Global evolutionary dynamics of virulence genes in ST11-KL47 carbapenem-resistant Klebsiella pneumoniae.

ST11-KL47 is a hypervirulent carbapenem-resistant Klebsiella pneumoniae (CRKP) that is highly prevalent in China and poses a major public health risk. To investigate the evolutionary dynamics of virulence genes in this subclone, we analysed 78 sequenced isolates obtained from a long-term study across 29 centres from 17 cities in China. Virulence genes were located in large hybrid pNDM-Mar-like plasmids (length: ∼266 kilobases) rather than in classical pK2044-like plasmids. These hybrid plasmids, derived from the fusion of pK2044 and pNDM-Mar plasmids mediated by insertion sequence (IS) elements (such as ISKpn28 and IS26), integrated virulence gene fragments into the chromosome. Analysis of 217 sequences containing the special IncFIB(pNDM-Mar) replicon using public databases indicated that these plasmids typically contained T4SS-related and multiple antimicrobial resistance genes, were present in 24 countries, and were found in humans, animals, and the environment. Notably, the chromosomal integration of virulence genes was observed in strains across five countries across two continents. In vivo and in vitro models showed that the large hybrid plasmid increased the host fitness cost while increasing virulence. Conversely, virulence genes transferred to chromosomes resulted in increased fitness and lower virulence. In conclusion, virulence genes in the plasmids of ST11-KL47 CRKP are evolving, driven by adaptive negative selection, to enable vertical chromosomal inheritance along with conferring a survival advantage and low pathogenicity.

Expansion and transmission dynamics of high risk carbapenem-resistant Klebsiella pneumoniae subclones in China: An epidemiological, spatial, genomic analysis.

AIMS: Carbapenem-resistant Klebsiella pneumonia (CRKP) is a global threat that varies by region. The global distribution, evolution, and clinical implications of the ST11 CRKP clone remain obscure. METHODS: We conducted a multicenter molecular epidemiological survey using isolates obtained from 28 provinces and municipalities across China between 2011 and 2021. We integrated sequences from public databases and performed genetic epidemiology analysis of ST11 CRKP. RESULTS: Among ST11 CRKP, KL64 serotypes exhibited considerable expansion, increasing from 1.54% to 46.08% between 2011 and 2021. Combining our data with public databases, the phylogenetic and phylogeography analyses indicated that ST11 CRKP appeared in the Americas in 1996 and spread worldwide, with key clones progressing from China's southeastern coast to the inland by 2010. Global phylogenetic analysis showed that ST11 KL64 CRKP has evolved to a virulent, resistant clade with notable regional spread. Single-nucleotide polymorphism (SNP) analysis identified BMPPS (bmr3, mltC, pyrB, ppsC, and sdaC) as a key marker for this clade. The BMPPS SNP clade is associated with high mortality and has strong anti-phagocytic and competitive traits in vitro. CONCLUSIONS: The high-risk ST11 KL64 CRKP subclone showed strong expansion potential and survival advantages, probably owing to genetic factors.

Increase in antioxidant capacity associated with the successful subclone of hypervirulent carbapenem-resistant Klebsiella pneumoniae ST11-KL64.

The acquisition of exogenous mobile genetic material imposes an adaptive burden on bacteria, whereas the adaptational evolution of virulence plasmids upon entry into carbapenem-resistant Klebsiella pneumoniae (CRKP) and its impact remains unclear. To better understand the virulence in CRKP, we characterize virulence plasmids utilizing a large genomic data containing 1219 K. pneumoniae from our long-term surveillance and publicly accessible databases. Phylogenetic evaluation unveils associations between distinct virulence plasmids and serotypes. The sub-lineage ST11-KL64 CRKP acquires a pK2044-like virulence plasmid from ST23-KL1 hypervirulent K. pneumoniae, with a 2698 bp region deletion in all ST11-KL64. The deletion is observed to regulate methionine metabolism, enhance antioxidant capacity, and further improve survival of hypervirulent CRKP in macrophages. The pK2044-like virulence plasmid discards certain sequences to enhance survival of ST11-KL64, thereby conferring an evolutionary advantage. This work contributes to multifaceted understanding of virulence and provides insight into potential causes behind low fitness costs observed in bacteria.

