Occurrence of Clostridium difficile infections in Serbia and high proportion of PCR ribotype 027 strains in two hospitals in Belgrade.

BACKGROUND: The incidence of Clostridium difficile infections (CDI) in the Clinical Center of Serbia (CCS) and the entire Serbia has been constantly rising in the previous 5 years. We aimed to study C. difficile PCR-ribotypes isolated from patients hospitalized at two healthcare institutions: CCS and Specialized Hospital for Cerebrovascular Diseases "Sveti Sava" (SS), both of them from Belgrade, and to investigate the incidence rates of CDI in hospital settings in Serbia, from 2009 to 2013. METHODS: The Bacteriology laboratory database at Clinic for Infectious and Tropical Diseases of CCS was queried from January 1, 2009 to December 31, 2013 for all patients who underwent immunochromatographic toxin A and/or toxin B stool testing and C. difficile stool culture for suspected infection caused by this bacterium. Toxigenic culture was not performed. Ninety- six C. difficile isolates were then selected and characterized by PCR-ribotyping. These were obtained from 94 patients hospitalized in different clinics of CCS and SS from November 2011 to December 2013. RESULTS: Among 6164 stool samples sent to Bacteriology laboratory for culture of C. difficile and toxin detection during the study period, 1775 (28.8%) were positive, displaying linear trend of growth. From 96 isolates, typed by PCR-ribotyping, majority (85; 88.54%) belonged to PCR-ribotype 027. The remaining 11 isolates belonged to PCR-ribotypes 014/020 (3 isolates), 015, SLO 191 (two isolates each), 017, 018, 070 and 001/072 (one isolate each). CONCLUSION: Our results demonstrated that C. difficile PCR-ribotype 027 is by far predominant in two hospital settings in Belgrade, at least since 2011.

Serotype distribution, antimicrobial susceptibility and molecular epidemiology of invasive Streptococcus pneumoniae in the nine-year period in Serbia.

Streptococcus pneumoniae is one of the leading bacterial pathogens that can cause severe invasive diseases. The aim of the study was to characterize invasive isolates of S. pneumoniae obtained during the nine-year period in Serbia before the introduction of the pneumococcal conjugate vaccines (PCVs) into routine vaccination programs by determining: serotype distribution, the prevalence and genetic basis of antimicrobial resistance, and genetic relatedness of the circulating pneumococcal clones. A total of 490 invasive S. pneumoniae isolates were included in this study. The serotype, antimicrobial susceptibility, and ST of the strains were determined by the Quellung reaction, disk- and gradient-diffusion methods, and multilocus sequence typing (MLST), respectively. The most common serotypes in this study were 3, 19F, 14, 6B, 6A, 19A, and 23F. The serotype coverages of PCV10 and PCV13 in children less than 2 years were 71.3 and 86.1%, respectively, while PPV23 coverage in adults was in the range of 85-96%, depending on the age group. Penicillin and ceftriaxone-non-susceptible isolates account for 47.6 and 16.5% of all isolates, respectively. Macrolide non-susceptibility was detected in 40.4% of isolates, while the rate of multidrug- and extensive-drug resistance was 20.0 and 16.9%, respectively. The MLST analysis of 158 pneumococci identified 60 different STs belonging to the 16 Clonal Complexes (CCs) (consisting of 42 STs) and 18 singletons. The most common CC/ST were ST1377, CC320, CC15, CC273, CC156, CC473, CC81, and CC180. Results obtained in this study indicate that the pre-vaccine pneumococcal population in Serbia is characterized by high penicillin and macrolides non-susceptibility, worrisome rates of MDR and XDR, as well as a high degree of genetic diversity. These findings provide a basis for further investigation of the changes in serotypes and genotypes that can be expected after the routine introduction of PCVs.

Clostridioides difficile ribotype distribution in a large teaching hospital in Serbia.

