Genetic Characterization of Plasmid-Borne blaOXA-58 and blaOXA-72 in Acinetobacter pittii in Shaanxi, China.

BACKGROUND: Acinetobacter pittii has emerged as an opportunistic nosocomial pathogen associated with hospital-acquired infections. The purpose of this study was to investigate the genetic structures of plasmids carrying carbapenemase genes blaOXA-58 and blaOXA-72 in A. pittii strains AR3676 and AR3651 isolated from patients. METHODS: Antimicrobial susceptibility testing was performed using broth microdilution. Whole-genome sequencing and bioinformatics analysis were performed to characterize the genome of A. pittii AR3676 and AR3651. Conjugation experiments were conducted to evaluate plasmids transferability. Phylogenetic and comparative genomic analysis were performed to explore the characteristics of carbapenem-resistant A. pittii isolates worldwide. RESULTS: The AR3676 strain exhibited resistance to imipenem. The 19,700-bp plasmid pAR3676-OXA-58 harbored blaOXA-58 with genetic contexts consisting of a truncated ISAba3-like-blaOXA58-ISAba3. Additionally, the AR3651 strain exhibited resistance to imipenem and meropenem. The genome of AR3651 comprised one 9,837-bp RepA_AB plasmid pAR3651-OXA-72 harboring blaOXA-72. The blaOXA-72 was flanked by XerC/XerD recombination sites. The conjugation of plasmids pAR3676-OXA-58 and pAR3651-OXA-72 from A. pittii to A. baumannii ATCC 17978RIFR failed three independent times. Phylogenetic analysis of A. pittii strains AR3676, AR3651 and other 504 A. pittii strains collected between 1966 and 2022 from various geographic localities, revealed genetic diversity with a heterogeneous distribution of carbapenemase genes. CONCLUSION: A. pittii strains with plasmid carrying blaOXA-58 or blaOXA-72 may serve as an important reservoir of carbepenemase genes. The carbepenemase genes on a single plasmid may facilitate their dissemination and persistence. Additionally, the pdif sites and mobile elements play an important role in the mobilization of resistance genes and plasmid evolution.

Dissemination of Ceftriaxone-Resistant Salmonella Enteritidis Harboring Plasmids Encoding blaCTX-M-55 or blaCTX-M-14 Gene in China.

Salmonella Enteritidis was the primary foodborne pathogen responsible for acute gastroenteritis. The growing ceftriaxone resistance poses a significant threat to public health. Infection with S. Enteritidis has emerged as a major public health concern, particularly in developing countries. However, research on ceftriaxone-resistant S. Enteritidis (CRO-RSE) remains limited, particularly concerning its resistance mechanism, plasmid structure, and transmission characteristics. This study aims to address these gaps comprehensively. We collected 235 S. Enteritidis isolates from Hangzhou First People's Hospital between 2010 and 2020. Among these, 8.51% (20/235) exhibited resistance to ceftriaxone. Whole-genome analysis revealed that 20 CRO-RSE isolates harbored blaCTX-M-55 or blaCTX-M-14 on the plasmid. Moreover, the dissemination of the blaCTX-M-type gene was associated with IS26 and ISEcp1. Plasmid fusion entailing the integration of the p1 plasmid with antibiotic resistance genes and the p2 (pSEV) virulence plasmid was observed in certain CRO-RSE. Additionally, the structural analysis of the plasmids unveiled two types carrying the blaCTX-M-type gene: type A with multiple replicons and type B with IncI1 (Alpha) replicon. Type B plasmids exhibited superior adaptability and stability compared to type A plasmids within Enterobacteriaceae. Interestingly, although the type B (S808-p1) plasmid displayed the potential to spread to Acinetobacter baumannii, it failed to maintain stability in this species.

Characteristics and phylogenetic distribution of megaplasmids and prediction of a putative chromid in Pseudomonas aeruginosa.

