Incidence Rate, Pathogens and Economic Burden of Catheter-Related Bloodstream Infection: A Single-Center, Retrospective Case-Control Study.

Purpose: Indwelling central venous catheters (CVCs) can cause catheter related bloodstream infection (CRBSI). CRBSI occurring in intensive care unit (ICU) patients may lead to the worse outcomes and extra medical costs. The present study aimed to assess the incidence and incidence density, pathogens and economic burden of CRBSI in ICU patients. Patients and Methods: A case-control study was retrospectively carried out in six ICUs of one hospital between July 2013 and June 2018. The Department of Infection Control performed routinely surveillance for CRBSI on these different ICUs. Data of the clinical and microbiological characteristics of patients with CRBSI, the incidence and incidence density of CRBSI in ICUs, the attributable length of stay (LOS), and the costs among patients with CRBSI in ICU were collected and assessed. Results: A total of 82 ICU patients with CRBSI were included into the study. The CRBSI incidence density was 1.27 per 1000 CVC-days in all ICUs, in which the highest was 3.52 per 1000 CVC-days in hematology ICU and the lowest was 0.14 per 1000 CVC-days in Special Procurement ICU. The most common pathogen causing CRBSI was Klebsiella pneumoniae (15/82, 16.67%), in which 12 (80%) were carbapenem resistant. Fifty-one patients were successfully matched with control patients. The average costs in the CRBSI group were $ 67,923, which were significantly higher (P < 0.001) than the average costs in the control group. The total average costs attributable to CRBSI were $33, 696. Conclusion: The medical costs of ICU patients were closely related to the incidence of CRBSI. Imperative measures are needed to reduce CRBSI in ICU patients.

Chronic Splenic Melioidosis in a Patient with Fever of Unknown Origin Diagnosed by Metagenomics Next-Generation Sequencing: An Emerging Cause and Literature Review.

Introduction: Human melioidosis is an emerging infectious disease in tropical areas of China, and chronic melioidosis can be a rare cause of fever of unknown origin (FUO). Timely diagnosis may improve the prognosis of melioidosis. Case Presentation: We report a case of melioidosis with splenic abscesses caused by Burkholderia pseudomallei in a 57-year-old man, who presented with FUO. Positron emission tomography/computed tomography (PET/CT) revealed multiple hypermetabolic lesions in the spleen. The spleen biopsy was conducted and metagenomics next-generation sequencing (mNGS) of the spleen specimen identified the presence of B. pseudomallei, confirming the diagnosis of melioidosis. Antimicrobial treatment was initiated with intravenous meropenem, followed by oral faropenem. During the follow-up, the patient was in good condition except having a low-grade fever occasionally. A splenectomy was performed, and subsequent culture and mNGS of the spleen pus were both positive for B. pseudomallei. Histopathological characteristics of chronic splenic melioidosis were noted. Conclusion: Melioidosis is a serious endemic disease, and it is critical to raise awareness about this disease.

Clinical and Microbiological Characteristics of Klebsiella pneumoniae Bloodstream Infection in a Chinese Hospital: Hypervirulent and Multiclonal.

Purpose: Hypervirulent Klebsiella pneumoniae (hvKP) is emerging globally and can cause various infections. This study aimed to investigate the clinical and microbiological characteristics of bloodstream infection (BSI) caused by hvKP. Patients and Methods: The clinical data of hospitalized patients with K. pneumoniae BSI were retrospectively analyzed. The K. pneumoniae strains were collected and re-identified, and antimicrobial susceptibility testing was performed using the broth microdilution method. Capsular serotypes and virulence genes were detected using polymerase chain reaction, and hvKP was defined as aerobactin positive. Molecular typing was done by multilocus sequence typing. The hvKP and classic K. pneumoniae (cKP) subgroups were compared. Results: Of the 66 nonrepetitive BSI K. pneumoniae strains included, 29 (43.9%) were hvKP. In these BSI hvKP strains, salmochelin and yersiniabactin accounted for 86.2% and 72.4%, respectively. The prevalence of rmpA, iroBCD cluster, ybtS, clbA, and allS was 89.7%, 86.2%, 72.4%, 51.7%, and 41.4%, respectively, which were all significantly different between the hvKP and cKP subgroups. Serotypes K1 and K2 were strongly associated with hypervirulence (P < 0.05). Nineteen sequence types were scattered in the 29 hvKP strains, and the most common was ST23 (24.1%). None of the hvKP strains were carbapenem resistant. Compared with cKP, hvKP was more capable of developing a liver abscess. However, the 30-day mortality rate was lower (13.8% vs 21.6%) in the hvKP subgroup than in the cKP subgroup. Conclusion: This study demonstrated a high proportion of hvKP in BSI K. pneumoniae, most of which were RmpA and siderophore producing, and of multiclonal origin.

