Antibiotic Resistance In Clinical Isolates Research Articles

THE COMBINATORY EFFECTS OF ESSENTIAL OIL FROM Lippia macrophylla ON MULTIDRUG RESISTANT Acinetobacter baumannii CLINICAL ISOLATES.

To assess the antibacterial effectiveness of Lippia macrophylla essential oil (LMEO) against multidrug-resistant Acinetobacter baumannii isolates, both as a standalone treatment and in combination with conventional antibiotics. LMEO demonstrated a significant inhibitory effect on the growth of A. baumannii, with a minimum inhibitory concentration (MIC) below 500μg/mL. Notably, LMEO was capable of reversing the antibiotic resistance of clinical isolates or reducing their MIC values when used in combination with antibiotics, showing synergistic (FICI ≤ 0.5) or additive effects. The combination of LMEO and imipenem was particularly effective, displaying synergistic interactions for most isolates. Ultrastructural analyses supported these findings, revealing that the combination of LMEO + ceftazidime compromised the membrane integrity of the Acb35 isolate, leading to cytoplasmic leakage and increased formation of Outer Membrane Vesicles (OMVs). Taken together our results point for the use of LMEO alone or in combination as an antibacterial agent against A. baumannii. These findings offer promising avenues for utilizing LMEO as a novel antibacterial strategy against drug-resistant infections in healthcare settings, underscoring the potential of essential oils in enhancing antibiotic efficacy.

Dry and liquid formulations of IBT-V02, a novel multi-component toxoid vaccine, are effective against Staphylococcus aureus isolates from low-to-middle income countries.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) in the U.S. as well as more serious invasive diseases, including bacteremia, sepsis, endocarditis, surgical site infections, osteomyelitis, and pneumonia. These infections are exacerbated by the emergence of antibiotic-resistant clinical isolates such as methicillin-resistant S. aureus (MRSA), highlighting the need for alternatives to antibiotics to treat bacterial infections. We have previously developed a multi-component toxoid vaccine (IBT-V02) in a liquid formulation with efficacy against multiple strains of Staphylococcus aureus prevalent in the industrialized world. However, liquid vaccine formulations are not compatible with the paucity of cold chain storage infrastructure in many low-to-middle income countries (LMICs). Furthermore, whether our IBT-V02 vaccine formulations are protective against S. aureus isolates from LMICs is unknown. To overcome these limitations, we developed lyophilized and spray freeze-dried formulations of IBT-V02 vaccine and demonstrated that both formulations had comparable biophysical attributes as the liquid formulation, including similar levels of toxin neutralizing antibodies and protective efficacy against MRSA infections in murine and rabbit models. To enhance the relevancy of our findings, we then performed a multi-dimensional screen of 83 S. aureus clinical isolates from LMICs (e.g., Democratic Republic of Congo, Palestine, and Cambodia) to rationally down-select strains to test in our in vivo models based on broad expression of IBT-V02 targets (i.e., pore-forming toxins and superantigens). IBT-V02 polyclonal antisera effectively neutralized toxins produced by the S. aureus clinical isolates from LMICs. Notably, the lyophilized IBT-V02 formulation exhibited significant in vivo efficacy in various preclinical infection models against the S. aureus clinical isolates from LMICs, which was comparable to our liquid formulation. Collectively, our findings suggested that lyophilization is an effective alternative to liquid vaccine formulations of our IBT-V02 vaccine against S. aureus infections, which has important implications for protection from S. aureus isolates from LMICs.

Duplicated antibiotic resistance genes reveal ongoing selection and horizontal gene transfer in bacteria

Horizontal gene transfer (HGT) and gene duplication are often considered as separate mechanisms driving the evolution of new functions. However, the mobile genetic elements (MGEs) implicated in HGT can copy themselves, so positive selection on MGEs could drive gene duplications. Here, we use a combination of modeling and experimental evolution to examine this hypothesis and use long-read genome sequences of tens of thousands of bacterial isolates to examine its generality in nature. Modeling and experiments show that antibiotic selection can drive the evolution of duplicated antibiotic resistance genes (ARGs) through MGE transposition. A key implication is that duplicated ARGs should be enriched in environments associated with antibiotic use. To test this, we examined the distribution of duplicated ARGs in 18,938 complete bacterial genomes with ecological metadata. Duplicated ARGs are highly enriched in bacteria isolated from humans and livestock. Duplicated ARGs are further enriched in an independent set of 321 antibiotic-resistant clinical isolates. Our findings indicate that duplicated genes often encode functions undergoing positive selection and horizontal gene transfer in microbial communities.

