Short-term exposure to antibiotics begets long-term disturbance in gut microbial metabolism and molecular ecological networks.

BACKGROUND: Antibiotic exposure can occur in medical settings and from environmental sources. Long-term effects of brief antibiotic exposure in early life are largely unknown. RESULTS: Post a short-term treatment by ceftriaxone to C57BL/6 mice in early life, a 14-month observation was performed using 16S rRNA gene-sequencing technique, metabolomics analysis, and metagenomics analysis on the effects of ceftriaxone exposure. Firstly, the results showed that antibiotic pre-treatment significantly disturbed gut microbial α and ß diversities (P < 0.05). Both Chao1 indices and Shannon indices manifested recovery trends over time, but they didn't entirely recover to the baseline of control throughout the experiment. Secondly, antibiotic pre-treatment reduced the complexity of gut molecular ecological networks (MENs). Various network parameters were affected and manifested recovery trends over time with different degrees, such as nodes (P < 0.001, R2 = 0.6563), links (P < 0.01, R2 = 0.4543), number of modules (P = 0.0672, R2 = 0.2523), relative modularity (P = 0.6714, R2 = 0.0155), number of keystones (P = 0.1003, R2 = 0.2090), robustness_random (P = 0.79, R2 = 0.0063), and vulnerability (P = 0.0528, R2 = 0.28). The network parameters didn't entirely recover. Antibiotic exposure obviously reduced the number of key species in gut MENs. Interestingly, new keystones appeared during the recovery process of network complexity. Changes in network stability might be caused by variations in network complexity, which supports the ecological theory that complexity begets stability. Besides, the metabolism profiles of the antibiotic group and control were significantly different. Correlation analysis showed that antibiotic-induced differences in gut microbial metabolism were related to MEN changes. Antibiotic exposure also caused long-term effects on gut microbial functional networks in mice. CONCLUSIONS: These results suggest that short-term antibiotic exposure in early life will cause long-term negative impacts on gut microbial diversity, MENs, and microbial metabolism. Therefore, great concern should be raised about children's brief exposure to antibiotics if the results observed in mice are applicable to humans. Video Abstract.

Clinical distribution of carbapenem genotypes and resistance to ceftazidime-avibactam in Enterobacteriaceae bacteria.

Introduction: Bacterial resistance is a major threat to public health worldwide. To gain an understanding of the clinical infection distribution, drug resistance information, and genotype of CRE in Dongguan, China, as well as the resistance of relevant genotypes to CAZ-AVI, this research aims to improve drug resistance monitoring information in Dongguan and provide a reliable basis for the clinical control and treatment of CRE infection. Methods: VITEK-2 Compact automatic analyzer was utilized to identify 516 strains of CRE collected from January 2017 to June 2023. To determine drug sensitivity, the K-B method, E-test, and MIC methods were used. From June 2022 to June 2023, 80 CRE strains were selected, and GeneXpert Carba-R was used to detect and identify the genotype of the carbapenemase present in the collected CRE strains. An in-depth analysis was conducted on the CAZ-AVI in vitro drug sensitivity activity of various genotypes of CRE, and the results were statistically evaluated using SPSS 23.0 and WHONET 5.6 software. Results: This study identified 516 CRE strains, with the majority (70.16%) being K.pneumoniae, followed by E.coli (18.99%). Respiratory specimens had highest detection rate with 53.77% identified, whereas urine specimens had the second highest detection rate with 17.99%. From June 2022 to June 2023, 95% of the strains tested using the CRE GeneXpert Carba-R assay possessed carbapenemase genes, of which 32.5% were blaNDM strains and 61.25% blaKPC strains. The results showed that CRE strains containing blaKPC had a significantly higher rate of resistance to amikacin, cefepime, and aztreonam than those harboring blaNDM. Conclusions: The CRE strains isolated from Dongguan region demonstrated a high resistance rate to various antibiotics used in clinical practice but a low resistance rate to tigecycline. These strains produce Class A serine carbapenemases and Class B metals ß-lactamases, with the majority of them carrying blaNDM and blaKPC. Notably, CRE strains with blaKPC and blaNDM had significantly lower resistance rates to tigecycline. CAZ-AVI showed a good sensitivity rate with no resistance to CRE strains carrying blaKPC. Therefore, CAZ-AVI and tigecycline should be used as a guide for rational use of antibiotics in clinical practice to effectively treat CRE.

