Gram-positive bacteria can develop resistance to linezolid through mutations in the 50S large-subunit ribosomal proteins, such as L3. However, this mechanism is rarely reported in clinical enterococcal strains. Here, we describe a pair of Enterococcus faecium strains, 1505efm and 1583efm, isolated from a meningioma patient at different stages of linezolid treatment. Strain 1583efm, which was obtained after 17 days of linezolid treatment, exhibited an eightfold higher linezolid MIC (64 mg/L) than the initially isolated strain 1505efm (8 mg/L). Meanwhile, 1583efm became susceptible to erythromycin, tetracycline, and high-level streptomycin due to the loss of plasmid-borne resistance genes erm(A), erm(B), tet(M), ant(6)-Ia, and aph(3')-III. Whole-genome comparison revealed a deletion in the rplC gene of 1583efm, resulting in the loss of 21 amino acids (∆Tyr139-Arg159) in ribosomal protein L3. Structural modeling showed that this deletion disrupts the linezolid hydrogen bond network near the ribosomal peptidyl transferase center, reducing its binding affinity. The mutation, which remained stable over 50 generations without antibiotics, imposed a significant fitness cost on the bacterial cells and led to an increase in the expression of the truncated rplC gene. Our study uncovers a novel linezolid resistance mechanism involving the deletion of ribosomal protein L3 in clinical E. faecium strains within the host.

Vancomycin heteroresistance is prone to missed detection and poses a risk of clinical treatment failure. We encountered one clinical Enterococcus faecium strain, SRR12, that carried a complete vanM gene cluster but was determined as susceptible to vancomycin using the broth microdilution method. However, distinct subcolonies appeared within the clear zone of inhibition in the E-test assay, one of which, named SRR12-v1, showed high-level resistance to vancomycin. SRR12 was confirmed as heteroresistant to vancomycin using population analysis profiling and displayed "revive" growth curves with a lengthy lag phase of over 13 hours when exposed to 2-32 mg/L vancomycin. The resistant subcolony SRR12-v1 was found to carry an identical vanM gene cluster to that of SRR12 but a significantly increased vanM copy number in the genome. Long-read whole genome sequencing revealed that a one-copy vanM gene cluster was located on a pELF1-like linear plasmid in SRR12. In comparison, tandem amplification of the vanM gene cluster jointed with IS1216E was seated on a linear plasmid in the genome of SRR12-v1. These amplifications of the vanM gene cluster were demonstrated as unstable and would decrease accompanied by fitness reversion after serial passaging for 50 generations under increasing vancomycin pressure or without antibiotic pressure but were relatively stable under constant vancomycin pressure. Further, vanM resistance in resistant variants was verified to be carried by conjugative plasmids with variable sizes using conjugation assays and S1-pulsed field gel electrophoresis blotting, suggesting the instability/flexibility of vanM cluster amplification in the genome and an increased risk of vanM resistance dissemination.

Purpose:We aimed to characterize a novel blaNDM-5 and blaKPC-2 co-carrying hybrid plasmid from a clinical carbapenem-resistant Klebsiella pneumoniae (CRKP) strain. Methods:Antimicrobial susceptibility was determined by the broth microdilution method. Plasmid size and localization were estimated using S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting. Plasmid transfer ability was evaluated by conjugation experiments. Whole genome sequencing (WGS) was performed using Illumina NovaSeq6000 and Oxford Nanopore MinION platforms. Genomic characteristics were analyzed using bioinformatics methods. Results:Strain ZY27320 was a multidrug-resistant (MDR) clinical ST11 K. pneumoniae strain that confers high-level resistance to carbapenems (meropenem, MIC 128 mg/L; imipenem, MIC 64 mg/L) and ceftazidime/avibactam (MIC >128/4 mg/L). Both S1-PFGE-Southern blotting and whole genome sequencing revealed that the carbapenemase genes blaKPC-2 and blaNDM-5 were carried by the same IncFIIpHN7A8:IncR:IncN hybrid plasmid (pKPC2_NDM5). Conjugation experiments indicated that pKPC2_NDM5 was a non-conjugative plasmid. Conclusion:This is the first report of a hybrid plasmid carrying both carbapenemase genes blaNDM-5 and blaKPC-2 in a clinical K. pneumoniae ST11 isolate that confers resistance to both ceftazidime/avibactam and carbapenems, thereby presenting a serious threat to treatment in clinical practice.

