Dissemination of Metallo-ß-Lactamase-Producing Pseudomonas aeruginosa in Serbian Hospital Settings: Expansion of ST235 and ST654 Clones.

This nationwide study aimed to investigate the molecular characteristics of metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa in Serbia, underlying resistance mechanisms, the genetic context of detected MBL genes, and the clonal relationship between isolates harboring genes-encoding MBL. Overall, 320/5334 isolates collected from 2018 to 2021 were identified as P. aeruginosa. Carbapenem-resistant P. aeruginosa (CRPA) were screened for the presence of blaVIM, blaIMP, and blaNDM, genes whereas MBL-positive isolates were tested for the presence of the blaCTX-M-2, blaPER, blaTEM, blaSHV, blaVEB, and blaGES. Multilocus sequence typing and phylogenomic analysis were performed for P. aeruginosa-producing MBL. The majority of the P. aeruginosa isolates were recovered from the lower respiratory tract (n = 120; 37.5%) and wound specimens (n = 108; 33.75%). CRPA isolates accounted for 43.1% (n = 138) of the tested isolates, 31 out of them being blaNDM-1-positive (22.5%). The colistin resistance rate was 0.3%. MLST analysis revealed the occurrence of ST235 (n = 25) and ST654 (n = 6), mostly confined to Serbia. The distribution of beta-lactamase-encoding genes in these isolates suggested clonal dissemination and possible recombination: ST235/blaNDM-1, ST235/blaNDM-1/blaPER-1, ST654/blaNDM-1, ST654/blaNDM-1/blaPER-1, and ST654/blaNDM-1/blaGES-5. High-risk clones ST235 and ST654 identified for the first time in Serbia, are important vectors of acquired MBL and ESBL and their associated multidrug resistance phenotypes represent a cause for considerable concern.

Biofilm Production and Antimicrobial Resistance of Clinical and Food Isolates of Pseudomonas spp.

Due to its ubiquity, ability to form biofilms, and acquire resistance mechanisms, Pseudomonas spp. become one of the major challenge for healthcare settings and food industry. The aims of this study were to assess the biofilm production of Pseudomonas spp. recovered from clinical and food specimens and to evaluate their antimicrobial resistance. A total of 108 isolates of Pseudomonas spp. were included in the study, 48 being clinical isolates recovered from patients admitted to four tertiary care hospitals throughout Serbia and 60 were isolated from the bulk tank milk samples and meat carcasses. Biofilm production was analyzed by microtiter plate assay. Antimicrobial susceptibility was evaluated by disk diffusion method according to the CLSI guidelines, while class A and B ß-lactamases encoding genes were screened by PCR. A total of 98 (90.7%) strains were biofilm producers (moderate producer: 68, 69.4%; strong producer: 8, 8.2%). Although a slightly higher percentage of clinical isolates were biofilm producers (91.7%) compared to food isolates (90%), statistical significance was not observed (P > 0.05). The proportion of carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates was significantly higher among clinical (42%) isolates compared to food (1.7%) Pseudomonads (P < 0.05). The blaPER and blaNDM genes were found in ESBL (seven isolates) and MBL (two isolates) production, respectively. In the present study, we confirmed that biofilm formation was highly present in both clinical and food Pseudomonas spp. irrespective of the prior existence of resistance genes. Additionally, clinical settings pose a major reservoir of MDR Pseudomonas spp. and especially CRPA isolates.

Tigecycline susceptibility of multidrug-resistant Acinetobacter baumannii from intensive care units in the western Balkans.

