End 2023, the UK Health Security Agency sent an alert about a new hypervirulent Clostridioides difficile PCR ribotype, ribotype 955 (RT955), causing slowly progressing infection clusters in hospitals in the Midlands. Between March 2018 and February 2022, surveillance of Clostridioides difficile infections (CDI) was performed in southern Serbia with centres providing medical services for approximately 750,000 inhabitants. Using the ECDC recommended protocol, clinical, epidemiological and microbiological data were collected. C. difficile RT955 was identified in 27 (7%) of 383 surveyed patients with CDI. Of 27 patients, 16 (59%) was older than 60 years and 19 (70%) were male. CDI was always associated with previous antibiotic therapy and had a hospital onset in 23 (85%) patients. The clinical presentation was milder than reported in UK. All sequenced strains belonged to multilocus sequence type (ST) 1 and were highly similar, with 0-1 alleles differences in a core genome multilocus sequence typing analysis. The strains differed clearly from the UK RT955 outbreak strain by whole genome sequencing and phenotypic susceptibility to metronidazole, lincosamides and rifampicin. Interestingly, a high-level erythromycin resistance was observed associated with the presence of the mrmA gene. Both the UK and Serbian RT955 strains contained gyrA_p.T82I associated with resistance to fluoroquinolone antimicrobials and carried the PnimBG promoter mutation, suggestive for haem-dependent metronidazole resistance. We conclude that C. difficile RT955 is present in southern Serbia since 2018. The Serbian RT955 strains differed clearly from a representative UK cluster strain.

There is growing interest in developing monotherapy drugs that treat inflammation caused by microbial infections, focusing on dual antimicrobial and anti-inflammatory agents with minimal side effects and high safety margins. This study synthesized and characterized a library of novel cis-4-ferrocenylazetidin-2-ones, evaluating their antimicrobial and anti-inflammatory activities. These organometallic monocyclic β-lactams showed moderate in vitro antimicrobial activity against various standard microbial strains, including yeasts and Gram-negative and Gram-positive bacteria. Some compounds overcame the resistance of clinical Staphylococcus aureus isolates. Traditionally, monocyclic β-lactams target Gram-negative bacilli, but adding a ferrocene moiety and substituting the COOH group near the N-1 position with a non-ionizable ester group (COOR) extended their activity spectrum. The anti-inflammatory properties were assessed in macrophage-based models, revealing non-cytotoxicity below 10 μM. Two compounds were shown to be strong and selective arginase inhibitors, while five others effectively suppressed excessive NO formation without affecting basal NO production. The presence of a phenoxy group at C-3 of the β-lactam ring appeared to be crucial for selective NO inhibition. These hybrids did not scavenge NO but inhibited NO synthesis by suppressing iNOS expression. Overall, two novel hybrids were identified as promising hit candidates for treating infection-induced inflammatory reactions.

There is growing interest in developing monotherapy drugs that treat inflammation caused by microbial infections, focusing on dual antimicrobial and anti-inflammatory agents with minimal side effects and high safety margins. This study synthesized and characterized a library of novel cis-4-ferrocenylazetidin-2-ones, evaluating their antimicrobial and anti-inflammatory activities. These organometallic monocyclic (3-lactams showed moderate in vitro antimicrobial activity against various standard microbial strains, including yeasts and Gramnegative and Gram-positive bacteria. Some compounds overcame the resistance of clinical Staphylococcus aureus isolates. Traditionally, monocyclic (3-lactams target Gram-negative bacilli, but adding a ferrocene moiety and substituting the COOH group near the N-1 position with a non-ionizable ester group (COOR) extended their activity spectrum. The anti-inflammatory properties were assessed in macrophage-based models, revealing noncytotoxicity below 10 mu M. Two compounds were shown to be strong and selective arginase inhibitors, while five others effectively suppressed excessive NO formation without affecting basal NO production. The presence of a phenoxy group at C-3 of the (3-lactam ring appeared to be crucial for selective NO inhibition. These hybrids did not scavenge NO but inhibited NO synthesis by suppressing iNOS expression. Overall, two novel hybrids were identified as promising hit candidates for treating infection-induced inflammatory reactions.

