Surveillance of antimicrobial susceptibilities reveals high proportions of multidrug resistance in toxigenic Clostridium difficile strains in different areas of Poland.

Two hundred and fifty-three non-duplicate toxigenic Clostridium difficile isolates, collected from February 2012 to December 2014, were evaluated for phenotypic resistance to ten antimicrobial drugs with the E-test gradient diffusion method. All strains of C. difficile were susceptible to metronidazole, vancomycin, and tigecycline. The metronidazole MIC values of the hyperepidemic PCR-ribotypes RT027 and RT176 were higher than those of non-epidemic PCR-ribotypes (p < 0.05, as evidenced by Mann-Whitney U test). In contrast, vancomycin susceptibility did not differ between hyperepidemic and non-epidemic strains, although the difference was almost significant (p = 0.065). Clostridium difficile RT027 and RT176 isolates could be assessed to five and four different susceptibility patterns, respectively, representing various combinations of resistance to different antimicrobial classes. A single point mutation (Thr82Ile) in the gyrA gene was detected in 11 (78.6%) of 14 isolates with high level of resistance to ciprofloxacin and moxifloxacin and four different types of single point mutations (Arg447Lys, Ser416Ala, Asp426Val, Asp426Asn) in the gyrB gene were detected in 4 strains, also with high level of resistance to ciprofloxacin and moxifloxacin. Four different point mutations were detected in the rpoB gene in 21 rifampicin-resistant strains of which one has not been reported previously, Gln489Leu. This study demonstrates the presence of multidrug-resistant C. difficile strains in Polish hospitals over the study period, irrespective of geographical location or reference level of the hospital.

Antimicrobial effects of Manuka honey on in vitro biofilm formation by Clostridium difficile.

Clostridium difficile is the cause of the nosocomial C. difficile infection (CDI). The conventional antibiotics used in CDI therapy are often unsuccessful, and recurrent infections may occur. Biofilm formation by C. difficile is associated with chronic or recurrent infections; biofilms may contribute to virulence and impaired antimicrobial efficacy. Manuka honey, derived from the Manuka tree (Leptospermum scoparium), is known to exhibit antimicrobial properties that are associated with its significant content of methylglyoxal, a natural antibiotic. The aim of the present study was to determine the antimicrobial effect of Manuka honey on clinical C. difficile strains belonging to four prominent polymerase chain reaction (PCR) ribotypes (RTs) (RT017, RT023, RT027 and RT046) and on their biofilm formation in vitro. Minimal inhibitory and bactericidal concentrations (MICs and MBCs, respectively) were determined using the broth dilution method. The biomass of the biofilm and the clearance of C. difficile biofilms by Manuka honey were determined using the crystal violet staining method. The MIC and MBC of Manuka honey for C. difficile strains were equal at 6.25% (v/v). PCR RT027 strains produced more biofilm in vitro than the other examined strains. Manuka honey effectively inhibited biofilm formation by C. difficile strains of different PCR RTs.

First Polish outbreak of Clostridium difficile ribotype 027 infections among dialysis patients.

This report describes an outbreak of Clostridium difficile infection (CDI) in a nephrology ward in 2012, caused by the fluoroquinolone- and clindamycin-resistant polymerase chain reaction (PCR) ribotype 027 strains. An increase in the number of cases of diarrhoea was noted among patients hospitalised between 26 November 2012 and 17 December 2012 in a hospital in North Poland. Eight patients were on haemodialysis in the outpatient dialysis facility, while one patient was receiving peritoneal dialysis. The 027 strain could be detected in eight haemodialysis patients. One strain, isolated from the patient receiving peritoneal dialysis, belonged to PCR ribotype 001. In this study, we documented the first outbreak of CDI caused by a fluoroquinolone-resistant (FQR) C. difficile PCR ribotype 027 strain in Polish dialysis patients.

Emergence of Clostridium difficile infection in tuberculosis patients due to a highly rifampicin-resistant PCR ribotype 046 clone in Poland.

Clostridium difficile infection (CDI) is a major cause of nosocomial diarrhea. CDI is known to develop after antibiotic administration, but anti-tuberculosis agents have rarely been implicated. We documented an outbreak caused by a highly rifampicin-resistant C. difficile strain of polymerase chain reaction (PCR) ribotype 046 in patients with active tuberculosis.

Clostridium difficile infection in Polish pediatric outpatients with inflammatory bowel disease.

