Antibiotic Efflux Pump Research Articles

Resistome analysis of wastewater treatment plants in Agadir city, Morocco, using a metagenomics approach

Water scarcity has evolved into a pressing global issue, significantly impacting numerous regions worldwide. The use of treated wastewater stands out as a promising solution to this problem. However, the proliferation of various contaminants, primarily Antimicrobial Resistance Genes (ARGs), poses a significant challenge to its safe and sustainable use. In this study, we assessed the composition and abundance of 373 ARGs, corresponding to 31 different classes of antibiotics, in six wastewater treatment plants (WWTP) in Agadir city of Morocco. Influent and effluent samples were collected during the months of February and July in 2020, in addition to samples from the Atlantic Ocean. In total, 223 ARGs were uncovered, highlighting in particular resistance to aminoglycoside, macrolide lincosamide, beta-lactamase, chloramphenicol, sulfonamide, tetracycline, and other antibiotics. The mechanisms of action of these ARGs were mainly antibiotic inactivation, antibiotic target alteration, efflux pump and cellular protection. Mobile genetic elements (MGEs) were detected at high levels their co-occurrence with ARGs highlights their involvement in the acquisition and transmission of ARGs in microbial communities through horizontal gene transfer. While many wastewater treatment methods effectively reduce a large proportion of gene material and pathogens, a substantial fraction of ARGs and other contaminants persist in treated wastewater. This persistence poses potential risks to both human health and the environment, warranting the need of more effective treatment strategies.

Heterogeneity and Genomic Plasticity of Acinetobacter baumannii and Acinetobacter nosocomialis Isolates Recovered from Clinical Samples in India.

Acinetobacter baumannii and Acinetobacter nosocomialis are the imperious pathogens in the intensive care units. We aimed to explore the genomic features of these pathogens to understand the factors influencing their plasticity. Using next-generation sequencing, two carbapenem-resistant A. baumannii (AbaBS-3, AbaETR-4) isolates and a pan-susceptible A. nosocomialis (AbaAS-5) isolate were characterised. All genomes exhibited 94% similarity with a degree of heterogeneity. AbaBS-3 and AbaETR-4 harboured antibiotic resistance gene (ARG) repertoire to most antibiotic classes. Carbapenem resistance was due to blaOXA-23 and blaOXA-66 besides the antibiotic efflux pumps. Diverse mobile genetic elements (MGE), insertion sequences (IS), prophages and virulence determinants with a plethora of stress response genes were identified in all three genomes. Class-1 integron in AbaETR-4, encoded genes that confer resistance to aminoglycosides, phenicol, sulfonamides and disinfectants. Substitutions in LpxACD and PmrCAB of AbaETR-4 confirmed the colistin resistance in vitro. Novel mutations in piuA, responsible for transporting cefiderocol, were found in AbaBS-3 and AbaETR-4. Plasmids carrying toxin-antitoxin systems, ARGs and ISs were present in these genomes. All three genomes harboured diverse protein secretion systems, virulence determinants related to immune evasion, adherence, biofilm formation and iron acquisition systems. AbaAS-5 exclusively harboured serine protease pkf, and CpaA substrate of type-II secretion system but lacked the acinetobactin-iron acquisition system. Our work delivers a holistic genome characterization of A. baumannii, coupled with a trailblazing attempt to study A. nosocomialis from India. The presence of ARGs and potential virulence factors interspersed with MGE is a cause for concern, depicting the dynamic adaptability mediated by genetic recombination.

Decoding Pseudomonas aeruginosa: Genomic insights into adaptation, antibiotic resistance, and the enigmatic role of T6SS in interbacterial dynamics

Pseudomonas aeruginosa demonstrates a remarkable capacity for adaptation and survival in diverse environments. Furthermore, its clinical importance is underscored by its intrinsic and acquired resistance to a wide range of antimicrobial agents, posing a substantial challenge in healthcare settings. Amidst this complex landscape of resistance, the Type VI Secretion System (T6SS) in P. aeruginosa adds yet another layer of intricacy and allows bacteria to engage in interbacterial competition, potentially influencing their resilience and pathogenicity. Whole genome sequencing (WGS) was conducted on the five isolates under investigation, enabling the identification of antibiotic resistance genes (ARGs) and mutations associated with resistance. All isolates exhibit class C and D β-lactamases, displaying variant differences. The Resistance-nodulation-division (RND) antibiotic efflux pumps, crucial for multidrug resistance, have been encoded chromosomally. When exploring the role of the T6SS in urinary tract infections involving other bacteria, it was noted that P. aeruginosa isolates exhibited reduced counts when co-cultivated with other bacteria. The downregulation of the tssJ gene, associated with the T6SS under bacterial stress, and the exclusion of several cluster genes in this study suggest the hypothesis of a basal state rather than an attack/defence mechanism in the initial contact.