Antibiotic Exposure during the Preceding Six Months Is Related to Intestinal ESBL-Producing Enterobacteriaceae Carriage in the Elderly.

Intestinal carriage of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE carriage) poses a health risk to the elderly. It was aimed to study the prevalence and the risk factors of intestinal ESBL-PE carriage in the elderly. An observational study of a 921-elderly cohort was examined at health checkup for intestinal ESBL-PE carriage at a tertiary medical center in Shanghai. The prevalence and risk factors of intestinal ESBL-PE carriage, especially antimicrobial use in the preceding 9 months, were studied. The prevalence of intestinal ESBL-PE carriage was 53.3% (491/921) in community-dwelling elderly people. A total of 542 ESBL-producing isolates, including E. coli (n = 484) and K. pneumoniae (n = 58), were obtained. On genotyping, the CTX-M-9 ESBL was the most prevalent for 66.0% (358/542) of all isolates. Multivariate analysis showed that antibiotic exposure, age (61-70 years), and nursing home residence were independent risk factors of the ESBL-PE carriage. The analysis on the monthly use of antimicrobials showed that antibiotic exposure during the 6 months prior to sample collection contributed to the high prevalence of ESBL-PE carriage. A single exposure to an antimicrobial increased the risk of the carriage significantly, and the risk increased with the frequency of antimicrobial exposure (RR, 1.825 to 5.255). Prior use of second or third generation cephalosporins, fluoroquinolones, and macrolides increased the risk of the carriage. The results of this study indicate the importance of using antimicrobials judiciously in clinical settings to reduce antimicrobial resistance. Further studies with multiple center surveillance and with comparison of ESBL-PE carriage in the elderly and in the general population simultaneously are needed.

COVID-19 vaccines in patients with decompensated cirrhosis: a retrospective cohort on safety data and risk factors associated with unvaccinated status.

BACKGROUND: Safety data reported from the large-scale clinical trials of the coronavirus disease 2019 (COVID-19) vaccine are extremely limited in patients with decompensated cirrhosis. The vaccination campaign in this specific population could be difficult due to uncertainty about the adverse events following vaccination. We aimed to assessed the COVID-19 vaccination rate, factors associated with unvaccinated status, and the adverse events following vaccination in patients with decompensated cirrhosis. METHODS: This is a retrospective study from Ruijin Hospial (Shanghai, China) on an ongoing prospective cohort designed for long-term survival analysis of decompensated cirrhotic patients who recovered from decompensating events or acute-on-chronic liver failure (ACLF) between 2016 and 2018. We assessed the COVID-19 vaccination rate, the number of doses, type of vaccine, safety data, patient-reported reasons for remaining unvaccinated, factors associated with unvaccinated status, and the adverse events of COVID-19 vaccine. Binary logistic regression was used for identifying factors associated with unvaccinated status. RESULTS: A total of 229 patients with decompensated cirrhosis without previous SARS-CoV-2 infection participated (mean age, 56 ± 12.2 years, 75% male, 65% viral-related cirrhosis). Mode of decompensation were grade II‒III ascites (82.5%), gastroesophageal varices bleeding (7.9%), hepatic encephalopathy (7.9%). Eighty-five participants (37.1%) received at least one dose of vaccination (1 dose: n = 1, 2 doses: n = 65, 3 doses: n = 19) while 62.9% remained unvaccinated. Patient-reported reasons for remaining unvaccinated were mainly fear of adverse events (37.5%) and lack of positive advice from healthcare providers (52.1%). The experience of hepatic encephalopathy (OR = 5.61, 95% CI: 1.24-25.4) or ACLF (OR = 3.13, 95% CI: 1.12-8.69) and post-liver transplantation status (OR = 2.47, 95% CI: 1.06-5.76) were risk factors of remaining unvaccinated independent of residential areas. The safety analysis demonstrated that 75.3% had no adverse events, 23.6% had non-severe reactions (20% injection-site pain, 1.2% fatigue, 2.4% rash) and 1.2% had a severe event (development of acute decompensation requiring hospitalization). CONCLUSIONS: Patients with decompensated cirrhosis in eastern China are largely remained at unvaccinated status, particularly those with previous episodes of ACLF or hepatic encephalopathy and liver transplantation recipients. Vaccination against COVID-19 in this population is safe.