Commentary: Global prevalence and antibiotic resistance in clinical isolates of Stenotrophomonas maltophilia: a systematic review and meta-analysis.

Commentary: Global prevalence and antibiotic resistance in clinical isolates of Stenotrophomonas maltophilia: a systematic review and meta-analysis.

Detection of the effect of synthetic siRNA on efflux pump MexA gene expression and antibiotic resistance in clinical isolates of Pseudomonas aeruginosa

Introduction and Aim: Pseudomonas aeruginosa is a nosocomial infection with an ability to develop high levels of antibiotic resistance. The efflux pump system is one of the mechanisms that is linked to multidrug resistance in P. aeruginosa. In this study, we employed siRNA loaded on gold nanoparticles against the MexA efflux pump gene to decrease the MexA gene expression in P. aeruginosa and estimated antibiotic resistance after gene silencing. Materials and Methods: This study examined four strains of P. aeruginosa isolated from patients in various hospitals in Baghdad. Bacteria isolated were identified by biochemical tests and Vitek compact 2 system. Single-stranded siRNA (33bp) designed in this study was loaded onto gold nanoparticles (AuNPls). Detection of the MexA gene was carried out by conventional PCR technique. The expression of MexA gene was examined by qRT-PCR in order to determine if the siRNA have impacted on MexA gene expression and on the antibiotic resistance in aeruginosa Results: This study showed that the mRNA expression level of the MexA gene exhibited a decrease in fold change CT -2 in P. aeruginosa (isolates numbers 67, 66,49, and PDR(5p)) when examined in vitro. The specific fold change values observed were (0.202, 0.040, 0.063, and 0.163) respectively. The resistance percentages of antibiotics tested was observed to increase after MexA gene silencing. Conclusion: Targeting the MexA gene with synthetic siRNA may be a unique approach to diminish P. aeruginosa resistance to antibiotics. However, many unexpected consequences may occur when utilizing any genetic manipulation in bacteria.

Inhibition of the type IV secretion system from antibiotic-resistant Helicobacter pylori clinical isolates supports the potential of Cagα as an anti-virulence target.

Helicobacter pylori resistance to antibiotics is a growing problem and it increasingly leads to treatment failure. While the bacterium is present worldwide, the severity of clinical outcomes is highly dependent on the geographical origin and genetic characteristics of the strains. One of the major virulence factors identified in H. pylori is the cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS) used to translocate effectors into human cells. Here, we investigated the genetic variability of the cagPAI among 13 antibiotic-resistant H. pylori strains that were isolated from patient biopsies in Québec. Seven of the clinical strains carried the cagPAI, but only four could be readily cultivated under laboratory conditions. We observed variability of the sequences of CagA and CagL proteins that are encoded by the cagPAI. All clinical isolates induce interleukin-8 secretion and morphological changes upon co-incubation with gastric cancer cells and two of them produce extracellular T4SS pili. Finally, we demonstrate that molecule 1G2, a small molecule inhibitor of the Cagα protein from the model strain H. pylori 26695, reduces interleukin-8 secretion in one of the clinical isolates. Co-incubation with 1G2 also inhibits the assembly of T4SS pili, suggesting a mechanism for its action on T4SS function.

Systematic identification and repurposing of FDA-approved drugs as antibacterial agents against Streptococcus pyogenes: In silico and in vitro studies

Streptococcus pyogenes (Group A Streptococcus - GAS) is a human pathogen causing wide range of infections and toxin-mediated diseases in human beings of all age groups with fatality of 10–30 %. The limited success of antibiotics and the non-availability of vaccines makes GAS a global burden. The multi-subunit RNA polymerase (RNAP) is a validated bacterial therapeutic target as it is involved in transcription and can arrest growth. Of the five subunits of this enzyme complex, the β-subunit (RpoC) has attracted specific attention as a drug target, particularly in the switch region. Here we attempt to repurpose non-antimicrobial drugs to act as RpoC inhibitors against S. pyogenes. In this study, 1826 FDA approved drugs have been identified through high-throughput virtual screening. Free Energy Perturbation (FEP) based binding free energy calculations have been performed at the final step of the virtual screening funnel to ensure high accuracy in silico results. Three compounds identified have been tested for susceptibility of S. pyogenes MTCC 442 strain and two antibiotic-resistant clinical isolates of S. pyogenes using microdilution assay. Among the three, two drugs Amlodipine Besylate (Amd) and Ranitidine hydrochloride (Rnt) have shown inhibition against all the tested strains and its mechanism of interaction with RpoC has been studied. The docked complexes were analyzed to understand the binding mode of the drugs to the target. Classical Molecular Dynamics studies for RpoC-Rnt complex and the two stable conformations of RpoC-Amd complex was carried out. Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of Gyration (RoG) and Solvent Accessible Surface Area (SASA) of the complexes were plotted and studied. The thermodynamic parameters of protein-drug were experimentally determined using Isothermal Titration Calorimetry (ITC). Infrared spectroscopic studies and Fluorescence quenching studies provided insights into the secondary structural changes in RpoC on binding to the drugs.