The prevalence of Clostridium difficile infection (CDI) in pediatric patients with inflammatory bowel disease (IBD) is still not sufficiently recognized. We assessed the prevalence of CDI and recurrences in outpatients with IBD. In addition, the influence of IBD therapy on CDI and antimicrobial susceptibility of the potentially causative C. difficile strains was assessed. This was a prospective, single-center, observational study. All specimens were obtained between January 2005 and January 2007 from the IBD outpatient service and screened for C. difficile and its toxins. C. difficile isolates were genotyped by PCR ribotyping. Diagnosis of Crohn's disease (CD) and ulcerative colitis (UC) was based on Porto criteria. Severity of disease was assessed using the Hyams scale (for Crohn's disease) and the Truelove-Witts scale (for ulcerative colitis). One hundred and forty-three fecal samples from 58 pediatric IBD patients (21 with Crohn's disease and 37 with ulcerative colitis) were screened. The risk of C. difficile infection was 60% and was independent of disease type (CD or UC) (χ2 = 2.5821, df = 3, p = 0.4606). About 17% of pediatric IBD patients experienced a recurrence of CDI. All C. difficile strains were susceptible to metronidazole, vancomycin and rifampin. A high prevalence of C. difficile infection and recurrences in pediatric outpatients with IBD was observed, independent of disease type. There was no significant correlation between C. difficile infection and IBD therapy. PCR ribotyping revealed C. difficile re-infection and relapses during episodes of IBD in pediatric outpatients.

Clostridium difficile and enterotoxigenic Bacteroides fragilis strains isolated from patients with antibiotic associated diarrhoea.

From the fecal samples of 332 patients with a clinical diagnosis of antibiotic associated diarrhoea (AAD), 131 Clostridium difficile strains were isolated. For detection of toxin A in the isolated strains the enzymatic immunoassay was used. The cytopathic effect was determined on McCoy cell line. PCR was used for the detection of non-repeating and repeating sequences of toxin A gene and non-repeating sequences of toxin B gene. One hundred and six isolated C. difficile strains were TcdA(+)TcdB(+), 10 strains TcdA(-)TcB(+) and 15 were non-toxigenic TcdA(-)TcdB(-). Out of the same fecal samples 50 Bacteroides fragilis strains were isolated. All B. fragilis strains were tested in PCR reaction for fragilysine gene detection (bft). In 9 strains (18%) this gene was detected and the strains could be assumed as enterotoxigenic Bacteroides fragilis (ETBF). In 4 fecal samples toxigenic C. difficile (TcdA(+)TcdB(+)) was found simultaneously with ETBF. One sample contained C. difficile (TcdA(-)TcdB(+)) and ETBF. Out of 4 fecal samples only ETBF was isolated. The cytotoxicity of ETBF strains was tested on HT29/C1 human colon carcinoma cell line. The cytotoxicity titer in the range of 20 and 80 was observed.

Clonal dissemination of a toxin-A-negative/toxin-B-positive Clostridium difficile strain from patients with antibiotic-associated diarrhea in Poland.

OBJECTIVE: To determine the incidence of toxin-A-negative/toxin-B-positive Clostridium difficile strains and their genetic relatedness in the feces of patients suffering from antibiotic-associated diarrhea (AAD) in Polish hospitals. METHODS: C. difficile strains were cultured from patients' stool samples. The present study characterises these strains with respect to their cytopathogenicity on McCoy cells and the absence of toxin A despite a functional toxin B as determined with commercial test kits (Culturette Brand Toxin CD-TCD toxin A test and C. difficile Tox A/B test). In addition, PCR using different primer pairs aiming at non-repeating or repeating regions of the toxin A and B genes were used to confirm the findings. All toxin A(-)B(+) strains were genetically characterised by random amplification of polymorphic DNA (RAPD) analysis, PCR ribotyping and, in part, pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. RESULTS: We here present the presence of 17 toxin A(-)B(+) strains among 159 C. difficile strains (11%) isolated from fecal samples from 413 patients with antibiotic-associated diarrhea. All 17 strains possessed the toxin B gene, demonstrated a cytopathogenic effect on the McCoy cells, and were positive in the Tox A/B test. Molecular typing of these 17 C. difficile strains revealed that 7 of 17 (41%) toxin A(-)/B(+) C. difficile strains could not be discriminated. It appeared that these strains had a genotype that could not be distinguished from that of a Japanese control strain. CONCLUSION: Our observations imply that a particular genotype of toxin A(-)B(+) C. difficile has spread extensively, not only in Poland but possibly even worldwide.

Acute appendicitis: the role of enterotoxigenic strains of Bacteroides fragilis and Clostridium difficile.

