Mechanisms of reduced susceptibility to ciprofloxacin in Escherichia coli isolates from Canadian hospitals

OBJECTIVE:

To determine whether plasmid-mediated quinolone resistance (PMQR) determinants play a role in the increasing resistance to fluoroquinolones among Escherichia coli isolates in Canadian hospitals, and to determine the mechanisms of reduced susceptibility to ciprofloxacin in a recent collection of 190 clinical E coli isolates.

METHODS:

E coli isolates (n=1702) were collected as part of the 2007 Canadian Hospital Ward Antibiotic Resistance Surveillance (CANWARD) study. Antimicrobial susceptibility testing was performed by Clinical and Laboratory Standards Institute (CLSI) broth microdilution. Using a representative subset of isolates (n=190), the mechanisms of reduced susceptibility to ciprofloxacin were detected by polymerase chain reaction and sequencing of the quinolone resistance-determining regions (QRDR) of chromosomal gyrA and parC genes, and by polymerase chain reaction for the PMQR genes: qnr, aac(6′) Ib-cr and qepA.

RESULTS:

2.1% and 1.1% of E coli harboured aac(6′)Ib-cr and qnrB, respectively. Single amino acid substitutions in the QRDR of gyrA were observed among isolates with ciprofloxacin minimum inhibitory concentrations as low as 0.12 μg/mL. As the ciprofloxacin minimum inhibitory concentration increased to 1 μg/mL (which is still considered to be susceptible by the CLSI), the vast majority of isolates demonstrated both gyrA and parC mutations.

CONCLUSION:

PMQR determinants and QRDR mutants among clinical E coli isolates with reduced susceptibility to ciprofloxacin demonstrates the need for increased surveillance and the need to re-evaluate the current CLSI breakpoints to prevent further development of fluoroquinolone resistance.     

Cotrimoxazole-resistant Escherichia coli bacteremia in neutropenic patients at a regional oncology hospital

In a regional oncology hospital using cotrimoxazole (trimethoprim-sulphamethoxazole) prophylaxis during chemotherapy-induced neutropenia, a single strain of Escherichia coli (indole negative) caused 15 of 27 episodes of Gram-negative rod bacteremia in 1987, and four of 32 such episodes in 1988. This biotype had not been recovered in 1986. Investigations during this ‘outbreak’ of bacteremias revealed enteric colonization with this strain of E coli in 37% of patients on leukemia or bone marrow transplant wards and in several staff members in July 1987. In 1988, 11 of 32 Gram-negative rod bacteremias were secondary to other strains of indole positive E coli of several different biotypes and plasmid profiles. Indole negative strains all exhibited low level trimethoprim resistance, whereas indole positive strains which subsequently appeared exhibited high level trimethoprim resistance. Failure of cotrimoxazole prophylaxis was initially due to the clonal dissemination of a single strain of E coli within the institution, with the subsequent appearance of multiple E coli strains with probable differing genetic bases for their resistance.     

Lactobacillus reuteri and Escherichia coli in the human gut microbiota may predict weight gain associated with vancomycin treatment

Background:

Antibiotics, used for 60 years to promote weight gain in animals, have been linked to obesity in adults and in children when administered during early infancy. Lactobacillus reuteri has been linked to obesity and weight gain in children affected with Kwashiorkor using ready-to-use therapeutic food. In contrast, Escherichia coli has been linked with the absence of obesity. Both of these bacteria are resistant to vancomycin.

Objectives and methods:

We assessed vancomycin-associated weight and gut microbiota changes, and tested whether bacterial species previously linked with body mass index (BMI) predict weight gain at 1 year. All endocarditis patients treated with vancomycin or amoxicillin in our center were included from January 2008 to December 2010. Bacteroidetes, Firmicutes, Lactobacillus and Methanobrevibacter smithii were quantified using real-time PCR on samples obtained during the 4–6 weeks antibiotic regimen. L. reuteri, L. plantarum, L. rhamnosus, Bifidobacterium animalis and E. coli were quantified on stool samples obtained during the first week of antibiotics.

Results:

Of the193 patients included in the study, 102 were treated with vancomycin and 91 with amoxicillin. Vancomycin was associated with a 10% BMI increase (odds ratio (OR) 14.1; 95% confidence interval (CI; 1.03–194); P=0.047) and acquired obesity (4/41 versus 0/56, P=0.01). In patients treated with vancomycin, Firmicutes, Bacteroidetes and Lactobacillus increased, whereas M. smithii decreased (P<0.05). The absence of E. coli was an independent predictor of weight gain (OR=10.7; 95% CI (1.4–82.0); P=0.02). Strikingly, a patient with an 18% BMI increase showed a dramatic increase of L. reuteri but no increase of E. coli.

