Resistant Clinical Strains Research Articles

Identification and phylogenetic analysis of carbapenemase genes from clinical strains of Klebsiella pneumoniae.

Klebsiella pneumoniae is an encapsulated Gram-negative bacterium that is responsible for numerous infections in healthcare facilities worldwide and is frequently isolated. The World Health Organization has listed K. pneumoniaeas as a critical antibiotic resistant bacterial pathogen for which new antibiotics are urgently needed. This study aimed to use molecular tools to identify and examine antibiotic resistance in clinical strains of K. pneumoniae. A total of 15 unduplicated K. pneumoniae strains isolated from patient samples with multidrug-resistant (MDR) profiles were subjected to polymerase chain reaction (PCR) to amplify the most common carbapenem resistance genes. (GTG)5 PCR and phylogenetic analysis were performed to identify the genetic relationship between the strains. All strains yielded a (GTG)5-PCR profile, and this allowed us to group these strains into 8 groups according to the size and number of characteristic bands. Phylogenetic analysis was done using the free software UPGMA and a single bacterial clone with a correlation coefficient of over 97% was identified. New Delhi metallo-beta-lactamase NDM-like (blaNDM) carbapenem resistance genes were detected in three strains of K. pneumoniae, which represented a resistance rate of 20%. However, carbapenemases type A [Klebsiella pneumoniae carbapenemase (KPC) and imipenem-hydrolysing beta-lactamase (IMI), type D [oxacillinase-48 (OXA-48)], and other metallo-β-lactamase [Verona integron-encoded metallo-beta-lactamase (VIM), and enzyme active on imipenem (IMP)] were not detected. We identified and grouped the blaNDM resistance genes of Klebsiella pneumonia strains.

Comparative proteomic analysis of metronidazole-sensitive and resistant Trichomonas vaginalis suggests a novel mode of metronidazole action and resistance

The microaerophilic parasite Trichomonas vaginalis occurs worldwide and causes inflammation of the urogenital tract, especially in women. With 156 million infections annually, trichomoniasis is the most prevalent non-viral sexually transmitted disease. Trichomoniasis is treated with 5-nitroimidazoles, especially metronidazole, which are prodrugs that have to be reduced at their nitro group to be activated. Resistance rates to metronidazole have remained comparably low, but they can be higher in certain areas leading to an increase of refractory cases. Metronidazole resistance in T. vaginalis can develop in vivo in clinical isolates, or it can be induced in the laboratory. Both types of resistance share certain characteristics but differ with regard to the dependence of ambient oxygen to become manifest. Although several candidate factors for metronidazole resistance have been described in the past, e.g. pyruvate:ferredoxin oxidoreductase and ferredoxin or thioredoxin reductase, open questions regarding their role in resistance have remained.In order to address these questions, we performed a proteomic study with metronidazole-sensitive and –resistant laboratory strains, as well as with clinical strains, in order to identify factors causative for resistance. The list of proteins consistently associated with resistance was surprisingly short. Resistant laboratory and clinical strains only shared the downregulation of flavin reductase 1 (FR1), an enzyme previously identified to be involved in resistance. Originally, FR1 was believed to be an oxygen scavenging enzyme, but here we identified it as a ferric iron reductase which produces ferrous iron. Based on this finding and on further experimental evidence as presented herein, we propose a novel mechanism of metronidazole activation which is based on ferrous iron binding to proteins, thereby rendering them susceptible to complex formation with metronidazole. Upon resolution of iron-protein-metronidazole complexes, metronidazole radicals are formed which quickly react with thiols or proteins in the direct vicinity, leading to breaks in the peptide backbone.