Assessing the impact of low organic loading on effluent safety in wastewater treatment: Insights from an activated sludge reactor study.

In this study, an organic loading (OL) of 300 mg/(L d) was set as the relative normal condition (OL-300), while 150 mg/(L d) was chosen as the condition reflecting excessively low organic loading (OL-150) to thoroughly assess the associated risks in the effluent of the biological wastewater treatment process. Compared with OL-300, OL-150 did not lead to a significant decrease in dissolved organic carbon (DOC) concentration, but it did improve dissolved organic nitrogen (DON) levels by ∼63 %. Interestingly, the dissolved organic matter (DOM) exhibited higher susceptibility to transformation into chlorinated disinfection by-products (Cl-DBPs) in OL-150, resulting in an increase in the compound number of Cl-DBPs by ∼16 %. Additionally, OL-150 induced nutrient stress, which promoted engendered human bacterial pathogens (HBPs) survival by ∼32 % and led to ∼51 % increase in the antibiotic resistance genes (ARGs) abundance through horizontal gene transfer (HGT). These findings highlight the importance of carefully considering the potential risks associated with low organic loading strategies in wastewater treatment processes.

Emergency of the plasmid co-carrying blaKPC-2 and blaNDM-1 genes in carbapenem-resistant hypervirulent Klebsiella pneumoniae.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a prevalent issue in China, with its spread primarily attributed to the presence of the plasmid-borne carbapenemase genes, blaKPC and blaNDM. However, instances of plasmids containing both blaKPC-2 and blaNDM-1have never been reported. METHODS: In this study, the genomic and microbiological characteristics of hybrid plasmids containing both blaKPC-2 and blaNDM-1 were identified in Chinese clinical CRKP isolates by Illumina combined with ONT nanopore sequencing technology. RESULTS: The newly identified plasmid was formed via IS26-mediated recombination and has been shown to be transferable to Escherichia coli. It substantially elevates the minimum inhibitory concentration (MIC) of meropenem by 4000-fold in E. coli, surpassing the MIC values observed in E. coli strains that carry either blaKPC-2 and blaNDM-1 alone, as previously demonstrated in our study. Notably, the co-occurrence of the KPC-NDM fusion plasmid and a pLVPK-like virulence plasmid was observed in these organisms. In vivo experiments revealed that the isolates harbouring the pLVPK-like virulence plasmid exhibited a significantly higher lethality rate in Galleria mellonella. CONCLUSIONS: The increased antibiotic resistance brought by this novel fusion plasmid and its accompanying virulence factors pose a serious potential threat to human health and deserve our vigilance.

A dual-process of targeted and unbiased Nanopore sequencing enables accurate and rapid diagnosis of lower respiratory infections.

BACKGROUND: Nanopore metagenomics has been used for infectious disease diagnosis for bacterial pathogens. However, this technology currently lacks comprehensive performance studies in clinical settings for simultaneous detection of bacteria, fungi, and viruses. METHODS: We developed a dual-process of Nanopore sequencing for one sample, with unbiased metagenomics in Meta process and target enrichment in Panel process (Nanopore Meta-Panel process, NanoMP) and prospectively enrolled 450 respiratory specimens from multiple centers. The filter system of pathogen detection was established with machine learning and receiver operator characteristic (ROC) curve to optimize the detection accuracy based on orthogonal test of 21 species. Antimicrobial resistance (AMR) genes were identified based on the Comprehensive Antibiotic Resistance Database (CARD) and single-nucleotide polymorphism matrix. FINDINGS: Our approach showed high sensitivity in Meta process, with 82.9%, 88.7%, and 75.0% for bacteria, fungi (except Aspergillus), and Mycobacterium tuberculosis groups, respectively. Moreover, target amplification improved the sensitivity of virus (>80.0% vs. 39.4%) and Aspergillus (81.8% vs. 42.3%) groups in Panel process compared with Meta process. Overall, NanoMP achieved 80.2% sensitivity and 98.8% specificity compared with the composite reference standard, and we were able to accurately detect AMR genes including blaKPC-2, blaOXA-23 and mecA and distinguish their parent organisms in patients with mixed infections. INTERPRETATION: We combined metagenomic and enriched Nanopore sequencing for one sample in parallel. Our NanoMP approach simultaneously covered bacteria, viruses and fungi in respiratory specimens and demonstrated good diagnostic performance in real clinical settings. FUNDING: National Key Research and Development Program of China and National Natural Science Foundation of China.

