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In an era of increasing concerns about antimicrobial resistance, tigecycline is likely to have a critically important role in the treatment of carbapenem-resistant Enterobacteriaceae , the most problematic pathogens in human clinical settings—especially carbapenem-resistant K. pneumoniae . Here, we identified a new plasmid-borne RND-type tigecycline resistance determinant, TMexCD1-TOprJ1, which is widespread among K. pneumoniae isolates from food animals. tmexCD1-toprJ1 appears to have originated from the chromosome of a Pseudomonas species and may have been transferred onto plasmids by adjacent site-specific integrases. Although tmexCD1-toprJ1 still appears to be rare in human clinical isolates, considering the transferability of the tmexCD1-toprJ1 gene cluster and the broad substrate spectrum of TMexCD1-TOprJ1, further dissemination of this mobile tigecycline resistance determinant is possible. Therefore, from a “One Health” perspective, measures are urgently needed to monitor and control its further spread. The current low prevalence in human clinical isolates provides a precious time window to design and implement measures to tackle this. Transporters belonging to the chromosomally encoded resistance-nodulation-division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria. However, the cotransfer of large gene clusters encoding RND-type pumps from the chromosome to a plasmid appears infrequent, and no plasmid-mediated RND efflux pump gene cluster has yet been found to confer resistance to tigecycline. Here, we identified a novel RND efflux pump gene cluster, designated tmexCD1-toprJ1 , on plasmids from five pandrug-resistant Klebsiella pneumoniae isolates of animal origin. TMexCD1-TOprJ1 increased (by 4- to 32-fold) the MICs of tetracyclines (including tigecycline and eravacycline), quinolones, cephalosporins, and aminoglycosides for K. pneumoniae , Escherichia coli , and Salmonella . TMexCD1-TOprJ1 is closely related (64.5% to 77.8% amino acid identity) to the MexCD-OprJ efflux pump encoded on the chromosome of Pseudomonas aeruginosa . In an IncFIA plasmid, pHNAH8I, the tmexCD1-toprJ1 gene cluster lies adjacent to two genes encoding site-specific integrases, which may have been responsible for its acquisition. Expression of TMexCD1-TOprJ1 in E. coli resulted in increased tigecycline efflux and in K. pneumoniae negated the efficacy of tigecycline in an in vivo infection model. Expression of TMexCD1-TOprJ1 reduced the growth of E. coli and Salmonella but not K. pneumoniae . tmexCD1-toprJ1 -positive Enterobacteriaceae isolates were rare in humans (0.08%) but more common in chicken fecal (14.3%) and retail meat (3.4%) samples. Plasmid-borne tmexCD1-toprJ1 -like gene clusters were identified in sequences in GenBank from Enterobacteriaceae and Pseudomonas strains from multiple continents. The possibility of further global dissemination of the tmexCD1 - toprJ1 gene cluster and its analogues in Enterobacteriaceae via plasmids may be an important consideration for public health planning. IMPORTANCE In an era of increasing concerns about antimicrobial resistance, tigecycline is likely to have a critically important role in the treatment of carbapenem-resistant Enterobacteriaceae , the most problematic pathogens in human clinical settings—especially carbapenem-resistant K. pneumoniae . Here, we identified a new plasmid-borne RND-type tigecycline resistance determinant, TMexCD1-TOprJ1, which is widespread among K. pneumoniae isolates from food animals. tmexCD1-toprJ1 appears to have originated from the chromosome of a Pseudomonas species and may have been transferred onto plasmids by adjacent site-specific integrases. Although tmexCD1-toprJ1 still appears to be rare in human clinical isolates, considering the transferability of the tmexCD1-toprJ1 gene cluster and the broad substrate spectrum of TMexCD1-TOprJ1, further dissemination of this mobile tigecycline resistance determinant is possible. Therefore, from a “One Health” perspective, measures are urgently needed to monitor and control its further spread. The current low prevalence in human clinical isolates provides a precious time window to design and implement measures to tackle this.

