Ivana Cirkovic

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.

Origin and Evolution of European Community-Acquired Methicillin-Resistant Staphylococcus aureus

ABSTRACT Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations. IMPORTANCE With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA. With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA.

Carriage and Genetic Diversity of Methicillin-Resistant Staphylococcus aureus among Patients and Healthcare Workers in a Serbian University Hospital

Objectives There is a paucity of data on methicillin-resistant Staphylococcus aureus (MRSA) epidemiology among Balkan countries. The aim of our study was to determine the prevalence of nasal and pharyngeal carriages and diversity of MRSA among patients and healthcare workers (HCWs) in the major referral centre in Serbia, and to evaluate performance of three different media for MRSA screening. Methods Nasal and pharyngeal swabs were obtained from 195 patients and 105 HCWs in Emergency Department (ED), Surgical Department (SD) and Medical Department (MD). After broth enrichment, samples were inoculated onto MRSA-ID, ORSA and oxacillin-MSA and incubated for 24/48 hours. Characterisation of isolated MRSA strains was determined by MLVA, spa, SCCmec and agr typing, PVL genes detection and antimicrobial susceptibility. Results MRSA carriage prevalence was 11.8% in patients and 7.6% in HCWs. Introduction of pharyngeal swabs in screening procedure increased MRSA carriage rate by over 30%. Variable found to be independently associated with an increased risk for MRSA carriage was ED (odd ratio (OR) = 4.45, 95% confidence interval (CI) 1.78-11.14). A higher risk of multidrug-resistant MRSA carriage was observed among patients (OR = 22; 95% CI 1.92-251.54). CC5-MRSA-SCCmecI was the dominant clone among patients and HCWs in ED and MD, while high genetic diversity of community-associated MRSA (CA-MRSA) was shown in SD especially among HCWs. MRSA-ID was superior to the other tested media with a sensitivity/specificity of 95.2% and 99.6% after 48 hours of incubation. Conclusions These results indicate high MRSA carriage rate in the hospital and emergence of CA-MRSA through HCWs in these settings. MRSA-ID was the optimal available choice for MRSA screening.

Newly-synthesized chalcones-inhibition of adherence and biofilm formation of methicillin-resistant Staphylococcus aureus

Biofilm formation and adherence of bacteria to host tissue are one of the most important virulence factors of methicillin-resistant strains of Staphylococcus aureus (MRSA). The number of resistant strains is seriously increasing during the past years and bacteria have become resistant, not only to methicillin, but also to other commonly used antistaphylococcal antibiotics. There is a great need for discovering a novel antimicrobial agent for the treatment of staphylococcal infections. One of the most promising groups of compounds appears to be chalcones. In present study we evaluated the in vitro effect of three newly synthesized chalcones: 1,3- Bis-(2-hydroxy-phenyl)-propenone, 3-(3-Hydroxy-phenyl)-1-(2-hydroxy-phenyl)-propenone and 3-(4-Hydroxy-phenyl)-1-(2-hydroxy-phenyl)-propenone on glycocalyx production, biofilm formation and adherence to human fibronectin of clinical isolates and laboratory control strain of MRSA (ATCC 43300). Subinhibitory concentrations of the tested compounds reduced the production of glycocalyx, biofilm formation and adherence to human fibronectin of all MRSA strains. Inhibition of biofilm formation was dose dependent and the most effective was 1,3- Bis-(2-hydroxy-phenyl)-propenone. In our study we demonstrated that three newly-synthesized chalcones exhibited significant effect on adherence and biofilm formation of MRSA strains. Chalcones may be considered as promising new antimicrobial agents that can be used for prevention of staphylococcal infections or as adjunct to antibiotics in conventional therapy.

National surveillance reveals findings of Panton-Valentine leukocidin positive meticillin-resistant Staphylococcus aureus in Serbia.

Panton–Valentine leukocidin (PVL) has been the subject of worldwide attention due to its epidemiological linkage to community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) (Tristan et al., 2007). Surveillance data for meticillin-resistant S. aureus (MRSA) has not been reported in Serbia before as the last report was before the splitting of Yugoslavia. We initiated MRSA surveillance in 2008 and report here what is to the best of our knowledge the first finding of PVL-positive MRSA in Serbia. The phenotypic and genotypic characteristics of the isolates and the clinical data related to them are reported in this article and compared to findings in other European and non-European countries.

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.

Licheniocin 50.2 and Bacteriocins from Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 Inhibit Biofilms of Coagulase Negative Staphylococci and Listeria monocytogenes Clinical Isolates.