Research on megaplasmids that contribute to the spread of antimicrobial resistance (AMR) in Pseudomonas aeruginosa strains has grown in recent years due to the now widely used technologies allowing long-read sequencing. Here, we systematically analyzed distinct and consistent genetic characteristics of megaplasmids found in P. aeruginosa. Our data provide information on their phylogenetic distribution and hypotheses tracing the potential evolutionary paths of megaplasmids. Most of the megaplasmids we found belong to the IncP-2-type, with conserved and syntenic genetic backbones carrying modules of genes associated with chemotaxis apparatus, tellurite resistance and plasmid replication, segregation, and transmission. Extensively variable regions harbor abundant AMR genes, especially those encoding ß-lactamases such as VIM-2, IMP-45, and KPC variants, which are high-risk elements in nosocomial infection. IncP-2 megaplasmids act as effective vehicles transmitting AMR genes to diverse regions. One evolutionary model of the origin of megaplasmids claims that chromids can develop from megaplasmids. These chromids have been characterized as an intermediate between a megaplasmid and a chromosome, also containing core genes that can be found on the chromosome but not on the megaplasmid. Using in silico prediction, we identified the "PABCH45 unnamed replicon" as a putative chromid in P. aeruginosa, which shows a much higher similarity and closer phylogenetic relationship to chromosomes than to megaplasmids while also encoding plasmid-like partition genes. We propose that such a chromid could facilitate genome expansion, allowing for more rapid adaptations to novel ecological niches or selective conditions, in comparison to megaplasmids.

Precise CRISPR/Cpf1 genome editing system in the Deinococcus radiodurans with superior DNA repair mechanisms.

Deinococcus radiodurans, with its high homologous recombination (HR) efficiency of double-stranded DNA breaks (DSBs), is a model organism for studying genome stability maintenance and an attractive microbe for industrial applications. Here, we developed an efficient CRISPR/Cpf1 genome editing system in D. radiodurans by evaluating and optimizing double-plasmid strategies and four Cas effector proteins from various organisms, which can precisely introduce different types of template-dependent mutagenesis without off-target toxicity. Furthermore, the role of DNA repair genes in determining editing efficiency in D. radiodurans was evaluated by introducing the CRISPR/Cpf1 system into 13 mutant strains lacking various DNA damage response and repair factors. In addition to the crucial role of RecA-dependent HR required for CRISPR/Cpf1 editing, D. radiodurans showed higher editing efficiency when lacking DdrB, the single-stranded DNA annealing (SSA) protein involved in the RecA-independent DSB repair pathway. This suggests a possible competition between HR and SSA pathways in the CRISPR editing of D. radiodurans. Moreover, off-target effects were observed during the genome editing of the pprI knockout strain, a master DNA damage response gene in Deinococcus species, which suggested that precise regulation of DNA damage response is critical for a high-fidelity genome editing system.

High-Throughput Host-Microbe Single-Cell RNA Sequencing Reveals Ferroptosis-Associated Heterogeneity during Acinetobacter baumannii Infection.

Interactions between host and bacterial cells are integral to human physiology. The complexity of host-microbe interactions extends to different cell types, spatial aspects, and phenotypic heterogeneity, requiring high-resolution approaches to capture their full complexity. The latest breakthroughs in single-cell RNA sequencing (scRNA-seq) have opened up a new era of studies in host-pathogen interactions. Here, we first report a high-throughput cross-species dual scRNA-seq technology by using random primers to simultaneously capture both eukaryotic and bacterial RNAs (scRandom-seq). Using reference cells, scRandom-seq can detect individual eukaryotic and bacterial cells with high throughput and high specificity. Acinetobacter baumannii (A.b) is a highly opportunistic and nosocomial pathogen that displays resistance to many antibiotics, posing a significant threat to human health, calling for discoveries and treatment. In the A.b infection model, scRandom-seq witnessed polarization of THP-1 derived-macrophages and the intracellular A.b-induced ferroptosis-stress in host cells. The inhibition of ferroptosis by Ferrostatin-1 (Fer-1) resulted in the improvement of cell vitality and resistance to A.b infection, indicating the potential to resist related infections. scRandom-seq provides a high-throughput cross-species dual single-cell RNA profiling tool that will facilitate future discoveries in unraveling the complex interactions of host-microbe interactions in infection systems and tumor micro-environments.

CRISPR/Cas9-Mediated Editing of BmEcKL1 Gene Sequence Affected Silk Gland Development of Silkworms (Bombyx mori).

