Insight into the Mechanism of Lysogeny Control of phiCDKH01 Bacteriophage Infecting Clinical Isolate of Clostridioides difficile.

Clostridioides difficile is a causative agent of antibiotic-associated diarrhea as well as pseudomembranous colitis. So far, all known bacteriophages infecting these bacteria are temperate, which means that instead of prompt lysis of host cells, they can integrate into the host genome or replicate episomally. While C. difficile phages are capable of spontaneous induction and entering the lytic pathway, very little is known about the regulation of their maintenance in the state of lysogeny. In this study, we investigated the properties of a putative major repressor of the recently characterized C. difficile phiCDKH01 bacteriophage. A candidate protein belongs to the XRE family and controls the transcription of genes encoding putative phage antirepressors, known to be involved in the regulation of lytic development. Hence, the putative major phage repressor is likely to be responsible for maintenance of the lysogeny.

Infections caused by Slackia exigua: A single-center experience and literature review. / Infekcje wywolane przez Slackia exigua. Przeglad literatury i obserwacje wlasne.

Slackia exigua, originally classified as Eubacterium exiguum, is a Gram-positive, asaccharolytic, rod-shaped anaerobic bacterium. The virulence factors of S. exigua have not been accurately identified. The objective of the study is to evaluate the pathogenic potential of S. exigua by presenting the cases of infections diagnosed at our hospital laboratory. Additionally, we reviewed the literature to summarize the experience with S. exigua infections to clarify, in the light of current knowledge, the clinical picture, diagnostic, and therapeutic issues related to this anaerobic bacterium. We reported eleven severe human infections caused by S. exigua. All patients required hospitalization. Nine of the cases involved chronic infections in the stomatognathic system, in two patients, skin infections were diagnosed. As it is known, S. exigua is a component of the human microbiota; however, it can cause opportunistic infections, particularly in the case of translocation outside its natural habitat. A critical literature analysis revealed that S. exigua can be responsible for bacteremia, meningitis, tissue necrosis, periprosthetic joint infection, and osteomyelitis. Several studies have been published regarding the determination of drug susceptibility of S. exigua. The isolated strains were susceptible to most antibiotics used for the treatment of anaerobic infections. The interpretation of antimicrobial susceptibility testing for some slow-growing in vitro, infrequently causing infections anaerobic bacteria, such as S. exigua, is based on The European Committee on Antimicrobial Susceptibility Testing (EUCAST) additional guidance taking into account the determination of drug susceptibility for groups of microorganisms for which cut-off values have not been developed.

Phenotypic and genotypic identification of carbapenem resistance in Bacteroides fragilis clinical strains.

Bacteroides fragilis is an important etiological agent of serious infections in humans. Rapid methods, readily adaptable to use in medical laboratories, are needed to detect antibiotic resistance and decrease the likelihood of therapy failure. The aim of this study was to determine the prevalence of B. fragilis cfiA-positive isolates. The second purpose was to investigate the carbapenemase activity in B. fragilis strains by Carba NP test. In the study, 5.2% of B. fragilis isolates are phenotypically resistant to meropenem. The cfiA gene was identified in 6.1% of B. fragilis isolates. The MICs of meropenem were significantly higher in cfiA-positive strains. The presence of the cfiA gene along with the IS1186 was detected in one B. fragilis strain which was resistant to meropenem (MIC 1.5 mg/L). The Carba NP test results were positive for all the cfiA-positive strains, including those susceptible to carbapenems based on their MIC values. A review of the literature revealed that the rate of B. fragilis with the cfiA gene varies from 7.6 to 38.9% worldwide. Presented results are in line with the other European studies. Phenotypic testing with the Carba NP test, it seems to be a viable alternative for the cfiA gene detection in B. fragilis isolates. The positive result obtained is of greater clinical importance than the detection of the gene cfiA.

Motility and the genotype diversity of the flagellin genes fliC and fliD among Clostridioides difficile ribotypes.