Comprehensive Genomic and Evolutionary Analysis of Biofilm Matrix Clusters and Proteins in the Vibrio Genus.

Vibrio cholerae pathogens cause cholera, an acute diarrheal disease resulting in significant morbidity and mortality worldwide. Biofilm formation by V. cholerae enhances its survival in natural ecosystems and facilitates transmission during cholera outbreaks. Critical components of the biofilm matrix are the Vibrio polysaccharide (VPS) produced by the vps-1 and vps-2 gene clusters, and biofilm matrix proteins encoded in the rbm cluster. However, the biofilm matrix clusters and associated matrix proteins in other Vibrio species remain under investigated, and their evolutionary patterns are largely unknown. In this study, we systematically annotated the biofilm matrix clusters across 6,121 Vibrio genomes, revealing their distribution, diversity, and evolution. We found that biofilm matrix clusters not only exist in V. cholerae but also in phylogenetically distant Vibrio species. Additionally, vps-1 clusters tend to co-locate with rbmABC genes, while vps-2 clusters are often adjacent to rbmDEF genes in various Vibrio species, which helps explain the separation of these clusters by the rbm cluster in well-characterized V. cholerae strains. Evolutionary analysis of RbmC and Bap1 reveals that these two major biofilm matrix proteins are sequentially and structurally related and have undergone domain/modular alterations during their evolution. RbmC genes are more prevalent, while bap1 likely resulted from an ancient duplication event of rbmC and is only present in a major clade of species containing rbmC counterparts. Notably, a novel loop-less Bap1 variant, identified in two subspecies clades of V. cholerae, was found to be associated with altered biofilm formation and the loss of antibiotic efflux pumps and chemotaxis. Another rbm cluster gene, rbmB, involved in biofilm dispersal, was found to share a common ancestor with Vibrio prophage pectin lyase-like tail proteins, indicating its functional and evolutionary linkages to Vibriophage proteins. In summary, our findings establish a foundational understanding of the proteins and gene clusters that contribute to Vibrio biofilm formation from an evolutionary perspective across a broad taxonomic scale. This knowledge paves the way for future strategies aimed at engineering and controlling biofilms through genetic modification.

The Effect of Manure Application Rates on the Vertical Distribution of Antibiotic Resistance Genes in Farmland Soil

Manure application is the primary input route for antibiotic resistance genes (ARGs) in farmland soil. This study investigated the effects of varying the rates of five chicken manure applications on the accumulation and distribution of ARGs across different soil depths (0–20, 20–40, and 40–60 cm) using metagenomic sequencing. The results revealed that the distribution of ARGs in farmland soil was closely linked to soil depth and influenced to some extent by the fertilizer quantity after 30 days of fertilization. ARGs were predominantly concentrated in the surface soil and exhibited a significant decrease in type and abundance with an increased soil depth. Compared with soil treated with chemical fertilizers alone, chicken manure-treated surface soil presented a higher diversity and abundance of ARGs. However, the diversity and abundance of ARGs did not increase proportionally with the increasing ratios of chicken manure application (0, 25, 50, 75, and 100%). ARGs in soil primarily conferred resistance to host bacteria through antibiotic efflux pumps (~33%), antibiotic target alteration (~31%), antibiotic inactivation (~20%), and antibiotic target protection (~8%). Correlation analysis involving ARGs and soil microorganisms revealed widespread multidrug resistance among soil microorganisms. Furthermore, two genera of human pathogenic bacteria (Pseudomonas sp. and Listeria sp.) were identified as potential microbial hosts of ARGs in all treatments. Correlation analysis involving ARGs and environmental factors indicated that soil ARGs are predominantly influenced by heavy metals and microorganisms. This paper offers valuable insights for environmental risk assessments regarding the utilization of livestock manure resources. Additionally, it furnishes a scientific foundation for farmland application strategies pertaining to livestock manure.

