Innate immune surveillance in the blood is executed mostly by circulating monocytes, which recognise conserved bacterial molecules such as peptidoglycan and lipopolysaccharide. Toll-like receptors (TLR) play a central role in microbe-associated molecular pattern detection. Here, we compared the differences in TLR expression and cytokine production after stimulation of peripheral blood cells with heat-killed Gram-negative and Gram-positive human pathogens Neisseria meningitidis, Escherichia coli, Staphylococcus aureus and Streptococcus pneumoniae. We found that TLR2 expression is up-regulated on monocytes after stimulation with S. aureus, S. pneumoniae, E. coli and N. meningitidis. Moreover, TLR2 up-regulation was positively associated with increasing concentrations of Gram-positive bacteria, whereas higher concentrations of Gram-negative bacteria, especially E. coli, caused a milder TLR2 expression increase compared with low doses. Cytokines were produced in similar dose-dependent profiles regardless of the stimulatory pathogen; however, Gram-negative pathogens induced higher cytokine levels than Gram-positive ones at same concentrations. These results indicate that Gram-positive and Gram-negative bacteria differ in their dose-dependent patterns of induction of TLR2 and TLR4, but not in cytokine expression.
- MeSH
- Transcriptional Activation MeSH
- Cytokines biosynthesis MeSH
- Gram-Negative Bacteria genetics immunology MeSH
- Gram-Positive Bacteria genetics immunology MeSH
- Humans MeSH
- Monocytes immunology MeSH
- Immunity, Innate MeSH
- Gene Expression Regulation MeSH
- Signal Transduction immunology MeSH
- Toll-Like Receptor 2 biosynthesis genetics immunology MeSH
- Toll-Like Receptor 4 biosynthesis genetics immunology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Among the non-traditional antibacterial agents in development, only a few targets critical Gram-negative bacteria such as carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii or cephalosporin-resistant Enterobacteriaceae. Endolysins and their genetically modified versions meet the World Health Organization criteria for innovation, have a novel mode of antibacterial action, no known bacterial cross-resistance, and are being intensively studied for application against Gram-negative pathogens. METHODS: The study presents a multidisciplinary approach, including genetic engineering of LysECD7-SMAP and production of recombinant endolysin, its analysis by crystal structure solution following molecular dynamics simulations and evaluation of antibacterial properties. Two types of antimicrobial dosage forms were formulated, resulting in lyophilized powder for injection and hydroxyethylcellulose gel for topical administration. Their efficacy was estimated in the treatment of sepsis, and pneumonia models in BALB/c mice, diabetes-associated wound infection in the leptin receptor-deficient db/db mice and infected burn wounds in rats. RESULTS: In this work, we investigate the application strategies of the engineered endolysin LysECD7-SMAP and its dosage forms evaluated in preclinical studies. The catalytic domain of the enzyme shares the conserved structure of endopeptidases containing a putative antimicrobial peptide at the C-terminus of polypeptide chain. The activity of endolysins has been demonstrated against a range of pathogens, such as Klebsiella pneumoniae, A. baumannii, P. aeruginosa, Staphylococcus haemolyticus, Achromobacter spp, Burkholderia cepacia complex and Haemophylus influenzae, including those with multidrug resistance. The efficacy of candidate dosage forms has been confirmed in in vivo studies. Some aspects of the interaction of LysECD7-SMAP with cell wall molecular targets are also discussed. CONCLUSIONS: Our studies demonstrate the potential of LysECD7-SMAP therapeutics for the systemic or topical treatment of infectious diseases caused by susceptible Gram-negative bacterial species and are critical to proceed LysECD7-SMAP-based antimicrobials trials to advanced stages.