Effects of dietary arsenic exposure on liver metabolism in mice.

Arsenic, a ubiquitous environmental toxicant with various forms and complex food matrix interactions, can reportedly exert differential effects on the liver compared to drinking water exposure. To examine its specific liver-related harms, we targeted the liver in C57BL/6 J mice (n=48, 8-week-old) fed with arsenic-contaminated food (30 mg/kg) for 60 days, mimicking the rice arsenic composition observed in real-world scenarios (iAsV: 7.3%, iAsIII: 72.7%, MMA: 1.0%, DMA: 19.0%). We then comprehensively evaluated liver histopathology, metabolic changes, and the potential role of the gut-liver axis using human hepatocellular carcinoma cells (HepG2) and microbiota/metabolite analyses. Rice arsenic exposure significantly altered hepatic lipid (fatty acids, glycerol lipids, phospholipids, sphingolipids) and metabolite (glutathione, thioneine, spermidine, inosine, indole-derivatives, etc.) profiles, disrupting 33 metabolic pathways (bile secretion, unsaturated fatty acid biosynthesis, glutathione metabolism, ferroptosis, etc.). Pathological examination revealed liver cell necrosis/apoptosis, further confirmed by ferroptosis induction in HepG2 cells. Gut microbiome analysis showed enrichment of pathogenic bacteria linked to liver diseases and depletion of beneficial strains. Fecal primary and secondary bile acids, short-chain fatty acids, and branched-chain amino acids were also elevated. Importantly, mediation analysis revealed significant correlations between gut microbiota, fecal metabolites, and liver metabolic alterations, suggesting fecal metabolites may mediate the impact of gut microbiota and liver metabolic disorders. Gut microbiota and its metabolites may play significant roles in arsenic-induced gut-liver injuries. Overall, our findings demonstrate that rice arsenic exposure triggers oxidative stress, disrupts liver metabolism, and induces ferroptosis.

Comparison of the adherence of nontypeable haemophilus influenzae to lung epithelial cells.

OBJECTIVE: Nontypeable Haemophilus influenzae (NTHi) plays an important role in respiratory tract infections, and adherence to lung epithelial cells is the first step in lung infections. To explore the role of NTHi in childhood lung infections, a comparative study was conducted on the adherence of strains isolated from sputum culture and bronchoalveolar lavage fluid to A549 lung epithelial cells. METHODS: Haemophilus influenzae strains were obtained from the sample bank of Shenzhen Children's Hospital, and identified as NTHi via PCR detection of the capsule gene bexA. NTHi obtained from healthy children's nasopharyngeal swabs culture were selected as the control group, and a comparative study was conducted on the adherence of strains isolated from sputum culture or bronchoalveolar lavage fluid of patients to A549 cells. RESULTS: The adherence bacterial counts of NTHi isolated from the nasopharyngeal cultures of healthy children to A549 cells was 58.2 CFU. In patients with lung diseases, NTHi isolated from bronchoalveolar lavage fluid was 104.3 CFU, and from sputum cultures was 115.1 CFU, both of which were significantly higher in their adherence to A549 cells compared to the strains isolated from the healthy control group. There was no significant difference in adherence between the strains isolated from sputum cultures and bronchoalveolar lavage fluid (t = 0.5217, p = 0.6033). CONCLUSION: NTHi played an important role in childhood pulmonary infections by enhancing its adherence to lung epithelial cells.

Investigating the ibrutinib resistance mechanism of L528W mutation on Bruton's tyrosine kinase via molecular dynamics simulations.