Antimicrobial blue light inactivation of Pseudomonas aeruginosa: Unraveling the multifaceted impact of wavelength, growth stage, and medium composition

Pseudomonas aeruginosa, a notable pathogen frequently associated with hospital-acquired infections, displays diverse intrinsic and acquired antibiotic resistance mechanisms, posing a significant challenge in infection management. Antimicrobial blue light (aBL) has been demonstrated as a potential alternative for treating P. aeruginosa infections. In this study, we investigated the impact of blue light wavelength, bacterial growth stage, and growth medium composition on the efficacy of aBL. First, we compared the efficacy of light wavelengths 405 nm, 415 nm, and 470 nm in killing three multidrug resistant clinical strains of P. aeruginosa. The findings indicated considerably higher antibacterial efficacy for 405 nm and 415 nm wavelength compared to 470 nm. We then evaluated the impact of the bacterial growth stage on the efficacy of 405 nm light in killing P. aeruginosa using a reference strain PAO1 in exponential, transitional, or stationary phase. We found that bacteria in the exponential phase were the most susceptible to aBL, followed by the transitional phase, while those in the stationary phase exhibited the highest tolerance. Additionally, we quantified the production of reactive oxygen species (ROS) in bacteria using the 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe and flow cytometry, and observed a positive correlation between aBL efficacy and ROS production. Finally, we determined the influence of growth medium on aBL efficacy. PAO1 was cultivated in brain heart infusion (BHI), Luria-Bertani (LB) broth or Casamino acids (CAA) medium, before being irradiated with aBL at 405 nm. The CAA-grown bacteria exhibited the highest sensitivity to aBL, followed by those grown in LB broth, and the BHI-grown bacteria demonstrated the lowest sensitivity. By incorporating FeCl3, MnCl2, ZnCl2, or the iron chelator 2,2′-bipyridine (BIP) into specific media, we discovered that aBL efficacy was affected by the iron levels in culture media.

Synergistic Effect of Essential Oils and Antifungal Agents in Fighting Resistant Clinical Isolates of Candida auris.

Recently, a large number of nosocomial infections have been caused by an emerging pathogen that is rising as a worldwide issue in human health: Candida auris. This yeast is considered resistant to antifungals of the first-line therapies, and consequently it is related to morbidity and mortality. Therefore, the aim of this research was to determine the in vitro anti-C. auris activity against twenty-three resistant clinical strains of different essential oils (EOs), pure or in combination with traditional antifungal agents, mainly caspofungin, fluconazole, micafungin and 5-flucytosine. Broth dilution assay was performed to evaluate the fungistatic and fungicidal effectiveness of fifteen EOs towards all the C. auris isolates. The data demonstrated that EOs were able to prevent C. auris growth, with MIC values ranging from 0.03 to 1% for the efficacious EOs (thyme, cinnamon, geranium, clove bud, lemongrass and mentha of Pancalieri), whereas the MICs were >1% for the ineffective ones. Thereafter, the six most effective EOs were used to perform the checkerboard experiments by assaying simultaneously the activity of EOs and traditional antifungals towards two selected strains. The most promising synergic combinations towards C. auris, depending on the isolate, were those with micafungin and geranium, thyme, cinnamon, lemongrass or clove bud EOs, with fluconazole and mentha of Pancalieri EO, and with 5-flucytosine and mentha of Pancalieri EO. These EOs and their combinations with antifungal drugs may provide a useful therapeutic alternative that could reduce the dose of the individual components, limiting the overall side effects. These associations might be a prospective option for the future treatment of infections, thus helping to overcome the challenging issue of resistance in C. auris.

Rapid evolution of colistin resistance in a bioreactor model of infection of Klebsiella pneumoniae

Colistin remains an important antibiotic for the therapeutic management of drug-resistant Klebsiella pneumoniae. Despite the numerous reports of colistin resistance in clinical strains, it remains unclear exactly when and how different mutational events arise resulting in reduced colistin susceptibility. Using a bioreactor model of infection, we modelled the emergence of colistin resistance in a susceptible isolate of K. pneumoniae. Genotypic, phenotypic and mathematical analyses of the antibiotic-challenged and un-challenged population indicates that after an initial decline, the population recovers within 24 h due to a small number of “founder cells” which have single point mutations mainly in the regulatory genes encoding crrB and pmrB that when mutated results in up to 100-fold reduction in colistin susceptibility. Our work underlines the rapid development of colistin resistance during treatment or exposure of susceptible K. pneumoniae infections having implications for the use of cationic antimicrobial peptides as a monotherapy.