Acquisition of the Conjugative Virulence Plasmid From a CG23 Hypervirulent Klebsiella pneumoniae Strain Enhances Bacterial Virulence.

The emergence of hypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has become a hot topic and confounding problem for clinicians and researchers alike. Conjugative virulence plasmids have the potential to cause more threatening dissemination of hv-CRKP strains. We previously identified K2606, a CG23 clinical hypervirulent strain of Klebsiella pneumoniae harboring a conjugative virulence plasmid designated pK2606. In this study we examined hypervirulence levels using assays of biofilm formation, serum resistance, and wax larvae and mouse in vivo infection models. Moreover, to define the transfer ability of pK2606 and whether this confers hypervirulence to other strains we performed plasmid transconjugation experiments between K2606 and the ST11 CRKP strain HS11286 along with E. coli J53. We found that although biofilm formation and serum resistance were not significantly increased, the transconjugants acquired the ability of produce high level of siderophores and also caused high mortality of wax larvae and mice. Furthermore, we identified pK2606-like conjugative virulence plasmids in GenBank, providing evidence that such plasmids may have begun to spread throughout China. These findings provide an evidence base for the possible mechanisms of the emergence of hv-CRKP strains and highlight the potential of pK2606-like conjugative virulence plasmids to spread worldwide.

Laboratory-based Surveillance and Clinical Profile of Sporadic HEV Infection in Shanghai, China.

The study aimed to describe the epidemiological, virological and clinical features of sporadic HEV infection in eastern China. A total of 6112 patient sera were tested for anti-HEV IgG or anti-HEV IgM during one consecutive year (between August 2018 and July 2019). HEV RNA presence was evaluated by RT-PCR and HEV sequences were phylogenetically analyzed. Clinical features of confirmed HEV-infected patients were delineated. The sero-positivity rate of anti-HEV IgG maintained stable around 40%, while an obvious winter spike of anti-HEV IgM prevalence was observed. A total of 111 patients were confirmed of HEV viremia by molecular diagnosis. Subtype 4d was predominant. Phylogenetic analyses suggest that certain strains circulate across species and around the country. Subjects with confirmed current HEV infection had a high median age (58 years) and males were predominant (62.2%). Most patients presented with jaundice (75.7%) and anorexia (68.0%). Significantly elevated levels of liver enzymes and bilirubin were observed. Remarkably, the baseline bilirubin level was positively correlated with illness severity. Pre-existing HBV carriage may deteriorate illness. The clinical burden caused by locally acquired HEV infection is increasing. Surveillance should be enforced especially during the transition period from winter to spring. Patients with higher level of bilirubin at disease onset had slower recovery from HEV infection.

RamA upregulates multidrug resistance efflux pumps AcrAB and OqxAB in Klebsiella pneumoniae.