Effects of groundwater depth on groundwater recharge and soybean growth dynamics in Northeast China Plain.

To explore the groundwater recharge rate and soybean growth dynamics under different groundwater depths, we conducted a field experiment with four groundwater depth treatments (1 m, D1; 2 m, D2; 3 m, D3; 4 m, D4) through the groundwater simulation system in 2021 and 2022 and explored the relationships between groundwater depth and groundwater recharge, irrigation, growth dynamics of soybean plants, and yield. We used the Logistic regression model to simulate the dynamics of soybean growth indices, including plant height, leaf area index, and dry matter accumulation. The results showed that compared with D1 treatment, the amount of groundwater recharge under D2, D3, and D4 treatments decreased by 81.1%, 96.8%, 97.5% and 80.7%, 96.7%, 97.3% in the two years, respectively. The groundwater in D1 treatment could meet water needs of soybean throughout the whole growth period, except that irrigation was needed in the sowing stage. The amount of irrigation under D1 treatment was decreased by 91.7%, 93.0%, 94.2%, and 90.9%, 92.9%, 94.0% in the two years, respectively, compared with D2, D3, D4 treatments. Among the four treatments, D1 treatment took the shortest time for entering the rapid growth stage and reach the maximum growth rate, which had the highest maximum growth rate. At the mature stage of soybean, the dry matter distribution ratio of stem in D1 treatment was the highest. D1 treatment promoted the translocation of post-flowering assimilates in soybean, and its post-flowering assimilate contribution to seeds increased by 15.5%, 16.2%, 32.6% and 45.5%, 48.7%, 63.3% in the two years, respectively, compared with D2, D3, D4 treatments. D1 treatment had the highest plant height, leaf area index, and dry matter accumulation, follo-wed by D4 treatment, while D3 treatment had the lowest. Soybean yield, number of pods per plant, number of grains per plant, and 100-grain weight all decreased and then increased with increasing groundwater depth, following an order of D1>D4>D2>D3. Soybean yield was significantly positively correlated with groundwater recharge, which was positively correlated with plant height, leaf area index, and dry matter accumulation. Our results indicated that the D1 treatment with adequate groundwater recharge increased plant height, leaf area index, and dry matter accumulation, coordinated the distribution and translocation of dry matter among all plant parts in the late soybean growth period, and ultimately achieved the highest yield. When groundwater depth was deep (D4), groundwater recharge was small. In such case, the growth and development status and yield of soybean could also reach a high level if there was sufficient water supply.

Phenotypic and Genomic Comparison of Staphylococcus aureus Highlight Virulence and Host Adaptation Favoring the Success of Epidemic Clones.

Methicillin-resistant Staphylococcus aureus (MRSA) of the sequence type 59 (ST59) and ST398 lineages has emerged in hospitals and displayed a higher virulent potential than its counterparts ST5 and ST239. However, the mechanism of the host cell-pathogen interaction and specific determinates that contribute to the success of epidemic clones remain incompletely understood. In the present study, 142 S. aureus strains (ST59, ST398, ST239, and ST5) were selected from our 7-year national surveillance of S. aureus bloodstream infections (n = 983). We revealed that ST59 and ST398 had a higher prevalence of the protease-associated genes hysAVSaß, paiB, and cfim and enhanced proteolytic activity than the other lineages. ST59 and ST398 showed a higher expression of RNAIII and psmα and greater proficiency at causing cell lysis than other lineages. Furthermore, ST59 and ST398 were strongly recognized by human neutrophils and caused more cell apoptosis and neutrophil extracellular trap degradation than the other lineages. In addition, these strains differed substantially in their repertoire and composition of intact adhesion genes. Moreover, ST398 displayed higher adaptability to human epidermal keratinocytes and a unique genetic arrangement inside the oligopeptide ABC transport system, indicating functional variations. Overall, our study revealed some potential genomic traits associated with virulence and fitness that might account for the success of epidemic clones. IMPORTANCE Considerable efforts have been exerted to identify factors contributing to the success of epidemic Staphylococcus aureus clones, however, comparative phenotypic studies lack representation owing to the small number of strains. Large-scale strain collections focused on the description of genomic characteristics. Moreover, methicillin-resistant S. aureus infections constitute 30% to 40% of S. aureus bloodstream infections, and recent research has elucidated highly virulent methicillin-susceptible S. aureus strains. However, comprehensive research on the factors contributing to the success of epidemic S. aureus clones is lacking. In this study, 142 S. aureus strains were selected from our 7-year national surveillance of S. aureus bloodstream infections (n = 983) accompanied by a rigorous strain selection process. A combination of host cell-pathogen interactions and genomic analyses was applied to the represented strains. We revealed some potential genomic traits associated with virulence and fitness that might account for the success of epidemic clones.