Conclusion:

The acquired obesity observed in patients treated with vancomycin may be related to a modulation of the gut microbiota rather than a direct antibiotic effect. L. reuteri, which is resistant to vancomycin and produces broad bacteriocins, may have an instrumental role in this effect.     

Antimicrobial activity of bismuth subsalicylate on Clostridium difficile,Escherichia coli O157:H7, norovirus,and other common enteric pathogens

Previous studies have shown bismuth subsalicylate (BSS) has antimicrobial properties, but few studies have addressed the mechanism of action. Furthermore, following BSS ingestion other bismuth salts form throughout the gastrointestinal tract including bismuth oxychloride (BiOCl) that also act upon enteric pathogens. To further understand the antimicrobial activity of bismuth in infectious diarrhea, the antimicrobial effect of BSS and BiOCl on Clostridium difficile, Salmonella, Shigella, Shiga toxin-producing Escherichia coli strains and norovirus (NoV) were measured. Bacterial enteric pathogens in pure culture or in human fecal material were exposed to 35mg/ml BSS or BiOCl with or without a vehicle suspension. BSS and BiOCl treated samples were quantified and visualized by transmission electron microscopy. To measure the effect on NoV, reduction of infectious murine NoV (MNV), a surrogate for human NoV, and Norwalk virus RNA levels were measured by viral plaque assay and RT-qPCR, respectively. BSS and BiOCl reduced bacterial growth by 3–9 logs in all strains with majority resulting in populations of <10 cfu/ml within 24 h. Similar results were found when fecal material was included. Microscopy images detected bismuth on bacterial membranes and within the bacterial organisms at 30 min post-treatment. At 8.8mg/ml BSS and BiOCl reduced infectivity of MNV significantly by 2.7 and 2.0 log after 24 h of exposure. In addition, both BSS and BiOCl slightly reduced the level of Norwalk replicon-bearing cells suggesting that bismuth may inhibit NoV in vivo. Collectively, our results confirm and build on existing data that BSS has antimicrobial properties against a wide-range of diarrhea-causing pathogens.     

Cancerous patients and outbreak of Escherichia coli: an important issue in oncology

The widespread of the Escherichia coli outbreak in Europe becomes an important public concern at global level. The infection can be serious and might result in death. The retrospective literature review on this specific topic is performed. In this specific brief article, the author presented and discussed on the problem of Escherichia coli infection in the cancerous patients. This is an actual important issue in medical oncology for the scenario of Escherichia coli epidemic.     

Prevalence of plasmid-mediated quinolone resistance genes among ciprofloxacin-nonsusceptible Escherichia coli and Klebsiella pneumoniae isolated from blood cultures in Korea

OBJECTIVES:

To analyze the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants in ciprofloxacin-nonsusceptible Escherichia coli and Klebsiella pneumoniae isolated from patients at a tertiary care hospital in Korea.

METHODS:

A total of 102 nonduplicate isolates of ciprofloxacin-intermediate or ciprofloxacin-resistant E coli (n=80) and K pneumoniae (n=22) from blood cultures were obtained. The qnr (qnrA, qnrB, qnrS), aac(6′)-Ib-cr, qepA and oqxAB genes were detected using polymerase chain reaction (PCR) and confirmed using direct sequencing. To determine whether the PMQR-positive plasmid was horizontally transferable, conjugation experiments were performed.

RESULTS:

Of the 102 isolates, 81 (79.4%) had one or more PMQR genes; these consisted of 59 (73.8%) E coli and 22 (100%) K pneumoniae isolates. The qnr genes were present in 15 isolates (14.7%): qnrB4 was detected in 10.8% and qnrS1 was detected in 3.9%. The aac(6′)-Ib-cr, qepA and oqxAB genes were detected in 77.5%, 3.9% and 10.8%, respectively. In conjugation experiments, PMQR genes were successfully transferred from seven (8.6%) isolates. The range of minimum inhibitory concentrations of ciprofloxacin for these seven transconjugants increased to 0.5 mg/L to 1 mg/L, which was 16- to 33-fold that of the recipient E coli J53 bacteria.

CONCLUSIONS:

PMQR genes were highly prevalent among ciprofloxacin-nonsusceptible E coli and K pneumoniae from blood cultures in the authors’ hospital. Therefore, it is necessary to monitor for the spread of PMQR genes of clinical isolates and to ensure careful antibiotic use in a hospital setting.     