Network meta-analysis of antibiotic resistance patterns in gram-negative bacterial infections: a comparative study of carbapenems, fluoroquinolones, and aminoglycosides

IntroductionAntimicrobial resistance poses a grave global threat, particularly with the emergence of multidrug-resistant gram-negative bacterial infections, which severely limit treatment options. The increasing global threat of antimicrobial resistance demands rigorous investigation, particularly concerning multidrug-resistant gram-negative bacterial infections that present limited therapeutic options. This study employed a network meta-analysis, a powerful tool for comparative effectiveness assessment of diverse antibiotics. The primary aim of this study was to comprehensively evaluate and compare resistance patterns among widely used antibiotic classes, namely carbapenems, fluoroquinolones, and aminoglycosides, for combating gram-negative pathogens.MethodsWe searched PubMed, Web of Sciences, Scopus, Scholarly, Medline, Embase, and Cochrane databases up to August 27, 2023. Studies showing antibiotic resistance in clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii exposed to carbapenems, fluoroquinolones, and aminoglycosides were included. This study determined treatment-specific resistance percentages and ranked these treatments based on resistance using a random-effects network meta-analysis technique. To investigate the impact of the study and pathogen features, subgroup and meta-regression analyses were performed. Risk ratios and 95% confidence intervals (CIs) were calculated using a network meta-analysis (NMA) incorporating both direct and indirect evidence. Clinical improvement, cure, microbiological eradication, and death from any cause were the primary outcomes. Nephrotoxicity was a secondary result.ResultsThe analysis included 202 publications and 365,782 gram-negative isolates. The NMA included data from 20 studies and 4,835 patients. Carbapenems had the lowest resistance rates throughout the pathogen spectrum, with resistance percentages of 17.1, 22.4, and 33.5% for Enterobacteriaceae, P. aeruginosa, and A. baumannii, respectively. For the same infections, aminoglycosides showed resistance rates of 28.2, 39.1, and 50.2%, respectively. Fluoroquinolones had the highest resistance rates at 43.1, 57.3, and 65.7%, respectively. Unexpectedly, resistance to all three antibiotic classes has increased over time, with multidrug resistance being the most prevalent.ConclusionThis extensive network meta-analysis provides an overview of the patterns of resistance throughout the world and how they are changing. The most effective choice is still carbapenems, but the increasing resistance highlights the critical need for multimodal therapies to protect antibiotic effectiveness against these powerful gram-negative infections.

The Relationship between the Biofilm Genes and Antibiotic Resistance in Stenotrophomonas maltophilia.

Today, Stenotrophomonas maltophilia (S. maltophilia) is a major opportunistic pathogen among hospitalized or immunocompromised patients. Antibiotic-resistant clinical isolates are increasing in several parts of the world. Various antibiotic-resistance and biofilm-forming genes are identified in this bacterium. Its capacity to form biofilms is an important virulence factor that may impact antibiotic-resistance patterns. In the current study, we evaluated the biofilm-formation capacity, antibiotic-resistance profile, and prevalence of biofilm-forming genes as well as antibiotic resistance genes among S. maltophilia isolates. In this cross-sectional study, 94 clinical S. maltophilia isolates were recovered from four tertiary-care hospitals in Iran between 2021 and 2022. The presence of the selected antibiotic-resistance genes and biofilm-forming genes was examined by polymerase chain reaction (PCR). The ability of biofilm formation was examined by microtiter plate assay. The Kirby-Bauer disc diffusion method was used to evaluate the trimethoprim-sulfamethoxazole (TMP-SMX), levofloxacin, and minocycline resistance. S. maltophilia is mainly isolated from bloodstream infections. Notably, 98.93% of isolates were biofilm producers, of which 19.35%, 60.22%, and 20.43% produced strong, moderate, and weak biofilm, respectively. The frequency of biofilm genes was 100%, 97.88%, 96.80%, and 75.53% for spgM, rmlA, smf-1, and rpfF, respectively. Isolates with the genotype of smf-1+/rmlA+/spgM+/rpfF+ were mostly strong biofilm producers. Among the antibiotic-resistance genes, the Smqnr, L1, and sul1 had the highest prevalence (76.59%, 72.34%, and 64.89), respectively. Antimicrobial susceptibility evaluation showed 1.06%, 3.19%, and 6.3% resistance to minocycline, TMP-SMX, and levofloxacin. The results of the current study demonstrated that S. maltophilia isolates differ in biofilm-forming ability. Moreover, smf-1, rmlA, and spgM genes were presented in all strong biofilm producers. Although the overall resistance rate to the evaluated antibiotics was high, there was no statistically significant relation between antibiotic resistance and the type of biofilm.