The silkworm (Bombyx mori) has served humankind through silk protein production. However, traditional sericulture and the silk industry have encountered considerable bottlenecks and must rely on major technological breakthroughs to keep up with the current rapid developments. The adoption of gene editing technology has nevertheless brought new hope to traditional sericulture and the silk industry. The long period and low efficiency of traditional genetic breeding methods to obtain high silk-yielding silkworm strains have hindered the development of the sericulture industry; the use of gene editing technology to specifically control the expression of genes related to silk gland development or silk protein synthesis is beneficial for obtaining silkworm strains with excellent traits. In this study, BmEcKL1 was specifically knocked out in the middle (MSGs) and posterior (PSGs) silk glands using CRISPR/Cas9 technology, and ΔBmEcKL1-MSG and ΔBmEcKL1-PSG strains with improved MSGs and PSGs and increased silk production were obtained. This work identifies and proves that BmEcKL1 directly or indirectly participates in silk gland development and silk protein synthesis, providing new perspectives for investigating silk gland development and silk protein synthesis mechanisms in silkworms, which is of great significance for selecting and breeding high silk-yielding silkworm varieties.

Genome sequencing unveils blaKPC-2-harboring plasmids as drivers of enhanced resistance and virulence in nosocomial Klebsiella pneumoniae.

The threat posed by Klebsiella pneumoniae in healthcare settings has worsened due to the evolutionary advantages conferred by blaKPC-2-harboring plasmids (pKPC-2). However, the specific evolutionary pathway of nosocomial K. pneumoniae carrying pKPC-2 and its transmission between patients and healthcare environments are not yet well understood. Between 1 August and 31 December 2019, 237 ST11 KPC-2-producing-carbapenem-resistant K. pneumoniae (CRKP) (KPC-2-CRKP) were collected from patient or ward environments in an intensive care unit and subjected to Illumina sequencing, of which 32 strains were additionally selected for Nanopore sequencing to obtain complete plasmid sequences. Bioinformatics analysis, conjugation experiments, antimicrobial susceptibility tests, and virulence assays were performed to identify the evolutionary characteristics of pKPC-2. The pKPC-2 plasmids were divided into three subgroups with distinct evolutionary events, including Tn3-mediated plasmid homologous recombination, IS26-mediated horizontal gene transfer, and dynamic duplications of antibiotic resistance genes (ARGs). Surprisingly, the incidence rates of multicopy blaKPC-2, blaSHV-12, and blaCTX-M-65 were quite high (ranging from 27.43% to 67.01%), and strains negative for extended-spectrum ß-lactamase tended to develop multicopy blaKPC-2. Notably, the presence of multicopy blaSHV-12 reduced sensitivity to ceftazidime/avibactam (CZA), and the relative expression level of blaSHV-12 in the CZA-resistant group was 6.12 times higher than that in the sensitive group. Furthermore, a novel hybrid pKPC-2 was identified, presenting enhanced virulence levels and decreased susceptibility to CZA. This study emphasizes the notable prevalence of multicopy ARGs and provides a comprehensive insight into the intricate and diverse evolutionary pathways of resistant plasmids that disseminate among patients and healthcare environments.IMPORTANCEThis study is based on a CRKP screening program between patients and ward environments in an intensive care unit, describing the pKPC-2 (blaKPC-2-harboring plasmids) population structure and evolutionary characteristics in clinical settings. Long-read sequencing was performed in genetically closely related strains, enabling the high-resolution analysis of evolution pathway between or within pKPC-2 subgroups. We revealed the extremely high rates of multicopy antibiotic resistance genes (ARGs) in clinical settings and its effect on resistance profile toward novel ß-lactam/ß-lactamase inhibitor combinations, which belongs to the last line treatment choices toward CRKP infection. A novel hybrid pKPC-2 carrying CRKP with enhanced resistance and virulence level was captured during its clonal spread between patients and ward environment. These evidences highlight the threat of pKPC-2 to CRKP treatment and control. Thus, surveillance and timely disinfection in clinical settings should be practiced to prevent transmission of CRKP carrying threatful pKPC-2. And rational use of antibiotics should be called for to prevent inducing of pKPC-2 evolution, especially the multicopy ARGs.

Correlation analysis between Raman spectral signature and transcriptomic features of carbapenem-resistant Klebsiella pneumoniae.