Tigecycline can be effective to treat infections of carbapenem-resistant Acinetobacter baumannii (CRAB) however, no interpretive criteria have been approved so far. The objectives of this study were to evaluate the proportion of CRAB isolates and to compare gradient test with a broth microdilution (BMD) method for tigecycline susceptibility testing of A. baumannii.This study included 349 multidrug-resistant (MDR) Acinetobacter spp. collected from Serbia, Montenegro, Bosnia and Herzegovina in 2016 and 2017. Antibiotic susceptibility testing was performed by disk diffusion, VITEK2, gradient, ComASP Colistin. Tigecycline susceptibilities were interpreted according to breakpoints of European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Food and Drug Administration (FDA).Majority of the tested isolates were CRAB (92.8%). Tigecycline MIC50/MIC90 values were 4/8 µg/mL by BMD and 0.5/4 µg/mL by gradient test. Essential agreement for BMD and gradient test amounted to 65.1%. With EUCAST breakpoints, categorical agreement (CA) was achieved in 38% isolates. Major discordance (MD-false susceptibility/resistance) and minor discordance (mD-false categorization involving intermediate results) were observed in 10% and 57% A. baumannii, respectively. With FDA breakpoints, CA, MD and mD were observed in 44%, 16% and 47% isolates, respectively. Colistin resistance was 2.1%.The study highlights a high proportion of CRAB and several discordances between BMD and gradient test which may lead to inappropriate therapy.

Molecular epidemiology of invasive and non-invasive group B Streptococcus circulating in Serbia.

Streptococcus agalactiae (group B Streptococcus, GBS) remains the leading cause of invasive diseases in neonates and an important cause of infections in the elderly. The aim of this study was to access the prevalence of GBS genito-rectal colonisation of pregnant women and to evaluate the genetic characteristics of invasive and non-invasive GBS isolates recovered throughout Serbia. A total of 432 GBS isolates were tested for antimicrobial susceptibility, capsular polysaccharide (CPS) types and the presence of the hvgA gene. One hundred one randomly selected isolates were further characterized by clustered regularly interspaced short palindromic repeats (CRISPRs) analysis and/or multilocus sequence typing (MLST). The prevalence of GBS colonization in pregnant women was 15%. Overall, six capsular types (Ia, Ib, II to V) were identified, the most common being III (32.2%) and V (25.2%). The hiper-virulent clone type III/ST17 was present in 43.1% and 6.3% (p < 0.05) of paediatric and adults isolates, respectively. Comparative sequence analysis of the CRISPR1 spacers content indicated that a few clones comprised the vast majority of the tested GBS isolates. Thus, it was estimated that dominant clones recovered from infants were CPS III/ST17 in late-onset infections (19/23; 82.6%), and Ia/ST23 in early-onset disease (44.4%). Conversely, genotype CPS V/ST1 was the most prevalent in adults (4/9; 25.4%). All isolates were susceptible to penicillin. Macrolide resistance (23.1%) was strongly associated with the ermB gene and constitutive resistance to clindamycin (63.9%). The majority of strains was resistant to tetracycline (86.6%), mostly mediated by the tetM gene (87.7%). GBS isolates of CPS V/ST1 and CPS III/ST23 were significantly associated with macrolide and tetracycline resistance, respectively. In conclusion, hyper-virulent CPS III/ST17 and V/ST1 were recognized as dominant GBS clones in this study.

Effect of Childhood Pneumococcal Conjugate Vaccination on Invasive Disease Serotypes in Serbia.

In Serbia, PCV10 was introduced into the routine immunization for children under 2 in 2018 and replaced by PCV13 in 2022. We evaluated their impact on the distribution of invasive pneumococcal disease (IPD) serotypes across all age groups. Overall, 756 isolates were obtained from patients with IPD between 2010 and 2023 through laboratory surveillance. In the post-vaccination period, serotypes 14, 19F, 23F, and 6A significantly declined, while 3 and 19A considerably increased. This was especially evident in the ≤2 years group, making these serotypes the most prevalent among them. Serotype 3 dominated, representing 19.1% of all invasive isolates prior to 2018 and 33.1% thereafter. While serotype coverage of PCV10 has significantly decreased in the ≤2 years group (from 74.2% before 2018 to 29.5% after 2018), PCV13 coverage was 63.9% after 2018. In the post-PCV period, non-PCV13 serotypes, such as 9N, 10A, 15A, 15B, 15C, 22F, 6C, 6D, and 7C, increased across all isolates. Antibiotic non-susceptibility considerably decreased after 2018. MLST analysis showed shifts in sequence type prevalence, with pre-PCV lineages replaced and ongoing serotype 3 persistence, alongside potential capsule-switching events. These findings emphasize a noticeable shift in the distribution of serotypes and adaptability of pneumococcal populations, highlighting the importance of ongoing surveillance and the requirement for the urgent introduction of higher valent vaccines into the National Immunization Program.