Correlation analysis of whole genome sequencing of a pathogenic Escherichia coli strain of Inner Mongolian origin

Anal swabs of 1-month-old Holstein calves with diarrhea were collected from an intensive cattle farm, and a highly pathogenic Escherichia coli strain was obtained by isolation and purification. To study the virulence and resistance genes of pathogenic E.coli that cause diarrhea in calves, a strain of E. coli E12 isolated from calf diarrhea samples was used as experimental material in this experiment, and the virulence of the E12 strain were identified by the mouse infection test, and the whole genome map of the E12 strain were obtained by whole-genome sequencing and analyzed for genome characterization. The results showed that the lethality of strain E12 was 100%, the total length of E12-encoded genes was 4,294,530 bp, Cluster of Orthologous Groups of proteins (COG) annotated to 4,194 functional genes, and the virulence genes of sequenced strain E12 were compared with the virulence genes of sequenced strain E12 from the Virulence Factors of Pathogenic Bacteria (VFDB), which contained a total of 366 virulence genes in sequenced strain E12. The analysis of virulence genes of E12 revealed a total of 52 virulence genes in the iron transferrin system, 56 virulence genes in the secretory system, 41 virulence genes in bacterial toxins, and a total of 217 virulence genes in the Adhesin and Invasins group. The antibiotic resistance genes of sequenced strain E12 were identified through the Antibiotic Resistance Genes Database (ARDB) and Comprehensive Antibiotic Research Database, and it was found that its chromosome and plasmid included a total of 127 antibiotic resistance genes in four classes, and that E12 carried 71 genes related to the antibiotic efflux pumps, 36 genes related to antibiotic inactivation, and 14 antibiotic target alteration and reduced penetration into antibiotics, and 6 antibiotic resistance genes, and the resistance phenotypes were consistent with the genotypes. The pathogenic E. coli that causes diarrhea in calves on this ranch contains a large number of virulence and resistance genes. The results provide a theoretical basis for the prevention and treatment of diarrhea and other diseases caused by E. coli disease.

Comprehensive analysis of distribution characteristics and horizontal gene transfer elements of blaNDM-1-carrying bacteria

The worldwide dissemination of New Delhi metallo-β-lactamase-1 (NDM-1), which mediates resistance to almost all clinical β-lactam antibiotics, is a major public health problem. The global distribution, species, sources, and potential transfer risk of blaNDM-1-carrying bacteria are unclear. Results of a comprehensive analysis of literature in 2010–2022 showed that a total of 6002 blaNDM-1 carrying bacteria were widely distributed around 62 countries with a high trend in the coastal areas. Opportunistic pathogens or pathogens like Klebsiella sp., Escherichia sp., Acinetobacter sp. and Pseudomonas sp. were the four main species indicating the potential microbial risk. Source analysis showed that 86.45 % of target bacteria were isolated from the source of hospital (e.g., Hospital patients and wastewater) and little from surface water (5.07 %) and farms (3.98 %). A plasmid-encoded blaNDM-1Acinetobacter sp. with the resistance mechanisms of antibiotic efflux pump, antibiotic target change and antibiotic degradation was isolated from the wastewater of a typical tertiary hospital. Insertion sequences (IS3 and IS30) located in the adjacent 5 kbp of blaNDM-1-bleMBL gene cluster indicating the transposon-mediated horizontal gene transfer risk. These results showed that the worldwide spread of blaNDM-1-carrying bacteria and its potential horizontal gene transfer risk deserve good control.

Discovery and Characterization of Two Folded Intermediates for Outer Membrane Protein TolC Biogenesis

TolC is the outer membrane protein responsible for antibiotic efflux in E. coli. Compared to other outer membrane proteins it has an unusual fold and has been shown to fold independently of commonly used periplasmic chaperones, SurA and Skp. Here we find that the assembly of TolC involves the formation of two folded intermediates using circular dichroism, gel electrophoresis, site-specific disulfide bond formation and radioactive labeling. First the TolC monomer folds, and then TolC assembles into a trimer both in detergent-free buffer and in the presence of detergent micelles. We find that a TolC trimer also forms in the periplasm and is present in the periplasm before it inserts in the outer membrane. The monomeric and trimeric folding intermediates may be used in the future to develop a new approach to antibiotic efflux pump inhibition by targeting the assembly pathway of TolC.

