Synergistic Activity of Imipenem in Combination with Ceftazidime/Avibactam or Avibactam against Non-MBL-Producing Extensively Drug-Resistant Pseudomonas aeruginosa.

Extensively drug-resistant Pseudomonas aeruginosa (XDRPA) infection is a significant public health threat due to a lack of effective therapeutic options. New ß-lactam-ß-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-ß-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.

Clinical factors associated with composition of lung microbiota and important taxa predicting clinical prognosis in patients with severe community-acquired pneumonia.

Few studies have described the key features and prognostic roles of lung microbiota in patients with severe community-acquired pneumonia (SCAP). We prospectively enrolled consecutive SCAP patients admitted to ICU. Bronchoscopy was performed at bedside within 48 h of ICU admission, and 16S rRNA gene sequencing was applied to the collected bronchoalveolar lavage fluid. The primary outcome was clinical improvements defined as a decrease of 2 categories and above on a 7-category ordinal scale within 14 days following bronchoscopy. Sixty-seven patients were included. Multivariable permutational multivariate analysis of variance found that positive bacteria lab test results had the strongest independent association with lung microbiota (R2 = 0.033; P = 0.018), followed by acute kidney injury (AKI; R2 = 0.032; P = 0.011) and plasma MIP-1ß level (R2 = 0.027; P = 0.044). Random forest identified that the families Prevotellaceae, Moraxellaceae, and Staphylococcaceae were the biomarkers related to the positive bacteria lab test results. Multivariable Cox regression showed that the increase in α-diversity and the abundance of the families Prevotellaceae and Actinomycetaceae were associated with clinical improvements. The positive bacteria lab test results, AKI, and plasma MIP-1ß level were associated with patients' lung microbiota composition on ICU admission. The families Prevotellaceae and Actinomycetaceae on admission predicted clinical improvements.

Characterization of an NDM-5-producing hypervirulent Klebsiella pneumoniae sequence type 65 clone from a lung transplant recipient.

The emergence of New Delhi Metallo-ß-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.

Risk Factors for Mortality of Inpatients with Pseudomonas aeruginosa Bacteremia in China: Impact of Resistance Profile in the Mortality.

PURPOSE: Pseudomonas aeruginosa bacteremia presents a severe challenge to hospitalized patients. However, to date, the risk factors for mortality among inpatients with P. aeruginosa bacteremia in China remain unclear. PATIENTS AND METHODS: This retrospective multicenter study was performed to analyze 215 patients with culture-confirmed P. aeruginosa bacteremia in five healthcare centers in China during the years 2012-2019. RESULTS: Of 215 patients with P. aeruginosa bacteremia, 61 (28.4%) died during the study period. Logistic multivariable analysis revealed that cardiovascular disease (OR=3.978, P=0.001), blood transfusion (OR=5.855, P<0.001) and carbapenem-resistant P. aeruginosa (CRPA) phenotype (OR=4.485, P=0.038) constituted the independent risk factors of mortality. Furthermore, both CRPA and multidrug-resistant P. aeruginosa (MDRPA) phenotypes were found to be significantly associated with 5-day mortality (Log-rank, P<0.05). CONCLUSION: This study revealed a high mortality rate amongst hospitalized patients with P. aeruginosa bacteremia, and those with cardiovascular diseases, CRPA and MDRPA phenotypes, should be highlighted and given appropriate management in China.

Severity of influenza virus and respiratory syncytial virus coinfections in hospitalized adult patients.

BACKGROUND: With the introduction of molecular diagnostic techniques over the past decades, different kinds of viral pathogens in the same sample are detected simultaneously more frequently. Nevertheless, influenza virus (Flu) and respiratory syncytial virus (RSV) coinfection in adults was reported only occasionally. Moreover, the clinical implications of Flu/RSV coinfection in the respiratory tract of adults remain unclear. METHODS: This retrospective study analyzed adult patients with acute respiratory infection from January 2017 to June 2019 in China-Japan Friendship Hospital. RESULTS: A total of 574, 235 and 113 patients were positive for influenza A-only (FA-only), influenza B-only (FB-only) and RSV-only in influenza seasons (from Nov 2017 to Mar 2018 and from Nov 2018 to Mar 2019), respectively. Of these, 19 cases were coinfected by Flu and RSV and admitted to this hospital. Compared with 809 Flu-only infected patients and 113 RSV-only infected patients, both the rates of intensive care unit(ICU) admission and use of invasive mechanical ventilation in Flu/RSV coinfected patients were higher (ICU admission: 47.4% vs. 20.1%, P=0.004; 47.4% vs. 22.1%, P=0.020; invasive mechanical ventilation: 47.4% vs.13.2%, P<0.001; 47.4% vs. 17.7%, P=0.004). Furthermore, 60-day all-cause mortality attributed to Flu/RSV coinfections was significantly greater than that for Flu and RSV mono-infected patients (36.8% vs. 8.0%,P<0.001; 36.8% vs. 11.5%, P=0.004. CONCLUSION: The findings of this study suggest that coinfection of Flu/RSV in adults is associated with a high adverse outcome. Thus, Flu/RSV coinfections should be increasingly appreciated and given appropriate management.

