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Background: The pre-analytical (PA) phase is the most vulnerable phase of the laboratory testing procedure, with critical procedures-collection, handling, sample transport, and time and temperature of sample storage. This study aimed to examine the stability of basic biochemical parameters depending on the samples' storage conditions and the number of freeze-thaw cycles (FTCs). In parallel, the presence of sample bacterial contamination during routine laboratory work was examined. Methods: Two plasma pools (ethylenediaminetetraacetic acid (EDTA), and sodium-fluoride/potassium oxalate plasma (NaF)) were stored at +4 ËC/-20 ËC. Total chole - sterol (TC), glucose, triglycerides (TG), urea, and albumin concentrations were measured using BioSystems reagents (cholesterol oxidase/peroxidase, glucose oxidase/per - oxidase, glycerol phosphate oxidase/peroxidase, urease/ salicylate, and bromcresol green method, respectively) on Ilab 300+. Sample bacterial contamination was determined by 16S rRNA sequence analysis. The expe - riment encompassed a 5 day-period: Day 1-fresh sample, Day 2-1st FTC, Day 3-2nd FTC, Day 4-3rd FTC, Day 5-4th FTC. The appearance of bacteria in two consecutive samples was the experiment's endpoint.
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Antimicrobial resistance is a global threat, leading to an alarming increase in the prevalence of bacterial infections that can no longer be treated with available antibiotics. The World Health Organization estimates that by 2050 up to 10 million deaths per year could be associated with antimicrobial resistance, which would equal the annual number of cancer deaths worldwide. To overcome this emerging crisis, novel anti-bacterial compounds are urgently needed. There are two possible approaches in the fight against bacterial infections: a) targeting structures within bacterial cells, similar to existing antibiotics; and/or b) targeting virulence factors rather than bacterial growth. Here, for the first time, we provide a comprehensive overview of the key steps in the evaluation of potential new anti-bacterial and/or anti-virulence compounds. The methods described in this review include: a) in silico methods for the evaluation of novel compounds; b) anti-bacterial assays (MIC, MBC, Time-kill); b) anti-virulence assays (anti-biofilm, anti-quorum sensing, anti-adhesion); and c) evaluation of safety aspects (cytotoxicity assay and Ames test). Overall, we provide a detailed description of the methods that are an essential tool for chemists, computational chemists, microbiologists, and toxicologists in the evaluation of potential novel antimicrobial compounds. These methods are cost-effective and have high predictive value. They are widely used in preclinical studies to identify new molecular candidates, for further investigation in animal and human trials.
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Antiinfecciosos , Infecciones Bacterianas , Animales , Humanos , Antibacterianos/farmacología , Antibacterianos/química , Biopelículas , Percepción de Quorum , Bacterias , Antiinfecciosos/farmacología , Infecciones Bacterianas/tratamiento farmacológico , Factores de Virulencia/farmacología , Pseudomonas aeruginosaRESUMEN
Ever since the development of the first vaccine, vaccination has had the great impact on global health, leading to the decrease in the burden of numerous infectious diseases. However, there is a constant need to improve existing vaccines and develop new vaccination strategies and vaccine platforms that induce a broader immune response compared to traditional vaccines. Modern vaccines tend to rely on certain nanotechnology platforms but are still expected to be readily available and easy for large-scale manufacturing and to induce a durable immune response. In this review, we present an overview of the most promising nanoadjuvants and nanoparticulate delivery systems and discuss their benefits from tehchnological and immunological standpoints as well as their objective drawbacks and possible side effects. The presented nano alums, silica and clay nanoparticles, nanoemulsions, adenoviral-vectored systems, adeno-associated viral vectors, vesicular stomatitis viral vectors, lentiviral vectors, virus-like particles (including bacteriophage-based ones) and virosomes indicate that vaccine developers can now choose different adjuvants and/or delivery systems as per the requirement, specific to combatting different infectious diseases.