Clonal complex 4821 (CC4821) Neisseria meningitidis, usually resistant to quinolones but susceptible to penicillin and third-generation cephalosporins, is increasing worldwide. To characterize the penicillin-nonsusceptible (Pen ) meningococci, we analyzed 491 meningococci and 724 commensal Neisseria isolates in Shanghai, China, during 1965-2020. The Pen proportion increased from 0.3% in 1965-1985 to 7.0% in 2005-2014 and to 33.3% in 2015-2020. Of the 26 Pen meningococci, 11 (42.3%) belonged to the CC4821 cluster; all possessed mutations in penicillin-binding protein 2, mostly from commensal Neisseria. Genetic analyses and transformation identified potential donors of 6 penA alleles. Three Pen meningococci were resistant to cefotaxime, 2 within the CC4821 cluster. With 96% of the Pen meningococci beyond the coverage of scheduled vaccination and the cefotaxime-resistant isolates all from toddlers, quinolone-resistant CC4821 has acquired penicillin and cefotaxime resistance closely related to the internationally disseminated ceftriaxone-resistant gonococcal FC428 clone, posing a greater threat especially to young children.

We explored the role of commensal Neisseria in the emergence of third-generation cephalosporin-resistant N. meningitidis. Cefotaxime resistance-conferring penA795 was prevalent among commensal Neisseria isolates in Shanghai, China, and was acquired by a serogroup C quinolone-resistant sequence type 4821 N. meningitidis, Nm507, causing fulminant meningitis in an unvaccinated 2-year-old child.

Background Soybean ( Glycine max (L.) Merr.) is a crucial crop due to its high plant protein and oil content. Previous studies have shown that soybeans exhibit significant heterosis in terms of yield and protein content However, the practical application of soybean heterosis remains difficult, as the molecular mechanisms underlying photoperiod-sensitive genic male sterile (PGMS) is still unclear. Results This study characterized the PGMS line 88-428BY, which is sterile under short-day (SD) conditions and fertile under long-day (LD) conditions. To elucidate the genetic basis for this trait, we collected anthers, from 88-428BY under SD and LD conditions at three developmental stages, resulting in the identification of differentially expressed genes (DEGs) (2333, 2727 and 7282 DEGs, respectively) using Illumina transcriptome analysis. Using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, we fund that among the DEGs, enriched genes were associated with photoperiod stress, light stimulus, oxidation-reduction processes, multicellular organism development and protein phosphorylation. Additionally, weighted correlation network analysis identified four modules (blue, brown, red, and yellow) that were significantly correlated with PGMS, revealing co-expressed hub genes with potential regulatory roles. Functional annotation of 224 DEGs with|KME| >0.9 across the four modules in seven databases highlighted their involvement in light stimulus, oxidation-reduction processes, multicellular organism development, and protein phosphorylation, suggesting their importance in soybean PGMS. By integrating fertility-related genes previously identified by other studies with the DEGs from our analysis, we identified eight candidate genes associated with the photosensitive sterility in soybeans. Conclusions This study enhances the understanding of PGMS in soybean and establishes the genetic basis for a two-line hybrid seed production system in soybean.

The presence of adhesins is arguably an important determinant of pathogenicity for Uropathogenic Escherichia coli (UPEC). Antimicrobial susceptibilities were tested by agar dilution method, fifteen adhesin genes were detected by polymerase chain reaction, and multilocus sequence typing (MLST) was analyzed in 70 UPEC isolates and 41 commensal E. coli strains. Extended-spectrum β-lactamase (ESBL) was determined with confirmatory test. The prevalence of ESBL-producers in UPEC (53%, 37/70) was higher than the commensal intestinal isolates (7%, 3/41), and 97% (36/37) of the ESBL-producing UPEC harbored bla CTX-M genes. afa was present in 36% (10/28) UPEC isolates from recurrent lower urinary tract infection (UTI), and none in the acute pyelonephritis, acute uncomplicated cystitis or commensal strains (P<0.0001). papG was detected in 28% (20/70) of UPEC isolates, while 5% (2/41) of the commensal strains were papG positive (P = 0.0025), and the prevalence of papG was significantly higher in acute pyelonephritis group (71%) than the other two UTI groups (P<0.0001). The prevalence of flu, yqi, yadN and ygiL was significantly higher in UPEC isolates than in the commensal strains. ESBL-producing UPEC showed a lower prevalence of adhesin genes compared with non-ESBL-producing strains. The MLST profiles were different between UPEC and commensal strains, with ST131 (19%, 13/70) and ST10 (20%, 8/41) being the most common MLSTs, respectively. This study demonstrated that several adhesin genes were more prevalent in UPEC isolates than in commensal E. coli, and afa may be associated with recurrent lower UTI whereas papG is more frequently associated with acute pyelonephritis.

