Phenotypic and genotypic Characterization and clonal Relatedness of pharyngeal Isolates of group a Streptococci resistant to Macrolides in Serbia

Uvod: S. pyogenes je najčešći bakterijski uzročnik akutnih tonzilofaringitisa. Iako su penicilinski preparati prva terapijska linija u lečenju streptokoknih faringitisa, u slučajevima preosetljivosti na penicilin, propisuju se makrolidi. Početkom 21. veka, uočen je porast rezistencije grupe A streptokoka (GAS) na eritromicin, širom sveta. Dva glavna mehanizma rezistencije S. pyogenes na makrolide su aktivni efluks antibiotika i izmena ciljnog mesta delovanja. Gen mefA, kodira proteine efluksne pumpe, koji dovode do umerenog stepena rezistencije na 14-člane i 15-člane makrolide (M fenotip). Drugi mehanizam rezistencije je metilacija ciljnog mesta delovanja makrolida. On se odlikuje ukrštenom rezistencijom na makrolide, linkozamide i streptogramine (MLS fenotip). Inducibilan MLS fenotip (iMLS) najčešće determiniše ermA, a konstitutivan MLS fenotip (cMLS) ermB gen. Najčešće korišćena metoda za tipizaciju GAS je emm tipizacija. Ova metoda se zasniva na sekvenciranju hipervarijabilnog dela emm gena, koji kodira M protein, glavni faktor virulencije GAS. MLST (engl. multilocus sequence typing) je metoda genotipizacije, koja se bazira na umnožavanju i sekvenciranju 7 visoko konzerviranih, tzv. „house-keeping” gena, koji kodiraju enzime od vitalnog značaja. RAPD (engl. random amplified polymorphic DNA) metoda se bazira na nasumičnom umnožavanju fragmenata DNK i elektroforetskom razdvajanju dobijenih produkata. Cilj ove studije je bio da se proceni učestalost rezistencije grupe A streptokoka na makrolide u Srbiji, da se odrede fenotipovi rezistencije na makrolide, da se odredi distribucija gena koji kodiraju rezistenciju na makrolide i tetracikline, da se utvrdi klonska distribucija i eventualna klonska veza među sojevima GAS rezistentnih na makrolide (MRGAS), kao i da se proceni osetljivost MRGAS sojeva na druge klase antibakterijskih lekova. Materijal i metode: Analizirani su podaci o rezistenciji 3893 sojeva izolovanih od pacijenata sa faringitisom, širom Srbije, u periodu od decembra 2007. do decembra 2008. godine. Sedamdeset sedam MRGAS izolata je poslato u Nacionalnu referentnu laboratoriju za streptokok radi daljih ispitivanja. Identifikacija je vršena na osnovu mikroskopskih, kulturelnih i biohemijskih osobina. Konzervacija je vršena u Todd Hewitt bujonu sa 10% sadržajem glicerola na -80°C...Introduction: S. pyogenes is the most common causative agent of bacterial tonsillopharyngitis. Penicillin is a first choice therapy for infections caused by GAS, since penicillin resistance in streptococci has not yet emerged. Macrolides are preferred for treatment of GAS infections in patients with beta-lactam hypersensitivity. At the beginning of 2000s, significant increase in macrolide resistance has been reported from many countries. Two main well-described molecular mechanisms are responsible for macrolide resistance among streptococci: target site modification and antibiotic efflux. Target site modification due to methylase activity has been linked to the presence of erm gene. This mechanism confers cross-resistance to macrolides, lincosamides and streptogramin (MLS resistance). It can be constitutive (cMLS), usually mediated by the ermB gene or inducible (iMLS), mediated by the ermA gene. The other mechanism of resistance, macrolide efflux is encoded by mefA gene and confers low-level resistance to 14- and 15-membered macrolides. The most common tool used to characterize isolates of S. pyogenes today is emm typing, which is based on sequence at the 5’ end of emm gene that encode M protein, one of the major virulence factor in GAS. Multilocus sequence typing (MLST) is genotyping scheme based on nucleotide sequences of internal fragments of seven selected housekeeping loci. Randomly amplification of polymorphic DNA (RAPD) analysis is genotyping gel based method, with relatively high discriminatory index. The aim of this study was to asses prevalence of macrolide resistance group A streptococci (MRGAS) in Serbia, to determine genotypes and phenotypes of macrolide resistance, as well as to evaluate resistance to tetracycline and to determine tetracycline resistance genes. We were also investigated the clonal relatedness of macrolide resistance strains and their susceptibility to other antimicrobial agents. Material and methods: We evaluated resistance rate of MRGAS in Serbia by analyzing data of 3893 pharyngeal isolates of GAS. A total of 77 MRGAS isolates, originated from patients with pharyngitis, were collected from 7 regional laboratories between December 2007 and 2008. Strains were sent to the National Reference Laboratory for streptococci for further testing..

