Detection of enterotoxin and protease genes among Hungarian clinical Bacteroides fragilis isolates

Accepted Manuscript Detection of enterotoxin and protease genes among Hungarian clinical Bacteroides fragilis isolates Károly Péter Sárvári, József Sóki, Miklós Iván, Cecilia Miszti, Krisztina Latkóczy, Szilvia Zsóka Melegh, Edit Urbán PII:

S1075-9964(17)30149-X

DOI:

10.1016/j.anaerobe.2017.07.005

Reference:

YANAE 1777

To appear in:

Anaerobe

Received Date: 10 May 2017 Revised Date:

19 July 2017

Accepted Date: 23 July 2017

Please cite this article as: Péter Sárvári Ká, Sóki Jó, Iván Mikló, Miszti C, Latkóczy K, Zsóka Melegh S, Urbán E, Detection of enterotoxin and protease genes among Hungarian clinical Bacteroides fragilis isolates, Anaerobe (2017), doi: 10.1016/j.anaerobe.2017.07.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Research article

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Detection of enterotoxin and protease genes among Hungarian clinical Bacteroides

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fragilis isolates

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Károly Péter Sárvária, József Sókia, Miklós Ivánb, Cecilia Misztic, Krisztina Latkóczyd,

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Szilvia Zsóka Meleghe, Edit Urbána

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Institute of Clinical Microbiology, University of Szeged, Szeged, Hungary

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Institute of Laboratory Medicine, Semmelweis University, Budapest, Hungary

SYNLAB Ltd., Budapest, Hungary

Institute of Medical Microbiology and Immunology, University of Pécs, Pécs, Hungary

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Running title: Investigation of enterotoxin and protease genes of Hungarian B. fragilis strains

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Keywords: bft, cfiA, bfp1-4, fpn, PCR

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Institute of Medical Microbiology, University of Debrecen, Debrecen, Hungary

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Correspondence:

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Edit Urbán PharmD, PhD, Professor

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Department of Clinical Microbiology

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University of Szeged, Szeged, Hungary

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H-6725 Szeged, Semmelweis Str. 6.

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Tel./Fax: +36 62 54 5712 1

ACCEPTED MANUSCRIPT 1

Abstract Bacteroides fragilis as a commensal bacterium is a member of the human intestinal

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flora, but as an opportunistic pathogen it can cause serious infections as well. Some of them,

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harbouring an enterotoxin gene (bft), may cause diarrhoea mainly in young children. Recently

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it has been shown that a member of C11 proteases called fragipain (fpn) can activate the

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enterotoxin, while C10 protease (bfp) is suspected of playing an important role in the

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invasiveness of the B. fragilis isolates. The objective of this study was to investigate the

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prevalence and distribution of the bft isotypes in 200 Hungarian B. fragilis isolates collected

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recently; and in a subset of 72 strains, we wanted to determine the prevalence of bfp1-4 and

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fpn genes in bft-positive and bft-negative strains. Using the MALDI-TOF MS cfiA

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identification project file, 19 B. fragilis strains belonging to Division II were identified and

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the presence of the cfiA gene was confirmed by RT-PCR. Twenty two (13.0%) B. fragilis

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isolates turned out to be bft gene positive by RT-PCR; 20 isolates harboured bft-1 and six bft-

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2 isotypes, but no bft-3 isotype containing strains were found. A melting curve analysis and

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the PCR-RFLP were performed to differentiate between the bft-1 and bft-2 isotypes confirmed

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by sequencing. Thirty eight strains harboured bfp1, 58 isolates contained bfp2 gene, while 17

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isolates proved positive for bfp3. Morever, no bfp4 positive isolate was found, and some of

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the B. fragilis strains tested harboured two or three bfp isotypes simultaneously. Among the

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26 bft-positive strains, 24 contained the fpn gene, which confirms the role of fragipain in the

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activation of B. fragilis enterotoxin. In experiments, a significant negative correlation between

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fpn and cfiA was demonstrated (p<0.000), a positive correlation was found between bfp2 and

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fpn genes (p=0.0000803), and a negative correlation between bfp2 and cfiA genes (p=0.011).

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Introduction

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ACCEPTED MANUSCRIPT In the normal human gut flora, one well-known constituent is the Bacteroides genus.