The Raman microspectroscopy technology has been successfully applied to evaluate the molecular composition of living cells for identifying cell types and states, but the rationale behind it was not well investigated. In this study, we acquired single-cell Raman spectra (SCRS) of three Klebsiella pneumoniae (K. pneumoniae) strains with different Carbapenem resistant mechanisms and analyzed them with machine learning algorithm. Two carbapenem resistant Klebsiella pneumoniae (CRKP) strains can be successfully distinguished from susceptible strain and CRKP with KPC or IMP carbapenemases can be classified with an overall accuracy achieving 100 %. Furthermore, we performed a correlation analysis between transcriptome and Raman spectra, and found that Raman shifts such as 752 and 1039 cm-1 highly correlated with drug resistance genes expression and could be regarded as Raman biomarkers for CRKP with different mechanisms. The findings of the study provide a theoretical basis for identifying the relationship between Raman spectra and transcriptome of bacteria, as well as a novel method for rapid identification of CRKP and their carbapenemases types.

bla KPC-2 overexpression and bla GES-5 carriage as major imipenem/relebactam resistance mechanisms in Pseudomonas aeruginosa high-risk clones ST463 and ST235, respectively, in China.

Pseudomonas aeruginosa high-risk clones pose severe threats to public health. Here, we characterize the imipenem/relebactam (IR) resistance mechanisms in P. aeruginosa high-risk clones sequence type 235 (ST235) and ST463 in China. Minimum inhibitory concentrations (MICs) were determined, and Illumina short-read sequencing was performed for 1,168 clinical carbapenem-resistant P. aeruginosa (CRPA) isolates. The gene copy number and expression level were analyzed by Illumina sequencing depth and reverse transcription-quantitative PCR, respectively. Resistance conferred by bla GES-5 was evaluated by cloning experiments. ST463 and ST235 accounted for 9.8% (115/1,168) and 4.5% (53/1,168) of total isolates, respectively, and showed high frequencies of extensively drug-resistant and difficult-to-treat resistant phenotypes. The overall IR-resistant rate in CRPA was 21.0% (245/1,168). However, the IR resistance rate was 81.7% (94/115) in ST463-PA and 52.8% (28/53) in ST235-PA. Of the ST463 isolates, 92.2% (106/115) were Klebsiella pneumoniae carbapenemase-producing P. aeruginosa (KPC-PA), and all 94 IR-resistant ST463-PA produced KPC-2. Compared to IR-susceptible ST463 KPC-2-PA, IR-resistant ST463 KPC-2-PA exhibited significantly higher bla KPC-2 copy numbers and expression levels. In ST463 KPC-2-PA, 16 mg/L relebactam resulted in additional fourfold reductions in imipenem MIC50/90 values compared to 4 mg/L relebactam. In ST235, 1.9% (1/53) carried bla IMP carbapenemase and 54.7% (29/53) carried bla GES carbapenemase. Other than the IMP producer, all 27 IR-resistant ST235-PA produced GES-5. Cloning experiments revealed that imipenem resistance in bla GES-5-carrying PAO1 transformants was generally unaffected by relebactam. In conclusion, IR-resistant CRPA isolates in China were mainly distributed in P. aeruginosa high-risk clones ST463 and ST235. The major underlying IR resistance mechanisms were bla KPC-2 overexpression and bla GES-5 carriage.

IS26 mediated blaCTX-M-65 amplification in Escherichia coli increase the antibiotic resistance to cephalosporin in vivo.

OBJECTIVES: To characterize two Escherichia coli strains isolated from a patient pre- and post-treatment, using ß-lactams and ß-lactam/ß-lactamase inhibitor combinations (BLBLIs). METHODS: A combination of antibiotic susceptibility testing (AST) with whole genome sequencing using Illumina and Oxford Nanopore platforms. Long-read sequencing and reverse transcription-quantitative PCR were performed to determine the copy numbers and expression levels of antibiotic resistance genes (ARGs), respectively. Effect on fitness costs were assessed by growth rate determination. RESULTS: The strain obtained from the patient after the antibiotic treatment (XH989) exhibited higher resistance to cefepime, BLBLIs and quinolones compared with the pre-treatment strain (XH987). Sequencing revealed IS26-mediated duplications of a IS26-fosA3-blaCTX-M-65 plasmid-embedded element in strain XH989. Long-read sequencing (7.4 G data volume) indicated a variation in copy numbers of blaCTX-M-65 within one single culture of strain XH989. Increased copy numbers of the IS26-fosA3-blaCTX-M-65 element were correlated with higher CTX-M-65 expression level and did not impose fitness costs, while facilitating faster growth under high antibiotic concentrations. CONCLUSION: Our study is an example from the clinic how BLBLIs and ß-lactams exposure in vivo possibly promoted the amplification of an IS26-multiple drug resistance (MDR) region. The observation of a copy number variation seen with the blaCTX-M-65 gene in the plasmid of the post-treatment strain expands our knowledge of insertion sequence dynamics and evolution during treatment.