Background: Data from the past decade indicates that Clostridioides difficile infection (CDI) is not only a nosocomial infection but is also increasingly recognized as a disease in the community. Objective: We aimed to study community-onset (CO) CDI in the various age groups in south Serbia with its clinical characteristics, risk factors and microbiological characterization. Methods: The study group included 93 patients with CO-CDI (median age 62). The control group consisted of 186 patients with community-onset diarrhea and stool samples negative tested for CDI. Results: Of all CDI cases diagnosed with a community onset, 74.19% had a previous contact with a healthcare facility in the previous 12 weeks, but 34.40% have no record on hospitalization in the previous 12 months. Using a multivariate statistical regression model, the following risk factors for CO-CDI development were found; antacid usage (OR 1/4 0.267, 95%C.I.:0.10e0.291, p < 0.01), chronic kidney disease (OR = 0.234, 95%C.I.:0.10e0.51, p < 0.01) and antibiotic use during the prior 2 months (OR = 0.061, 95%C.I.:0.02e0.17, p < 0.01), especially tetracycline's (OR = 0.146, 95% C.I.:0.07e0.22, p < 0.01) and cephalosporin's (OR = 0.110, 95%C.I.:0.14e0.42, p < 0.01). The most common ribotypes (RTs) detected in patients with CO-CDI were RT001 (32.3%) and RT027 (24.7%). All tested toxin producing C. difficile isolates were sensitive to metronidazole, vancomycin and tigecycline. A high rate of resistance to moxifloxacin (73.11%) and rifampicin (23.65%) was found. Conclusion: Patients with CO-CDI had frequently contact with healthcare facility in the previous 12 weeks. Restriction of antacid usage and of high-risk antibiotics in the community may help reduce the incidence of CO-CDI. (c) 2022 Elsevier Ltd. All rights reserved.

Background: Clostridioides difficile is the most common causative agent of antibiotic-acquired diarrhea in hospitalized patients associated with substantial morbidity and mortality. The global epidemic of CDI (Clostridioides difficile infection) began in the early 20th century with the emergence of the hypervirulent and resistant ribotype 027 strains, and requires an urgent search for new therapeutic agents.Objective: The aim of this study is to investigate the antibacterial activity of the three essential oils isolated from spice herbs (wild oregano, garlic and black pepper) against C. difficile clinical isolates belonging to 6 different PCR ribotypes and their potential inhibitory effect on the biofilm production in in vitro conditions.Results: Wild oregano essential oil showed strong inhibitory activity in concentrations 0.02-1.25 mg/mL and bactericidal activity in concentrations from 0.08 to 10 mg/mL. Garlic essential oil was effective in the concentration range of 0.02-40 mg/mL, and 0.16 -> 40 mg/mL. MIC and MBC for black pepper oil ranged from 0.04 to 40 mg/mL, and 0.08 -> 40 mg/mL, respectively. All the tested oils reduced in vitro biofilm production, with the best activity of oregano oil.Conclusion: Essential oils of wild oregano, black pepper and garlic are candidates for adjunctive therapeutics in the treatment of CDI. Oregano oil should certainly be preferred due to the lack of selectivity of action in relation to the ribotype, the strength of the produced biofilm and/or antibiotic-susceptibility patterns.(c) 2022 Elsevier Ltd. All rights reserved.

Platelet-rich fibrin (PRF) represents second generation of platelet concentrates, which has gained increasing awareness in recent years for regenerative procedures. This biologic additive is completely autologous, easy to prepare, has minimal expense, and possesses prolonged growth factor release, together with several other advantages over traditionally prepared platelet concentrates. Since its introduction, various protocols for PRF preparation have been proposed with different amounts of growth factors and other biomolecules necessary for wound healing. However, reference data about potential effect of some PRF components on hard and soft tissue healing are still conflicting. The current article intends to clarify the relevant advances about physiological role of certain PRF components and to provide insight into the new developmental approach. Also, this review summarizes the evolution of platelet concentrates and biologic properties of different modifications of PRF procedure.