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Since the WHO declared the COVID-19 pandemic in March 2020, the disease has spread rapidly leading to overload of the health system and many of the patients infected with SARS-CoV-2 needed to be admitted to the intensive care unit (ICU). Around 10% of patients with the severe manifestation of COVID-19 need noninvasive or invasive mechanical ventilation, which represent a risk factor for Acinetobacter baumannii superinfection. The 64 A. baumannii isolates were recovered from COVID-19 patients admitted to ICU at General Hospital "Dr Laza K. Lazarevic" Sabac, Serbia, during the period from December 2020 to February 2021. All patients required mechanical ventilation and mortality rate was 100%. The goal of this study was to evaluate antibiotic resistance profiles and virulence potential of A. baumannii isolates recovered from patients with severe form of COVID-19 who had a need for mechanical ventilation. All tested A. baumannii isolates (n = 64) were sensitive to colistin, while resistant to meropenem, imipenem, gentamicin, tobramycin, and levofloxacin according to the broth microdilution method and MDR phenotype was confirmed. In all tested isolates, representatives of international clone 2 (IC2) classified by multiplex PCR for clonal lineage identification, bla AmpC, bla OXA-51, and bla OXA-23 genes were present, as well as ISAba1 insertion sequence upstream of bla OXA-23. Clonal distribution of one dominant strain was found, but individual strains showed phenotypic differences in the level of antibiotic resistance, biofilm formation, and binding to mucin and motility. According to PFGE, four isolates were sequenced and antibiotic resistance genes as well as virulence factors genes were analyzed in these genomes. The results of this study represent the first report on virulence potential of MDR A. baumannii from hospital in Serbia.
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Trimethoprim-sulfamethoxazole (SXT) is the preferable treatment option of the infections caused by Achromobacter spp. Our study aimed to analyze the SXT resistance of 98 Achromobacter spp. isolates from pediatric patients, among which 33 isolates were SXT-resistant. The presence of intI1 was screened by PCR and genome sequence analyses. The intI1 gene was detected in 10 of SXT-resistant isolates that had shorter intI1 PCR fragments named intI1S. Structural changes in intI1S were confirmed by genome sequencing and analyses which revealed 86 amino acids deletion in IntI1S protein compared to canonical IntI1 protein. All IntI1S isolates were of non-CF origin. Pan-genome analysis of intI1S bearing A. xylosoxidans isolates comprised 9052 genes, with the core genome consisting of 5455 protein-coding genes. Results in this study indicate that IntI1S isolates were derived from clinical settings and that cystic fibrosis (CF) patients were potential reservoirs for healthcare-associated infections that occurred in non-CF patients.
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Achromobacter denitrificans , Achromobacter , Fibrosis Quística , Infecciones por Bacterias Gramnegativas , Humanos , Niño , Combinación Trimetoprim y Sulfametoxazol , Achromobacter denitrificans/genética , Antibacterianos/uso terapéutico , Integrasas/uso terapéutico , Integrones/genética , Serbia , Genómica , Pruebas de Sensibilidad MicrobianaRESUMEN
AIMS: To investigate the synergistic activity of colistin and selenium nanoparticles (SeNPs) against pandrug-resistant (PDR) Ac. baumannii. METHODS AND RESULTS: Chequerboard and time-kill assays were employed to explore the potential synergistic interactions between colistin and SeNPs against Ac. baumannii isolates (8), previously determined as colistin-resistant (MIC range 16-256 µg ml-1 ). Also, whole-genome sequencing (WGS) and gene expression analyses were used to elucidate the mechanisms of colistin resistance. Exceptionally strong synergistic activity (FICI range 0.004-0.035) of colistin and SeNPs against colistin-resistant isolates was revealed. Colistin (0.5 or 1 µg ml-1 ) used in combination with SeNPs (0.5 µg ml-1 ) was able to reduce initial inoculum during the first 4 h of incubation, in contrast to colistin (0.5, 1 or 2 µg ml-1 ) alone. CONCLUSIONS: These findings propose colistin/SeNPs combination as a new option to fight PDR Ac. baumannii, the therapeutic possibilities of which should be proved in future in vivo studies. SIGNIFICANCE AND IMPACT OF STUDY: Here we present the first evidence of synergy between colistin and selenium compounds against bacteria in general. Also, WGS and gene expression analyses provide some new insights into Ac. baumannii colistin resistance mechanisms.