Peptaibol Analogs Show Potent Antibacterial Activity against Multidrug Resistant Opportunistic Pathogens.

New classes of antibacterial drugs are urgently needed to address the global issue of antibiotic resistance. In this context, peptaibols are promising membrane-active peptides since they are not involved in innate immunity and their antimicrobial activity does not involve specific cellular targets, therefore reducing the chance of bacterial resistance development. Trichogin GA IV is a nonhemolytic, natural, short-length peptaibol active against Gram-positive bacteria and resistant to proteolysis. In this work, we report on the antibacterial activity of cationic trichogin analogs. Several peptides appear non-hemolytic and strongly active against many clinically relevant bacterial species, including antibiotic-resistant clinical isolates, such as Staphylococcus aureus, Acinetobacter baumannii, and extensively drug-resistant Pseudomonas aeruginosa, against which there are only a limited number of antibiotics under development. Our results further highlight how the modification of natural peptides is a valuable strategy for obtaining improved antibacterial agents with potential therapeutic applications.

Tackling antibiotic resistance by inducing transient and robust collateral sensitivity

Collateral sensitivity (CS) is an evolutionary trade-off traditionally linked to the mutational acquisition of antibiotic resistance (AR). However, AR can be temporally induced, and the possibility that this causes transient, non-inherited CS, has not been addressed. Mutational acquisition of ciprofloxacin resistance leads to robust CS to tobramycin in pre-existing antibiotic-resistant mutants of Pseudomonas aeruginosa. Further, the strength of this phenotype is higher when nfxB mutants, over-producing the efflux pump MexCD-OprJ, are selected. Here, we induce transient nfxB-mediated ciprofloxacin resistance by using the antiseptic dequalinium chloride. Notably, non-inherited induction of AR renders transient tobramycin CS in the analyzed antibiotic-resistant mutants and clinical isolates, including tobramycin-resistant isolates. Further, by combining tobramycin with dequalinium chloride we drive these strains to extinction. Our results support that transient CS could allow the design of new evolutionary strategies to tackle antibiotic-resistant infections, avoiding the acquisition of AR mutations on which inherited CS depends.

Role of AlgC and GalU in the Intrinsic Antibiotic Resistance of Helicobacter pylori.

Helicobacter pylori is associated with the development of gastrointestinal diseases. However, its eradication is challenged by an increased rate of drug resistance. AlgC and GalU are important for the synthesis of UDP-glucose, which is a substrate for the synthesis of lipopolysaccharide (LPS) in H. pylori. In this study, we investigated the role of UDP-glucose in the intrinsic drug resistance in H. pylori. Gene knockout strains or complementation strains, including ΔalgC, ΔgalU, ΔgalE, Δhp0045, ΔalgC/algC* and ΔgalU/galU* were constructed in Hp26695; and ΔalgC and ΔgalU were also constructed in two clinical drug-resistant strains, Hp008 and Hp135. The minimum inhibitory concentrations (MIC) of H. pylori to amoxicillin (AMO), tetracycline (TET), clarithromycin (CLA), metronidazole (MNZ), levofloxacin (LEV), and rifampicin (RIF) were measured using MIC Test Strips. Silver staining was performed to examine the role of AlgC and GalU in LPS synthesis. Ethidium bromide (EB) accumulation assay was performed to assess the outer membrane permeability of H. pylori strains. Knockout of algC and galU in H. pylori resulted in increased drug sensitivity to AMO, MNZ, CLA, LEV, and RIF; whereas knockout of hp0045 and galE, which are involved in GDP-fucose and UDP-galactose synthesis, respectively, did not significantly alter the drug sensitivity of H. pylori. Knockout of algC and galU in clinically drug-resistant strains resulted in significantly increased drug sensitivity to all the antibiotics, except MNZ. The lipid A-core structure was altered in ΔalgC and ΔgalU when their EB accumulation was higher than that in the wild type and complementation strains. UDP-glucose may play an important role in increasing drug resistance to AMO, MNZ, CLA, LEV, TET, and RIF by maintaining the lipid A-core structure and decreasing membrane permeability. AlgC and GalU may serve as potential drug targets for decreasing antibiotic resistance in clinical isolates.