Genetic Determinants of Clarithromycin and Fluoroquinolones Resistance in Helicobacter pylori in Serbia.

BACKGROUND/OBJECTIVES: Stomach infections by Helicobacter pylori can cause acute or chronic gastritis, peptic ulcers, and gastric cancer. The rise in antibiotic resistance is a significant health issue highlighted by the World Health Organization. The increasing number of treatment failures underscores the necessity for antibiotic susceptibility testing (AST). The study aimed to investigate the current prevalence and resistance to fluoroquinolones and clarithromycin with their detected mutations. METHODS: Stomach biopsies from symptomatic patients were subjected to molecular testing by GenoType Helico DR kit (Hain Lifescience GmbH, Nehren, Germany). RESULTS: Positive findings on the presence of H. pylori were detected in 42.4% of symptomatic patients, with the significant majority of patients (69%) having previously failed treatments. The resistance rates to fluoroquinolones and clarithromycin were 53.9% and 58.5%, respectively, with significantly higher rates in secondary resistant strains. The main resistance markers in fluoroquinolones and clarithromycin were N87K (27.4%) and A2147G (78.6%), respectively. Hetero-resistance or mixed genotypes were detected in over 20% of tested patients. During the study period, a significant increase in trends in both fluoroquinolones and clarithromycin resistance rates was observed. CONCLUSIONS: Results indicate the need for the implementation of the latest Maastricht VI Consensus recommendations for both AST whenever possible and the use of tailored guided therapy options due to high resistance rates and possible treatment failures. The GenoType Helico DR kit is a useful tool for AST, especially in cases of mixed H. pylori genotypes.

Comparative genomics and molecular epidemiology of colistin-resistant Acinetobacter baumannii.

This study aimed to investigate the prevalence and resistance mechanisms of colistin-resistant Acinetobacter baumannii (ColRAB) isolates in Serbia, assess their genetic relatedness to other circulating A. baumannii isolates in the neighbouring European countries, and analyse the global genomic epidemiology of ColRAB isolates. A total of 784 isolates of A. baumannii were recovered from hospitalised patients in Serbia between 2018 and 2021. The antimicrobial susceptibility testing was performed using disk diffusion and broth microdilution. All ColRAB isolates were subjected to DNA isolation and whole-genome sequencing (WGS). Overall, 3.94 % (n = 30) isolates were confirmed as ColRAB. Results of mutational and transcriptional analysis of genes associated with colistin resistance indicate the central role of the two-component regulating system, PmrAB, and increased expression of the pmrC gene in ColRAB. Most of the isolates (n = 29, 96.6 %) belonged to international clone II, with the most common sequence type being STPas2 (n = 23, 76.6 %). Based on the WGS analysis, ColRAB isolates belonging to the same ST isolated in various countries were grouped into the same clusters, indicating the global dissemination of several high-risk clonal lineages. Phylogenomic analysis of ColRAB isolates, together with all previously published A. baumannii genomes from South-Eastern European countries, showed that colistin resistance arose independently in several clonal lineages. Comparative genomic analysis revealed multiple genes with various roles (transcriptional regulation, transmembrane transport, outer membrane assembly, etc.), which might be associated with colistin resistance in A. baumannii. The obtained findings serve as the basis for further studies, contributing to a better understanding of colistin resistance mechanisms in A. baumannii.

Potential influence of antimicrobial resistance gene content in probiotic bacteria on the gut resistome ecosystems.

Antimicrobial resistance (AMR) poses a substantial threat to human health. The commensal bacteria of the gut microbiome were shown to serve as a reservoir of antibiotic resistance genes (ARGs), termed the gut resistome, which has the potential to transfer horizontally to pathogens and contribute to the emergence of drug-resistant bacteria. Namely, AMR traits are generally linked with mobile genetic elements (MGEs), which apart from disseminating vertically to the progeny, may cross horizontally to the distantly related microbial species. On the other hand, while probiotics are generally considered beneficiary to human health, and are therefore widely consumed in recent years most commonly in conjunction with antibiotics, the complexities and extent of their impact on the gut microbiome and resistome have not been elucidated. By reviewing the latest studies on ARG containing commercial probiotic products and common probiotic supplement species with their actual effects on the human gut resistome, this study aims to demonstrate that their contribution to the spread of ARGs along the GI tract merits additional attention, but also indicates the changes in sampling and profiling of the gut microbiome which may allow for the more comprehensive studying of the effects of probiotics in this part of the resistome.