Adherence and Biofilm Production of Streptococcus pyogenes

Streptococcus pyogenes (group A streptococcus – GAS) can cause numerous human infections, varying from mild skin infections to life‐threatening, e.g. necrotizing fasciitis. Adherence and biofilm production are important in streptoccocal pathogenesis. GAS adhesins are numerous and diverse, with the ability to bind to several different receptors at the same time, which leads to difficulties in their precise identification and classification. Biofilm production is one of the most probable explanation for therapeutic failure in the treatment of GAS infections. Most researchers agreed that biofilm formation is a trait of individual strains rather than a general serotype attribute. The aim of our study is to investigate differences in adherence to laminin and biofilm production between invasive and non‐invasive isolates (NI) of GAS. In this study the correlation between adherence to laminin and invasiveness in GAS isolates is noticed. The strains isolated from GAS carriers and highly invasive (HI) GAS strains have excellent capacity for binding to laminin. When testing biofilm production, there was noticeable positive correlation between adherence and biofilm production among non‐invasive isolates. Non‐invasive isolates were stable biofilm productors. There was no correlation between adherence and biofilm production among invasive isolates. Invasive isolates were also unstable biofilm productors

Phenotypes and genotypes of macrolide-resistant Streptococcus pneumoniae in Serbia

Although macrolides are widely used for treating pneumococcal infections, an increase in macrolide resistance might compromise their use. The objective of this study was to determine the prevalence of macrolide-resistant phenotypes and genotypes in macrolide-resistant S. pneumoniae isolates in Serbia. A total of 228 macrolide-resistant strains isolated during the period of 2009-2012, were analyzed. Macrolide resistance phenotypes were determined by a double disk diffusion test. The presence of macrolide resistance genes was detected by PCR. Antibiotics susceptibilities were tested using the VITEK2 system and E test. Among the examined isolates, the MLSB phenotype which is linked to the presence of the erm(B) gene dominated (83.3%), while the mef(A) gene which is associated with the M phenotype, was identified in 16.7% isolates. Over 40% of isolates expressed co-resistance to penicillin. A multiple-resistant pattern was found in 36.4% strains, more frequently in children. However, all strains were susceptible to telithromycin, vancomycin, linezolid, fluoroquinolones and rifampicin. [Projekat Ministarstva nauke Republike Srbije, br. 175039

Distribution of macrolide-resistant genes among isolates of macrolideresistant Streptococcus pyogenes and Streptococcus pneumoniae in Serbia

