Antibiotic Resistance-related Genes Research Articles

Characterization and Genome Analysis of the Delftia lacustris Strain LzhVag01 Isolated from Vaginal Discharge

Delftia has been separated from freshwater, sludge, and soil and has emerged as a novel opportunistic pathogen in the female vagina. However, the genomic characteristics, pathogenicity, and biotechnological properties still need to be comprehensively investigated. In this study, a Delftia strain was isolated from the vaginal discharge of a 43-year-old female with histologically confirmed cervical intraepithelial neoplasm (CIN III), followed by whole-genome sequencing. Phylogenetic analysis and average nucleotide identity (ANI) analysis demonstrated that it belongs to Delftia lacustris, named D. lacustris strain LzhVag01. LzhVag01 was sensitive to β-lactams, macrolides, and tetracyclines but exhibited resistance to lincoamines, nitroimidazoles, aminoglycosides, and fluoroquinolones. Its genome is a single, circular chromosome of 6,740,460 bp with an average GC content of 66.59%. Whole-genome analysis identified 16 antibiotic resistance-related genes, which match the antimicrobial susceptibility profile of this strain, and 11 potential virulence genes. These pathogenic factors may contribute to its colonization in the vaginal environment and its adaptation and accelerate the progression of cervical cancer. This study sequenced and characterized the whole-genome of Delftia lacustris isolated from vaginal discharge, which provides investigators and clinicians with valuable insights into this uncommon species.

Genomic and Transcriptomic Analyses Reveal Multiple Strategies for Vibrio parahaemolyticus to Tolerate Sub-Lethal Concentrations of Three Antibiotics.

Vibrio parahaemolyticus can cause acute gastroenteritis, wound infections, and septicemia in humans. The overuse of antibiotics in aquaculture may lead to a high incidence of the multidrug-resistant (MDR) pathogen. Nevertheless, the genome evolution of V. parahaemolyticus in aquatic animals and the mechanism of its antibiotic tolerance remain to be further deciphered. Here, we investigated the molecular basis of the antibiotic tolerance of V. parahaemolyticus isolates (n = 3) originated from shellfish and crustaceans using comparative genomic and transcriptomic analyses. The genome sequences of the V. parahaemolyticus isolates were determined (5.0-5.3 Mb), and they contained 4709-5610 predicted protein-encoding genes, of which 823-1099 genes were of unknown functions. Comparative genomic analyses revealed a number of mobile genetic elements (MGEs, n = 69), antibiotic resistance-related genes (n = 7-9), and heavy metal tolerance-related genes (n = 2-4). The V. parahaemolyticus isolates were resistant to sub-lethal concentrations (sub-LCs) of ampicillin (AMP, 512 μg/mL), kanamycin (KAN, 64 μg/mL), and streptomycin (STR, 16 μg/mL) (p < 0.05). Comparative transcriptomic analyses revealed that there were significantly altered metabolic pathways elicited by the sub-LCs of the antibiotics (p < 0.05), suggesting the existence of multiple strategies for antibiotic tolerance in V. parahaemolyticus. The results of this study enriched the V. parahaemolyticus genome database and should be useful for controlling the MDR pathogen worldwide.

Growth and Genome Features of Non-O1/O139 Vibrio cholerae Isolated from Three Species of Common Freshwater Fish