Microbiological and molecular studies on a multidrug-resistant Pseudomonas aeruginosa from a liver transplant patient with urinary tract infection in Egypt.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for complicated UTIs and exhibits high antibiotic resistance, leading to increased mortality rates, especially in cases of multidrug-resistant strains. This study aimed to investigate the antibiotic susceptibility patterns and genomic characterization of XDR strains identified in end-stage liver disease patients who underwent liver transplants. In this study, a number of 30 individuals who underwent liver transplants were registered. Ninety urine and 60 wound site swab samples were collected and processed for culturing, identification, and antimicrobial sensitivity. Extensively drug-resistant strain EMARA01 was confirmed through Sanger sequencing and was then processed for whole genome sequencing to characterize the genomic pattern. Sequencing data were processed for de novo assembly using various tools and databases, including genome annotation, serotype identification, virulence factor genes, and antimicrobial resistance gene. Pangenome analysis of randomly selected 147 reference strains and EMAR01 sequenced strain was performed using the Bacterial Pan Genome Analysis (BPGA) software. Of these total examined samples, nosocomial infection due to P. aeruginosa was detected in twelve patients' samples. AST analysis showed that P. aeruginosa strains exhibit resistance to tobramycin, erythromycin, and gentamicin, followed by piperacillin and ofloxacin, and no strains exhibit resistance to meropenem and imipenem. The CARD database identified 59 AMR genes similar to the EMAR01 strain genome and mostly belong to the family involved in the resistance-nodulation-cell division (RND) antibiotic efflux pump. Five genes; nalC, nalD, MexR, MexA, and MexB, exhibit resistance to 14 classes of antibiotics, while two AMR; CpxR, and OprM, exhibit resistance to 15 classes of drugs. Pangenome analysis revealed that the pan-genome remained open, suggesting the potential for acquiring accessory and unique genes. Notably, the genes predominantly involved in amino acid transport metabolism were identified using the KEGG database. This study provides valuable insights into the antimicrobial resistance profile, genetic features, and genomic evolution of P. aeruginosa strains causing UTIs in liver transplant patients. The findings emphasize the significance of comprehending AMR mechanisms and genetic diversity in P. aeruginosa for developing effective treatment strategies and infection control measures.

Influence of helicobacter pylori on composition and function of gastric microbiota in patients with chronic non-atrophic gastritis

ObjectiveHelicobacter pylori (H. pylori) plays a major role in causing and advancing gastrointestinal illnesses. Our aim is to analyze the unique makeup and functional changes in the gastric microbiota of patients with chronic non-atrophic gastritis (CNAG), regardless of the presence of H. pylori, and to determine the potential signaling pathways. MethodsWe performed metagenomic sequencing on gastric mucosa samples collected from 17 individuals with non-atrophic gastritis, comprising 6 cases were infected with H. pylori (H. pylori-infected case group) and 11 cases without (control group). The species composition was evaluated with DIAMOND software, and functional enrichment was assessed utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We analyzed antibiotic resistance patterns using the Comprehensive Antibiotic Resistance Database as a reference (CARD). ResultsThe presence of H. pylori colonization in CNAG patients was associated with increased diversity in the gastric microbiota. The Phylum Firmicutes was found to be less prevalent, while the Phylum Proteobacteria showed an increase. Functionally, pathways associated with metabolic pathways, including vitamins, auxiliaries, amino acid residue, carbon hydrate, and metabolic energy pathways, were enriched in CNAG patients with H. pylori infection. Additionally, antibiotic resistance genes correlated with antibiotic efflux pump were enriched. ConclusionsFrom a holistic genomic perspective, our findings offer fresh perspectives into the gastric microbiome among CNAG patients carrying H. pylori, which is valuable for future research on CNAG.

Antibiotic influx and efflux in Pseudomonas aeruginosa: Regulation and therapeutic implications.