Drug resistance to Bruton's Tyrosine Kinase (BTK) inhibitors presents a challenge in treating B-cell malignancies, and the mechanism behind drug resistance remains unclear. In this study, we focused on the BTK L528W mutation and investigated the underlying mechanisms of resistance to ibrutinib (including prototype and its active metabolite from, PCI-45227) using a combination of bioinformatics analysis, and molecular dynamics (MD) simulations. Protein stability of wild type (WT) BTK and L528W mutant was predicted using DUET, PoPMuSiC, and I-Mutant2.0. We performed MD simulations of six systems, apo-WT, metabolite-WT, prototype-WT and their mutants, to analyze the significant conformational and BTK-inhibitor binding affinity changes induced by the L528W mutation. Results show that the L528W mutation reduces the conformational stability of BTK compared to the WT. Principal component analysis (PCA) based free energy landscape (FEL) analysis shows that the L528W mutant ensemble tends to form more conformation clusters and exhibit higher levels of local minima than the WT counterpart. The interaction analysis reveal that the L528W mutation disrupts the strong hydrogen bond between Cys481 and inhibitors and reduces the number of hydrogen bonds between inhibitors and BTK in the L528W mutant complex structures compared to the WT. Porcupine plot analysis in association with cross-correlation analysis show the high-intensity flexible motion exhibited by the P-loop region. MM/GBSA calculations show that the L528W mutation in metabolite-BTK and prototype-BTK complexes increases binding free energy compared to the WT, with a reduction in binding affinity confirmed by per-residue energy decomposition. Specifically, the binding free energy increases from -57.86 kcal/mol to -48.26 kcal/mol for the metabolite-BTK complex and from -62.04 kcal/mol to -50.55 kcal/mol for the prototype-BTK complex. Overall, our study finds that the L528W mutation reduces BTK stability, decreases binding affinity, and leads to drug resistance and potential disease recurrence.

Study on the Genome and Mechanism of Tigecycline Resistance of a Clinical Chryseobacterium indologenes Strain.

Purpose: Chryseobacterium indologenes is a clinically relevant microorganism that has been on the rise, with multidrug-resistant (MDR) strains being reported. C. indologenes carrying tet(X2) has been demonstrated to be resistant to the antibiotic tigecycline, yet, sensitive to all other members of the tetracycline family. This inconsistency in resistance prompts an inquiry into the contribution of tet(X2) to tigecycline resistance in C. indologenes. Materials and Methods: In this study, we report on a comprehensive analysis of the genomic mechanisms underlying tigecycline resistance in a MDR C. indologenes strain (CI3125) that was resistant to tigecycline but sensitive to tetracycline, doxycycline, and minocycline. We used whole-genome sequencing, quantitative reverse transcription PCR, Western blot, antibiotic-degrading tests, and efflux pump inhibiting tests to reveal the mechanism of tigecycline resistance in C. indologenes and elucidate the inconsistency in the antibiotic resistance mechanism for the tetracycline family. Results: Our findings demonstrate that CI3125 carries 60 antibiotic resistance genes distributed on 6 different genetic islands (GIs), with the potential for horizontal transfer. Notably, the tet(X2) gene is located on GI06 of CI3125. Genetic environment analysis of tet(X2) showed that all tet(X2) genes in Flavobacterium and Bacteroides share a conservative and functional ribosome-binding site upstream. Contrary to expectation, our RT-qPCR showed that tet(X2) was not transcribed in CI3125, and Western blot suggested the absence of tet(X2) protein in CI3125. Rather, we demonstrate that minimum inhibitory concentration values for tigecycline decreased two- to eight-folds in the presence of five different efflux pump inhibitors [1-(1-naphthyl- methyl)-piperazine, phenyl-arginine-ß-naphthylamide, verapamil, reserpine, and carbonyl cyanide 3-chlorophenylhydrazone]. This finding provides evidence for the involvement of efflux pumps in tigecycline resistance, which is likely to be a universal mechanism among C. indologenes. Our study proposes that the inconsistency in resistance to the tetracycline family in CI3125 may be ascribed to the silence of tet(X2) and the functions of efflux pumps for tigecycline. Conclusions: Overall, our results highlight the importance of genomic approaches in understanding the underlying mechanisms of antibiotic resistance in clinically relevant microorganisms. While tet(X2) in CI3125 is silent, our findings suggest that it may be horizontally spread through GIs. Hence, our findings have significant implications for the management of C. indologenes infections in clinical settings.

