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

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

Characterization of ST11 and ST15 Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae from Patients with Ventilator-Associated Pneumonia.

Background: The prevalence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (hv-CRKP) is a serious public threat globally. Here, we performed clinical, molecular, and phenotypic monitoring of hv-CRKP strains isolated from the intensive care unit (ICU) to offer evidence for prevention and control in hospitals. Methods: Data analysis of ICU patients suffering from ventilator-associated pneumonia (VAP) because of hv-CRKP infection, admitted at the Chinese Teaching Hospital between March 2019 and September 2021 was performed. Patients' antibiotic-resistance genes, virulence-associated genes, and capsular serotypes of these isolates were detected. Homology analysis of the strains was performed by MLST and PFGE. Six different strains were tested for their virulence traits using the serum killing test and the Galleria mellonella infection assay. For whole genome sequencing, KP3 was selected as a representative strain. Results: Clinical data of 19 hv-CRKP-VAP patients were collected and their hv-CRKP were isolated, including 10 of ST11-KL64, 4 of ST15-KL112, 2 of ST11-KL47, 1 of ST15-KL19, 1 of ST17-KL140, and 1 of ST48-KL62. Four ST15 and 8 ST11 isolates revealed high homology, respectively. Most strains carried the carbapenemase gene blaKPC-2 (14/19, 73.68%), followed by blaOXA-232 (4/19, 21.05%). All strains were resistant to almost all the antibiotics except polymyxin and tigacycline. Ten patients were treated with polymyxin or tigacycline based on their susceptibility results, and unfortunately 6 patients died. All strains exhibited a hyper-viscous phenotype, and the majority (17/19, 89.47%) of them contained rmpA and rmpA2. The serum killing test showed that KP9 was resistant to normal healthy serum, others were intermediately or highly sensitive. G. mellonella larvae infection assay suggested that the strains in this study were hypervirulent. Conclusion: This study highlights the dominant strain and molecular epidemiology of hv-CRKP in a hospital in China. We should pay more attention to the effect of hv-CRKP on VAP, strengthen monitoring and control transmission.

Identification of Bacterial Pathogens at Genus and Species Levels through Combination of Raman Spectrometry and Deep-Learning Algorithms.

The rapid and accurate identification of the causing agents during bacterial infections would greatly improve pathogen transmission, prevention, patient care, and medical treatments in clinical settings. Although many conventional and molecular methods have been proven to be efficient and reliable, some of them suffer technical biases and limitations that require the development and application of novel and advanced techniques. Recently, due to its cost affordability, noninvasiveness, and label-free feature, Raman spectroscopy (RS) is emerging as a potential technique for fast bacterial detection. However, the method is still hampered by many technical issues, such as low signal intensity, poor reproducibility, and standard data set insufficiency, among others. Thus, it should be cautiously claimed that Raman spectroscopy could provide practical applications in real-world settings. In order to evaluate the implementation potentials of Raman spectroscopy in the identification of bacterial pathogens, we investigated 30 bacterial species belonging to 9 different bacterial genera that were isolated from clinical samples via surfaced enhanced Raman spectroscopy (SERS). A total of 17,149 SERS spectra were harvested from a Raman spectrometer and were further analyzed via machine learning approaches, which showed that a convolutional neural network (CNN) deep learning algorithm achieved the highest prediction accuracy for recognizing pathogenic bacteria at both the genus and species levels. In summary, the SERS technique holds a promising potential for fast bacterial pathogen identification in clinical laboratories with the integration of machine learning algorithms, which might be further developed and sharpened for the direct identification and prediction of bacterial pathogens from clinical samples. IMPORTANCE In this study, we investigated 30 bacterial species belonging to 9 different bacterial genera that were isolated from clinical samples via surfaced enhanced Raman spectroscopy (SERS). A total of 17,149 SERS spectra were harvested from a Raman spectrometer and were further analyzed via machine learning approaches, the results of which showed that the convolutional neural network (CNN) deep learning algorithm could achieve the highest prediction accuracy for recognizing pathogenic bacteria at both the genus and species levels. Taken together, we concluded that the SERS technique held a promising potential for fast bacterial pathogen diagnosis in clinical laboratories with the integration of deep learning algorithms, which might be further developed and sharpened for the direct identification and prediction of bacterial pathogens from clinical samples.

Liposomes trigger bone marrow niche macrophage "foam" cell formation and affect hematopoiesis in mice.

