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Abstract Background Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a significant threat to immunocompromised populations, including lung transplant recipients. This study investigates mixed CRKP strains carrying either bla KPC−2 or bla KPC−33 following ceftazidime-avibactam (CAZ/AVI) exposure, particularly in the context of lung transplantation. Mixed CRKP strains with shifting resistance phenotypes were frequently identified in patients exposed to CAZ/AVI. We aimed to elucidate the transitional state of bla KPC variants by selecting CAZ/AVI-sensitive and -resistant CRKP strains from a lung transplantation patient. Methods The bla KPC-variant-carrying CRKP strains were collected from lung transplant recipients exposed to CAZ/AVI in less than two years. Antibiotic susceptibility testing (AST) was conducted using microbroth dilution, and whole-genome sequencing (WGS) was used to identify genotypes and resistance mechanisms. Limiting dilution, drop-plate, and in vitro induction experiments determined bla KPC-variant changes during CAZ/AVI administration. qPCR primers/probes were designed to identify bla KPC−2 mutations. Results Among 104 lung transplant recipients infected by bla KPC-harboring CRKP strains and receiving CAZ/AVI, 10 (9.6%) experienced changing resistance phenotypes. The limiting dilution method found that Patient 10’s CRKP strains carried either bla KPC−2 or bla KPC−33. The drop-plate experiment showed differing growth patterns on CAZ/AVI mediums. The in vitro induction experiment demonstrated shifting from bla KPC−2 to bla KPC−33. Conclusions The study identified a “transitional state” of the mixed CRKP strains carrying either bla KPC−2 or bla KPC−33 in CAZ/AVI-exposed patients. Molecular diagnostics are crucial for identifying mixed strains and the transitional state of bla KPC variants, guiding treatment decisions in this complex landscape.

Abstract Background Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections poses global challenges, with limited options available for targeted therapy. Polymyxin was been regarded as one of the most important last-resort antimicrobial agents. Many factors could accelerate the resistance evolution of polymyxin. Insertion sequence (IS) inserted into mgrB is the main polymyxin resistance mechanism in K. pneumoniae. In this study, two CRKPs (KP31157 and KP31311) were isolated from the urine of a patient, shifting from susceptible to resistant as the mgrB inserted by ISkpn14. We intended to explore the origin of the IS and underlying mechanisms resulting in polymyxin resistance. Methods The within-host evolution relationship and molecular features of both CRKPs were determined by pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). pKP31311_KPC-2 plasmid genome structures contained in the above two CRKPs were aligned with the homologic plasmids, retrieved from the NCBI genome database via comparative genomic analysis. The plasmids encoding ISkpn14 elements flanked by direct repeat (DR) or not were analyzed. The mRNA expression, plasmid curing and in vitro antibiotics inducing experiment were employed to understand the potential mechanism of polymyxin resistance. Results Both strains, sharing homology, exhibited polymyxin resistance due to the insertion of ISkpn14 into the mgrB gene, influenced by minocycline exposure. Minocycline and tigecycline could accelerate polymyxin resistance (P

Objectives: The emergence of pathogens co-harbouring multiple mobile resistance and virulence elements is of great concern in clinical settings. Herein, we report an O101: H10-ST167 Escherichia coli Hu106 strain isolated from the urinary tract of a female in China. Methods: Antibiotic susceptibility testing was used to present the antimicrobial resistance spectrum. Whole-genome sequencing (WGS) and bioinformatic analysis were used to clarify the virulent and resistance mechanisms. Furthermore, the virulence of this strain was tested by the Greater wax moth larvae and siderophore production experiment. Results: The strain E. coli Hu106 was resistant to almost all antimicrobials tested, and only susceptible to aztreonam, amikacin, and tigecycline. WGS analysis revealed that the strain Hu106 co-harboured blaNDM-9 and mcr-1 on p2-Hu106, belonging to IncHI2/IncHI2A (256,000 bp). The co-existence of both resistance genes, blaNDM-9 and mcr-1, on the plasmid p2-Hu106 was mainly acquired by transposition recombination of mobile antibiotic elements mediated by IS26 and/or IS1 on IncHI2/IncHI2A type plasmid. In addition, the virulence clusters aerobactin (iutA-iucABCD) and salmochelin (iroBCDEN) were identified on an IncFIB/IncFIC(IncFII) type plasmid p1-Hu106, flanked by small mobile elements such as IS1A, ISkpn28, and IS3, respectively. After performing genomic comparison of p1-Hu106 with the WGS in NCBI, we identified that the virulent plasmid p1-Hu106-like could spread in different clones of E. coli and Klebsiella pneumoniae, revealing its underlying dissemination mechanism between Enterobacterales. Furthermore, the strain caused a decreased survival rate of larvae and produced high siderophore units (62.33%), similar to hypervirulent K. pneumoniae NTUH-K2044. Conclusions: The strains co-carrying the multidrug-resistant plasmid p2-Hu106 and virulent plasmid p1-Hu106 should be closely monitored to prevent its further spreading.

ABSTRACTObjectives This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp).Methods Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted.Results Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes blaCTX-M-15 and blaCTX-M-71, respectively, but KP30086 lost both. Cloning of gene blaCTX-M-71 and conjugation experiment of blaCTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105.Conclusions In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying blaCTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.

BackgroundBloodstream infections (BSI) are one of the most severe healthcare-associated infections in intensive care units (ICU). However, there are few studies on pneumonia-related BSI (PRBSI) in the ICU. This study aimed to investigate the clinical and prognostic characteristics of patients with PRBSI in the ICU and to provide a clinical basis for early clinical identification.MethodsWe retrospectively collected data from patients with bacterial BSI in a single-center ICU between January 1, 2017, and August 31, 2020. Clinical diagnosis combined with whole-genome sequencing (WGS) was used to clarify the diagnosis of PRBSI, and patients with PRBSI and non-PRBSI were analyzed for clinical features, prognosis, imaging presentation, and distribution of pathogenic microorganisms.ResultsOf the 2,240 patients admitted to the MICU, 120 with bacterial BSI were included in this study. Thirty-two (26.7%) patients were identified as having PRBSI based on the clinical diagnosis combined with WGS. Compared to patients without PRBSI, those with PRBSI had higher 28-day mortality (81.3 vs.51.1%, p = 0.003), a higher total mortality rate (93.8 vs. 64.8%, p = 0.002), longer duration of invasive mechanical ventilation (median 16 vs. 6 days, p = 0.037), and prolonged duration of ICU stay (median 21 vs. 10 days, p = 0.004). There were no differences in other baseline data between the two groups, but patients with PRBSI had extensive consolidation on chest radiographs and significantly higher Radiographic Assessment of Lung Edema scores (mean 35 vs. 24, p