Background Vancomycin-variable enterococci (VVE) are a potential risk factor for vancomycin resistance gene dissemination and clinical treatment failure. vanM has emerged as a new prevalent resistance determinant among clinical enterococci in China. A total of 54 vancomycin-susceptible enterococci (VSE) isolates carrying incomplete vanM gene clusters were isolated in our previous study. Objectives To determine the potential of vanM-carrying VSE to develop vancomycin resistance and investigate the mechanism of alteration of the resistance phenotype. Methods Fifty-four vanM-positive VSE strains were induced in vitro by culturing in increasing concentrations of vancomycin. Genetic changes between three parent VVE strains and their resistant variants were analysed using Illumina and long-read sequencing technologies, quantitative PCR and Southern blot hybridization. Changes in expression level were determined by quantitative RT-PCR. Results Twenty-five of the 54 VSE strains carrying vanM became resistant upon vancomycin exposure. A significant increase in vanM copy number was observed ranging from 5.28 to 127.64 copies per cell in induced resistant VVE strains. The vanM transposon was identified as tandem repeats with IS1216E between them, and occurred in either the plasmid or the chromosome of resistant VVE cells. In addition, an increase in vanM expression was observed after resistance conversion in VVE. Conclusions This study identified tandem amplification of the vanM gene cluster as a new mechanism for vancomycin resistance in VVE strains, offering a competitive advantage for VVE under antibiotic pressure.

Here we report the prevalence of the suppressed vanM gene cluster as a reservoir of vancomycin resistance genes. Among 1284 clinical isolates of enterococci from four hospitals in Hangzhou, China, 55 isolates of Enterococcus faecium and one isolate of Enterococcus faecalis were screened positive for the vanM genotype. Antimicrobial susceptibility testing showed that 55 of the 56 vanM-positive isolates were susceptible to vancomycin and teicoplanin. Most of them (54/56) belonged to the main epidemic lineage CC17, mostly the ST78 type. The vanM gene clusters in the 55 vancomycin-susceptible isolates showed sequence diversity owing to different insertion locations of IS1216E. The vanM transposons could be classified into five types and they all carried two or more IS1216E elements, leading to complete or partial deletions of vanR, vanS, or vanX. Quantitative reverse transcription polymerase chain reaction showed that the expression level of vanM was significantly lower in the vancomycin-susceptible isolates than in the vancomycin-resistant isolate. Considering the prevalence of the vanM genotype and the potential for conversion to a resistant phenotype, vanM might act as an important determinant of glycopeptide resistance in the future. It is essential to strengthen the surveillance of vanM-containing enterococci to control the dissemination of vancomycin resistance.

To further characterize the fosB-carrying plasmids of 19 vancomycin-resistant enterococci, the complete sequences of the fosB- and vanA-containing plasmids of Enterococcus faecium (pEMA120) and E. avium (pEA19081) were obtained by single-molecule, real-time sequencing. We found that these two plasmids are essentially identical (99.99% nucleotide sequence identity), which proved the possibility of interspecies transmission. Comparative analysis of the plasmids revealed that the backbone of pEMA120 is 99% similar to a conjugative fosB-negative E. faecium plasmid, pZB18. There is a traE disrupted in the transfer region of pEMA120, in comparison to pZB18 with an intact traE. The difference of their transfer frequencies between pEMA120 and pZB18 suggests this interruption of traE might affect conjugative transfer. Two copies of the fosB gene linked to a tnpA gene, forming an ISL3-like transposon, were found at separate locations within pEMA120, which had not been reported previously. These two fosB-carrying transposons were confirmed to form circular intermediates by inverse PCR. The hybridization of plasmid DNA digested by BsaI, having restriction site within the fosB sequence, demonstrated that the presence of multiple copies of fosB per plasmid is common. The total copy number of the fosB gene as revealed by qRT-PCR did not correlate with fosfomycin MICs or growth rates at sub-MICs of fosfomycin in different transconjugants. From susceptibility tests, the fosB gene, regardless of the copy number, conferred high fosfomycin MICs that ranged from 16384 to 65536 μg/ml. This first complete nucleotide sequence of a plasmid carrying two copies of fosB in VRE suggests that the fosB gene can transfer to multiple loci of plasmids by the ISL3 family transposase TnpA, possibly in the form of circular intermediates, leading to the dissemination of high fosfomycin resistance in VRE.