Overexpression of the acrAB genes regulated by RamA and overexpression of oqxAB regulated by RarA have been reported to mediate multidrug resistance in Gram-negative bacilli. In this study, regulation of acrAB and oqxAB simultaneously by the global regulator RamA was investigated in a multidrug-resistant Klebsiella pneumoniae clinical isolate (KP22) resistant to tigecycline and other antimicrobials. KP22 overexpressed ramA due to a ramR mutation, along with an unexpected overexpression of oqxB. Deletion of ramA led to a 16-fold decrease in the tigecycline minimum inhibitory concentration (MIC) with decreased expression of acrB (4.3-fold) and oqxB (7.1-fold) compared with KP22. Transcomplementation of KP22ΔramA with the wild-type ramA gene restored the tigecycline MIC and upregulation of the acrB (3.9-fold) and oqxB (4.0-fold) genes compared with KP22. When oqxB was knocked out, MICs of ciprofloxacin, olaquindox and nitrofurantoin were considerably decreased, while deletion of acrB led to MIC decreases for cefepime, piperacillin/tazobactam and tigecycline in addition to the above three antimicrobials. The results of electrophoretic mobility shift assay showed that RamA could bind the promoter regions of both the acrAB and oqxAB operons. This study demonstrates for the first time that RamA can directly regulate multidrug resistance efflux pumps AcrAB and OqxAB in K. pneumoniae.

Application of Fatty Liver Inhibition of Progression Algorithm and Steatosis, Activity, and Fibrosis Score to Assess the Impact of Non-Alcoholic Fatty Liver on Untreated Chronic Hepatitis B Patients.

BACKGROUNDS AND PURPOSE: Concurrent non-alcoholic fatty liver disease (NAFLD) in chronic hepatitis B (CHB) patients is a frequent and increasingly concerning problem because of the NAFLD pandemic. Admittedly, NAFLD can progress to non-alcoholic steatohepatitis (NASH) and severe fibrosis. Direct evidence of the fibrotic effect of NAFLD or NASH in chronic hepatitis B virus (HBV) infection remains lacking. We aimed to reveal the influence of concurrent histologically proven fatty liver diseases in fibrogenesis with chronic HBV infection. METHODS: We performed a retrospective cross-sectional study on a liver biopsy population of CHB patients without excessive alcohol intake to evaluate the prevalence of concurrent histologically proven NAFLD or NASH according to the fatty liver inhibition of progression (FLIP) algorithm and its association with the liver fibrosis stage. RESULTS: Among 1,081 CHB patients, concurrent NAFLD was found in 404 patients (37.4%), among whom 24.0% (97/404) have NASH. The presence of NASH was an independent predictor of significant fibrosis (odds ratio (OR), 2.53; 95% CI, 1.52-4.21; p < 0.001) and severe fibrosis (OR, 1.83; 95% CI, 1.09-3.09; p = 0.023) in all patients, as well as in patients with normal alanine aminotransferase (ALT) (predicting significant fibrosis, OR, 2.86, 95% CI, 1.34-6.10; p = 0.007). The presence of lobular inflammation (p < 0.001) or presence of cytological ballooning (p < 0.001), rather than presence of steatosis (p = 0.419), was related with severity of fibrosis in Spearman's correlation analysis. CONCLUSIONS: Concurrent NAFLD is common in CHB patients, and NASH is an independent risk factor potentiating significant fibrosis by 2.53-fold and severe fibrosis by 1.83-fold. While coping with chronic HBV infection, routine assessment of co-existing NAFLD or NASH is also important.

Efflux Pump AcrAB Confers Decreased Susceptibility to Piperacillin-Tazobactam and Ceftolozane-Tazobactam in Tigecycline-Non-Susceptible Klebsiella pneumoniae.