Open-Cage Fullerene as Molecular Container for F- , Cl- , Br- and I.

A 19-membered open-cage fullerene derivative was prepared from C60 in 7 steps and 5.5 % yield through the peroxide-mediate pathway. There are four carbonyl groups, an ether oxygen and a quinoxaline moiety on the rim of the orifice. A chloride anion could be inserted into its cavity by heating with hydrochloric acid at 60 °C for 4 h. Encapsulation of fluoride, bromide and iodide anions was also achieved at slightly more forcing conditions, 90 °C for 14 h. Single crystal X-ray structures of the sodium salt of the chloride and the bromide encapsulated derivatives were obtained, which showed the halide anion in the center of the cavity and two sodium cations connecting two cages through coordination to the oxygen atoms on the rim of the orifices. The halide encapsulation ratio is quantitative in the isolated products.

ST11 Carbapenem-Resistant Klebsiella pneumoniae Clone Harboring blaNDM Replaced a blaKPC Clone in a Tertiary Hospital in China.

The nosocomial spread of carbapenem-resistant Enterobacterales (CRE) is extremely common, resulting in severe burdens on healthcare systems. In particular, the high-risk Klebsiella pneumoniae ST11 strain has a wide endemic area in China. The current study describes the results of continuous monitoring of CRE genotypes and phenotypes in a tertiary hospital in North China from 2012 to 2020. A total of 160 isolates were collected, including 109 Klebsiella. pneumoniae (68.13%), 29 Escherichia coli (26.60%), 12 Enterobacter cloacae (7.50%), and 10 other strains (6.25%). A total of 149 carbapenemase genes were detected, of which blaKPC-2 (51.0%) was the most common, followed by blaNDM-1 (22.82%), and blaNDM-5 (23.49%). Based on multi-locus sequence typing, the ST11 strain (66.1%) dominates K. pneumoniae, followed by ST15 (13.8%). Interestingly, the proportion of blaNDM (22.2%, 16/72) in ST11 K. pneumoniae was significantly increased in 2018−2019. Hence, whole-genome sequencing was performed on ST11 K. pneumoniae. Growth curves and in vitro competition experiments showed that K. pneumoniae carrying blaNDM exhibited a stronger growth rate (p < 0.001) and competition index (p < 0.001) than K. pneumoniae carrying blaKPC. Moreover, K. pneumoniae carrying blaNDM had a stronger biofilm-forming ability than K. pneumoniae carrying blaKPC (t = 6.578; p < 0.001). K. pneumoniae carrying blaKPC exhibited increased defense against bactericidal activity than K. pneumoniae carrying blaNDM. Thus, ST11 K. pneumoniae carrying blaNDM has strong adaptability and can locally replace K. pneumoniae carrying blaKPC to become an epidemic strain. Based on these findings, infection control and preventive measures should focus on the high-risk ST11-K. pneumoniae strain.

Dry Matter Accumulation in Maize in Response to Film Mulching and Plant Density in Northeast China.