An efficient method for knocking out genes on the virulence plasmid of hypervirulent Klebsiella pneumoniae.

Currently, the infection of hypervirulent Klebsiella pneumoniae (hvKp) is becoming increasingly serious and the virulent mechanisms of hvKp are still not very clear. An effective gene-editing method for genes on hvKp virulence plasmid can help us reveal related virulent mechanisms. There are a few reports focusing on the methods mentioned above, however with certain limitations. In this work, we first constructed the pRE112-basing recombinant suicide plasmid to knock out or replace the genes in the hvKp virulence plasmid based on the principle of homology recombination. Results showed that the target virulent genes iucA, iucB, iroB, and rmpA2 on the hvKp virulence plasmid were scarlessly knocked out or replaced by marker genes, and mutant hvKp strains with the expected phenotypes were obtained. These indicated that we established an efficient gene-editing method for genes on hvKp virulence plasmid, which could help us explore the functions of these genes and reveal the virulent mechanisms of hvKp.

Genome-Based Analysis of a Multidrug-Resistant Hypervirulent Klebsiella pneumoniae.

Reports on multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKP) in recent years indicate the wide-spreading trend of MDR-hvKP. The co-occurrence of hypervirulence and drug resistance poses a great challenge to clinical treatment. In this study, molecular characteristics of an MDR strain hvKP247 and 30 clinically isolated hvKP strains were characterized. The whole genome of hvKP247 belonging to sequence type (ST) 5214 and capsule serotype K1 was sequenced and analyzed. Conjugation experiments were performed to evaluate transferability of the plasmids in hvKP247. We found two new STs among our isolates, ST5570 and ST5571. The ST5214 hvKP247 contained two transferable plasmids: a hybrid virulence plasmid (pHvKP247-vir) carrying transfer-related modules that had self-transferable ability, and a drug-resistant plasmid (pHvKP247-MDR) that could be indirectly transferred with the help of pHvKP247-vir. The virulence-related genes were located on the pHvKP247-vir and chromosomal ICEKp1 mobile genetic element. In conclusion, the hybrid virulence plasmid and the drug-resistant plasmid are co-transferred, which emphasizes the importance of raising public awareness of the simultaneous spread of virulence and resistance genes of MDR-hvKP strains.

Binding mechanism of oseltamivir and influenza neuraminidase suggests perspectives for the design of new anti-influenza drugs.

Oseltamivir is a widely used influenza virus neuraminidase (NA) inhibitor that prevents the release of new virus particles from host cells. However, oseltamivir-resistant strains have emerged, but effective drugs against them have not yet been developed. Elucidating the binding mechanisms between NA and oseltamivir may provide valuable information for the design of new drugs against NA mutants resistant to oseltamivir. Here, we conducted large-scale (353.4 µs) free-binding molecular dynamics simulations, together with a Markov State Model and an importance-sampling algorithm, to reveal the binding process of oseltamivir and NA. Ten metastable states and five major binding pathways were identified that validated and complemented previously discovered binding pathways, including the hypothesis that oseltamivir can be transferred from the secondary sialic acid binding site to the catalytic site. The discovery of multiple new metastable states, especially the stable bound state containing a water-mediated hydrogen bond between Arg118 and oseltamivir, may provide new insights into the improvement of NA inhibitors. We anticipated the findings presented here will facilitate the development of drugs capable of combating NA mutations.

Overexpression of Efflux Pumps Mediate Pan Resistance of Klebsiella pneumoniae Sequence Type 11.

