In vitro and in vivo enhancement effect of glabridin on the antibacterial activity of colistin, against multidrug resistant Escherichia coli strains.

BACKGROUND: The increase in antimicrobial resistance leads to complications in treatments, prolonged hospitalization, and increased mortality. Glabridin (GLA) is a hydroxyisoflavan from Glycyrrhiza glabra L. that exhibits multiple pharmacological activities. Colistin (COL), a last-resort antibiotic, is increasingly being used in clinic against Gram-negative bacteria. Previous reports have shown that GLA is able to sensitize first line antibiotics such as norfloxacin and vancomycin on Staphylococcus aureus, implying that the use of GLA as an antibiotic adjuvant is a promising strategy for addressing the issue of drug resistance. However, the adjuvant effect on other antibiotics, especially COL, on Gram-negative bacteria such as Escherichia coli has not been studied. PURPOSE: The objective of our study was to investigate the targets of GLA and the synergistic effect of GLA and COL in E. coli, and to provide further evidence for the use of GLA as an antibiotic adjuvant to alleviate the problem of drug resistance. METHODS: We first investigated the interaction between GLA and enoyl-acyl carrier protein reductase, also called "FabI", through enzyme inhibition assay, differential scanning fluorimetry, isothermal titration calorimetry and molecular docking assay. We tested the transmembrane capacity of GLA on its own and combined it with several antibiotics. The antimicrobial activities of GLA and COL were evaluated against six different susceptible and resistant E. coli in vitro. Their interactions were analyzed using checkerboard assay, time-kill curve and CompuSyn software. A series of sensitivity tests was conducted in E. coli overexpressing the fabI gene. The development of COL resistance in the presence of GLA was tested. The antimicrobial efficacy of GLA and COL in a mouse model of urinary tract infection was assessed. The anti-biofilm effects of GLA and COL were investigated. RESULTS: In this study, enzyme kinetic analysis and thermal analysis provided evidence for the interaction between GLA and FabI in E. coli. GLA enhanced the antimicrobial effect of COL and synergistically suppressed six different susceptible and resistant E. coli with COL. Overexpression experiments showed that targeted inhibition of FabI was a key mechanism by which GLA synergistically enhanced COL activity. The combination of GLA and COL slowed the development of COL resistance in E. coli. Combined GLA and COL treatment significantly reduced bacterial load and mitigated urinary tract injury in a mouse model of E. coli urinary tract infection. Additionally, GLA + COL inhibited the formation and eradication of biofilms and the synthesis of curli. CONCLUSION: Our findings indicate that GLA synergistically enhances antimicrobial activities of COL by targeting inhibition of FabI in E. coli. GLA is expected to continue to be developed as an antibiotic adjuvant to address drug resistance issues.

Fighting against Clostridioides difficile infection: Current medications.

Clostridioides difficile (formerly Clostridium difficile) has been regarded as an 'urgent threat' and a significant global health problem, as life-threatening diarrhoea and refractory recurrence are common in patients with C. difficile infection (CDI). Unfortunately, the available anti-CDI drugs are limited. Recent guidelines recommend fidaxomicin and vancomycin as first-line drugs to treat CDI, bezlotoxumab to prevent recurrence, and faecal microbiota transplantation for rescue treatment. Currently, researchers are investigating therapeutic antibacterial drugs (e.g. teicoplanin, ridinilazole, ibezapolstat, surotomycin, cadazolid, and LFF571), preventive medications against recurrence (e.g. Rebyota, Vowst, VP20621, VE303, RBX7455, and MET-2), primary prevention strategies (e.g. vaccine, ribaxamase, and DAV132) and other anti-CDI medications in the preclinical stage (e.g. Raja 42, Myxopyronin B, and bacteriophage). This narrative review summarises current medications, including newly marketed drugs and products in development against CDI, to help clinicians treat CDI appropriately and to call for more research on innovation.

Safety and efficacy of phage application in bacterial decolonisation: a systematic review.