Pigeon paramyxovirus type 1 (PPMV-1), an antigenic variant of avian paramyxovirus type 1 (APMV-1), mainly infects pigeons. PPMV-1 genotype VI is the dominant genotype infecting pigeons in China. Human infection of avian paramyxovirus was rarely reported, and usually developed mild symptoms, such as conjunctivitis. We detected PPMV-1 in the lower respiratory sample from a fatal case with severe pneumonia; this patient aged 64 years presented cough, fever, and haemoptysis for 8 days and was admitted to hospital on Dec 26, 2020. He developed acute respiratory distress syndrome and sepsis in the following days and died of multiple organ failure on Jan 7, 2021. Sputum and blood culture reported multidrug-resistant Acinetobacter baumannii (ABA) for samples collected on days 22 and 19 post-illness, respectively. However, clinical metagenomic sequencing further reported PPMV-1 besides ABA in the bronchoalveolar lavage fluid. The PPMV-1 genome showed 99.21% identity with a Chinese strain and belonged to VI genotype by BLAST analysis. Multiple basic amino acids were observed at the cleavage site of F protein (113RKKRF117), which indicated high virulence of this PPMV-1 strain to poultry. The patient had close contact with pigeons before his illness, and PPMV-1 nucleic acid was detected from the pigeon feather. PPMV antibody was also detected in the patient serum 20 days after illness. In conclusion, concurrent PPMV-1 genotype VI.2.1.1.2.2 and ABA infection were identified in a fatal pneumonia case, and cross-species transmission of PPMV-1 may occur between infected pigeons and the human being.

BackgroundColistin, as the antibiotic of “last resort” for carbapenem-resistant Klebsiella, develop resistance during administration of this antimicrobial agent. We identified an NDM-1-producing Klebsiella quasipneumonuae subsp. similipneumoniae (KQSS) strain KQ20605 recovered from a child, which developed resistance to colistin (KQ20786) through acquiring an IS903B element between the -27th and -26th bp of mgrB promoter region after 6-day colistin usage.ObjectivesThe aim of this study is to explore the source of IS903B in the disruptive mgrB gene and its underlying mechanisms.Materials and methodsAntibiotics susceptibility testing was conducted via microbroth dilution method. The in vitro colistin-induced experiment of KQ20605 was performed to mimic the in vivo transition from colistin-sensitive to resistant. Whole-genome sequencing was used to molecular identification of colistin resistance mechanism.ResultsThe IS903B element integrated into mgrB gene of KQ20786 had a 100% nucleotide identity and coverage match with one IS903B on plasmid IncR, and only 95.1% (1005/1057) identity to those on chromosome. In vitro, upon the pressure of colistin, KQ20605 could also switch its phenotype from colistin-sensitive to resistant with IS elements (e.g., IS903B and IS26) frequently inserted into mgrB gene at “hotspots”, with the insertion site of IS903B nearly identical to that of KQ20786. Furthermore, IS26 elements in this isolate were only encoded by plasmids, including IncR and conjugative plasmid IncN harboring blaNDM.ConclusionMobilizable IS elements on plasmids tend to be activated and integrated into mgrB gene at “hotspots” in this KQSS, thereby causing the colistin resistance emergence and further dissemination.

IntroductionThe increase in clinical Enterobacteriaceae with dual carbapenemase has become a serious healthcare concern. It is essential to characterize the transferability and potential dissemination of blaKPC−2- and blaNDM−1-coharboring carbapenem-resistant Citrobacter freundii (CRCF).MethodsFour blaKPC−2- and blaNDM−1-coharboring CRCF strains were collected from our surveillance of the prevalence of carbapenem-resistant Enterobacteriaceae. The isolates were assessed using species identification, antimicrobial susceptibility testing, conjugation assays, whole-genome sequencing, plasmid stability, and fitness costs. Clonality, genome, plasmidome, and phylogeny were analyzed to reveal potential dissemination.ResultsThree ST523 blaKPC−2- and blaNDM−1-coharboring CRCF strains, collected from the same hospital within 1 month, exhibited high homology (both identity and coverage >99%), implying clonal dissemination and a small-scale outbreak. Moreover, the blaKPC−2 and blaNDM−1 genes were coharbored on an IncR plasmid, probably generated by a blaKPC−2-harboring plasmid acquiring blaNDM−1, in these three strains. Importantly, the IncR plasmid may form a transferable hybrid plasmid, mediated by IS6100 via transposition, with another IncFII plasmid included in the same C. freundii strain. Furthermore, the blaKPC−2 and blaNDM−1 of the fourth CRCF strain are located on two different non-transferable plasmids lacking complete transfer elements. Additionally, throughout the course of the 10-day continuous passage, the genetic surroundings of blaNDM−1 in four CRCF strains were gradually excised from their plasmids after the 8th day, whereas they maintained 100% retention for blaKPC−2. Genome and plasmidome analyses revealed that blaKPC−2- or blaNDM−1-harboring C. freundii were divergent, and these plasmids have high homology to plasmids of other Enterobacteriaceae.ConclusionClonal dissemination of ST523 blaKPC−2- and blaNDM−1-coharboring CRCF strains was detected, and we first reported blaKPC−2 and blaNDM−1 concomitantly located on one plasmid, which could be transferred with mediation by IS6100 via transposition. Continued surveillance should urgently be implemented.

ABSTRACT Inappropriate antibiotic prescriptions are common for patients with upper respiratory tract infections (URTIs). Few data exist regarding the effects of antibiotic administration on airway microbiota among healthy adults. We conducted a randomized, double-blind, placebo-controlled trial to characterize the airway microbiota longitudinally in healthy adults using 16S rRNA gene sequencing and quantification. Both the induced sputum and oral wash samples were collected over a 60-day period following a 3-day intervention with 500 mg azithromycin or placebo. Environmental information, including air quality data (particulate matter [PM2.5] and PM10, air quality index [AQI] values), were also collected during the study. A total of 48 healthy volunteers were enrolled and randomly assigned into two groups. Azithromycin did not alter bacterial load but significantly reduced species richness and Shannon index. Azithromycin exposure resulted in a decrease in the detection rate and relative abundance of different genera belonging to Veillonellaceae, Leptotrichia, Fusobacterium, Neisseria, and Haemophilus. In contrast, the relative abundance of taxa belonging to Streptococcus increased immediately after azithromycin intervention. The shifts in the diversity of the microbiology composition took between 14 and 60 days to recover, depending on the measure used: either UniFrac phylogenetic distance or α-diversity. Outdoor environmental perturbations, especially the high concentration of PM2.5, contributed to novel variability in microbial community composition of the azithromycin group at D30 (30 days after baseline). The network analysis found that azithromycin altered the microbial interactions within airway microbiota. The influence was still obvious at D14 when the relative abundance of most taxa had returned to the baseline level. Compared to the sputum microbiota, oral cavity microbiota had a different pattern of change over time. The induced sputum microbial data can represent the airway microbiota composition in healthy adults. Azithromycin may have transient effects in the airway microbiota of healthy adults and decrease the airway microbiota resilience against outdoor environmental stress. The influence of azithromycin on microbial interactions is noteworthy, although the airway microbiota has returned to a near-baseline level. IMPORTANCE The influence of antibiotic administration on the airway microbiota of healthy adults remains unknown. This study is a randomized, double-blind, placebo-controlled trial aiming to investigate the microbial shifts in airways after exposure to azithromycin among heathy adults. We find that azithromycin changes the airway microbial community composition of healthy adults and decreases the airway microbiota resilience against outdoor environmental stress. This study depicts the longitudinal recovery trajectory of airway microbiota after the antibiotic perturbation and may provide reference for appropriate antibiotic prescription.