OBJECTIVE: The motility and genotype of the flagellin fliC and fliD genes were investigated in 82 Clostridioides difficile isolates belonging to the ribotypes (RTs): 027 (n = 41), 176 (n = 17), 023 (n = 8), 017 (n = 6) and 046 (n = 10). The reference C. difficile strains 630 and M120 were included as controls for the motility assay. METHODS: A Multiple Locus Variable-number Tandem Repeat Analysis (MLVA) was used to exclude the genetic relatedness of C. difficile isolates belonging to the same RT. The variability of the fliC and fliD genes was determined by PCR-restriction fragment length polymorphism (RFLP) analysis and Sanger sequencing. The motility assay was carried out with 0.175% BHI agar tubes and BHI solid media plates with 0.4% agar. RESULTS: The highest motility was observed in C. difficile RT023 isolates (p < 0.01), followed by RTs 027 and 176. C. difficile isolates of RTs 017 and 046 were less motile than RTs 027, 176 and 023 (p < 0.01). The fliC and fliD genes were present in all clinical isolates irrespective of the motility results. In the fliC gene analysis, four different RFLP groups were identified (I, II, VII, X). The fliC group VII was identified in two RTs (027 and 176), whereas the remaining three groups (I, II and X) belonged to a single RT 046, 017 and 023, respectively. The fliD gene analysis identified four new RFLP groups (a, b, c and d). CONCLUSIONS: C. difficile RT023 is highly motile and its motility is comparable to the hypervirulent RT027 and its genetic relative RT176.

The Effect of Selected Bee Products on Adhesion and Biofilm of Clostridioides difficile Strains Belonging to Different Ribotypes.

There is an ongoing search for alternative treatments for Clostridioides difficile infections. The aim of the study was to investigate the antibacterial and antibiotic activity of bee products against C. difficile strains with different polymerase chain reaction ribotypes (RTs). The minimum inhibitory concentration (MICs) of Manuka honey 550+, goldenrod honey, pine honey, and bee bread were determined by the broth dilution method. C. difficile adhesion to HT-29, HT-29 MTX, and CCD 841 CoN cell lines was assessed. Biofilm was cultured in titration plates and visualized by confocal microscopy. The MICs of Manuka honey for C. difficile 630 and ATCC 9689 strains and control strain, M 120, were 6.25%, 6.25%, and 1.56% (v/v), respectively; of goldenrod honey, 50%, 50%, and 12.5%, respectively; of pine honey, 25%, 25%, and 25%, respectively; and of bee bread, 100 mg/L, 50 mg/L, and 100 mg/L, respectively. Manuka honey (1%) increased adhesion of C. difficile RT176 strains, and one strain of RT023, to the CCD 841 cell line. Pine honey (1%) increased RT027 adhesion to the HT-29 cell line. Manuka honey, pine honey, and bee bread at subinhibitory concentrations increased the adhesion of C. difficile. Our research proved that bee products are active against the tested strains of C. difficile.

The effect of berberine chloride and/or its combination with vancomycin on the growth, biofilm formation, and motility of Clostridioides difficile.

This study aims to investigate the antimicrobial and antibiofilm activity of berberine chloride (BBR) and vancomycin (VAN) as well as synergistic combinations of BBR with VAN against Clostridioides difficile strains. The effect of different concentrations of BBR on strain motility was also assessed. Twelve C. difficile strains (two reference C. difficile 630, ATCC 9689, and one control M120, and 9 clinical C. difficile strains belonging to the PCR-ribotype (RT027)) were collected and investigated for their susceptibility to BBR and VAN in planktonic and biofilm forms. Both the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of BBR for the C. difficile strains were found to vary over a broad range (256-1.024 mg/L and 256-16.384 mg/L, respectively). The MIC and MBC of VAN also varied greatly, ranging from 0.25 to 4.0 mg/L for MIC and 0.25 to 64.0 mg/L for MBC. The synergistic effect of the sub-MIC (1/2 MIC) BBR with VAN reduced of MICs of VAN against the planktonic forms of ten C. difficile strains. The sub-MIC of BBR enhanced the biofilm formation of one strain and was found to be statistically significant. In addition, the sub-MIC of BBR with VAN surprisingly enhanced the biofilm formation of one C. difficile strain. The effect of inhibition of motility in the presence of BBR was statistically significant for 3 clinical strains (p < 0.05). Altogether, BBR exhibited strong antimicrobial activity against C. difficile, and the analysis of the combination of BBR with VAN showed a synergistic effect.