BACKGROUND: The global epidemic of nosocomial diarrhea caused by Clostridioides (Clostridium) difficile started in 2000, with high mortality rates and emergence of a new hypervirulent strain NAP1/BI/027. The aim of this study was to assess the presence of ribotype 027 and other C. difficile ribotypes in a Serbian University Hospital, compare the temporal variability of ribotypes 3 years apart, as well as to compare clinical, demographic and laboratory characteristics and disease outcome among patients infected with 027 and non-027 ribotype. This was a prospective observational cohort study addressing 4-month intervals during 2014/2015 and 2017/2018. RESULTS: Ribotyping was performed in 64 non-duplicate C. difficile strains. Ribotype 027 was the most prevalent, and was detected in 53 (82.8%) patients (43/45 and 10/19 patients in 2014-2015 and 2017/2018, respectively). Other detected ribotypes were 001/072 in 4 (6.3%), 002 in 4 (6.3%), 014/020 in 2 (3.1%) and 176 in 1 (1.5%) patient. The percentage of the patients infected with ribotype 027 significantly decreased during the 3-year period, from 95.6 to 52.6% (p < 0.001). Ribotype 027 infection was associated with fluoroquinolone treatment more frequently than infection with other ribotypes [33 (62.3%) vs. 2 (18.2%), p = 0.010)]. A severe C. difficile infection was diagnosed more often in patients with the detected ribotype 027 compared to those infected with non-027 ribotypes (p = 0.006). No significant difference in the mortality and recurrence rates was found between the patients infected with ribotype 027 and those infected with other ribotypes [10/53 (18.8%) vs. 2/11 (18.2%), p = 0.708, and 10/35 (28.6%) vs. 0/2 (0%), p = 1.000, respectively]. CONCLUSION: Clostridium difficile ribotype 027 was the most prevalent ribotype among patients in a large Serbian hospital, but there is a clear decreasing trend.

Presence of the esp gene in Enterococcus faecium derived from oropharyngeal microbiota of haematology patients.

OBJECTIVES: Antibiotic use and immunocompromised status in haematology patients have been shown to determine the constituents of commensal microbiota with highly increased resistance, including vancomycin resistant enterococci. We compared the carriage of virulence factor genes and the capacity for biofilm formation in vancomycin resistant enterococci (VRE) originating from the oropharyngeal and stool cultures of haematology patients. DESIGN: PCR tests were used to investigate the presence of genes encoding pathogenicity factors (esp and hyl) in VRE isolates. The genotype of vancomycin resistance was investigated by multiplex PCR tests for vanA and vanB genes. PFGE typing was conducted to exclude the duplicate isolates. RESULTS: Of 3310 pharyngeal swabs taken from inpatients at a clinic for haematology, Enterococcus species were recovered in 6.46%. All VRE investigated were identified as Enterococcus faecium and were highly vancomycin resistant. VanA genotype was confirmed in all. In the group of oropharyngeal carriers (n = 8 patients), 15 strains were recovered from oropharyngeal specimens and PFGE typing revealed 5 types and 3 subtypes. Identical types of VRE in the oropharynx and stool cultures were found in three patients from this group. In the group of stool carriers (n = 24 patients) VRE were obtained from stools only and placed in 21 macro-restriction patterns. The esp gene was more common in VRE isolated from the oropharynx than in isolates from stools (p = 0.014). Results were not significant when we compared the presence of hyl genes in oropharyngeal isolates with those from stool cultures (p = 0.66) or when we investigated the association between esp and hyl gene carriage and capability of biofilm formation in non-repeated VRE. CONCLUSIONS: In the present study, isolation of VRE from the oropharynx in haematology patients was associated with esp gene carriage. Further research is needed to investigate the clinical and long-term effects of this finding.

Differences in MRSA prevalence and resistance patterns in a tertiary center before and after joining an international program for surveillance of antimicrobial resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) emerged as one of the most important causes of hospital-acquired bloodstream infections (BSIs), especially the multidrug resistant clones. The aim of the present study was to compare prevalence and resistance patterns of MRSA bacteremia in the major tertiary-care academic and referral center in Serbia before and after implementing an active antimicrobial resistance (AMR) surveillance. Laboratory-based before-after study was conducted during a two-year period (January 2012 to December 2013) in Clinical Centre of Serbia. Isolation and identification of bacterial strains were done following standard microbiological procedures. During the AMR surveillance, nearly twice more bloodstream samples were collected compared to the year without surveillance (1,528 vs. 855). In total, 43 isolates of MRSA were identified. MRSA was significantly more prevalent during the AMR surveillance compared to the previous year [14 (66.7%) to 29 (76.3%); P = 0.046]. During the AMR surveillance, MRSA more frequently originated from medical departments compared to intensive care unit, surgical department, and internal medicine (P = 0.027) indicating increasing MRSA infections in patients with less severe clinical condition and no apparent risk factors. Higher prevalence of MRSA and its lower susceptibility to erythromycin were revealed by implementation of active AMR surveillance, which may reflect more thoughtful collection of bloodstream samples from patients with suspected BSI.