ABSTRACT Tuberculosis remains a major global public concern as a leading cause of health care-associated infections. The detection of Mycobacterium tuberculosis (MTB) is challenging due to the paucibacillary nature of the pathogen. For suspected pulmonary and extrapulmonary tuberculosis patients, if sputum, bronchoalveolar lavage fluid (BALF), related samples are negative for MTB, or suspected tumors, biopsy tissues may provide a better diagnostic yield. This study was aimed at comparing the performances of three methods in identifying MTB in biopsy tissues, including the Bactec mycobacterial growth indicator tube 960 (MGIT 960) system, the GeneXpert MTB/RIF assay (GeneXpert), and the Bactec Myco/F lytic culture (Myco/F) system. Biopsy samples from 3,209 nonduplicated patients were retrospectively enrolled between January 2018 and September 2021, of which 180 (5.6%) were positive for MTB by at least one method. GeneXpert revealed the highest recovery rate (134/162, 82.7%), followed by MGIT 960 (99/135, 73.3%) and Myco/F (26/143, 18.1%), and the composite positive rate for GeneXpert and MGIT 960 was 96.6% (173/179). Pairwise comparisons were conducted after completion of both tests, and the results showed that Myco/F had significantly lower detection rates than GeneXpert and MGIT 960 (16.4% versus 82.8%, P

Background: Porphyromonas pogonae was first reported in 2011 from polymicrobial infections in central bearded dragons. Human infections caused by P. pogonae remain rare, with only four cases published in the PubMed database. Herein, we present an unusual Chinese case of the thoracoabdominal wall abscess with sepsis caused by P. pogonae and review the clinical manifestations, diagnostic evaluation, and clinical outcome of the case. Case presentation: A 62-year-old female with sinusitis but no diabetes mellitus presented with fever, tenderness under an abdominal mass, and paroxysmal stabbing pain in the chest after receiving augmentation mammoplasty. Cultures of blood and pus yielded P. pogonae, and a diagnosis of sepsis with an abscess in the chest and abdominal wall was made. After repeated debridement and appropriate meropenem antibiotic treatment, the patient was successfully treated and discharged home. Conclusions: We report the first human case of P. pogonae causing sepsis in a patient with an abscess in China. The identification of P. pogonae should be considered if the strain grows well under either anaerobic or microaerobic conditions and exhibits strong β-hemolysis with fluorescence. This study retrospectively reviewed patients' infection diagnosis, clinical treatment, and prognosis to enhance awareness of the risk of P. pogonae infection.

Abstract Background The diagnosis and treatment of patients with bronchiectasis and nontuberculous mycobacterium (NTM) pulmonary disease are challenging issues and the treatment is also prolonged and depends on the species. There is limited information on patients with bronchiectasis and NTM pulmonary disease in Mainland China. Methods This cross-sectional study was conducted at the China–Japan Friendship Hospital, Beijing, China. Those adult patients who met the diagnostic criteria for bronchiectasis and obtained a culture result of mycobacteria from lower respiratory tract specimens or lung tissue were included in this study. A logistic regression model was used to identify the related factors in patients with NTM pulmonary disease. Results A total of 202 patients with bronchiectasis from 19 cities, 155 without and 47 (23.3%) with NTM pulmonary disease, were included. In all the 47 patients with NTM pulmonary disease, Mycobacterium avium complex was the most common species (66.0%), and 72.3% of them were initiated on standard anti-NTM treatment within 3 months after the diagnosis of NTM pulmonary disease. A larger proportion of patients with NTM pulmonary disease had acute exacerbations of ≥ 3 times within 1 year and were diagnosed bronchiectasis ≥ 50 years among patients with NTM pulmonary disease. The HRCT chest images revealed higher proportions of nodular shadow (100% vs. 35.3%), tree-in-bud sign (97.9% vs. 29.0%), cavities (29.8% vs. 5.8%), and airway dilation of the right middle lobe or the left lingular lobe (63.8% vs. 23.9%) in patients with NTM pulmonary disease than in those without NTM pulmonary disease (all P values = 0.001). The multivariable logistic regression model indicated that three and more abnormal features (OR 33.8; 95% CI 11.1–102.8) and main lesions of bronchial expansion in the middle or lingual lobe (OR 6.4; 95% CI 2.4–16.6) in HRCT chest images were independently associated with NTM pulmonary disease (P values = 0.001). Conclusion In a single center of Mainland China, > 23% of patients with bronchiectasis had NTM pulmonary disease, and most patients were started on standard treatment within 3 months after the diagnosis of NTM pulmonary disease. These findings suggest that patients with bronchiectasis should be thoroughly examined for the presence of NTM pulmonary disease. Trial registration NCT03594032.

We characterized the first NDM-5 and MCR-8.2 co-harboring ST656 Klebsiella pneumoniae clinical isolate, combining with chromosomal gene-mediated resistance to colistin and tigecycline. The K. pneumoniae KP32558 was isolated from the bronchoalveolar lavage fluid from a lung transplant patient. Complete genome sequences were obtained through Illumina HiSeq sequencing and nanopore sequencing. The acquired resistance genes and mutations in chromosome-encoded genes associated with colistin and tigecycline resistance were analyzed. Comparative genomic analysis was conducted between mcr-8.2-carrying plasmids. The K. pneumoniae KP32558 was identified as a pan-drug resistant bacteria, belonging to ST656, and harbored plasmid-encoded blaNDM-5 and mcr-8.2 genes. The blaNDM-5 gene was located on an IncX3 type plasmid. The mcr-8.2 gene was located on a conjugative plasmid pKP32558-2-mcr8, which had a common ancestor with another two mcr-8.2-carrying plasmids pMCR8_020135 and pMCR8_095845. The MIC of KP32558 for colistin was 256 mg/L. The mcr-8.2 gene and mutations in the two-component system, pmrA and crrB, and the regulator mgrB, had a synergistic effect on the high-level colistin resistance. The truncation in the acrR gene, related to tigecycline resistance, was also identified. K. pneumoniae has evolved a variety of complex resistance mechanisms to the last-resort antimicrobials, close surveillance is urgently needed to monitor the prevalence of this clone.

The emergence of New Delhi Metallo-β-lactamase (NDM)-producing Klebsiella pneumoniae has aroused critical concern worldwide. Herein, we reported the first emergence of NDM-5-producing K. pneumoniae isolates in a 68-year-old lung transplant recipient, who died of septic shock 13 days after surgery. The K. pneumoniae strain KP22937 isolated from the bloodstream of the patient was analyzed for phenotypes and genotypes. KP22937 belonged to sequence type (ST) 65 and capsule serotype K2, contained iucABCDiutA and iroBCDN virulence clusters, showed high virulence to mice, and was therefore considered a hypervirulent K. pneumoniae. The blaNDM-5 gene was located on a genomic island region of the IncX3-type plasmid pNDM22937, which was successfully transferred to Escherichia coli EC600 with insignificant fitness costs. The transconjugant demonstrated similar antimicrobial susceptibility and growth kinetics to the recipient E. coli EC600. The plasmid pNDM22937 was almost identical to the blaNDM-5-carrying IncX3 plasmids previously reported in K. pneumoniae strains with different ST types and in other species. Our findings raise concerns about the horizontal spread of blaNDM-5 gene mediated by IncX3 plasmid, where hypervirulent K. pneumoniae strains are also involved. Stricter control measures are needed to prevent the dissemination of the novel clone in hospital settings.