Vibrio cholerae is the etiological agent of cholera in humans. The bacterium is frequently detected in aquatic products worldwide. However, the current literature on the genome evolution of V. cholerae of aquatic animal origins is limited. Here, we firstly characterized the growth and genome features of V. cholerae isolates with different resistance phenotypes from three species of common freshwater fish. The results revealed that the non-O1/O139 V. cholerae isolates (n = 4) were halophilic and grew optimally at 2% NaCl and pH 8.0. Their draft genome sequences were 3.89 Mb–4.15 Mb with an average GC content of 47.35–47.63%. Approximately 3366–3561 genes were predicted to encode proteins, but 14.9–17.3% of them were of an unknown function. A number of strain-specific genes (n = 221–311) were found in the four V. cholerae isolates, 3 of which belonged to none of any of the known sequence types (STs). Several putative mobile genetic elements (MGEs) existed in the V. cholerae isolates, including genomic islands (n = 4–9), prophages (n = 0–3), integrons (n = 1–1), and insertion sequences (n = 0–3). Notably, CRISPR-Cas system arrays (n = 2–10) were found in the V. cholerae genomes, whereby the potential immunity defense system could be active. Comparative genomic analyses also revealed many putative virulence-associated genes (n = 106–122) and antibiotic resistance-related genes (n = 6–9). Overall, the results of this study demonstrate the bacterial broader-spectrum growth traits and fill prior gaps in the genomes of V. cholerae originating from freshwater fish.

Epidemiological and Genomic analysis of Vibrio parahaemolyticus isolated from imported travelers at the port of Shanghai, China (2017-2019)

BackgroundVibrio parahaemolyticus is the predominant etiological agent of seafood-associated foodborne illnesses on a global scale. It is essential to elucidate the mechanisms by which this pathogen disseminates. Given the existing research predominantly concentrates on localized outbreaks, there is a pressing necessity for a comprehensive investigation to capture strains of V. parahaemolyticus cross borders.ResultsThis study examined the frequency and genetic attributes of imported V. parahaemolyticus strains among travelers entering Shanghai Port, China, between 2017 and 2019.Through the collection of 21 strains from diverse countries and regions, Southeast Asia was pinpointed as a significant source for the emergence of V. parahaemolyticus. Phylogenetic analysis revealed clear delineation between strains originating from human and environmental sources, emphasizing that underlying genome data of foodborne pathogens is essential for environmental monitoring, food safety and early diagnosis of diseases. Furthermore, our study identified the presence of virulence genes (tdh and tlh) and approximately 120 antibiotic resistance-related genes in the majority of isolates, highlighting their crucial involvement in the pathogenesis of V. parahaemolyticus.ConclusionsThis research enhanced our comprehension of the worldwide transmission of V. parahaemolyticus and its antimicrobial resistance patterns. The findings have important implications for public health interventions and antimicrobial stewardship strategies, underscoring the necessity for epidemiological surveillance of pathogen at international travel hubs.

Unveiling a Listeria monocytogenes Outbreak in a Rabbit Farm: Clinical Manifestation, Antimicrobial Resistance, Genomic Insights and Environmental Investigation.

Listeria monocytogenes poses a threat to both human and animal health. This work describes an L. monocytogenes outbreak in a Portuguese rabbit farm, detailing the isolates' clinical manifestations, necropsy findings, and phenotypic and genomic profiles. Clinical signs, exclusively observed in does, included lethargy and reproductive signs. Post-mortem examination of does revealed splenomegaly, hepatomegaly with a reticular pattern, pulmonary congestion, and haemorrhagic lesions in the uterus, with thickening of the uterine wall and purulent greyish exudates. Positive L. monocytogenes samples were identified in fattening and maternity units across different samples, encompassing does and environmental samples. Core-genome Multi Locus Sequence Typing (cgMLST) analysis confirmed the outbreak, with the 16 sequenced isolates (lineage II, CC31, and ST325) clustering within a ≤2 allelic difference (AD) threshold. Antimicrobial susceptibility testing for five antibiotics revealed that 15 out of 19 outbreak isolates were resistant to sulfamethoxazole-trimethoprim (SXT). Concordantly, all SXT-resistant sequenced isolates were found to exclusively harbour a plasmid containing a trimethoprim-resistance gene (dfrD), along with loci linked to resistance to lincosamides (lnuG), macrolides (mphB), and polyether ionophores (NarAB operon). All sequenced outbreak isolates carried the antibiotic resistance-related genes tetM, fosX, lin, norB, lmrB, sul, and mprF. The outbreak cluster comprises isolates from does and the environment, which underscores the ubiquitous presence of L. monocytogenes and emphasizes the importance of biosecurity measures. Despite limited data on listeriosis in rabbit farming, this outbreak reveals its significant impact on animal welfare and production.

