Correlation between the sulfamethoxazole-trimethoprim resistance of Shigella flexneri and the sul genes

The aim of this study was to discuss the correlation between the sulfamethoxazole-trimethoprim resistance of Shigella flexneri (S. flexneri) and the antibiotic resistance genes sul1, sul2, and sul3 and SXT element.From May 2013 to October 2018, 102 isolates of S. flexneri were collected from the clinical samples in Jinan. The Kirby–Bauer (K-B) test was employed to determine the antibiotic susceptibility of the S. flexneri isolates. The antibiotic resistance rate was analyzed with the WHONET5.4 software. The isolates were subject to the PCR amplification of the sul genes (sul1, sul2, and sul3) and the SXT element. On the basis of the sequencing results, the correlation between the sulfamethoxazole-trimethoprim resistance of the S. flexneri isolates and the sul genes was analyzed.The antibiotic resistance rates of the 102 S. flexneri isolates to ampicillin, streptomycin, chloramphenicol, tetracycline, and sulfamethoxazole-trimethoprim were 90.2%, 90.2%, 88.2%, 88.2%, and 62.7%, respectively. The antibiotic resistance rates of these isolates to cefotaxime, ceftazidime, and ciprofloxacin varied between 20% and 35%. However, these isolates were 100% susceptible to cefoxitin. Positive fragments were amplified from 59.8% (61/102) of the 102 S. flexneri isolates, the sizes of the sul1 and sul2 genes being 338 bp and 286 bp, respectively. The sequence alignment revealed the presence of the sul1 and sul2 genes encoding for dihydrofolate synthase. The carrying rate of the sul1 gene was 13.7% (14/102), and that of the sul2 gene was 48.0% (49/102). No target gene fragments were amplified from the 3 isolates resistant to sulfamethoxazole-trimethoprim. The sul3 gene and SXT element were not amplified from any of the isolates. The testing and statistical analysis showed that the resistance of the S. flexneri isolates to sulfamethoxazole-trimethoprim correlated to the sul1 and sul2 genes.The acquired antibiotic resistance genes sul1 and sul2 were closely associated with the resistance of the 102 S. flexneri isolates to sulfamethoxazole-trimethoprim.     

Bioimage analysis of Shigella infection reveals targeting of colonic crypts

Few studies within the pathogenic field have used advanced imaging and analytical tools to quantitatively measure pathogenicity in vivo. In this work, we present a novel approach for the investigation of host–pathogen processes based on medium-throughput 3D fluorescence imaging. The guinea pig model for Shigella flexneri invasion of the colonic mucosa was used to monitor the infectious process over time with GFP-expressing S. flexneri. A precise quantitative imaging protocol was devised to follow individual S. flexneri in a large tissue volume. An extensive dataset of confocal images was obtained and processed to extract specific quantitative information regarding the progression of S. flexneri infection in an unbiased and exhaustive manner. Specific parameters included the analysis of S. flexneri positions relative to the epithelial surface, S. flexneri density within the tissue, and volume of tissue destruction. In particular, at early time points, there was a clear association of S. flexneri with crypts, key morphological features of the colonic mucosa. Numerical simulations based on random bacterial entry confirmed the bias of experimentally measured S. flexneri for early crypt targeting. The application of a correlative light and electron microscopy technique adapted for thick tissue samples further confirmed the location of S. flexneri within colonocytes at the mouth of crypts. This quantitative imaging approach is a novel means to examine host–pathogen systems in a tailored and robust manner, inclusive of the infectious agent.The light microscope is an important tool in resolving the interactions between microbes and their hosts. With the advancement of both optical and computational techniques, quantification of biological events is not only possible, but essential in deciphering the complex interplays between host and pathogen. Acquired images are multidimensional datasets that capture complex biological phenomena, and a major task is to computationally extract statistically relevant data from those images. Such techniques have been readily applied in Cellular Microbiology, a discipline at the interface between cellular biology and microbiology (1). This field is dominated by studies focusing on microbe–host interactions at the in vitro, cellular scale, and a multitude of bioimage analysis tools have been developed and used to decipher pathogenic strategies at the cellular, as well as the molecular, level in high resolution in both space and time (2).The next imaging challenge exists on a much larger scale in the growing field of Tissue Microbiology, which places the pathogen in its native in vivo environment, the host (3). Biologically speaking, this environment is essential for the full understanding of a given pathogen’s invasive strategies and the complex host response; however, this also places additional complexity and limitations in regards to imaging, particularly in maintaining cellular and molecular resolution. Novel tools and studies addressing bacterial infection in vivo are frequently reported in the literature (4, 5). However, emphasis has been traditionally placed on qualitative observations at the expense of extensive quantitative efforts. The importance of bioimage analyses to automatically extract data from medium- to large-scale image sampling of infected tissue is often underappreciated as a method to expand our understanding of pathogens’ strategies and disease progression. In this work, we aim to bring quantitative image analysis into the realm of host–pathogen interactions, specifically examining the progression of tissue invasion by a model enteropathogenic bacterium, Shigella flexneri.S. flexneri is the causative agent of bacillary dysentery, an infectious rectocolitis, which remains a major pediatric public health concern in developing countries. This human-specific pathogen is transmitted via the fecal–oral route, and namely targets the large intestine, resulting in acute inflammation, tissue edema, and erosion of the colonic epithelium (6). The infection strategy of S. flexneri relies on (i) the transfer of bacterial proteins, termed “effectors,” into targeted host cells through the type III secretion apparatus (T3SA), which induces the uptake of the bacteria and perturbs host cellular processes, and (ii) the capacity of the intracellular bacteria to spread from cell to cell using actin microfilament-mediated cytoplasmic movement and reactivation of the T3SA (7–10).Herein, we present a straightforward approach that relies on the combination of light microscopy and computer analyses to study the mucosal invasion of a newly developed in vivo model for shigellosis, S. flexneri intrarectal inoculation of the guinea pig colon (11). Through the application of simple, open-source image analysis tools, we built up a medium-throughput analysis that allows observing and robustly measuring host–pathogen interactions. Most importantly, this approach can be adapted to other host–pathogen systems, thereby providing generic tools that bridge Cellular and Tissue Microbiology.     

