Analysis of Etiology of Community-Acquired and Nosocomial Urinary Tract Infections and Antibiotic Resistance of Isolated Strains: Results of a 3-Year Surveillance (2020-2022) at the Pediatric Teaching Hospital in Warsaw.

Urinary tract infections (UTIs) remain the most common infections diagnosed in outpatients and hospitalized patients. This study was designed to determine the patterns of antibiotic resistance and the prevalence of uropathogens causing UTIs in pediatric patients hospitalized between 1 January 2020 and 31 December 2022 at Teaching Hospital in Warsaw. The most frequent species isolated from urine samples were E. coli (64.5%), Klebsiella spp. (11.6%), and Enterococcus spp. (6.1%). UTIs caused by Enterobacter spp., Enterococcus spp., and Klebsiella spp. were significantly more common in children younger than three months of age than in children older than three months (p < 0.001). Trimethoprim and trimethoprim-sulfamethoxazole were the least active compounds against Enterobacterales with the resistance of E. coli, Klebsiella spp., P. mirabilis, and Enterobacter spp. in the range of 26.7/25.2%, 48.4/40.4%, 51.1/40.4%, and 15.8/13.2% respectively. Ampicillin was also found to have resistance rates for E. coli of 54.9% and P. mirabilis of 44.7%. Cefalexin and cefuroxime were highly active towards Enterobacterales except for Klebsiella spp., in which the resistance level reached 40%. Regarding third- and fourth- generation cephalosporins, resistance in E. coli and P. mirabilis was observed in approximately 2-10% of the isolates, but in Klebsiella spp. and Enterobacter spp. ranged over 30%. The resistance of Enterobacterales to carbapenems, nitrofurantoin, and fosfomycin was below 1%. The quinolones resistance was very high for Klebsiella spp. (31.1%) and P. mirabilis (29.8%) and three times lower for E. coli (11.9%), P. aeruginosa (9.3%), Enterobacter spp. (2.6%), and E. faecalis (4.6%). Resistance to multiple antibiotic classes was identified in 396 Enterobacterales strains, 394 of which were multi-drug resistant (MDR) and 2 were extensive drug-resistant (XDR). In the case of E. coli, 30% of isolates were MDR, with the proportion of strains having this exact resistance pattern similar in all of the analyzed years; no E. coli XDR strains were isolated. The number of Klebsiella spp. MDR strains was much higher in 2022 (60%) than in 2021 (47.5%). In the analyzed time, only one strain of K. pneumonia XDR, producing New Delhi metallo-ß-lactamase, was isolated. Monitoring infection trends is essential to improve control and limit the rise of bacterial resistance.

Corynebacterium coyleae as potential urinary tract pathogen.

Corynebacterium coyleae is part of the commensal microflora of the skin, urethra, mucous membranes, and genital tract. Isolates from patients with urinary tract infection (UTI) were reported, but the pathogenic potential of this species has not been defined yet. The aim of the study is to determine whether C. coyleae could be the etiological agent of UTI and to analyze its antibiotic susceptibility. Urine samples were cultured quantitatively according to accepted laboratory procedures. The identification of bacterial isolates was carried out using the Vitek MS (bioMérieux) and antibiotic susceptibility was tested using disc diffusion according to EUCAST guidelines. Between 1 January 2017 and 30 October 2018, a total of 39 C. coyleae strains were isolated. This represented 0.32% of all urine samples cultured in the laboratory during the collection period. The strains were isolated from samples obtained from 35 women and 3 men (age median for all-64 years). One female patient presented with C. coyleae in her urine twice at an interval of 21 months. In six cases of UTI, C. coyleae was isolated in monoculture. The isolates had the same resistance pattern. A total of 11 strains were obtained from cases with a clinical diagnosis of UTI. In 13 cases, the strain was cultured in a monoculture and in 28 cases with accompanying species. All strains were susceptible to vancomycin. However, resistance to ciprofloxacin was observed for 58.4% of the strains. Urine isolates of C. coyleae must be considered as contamination or normal flora in most cases (28/39, 72%). In the remaining cases, it can be considered as potential etiologic agents, mostly in women and especially in the 6 UTI cases where C. coyleae was found as the single culture-positive species. Several of these isolates demonstrate resistance to antibiotics commonly used in empiric treatment of urinary tract infections.

Prevalence of Clostridium difficile infection in hospitalized patients with diarrhoea: Results of a Polish multicenter, prospective, biannual point-prevalence study.