ABSTRACT Extensively drug-resistant Pseudomonas aeruginosa (XDRPA) infection is a significant public health threat due to a lack of effective therapeutic options. New β-lactam-β-lactamase inhibitor combinations, including ceftazidime-avibactam (CZA), have shown a high resistance rate to XDRPA. This study was therefore conducted to describe the underlying genomic mechanism of resistance for CZA nonsusceptible XDRPA strains that are non-metallo-β-lactamase (MBL) producers as well as to examine synergism of CZA and other antipseudomonal agents. Furthermore, the synergistic antibacterial activity of the most effective antimicrobial combination against non-MBL-producing XDRPA was evaluated through in vitro experiments. The resistance profiles of 15 CZA-resistant XDRPA strains isolated from clinical specimens in China-Japan Friendship Hospital between January 2017 to December 2020 were obtained by whole-genome sequencing (WGS) analysis. MBL genes blaIMP-1 and blaIMP-45 were found in 2 isolates (2/15, 13.3%); the other underlying CZA-resistance mechanisms involved the decreased OprD porin (13/13), blaAmpC overexpression (8/13) or mutation (13/13), and upregulated efflux pumps (13/13). CZA-imipenem (CZA-IPM) combination was identified to be the most effective against non-MBL-producing XDRPA according to the results of WGS analysis and combined antimicrobial susceptibility tests, with an approximately 16.62-fold reduction in MICs compared to CZA alone. Furthermore, the results of checkerboard analysis and growth curve displayed the synergistic antimicrobial activity of CZA and IPM against non-MBL-producing XDRPA. Electron microscopy also revealed that CZA-IPM combination might lead to more cellular structural alterations than CZA or IPM alone. This study suggested that the CZA-IPM combination has potential for non-MBL-producing XDRPA with blaAmpC overexpression or mutation, decreased OprD porin, and upregulated efflux pumps. IMPORTANCE Handling the infections by extensively drug-resistant Pseudomonas aeruginosa (XDRPA) strains is challenging due to their complicated antibiotic resistance mechanisms in immunosuppressed patients with pulmonary diseases (e.g., cystic fibrosis, chronic obstructive pulmonary disease, and lung transplant), ventilator-associated pneumonia, and bloodstream infections. The current study suggested the potentiality of the ceftazidime-avibactam-imipenem combination against XDRPA with blaAmpC overexpression or mutation, decreased OprD porin, and/or upregulated efflux pumps. Our findings indicate the necessity of combined drug sensitivity tests against XDRPA and also lay a foundation for the development of prevention, control, and treatment strategies in XDRPA infections.

ABSTRACT The genus Nocardia includes ubiquitous environmental saprophytes and the most frequently isolated aerobic actinomycete human pathogen responsible for localized or disseminated infection. Herein, the species distribution and antimicrobial susceptibility profiles of 441 nonrepetitive Nocardia strains are reported, collected from 21 provinces/cities in China over 13 years (from 2009 to 2021). These isolates were identified to species level by mass spectrometry or targeted DNA sequencing. The susceptibility profiles of Nocardia species for 15 antibiotics were determined by the broth microdilution method. Among these Nocardia isolates, Nocardia farcinica was the most commonly isolated species (39.9%, 176 of 441), followed by Nocardia cyriacigeorgica (28.6%, 126), Nocardia abscessus (6.6%, 29), and Nocardia otitidiscaviarum (5.9%, 26). Furthermore, 361 Nocardia strains (81.9%) were collected from lower respiratory tract (sputum, lung tissue, and bronchoalveolar lavage fluid), 50 (11.3%) were collected from skin and soft tissues, 9 were collected from blood, 9 were collected from eye, 4 were collected from cerebrospinal fluid and brain abscesses, and 2 were collected from pleural effusion. All of the Nocardia strains were susceptible to linezolid, followed by amikacin (99.3%) and trimethoprim-sulfamethoxazole (TMP-SMX) (99.1%). The antibiotic resistance profiles of other antibiotics varied tremendously among different Nocardia species. This demonstrated that accurate species identification and/or antibiotic susceptibility testing should be performed before the usage of these antibiotics. In summary, this is the largest study on the species and antibiotic resistance profiles of the genus Nocardia circulating in China, and our data will contribute to a better understanding of clinical nocardiosis. IMPORTANCE The genus Nocardia has the potential to cause nocardiosis, which might be underrecognized and underdiagnosed. Herein, the demographical features of 441 nonrepetitive nocardiosis cases and species distribution of their Nocardia strains in China, 2009 to 2021, are summarized. The susceptibility profiles for 15 antibiotics against all of the above Nocardia strains were also determined by the broth microdilution method. To date, this is the largest study on the genus Nocardia contributing to nocardiosis in China. Our study will be helpful for understanding the species diversity of Nocardia isolates distributed in China and for decision-making in the context of nocardiosis diagnosis and treatment.

Objectives: ST11 Klebsiella pneumoniae is among the most important clinical pathogens in China, and KL47 and KL64 are the dominant K types of these strains. Understanding the genomic characteristics of these strains would be critical to their anti-infection treatment. Methods: There were 364 genome sequences of ST11 K. pneumoniae strains isolated and collected from 13 countries from 2003 to 2018. These genome sequences included 338 downloaded from the National Center for Biotechnology Information (NCBI) database and 26 newly sequenced. Phylogenetic analyses of pan-genome and unique genes, and resistance and virulence gene analyses, were carried out to elucidate the molecular characteristics of these strains. Results: A total of 19 732 genes were identified from the 364 ST11 strains, and the pan-genome was open, indicating the genetic diversity of ST11 K. pneumoniae. These strains were clustered into three clades. Clade 1 contained the most various K types (14/15, 93.3%) and unique genes. KL47 and KL64 were the dominant K types of clades 2 and 3, accounting for 100% and 99.4% of strains in each clade, respectively. KL64 strains contained the most virulence genes, including iucA and rmpA, and the two genes tend to coexist. In addition, strains in clade 1 were isolated from all 13 countries; the strains in clades 2 and 3 were isolated mainly from China. Conclusions: The ST11 K. pneumoniae strain of KL64 is a newly emerging superbug, with more resistance and virulence genes in China; this was significantly different from other countries, and we should be alert to the dissemination of this subclone.