- MeSH
- Anti-Bacterial Agents pharmacology administration & dosage MeSH
- Endopeptidases * pharmacology administration & dosage MeSH
- Gram-Negative Bacterial Infections * drug therapy MeSH
- Gram-Negative Bacteria * drug effects MeSH
- Rats MeSH
- Mice, Inbred BALB C * MeSH
- Mice MeSH
- Protein Engineering methods MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Cíl práce: Zjištění výskytu gramnegativních patogenů izolovaných od pacientů hospitalizovaných na jlP velkých nemocnic v CR a jejich rezistence k hlavním skupinám antibiotik. Zjištění výskytu producentů širokospektrých β-laktamáz (ESBL) v nemocnicích CR u kmenů Klebsiella spp. E. coli. Materiál a metody: U 100 po sobě následujících kmenů izolovaných z klinických vzorků odebraných nemocným z jednotek intenzivní péče (JIP) byla testována citlivost k definované sestavě antibiotik metodou E-testu. Kmeny byly identifikovány standardními postupy. Data byla zaznamenavána do standardního protokolu a po použiti vylučovacích kritérií vyhodnocena. K hodnocení rczistence/citlivosti byly použity hraniční koncentrace doporučené Národní referenční laboratoří pro antibiotika. Byl stanoven celkový výskyt rezistence v procentech a lokální výskyt rezistentních kmenů v jednoUivých nemocnicích. Současně byl zjištěn celkový i lokální výskyt kmenů produkujících ESBL. Výsledky: Celkem bylo hodnoceno 1 576 izolátů gramnegativních tyčinek izolovaných ze vzorků odebraných 1 051 pacientům v období od 23. 1. 1997 do 10. 7. 1998. Byly zjištěny následující frekvence nejdůležitějších patogenů: Enterobarleriacae (67 %), Pseudomonas aeruginosa (21 %), Adndobader spp. (6 %), Stenolrophomonas mnllophilia (2 %), Burkholderia cepacia (0,5 %). 34 % kmenů bylo izolováno z dolních cest dýchacích, 21 % z moči, 13 % z krve, 8 % z horních cest dýchacích a 24 % ze vzorků jiného původu. Celkový výskyt rezistence hlavních gramnegativních patogenů k lékům volby se pohyboval až v desítkách procent. Celková rezistence všech kmenů, resp. kmenů izolovaných z krve byla tato: imipenem 10 % (resp. 5 %), cefuroxim 77 % (74 %), cefotaxim 48 % (45 %), ceftriaxon 50 % (46 %), ceftazidim 36 % (29 %), gentamicin 39 % (31 %), amikacin 20 % (12 %), ciprofloxacin 31 % (22 %), amoxicilin klavulanát 71 % (69 %), piperacilin tazobaktam 30 % (20 %), piperacilin 53 % (45 %), kotrimoxazol 35 % (31 %). Byly zjištěny velké lokální rozdíly mezi jednotlivými pracovišti. Závěr: Byla zjištěna překvapivě vysoká rezistence hlavních gramnegativních patogenů na jlP v nemocnicích České republiky. Na většině pracovišť byl zaznamenán výskyt kmenů produkujících ESBL a častá byla rezistence Pseudomonas aeruginosa k lékům volby. Systematické sledování rezistence k antibiotikům na JIP je nezbytným předpokladem účinné antimikrobní terapie infekčních komplikací kriticky nemocných.
Objectives: To ascertain the incidence of gram-negative pathogens isolated from patients in intensive care units (ICU) of large hospitals in the Oech Republic, and assess their resistance to the main antibiofic groups. To ascertain the incidence of broad-spectrum B-lactamase producers (ESBL) among strains of Klebsiella spp. and E. coli in Czech hospitals. Materials and methods: E-test was employed for testing of sensitivity to a defined series of anfibiofics by a total of 100 strains isolated from clinical material from ICU patients. Strains were idenfified by standard procedures. Standard protocol was used for recording data which were evaluated after use of eliminafion criteria. Evaluafion of resistance/susceptibility was based on concentration limits recommended by the Nafional Reference Laboratory for Anfibiofics. Local incidence of resistant strains in individual hospitals was determined as well as overall resistance as a percentage. Further, the overall and local incidence of ESBL-producing strains was recorded. Results: A total of 1 576 isolates of gram-negative rods from 1 051 patients were evaluated over a period from 23. 1. 1997 to 10. 7. 1998. The following frequency of the most significant pathogens was recorded: Enterobacteriacae (67 %), Pseudomonas aeruginosa (21 %), Acinetobacter spp. (6 %), Stenotrophyhomonas maltophilia (2 %), Burkholderia cepacia (0.5 %). 34 % of strains were isolated from the lower respiratory tract, 21 % from urine, 13 % from blood, 8 % from the upper respiratory tract and 24 % from other samples. The percentage of incidence of overall resistance by the major gram-negative pathogens to drugs of choice was in double figures. Overall resistance of all strains and strains isolated from blood was as follows: imipenem 10 % (5 %), cefuroxim 77 % (74 %), cefotaxime 48 % (45 %), ceftriaxone 50 % (46 %), ceftazidim 36 % (29 %), gentamicin 39 % (31 %), amikacin 20 % (12 %), ciprofioxacin 31 % (22 %), amoxicilin clavulanate 71 % (69 %), piperacilin tazobactam 30 % (20 %), piperacihn 53 % (45 %) and co-trimoxazole 35 % (31 %). Creat local differences were apparent amongst the different sites involved. Conclusion: A surprisingly high resistance of the major gram-negative pathogens was detected in ICU from hospitals throughout the Czech Republic. Most sites had an incidence of ESBL-producing strains, and resistance of Pseudomonas aeruginosa to drugs of choice was frequent. Systematic monitoring of anfibiofic resistance in ICU is essential for achieving effective anfimicrobial therapy of infecfions complicafions in critically ill patients.
- MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Drug Resistance, Microbial MeSH
- Gram-Negative Bacteria isolation & purification pathogenicity drug effects MeSH
- Cross Infection epidemiology drug therapy microbiology MeSH
- Intensive Care Units MeSH
- Humans MeSH
- Check Tag
- Humans MeSH
- Publication type
- Multicenter Study MeSH
- Geographicals
- Czech Republic MeSH
Innate immune surveillance in the blood is executed mostly by circulating monocytes, which recognize conserved bacterial molecules such as peptidoglycan and lipopolysaccharide. Toll-like receptors (TLR) play a central role in microbe-associated molecular pattern detection. The aim of this study was to compare the differences in TLR expression and cytokine production after stimulation of peripheral blood cells with heat-killed gram-negative and gram-positive human pathogens: Neisseria meningitidis, Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. We found that TLR2 expression is up-regulated on monocytes after stimulation with S. aureus, S. pneumoniae, E. coli, and N. meningitidis. Moreover, TLR2 up-regulation was positively associated with increasing concentrations of gram-positive bacteria, whereas higher concentrations of gram-negative bacteria, especially E. coli, caused a milder TLR2 expression increase when compared to low doses. Cytokines were produced in similar dose-dependent profiles regardless of the stimulatory pathogen; however, gram-negative pathogens induced higher cytokine levels when compared to gram-positive bacteria at the same density. These results indicate that gram-positive and gram-negative bacteria differ in their dose-dependent patterns of induction of TLR2 and TLR4, but not cytokine expression.
- MeSH
- Cytokines secretion MeSH
- Gram-Negative Bacteria immunology MeSH
- Gram-Positive Bacteria immunology MeSH
- Cells, Cultured MeSH
- Leukocytes, Mononuclear immunology MeSH
- Humans MeSH
- Toll-Like Receptor 2 biosynthesis MeSH
- Toll-Like Receptor 4 biosynthesis MeSH
- Hot Temperature MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Against a background of rapid increase of β-lactamase-producing or multi-resistant pathogenic bacteria and the resulting lack of effective antibiotic treatment, some older antibiotics have been tested for new therapeutic uses. One of these is fosfomycin, to which according to studies these resistant bacteria are very sensitive. Our study was designed because there is no data on the fosfomycin susceptibility rate in the Czech Republic. METHOD: In this study from January 2013 to June 2014 3295 unique isolates of Gram-negative bacteria which had caused urinary tract infections were examined. The antibiotic susceptibility was measured by disk diffusion test. Both EUCAST and CLSI guidelines criteria (for fosfomycin only) were used for the antibiotic susceptibility evaluation. RESULTS: The most frequently tested bacterial isolates were Escherichia coli (51.3%, n = 1703), Klebsiella pneumoniae (19.4%, n = 643) and Proteus spp. (11.8%, n = 392). Among all isolates 29.0% (n = 963) were resistant to fluoroquinolones, 11.3% (n = 374) produced extended spectrum β-lactamase and 4.2% (n = 141) produced AmpC β-lactamase. The overall in vitro susceptibility was significantly higher for fosfomycin compared to the other tested per-oral antibiotics (nitrofurantoin, ampicillin, co-trimoxazole, ciprofloxacin and cefuroxime) against all tested Gram-negative rod isolates (excluding Morganella morgani and Acinetobacter spp. isolates). Fosfomycin also remained highly active against those isolates with extended spectrum β-lactamase (ESBL) production (95.8% in Escherichia coli isolates and 85.3% in Klebsiella pneumoniae isolates), unlike other tested per-oral antibiotics, which showed significant (p < 0.0001) susceptibility decrease. CONCLUSION: We have confirmed in the Czech Republic the very high susceptibility to fosfomycin trometamol of urinary tract infection pathogens, particularly Gram-negative rods including those producing β-lactamase.