Isolation, characterization, therapeutic potency, and genomic analysis of a novel bacteriophage vB_KshKPC-M against carbapenemase-producing Klebsiella pneumoniae strains (CRKP) isolated from Ventilator-associated pneumoniae (VAP) infection of COVID-19 patients

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant clinical problem, given the lack of therapeutic options. The CRKP strains have emerged as an essential worldwide healthcare issue during the last 10 years. Global expansion of the CRKP has made it a significant public health hazard. We must consider to novel therapeutic techniques. Bacteriophages are potent restorative cases against infections with multiple drug-resistant bacteria. The Phages offer promising prospects for the treatment of CRKP infections.ObjectiveIn this study, a novel K. pneumoniae phage vB_KshKPC-M was isolated, characterized, and sequenced, which was able to infect and lyse Carbapenem-resistant K. pneumoniae host specifically.MethodsOne hundred clinical isolates of K. pneumoniae were collected from patients with COVID-19 associated with ventilator-associated acute pneumonia hospitalized at Shahid Beheshti Hospital, Kashan, Iran, from 2020 to 2021. Initially, all samples were cultured, and bacterial isolates identified by conventional biochemical tests, and then the ureD gene was used by PCR to confirm the isolates. The Antibiotic susceptibility test in the disc diffusion method and Minimum inhibitory concentrations for Colistin was done and interpreted according to guidelines. Phenotypic and molecular methods determined the Carbapenem resistance of isolates. The blaKPC, blaNDM, and blaOXA-23 genes were amplified for this detection. Biofilm determination of CRKP isolates was performed using a quantitative microtiter plate (MTP) method. The phage was isolated from wastewater during the summer season at a specific position from Beheshti Hospital (Kashan, Iran). The sample was processed and purified against the bacterial host, a CRKP strain isolated from a patient suffering from COVID-19 pneumoniae and resistance to Colistin with high potency for biofilm production. This isolate is called Kp100. The separated phages were diluted and titration by the double overlay agar plaque assay. The separate Phage is concentrated with 10% PEG and stored at −80 °C until use. The phage host range was identified by the spot test method. The purified phage morphology was determined using a transmission electron microscope. The phage stability tests (pH and temperature) were analyzed. The effect of cationic ions on phage adsorption was evaluated. The optimal titer of bacteriophage was determined to reduce the concentration of the CRKP strain. One-step growth assays were performed to identify the purified phage burst’s latent cycle and size. The SDS-PAGE was used for phage proteins analysis. Phage DNA was extracted by chloroform technique, and the whole genome of lytic phage was sequenced using Illumina HiSeq technology (Illumina, San Diego, CA). For quality assurance and preprocessing, such as trimming, Geneious Prime 2021.2.2 and Spades 3.9.0. The whole genome sequence of the lytic phage is linked to the GenBank database accession number. RASTtk—v1.073 was used to predict and annotate the ORFs. Prediction of ORF was performed using PHASTER software. ResFinder is used to assess the presence of antimicrobial resistance and virulence genes in the genome. The tRNAs can-SE v2.0.6 is used to determine the presence of tRNA in the genome. Linear genome comparisons of phages and visualization of coding regions were performed using Easyfig 2.2.3 and Mauve 2.4.0. Phage lifestyles were predicted using the program PHACTS. Phylogenetic analysis and amino acid sequences of phage core proteins, such as the major capsid protein. Phylogenies were reconstructed using the Neighbor-Joining method with 1000 bootstrap repeat. HHpred software was used to predict depolymerase. In this study, GraphPad Prism version 9.1 was used for the statistical analysis. Student’s t-test was used to compare the sets and the control sets, and the significance level was set at P ≤ 0.05.ResultsPhage vB_KshKPC-M is assigned to the Siphoviridae, order Caudovirales. It was identified as a linear double-stranded DNA phage of 54,378 bp with 50.08% G + C content, had a relatively broad host range (97.7%), a short latency of 20 min, and a high burst size of 260 PFU/cell, and was maintained stable at different pH (3–11) and temperature (45–65 °C). The vB_KshKPC-M genome contains 91 open-reading frames. No tRNA, antibiotic resistance, toxin, virulence-related genes, or lysogen-forming gene clusters were detected in the phage genome.Comparative genomic analysis revealed that phage vB_KshKPC-M has sequence similarity to the Klebsiella phages, phage 13 (NC_049844.1), phage Sushi (NC_028774.1), phage vB_KpnD_PeteCarol (OL539448.1) and phage PWKp14 (MZ634345.1).ConclusionThe broad host range and antibacterial activity make it a promising candidate for future phage therapy applications. The isolated phage was able to lyse most of the antibiotic-resistant clinical isolates. Therefore, this phage can be used alone or as a phage mixture in future studies to control and inhibit respiratory infections caused by these bacteria, especially in treating respiratory infections caused by resistant strains in sick patients.