Comparison of testing methods assessing the in vitro efficacy of the combination of aztreonam with avibactam on multidrug-resistant Gram-negative bacilli

BackgroundAztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches.Methods96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher).ResultsAccording to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%).ConclusionsThe ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.

2‐Thiophenyl–Isoquinoline Ir(III) Complex: A Promising Tool in Antipseudomonal Photodynamic Therapy under Red Irradiation

AbstractInnovative therapeutic strategies are more than ever needed to counter the rise of antibiotic–resistant bacterial pathogens worldwide. The use of light, and especially photodynamic therapy (PDT) appears as a promising alternative or complement to antibiotic treatments, fostered by the development of new photosensitizers. In this study, eight Ir(III) complexes were synthesized and evaluated for their photoactivation properties and capacity to generate radical species under blue (452 nm), green (525 nm), and red (631 nm) LED light, respectively. Their antibacterial properties were assessed on Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, and Staphylococcus aureus with most of these complexes exhibiting potentially useful activities upon light irradiation, at concentrations below 10 mg/L. A complex of Ir(III) cyclometallated to thiophenyl–isoquinoline (tiq) and bearing 2,2’‐bipyridine (bipy) as ancillary ligand was further investigated. This latter showed a concentration‐ and light intensity–dependent bactericidal activity on P. aeruginosa when irradiated under blue to red lights, proving that such complexes would be suitable candidates for PDT. Importantly, this lead complex remained active against antibiotic resistant clinical strains and was unaffected by active efflux systems. These data open interesting perspectives for the development of new treatments to tackle antibiotic resistant Gram–negative bacteria.

Limonene synergistically augments fluconazole susceptibility in clinical Candida isolates from cleft lip and palate patients.

Cleft lip and palate (CLP) patients are prone to Candida infections (oral thrush) mainly due to poor oral hygiene, repetitive surgeries, and orthodontic procedures. This study was undertaken to evaluate the antifungal efficacy of limonene against clinical Candida isolates from CLP patients. The antifungal efficacy of limonene was studied alone and in combination with fluconazole (FLC) against six standards, twenty nine FLC sensitive, and three FLC resistant clinical strains using broth dilution, checkerboard microdilution, agar disk diffusion, growth curves, and spot assays. This nontoxic monoterpene gave low minimum inhibitory concentration (MIC) values of 300-375 µg/mL and 500-520 µg/mL for FLC susceptible and FLC resistant strains, respectively. It showed synergistic interaction with FLC in all clinical and standard Candida strains (fractional inhibitory concentration (FIC) index ≤0.5). Significant chemosensitization of FLC was observed even against resistant clinical isolates. Complete suppression of fungal growth was observed when using combinations. Negligible toxicity, easy availability, and potent antifungal properties suggest that limonene and FLC combinations in appropriate doses can make excellent antifungal mouthwashes during CLP treatment pre and post surgery. Impending in vivo studies are needed to validate the present data.

Structural and Functional Alterations Caused by Aureobasidin A in Clinical Resistant Strains of Candida spp.

Candida species are one of the most concerning causative agents of fungal infections in humans. The treatment of invasive Candida infections is based on the use of fluconazole, but the emergence of resistant isolates has been an increasing concern which has led to the study of alternative drugs with antifungal activity. Sphingolipids have been considered a promising target due to their roles in fungal growth and virulence. Inhibitors of the sphingolipid biosynthetic pathway have been described to display antifungal properties, such as myriocin and aureobasidin A, which are active against resistant Candida isolates. In the present study, aureobasidin A did not display antibiofilm activity nor synergism with amphotericin B, but its combination with fluconazole was effective against Candida biofilms and protected the host in an in vivo infection model. Alterations in treated cells revealed increased oxidative stress, reduced mitochondrial membrane potential and chitin content, as well as altered morphology, enhanced DNA leakage and a greater susceptibility to sodium dodecyl sulphate (SDS). In addition, it seems to inhibit the efflux pump CaCdr2p. All these data contribute to elucidating the role of aureobasidin A on fungal cells, especially evidencing its promising use in clinical resistant isolates of Candida species.

