Milica Jovanović

Brain abscess due to Actinobacillus actinomycetemcomitans Case report

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.

Vancomycin-resistant Enterococcus faecalis in Serbia.

To the Editor: First isolated in France (1), vancomycin-resistant enterococci (VRE) have become pathogens of major importance, particularly in the United States (2). Infections due to VRE are still uncommon in most European countries (3). We report the first isolation of high-level vancomycin-resistant Enterococcus faecalis in Serbia. n nA 55-year-old woman was admitted to the Clinic for Cardiovascular Diseases, Belgrade, on April 1, 2002, for aortobifemoral bypass surgery. Three weeks after she was admitted to the hospital, an infection developed in the surgical wound and treatment with sulfamethoxazole-trimethoprim (160/800 mg q 12 h) was empirically introduced. Bacteriologic analysis of the wound swab sample showed a methicillin-resistant strain of Staphylococcus aureus, a multiresistant strain of Acinetobacter sp., a commonly susceptible strain of Enterococcus sp., and a VRE strain. According to the results of susceptibility testing, imipenem (1 g q 6 h) was added to the patient’s treatment protocol. VRE were not isolated from subsequent wound samples or any other sample submitted for microbiologic analysis. The patient was discharged at the end of the 14-day treatment period. n nThe isolate was identified as E. faecalis by biochemical characterization, as recommended by Facklam and Collins (4) and confirmed by API 20 Strep (bioMerieux, Marcy-l’Etoile, France). Susceptibility testing, performed by the disk diffusion method, showed that the isolate was resistant to vancomycin, teicoplanin, gentamicin, streptomycin, tetracycline, and ciprofloxacin, while susceptible to ampicillin, amoxicillin, amoxicillin and clavulanic acid, and imipenem. Resistance to vancomycin, teicoplanin, gentamicin, and streptomycin was confirmed by the broth dilution method, according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations (5). The obtained MICs were 256 μg/mL for vancomycin, 64 μg/mL for teicoplanin, >4,000 μg/mL for gentamicin, and >2,000 μg/mL for streptomycin. This phenotype, with high-level resistance to vancomycin and teicoplanin, is typical for the vanA genotype (2). The strain was subsequently genotyped by pulsed-field gel electrophoresis, using previously described methods (6). The presence of the vanA gene was confirmed by polymerase chain reaction assay, according to a previously described procedure (7). E. faecium EF228 was used as the positive control. n nThe enterococci are among the most frequent causes of nosocomial infections, particularly in intensive care units, and present a major therapeutic challenge (2). While the emergence of VRE strains in the United States is probably associated with extensive use of vancomycin, the occurrence of VRE in Europe is possibly due to application of avoparcin (glycopeptide analog) as a growth promoter in animal husbandry (3). However, avoparcin has not been used in Serbia, and vancomycin application has been restricted to hospitalized patients and quite limited due to its high cost. Thus, emergence of VRE strains in Serbia has not been likely. n nThe origin of this VRE isolate is unknown: the strain may have been imported or may have originated from the hospital environment. The first prospective pan-European VRE surveillance study (January–April 1997) showed VanA-VRE strains in only eight European countries, with isolates numbering from one to four per country (3). No epidemiologic relations were established among the VanA isolates, and only 2 out of 18 isolates (11%) were identified as E. faecalis (3). Since our patient-case had no history of travel outside Serbia, we assumed that the VRE isolate originated from the hospital environment. However, a study investigating the occurrence of VRE strains in Belgrade, the capital of Serbia, detected no such isolates in five different hospitals (8). Although the study did not analyze samples from the Clinic for Cardiovascular Diseases, it did include samples from the Clinic for General Surgery, which is located within the same building. The susceptibility of 191 isolates of enterococci to vancomycin was tested by agar dilution method according to NCCLS recommendations. Of the 191 isolates, 159 were classified as susceptible and 32 as intermediately susceptible. n nThis report of the first isolation of VRE in Serbia, as well as the previously shown presence of enterococci displaying intermediary susceptibility to vancomycin, provides the rationale for future active screening for VRE in hospital environments in the region.

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

BACKGROUND/AIMnVancomycin-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.nnnMETHODSnA 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.nnnRESULTSnAnalysing 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.nnnCONCLUSIONnVRE 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 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.

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.

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

OBJECTIVESnAntibiotic 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.nnnDESIGNnPCR 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.nnnRESULTSnOf 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 (nu202f=u202f8 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 (nu202f=u202f24 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 (pu202f=u202f0.014). Results were not significant when we compared the presence of hyl genes in oropharyngeal isolates with those from stool cultures (pu202f=u202f0.66) or when we investigated the association between esp and hyl gene carriage and capability of biofilm formation in non-repeated VRE.nnnCONCLUSIONSnIn 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.

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

BACKGROUNDnThe 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.xa0difficile 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.nnnMETHODSnThe 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.xa0difficile stool culture for suspected infection caused by this bacterium. Toxigenic culture was not performed. Ninety- six C.xa0difficile 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.nnnRESULTSnAmong 6164 stool samples sent to Bacteriology laboratory for culture of C.xa0difficile 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).nnnCONCLUSIONnOur results demonstrated that C.xa0difficile PCR-ribotype 027 is by far predominant in two hospital settings in Belgrade, at least since 2011.

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%); Pu2009=u20090.046]. During the AMR surveillance, MRSA more frequently originated from medical departments compared to intensive care unit, surgical department, and internal medicine (Pu2009=u20090.027) 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.

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

INTRODUCTIONnWe aimed to report the distribution and resistance patterns of eight invasive clinically relevant bacteria surveyed in the Clinical Center of Serbia (CCS) in Belgrade.nnnMETHODOLOGYnA 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.nnnRESULTSnAcinetobacter 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.nnnCONCLUSIONSnThe 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.

Antimicrobial resistance in patients with urinary tract infections and the impact on empiric therapy in Serbia.

INTRODUCTIONnSurveillance of antimicrobial resistance is essential in establishing treatment guidelines for urinary tract infections. The aim of this pilot study was to analyse resistance rates of pathogens, across different demographics and determine whether adjustments in empiric therapy should be considered for different age and gender groups.nnnMETHODOLOGYnA 5-year retrospective study included 256 patients hospitalised, under the initial diagnosis of Fever of Unknown Origin who were then subsequently diagnosed with a urinary tract infection at the Clinic for Infectious and Tropical Diseases, Clinical Centre of Serbia. Patients were evaluated using demographic, clinical, and antimicrobial resistance data with appropriate statistical analysis including ANOVA significance testing, univariate, and multivariate analysis.nnnRESULTSnResistance rates were above the threshold of 20% for the majority of the antimicrobials tested, the only exception being carbapenems. Amikacin, cefepime, and norfloxacin were agents that could be effectively used as empiric therapy in younger adults with resistance rates of 4.2, 8.0, and 10.0%, respectively. Moderate resistance rates of 17.4% for amikacin and 19.1% for cefepime were observed in the age group 35-64 years. High resistance rates were observed for all antimicrobials among patients 65 years and over. Among male patients, resistance rates to most antimicrobials were high. In female patients, amikacin and cefepime had resistance rates less than 20%. Younger age presented as a negative risk factor for infection by a multi-drug resistant pathogen.nnnCONCLUSIONnAge and gender demonstrated to be significant factors for determining proper empiric therapy; large-scale studies from Serbia are needed to solidify these findings.