Pseudomonas aeruginosa is a notorious multidrug-resistant pathogen that poses a serious and growing threat to the worldwide public health. The expression of resistance determinants is exquisitely modulated by the abundant regulatory proteins and the intricate signal sensing and transduction systems in this pathogen. Downregulation of antibiotic influx porin proteins and upregulation of antibiotic efflux pump systems owing to mutational changes in their regulators or the presence of distinct inducing molecular signals represent two of the most efficient mechanisms that restrict intracellular antibiotic accumulation and enable P. aeruginosa to resist multiple antibiotics. Treatment of P. aeruginosa infections is extremely challenging due to the highly inducible mechanism of antibiotic resistance. This review comprehensively summarizes the regulatory networks of the major porin proteins (OprD and OprH) and efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY) that play critical roles in antibiotic influx and efflux in P. aeruginosa. It also discusses promising therapeutic approaches using safe and efficient adjuvants to enhance the efficacy of conventional antibiotics to combat multidrug-resistant P. aeruginosa by controlling the expression levels of porins and efflux pumps. This review not only highlights the complexity of the regulatory network that induces antibiotic resistance in P. aeruginosa but also provides important therapeutic implications in targeting the inducible mechanism of resistance.

M1UKStreptococcus pyogenes causing community-acquired pneumonia, pleural empyema and streptococcal toxic shock syndrome

Streptococcus pyogenes causes superficial infections but can also cause deep-seated infections and toxin-mediated diseases. In the present study, phylogenetic and in silico prediction analyses were performed on an antimicrobial resistant M1UKS. pyogenes strain causing severe clinical manifestations during the current surge of invasive group A Streptococcus (iGAS) disease. A 40-year-old patient was admitted to the hospital with fever, chest pain and fatigue. Based on the clinical and laboratory findings, a diagnosis of sepsis with disseminated intravascular coagulation, community-acquired pneumonia, pleural empyema and streptococcal toxic shock syndrome was made. Microbial identification was performed by multiplex PCR and conventional culturing. Furthermore, antimicrobial susceptibility testing, whole genome sequencing, phylogenomic analysis and in silico prediction analysis of antimicrobial resistance genes and virulence factors were performed. S. pyogenes isolates were detected in pleural fluid and sputum of the patient. Both isolates belonged to the M1UK lineage of the emm1/ST28 clone, being closely related with an M1UK GAS strain from Australia. They exhibited resistance to erythromycin and clindamycin and susceptibility-increased exposure to levofloxacin and carried genes encoding for protein homologues of antibiotic efflux pumps. Moreover, several virulence factors, and a previously described single-nucleotide polymorphism in the 5' transcriptional leader sequence of the ssrA gene, which enhances expression of SpeA, were detected. The present antimicrobial-resistant M1UKS. pyogenes strain represents the first report of this emerging lineage associated with such manifestations of iGAS disease.

Genome-Wide Investigation Reveals Potential Therapeutic Targets in Shigella spp.

Shigella stands as a major contributor to bacterial dysentery worldwide scale, particularly in developing countries with inadequate sanitation and hygiene. The emergence of multidrug-resistant strains exacerbates the challenge of treating Shigella infections, particularly in regions where access to healthcare and alternative antibiotics is limited. Therefore, investigations on how bacteria evade antibiotics and eventually develop resistance could open new avenues for research to develop novel therapeutics. The aim of this study was to analyze whole genome sequence (WGS) of human pathogenic Shigella spp. to elucidate the antibiotic resistance genes (ARGs) and their mechanism of resistance, gene-drug interactions, protein-protein interactions, and functional pathways to screen potential therapeutic candidate(s). We comprehensively analyzed 45 WGS of Shigella, including S. flexneri (n = 17), S. dysenteriae (n = 14), S. boydii (n = 11), and S. sonnei (n = 13), through different bioinformatics tools. Evolutionary phylogenetic analysis showed three distinct clades among the circulating strains of Shigella worldwide, with less genomic diversity. In this study, 2,146 ARGs were predicted in 45 genomes (average 47.69 ARGs/genome), of which only 91 ARGs were found to be shared across the genomes. Majority of these ARGs conferred their resistance through antibiotic efflux pump (51.0%) followed by antibiotic target alteration (23%) and antibiotic target replacement (18%). We identified 13 hub proteins, of which four proteins (e.g., tolC, acrR, mdtA, and gyrA) were detected as potential hub proteins to be associated with antibiotic efflux pump and target alteration mechanisms. These hub proteins were significantly (p < 0.05) enriched in biological process, molecular function, and cellular components. Therefore, the finding of this study suggests that human pathogenic Shigella strains harbored a wide range of ARGs that confer resistance through antibiotic efflux pumps and antibiotic target modification mechanisms, which must be taken into account to devise and formulate treatment strategy against this pathogen. Moreover, the identified hub proteins could be exploited to design and develop novel therapeutics against MDR pathogens like Shigella.

