Ivana Dakić

A modified microtiter-plate test for quantification of staphylococcal biofilm formation

The tube test and the microtiter-plate test are the most frequently used techniques for quantifying biofilm formation, an important indicator for the pathogenicity of staphylococci. The purpose of the present study was to develop a modified microtiter-plate technique for quantification of biofilm formation. This technique involves fixing the bacterial film with methanol, staining with crystal violet, releasing the bound dye with 33% glacial acetic acid, and measuring the optical density (OD) of the solution at 570 nm by using an enzyme immunosorbent assay reader. Biofilm formation of 30 Staphylococcus strains was estimated by the tube test, the standard microtiter-plate test and the modified microtiter-plate test. The modified microtiter-plate test, as a quantitative assay, is superior to the tube test in terms of objectivity and accuracy. It is also superior to the standard microtiter-plate test because it enables indirect measuring of bacteria attached both to the bottom and to the walls of the wells, while in the standard test only the dye bound to the bacteria adhered to the bottom of the wells is spectrophotometrically registered. Highly significant differences between OD values obtained by the standard microtiter-plate test and those obtained by the modified test suggest that large number of bacteria were attached to the walls of the wells. Therefore, the modification of the standard microtiter-plate test by introduction of an additional step of decolorization by acetic acid seems to be a useful improvement of the technique.

In vitro antimicrobial activity of propolis and synergism between propolis and antimicrobial drugs

The aim of this study was to investigate antimicrobial properties of ethanolic extract of 13 propolis (EEP) samples from different regions of Serbia against 39 microorganisms (14 resistant or multiresistant to antibiotics), and to determine synergistic activity between antimicrobials and propolis. Antimicrobial activity of propolis samples was evaluated by agar diffusion and agar dilution method. The synergistic action of propolis with antimicrobial drugs was assayed by the disc diffusion method on agar containing subinhibitory concentrations of propolis. Obtained results indicate that EEP, irrespectively of microbial resistance to antibiotics, showed significant antimicrobial activities against Gram-positive bacteria (MIC 0.078%-1.25% of EEP) and yeasts (0.16%-1.25%), while Gram-negative bacteria were less susceptible (1.25%-->5%). Enterococcus faecalis was the most resistant Gram-positive bacterium, Salmonella spp. the most resistant Gram-negative bacteria, and Candida albicans the most resistant yeast. EEP showed synergism with selected antibiotics, and displayed ability to enhance the activities of antifungals. The shown antimicrobial potential of propolis alone or in combination with certain antibiotics and antifungals is of potential medical interest.

Isolation of Members of the Staphylococcus sciuri Group from Urine and Their Relationship to Urinary Tract Infections

ABSTRACT During a 3-year study period, 32,741 urine samples were analyzed for the presence of members of the Staphylococcus sciuri group (S. sciuri, S. lentus, and S. vitulinus), and 13 isolates were identified. They presented 0.79% of the total number of coagulase-negative staphylococci isolated. One case of symptomatic urinary tract infection and five possible cases of asymptomatic bacteriuria caused by these bacteria were established. It is noteworthy, however, that over 50% of the isolates originated from hospitalized patients.

Identification and Characterization of Clinical Isolates of Members of the Staphylococcus sciuri Group

ABSTRACT A total of 28 staphylococcal isolates from human clinical specimens belonging to the Staphylococcus sciuri group were identified and characterized. The API Staph and ID32 STAPH correctly identified S. sciuri and S. lentus but not S. vitulinus strains. Identification to the subspecies level was possible only by a PCR-based method.

Staphylococcus sciuri as a part of skin, nasal and oral flora in healthy dogs

The coagulase-negative species Staphylococcus sciuri is widespread in nature and is associated with a variety of domestic and wild animals. However, the occurrence of S. sciuri in dogs has received little attention so far. In the present study, we established the prevalence of S. sciuri in a large population of healthy dogs, and characterized isolated strains. Samples from two mucous membrane sites (anterior nares and mouth), and two hair-coated sites (head and withers) were taken from 122 dogs and inoculated into STS agar, a novel selective medium that was introduced and tested in the study. In total, 116 isolates of S. sciuri were obtained from 488 specimens. S. sciuri was isolated from 56 out of 122 (46%) dogs. The occurrence of S. sciuri in the anterior nares and mouth were significantly higher than those in withers and head. No significant association of S. sciuri occurrence in dogs and factors such as sex, age, and living environment (indoor/outdoor) was found. Out of 56 dogs, which tested positive for S. sciuri, 30 (54%) would have it as a resident flora. Thus, we showed that S. sciuri was frequently present as a part of skin, nasal and oral flora in healthy dogs both as a resident and transient carriage.