Liposomes are the most widely used nanocarrier platform for the delivery of therapeutic and diagnostic agents, and a number of liposomes have been approved for use in clinical practice. After systemic administration, most liposomes are cleared by macrophages in the mononuclear phagocyte system, such as the liver and bone marrow (BM). However, the majority of studies have focused on investigating the therapeutic results of liposomal drugs, and too few studies have evaluated the potential side effects of empty nanocarriers on the functions of macrophages in the mononuclear phagocyte system. Here, we evaluate the potential effects of empty liposomes on the functions of BM niche macrophages. Following liposome administration, we observed lipid droplet (LD) accumulation in cultured primary macrophages and BM niche macrophages. We found that these LD-accumulating macrophages, similar to foam cells, exhibited increased expression of inflammatory cytokines, such as IL-1ß and IL-6. We further provided evidence that liposome deposition and degradation induced LD biogenesis on the endoplasmic reticulum membrane and subsequently disturbed endoplasmic reticulum homeostasis and activated the inositol-requiring transmembrane kinase/endoribonuclease 1α/NF-κB signaling pathway, which is responsible for the inflammatory activation of macrophages after liposome engulfment. Finally, we also showed the side effects of dysfunctional BM niche macrophages on hematopoiesis in mice, such as the promotion of myeloid-biased output and impairment of erythropoiesis. This study not only draws attention to the safety of liposomal drugs in clinical practice but also provides new directions for the design of lipid-based drug carriers in preclinical studies.

Preliminary exploration on the serum biomarkers of bloodstream infection with carbapenem-resistant Klebsiella pneumoniae based on mass spectrometry.

BACKGROUND: Carbapenem-resistant K. pneumoniae (CRKP) bloodstream infections (BSI) must be rapidly identified to improve patient survival rates. This study investigated a new mass spectrometry-based method for improving the identification of CRKP BSI and explored potential biomarkers that could differentiate CRKP BSI from sensitive. METHODS: Mouse models of BSI were first established. MALDI-TOF MS was then used to profile serum peptides in CRKP BSI versus normal samples before applying BioExplorer software to establish a diagnostic model to distinguish CRKP from normal. The diagnostic value of the model was then tested against 32 clinical CRKP BSI and 27 healthy serum samples. Finally, the identities of the polypeptides used to establish the diagnostic model were determined by secondary mass spectrometry. RESULTS: 107 peptide peaks were shared between the CRKP and normal groups, with 18 peaks found to be differentially expressed. Five highly expressed peptides in the CRKP group (m/z 1349.8, 2091.3, 2908.2, 4102.1, and 8129.5) were chosen to establish a diagnostic model. The accuracy, specificity and sensitivity of the model were determined as 79.66%, 81.48%, and 78.12%, respectively. Secondary mass spectrometry identified the Fibrinogen alpha chain (FGA), Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) and Serum amyloid A-2 protein (SAA2) as the source of the 5 serum peptides. CONCLUSIONS: We successfully established a serum peptide-based diagnostic model that distinguished clinical CRKP BSI samples from normal healthy controls. The application of MALDI-TOF MS to measure serum peptides, therefore, represents a promising approach for early BSI diagnosis of BSI, especially for multidrug-resistant bacteria where identification is urgent.

Rapid Detection Method for Pathogenic Candida Captured by Magnetic Nanoparticles and Identified Using SERS via AgNPs.

PURPOSE: Candidemia infection is common in the clinic and has a high mortality rate. Candida albicans, Candida tropicalis, and Candida krusei are very important and common pathogenic species. Candida is difficult to isolate from clinical samples and culture, and immunological detection cannot distinguish these related strains. Furthermore, Candida has a complex cell wall, which causes difficulties in the extraction of DNA for nucleic acid detection. The purpose of this study was to establish a protocol for the direct identification of Candida from serum. MATERIALS AND METHODS: We synthesized Fe3O4@PEI (where PEI stands for polyethylenimine) magnetic nanoparticles to capture Candida and prepared positively charged silver nanoparticles (AgNPs+) as the substrate for surface-enhanced Raman scattering (SERS). Candida was directly identified from serum by SERS detection. RESULTS: Orthogonal partial least squares discriminant analysis (OPLS-DA) was used as the multivariate analysis tool. Principal component analysis confirmed that this method can clearly distinguish common Candida. After 10-fold cross-validation, the accuracy of training data in this model was 100% and the accuracy of test data was 99.8%, indicating that the model has good classification ability. CONCLUSION: The detection could be completed within 40 minutes using Fe3O4@PEI and AgNPs+ prepared in advance. This is the first time that Fe3O4@PEI was used in the detection of Candida by SERS. We report the first rapid method to identify fungi directly from serum without breaking the cell wall to extract DNA from the fungi.