8省市鸡肉源性大肠杆菌分离鉴定及其耐药性检测

目的 了解中国不同地区市售鸡肉源大肠杆菌耐药性和耐药特征,为抗生素的合理使用提供参考。方法 对全国8个省市的超市或农贸市场随机采集的1 152份鸡肉样品进行大肠杆菌的分离鉴定,采用琼脂稀释法对15种抗生素进行药敏试验。结果 鸡肉大肠杆菌的分离率为65.97%;大肠杆菌对15种抗生素具有不同程度的耐药,萘啶酮酸、阿莫西林-克拉维酸、四环素、甲氧苄啶-新诺明和氨苄西林的耐药率高达60%以上;而对加替沙星、头孢哌酮和阿米卡星的抗性均低于20%。大肠杆菌多重耐药为70.53%,其中以8重耐药最多10.26%。结论 大肠杆菌存在区域性耐药差异,并且多重耐药现象严重,存在通过食物链传播给人的风险。     

Genotoxicity of Escherichia coli Nissle 1917 strain cannot be dissociated from its probiotic activity

Oral administration of the probiotic bacterium Escherichia coli Nissle 1917 improves chronic inflammatory bowel diseases, but the molecular basis for this therapeutic efficacy is unknown. E. coli Nissle 1917 harbors a cluster of genes coding for the biosynthesis of hybrid nonribosomal peptide-polyketide(s). This biosynthetic pathway confers the ability for bacteria to induce DNA double strand breaks in eukaryotic cells. Here we reveal that inactivation of the clbA gene within this genomic island abrogated the ability for the strain to induce DNA damage and chromosomal abnormalities in non-transformed cultured rat intestinal epithelial cells but is required for the probiotic activity of E. coli Nissle 1917. Thus, evaluation of colitis severity induced in rodent fed with E. coli Nissle 1917 or an isogenic non-genotoxic mutant demonstrated the need for a functional biosynthetic pathway both in the amelioration of the disease and in the modulation of cytokine expression. Feeding rodents with a complemented strain for which genotoxicity was restored confirmed that this biosynthetic pathway contributes to the health benefits of the probiotic by modulating its immunomodulatory properties. Our data provide additional evidence for the benefit of this currently used probiotic in colitis but remind us that an efficient probiotic may also have side effects as any other medication.     

High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease

BACKGROUND & AIMS: Adherent-invasive Escherichia coli (AIEC) pathovar has been identified in the intestinal mucosa of patients with Crohn's disease (CD). AIEC reference strain LF82 is able to adhere to intestinal epithelial cells, to invade epithelial cells via a mechanism involving actin polymerization and microtubules, and to survive and replicate within macrophages. This study was performed to assess the prevalence of AIEC associated with intestinal mucosa of patients with CD, ulcerative colitis (UC), and of controls. METHODS: A search for E. coli strains was performed with ileal specimens of 63 patients with CD and 16 controls without inflammatory bowel disease (IBD), and with colonic specimens of 27 patients with CD, 8 patients with UC, and 102 controls. The abilities of E. coli strains to invade epithelial cells and to survive and replicate within macrophages were assessed using the gentamicin protection assay. Bacterial uptake by epithelial cells was analyzed using cytoskeletal inhibitors. Bacterial adhesion was quantified with Caco-2 and Intestine-407 cells. The presence of known E. coli virulence genes was assessed by polymerase chain reaction and DNA hybridization. RESULTS: In ileal specimens, AIEC strains were found in 21.7% of CD chronic lesions vs. in 6.2% of controls. In neoterminal ileal specimens, AIEC strains were found in 36.4% of CD early lesions (P = 0.034 vs. controls) and 22.2% of healthy mucosa of CD patients. In colonic specimens, AIEC strains were found in 3.7% of CD patients, 0% of UC patients, and 1.9% of controls. CONCLUSIONS: AIEC strains are associated specifically with ileal mucosa in CD.     

Diarrhea caused by enterotoxigenic Escherichia coli infection of humans is inhibited by dietary calcium