Objective Healthcare-associated Gram-negative bacterial meningitis is a substantial clinical issue with poor outcomes, especially for neurosurgical patients. Here, we aimed to study the characteristics and treatment options of patients with healthcare-associated carbapenem-non-susceptible (Carba-NS) Gram-negative bacterial meningitis. Methods This observational cohort study was conducted at a teaching hospital from 2004 to 2019. The clinical characteristics of patients with meningitis with Carba-NS and carbapenem-susceptible (Carba-S) bacilli were compared, and the antimicrobial chemotherapy regimens and outcomes for Carba-NS Gram-negative bacterial meningitis were analyzed. Results A total of 505 patients were included, of whom 83.8% were post-neurosurgical patients. The most common isolates were Acinetobacter spp. and Klebsiella spp., which had meropenem-resistance rates of 50.6% and 42.5%, respectively, and showed a markedly growing carbapenem-resistance trend. Kaplan–Meier curve analysis revealed that Carba-NS Gram-negative bacilli were associated with a significantly higher in-hospital mortality rate (18.8%, 35/186) compared to the Carba-S group (7.4%, 9/122; P  = 0.001). For Carba-NS Enterobacterales meningitis, aminoglycoside-based and trimethoprim-sulfamethoxazole-based regimens yielded significantly higher clinical efficacy rates than non-aminoglycoside-based and non-trimethoprim-sulfamethoxazole-based regimens (69.0% vs. 38.7%, P  = 0.019 and 81.8% vs. 46.9%, P  = 0.036, respectively). For Carba-NS A. baumannii complex meningitis, tetracycline-based (including doxycycline, minocycline, or tigecycline) therapy achieved a significantly higher clinical efficacy rate (62.9%, 22/35) than the non-tetracycline-based therapy group (40.4%, 19/47; P  = 0.044). Conclusions Our findings revealed that Carba-NS Gram-negative bacilli are associated with higher in-hospital mortality in patients with healthcare-associated meningitis. The combination therapies involving particular old antibiotics may improve patients’ outcome. Trial registration This study was registered on the Chinese Clinical Trial Register under ChiCTR2000036572 (08/2020).

Background Hypervirulent Klebsiella pneumoniae (hvKP) infection is characterized by its potential severity and metastatic propensity, underlying the importance to employ bactericidal treatment promptly for infection source control. The aim of this study is to investigate the bactericidal activity of antibacterial combination against hvKP, with a particular focus on whether levofloxacin-based combinations can suppress levofloxacin-induced efflux pump related drug resistance. Methods We conducted in vitro antimicrobial susceptibility tests on 12 hvKP clinical isolates and the reference strain NTUH-K2044. Checkerboard assays were conducted to evaluate the combination activity of levofloxacin with amikacin or ceftazidime. Time-kill experiments were performed to determining the bactericidal activities of levofloxacin, amikacin, ceftazidime, and the levofloxacin-based combinations against NTUH-K2044. Additionally, the mechanisms underlying induced quinolone resistance were investigated. Results Twelve clinical hvKP isolates were highly susceptible to levofloxacin, amikacin, and ceftazidime. The levofloxacin–amikacin and levofloxacin–ceftazidime combinations both demonstrated antibacterial activities as indifference. Time-kill experiments showed that an eight-fold or lower minimal inhibitory concentration (≤ 8×MIC) of levofloxacin, ≤ 1×MIC of amikacin and of ceftazidime, exhibited bactericidal activity against NTUH-K2044 during the initial 1–4 h with various levels of bacterial counts decreasing followed by regrowth. The regrowth samples had high levofloxacin MICs indicating the occurrence of induced resistance. Real-time PCR and wild-type oqxR complementation demonstrated point mutations in oqxR of NTUH-K2044, resulting in an overexpression of the efflux pump encoding gene of oqxAB . Both the levofloxacin–amikacin and levofloxacin–ceftazidime combinations exhibited bactericidal synergy without bacterial regrowth when they were tested with 2×MIC of levofloxacin combined with either 1/2×MIC of amikacin or 1×MIC of ceftazidime. Conclusions These results demonstrated that levofloxacin combined with amikacin or ceftazidime exhibited superior bactericidal activities compared to levofloxacin against hvKP and might prevent levofloxacin-induced resistance related to oqxAB overexpression.