Colonisation by bacterial pathogens typically precedes invasive infection and seeds transmission. Thus, effective decolonisation strategies are urgently needed. The literature reports attempts to use phages for decolonisation. To assess the in-vivo efficacy and safety of phages for bacterial decolonisation, we performed a systematic review by identifying relevant studies to assess the in-vivo efficacy and safety of phages for bacterial decolonisation. We searched PubMed, Embase (Ovid), MEDLINE (Ovid), Web of Science, and the Cochrane Library to identify relevant articles published between Jan 1, 1990, and May 12, 2023, without language restrictions. We included studies that assessed the efficacy of phage for bacterial decolonisation in humans or vertebrate animal models. This systematic review is registered with PROSPERO, CRD42023457637. We identified 6694 articles, of which 56 (51 animal studies and five clinical reports) met the predetermined selection criteria and were included in the final analysis. The gastrointestinal tract (n=49, 88%) was the most studied bacterial colonisation site, and other sites were central venous catheters, lung, nose, skin, and urinary tract. Of the 56 included studies, the bacterial load at the colonisation site was reported to decrease significantly in 45 (80%) studies, but only five described eradication of the target bacteria. 15 studies reported the safety of phages for decolonisation. No obvious adverse events were reported in both the short-term and long-term observation period. Given the increasing life-threatening risks posed by bacteria that are difficult to treat, phages could be an alternative option for bacterial decolonisation, although further optimisation is required before their application to meet clinical needs.

The draft genome sequence of blaCTX-M-270-carrying Kluyvera ascorbata isolated from human sputum.

Kluyvera ascorbata is recognized as an opportunistic pathogen of the Enterobacteriaceae family but remains less studied. We report the draft genome of a K. ascorbata clinical strain recovered from human sputum, comprising approximately 5.18 million bases and harboring an intrinsic gene encoding the extended-spectrum ß-lactamase CTX-M-270.

Safety and efficacy of a phage cocktail on murine wound infections caused by carbapenem-resistant Klebsiella pneumoniae.

OBJECTIVES: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a leading pathogen causing difficult-to-treat, healthcare-associated wound infections. Phages are an alternative approach against CRKP. This study established murine wound infection models with a CRKP clinical strain of sequence type 11 and capsular type KL64, which is the dominant type in China, carrying genes encoding KPC-2 and NDM-1 carbapenemases. METHODS: A cocktail was made comprising three lytic phages of different viral families against the strain. The phage cocktail restricted bacterial growth for 10 hours in vitro. The efficacy and safety of the phage cocktail in treating a murine wound CRKP infection were then evaluated. Mice were randomly assigned into four groups (16 for each) comprising a phage treatment group, infected with bacteria and 30 minutes later with phages, and three control groups administered with PBS (negative control), bacteria (infection control), or phages (phage control) on the wound. Wound tissues were processed for counting bacterial loads on days 1, 3, and 7 post-infection and examined for histopathological change on days 3 and 7. Two remaining mice in each group were monitored for wound healing until day 14. RESULTS: Compared with the infection control group, the wound bacterial load in the phage treatment group decreased by 4.95 × 102 CFU/g (> 100-fold; P < 0.05) at day 7 post-treatment, and wounds healed on day 10, as opposed to day 14 in the infection control group. No adverse events associated with phages were observed. CONCLUSION: The phage cocktail significantly reduced the wound bacterial load and promoted wound healing with good safety.

Draft genome sequence of an Atlantibacter hermannii clinical strain isolated from human muscle tissue.

Atlantibacter hermannii is a species of the family Enterobacteriaceae and a rare opportunistic pathogen. The draft genome sequence of an Atlantibacter hermannii clinical strain that had been isolated from infected muscle tissue was obtained. The genome contains about 4.4 million bases and has no known plasmid replicons.

The draft genome sequence of bla IMP-4-carrying Enterobacter soli isolated from human blood.

Enterobacter soli is a Gram-negative rod characterized by its motile, non-spore-forming, and facultatively anaerobic nature. In this study, strain 140044, identified as Enterobacter soli, was isolated from human blood. The strain was sequenced, revealing a 5.37-Mb draft genome harboring the carbapenemase gene bla IMP-4.