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Different members of the genus are frequently isolated in clinical specimens as pathogenic

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bacteria as well. The most important species of this genus is Bacteroides fragilis. Enterotoxin

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producing B. fragilis (ETBF) strains were first isolated from the faeces of neonatal lambs with

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diarrhoea in 1984 by Myers et al. [1]. In humans, ETBF was also defined as one of the

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possible causative agents of diarrhoea in children aged one to five years and to a lesser extent

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in adults [2]. The prevalence of ETBF strains among B. fragilis isolates from extraintestinal

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clinical samples has also been shown to lie between 9.0% and 20.3% [3,4]. The enterotoxin,

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also known as fragilysin, is a zinc-dependent metalloprotease of 20 kDa, which cleaves E-

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cadherin protein of tight junctions [5,6]. The first diagnostic method for the detection of toxin

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production of B. fragilis isolates was the use of the HT29/C1 intestinal immortalised cell line

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on which toxigenic strains can induce a cytopathic effect [7]. Three isotypes of fragilysin

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gene (bft) have been recognised so far, namely bft-1, bft-2 and bft-3, the latter of which is

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typically found in the Far East [8]. Some studies demonstrated that the increased prevalence

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of ETBF strains is associated with inflammatory bowel diseases (Crohn’s disease, ulcerative

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colitis), while coeliac disease and the possible pathogenic role of these isolates has also been

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suggested in the aetiology of colorectal cancer [9,10]. Some B. fragilis harbour class B zinc

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metallo-beta-lactamase gene (cfiA). Not all cfiA-positive B. fragilis strains express

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phenotypical carbapenem resistance, because appropriate insertion element (IS) is needed for

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full expression of the cfiA gene [11]. Members of the cysteine peptidase families are widely

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distributed in pro- and eukaryotes as well. Two cytein peptidase types with pathogenetic role

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in B. fragilis have been recently described: bfp1-4 genes encoding C10 and fpn gene encoding

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C11 peptidase (fragipain). The C10 peptidase is suspected of having a potential role in the

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pathogenesis in immune dysregulation, inflammatory bowel disease, irritable bowel

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syndrome. The fragipain activates B. fragilis enterotoxin and may play a role in the

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ACCEPTED MANUSCRIPT progression of sepsis [12,13]. The objectives of this study were the followings: (1) to

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investigate the prevalence of the cfiA gene, the bft gene and their isotypes in recently collected

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B. fragilis isolates, (2) to compare these data with previous findings, (3) in a subset of 72

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strains to look for the prevalence of C10 (bfp1-4) and C11 peptidase (fpn), (4) comparing the

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prevalence data of cfiA, bft, bfp1-4 and fpn genes.

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Material and methods

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Strains involved in this study

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In a multicentre study involving five Hungarian microbiological laboratories, 200

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B. fragilis isolates, originating from clinically relevant extraintestinal samples, were collected

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between January and June 2016. The strains were stored in BHI (Brain Heart Infusion) broth

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containing 20% glycerol at -80 °C. In our investigations, isolates were cultured on Schaedler

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blood agar (bioMérieux, France) for 48 h, at 37 °C in an anaerobic chamber (Perkin Elmer,

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UK) in an atmosphere containing 85% N2, 10% CO2, 5% H2. The identification was

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confirmed by a MALDI-TOF MS analysis (Bruker Biotyper, Germany), using Biotyper

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Version 3.0 software. All the isolates produced a high confidence species level identification

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(log(score) of ≥2.000) as B. fragilis. Using a typing scheme described earlier, all the isolates

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were placed into two categories, namely Division II (exclusively positive for the cfiA

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carbapenemase gene) and Division I using the MALDI-TOF Biotyper 3.1 software containing

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„cfiA identification project file” made at Bruker Daltonik (Bremen, Germany) [14,15].

