Haiyan Yang

The AcrAB-TolC Pump Is Involved in Multidrug Resistance in Clinical Shigella flexneri Isolates

The two multiple-antibiotic resistance (Mar) phenotypes (n = 8, respectively) and susceptible isolates (n = 4) of Shigella flexneri from China were characterized involving the efflux pump AcrAB-TolC. The accumulation of ciprofloxacin, acrAB-tolC PCR, and levels of mRNA with northern blots were performed in three groups. Sequencing of acrAB-tolC was performed in selected isolates. An efflux inhibition was performed with Phe-Arg-beta-naphthylamide. The accumulation of ciprofloxacin at steady state in susceptible isolates was significantly higher than that in the two Mar groups (p < 0.05). The level of accumulation in the Mar strains was increased upon the addition of the protonophore carbonyl cyanidem-chlorophenylhydrazone. The expression level of acrA mRNA in the Mar isolates was significantly higher than that in the susceptible isolates (p < 0.05). Mar strain H26 had a single-nucleotide substitution in locus 322(G-->T) of acrA, and Mar 0008 in locus 171(C-->A). The susceptible strain N15 had a base deletion in locus 36 (C) of tolC gene. The role of the inhibitors of efflux pumps was significant in some isolates with the high expression of Mar pump genes. In conclusion, overexpression of acrA gene leads to Mar in clinical isolates of S. flexneri, and ciprofloxacin acquired susceptible to S. flexneri with Phe-Arg-beta-naphthylamide.

Multi-drug Resistance and Characteristic of Integrons in Shigella spp.Isolated from China

OBJECTIVE To investigate the characteristic of integrons and the relationship between integrons and antimicrobial resistance in Shigella spp. METHODS Ninety Shigella strains (83 S. flexneri and 7 S. sonnei) were isolated from the stools of patients in China. Susceptibility to 8 antimicrobials was tested for all isolated strains. PCR, RFLP and sequencing analysis of integrons were applied to all of them. RESULTS High prevalence of multi-drug resistance (95.6%) was identified. Of the isolates 79 (87.8%) carried integrase genes of class 1 integron (3.3%), class 2 integron (10.0%) or both (74.4%). No intI3 was detected in the tested isolates. The prevalence of intI2 was significantly higher in isolates with multi-drug resistance to at least 3 antibiotics than that in isolates with resistance to 2 and less antibiotics (P<0.05). Gene cassettes dfrA17-aadA5, dfrA12-orfF-aadA2 of class 1 integron and dfrA1-sat1-aadA1 of class 2 integron were identified. CONCLUSION The class 2 integron may play a role in the emergence of multi-drug resistance in Shigella spp.

Serum inflammatory cytokine levels correlate with Hand-Foot-Mouth disease severity: A nested serial case-control study

Background Hand-food-mouth disease (HFMD) cases can be fatal. These cases develop rapidly, and it is important to predict the severity of HFMD from mild to fatal and to identify risk factors for mild HFMD. The objective of this study was to correlate the levels of serum inflammatory cytokines with HFMD severity. Methods This study was designed as a nested serial case-control study. The data collected included general information, clinical symptoms and signs, laboratory findings and serum cytokine levels. Results The levels of IL-4, IL-6, IL-10, TNF-α and IFN-γ in patients with severe HFMD were significantly higher than in mild patients during the 2nd to 5th day after disease onset. The levels of IL-4, IL-6, IL-10 and IFN-γ increased from the 2nd day to the 4th day and later decreased. The levels of TNF-α were high on the first two days and subsequently decreased. The changes of IL-10, TNF-α and IFN-γ in the controls were similar for all cases. The levels of IL-4, IL-6 and IL-17 in the controls were not significantly different with the progression of HFMD. Conclusions Our findings indicate that the IL-4, IL-6, IL-10, TNF-α and IFN-γ levels correlate with HFMD severity.

Transposition of ISEcp1 modulates blaCTX-M-55-mediated Shigella flexneri resistance to cefalothin

The aim of this study was to uncover the mechanisms underlying Shigella flexneri resistance to cefalothin. In this study, a resistance-related S. flexneri isolate, S. flexneri YDC, was obtained from S. flexneri mel-1998023/zz pre-incubated with cefalothin at a dose of 0.5 × the minimum inhibitory concentration. The ISEcp1 coding element was identified upstream of bla(CTX-M-55) in S. flexneri YDC. To further determine the role of ISEcp1 in S. flexneri resistance, plasmids containing bla(CTX-M-55) recombinant with or without the ISEcp1 sequence were constructed and named as pCTX and pISECTX, respectively. It was shown that Escherichia coli DH5α(pISECTX) was resistant to all β-lactams tested. In contrast, E. coli DH5α(pCTX) was sensitive to all except β-lactams cefazolin and cefalothin. In addition, reverse transcription PCR showed that expression levels of bla(CTX-M-55) were higher in E. coli DH5α(pISECTX). The Clinical and Laboratory Standards Institute (CLSI) assay demonstrated that extended-spectrum β-lactamase was only positively detected in E. coli DH5α(pISECTX) but not in E. coli DH5α(pCTX). Taken together, these results suggest that the translocated ISEcp1 element upstream of bla(CTX-M-55) is required for overexpression of bla(CTX-M-55), leading to cephalosporin resistance.