Liposomal nanoformulations with trans-caryophyllene and caryophyllene oxide: do they have an inhibitory action on the efflux pumps NorA, Tet(K), MsrA, and MepA?

This study aimed to evaluate the antibacterial and inhibitory action of NorA, Tet(K), MsrA and MepA efflux pumps in S. aureus strains using the sesquiterpenes named trans-caryophyllene and caryophyllene oxide, both isolated and encapsulated in liposomes. The antibacterial and inhibitory action of these efflux pumps was evaluated through the serial microdilution test in 96-well microplates. Each sesquiterpene and liposome/sesquiterpene was combined with antibiotics and ethidium bromide (EtBr). The antibiotics named norfloxacin, tetracycline and erythromycin were used. The 1199 B, IS-58, RN4220 and K2068 S. aureus strains carrying NorA, Tet(K), MsrA and MepA, respectively, were tested. In the fluorescence measurement test, K2068 S. aureus was incubated with the sesquiterpenes and EtBr, and the fluorescence emission by EtBr was measured. The tested substances did not show direct antibacterial activity, with MIC >1024 μg/mL. Nonetheless, the isolated trans-caryophyllene and caryophyllene oxide reduced the MIC of antibiotics and EtBr, indicating inhibition of NorA, Tet(K) and MsrA. In the fluorescence test, these same sesquiterpenes increased fluorescence emission, indicating inhibition of MepA. Therefore, the sesquiterpenes named trans-caryophyllene and caryophyllene oxide did not show direct antibacterial action; however, in their isolated form, they showed possible inhibitory action on NorA, Tet(K), MsrA and MepA efflux pumps. They may also act in antibiotic potentiation. Further studies are needed to identify the mechanisms involved in antibiotic potentiation and efflux pump inhibitory action.

Genomic Features and Pathophysiological Impact of a Multidrug-Resistant Staphylococcus warneri Variant in Murine Mastitis

Non-aureus staphylococci (NAS) represent a major etiological factor in dairy animal mastitis, yet their role and impact remain insufficiently studied. This study aimed to elucidate the genomic characteristics of a newly identified multidrug-resistant NAS strain, specifically Staphylococcus warneri G1M1F, isolated from murine feces in an experimental mastitis model. Surprisingly, NAS species accounted for 54.35% of murine mastitis cases, with S. warneri being the most prevalent at 40.0%. S. warneri G1M1F exhibited resistance to 10 major antibiotics. Whole-genome sequencing established a genetic connection between G1M1F and S. warneri strains isolated previously from various sources including mastitis milk in dairy animals, human feces and blood across diverse geographical regions. Genomic analysis of S. warneri G1M1F unveiled 34 antimicrobial resistance genes (ARGs), 30 virulence factor genes (VFGs), and 278 metabolic features. A significant portion of identified ARGs (64%) conferred resistance through antibiotic efflux pumps, while VFGs primarily related to bacterial adherence and biofilm formation. Inoculation with G1M1F in mice resulted in pronounced inflammatory lesions in mammary and colon tissues, indicating pathogenic potential. Our findings highlight distinctive genomic traits in S. warneri G1M1F, signifying the emergence of a novel multidrug-resistant NAS variant. These insights contribute to understanding NAS-related mastitis pathophysiology and inform strategies for effective treatment in dairy animals.