Information on recurrent Clostridium difficile infections (rCDI) in children is rare and limited, especially community acquired (CA-CDI).This study was designed to identify risk factors for rCA-CDI in Serbian pediatric population. The study group included 71 children (aged from 1 to 14 years) with a first episode of CDI. Data were collected from 56 (78.87%) children with only one episode of CA-CDI and from 15 (21.13%) children with rCA-CDI were mutually compared. The following parameters were found to be statistically significantly more frequent in the children with rCA-CDI group (p < 0.05); leukemia as underlying disease, treatment with immunosuppressive and-or cytostatic drugs, and treatment with antibiotics. Similarly, previously visits to outpatient facilities, daycare hospitals and hospitals were also associated with rCDI. Analysis of clinical symptoms and laboratory parameters, revealed a statistically significant association of the severity of the first episode of CDI (determined by an increase in body temperature, higher maximum WBC and higher CRP) with development of a rCDI. Ribotype (RT) 027 was more common in children with rCA-CDI (66.7%, p = 0.006). During the seven-year research period, we found a rate of rCA-CDI rate in children of 21.13%. Our study identified several parameters statistically significantly more frequently in children with rCA-CDI. The obtained results will serve as a basis for future larger studies, but new prospective, studies are necessary to build a prediction model of rCDI in children that can be used to guide the treatment to prevent rCDI.

The aim of the study was to analyze the characteristics (sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and credibility} of tests for microbiological diagnosis of infections caused by Clostridium (C. difficile). The research was done at the Public Health LnstituteNig, the Center for Microbiology during the period 2016-2019. The study included 25 patients with Clostridium difficile infection (CDI) (25 fecal samples) and 50 patients with diarrhea who were not diagnosed with CDI (50 faecal samples). The samples were examined by various tests for the detection of toxin in the stool and with two tests to prove the produced glutamate dehydrogenase enzyme - GDH. RIDA GENE Clostridium difficile test (Real Time Multiplex PCR, R-Biofarm, Damstadt, Germany) was used as a reference test. Among the examined tests, the highest sensitivity in toxin detection was the ELISA-ridascreen C. difficile Toxin A / B (R - Biopharm AG, Darmstadt, Germany) (88%, PPV = 85.71%, NPV = 97.87%) and the lowest C. difficile TOXIN A& B - CHEK-1 (VEDALAB, ALENCON CEDEX, France) (68%, PPV = 85%, NPV = 85.45%). All toxin detection tests had a high specificity that was not less than 92%. In detecting the GDH antigen, the RideQUICK C. difficile GDH test (R - Biophann AG, Darmstadt, Germany) showed better performance (sensitivity 100%, specificity 92%, PPV = 89.28%, NPV =100% and authenticity 94.66%). The results of the research conducted in Nig showed high values of the characteristics for certain toxin-detecting tests in feces (sensitivity up to 88%, specificity to 98%, etc.). Research results in other studieshave shown that the values of the parameters that determine the characteristics of the tests are lower by 10% - 20%, although there is a study whose results are consistent with ours. The reasons for relatively high values should be sought in strict selection criteria for samples that are dose to criteria present in factory conditions when designing such tests. Although the results of this study confinn that there is no ideal diagnostic test in the microbiological diagnosis of CDI, the high sensitivity, specificity, PPV and NPV values make GDH tests suitable for the first screening of sample stools in everyday work.