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PCR detection of cfiA, bft, bfp1-4, and fpn genes

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The presence of the cfiA gene in B. fragilis strains belonging to Division II was

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confirmed by RT-PCR, as described by Eitel et al. [16]. Using the procedure outlined by Soki 4

ACCEPTED MANUSCRIPT et al., a RT-PCR was performed for the detection of bft gene in the case of all 200 B. fragilis,

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using bftF and bftR primers [11]. For the typing of the bft gene, an internal fragment of bft

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gene was amplified with BTT1 and BTT2 primers and a melting point analysis was

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performed. These PCR products were purified using the Gel/PCR DNA Fragment Extraction

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Kit (Geneaid Biotech Ltd., Taiwan) and investigated with RFLP as well to differentiate the

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isotypes of the gene [10]. During the RFLP analysis, the following positive controls were

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used: B. fragilis R19811 (bft-1), B. fragilis ATCC 43858 (bft-2), and B. fragilis GAI 96462

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(bft-3). The expected lengths of the restricted fragments are 839 and 310 bp for bft-1, 575,

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453 and 111 bp for bft-2 and 839, and 189 and 111 bp for bft-3 [3,13]. An internal fragment of

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three bft-1 and three bft-2 harbouring B. fragilis isolates was sequenced with the ABI

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BigDye® Terminator Version 3.1 kit in the Series Genome Analyser 3500 (Life

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Technologies, USA) to confirm the possible separation of bft-1 and bft-2 harbouring isolates

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based on the melting-point analysis using the RT-PCR. The prevalence of the bfp1-4 and fpn

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genes in the C10 and C11 proteases, respectively, was investigated in a subset of 26 bft-

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positive and 46 bft–negative B. fragilis strains by RT-PCR using the following control strains:

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B. fragilis 638R (bfp1-4) and B. fragilis ATCC 43859 (fpn) [3,12,13]. All of the RT-PCR tests

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were carried out using the StepOne RT-PCR machine (Applied Biosystems, USA). The PCR

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set-up for the detection of cfiA, bft, bfp1-4 and fpn genes are summarized in Table 1.

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The data were analysed by Fischer’s Exact and Spearman correlation tests in order to

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look for differences using SigmaPlot 12, and the significance level was set to 0.05 (i.e.

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p<0.05).

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Statistical evaluation

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ACCEPTED MANUSCRIPT 1

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Results

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Here, we investigated 200 B. fragilis strains which were collected from five

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microbiological centres. The distribution of the clinical samples from which the B. fragilis

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strains were isolated, can be seen in Table 2. Most of the strains originated from superficial or

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deep wounds (51.0%), while intra-abdominal samples (36.5%) and just 12 (6%) strains were

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isolated from a septic patient’s blood culture. Based on the results of the MALDI-TOF MS

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analysis, 19 (9.5%) belonged to B. fragilis Division II, and all of these strains harboured the

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cfiA gene confirmed by RT-PCR (Table 2). Out of the 200 B. fragilis isolates, 26 (13.0%)

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turned out to harbour the bft gene detected by RT-PCR. Twenty proved to be bft-1 and 6 bft-2

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isotypes after performing PCR-RFLP. We did not find any isolate carrying the bft-3 isotype

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among the ETBF strains. A melting curve analysis also differentiated between the bft-1 and

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bft-2 isotypes here; and the average temperature of the bft-1-positive strains was 80.1±0.4°C

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and that of the bft-2-positive strains was 81.2±0.2 °C) ( Fig. 1). The separation of bft-1 and

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bft-2 strains via obtained from by the melting-cure analysis was confirmed by sequencing of

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three bft-1 and three bft-2 positive B. fragilis isolates, which were randomly selected during

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the experiments. A good correlation was observed between the results obtained by the melting

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curve analysis to differentiate bft-1 and bft-2 and the search for the typical bands by PCR-

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RFLP to differentiate between the bft-1 and bft-2 isotypes. During this study, a rare B. fragilis

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isolate was also found that originated from an abscess sample (B. fragilis SZ54) which

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contained the cfiA and the bft-1 allele simultaneously (not shown).

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To investigate the presence of bfp1-4 (the C10 protease gene) and fpn (the C11 protease

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gene), a subset of 72 B. fragilis isolates (26 ETBF strains and 46 non-ETBF strains) was 6

ACCEPTED MANUSCRIPT analysed via RT-PCR. Here, the distribution of the C10 protease genes was the following: 38

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strains harboured bfp1, 58 isolates contained bfp2 gene; while 17 isolates proved positive for

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bfp3 and no bfp4 positive strain was detected. Nine strains simultaneously harboured bfp1,

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bfp2 and bfp3 genes; 22 proved positive for bfp1 and bfp2; while five isolates contained bfp2

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and bfp3; and 1 isolate proved positive for bfp1 and bfp3 (Table 3). Among the 24 of the 26

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bft-positive strains (92.3%) containing the fpn gene; while 36 of the 46 bft-negative isolates

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(78.3%) did harbour the fpn gene ass well (Table 3). Among the cfiA-positive isolates, three

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harbouring bfp-1 and two bfp-2 were identified; while among the cfiA-negative strains 35

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proved positive for bfp-1, 56 for bfp-2 and 17 for bfp-3 (Table 4). Looking for significant

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positive or negative correlations among the genes investigated among the 72 selected B.