INTRODUCTION: Drug efflux pumps are critical for resistance in Gram-negative organisms, but there are limited data on the role they play in decreased susceptibility to ß-lactam/ß-lactamase inhibitor combinations. In this study, we aimed to investigate the impact of efflux pump AcrAB on piperacillin-tazobactam (TZP) and ceftolozane-tazobactam (C/T) susceptibility in tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) strains. METHODS: A tigecycline gradient was used to obtain various TNSKP strains, and in conjunction with the gradient derived strains, a TNSKP clinical strain (TNSKP24) was also included. Minimum inhibitory concentrations (MICs) of antibiotics were determined by the broth microdilution method, and whole-genome sequencing (WGS) was carried out to analyze genomic changes. PCR and sequencing were performed to confirm mutations in ramR, acrR, and the intergenic region of ramR-romA, and qRT-PCR was applied to evaluate levels of gene expression. In-frame acrB knockout and complementation were performed in 3 TNSKP strains. RESULTS: Two derivatives of K. pneumoniae K2606 (K2606-4 and K2606-16) and TNSKP24 overexpressed efflux pump AcrAB were obtained for further study. The MICs of TZP and C/T exhibited a 4- to 8-fold increase in K2606-4 and K2606-16, respectively, when compared with K2606 (TZP, 2/4 µg/mL; C/T, 0.25/4 µg/mL). Deletion of acrB decreased the MICs of TZP and C/T by 4- to 16-fold in TNSKP24, K2606-4, and K2606-16, respectively, and complementation of acrB increased the MICs of these agents. MICs of clavulanate, sulbactam, and avibactam in the presence of ß-lactam compounds did not change after acrB deletion and subsequent introduction of complementation mutants. CONCLUSION: This study highlights that decreased susceptibility to TZP and C/T could be caused by the multidrug efflux pump AcrAB in TNSKP strains.

Serotype Distribution, Antimicrobial Susceptibility, and Multilocus Sequencing Type (MLST) of Streptococcus pneumoniae From Adults of Three Hospitals in Shanghai, China.

Background:Streptococcus pneumoniae, a main causative agent associated with invasive and non-invasive infection in elderly population, is a major global health problem. After pneumococcal conjugate vaccines (PCV) and pneumococcal polysaccharide vaccines (PPV) were introduced, the distribution of S. pneumoniae serotypes has changed. There was currently limited data on epidemiology and status of antimicrobial resistance of S. pneumoniae in Shanghai. Objective: To determine the serotype distribution, antimicrobial susceptibility and molecular epidemiology of S. pneumoniae isolated from adults in Shanghai. Method: A total of 75 S. pneumoniae isolates consecutively collected from 2015 through 2017 were serotyped by conventional multiplex-PCR. The antimicrobial susceptibility was determined by broth microdilution method. The multilocus sequence type (MLST) was performed to estimate the molecular epidemiology. Results: The predominant serotypes among the isolates were 19F (20.00%), 3 (16.00%), 23F (9.33%), 14 (8.00%), and19A (5.33%). The prevalence of pneumococcal strains with serotypes targeted by vaccines PCV7, PCV10, PCV13, and PPV23 was 44, 45.33, 66.67, and 80%, respectively. Penicillin non-susceptible S. pneumoniae (PNSSP) accounted for 16% of the isolates examined and resistance to erythromycin, azithromycin, tetracycline, clindamycin, cefaclor and trimethoprim-sulfamethoxazole were found in 92.00, 90.67, 86.67, 81.33, 54.67, and 54.67% of isolates, with most isolates (78.67%) presenting multidrug-resistance. The top three sequence types (STs) were ST271 (17.33%), ST180 (9.33%), and ST81 (8.00%). The international resistance clone complexes Spain23F-1 (n = 4), Netherland3-31 (n = 8), and Taiwan19F-14 (n = 14) were identified. Conclusions: The S. pneumoniae isolates showed high genetic diversity in Shanghai and the prevalence of antimicrobial resistance was also high among S. pneumoniae isolates, most of which were multidrug-resistant. The spread of international resistance clones might contribute to the increase of resistant isolates. The PPV23 could protect against most pneumococcal capsular serotypes causing infection of adults in Shanghai.

Efflux pumps AcrAB and OqxAB contribute to nitrofurantoin resistance in an uropathogenic Klebsiella pneumoniae isolate.