Clinical Outcomes and Bacterial Characteristics of Carbapenem-resistant Acinetobacter baumannii Among Patients From Different Global Regions.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAb) is 1 of the most problematic antimicrobial-resistant bacteria. We sought to elucidate the international epidemiology and clinical impact of CRAb. METHODS: In a prospective observational cohort study, 842 hospitalized patients with a clinical CRAb culture were enrolled at 46 hospitals in five global regions between 2017 and 2019. The primary outcome was all-cause mortality at 30 days from the index culture. The strains underwent whole-genome analysis. RESULTS: Of 842 cases, 536 (64%) represented infection. By 30 days, 128 (24%) of the infected patients died, ranging from 1 (6%) of 18 in Australia-Singapore to 54 (25%) of 216 in the United States and 24 (49%) of 49 in South-Central America, whereas 42 (14%) of non-infected patients died. Bacteremia was associated with a higher risk of death compared with other types of infection (40 [42%] of 96 vs 88 [20%] of 440). In a multivariable logistic regression analysis, bloodstream infection and higher age-adjusted Charlson comorbidity index were independently associated with 30-day mortality. Clonal group 2 (CG2) strains predominated except in South-Central America, ranging from 216 (59%) of 369 in the United States to 282 (97%) of 291 in China. Acquired carbapenemase genes were carried by 769 (91%) of the 842 isolates. CG2 strains were significantly associated with higher levels of meropenem resistance, yet non-CG2 cases were over-represented among the deaths compared with CG2 cases. CONCLUSIONS: CRAb infection types and clinical outcomes differed significantly across regions. Although CG2 strains remained predominant, non-CG2 strains were associated with higher mortality. Clinical Trials Registration. NCT03646227.

Proposal for Acinetobacter higginsii sp. nov. to accommodate organisms of human clinical origin previously classified as Acinetobacter genomic species 16.

In 1989, Bouvet and Jeanjean delineated five proteolytic genomic species (GS) of Acinetobacter, each with two to four human isolates. Three were later validly named, whereas the remaining two (GS15 and GS16) have been awaiting nomenclatural clarification. Here we present the results of the genus-wide taxonomic study of 13 human strains classified as GS16 (n=10) or GS15 (n=3). Based on core genome phylogenetic analysis, the strains formed two respective but closely related phylogroups within the Acinetobacter haemolytic clade. The intraspecies genomic average nucleotide identity based on blast (ANIb) values for GS16 and GS15 reached ≥94.9 % and ≥98.7, respectively, whereas ANIb values between them were 92.5-93.5% and those between them and the known species were ≤91.5 %. GS16 and GS15 could be differentiated from the other Acinetobacter species by their ability to lyse gelatin and sheep blood and to assimilate d,l-lactate, along with their inability to acidify d-glucose and assimilate glutarate. In contrast, GS16 and GS15 were indistinguishable from one another by metabolic/physiological features or whole-cell MALDI-TOF mass spectra. All the GS15/GS16 genomes contained genes encoding a class D ß-lactamase, Acinetobacter-derived cephalosporinase and aminoglycoside 6'-N-acetyltransferase. Searching NCBI databases revealed genome sequences of three additional isolates of GS16, but none of GS15. We conclude that our data support GS16 as representing a novel species, but leave the question of the taxonomic status of GS15 open, given its close relatedness to GS16 and the small number of available strains. We propose the name Acinetobacter higginsii sp. nov. for GS16, with the type strain NIPH 1872T (CCM 9243T=CIP 70.18T=ATCC 17988T).

Hijacking a small plasmid to confer high-level resistance to aztreonam-avibactam and ceftazidime-avibactam.