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fragilis isolates, none of the 63 fpn-positive isolates harboured the cfiA gene, so a significant

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negative correlation was demonstrated between cfiA and fpn (p<0.000) genes. A significant

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positive correlation was observed between the bfp2 and fpn genes (p=0.0000803) and a

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negative correlation was found between the bfp2 and cfiA genes (p=0.011) (data not shown).

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These new findings were slightly unexpected and in the literature very little data can be found

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concerning the prevalence or distribution of these genes together.

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Discussion

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The asymptomatical carrying rate of ETBF strains in the gut has been shown to be

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between 6.2% and 20.0% [4,17,18,19], while the prevalence of ETBF isolates among

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extraintestinal B. fragilis strains lies between 9.0% and 38.2% [3,4,8,20]. The percentage of

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enterotoxin producing B. fragilis strains isolated from blood cultures was also significant.

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Claros et al. investigated 63 B. fragilis isolates obtained from septic patients and the rate of

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the ETBF strains was found to be 19.0% [21]; however, Kato et al. reported a 28.1% rate of 7

ACCEPTED MANUSCRIPT fragilysin production among blood culture isolates [22]. In our study, 200 B. fragilis

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extraintestinal clinical isolates were investigated and 26 (13.0%) harboured the bft gene,

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which is much less than that found in a previous study (25.3%) for Hungarian isolates [4].

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Our data showed that the majority of the isolates contained the bft-1 allele (20/26, 76.9%),

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while 23.1% contained the bft-2 (6/26) allele and there no bft-3 harbouring strain was

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detected. Scotto d’Abusco et al. [23] investigated intestinal and extraintestinal ETBF strains

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and reported that the most common isotype was bft-1 (10/16, 62.5%), while 25.0% harboured

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the bft-2 isotype (4/16) and 12.5% harboured the bft-3 (2/16) isotype. Our data revealed a

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quite similar distribution of the different isotypes, but the apparent lack of bft-3 might be due

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to the different geographical distribution of the alleles. The metalloprotease activity of these

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isolates on the cell culture of HT29/C1 cells (not tested in this study) suggests that there is a

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potential invasivity of the bft-positive B. fragilis isolates in different types of infections [24].

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In order to identify the three bft isotypes and separate them, PCR-RFLP was applied.

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What is more, using the RT-PCR to detect the genes, we found that a melting curve analysis

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was also able to distinguish between the bft-1 and bft-2 isotypes. To confirm the validity of

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the melting analysis, the sequencing of randomly chosen three bft-1 and three bft-2 harbouring

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B. fragilis isolates was performed. The complete identity using the published sequences in the

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Genbank of the bft subtypes helped confirm the reliability of this technique. A good

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correlation was also observed between the results obtained from the melting curve analysis

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and those got via the PCR-RFLP, which were then used to differentiate between the bft-1 and

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bft-2 isotypes.

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The high discrimination power of MALDI-TOF MS between B. fragilis Division I and

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II was demonstrated by Nagy et al. and Wybo et al. [14,25]. To verify earlier results, all the

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200 B. fragilis isolates were investigated with the MALDI-TOF Biotyper 3.1 software cfiA

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identification project file developed by Bruker Daltonik and used by Fenyvesi et al. [15].

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ACCEPTED MANUSCRIPT Nineteen isolates (9.5%) were placed into Division II. RT-PCR confirmed the presence of the

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cfiA gene in all of the B. fragilis strains belonging to Division II. Our results indicated a

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slightly higher rate of cfiA-harbouring B. fragilis strains compared to the baseline range of

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2.0-8.85% [16,25,26]. Among the 200 isolates we were able to identify a strain called B.