Development of ionic liquid-capped carbon dots derived from Tecoma stans (L.) Juss. ex Kunth: Combatting bacterial pathogens in diabetic foot ulcer pus swabs, targeting both standard and multi-drug resistant strains

Bacterial infection is rapidly growing and is a severe danger to global welfare worldwide. Consequently, there is a growing demand for novel antibacterial medications. As an outcome, the evaluation of antibacterial assets of carbon dots (CDs) including a variety of ionic liquids (ILs) were employed to reside advance notice of the structure tunability of ILs along with abundant surface functional group of designed amine-modified CDs, have recently sparked a lot of interest. In this study for the first time, highly efficient ionic-liquid decorated CDs were synthesized to assess the impact of capped-ionic liquid on the degree of antibacterial activity. Fluorescent amine-modified CDs (CDs-NH2) were obtained from the Tecoma stans (yellow elder flower) extract by a single-step carbonization method in presence of ethylenediamine, and then capped with ionic liquids; 3-(carboxymethyl)-1-methyl-1-H-imidazol-3-ium bromide (ILIM) and 3-(carboxymethyl)-1-methyl-1-H-benzo[d]imidazole-3-ium bromide (ILBIM) by a simple carbodiimide chemistry. In-vitro antibacterial activity of CDs-NH2, ILIM@CDs and ILBIM@CDs were evaluated against common pathogens, Escherichia coli (E. coli) as Gram(-ive) bacteria and Staphylococcus aureus (S. aureus) as Gram(+ive) bacteria as well as against clinical multi-drug resistant strain obtained from pus swab of diabetic foot ulcer, by microdilution method as well as well-diffusion method. The results showed that functional ILs-capped CDs have enhanced antibacterial activity against standard (E. coli, S. aureus) and clinical resistant bacterial strains, with minimal inhibitory concentrations (MICs) values range from 0 to 64 μg/mL for standard E. coli/ S. aureus and 250 μg/mL for drug resistant clinical strains, respectively. Structure activity analysis and molecular docking studies revealed that the antibacterial activity of ILBIM@CDs/ILIM@CDs is particularly better compared to CDs-NH2 and ionic-liquids.

Evaluating the incidence of ampC-lactamase genes, biofilm formation, and antibiotic resistance among hypervirulent and classical Klebsiella pneumoniae strains.

Both immunocompetent and healthy individuals can become life-threateningly ill when exposed to the hypervirulent (hvKp) strains of Klebsiella pneumoniae (Kp). The main objectives of this study were to evaluate the presence of ampC-lactamase genes, biofilm formation, and antibiotic resistance in clinical strains of hvKp and cKp (classical K. pneumoniae). Kp strains were collected from patients referred to Shahidzadeh Hospital in Behbahan city, Khuzestan Province, Iran. Several techniques were used to identify hvKp. The hypermucoviscosity phenotype was determined using the string test. Isolates that developed dark colonies on tellurite agar were assumed to be hvKp strains. If any of the iucA, iutA, or peg-344 genes were detected, the isolates were classified as hvKp. Phenotypic and genotypic detection of AmpC β-lactamases of hvKp strains was performed by combined disk method and PCR, respectively. In addition, crystal violet staining was used to determine the biofilm formation of these isolates. For this study, 76 non-duplicative isolates of Kp were collected. Overall, 22 (28.94%) strains had positive string test results, and 31 (40.78%) isolates were grown in tellurite-containing medium. The genes iucA and iutA or peg-344 were found in 23.68% of all Kp strains and in 50% of tellurite-resistant isolates, respectively. The most effective antibiotics against hvKp isolates were tetracycline (85.52%) and chloramphenicol (63.15%). Using the cefoxitin disc diffusion method, we observed that 56.57% (43/76) of the strains were AmpC producer. A total of 30.26% (n=23/76) of the isolates tested positive for at least one ampC gene, including blaDHA (52.63%, n=40), blaCIT (40.78%, n=31), blaACC (19.76%, n=15), blaMOX (25%, n=19), and blaFOX (43.42%, n=33). Biofilm formation analysis revealed that most hvKp isolates were weak (n=6, 40%) and moderate (n=5, 33.33%) biofilm producers. Healthcare practitioners should consider the possibility of the existence and acquisition of hvKp everywhere. The exact mechanisms of bacterial acquisition are also unknown, and it is unclear whether the occurrence of infections is related to healthcare or not. Thus, there are still many questions about hvKp that need to be investigated.