A clinically isolated pan-resistant Klebsiella pneumoniae strain (ST11), KPN142 was subjected to whole-genome sequencing. Genomic sequence of KPN142 showed that limited antibiotic resistances (ß-lactams [blashv-11], sulfonamides [sul1 and dfrA22], bacitracin [bacA], tetracycline [tet34], aminoglycosides [ksgA, kdpE, aph(3)Ia, aac(3)III, and ant(3)Ia], and chloramphenicol [catA1]) were mediated by enzymes, and efflux pumps contributed most to pan resistance. Five types of multidrug resistance efflux pump families were identified, including the resistance nodulation division superfamily (AcrAB-TolC, AcrD, MdtABC, and KexD), the ATP-binding cascade superfamily (MacAB), the small multidrug resistance family (KpnEF), the multidrug and toxic compound extrusion family (KdeA), and the major facilitator superfamily (EmrAB). There was an AcrAB-TolC efflux pump system, and inhibitory regulatory gene acrR and ramR of system carried deletion mutation, which lead to overexpression of AcrAB-TolC efflux pump, and in turn plays key role in the pan resistance of KPN142. Moreover, we did not find mgrb, a suppressor in the expression of phoPQ, overexpression of which may confer the resistance of KPN142 to colistin B. In addition, K. pneumoniae KPN142 carries IS1, IS3, and IntI1, which means that KPN142 is able to transfer drug-resistance genes. Of note, we detected the overexpression of acrB, ramA, phoP, and phoQ by real-time quantitative reverse transcription-polymerase chain reaction, and carbonyl cyanide chlorophenylhydrazone was able to reverse the resistance patterns of K. pneumoniae KPN142. In conclusion, we consider that the overexpression of AcrAB-TolC efflux pump mediates the resistance to most common clinical antimicrobial agents, and the overexpression of phoPQ mediates the resistance to colistin B in K. pneumoniae KPN142.

Antibacterial potential of Forsythia suspensa polysaccharide against resistant Enterobacter cloacae with SHV-12 extended-spectrum ß-lactamase (ESBL).

In this study, a homogenous polysaccharide (FSP), with an average molecular weight of 9.08 × 104  Da, was isolated from Forsythia suspense and its antibacterial potential against Enterobacter cloacae producing SHV-12 ESBL was investigated. Growth kinetics, in vitro competition and biofilm formation experiments demonstrated that SHV-12 ESBL contributed to a fitness benefit to E cloacae strain. The antibacterial activity of FSP (2.5, 5.0 and 10.0 µg/mL) was tested against E cloacae bearing SHV-12 ESBL gene using bacterial sensitivity, agar bioassay and agar well diffusion assays. It was found that the addition of FSP demonstrated potent antibacterial activities against this bacterial as showed by the decrease of bacterial growth and the increase of the inhibition zone diameter. Furthermore, SHV-12 ESBL gene expression was decreased in E cloacae strain following different FSP treatment in a concentration-dependent manner. In conclusion, these data showed that FSP exhibited potent good antibacterial activity against E cloacae producing SHV-12 ESBL via inhibition of SHV-12 ESBL gene expression, which may promote the development of novel natural antibacterial agents to treat infections caused by this drug-resistant bacterial pathogen.

The impacts of viral inactivating methods on quantitative RT-PCR for COVID-19.

OBJECTIVE: This paper aims to explore the effect of four virus inactivation methods on the rapid detection results of COVID-19 nucleic acid. METHODS: Collected samples of nasopharyngeal swabs from 2 patients diagnosed with COVID-19 at the First People's Hospital of Zhaoqing City, each of sample was divided into 5 groups (groupA∼E): A:Non-inactivated raw sample; B:75 % ethanol inactivation; C:56 ℃ incubation for 30 min inactivation; D:65 ℃ incubation for 10 min inactivation; E:Pre-inactivation using RNA virus special preservation fluid added into the sampling tube to treated the nasopharyngeal swab sample separately, using real-time fluorescent RT-PCR to detect the N gene of COVID-19 and the ORF1ab gene simultaneously. All the groups are diluted in 1:2, 1:4, 1:8 ratios. The objectives are to compare the effect of the varied inactivation method on CT(Cycle Threshold)results in PCR, conduct correlation and Bland-Altman analysis. RESULTS: For the N gene and ORF1ab gene, the CT values of 4 inactivated and Non-inactivated treatment were correlated (P＜0.001). The results of the four treatment methods and specimens without inactivated treatment have shown good consistency. CONCLUSION: The treatment of nasopharyngeal swab specimens using mentioned four inactivated methods had no significant effect on the subsequent detection of the new COVID-19 nucleic acid test. Lab test-persons can flexibly adopt pre-inactivation methods to ensure the accuracy of virus nucleic acid test results, meanwhile guarantee the safety of lab test-persons.