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fragilis SZ54 that harboured the cfiA and bft genes simultaneously, which is a rare finding,

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and it was described earlier by Soki et al. [27].

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In the cysteine peptidase families, seventy two cystein peptidase families have been

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identified so far of these, 43 families belong to nine exclusive cysteine peptidase

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superfamilies (clans) namely, CA, CD, CE, CF, CH, CL, CM, CN and CO [28]. These

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cysteine peptidases can be found in plants, animals, fungi, humans, bacteria and parasites as

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well. Four members of the C10 family cysteine proteases (bfp1-4) belonging to the CA

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superfamily were discovered in B. fragilis strains, and two of these were homologues of

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streptococcal pyrogenic exotoxin B. It has been suggested that these enzymes might be

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involved in the pathogenesis of inflammatory bowel disease or irritable bowel syndrome [12].

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In our study, 34 strains harboured two bfp isotypes, while nine isolates contained

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simultaneously three bfp isotypes. Among the bft-positive and –negative B. fragilis strains

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investigated, the bfp2 isotype was the most prevalent. Also a positive correlation was found

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between the bfp2 gene and fpn gene. The significance of these findings in the pathogenesis of

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B. fragilis requires further investigation.

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Recently it has been demonstrated that B. fragilis strains produce a special protease

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called fragipain (Fpn), which is a member of the C11 family (belonging to the CD clan of

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cysteine peptidase). Fragipain is required for the activation of fragilysin, and Choi et al. also

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hypothesised that activation of fragilysin may play a role in the progression of sepsis caused

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by ETBF srains [13]. According to our results, amongst the 26 bft-positive strains 24

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contained the fpn gene, which confirms the key role of fragipain in the activation of B. fragilis

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ACCEPTED MANUSCRIPT enterotoxin. Nevertheless, 36 bft-negative B. fragilis isolates also contained the fpn gene.

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Moreover, fragipain might have a further role in the cell function and pathogenesis in the

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sepsis, because members of the CD clan have several functions such as cell proliferation, the

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regulation of cell death pathways, inflammation, the clearance of insoluble aggregates,

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virulence, cell-cycle progression, and endoplasmic reticulum stress [29].

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In a study, performed in 1997 by our institute, 25.3% of the B. fragilis strains were bft-

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positive, determined by morphological changes on the HT-29 cell line [4]. In 2006 Nagy et al.

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reported of 275 B. fragilis strains 8.7% bft-harbouring isolates investigated by RT-PCR [30].

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During the last one and half decades the number of Bacteroides fragilis isolates in our

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institute has increased almost threefold (2002: 63, 2016: 166 isolates). Considered this fact, a

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comprehensive epidemiological survey was performed, which was the only in the last decade

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that investigated the prevalence of the bft gene and its isotypes and their correlation with

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bfp1-4, fpn and cfiA genes in Hungary. In the literature very little data can be achived

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concerning the prevalence or distribution of these genes together so far.

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Conflict of interest declaration: No.

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Funding: None.

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Competing interest: None.

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Ethical approval: Not required.

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References

2

[1]

Myers L. L., Firehammer B. D., Shoop D. S., Border M. M.: Bacteroides fragilis: a

3

possible cause of acute diarrheal disease in newborn lambs Infect. Immun.

4

1984;44:241-244 [2]

Akpinar M., Aktaş E., Cömert F., Külah C., Sümbüloğlu: Evaluation of the prevalence

RI PT

5 6

of enterotoxigenic Bacteroides fragilis and the distribution bft gene subtypes in

7

patients with diarrhoea Anaerobe 2010;16:505-509 [3]

Claros M. C., Claros Z. C., Tang Y. J., Cohen S. H., Silva J. Jr., Goldstein E. J. C., Rodloff A. C.: Occurence of Bacteroides fragilis enterotoxin gene-carrying strains in

9

SC

8

Germany and the United States J. Clin. Microbiol. 2000;38:1996-1997

10

[4]

Szoke I., Dósa E., Nagy E.: Enterotoxigenic Bacteroides fragilis in Hungary Anaerobe 1997;3:87-89

13

[5]

Obiso R. J., Azghani A. O., Wilkins T. D.: The Bacteroides fragilis toxin fragilysin disrupts the paracellular barrier of epithelial cells Infect. Immun. 1997;65:1431-1439

14 15

M AN U

11 12

[6]