Comparative genomics of four lactic acid bacteria identified with Vitek MS (MALDI-TOF) and whole-genome sequencing

Lactic acid bacteria (LAB) can be used as a probiotic or starter culture in dairy, meat, and vegetable fermentation. Therefore, their isolation and identification are essential. Recent advances in omics technologies and high-throughput sequencing have made the identification and characterization of bacteria. This study firstly aimed to demonstrate the sensitivity of the Vitek MS (MALDI-TOF) system in the identification of lactic acid bacteria and, secondly, to characterize bacteria using various bioinformatics approaches. Probiotic potency-related genes and secondary metabolite biosynthesis gene clusters were examined. The Vitek MS (MALDI-TOF) system was able to identify all of the bacteria at the genus level. According to whole genome sequencing, the bacteria were confirmed to be Lentilactobacillus buchneri, Levilactobacillus brevis, Lactiplantibacillus plantarum, Levilactobacillus namurensis. Bacteria had most of the probiotic potency-related genes, and different toxin-antitoxin systems such as PemIK/MazEF, Hig A/B, YdcE/YdcD, YefM/YoeB. Also, some of the secondary metabolite biosynthesis gene clusters, some toxic metabolite-related genes, and antibiotic resistance-related genes were detected. In addition, Lentilactobacillus buchneri Egmn17 had a type II-A CRISPR/Cas system. Lactiplantibacillus plantarum Gmze16 had a bacteriocin, plantaricin E/F.

Effect of Temperature on Carbapenemase-Encoding Plasmid Transfer in Klebsiella pneumoniae.

Bacteria causing human infections can develop antibiotic resistance due to various factors. Temperature affects bacterial growth and gene transfer; however, studies exploring the association between the changes in local temperature and antibiotic resistance are limited. Here, we investigated the effects of local temperatures on the distribution of antibiotic resistance and transmission of carbapenemase-producing Enterobacterales using the data on Klebsiella pneumoniae from sentinel hospitals in eight regions included in the Korea Global Antimicrobial Resistance Surveillance System between 2017 and 2021. The resistance rates to most antibiotics, including carbapenems, varied significantly according to local temperature (p < 0.047), except for aminoglycosides. Conjugation experiments at various temperatures for strains encoding the carbapenemase gene on a plasmid revealed significant variation in the optimal conjugation temperatures for plasmids carrying blaKPC and blaNDM genes. The optimal conjugation temperatures demonstrating the highest stability for blaKPC- and blaNDM-carrying plasmids were 25 °C (p = 0.030) and 30 °C (p = 0.007), respectively. The stability of blaKPC-IncF was higher at 25 °C than that at 30 °C (p = 0.032) or 37 °C (p = 0.047), while blaKPC-IncX3 exhibited the lowest stability at 37 °C (p = 0.047). blaNDM-IncX3 was more stable at 30 °C than at 37 °C (p = 0.049). These findings suggest that the optimal temperature for carbapenemase gene transmission varied between 25 °C and 30 °C, indicating that warmer seasons promote the transfer of more antibiotic resistance-related genes and highlighting the importance of local temperature in the spread and transmission of plasmids carrying carbapenemases.

Complete genome sequence and comparative analysis of a Vibrio vulnificus strain isolated from a clinical patient.