Serotype distribution and characteristics of antimicrobial resistance in Shigella isolated from Henan province, China, 2001–2008

The serotype distribution and susceptibility to 14 antimicrobial agents of 526 isolates of Shigella spp. from four hospitals in Sun county, Henan province, China during 2001–2008, were analysed to identify associations of serotypes with resistance trends. S. flexneri was the most frequent species (92·4%), the remainder was S. sonnei. The prevalent serotype of S. flexneri was 2a (26·7%). Almost all (>99%) isolates were resistant to tetracycline, nalidixic acid and pipemidic acid; >80% were resistant to chloramphenicol, amoxicillin and co-trimoxazole but less than 5% were resistant to polymyxin B, furazolidone, cefotaxime and gentamicin. S. flexneri showed statistically significant higher resistance than S. sonnei to amoxicillin, ampicillin, chloramphenicol and ciprofloxacin but resistance to co-trimoxazole was more common in S. sonnei than in S. flexneri. These results emphasize that monitoring of emerging resistance in Shigella isolates is essential for timely and appropriate recommendations for antimicrobial therapy.

Detection and Analysis of CRISPRs of Shigella

The recently discovered CRISPRs (Clustered regularly interspaced short palindromic repeats) and Cas (CRISPR-associated) proteins are a novel genetic barrier that limits horizontal gene transfer in prokaryotes and the CRISPR loci provide a historical view of the exposure of prokaryotes to a variety of foreign genetic elements. The aim of study was to investigate the occurrence and distribution of the CRISPRs in Shigella. A collection of 61 strains of Shigella were screened for the existence of CRISPRs. Three CRISPR loci were identified among 61 shigella strains. CRISPR1/cas loci are detected in 49 strains of shigella. Yet, IS elements were detected in cas gene in some strains. In the remaining 12 Shigella flexneri strains, the CRISPR1/cas locus is deleted and only a cas3’ pseudo gene and a repeat sequence are present. The presence of CRISPR2 is frequently accompanied by the emergence of CRISPR1. CRISPR3 loci were present in almost all strains (52/61). The length of CRISPR arrays varied from 1 to 9 spacers. Sequence analysis of the CRISPR arrays revealed that few spacers had matches in the GenBank databases. However, one spacer in CRISPR3 loci matches the cognate cas3 genes and no cas gene was present around CRISPR3 region. Analysis of CRISPR sequences show that CRISPR have little change which makes CRISPR poor genotyping markers. The present study is the first attempt to determine and analyze CRISPRs of shigella isolated from clinical patients.

Pulmonary edema following central nervous system lesions induced by a non- mouse-adapted EV71 strain in neonatal BALB/c mice

BackgroundEnterovirus (EV) infection has been a serious health issue in Asia-Pacific region. It has been indicated that the occurrence of fatal hand foot and mouth disease (HFMD) cases following EV71 infection is mainly attributed to pulmonary edema. However, the development of pulmonary disorders after EV71 infection remains largely unknown. To establish an EV71-infected animal model and further explore the underlying association of central nervous system (CNS) invasion with pulmonary edema, we isolated a clinical source EV71 strain (ZZ1350) from a severe case in Henan Province.MethodsWe evaluated the cytotoxicity of ZZ1350 strain and the susceptibility in 3-day-old BALB/c mice with intraperitoneal, intracerebral and intramuscular inoculation. Various histopathological and immunohistochemical techniques were applied to determine the target organs or tissue damage after infection. Correlation analysis was used to identify the relationship between CNS injury and pulmonary disorders.ResultsOur experimental results suggested that ZZ1350 (C4 subtype) had high cytotoxicity against African green monkey kidney (Vero) cells and human rhabdomyosarcoma (RD) cells and neonatal BALB/c mice were highly susceptible to the infection with ZZ1350 through three different inoculation routes (2 × 106 pfu/mouse) exhibiting severe neurological and respiratory symptoms that were similar to clinical observation. Viral replication was found in brain, spinal cord, skeletal muscle, lung, spleen, liver, heart of infected mice and these sections also showed histopathological changes. We found that brain histology score was positive correlated with lung histology score in total experimental mice and mice under the three inoculation routes (P < 0.05). At the same time, there were positive correlations between spinal cord score and lung score in total experimental mice and mice with intracerebral inoculation (P < 0.05).ConclusionsZZ1350 strain is effective to establish animal model of EV71 infection with severe neurological and respiratory symptoms. The development of pulmonary disorders after EV71 infection is associated with severity of CNS damage.