Wu S., Lim K. C., Huang J., Saidi R. F., Sears C. L.: Bacteroides fragilis enterotoxin cleaves the zonula adherens protein, E-cadherin Proc.Natl. Acad. Sci. USA

17

1998;95:14979-14984

18

[7]

TE D

16

Weikel C. L., Grieco F. D., Reuben J., Myers L. L., Sack R. B.: Human colonic epithelial cells, HT29/C1, treated with crude Bacteroides fragilis enterotoxin

20

dramatically alter their morphology Infect. Immun. 1992;60:321-327 [8]

Identification of the third metalloprotease toxin gene in extraintestinal isolates of

22

Bacteroides fragilis Infect. Immun. 1999;67:4945-4949

23 24

Chung G. T., Franco A. A., Wu S., Rhie G. E., Cheng R., Oh H. B., Sears C. L.:

AC C

21

EP

19

[9]

Ulger Toprak N., Yagci A., Gulluoglu B. M., Akin M. L. Demirkalem P., Celenk T.,

25

Soyletir G.: A possible role of Bacteroides fragilis enterotoxin in the aetiology of

26

colorectal cancer Clin. Microbiol. Infect. 2006;12:782-786

27

[10]

Prindiville T., Sheikh R., Cohen S., Tang Y., Cantrell M., Silva J.: Bacteroides fragilis

28

enterotoxin gene sequences in patients with inflammatory bowel disease Emerg.

29

Infect. Dis. 2000;6:171-174 11

ACCEPTED MANUSCRIPT 1

[11]

Sóki J., Edwards R., Hedberg M., Fang H., Nagy E., Nord C. E.: Examination of

2

cfiA-mediated carbapenem resistance in Bacteroides fragilis strains from a European

3

antibiotic susceptibility survey Int. J. Antimicrob. Ag. 2006;28:497-502

4

[12]

Choi V. M., Herrou J., Hecht A. L., Teoh W. P., Turner J., R., Crosson S., Wardenburg J. B.: Activation of Bacteroides fragilis toxin by a novel bacterial

6

protease contributes to anaerobic sepsis Nat. Med. 2016;22(5):563-567

7

[13]

RI PT

5

Thornton R. F., Kagawa T. F., O’Toole P. W., Cooney J. C.: The dissemination of C10 cysteine protease genes in Bacteroides fragilis by mobile genetic elements

9

BMC Microbiol. 2010;10:122-137

10

[14]

SC

8

Nagy E., Becker S., Sóki J., Urbán E., Kostrzewa M.: Differentiation of division I (cfiA-negative) and division II (cfiA-positive) Bacteroides fragilis strains by matrix-

12

assisted laser desorption/ionization time-of-flight mass spectrometry J. Med.

13

Microbiol. 2011;60:1584-90

14

[15]

M AN U

11

Fenyvesi V. S., Urbán E., Bartha N., Ábrók M., Kostrzewa M.: Use of MALDITOF/MS for routine detection of cfiA gene-positive Bacteroides fragilis strains

16

Anaerobe 2014;44(5):474-475

17

[16]

TE D

15

Eitel Zs., Sóki J., Urbán E., Nagy E.: The prevalence of antibiotic resistance genes in Bacteroides fragilis group strains isolated in different European countries

19

Anaerobe 2013;21:43-49

20

[17]

EP

18

Nurver U. , Rajdendarm D., Yagci A., Gharbia S., Shah H., N., Gulluoglu B. M., Akin L. M., Demirkalem P., Celenk T., Soyletir: The distribution of the bft alleles among

22

enterotoxigenic Bacteroides fragilis from stool specimens and extraintestinal sites Anaerobe 2006;12:71-74

23 24

[18]

Pantosi A., Menozzi M. G., Frate A., Sanfilippo L., D’Ambrosio F., Malpeli M.:

Detection of enterotoxigenic Bacteroides fragilis and its toxin in stool samples from

25

adults in children from Italy Clin. Infect. Dis. 1997;24:12-16

26 27

AC C

21

[19]

Luczak M., Obuch-Woszczatyński P., Pituch H., Leszczyński P., Martirosian G.,

28

Patrick S., Paxton I., Wintermans R. G. F., Dubreuil G., Meisel-Mikołajczyk: Search

29

for enterotoxin gene in Bacteroides fragilis isolated from clinical specimens in Poland,

30

Great Britain, the Netherlands and France Med. Sci. Monit. 2001;7:222-225 12

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[20]

Mundy L. M., Sears C. L.: Detection of toxin production by Bacteroides fragilis: assay

2

development and screening of extraintestinal clinical isolates Clin. Infect. Dis.