Disparate Regions of the Human Chemokine CXCL10 Exhibit Broad-Spectrum Antimicrobial Activity against Biodefense and Antibiotic-Resistant Bacterial Pathogens.

CXCL10 is a pro-inflammatory chemokine produced by the host in response to microbial infection. In addition to canonical, receptor-dependent actions affecting immune-cell migration and activation, CXCL10 has also been found to directly kill a broad range of pathogenic bacteria. Prior investigations suggest that the bactericidal effects of CXCL10 occur through two distinct pathways that compromise the cell envelope. These observations raise the intriguing notion that CXCL10 features a separable pair of antimicrobial domains. Herein, we affirm this possibility through peptide-based mapping and structure/function analyses, which demonstrate that discrete peptides derived from the N- and C-terminal regions of CXCL10 mediate bacterial killing. The N-terminal derivative, peptide P1, exhibited marked antimicrobial activity against Bacillus anthracis vegetative bacilli and spores, as well as antibiotic-resistant clinical isolates of Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecium, and Staphylococcus aureus, among others. At bactericidal concentrations, peptide P1 had a minimal degree of chemotactic activity, but did not cause red blood cell hemolysis or cytotoxic effects against primary human cells. The C-terminal derivative, peptide P9, exhibited antimicrobial effects, but only against Gram-negative bacteria in low-salt medium─conditions under which the peptide can adopt an α-helical conformation. The introduction of a hydrocarbon staple induced and stabilized α-helicity; accordingly, stapled peptide P9 displayed significantly improved bactericidal effects against both Gram-positive and Gram-negative bacteria in media containing physiologic levels of salt. Together, our findings identify and characterize the antimicrobial regions of CXCL10 and functionalize these novel determinants as discrete peptides with potential therapeutic utility against difficult-to-treat pathogens.

Antimicrobial resistance patterns and virulence gene profiles of Salmonella enteritidis and Salmonella typhimurium recovered from patients with gastroenteritis in three cities of Iran.

This study evaluated distribution of virulence factors and antibiotic resistance in clinical isolates of Salmonella enteritidis and Salmonella typhimurium in three cities of Iran. Altogether 48 S. enteritidis and S. typhimurium isolates were collected from patients at certain Iranian hospitals between May 2018 and September 2021. Antimicrobial susceptibility testing was performed by disk diffusion and broth microdilution methods. The presence of antibiotic-resistance genes (blaTEM,blaSHV,blaCTX-M,blaNDM,strA, strB, aadA1, tetA, tetB, floR, sul1, sul2, dfrA), integrons (classe 1 and 2), and virulence-associated genes (invA, stn, sopB, spvC, rck, phoPQ) was investigated by PCR and sequencing. Antimicrobial agents like trimethoprim-sulfamethoxazole and imipenem represent highly efficient agents with 97% susceptibility. S. enteritidis and S. typhimurium exhibited high resistance to ciprofloxacin (n = 20, 71.43%) and ceftazidime (n = 9, 45%), respectively. Overall, 3 (6.25%), 13 (27.08%), and 6 (12.5%) isolates were divided into strong, moderate, and weak biofilm producers, respectively. Moreover, blaCTX-M,blaTEM, blaSHV, sul1, sul2, tetA, tetB, floR, strA, and strB resistant genes were detected in 10 (20.8%), 5 (10.4%), 1(2.08%), 7 (14.58%), 1 (2.08%), 3 (6.25%), 2 (4.1%), 1 (2.08%), 2 (4.1%), 2 (4.1%), respectively. Furthermore, 7 (14.58%) strains had classe 1 integron. All tested S. enteritidis strains had invA and sopB, and all S. typhimurium strains had invA and phoPQ. However, spvC remained undetected in all isolates. Extensive surveillance and efficient control measures against infection help to stop the upsurge of various antibiotic-resistant isolates.