Klebsiella pneumoniae is a common cause of urinary tract infections (UTIs). Nitrofurantoin (NIT), with high therapeutic concentrations in urine, is recommended as the first-line drug for both empiric treatment and chemoprophylaxis of UTIs. Although NIT resistance in K. pneumoniae is relatively high, the resistance mechanism is not well understood. This study collected a NIT-resistant K. pneumoniae [NRKP, minimum inhibitory concentration (MIC)=128 mg/L] and investigated the resistance mechanism. Addition of efflux pump inhibitors increased the susceptibility of NRKP to NIT (MIC decreased from 128 to 32 mg/L), implying the important role of efflux pumps in NIT resistance. Quantitative reverse transcriptase polymerase chain reaction analysis showed that NRKP had >100-fold increased expression of ramA, which was demonstrated to be caused by ramR mutation. Deletion of ramA led to a four-fold decrease in the MIC of NIT, and the expression levels of efflux pumps acrB and oqxB were downregulated by four- to seven-fold. Complementation of ramA restored both the MIC value and the expression level of acrB and oqxB in the ramA mutant strain. In order to confirm the role of acrB and oqxB in NIT resistance, gene knockout strains were constructed. Deletion of acrB or oqxB alone led to a four-fold decrease in the MIC of NIT, and deletion of acrB and oqxB simultaneously led to a 16-fold decrease in the MIC of NIT. These results demonstrate that AcrAB and OqxAB contribute to NIT resistance in K. pneumoniae.

Comparison of FibroTouch and FibroScan for staging fibrosis in chronic liver disease: Single-center prospective study.

BACKGROUND: The aim of this study was to compare the diagnostic accuracy of the FibroTouch and FibroScan in patients with chronic liver disease (CLD) for staging fibrosis. METHODS: A prospective study was conducted in 435 CLD patients between 2014 and 2017. Index tests (FibroTouch, FibroScan, APRI, and FIB-4 score) and a reference standard (liver biopsy) were performed within one week. RESULTS: The area under the receiver operating curve (AUROC) of the FibroTouch was similar with that of the FibroScan for the diagnosis of significant fibrosis, severe fibrosis, or cirrhosis; however, the AUROC of the FibroTouch was higher than that of APRI or FIB-4 (p < 0.001). There was a significant correlation (rho = 0.85, p < 0.001) between the FibroTouch and FibroScan for liver stiffness. The overall diagnostic accuracy of FibroTouch for significant fibrosis, severe fibrosis, or cirrhosis was 73.3%, 83.2%, or 84.1%, respectively. No significant differences between the FibroTouch and FibroScan were detected regarding the sensitivity, specificity, negative predictive value, positive predictive value, and accuracy. The optimal cut-off values for each stage of fibrosis were similar between the FibroTouch and FibroScan. CONCLUSION: The FibroTouch is a valuable diagnostic tool for diagnosing liver fibrosis with good diagnostic accuracy which was comparable with that of the FibroScan, but superior to that of the APRI and FIB-4.

Long Non-coding RNA NEAT1 Alleviates Acute-on-Chronic Liver Failure Through Blocking TRAF6 Mediated Inflammatory Response.

BACKGROUND: Long non-coding RNAs (lncRNAs) have recently been tightly linked to plenty of human diseases. However, knowledge of acute-on-chronic liver failure (ACLF) related lncRNAs remains insufficient. In this work, we studied the role of the lncRNA nuclear enriched abundant transcript 1 (NEAT1) in the pathogenesis of ACLF. METHODS: ACLF model was established by challenging D-galactosamine (D-GalN)/ lipopolysaccharide (LPS) i.p. in rats with cirrhosis. The serum levels of IL-1, IL-6, and HMGB1 were determined using ELISA. Quantitative real time-PCR and western blot were performed to evaluate RNA and protein levels of inflammatory response. RNA immunoprecipitation assay was performed to confirm protein that interacts with NEAT1. FINDINGS: Over-expression of NEAT1 could interact with TRAF6 and decrease its ubiquitination level, and significantly reduced the expression levels of IL-6, IL-22. Importantly, in ACLF rat model, NEAT1 over-expression reduced several cytokines expression and alleviated the pathological status in contrast to the control group. Additionally, NEAT1 was increased and positively correlated with IL-22 and IL-6 levels in PBMCs from the ACLF patients. INTERPRETATION: NEAT1 can suppress inflammatory response through blockade of TRAF6 ubiquitination in ACLF rat model, suggesting that lncRNA NEAT1 might play protective roles in the pathogenesis of ACLF and provide promising novel target for pharmacological intervention.