3

1996;23:269

4

[21]

Claros M. C., Claros Z. C., Hecht D. W., Citron D. M., Goldstein E. J. C., Silva J. Jr., Tang-Feldman Y., Rodloff A. C.: Characterisation of the Bacteroides fragilis

6

pathogenicity island in human blood culture isolates Anaerobe 2006;12:17-22 [22]

fragilis with bacteremia Clin. Infect. Dis. 1996;83-86

8 9

Kato N., Kato H., Watanabe K., Ueno K.: Association of enterotoxigenic Bacteroides

[23]

Scotto d’Abusco A., del Grosso M., Censini S., Covacci A., Pantosti A.: The alleles of

SC

7

RI PT

5

the bft gene are distributed differently among enterotoxigenic Bacteroides fragilis

11

strains from human sources and can be present in double copies J. Clin. Microbiol.

12

2000;38:607-612

13

[24]

M AN U

10

Mocrief J. S., Obiso R., Jr., Barroso L. A., Kling J. J., Wright R. L., van Tassell R. L.,

14

Lyerly D. M., Wilkins T. D.: The enterotoxin of Bacteroides fragilis is a

15

metalloprotease Infect. Immun. 1995;63:175-181 [25]

Wybo I., De Bel A., Soetens O., Echahidi F., Vandoorslaer K., Van Cauwenbergh M.,

TE D

16

Piérard D.: Differentiation of cfiA-Negative and cfiA-Positive Bacteroides fragilis

18

Isolates by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass

19

Spectrometry J. Clin. Microbiol. 2011;49:1961-1964

20

[26]

EP

17

Soki J., Eitel Zs., Urban E., Nagy E.: Molecular analysis of the carbapenem and metronidazole resistance mechanisms of Bacteroides strains reported in a Europe-wide

22

antibiotic resistance survey Int. J. Antimicrob. Ag. 2013;41:122-125

23

[27]

Soki J., Eitel Zs., Terhes G., Nagy E., Urbán E.: Occurrence and analysis of rare cfiAbft doubly posiitive Bacteroides fragilis strains Anaerobe 2013;23:70-73

24 25

AC C

21

[28]

Cambra I., Garcia F. J., Martinez M.: Clan CD of cysteine peptidases as an example of

26

evolutionary divergencies in related protein families across plant clades Gene

27

2010;449:59-69

28 29

[29]

McLuskey K., Mottram J. C.: Comparative structural analysis of the caspase family with other clan CD cysteine peptidases Biochem. J. 2015;466:219-232 13

ACCEPTED MANUSCRIPT 1

[30] Nagy E., Urbán E., Sóki J., Terhes G., Nagy K.: The place of molecular methods in the diagnostics of human pathogenic anaerobic bacteria. A minireview Acta Microbiol.

3

Imm. H. 2006;53:183-194

AC C

EP

TE D

M AN U

SC

RI PT

2

14

ACCEPTED MANUSCRIPT Table 1.

Primers and PCR conditions of cfiA, bft, fpn and bfp1-4 genes

Gene

Primer

AATCGAAGGATGGGGTATGG

cfiA-R

CGGTCAGTGAATCGGTGAAT

bftF

CGAACTCGGTTTATGCAGTT

(RT-PCR) bftR

GGATACATCAGCTGGGTTGT

BTT1

CATGTTCTAATGAAGCTGATTC

(RFLP)

BTT2

ATCGCCATCTGCTGTTTCCC

fpn

C11_protease_F ATTCGGCCGATGCAAATGTG

95°C 5 min; 35 cycles 95°C for 15 sec; 56 °C for 1 min; 72 °C for 30 sec; melting 72-95 C°

95°C 5 min; 35 cycles 95°C for 15 sec; 56 °C for 1 min; 72 °C for 30 sec; melting 72-95 C°

M AN U

bft

95°C 10 min; 35 cycles 95°C for 15 sec; 59 °C for 30 sec; 72 °C for 75 sec; melting 72-95 C°