Vibrio vulnificus is an opportunistic, global pathogen that naturally inhabits sea water and is responsible for most vibriosis-related deaths. We investigated the genetic characteristics of V. vulnificus isolated from the clinical blood culture specimen of a patient with hepatitis B virus cirrhosis in 2018 (named as V. vulnificus VV2018) by whole genome sequencing (WGS). VV2018 belonged to a novel sequencing type 620 (ST620) and comprised two circular chromosomes, containing 4,389 potential coding sequences (CDSs) and 152 RNA genes. The phylogenetic tree of single nucleotide polymorphisms (SNPs) using 26 representative genomes revealed that VV2108 grouped with two other V. vulnificus strains isolated from humans. The pan-genome of V. vulnificus was constructed using 26 representative genomes to elucidate their genetic diversity, evolutionary characteristics, and virulence and antibiotic resistance profiles. The pan-genome analysis revealed that VV2018 shared a total of 3,016 core genes (≥99% presence), including 115 core virulence factors (VFs) and 5 core antibiotic resistance-related genes, and 309 soft core genes (≥95 and <99% presence) with 25 other V. vulnificus strains. The varG gene might account for the cefazolin resistance, and comparative analysis of the genetic context of varG revealed that two genes upstream and downstream of varG were conserved. The glycosylation (pgl) like genes were found in VV2018 compared with Pgl-related proteins in Neisseria that might affect the adherence of the strain in hosts. The comparative analysis of VV2018 would contribute to a better understanding of the virulence and antibiotic resistance profiles of V. vulnificus. Meanwhile much work remains to be done to better understand the function of pgl-like genes in V. vulnificus.

Impacts of norfloxacin on sewage sludge anaerobic digestion: Bioenergy generation and potential environmental risks

Norfloxacin is a broad-spectrum antibiotic which has been frequently detected in the sewage sludge. However, whether norfloxacin in sewage sludge would affect the anaerobic digestion for bioenergy generation and induce environmental risks remained unclear. Therefore, this study investigated the impact of norfloxacin on sewage sludge anaerobic digestion and evaluated potential environmental risks by exploring related antibiotic resistance genes (ARGs) and norfloxacin transformation pathways. The results showed that norfloxacin at environmental relevant concentration ranges would not significantly affect methane production (p < 0.05) during the sludge anaerobic digestion, as the sludge solubilization and hydrolysis is unaffected. However, the individual methanogenesis process was enhanced with the increase of Methanosaeta abundances. Meanwhile, the increase of norfloxacin concentration will not decrease the removal efficiencies of fluoroquinolones-family ARGs by the anaerobic digestion process. However, the norfloxacin concentration is found to be increased during the anaerobic digestion process, which could probably be due to the desorption from the sludge due to cell lysis. In addition, 13 norfloxacin transformation metabolites were identified with 5 potential norfloxacin degradation pathways being constructed. The metabolites of levofloxacin/ofloxacin could be generated through Eschweiler-Clarke reaction, which may be more toxic than norfloxacin. Thus, the results of this study suggested that although norfloxacin would not affect the bioenergy generation and ARG removal during sludge anaerobic digestion, measures should be taken to deal with the potential environmental risks caused by norfloxacin release and formation of more toxic metabolites.

Mesomycoplasma ovipneumoniae from goats with respiratory infection: pathogenic characteristics, population structure, and genomic features

BackgroundMycoplasma ovipneumoniae is a critical pathogen that causes respiratory diseases that threaten Caprini health and cause economic damage. A genome-wide study of M. ovipneumoniae will help understand the pathogenic characteristics of this microorganism.ResultsToxicological pathology and whole-genome sequencing of nine M. ovipneumoniae strains isolated from goats were performed using an epidemiological survey. These strains exhibited anterior ventral lung consolidation, typical of bronchopneumonia in goats. Average nucleotide identity and phylogenetic analysis based on whole-genome sequences showed that all M. ovipneumoniae strains clustered into two clades, largely in accordance with their geographical origins. The pan-genome of the 23 M. ovipneumoniae strains contained 5,596 genes, including 385 core, 210 soft core, and 5,001 accessory genes. Among these genes, two protein-coding genes were annotated as cilium adhesion and eight as paralog surface adhesins when annotated to VFDB, and no antibiotic resistance-related genes were predicted. Additionally, 23 strains carried glucosidase-related genes (ycjT and group_1595) and glucosidase-related genes (atpD_2), indicating that M. ovipneumoniae possesses a wide range of glycoside hydrolase activities.ConclusionsThe population structure and genomic features identified in this study will facilitate further investigations into the pathogenesis of M. ovipneumoniae and lay the foundation for the development of preventive and therapeutic methods.