- MeSH
- Anti-Bacterial Agents pharmacology MeSH
- beta-Lactamases MeSH
- Fosfomycin pharmacology MeSH
- Gram-Negative Bacteria drug effects isolation & purification MeSH
- Urinary Tract Infections microbiology MeSH
- Humans MeSH
- Microbial Sensitivity Tests MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
INTRODUCTION: Red blood cells (RBCs), also known as erythrocytes, are underestimated in their role in the immune system. In mammals, erythrocytes undergo maturation that involves the loss of nuclei, resulting in limited transcription and protein synthesis capabilities. However, the nucleated nature of non-mammalian RBCs is challenging this conventional understanding of RBCs. Notably, in bony fishes, research indicates that RBCs are not only susceptible to pathogen attacks but express immune receptors and effector molecules. However, given the abundance of RBCs and their interaction with every physiological system, we postulate that they act in surveillance as sentinels, rapid responders, and messengers. METHODS: We performed a series of in vitro experiments with Cyprinus carpio RBCs exposed to Aeromonas hydrophila, as well as in vivo laboratory infections using different concentrations of bacteria. RESULTS: qPCR revealed that RBCs express genes of several inflammatory cytokines. Using cyprinid-specific antibodies, we confirmed that RBCs secreted tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). In contrast to these indirect immune mechanisms, we observed that RBCs produce reactive oxygen species and, through transmission electron and confocal microscopy, that RBCs can engulf particles. Finally, RBCs expressed and upregulated several putative toll-like receptors, including tlr4 and tlr9, in response to A. hydrophila infection in vivo. DISCUSSION: Overall, the RBC repertoire of pattern recognition receptors, their secretion of effector molecules, and their swift response make them immune sentinels capable of rapidly detecting and signaling the presence of foreign pathogens. By studying the interaction between a bacterium and erythrocytes, we provide novel insights into how the latter may contribute to overall innate and adaptive immune responses of teleost fishes.
- MeSH
- Aeromonas hydrophila * immunology MeSH
- Cytokines * metabolism immunology MeSH
- Erythrocytes * immunology metabolism MeSH
- Phagocytosis immunology MeSH
- Gram-Negative Bacterial Infections * immunology MeSH
- Carps * immunology microbiology MeSH
- Fish Diseases * immunology microbiology MeSH
- Pathogen-Associated Molecular Pattern Molecules immunology MeSH
- Immunity, Innate MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Gram negativní bakterie jsou nejčastější patogeny způsobující infekce močových cest Celosvětově dochází k nárůstu incidence rezistentních bakterií. Prevence a léčba infekcí způsobených rezistentními bakteriemi se stává závažným medicínským, sociálním a finančním problémem Jedním z největších problémů zůstávají nozokomiální infekce a infekce spojené s invazivními vstupy. Znalost mechanismu rezistence a lokální epidemiologické situace napomáhá ke správné volbě empirické antibiotické terapie v situaci, kdy nejsou k dispozici výsledky kultivačního vyšetření.
Gram-negative bacteria are the most common pathogens causing urinary tract infections. The incidence of bacterial resistance is increasing worldwide. Prevention and treatment of infections caused by resistant bacteria are becoming a vast medical, social and financial problem. The biggest problems are health care-associated infections and infections related to invasive lines. Knowledge of resistance mechanisms and local epidemiological situation help with effective initiation of empirical antibiotic therapy.
Outer membrane vesicles secreted by gram-negative bacteria play an important role in bacterial physiology as well as in virulence and host-pathogen interaction. Isolated vesicles of some bacteria have also been studied for their immunomodulatory potential in the vaccine development. However, the production of vesicles in sufficient amount, purity and reproducibility remains a critical challenge for subsequent analyses in most bacteria. In the present review methods of production, isolation, purification and quantification of outer membrane vesicles are summarized and discussed.