Abstract Background Members of the genus Proteus are mostly opportunistic pathogens that cause a variety of infections in humans. The molecular evolutionary characteristics and genetic relationships among Proteus species have not been elucidated to date. In this study, we developed a multilocus sequence analysis (MLSA) approach based on five housekeeping genes (HKGs) to delineate phylogenetic relationships of species within the genus Proteus. Results Of all 223 Proteus strains collected in the current study, the phylogenetic tree of five concatenated HKGs (dnaJ, mdh, pyrC, recA and rpoD) divided 223 strains into eleven clusters, which were representative of 11 species of Proteus. Meanwhile, the phylogenetic trees of the five individual HKGs also corresponded to that of the concatenated tree, except for recA, which clustered four strains at an independent cluster. The evaluation of inter- and intraspecies distances of HKG concatenation indicated that all interspecies distances were significantly different from intraspecies distances, which revealed that these HKG concatenations can be used as gene markers to distinguish different Proteus species. Further web-based DNA-DNA hybridization estimated by genome of type strains confirmed the validity of the MLSA, and each of eleven clusters was congruent with the most abundant Proteus species. In addition, we used the established MLSA method to identify the randomly collected Proteus and found that P. mirabilis is the most abundant species. However, the second most abundant species is P. terrae but not P. vulgaris. Combined with the genetic, genomic and phenotypic characteristics, these findings indicate that three species, P. terrae, P. cibarius and Proteus genospecies 5, should be regarded as heterotypic synonyms, and the species should be renamed P. terrae, while Proteus genospecies 5 has not been named to date. Conclusions This study suggested that MLSA is a powerful method for the discrimination and classification of Proteus at the species level. The MLSA scheme provides a rapid and inexpensive means of identifying Proteus strains. The identification of Proteus species determined by the MLSA approach plays an important role in the clinical diagnosis and treatment of Proteus infection.

ABSTRACTTo identify the association between the kinetics of viral load and clinical outcome in severe coronavirus disease 2019 (COVID-19) patients, a retrospective study was performed by involved 188 hospitalized severe COVID-19 patients in the LOTUS China trial. Among the collected 578 paired throat swab (TS) and anal swab (AS) samples, viral RNA was detected in 193 (33.4%) TS and 121 (20.9%) AS. A higher viral RNA load was found in TS than that of AS, with means of 1.0 × 106 and 2.3 × 105 copies/ml, respectively. In non-survivors, the viral RNA in AS was detected earlier than that in survivors (median of 14 days vs 19 days, P = 0.007). The positivity and viral load in AS were higher in non-survivors than that of survivors at week 2 post symptom onset (P = 0.006). A high initial viral load in AS was associated with death (OR 1.368, 95% CI 1.076–1.741, P = 0.011), admission to the intensive care unit (OR 1.237, 95% CI 1.001–1.528, P = 0.049) and need for invasive mechanical ventilation (OR 1.340, 95% CI 1.076–1.669, P = 0.009). Our findings indicated viral replication in extrapulmonary sites should be monitored intensively during antiviral therapy.

BackgroundThe diagnosis of bacterial pathogens in lower respiratory tract infections (LRI) using conventional culture methods remains challenging and time-consuming.ObjectivesTo evaluate the clinical performance of a rapid nanopore-sequencing based metagenomics test for diagnosis of bacterial pathogens in common LRIs through a large-scale prospective study.MethodsWe enrolled 292 hospitalized patients suspected to have LRIs between November 2018 and June 2019 in a single-center, prospective cohort study. Rapid clinical metagenomics test was performed on-site, and the results were compared with those of routine microbiology tests.Results171 bronchoalveolar lavage fluid (BAL) and 121 sputum samples were collected from patients with six kinds of LRIs. The turnaround time (from sample registration to result) for the rapid metagenomics test was 6.4 ± 1.4 hours, compared to 94.8 ± 34.9 hours for routine culture. Compared with culture and real-time PCR validation tests, rapid metagenomics achieved 96.6% sensitivity and 88.0% specificity and identified pathogens in 63 out of 161 (39.1%) culture-negative samples. Correlation between enriched anaerobes and lung abscess was observed by Gene Set Enrichment Analysis. Moreover, 38 anaerobic species failed in culture was identified by metagenomics sequencing. The hypothetical impact of metagenomics test proposed antibiotic de-escalation in 34 patients compared to 1 using routine culture.ConclusionsRapid clinical metagenomics test improved pathogen detection yield in the diagnosis of LRI. Empirical antimicrobial therapy could be de-escalated if rapid metagenomics test results were hypothetically applied to clinical management.

We describe in vivo evolution of carbapenem and ceftazidime-avibactam resistance by analyzing four longitudinal Klebsiella pneumoniae clinical isolates from a patient with pneumonia following antimicrobial treatment. The patient had fever, cough associated with expectoration, and new infiltration was found on the chest CT. Antimicrobial susceptibility was determined, and whole genome sequencing (WGS) was performed to investigate its dynamic change of resistance phenotype. Population analysis profile was performed to investigate the population of Klebsiella pneumoniae. The infection started with a KPC-2-producing K. pneumoniae (ZRKP01, ceftazidime-avibactam-S/carbapenem-R). Then, after ceftazidime-avibactam treatment, the strain switched to D179Y mutant that is KPC-33 (ZRKP02, ceftazidime-avibactam-R/carbapenem-S), which restored carbapenem susceptibility. However, the restored carbapenem susceptibility in vivo was not stable and the subsequent use of imipenem against KPC-33-producing K. pneumoniae infection resulted in a reversion of KPC-2 producers (ZRKP03 and ZRKP04, ceftazidime-avibactam-S/carbapenem-R). Genetic analysis demonstrated that all four K. pneumoniae isolates belonged to sequence type 11and had identical capsular polysaccharide (KL47), identical porin genes, and same plasmid replicon types. Phylogenetic analysis indicated that four K. pneumoniae isolates showed a high degree of relatedness. Single nucleotide polymorphisms analysis indicated that the number of mutations observed in the KPC-33 isolate was more than in the wild-type KPC-2 isolates and the four KPC-Kp isolates evolved from a longitudinal evolution of K. pneumoniae harboring blaKPC-2 gene. This is the first report to observe the in vivo evolution of wild-type KPC-2 to KPC-33 and then the reversion to its original wild-type KPC-2. Through WGS, we demonstrated the role of selective pressure of antibiotic in the mutation and reversion of blaKPC genes, which leading to the dynamic change of KPC enzymes and the dynamic emergence of resistance to ceftazidime-avibactam and carbapenems.Statement: Recently, studies reported the emergence of ceftazidime-avibactam-resistant strains. The KPC mutations mediating ceftazidime-avibactam resistance are generally associated with the restoration of carbapenem susceptibility. However, clinical significance of this observation is unclear. In this manuscript, we demonstrate the role of selective pressure of antibiotic in the mutation and reversion of blaKPC genes, which leading to the dynamic change of KPC enzymes and the dynamic emergence of resistance to ceftazidime-avibactam and carbapenems. To the best of our knowledge, this is the first report to observe the in vivo evolution of wild-type KPC-2 to KPC-33 and then the reversion to its original wild-type KPC-2. It should be noted that understanding the clinical significance of this observation is of critical importance, and reversion to carbapenem susceptibility would not imply a potential role for carbapenems monotherapy. We hope our study will draw attention to clinicians, so that this agent can be used most effectively for the longest period of time.

We report co-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus in a patient with pneumonia in China. The case highlights possible co-detection of known respiratory viruses. We noted low sensitivity of upper respiratory specimens for SARS-CoV-2, which could further complicate recognition of the full extent of disease.