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Infecciones por Acinetobacter , Acinetobacter baumannii , Nanopartículas , Selenio , Infecciones por Acinetobacter/microbiología , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Colistina/farmacología , Colistina/uso terapéutico , Farmacorresistencia Bacteriana Múltiple/genética , Sinergismo Farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Selenio/farmacologíaRESUMEN
Screening for producers of potent antimicrobial peptides, resulted in the isolation of Bacillus cereus BGNM1 with strong antimicrobial activity against Listeria monocytogenes. Genome sequence analysis revealed that BGNM1 contains the gene cluster associated with the production of the lantibiotic, thusin, previously identified in B. thuringiensis. Purification of the antimicrobial activity confirmed that strain BGMN1 produces thusin. Both thusin sensitive and resistant strains were detected among clinical isolates of Streptococcus agalactiae. Random mutagenesis of a thusin sensitive strain, S. agalactiae B782, was performed in an attempt to identify the receptor protein for thusin. Three independent thusin resistant mutants were selected and their complete genomes sequenced. Comparative sequence analysis of these mutants with the WT strain revealed that duplication of a region encoding a 79 amino acids repeat in a C-protein α-antigen was a common difference, suggesting it to be responsible for increased resistance to thusin. Since induced thusin resistant mutants showed higher level of resistance than the naturally resistant B761 strain, complete genome sequencing of strain B761 was performed to check the integrity of the C-protein α-antigen-encoding gene. This analysis revealed that this gene is deleted in B761, providing further evidence that this protein promotes interaction of the thusin with receptor.
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Bacteriocinas , Listeria monocytogenes , Antibacterianos/farmacología , Bacteriocinas/genética , Familia de Multigenes , Streptococcus agalactiae/genéticaRESUMEN
Plasmids are autonomous episomally replicating genetic elements, which carry backbone genes important for the replication and maintenance within their host, and accessory genes that might confer an advantage to their host under specific selective pressure in its ecological niche. The genome of dairy isolate L. lactis subsp. lactis bv. diacetylactis S50 was sequenced using the PacBio SMRT Cell Seq-RSII platform and revealed to possess one of the largest plasmidomes among L. lactis strains studied so far, harboring six plasmids: pS6 (5553 bp), pS7a (7308 bp), pS7b (7266 bp), pS19 (19,027 bp), pS74 (74,256 bp) and pS127 (127,002 bp) in total representing 8.9% of genome size (240,412 bp). Based on predicted plasmid replication proteins and origins it appears that all six plasmids replicate via the theta-type mechanism. The two the largest plasmids (pS74 and pS127), carry a number of genes known to be important for growth and survival in the dairy environment. These genes encode technological functions such as bacteriocin production, protein degradation, magnesium and cobalt/nickel transporters, selenium binding, exopolysaccharides (EPS) production, bacteriophage and stress resistance. Beside genes for replication, the small plasmids (pS6, pS7a, pS7a, and pS19) also carry genes important for mobilization and host survival such as type I restriction-modification (R-M) system, metal transporters, enzymes and transcriptional regulators. All plasmids in S50 strain are mobilizable, containing an oriT sequences, while pS127 is self-conjugative and allows for mobilization of the other plasmids. Small plasmids are prone to structural and segregational instability, while pS127 appeared to be segregationally stable thanks to the possession of two partition systems. The main characteristic of plasmid pS74 is EPS production, while plasmid pS127 is characterized by proteinase and multiple bacteriocins, tra locus, phage abortive systems and metal transporters. In addition to LcnA and LcnB, plasmid pS127 encodes several bacteriocin-pheromone molecules and a new bacteriocin named LcnS50, with narrow spectrum of action limited to lactococci, that has been successfully cloned and heterologously expressed.
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Plásmidos de Bacteriocinas/genética , Bacteriocinas/genética , Biotecnología , Lactococcus lactis/genética , Microbiología IndustrialRESUMEN
The antimicrobial susceptibility testing was conducted on 174 single isolates from poultry farms in Serbia and it was determined that seven Salmonella spp. were multidrug resistant. Sixteen serotypes were detected, but only serotype Infantis confirmed reduced susceptibility to colistin. Seven colistin resistant Salmonella Infantis were studied in detail using the WGS approach. Three sequence types were identified corresponding to different epizootiology region. The isolate from the Province of Vojvodina 3842 and isolates from Jagodina (92 and 821) are represented by the sequence type ST413 and ST11, respectively. Four isolates from Kraljevo are ST32, a common S. Infantis sequence type in humans, poultry and food. The fosfomycin resistance gene fosA7 in isolate 3842 and the vgaA gene in isolate 8418/2948 encoding resistance to pleuromutilins were reported for the first time in serovar Infantis. The changes in relative expression of the phoP/Q, mgrB and pmrA/B genes were detected. Single nucleotide polymorphisms of the pmrB gene, including transitions Val164Gly or Val164Met, and Arg92Pro are described. Analyses of quinolone resistance determining region revealed substitutions Ser83Tyr in GyrA protein and Thr57Ser and Ser80Arg in ParC protein. Based on WGS data, there are two major clusters among analyzed Salmonella Infantis isolates from central Serbia.