Decoding the enigma: unveiling the molecular transmission of avian-associated tet(X4)-positive E. coli in Sichuan Province, China

Tigecycline is considered one of the "last resort antibiotics" for treating complex infections caused by multidrug-resistant (MDR) bacteria, especially for combating clinical resistant strains that produce carbapenemases. However, the tet(X4) gene, which carried by different plasmids can mediate high levels of bacterial resistance to tigecycline, was first reported in 2019. Here, we report the emergence of the plasmid-mediated tet(X4) in avian environment of Sichuan Province. A total of 21 tet(X4)-positive Escherichia coli (E. coli) strains were isolated and identified from avian samples in selected regions, with an isolation rate of 1.6% (21/1,286), and all of them were MDR strains. Multilocus Sequence Typing (MLST) method was used to classify the 21 tet(X4)-positive E. coli into the ST206, ST761, ST155, ST1638, ST542, and ST767 types, which also belong to the 3 phylogenetic subgroups A, B1, and C. Tet(X4) is located on mobile plasmids that can be efficiently and stably propagated. The results of fitness cost experiments showed that tet(X4)-positive plasmids may incur some fitness cost to host bacteria, but different tet(X4)-positive plasmids bring about differential fitness costs. Whole-genome sequencing further confirmed the tet(X4) gene can be located on IncX1-type plasmids and the core genetic structures are ISVsa3-rdmc-tet(X4) or rdmc-tet(X4)-ISVsa3, the former is a 7 copies tandem repeat structure. In this study, we isolated and identified tet(X4)-positive E. coli from the avian origin in Sichuan, analyzed the mobility of the tet(X4) by conjugational transfer and S1-PFGE, and evaluated the biological characteristics of the tet(X4)-positive plasmid using the results of conjugational frequency, plasmid stability, and fitness costs. Finally, combined with the third-generation whole-genome sequencing analysis, the molecular transmission characteristics of the tet(X4) were preliminarily clarified, providing a scientific basis for guiding veterinary clinical use in this area, as well as risk assessment and prevention of the transfer and spread of tigecycline resistant strains or genes.

Disrupting iron homeostasis can potentiate colistin activity and overcome colistin resistance mechanisms in Gram-Negative Bacteria

Acinetobacter baumannii is a Gram-negative priority pathogen that can readily overcome antibiotic treatment through a range of intrinsic and acquired resistance mechanisms. Treatment of carbapenem-resistant A. baumannii largely relies on the use of colistin in cases where other treatment options have been exhausted. However, the emergence of resistance against this last-line drug has significantly increased amongst clinical strains. In this study, we identify the phytochemical kaempferol as a potentiator of colistin activity. When administered singularly, kaempferol has no effect on growth but does impact biofilm formation. Nonetheless, co-administration of kaempferol with sub-inhibitory concentrations of colistin exposes bacteria to a metabolic Achilles heel, whereby kaempferol-induced dysregulation of iron homeostasis leads to bacterial killing. We demonstrate that this effect is due to the disruption of Fenton’s reaction, and therefore to a lethal build-up of toxic reactive oxygen species in the cell. Furthermore, we show that this vulnerability can be exploited to overcome both intrinsic and acquired colistin resistance in clinical strains of A. baumannii and E. coli in vitro and in the Galleria mellonella model of infection. Overall, our findings provide a proof-of-principle demonstration that targeting iron homeostasis is a promising strategy for enhancing the efficacy of colistin and overcoming colistin-resistant infections.