Distribution characteristics of antibiotic resistance in direct-eating food and analysis of Citrobacter freundii genome and pathogenicity

Purpose This study aims to study the distribution characteristics of antibiotic resistance in direct-eating food and analysis of Citrobacter freundii genome and pathogenicity. Residual antibiotics and antibiotic resistance genes (ARGs) in the environment severely threaten human health and the ecological environment. The diseases caused by foodborne pathogenic bacteria are increasing daily, and the enhancement of antibiotic resistance of pathogenic bacteria poses many difficulties in the treatment of disease. Design/methodology/approach In this study, six fresh fruits and vegetable samples were selected for isolation and identification of culturable bacteria and analysis of antibiotic resistance. The whole genome of Citrobacter freundii isolated from cucumber was sequenced and analyzed by Oxford Nanopore sequencing. Findings The results show that 270 strains of bacteria were identified in 6 samples. From 12 samples of direct food, 2 kinds of probiotics and 10 kinds of opportunistic pathogens were screened. The proportion of Citrobacter freundii screened from cucumber was significantly higher than that from other samples, and it showed resistance to a variety of antibiotics. Whole genome sequencing showed that Citrobacter freundii was composed of a circular chromosome containing signal peptides, transmembrane proteins and transporters that could induce antibiotic efflux, indicating that Citrobacter freundii had strong adaptability to the environment. The detection of genes encoding carbohydrate active enzymes is more beneficial to the growth and reproduction of Citrobacter freundii in crops. A total of 29 kinds of ARGs were detected in Citrobacter freundii, mainly conferring resistance to fluoroquinolones, aminoglycosides, carbapenem, cephalosporins and macrolides. The main mechanisms are the change in antibiotic targets and efflux pumps, the change in cell permeability and the inactivation of antibiotics and the detection of virulence factors and ARGs, further indicating the serious risk to human health. Originality/value The detection of genomic islands and prophages increases the risk of horizontal transfer of virulence factors and ARGs, which spreads the drug resistance of bacteria and pathogenic bacteria more widely.

Escherichia coli resistance mechanism AcrAB-TolC efflux pump interactions with commonly used antibiotics: a molecular dynamics study

While antibiotic resistance poses a threat from both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB), GNB pose a more imminent public health hazard globally. GNB are a threat to growing antibiotic resistance because of the complex makeup of the membrane. The AcrAB-TolC efflux pump is a known resistance mechanism of Escherichia coli (E. coli) cells. This study utilized molecular dynamics modeling to visualize some of the changes occurring at a molecular level when airborne bacteria are exposed to stress and antibiotics. This study was conducted to build upon previous experimental research showing that there is an increase in antibiotic resistance and efflux pump activity when exposed to aerosolization. AcrB and AcrAB-TolC proteins were simulated under standard and increased pressure to compare the effect of aerosolization on the binding to the three different antibiotics (puromycin (PUY), ampicillin (AMP) and sulfamethoxazole-trimethoprim (SXT)) to the AcrB binding site. Analysis such as root-mean-square deviation of atomic positions and root-mean-square fluctuation, the opening of TolC, and the significant molecular mechanics with generalized Born and surface area solvation (MM-GBSA) scores associated with specific ligands were recorded. Resistance in experimental data indicated a relationship between the docking scores and some ligand–protein interactions. Results showed that there was more flexibility in the proteins within simulations conducted under standard pressure for the AcrB protein and the full tripartite complex AcrAB-TolC, showing that increased pressure causes more rigidity. MM-GBSA scores, used to calculate the free energy of ligand–protein binding, did not show a significant change, but interestingly, the strongest MM-GBSA scores were for ligands that moved to another binding pocket and did not result in resistance or opening of the efflux pump. However, the ligand moved from the binding site and did not cause the opening of TolC to increase significantly, whereas PUY and AMP were bound to the binding site for the duration of all simulations. AMP ligands under increased pressure showed the largest change in opening of the TolC efflux pump and aligns with experimental data showing E. coli cells had the most resistance to AMP after aerosolization. These results, in addition to other real-time changes such as OM proteins and mutations of targets within the cell, could be used to delineate and mitigate antibiotic resistance mechanisms.

Stenotrophomonas maltophilia complex: insights into evolutionary relationships, global distribution and pathogenicity.