Endemicity and diversification of carbapenem-resistant Acinetobacter baumannii in an intensive care unit.

Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major public health concern globally. Often studied in the context of hospital outbreaks, little is known about the persistence and evolutionary dynamics of endemic CRAB populations. Methods: A three-month cross-sectional observational study was conducted in a 28-bed intensive care unit (ICU) in Hangzhou, China. A total of 5068 samples were collected from the hospital environment (n = 3985), patients (n = 964) and staff (n = 119). CRAB isolates were obtained from 10.5% of these samples (n = 532). All of these isolates, plus an additional 19 from clinical infections, were characterised through whole-genome sequencing. Findings: The ICU CRAB population was dominated by OXA-23-producing global clone 2 isolates (99.3% of all isolates) that could be divided into 20 distinct clusters, defined through genome sequencing. CRAB was persistently present in the ICU, driven by regular introductions of distinct clusters. The hospital environment was heavily contaminated, with CRAB isolated from bed units on 183/335 (54.6%) sampling occasions but from patients on only 72/299 (24.1%) occasions. CRAB was spread to adjacent bed units and rooms, and following re-location of patients within the ICU. We also observed three horizontal gene transfer events between CRAB strains in the ICU, involving three different plasmids. Interpretation: The epidemiology of CRAB in this setting contrasted with previously described clonal outbreaks in high-income countries, highlighting the importance of environmental CRAB reservoirs in ICU epidemiology and the unique challenges in containing the spread of CRAB in ICUs where this important multidrug-resistant pathogen is endemic. Funding: This work was undertaken as part of the DETECTIVE research project funded by the Medical Research Council (MR/S013660/1), National Natural Science Foundation of China (81861138054, 32011530116, 31970128, 31770142), Zhejiang Province Medical Platform Backbone Talent Plan (2020RC075), and the National Key Research and Development Program of China grant (2018YFE0102100). W.v.S was also supported by a Wolfson Research Merit Award (WM160092).

Characterization of two novel VIM-type metallo-ß-lactamases, VIM-84 and VIM-85, associated with the spread of IncP-2 megaplasmids in Pseudomonas aeruginosa.

This study aimed to characterize two novel VIM-type metallo-ß-lactamases, VIM-84 and VIM-85, and reveal the important role of the IncP-2 type megaplasmids in the spread of antimicrobial resistance (AMR) genes. VIM-84 and VIM-85 were encoded by two novel genes bla VIM-84 and bla VIM-85 which showed similarity to bla VIM-24. Both bla VIM-84 and bla VIM-85 are harbored into class 1 integrons embedded into the Tn1403 transposon. The bla VIM-85 gene was identified in a megaplasmid, which was related to 17 megaplasmid sequences with sizes larger than 430 kb, deposited previously in Genbank. A comparative analysis of complete plasmid sequences showed highly similar backbone regions and various AMR genes. A phylogenetic tree revealed that these megaplasmids, which were widely distributed globally, were vehicles for the spread of AMR genes. The bla VIM-24, bla VIM-84, and bla VIM-85 genes were cloned into pGK1900, and the recombinant vectors were further transformed into Escherichia coli DH5α and Pseudomonas aeruginosa PAO1. The antimicrobial susceptibility test of the cloning strains showed high levels of resistance to ß-lactams while they remained susceptible to aztreonam. Enzymatic tests revealed that both, VIM-84 and VIM-85, exhibited higher activity in hydrolyzing ß-lactams compared to VIM-24. A D117N mutation found in VIM-24 affected binding to the antibiotics. IMPORTANCE The metallo-ß-lactamases-producing Pseudomonas aeruginosa strains play an important role in hospital outbreaks and the VIM-type enzyme is the most prevalent in European countries. Two novel VIM-type enzymes in our study, VIM-84 and VIM-85, have higher levels of resistance to ß-lactams and greater hydrolytic activities for most ß-lactams compared with VIM-24. Both bla VIM-84 and bla VIM-85 are harbored into class 1 integrons embedded into the Tn1403 transposon. Notably, the genes bla VIM-85 are carried by three different IncP-2-type megaplasmids which are distributed locally and appear responsible for the spread of antimicrobial resistance genes in hospital settings.