BACKGROUND & AIMS: In several rat infection experiments, we have shown that dietary calcium inhibits intestinal colonization and translocation of invasive salmonella. The aim of the present study was to find out whether calcium is also protective against enterotoxigenic Escherichia coli (ETEC) infection. This was first tested in our rat model and subsequently verified in a human infection study. METHODS: Rats were fed a purified diet with either a low or a high amount of calcium phosphate and orally infected with ETEC. In addition, a parallel, double-blind, placebo-controlled intervention study of 3 weeks was performed with 32 healthy men. Subjects largely maintained their habitual diet and consumed either regular milk products (calcium supply, 1100 mg/day) or placebo milk products (calcium supply, 60 mg/day). On day 10, subjects ingested a live but attenuated ETEC strain (strain E1392/75-2A), able to induce mild although short-lived symptoms. Primary outcomes studied were infection-induced diarrhea (total fecal output and relative fecal dry weight) and fecal mucin excretion. RESULTS: In humans, ETEC induced diarrhea in both groups, in that total fecal output doubled and mean relative fecal dry weight dropped from 25% to 20%. Additionally, fecal mucin excretion was increased in both groups. All these fecal parameters were completely normalized in the calcium group on the second infection day, in contrast to the placebo group, which recovered on the third infection day. Likewise, supplemental calcium inhibited ETEC colonization and diarrhea in rats. CONCLUSIONS: Calcium in milk products improves human resistance to ETEC infection as it inhibits infectious diarrhea.     

A structural and biochemical basis for PAPS-independent sulfuryl transfer by aryl sulfotransferase from uropathogenic Escherichia coli

Sulfotransferases are a versatile class of enzymes involved in numerous physiological processes. In mammals, adenosine 3′-phosphate-5′-phosphosulfate (PAPS) is the universal sulfuryl donor, and PAPS-dependent sulfurylation of small molecules, including hormones, sugars, and antibiotics, is a critical step in hepatic detoxification and extracellular signaling. In contrast, little is known about sulfotransferases in bacteria, which make use of sulfurylated molecules as mediators of cell–cell interactions and host–pathogen interactions. Bacterial arylsulfate sulfotransferases (also termed aryl sulfotransferases), in contrast to PAPS-dependent sulfotransferases, transfer sulfuryl groups exclusively among phenolic compounds in a PAPS-independent manner. Here, we report the crystal structure of the virulence factor arylsulfate sulfotransferase (ASST) from the prototypic, pyelonephritogenic Escherichia coli strain CFT073 at 2.0-Å resolution, and 2 catalytic intermediates, at 2.1-Å and 2.4-Å resolution, with substrates bound in the active site. ASST is one of the largest periplasmic enzymes and its 3D structure differs fundamentally from all other structurally characterized sulfotransferases. Each 63.8-kDa subunit of the ASST homodimer comprises a 6-bladed β-propeller domain and a C-terminal β-sandwich domain. The active sites of the dimer are situated at the center of the channel formed by each β-propeller and are defined by the side chains of His-252, His-356, Arg-374, and His-436. We show that ASST follows a ping-pong bi–bi reaction mechanism, in which the catalytic residue His-436 undergoes transient sulfurylation, a previously unreported covalent protein modification. The data provide a framework for understanding PAPS-independent sulfotransfer and a basis for drug design targeting this bacterial virulence factor.     

广州市鼠形动物肠道产ESBL大肠埃希菌分布及病原特征分析

目的 获得鼠形动物肠道产ESBL大肠埃希菌分布特征、菌型特点及相关耐药机制。方法 2016年9月-11月,在广州市城区内某居民区捕捉鼠形动物。从鼠形动物肠道内容物中分离耐头孢噻肟大肠埃希菌并进行产ESBL表型确认实验。Vitek2 compact进行产ESBL大肠埃希菌的抗生素敏感实验。PCR检测产ESBL大肠埃希菌的CTX-M、TEM、SHV和OXA等ESBL基因,并测序鉴定CTX-M亚型。对CTX-M大肠埃希菌进行MLST和PFGE分析。结果 共捕获鼠形动物123只,包括57只黄胸鼠、54只褐家鼠、11只臭鼩鼱和1只黑毛鼠。分离到35株耐头孢噻肟大肠埃希菌,经表型确认实验证实全部是产ESBL菌株,检出率为28.5%(35/123)。35株产ESBL菌株对氨苄西林、氨苄西林-舒巴坦、哌拉西林、头孢噻肟、头孢曲松、头孢吡肟、环丙沙星以及左氧氟沙星的耐药率很高,分别为100%(35/35)、91.4%(32/35)、97.1%(34/35)、100%(35/35)、100%(35/35)、88.6%(31/35)、80%(28/35)和74.3%(26/35)。 35株菌一共检出CTX-M耐药基因37个,有2株菌各携带2种CTX-M耐药基因,其中bla_ctx-m-79 23株,bla_ctx-m-55 6株,bla_ctx-m-123 4株,bla_ctx-m-15 2株,bla_ctx-m-65和bla_ctx-m-27各1株。MLST结果发现鼠形动物肠道分离的产ESBL大肠埃希菌具有遗传多样性,无优势克隆。结论 鼠形动物产ESBL大肠埃希菌的耐药率很高,产ESBL耐药机制主要是菌株携带CTX-M型基因。本研究检出的几种CTX-M型ESBL基因也见于人临床或健康人群菌株。MLST和PFGE的结果发现产ESBL大肠埃希菌具有遗传多样性。本研究结果表明鼠型动物是产ESBL大肠埃希菌的重要储存宿主。     

