The emergence of CRE strains resistant to multiple antibiotics is considered a substantial threat to human health. Therefore, all the efforts to provide a detailed molecular transmission mechanism of specific drug resistance can contribute positively to prevent the further spread of multidrug-resistant bacteria. Although the new superbug harboring bla _NDM-5 has been reported in many countries, it was mostly identified among E. coli strains, and the gene transfer mechanism has not been fully recognized and studied. In this work, we identified 22 bla _NDM-5 -positive strains in different species of Enterobacteriaceae , including 16 Klebsiella pneumoniae strains, four Klebsiella aerogenes strains, and two Escherichia coli strains, which indicated the horizontal gene transfer of bla _NDM-5 among Enterobacteriaceae strains in pediatric patients. Moreover, bla _NDM-5 was located on a 46-kb IncX3 plasmid, which is possibly responsible for this widespread horizontal gene transfer. The different genetic contexts of the bla _NDM-5 gene indicated some minor evolutions of the plasmid, based on the complete sequences of the bla _NDM-5 plasmids. These findings are of great significance to understand the transmission mechanism of drug resistance genes, develop anti-infection treatment, and take effective infection control measures.

We investigated the epidemiological characteristics of drug resistance and virulence factors of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates from paediatric patients in Shanghai. CRKP strains were consecutively collected between January and December in 2018. Antimicrobial susceptibility was determined by VITEK 2 compact. Polymerase chain reaction (PCR) was used to analyse drug resistance determinants, virulence genes and plasmid types. wzi sequencing and multilocus sequence typing was used to determine clonal relatedness. Among 172 CRKP strains, blaKPC-2 and blaNDM-5 were the predominant carbapenemase genes. Compared with NDM-5, KPC-2 producers showed higher resistance rates to fluoroquinolones and aminoglycosides. The majority of KPC-2 producers belonged to KL64-ST11 background, while NDM-5 producers were mainly identified as KL62-ST48. Plasmid typing shown that IncF and IncFIB were the most prevalent plasmids in KPC-2 producers and IncX3 was widely spread in NDM-5-KP. Thirty-seven isolates carried various hypervirulence genes and the profiles of these genes showed high diversity. The predominant carbapenemase of CRKP strains from paediatric patients in Shanghai were KPC-2 and NDM-5. KL47-ST11 KPC-2-KP and KL62-ST48 NDM-5-KP were representative clonal lineages. Although not prevalent, hypervirulence associated genes have begun to spread. Active long-term surveillance should be performed in both drug resistance characteristics and virulence factors.

Background: Streptococcus pneumoniae is the most common pathogen causing death in children under 5 years old. This retrospective surveillance aimed to analyze serotype distribution, drug resistance, virulence factors, and molecular characteristics of pneumonia isolates from children in Shanghai, China. Methods: A total of 287 clinical pneumococcal isolates were collected from January to December in 2018 and were divided into community-acquired pneumonia (CAP) and healthcare-associated pneumonia (HAP) two groups according to where someone contracts the infection. All isolates were serotyped by multiplex sequential PCR and antimicrobial susceptibility testing was performed using E-test or disk diffusion method. The molecular epidemiology was analyzed using multilocus sequence typing and seven housekeeping genes were sequenced to identified the sequence types (STs). In addition, we investigated the presence of virulence genes via PCR. Results: The most common serotypes were 19F, 6A, 19A, 23F, 14, and 6B, and the coverage rates of the 7-, 10- and 13-valent pneumococcal conjugate vaccines were 58.9, 58.9, and 80.5%, respectively. More PCV13/non-PCV7 serotypes and higher rate of penicillin non-susceptible S. pneumoniae were seen in HAP. Molecular epidemiological typing showed a high level of diversity and five international antibiotic-resistant clones were found, including Taiwan^19F-14, Spain^23F-1, Spain^6B-2, Taiwan^23F-15 and Sweden^15A-25. No significant difference was observed in the presence of virulence genes among the isolates obtained from CAP and HAP. All of the S. pneumoniae isolates carried lytA, ply, psaA, pavA, spxB, htrA, and clpP, and the carriage rate of nanA and piaA were 96.2 and 99.0%. Conversely, cps2A, cbpA, and pspA were present in 33.8–44.3% of the isolates. Conclusions: Serotype changes and emerging multidrug-resistant international clones were found in current study. lytA, ply, psaA, pavA, spxB, htrA, and clpP may be good protein vaccine candidates. Long-term high-quality surveillance should be conducted to assess impact and effectiveness brought by vaccines, and provide a foundation for prevention strategies and vaccine policies.