Prevalence and epidemiological investigation of mgrB-dependent colistin resistance in extensively drug resistant Klebsiella pneumoniae in Iran

Carbapenemases-producing K. pneumoniae are challenging antimicrobial therapy of hospitalised patients, which is further complicated by colistin resistance. The aim of this study was to investigate the molecular epidemiological insights into carbapenemases-producing and colistin-resistant clinical K. pneumoniaeA total of 162 colistin resistant clinical strains of K. pneumoniae were collected during 2017–2019. Antimicrobial susceptibility and the colistin minimum inhibitory concentration were determined. Using PCR assay, the prevalence of resistance-associated genes including blaKPC, blaIMP, blaVIM, blaOXA-48, blaNDM-1 and mcr-1 to -9 was examined. Additionally, a PCR assay was used to examine the mgrB gene in colistin-resistant bacteria. 94.4% of the tested strains were resistant to imipenem and 96.3% were resistant to meropenem. Colistin resistance (MIC > 4 µg/L) was observed in 161 isolates (99.4%) by Colistin Broth Disk Elution method. The KPC enzyme was the most common carbapenemase and was identified in 95 strains (58.6%), followed by the IMP, VIM and OXA-48 detected in 47 (29%), 23 (14.2%) and 12 (7.4%) isolates, respectively. However, no NDM-1 gene was detected. Additionally, none of the studied isolates harbored mcr variants, while mgrB gene was observed in 152 (92.6%) isolates. Colistin resistance of K. pneumoniae isolates may be associated with mgrB gene mutation. To stop the spread of resistant K. pneumoniae, surveillance must be improved, infection prevention protocols must be followed, and antibiotic stewardship must be practised.

Isolation and characterization of novel lytic bacteriophages that infect multi drug resistant clinical strains of Escherichia coli.

The pathogenic strains of Escherichia coli (E. coli) are frequent cause of urinary tract infections including catheter-associated, soft tissue infections and sepsis. The growing antibiotic resistance in E. coli is a major health concern. Bacteriophages are specific for their bacterial host, thus providing a novel and effective alternatives. This study focuses on isolation of bacteriophages from urban sewage treatment plants. Initially 50 different bacteriophages have been isolated against non-resistant reference E. coli strain and fifty multidrug resistant clinical isolates of extraintestinal infections. Out of which only thirty-one lytic phages which gave clear plaques were further analysed for different physico-chemical aspects such as thermal inactivation, pH, effect oforganic solvents and detergents. Two bacteriophages, ASEC2201 and ASEC2202, were selected for their ability to withstand temperature fluctuation from -20 to 62 °C and a pH range from 4 to 10. They also showed good survival (40-94%) in the presence of organic solvents like ethanol, acetone, DMSO and chloroform or ability to form plaques even after the treatment with detergents like SDS, CTAB and sarkosyl. Both efficiently killed reference strain and 40-44% of multidrug resistant clinical isolates of E. coli. Later ASEC2201 and ASEC2202 were subjected to morphological characterisation through transmission electron microscopy, which revealed them to be tailed phages. The genomic analysis confirmed them to be Escherichia phages which belonged to family Drexlerviridae of Caudovirales.

Effect of Indole-Containing Pyrazino[2,1-b]quinazoline-3,6-diones in the Virulence of Resistant Bacteria.

Drug resistance is rising to alarming levels, constituting one of the major threats to global health. The overexpression of efflux pumps and the formation of biofilms constitute two of the most common resistance mechanisms, favoring the virulence of bacteria. Therefore, the research and development of effective antimicrobial agents that can also counteract resistance mechanisms are extremely important. Pyrazino[2,1-b]quinazoline-3,6-diones, from marine and terrestrial organisms and simpler synthetic analogues, were recently disclosed by us as having relevant antimicrobial properties. In this study, using a multi-step approach, it was possible to synthesize new pyrazino[2,1-b]quinazoline-3,6-diones focusing on compounds with fluorine substituents since, to the best of our knowledge, the synthesis of fluorinated fumiquinazoline derivatives had not been attempted before. The new synthesized derivatives were screened for antibacterial activity and, along with previously synthetized pyrazino[2,1-b]quinazoline-3,6-diones, were characterized for their antibiofilm and efflux-pump-inhibiting effects against representative bacterial species and relevant resistant clinical strains. Several compounds showed relevant antibacterial activity against the tested Gram-positive bacterial species with MIC values in the range of 12.5-77 μM. Furthermore, some derivatives showed promising results as antibiofilm agents in a crystal violet assay. The results of the ethidium bromide accumulation assay suggested that some compounds could potentially inhibit bacterial efflux pumps.