Film mulching in combination with high plant density (PD) is a common agronomic technique in rainfed maize (Zea mays L.) production. However, the effects of combining colored plastic film mulching and PD on dry matter accumulation (DMA) dynamics and yield of spring maize have not been thoroughly elucidated to date. Thus, a 2-year field experiment was conducted with three mulching treatments (no mulching (M0), transparent plastic film mulching (M1), and black plastic film mulching (M2)) and five plant densities (60,000 (D1), 67,500 (D2), 75,000 (D3), 82,500 (D4), and 90,000 plants ha-1 (D5)). A logistic equation was used to simulate the DMA process of spring maize by taking the effective accumulated air temperature compensated by effective accumulated soil temperature as the independent variable. The results showed that compared with M0 treatment, the growth period of M1 and M2 treatments was preceded by 10 and 4 days in 2016, and 10 and 7 days in 2017, respectively. The corrected logistic equation performed well in the characterization of maize DMA process with its characteristic parameter (final DMA, a; maximum growth rate of DMA, GRmax; effective accumulated temperature under maximum growth rate of DMA, xinf; effective accumulated temperature when maize stops growing, xmax; effective accumulated temperature when maize enters the fast-growing period, x1). Plastic film color mainly affected DMA by influencing xinf. PD mainly affected DMA by affecting GRmax and x1. During the first slow growing period, the DMA of M1 treatment was the largest among the three mulching treatments, however, during the fast growing period, the DMA of M2 treatment accelerated and exceeded that of M1 treatment, resulting in the largest final DMA(a) and yield. When the PD was increased from D1 to D4, the maximum growth rate (GRmax) continued to increase, and the effective accumulated temperature when maize enters the fast growing period (x1) continued to decrease, which substantially increased the final DMA(a) and yield. The application of M2D4 treatment can harmonize the relevant factors to improve the DMA and yield of spring maize in rainfed regions of Northeast China.

Photophysical Properties of Naphthalene-oxacalix[m]arene and Recognition of Fullerene C60.

Three different pore sizes of oxacalix[m]arene[n]pyrimidines modified with a naphthalene substituent were synthesized and characterized by HRMS, 1H NMR, and single-crystal analysis (8OA and 8OA-N). Steady-state spectroscopy indicates these naphthalene-oxacalix[m]arenes exhibit good fluorescence properties, which isattributed to the locally excited (LE) state emission, and electrochemical results show that the photoinduced electron transfer (PET) process occurs from the naphthalene substituent to the linked pyrimidine. Nanosecond transient absorption spectra, singlet oxygen quantum yields (ΦΔ4OA-N = 45.1%, ΦΔ6OA-N = 56.6%, and ΦΔ8OA-N = 65.7%) and theoretical calculations demonstrate that the torsion angle between the donor (naphthalene) and the acceptor (pyrimidine) promotes intersystem crossing (ISC), and the lifetime of the triplet state reaches ca. 8 ms. Interestingly, all three host molecules (4OA-N, 6OA-N, and 8OA-N) showed a high affinity for fullerene C60, and significant binding constants in the range of 4.10-6.68 × 104 M-1 were obtained by fluorescence titration; in contrast, previous reports indicated that the similar oxacalix[m]arene[n]pyrimidine scaffold could not efficiently complex with C60. In the frontier molecular orbital theory calculations of the supramolecular system of 4OA-N@C 60 , the HOMO is distributed on 4OA-N and the LUMO is localized on fullerene. The calculation results further demonstrated that there are strong interactions between the host and the fullerene guest, which is consistent with the result of the experiments. The characteristic photophysical properties of these novel naphthyl-decorated oxacalix[m]arene[n]pyrimidines broaden their application field, and the stable host-guest system with fullerene can be applied to supramolecular chemistry.

Amphotericin B Tamed by Salicylic Acid.

Although Amphotericin B (AmB) is considered as the "gold standard" treatment for deep fungal infections, owing to its excellent antifungal effect, it often causes severe hemolytic toxicity and nephrotoxicity, which limits its clinical use. We designed and synthesized AmB derivatives by attaching salicylic acid (SA) to the carboxyl group and confirmed their structures using 1H NMR, 13C NMR, HR-MS, and IR. We evaluated its biological activity in vitro and measured its ultraviolet-visible (UV-vis) absorption spectrum. The AmB-SA conjugates exhibited good antifungal effects against Candida albicans, Candida glabrata, and Cryptococcus neoformans compared with AmB, and the renal cytotoxicity toward HEK 293T cells in vitro was significantly reduced, with almost no nephrotoxicity in the therapeutic window of the drug. At the same time, the hemolytic toxicity was significantly reduced. Therefore, modification of AmB by introducing SA is an effective strategy to maintain the broad antifungal activity of AmB and reduce its cytotoxicity. These AmB derivatives could be applied in clinical therapy in the future.