Acquired ß-lactamase-encoding genes are typically carried by large plasmids in Gram-negative bacteria, which also commonly carry multi-copy small plasmids. This study found that mobile genetic elements carrying antimicrobial resistance genes are capable of hijacking small plasmids. This study focused on aztreonam-avibactam (ATM-AVI) as this combination can be used to effectively counter almost all ß-lactamases produced by bacteria, and has been recommended against carbapenem-resistant Enterobacterales. A clinical strain (085003) of carbapenem-resistant Escherichia coli was investigated, and mutants (085003R32 and 085003R512) able to grow under 32/4 and 512/4 mg/L of ATM-AVI were obtained as representatives of low- and high-level resistance, respectively, by induction. Comparative genomics showed that 085003R32 and 085003R512 had a single nucleotide mutation of ß-lactamase gene blaCMY-2, encoding a novel CMY with a Thr319Ile substitution, assigned 'CMY-2R'. Cloning and enzyme kinetics were used to verify that CMY-2R conferred ATM-AVI resistance by compromising binding of AVI and subsequent protection of ATM. Mechanisms for the discrepant resistance between 085003R32 and 085003R512 were investigated. Three tandem copies of blaCMY-2R were identified on a self-transmissible IncP1 plasmid of 085003R32 due to IS1294 misrecognizing its end terIS and rolling-circle replication. 085003R512 had only a single copy of blaCMY-2R on the IncP1 plasmid, but possessed anther blaCMY-2R on an already present 4-kb small plasmid. IS1294-mediated mobilization on to this multi-copy small plasmid increased the copy number of blaCMY-2R significantly, rendering higher resistance. This study shows that bacteria can employ multiple approaches to accommodate selection pressures imposed by exposure to varied concentrations of antimicrobial agents.

Prevalence and clonal diversity of carbapenem-resistant Klebsiella pneumoniae causing neonatal infections: A systematic review of 128 articles across 30 countries.

BACKGROUND: Klebsiella pneumoniae is the most common pathogen causing neonatal infections, leading to high mortality worldwide. Along with increasing antimicrobial use in neonates, carbapenem-resistant K. pneumoniae (CRKP) has emerged as a severe challenge for infection control and treatment. However, no comprehensive systematic review is available to describe the global epidemiology of neonatal CRKP infections. We therefore performed a systematic review of available data worldwide and combined a genome-based analysis to address the prevalence, clonal diversity, and carbapenem resistance genes of CRKP causing neonatal infections. METHODS AND FINDINGS: We performed a systematic review of studies reporting population-based neonatal infections caused by CRKP in combination with a genome-based analysis of all publicly available CRKP genomes with neonatal origins. We searched multiple databases (PubMed, Web of Science, Embase, Ovid MEDLINE, Cochrane, bioRxiv, and medRxiv) to identify studies that have reported data of neonatal CRKP infections up to June 30, 2022. We included studies addressing the prevalence of CRKP infections and colonization in neonates but excluded studies lacking the numbers of neonates, the geographical location, or independent data on Klebsiella or CRKP isolates. We used narrative synthesis for pooling data with JMP statistical software. We identified 8,558 articles and excluding those that did not meet inclusion criteria. We included 128 studies, none of which were preprints, comprising 127,583 neonates in 30 countries including 21 low- and middle-income countries (LMICs) for analysis. We found that bloodstream infection is the most common infection type in reported data. We estimated that the pooled global prevalence of CRKP infections in hospitalized neonates was 0.3% (95% confidence interval [CI], 0.2% to 0.3%). Based on 21 studies reporting patient outcomes, we found that the pooled mortality of neonatal CRKP infections was 22.9% (95% CI, 13.0% to 32.9%). A total of 535 neonatal CRKP genomes were identified from GenBank including Sequence Read Archive, of which 204 were not linked to any publications. We incorporated the 204 genomes with a literature review for understanding the species distribution, clonal diversity, and carbapenemase types. We identified 146 sequence types (STs) for neonatal CRKP strains and found that ST17, ST11, and ST15 were the 3 most common lineages. In particular, ST17 CRKP has been seen in neonates in 8 countries across 4 continents. The vast majority (75.3%) of the 1,592 neonatal CRKP strains available for analyzing carbapenemase have genes encoding metallo-ß-lactamases and NDM (New Delhi metallo-ß-lactamase) appeared to be the most common carbapenemase (64.3%). The main limitation of this study is the absence or scarcity of data from North America, South America, and Oceania. CONCLUSIONS: CRKP contributes to a considerable number of neonatal infections and leads to significant neonatal mortality. Neonatal CRKP strains are highly diverse, while ST17 is globally prevalent and merits early detection for treatment and prevention. The dominance of blaNDM carbapenemase genes imposes challenges on therapeutic options in neonates and supports the continued inhibitor-related drug discovery.

Resistance to aztreonam-avibactam due to a mutation of SHV-12 in Enterobacter.