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Long-term overuse of antibiotics has driven the propagation and spreading of antibiotic resistance genes (ARGs) such as efflux pumps in the environment, which can be transferred to clinically relevant pathogens. This study explored the abundance and diversity of ARGs and mobile genetic elements within bacterial communities from sediments of three Western Balkans glacial lakes: Plav Lake (high impact of human population), Black Lake (medium impact of human population) and Donje Bare Lake (remote lake, minimal impact of human population) via shotgun metagenomics. Assembled metagenomic sequences revealed that Resistance-Nodulation-Division (RND) efflux pumps genes were most abundant in metagenome from the Plav Lake. The Integron Finder bioinformatics tool detected 38 clusters of attC sites lacking integron-integrases (CALIN) elements: 20 from Plav Lake, four from Black Lake and 14 from Donje Bare Lake. A complete integron sequence was recovered only from the assembled metagenome from Plav Lake. Plasmid contents within the metagenomes were similar, with proportions of contigs being plasmid-related: 1.73% for Plav Lake, 1.59% for Black Lake and 1.64% for Donje Bare Lake. The investigation showed that RNDs and mobile genetic elements content correlated with human population impact.
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Farmacorresistencia Microbiana/genética , Genes Bacterianos , Lagos/microbiología , Metagenómica , Antibacterianos , Peninsula Balcánica , HumanosRESUMEN
Pseudomonas aeruginosa is one of the most common opportunistic pathogens that use quorum sensing (QS) system to regulate virulence factors expression and biofilm development. Delftia sp. 11304 was selected among 663 Gram-negative clinical isolates based on its QS inhibitory activity against P. aeruginosa MMA83 clinical isolate. Whole genome sequencing identified this isolate as D. tsuruhatensis and revealed genetic armamentarium of virulence factors and antibiotic resistance determinants. Ethyl acetate extract of D. tsuruhatensis 11304 culture supernatant (QSI extract) prevented biofilm formation of P. aeruginosa MMA83, but was unable to cause biofilm decomposition. QSI extract showed a synergistic effect in combination with meropenem and gentamycin, against P. aeruginosa MMA83. A dose-dependent reduction of the virulence factors: elastase, rhamnolipid and pyocyanin production by P. aeruginosa MMA83 and significant downregulation of lasI, lasR, rhlI, rhlR, pqs and mvfR expression were observed. Matrix-assisted Laser Desorption Ionization (MALDI) mass spectrometry of D. tsuruhatensis 11304 QSI extract revealed the presence of N-acyl homoserine lactones (AHL) with chain lengths of C12 to C18. The main ion peak was identified as N-octadecanoylhomoserine lactone (C18-HSL). Commercial C18-HSL (20 µM) reduced pyocyanin production as well as mRNA level of the lasI gene. A novel AHL species, dihydroxy-N-octadecanoylhomoserine lactone, was also described.
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Acil-Butirolactonas/farmacología , Antibacterianos/farmacología , Biopelículas/crecimiento & desarrollo , Delftia/aislamiento & purificación , Pseudomonas aeruginosa/crecimiento & desarrollo , Percepción de Quorum , Factores de Virulencia/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Humanos , Pseudomonas aeruginosa/efectos de los fármacos , Factores de Virulencia/genética , Secuenciación Completa del GenomaRESUMEN
Bacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37°C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects.
