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.

The recent emergence of a highly related virulent Clostridium difficile clade with unique characteristics.

OBJECTIVES: Clostridium difficile is a major global human pathogen divided into five clades, of which clade 3 is the least characterized and consists predominantly of PCR ribotype (RT) 023 strains. Our aim was to analyse and characterize this clade. METHODS: In this cohort study the clinical presentation of C. difficile RT023 infections was analysed in comparison with known 'hypervirulent' and non-hypervirulent strains, using data from the Netherlands national C. difficile surveillance programme. European RT023 strains of diverse origin were collected and whole-genome sequenced to determine the genetic similarity between isolates. Distinctive features were investigated and characterized. RESULTS: Clinical presentation of C. difficile RT023 infections show severe infections akin to those seen with 'hypervirulent' strains from clades 2 (RT027) and 5 (RT078) (35%, 29% and 27% severe CDI, respectively), particularly with significantly more bloody diarrhoea than RT078 and non-hypervirulent strains (RT023 8%, other RTs 4%, p 0.036). The full genome sequence of strain CD305 is presented as a robust reference. Phylogenetic comparison of CD305 and a further 79 previously uncharacterized European RT023 strains of diverse origin revealed minor genetic divergence with >99.8% pairwise identity between strains. Analyses revealed distinctive features among clade 3 strains, including conserved pathogenicity locus, binary toxin and phage insertion toxin genotypes, glycosylation of S-layer proteins, presence of the RT078 four-gene trehalose cluster and an esculinase-negative genotype. CONCLUSIONS: Given their recent emergence, virulence and genomic characteristics, the surveillance of clade 3 strains should be more highly prioritized.

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.

Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages.

The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents.

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.

Update of Clostridium difficile infection due to PCR ribotype 027 in Europe, 2008.

Outbreaks of Clostridium difficile infections (CDI) with increased severity, high relapse rate and significant mortality have been related to the emergence of a new, hypervirulent C. difficile strain in North America and Europe. This emerging strain is referred to as PCR ribotype 027 (Type 027). Since 2005, individual countries have developed surveillance studies about the spread of type 027.C. difficile Type 027 has been reported in 16 European countries. It has been responsible for outbreaks in Belgium, Germany, Finland, France, Ireland, Luxembourg, The Netherlands, Switzerland and the United Kingdom (England, Wales, Northern Ireland and Scotland). It has also been detected in Austria, Denmark, Sweden, Norway, Hungary, Poland and Spain. Three countries experienced imported patients with CDI due to Type 027 who acquired the infection abroad.The antimicrobial resistance pattern is changing, and outbreaks due to clindamycin-resistant ermB positive Type 027 strains have occurred in three European countries. Ongoing epidemiological surveillance of cases of CDI, with periodic characterisation of the strains involved, is required to detect clustering of cases in time and space and to monitor the emergence of new, highly virulent clones.

Update of Clostridium difficile-associated disease due to PCR ribotype 027 in Europe.

Recent outbreaks of Clostridium difficile-associated diarrhoea (CDAD) with increased severity, high relapse rate and significant mortality have been related to the emergence of a new, hypervirulent C. difficile strain in North America, Japan and Europe. Definitions have been proposed by the European Centre of Disease Prevention and Control (ECDC) to identify severe cases of CDAD and to differentiate community-acquired cases from nosocomial CDAD (http://www.ecdc.europa.eu/documents/pdf/Cl_dif_v2.pdf). CDAD is mainly known as a healthcare-associated disease, but it is also increasingly recognised as a community-associated disease. The emerging strain is referred to as North American pulsed-field type 1 (NAP1) and PCR ribotype 027. Since 2005, individual countries have developed surveillance studies to monitor the spread of this strain. C. difficile type 027 has caused outbreaks in England and Wales, Ireland, the Netherlands, Belgium, Luxembourg, and France, and has also been detected in Austria, Scotland, Switzerland, Poland and Denmark. Preliminary data indicated that type 027 was already present in historical isolates collected in Sweden between 1997 and 2001.

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.

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.