Macrolide resistance in Streptococcus pneumoniae and in group A streptococci (GAS) is a significant problem worldwide. In Serbia, data on the mechanisms of resistance and the corresponding resistance genes in streptococci are largely lacking. Therefore, we analyzed the distribution of macrolide resistance phenotypes and genotypes in 44 macrolideresistant GAS (MRGAS) and 50 macrolide-resistant S. pneumoniae (MRSP) isolates collected in the same period. The double disk diffusion test and PCR were used to analyze resistance phenotypes and resistance genes, respectively. Among MRSP, the MLSB phenotype dominated, whereas the M phenotype was the most prevalent among MRGAS isolates. Consequently, in MRSP, the ermB gene was the most common (n=40, 80%), followed by the mefA gene (n=7,14%). In MRGAS strains, mefA dominated (n=27,61%), followed by ermA (n=15, 33%) and ermB (n=3, 7%). In 3 MRSP isolates no resistance genes were detected, while one MRGAS strain with iMLSB phenotype harbored both ermA and mefA genes

Antimicrobial Susceptibility Testing: A Comprehensive Review of Currently Used Methods

Antimicrobial resistance (AMR) has emerged as a major threat to public health globally. Accurate and rapid detection of resistance to antimicrobial drugs, and subsequent appropriate antimicrobial treatment, combined with antimicrobial stewardship, are essential for controlling the emergence and spread of AMR. This article reviews common antimicrobial susceptibility testing (AST) methods and relevant issues concerning the advantages and disadvantages of each method. Although accurate, classic technologies used in clinical microbiology to profile antimicrobial susceptibility are time-consuming and relatively expensive. As a result, physicians often prescribe empirical antimicrobial therapies and broad-spectrum antibiotics. Although recently developed AST systems have shown advantages over traditional methods in terms of testing speed and the potential for providing a deeper insight into resistance mechanisms, extensive validation is required to translate these methodologies to clinical practice. With a continuous increase in antimicrobial resistance, additional efforts are needed to develop innovative, rapid, accurate, and portable diagnostic tools for AST. The wide implementation of novel devices would enable the identification of the optimal treatment approaches and the surveillance of antibiotic resistance in health, agriculture, and the environment, allowing monitoring and better tackling the emergence of AMR

The first nationwide multicenter study ofAcinetobacter baumanniirecovered in Serbia: emergence of OXA-72, OXA-23 and NDM-1-producing isolates

Background The worldwide emergence and clonal spread of carbapenem-resistantAcinetobacter baumannii(CRAB) is of great concern. The aim of this nationwide study was to investigate the prevalence of CRAB isolates in Serbia and to characterize underlying resistance mechanisms and their genetic relatedness. Methods Non-redundant clinical samples obtained from hospitalized patients throughout Serbia were included in the prospective, observational, multicenter study conducted from January to June 2018. Samples were initially screened for the presence ofAcinetobacter baumannii-calcoaceticus(Acb) complex using conventional bacteriological techniques. Acb complexes recovered from clinical samples obtained from inpatients with confirmed bacterial infections were further evaluated for the presence ofA. baumannii. Identification to the species level was done by the detection of thebla(OXA-51)gene andrpoBgene sequence analysis. Susceptibility testing was done by disk diffusion and broth microdilution method. CRAB isolates were tested for the presence of acquired carbapenemases(bla(OXA-24-like),bla(OXA-23-like,)bla(OXA-58-like),bla(OXA-143-like),bla(IMP),bla(VIM),bla(GIM),bla(SPM),bla(SIM),bla(NDM)) by PCR. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results Acb complex was isolated in 280 out of 2401 clinical samples (11.6%). Overall,A. baumanniiwas identified in 237 out of 280 Acb complex (84.6%). CRAB prevalence was found to be 93.7% (237/222). The MIC50/MIC(90)for imipenem and meropenem were 8/ gt 32 mu g/mL and 16/ gt 32 mu g/mL, respectively. Although susceptibility was high for colistin (95.7%;n = 227) and tigecycline (75.1%;n = 178), ten isolates (4.3%) were classified as pandrug-resistant. The following carbapenemases-encoding genes were found: 98 (44.2%)bla(OXA-24-like), 76 (34.5%)bla(OXA-23-like), and 7 (3.2%)bla(NDM-1). PFGE analysis revealed six different clusters. MLST analysis identified three STs: ST2 (n = 13), ST492 (n = 14), and ST636 (n = 10). Obtained results evaluated that circulating CRAB clones in Serbia were as follows:bla(OXA66)/bla(OXA23)/ST2 (32.4%),bla(OXA66)/bla(OXA23)/bla(OXA72)/ST2 (2.7%),bla(OXA66)/bla(OXA72)/ST492 (37.8%), andbla(OXA66)/bla(OXA72)/ST636 (27.1%). Conclusion This study revealed extremely high proportions of carbapenem resistance amongA. baumanniiclinical isolates due to the emergence ofbla(OXA-72),bla(OXA-23), andbla(NDM-1)genes among CRAB isolates in Serbia and their clonal propagation