- MeSH
- Cell Wall * immunology metabolism MeSH
- Cell Fractionation methods MeSH
- Stress, Physiological MeSH
- Genetic Engineering MeSH
- Gram-Negative Bacteria * genetics immunology metabolism MeSH
- Secretory Vesicles * immunology metabolism MeSH
- Subcellular Fractions MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Laribacter hongkongensis is relatively a new name in the list of bacterial pathogens for gastroenteritis and travelers' diarrhea. Addition of another name increases burden on the enteric infections as a whole. L. hongkongensis belongs to Neisseriaceae family of β subclass Proteobacteria. L. hongkongensis was initially isolated in Hong Kong from blood and empyema of an alcoholic cirrhotic patient in 2001, followed by reports from Korea and China, representing a total of 38 articles in PubMed until April 2013. As of now, there is no report from Indian subcontinent where infectious diarrhea is very much prevalent and a major burden. This review provides information about the microbiological characteristics, consideration of an emerging pathogen, relative pathogenicity, genome and proteome content, resistance toward multiple antibiotics, adaptability to different stress, and other features since its time of discovery. Investigation for this bacterium may avoid misidentification as other microbial flora. Further studies like the geographical distribution, type of infection, disease burden, pathogenicity, or genomic exploration of this bacterium will be useful in characterizing them properly. This bacterium may possibly be the emerging threat to public health.
- MeSH
- Gastroenteritis microbiology MeSH
- Genome, Bacterial MeSH
- Gram-Negative Bacterial Infections drug therapy genetics microbiology MeSH
- Humans MeSH
- Drug Resistance, Multiple, Bacterial MeSH
- Neisseriaceae genetics isolation & purification pathogenicity MeSH
- Food Microbiology * MeSH
- Proteome MeSH
- Public Health MeSH
- Virulence MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Geographicals
- Hong Kong MeSH
Bacterial resistance surveillance is one of the main outputs of microbiological laboratories and its results are important part of antimicrobial stewardship (AMS). In this study, the susceptibility of specific bacteria to selected antimicrobial agents was tested. The susceptibility of 90 unique isolates of pathogens of critical priority obtained from clinically valid samples of ICU patients in 2017-2021 was tested. 50% of these fulfilled difficult-to-treat resistance (DTR) criteria and 50% were susceptible to all antibiotics included in the definition. 10 Enterobacterales strains met DTR criteria, and 2 (20%) were resistant to colistin (COL), 2 (20%) to cefiderocol (FCR), 7 (70%) to imipenem/cilastatin/relebactam (I/R), 3 (30%) to ceftazidime/avibactam (CAT) and 5 (50%) to fosfomycin (FOS). For Enterobacterales we also tested aztreonam/avibactam (AZA) for which there are no breakpoints yet. The highest MIC of AZA observed was 1 mg/l, MIC range in the susceptible cohort was 0.032-0.064 mg/l and in the DTR cohort (incl. class B beta-lactamase producers) it was 0.064-1 mg/l. Two (13.3%) isolates of Pseudomonas aeruginosa (15 DTR strains) were resistant to COL, 1 (6.7%) to FCR, 13 (86.7%) to I/R, 5 (33.3%) to CAT, and 5 (33.3%) to ceftolozane/tazobactam. All isolates of Acinetobacter baumannii with DTR were susceptible to COL and FCR, and at the same time resistant to I/R and ampicillin/sulbactam. New antimicrobial agents are not 100% effective against DTR. Therefore, it is necessary to perform susceptibility testing of these antibiotics, use the data for surveillance (including local surveillance) and conform to AMS standards.
- MeSH
- Anti-Bacterial Agents * pharmacology therapeutic use MeSH
- Azabicyclo Compounds * MeSH
- Aztreonam MeSH
- Cephalosporins * MeSH
- Cefiderocol MeSH
- Gram-Negative Bacteria MeSH
- Colistin pharmacology MeSH
- Humans MeSH
- Microbial Sensitivity Tests MeSH
- Pseudomonas aeruginosa MeSH
- Retrospective Studies MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