To investigate the prevalence, location and genetic environments of fosfomycin-resistance (fos) genes in methicillin-resistant Staphylococcus aureus (MRSA) clinical strains, 67 fosfomycin-resistant MRSA strains were isolated from the blood and cerebrospinal fluid samples at a teaching hospital in Shanghai. The presence of fos genes in these clinical strains was detected by PCR and sequencing. The locations of fos genes were determined by Southern blotting and genetic environments were analyzed by primer walking sequencing. Multiple locus sequence typing (MLST) was used to characterize genetic diversity. Conjugation was performed to evaluate the transferability of fos genes. Among 67 fosfomycin-resistant MRSA strains, nine high level fosfomycin resistant strains (≥128 μg/ml) were fosB-positive. Three new subtypes of fosB, designated as fosB4, fosB5, and fosB6, were identified. fosB1, fosB4 or fosB6 genes were located on small plasmids (ca. 2.5 kb) and flanked by an analogous replication gene (rep). Differently, the fosB5 gene was surrounded by a shorter rep gene and two copies of a transposon gene (tnp) that shared high identity with the IS257-like transposon. Four MLST types were found among the nine fosB-positive strains. Transconjugants with the fosB genes were resistant to fosfomycin with MIC 64 or 128 μg/ml. In conclusion, different subtypes and genetic environment of fosB genes indicate that gene heterogeneity for fosfomycin resistance in MRSA isolates.

Hypervirulent variants of (hvKp) that cause invasive community-acquired pyogenic liver abscess (PLA) have emerged globally. Little is known about the virulence determinants associated with hvKp, except for the virulence genes and siderophores ( ) carried by the pK2044-like large virulence plasmid. Here, we collected most recent clinical isolates of hvKp from PLA samples in China, and performed clinical, molecular, and genomic sequencing analyses. We found that 90.9% (40/44) of the pathogens causing PLA were . Among the 40 LA-Kp, K1 (62.5%), and K2 (17.5%) were the dominant serotypes, and ST23 (47.5%) was the major sequence type. S1-PFGE analyses demonstrated that although 77.5% (31/40) of the LA-Kp isolates harbored a single large virulence plasmid varied in size, 5 (12.5%) isolates had no plasmid and 4 (10%) had two or three plasmids. Whole genome sequencing and comparative analysis of 3 LA-Kp and 3 non-LA-Kp identified 133 genes present only in LA-Kp. Further, large scale screening of the 133 genes in 45 LA-Kp and 103 non-LA-Kp genome sequences from public databases identified 30 genes that were highly associated with LA-Kp, including and and 21 new genes. Then, these 21 new genes were analyzed in 40 LA-Kp and 86 non-LA-Kp clinical isolates collected in this study by PCR, showing that new genes were present 80-100% among LA-Kp isolates while 2-11% in isolates from sputum and urine. Several of the 21 genes have been proposed as virulence factors in other bacteria, such as the gene encoding SAM-dependent methyltransferase and which protects bacteria from phagocytosis. Taken together, these genes are likely new virulence factors contributing to the hypervirulence phenotype of hvKp, and may deepen our understanding of virulence mechanism of hvKp.