Nasal Delivery of Hesperidin/Chitosan Nanoparticles Suppresses Cytokine Storm Syndrome in a Mouse Model of Acute Lung Injury.

The cytokine storm or cytokine storm syndrome (CSS) is associated with high mortality in patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), for example following sepsis or infectious diseases including COVID-19. However, there are no effective treatments for CSS-associated ALI or ALI/ARDS. Thus, there remains an urgent need to develop effective drugs and therapeutic strategies against CSS and ALI/ARDS. Nasal and inhaled drug delivery methods represent a promising strategy in the treatment of inflammatory lung disease as a result of their ability to improve drug delivery to lungs. Improving the nasal mucosa absorption of poorly water-soluble drugs with poor mucosa bioavailability to a therapeutically effective level is another promising strategy in the fight against ALI/ARDS. Here, chitosan nanoparticles loaded with hesperidin (HPD/NPs) were developed for nasal delivery of the anti-inflammatory HPD compound to inflammatory lungs. In vitro and in vivo, HPD/NPs exhibited enhanced cellular uptake in the inflammatory microenvironment compared with free HPD. In a mouse model of inflammatory lung disease, the HPD/NPs markedly inhibited lung injury as evidenced by reduced inflammatory cytokine levels and suppressed vascular permeability compared with free HPD. Collectively, our study demonstrates that nasal delivery of HPD/NPs suppresses CSS and ALI/ARDS in a murine model of inflammatory lung disease, and that nanoparticle-based treatment strategies with anti-inflammatory effects could be used to reduce CSS and ALI in patients with inflammatory lung injury.

Whole-genome analysis of New Delhi Metallo-Beta-Lactamase-1-producing Acinetobacter haemolyticus from China.

OBJECTIVE: Infections caused by multidrug-resistant Acinetobacter spp. have generated worldwide attention. With the increasing isolation of non-baumannii Acinetobacter, the nature of associated infection and resistance needs to be explored. This study aimed to analyse the characteristics of New Delhi Metallo-Beta-Lactamase-1 (NDM-1)-producing Acinetobacter haemolyticus (named sz1652) isolated from Shenzhen city, China. METHODS: The antibiotic spectrum was analysed after antimicrobial susceptibility testing. Combined disk test (CDT) was used to detect the metallo-beta-lactamases (MBLs). Transferability of carbapenem resistance was tested by filter mating experiments and plasmid transformation assays. Whole-genome sequencing (WGS) was performed using HiSeq 2000 and PacBio RS system. RESULTS: The Acinetobacter haemolyticus strain sz1652 was resistant to carbapenems and other tested agents except for amikacin, tigecycline and colistin. Production of MBLs was confirmed by CDT. Transfer of carbapenem resistance was unsuccessful. WGS analysis showed that the genome of sz1652 comprised a chromosome and two plasmids; 16 genomic islands (GIs) were predicted. Genes associated with resistance were found in this strain, including the beta-lactamase genes blaNDM-1, blaOXA-214 and blaLRA-18, the fluoroquinolone resistant-related mutations [GyrA subunits (Ser81Ile) and ParC subunits (Ser84Tyr)], and efflux pump genes related to tetracycline and macrolide resistance. Analysis of the genetic environment showed that blaNDM-1 was embedded in Tn125 transposon. The Tn125 structure was chromosomally located and shared > 99% sequence identity with the previously reported blaNDM-1 carrying region. CONCLUSION: The NDM-1-producing Acinetobacter haemolyticus coexisted with multiple drug-resistant determinants. The acquisition of the blaNDM-1 gene was probably facilitated by Tn125 in this strain. Non-Acinetobacter baumannii species also contained GIs.

Yunnan Baiyao reduces hospital-acquired pressure ulcers via suppressing virulence gene expression and biofilm formation of Staphylococcus aureus.