Antibacterial Properties and Efficacy of LL-37 Fragment GF-17D3 and Scolopendin A2 Peptides Against Resistant Clinical Strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii In Vitro and In Vivo Model Studies.

Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumanniihave emerged as major clinical threats owing to the increasing prevalence of ventilator-associated pneumonia caused by multidrug-resistant or extensively drug-resistant strains. The present study aimed to assess the antibacterial effects and efficacy of LL-37 fragment GF-17D3 and synthetic Scolopendin A2 peptides against resistant clinical strains in vitro and in vivo models.P. aeruginosa,S. aureus, andA. baumanniiwere isolated from clinical infections. Their antibiotic resistance and minimum inhibitory concentration were assessed. LL-37 fragment GF-17D3 peptide was selected from available databases. Scolopendin A2 peptide's 6th amino acid (proline) was substituted with lysine and peptides and MICs were determined. The biofilm inhibitory activity was quantified at sub MIC concentrations. Synergetic effects of Scolopendin A2 and imipenem were assessed by checkerboard. After mice nasal infection withP. aeruginosa, peptides LD50 was determined. Isolates harbored complete resistance toward the majority of antibiotics and MIC values ranged between 1 and > 512µg/ml. The majority of isolates exhibited strong biofilm activity. Synthetic peptides showed lower MIC values than antibiotic agents and the lowest MIC values were obtained for synthetic peptides in combination with antibiotics. The Synergisms effect of Scolopendin A2 with imipenem was also determined. Scolopendin A2was found to have antibacterial efficacy againstP. aeruginosa, S. aureus, and A. baumanniiwith MIC 64µg/ml, 8µg/ml, and 16µg/ml, respectively, and LL37 showed antibacterial efficacy againstP. aeruginosa, S. aureus, and A. baumanniiwith MIC 128µg/ml, 32µg/ml, and 32µg/ml, respectively. Both AMPs decreased biofilms by ≥ 96% at 1 × MIC. The biofilm inhibitory activity was measured at sub MIC concentrations of the peptides and the results demonstrated that Scolopendin A2 exhibited anti-biofilm activity at 1/4 × MIC and 1/2 × MIC concentrations was 47.9 to 63.8%, although LL37 among 1/4 × MIC and 1/2 × MIC concentrations was 21.3 to 49.6% against three pathogens. The combination of Scolopendin A2 and antibiotics demonstrated synergistic activity-resistant strains with FIC values ≤ 0.5 for three pathogens, while LL37 and antibiotics showed synergistic activity FIC values ≤ 0.5 for onlyP. aeruginosa.Infection model Scolopendin A2 with Imipenem (2 × MIC) was efficacious in vivo, with a 100% survival rate following treatment at 2 × MIC after 120h. The mRNA expression of biofilm-related genes was decreased for both peptides. Synthesis Scolopendin A2 decreased the expression of biofilm formation genes compared to the control group. Synthetic Scolopendin A2 exhibits antimicrobial activity without causing toxicity on the human epithelial cell line. Based on our findings, it seems that synthetic Scolopendin A2 is an appropriate antimicrobial source. That could be a promising option in combination with antibiotics for a topical medication and in the prevention of acute and chronic infections caused by multidrug-resistant bacteria. Nevertheless, additional experiments are required to assess another potential of this novel AMP.

Untargeted proteomic differences between clinical strains of methicillin-sensitive and methicillin-resistant Staphylococcusaureus

Staphylococcus aureus is a common disease-causing bacterium that has developed resistances to a wide variety of antibiotics. This increasing antibiotic resistance has made management of these infections difficult. A better understanding of the general differences among clinical S. aureus strains beyond the well characterized resistance mechanisms may help in identifying new anti-microbial targets. This study aimed to identify and compare the general differences in protein profiles among clinical strains of S. aureus sensitive and resistant to methicillin. The proteomic profiles of five methicillin sensitive (MSSA) and five methicillin resistant (MRSA) S. aureus strains were analyzed by ultra-performance liquid chromatography-mass spectrometry. Protein identification was done using Progenesis QI for Proteomics and the UniProt S. aureus database. Proteins that play roles in virulence, metabolism, and protein synthesis were found to be present at different abundances between MSSA and MRSA (Data available via ProteomeXchange with identifier PXD021629). This study shows differences in protein profiles between antibiotic sensitive and antibiotic resistant clinical strains of S. aureus that may affect the resistance mechanism. Further research on these differences may identify new drug targets against methicillin resistant S. aureus strains.