Microbiota That Affect Risk for Shigellosis in Children in Low-Income Countries

Pathogens in the gastrointestinal tract exist within a vast population of microbes. We examined associations between pathogens and composition of gut microbiota as they relate to Shigella spp./enteroinvasive Escherichia coli infection. We analyzed 3,035 stool specimens (1,735 nondiarrheal and 1,300 moderate-to-severe diarrheal) from the Global Enteric Multicenter Study for 9 enteropathogens. Diarrheal specimens had a higher number of enteropathogens (diarrheal mean 1.4, nondiarrheal mean 0.95; p<0.0001). Rotavirus showed a negative association with Shigella spp. in cases of diarrhea (odds ratio 0.31, 95% CI 0.17–0.55) and had a large combined effect on moderate-to-severe diarrhea (odds ratio 29, 95% CI 3.8–220). In 4 Lactobacillus taxa identified by 16S rRNA gene sequencing, the association between pathogen and disease was decreased, which is consistent with the possibility that Lactobacillus spp. are protective against Shigella spp.–induced diarrhea. Bacterial diversity of gut microbiota was associated with diarrhea status, not high levels of the Shigella spp. ipaH gene.     

2011—2013年北京某城区感染性腹泻患者致病菌检测分析

目的分析北京某城区2011—2013年感染性腹泻患者致病菌分布及脉冲场凝胶电泳(PFGE)图谱特征,为感染溯源提供依据。方法收集2011年1月—2013年12月该区域肠道门诊感染性腹泻患者粪便标本1 179份,对其培养分离的致病菌进行血清分型及PFGE分析。结果 1 179份标本,共培养肠道病原菌330株,其中检出居前4位的肠道致病菌依次为志贺菌属93株(28.18%)、沙门菌属69株(20.91%)、副溶血性弧菌44株(13.33%)及致泻性大肠埃希菌11株(3.33%)。18株宋内志贺菌PFGE分为8个型别,聚类相似度接近88%;69株沙门菌属细菌分为18个血清型,共41种PFGE型别,其中山夫登堡沙门菌和肠炎沙门菌具有明显的优势带型;23株副溶血性弧菌PFGE型别较为分散。结论 3年间感染性腹泻患者致病菌的血清型和PFGE带型分布较为广泛,应注意沙门菌属和志贺菌属细菌的PFGE优势菌型,警惕其引发的感染性腹泻暴发流行。     

Distribution and antibiotic susceptibility of Shigella isolates in Bangui, Central African Republic

We conducted a prospective study in four urban health care centres between January 2004 and November 2005 to determine the distribution and susceptibility patterns of Shigella strains causing invasive diarrhoea in Bangui, Central African Republic. Of the 155 Shigella isolated, Shigella flexneri (51%) and Shigella dysenteriae (30%) were the most common and the most resistant to usual antibiotics, including amoxicillin, sulphamethoxazole-trimethoprim and chloramphenicol. Though multi-drug resistance was common, no strains were resistant to quinolone and fluoroquinolones. We conclude that short-course treatments with ciprofloxacin can be recommended in Bangui for treatment of invasive diarrhoea.     

应用脉冲场凝胶电泳技术对浙江省福氏4c型痢疾志贺菌的分型研究

目的 通过应用脉冲场凝胶电泳技术对浙江省痢疾志贺菌福氏4c(F4c)型菌株进行分子流行病学研究,耐药谱分析,为了解本地区菌型的变化提供资料.方法 60株F4c型志贺菌均按GB16002-1995标准鉴定分型并采用kirby-Bauer法检测其对13种抗生素的敏感性.参照美国疾病预防控制中心PulseNet USA的统一方法进行脉冲场凝胶电泳(PFGE)分型并做同源性分析.结果 60株F4c型菌株都存在多重耐药,其中红霉素、利福平的耐药率最高,达100.0%,其次是奈定酸、氨苄西林、多西环素,耐药率均在96.2%以上;丁胺卡那、庆大霉素最敏感,其敏感率均在86.5%以上.脉冲场凝胶电泳分型60株试验菌株共分为24个型,1型36株占60.0%,且所有的暴发型菌株都为1型,2型2株占3.3%,其余22个型各为1株,各占1.7%.结论 F4c型志贺菌是2004年在浙江首次分离到,PFGE分型1型菌株占60.0%,有明显的优势,并且容易引起暴发,其余各型均为少见菌型.此次检测发现其多重耐药现象严重,这可能与浙江省F4c型菌痢病人迅速增多和高度散发有关,应引起临床和疾病控制部门的高度重视.     