Yunnan Baiyao (YB) as a kind of famous Chinese herbal medicine, possessed hemostatic, invigorating the circulation of blood, and anti-inflammatory effects. Identifying strategies to protect patients at risk for hospital-acquired pressure ulcers (HAPU) is essential. Herein, our results showed that YB treatment can effectively reduce the acne wound area and improve efficacy in a comparative study of 60 cases HAPU patients with S. aureus positive of acne wound pathogens. Furthermore, YB inhibited HIa expression and suppressed accessory gene regulator (agr) system controlled by regulatory RNA II and RNA III molecule using pALC1740, pALC1742 and pALC1743 S. aureus strain linked to gfpuvr reporter gene. Moreover, YB downregulated cao mRNA expression and inhibited coagulase activity by RT-PCR, slide and tube coagulase test. Additionally, YB downregulated seb, sec, sed, and tsst-1 mRNA expression to suppress enterotoxin and tsst-1 secretion and adhesion function related genes sarA, icaA, and cidA mRNA expression. Taken together, the data suggest that YB may reduce HAPU via suppressing virulence gene expression and biofilm formation of S. aureus.

Inhibition of bleomycin-induced pulmonary fibrosis in mice by the novel peptide EZY-1 purified from Eucheuma.

For the first time, a new 16-amino-acid peptide was isolated from Eucheuma, an edible seaweed, and named EZY-1. EZY-1 was used to interfere with bleomycin-induced mice pulmonary fibrosis. The target proteins of EZY-1 were screened by an in vitro pull-down method combined with LC-MS/MS. The results showed that EZY-1 can inhibit the idiopathic pulmonary fibrosis (IPF) induced by bleomycin. The potency and safety of EZY-1 are superior to those of the drug used for clinical treatment, pirfenidone. The results showed that EZY-1 suppresses the TGF-ß/Smad, PI3K-Akt-mTOR, Rac1-PAK2-cAb1 and MAPK signal transduction pathways. Proteins such as ERK, Akt, PDGF receptor ß, vitronectin, raptor and SHP2 exhibited binding to EZY-1 in an in vitro pull-down assay combined with LC-MS/MS analysis. EZY-1 was confirmed to be an effective component of Eucheuma in the inhibition of IPF. The signalling pathways and target proteins of EZY-1 were preliminarily predicted. This study lays the foundation for the development of new drugs from Eucheuma for the treatment of IPF.

Molecular epidemiology and genetic characterisation of carbapenem-resistant Acinetobacter baumannii isolates from Guangdong Province, South China.

OBJECTIVES: Carbapenem-resistant Acinetobacter baumannii (CRAB) has become a worldwide issue. This study aimed to characterise the epidemiology and genetic relationships of A. baumannii isolates in Guangdong Province, China. METHODS: CRAB isolates were collected from five municipal hospitals from June-December 2017. The 16S-23S rRNA intergenic spacer region was used for confirmation of strain identity. Antimicrobial susceptibility testing and the CarbAcineto NP test were performed to analyse the resistance spectrum and carbapenemase production of the isolates. PCR-based assays were used to detect ß-lactamase genes and related mobile genetic elements. Genetic diversity among the isolates was analysed by enterobacterial repetitive intergenic consensus (ERIC)-PCR, multilocus sequence typing (MLST) and multiplex PCR. RESULTS: A total of 122 isolates were confirmed as A. baumannii; all were resistant to the tested antibiotics except for tigecycline and colistin. The CarbAcineto NP test showed that 93.4% of the isolates produced a carbapenemase. blaOXA-23-like and extended-spectrum ß-lactamase-encoding genes were found by PCR in 94.3% and 91.8% of the isolates, respectively. Furthermore, the genetic environment of blaOXA-23-like was mainly associated with transposons Tn2008 (46.1%), Tn2006 (27.0%) and Tn2009 (20.9%). MLST identified six existing sequence types (STs) and three novel STs, of which ST195 (35.7%) and ST208 (32.1%) were the most common, belonging to clonal group 92 and European clone II. CONCLUSION: This study suggests that co-production of ß-lactamases was the major resistance mechanism of CRAB isolates. Dissemination of blaOXA-23-like may be facilitated by transposable elements. ST195 and ST208 were the predominant epidemic types of A. baumannii in Guangdong Province.