Stenotrophomonas maltophilia complex (Smc) comprises opportunistic Gram-negative bacilli responsible for various nosocomial infections. Limited data exists concerning its evolutionary lineage, global prevalence and pathogenicity. We conducted an extensive genomic analysis on 734 Smc genomes, of which 90 were newly sequenced and isolated from different patients. The species composition and evolutionary relationships of Smc were examined using core protein sequence analysis. Pathogenicity evaluation was used by assays for swimming motility, biofilm formation and identification of virulence factors. The broth microdilution method was used to evaluate the drug resistance spectrum of clinical isolates. Phylogenetic analyses delineated 24 species-level clades, dominated by S. maltophilia (42.8%), S. sepilia (13.6%) and S. geniculata (9.9%). Geographically, strains were primarily distributed in Europe (34.2%), Asia (33.7%) and North America (24.0%), with intricate global distribution patterns. Meanwhile, 154 virulence-associated genes and 46 antimicrobial resistance genes within Smc were identified. These genes encoded span various functions, including motility, adherence, toxin, RND antibiotic efflux pumps, beta-lactamases and aminoglycoside-modifying enzymes. Moreover, significant variations were indicated in swimming motility and biofilm-forming capability across the different species, with S. sepilia exhibiting superior levels of both traits. Additionally, no statistically significant discrepancy was detected among Smc species to other antibiotics, despite the fact that all S. geniculata isolates were resistant to Ceftazidime and much higher than other species. Our findings indicate the need to pay increased attention to other mainstream species of Smc besides S. maltophilia in order to better manage Smc-related infections and tailor effective treatment strategies.

Metatranscriptomic analysis shows functional alterations in subgingival biofilm in young smokers with periodontitis: a pilot study.

This study aimed to assess the influence of smoking on the subgingival metatranscriptomic profile of young patients affected by stage III/IV and generalized periodontal disease. In total, six young patients, both smokers and non-smokers (n=3/group), who were affected by periodontitis were chosen. The STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines for case-control reporting were followed. Periodontal clinical measurements and subgingival biofilm samples were collected. RNA was extracted from the biofilm and sequenced via Illumina HiSeq. Differential expression analysis used Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and differentially expressed genes were identified using the Sleuth package in R, with a statistical cutoff of ≤0.05. This study found 3351 KEGGs in the subgingival biofilm of both groups. Smoking habits altered the functional behavior of subgingival biofilm, resulting in 304 differentially expressed KEGGs between groups. Moreover, seven pathways were modulated: glycan degradation, galactose metabolism, glycosaminoglycan degradation, oxidative phosphorylation, peptidoglycan biosynthesis, butanoate metabolism, and glycosphingolipid biosynthesis. Smoking also altered antibiotic resistance gene levels in subgingival biofilm by significantly overexpressing genes related to beta-lactamase, permeability, antibiotic efflux pumps, and antibiotic-resistant synthetases. Due to the limitations of a small sample size, our data suggest that smoking may influence the functional behavior of subgingival biofilm, modifying pathways that negatively impact the behavior of subgingival biofilm, which may lead to a more virulent community.

Comparative molecular profiling of multidrug-resistant Pseudomonas aeruginosa identifies novel mutations in regional clinical isolates from South India.

We sought to analyse the antibiotic susceptibility profiles and molecular epidemiology of MDR clinical Pseudomonas aeruginosa isolates from South India using non-MDR isolates as a reference. We established a comprehensive clinical strain library consisting of 58 isolates collected from patients across the South Indian state of Kerala from March 2017 to July 2019. The strains were subject to antibiotic susceptibility testing, modified carbapenem inactivation method assay for carbapenemase production, PCR sequencing, comparative sequence analysis and quantitative PCR of MDR determinants associated with antibiotic efflux pump systems, fluoroquinolone resistance and carbapenem resistance. We performed in silico modelling of MDR-specific SNPs. Of our collection of South Indian P. aeruginosa clinical isolates, 74.1% were MDR and 55.8% were resistant to the entire panel of antibiotics tested. All MDR isolates were resistant to levofloxacin and 93% were resistant to meropenem. We identified seven distinct, MDR-specific mutations in nalD, three of which are novel. mexA was significantly overexpressed in strains that were resistant to the entire test antibiotic panel while gyrA and gyrB were overexpressed in MDR isolates. Mutations in fluoroquinolone determinants were significantly associated with MDR phenotype and a novel GyrA Y100C substitution was observed. Carbapenem resistance in MDR isolates was associated with loss-of-function mutations in oprD and high prevalence of NDM (blaNDM-1) within our sample. This study provides insight into MDR mechanisms adopted by P. aeruginosa clinical isolates, which may guide the potential development of therapeutic regimens to improve clinical outcomes.