105株志贺菌流行菌株的血清分型及耐药性分析

目的　调查志贺菌在上海地区流行的主要血清型和耐药性。方法　收集临床分离的志贺菌 1 0 5株进行生化鉴定、血清学分型和抗生素敏感性试验。结果　 1 0 5株志贺菌中 5 1株为宋氏志贺菌 ,占 4 8.6 % ;5 4株为福氏志贺菌 ,占 5 1 .4 % (包括 5株福氏变异株 ,占总数的 4 .7% )。宋氏志贺菌对抗菌药物的敏感率依次为氟喹诺酮类 1 0 0 %、头孢噻肟 94 .1 %。而福氏志贺菌对抗菌药物的敏感率依次为头孢噻肟 98.1 %、氟喹诺酮类70 %。结论　上海地区志贺菌感染以福氏志贺菌和宋氏志贺菌为主 ,福氏志贺菌中以福氏 2型为主 ,占 6 6 .7% ,其次为福氏 1型 ,占 2 0 .4 %。福氏志贺菌对各种抗生素耐药性有增高趋势 ,必须引起临床重视     

Outer membrane protein A (OmpA) from Shigella flexneri 2a: A promising subunit vaccine candidate

Shigellosis is the leading cause of childhood mortality and morbidity. Despite many years of extensive research a practical vaccine is not yet available against the disease. Recent studies illustrate that bacterial outer membrane proteins are budding target as vaccine antigen. Outer membrane proteins A (OmpA) are among the most immunodominant antigens in the outer membrane of gram negative bacteria and possess many characteristics desired of a vaccine candidate. We observe that OmpA of Shigella flexneri 2a is crossreactive and common antigen among Shigella spp. and the epitope is widely exposed on the cell surface as well as capable of evoking protective immunity in mice. The protective immunity involves participation of both the humoral and cellular immune responses, since OmpA boosts rapid induction of IgG and IgA in both the systemic and mucosal compartments and also activates Th1 cells. The immunopotentiating activity of OmpA is mediated by its ability to bind and stimulate macrophages and up-regulate the surface expression of MHCII, CD80 and CD40, leading to activation of CD4⁺ T cells to secrete cytokines and express chemokine receptor and IL-12Rβ2, thereby orchestrating the bridge between innate and adaptive immune responses. This ability is dependent on Toll-like receptor 2 (TLR2), as demonstrated by lack of response by TLR2 knockdown macrophages to OmpA. Hence this property of OmpA to link innate and adaptive immunity via TLR2 offers a novel vista to develop vaccine against shigellosis.     

Vaccines against gastroenteritis,current progress and challenges

ABSTRACT

Enteric viral and bacterial infections continue to be a leading cause of mortality and morbidity in young children in low-income and middle-income countries, the elderly, and immunocompromised individuals. Vaccines are considered an effective and practical preventive approach against the predominantly fecal-to-oral transmitted gastroenteritis particularly in the resource-limited countries or regions where implementation of sanitation systems and supply of safe drinking water are not quickly achievable. While vaccines are available for a few enteric pathogens including rotavirus and cholera, there are no vaccines licensed for many other enteric viral and bacterial pathogens. Challenges in enteric vaccine development include immunological heterogeneity among pathogen strains or isolates, a lack of animal challenge models to evaluate vaccine candidacy, undefined host immune correlates to protection, and a low protective efficacy among young children in endemic regions. In this article, we briefly updated the progress and challenges in vaccines and vaccine development for the leading enteric viral and bacterial pathogens including rotavirus, human calicivirus, Shigella, enterotoxigenic Escherichia coli (ETEC), cholera, nontyphoidal Salmonella, and Campylobacter, and introduced a novel epitope- and structure-based vaccinology platform known as MEFA (multiepitope fusion antigen) and the application of MEFA for developing broadly protective multivalent vaccines against heterogenous pathogens.     

志贺菌依赖解旋酶DNA恒温扩增技术建立

目的利用依赖解旋酶DNA恒温扩增技术(HDA),建立一种快速检测志贺菌(Shigella)的新方法。方法根据志贺菌的ipaH基因序列设计特异性引物,优化反应体系和反应条件;并对方法进行特异性和灵敏度评价。结果对21株实验菌株检测,3株志贺菌均为阳性,其余18株非志贺菌均为阴性,灵敏度为5.1×103cfu/mL,与普通PCR方法检测结果相当。结论 HDA法检测志贺菌具有特异、灵敏及仪器要求低等特点,具有广阔的应用前景。