Molecular characterization of antibiotic resistance, aminoglycoside and PmrA genes among foodborne and clinical Acinetobacter spp.

The assessment of antibiotic resistance and related genes of foodborne Acinetobacter spp. and the analysis of whether they are genetically related to clinical infection-agent strains are crucial in terms of sustainability of food safety. The study at hand investigated antibiotic resistance, aminoglycoside-modifying enzyme (AME), and colistin resistance (PmrA) genes, clonal relationships while evaluating a possible correlation between antibiotic resistance and related genes between 27 foodborne and 50 clinical Acinetobacter spp. in Turkey. Antimicrobial susceptibilities, (AME), PmrA genes, and clonal relatedness of the strains were performed by disc diffusion, PCR, and Pulsed Field gel Electrophoresis (PFGE) methods, respectively. The aph-AI, aph-6, anth (3’’)-I, aadA1, aadB and PmrA genes were found as (24, 48%), (11, 22%), (7, 14%), (1, 2%), (2, 4%), and (46, 92%) respectively in clinical strains. This rate was found as (14, 51.9%), (16, 59.3%), (19, 70.4%), (2, 7.4%), (0, 0%), and (27, 100%) respectively in foodborne isolates. A positive correlation existed between the number of aph-AI gene positivity and trimethoprim-sulfamethoxazole and gentamycin resistance; anth (3’’)-I gene positivity, and colistin resistance; PmrA gene positivity and piperacillin-tazobactam, ceftazidime, meropenem, amikacin, and imipenem resistance in clinical strains (P

Clinical Resistant Strains of Enterococci and Their Correlation to Reduced Susceptibility to Biocides: Phenotypic and Genotypic Analysis of Macrolides, Lincosamides, and Streptogramins.

Enterococci are troublesome nosocomial, opportunistic Gram-positive cocci bacteria showing enhanced resistance to many commonly used antibiotics. This study aims to investigate the prevalence and genetic basis of antibiotic resistance to macrolides, lincosamides, and streptogramins (MLS) in Enterococci, as well as the correlation between MLS resistance and biocide resistance. From 913 clinical isolates collected from King Khalid Hospital, Hail, Saudi Arabia, 131 isolates were identified as Enterococci spp. The susceptibility of the clinical enterococcal isolates to several MLS antibiotics was determined, and the resistance phenotype was detected by the triple disk method. The MLS-involved resistance genes were screened in the resistant isolates. The current results showed high resistance rates to MLS antibiotics, and the constitutive resistance to all MLS (cMLS) was the most prevalent phenotype, observed in 76.8% of resistant isolates. By screening the MLS resistance-encoding genes in the resistant isolates, the erythromycin ribosome methylase (erm) genes that are responsible for methylation of bacterial 23S rRNA were the most detected genes, in particular, ermB. The ereA esterase-encoding gene was the most detected MLS modifying-encoding genes, more than lnuA (adenylation) and mphC (phosphorylation). The minimum inhibitory concentrations (MICs) of commonly used biocides were detected in resistant isolates and correlated with the MICs of MLS antibiotics. The present findings showed a significant correlation between MLS resistance and reduced susceptibility to biocides. In compliance with the high incidence of the efflux-encoding genes, especially mefA and mefE genes in the tolerant isolates with higher MICs to both MLS antibiotics and biocides, the efflux of resistant isolates was quantified, and there was a significant increase in the efflux of resistant isolates with higher MICs as compared to those with lower MICs. This could explain the crucial role of efflux in developing cross-resistance to both MLS antibiotics and biocides.