Fructooligosaccharides and mannose affect Clostridium difficile adhesion and biofilm formation in a concentration-dependent manner.

The aim of this study was to investigate the effects that prebiotic and candidates for prebiotics on Clostridium difficile strains to adhere to various human epithelial cell lines and to compare the adhesive properties of specific C. difficile strains. We also sought to examine the effect of different concentrations of fructooligosaccharides and mannose on the formation of biofilms by C. difficile strains. The influence of cellobiose, fructooligosaccharides, inulin, mannose, and raffinose on the adherence properties of various C. difficile strains, including motile 630, non-motile M120, and 10 clinical motile ribotype 027 strains, to non-mucous secreting HT-29, mucous secreting HT-29 MXT, and CCD 841 CoN cells lines. The most effective prebiotics were used in biofilm formation assays. We demonstrated that all C. difficile strains adhered to all cell lines. However, the C. difficile M120 non-motile strain was statistically more likely to adhere to all three cell lines (CFU median, 40) compared to the motile strains (CFU median, 3; p < 0.001). Furthermore, among the carbohydrates examined, only fructooligosaccharides and mannose were found to significantly decrease adhesion (p < 0.001) of C. difficile strains. Alternatively, using a biofilm assay, we observed, via confocal laser scanning microscopy, that sub-inhibitory concentrations (1%) of fructooligosaccharides and mannose functioned to increase biofilm formation by C. difficile. We demonstrated that specific prebiotics and candidate prebiotics exhibit varying anti-adhesive properties towards C. difficile in vitro and that treatment with sub-inhibitory concentrations of prebiotics can cause an increase in biofilm formation by C. difficile.

Clostridium difficile infection: review.

Clostridium difficile (C. difficile) is a Gram-positive, spore-forming, anaerobic bacillus, which is widely distributed in the intestinal tract of humans and animals and in the environment. In the last decade, the frequency and severity of C. difficile infection has been increasing worldwide to become one of the most common hospital-acquired infections. Transmission of this pathogen occurs by the fecal-oral route and the most important risk factors include antibiotic therapy, old age, and hospital or nursing home stay. The clinical picture is diverse and ranges from asymptomatic carrier status, through various degrees of diarrhea, to the most severe, life threatening colitis resulting with death. Diagnosis is based on direct detection of C. difficile toxins in feces, most commonly with the use of EIA assay, but no single test is suitable as a stand-alone test confirming CDI. Antibiotics of choice are vancomycin, fidaxomicin, and metronidazole, though metronidazole is considered as inferior. The goal of this review is to update physicians on current scientific knowledge of C. difficile infection, focusing also on fecal microbiota transplantation which is a promising therapy.

The level of fecal calprotectin significantly correlates with Clostridium difficile infection severity.