Characterization of bacteriophage BUCT631 lytic for K1 Klebsiella pneumoniae and its therapeutic efficacy in Galleria mellonella larvae

Severe infections caused by multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) highlight the need for new therapeutics with activity against this pathogen. Phage therapy is an alternative treatment approach for multidrug-resistant K. pneumoniae infections. Here, we report a novel bacteriophage (phage) BUCT631 that can specifically lyse capsule-type K1 K. pneumoniae. Physiological characterization revealed that phage BUCT631 could rapidly adsorb to the surface of K. pneumoniae and form an obvious halo ring, and it had relatively favorable thermal stability (4–50 ​°C) and pH tolerance (pH ​= ​4–12). In addition, the optimal multiplicity of infection (MOI) of phage BUCT631 was 0.01, and the burst size was approximately 303 ​PFU/cell. Genomic analysis showed that phage BUCT631 has double-stranded DNA (total length of 44,812 bp) with a G ​+ ​C content of 54.1%, and the genome contains 57 open reading frames (ORFs) and no virulence or antibiotic resistance related genes. Based on phylogenetic analysis, phage BUCT631 could be assigned to a new species in the genus Drulisvirus of the subfamily Slopekvirinae. In addition, phage BUCT631 could quickly inhibit the growth of K. pneumoniae within 2 ​h in vitro and significantly elevated the survival rate of K. pneumoniae infected Galleria mellonella larvae from 10% to 90% in vivo. These studies suggest that phage BUCT631 has promising potential for development as a safe alternative for control and treatment of multidrug-resistant K. pneumoniae infection.

Occurrence and distribution of antibiotics and antibiotic resistance genes in the different croplands along the Yellow River shoreline

Many antibiotics and their related antibiotic resistance genes (ARGs) are found in agricultural soil, raising concerns for public and environmental health. As a result, it is crucial to examine the abundance of sulfonamides, fluoroquinolones, tetracyclines, macrolides, and β-lactam antibiotics and their corresponding ARGs in agricultural soils. We investigted the abundance of aadA, sul1, aac(6’)-Ib, tetX, tetA, ermB, mphB, qnrA, intI1, intI2, and ISCR1 genes and explore their relationships with antibiotics in rice, peach orchard, maize, peanut, and garlic croplands along the Yellow River in Zhongmu and Yuanyang, Henan Province. There was no significant difference across the five croplands (P > 0.05), however, the paddy field had the highest average concentration (>10 μg kg−1) of oxytetracycline, tetracycline, ciprofloxacin and norfloxacin antibiotics. The predominant ARGs were aadA, sul1, with the the highest number of ARGs and MGEs detected in paddy rice, followed by maize, peanut, garlic, and peach soils. Integrase gene intI1 was significantly greater than intI2 and ISCR1, which was positively linked with sul1, aadA, aac(6′)-Ib, tetA, tetX, and ermB genes (P < 0.05). According to redundancy analysis, antibiotics may accelerated the prevalence of ARGs. Redundancy analysis demonstrated that antibiotics contributed 78.7% of the total ARG variations. Notably, the concentration of oxytetracycline in all the croplands ranged from 10.33 to 14.42 μg kg−1, which had impacted the frequency of ermB, bla CTX-M, intI2, and mphB genes as demonstrated by RDA analysis. Results revealed that effective techniques are required to lower the prevalence of ARGs in the agroecosystems.