Abstract Rotavirus is the important cause of acute gastroenteritis in pediatric patients. The aim of the present research was to determine the incidence of rotavirus infections in infants and children up to seven years of age in the town of Niš. Seasonal prevalence of rotavirus-associated acute gastroenteritis was also evaluated. An enzyme immunoassay (RIDASCREEN® Rotavirus; R-Biopharm AG, Darmstadt, Germany) was used to detect rotavirus in the stool specimens of 1,156 patients (newborns up to 7 years of age) presenting with gastroenteritis. Identification of bacteria and yeasts was performed by classical methods. The overall incidence of rotavirus in examined children was 5.97%. Among 144 hospitalized children, rotavirus infection was diagnosed in 28 (19.44%). In 1,012 children treated in outpatient setting for diarrheal diseases, rotaviruses were found in 41 (4.05%). The highest incidence of rotavirus infection was among the patients of one year of age. Among 1,156 pediatric children tested, bacterial pathogens were found in 6.31% and the most frequently isolated pathogens were Campylobacter spp. and Salmonella enteritidis. The highest prevalence of GE was recorded in the colder season, peaking in April (15.94%). Rotaviruses are an important factor in the etiology of the acute diarrheal diseases, especially in children hospitalized during the winter/spring season.

Carriage of Clostridium (C.) difficile in the intestinum of children, as well as its role in the disease (diarrhea) onset, is still controversial. The aim of this study is to investigate the community-acquired Clostridium difficile infection (CA-CDI) in Serbian pediatric population and to describe the basic clinical characteristics and risk factors for CA-CDI occurrence in Serbian pediatric population. The data obtained from 63 Serbian pediatric patients with CA-CDI and from control group of 126 children with community-acquired diarrhea, whose stool specimens were negative for C. difficile and toxins A/B, were mutually compared. In the current work, we found that children with CA-CDI display a significantly less severe disease clinical presentation than children with diarrheas of other origin. Lethal outcome was noted in two cases, but in children with severe underlying diseases (Crohn’s disease and leukemia). By using the multivariate statistical regression model, the following statistically significant risk factors for community-acquired C. difficile-associated diarrhea development were determined: previous application of laxatives (OR = 0.199, CI 0.55–0.79, p = 0.015), general antibiotic use during the previous 2 months (OR = 0.05, CI 0.02–0.17, p < 0.001), and specifically the use of penicillins (OR = 0.112, CI 0.04–0.31, p < 0.0001) and cephalosporins (OR = 0.16, CI 40.06–0.44, p < 0.0001). Antibiotics from the groups of cephalosporins and penicillins were found to be the most important independent risk factors. Laxative application plays a significant role in the community-acquired Clostridium difficile infections in children, with mechanisms that are not completely understood.

Pathogens from the genus Salmonella belong to the group of the most common food poisoning causative agents. The present paper investigated the effect of the basil and rosemary essential oils as well as their combination on the growth of Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis) in chicken meat, together with their spoilage protective potential at the two storage temperatures. Food model experiments included investigation of their effect to accompanying microbial flora on fresh meat, while anti-salmonella activity was evaluated on artificially inoculated raw and thermally processed meat. The tests were performed in two storage conditions, +4 degrees C (usual meat storage temperature) and +18 degrees C (room temperature, which favors the spoilage of investigated meat samples and development of food pathogens). Beside evaluation of microbiological status, physico chemical tests relevant as meat quality indicators (pH, colour, texture, thiobarbituric acid, cooking loss), as well as sensory evaluation of the studied meat samples were performed. Changes in normal flora pointed to significant effect of both oils against microbial meat spoilage, where various groups were affected by different treatments. In addition, all the treatments reduced the number of salmonella cells in comparison to the control samples. Obtained results on the physico-chemical parameters highlight their applicative value since they showed either beneficial effect either did not caused any notable meat quality changes. (C) 2018 Elsevier Ltd. All rights reserved.