In Silico Typing and Comparative Genomic Analysis of IncFIIK Plasmids and Insights into the Evolution of Replicons, Plasmid Backbones, and Resistance Determinant Profiles.

IncFIIK plasmids are associated with the acquisition and dissemination of multiple-antimicrobial resistance in Klebsiella pneumoniae and often encountered in clinical isolates of this species. Since the phylogeny and evolution of IncFIIK plasmids remain unclear, here we performed large-scale in silico typing and comparative analysis of these plasmids in publicly available bacterial/plasmid genomes. IncFIIK plasmids are prevalent in K. pneumoniae, being found in 69% of sequenced genomes, covering 66% of sequenced STs (sequence types), but sparse in other Enterobacteriaceae IncFIIK replicons have three lineages. One IncFIIK allele could be found in distinct K. pneumoniae STs, highlighting the lateral genetic flow of IncFIIK plasmids. A set of 77 IncFIIK plasmids with full sequences were further analyzed. A pool of 327 antibiotic resistance genes or remnants were annotated in 75.3% of these plasmids. Plasmid genome comparison reiterated that they often contain other replicons belonging to IncFIA, IncFIB, IncFIIYp, IncFIIpCRY, IncR, IncL, and IncN groups and that they share a conserved backbone featuring an F-like conjugation module that has divergent components responsible for regulation and mating pair stabilization. Further epidemiological studies of IncFIIK plasmids are required due to the sample bias of K. pneumoniae genomes in public databases. This study provides insights into the evolution and structures of IncFIIK plasmids.

Emergence of tigecycline- and carbapenem-nonsusceptible Klebsiella pneumoniae ST11 clone in patients without exposure to tigecycline.

BACKGROUND/PURPOSE: Currently, tigecycline-nonsusceptible Klebsiella pneumoniae (TNSKP) is mainly reported to emerge following clinical use of tigecycline and is usually polyclonal. This study aimed to characterize TNSKP isolated from patients without prior tigecycline use. METHODS: Twenty-six TNSKP clinical isolates were collected, and carbapenemase and 16S rRNA methylase genes were identified by polymerase chain reaction and sequencing. Molecular typing was conducted by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Clinical data of patients in the carbapenem-susceptible TNSKP group and the tigecycline- and carbapenem-nonsusceptible K. pneumoniae (TCNSKP) group were compared. RESULTS: Of the 26 TNSKP isolates, eight contained both blaKPC-2 and 16S rRNA methylase genes. In the remaining 18 TNSKP isolates, no carbapenemase gene was detected, and only three had the 16S rRNA methylase gene. Among the 26 isolates, 24 distinct pulsotypes and 19 sequence types (STs) were identified by PFGE and MLST, respectively. Six of the eight TCNSKP were ST11, whereas the remaining 18 TNSKP isolates were assigned to 17 different STs. No patient received tigecycline prior to the isolation of TNSKP. By comparison, intensive care unit exposure, mechanical ventilation, prior ß-lactam/ß-lactamase use, and longer hospitalization were more common for the TCNSKP group than for the carbapenem-susceptible TNSKP group. CONCLUSION: TNSKP can occur without tigecycline use, and TCNSKP ST11 is predominant among them. Further, this report proposes potential risk factors for the occurrence of carbapenem-nonsusceptibility in TNSKP.

Complete Sequence of a Novel IncR-F33:A-:B- Plasmid, pKP1034, Harboring fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12, and rmtB from an Epidemic Klebsiella pneumoniae Sequence Type 11 Strain in China.