Abstract Background Burkholderia pseudomallei is a gram-negative bacterium and the causative pathogen of melioidosis, which manifests a variety ranges of infection symptoms. However, deep venous thrombosis (DVT) and pulmonary embolism (PE) secondary to bacteremic melioidosis are rarely documented in the literature. Herein, we reported a fatal case of melioidosis combined with DVT and PE. Case presentation A 54-year-old male construction worker and farmer with a history of diabetes was febrile, painful in left thigh, swelling in left lower limb, with chest tightness and shortness of breath for 4 days. He was later diagnosed as DVT of left lower extremity and PE. The culture of his blood, sputum and bone marrow samples grew B. pseudomallei. The subject was administrated with antibiotics (levofloxacin, cefoperazone/tazobactam, and imipenem) according to antimicrobial susceptibility testing and low molecular heparin for venous thrombosis. However, even after appropriate treatment, the patient deteriorated rapidly, and died 2 weeks after admission. Conclusions This study enhanced awareness of the risk of B. pseudomallei bloodstream infection in those with diabetes. If a patient has predisposing factors of melioidosis, when DVT is suspected, active investigation and multiple therapeutic interventions should be implemented immediately to reduce mortality rate.

Abstract Background Infections by Streptococcus gallolyticus subsp. pasteurianus (SGSP) is often underestimated. Herein, the epidemiological features and resistant characteristics of SGSP in mainland China are characterized to enable a better understanding of its role in clinical infections. Methods In the present work, 45 SGSP isolates were collected from the samples of bloodstream, urine, aseptic body fluid, and fetal membrane/placenta from patients in 8 tertiary general hospitals of 6 cities/provinces in China from 2011 to 2017. The identification of all isolates was performed using traditional biochemical methods, 16S rRNA and gyrB sequencing, followed by the characterization of their antibiotic resistance profiling and involved genes. Results Among 34 non-pregnancy-related patients, 4 (4/34,11.8%) patients had gastrointestinal cancer, 10 (10/34, 29.4%) patients had diabetes, and one patient had infective endocarditis. Moreover, 11 cases of pregnant women were associated with intrauterine infection (9/11, 81.2%) and urinary tract infection (1/11, 9.1%), respectively. Except one, all other SGSP isolates were correctly identified by the BD Phoenix automated system. We found that all SGSP isolates were phenotypically susceptible to penicillin, ampicillin, cefotaxime, meropenem, and vancomycin. Forty strains (40/45, 88.9%) were both erythromycin and clindamycin-resistant, belonging to the cMLSB phenotype, and the majority of them carried erm(B) gene (39/40, 97.5%). Although the cMLSB/erm(B) constituted the most frequently identified phenotype/genotype combination (25/40, 62.5%) among all erythromycin-resistant cMLSB isolates, erm(B)/erm(A), erm(B)/mef(A/E), and erm(B)/erm(T) was detected in 7, 4, and 3 isolates, respectively. Furthermore, 43 strains (43/45, 95.6%) were tetracycline-resistant, and out of these, 39 strains (39/45, 86.7%) carried tet(L), 27(27/45, 60.0%) strains carried tet(O), and 7 (7/45, 15.6%) strains carried tet(M), alone or combined, respectively. All erythromycin-resistant isolates were also resistant to tetracycline. Conclusions It is important to study and draw attention on SGSP, an underreported opportunistic pathogen targeting immunodeficient populations, notably elderly subjects, pregnant women and neonates.

Background: The Xpert Xpress Flu/RSV assay is released by FDA for rapid detection of influenza A (FluA), influenza B (FluB), and respiratory syncytial virus (RSV). This study aimed to evaluate its clinical performance in comparison to that of the RT-PCR assay cleared by China FDA (CFDA-PCR). Methods: Nasopharyngeal specimens were collected from patients and tested by the two assays side by side. Discordant results were tested with a laboratory-developed real-time PCR for resolution. Viral load in the sample was quantified with a droplet digital PCR. Results: A total of 658 specimens were involved and gave 94.7%–99.1% agreement between the two assays. The Xpert assay showed higher sensitivity for FluA (100% vs. 89.8%) and FluB detection (100% vs. 95.3%), and also higher accuracy (98.9% vs. 95.7%) for FluA than the CDFA-PCR. The positive and negative predictive values (NPV) for the three viruses ranged from 90.5% to 100% in the two assays, with higher NPV for FluA and FluB in Xpert assay. Moreover, the Xpert Ct values showed a linear correlation with virus titer in specimens tested. Conclusion: Overall, the Xpert assay is a reliable and sensitive tool for the detection of FluA, FluB and RSV in our clinical settings. Keywords: Xpert Xpress Flu/RSV, RT-PCR, Digital PCR, Sensitivity

Abstract Background Hip replacement is generally conducted in those with prolonged arthritis pain or hip fractures, and postoperative infection is a serious complication. Mycoplasma hominis, belonging to mycoplasma species, exists mainly in the genitourinary tract. M. hominis infection after total hip replacement was rarely documented in literature. Case presentation A 59-year-old male was febrile after left total hip replacement. Empiric therapy with cefepime for suspected infection was ineffective. Specimens at the infection site were collected for culture, and pinpoint colonies grew after incubation at 35 °C for 48 h on blood agar plate. They grew to approximately 0.5 mm colonies in diameter after 7-day incubation, and were identified as M. hominis. Sequentially, combination therapy with clindamycin hydrochloride and moxifloxacin was initiated, and the patient defervesced within 3 days and was discharged home. Conclusions The study highlighted the potential pathogenicity of M. hominis in postoperative infection. The possibility of this microorganism involvement should be valued if the patients who experienced the hip or joint replacement had inexplicable fever.

Burkholderia pseudomallei is the causative agent of melioidosis, endemic mainly in tropical and subtropical areas. Its clinical manifestation is broad ranging from a localized skin lesion to a life-threatening systemic disease. Osteomyelitis and septic arthritis caused by B. pseudomallei are a rare, fatal illness, whose clinical features have not been illustrated in mainland China. Over 10 years (2010 to 2019), of 334 culture-confirmed melioidosis in Hainan province, China, 44 patients (13.2%) were confirmed to have osteomyelitis and septic arthritis through the combination of clinical features, imaging examination and microbiological culture. Herein, we summarized these 44 patients’ clinical manifestations, demographical features, antibiotic treatment, and outcomes. Of them, osteomyelitis and septic arthritis accounted for 25 (56.8%) and 15 (34.1%), respectively, and 4 patients (9.1%) had both. The gender ratio of male/female was approximately 13.7:1; diabetes mellitus was the most common risk factor (38/44, 86.4%); imipenem and trimethoprim/sulfamethoxazole were the most frequently used antibiotics. Most B. pseudomallei strains were isolated from blood samples (41/44, 93.2%). After surgical handling, antibiotic treatment, or both, 9 patients died, with a mortality rate of 20.5%. In summary, in melioidosis endemic areas, for patients with both localized manifestations of joint and bone and a positive B. pseudomallei blood culture, increased awareness is required for melioidotic osteomyelitis and septic arthritis.