Droplet-based high-throughput single microbe RNA sequencing by smRandom-seq.

Bacteria colonize almost all parts of the human body and can differ significantly. However, the population level transcriptomics measurements can only describe the average bacteria population behaviors, ignoring the heterogeneity among bacteria. Here, we report a droplet-based high-throughput single-microbe RNA-seq assay (smRandom-seq), using random primers for in situ cDNA generation, droplets for single-microbe barcoding, and CRISPR-based rRNA depletion for mRNA enrichment. smRandom-seq showed a high species specificity (99%), a minor doublet rate (1.6%), a reduced rRNA percentage (32%), and a sensitive gene detection (a median of ~1000 genes per single E. coli). Furthermore, smRandom-seq successfully captured transcriptome changes of thousands of individual E. coli and discovered a few antibiotic resistant subpopulations displaying distinct gene expression patterns of SOS response and metabolic pathways in E. coli population upon antibiotic stress. smRandom-seq provides a high-throughput single-microbe transcriptome profiling tool that will facilitate future discoveries in microbial resistance, persistence, microbe-host interaction, and microbiome research.

Mutation in the two-component regulator BaeSR mediates cefiderocol resistance and enhances virulence in Acinetobacter baumannii.

Acinetobacter baumannii has become one of the most challenging pathogens in many countries with limited treatment options available. Cefiderocol, a novel siderophore-conjugated cephalosporin, shows potent in vitro activity against A. baumannii, including isolates resistant to carbapenems. To date, few reports on the mechanisms of cefiderocol resistance are available. In order to investigate potential mechanisms of cefiderocol resistance in A. baumannii, we performed in vitro evolution experiments at sub-lethal concentrations of the antibiotic. All four cefiderocol-resistant strains obtained harbored mutations in two-component system BaeS-BaeR. When we engineered the mutations of BaeS (D89V) and BaeR (S104N) into the genome of ATCC 17978, these mutations increased cefiderocol minimum inhibitory concentrations (MICs) by 8-fold to 16-fold. Transcriptome analyses showed that the expression of MacAB-TolC and MFS transporters was up-regulated in BaeSR mutants. Strains over-expressing MFS transporter and MacAB-TolC displayed higher MICs and higher median inhibition concentration (IC50) values, while MICs and IC50 decreased when efflux pump genes were knocked out. In a BaeR mutant with up-regulated csu operon, we observed a higher number of pili, enhanced surface motility, and increased biofilm formation compared to wild-type ATCC 17978. Using the Galleria mellonella infection model, we found that the BaeS mutant in which paa operon was up-regulated exhibited increased virulence. In conclusion, the mutations in BaeSR decreased cefiderocol susceptibility of A. baumannii through up-regulating efflux pumps gene expression. BaeS or BaeR also controls the expression of csu and paa, influencing biofilm formation, surface motility, and virulence in A. baumannii. IMPORTANCE The widespread prevalence of multi-drug-resistant A. baumannii (MDRAB) poses a significant therapeutic challenge. Cefiderocol is considered a promising antibiotic for the treatment of MDRAB infections. Therefore, it is necessary to study the potential resistance mechanisms of cefiderocol to delay the development of bacterial resistance. Here, we demonstrated that mutations in baeS and baeR reduced the susceptibility of A. baumannii to cefiderocol by up-regulating the expression of the MFS family efflux pump and MacAB-TolC efflux pump. We propose that BaeS mutants increase bacterial virulence by up-regulating the expression of the paa operon. This also reports the regulatory effect of BaeSR on csu operon for the first time. This study provides further insights into the role of BaeSR in developing cefiderocol resistance and virulence in A. baumannii.

Resistance mechanisms of tigecycline in Acinetobacter baumannii.

Acinetobacter baumannii is widely distributed in nature and in hospital settings and is a common pathogen causing various infectious diseases. Currently, the drug resistance rate of A. baumannii has been persistently high, showing a worryingly high resistance rate to various antibiotics commonly used in clinical practice, which greatly limits antibiotic treatment options. Tigecycline and polymyxins show rapid and effective bactericidal activity against CRAB, and they are both widely considered to be the last clinical line of defense against multidrug resistant A. baumannii. This review focuses with interest on the mechanisms of tigecycline resistance in A. baumannii. With the explosive increase in the incidence of tigecycline-resistant A. baumannii, controlling and treating such resistance events has been considered a global challenge. Accordingly, there is a need to systematically investigate the mechanisms of tigecycline resistance in A. baumannii. Currently, the resistance mechanism of A. baumannii to tigecycline is complex and not completely clear. This article reviews the proposed resistance mechanisms of A. baumannii to tigecycline, with a view to providing references for the rational clinical application of tigecycline and the development of new candidate antibiotics.