A Novel bla CTX-M-65-Harboring IncHI2 Plasmid pE648CTX-M-65 Isolated from a Clinical Extensively-Drug-Resistant Escherichia coli ST648.

BACKGROUND: An ESBL, carbapenemase- and MCR-1-producing Escherichia coli ST648 strain was isolated from the urine sample of a patient in a Chinese tertiary hospital in 2016. METHODS: The strain was fully sequenced by GridION X5 platform of Oxford Nanopore Technology. RESULTS: The sequence analysis showed that the extended-spectrum ß-lactamases CTX-M-65 and OXA-1, the carbapenemase NDM-5, the MCR-1 were encoded, respectively, by three different resistance plasmids. The pE648CTX-M-65-carrying bla CTX-M-65 was a novel conjugative plasmid belonging to IncHI2 type; except for the bla CTX-M-65, it also carried resistance genes ble, floR, sul1, aph(4)-Ia, aac(3)-VI, aac(6')-II, bla OXA-1, catB, arr3 and tetA. Besides, an IncX4 plasmid pE648MCR-1-carrying mcr-1 and an IncX3 plasmid pE648NDM-5-carrying bla NDM-5 were also identified. CONCLUSION: The three transferable resistance plasmids coexisting in the E. coli ST648 isolate indicated the high risk to disseminate the extensively-drug-resistance among Enterobacteriaceae.

Comparative analysis of laboratory indexes of severe and non-severe patients infected with COVID-19.

BACKGROUND: The pandemic coronavirus disease 2019 (COVID-19) has threaten the global health. The characteristics of laboratory findings of coronavirus are of great significance for clinical diagnosis and treatment. We found indicators that may most effectively predict a non-severe COVID-19 patient develop into a severe patient. METHODS: We conducted a meta-analysis to compare the laboratory findings of severe patients with non-severe patients with COVID-19 from searched articles. RESULTS: Through the analysis of laboratory examination information of patients with COVID-19 from 35 articles (5912 patients), we demonstrated that severe cases possessed higher levels of leukocyte (1.20-fold), neutrophil (1.33-fold), CRP (3.04-fold), PCT (2.00-fold), ESR (1.44-fold), AST (1.40-fold), ALT (1.34-fold), LDH (1.54-fold), CK (1.44-fold), CK-MB (1.39-fold), total bilirubin (1.14-fold), urea (1.28-fold), creatine (1.09-fold), PT (1.03-fold) and D-dimer (2.74-fold), as well as lower levels of lymphocytes (1.44-fold), eosinophil (2.00-fold), monocyte (1.08-fold), Hemoglobin (1.53-fold), PLT (1.15-fold), albumin (1.15-fold), and APTT (1.02-fold). Lymphocyte subsets and series of inflammatory cytokines were also different in severe cases with the non-severe ones, including lower levels of CD4 T cells (2.10-fold) and CD8 T cells (2.00-fold), higher levels of IL-1ß (1.02-fold), IL-6 (1.93-fold) and IL-10 (1.55-fold). CONCLUSIONS: Some certain laboratory inspections could predict the progress of the COVID-19 changes, especially lymphocytes, CRP, PCT, ALT, AST, LDH, D-dimer, CD4 T cells and IL6, which provide valuable signals for preventing the deterioration of the disease.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry combined with UF-5000i urine flow cytometry to directly identify pathogens in clinical urine specimens within 1 hour.