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Antibacterianos/farmacología , Brevibacillus/aislamiento & purificación , Ensilaje/microbiología , Bacteriocinas/genética , Brevibacillus/efectos de los fármacos , Brevibacillus/genética , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Calor , Concentración de Iones de Hidrógeno , Cinética , Pruebas de Sensibilidad MicrobianaRESUMEN
Pseudomonas aeruginosa, which is a clinically important representative of Pseudomonas spp., has been recognized as causative agent of severe nosocomial infections worldwide. An increase in antibiotic resistance of P. aeruginosa clinical strains could be attributed to their capacity to acquire resistance through mobile genetic elements such as mobile integrons that are present in one-half of multidrug-resistant P. aeruginosa strains. Mobile class 1 integrons are recognized as genetic elements involved in the rapid dissemination of multiple genes encoding for antibiotic resistance. The LexA protein is a major repressor of integrase transcription, but differences in transcription regulation among bacterial species have also been noted. In this study, the promoter activity of class 1 integron integrase gene (intI1) and its variant lacking the LexA binding site in Pseudomonas putida WCS358 wild type, ΔrpoS and ΔpsrA was analysed. The results show that the activity of the intI1 gene promoter decreased in the rpoS and psrA mutants in the stationary phase of growth compared to the wild type, which indicates the role of RpoS and PsrA proteins in the positive regulation of integrase transcription. Additionally, it was determined that the activity of the lexA gene promoter decreased in ΔrpoS and ΔpsrA, and thus, we propose that PsrA indirectly regulates the intI1 gene promoter activity through regulation of lexA gene expression in co-operation with some additional regulators. In this study, intI1 gene expression was shown to be controlled by two major stress response (SOS and RpoS) regulons, which indicates that integrase has evolved to use both systems to sense the cell status.
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Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Integrasas/genética , Pseudomonas/fisiología , Serina Endopeptidasas/genética , Factores de Transcripción/metabolismo , Sitios de Unión , Fenómenos Fisiológicos Celulares , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Modelos Genéticos , Regiones Promotoras Genéticas , Pseudomonas/genética , Pseudomonas/crecimiento & desarrollo , Regulón , Eliminación de Secuencia , Serina Endopeptidasas/metabolismo , Factor sigma/deficiencia , Factor sigma/genética , Factor sigma/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
The aim of this study was to evaluate the contribution of plasmid-mediated genes and efflux to fluoroquinolone resistance in collection of Achromobacter spp. gathered during a 3-year period. Susceptibility to ciprofloxacin and levofloxacin was tested by disk diffusion and microdilution tests for a collection of 98 Achromobacter spp. clinical isolates. Identification of fluoroquinolone-resistant isolates was performed by sequencing and phylogenetic analyses of the nrdA gene. Genetic relatedness among resistant isolates was determined by pulsed-field gel electrophoresis (PFGE) analysis. The influence of an H+ conductor cyanide m-chlorophenyl hydrazone (CCCP) and a resistance-nodulation-division-type efflux pump inhibitor phenylalanine-arginine beta-naphthylamide (PAßN) on minimal inhibitory concentration (MIC) value was evaluated by broth microdilution. The presence of the plasmid-mediated qnrA, qnrB, qnrC, qnrS, and aac-(6')-Ib-cr genes was investigated by PCR and sequencing. Achromobacter spp. isolates that were resistant or intermediately resistant to fluoroquinolones in disk diffusion tests (44/98) were subjected to microdilution. As a result, 20/98 isolates were confirmed to be resistant to ciprofloxacin while 10/98 was resistant to levofloxacin. CCCP decreased twofold MIC value for ciprofloxacin in six isolates and more than 16 times in one isolate, while MIC value for levofloxacin was decreased in all isolates (twofold to more than eightfold). Fluoroquinolone-resistant isolates were identified as A. xylosoxidans with the nrdA gene sequencing. PFGE revealed that resistant isolates belonged to seven different genotypes. Ten isolates belonging to four genotypes were positive for the aac-(6')-Ib-cr gene. Although resistance to fluoroquinolones was not widespread among analyzed isolates, detected contribution of efflux pumps and the presence of the aac-(6')-Ib-cr gene present a platform for emergence of more resistant strains.