Serotype distribution, antimicrobial susceptibility and molecular epidemiology of invasive Streptococcus pneumoniae in the nine-year period in Serbia.

Streptococcus pneumoniae is one of the leading bacterial pathogens that can cause severe invasive diseases. The aim of the study was to characterize invasive isolates of S. pneumoniae obtained during the nine-year period in Serbia before the introduction of the pneumococcal conjugate vaccines (PCVs) into routine vaccination programs by determining: serotype distribution, the prevalence and genetic basis of antimicrobial resistance, and genetic relatedness of the circulating pneumococcal clones. A total of 490 invasive S. pneumoniae isolates were included in this study. The serotype, antimicrobial susceptibility, and ST of the strains were determined by the Quellung reaction, disk- and gradient-diffusion methods, and multilocus sequence typing (MLST), respectively. The most common serotypes in this study were 3, 19F, 14, 6B, 6A, 19A, and 23F. The serotype coverages of PCV10 and PCV13 in children less than 2 years were 71.3 and 86.1%, respectively, while PPV23 coverage in adults was in the range of 85-96%, depending on the age group. Penicillin and ceftriaxone-non-susceptible isolates account for 47.6 and 16.5% of all isolates, respectively. Macrolide non-susceptibility was detected in 40.4% of isolates, while the rate of multidrug- and extensive-drug resistance was 20.0 and 16.9%, respectively. The MLST analysis of 158 pneumococci identified 60 different STs belonging to the 16 Clonal Complexes (CCs) (consisting of 42 STs) and 18 singletons. The most common CC/ST were ST1377, CC320, CC15, CC273, CC156, CC473, CC81, and CC180. Results obtained in this study indicate that the pre-vaccine pneumococcal population in Serbia is characterized by high penicillin and macrolides non-susceptibility, worrisome rates of MDR and XDR, as well as a high degree of genetic diversity. These findings provide a basis for further investigation of the changes in serotypes and genotypes that can be expected after the routine introduction of PCVs.

Carbapenem-Resistant Acinetobacter baumannii: Biofilm-Associated Genes, Biofilm-Eradication Potential of Disinfectants, and Biofilm-Inhibitory Effects of Selenium Nanoparticles.

This study aimed to investigate the biofilm-production ability of carbapenem-resistant Acinetobacter baumannii (CRAB), the biofilm-eradication potential of 70% ethanol and 0.5% sodium hypochlorite, the effects of selenium nanoparticles (SeNPs) against planktonic and biofilm-embedded CRAB, and the relationship between biofilm production and bacterial genotypes. A total of 111 CRAB isolates were tested for antimicrobial susceptibility, biofilm formation, presence of the genes encoding carbapenemases, and biofilm-associated virulence factors. The antibiofilm effects of disinfectants and SeNPs against CRAB isolates were also tested. The vast majority of the tested isolates were biofilm producers (91.9%). The bap, ompA, and csuE genes were found in 57%, 70%, and 76% of the CRAB isolates, with the csuE being significantly more common among biofilm producers (78.6%) compared to non-biofilm-producing CRAB (25%). The tested disinfectants showed a better antibiofilm effect on moderate and strong biofilm producers than on weak producers (p < 0.01). The SeNPs showed an inhibitory effect against all tested planktonic (MIC range: 0.00015 to >1.25 mg/mL) and biofilm-embedded CRAB, with a minimum biofilm inhibitory concentration of less than 0.15 mg/mL for 90% of biofilm producers. In conclusion, SeNPs might be used as promising therapeutic and medical device coating agents, thus serving as an alternative approach for the prevention of biofilm-related infections.

Current Susceptibility Surveillance and Distribution of Antimicrobial Resistance in N. gonorrheae within WHO Regions.