Surgical Wound Infection Associated with Staphylococcus sciuri

We describe a case of surgical wound infection due to Staphylococcus sciuri. The isolated strain was susceptible to trimethoprim–sulfamethoxazole, erythromycin, chloramphenicol, ciprofloxacin and vancomycin and resistant to gentamicin, clindamycin, rifampicin, methicillin, ampicillin and ceftriaxone. The multiresistance of the strain had a serious impact on the prolonged course of the infection. Although this bacterium is principally found in animals, our strain was probably of nosocomial origin.

Isolation and Molecular Characterization of Staphylococcus sciuri in the Hospital Environment

ABSTRACT Staphylococcus sciuri is a principally animal-associated bacterial species, but its clinical relevance for humans is increasing. Our study aimed to provide the first insight into the prevalence of this bacterium in a hospital environment. A 3-month surveillance was conducted in a hospital located in Belgrade, Serbia, and 1,028 samples taken from hands of medical personnel, medical devices, and various hospital surfaces were screened for S. sciuri presence. In total, 108 isolates were obtained, which resulted in a relatively high rate of colonization (10.5%). These isolates, along with 7 S. sciuri strains previously isolated in the same hospital (n = 115), were phenotypically and genotypically characterized. Antimicrobial susceptibility testing revealed that 73% of the strains were resistant to one or more antibiotics, with 4.3% strains displaying multiresistance. Examination of 16S-23S ribosomal DNA intergenic spacer length polymorphism identified the strains at the subspecies level, and 74 (64.3%) strains of S. sciuri subsp. sciuri, 37 (32.2%) strains of S. sciuri subsp. rodentium, and 4 (3.5%) strains of S. sciuri subsp. carnaticus were established. Pulsed-field gel electrophoresis (PFGE) analysis showed 21 distinct pulsotypes, including 17 main types and 4 subtypes. One dominant cluster with 62 strains was found, while 19 (90.5%) of the PFGE types and subtypes identified had 5 or fewer strains. The predominance of small PFGE clusters suggests that the ubiquitous presence of S. sciuri in the outside environment presents the continuous source for colonization of the hospital environment. The presence of one dominant PFGE cluster of strains indicates that some S. sciuri strains may be capable for adaptation to hospital environment conditions and continuous existence in this environment.

Evaluation of Phenotypic and Molecular Methods for Detection of Oxacillin Resistance in Members of the Staphylococcus sciuri Group

ABSTRACT In this paper we report on an experimental evaluation of phenotypic and molecular methods as means for the detection of oxacillin resistance in members of the Staphylococcus sciuri group. A total of 109 S. sciuri group member isolates (92 S. sciuri isolates, 9 S. lentus isolates, and 8 S. vitulinus isolates) were tested by the disk diffusion method, the agar dilution method, the oxacillin salt-agar screening method, slide latex agglutination for PBP 2a, and PCR assay for mecA as the reference method. The mecA gene was detected in 29 S. sciuri isolates, and the true-positive and true-negative results of the other tests were defined on the basis of the presence or the absence of the mecA gene. For the different methods evaluated, the sensitivities and specificities were as follows: for the disk diffusion test with a 1-μg oxacillin disk, 100% and 55.9%, respectively; for the disk diffusion test with a 30-μg cefoxitin disk, 93.5% and 100%, respectively; for the agar dilution method, 100% and 50%, respectively; for the oxacillin salt-agar screen test (with 6 μg of oxacillin per ml and 4% NaCl) 100% and 100%, respectively; and for the slide latex agglutination test for PBP 2a, 100% and 100%, respectively. The disk diffusion test with various β-lactam antibiotics was performed to evaluate their use for the prediction of oxacillin resistance. The results indicate that meropenem, cefazolin, cefamandole, cefuroxime, cefotetan, cefoperazone, cefotaxime, ceftriaxone, moxalactam, cefaclor, and cefprozil may be used as surrogate markers of oxacillin resistance, although further studies of their use for the detection of oxacillin resistance are required.