Structural investigation of a polysaccharide from the mycelium of Enterobacter cloacae and its antibacterial activity against extensively drug-resistant E. cloacae producing SHV-12 extended-spectrum ß-lactamase.

In this study, a polysaccharide (ECP) was isolated from the mycelium of Enterobacter cloacae and was found to exhibit strong antibacterial activities against E. cloacae producing SHV-12 ESBL with the increase of the inhibition zone diameter. Its minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were 12.5 mg/L and 25 mg/L, respectively. ECP at these concentrations immediately inhibited planktonic growth of the bacteria especially at the time from 2 to 10 h. Flow cytometry analysis further revealed that almost all the bacterial cells were damaged following ECP treatment. The permeability of the cytoplasmic membrane of E. cloacae was increased when ECP concentrations increasing, as evidenced by an influx of Na and an efflux of K, P or S, the leakage of intracellular ATP and the UV-absorbing substances, as well as the depolarization of the cytoplasmic membrane, indicating that bactericidal activity of ECP was achieved by inducing cell membrane damage.

A molecular dynamics study of the complete binding process of meropenem to New Delhi metallo-ß-lactamase 1.

The mechanism of substrate hydrolysis of New Delhi metallo-ß-lactamase 1 (NDM-1) has been reported, but the process in which NDM-1 captures and transports the substrate into its active center remains unknown. In this study, we investigated the process of the substrate entry into the NDM-1 activity center through long unguided molecular dynamics simulations using meropenem as the substrate. A total of 550 individual simulations were performed, each of which for 200 ns, and 110 of them showed enzyme-substrate binding events. The results reveal three categories of relatively persistent and noteworthy enzyme-substrate binding configurations, which we call configurations A, B, and C. We performed binding free energy calculations of the enzyme-substrate complexes of different configurations using the molecular mechanics Poisson-Boltzmann surface area method. The role of each residue of the active site in binding the substrate was investigated using energy decomposition analysis. The simulated trajectories provide a continuous atomic-level view of the entire binding process, revealing potentially valuable regions where the enzyme and the substrate interact persistently and five possible pathways of the substrate entering into the active center, which were validated using well-tempered metadynamics. These findings provide important insights into the binding mechanism of meropenem to NDM-1, which may provide new prospects for the design of novel metallo-ß-lactamase inhibitors and enzyme-resistant antibiotics.

Association of antibiotic resistance with SHV-12 extended-spectrum ß-lactamase in Enterobacter cloacae.

The association between antibiotic resistance and SHV-12 extended-spectrum ß-lactamase (ESBL) in Enterobacter cloacae remains unknown. The aim of the present study was to investigate the prevalence of both chromosome- and plasmid-borne SHV-12 ESBL genes in Enterobacter cloacae. Transmission of the SHV-12 ESBL gene was explored, and the risk factors for antibiotic resistance in E. cloacae were analyzed. Polymerase chain reaction (PCR) results showed that 58 out of the 100 isolates carried the SHV-12 ESBL gene: 34.48% of them occurred in the chromosome, 48.28% were plasmid-borne and 17.24% appeared in both. Enterobacterial repetitive intergenic consensus-PCR tests detected 82 chromosomal genotypes. Conjugation assays showed that 70.00% of plasmid-borne SHV-12 ESBL genes were successfully transconjugated into E. coli C600 and that the antibiotic resistance phenotype of E. cloacae was partially (84%) or completely (10%) transferred. A significantly higher SHV-12 ESBL detection rate was found in patients with underlying conditions and/or complications compared with those without (P<0.05). The detection of SHV-12 ESBL-producing E. cloacae from vertical transmission varied significantly across clinical departments and age groups (P<0.05), with the highest rates in the intensive care unit and the group of patients aged ≥60 years. The present results indicate that the location and transmission efficiency of SHV-12 ESBL are closely correlated with the antibiotic resistance of E. cloacae.