Occurrence of High Levels of Cefiderocol Resistance in Carbapenem-Resistant Escherichia coli before Its Approval in China: a Report from China CRE-Network.

Cefiderocol has been approved in the United States and Europe but not in China. We aim to evaluate carbapenem-resistant Enterobacterales (CRE) susceptibility to cefiderocol to provide baseline data and investigate the resistance mechanism. From 2018 to 2019, 1,158 CRE isolates were collected from 23 provinces and municipalities across China. The MICs of antimicrobials were determined via the agar dilution and broth microdilution methods. Whole-genome sequencing was performed for 26 cefiderocol-resistant Escherichia coli isolates to investigate the resistance mechanism. Clone transformations were used to explore the function of cirA, pbp3, and blaNDM-5 in resistance. Among the 21 antimicrobials tested, aztreonam-avibactam had the highest antibacterial activity (98.3%), followed by cefiderocol (97.3%) and colistin (95.3%). A total of 26 E. coli isolates harboring New Delhi metallo-beta-lactamase 5 (NDM-5) showed high levels of cefiderocol resistance, of which sequence type 167 (ST167) accounted for 76.9% (20/26). We found 4 amino-acid insertions (YRIN/YRIK) at position 333 of penicillin-binding protein 3 (PBP3) in the 26 E. coli isolates, and 22 isolates had a siderophore receptor cirA premature stop codon. After obtaining the wild-type cirA supplementation, the MIC of the transformants decreased by 8 to 16 times in two cefiderocol-resistant isolates. A cefiderocol-susceptible isolate harboring NDM-5 has an MIC increased from 1 µg/mL to 64 µg/mL after cirA deletion, and the MIC decreased from 64 µg/mL to 0.5 µg/mL after blaNDM-5 deletion. The MIC of the E. coli DH5α, from which the pbp3 mutant was obtained, increased from 0.064 µg/mL to 0.25 µg/mL. Cefiderocol showed activity against most CRE in China. The resistance of ST167 E. coli to cefiderocol is a combination of the premature stop codon of cirA, pbp3 mutation, and blaNDM-5 existence. IMPORTANCE Cefiderocol, a new siderophore cephalosporin, has been approved in the United States and Europe but not in China. At present, there are almost no antimicrobial susceptibility evaluation data on cefiderocol in China. We evaluated the in vitro susceptibility of 1,158 strains of carbapenem-resistant Enterobacterales to cefiderocol and other antibiotics. We found that a high proportion of Escherichia coli showed high-level resistance to cefiderocol. Whole-genome sequencing (WGS) and molecular cloning experiments confirmed that the synergistic effect of the cirA gene premature stop codon, blaNDM-5 existence, and the pbp3 mutation is associated with high levels of cefiderocol resistance.

Role of mobile genetic elements in the global dissemination of the carbapenem resistance gene blaNDM.

The mobile resistance gene blaNDM encodes the NDM enzyme which hydrolyses carbapenems, a class of antibiotics used to treat some of the most severe bacterial infections. The blaNDM gene is globally distributed across a variety of Gram-negative bacteria on multiple plasmids, typically located within highly recombining and transposon-rich genomic regions, which leads to the dynamics underlying the global dissemination of blaNDM to remain poorly resolved. Here, we compile a dataset of over 6000 bacterial genomes harbouring the blaNDM gene, including 104 newly generated PacBio hybrid assemblies from clinical and livestock-associated isolates across China. We develop a computational approach to track structural variants surrounding blaNDM, which allows us to identify prevalent genomic contexts, mobile genetic elements, and likely events in the gene's global spread. We estimate that blaNDM emerged on a Tn125 transposon before 1985, but only reached global prevalence around a decade after its first recorded observation in 2005. The Tn125 transposon seems to have played an important role in early plasmid-mediated jumps of blaNDM, but was overtaken in recent years by other elements including IS26-flanked pseudo-composite transposons and Tn3000. We found a strong association between blaNDM-carrying plasmid backbones and the sampling location of isolates. This observation suggests that the global dissemination of the blaNDM gene was primarily driven by successive between-plasmid transposon jumps, with far more restricted subsequent plasmid exchange, possibly due to adaptation of plasmids to their specific bacterial hosts.