Host receptor identification of a polyvalent lytic phage GSP044, and preliminary assessment of its efficacy in the clearance of Salmonella

Salmonella and pathogenic Escherichia coli are important foodborne pathogens. Phages are being recognized as potential antibacterial agents to control foodborne pathogens. In the current study, a polyvalent broad-spectrum phage, GSP044, was isolated from pig farm sewage. It can simultaneously lyse many different serotypes of Salmonella and E. coli, exhibiting a broad host range. Using S. Enteritidis SE006 as the host bacterium, phage GSP044 was further characterized. GSP044 has a short latent period (10 min), high stability at different temperatures and pH, and good tolerance to chloroform. Genome sequencing analysis revealed that GSP044 has a double-stranded DNA (dsDNA) genome consisting of 110,563 bp with G + C content of 39%, and phylogenetic analysis of the terminase large subunit confirmed that GSP044 belonged to the Demerecviridae family, Epseptimavirus genus. In addition, the genomic sequence did not contain any lysogenicity-related, virulence-related, or antibiotic resistance-related genes. Analysis of phage-targeted host receptors revealed that the outer membrane protein (OMP) BtuB was identified as a required receptor for phage infection of host bacteria. The initial application capability of phage GSP044 was assessed using S. Enteritidis SE006. Phage GSP044 could effectively reduce biofilm formation and degrade the mature biofilm in vitro. Moreover, GSP044 significantly decreased the viable counts of artificially contaminated S. Enteritidis in chicken feed and drinking water. In vivo tests, a mouse model of intestinal infection demonstrated that phage GSP044 was able to reduce the number of colonized S. Enteritidis in the intestine. These results suggest that phage GSP044 may be a promising candidate biologic agent for controlling Salmonella infections.

Comparative Genomics of Pseudomonas aeruginosa Strains Isolated from Different Ecological Niches.

The Pseudomonas aeruginosa genome can change to adapt to different ecological niches. We compared four genomes from a Mexican hospital and 59 genomes from GenBank from different niches, such as urine, sputum, and environmental. The ST analysis showed that high-risk STs (ST235, ST773, and ST27) were present in the genomes of the three niches from GenBank, and the STs of Mexican genomes (ST167, ST2731, and ST549) differed from the GenBank genomes. Phylogenetic analysis showed that the genomes were clustering according to their ST and not their niche. When analyzing the genomic content, we observed that environmental genomes had genes involved in adapting to the environment not found in the clinics and that their mechanisms of resistance were mutations in antibiotic resistance-related genes. In contrast, clinical genomes from GenBank had resistance genes, in mobile/mobilizable genetic elements in the chromosome, except for the Mexican genomes that carried them mostly in plasmids. This was related to the presence of CRISPR-Cas and anti-CRISPR; however, Mexican strains only had plasmids and CRISPR-Cas. blaOXA-488 (a variant of blaOXA50) with higher activity against carbapenems was more prevalent in sputum genomes. The virulome analysis showed that exoS was most prevalent in the genomes of urinary samples and exoU and pldA in sputum samples. This study provides evidence regarding the genetic variability among P. aeruginosa isolated from different niches.

Metagenomics insights into gentamicin degradation and the dynamic of antibiotic resistance genes during co-composting of the gentamicin myelial residues with addition of various organic wastes

Increasing antibiotic mycelial residues (AMRs) and related antibiotic resistance genes (ARGs) pose a significant threat to ecosystems and public health. Composting is a crucial method for recycling AMRs. However, the variation in ARGs and gentamicin degradation in the composting process of gentamicin mycelial residues (GMRs) has received little attention on an actual industrial scale. This research investigated the metabolic pathways and functional genes on the gentamicin and ARGs removal during the co-composting of GMRs with addition of various organic wastes (rice chaff, mushroom residue, etc.) under various C/N ratios (15:1, 25:1, 35:1). The results showed that the removal efficiencies of gentamicin and the total ARGs were 98.23 % and 53.20 %, respectively, with the C/N ratio of 25:1. Moreover, metagenomics and LS-MS/MS analysis demonstrated that the acetylation was the primary pathway for gentamicin biodegradation and the corresponding degrading genes were the categories of aac(3) and aac(6′). However, the relative abundance of aminoglycoside resistance genes (AMGs) were increased after 60 days composting. The partial least squares path modeling analysis demonstrated that the AMG abundance was directly influenced by the predominant mobile gene elements intI1 (p < 0.05) which was closely related to the bacterial community composition. Therefore, the ecological environmental risks should be assessed in the future application of GMRs composting products.