Aztreonam-avibactam is an important option against Enterobacterales producing metallo-ß-lactamases (MBLs). We obtained an aztreonam-avibactam-resistant mutant of an MBL-producing Enterobacter mori strain by induced mutagenesis. Genome sequencing revealed an Arg244Gly (Ambler position) substitution of SHV-12 ß-lactamase in the mutant. Cloning and susceptibility testing verified that the SHV-12 Arg244Gly substitution led to significantly reduced susceptibility to aztreonam-avibactam (MIC, from 0.5/4 to 4/4 mg/L) but with the loss of resistance to cephalosporins as tradeoff. Arg244 of SHV involves in the binding of avibactam by forming an arginine-mediated salt bridge and is a critical residue to interact with ß-lactams. Molecular modeling analysis demonstrated that the Arg244Gly substitution hindered the binding of avibactam to SHV with higher binding energy (from - 5.24 to -4.32 kcal/mol) and elevated inhibition constant Ki (from 143.96 to 677.37 µM) to indicate lower affinity. This substitution, however, resulted in loss of resistance to cephalosporins as tradeoff by impairing substrate binding. This represents a new aztreonam-avibactam resistance mechanism.

Complete Genome Sequence of Mycobacterium marinum Strain 050012 Isolated from Infected Skin Tissue.

We report the complete genome sequence of a Mycobacterium marinum strain, which was isolated from skin tissue of a wound infection. This strain was subjected to short- and long-read sequencing. Its complete genome contains a single 6,393,703-bp circular chromosome. Phylogenomic analysis of all M. marinum genomes assigned this strain to cluster I.

Worldwide transmission of ST11-KL64 carbapenem-resistant Klebsiella pneumoniae: an analysis of publicly available genomes.

ST11-KL64 is an internationally distributed lineage of carbapenem-resistant Klebsiella pneumoniae and is the most common type in China. The international and interprovincial (in China) transmission of ST11-KL64 CRKP remains to be elucidated. We used both static clusters defined based on a fixed cutoff of ≤21 pairwise single-nucleotide polymorphisms and dynamic groups defined by modeling the likelihood to be linked by a transmission threshold to investigate the transmission of ST11-KL64 strains based on genome sequences mining. We analyzed all publicly available genomes (n = 730) of ST11-KL64 strains, almost all of which had known carbapenemase genes with KPC-2 being dominant. We identified 4 clusters of international transmission and 14 clusters of interprovincial transmission across China of ST11-KL64 strains. We found that dynamic grouping could provide further resolution for determining clonal relatedness in addition to the widely adopted static clustering and therefore increases the confidence for inferring transmission.IMPORTANCECarbapenem-resistant Klebsiella pneumoniae (CRKP) is a serious challenge for clinical management and is prone to spread in and between healthcare settings. ST11-KL64 is the dominant CRKP type in China with a worldwide distribution. Here, we used two different methods, the widely used clustering based on a fixed single-nucleotide polymorphism (SNP) cutoff and the recently developed grouping by modeling transmission likelihood, to mine all 730 publicly available ST11-KL64 genomes. We identified international transmission of several strains and interprovincial transmission in China of a few, which warrants further investigations to uncover the mechanisms for their spread. We found that static clustering based on ≤21 fixed SNPs is sensitive to detect transmission and dynamic grouping has higher resolutions to provide complementary information. We suggest the use of the two methods in combination for analyzing transmission of bacterial strains. Our findings highlight the need of coordinated actions at both international and interprovincial levels for tackling multi-drug resistant organisms.

Enterobacter pseudoroggenkampii sp. nov. carrying quinolone-resistant gene qnrE recovered from clinical samples in China.