Enterococcus faecium was considered an important nosocomial pathogen that could incur a substantial health burden owing to its high drug resistance and its ability to adapt to the hospital niche. Here we presented the draft genome sequence of multidrug-resistant E. faecium XH877. The draft genome sequence was composed of 272 contigs covering 2984730bp and 2809 protein-coding genes. An in silico genomic analysis was performed to determine the drug resistance genes and the pathogenic potential of the strain. Drug resistance and virulence genes detected in this strain will be helpful in understanding how E. faecium emerged as a leading hospital pathogen.

Gluconobacter oxydans is widely used in industrial application for its dehydrogenase system locating on cell membrane. These dehydrogenases have a character to oxidize sugars and sugar alcohols incompletely. There are two potential pathways known for glucose oxidization in G. oxydans: More than 90% of glucose is transformed into gluconate in the periplasmic space; only a minority of glucose (about 5%) is phosphorylated and taken into functional central metabolic pathways such as Entner-Doudoroff pathway (EDP) and pentose phosphate pathway (PPP) in the cytoplasmic compartment. In previous study, the Embden-Meyerhof-Parnas pathway (EMP) was found inactive in G. oxydans due to its lack of phosphofructokinase.In this study, a G. oxydans strain named DHA3-9 was screened which produced dihydroxyacetone (DHA) during glucose degradation. But DHA was not a product in EDP or PPP. A mutant strain of G. oxydans DHA3- 9 lacking of glucose dehydrogenase in cell membrane was constructed to study the possibility of other pathway of glucose metabolism in G. oxydans.A mgdh gene-disrupted mutant of G. oxydans DHA3-9 was constructed by the way of homologous recombination and its characteristic changes of the cells growth on glucose, glucose degradation, gluconate transformation, intermediate products and growth inhibition on acetate were studied.The results indicated that the growth of mutant strain on glucose showed an obvious delay and pH dropped much slower than that of wild type. The mutant lost most of its ability of glucose degradation and produced little gluconate. Instead, DHA formation of the mutant was recorded four times as that of wild type. Pyruvate and acetate were detected in the products of mutant whereas none of such products were found in wild type culture. Under the condition with glucose as the sole carbon source, 50 mmol/L acetate completely inhibited the growth of mutant, whereas this effect was remarkably low on wild type.These results prove that in G. oxydans DHA3-9 mutant strain, glucose is utilized in cytoplasmic compartment primarily through EMP and acetate can be produced by activities of pyruvate decarboxylase and acetaldehyde dehydrogenase.

Response surface methodology was applied to optimise the enzymatic transformation process for enhancement of the dihydroxyacetone(DHA) fermentation concentration.It was demonstrated that with the parameters of a pH of 4.61,a temperature of 33.3 ℃ and the concentration of glycerol at 80.0 g·L-1,the glycerol dehydrogenase(GDH) had an optimum activity.GDH was stable in a narrow pH range,from 4.0 to 5.0,and at temperatures below 25 ℃;further,100 g·L-1 glycerol or 50 g·L-1 DHA were best to stabilise GDH.As the parameters for optimal microbial fermentation and GDH catalysis differed markedly,a two-stage process of enzymatic transformation was developed in a 5-L bioreactor that separated the growth of G.oxydans and the biotransformation process.This methodology showed high stability of GDH in a 136-hour transformation process and elevated DHA production to as high as 286.2 g·L-1.

We purified a sarcosine oxidase from Bacillus sp. strain BSD-8 isolated from soil. We purified the enzyme by ammonium sulfate precipitation, DEAE-cellulose, Toyopearl hydrophobic and Sephadex G-75 molecular sieve chromatography and characterized the purified sarcosine oxidase. This sarcosine oxidase was a flavin enzyme containing a noncovalently bound flavin with the subunit molecular mass of 51 kDa. The optimal temperature for this enzyme was 60 degrees C and it showed its highest activity at pH 8.5. It was stable in the pH range of 8.0-10.0 and at the temperature of 60 degrees C. Estimated by Lineveaver-Burk plots, the K(m) of the enzyme was 3.1 mmol/L. Ag+, Hg2+, SDS and Tween 80 dramatically inhibted the enzyme activity, whereas Tween 20 and Triton X-100 had no effect on enzyme activity. The thermostability of this enzyme was better than reported sarcosine oxidases, and it could be applied in enzymatic measuring of creatinine.