Bioinformatics analyses of Shigella CRISPR structure and spacer classification

Clustered regularly interspaced short palindromic repeats (CRISPR) are inheritable genetic elements of a variety of archaea and bacteria and indicative of the bacterial ecological adaptation, conferring acquired immunity against invading foreign nucleic acids. Shigella is an important pathogen for anthroponosis. This study aimed to analyze the features of Shigella CRISPR structure and classify the spacers through bioinformatics approach. Among 107 Shigella, 434 CRISPR structure loci were identified with two to seven loci in different strains. CRISPR-Q1, CRISPR-Q4 and CRISPR-Q5 were widely distributed in Shigella strains. Comparison of the first and last repeats of CRISPR1, CRISPR2 and CRISPR3 revealed several base variants and different stem-loop structures. A total of 259 cas genes were found among these 107 Shigella strains. The cas gene deletions were discovered in 88 strains. However, there is one strain that does not contain cas gene. Intact clusters of cas genes were found in 19 strains. From comprehensive analysis of sequence signature and BLAST and CRISPRTarget score, the 708 spacers were classified into three subtypes: Type I, Type II and Type III. Of them, Type I spacer referred to those linked with one gene segment, Type II spacer linked with two or more different gene segments, and Type III spacer undefined. This study examined the diversity of CRISPR/cas system in Shigella strains, demonstrated the main features of CRISPR structure and spacer classification, which provided critical information for elucidation of the mechanisms of spacer formation and exploration of the role the spacers play in the function of the CRISPR/cas system.

Antimicrobial susceptibility and mechanism of resistance in Shigella isolates from rural China

This study was set up to assess the patterns of antimicrobial susceptibility and mutations in acrA-tolC genes of Shigella isolates and its association. One hundred and three isolates of Shigella spp. were tested to evaluate the antimicrobial susceptibility and mutation on acrA and tolC genes. The antimicrobial resistance profiles were: tetracycline (99%), ampicillin (85.4%), chloramphenicol (83.5%), trimethoprim (85.4%), ciprofloxacin (25.2%), cefazolin (3.9%), cefotaxime (3.9%), and gentamicin (2.9%). The rate of mutation in tolC was 90% (S. flexneri) and 30.8% (S. sonnei). Shigella flexneri isolates were more resistant than those of Shigella sonnei to chloramphenicol (97.8 versus 0.0%, p < 0.001) and ciprofloxacin (27.8 versus 7.7%, p = 0.106). High frequency of mutation was found in gene tolC (82.5%), but relatively less in acrA (22.3%). Shigella flexneri isolates were more mutated in tolC gene than S. sonnei (90 versus 30.8%, p < 0.001). Our study suggested that mutation of acrA and tolC may play major role in multiple antimicrobial resistance in Shigella spp. Shigella isolates are emerging which are resistant to first and third generation cephalosporin like cefazolin and cefotaxime, which is a matter of concern in terms of shigellosis treatment.

Management of ST-segment elevation myocardial infarction in predominantly rural central China: A retrospective observational study

Abstract The degree of adherence to current guidelines for clinical management of ST-segment elevation myocardial infarction (STEMI) is known in developed countries and large Chinese cities, but in predominantly rural areas information is lacking. We assessed the application of early reperfusion therapy for STEMI in secondary and tertiary hospitals in Henan province in central China. Data were retrospectively collected from 5 secondary and 4 tertiary hospitals in Henan concerning STEMI patients treated from January 2011 to January 2012, including management strategy, delay time, and inhospital mortality. Among 1311 STEMI patients, 613 and 698 were treated at secondary and tertiary hospitals, respectively. Overall, 460 (35.1%) patients received early reperfusion therapy including thrombolysis in 383 patients and primary percutaneous coronary intervention in 77. Compared with secondary centers, early (37.2% vs 32.6%) and successful reperfusion (34.5% vs 25.1%) was significantly higher, whereas thrombolysis was lower in the tertiary hospitals (26.4% vs 32.5%). Median symptom onset-to-first medical contact, and door-to-needle and door-to-balloon time was 168, 18, and 60 minutes, respectively. Delay times closely approached recommended guidelines, especially in secondary centers. Use of recommended pharmacotherapy was low, particularly in secondary hospitals. Inhospital mortality was 5.8%, and similar between secondary and tertiary hospitals (6.0% vs 5.6%; P = 0.183). Two-thirds of STEMI patients did not receive early reperfusion, and tertiary hospitals mostly failed to take advantage of around-the-clock primary percutaneous coronary intervention. Actions such as referrals are warranted to shorten prehospital delay, and the concerns of patients and doctors regarding reperfusion risk should be addressed.