INTRODUCTION: Fecal calprotectin (FC) rises significantly in intestinal inflammation accompanied by neutrophil activation - such as Clostridium difficile infection (CDI). The aim of the study was to evaluate the benefit of FC testing in assessing the severity of CDI. MATERIALS AND METHODS: The study group included 76 patients with CDI hospitalized in the Jagiellonian University Hospital in Krakow from July 2017 till January 2018. FC levels were measured using an EIA (Enzyme Immunoassay). Demographic, clinical information and blood tests were recorded using standardized data collection forms. The selection of patients into non-severe and severe groups was carried out in accordance with the ESCMID criteria (European Society of Clinical Microbiology and Infectious Diseases) and some modi cations to those criteria were proposed. RESULTS: the studied population included 76 patients (39 men and 37 women) with CDI aged from 24 to 98 years (mean: 72). Median calprotectin level was 739 (Q25-Q75: 612-799 µg/g), characteristic of patients with colitis. A statistically significant difference in FC concentration in patients with severe vs non-severe CDI was observed (severe - 770 vs non-severe - 659 µg/g, p = 0.009). FC directly correlated with platelets level; however, no correlation between FC level and the blood parameters prognostic for CDI (leukocyte, neutrophil count, albumin, creatinine levels) was found. CONCLUSION: FC level is an indication of ongoing intestinal inflammation in CDI patients. FC level significantly correlated with CDI severity, which demonstrates that FC could serve as a predictive marker for assessing CDI severity.

Standardised surveillance of Clostridium difficile infection in European acute care hospitals: a pilot study, 2013.

Clostridium difficile infection (CDI) remains poorly controlled in many European countries, of which several have not yet implemented national CDI surveillance. In 2013, experts from the European CDI Surveillance Network project and from the European Centre for Disease Prevention and Control developed a protocol with three options of CDI surveillance for acute care hospitals: a 'minimal' option (aggregated hospital data), a 'light' option (including patient data for CDI cases) and an 'enhanced' option (including microbiological data on the first 10 CDI episodes per hospital). A total of 37 hospitals in 14 European countries tested these options for a three-month period (between 13 May and 1 November 2013). All 37 hospitals successfully completed the minimal surveillance option (for 1,152 patients). Clinical data were submitted for 94% (1,078/1,152) of the patients in the light option; information on CDI origin and outcome was complete for 94% (1,016/1,078) and 98% (294/300) of the patients in the light and enhanced options, respectively. The workload of the options was 1.1, 2.0 and 3.0 person-days per 10,000 hospital discharges, respectively. Enhanced surveillance was tested and was successful in 32 of the hospitals, showing that C. difficile PCR ribotype 027 was predominant (30% (79/267)). This study showed that standardised multicountry surveillance, with the option of integrating clinical and molecular data, is a feasible strategy for monitoring CDI in Europe.

Hospital-based Clostridium difficile infection surveillance reveals high proportions of PCR ribotypes 027 and 176 in different areas of Poland, 2011 to 2013.

As part of the European Clostridium difficile infections (CDI) surveillance Network (ECDIS-Net), which aims to build capacity for CDI surveillance in Europe, we constructed a new network of hospital-based laboratories in Poland. We performed a survey in 13 randomly selected hospital-laboratories in different sites of the country to determine their annual CDI incidence rates from 2011 to 2013. Information on C. difficile laboratory diagnostic testing and indications for testing was also collected. Moreover, for 2012 and 2013 respectively, participating hospital-laboratories sent all consecutive isolates from CDI patients between February and March to the Anaerobe Laboratory in Warsaw for further molecular characterisation, including the detection of toxin-encoding genes and polymerase chain reaction (PCR)-ribotyping. Within the network, the mean annual hospital CDI incidence rates were 6.1, 8.6 and 9.6 CDI per 10,000 patient-days in 2011, 2012, and 2013 respectively. Six of the 13 laboratories tested specimens only on the request of a physician, five tested samples of antibiotic-associated diarrhoea or samples from patients who developed diarrhoea more than two days after admission (nosocomial diarrhoea), while two tested all submitted diarrhoeal faecal samples. Most laboratories (9/13) used tests to detect glutamate dehydrogenase and toxin A/B either separately or in combination. In the two periods of molecular surveillance, a total of 166 strains were characterised. Of these, 159 were toxigenic and the majority belonged to two PCR-ribotypes: 027 (n=99; 62%) and the closely related ribotype 176 (n=22; 14%). The annual frequency of PCR-ribotype 027 was not significantly different during the surveillance periods (62.9% in 2012; 61.8% in 2013). Our results indicate that CDIs caused by PCR-ribotype 027 predominate in Polish hospitals participating in the surveillance, with the closely related 176 ribotype being the second most common agent of infection.