Background Coagulase negative staphylococci (CoNS) and Listeria monocytogenes have important roles in pathogenesis of various genital tract infections and fatal foetomaternal infections, respectively. The aim of our study was to investigate the inhibitory effects of two novel bacteriocins on biofilms of CoNS and L. monocytogenes genital isolates. Methods The effects of licheniocin 50.2 from Bacillus licheniformis VPS50.2 and crude extract of bacteriocins produced by Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 (BGBU1-4 crude extract) were evaluated on biofilm formation and formed biofilms of eight CoNS (four S. epidermidis, two S. hominis, one S. lugdunensis and one S. haemolyticus) and 12 L. monocytogenes genital isolates. Results Licheniocin 50.2 and BGBU1-4 crude extract inhibited the growth of both CoNS and L. monocytogenes isolates, with MIC values in the range between 200–400 AU/ml for licheniocin 50.2 and 400–3200 AU/ml for BGBU1-4 crude extract. Subinhibitory concentrations (1/2 × and 1/4 × MIC) of licheniocin 50.2 inhibited biofilm formation by all CoNS isolates (p < 0.05, respectively), while BGBU1-4 crude extract inhibited biofilm formation by all L. monocytogenes isolates (p < 0.01 and p < 0.05, respectively). Both bacteriocins in concentrations of 100 AU/mL and 200 AU/mL reduced the amount of 24 h old CoNS and L. monocytogenes biofilms (p < 0.05, p < 0.01, p < 0.001). Conclusions This study suggests that novel bacteriocins have potential to be used for genital application, to prevent biofilm formation and/or to eradicate formed biofilms, and consequently reduce genital and neonatal infections by CoNS and L. monocytogenes.

Antibiofilm effects of topical corticosteroids and intranasal saline in patients with chronic rhinosinusitis with nasal polyps depend on bacterial species and their biofilm-forming capacity

Microbial biofilms have been implicated in the pathogenesis of chronic rhinosinusitis with nasal polyposis (CRSwNP). Intranasal application of corticosteroids and saline is a reliable option for their management. The aim of our study was to evaluate in vitro antibiofilm effects of corticosteroids and isotonic and hypertonic nasal saline in CRSwNP patients. The sinus mucosal specimens were harvested from the ethmoid cavity of 48 patients with CRSwNP and further subjected to hematoxylin–eosin staining and microbiology analysis. The biofilm-forming capacity of isolated bacterial strains was detected by microtiter-plate method and the effects of therapeutic doses of mometasone, fluticasone, isotonic and hypertonic saline on biofilm production were investigated. Bacterial strains were isolated in 42 (87.5%) patients: one organism in 34 (80.9%) and two organisms in 8 (19.1%). Staphylococcus epidermidis (34%) and Staphylococcus aureus (28%) were the most prevalent bacteria in biofilms of CRSwNP patients. Corticosteroids and saline solutions significantly reduced biofilm formation (p < 0.01 and p < 0.05, respectively) with better efficacy of fluticasone and isotonic nasal saline. Treatment with fluticasone, mometasone, isotonic and hypertonic nasal saline completely prevented biofilm production in 66, 50, 84 and 38% of bacterial strains, respectively. The most significant density reduction was observed in biofilm formed by Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pneumoniae compared to other bacterial species (p < 0.01, p < 0.05, p < 0.05, respectively). The antibiofilm effects of corticosteroids and saline solutions also greatly depended on bacterial biomass (p < 0.05), with the most significant effect on high compared to small amount of formed biofilm. The topical steroids and nasal saline are shown to be potent antibiofilm agents in patients with CRSwNP. The effects of tested compounds depend on bacterial species and volume of formed biofilm.

Molecular Epidemiology of Colistin-Resistant, Carbapenemase-Producing Klebsiella pneumoniae in Serbia from 2013 to 2016

ABSTRACT Twenty-seven colistin-resistant, carbapenemase-producing Klebsiella pneumoniae isolates were identified from hospitals in Serbia. All isolates were blaCTX-M-15 positive; ST101, ST888, ST437, ST336, and ST307 were blaOXA-48 positive; and ST340 was blaNDM-1 positive. ST307 had an insertion, and ST336 had a premature stop codon in the mgrB gene. Amino acid substitutions were detected in PmrAB of isolates ST101, ST888, ST336, and ST307. The mcr-1 and mcr-2 were not detected. An increase in phoP, phoQ, and pmrK gene transcription was detected for all sequence types.

High Prevalence and Resistance Patterns of Community-Associated Methicillin-Resistant Staphylococcus Aureus in the Pomoravlje Region, Serbia.

With a view to estimating the prevalence and resistance patterns of CA-MRSA in one region of Serbia, we performed an analysis of MRSA isolates from healthy people and hospitalised patients. The detection of CA-MRSA was carried out by SCCmec typing. In MRSA isolates from hospitalised patients SCCmec types IV and V were found in 76% of the strains. Similar percentage (80%) of CA-MRSA genotypes was present in healthy people. SCCmec type V harbouring MRSA was the most successful clone. Higher prevalence of type V in hospitalised patients to that in healthy people (70% vs 54%) may indicate nosocomial transmissions in at least some hospital units. All MRSA strains from hospitalised patients were resistant to one or more non-β-lactam antibiotics while 52% were multi-resistant. In isolates from healthy people, 16% were sensitive to all non-β-lactam antibiotics and 40% were multi-resistant. Similar percentage of multi-resistant CA- and HA-genotypes occurred in a particular environment (53% vs 50% in hospitalised patients, and 37.5% vs 37.5% in healthy people) indicating selective pressure of antibiotics as a leading force conferring antibiotic resistance. High prevalence of CA-MRSA and high resistance rate both in hospitals and the community suggest that this pathogen has been present in the Pomoravlje Region, central Serbia for years.