[Antagonistic activity of Lactobacillus bacteria strains against anaerobic gastrointestinal tract pathogens (Helicobacter pylori, Campylobacter coli, Campylobacter jejuni, Clostridium difficile)]. / Antagonistyczne dzialanie bakterii z rodzaju Lactobacillus wobec beztlenowych i mikroaerofilnych czynników zakazen przewodu pokarmowego (Helicobacter pylori, Campylobacter coli, Campylobacter jejuni, Clostridium difficile).

Antagonistic activity of Lactobacillus strains has been known for some time. This property is connected with production of many active substances by lactobacilli e.g., organic acids and bacteriocin-like substances which interfere with other indigenous microorganisms inhabiting the same ecological niche, including also anaerobic gastrointestinal tract pathogens. Growing interest of clinical medicine in finding new approaches to treatment and prevention of common inflammatory infections of the digestive tract resulted in studies on a possible usage of lactic acid bacteria. Last years, several in vitro and in vivo experiments on antagonism of different Lactobacillus strains against Helicobacter pylori and Clostridium difficile were performed. These observations had been done on already established, well known probiotic Lactobacillus strains. We tested antibacterial activities of Lactobacillus strains isolated from human digestive tract. As indicator bacteria, four species known as anaerobic bacterial etiologic agents of gastroenteric infections: Helicobacter pylori, Campylobacter jejuni, C. coli and Clostridium difficile were used. Some of them were obtained from international collections, others were clinical isolates from specimens taken from patients with different defined gastrointestinal infections. We used a slab method of testing inhibitory activity described in details previously. Following conclusions were drawn from our study: All tested human Lactobacillus strains were able to inhibit the growth of all strains of anaerobic human gastrointestinal pathogens used in this study. Inhibitory activities of tested Lactobacillus strains against Helicobacter pylori, Campylobacter spp., and Clostridium difficile as measured by comparing mean diameters of the inhibition zones were similar. Differences in susceptibility of individual indicator strains of Campylobacter spp. and Clostridium difficile to inhibitory activity of Lactobacillus strains were small. A similar mechanism of inhibition of anaerobic bacteria by lactobacilli is postulated.

[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.

[Beta-lactamases with a wide substrate spectrum in gram negative strictly anaerobic rods]. / Beta-laktamazy o rozszerzonym spektrum substratowym u Gram-ujemnych paleczek rosnacych w warunkach bezwzglednie beztlenowych.

This study was performed to determine the susceptibility of the clinical strains of Gram-negative strictly anaerobic rods to newer beta-lactam antibiotics. Also, the trial was undertaken to detect strains producing extended-spectrum beta-lactamases (ESBLs) and inducible beta-lactamases (IBLs) among Bacteroides spp. and Prevotella spp. rods isolated from hospitalized patients. One hundred strains of Gram-negative, obligatory anaerobic rods were applied in the study. The strains were identified in automatic ATB system using API 20 A strips. beta-lactamase-positive strains were determined with disc nitrocefin test. ESBL-producing strains were detected with double disc test according to Jarlier et al. (1988). Clavulanate was applied as the inhibitor of these beta-lactamases (AMO/CLAV disc). ESBL-positive strains were confirmed with the use of E test (TZ/TZL strip). Inducible beta-lactamases were determined by double disc method according to Sanders and Sanders (1979). Cefoxitin was the inducer of these beta-lactamases (FOX disc). Among 93 Bacteroides spp. strains and 7 Prevotella spp. strains, 91 strains (91%) produced beta-lactamases. Two ESBL-producing strains (2%) were detected. Strains producing inducible beta-lactamases (IBL) were not found. A high activity of the examined beta-lactam antibiotics against strains of Gram-negative anaerobes was found. The majority of strains were susceptible to piperacillin (95%), piperacillin combined with tazobactam (99%), ticarcillin combined with clavulanic acid (99%), meropenem (97%) and imipenem (99%). The obtained results indicate the necessity of ESBL determination among strains of the genus Bacteroides, isolated from clinical specimens. Newer beta-lactam antibiotics, especially penicillins in combination with beta-lactamase inhibitors and carbapenems, are useful in empiric therapy of infections caused by Bacteroides spp. and Prevotella spp. anaerobic rods.

[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.

[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.