Neisseria gonorrhoeae (N. gonorrhoeae) is the etiological agent of the second most common sexually transmitted disease in the world, gonorrhoea. Currently recommended and last available first-line therapy is extended-spectrum cephalosporins most often combined with azitromycin. However, misuse of antibiotics and the abilities of N. gonorrhoeae to acquire new genetic and plasmid-borne resistance determinants has gradually led to the situation where this bacterium has become resistant to all major classes of antibiotics. Together with a generally slow update of treatment guidelines globally, as well as with the high capacity of gonococci to develop and retain AMR, this may lead to the global worsening of gonococcal AMR. Since effective vaccines are unavailable, the management of gonorrhoea relies mostly on prevention and accurate diagnosis, together with antimicrobial treatment. The study overviews the latest results of mostly WHO-initiated studies, primarily focusing on the data regarding the molecular basis of the resistance to the current and novel most promising antibacterial agents, which could serve to establish or reinforce the continual, quality-assured and comparable AMR surveillance, including systematic monitoring and treatment with the use of molecular AMR prediction methods.

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.

Trends in molecular characteristics and antimicrobial resistance of group B streptococci: a multicenter study in Serbia, 2015-2020.

Group B Streptococcus (GBS) is a major cause of neonatal morbidity and mortality. Serbia has not fully implemented preventive measures against GBS neonatal diseases. Therefore, we aimed to assess the maternal GBS colonisation and invasive neonatal disease rate, to reveal the trends of antimicrobial resistance and serotype distribution of GBS from various patient groups. Randomly selected non-invasive (n = 991) and all invasive GBS (n = 80) collected throughout Serbia from 2015 to 2020 were tested for antimicrobial susceptibility, capsular typing, and hvgA detection. Overall, 877/5621 (15.6%) pregnant women were colonised with GBS. Invasive GBS infections incidence in infants (0.18/1000 live births) showed a decreasing trend (0.3 to 0.1/1000 live births). Type III was overrepresented in infants with invasive infections (n = 35, 58.3%), whereas type V predominated among colonised adults (n = 224, 25.5%) and those with noninvasive (n = 37, 32.5%) and invasive infections (n = 8, 40%). The hypervirulent clone III/ST17 was highly associated with invasive infections (n = 28, 35%), particularly late-onset disease (n = 9, 47.4%), showing an increase from 12.3 to 14.8%. The GBS resistance to erythromycin and clindamycin was 26.7% and 22.1%, respectively, with an upward trend. The emergence of the hypervirulent clone III/ST17 and the escalation in GBS resistance highlight an urgent need for continuous monitoring of GBS infections.

A Laboratory-Based Surveillance Study of Invasive Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae Diseases in a Serbian Pediatric Population-Implications for Vaccination.

The aim of this study was to present the epidemiology of invasive diseases caused by Neisseria meningitidis and Streptococcus pneumoniae in the pre-vaccine period, and Haemophilus influenzae in the post-vaccine period in a pediatric population from Serbia. Among the meningococci, serogroup B dominated (83%), followed by serogroup C (11.3%). High antigenic diversity was found, with fine type P1.5-1,10-4 being the most frequent. Moderate susceptibility to penicillin was common (55%). Within pneumococci, serotypes 19F, 14, 6B, 6A, 18C, 23F, 3, and 7F prevailed, while 19A was rare (3.6%). The coverages of PCV10 and PCV13 were 68% and 84%, respectively. Major sequence types were ST320, ST15, ST273, ST271, and ST81. Non-susceptibility to penicillin (66.7%), cefotaxime (37%), and macrolides (55%) was predominantly detected in vaccine-related serotypes. Among the 11 invasive H. influenzae isolates collected, there were six Hib, three non-type b, and two non-typeable strains (ntHi) that were antibiotic susceptible. These results imply a potential benefit of future Men-B vaccine implementations. For pneumococci, as PCV10 was recently introduced, a significant reduction of morbidity and antibiotic resistance might be expected. The efficiency of Hib vaccination is evident, but a shift towards non-type b and ntHi strains may be anticipated.

The first nationwide multicenter study of Acinetobacter baumannii recovered in Serbia: emergence of OXA-72, OXA-23 and NDM-1-producing isolates.