RI PT

bft

cfiA-F

PCR conditions

SC

cfiA

Primer sequence (5’ → 3’)

94°C 5 min; 30 cycles 94°C for 1 min; 54 °C for 1 min; 72 °C for 1min

C11_protease_R CGGAATCTCGGTAGGGAAC

bfp1

C10_protease_F1 GCGGTGAACAAAGAACGACA

bfp2

TE D

C10_protease_R1 TCGCCTGAGCAACTGCAATA

C10_protease_F2 CGTACCAATTGCAATTGCGC C10_protease_R2 AGCTCCCGTGGCTTTATCTT

C10_protease_F3 TTTGGAGTAGCAGCAGCAGA

EP

bfp3

bfp4

AC C

C10_protease_R3 TTTCTGGTTTCGGGTGTTTC

C10_protease_F4 TACAACGGTGTTGGTGCAAG

C10_protease_R4 ACACAAATGCGCCACTTCAT

95°C 10 min; 35 cycles 95°C for 15 sec; 59 °C for 30 sec; 72 °C for 75 sec; melting 72-95 C°

ACCEPTED MANUSCRIPT Table 2.

Source of the 200 B. fragilis isolates tested for the presence of cfiA and bft isotypes

Number of strains

Wound

Intraabdominal samples

2

Abscess other than intraabdominal Blood culture 4

Others Total

3

bft-positive

bft-1-positive

cfiA-positive

102 (51.0%)

16

11

5

8

73 (36.5%)

9

8

1

2

5 (2.5%)

0

0

12 (6.0%)

1

1

8 (4.0%)

0

200 (100.0%)

26 (13.0%)

1: superficial, surgical, ulcus cruris, decubitus, burn, fistula

20 (10.0%)

M AN U

3: maxillofacial surgery, urological, dermatological

TE D

4: gynaecological samples, middle ear, cerebrospinal fluid, pericardial fluid

EP

5

4

2: intraabdominal fluid, intraabdominal abscess, ascites, abdominal wound, bile

AC C

bft-2-positive

RI PT

1

6 (3.0%)

SC

Sample types

19 (9.5%)

ACCEPTED MANUSCRIPT Table 3.

Distribution of fpn and bfp1-4genes among a subset of 26 bft-positive and 46 bftnegative B. fragilis isolates

Number of bft-

Number of bft-

positive strains

negative strains

(n=26)

(n=46)

12

26

bfp2-posititve

19

39

bfp3-positive

7

10

bfp4-positive

0

0

bfp1- and bfp2-positive

3

19

bfp1- and bfp3-positive

0

bfp2- and bfp-3-positive

0

bfp1-, bfp2- and bfp3-positive

6

1 5

TE D EP AC C

3

M AN U

24

fpn-positive

SC

bfp1-positive

RI PT

Strains

36

ACCEPTED MANUSCRIPT Table 4.

Distribution of fpn and bfp1-4 genes in 6 cfiA-positive and 66 cfiA–negative B. fragilis strains

Strains

cfiA-positive

(n=66) 35

RI PT

(n=6) 3

bfp1-positive

cfiA-negative

2

56

bfp3-positive

0

17

bfp4-positive

0

0

bfp1- and bfp2-positive

0

22

bfp1- and bfp3-positive

0

1

bfp2- and bfp-3-positive

0

bfp1-, bfp2- and bfp3-positive

0

fpn-positive

0

SC

bfp2-posititve

5 9

AC C

EP

TE D

M AN U

63

ACCEPTED MANUSCRIPT Figure 1.

Melting curve analysis of bft-1 and bft-2 positive B. fragilis strains

↓

↓ Tm of bft-2 positive strains

AC C

EP

TE D

M AN U

SC

RI PT

Tm of bft-1 positive strains

ACCEPTED MANUSCRIPT

Highlights

1. The prevalence of enterotoxin gene (bft) and its subtypes (bft1-3) were tested among 200 Hungarian clinical B. fragilis strains.

RI PT

2. The distribution of cfiA and two cysteine peptidase genes (bfp-1-4 and fpn) were investigated in a subset of 72 bft-positive and –negative B. fragilis isolates by molecular methods.

AC C

EP

TE D

M AN U

SC

3. A statistical analysis was performed with the data of these genes.