BACKGROUND: Urinary tract infection (UTI) is one of the most common hospital-associated infectious. The traditional laboratory diagnosis method for UTI requires at least 24 hours, and it cannot provide the etiology basis for the clinic in time. The aim of our study is to develop a new method for pathogenic diagnosis of UTI by combining matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and UF-5000i from urine samples directly within 1 hour. METHODS: A total of 1,503 urine samples were collected from patients suggesting symptoms of UTI from August 2018 to January 2019. Each of these samples was divided into three aliquots. The first aliquot was used for conventional cleaning mid-stream urine culture; the second one for UF-5000i analysis to screen out the bacterial counts, which were more than 1×105 bacteria/mL. The third one was processed to bacterial purification and directly identified by the MALDI-TOF MS. RESULTS: In our study, 296 of 1,503 urine specimens were screened out by UF-5000i (bacterial pellets counts ≥105/mL). Compared the conventional culture-dependent method, the results of our methods were consistent in 249 of 263 (94.7%) cases, and they were both single-microorganism. Among 249 credible results, species-level identification (score ≥2.0) was contained 233 (233/249. 93.6%), 16 (16/249, 6.4%) samples scored between 1.7 and 1.99, and 14 (14/249, 5.6%) samples scored <1.7 or no peaks found. When there were 2 different kinds of bacteria in the urine, the result of MALDI-TOF MS was unreliable. CONCLUSIONS: MALDI-TOF MS combined with UF-5000i to identify the pathogenic bacteria in urine directly is a novel and reliable method and saves at least 23 hours relative to the current routine conventional method. Thus its rapid and accurate detection may provide the basis of etiology for clinical diagnosis of UTIs efficiently.

Disease burden and molecular epidemiology of carbapenem-resistant Klebsiella pneumonia infection in a tertiary hospital in China.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become an urgent global public health issue, but its distribution has obvious regional differences. The purpose of this study was to investigate the patient-based disease burden and molecular epidemiology of CRKP infections in a tertiary hospital in northern Jiangsu Province in China. METHODS: A retrospective, epidemiological survey of CRKP infections in our hospital from January to December 2016 was conducted to collect clinical and epidemiologic data. Non-duplicated clinical CRKP isolates were collected for the resistance-associated genes and clonal correlation analysis by PCR, sequencing and multilocus sequence typing (MLST). RESULTS: 252 CRKP infection cases were collected, and the annual CRKP infection incidence of the hospital during 2016 was 14.64 per 10,000 hospital discharges (252/172,112*10,000) and 13.78 per 100,000 patient days (252/1,829,190*100,000). The patient-based disease burden concentrated on antimicrobial exposure history (133/224, 59.37%)-the most dominant STs. KPC-2 (120/128, 93.8%) was the predominant carbapenemase and ST11 (98/128, 76.5%) was the dominant STs. One isolate was detected with harboring bla KPC-2 and bla MCR-1 simultaneously. CONCLUSIONS: Patient-based disease burden and KPC-2-producing ST11 Klebsiella pneumonia caused in higher CRKP incidence in the hospital. The emergence of CRKP with bla KPC-2 and bla MCR-1 should be of concern.

First Reported Nosocomial Outbreak Of NDM-5-Producing Klebsiella pneumoniae In A Neonatal Unit In China.

PURPOSE: Carbapenem-resistant Klebsiella pneumoniae (CRKP) have emerged worldwide and also being a major threat to children and neonate. In this study, we describe a nosocomial outbreak of NDM-5-producing Klebsiella pneumoniae in neonatal unit of a teaching hospital in China from September 2015 to September 2016. PATIENTS AND METHODS: We collected 12 carbapenem-resistant K. pneumoniae outbreak strains from 12 newborns and characterized these isolates for their antimicrobial susceptibility, clone relationships, and multi-locus sequence types using vitek-2 compact system, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Resistant genes were detected by using PCR and sequencing. Plasmid conjugation experiment was carried out to determine the transferability of carbapenem resistance. PCR-based replicon typing (PBRT), S1 nuclease-PFGE, and southern blotting were conducted for plasmid profiling. RESULTS: All 12 K. pneumoniae isolates were resistant to carbapenems and carried bla NDM-5, bla TEM-1 and bla SHV-11. Furthermore, PFGE analysis showed that NDM-5-producing K. pneumoniae were clonally related and MLST assigned them to sequence type 337. Conjugative assays showed that plasmids harboring bla NDM-5 gene were self-transmissible. Plasmid analysis suggested that all bla NDM-5 gene located on a ~45 kb IncX3 type plasmid. CONCLUSION: To the best of our knowledge, this is the first report of a clone outbreak of bla NDM-5-carrying K. pneumoniae isolates from neonates. There is an urgent need for effective infection control measures to prevent bla NDM-5 variants from becoming epidemic in the neonates in the future.

Overestimated discriminatory power of MALDI-TOF mass spectrometry for typing of carbapenem-resistant Klebsiella pneumoniae clones.