Local spread of Tn1546-like element among three species of vancomycin resistant enterococci in an intensive care unit.

INTRODUCTION: In an intensive care unit (ICU) of the Emergency Center in the Clinical Center of Serbia, four species of vancomycin resistant enterococci (VRE) were isolated in a 17-month period mostly from blood cultures, including E. faecalis, E. faecium, E. raffinosus and E.gallinarum. METHODOLOGY: The relationship between isolates from each species was investigated by PFGE, and PCR experiments for detection of pathogenicity factor genes and van genes to determine the nature of each clone. A PCR-based method, using 10 primer pairs (p1/2-p19/20), was used to investigate the presence of the Tn1546-like structure. RESULTS: PFGE indicated the presence of two different E. faecium clones, while the three other enterococcal species belonged to one clone each. Transposon typing revealed that isolates of E. raffinosus (4), E. gallinarum (4) and E. faecalis (3) yielded gene sequences identical to 10 primer pairs (p1/2-p19/20), suggesting the possibility of identical transposon-like structure in these species. CONCLUSIONS: The results of the study indicate probable horizontal spread of Tn1546-like structure in three species of VRE obtained from the same ICU.

Antimicrobial resistance of selected invasive bacteria in a tertiary care center: results of a prospective surveillance study.

INTRODUCTION: We aimed to report the distribution and resistance patterns of eight invasive clinically relevant bacteria surveyed in the Clinical Center of Serbia (CCS) in Belgrade. METHODOLOGY: A total of 477 clinical blood stream isolates of Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter spp. were collected in the period from January to December 2013. Antimicrobial susceptibility testing was performed using standard methods and interpreted using the Clinical and Laboratory Standards Institute (CLSI) breakpoint criteria. RESULTS: Acinetobacter spp. was the most prevalent bacteria encountered (37%), followed by K. pneumoniae (25.7%). Multidrug resistance was observed in 92.5% of all isolates. Out of 177 strains of Acinetobacter spp., 97.7% were resistant to fluoroquinolones and carbapenems. Resistance to aminoglycosides, fluoroquinolones, and third-generation cephalosporins was 97.1%, 95.4%, and 95.8% among K. pneumoniae and 21.4%, 21.7%, and 31% among E. coli isolates, respectively. In total, 65.1% of K. pneumoniae and 12.1% of E. coli isolates were determined to be extended-spectrum beta-lactamase (ESBL) positive. High-level aminoglycoside resistance of E. faecalis was 71.4%, and glycopeptide resistance of E. faecium was 95%. Out of 66 strains of S. aureus, 63.4% were methicillin resistant. CONCLUSIONS: The majority of bloodstream isolates of clinically relevant bacteria in CCS were multidrug resistant. The biggest concerns are carbapenem-resistant Acinetobacter spp., K. pneumoniae, and P. aeruginosa; third-generation cephalosporin-resistant E. coli; vancomycin-resistant E. faecium; and methicillin-resistant S. aureus. Stricter measures of infection control and antibiotic use are needed.

Brain abscess due to Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, a constituent of the oral flora, is a rare cause of brain abscesses. We report the case of a 47-year-old male who presented with multiple brain abscesses due to this organism, presumably originating from his poor dentition. Problems met in isolating and identifying A. actinomycetemcomitans suggest that its true rate of isolation from non-oral samples may have been underestimated.

Molecular typing, pathogenicity factor genes and antimicrobial susceptibility of vancomycin resistant enterococci in Belgrade, Serbia.

In this study the distribution of species and antimicrobial resistance among vancomycin resistant enterococci (VRE) recovered from clinical specimens obtained from five hospitals in Belgrade was analyzed. Strains were further characterized by pulsed-field gel electrophoresis (PFGE). Polymerase chain reaction (PCR) was used to investigate the presence of vanA and vanB genes and pathogenicity factor genes. Identification of 194 VRE isolates revealed 154 Enterococcus faecium, 21 Enterococcus faecalis, 10 Enterococcus raffinosus and 9 Enterococcus gallinarum. This study revealed existence of 8 major clones of VRE. PCR determined vanA gene to be present in all of the VRE studied. Esp and hyl genes were present in 29.22% and 27.92% of E. faecium, respectively, and in 76.19% and 0 of E. faecalis, respectively. Esp and hyl genes were not found more frequently in members of predominant clones of E. faecium than in single isolates; nor was their presence connected to invasiveness.

