Dragana Vuković

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.

Evolutionary history and global spread of the Mycobacterium tuberculosis Beijing lineage

Mycobacterium tuberculosis strains of the Beijing lineage are globally distributed and are associated with the massive spread of multidrug-resistant (MDR) tuberculosis in Eurasia. Here we reconstructed the biogeographical structure and evolutionary history of this lineage by genetic analysis of 4,987 isolates from 99 countries and whole-genome sequencing of 110 representative isolates. We show that this lineage initially originated in the Far East, from where it radiated worldwide in several waves. We detected successive increases in population size for this pathogen over the last 200 years, practically coinciding with the Industrial Revolution, the First World War and HIV epidemics. Two MDR clones of this lineage started to spread throughout central Asia and Russia concomitantly with the collapse of the public health system in the former Soviet Union. Mutations identified in genes putatively under positive selection and associated with virulence might have favored the expansion of the most successful branches of the lineage.

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.

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.

Molecular Epidemiology of Pulmonary Tuberculosis in Belgrade, Central Serbia

ABSTRACT In order to gain precise data on the actual epidemiology of tuberculosis (TB) in Belgrade, central Serbia, we conducted the molecular epidemiological investigation described herein. IS6110 restriction fragment length polymorphism (RFLP) typing of 176 Mycobacterium tuberculosis isolates was performed. These strains were obtained from 48.4% of all patients diagnosed with culture-proven pulmonary TB from April through September 1998 and from May through October 1999. Clusters containing strains with identical RFLP IS6110 patterns were assumed to have arisen from recent transmission. Of the 176 cases, 55 (31.2%) were grouped into 23 clusters ranging in size from two to six patients. Nearly 80% of clustered patients were directly interviewed, and transmission between family-unrelated contacts was found to be predominant in the study population. Classical contact investigation identified only 2 (3.6%) of the 55 clustered patients. The clustering of TB patients was not associated with any demographic or clinical characteristic other than infection with multidrug-resistant (MDR) M. tuberculosis strains. Nearly 70% of MDR strains were clustered, which indicates active transmission of MDR TB in Belgrade. However, this was not observed by conventional epidemiologic surveillance. In conclusion, the first molecular epidemiologic analysis of TB in the region revealed frequent recent transmission of TB and pointed out significant shortcomings of the current concept for conventional contact tracing. The results presented also demonstrate that transmission of MDR TB in Belgrade is not optimally controlled, and they provide support for the development of improved control strategies, including application of molecular methods.

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.