Genotypic characterization of a Proteus mirabilis strain harboring blaKPC-2 on the IncN plasmid isolated from a patient with bloodstream infection in China.

BACKGROUND: Carbapenemase is the predominant enzyme in the mechanism leading to Enterobacterales resistance to carbapenems, and the rapid spread of the blaKPC gene is a major public health concern. Here, we describe a carbapenem-resistant Proteus mirabilis strain XH983, which harbored a blaKPC-2-producing IncN plasmid, isolated from a bloodstream infection. METHODS: Whole-genome sequencing and bioinformatics analysis were performed to assess the genetic environment of P. mirabilis XH983. Conjugation and transfer experiments were performed and the corresponding strains were confirmed by antimicrobial susceptibility testing. Phylogenetic and comparative genomic analysis were performed to explore the characteristics of carbapenem-resistant P. mirabilis isolates worldwide. RESULTS: P. mirabilis XH983 was isolated from the blood of a patient in Hangzhou, China. The genome of XH983 contained one 4128,916 bp circular chromosome and one 24,225 bp IncN plasmid harboring blaKPC-2. P. mirabilis XH983 had multiple resistance genes, conferring resistance to aminoglycosides [aph(3')-Ia, aph(3'')-Ib, aph(6)-Id, aac(3)-IId, aadA5, aadA1], ß-lactams (blaKPC-2, blaTEM-1B), phenicol (cat, catA1), sulphonamide/trimethoprim (drfA1, drfA17, sul1, sul2) and tetracycline [tet(J)]. The phylogenetic tree showed that XH983 was present in a cluster of 30 isolates, all of which carried blaKPC-2 and most of them came from the same hospital as XH983, indicating the clonal spread of the cluster. CONCLUSION: We characterized carbapenem-resistant P. mirabilis clinical isolate XH983. The genome sequence of P. mirabilis XH983 provides information about resistance mechanisms of P. mirabilis carrying the blaKPC-2 plasmid and the potential spread of blaKPC-2.

Alteration of adeS Contributes to Tigecycline Resistance and Collateral Sensitivity to Sulbactam in Acinetobacter baumannii.

The treatment of extensively drug-resistant (XDR) A. baumannii has emerged as a major problem. Tigecycline (TGC) and sulbactam (SUL) are both effective antibiotics against XDR A. baumannii. Here, we investigated the in-host evolution and mechanism of collateral sensitivity (CS) phenomenon in development of tigecycline resistance accompanied by a concomitant increase of sulbactam susceptibility. A total of four XDR A. baumannii strains were sequentially isolated from the same patient suffering from bacteremia. Core-genome multilocus sequence typing separated all the strains into two clusters. Comparative analysis of isolate pair 1 revealed that multiplication of blaOXA-23 within Tn2006 on the chromosome contributed to the change in the antimicrobial susceptibility phenotype of isolate pair 1. Additionally, we observed the emergence of CS to sulbactam in isolate pair 2, as demonstrated by an 8-fold increase in the TGC MIC with a simultaneous 4-fold decrease in the SUL MIC. Compared to the parental strain Ab-3557, YZM-0406 showed partial deletion in the two-component system sensor adeS. Reconstruction of the adeS mutant in Ab-3557 in situ suggested that TGC resistance and CS to SUL were mainly caused by the mutation of adeS. Overall, our study reported a novel CS combination of TGC and SUL in A. baumannii and further revealed a mechanism of CS attributed to the mutation of adeS. This study provides a valuable foundation for developing effective regimens and sequential combinations of tigecycline and sulbactam against XDR A. baumannii. IMPORTANCE Collateral sensitivity (CS) has become an increasingly common evolutionary trade-off during adaptive bacterial evolution. Here, we report a novel combination of tigecycline (TGC) resistance and CS to sulbactam (SUL) in A. baumannii. TGC and SUL are both effective antibiotics against XDR A. baumannii, and it is essential to reveal the mechanism of CS between TGC and SUL. In our study, the partial deletion of adeS, a two-component system sensor, was confirmed to be the key factor contributing to this CS phenomenon. This study provides a valuable foundation for developing effective regimens and sequential combinations of tigecycline and sulbactam against XDR A. baumannii.