Risk factors for vancomycin-resistant Enterococcus colonization in hematologic patients.

BACKGROUND/AIM: Vancomycin-resistant Enterococci (VRE) is one of the most important hospital pathogens. The aim of the study was to evaluate VRE colonization in patients hospitalized at the Hematology Intensive Care Unit, as well as the associated risk factors. METHODS: A prospective cohort study involved 70 patients hospitalized at the Intensive Care Unit (ICU), Clinic for Hematology, Clinical Center of Serbia, Belgrade, during 3 months. Baseline demographic data, data about antibiotic usage and other risk factors for VRE colonization during the present and previous hospitalizations (within 6 months) were recorded for each patient using the questionnaire. Feces or rectal swab was collected for culture from patients on admission and at discharge in case when VRE was not isolated on admission. Enterococci were isolated by standard microbiological methods. Isolate sensitivity was tested by disk-diffusion test using 30 microg/mL (BBL) Vancomycin plates according to the Clinical and Laboratory Standards Institute (CLSI) standard. RESULTS: Analysing results showed that 7% of the patients had been already colonized with VRE upon ICU admission. The rate of VRE colonization during present hospitalization was 41.5%. Univariate logistic regression demonstrated the statistically significant differences in diagnosis, length of present stay, use of aminoglycosides and piperacillin/tazobactam in present hospitalization, duration of use of carbapenem and piperacillin/tazobactam in present hospitalization between the VRE-colonized and non-colonized patients. Acute myeloid leukemia (AML), use of carbapenem in previous hospitalization and duration of use of piperacillin/tazobactam in present hospitalization were independent risk factors for VRE-colonized patients according to multivariate logistic regression. CONCLUSION: VRE colonization rate was high among the patients admitted to hematology ICU. Rational use of antibiotics and active surveillance may be helpful preventive measures against the development of bacterial resistance to antimicrobial agents.

Molecular characterization of vancomycin-resistant enterococci in Serbia: intensive care unit as the source.

The purpose of this study was to evaluate the molecular relatedness of clinical isolates of vancomycin-resistant enterococci (VRE) collected from patients of the Clinic for Infectious and Tropical Diseases in Belgrade. Among 40 isolates available for the investigation, 36 were identified as Enterococcus faecium, whereas 2 were Enterococcus faecalis and Enterococcus raffinosus, respectively. Pulsed-field gel electrophoresis (PFGE) typing revealed 21 strain types, comprising 7 clusters which contained at least two isolates and 14 unique PFGE patterns. Although we searched for pathogenicity factor genes (gelE, cylB, asa1, efaAfs, esp, cpd, cob) in representatives of all macro-restriction patterns, they have been confirmed in only one clone of E. faecalis. Genes esp and hyl, commonly found in E. faecium, were yilded in 10 macro-restriction patterns of this species, and their presence could not be ascribed to clonally related strains (p = 0.05). All VRE isolates were multiresistant and positive for vanA gene. Twenty strains of VRE and 6 clusters obtained from Intensive care unit (ICU) are proof of intensive transmission of these microorganisms at this department. The results of this study suggest wide genotypic variability among the clinical VRE isolates, but also intrahospital dissemination of some of them.

Bilateral pulmonary shadows and incapsulated mediastinal effusion in a patient with respiratory blastoschizomycosis.

INTRODUCTION: Invasive blastoschizomycosis is an emerging and fatal infection of severely immunocompromised patients. Pneumonia caused by this fungus has been reported as a post mortem diagnosis in a patient with haematologic malignancy. CASE REPORT: A 46-year-old unemployed Caucasian man, smoker, was admitted on emergency in a serious condition, with bilateral pulmonary infiltrates (chest x-ray) and incapsulated mediastinal pleural effusions (computed tomography scans). Blastoschizomyces capitatus (Trichosporon capitatum) has been isolated from two sputum samples and bronchial secretion aspirate obtained by bronchoscopy. CONCLUSION: Pneumonia caused by Blastoschizomyces capitatus can develop in subjects without underlying malignancy but with history of heavy alcohol intake, tobacco smoking and malnutrition and may have favorable disease outcome.