BACKGROUND: The aim of this study was to investigate whether there is a relationship between enterotoxin-producing B. fragilis strains and toxigenic C. difficile strains and the pathogenesis of acute appendicitis. MATERIAL AND METHODS: Post-appendectomy tissues from 34 patients with histopathologically confirmed phlegmonous or gangrenous appendicitis were studied. RESULTS: Among 86 anaerobes isolated, the B. fragilis group was most frequently isolated: 34 B. fragilis strains were cultured from 21 post-appendectomy tissues. Two enterotoxin-producing B. fragilis strains were found. Enterotoxin titers (1:10 and 1:160, respectively) were measured on HT29/C cells. The presence of the enterotoxin gene was confirmed by PCR in DNA extracted from both strains. Among 21 DNA samples isolated from those post-appendectomy tissues from which B. fragilis strains were cultured, the presence of the enterotoxin gene was confirmed in only one case (the corresponding B. fragilis strain enterotoxin titer was 1:160). A unique toxigenic C. difficile strain was also cultured from the tissue of an adult patient with gangrenous non-perforated appendicitis. The presence of toxin A and toxin B genes was confirmed by PCR in DNA extracted from the C. difficile strain, but these genes were not found in the DNA extracted from the corresponding tissue. CONCLUSION: The presence of enterotoxigenic B. fragilis and toxigenic C. difficile strains was shown in post-appendectomy tissue from patients with phlegmonous and gangrenous appendicitis, and the B. fragilis enterotoxin gene was detected directly in the corresponding tissue. Further investigations (including immunologic aspects) require to confirm the role of these toxins in pathogenesis of acute appendicitis.

Search for enterotoxin gene in Bacteroides fragilis strains isolated from clinical specimens in Poland, Great Britain, The Netherlands and France.

BACKGROUND: Bacteroides fragilis is a member of normal human flora and well known pathogenic agent. This bacterium produces many virulence factors. In 1984 new virulence factor--enterotoxin was described. The aim of the study was to search for enterotoxin gene in B. fragilis strains isolated from clinical specimens. MATERIAL AND METHODS: Strains isolated in Poland, Great Britain, France and the Netherlands were cultured on BBE medium. For DNA isolation Genomic DNA PREP PLUS isolation kit manufactured by A&A Biotechnology (Poland) was used. In order to detect enterotoxin (fragilysin) gene, polymerase chain reaction (PCR) was applied utilizing the following primers: 404 (GAG CGG AAG ACG GTG TAT GTG ATT TGT) and 407 (TGC TCA GCG CCC AGT ATA TGA CCT AGT). DNA obtained from bacterial cells was amplified in thermocycler Techne. The amplification products were detected by the electrophoresis in 1% agarose gel. RESULTS: Among 65 investigated B. fragilis strains, the enterotoxin gene was detected in DNA isolated from 12 strains. CONCLUSION: The enterotoxin producing B. fragilis strains were detected among strains isolated from different clinical specimens in Poland, Great Britain, the Netherlands and France.

[Enterotoxin-producing Bacteroides fragilis strains isolated from horses]. / Enterotoksynotwórcze szczepy Bacteroides fragilis izolowane od koni.

Seven Bacteroides fragilis strains were cultured from samples collected from horses. From all the tested strains, as well as from the reference B. fragilis strains: enterotoxigenic NCTC 11925 and nonenterotoxigenic IPL 323 strain, DNA was isolated using Genomic DNA PREP PLUS isolation kit manufactured by A&A Biotechnology (Poland). To detect the enterotoxin (fragilysin) gene, polymerase chain reaction (PCR) was applied, using the following starters: 404 (GAG CCG AAG ACG GTG TAT GTG ATT TGT) and 407 (TGC TCA GCG CCC AGT ATA TGA CCT AGT). DNA obtained from bacterial cells was amplified in a thermocycler (Techne). The temperature profile was as follows: 1 cycle (4 min. 94 degrees C), 40 cycles (1 min. 94 degrees C, 1 min. 52 degrees C, 1 min. 74 degrees C). Amplification products were detected by electrophoresis in agarose gel (1%) with ethidium bromide added. The presence of the fragilysin gene was detected in two strains. Among the strains isolated from horses enterotoxin gene-possessing Bacteroides fragilis strains (ETBF) can be detected.

[Intestinal flora of patients with suspected antibiotic associated diarrhea (AAD). I. Clostridium perfringens]. / Flora przewodu pokarmowego chorych z podejrzeniem biegunki poantybiotykowej (AAD). I. Clostridium perfringens.