Listeriosis, caused by Listeria monocytogenes, is a severe food-borne infection. The nationwide surveillance in China concerning listeriosis is urgently needed. In the present study, 144 L. monocytogenes isolates were collected from the samples of blood, cerebrospinal fluid (CSF), and fetal membrane/placenta in China for 12 years from 2008 to 2019. We summarized these listeriosis patients’ demographical and clinical features and outcomes. The susceptibility profile for 12 antibiotics was also determined by the broth microdilution method. Multilocus sequence typing (MLST) and serogroups of these listeria isolates were analyzed to designate epidemiological types. We enrolled 144 cases from 29 healthcare centers, including 96 maternal-neonatal infections, 33 cases of bacteremia, 13 cases of neurolisteriosis, and two cutaneous listeriosis. There were 31 (59.6%) fetal loss in 52 pregnant women and four (9.8%) neonatal death in 41 newborns. Among the 48 nonmaternal-neonatal cases, 12.5% (6/48) died, 41.7% (20/48) were female, and 64.6% (31/48) occurred in those with significant comorbidities. By MLST, the strains were distinguished into 23 individual sequence types (STs). The most prevalent ST was ST87 (49 isolates, 34.0%), followed by ST1 (18, 12.5%), ST8 (10, 6.9%), ST619 (9, 6.3%), ST7 (7, 4.9%) and ST3 (7, 4.9%). Furthermore, all L. monocytogenes isolates were uniformly susceptible to penicillin, ampicillin, and meropenem. In summary, our study highlights a high genotypic diversity of L. monocytogenes strains causing clinical listeriosis in China. Furthermore, a high prevalence of ST87 and ST1 in the listeriosis should be noted.

Background: The pharmacokinetics and appropriate dose regimens of favipiravir are unknown in hospitalized influenza patients; such data are also needed to determine dosage selection for favipiravir trials in COVID-19. Methods: In this dose-escalating study, favipiravir pharmacokinetics and tolerability were assessed in critically ill influenza patients. Participants received one of two dosing regimens; Japan licensed dose (1600 mg BID on day 1 and 600 mg BID on the following days) and the higher dose (1800 mg/800 mg BID) trialed in uncomplicated influenza. The primary pharmacokinetic endpoint was the proportion of patients with a minimum observed plasma trough concentration (Ctrough) ≥20 mg/L at all measured time points after the second dose. Results: Sixteen patients were enrolled into the low dose group and 19 patients into the high dose group of the study. Favipiravir Ctrough decreased significantly over time in both groups (p 80% of the duration of treatment of the two dose regimens evaluated (18.8% and 42.1% of patients for low and high dose regimen, respectively). Increasing the favipravir dosage predicted a higher proportion of patients reaching this threshold of 20 mg/L, suggesting that dosing regimens of ≥3600/2600 mg might be required for adequate concentrations. The two dosing regimens were well-tolerated in critical ill patients with influenza. Conclusion: The two dosing regimens proposed for uncomplicated influenza did not achieve our pre-defined treatment threshold.

Burkholderia pseudomallei is the causative agent of melioidosis, endemic in Southeast Asia and Northern Australia, and increasingly recognized in southern China, especially in Hainan Province. Mycotic aneurysm caused by B. pseudomallei is a rare but potentially severe illness with a high mortality rate. The clinical features of the mycotic aneurysm secondary to melioidosis have not been illustrated in China. Over a seven-year period (2013 to 2019), 159 patients with bacteremic melioidosis were retrospectively analyzed in Hainan province, China, of whom eight patients were confirmed to have mycotic aneurysm through the combination of imaging examination, pathologic examination and aneurysm tissue culture. We summarized these eight patients' clinical characteristics, demographical features, treatments and outcomes. The susceptibilities to five commonly-used antibiotics for these eight B. pseudomallei isolates were also determined by E-test strips. Furthermore, the mycotic aneurysm cases secondary to melioidosis retrieved from the literature were also reviewed. Of the eight cases, six had abdominal mycotic aneurysms, one had a left iliac aneurysm, and the other one had an infectious mesenteric aneurysm. They were aged from 48 to 69 years old, and had the underlying risk factors of diabetes mellitus (2 patients), long-term smoking (4 patients), hypertension (6 patients), and soil and water contact history (6 patients), respectively. The positive arterial aneurysm imaging was observed in all patients via computed tomography (CT) or angiography. Eight B. pseudomallei isolates collected from both blood and mycotic aneurysm tissues remained 100% susceptible to imipenem and ceftazidime. After surgery combined with antibiotic administration, six patients survived, with a mortality rate of 25%. In melioidosis endemic areas, the mycotic aneurysm secondary to melioidosis might be underdiagnosed, and increased awareness of predisposing risk factors and clinical features of the mycotic aneurysm is required. Following a positive B. pseudomallei blood culture, the diagnosis of mycotic aneurysm should be under consideration in those with abdominal pain and/or hypertension. Imaging by CT or angiography is indispensable for its timely diagnosis and management.

Cryptococcal meningitis, mainly caused by Cryptococcus neoformans/gattii species complexes, is a lethal infection in both immunosuppressive and immunocompetent populations. We characterized 110 Cryptococcus strains collected from Xiangya Hospital of Central South University in China during the 6-year study period between 2013 and 2018, and performed their antifungal susceptibility testing. Furthermore, the clinical features, laboratory and imaging data, treatment strategies and outcomes of the subjects were retrospectively analyzed. Of 110 Cryptococcus strains, C. neoformans species complexes accounted for 96.4% (106/110), including C. neoformans sensu stricto (VNI molecular type, 95.5%, 105/110) and Cryptococcus deneoformans (VNIV molecular type, 0.9%, 1/110), and Cryptococcus deuterogattii (VGII molecular type) accounted for 3.6% (4/110). The strains were further classified into 17 individual sequence types (STs) by using multilocus sequence typing (MLST). 89.1% (98/110) were represented by ST5; seven C. deuterogattii strains and one Cryptococcus deneoformans strain were assigned as ST7 and ST260, respectively. Antifungal minimal inhibitory concentrations above the epidemiological cutoff values (ECVs) were found mainly in C. neoformans species complexes strains (nine for amphotericin B, nine for fluconazole and seven for 5-fluorocytosine). Furthermore, 60.9% (67/110) of the subjects were male, and 40.0% (44/110) did not have underlying diseases. Hepatic diseases (hepatitis/HBV carrier status and cirrhosis) were the most common underlying health conditions (11.8%, 13/110), followed by autoimmune disorders (10.9%, 12/110) and chronic kidney disease (6.36%, 7/110). Only 4.5% (5/110) of the patients were HIV/AIDS positives. For clinical presentation, headache (77.3%, 85/110), fever (47.3%, 52/110), and stiff neck (40.9%, 45/110) were commonly observed. The mortality rate was 35.0% (36/103). In conclusion, our data were characterized by a high prevalence of the Cryptococcal meningitis patients without HIV/AIDS and other underlying health conditions, a relatively high non-wild-type rate of fluconazole and amphotericin B resistance, and low genetic diversity in Cryptococcus strains. The present study will provide evidence for further improvement of the diagnosis and treatment of cryptococcosis in China.