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Achromobacter denitrificans/genética , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Fluoroquinolonas/farmacología , Genes Bacterianos , Achromobacter denitrificans/clasificación , Achromobacter denitrificans/efectos de los fármacos , Achromobacter denitrificans/aislamiento & purificación , Electroforesis en Gel de Campo Pulsado , Genes Bacterianos/genética , Genotipo , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Filogenia , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , SerbiaRESUMEN
Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. Lactococcus raffinolactis BGTRK10-1 expresses LraI Type II restriction-modification enzyme, whose activity is similar to that shown for EcoRI; LraI methyltransferase protects DNA from EcoRI cleavage. The gene encoding LraI endonuclease was cloned and overexpressed in E. coli. Purified enzyme showed the highest specific activity at lower temperatures (between 13°C and 37°C) and was stable after storage at -20°C in 50% glycerol. The concentration of monovalent ions in the reaction buffer required for optimal activity of LraI restriction enzyme was 100 mM or higher. The recognition and cleavage sequence for LraI restriction enzyme was determined as 5'-G/AATTC-3', indicating that LraI restriction enzyme is an isoschizomer of EcoRI. In the reaction buffer with a lower salt concentration, LraI exhibits star activity and specifically recognizes and cuts another alternative sequence 5'-A/AATTC-3', leaving the same sticky ends on fragments as EcoRI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin of LraI restriction-modification system with previously described EcoRI-like restriction-modification systems.
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Proteínas Bacterianas/metabolismo , Lactococcus/enzimología , Lactococcus/genética , Proteínas Recombinantes/metabolismo , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración Osmolar , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/química , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genéticaRESUMEN
Achromobacter spp. are recognized as emerging pathogens in hospitalized as well as in cystic fibrosis (CF) patients. From 2012 to 2015, we collected 69 clinical isolates (41 patient) of Achromobacter spp. from 13 patients with CF (CF isolates, n = 32) and 28 patients receiving care for other health conditions (non-CF isolates, n = 37). Molecular epidemiology and virulence potential of isolates were examined. Antimicrobial susceptibility, motility, ability to form biofilms and binding affinity to mucin, collagen, and fibronectin were tested to assess their virulence traits. The nrdA gene sequencing showed that A. xylosoxidans was the most prevalent species in both CF and non-CF patients. CF patients were also colonized with A. dolens/A. ruhlandii, A. insuavis, and A. spiritinus strains while non-CF group was somewhat less heterogenous, although A. insuavis, A. insolitus, and A. piechaudii strains were detected beside A. xylosoxidans. Three strains displayed clonal distribution, one among patients from the CF group and two among non-CF patients. No significant differences in susceptibility to antimicrobials were observed between CF and non-CF patients. About one third of the isolates were classified as strong biofilm producers, and the proportion of CF and non-CF isolates with the ability to form biofilm was almost identical. CF isolates were less motile compared to the non-CF group and no correlation was found between swimming phenotype and biofilm formation. On the other hand, CF isolates exhibited higher affinity to bind mucin, collagen, and fibronectin. In generall, CF isolates from our study exhibited in vitro properties that could be of importance for the colonization of CF patients.
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Achromobacter/genética , Achromobacter/fisiología , Fibrosis Quística/complicaciones , Infecciones por Bacterias Gramnegativas/microbiología , Epidemiología Molecular , Virulencia/genética , Achromobacter/clasificación , Achromobacter/aislamiento & purificación , Adhesinas Bacterianas , Adolescente , Adulto , Antibacterianos/farmacología , Biopelículas/crecimiento & desarrollo , Niño , Preescolar , Farmacorresistencia Bacteriana , Femenino , Genes Bacterianos/genética , Genotipo , Infecciones por Bacterias Gramnegativas/complicaciones , Humanos , Lactante , Recién Nacido , Locomoción , Masculino , Pruebas de Sensibilidad Microbiana , Familia de Multigenes , Fenotipo , Filogenia , ARN Ribosómico 16S/genética , Adulto JovenRESUMEN