Stool samples of 158 patients suspected of antibiotic-associated diarrhoea (AAD) were studied. Toxin A of C. difficile and enterotoxin of C. perfringens were detected in stool samples by immunoenzymatic assays and PCR. In 35 stool samples toxin A of C. difficile was detected and in 48 cases (30%) C. difficile strains were cultured from 21 stool samples (13%). The presence of the cpe gene of C. perfringens, enabling the production of enterotoxin, could not be detected by PCR, both in stool samples and in isolated strains, using ent 1 and ent 2 primer pairs. C. difficile and C. perfringens were isolated from the same stool samples in 4 cases. From stool samples of two patients with AAD C. perfringens strains, thermoresistant spores were cultured.

Evaluation of different methods for detection of Clostridium difficile toxins in Poland.

The aim of this study was to compare different methods for C. difficile toxins detection. Fifty three stool samples taken from patients with antibiotic-associated diarrhoea were studied. TCD toxin A EIA (Becton Dickinson, USA), Tox A/B ELISA test (TechLab, USA), cytotoxicity and neutralization assay on McCoy cells and PCR for detection of both toxin A and B genes were performed in vivo (in stool samples) and in vitro (in isolated strains). Reference toxigenic and nontoxigenic and two Japanese toxin A-negative and toxin B-positive C. difficile strains were used as a controls. TCD toxin A EIA detected in vivo only 19 positive samples. Tox A/B test detected 52 positive samples out of 53 studied. All 53 stool samples were C. difficile culture positive (53 strains were cultured). Toxin B was detected in 52 strain-supernatants and in all controls (except the nontoxigenic one). Both toxin A and B genes were detected by PCR in all 53 isolated strains, Japanese and reference strain (except the nontoxigenic one). In vitro toxin A was detected by TCD toxin A EIA in 42 strains. These results were compared with those obtained in Tox A/B ELISA test. We observed 52 positive strains. Toxigenic reference strain and two Japanese toxA(-)/toxB(+) strains were also positive. Only 2 negative results were obtained with the nontoxigenic reference strain and unique nontoxigenic isolated strain. Tox A/B ELISA test seems to be the best for detection of C. difficile toxins in vivo and in vitro. Test avoids the false-negative results in the case of presence of toxin A-negative and toxin B-positive strain.

[Isolation of toxigenic strains of Clostridium difficile and enterotoxin producing Bacteroides fragilis from fecal specimens of patients suspected of antibiotic associated diarrhoea]. / Izolacja toksynotwórczych szczepów Clostridium difficile i enterotoksynotwórczych szczepów Bacteroides fragilis z kalu osób z podejrzeniem biegunki poantybiotykowej.

Fifty faecal samples from patients suspected of AAD (antibiotic associated diarrhoea) were studied for Clostridium difficile and enterotoxin producing Bacteroides fragilis (ETBF). Using TCD (Becton-Dickinson) and C. difficile Toxin A test (Oxoid) in 34% of specimens the presence of toxin A was detected. From all specimens 25 C. difficile strains were isolated. All isolated strains produced toxin B in vitro which was shown in Mc Coy cytotoxicity test. Eighteen strains only were toxin A positive in vitro. From all isolated C. difficile strains 28% were tox A (-) tox B (+). By means of PCR presence of toxin A and toxin B genes was tested directly in faecal samples and in strains. From the same 50 faecal samples 17 B. fragilis strains were isolated. Four of them produced the enterotoxin (fragilisin) which was detected on the HT 29/C1 cell line. Genes of fragilisin were found in strains and directly in faecal samples. Toxin producing C. difficile and B. fragilis (ETBF) together were found in 3 samples. From one faecal sample only ETBF was cultured.

[Detection of toxin producing strains of Clostridium difficile using rapid diagnostic methods]. / Wykrywanie toksynotwórczosci szczepów Clostridium difficile przy pomocy szybkich metod diagnostycznych.

Feces of 53 patients from different hospital wards suffering from long term post-antibiotic therapy diarrhea were tested. For direct detection of C. difficile toxin A, in samples TCD (Becton-Dickinson), and C. difficile Toxin A Test (Oxoid) tests were used. Toxin A was detected in 16 samples (29.6% tested). C. difficile strains were isolated from 40% of the fecal samples. Toxin A was detected in 25 Clostridium difficile strains with commercial tests and toxin B was detected using McCoy cell line. Toxin A was not produced by 3 C. difficile strains in vitro, but toxin B was produced by all strains. The polymerase chain reaction (PCR) test showed that all isolated strains possess genes of toxins A and B.