Graphene nano zinc oxide reduces the expression and release of antibiotic resistance-related genes and virulence factors in animal manure

Animal manure contains many antibiotic resistance genes (ARGs) and virulence factors (VFs), posing significant health threats to humans. However, the effects of graphene nano zinc oxide (GZnONP), a zinc bioaugmentation substitute, on bacterial chemotaxis, ARGs, and VFs in animal manure remain scanty. Herein, the effect of GZnONP on the in vivo anaerobic expression of ARGs and VFs in cattle manure was assessed using high-throughput sequencing. Results showed that GZnONP inhibited bacterial chemotaxis by reducing the zinc pressure under anaerobic fermentation, altering the microbial community structure. The expression of ARGs was significantly lower in GZnONP than in zinc oxide and nano zinc oxide (ZnONP) groups. The expression of VFs was lower in the GZnONP than in the zinc oxide and ZnONP groups by 9.85 % and 13.46 %, respectively. Co-occurrence network analysis revealed that ARGs and VFs were expressed by the Spirochaetes phylum, Paraprevotella genus, and Treponema genus et al. The ARGs-VFs coexistence was related to the expression/abundance of ARGs and VFs genes. GZnONP reduces the abundance of certain bacterial species by disrupting chemotaxis, minimizing the transfer of ARGs and VFs. These findings suggest that GZnONP, a bacterial chemotaxis suppressor, effectively reduces the expression and release of ARGs and VFs in animal manure.

Whole genome sequencing reveals novel genetic mutations of Helicobacter pylori associating with resistance to clarithromycin and levofloxacin.

Detection of mutations in one or a couple of genes may not provide enough data or cover all the genomic DNA variance related to antibiotic resistance of Helicobacter pylori to clarithromycin (CLA) and levofloxacin (LVX). We aimed to perform whole genome sequencing to explore novel antibiotic resistance-related genes to increase predictive accuracy for future targeted sequencing tests. Gastric mucosal biopsies were taken during upper endoscopy in 27 H. pylori-infected patients. According to culture-based antibacterial susceptibility test, H. pylori strains were divided into three groups, with nine strains in each group: CLA single-drug resistance (group C), LVX single-drug resistance (group L), and strains sensitive to all antibacterial drugs (group S). Based on whole genome sequencing with group S being the control, group C and group L group-specific single nucleotide variants and amino acid mutations were screened, and potential candidate genes related to CLA and LVX resistance were identified. The median age of study subjects was 35 years (IQR: 31-40), and 17 (63.0%) were male. All nine CLA-resistant strains had A2143G mutations in 23S rRNA, while none of nine sensitive strains had the mutation. Six of nine strains in group L and six of nine strains in group S had 87th or 91st mutation in gyrA. After comparing sequencing data of strains among the three groups, we identified five mutated positions belonging to four genes related to CLA resistance, and 31 mutated positions belonging to 20 genes related to LVX resistance. Novel genetic mutations were detected for CLA resistance (including fliJ and clpX) and LVX resistance (including fliJ, cheA, hemE, Val360Ile, and HP0568). Missense mutations in fliJ and cheA gene were mainly involved in chemotaxis and flagellar motility to facilitate bacterial escape of antibiotics, while the functions of other novel gene mutations underpinning antibiotic resistance remain to be investigated. Whole genome sequencing detected potential novel genetic mutations conferring resistance of H. pylori to CLA and LVX including fliJ and cheA. Further studies to correlate these findings with treatment outcome should be performed.

Characterization and genome analysis of phage vB_KpnS_SXFY507 against Klebsiella pneumoniae and efficacy assessment in Galleria mellonella larvae.