Abstract Background Bacterial species belonging to the genus Exiguobacterium are facultative anaerobic, non-spore-forming, Gram-positive bacilli, and rarely associated with human infections. Herein, we reported the first case of community-acquired pneumonia (CAP) and bacteremia due to Exiguobacterium spp. in China. Case presentation An adult male with severe CAP was hospitalized. The pathogen was isolated from his bloodstream and broncho-alveolar lavage fluid. The correct identification of the micro-organism was achieved using 16S rRNA sequencing, and its antibiotic susceptibility test was performed by microdilution method. The Whole Genome Sequencing (WGS) was used to characterize its genetic features and to elucidate its potential pathogenic mechanisms. Furthermore, its genome sequence was also compared with those of 3 publicly-available Exiguobacterium strains. A PubMed search was performed for further understanding the features of Exiguobacterium infections. Phylogenetic analysis of the 16S rRNA gene sequence showed that the strain GX59 was most closely related to Exiguobacterium AT1b (99.7%). The genome of GX59 was 2,727,929 bp in size, harbouring 2855 putative protein-coding genes, 5 rRNA operons, 37 tRNA genes and 1 tmRNA. The multiple genome comparison of 4 Exiguobacterium strains demonstrated that Exiguobacterium contained 37 genes of secretion systems, including sec, tat, FEA, Type IV Pili and competence-related DNA transformation transporter (Com). Virulence factors of the micro-organism included tlyC, NprR, MCP, Dam, which might play a critical role in causing lethal infection. Conclusions The study highlighted the potential pathogenicity of the genus Exiguobacterium for its unique genes encoding various virulence factors and those associated with antibiotic resistance, therefore, its clinical significance should be valued.

This study reports the simplified carbapenem inactivation method (sCIM) to detect carbapenemase-producing gram-negative bacilli in a simple and accurate manner. This method is based on the modified carbapenem inactivation method (mCIM) with the improvement of experimental procedures. Instead of incubating the antibiotic disk in the organism culture media, the organism to be tested was smeared directly onto the antibiotic disk in the sCIM. For evaluating the sensitivity and specificity of the method, a total of 196 Enterobacteriaceae, 73 Acinetobacter baumannii, and 158 Pseudomonas aeruginosa isolates were collected. Polymerase chain reaction (PCR) was used to detect the carbapenemase genes. Phenotypic evaluations were performed using both the sCIM and the mCIM. PCR results showed that, of the 196 Enterobacteriaceae strains, 147 expressed the carbapenemase genes blaKPC−2 (58.5%), blaIMP−4 (21.8%), blaIMP−2 (2.0%), blaVIM−1 (6.1%), blaNDM−1 (10.2%), and blaOXA−48 (1.4%). sCIM results had high concordance with PCR results (99.5%) and mCIM results (100%) with the exception of one Klebsiella pneumoniae strain, which had an minimal inhibitory concentration (MIC) for imipenem of 0.25 mg/L. PCR demonstrated that 53 of the 73 A. baumannii isolates expressed the carbapenemase genes blaOXA−23 (98.1%) and blaVIM−2 (1.8%). sCIM and PCR results corresponded but all A. baumannii isolates were carbapenemase negative by the mCIM. PCR demonstrated that 25 of the 158 P. aeruginosa isolates expressed carbapenemase genes blaVIM−1 (52%), blaVIM−2 (8%), blaVIM−4 (36%), and blaIMP−4 (4%). sCIM results had high concordance with PCR results (100%) and the mCIM results (99.4%) with the exception of one P. aeruginosa isolate that expressed the blaVIM−4 gene. The sCIM offers specificity and sensitivity comparable to PCR but has the advantage of being more user-friendly. This method is suitable for routine use in most clinical microbiology laboratories for the detection of carbapenemase-producing gram-negative bacilli.

Streptococcus pyogenes, or group A Streptococcus, is a pathogen responsible for a wide range of clinical manifestations, from mild skin and soft tissue infections and pharyngitis to severe diseases. Its epidemiological characteristics should be comprehensively under surveillance for regulating the national prevention and treatment practice. Herein, a total of 140 S. pyogenes, including 38 invasive and 102 noninvasive isolates, were collected from infected patients in 10 tertiary general hospitals from 7 cities/provinces in China during the years 2009–2016. All strains were characterized by classical and molecular techniques for its emm types/subtypes, virulent factors and antibiotic resistance profiling. Of 140 isolates, 15 distinct emm types and 31 subtypes were detected, dominated by emm12 (60 isolates, 42.9%), emm1(43, 30.7%), and emm89 (10, 7.1%), and 8 new emm variant subtypes were identified. All strains, invasive or not, harbored the superantigenic genes, speB and slo. The other virulence genes, smeZ, speF, and speC accounted for 96.4, 91.4, and 87.1% of collected isolates, respectively. Further multilocus sequence typing (MLST) placed all strains into 22 individual sequence types (STs), including 4 newly-identified STs (11, 7.9%). All isolates were phenotypically susceptible to penicillin, ampicillin, cefotaxime, and vancomycin, whereas 131(93.5%), 132(94.2%), and 121(86.4%) were resistant to erythromycin, clindamycin, and tetracycline, respectively. Our study highlights high genotypic diversity and high prevalence of macrolide resistance of S. pyogenes among clinical isolates circulating in China.

Introduction: The increase in clinical Enterobacteriaceae with dual carbapenemase has become a serious healthcare concern. It is essential to characterize the transferability and potential dissemination of blaKPC-2- and blaNDM-1-coharboring carbapenem-resistant Citrobacter freundii (CRCF). Methods: Four blaKPC-2- and blaNDM-1-coharboring CRCF strains were collected from our surveillance of the prevalence of carbapenem-resistant Enterobacteriaceae. The isolates were assessed using species identification, antimicrobial susceptibility testing, conjugation assays, whole-genome sequencing, plasmid stability, and fitness costs. Clonality, genome, plasmidome, and phylogeny were analyzed to reveal potential dissemination. Results: Three ST523 blaKPC-2- and blaNDM-1-coharboring CRCF strains, collected from the same hospital within 1 month, exhibited high homology (both identity and coverage >99%), implying clonal dissemination and a small-scale outbreak. Moreover, the blaKPC-2 and blaNDM-1 genes were coharbored on an IncR plasmid, probably generated by a blaKPC-2-harboring plasmid acquiring blaNDM-1, in these three strains. Importantly, the IncR plasmid may form a transferable hybrid plasmid, mediated by IS6100 via transposition, with another IncFII plasmid included in the same C. freundii strain. Furthermore, the blaKPC-2 and blaNDM-1 of the fourth CRCF strain are located on two di blaNDM-1-harboring C. freundii were divergent, and these plasmids have high homology to plasmids of other Enterobacteriaceae. Conclusion: Clonal dissemination of ST523 blaKPC-2- and blaNDM-1- coharboring CRCF strains was detected, and we first reported blaKPC-2 and blaNDM-1 concomitantly located on one plasmid, which could be transferred with mediation by IS6100 via transposition. Continued surveillance should urgently be implemented. [ABSTRACT FROM AUTHOR]