 28 healthy Ethiopian infants. The isolates belonged to Lactobacillus (81/150), Enterococcus (54/150) and Streptococcus (15/150) genera. Lactobacillus species were more abundant in the breast-fed infants while Enterococcus dominated the mixed-fed population. Bacteriocin-producing LAB species were isolated from eight of the infants. Many different bacteriocins were identified, including one new bacteriocin from Streptococcus salivarius, avicin A (class IIa) from Enterococcus avium, one class IIa bacteriocin from Enterococcus faecalis strains, one unknown bacteriocin from E. faecalis and two unknown bacteriocins from Lactobacillus fermentum strains and the two-peptide gassericin T from Lactobacillus gasseri isolate. Susceptibility tests performed for nine antibiotics suggest that some lactobacilli might have acquired resistance to erythromycin (3 %) and tetracycline (4 %) only. The streptococci were generally antibiotic sensitive except for penicillin, to which they showed intermediate resistance. All enterococci were susceptible to ampicillin while 13 % showed penicillin resistance. Only one E. faecalis isolate was vancomycin-resistant. Tetracycline (51 %) and erythromycin (26 %) resistance was prevalent among the enterococci, but multidrug resistance was confined to E. faecalis (47 %) and Enterococcus faecium (33 %). Screening of enterococcal virulence traits revealed that 2 % were ß-haemolytic. The structural genes of cytolysin were detected in 28 % of the isolates in five enterococcal species, the majority being E. faecalis and Enterococcus raffinosus. This study shows that bacteriocin production and antibiotic resistance is a common trait of faecal LAB of Ethiopian infants while virulence factors occur at low levels.

Small Bacillus cereus ATCC 14579 subpopulations are responsible for cytotoxin K production.

AIMS: Bacillus cereus diarrhoeal food poisoning can be caused by several potential enterotoxins, including the nonhaemolytic enterotoxin (Nhe), haemolysin BL (Hbl) and cytotoxin K (CytK). To get more insights into the CytK expression, a fluorescent reporter strain was created for CytK expression. METHODS: Bacillus cereus ATCC 14579 was used as the reporter strain that contained the cyan fluorescent protein (CFPopt) gene under control of the cytK promoter. Transcription of enterotoxin genes nheB, hblC and cytK was assessed by messenger RNA analysis (RT-qPCR), and their full expression was assessed by immunological protein detection in the case of Nhe and Hbl and fluorescence microscopy in the case of CytK, using the reporter gene CFPopt. RESULTS: Transcription of enterotoxins Nhe, Hbl and CytK showed similar kinetics with a peak during the late exponential growth phase. Toxin expression of the reporter strain was unaltered in comparison with the wild type. However, fluorescence, and thus CytK expression, only occurred in a small (1-2%) portion of the cell population. CONCLUSIONS: These results suggest that a small subpopulation of B. cereus ATCC 14579 is responsible for CytK production in a homogeneous monoculture. SIGNIFICANCE AND IMPACT OF THE STUDY: Future research is warranted to determine whether genetically homogeneous B. cereus populations utilize differential gene expression for other toxins and virulence genes than CytK and whether this also applies to other B. cereus strains. If so, differential expression of toxin genes could be used by these bacteria to increase the fitness and survival chances of their population by diversification and specialization into different subpopulations.

Cell vacuolation induced by Haemophilus influenzae supernatants in HEp-2 cells.

Haemophilus influenzae belongs to respiratory tract microbiota. We observed vacuoles formation in previous studies with H. influenzae culture supernatants, so in this work we characterised that cytotoxic effect. We observed an abundant production of acidic cytoplasmic vacuoles due to the presence of a "vacuolating factor" in H. influenzae supernatants which was characterised as thermolabile. Greatest vacuolating activity was observed when utilizing the fraction > 50 kDa. The presence of a large number of vacuoles in HEp-2 cells was verified by transmission electron microscopy and some vacuoles were identified with a double membrane and/or being surrounded by ribosomes. These results suggest similar behaviour to that of vacuolating effects described by autotransporter proteins an undescribed cytotoxic effect induced by H. influenzae.

Growth suppression of human hepatocellular carcinoma xenografts by a monoclonal antibody CH12 directed to epidermal growth factor receptor variant III.