Homology surveillance of carbapenem-resistant Klebsiella pneumoniae (CRKP) is critical to monitor and prevent outbreaks of nosocomial infections. In the present study, a matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF MS)-based method was evaluated as a rapid tool for typing CRKP in comparison with pulsed-field gel electrophoresis (PFGE) and multi locus sequence typing (MLST). Drug-resistant phenotypes and genotypes of 44 CRKP isolates were detected by microdilution broth method and polymerase chain reaction, and typed by PFGE, MLST and MALDI-TOF MS. Simpson's Index of Diversity was used to evaluate taxonomic diversity, Adjusted Rand Index (ARI) for congruence between the typing methods and Wallace coefficients (W) for the ability of either method to predict each other. Forty-four CRKP isolates of 15 sequence types (STs) produced either NDM-1 (n = 16), NDM-5 (n = 9) or KPC-2 (n = 19) carbapenemases. PFGE differentiated these isolates into 16 distinct types, and two deoxyribonucleic acid profiles were assigned to ST337 and ST11, respectively. MALDI-TOF MS failed to clearly delineate between clusters on dendrograms based on principal components analysis and main spectrum profile. The chosen parameters resulted in a maximum ARI of 0.310 (95% CI 0.088-0.531) between MALDI-TOF MS typing and the PFGE reference, indicating a low ability of the former to correctly identify related isolates. Likewise, the maximum W coefficient of 0.367 (95% CI 0.203-0.532) showed that MALDI-TOF MS had a lower predictive power than PFGE. We conclude that MALDI-TOF MS lacks the discriminatory power necessary for clone assignment of CRKP isolates and consequently cannot be considered as a rapid and creditable method for this purpose.

Dissemination of Multidrug-Resistant Shigella flexneri and Shigella sonnei with Class 1, Class 2, and Atypical Class 1 Integrons in China.

Background: Emergence of multidrug-resistant Shigella, a major causative agent of bacterial dysentery, has generated many concerns not only in China but also worldwide. However, the prevalence of Shigella resistance caused by integron in the nonpopular season of diarrhea is not clear. Materials and Methods: Thirty-one Shigella flexneri and 22 Shigella sonnei samples collected in December 2010 from 10 cities of China were characterized for antimicrobial susceptibility, gene cassettes, widespread of integrons, and pulsed-field gel electrophoresis (PFGE) profile. Results: Multidrug resistance (MDR) was detected in 29 (93.5%) S. flexneri and 20 (90.9%) S. sonnei isolates. Class 1 integrons were detected in 25 (80.6%) S. flexneri and in 13 (59.1%) S. sonnei isolates; class 2 integrons were detected in 26 (83.9%) S. flexneri and in 19 (86.4%) S. sonnei isolates. Interestingly, the atypical class 1 integrons were mostly detected in S. flexneri (45.2%) isolates, whereas in only 1 (4.5%) S. sonnei isolate. DNA sequencing revealed two novel cassette arrays, dfrA5 and aacA4-cmlA, of class 1 integrons in S. flexneri, and dfrA17-aadA5 in S. sonnei isolates. The cassette arrays, dfrA1-sat1-aadA1 of class 2 integron and blaoxa-30-aadA1 of atypical class 1 integron, were also identified. PFGE profiles demonstrated A6 subtype of S. flexneri strains prevalent in Shanghai, Changchun, Jinan, and Changsha; and F6 subtype of S. sonnei prevalent in Jinan, Changchun, and Shanghai. Conclusion: The dissemination of MDR Shigella strains with integrons makes it an increasing public health problem in China. Increased surveillance and the development of adequate prevention strategies are warranted.

High Prevalence of bla NDM Variants Among Carbapenem-Resistant Escherichia coli in Northern Jiangsu Province, China.