Separating overlapping bat calls with a bi-directional long short-term memory network.

Acquiring clear acoustic signals is critical for the analysis of animal vocalizations. Bioacoustics studies commonly face the problem of overlapping signals, which can impede the structural identification of vocal units, but there is currently no satisfactory solution. This study presents a bi-directional long short-term memory network to separate overlapping echolocation-communication calls of 6 different bat species and reconstruct waveforms. The separation quality was evaluated using 7 temporal-spectrum parameters. All the echolocation pulses and syllables of communication calls in the overlapping signals were separated and parameter comparisons showed no significant difference and negligible deviation between the extracted and original calls. Clustering analysis was conducted with separated echolocation calls from each bat species to provide an example of practical application of the separated and reconstructed calls. The result of clustering analysis showed high corrected rand index (82.79%), suggesting the reconstructed waveforms could be reliably used for species classification. These results demonstrate a convenient and automated approach for separating overlapping calls. The study extends the application of deep neural networks to separate overlapping animal sounds.

Identification of multiple transfer units and novel subtypes of tmexCD-toprJ gene clusters in clinical carbapenem-resistant Enterobacter cloacae and Klebsiella oxytoca.

OBJECTIVES: Tigecycline is a last-resort antibiotic used to treat lethal infections caused by carbapenem-resistant Enterobacterales; however, plasmid-borne tigecycline resistance tmexCD-toprJ gene clusters can confer tigecycline resistance. The aim of the study was to identify novel subtypes and the spread of tmexCD-toprJ. METHODS: Five non-duplicate isolates of different species, carrying tmexCD-toprJ gene clusters or novel subtypes, were isolated from patients across China between November 2018 and June 2019. WGS was performed using Illumina and Nanopore platforms. A phylogenetic tree was constructed using a dataset of 77 sequences carrying the tmexCD-toprJ gene clusters, 72 of which were downloaded from NCBI with a blastn identity cut-off of 95%. RESULTS: We detected six different transfer units and two novel subtypes (tmexC1D1.2-toprJ1 and tmexC2D2.2-toprJ2) of the tmexCD-toprJ gene clusters. Among the six transfer units, three were mediated by IS26, while the rest were presumably mediated by Tn5393, hypothetical integrases (xerD-hp clusters-umuC-integrases-tnfxB2-tmexC2D2-toprJ2-umuC) and hypothetical units (hp-hp-hp-tnfxB2-tmexC2D2.2-toprJ2-ΔTn5393-Tn6292). Moreover, two tmexCD-toprJ-like gene clusters co-located on the same plasmid with blaNDM in five isolates. Phylogenetic analysis revealed that tmexCD-toprJ gene clusters may have originated in Pseudomonas spp., being mainly distributed in Pseudomonas spp. and Klebsiella spp. (64/77). Most tmexCD-toprJ gene clusters in Enterobacterales were located on plasmids, indicating that the gene clusters have a high inter-species transfer risk after transfer to Enterobacterales. CONCLUSIONS: In summary, to the best of our knowledge, this is the first report of tmexCD-toprJ gene clusters being isolated from Enterobacter cloacae and Klebsiella oxytoca, revealing that these multiple transfer units should be further studied because of their clinical significance.

Emergence of Colistin Resistance in Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae Under the Pressure of Tigecycline.

Colistin and tigecycline are the last options against carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP). Intersecting resistance determinants have been detected between these antibiotics; however, there is only limited evidence of such association. Here, we describe a colistin-resistant CR-hvKP isolated from a patient with severe neonatal bacteremia treated with tigecycline as opposed to colistin before isolation of this strain, providing a clinical clue to colistin resistance under tigecycline pressure. Furthermore, an ST11-K64 KPC-2-producing, colistin-susceptible CR-hvKP strain was subjected to experimental evolution toward colistin resistance under tigecycline and colistin pressure to verify this phenomenon in vitro. The biological impact of acquiring colistin resistance on fitness and virulence was also studied. As expected, the parental strain rapidly developed colistin resistance under both tigecycline and colistin selection. However, different from the colistin resistance mechanism in the clinical strain that was due to an ISKpn26 insertion in the mgrB gene, the mutants in this study developed colistin resistance through a ∼4.4 or ∼4.6 kb deletion including the mgrB locus as well as the kdgR, yobH, yebO, yobF, cspC, ftsI, and rlmA genes. Although the virulence of the colistin-resistant mutants, as determined in the Galleria mellonella model, decreased compared with that of the parent strain, it was still higher than that of NTUH-K2044. This suggests a slight virulence cost when CR-hvKP develops colistin resistance under tigecycline or colistin pressure. Together, our results provide clinical and experimental evidence for the association between colistin resistance and tigecycline pressure in CR-hvKP, highlighting a critical issue in the clinical setting.