Human hepatocellular carcinoma (HCC) is considered difficult to cure because it is resistant to radio- and chemotherapy and has a high recurrence rate after curative liver resection. Epidermal growth factor receptor variant III (EGFRvIII) has been reported to express in HCC tissues and cell lines. This article describes the efficacy of an anti-EGFRvIII monoclonal antibody (mAb CH12) in the treatment of HCC xenografts with EGFRvIII expression and the underlying mechanism of EGFRvIII as an oncogene in HCC. The results demonstrated that CH12 bound preferentially to EGFRvIII with a dissociation constant (K(d)) of 1.346 nm/liter. In addition, CH12 induces strong antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in Huh7-EGFRvIII (with exogenous expression of EGFRvIII) and SMMC-7721 (with endogenous expression of EGFRvIII) cells. Notably, CH12 significantly inhibited the growth of Huh7-EGFRvIII and SMMC-7721 xenografts in vivo with a growth inhibition ratio much higher than C225, a U. S. Food and Drug Administration-approved anti-EGFR antibody. Treatment of the two HCC xenografts with CH12 significantly suppressed tumor proliferation and angiogenesis. Mechanistically, in vivo treatment with CH12 reduced the phosphorylation of constitutively active EGFRvIII, Akt, and ERK. Down-regulation of the apoptotic protectors Bcl-x(L), Bcl-2, and the cell cycle regulator cyclin D1, as well as up-regulation of the cell-cycle inhibitor p27, were also observed after in vivo CH12 treatment. Collectively, these results indicate that the monoclonal antibody CH12 is a promising therapeutic agent for HCC with EGFRvIII expression.

Unfolding distinguishes the Vibrio cholerae cytolysin precursor from the mature form of the toxin.

Vibrio cholerae cytolysin (VCC) is a potent cytolytic toxin that induces colloid osmotic lysis of its target eukaryotic cells by forming transmembrane oligomeric ß-barrel channels. VCC is secreted by the bacteria as an inactive precursor (Pro-VCC) and is subsequently activated by proteolytic removal of an N-terminal "Pro-domain", thus generating the active form of the toxin (Mature-VCC). Earlier studies have indicated an intramolecular chaperone-like role of the Pro-domain favoring efficient secretion of the toxin from the periplasm into the extracellular space. However, the exact role of the Pro-domain in the VCC structure--function mechanism remains unclear. Here, we have compared the Pro-VCC and Mature-VCC molecules in terms of their structural and conformational properties. We have studied unfolding of the two variants of the VCC molecule in response to an array of denaturing conditions, including low-pH, chemical denaturant and heat-induced unfolding. Pro-VCC shows a more profound tendency to unfold in response to such denaturing conditions compared to Mature-VCC. Biophysical characterization of the isolated Pro-domain further suggests that the increased unfolding propensity of Pro-VCC does not arise because of an increased level of unfolding of the Pro-region itself. Altogether, our results imply that a natively folded architecture of the Pro-VCC molecule with sufficient structural and conformational plasticity presumably allows it to adopt a suitable configuration that is possibly required for its efficient secretion and subsequent proteolytic maturation under physiological conditions.

The production and detoxification of a potent cytotoxin, nitric oxide, by pathogenic enteric bacteria.

The nitrogen cycle is based on several redox reactions that are mainly accomplished by prokaryotic organisms, some archaea and a few eukaryotes, which use these reactions for assimilatory, dissimilatory or respiratory purposes. One group is the Enterobacteriaceae family of Gammaproteobacteria, which have their natural habitats in soil, marine environments or the intestines of humans and other warm-blooded animals. Some of the genera are pathogenic and usually associated with intestinal infections. Our body possesses several physical and chemical defence mechanisms to prevent pathogenic enteric bacteria from invading the gastrointestinal tract. One response of the innate immune system is to activate macrophages, which produce the potent cytotoxin nitric oxide (NO). However, some pathogens have evolved the ability to detoxify NO to less toxic compounds, such as the neuropharmacological agent and greenhouse gas nitrous oxide (N2O), which enables them to overcome the host's attack. The same mechanisms may be used by bacteria producing NO endogenously as a by-product of anaerobic nitrate respiration. In the present review, we provide a brief introduction into the NO detoxification mechanisms of two members of the Enterobacteriaceae family: Escherichia coli and Salmonella enterica serovar Typhimurium. These are discussed as comparative non-pathogenic and pathogenic model systems in order to investigate the importance of detoxifying NO and producing N2O for the pathogenicity of enteric bacteria.

Evidence of isoprenoid precursor toxicity in Bacillus subtilis.

The mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways for isoprenoid biosynthesis both culminate in the production of the two-five carbon prenyl diphosphates: dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). These are the building blocks for higher isoprenoids, including many that have industrial and pharmaceutical applications. With growing interest in producing commercial isoprenoids through microbial engineering, reports have appeared of toxicity associated with the accumulation of prenyl diphosphates in Escherichia coli expressing a heterologous MVA pathway. Here we explored whether similar prenyl diphosphate toxicity, related to MEP pathway flux, could also be observed in the bacterium Bacillus subtilis. After genetic and metabolic manipulations of the endogenous MEP pathway in B. subtilis, measurements of cell growth, MEP pathway flux, and DMAPP contents suggested cytotoxicity related to prenyl diphosphate accumulation. These results have implications as to understanding the factors impacting isoprenoid biosynthesis in microbial systems.

Selection of phytotoxin producing rhizobacteria.

In order to select phytotoxin producing rhizobacteria to control weed plants, twenty five bacterial strains previously isolated from the rhizospheres of various plants were grown in a liquid medium and, after cell removal by centrifugation, the liquid phases were freeze-dried and the products were extracted with ethyl acetate/methanol. The extracts were concentrated to dryness under vacuum and dissolved in water and sucrose solution to be submitted to in vitro assays of lettuce (Lactuca sativa L.) seed germination and wheat (Triticum aestivum L.) coleoptile growth. Although most samples affected coleoptile growth, only those from four strains reduced lettuce seed germination. Two strains of Bacillus cereus, one strain of B. pumilus and one of Stenotrophoonas altophilia were the most promising microorganisms for producing phytotoxin and, consequently, for the development of new weed control products.

Optimization of L-asparaginase production from endophytic Fusarium proliferatum using OFAT and RSM and its cytotoxic evaluation.

L-asparaginase from endophytic Fusarium proliferatum (isolate CCH, GenBank accession no. MK685139) isolated from the medicinal plant Cymbopogon citratus (Lemon grass), was optimized for its L-asparaginase production and its subsequent cytotoxicity towards Jurkat E6 cell line. The following factors were optimized; carbon source and concentration, nitrogen source and concentration, incubation period, temperature, pH and agitation rate. Optimization of L-asparaginase production was performed using One-Factor-At-A-Time (OFAT) and Response surface methodology (RSM) model. The cytotoxicity of the crude enzyme from isolate CCH was tested on leukemic Jurkat E6 cell line. The optimization exercise revealed that glucose concentration, nitrogen source, L-asparagine concentration and temperature influenced the L-asparaginase production of CCH. The optimum condition suggested using OFAT and RSM results were consistent. As such, the recommended conditions were 0.20% of glucose, 0.99% of L-asparagine and 5.34 days incubation at 30.50 °C. The L-asparaginase production of CCH increased from 16.75 ± 0.76 IU/mL to 22.42 ± 0.20 IU/mL after optimization. The cytotoxicity of the crude enzyme on leukemic Jurkat cell line recorded IC50 value at 33.89 ± 2.63% v/v. To conclude, the enzyme extract produced from Fusarium proliferatum under optimized conditions is a potential alternative resource for L-asparaginase.

Cytotoxic effects of Klebsiella oxytoca strains isolated from patients with antibiotic-associated hemorrhagic colitis or other diseases caused by infections and from healthy subjects.

Antibiotic-associated hemorrhagic colitis (AAHC) is associated with Klebsiella oxytoca. This study analyzed whether cytotoxic properties are linked to specific subtypes of K. oxytoca. Klebsiella isolates from stools of AAHC patients, healthy carriers, and diarrhea patients as well as from infections of other organs were investigated. Cytotoxic effects on human epithelial cells were limited to the species K. oxytoca and were not detectable for any other Klebsiella species. Isolates from AAHC patients and from stools showed the highest proportion of cytotoxic strains. Urinary or respiratory tract isolates exhibited no cytotoxicity. Macrorestriction profiling of strains revealed no genetic relationships of AAHC isolates or the cytotoxic phenotype but identified that different K. oxytoca strains with different cytotoxic behaviors may be prevalent in the same AAHC patient. Under laboratory conditions, cytotoxicity was maximally effective after exponential bacterial growth and then declined despite the continued viability of K. oxytoca cells in culture. Given its capacity to induce AAHC and that a high proportion of stool isolates tested cytotoxin positive, we argue that K. oxytoca should be considered an opportunistic pathogen if detected in stools. The ability to induce disease after antibiotic treatment most likely represents an overgrowth of the toxin-producing bacterium due to an alteration of the normal colonic microflora.