Resistance phenotype and clinical molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae among pediatric patients in Shanghai Dongxing Tian, Fen Pan, Chun Wang, Yan Sun, Hong Zhang Department of Clinical Laboratory, Shanghai Children’s Hospital, Shanghai Jiaotong University, Shanghai, China Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) has caused wide global disseminations and serious clinical outcomes in pediatric patients, and the purpose of this study was to analyze drug resistance, molecular epidemiology, and clinical characteristics of CRKP from children in Shanghai, China.Methods: A retrospective study was conducted from January 2016 to December 2017, and a total of 170 CRKP isolates were collected. Antimicrobial susceptibility was determined by the broth microdilution method. MAST D73C and polymerase chain reaction were used for the analysis of carbapenemase types. Multilocus sequence typing of K. pneumoniae was performed for genetic relationship. Clinical data were also reviewed.Results: Of the 170 CRKP isolates, blaOXA-232 was mainly detected with a proportion of 42.35%, followed by blaNDM-1 (20.59%), blaKPC-2 (17.65%), blaNDM-5 (16.47%), and blaIMP-4 (1.18%). The predominant gene was blaOXA-232 in 2016 (54.46%; 55/101) and blaNDM-1 in 2017 (31.88%; 22/69). All these 170 CRKP isolates showed high resistance to cephalosporins and carbapenems (>95%), except for tigecycline and colistin. Sixteen distinct sequence types were observed with ST15 being mostly identified (41.76%). Most CRKP harboring OXA-232 type carbapenemase belonged to ST15, while NDM-1 type belonged to ST37 and KPC-2 type belonged to ST11. Furthermore, other β-lactamase genes including blaTEM, blaCTX-M, and DHA-1 were also found in this study. Clinical data reviewed that more than half of the patients produced clinical infections (112/170), mainly lower respiratory tract (58/112) and bloodstream (21/112) infections. A majority of these children had received therapy of antibiotics before CRKP isolation, especially for carbapenems (76/170) and β-lactam/β-lactamase inhibitor combinations (91/170).Conclusions: Our data revealed the increasing incidence of OXA-232-producing K. pneumoniae from pediatric patients in Shanghai, and infection control measures should be conducted to limit the spread of CRKP strains. Keywords: Klebsiella pneumoniae, carbapenemases, drug resistance, OXA-232, NDM-5, children

The recurrence of bacterial infectious diseases is closely associated with bacterial persisters. This subpopulation of bacteria can escape antibiotic treatment by entering a metabolic status of low activity through various mechanisms, for example, biofilm, toxin–antitoxin modules, the stringent response, and the SOS response. Correspondingly, multiple new treatments are being developed. However, due to their spontaneous low abundance in populations and the lack of research on in vivo interactions between persisters and the host’s immune system, microfluidics, high-throughput sequencing, and microscopy techniques are combined innovatively to explore the mechanisms of persister formation and maintenance at the single-cell level. Here, we outline the main mechanisms of persister formation, and describe the cutting-edge technology for further research. Despite the significant progress regarding study techniques, some challenges remain to be tackled.

Fecal colonization with carbapenem-resistant Enterobacteriaceae (CRE) is a risk factor for bacterial translocation resulting in subsequent endogenous infections. The purpose of this study is to investigate the prevalence of CRE strains colonization in stool samples of outpatient in a tertiary pediatric hospital of Shanghai, China. In a retrospective study, fecal samples were consecutively obtained from patients in 2016 and screening test for CRE was conducted by using home-made MacConkey agar. Antimicrobial susceptibility was determined by the broth microdilution method and β-lactamases were characterized by polymerase chain reaction (PCR) assays and DNA sequencing. Multilocus sequence typing (MLST) was performed for the genetic relationships of the isolates. A total of 880 fecal samples were included for this screening test and 32 CRE strains were identified in 32 non-duplicate fecal samples from 32 children (1.3 ± 1.5 years), with a carriage rate of 3.6%. These strains mainly distributed in Klebsiella pnuemoniae (37.5%) and Escherichia coli (37.5%). All CRE strains showed high resistance to most of the routinely used antibiotics (> 90%) except for polymyxin B and tigecycline. The blaNDM gene was the major carbapenemase gene harbored by gastrointestinal CRE strains, followed by blaKPC-2, blaIMP-26, and blaIMP-4. Other β-Lactamase genes including blaCTX-M, blaSHV, blaTEM-1, and blaDHA-1 were also detected. MLST analysis revealed that various sequence types (STs) were detected in these strains, with ST11 and ST37 being more prevalent in K.pneumoniae and ST101 in E.coli. This study revealed the prevalence of CRE fecal carriage in children from outpatient and urgent implementation of infection control measure should be conducted to limit the spread of CRE strains.