Antimicrobial susceptibility patterns of Clostridium difficile strains belonging to different polymerase chain reaction ribotypes isolated in Poland in 2012.

In the beginning of 2012, a study was conducted to obtain an overview of Clostridium difficile infections (CDIs) in Polish hospitals. The collection of 83 toxigenic C. difficile isolates obtained from this hospital-based survey was used to identify antimicrobial susceptibility patterns. Among the C. difficile isolates analyzed, 48 (57.8%) belonged to PCR ribotype 027, 21 (25.3%) to its closely related PCR ribotype 176, and 14 (16.9%) to different PCR ribotypes. Seventy one (85.5%) isolates were resistant to erythromycin, whereas 23 (27.7%) had high-level clindamycin resistance, having minimum inhibitory concentrations (MICs) greater than 256 mg/L. All strains were ciprofloxacin resistant and 69 (83.1%) were moxifloxacin resistant. Seventy-three (87.9%) strains were imipenem resistant, but only 2 (2.4%) strains were resistant to tetracycline. All strains were sensitive to tigecycline. Metronidazole and vancomycin were generally effective against the C. difficile isolates, both having an MIC90 value of 0.75 mg/L. Isolates belonging to PCR ribotype 027 and the closely related PCR ribotype 176, showed higher resistance. All ribotype 027 and 176 C. difficile isolates demonstrated high-level resistance to erythromycin (MIC ≥ 256 mg/L), and 95,2% of ribotype 176 isolates were co-resistant to erythromycin and clindamycin. The MIC of moxifloxacin for this epidemic strain was very high (≥32 mg/L). Resistance to erythromycin, moxifloxacin, and rifampicin was observed in 15 (18%) of the isolates, all of which belonged to PCR ribotype 027. Multidrug resistance (MDR), defined as resistance at least to three classes of antimicrobial agents was observed in 85.5% (n = 71) of toxigenic C. difficile strains.

[Evaluation of growth of clinical Clostridium difficile strains belonging to different PCR-ribotypes on chromID C. difficile Agar]. / Ocena wzrostu na podlozu chromID C. difficile Agar klinicznych szczepów Clostridium difficile nalezacych do róznych PCR-rybotypów.

INTRODUCTION: Clostridium difficile is main reason of antibiotic-associated diarrhea in hospitalized patients. Diagnostic method for detection of Clostridium difficile infection (CDI) are limited to an enzyme immunoassays (EIAs), while the culture of toxigenic strains is still seen as the gold standard for the laboratory diagnosis. The aim of this study was to compare growth of C. difficile strains belonging to different polymerase chain reaction (PCR) ribotypes on new ChromID C. difficile Agar (CDIFF, bioMérieux, Marcy l'Etoile, France). MATERIALS AND METHODS: One hundred thirty one of clinical C. difficile strains stored. in Anaerobic Laboratory were cultured on ChromID C. difficile Agar. Ten faecal samples were cultured on the same chromogenic medium and incubated at 37°C for 24 h under anaerobic conditions. Isolates were confirmed as C. difficile on the basis of well-known criteria. PCR-ribotyping was performed by visually comparison of patterns of PCR products of the 16S-23S rRNA intergenic spacer region. We examined the occurrence of beta-glucosidase gene, responsible for the dark color of the colony C. difficile on ChromID C.difficile Agar using a pair of primers: gluF (5'-AAGGT GTAAATTTAGGAGGTTGGTT-3') i gluR (5'-AGGTCCCAACTATCCC ATCC-3'). RESULTS: Among ten C. dfficile isolates obtained from stool specimens one formed colorless colonies. We received 8 colorless isolates from 131 additional examined strains. All C. difficile isolates forming colorless colonies belonged to PCR ribotype 023. The prevalence of PCR-ribotype 023 was about 6%. We detected lack of beta-glucosidase gene in PCR-ribotype 023 isolates. CONCLUSIONS: There are some C. difficile strains forming colorless colonies on ChromID C.difficile Agar. This appearance is important in routine diagnostic use this chromogenic culture medium.