A high fosfomycin resistance rate was observed in Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) in our previous study, but little is known about its mechanisms. In this study, we explored the prevalence of plasmid-mediated fosfomycin resistance determinants among fosfomycin-resistant KPC-KP strains from a Chinese university hospital and determined the complete sequence of a novel fosA3-carrying plasmid isolated from an epidemic K. pneumoniae sequence type (ST) 11 strain. A total of 97 KPC-KP strains were studied, of which 57 (58.8%) were resistant to fosfomycin, including 44 (45.4%) harboring fosA3 and 1 harboring fosA. All fosA3-positive strains belonged to the dominant ST11-pulse type (PT) A clone according to multilocus sequence typing and pulsed-field gel electrophoresis, suggesting clonal dissemination. The fosA-positive isolate belonged to ST11-PTE. The fosA3-carrying plasmid pKP1034 is 136,848 bp in length and is not self-transmissible. It is a multireplicon plasmid belonging to IncR-F33:A-: B-. Besides fosA3, a variety of other resistance determinants, including blaKPC-2, rmtB, blaCTX-M-65, and blaSHV-12, are identified in pKP1034, which would allow for coselection of fosA3 by most ß-lactams and/or aminoglycosides and facilitate its dissemination despite limited use of fosfomycin in China. Detailed comparisons with related plasmids revealed that pKP1034 is highly mosaic and might have evolved from alarming recombination of the blaKPC-2-carrying plasmid pKPC-LK30 from Taiwan and the epidemic fosA3-carrying plasmid pHN7A8 from mainland China.

Mapping the resistance-associated mobilome of a carbapenem-resistant Klebsiella pneumoniae strain reveals insights into factors shaping these regions and facilitates generation of a 'resistance-disarmed' model organism.

OBJECTIVES: This study aims to investigate the landscape of the mobile genome, with a focus on antibiotic resistance-associated factors in carbapenem-resistant Klebsiella pneumoniae. METHODS: The mobile genome of the completely sequenced K. pneumoniae HS11286 strain (an ST11, carbapenem-resistant, near-pan-resistant, clinical isolate) was annotated in fine detail. The identified mobile genetic elements were mapped to the genetic contexts of resistance genes. The blaKPC-2 gene and a 26 kb region containing 12 clustered antibiotic resistance genes and one biocide resistance gene were deleted, and the MICs were determined again to ensure that antibiotic resistance had been lost. RESULTS: HS11286 contains six plasmids, 49 ISs, nine transposons, two separate In2-related integron remnants, two integrative and conjugative elements (ICEs) and seven prophages. Sixteen plasmid-borne resistance genes were identified, 14 of which were found to be directly associated with Tn1721-, Tn3-, Tn5393-, In2-, ISCR2- and ISCR3-derived elements. IS26 appears to have actively moulded several of these genetic regions. The deletion of blaKPC-2, followed by the deletion of a 26 kb region containing 12 clustered antibiotic resistance genes, progressively decreased the spectrum and level of resistance exhibited by the resultant mutant strains. CONCLUSIONS: This study has reiterated the role of plasmids as bearers of the vast majority of resistance genes in this species and has provided valuable insights into the vital role played by ISs, transposons and integrons in shaping the resistance-coding regions in this important strain. The 'resistance-disarmed' K. pneumoniae ST11 strain generated in this study will offer a more benign and readily genetically modifiable model organism for future extensive functional studies.

Mechanisms of tigecycline resistance among Klebsiella pneumoniae clinical isolates.

Of 26 tigecycline-nonsusceptible Klebsiella pneumoniae (TNSKP) clinical isolates, 25 had nonsynonymous mutations in ramR and/or acrR (23 in ramR and 10 in acrR). Eight TNSKP isolates possessed overexpression of ramA, acrB, rarA, and oqxB simultaneously, while 8 and 1 TNSKP strains had upregulation of ramA and acrB and of rarA and oqxB, respectively. Thus, resistance mechanisms of 9 TNSKP isolates cannot be explained by the present pathways. This study underscores the role of RamA in TNSKP and suggests the presence of novel tigecycline resistance mechanisms.