Summary In Serbia, the first isolates of C. difficile were isolated in the Public Health Institute (PHI), Center for Microbiology in Niš, at the end of 2005. The National Reference Laboratory for Anaerobic Infections (NRLA) in PHI Niš confirmed the toxigenic strains that caused the first three registered hospital epidemics in Serbia, in 2006 at the Clinic of Neurology, Clinical Center Niš, in 2007 at the Clinical Center of Vojvodina in Novi Sad, and in 2009 in the General Hospital in Požarevac. In 2014, C. difficile species were isolated for the first time from 175 environment samples in the research studies which were conducted in NRLA of PHI Niš. In the samples of soil taken from the ground within the Clinical Center Niš, those taken from the parks at the territory of the Municipality of Niš, samples of mud and sand around the illegal sewage systems at the territory of the Municipality of Niška Banja, a small number of bacteria C. difficile producing the toxins (A+B+) as well as non-toxigenic isolates (A−B−) were found. Results of the first epidemiological investigations of cases of diarrhea associated with prior antibiotic treatment applied in hospitalized patients in a number of health centers in our country, microbiological investigations done in the Public Health Institute, valuable discussions at professional and scientific meetings influenced the general attitude that isolation and identification of C. difficile and/or detection of toxin produced by this bacteria should be part of the routine work in the Serbian microbiological laboratories.

INTRODUCTION:Candida spp. frequently cause hospital-acquired bloodstream infections (BSI) with a high mortality rate (up to 70%). We analyzed the frequency, infection characteristics, potential predisposing factors, susceptibility to antifungal drugs, biofilm production and other virulence characteristics of Candida spp. isolates obtained from a tertiary care hospital in Niš, Serbia, during a one year period.METHODS:Medical histories, characteristics of isolated strains and drug susceptibility, as well as the effect on the function of isolated macrophages and other virulence features were evaluated. The obtained results were subjected to student's t-test and multivariate statistical analyzes.RESULTS:Herein we report an annual incidence of 3.65 cases of C. albicans, C. lusitaniae and C. lipolytica infections per 105 population. Out of eight isolated strains, two (25%) were shown to be strong biofilm producers, one (12.5%) caused hemolysis on blood agar and in two (25%) cases macrophages were able to completely eliminate the yeast colonies. Chronic kidney disease, diabetes, malignant and other diseases were present in 37.5, 62.5, 50 and 75%, respectively, in the study group. All patients with Candida BSI received antifungal therapy (amphotericin B), however, hospital mortality was observed in 25% of patients.CONCLUSIONS:Evaluation of local Candida epidemiology, antifungal susceptibility and virulence factors, as well as personalized patient risk factors are important for the surveillance of Candida BSI, especially in intensive care unit patients and may contribute to the improved options and outcome for patients with Candida BSI.

Platelet rich plasma (PRP) represents a relatively new approach in regenerative medicine. It is obtained from patient’s own blood and contains different growth factors and other biomolecules necessary for wound healing. Since there are various protocols for PRP preparing, it usually results with PRP generation with different amounts of bioactive substances, which finally may modulate the intensity of wound healing. The reference data about potential effect of some PRP compounds on wound healing, in different tissues, are still controversial. This review summarizes recently known facts about physiological role of certain PRP components and guidance for further research. Also, this review discusses different procedure for PRP generation and potential effect of leukocytes on wound healing.

Despite the increasing of onychomycosis caused by Candida spp., in referent literature, there is still data insufficiency about this nail infection. The objectives of this retrospective study were to determine epidemiological characteristics of Candida onychomycosis, the antifungal susceptibility of isolated species in vitro, and to compare the results of antifungal susceptibility testing with conducted treatment in period from 2011 to the end of March 2015. Out of 761 patients who were underwent clinical and mycological examinations, 137 had Candida species isolated from nails. The dominant species was Candida albicans (C. albicans) (36.59%) followed by C. parapsilosis (23.78%), C. krusei (9.76%), and C. guilliermondii (6.71%). Antifungal susceptibility in vitro testing showed good susceptibility to antimycotics, except C. krusei, which was resistance to fluconazole (FCZ) and isolates of C. tropicalis and C. glabrata which were dose dependent to itraconazole (ITZ) and fluconazole. Evaluation of medical histories determined that combined therapy, which included pulsed systemic regimen of ITZ with topical application of clotrimazole, had better clinical outcomes regarding the proscribed only topical application of clotrimazole. Multidisciplinary approach of dermatologists and mycologists is required in solving the problem of onychomycosis, which is the dominant nail disease.