Occurrence of Clostridium difficile PCR-ribotype 027 and it's closely related PCR-ribotype 176 in hospitals in Poland in 2008-2010.

Since 2003, a rising incidence of Clostridium difficile infection (CDI) in North America and Europe has coincided with outbreaks of C. difficile PCR ribotype 027. This ribotype was not observed in Poland until 2008. In the period 2008-2010, outbreaks of antibiotic-associated diarrhoea occurred in three different hospitals in Poland. Of 30 C. difficile isolates available for microbiological characterisation, 17 (56%) were positive for binary toxin genes and belonged to PCR ribotype 027 (n = 7) and its closely related PCR ribotype 176 (n = 10). All 17 binary toxin-positive C. difficile strains demonstrated high-level resistance to fluoroquinolones (minimum inhibitory concentration (MIC) ≥ 32 mg/L), including ciprofloxacin, gatifloxacin, and moxifloxacin, as well as erythromycin and clindamycin (MIC ≥ 256 mg/L for both). Of 14 patients from whom clinical information was available, 50% had a severe form of CDI, defined by fever (>38.5 °C), decreased kidney function, and high leucocyte count. We conclude that outbreaks of CDI associated with hypervirulent strains belonging to PCR ribotypes 027 and 176 occurred in hospitals in Poland. Further studies evaluating the clinical impact of type 176 are urgently needed.

Three-step diagnostic algorithm in diagnosing patients suspected of Clostridium difficile-associated diarrhea.

UNLABELLED: Clostridium difficile is a predominant etiological agent of healthcare-associated infectious diarrhea. Immunoenzymatic tests for detecting toxins A/B from faecal samples are still used in routine diagnosis of Clostridium difficile-associated diseases in a number of healthcare centers in Poland. Recently, however, new diagnostic tests were introduced which allow for detecting toxigenic strains of C. difficile in a more effective and precise manner. It is of importance, especially in the light of hypervirulent strain occurrence. AIM: The aim of the present paper was to evaluate the efficacy of three-step algorithm in the diagnosis of Clostridium difficile-associated diseases (CDAD), considering the occurrence of false negative test results for toxins while using exclusively immunoenzymatic tests. MATERIALS AND METHODS: In the present study, faecal samples collected from patients presenting diarrhea were tested. Immunoenzymatic tests were used for detecting glutamate dehydrogenase (GDH) and toxins A/B. Culture and RT-PCR were also employed. RESULTS: Of 615 study participants, toxigenic strains GDH (+) TOX (+) were identified in 108 patients while for 67 patients, test results remained unspecified GDH (+) TOX (-). Further analysis of unspecified samples revealed 32 patients infected with toxigenic strains, i.e. 22.9% of all positive test results (n=140). CONCLUSION: Three-step diagnostic algorithm is an effective and reliable tool for diagnosing C.difficile- associated diseases.

Antimicrobial Effects of Some Natural Products on Adhesion and Biofilm Inhibition of Clostridioides difficile.

