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

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 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

Supplementary information for the article: Kabic, J., Novovic, K., Kekic, D., Trudic, A., Opavski, N., Dimkic, I., Jovcic, B., & Gajic, I. (2023). Comparative genomics and molecular epidemiology of colistin-resistant Acinetobacter baumannii. Computational and Structural Biotechnology Journal, 21, 574–585. https://doi.org/10.1016/j.csbj.2022.12.045

Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1784]Supplementary data associated with this article can be found in the online version at doi:[10.1016/j.csbj.2022.12.045.

Influence of subinhibitory antibiotic concentration on Streptococcus pyogenes adherence and biofilm production

In this study, the focus was on the effects of sub-MICs of the antibiotics on adherence, hydrophobicity, and biofilm formation by two groups of Streptococcus pyogenes strains, which were responsible for different clinical cases. The aim of this study was to explore the effects of sub-MICs of penicillin, ceftriaxone, erythromycin, and clindamycin on adherence, surface hydrophobicity, and biofilm biomass in two selected collections of group A streptococcus (GAS): strains isolated from carriers (CA) and strains isolated from patients with tonsillopharyngitis (TPh). Isolates were tested for hydrophobicity to xylene, adherence, and biofilm production in uncoated microtiter plates before and after treatment with 1/2 and 1/4 MICs of antibiotics. Penicillin reduced adherence and biofilm production in TPh strains, whereas ceftriaxone diminished adherence and biofilm formation in CA group. On the contrary, clindamycin enhanced adherence and biofilm production in both groups of strains. Erythromycin did not significantly alter adherence, but triggered biofilm production in both groups of isolates. Hydrophobicity of both groups of strains was significantly reduced after exposure to all antibiotics. Beta-lactams displayed anti-biofilm activity; penicillin diminished both adherence and biofilm production in TPh strains, whereas ceftriaxone reduced it in strains isolated from CA

Isolation, Characterization, Genome Analysis and Host Resistance Development of Two Novel Lastavirus Phages Active against Pandrug-Resistant Klebsiella pneumoniae

Klebsiella pneumoniae is a global health threat and bacteriophages are a potential solution in combating pandrug-resistant K. pneumoniae infections. Two lytic phages, LASTA and SJM3, active against several pandrug-resistant, nosocomial strains of K. pneumoniae were isolated and characterized. Their host range is narrow and latent period is particularly long; however, their lysogenic nature was refuted using both bioinformatic and experimental approaches. Genome sequence analysis clustered them with only two other phages into the new genus Lastavirus. Genomes of LASTA and SJM3 differ in only 13 base pairs, mainly located in tail fiber genes. Individual phages, as well as their cocktail, demonstrated significant bacterial reduction capacity in a time-dependent manner, yielding up to 4 log reduction against planktonic, and up to 2.59 log on biofilm-embedded, cells. Bacteria emerging from the contact with the phages developed resistance and achieved numbers comparable to the growth control after 24 h. The resistance to the phage seems to be of a transient nature and varies significantly between the two phages, as resistance to LASTA remained constant while resensitization to SJM3 was more prominent. Albeit with very few differences, SJM3 performed better than LASTA overall; however, more investigation is needed in order to consider them for therapeutic application