The Frequency of Antibiotic Resistance of Clinical Isolates of Clostridioides difficile to Commonly Used Antibiotics in Hospitalized Patients of Educational Hospitals of Kerman City During 2018-2020

The Frequency of Antibiotic Resistance of Clinical Isolates of Clostridioides difficile to Commonly Used Antibiotics in Hospitalized Patients of Educational Hospitals of Kerman City During 2018-2020

Genomic insights into antibiotic-resistance and virulence genes of Enterococcus faecium strains from the gut of Apis mellifera.

Enterococcus faecium is a lactic acid bacterium that confers beneficial health effects in humans. However, lately, a number of E. faecium strains have been linked to the spread of nosocomial infections in the hospital environment. Therefore, any potential commercial usage of E. faecium isolates should be preceded by an assessment of infection risk. In the current study, the genomes of two novel E. faecium strains Am1 (larval isolate) and Bee9 (adult bee isolate) isolated from the gut of Apis mellifera L. (honeybee) were sequenced to allow evaluation of their safety. In particular, their genomes were screened for antibiotic-resistance and virulence genes. In addition, their potential to spread resistance in the environment was evaluated. The analysis revealed that Am1 and Bee9 possess 2832 and 2844 protein-encoding genes, respectively. In each case, the genome size was 2.7 Mb with a G+C content of 37.9 mol%. Comparative analysis with probiotic, non-pathogenic and pathogenic enterococci revealed that there are variations between the two bee E. faecium isolates and pathogenic genomes. They were, however, closely linked to the probiotic comparison strains. Phenotypically, the Am1 and Bee9 strains were susceptible to most antibiotics tested, but showed intermediate sensitivity towards erythromycin, linezolid and trimethoprim/sulfamethoxazole. Notably, no genes associated with antibiotic resistance in clinical isolates (e.g. vancomycin resistance: vanA, vanB, vanS, vanX and vanY) were present. In addition, the insertion sequences (IS16, ISEfa11 and ISEfa5), acting as molecular pathogenicity markers in clinically relevant E. faecium strains, were also absent. Moreover, the analysis revealed the absence of three key pathogenicity-associated genes (acm, sgrA, ecbA) in the Am1 and Bee9 strains that are found in the prominent clinical isolates DO, V1836, Aus0004 and Aus0085. Overall, the findings of this investigation suggest that the E. faecium isolates from the bee gut have not suffered any recent clinically relevant antibiotic exposure. It also suggests that E. faecium Am1 and Bee9 are safe potential probiotic strains, because they lack the phenotypic and genetic features associated with strains eliciting nosocomial infections.

Serotype Distribution, Virulence Determinants and Antimicrobial Susceptibility of Streptococcus agalactiae Isolated from Young Infants