Understanding the potential antimicrobial properties of natural compounds and their impacts on Clostridioides difficile virulence factors may aid in developing alternative strategies for preventing and treating C. difficile infections (CDI). In this study, we investigated the bactericidal effects of ginger oil (GO), peppermint oil (PO), curcumin (CU), cinnamon aldehyde (CI), and trans-cinnamaldehyde (TCI) on the adhesion and biofilm disruption of C. difficile. We used three reference and five clinical C. difficile strains of different ribotypes. The bactericidal activity was assessed using the broth microdilution method. The adhesion was evaluated using human epithelial cell lines, and biofilm formation was visualized by confocal laser scanning microscopy. All tested strains exhibited susceptibility to CU, with minimum inhibitory concentration (MIC) values ranging from 128 µg/mL to 2048 µg/mL. Similarly, all strains were susceptible to CI and TCI, with MIC values ranging from 6.25% (v/v) to 25% (v/v). Most of the tested substances reduced the adhesion of C. difficile strains, while two tested strains showed significantly higher adhesion when co-incubated with the tested substances. Similar observations were made for biofilm formation, with observed density and morphology varied depending on the strain. In conclusion, the tested products demonstrated bactericidal activity and reduced the adhesion of C. difficile strains. They may be considered for further studies as potential antimicrobial agents targeting biofilm-related infections.

[The use of two-stage algorithm in the diagnosis of patients with low levels of Clostridium difficile toxins A/B in feces confirmed by using enzyme immunoassay]. / Zastosowanie dwustopniowego algorytmu w diagnostyce chorych z niskim poziomem toksyn A/B Clostridium difficile w kale potwierdzonym testem immunoenzymatycznym.

INTRODUCTION: Clostridium difficile infection (CDI) is a serious problem in hospitalized patients. Rapid and accurate laboratory diagnosis is the key to reducing of CDI. The suboptimal sensitivity and specificity of many commercial enzyme immunoassays have limited their utility. The aim of this study was analysis of faecal samples obtained from patients with clinical evidence of CDI, with non-detectable or questionable result of toxins A/B C. difficile recognized by toxins A/B EIA test. METHODS: A two-step algorithm for diagnostics of C. difficile infection (CDI) in patients with non-detectable or questionable result of toxins A/B C. difficile confirmed by C. difficile enzyme immunoassay (EIA) (Wampole, TOX A/B II, TechLab, USA) was used. Sixty nine faecal samples obtained from patients with nosocomial diarrhea were retested. All faecal samples were cultured on selective medium CLO C. difficile (BioMérieux, Francja). The positive samples on selective medium were tested by using Real Time-PCR (Xpert CD assay, Cepheid, Sunnyvale, CA, USA). Xpert CD assay is a real time multiplex PCR that can be used to detect toxigenic C. difficile strains and differentiate the C. difficile presumptive NAP1/BI/027 strain. All results when faecal samples were negative in culture growth on selective medium and result of EIA test were questionable was confirmed by use a RT-PCR test. RESULTS: Among 69 faecal samples 56 were negative for toxins A/B using EIA test and 13 gave questionable results. By anaerobic culture 60 of 69 specimens yielded C. difficile isolates. Among 69 faecal samples 55 were positive using RT-PCR. Thirty four (62%) of patients was infected by presumptive C. difficile NAP1/BI/027. CONCLUSIONS: C. difficile testing by use of culture and Real Time PCR (RT-PCR) increases diagnostic yield in a hospital patients with non-detectable or low level of toxins A/B in stool samples of patients infected by toxigenic C. difficile strains including presumptive C. difficile NAP1/BI/027.

The Effect of Subinhibitory Concentration of Metronidazole on the Growth and Biofilm Formation on Toxigenic Clostridioides difficile Strains Belonging to Different Ribotypes.

Clostridioides difficile is a predominant nosocomial pathogen within the healthcare setting able to produce biofilms. Sub-minimum inhibitory concentrations (sub-MICs) of antibiotics trigger mechanisms affecting bacterial virulence, including increased adhesion and biofilm formation. The aim of this study was to investigate how sub-MICs of metronidazole affect the biofilm formation of C. difficile strains. We tested 14 reference and clinical C. difficile strains, including hypervirulent strains of RT027. The MICs of metronidazole for the tested strains were determined using the broth microdilution method. Biofilm formation was evaluated using confocal laser scanning microscopy. The C. difficile strains belonging to RT027 produced the highest amounts of biofilm. The results of confocal laser scanning microscopy showed that all the tested C. difficile strains developed larger biofilms with diversified architectures upon exposure to sub-MICs of metronidazole. In our study, we reveal that sub-MIC concentrations of metronidazole affect the biofilm formation of clinical and reference strains of C. difficile. Importantly, metronidazole induces biofilm formation via hypervirulent RT027 strains.