The continuous emergence of carbapenem-resistant Escherichia coli (CRECO) presents a great challenge to public health. New Delhi metallo-lactamase (NDM) variants are widely disseminated in China, so the research on the prevalence and transmission of diverse bla NDM variants is urgently needed. In the present study, 54 CRECO isolates were collected from 1,185 Escherichia coli isolates in five hospitals in Northern Jiangsu Province, China from September 2015 to August 2016. Antimicrobial susceptibility tests, PCR detection of resistance determinants, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to characterize these strains. Plasmid conjugation experiments were carried out to determine the transferability of resistant genes from selected isolates. PCR-based replicon typing (PBRT), S1 nuclease-PFGE, and Southern blotting were conducted for plasmid profiling. Carbapenemase genes were detectable in all CRECO isolates, among which thirty-one CRECO isolates were found to carry bla NDM-5 (54.7%), while, bla NDM-1, bla NDM-7, bla NDM-4, bla NDM-9, and bla KPC-2 were identified in 14, five, two, one, and one isolates, respectively. MLST results revealed 15 different STs and four new STs were first reported to be linked with NDM-producing isolates. PFGE typing showed that no more than two isolates with the same ST appeared to the same band pattern except three ST410 isolates. Twenty-six selected NDM-producing isolates were successfully transferred to E. coli J53 by conjugation experiments. Notably, 50.0% (13/26) of blaNDM variants were found to be carried by ~55 kb IncX3 plasmid. Our study reported a high prevalence of blaNDM variants, especially bla NDM-5, in Northern Jiangsu province, China. Diverse bla NDM variants were mainly carried by ~55 kb IncX3 plasmids, suggesting that the fast evolution and high transferability of this kind of plasmid promote the high prevalence of bla NDM variants. Therefore, large-scale surveillance and effective infection control measures are also urgently needed to prevent diverse bla NDM variants from becoming epidemic in the future.

Two novel mutations in parE among Shigella flexneri isolated from Jiangsu Province of China, 2016.

BACKGROUND: The study was conducted to assess the resistance capacity of quinolone against Shigella flexneri, and to investigate the involved quinolone resistance mechanism. The data were collected from Jiangsu Province, China in 2016. METHODS: The number of 81 S. flexneri was obtained from 12 cities in Jiangsu Province of China during 2016. Slide agglutination was taken for serotyping, and susceptibility test was identified by the disc diffusion method. PCR aimed to amplify the quinolone resistance-determining region (QRDR) genes and screen for plasmid-mediated quinolone resistance (PMQR) determinants. Chromosomal mutation was confirmed by sequencing and Blast comparison. RESULTS: 2a was the commonest serotype, accounting for 40.7% (33/81) of the 81 S. flexneri. 70.4% (57/81) isolates expressed resistance against nalidixic acid, and the resistance against ciprofloxacin even reached up to a high proportion of 58.0% (47/81). A total of 8 point mutations were identified, including 2 novel mutations discovered in parE (Ser458Leu and Gly408Asp). The common mutation Ser83Leu in gyrA was still the most prevalent here with a percentage of 70.4% (57/81), followed by the approximate mutation of 69.1% (56/81) in parC (Ser80Ile) and His211Tyr in gyrA. Meanwhile, 35.8% (29/81) isolates were confirmed with mutation of Gln517Arg in gyrB. In addition, qnrS positive isolates occupied a proportion of 7.4% (6/81), but only 1 strain was observed with aac(6')-Ib-cr. All PMQR positive isolates were resistant to nalidixic acid. However, 5 of them didn't stay susceptible to ciprofloxacin any more. CONCLUSIONS: This is the first time that a study researches the occurrence of mutations in parE among S. flexneri, Ser458Leu and Gly408Asp included. The study indicates that the high resistance to fluoroquinolone remains a serious problem in Jiangsu, China. Thus, the prevention and control of current infection urge for a comprehensive and systematic surveillance based on persistent surveys.

Prevalence and characterisation of third-generation cephalosporin-resistant Shigella flexneri isolates from Jiangsu Province, China, 2013-2015.

OBJECTIVES: The aim of this study was to assess the prevalence of Shigella flexneri resistance to third-generation cephalosporins (3GCs) and to characterise the underlying resistance mechanisms. METHODS: A total of 282 S. flexneri strains isolated in 2013-2015 in Jiangsu Province, China, were identified, serotyped and analysed for their susceptibility to 3GCs. The blaTEM, blaSHV, blaOXA-1-like and blaCTX-M-type extended-spectrum ß-lactamase (ESBL) genes were amplified and sequenced by PCR. RESULTS: Of the 282 S. flexneri strains, 97 (34.4%) were resistant to cefotaxime, from which 68 (24.1%) were also resistant to ceftazidime. ESBL genes were detected in 73/97 isolates (75.3%), of which 66/73 (90.4%) showed resistance to 3GCs. Of the 73 ESBL-positive isolates, 32 (43.8%) were positive for CTX-M-1 group (17 for CTX-M-55, 4 for CTX-M-3, 1 for CTX-M-15, 3 for CTX-M-79 and 7 for CTX-M-123), 31 (42.5%) were positive for CTX-M-9 group (29 for CTX-M-14, 1 for CTX-M-24 and 1 for CTX-M-27), 25 (34.2%) were positive for TEM-types (21 for TEM-1 and 4 for TEM-1b) and 1 (1.4%) was positive for SHV-type (SHV-12); none were positive for CTX-M-2 group, CTX-M-8 group and OXA-type. CONCLUSION: ESBLs play an important role in Shigella resistance to 3GCs. CTX-M-14 and CTX-M-55 appeared to be the dominant ESBLs in 13 cities of Jiangsu Province. Therefore, it is time to regularly monitor resistance of S. flexneri to 3GCs and to take appropriate measures to manage this problem.