AdeABC efflux pump-mediated resistance to tigecycline in Acinetobacter baumannii isolates from Balkan hospitals

Introduction: Multidrug-resistant (MDR) Acinetobacter baumannii has been recognized as one of the most serious healthcare challenges worldwide. Although tigecycline represents one of the last resort therapies for MDR A. baumannii, resistance to this antibiotic has been reported and mostly is mediated by AdeABC efflux pump. The aim of our study was to investigate the molecular mechanism responsible for tigecycline resistance of thirty-seven A. baumannii isolates from Balkan medical settings (Serbia, Bosnia and Herzegovina and Montenegro) gathered in 2016 and 2022. Methods: Minimal inhibitory concentration (MIC) values for tigecycline were determined using microdilution method according to EUCAST guidelines. Inhibition of the efflux of tigecycline wastested by the same method using a combination of antibiotic and efflux pump inhibitor (CCCP). Amino acid alternations within AdeS and AdeR proteins were detected by comparing to sequences of referent isolates ATCC19606 and ATCC17978. Expression of the adeB gene ofselected isolates was monitored by RT-qPCR. Results: All tested isolates were resistant to tigecycline and showed significant decrease in tigecycline MIC values in presence of CCCP (≥16-fold reduction) indicating that antibiotic efflux is responsible for tigecycline resistance. The analysis of two-component system AdeRS, regulatory system of RND efflux pump AdeABC, revealed that most of the isolates have G186V and N268H alternations in AdeS (n=32), while most common changes in AdeR were V120I and A136V (n=29). In addition, RT-qPCR showed that selected isolates upregulate expression of the adeB gene (from 1,13- to 3-fold). Conclusion: This study revealed that AdeABC overexpression is the main mechanism of tigecycline resistance in A. baumannii isolated in Balkan hospitals

THE ROLE OF EFFLUX PUMPS IN TIGECYCLINE RESISTANCE OF ACINETOBACTER BAUMANNII ISOLATES FROM WESTERN BALKAN HOSPITALS

The increasing prevalence of multidrug-resistant (MDR) Acinetobacter baumannii limits effective therapeutic options, and tigecycline has been considered one of the last resort therapies for MDR A. baumannii infections. Nevertheless, A. baumannii isolates resistant to tigecycline are becoming increasingly reported, mostly due to overexpression of efflux pumps. The three major RND efflux systems conferring tigecycline resistance in A. baumannii are AdeABC, AdeFGH, and AdeIJK, and their expression is regulated by the two-component system AdeRS, the LysR-type regulator AdeL, and the TetR-type regulator AdeN, respectively. Following the above, we aimed to determine the role of efflux pumps in tigecycline resistance of thirty-seven A. baumannii isolates collected from Western Balkan healthcare settings (Serbia, Bosnia and Herzegovina and Montenegro) in 2016 and 2022. The majority of isolates belonged to the most prevalent international clonal lineage IC2 (n = 32), four isolates are members of IC1, while only one isolate is identified as IC3. All tested isolates demonstrated a significant decrease in tigecycline MIC in presence of efflux pump inhibitor CCCP (≥16-fold reduction) indicating that mechanism responsible for tigecycline resistance is antibiotic efflux. The comparison of target efflux pump regulatory proteins, translated from nucleotide sequences, to reference strains ATCC19606 and ATCC17978 revealed that most of the isolates have G186V and N268H alternations in AdeS (n = 32), while most common changes in AdeR were V120I and A136V (n = 29) as described in previous studies. Substitution Q262R was detected exclusively in AdeL proteins of IC1 isolates, while no mutations were observed within AdeN regulators. Expression of the adeB, adeG, and adeJ genes in six selected isolates was upregulated in four (1,4- to 3-fold), six (1,6- to 2,6-fold), and three isolates (1,7- to 4-fold), respectively. This study confirmed that overexpression of efflux pump encoding genes enables tigecycline resistance in clinical A. baumannii isolates.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi

NOVEL BACTERIOPHAGE ISOLATION FROM BELGRADE WASTEWATERS

Anti-microbial drug resistance (AMR) is one of the global health threats caused by the misuse of drugs typically used to treat microbial infections in humans, animals and plants. AMR in nosocomial infections not only significantly hinders treatment and endangers the patients’ lives, but also elevates the costs of healthcare. Multiple research approaches have been initiated to combat AMR, and one promising method is bacteriophage therapy. Bacteriophages (phages) are viruses that naturally exploit bacteria as their hosts for replication and can cause cell lysis, which makes them promising candidates for treating the infections that do not respond to conventional antibiotic therapies. In this study, we screened wastewater samples from four different collectors in Belgrade urban area for bacteriophages active against clinically isolated strains of two biofilm-producing bacteria that readily persist in hospital environment - Klebsiella pneumoniae (6 strains) and Pseudomonas aeruginosa (2 strains). Wastewaters were screened for phage presence through phage enrichment process, in which bacteria were grown in a mixture of water samples and nutrient-rich broth. Obtained cultures were screened for antimicrobial activity against the respective host strains, and candidates were subjected to a first-round plaque assay to detect the phages. Finally, the activity of all the candidates was tested against all strains of the same species to gain the first insight into their host range. We discovered 20 potentially distinct bacteriophages active against K. pneumoniae strains and two potentially different candidates targeting P. aeruginosa. Notably, one phage exhibited activity against all tested K. pneumoniae strains, and four were active against 5 out of 6 tested strains. Among 22 candidates in total, five showed depolymerizing activity, indicating promise in combating biofilm formation. Currently, isolation of new phages, as well as purification and host range analysis is underway for several candidates targeting K. pneumoniae and two targeting P. aeruginosa strains.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi

Resveratrol/Selenium Nanocomposite with Antioxidative and Antibacterial Properties

In this work, we synthesized a new composite material comprised of previously formulated resveratrol nanobelt-like particles (ResNPs) and selenium nanoparticles (SeNPs), namely ResSeNPs. Characterization was provided by FESEM and optical microscopy, as well as by UV-Vis and FTIR spectroscopy, the last showing hydrogen bonds between ResNPs and SeNPs. DPPH, TBA, and FRAP assays showed excellent antioxidative abilities with ResNPs and SeNPs contributing mainly to lipid peroxidation inhibition and reducing/scavenging activity, respectively. The antibacterial effect against common medicinal implant colonizers pointed to notably higher activity against Staphylococcus isolates (minimal inhibitory concentrations 0.75–1.5%) compared to tested gram-negative species (Escherichia coli and Pseudomonas aeruginosa). Antibiofilm activity against S. aureus, S. epidermidis, and P. aeruginosa determined in a crystal violet assay was promising (up to 69%), but monitoring of selected biofilm-related gene expression (pelA and algD) indicated the necessity of the involvement of a larger number of genes in the analysis in order to further establish the underlying mechanism. Although biocompatibility screening showed some cytotoxicity and genotoxicity in MTT and alkaline comet assays, respectively, it is important to note that active antioxidative and antibacterial/antibiofilm concentrations were non-cytotoxic and non-genotoxic in normal MRC-5 cells. These results encourage further composite improvements and investigation in order to adapt it for specific biomedical purposes