[Assessment of the usefulness of chromogenic medium for the isolation of Clostridium difficile from the gastrointestinal tract of children with diarrhoea]. / Ocena przydatnosci podloza chromogennego do izolacji Clostridium difficile z przewodu pokarmowego dzieci z biegunka.

INTRODUCTION: Clostridium difficile is well known as an important cause of nosocomial infection. Laboratory diagnostics have included bacterial culture or more commonly, direct detection of preformed toxin in stool samples using different assays. The aim of this study was to evaluate and compare two selecitve media to isolation of C. difficile from paediatric diarrhoeal stool samples. METHODS: Fifty nine stool samples, collected from 43 children with diarrhoea, were examined for routine laboratory diagnosis of C. difficile infection. Commercially available tests for detection of A/B toxins of C. difficile were performed. The same stool samples were cultured on two selective media for strain isolation: CLO and CDIFF (bioMerieux S.A., France) and incubated 48h and 24h respectively. RESULTS: Twenty two samples gave positive results for toxins A/B C. difficile. From 24 samples inoculated on selective media C. difficile strains were cultured: from 8 samples on CLO medium and from 16 samples on CDIFF medium. CONCLUSIONS: CDIFF medium is more effective for isolation of C. difficile strains from stool samples collected from children with diarrhoea.

[Assessment of antagonistic activity in vitro Lactobacillus spp. strains againts Clostridum difficile strains isolated from gastrointestinal tract of patients hospitalized in three hospitals in region Mazovia]. / Ocena antagonistycznego dzialania w warunkach in vitro szczepów z rodzaju Lactobacillus wobec toksynotwórczych szczepów Clostridium difficile wyhodowanych z przewodu pokarmowego pacjentów leczonych w szpitalach na terenie województwa mazowieckiego.

INTRODUCTION: The aim of this study was to evaluate the antagonistic activity of Lactobacillus strains against clinical C. difficile strains isolated from faecal samples of adults patients with diarrhea. A total 61 strains of C. difficile randomly selected isolated in the period 2007-2008 from the gastrointestinal tract of hospitalized patients in three hospitals province Mazovia, Poland. To determination of antagonistic activity ofprobiotic Lactobacillus spp. strains used four reference strains: Lactobacillus plantarum 2017405, Lactobacillus fermentum 353, Lactobacillus acidophilus DSM 21007 and Lactobacillus rhamnosus GG. METHODS: Isolation of C. difficile was performed on selective Columbia agar supplemented with cycloserine/cefoxitine and amphothericin B (CLO medium, bioMérieux, France). The plates were incubated in an anaerobic chamber for 48 h at 37 degrees C. Isolates were identified as C. difficile by the characteristic morphology of the colonies and horse-like odour, green yellow fluorescence under UV. Toxigenicity of of C. difficile strains was determined in PCR to detection of fragments of genes encoding toxin A (tcdA), toxin B (tcdB) and binary toxin (cdtA and cdtB). The study of antagonistic activity four Lactobacillus spp. strains against 61 clinical C. difficile strains was performed according to standard methods. Lactobacillus strains were inoculated on MRS medium and incubated in oxygen-free atmosphere and cut the bars of MRS agar and applied to the plate with cultures of C. difficile strains. RESULTS: Assessment of antagonist activity of Lactobacillus spp. strains was performed by measuring the zone of inhibition of grown of C. difficile strains. The study shows that of probiotic Lactobacillus spp. strains interacted antagonistically in vitro against all toxigenic (A+B+CDT- and A+B+CDT+) of C. difficile strains. CONCLUSIONS: The differences in the antagonistic activity of Lactobacillus spp. strains against different toxigenic clinical C. difficile strains were not observed.