Srdjan Stepanović

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.

Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci

The details of all steps involved in the quantification of biofilm formation in microtiter plates are described. The presented protocol incorporates information on assessment of biofilm production by staphylococci, gained both by direct experience as well as by analysis of methods for assaying biofilm production. The obtained results should simplify quantification of biofilm formation in microtiter plates, and make it more reliable and comparable among different laboratories.

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.

Influence of dynamic conditions on biofilm formation by staphylococci.

Abstract The modified microtiter plate test was used to investigate biofilm formation by staphylococci under both static and dynamic conditions. The quantity of biofilm produced under static conditions was used as a reference. Dynamic conditions, which were achieved by incubating microtiter plates on a horizontal shaker with and without the presence of glass beads in wells, either reduced biofilm formation or left it unchanged. Dynamic conditions particularly affected the capacity of certain species to produce biofilm: these species included the causative agents of infections associated with a foreign body (Staphylococcus epidermidis, Staphylococcus aureus). On the basis of these results, dynamic conditions should be included as a parameter for evaluating biofilm formation by staphylococci in vitro.

Effect of Environmental Factors on Biofilm Formation by Clinical Stenotrophomonas maltophilia Isolates

The influence of environmental factors (temperature, aerobiosis-anaerobiosis, static-dynamic conditions, pH) was determined on biofilm formation by 51S. maltophilia clinical isolates. The strains produced more biofilm at 32 °C than at 37 or 18 °C. Aerobic and 6 % CO2 atmosphere yielded comparable biofilm amounts, higher than under anaerobic conditions. Biofilm production was not affected by staticvs. agitated culture conditions. Biofilm production at pH 7.5 and 8.5 was comparable but significantly higher than at pH 5.5. The capacity of individual strains to form biofilm and thus contribute to the severity of some diseases is influenced by host traits and environmental conditions at the site of infection, and play an important role in the pathogenesis of biomaterial-related disease caused byS. maltophilia.

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.