Clostridium difficile is the leading cause of infectious diarrhoea in hospitalized patients. The aim of this study was to determine the risk factors important for the development of hospital-acquired Clostridium difficile-associated disease and clinical manifestations of Clostridium difficile-associated disease. The clinical trial group included 37 hospitalized patients who were selected according to the inclusion criteria. A control group of 74 hospitalized patients was individually matched with cases based on hospital, age (within 4 years), sex and month of admission.Clostridium difficile-associated disease most commonly manifested as diarrhoea (56.76%) and colitis (32%), while in 8.11% of patients, it was diagnosed as pseudomembranous colitis, and in one patient, it was diagnosed as fulminant colitis. Statistically significant associations (p < 0.05) were found with the presence of chronic renal failure, chronic obstructive pulmonary disease, cerebrovascular accident (stroke) and haemodialysis. In this study, it was confirmed that all the groups of antibiotics, except for tetracycline and trimethoprim-sulfamethoxazole, were statistically significant risk factors for Clostridium difficile-associated disease (p < 0.05). However, it was difficult to determine the individual role of antibiotics in the development of Clostridium difficile-associated disease. Univariate logistic regression also found that applying antibiotic therapy, the duration of antibiotic therapy, administration of two or more antibiotics to treat infections, administering laxatives and the total number of days spent in the hospital significantly affected the onset of Clostridium difficile-associated disease (p <0.05), and associations were confirmed using the multivariate model for the application of antibiotic therapy (p = 0.001), duration of antibiotic treatment (p = 0.01), use of laxatives (p = 0.01) and total number of days spent in the hospital (p = 0.001). In this study of patients with hospital-acquired diarrhoea, several risk factors for the development of Clostridium difficile-associated disease were identified. (C) 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda.

According to the literature data, Streptococcus pyogenes is the most common cause of infectious vulvovaginitis in prepubertal girls, but it is rarely described in adult women. In this paper we report the prevalence of S. pyogenes vulvovaginitis in adult women in the municipality of Nis, Serbia, in a five-year period. The total of 42.259 women with symptoms of vulvovaginal infection were enrolled in the study and their swabs from the vagina and cervix were examined using the standard microbiological procedures: direct microscopy of Gram stained preparations and sample culture. Women with positive S. pyogenes findings were later contacted to give anamnestic data and for post-treatment control examinations. S. pyogenes was isolated from the samples of 2 patients: in July 2012 in a 20-year-old woman with the anamnesis of a previously diagnosed tonsillopharyngitis, and in May 2015 in a 32-year-old breast-feeding woman. These patients had the signs and symptoms of acute inflammation: vaginal and vulvar pain, dyspareunia, burning sensation, pruritus, and a profuse, watery, yellow vaginal discharge. Gram staining revealed abundant segmented WBCs and gram-positive cocci in pairs and chains. After the administered penicillin therapy, the control microbiological examinations of the vaginal and cervical swabs were done, as well as the throat swab culture for the presence of S.pyogenes, and all the results were negative. Streptococcal vulvovaginitis in adult women was diagnosed in only two examinees in our study and was associated with a predisposing factors: personal history of a respiratory infection and lactational vaginal atrophy.