BACKGROUND: Burkholderia cenocepacia is considered one of the most problematic cystic fibrosis (CF) pathogens. Colonization prevalence in the Serbian CF population is high and virtually exclusively limited to a single highly transmissible clone of B. cenocepacia ST856 which is positive for both the B. cepacia epidemic strain marker (BCESM) and cable pilin, and is closely related to the epidemic strain CZ1 (ST32). METHODS: Biofilm formation for 182 isolates, and adhesion to components of the host extracellular matrix, proteolytic activity, mucoidy and motility of selected ST856 representatives, as well as B. cenocepacia ST858 and ST859, and B. stabilis ST857, novel STs isolated from Serbian CF patients, were investigated in this study. The presence of the cepI, cepR, fliG, llpE, wbiI, and bcscV genes was analyzed. RESULTS: Biofilm-formation ability of analyzed strains was poor under standard laboratory conditions, but changed in stress conditions (cold stress) and conditions that mimic CF milieu (increased CO2). All strains expressed ability to bind to collagen and fibronectin albeit with different intensity. Representatives of ST856 exhibited gelatinase activity. ST858, ST859 and 9/11 of ST856 genotypes were positive for swimming and twitching motility whereas ST857 was non-motile. Mucoidy was demonstrated in all ST856 genotypes, ST857 was semi-mucoid, and ST858 and ST859 were non-mucoid. Molecular analysis for major virulence factors revealed that ST856 and ST857 carried the six analyzed genes, while ST858 and ST859 were negative for the llpE gene. CONCLUSION: Variations in virulence phenotypes in different genotypes of epidemic B. cenocepacia ST856 clone, in vitro, could be a consequence of diversification driven by pathoadaptation. Diversity of epidemic clone genotypes virulence, could be challenging for accurate diagnosis and treatment, as well as for infection control.
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Enterococci represent the most controversial group of dairy bacteria. They are found to be the main constituent of many traditional Mediterranean dairy products and contribute to their characteristic taste and flavor. On the other hand, during the last 50 years antibiotic-resistant enterococci have emerged as leading causes of nosocomial infections worldwide. The aim of this study was to determine the diversity, technological properties, antibiotic susceptibility and virulence traits of 636 enterococci previously isolated from 55 artisan dairy products from 12 locations in the Western Balkan countries (WBC) of Serbia, Croatia and Bosnia and Herzegovina. All strains were identified both by microbiological and molecular methods. The predominant species was Enterococcus durans, followed by Enterococcus faecalis and Enterococcus faecium. Over 44% of the isolates were resistant to ciprofloxacin and erythromycin, while 26.2% of the isolates were multi-resistant to three or more antibiotics belonging to different families. 185 isolates (29.1%) were susceptible to all 13 of the antibiotics tested. The antibiotic-susceptible isolates were further tested for possible virulence genes and the production of biogenic amines. Finally, five enterococci isolates were found to be antibiotic susceptible with good technological characteristics and without virulence traits or the ability to produce biogenic amines, making them possible candidates for biotechnological application as starter cultures in the dairy industry.
RESUMEN
Carbapenem-resistant A. baumannii present a significant therapeutic challenge for the treatment of nosocomial infections in many European countries. Although it is known that the gradient of A. baumannii prevalence increases from northern to southern Europe, this study provides the first data from Serbia. Twenty-eight carbapenem-resistant A. baumannii clinical isolates were collected at a Serbian pediatric hospital during a 2-year period. The majority of isolates (67.68%) belonged to the sequence type Group 1, European clonal complex II. All isolates harbored intrinsic OXA-51 and AmpC cephalosporinase. OXA-23 was detected in 16 isolates (57.14%), OXA-24 in 23 isolates (82.14%) and OXA-58 in 11 isolates (39.29%). Six of the isolates (21.43%) harbored all of the analyzed oxacillinases, except OXA-143 and OXA-235 that were not detected in this study. Production of oxacillinases was detected in different pulsotypes indicating the presence of horizontal gene transfer. NDM-1, VIM and IMP were not detected in analyzed clinical A. baumannii isolates. ISAba1 insertion sequence was present upstream of OXA-51 in one isolate, upstream of AmpC in 13 isolates and upstream of OXA-23 in 10 isolates. In silico analysis of carO sequences from analyzed A. baumannii isolates revealed the existence of two out of six highly polymorphic CarO variants. The phylogenetic analysis of CarO protein among Acinetobacter species revised the previous classification CarO variants into three groups based on strong bootstraps scores in the tree analysis. Group I comprises four variants (I-IV) while Groups II and III contain only one variant each. One half of the Serbian clinical isolates belong to Group I variant I, while the other half belongs to Group I variant III.