Mycobacterium fortuitum Endocarditis Associated with Cardiac Surgery, Serbia

To the Editor: Mycobacterium fortuitum is a member of the group of rapidly growing nontuberculous mycobacteria. It is a well-known causative agent of skin and soft tissue infections, postsurgical wound infections, and other health care–associated infections (1). Only sporadic cases of endocarditis caused by this bacterium have been reported (2–4). We describe a cardiac surgery–related outbreak of endocarditis caused by M. fortuitum in 3 children. Over a 3-week period during 2009, eight children consecutively underwent surgery for correction of ventricular septal defect (VSD) by insertion of a bovine pericardial patch at the University Children’s Hospital in Belgrade, Serbia. None of them had previous cardiac surgery. The same patch, SJM Pericardial Patch with EnCap Technology (St. Jude Medical, St. Paul, MN, USA), was used as a source for smaller, tailored patches for all patients. Sterile scissors and forceps were used to tailor a piece of the patch needed for a corresponding VSD closure. During repeated performances of this procedure and between surgeries, the patch had been continuously stored in 2% propylene oxide (PO) provided by the manufacturer. Each tailored piece of the patch had been immersed into freshly prepared sterile saline for 6 min before defect patching. The postoperative course had been uneventful for all patients, and they were discharged 7 days after the procedure. However, 3 patients were readmitted to the hospital because of prolonged fever and increasing fatigue. Patients 1, 2, and 3 (Table) had been the fourth, sixth, and eighth patients undergoing VSD repair, respectively. Diagnosis of infective endocarditis in these patients was established by transthoracic echocardiography findings and blood cultures positive for acid-fast bacteria (Table). Acid-fast bacteria also were recovered from the patch and vegetation taken during reoperation in patient 3 (Table). The isolates were identified as M. fortuitum by the GenoType Mycobacterium CM assay (Hain Lifescience, Nehren, Germany) (5). Empiric treatment with vancomycin and ceftriaxone was switched to amikacin, ciprofloxacin, and imipenem. After 6 weeks of treatment, the patients were discharged, and all were asymptomatic 12 months later. Table Characteristics of patients in an outbreak of Mycobacterium fortuitum endocarditis, Serbia* The cultural characteristics and susceptibility patterns of all the isolates obtained were indistinguishable. To explore their possible clonal relatedness, we genotyped 3 M. fortuitum strains isolated from blood cultures (1 isolate per patient) and 2 M. fortuitum isolates recovered from samples taken during reoperation in 1 of the patients. The enterobacterial repetitive intergenic consensus PCR was used (6), and all isolates produced identical patterns. Nosocomially acquired M. fortuitum endocarditis has been reported but only sporadically in adults, and these cases usually were fatal (3,4,7). In contrast, we describe 3 related cases of M. fortuitum endocarditis in children who recovered. The relatedness of the cases is strongly supported by the following. First, epidemiologic links are obvious because the 3 patients underwent surgery in the same operating room, and the same patch was used in all of them. Second, M. fortuitum strains isolated from the 3 patients were phenotypically and genotypically identical. Repeated use of the same patch in multiple surgeries strongly suggests the contaminated patch was the source of M. fortuitum infection in the 3 patients. This possibility could not be corroborated by bacteriologic examination of the patch because the remaining unusable fragments had been discarded after the surgeries (i.e., ≈3 months before the outbreak became evident). Although contamination of the patch during manufacture is possible (8), it seems more reasonable to assume that the contamination occurred intraoperatively. The common factor in nosocomially acquired M. fortuitum infections is presumed to be exposure to a liquid contaminated with this organism (1,9). The patch was not exposed to solutions other than the PO in which it had been stored and the sterile saline used during the rinsing procedure. Because only a piece of the patch tailored for a particular patient was exposed to a saline freshly prepared for each surgery, contamination of the PO by M. fortuitum presumably led to contamination of the patch. Liquid PO is used as a chemical sterilant for bioprostheses intended for single use. However, multiple use of the same patch implied repeated exposure of the PO solution to the environment and prolonged storage at 4°C between surgeries. Because PO effectiveness is markedly reduced at temperatures <16°C (10), the specific circumstances could have compromised the sterilizing capacity of the PO solution and enabled contamination by ubiquitous M. fortuitum. We are well aware that the patch was intended for single use only and that application of the same patch in multiple patients is not a practice in industrialized countries. However, it is a practice in some resource-limited countries. The outbreak of M. fortuitum endocarditis we describe is a clear warning that such practice is associated with high risk and thus should be discontinued.

Survey of Genes Encoding Staphylococcal Enterotoxins, Toxic Shock Syndrome Toxin 1, and Exfoliative Toxins in Members of the Staphylococcus sciuri Group

ABSTRACT Genes encoding staphylococcal enterotoxins (sea to see, seg, and seh), toxic shock syndrome toxin 1 (tst), and exfoliative toxins (eta and etb) were not detected in a large panel of 48 Staphylococcus sciuri group isolates tested. This strongly suggests that production of the staphylococcal exotoxins by these bacteria is highly unlikely.