Drivers of methicillin-resistant Staphylococcus aureus (MRSA) lineage replacement in China.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen subdivided into lineages termed sequence types (STs). Since the 1950s, successive waves of STs have appeared and replaced previously dominant lineages. One such event has been occurring in China since 2013, with community-associated (CA-MRSA) strains including ST59 largely replacing the previously dominant healthcare-associated (HA-MRSA) ST239. We previously showed that ST59 isolates tend to have a competitive advantage in growth experiments against ST239. However, the underlying genomic and phenotypic drivers of this replacement event are unclear. METHODS: Here, we investigated the replacement of ST239 using whole-genome sequencing data from 204 ST239 and ST59 isolates collected in Chinese hospitals between 1994 and 2016. We reconstructed the evolutionary history of each ST and considered two non-mutually exclusive hypotheses for ST59 replacing ST239: antimicrobial resistance (AMR) profile and/or ability to colonise and persist in the environment through biofilm formation. We also investigated the differences in cytolytic activity, linked to higher virulence, between STs. We performed an association study using the presence and absence of accessory virulence genes. RESULTS: ST59 isolates carried fewer AMR genes than ST239 and showed no evidence of evolving towards higher AMR. Biofilm production was marginally higher in ST59 overall, though this effect was not consistent across sub-lineages so is unlikely to be a sole driver of replacement. Consistent with previous observations of higher virulence in CA-MRSA STs, we observed that ST59 isolates exhibit significantly higher cytolytic activity than ST239 isolates, despite carrying on average fewer putative virulence genes. Our association study identified the chemotaxis inhibitory protein (chp) as a strong candidate for involvement in the increased virulence potential of ST59. We experimentally validated the role of chp in increasing the virulence potential of ST59 by creating Δchp knockout mutants, confirming that ST59 can carry chp without a measurable impact on fitness. CONCLUSIONS: Our results suggest that the ongoing replacement of ST239 by ST59 in China is not associated to higher AMR carriage or biofilm production. However, the increase in ST59 prevalence is concerning since it is linked to a higher potential for virulence, aided by the carriage of the chp gene.

Metagenomic next-generation sequencing to identify pathogens and cancer in lung biopsy tissue.

BACKGROUND: Lung biopsy tissue samples can be used for infection detection and cancer diagnosis. Metagenomic next-generation sequencing (mNGS) has the potential to further improve diagnosis. METHODS: From July 2018 to May 2020, lung biopsy samples of 133 patients with suspected pulmonary infection or abnormal imaging findings were collected and subjected to clinical microbiological testing, Illumina and Nanopore sequencing to identify pathogens. The neural networks were pretrained by extracting features of human reads from 2,095 metagenomic next-generation sequencing results, and the human reads of lung biopsy samples were entered into the validated pipeline to predict the risk of cancer. FINDINGS: Based on the pathogen-cancer detection pipeline, the Illumina platform showed 77·6% sensitivity and 97·6% specificity compared to the composite reference standard for infection diagnosis. However, the Nanopore platform showed 34·7% sensitivity and 98·7% specificity. mNGS identified more fungi, which was confirmed by subsequent pathological examination. M. tuberculosis complex was weakly detected. For cancer detection, compared with histology, the Illumina platform showed 83·7% sensitivity and 97·6% specificity, diagnosing an additional 36 cancer patients, of whom half had abnormal imaging findings (pulmonary shadow, space-occupying lesions, or nodules). INTERPRETATION: For the first time, we have established a pipeline to simultaneously detect pathogens and cancer based on Illumina sequencing of lung biopsy tissue. This pipeline efficiently diagnosed cancer in patients with abnormal imaging findings. FUNDING: This work was supported by the National Key Research and Development Program of China and National Natural Science Foundation of China.