Background: Streptococcus agalactiae (Group B Streptococcus, GBS) is the most common cause of serious infections in the first 3 months of life worldwide. The pathogenicity of GBS is closely related to serotypes, surface proteins and virulence factors, and the distribution of them may vary temporally and geographically. However, data related to GBS surface proteins and virulence determinants in China are very few. The aim of this study is to investigate the genetic characteristics of clinical GBS isolates from infected infants. Methods: We recovered GBS isolates from infected infants younger than 3 months during 2017−2021 at Maternal and Child Health Hospital of Hubei Province in China. We assessed the GBS serotypes, surface proteins, virulence determinants and antibiotic resistance genes distribution, by Multilocus sequence typing (MLST) and whole-genome sequencing analysis. Results: Among 97 isolates (81 EOD and 16 LOD), 5 serotypes were detected. Serotype III was the most represented (49.5%), followed by type Ib (20.6%). The isolates belonged to 17 different sequence types (STs) that grouped into the 8 clonal complexes (CCs). The most frequently identified ST was ST17 (23.7%). The most predominant surface protein of alpha-protein-like (alp) family (one of the protein components of the GBS surface antigen, resistant to trypsin) present was Rib (41.2%), which was mainly detected in serotype III. The srr1, which encodes Srr1 protein, was identified in 54.6% of isolates. The hvgA encoding for hypervirulent GBS adhesin can be detected in all 24 serotype III GBS. Among the pilus islands genes, 50% and 58.8% of the isolates were positive for pi-1 and pi-2a genes, respectively. The presence of pi-2b was mainly associated with serotype III/CC17 strains; 56.7% of isolates carried tetM, tetO/tetL, ermB antibiotic resistant genes. Among all the virulence genes detected, the cfb-cylE-lmb-pavA pattern was the main virulence gene profile (81.4%), mainly in serotype III/CC17. Conclusions: The whole genomic sequencing data revealed the high variation in surface proteins, determining virulence and antibiotic resistance in clinical isolates from 97 GBS infected infants. These data provide insightful characteristics of genetic features of GBS. Constant epidemiological surveillance is warranted to provide information on the GBS pathogenic dynamics and antibiotic resistance profiles in the surveyed areas for improving therapeutic outcomes.

Association of CRISPR-Cas System with the Antibiotic Resistance and Virulence Genes in Nosocomial Isolates of Enterococcus.

This study aimed to investigate the prevalence of the CRISPR-Cas system in nosocomial isolates of Enterococcus and their possible association with antibiotic resistance and virulence genes. Identification and antimicrobial susceptibility of the microorganism were performed by the automatized VITEK 2 Compact system (bioMerieux, France). A total of 100 Enterococcus isolates were collected and identified by VITEK 2 Compact automatic microbial identification drug susceptibility analyzer. The prevalence of various CRISPR-Cas systems, antibiotic resistance genes and virulence genes were investigated by polymerase chain reaction (PCR). The prevalence of CRISPR-Cas systems associated with antibiotic resistance and virulence genes was performed by appropriate statistical tests. A total of 100 isolates of Enterococcus were identified and there were 62/100(62.0%) Enterococcus faecalis isolates and 38/100(38.0%) Enterococcus faecalis isolates. In total, 46 (46.0%) of 100 isolates had at least one CRISPR-Cas locus. CRISPR elements were more prevalent in Enterococcus faecalis isolates. The results of PCR demonstrated that CRISPR1-Cas, orphan CRISPR2, and CRISPR3-Cas were present in 23 (23.0%), 42 (42.0%) and 5 (5.0%) Enterococcus isolates, respectively. Compared with CRISPR-Casnegative isolates, the CRISPR-Cas positive isolates showed significant lower resistance rates against ampicillin, erythromycin, levofloxacin, tetracycline, vancomycin, gentamicin, streptomycin, and rifampicin. Presumably consistent with drug susceptibility, fewer CRISPR loci were identified in vanA, tetM, ermB, aac6'-aph(2"), aadE, and ant(6) positive isolates. There was a significant negative correlation between the CRISPR-Cas locus and the enterococcal virulence factors enterococcal surface protein (esp) gene. In conclusion, the results indicated that the absence of the CRISPR-Cas system was negatively associated with some antibiotic resistance in clinical isolates of Enterococcus faecalis and Enterococcus faecium. Also, there was a negative correlation with the carriage of antibiotic resistance genes. Furthermore, CRISPR-Cas may prevent some isolates from acquiring certain virulence factors.

Molecular Basis of Non-β-Lactam Antibiotics Resistance in Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most successful human pathogens with the potential to cause significant morbidity and mortality. MRSA has acquired resistance to almost all β-lactam antibiotics, including the new-generation cephalosporins, and is often also resistant to multiple other antibiotic classes. The expression of penicillin-binding protein 2a (PBP2a) is the primary basis for β-lactams resistance by MRSA, but it is coupled with other resistance mechanisms, conferring resistance to non-β-lactam antibiotics. The multiplicity of resistance mechanisms includes target modification, enzymatic drug inactivation, and decreased antibiotic uptake or efflux. This review highlights the molecular basis of resistance to non-β-lactam antibiotics recommended to treat MRSA infections such as macrolides, lincosamides, aminoglycosides, glycopeptides, oxazolidinones, lipopeptides, and others. A thorough understanding of the molecular and biochemical basis of antibiotic resistance in clinical isolates could help in developing promising therapies and molecular detection methods of antibiotic resistance.