Carbapenem-resistant Klebsiella pneumoniae is one of the primary bacterial pathogens that pose a significant threat to global public health because of the lack of available therapeutic options. Phage therapy shows promise as a potential alternative to current antimicrobial chemotherapies. In this study, we isolated a new Siphoviridae phage vB_KpnS_SXFY507 against KPC-producing K. pneumoniae from hospital sewage. It had a short latent period of 20 min and a large burst size of 246 phages/cell. The host range of phage vB_KpnS_SXFY507 was relatively broad. It has a wide range of pH tolerance and high thermal stability. The genome of phage vB_KpnS_SXFY507 was 53,122 bp in length with a G + C content of 49.1%. A total of 81 open-reading frames (ORFs) and no virulence or antibiotic resistance related genes were involved in the phage vB_KpnS_SXFY507 genome. Phage vB_KpnS_SXFY507 showed significant antibacterial activity in vitro. The survival rate of Galleria mellonella larvae inoculated with K. pneumoniae SXFY507 was 20%. The survival rate of K. pneumonia-infected G. mellonella larvae was increased from 20 to 60% within 72 h upon treatment with phage vB_KpnS_SXFY507. In conclusion, these findings indicate that phage vB_KpnS_SXFY507 has the potential to be used as an antimicrobial agent for the control of K. pneumoniae.

Response of submerged macrophytes and biofilms to coexisting azithromycin and tetracycline: Antibiotic resistance genes removal, toxicity assessment and microbial properties

Antibiotics, such as azithromycin (AZ), tetracycline (TC), and their related antibiotic resistance genes (ARGs), create serious ecological risks to aquatic organisms. This study examined the response mechanisms of submerged macrophytes and periphytic biofilms to a mixture of AZ and TC pollution and determined the antibiotic removal efficiencies and fate of ARGs. The results showed that the plant-biofilm system had a significant capacity for removing both single and combined antibiotics with removal efficiencies of 93.06% ∼99.80% for AZ and 73.35% ∼97.74% for TC. Higher ARG (tetA, tetC, tetW, ermF, ermX, and ermB) abundances were observed in the biofilm, and subsequent exposure to the antibiotic mixture increased the abundances of these genes. Both single and combined antibiotics triggered antioxidant stress, but antagonistic effects were induced only with mixed AZ and TC exposure. Furthermore, the antibiotics changed the structural characteristics of extracellular polysaccharides and induced alterations in the structure of the biofilm microbial community. Increased N-acylated-l-homoserine lactone confirmed alternations in microbial quorum-sensing. The results extend the understanding of the fate of antibiotics and ARGs when aquatic plants and biofilms are exposed to antibiotic mixtures, as well as the organism's response mechanisms.

Safety Evaluation by Phenotypic and Genomic Characterization of Four Lactobacilli Strains with Probiotic Properties.

Probiotic Lactobacillus species are known to exert health benefits in hosts when administered in adequate quantities. A systematic safety assessment of the strains must be performed before the Lactobacillus strains can be designated as probiotics for human consumption. In this study, we selected Lactobacillus fermentum IDCC 3901, L. gasseri IDCC 3101, L. helveticus IDCC 3801, and L. salivarius IDCC 3551 as representative Lactobacilli probiotic strains and investigated their probiotic properties and potential risks through phenotypic and genomic characterization. Various assays including antimicrobial resistance, biogenic amine production, L-/D-lactate production, acute oral toxicity, and antipathogenic effect were performed to evaluate the safety of the four Lactobacillus strains. Genomic analysis using whole genome sequencing was performed to investigate virulence and antibiotic resistance genes in the genomes of the selected probiotic strains. The phenotypes of the strains such as enzymatic activity and carbohydrate utilization were also investigated. As a result, antibiotic resistances of the four Lactobacillus species were detected; however, neither antibiotic resistance-related genes nor virulence genes were found by genomic analysis. Moreover, the four Lactobacillus species did not exhibit hemolytic activity or β-glucuronidase activity. The biogenic amine production and oral acute toxicity were not shown in the four Lactobacillus species, whereas they produced D-lactate with minor ratio. The four Lactobacillus species exhibited antipathogenic effect to five pathogenic microorganisms. This study provides a way to assess the potential risks of four different Lactobacillus species and validates the safety of all four strains as probiotics for human consumption.