Recombinant probiotics for treatment and prevention of enterotoxigenic Escherichia coli diarrhea

BACKGROUND & AIMS: We have developed a therapeutic strategy for gastrointestinal infections that is based on molecular mimicry of host receptors for bacterial toxins on the surface of harmless gut bacteria. The aim of this study was to apply this to the development of a recombinant probiotic for treatment and prevention of diarrheal disease caused by enterotoxigenic Escherichia coli strains that produce heat-labile enterotoxin. METHODS: This was achieved by expressing glycosyltransferase genes from Neisseria meningitidis or Campylobacter jejuni in a harmless Escherichia coli strain (CWG308), resulting in the production of a chimeric lipopolysaccharide capable of binding heat-labile enterotoxin with high avidity. RESULTS: The strongest heat-labile enterotoxin binding was achieved with a construct (CWG308:pLNT) that expresses a mimic of lacto-N-neotetraose, which neutralized > or = 93.8% of the heat-labile enterotoxin activity in culture lysates of diverse enterotoxigenic Escherichia coli strains of both human and porcine origin. When tested with purified heat-labile enterotoxin, it was capable of adsorbing approximately 5% of its own weight of toxin. Weaker toxin neutralization was achieved with a construct that mimicked the ganglioside GM2. Preabsorption with, or coadministration of, CWG308:pLNT also resulted in significant in vivo protection from heat-labile enterotoxin-induced fluid secretion in rabbit ligated ileal loops. CONCLUSIONS: Toxin-binding probiotics such as those described here have considerable potential for prophylaxis and treatment of enterotoxigenic Escherichia coli-induced travelers' diarrhea.     

NleH effectors interact with Bax inhibitor-1 to block apoptosis during enteropathogenic Escherichia coli infection

The human pathogens enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli and the related mouse pathogen Citrobacter rodentium subvert a variety of host cell signaling pathways via their plethora of type III secreted effectors, including triggering of an early apoptotic response. EPEC-infected cells do not develop late apoptotic symptoms, however. In this study we demonstrate that the NleH family effectors, homologs of the Shigella effector kinase OspG, blocks apoptosis. During EPEC infection, NleH effectors inhibit elevation of cytosolic Ca²⁺ concentrations, nuclear condensation, caspase-3 activation, and membrane blebbing and promote cell survival. NleH1 alone is sufficient to prevent procaspase-3 cleavage induced by the proapoptotic compounds staurosporine, brefeldin A, and tunicamycin. Using C. rodentium, we found that NleH inhibits procaspase-3 cleavage at the bacterial attachment sites in vivo. A yeast two-hybrid screen identified the endoplasmic reticulum six-transmembrane protein Bax inhibitor-1 (BI-1) as an NleH-interacting partner. We mapped the NleH-binding site to the N-terminal 40 amino acids of BI-1. Knockdown of BI-1 resulted in the loss of NleH’s antiapoptotic activity. These results indicate that NleH effectors are inhibitors of apoptosis that may act through BI-1 to carry out their cytoprotective function.     

Purification and application of C-terminally truncated hepatitis C virus E1 proteins expressed in Escherichia coli

AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli (E. coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally truncated E1 fragments were expressed in E. coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chromatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot. RESULTS: Full-length E1 protein proved difficult to express in E. coli. C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni(2+)-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 glycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization. CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E. coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.     

HPV16地方分离株E6基因的克隆与原核表达研究

目的克隆人乳头瘤病毒（HPV16E6）基因并在大肠埃希菌中表达，对表达产物进行鉴定。方法用PCR方法从克隆质粒pUC19-HPV16E6E7中扩增HPV16E6基因，采用定向克隆构建pQE30-HPV16E6原核表达质粒．利用酶切和序列测定鉴定重组质粒。将pQE30-HPV16E6转化大肠埃希菌BL21（DE3），建立重组工程菌pQE30-HPV16E6／BL21（DE3）。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导，SDS—PAGE分析蛋白表达情况，利用Western blot鉴定抗原特异性。结果PCR产物470bp，重组质粒经酶切和序列测定证实构建正确。SDS—PAGE分析在18×10^3处有蛋白条带出现，与预期一致。Western blot分析证实目的条带与HPV16E6抗体有特异性反应。结论成功构建了HPV16E6基因的基因工程菌株，能高效表达E6蛋白，表达蛋白具有良好的免疫反应性。