Severity and mortality of respiratory syncytial virus vs influenza A infection in hospitalized adults in China.

BACKGROUND: Respiratory syncytial virus (RSV) is an important cause of medically attended acute respiratory illnesses in older adults but awareness of the relevance of RSV in older people remains lower than that of influenza, which exhibits similar clinical characteristics to those of RSV. OBJECTIVES: This study was performed to assess the clinical significance of RSV in respiratory samples from hospitalized adults. METHODS: Characteristics and outcomes in adults (≥18 years) hospitalized for RSV infection (n = 51) were compared with a cohort hospitalized for influenza A infection (n = 279) in a single-center retrospective cohort study in Beijing, China. RESULTS: Respiratory syncytial virus patients were slightly older, with no significant differences in underlying chronic conditions. Lower respiratory tract infection and cardiovascular complications were more frequent (P < .05) in RSV patients. Rates of mortality in the RSV cohorts were significantly higher within 30 days (13.7% vs 5.0%, P = .019) and 60 days (17.6% vs 7.5%, P = .021). Bacterial co-infection in respiratory samples was associated with reduced survival among RSV patients (log rank, P = .013). CONCLUSIONS: Respiratory syncytial virus is a common cause of serious illness among hospitalized adults in China with greater mortality than influenza A. Increased awareness and the availability of antiviral agents might increase the scope for successful management.

The tcdA-negative and tcdB-positive Clostridium difficile ST81 clone exhibits a high level of resistance to fluoroquinolones: a multi-centre study in Beijing, China.

Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhoea worldwide. In order to gain a better understanding about the molecular epidemiology of C. difficile in Beijing, China, molecular typing, antimicrobial susceptibility testing and drug resistance gene sequencing were performed on 174 strains of C. difficile collected from four large tertiary hospitals in Beijing. In total, 31 sequence types (STs) were identified among the 174 strains. ST81 was found to be the most prevalent (26.4%, 46/174), followed by ST2 (16.7%, 29/174) and ST54 (9.8%, 17/174). All isolates were susceptible to metronidazole and vancomycin. The test strains displayed resistance rates of 97.1%, 44.3% and 44.3% for ciprofloxacin, levofloxacin and moxifloxacin, respectively. ST81 isolates displayed a drug resistance rate of 97.8% for levofloxacin and moxifloxacin, which was significantly higher than ST2 (0%), ST54 (17.6%) and ST42 (0%) isolates (P<0.05). An amino acid mutation (T82I) was identified in GyrA, and the total mutation rate of the C. difficile strains was 40.8% (71/174). The mutation rate of ST81 isolates was 95.7% (44/46). Three amino acid mutations (D426N, S366A and D426V) were identified in GyrB, and the total mutation rate of GyrB was 39.1%. A double-site mutation in GyrB (S366A+D426V) was identified in all ST81 (n=46) isolates. In conclusion, the C. difficile ST81 clone showed a high level of resistance to fluoroquinolones in Beijing, highlighting the need for nationwide surveillance of CDI.

Genomic characteristics of clinically important ST11 Klebsiella pneumoniae strains worldwide.

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.

Comparison of the Cepheid Xpert Xpress Flu/RSV assay and commercial real-time PCR for the detection of influenza A and influenza B in a prospective cohort from China.

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.

Transcriptional profiling of the two-component regulatory system VraSR in Staphylococcus aureus with low-level vancomycin resistance.

The objective of this study was to comprehensively identify the target genes regulated by the two-component regulatory system VraSR in Staphylococcus aureus and to clarify the role of VraSR in low-level vancomycin resistance. Expression of vraS was determined by real-time quantitative reverse transcriptase PCR (qRT-PCR). A clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) strain B6D and a vancomycin-intermediate S. aureus (VISA) strain D7 that was induced from a meticillin-resistant S. aureus strain were selected to construct vraSR null mutants by allelic replacement. The vraSR-complemented strain B6D_c was also constructed by allelic replacement. Genes differentially expressed in the wild-type, vraSR null mutant and complemented strains were detected using RNA-Seq and were validated by qRT-PCR. Compared with vancomycin-susceptible S. aureus strains, expression of vraS was upregulated in all four isogenic hVISA strains. Vancomycin minimum inhibitory concentrations (MICs) in the vraSR null mutants B6D-ΔvraSR and D7-ΔvraSR were significantly lower than in the wild-type strains B6D and D7 and the complemented strain B6D_c. RNA-Seq and qRT-PCR data showed that expression of genes encoding FmtA protein, foldase protein PrsA, capsular polysaccharide biosynthesis glycosyltransferase, TcaA, a putative membrane protein, and six hypothetical proteins was down regulated in both vraSR-null mutants B6D-ΔvraSR and D7-ΔvraSR. Most of these differentially expressed proteins are involved in cell wall biosynthesis, which is associated with vancomycin resistance in S. aureus. In conclusion, VraSR plays an important role in S. aureus strains with low-level vancomycin resistance. PrsA, FmtA, glycosyltransferase and TcaA are regulated directly or indirectly by VraSR.