BACKGROUND: The worldwide emergence and clonal spread of carbapenem-resistant Acinetobacter 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 of Acinetobacter 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 of A. baumannii. Identification to the species level was done by the detection of the blaOXA-51 gene and rpoB gene sequence analysis. Susceptibility testing was done by disk diffusion and broth microdilution method. CRAB isolates were tested for the presence of acquired carbapenemases (blaOXA-24-like, blaOXA-23-like,blaOXA-58-like, blaOXA-143-like, blaIMP, blaVIM, blaGIM, blaSPM, blaSIM, blaNDM) 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. baumannii was identified in 237 out of 280 Acb complex (84.6%). CRAB prevalence was found to be 93.7% (237/222). The MIC50/MIC90 for imipenem and meropenem were 8/> 32 µg/mL and 16/> 32 µg/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%) blaOXA-24-like, 76 (34.5%) blaOXA-23-like, and 7 (3.2%) blaNDM-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: blaOXA66/blaOXA23/ST2 (32.4%), blaOXA66/blaOXA23/blaOXA72/ST2 (2.7%), blaOXA66/blaOXA72/ST492 (37.8%), and blaOXA66/blaOXA72/ST636 (27.1%). CONCLUSION: This study revealed extremely high proportions of carbapenem resistance among A. baumannii clinical isolates due to the emergence of blaOXA-72, blaOXA-23, and blaNDM-1 genes among CRAB isolates in Serbia and their clonal propagation.

Changes in Macrolide Resistance Among Group A Streptococci in Serbia and Clonal Evolution of Resistant Isolates.

In Serbia, the frequency of macrolide-resistant group A streptococci (MRGASs) increased significantly from 2006 to 2009. MRGAS analysis in 2008 revealed the presence of three major clonal lineages: emm75/mefA, emm12/mefA, and emm77/ermTR. The aim of the present study was to determine the prevalence of macrolide resistance and to evaluate variations in the clonal composition of MRGASs. The study included 1,040 pharyngeal group A streptococci collected throughout Serbia, which were tested for antimicrobial susceptibility. MRGAS isolates were further characterized by the presence of resistance determinants, emm typing, and pulsed-field gel electrophoresis analysis. The prevalence of macrolide resistance was 9.6%, showing a slight decrease compared with the rate of 12.5% (2008). Tetracycline resistance was present in 6% of isolates, while norfloxacin nonsusceptibility detected for the first time in Serbia was 9.8%. The M phenotype dominated (84%), followed by the constitutive macrolides, lincosamides, and streptogramin B phenotype (12%). Five emm types were detected: emm75, emm12, emm1, emm28, and emm89. The emm75/mefA (62%), emm12/mefA (14%), and emm12/ermB/tetM (6%) were predominant clones and were found in both the present and the previous study periods at different frequencies. The major change was the loss of emm77/ermTR/tetO, which contributed to 15% of MRGASs in 2008.

Characterization of macrolide-resistant non-invasive pneumococci in the pre-vaccine era in Serbia.

Numerous reports have confirmed that increased macrolide use in the treatment of respiratory tract infection has contributed to the emergence of antibiotic resistance worldwide. Studies have also shown that pneumococcal vaccine can reduce pneumococcal resistance. The aim of this study was to determine the prevalence of co-resistance to penicillin and other antibiotics in macrolide-resistant (MR) non-invasive pneumococcal isolates and to evaluate serotype distribution in resistant strains in the pre-vaccine era in Serbia. About 80% of MR isolates expressed the MLS phenotype with very high resistance to both erythromycin and clindamycin. A total of 132 (84.1%) MR isolates were multiresistant, i.e., they were resistant to erythromycin, penicillin, tetracycline, and trimethoprim-sulfamethoxazole. Among 157 MR pneumococci, 11 different serotypes were found. Four serotypes, 19F, 14, 6B, and 23F, accounted for 77.7% of all MR pneumococcal isolates. Among isolates with the cMLS phenotype, serotypes 19F and 14 were predominant, whereas serotype 6A was the most common among those with the M phenotype, followed by 14. In conclusion, co-resistance to macrolides and penicillin in our non-invasive pneumococcal isolates is high. The majority of tested strains (∼80%) belonged to the four serotypes (19F, 14, 6B, and 23F) that are included in all conjugate vaccine formulations.