Dynamic chromatin conformation and accessibility changes mediate the spatial-specific gene regulatory network in Bombyx mori.

Silk gland genes of Bombyx mori can have strict spatial expression patterns, which impact their functions and silk quality. However, our understanding of their regulation mechanisms is currently insufficient. To address this, the middle silk gland (MSG) and posterior silk gland (PSG) of the silkworm were investigated. Gene ontology annotation showed that spatially specific expressed genes were involved in the formation of H3k9me and chromatin topology. Chromatin conformation data generated by Hi-C showed that the topologically associated domain boundaries around FibL and Sericin1 genes were significantly different between MSG and PSG. Changes in chromatin conformation led to changes in chromatin activity, which significantly affected the expression of nearby genes in silkworm. Chromatin accessibility regions of MSG and PSG were analyzed using FAIRE-seq, and 1006 transcription factor motifs were identified in open chromatin regions. Furthermore, the spatial-specific expression patterns of silk gland genes were mainly associated with homeobox-contained transcription factors, such as POU-M2, which was specifically bound and relatively highly expressed in the MSG. The regulatory network mediated by POU-M2 regulated most of the spatial-specific expressed genes in MSG, such as ADH1. These results can aid in improving silk performance, optimizing silkworm breeding, and improving the gene spatial regulatory model research for insects.

Artificial intelligence-aided rapid and accurate identification of clinical fungal infections by single-cell Raman spectroscopy.

Integrating artificial intelligence and new diagnostic platforms into routine clinical microbiology laboratory procedures has grown increasingly intriguing, holding promises of reducing turnaround time and cost and maximizing efficiency. At least one billion people are suffering from fungal infections, leading to over 1.6 million mortality every year. Despite the increasing demand for fungal diagnosis, current approaches suffer from manual bias, long cultivation time (from days to months), and low sensitivity (only 50% produce positive fungal cultures). Delayed and inaccurate treatments consequently lead to higher hospital costs, mobility and mortality rates. Here, we developed single-cell Raman spectroscopy and artificial intelligence to achieve rapid identification of infectious fungi. The classification between fungi and bacteria infections was initially achieved with 100% sensitivity and specificity using single-cell Raman spectra (SCRS). Then, we constructed a Raman dataset from clinical fungal isolates obtained from 94 patients, consisting of 115,129 SCRS. By training a classification model with an optimized clinical feedback loop, just 5 cells per patient (acquisition time 2 s per cell) made the most accurate classification. This protocol has achieved 100% accuracies for fungal identification at the species level. This protocol was transformed to assessing clinical samples of urinary tract infection, obtaining the correct diagnosis from raw sample-to-result within 1 h.

Bioinformatic analysis of structures and encoding genes of Escherichia coli surface polysaccharides sheds light on the heterologous biosynthesis of glycans.

BACKGROUND: Surface polysaccharides (SPs), such as lipopolysaccharide (O antigen) and capsular polysaccharide (K antigen), play a key role in the pathogenicity of Escherichia coli (E. coli). Gene cluster for polysaccharide antigen biosynthesis encodes various glycosyltransferases (GTs), which drive the process of SP synthesis and determine the serotype. RESULTS: In this study, a total of 7,741 E. coli genomic sequences were chosen for systemic data mining. The monosaccharides in both O and K antigens were dominated by D-hexopyranose, and the SPs in 70-80% of the strains consisted of only the five most common hexoses (or some of them). The linkages between the two monosaccharides were mostly α-1,3 (23.15%) and ß-1,3 (20.49%) bonds. Uridine diphosphate activated more than 50% of monosaccharides for glycosyltransferase reactions. These results suggest that the most common pathways could be integrated into chassis cells to promote glycan biosynthesis. We constructed a database (EcoSP, http://ecosp.dmicrobe.cn/ ) for browse this information, such as monosaccharide synthesis pathways. It can also be used for serotype analysis and GT annotation of known or novel E. coli sequences, thus facilitating the diagnosis and typing. CONCLUSIONS: Summarizing and analyzing the properties of these polysaccharide antigens and GTs are of great significance for designing glycan-based vaccines and the synthetic glycobiology.