Virulence-associated genes and molecular characteristics of non-O1/non-O139 Vibrio cholerae isolated from hepatitis B cirrhosis patients in China.

OBJECTIVES: We aimed to report virulence-associated genes and molecular characteristics of non-O1/non-O139 Vibrio cholerae isolated from hepatitis B cirrhosis patients in China. METHODS: Patient clinical data including course of disease, laboratory tests, antibiotic treatment and outcomes were collected. Antimicrobial susceptibility testing was performed and virulence-associated genes were detected by PCR. Genetic relatedness among non-O1/non-O139 V. cholerae strains was investigated by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS: All three strains in this study harbored pathogenicity related genes like rtxA, rtxC, toxR, hapA, hlyA and ompW whereas they lacked ctxA, ctxB, tcpA, ompU and zot genes. None of them showed resistance to any antibiotic detected. A new allele of gyrB was submitted to the MLST database and designated as 97. Two novel sequence types (ST518 and ST519) and ST271 were identified by multilocus sequence typing (MLST). PFGE indicated considerable diversity among three non-O1/non-O139 V. cholerae strains. CONCLUSIONS: Three sporadic cases highlight that non-O1/non-O139 V. cholerae can cause opportunistic invasiveness infection in cirrhosis patients. Pathogenicity may be related to virulence-associated genes. Timely detection and antibiotic therapy should be paid more attention to in clinic.

Label-free identification carbapenem-resistant Escherichia coli based on surface-enhanced resonance Raman scattering.

In this study, a surface-enhanced resonance Raman scattering (SERRS) method has been developed for the accurate detection and identification of carbapenem-resistant and carbapenem-sensitive Escherichia coli. A total of 89 human isolates of Enterobacteriaceae, comprising 41 strains of carbapenem-sensitive E. coli (CSEC) and 48 strains of carbapenem-resistant E. coli (CREC), were tested to assess the feasibility of our proposed SERRS method as a clinical tool, and the results showed almost 100% accuracy.

A 10-year surveillance of antimicrobial susceptibility patterns in Shigella sonnei isolates circulating in Jiangsu Province, China.

OBJECTIVES: The rapid emergence of drug-resistant Shigella sonnei is a serious public health problem. This study aimed to characterise the antimicrobial resistance patterns, molecular subtypes, and integron types and resistance gene cassettes in S. sonnei from Jiangsu Province, China. METHODS: In total, 340 S. sonnei were collected in 2002-2011 throughout Jiangsu Province. Antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), PCR amplification of integrons, restriction fragment length polymorphism (RFLP) and DNA sequencing of cassette regions were performed. RESULTS: Resistance rates to ampicillin (67.7%), nalidixic acid (75.2%), tetracycline (73.7%) and trimethoprim/sulfamethoxazole (68.7%) remained high. Strains from Centre and South Jiangsu showed higher resistance and multiresistance rates compared with the North. PFGE analysis indicated that large-scale clonal transmission among different cities occurred several times during 10 years. Among all strains, 55.9% (190/340) harboured class 1 integrons, 80.3% (273/340) harboured class 2 integrons and 49.4% (168/340) harboured an atypical class 1 integron. Resistance rates to nine antimicrobials in the class 1 integron-positive group were significantly higher than in the negative group (P<0.05). Seven different gene cassettes were detected in class 1 integrons. The most prevalent type was aacA4-cmlA1 (114/286). Class 2 integrons carried the gene cassette array dfrA1-sat1-aadA1, and the atypical class 1 integron carried blaOXA-30-aadA1. CONCLUSIONS: The increasing antimicrobial resistance and significant clonal transmission of S. sonnei circulating in Jiangsu were closely related to the high prevalence of integrons and gene cassettes. Long-term cross-regional monitoring of antimicrobial resistance is urgently required for S. sonnei.