PURPOSE: We aimed to measure the underdiagnosis of Clostridium difficile infection across Poland and the distribution of PCR-ribotypes of C. difficile. MATERIAL AND METHODS: Twenty seven Polish healthcare facilities (HCFs) participated in this prospective study. Each HCF systematically sent all diarrhoeal stools received from inpatients at their laboratories on two days (one in January 2013 and one in July 2013), independently of CDI test request, to the National Coordinating Laboratory (NCL) for standardized testing of CDI. Positive samples (using two-stage algorithm), had CDI, confirmed by qPCR and toxigenic culture. C. difficile isolates were characterized by PCR-ribotyping. Hospitals were questioned about their methods and testing policy for CDI during the study period: September 2011 to August 2013. RESULTS: During the study period, participating hospitals reported a mean of 33.2 tests for CDI per 10 000 patient-days and a mean of 8.4 cases of CDI per 10 000 patient-days. The overall prevalence of positive CDI patients at NCL was 16.5%. Due to absence of clinical suspicion, 19.1% of these patients were not diagnosed by the local diagnostic laboratory. We identified 23 different PCR-ribotypes among 87C. difficile strains isolated from patients. PCR-ribotype 027 (48%) was the most prevalent. CONCLUSIONS: The incidence of CDI in Poland in study period was very high. It should be noted however, that there is a lack of clinical suspicion and underestimation of the need to perform diagnostic tests for CDI in hospitalized patients. This will have an impact on the reported epidemiological status of CDI in Poland.

Metronidazole or Rifaximin for Treatment of Clostridium difficile in Pediatric Patients with Inflammatory Bowel Disease: A Randomized Clinical Trial.

BACKGROUND: Interestingly, Clostridium difficile infection (CDI) worsens the course of inflammatory bowel disease (IBD); however, there is a paucity of data regarding the treatment of CDI in this group of patients. METHODS: This was a prospective, single-blind trial. Children with flare of IBD and CDI were randomly assigned to receive metronidazole or rifaximin orally for 14 days. CDI was diagnosed based on a positive well-type enzyme immunoassay (EIA) toxins A/B stool test for C. difficile toxins A and/or B. The cure rate was defined as the percentage of patients with a negative EIA stool test for C. difficile toxins A/B measured 4 weeks after the end of treatment. Recurrence was defined as a repeat CDI within 2 to 8 weeks. RESULTS: In total, we included 31 patients with IBD including 12 patients with Crohn's disease and 19 with ulcerative colitis. Of them, 17 received metronidazole and 14 received rifaximin. There were no statistically significant differences between the 2 study groups including age, type of treatment, and disease activity. There was no statistically significant difference in the cure rate between patients treated with metronidazole and rifaximin (70.6% versus 78.6%, respectively, P = 0.5). We found no difference in recurrence rate between the 2 study treatment types (17% versus 0%, respectively, P = 0.3). We did not find an association between immunosuppressive therapy and CDI cure rate or CDI recurrence rate. CONCLUSIONS: Metronidazole and rifaximin were similarly effective treatments for CDI in pediatric patients with IBD.

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.

[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.

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.

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.

[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.

[Assessment of susceptibility of strictly anaerobic bacteria originated from different sources to fluoroquinolones and other antimicrobial drugs]. / Ocena wrazliwosci szczepów bezwzglednych beztlenowców pochodzacych z róznych zródel na fluorochinolony oraz inne leki stosowane w lecznictwie szpitalnym.

INTRODUCTION: During the past 20 years, several studies at a national level in different countries followed resistance trends for Bacteroides sp. and Clostridium difficile. This study analysed antimicrobial susceptibility 73 anaerobic bacteria strains of Bacteroides fragilis group (BFG) and C. difficile to fluoroquinolones and other antimicrobial drugs. METHODS: The strictly anaerobes strains isolated in different hospitals were sent to the Department of Medical Microbiology, Medical Uniwersity of Warsaw, where species determination was carried out with the API20 ANA (bioMerieux SA, Marcy-l'Etoile, France) system. Susceptibility to antimicrobials was determined using E-test. RESULTS: The rates of high resistance to ciprofloxacin and moxifloxacin of BFG was respectively 84% and 31% and among of C. difficile strains respectively 92% and 36%). The percentage of BFG strains resistant to erythromycin and clindamycin were respectively 84% and 46%. The percentage of C. difficile strains resistant to erythromycin and clindamycin was 52%. Reduced level of susceptibility of BFG strains to amoxicillin/clavulanic acid (8%) was confirmed. Resistance to cefoxitin was 16% of BFG strains. All tested strains as well as BFG and C. difficile were susceptible to metronidazole. Was observed reduced leve (EUCAST) of susceptibility of C. difficile strains to vancomycin (13%). CONCLUSIONS. Increasing resistance to various antimicrobial agents is a significant problem in Poland. This demonstrate the need to continue with antibiotic resistance testing and surveys in anaerobic bacteria.