Two Enterobacter strains 155092T and 170,225 were isolated from clinical samples, pus and sputum, from two hospitalised patients separately, in China. Preliminary identification using Vitek II microbiology system assigned the strains to the Enterobacter cloacae complex. The two strains were subjected to genome sequencing and genome-based taxonomy analysis with type strains of all Enterobacter species and those within closely related genera Huaxiibacter, Leclercia, Lelliottia, and Pseudoenterobacter. The average nucleotide identity (ANI) and in silico DNA-DNA hybridisation (isDDH) values between the two strains were 98.35% and 89.4%, respectively, suggesting that they belong to one species. The two strains had the highest ANI (95.02% and 95.04%) with the type strain of Enterobacter quasiroggenkampii. Their highest isDDH values, also seen with the type strain of E. quasiroggenkampii, were 59.5% and 59.8%, well below the 70% cutoff to define species. The two strains were also characterised for morphological and biochemical features by a set of experiments and observations. The abilities of metabolising gelatin and L-rhamnose could differentiate the two strains from all currently known Enterobacter species. Collectively, the two strains represent a novel Enterobacter species, for which we propose Enterobacter pseudoroggenkampii sp. nov. as the species name. The type strain of this novel species is155092T (= GDMCC 1.3415T = JCM 35646T). The two strains also carried multiple virulence factors comprising aerobactin-encoding iucABCD-iutA and salmochelin-encoding iroN. The two strains also had chromosomally located qnrE, a gene associated with reduced susceptibility to quinolones, suggesting that this species is a potential reservoir of qnrE genes.

Metallo-ß-lactamase-mediated antimicrobial resistance and progress in inhibitor discovery.

Resistance to ß-lactam antibiotics is rapidly growing, substantially due to the spread of serine-ß-lactamases (SBLs) and metallo-ß-lactamases (MBLs), which efficiently catalyse ß-lactam hydrolysis. Combinations of a ß-lactam antibiotic with an SBL inhibitor have been clinically successful; however, no MBL inhibitors have been developed for clinical use. MBLs are a worrying resistance vector because they catalyse hydrolysis of all ß-lactam antibiotic classes, except the monobactams, and they are being disseminated across many bacterial species worldwide. Here we review the classification, structures, substrate profiles, and inhibition mechanisms of MBLs, highlighting current clinical problems due to MBL-mediated resistance and progress in understanding and combating MBL-mediated resistance.

Analysis of risk factors associated with healthcare-associated carbapenem-resistant Klebsiella pneumoniae infection in a large general hospital: a case-case-control study.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection is a major public health threat in the world. To inform the prevention and control of CRKP infection in hospitals, this study analyzed the factors associated with CRKP infection and resistance to carbapenems in K. pneumoniae. This case-case-control study was carried out in a large general hospital in China from January 2016 to December 2018, comprising 494 hospitalized patients infected with CRKP (case group 1) and 2429 hospitalized patients infected with carbapenem-susceptible K. pneumoniae (CSKP, case group 2). We selected control groups from hospitalized patients without K. pneumoniae infections for the two case groups separately, with a 1:3 case-control ratio, to analyze the risk factors of the two case groups using the conditional logistic regression. Multivariate analysis showed that the risk factors of CRKP infection were intensive care unit (ICU) admission (odds ratio [OR], 6.85; 95% confidence interval [CI], 4.90-9.58; P < 0.001), respiratory failure (OR, 1.93; 95% CI, 1.34-2.77; P < 0.001), age-adjusted Charlson comorbidity index (aCCI; OR, 1.08; 95% CI, 1.02-1.15; P = 0.007), admission from the Emergency (OR, 1.37; 95% CI, 1.02-1.85; P = 0.036), and imipenem use (OR, 1.80; 95% CI, 1.30-2.49; P < 0.001). Among the aforementioned five risk factors, aCCI (OR, 1.09; 95% CI, 1.06-1.13; P < 0.001) was also identified as a risk factor of CSKP infections in multivariate analysis. The risk factors for resistance to carbapenems in K. pneumoniae were ICU admission, respiratory failure, admission from the Emergency, and imipenem use.

Complete Genome Sequence of an mcr-10-Carrying Enterobacter roggenkampii Strain Isolated from a Human Blood Culture.

The mcr-10 gene is a plasmid-borne colistin resistance gene. The complete genome sequence of an mcr-10-carrying Enterobacter roggenkampii clinical strain that had been isolated from a blood culture was obtained. The genome consists of a 4.95-Mbp chromosome and six plasmids, including a 195-kb plasmid harboring mcr-10 and two replicons, IncFIB(K) and IncFII(Yp).