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.

Hydrogen peroxide producing lactobacilli in women with vaginal infections.

OBJECTIVE: Hydrogen peroxide producing lactobacilli are isolated from the vaginas of a majority of healthy reproductive age women. Their toxic and inhibitory effect against the overgrowth of pathogens in the vagina is documented by in vitro studies. Clinical studies concerning the role of hydrogen peroxide producing lactobacilli in vaginal infections are controversial. STUDY DESIGN: The aim of this study was to isolate lactobacilli from women with major vaginal infections: bacterial vaginosis (BV), vulvovaginal candidiasis (VVC) and trichomoniasis (TV), and from women with normal flora (NF), to test their ability to produce hydrogen peroxide, to quantitate lactobacilli and to evaluate factors related to the colonization of vagina with hydrogen peroxide producing lactobacilli. The study group comprised 108 women: 27 with VVC, 26 with BV, 20 with TV and 35 with NF. Data was analysed with Fisher exact test, chi2-test, and ANOVA. RESULTS: Lactobacilli were isolated from 81.5% of women with VVC, 58% of women with BV (p=0019), 55% of women with TV (p=0.015) and from 86% of women with NF. The majority of isolates of lactobacilli from women with VVC, TV and NF produced hydrogen peroxide (77%, 63.5% and 80%, respectively), compared with women with BV where only 26.5% of isolates produced hydrogen peroxide (p=0.01). Quantitative analysis showed that in an overwhelming majority of women colonized with lactobacilli, their count was >10(7) CFU/ml. Upon evaluation of behavioural and demographic characteristics, we found that smokers had a lower incidence of isolation of hydrogen peroxide producing lactobacilli (p=0.018). Our results showed that women with BV lack lactobacilli, especially hydrogen peroxide producing ones. Women with TV had a lower rate of lactobacilli. In women with VVC, there was neither significant difference in the isolation of lactobacilli, nor in their ability to produce hydrogen peroxide, compared to women with NF. CONCLUSION: Our results point out that hydrogen peroxide producing lactobacilli could protect against the development of BV, but not against VVC and TV.

Streptococcus pneumoniae serotype distribution in Vojvodina before the introduction of pneumococcal conjugate vaccines into the National Immunization Program.

Introduction: Streptococcus pneumoniae is the most common causative agent of bacterial pneumonia and meningitis. Mandatory childhood immunization against pneumococcal diseases is introduced in the new Law on Protection of Population against Communicable Diseases in Serbia. Objective: The objective of this study was to determine the prevalence of pneumococcal serotype distribution in Vojvodina region before routine use of pneumococcal conjugate vaccine in Serbia. Methods: A total of 105 isolates of Streptococcus pneumoniae were collected in the period from January 2009 to April 2016. Based on the results of serotyping in the National Reference Laboratory, we analyzed distribution of circulating serotypes and coverage of conjugate and 23-valent polysaccharide pneumococcal vaccines in different age groups. Results: Among 105 isolates, a total of 21 different serotypes of Streptococcus pneumoniae were determined. The most frequent serotypes were 3 (21.9%), 19F (20.0%), and 14 (10.5%). The serotype coverage of pneumococcal conjugate vaccines (PCV7, PCV10, and PCV13) was 48.6%, 54.3%, and 84.8%, respectively, while pneumococcal polysaccharide vaccine (PPV23) covered 89.5% of the total number of isolates in all age groups. Serotypes included in PCV7, PCV10, and PCV13 represented 72.0%, 76.0%, and 88.0% of the total number of isolates in children ≤5 years, respectively. Vaccine serotype coverage of PCV13 and PPV23 ranged from 87.1% to 90.3% in adults 50­64 years of age, and 77.8% to 85.2% in adults ≥65 years old. Conclusion: Serotype distribution of Streptococcus pneumoniae in the population fairly overlaps with the serotypes contained in pneumococcal vaccines, so that implementation of childhood immunization is justified. The study was done in the Province of Vojvodina but the findings may be applied to Serbia as a whole.