Due to a confirmed antimicrobial activity of both natural and synthetic coumarin derivatives, the present study was envisaged to provide a further insight into the antimicrobial potential of coumarins through a screening of a designed library of nine 4-(alkylamino)-3-nitrocoumarins against a panel of 24 laboratory strains and resistant (isolates) bacterial and fungal pathogens. All compounds showed some degree of strain-selective activity, that was, in some cases, very pronounced, reaching the value of 0.04 nmol/ml (i.e. 12 ng/ml) for the minimal inhibitory concentration against Candida albicans. The observed activity was higher against Gram-negative strains, among which the most susceptible strain, among both ATCC strains and clinical isolates, was Salmonella enteritidis. These results point out to a high potential of these coumarins as antimicrobials for the treatment of gastrointestinal and other infections caused by highly resistant microbial strains. Finally, a multivariate statistical analysis of the herein obtained and previous results on the antimicrobial activity of related selected coumarins was performed to allow an easier structure-activity discussion.

Clostridium (C.) difficile is a typical representative of the genus Clostridium. After colonization of the intestinal tract, toxigenic C. difficile strains are capable to produce two exotoxins, enterotoxin (toxin A) and cytotoxin (toxin B), which cause diarrhea and colitis. Toxin A binds to specific carbohydrate receptors on the surface of intestinal cells and this is the beginning of damages in the intestinal tract which include destruction of the villi epithelium, limiting membrane, intercellular connections (zonula occludens) and surface of the mucosa. If only toxin B is injected into intestinal cells, it does not cause damage nor increased fluids secretion. Probably, the reason for this is the inability of the toxin to bind to the cell membrane receptor in the intestinal tract under normal physiological conditions. Toxigenic strains of C. difficile can be found in the intestines of healthy people, without any symptoms or clinical signs (asymptomatic colonization). However, in people with risk factors, they can cause diarrhea of varying severity and life-threatening pseudomembranous colitis. These diseases are known as C. difficile associated disease - CDAD.

The aim of this study was to fortify the clinical importance and representation of toxigenic and non-toxigenic Clostridium difficile isolated from stool samples of hospitalized patients. This survey included 80 hospitalized patients with diarrhea and positive findings of Clostridium difficile in stool samples, and 100 hospitalized patients with formed stool as a control group. Bacteriological examination of a stool samples was conducted using standard microbiological methods. Stool sample were inoculated directly on nutrient media for bacterial cultivation (blood agar using 5% sheep blood, Endo agar, selective Salmonella Shigella agar, Selenite-F broth, CIN agar and Skirrow's medium), and to selective cycloserine-cefoxitin-fructose agar (CCFA) (Biomedics, Parg qe tehnicologico, Madrid, Spain) for isolation of Clostridium difficile. Clostridium difficile toxin was detected by ELISA-ridascreen Clostridium difficile Toxin A/B (R-Biopharm AG, Germany) and ColorPAC ToxinA test (Becton Dickinson, USA). Examination of stool specimens for the presence of parasites (causing diarrhea) was done using standard methods (conventional microscopy), commercial concentration test Paraprep S Gold kit (Dia Mondial, France) and RIDA®QUICK Cryptosporidium/Giardia Combi test (R-Biopharm AG, Germany). Examination of stool specimens for the presence of fungi (causing diarrhea) was performed by standard methods. All stool samples positive for Clostridium difficile were tested for Rota, Noro, Astro and Adeno viruses by ELISA - ridascreen (R-Biopharm AG, Germany). In this research we isolated 99 Clostridium difficile strains from 116 stool samples of 80 hospitalized patients with diarrhea. The 53 (66.25%) of patients with diarrhea were positive for toxins A and B, one (1.25%) were positive for only toxin B. Non-toxigenic Clostridium difficile isolated from samples of 26 (32.5%) patients. However, other pathogenic microorganisms of intestinal tract cultivated from samples of 16 patients. Examination of cultivated colonies revealed that most of cultivated species belonged to genera of Campylobacter spp., Salmonella spp., and Candida spp.. In control group, toxigenic Clostridium difficile cultivated from stool samples of two patients (2%) and non-toxigenic Clostridium difficile from samples of five patients (5%). This research confirmed clinical importance of toxigenic Clostridium difficile found in liquid stool samples of hospitalized patient, and the possibility of asymptomatic carriage in 2% of patients with formed stool.