Genotyping of Bacteroides fragilis isolates from stool specimens by arbitrarily-primed-PCR

In order to determine genetic relatedness of Bacteroides fragilis isolates from different clinical sources, arbitrarily primed polymerase chain reaction (PCR) (AP-PCR) was used to compare 17 strains isolated from patients with inflammatory bowel disease (IBD) and 20 strains isolated from foals with diarrhea. Three reference ATCC strains were also analyzed. Eighteen unique types were identified with a 22-mer arbitrary primer (ERIC-2) among the 20 patient isolates. Types 1 (enterotoxigenic) and 9 (nonenterotoxigenic), were each found in the stools of two patients. All other isolates showed a distinct and unique DNA banding pattern indicating a high degree of genotypic variability. Eleven types were identified among the foal isolates. Type 20, a nonenterotoxigenic type, was present in 30% of the foals. No correlation was found between the human and horse isolates. No clear relationship between a disease state (diarrhea or IBD) and specific types was observed. AP-PCR will be useful as a rapid method to determine genetic relatedness and in future epidemiologic studies of diarrheal diseases due to B. fragilis.     

MRSA杀白细胞素基因检测和SCCmec分子流行病学调查

目的 了解我院临床分离的耐甲氧西林金葡菌(MRSA)葡萄球菌染色体mec基因盒(staphyloccoccal cassette chromosome mec,SCCmec)的分布和杀白细胞毒素(Panton-Valentine Leukocidin,PVL)基因的携带状况.方法 收集我院2005年10月-2006年6月从临床标本中分离的30株MRSA(mecA基因阳性).运用多重PCR检测SCCmec的基因型和亚型;PCR扩增PVL基因;纸片扩散法检测MRSA对10种抗菌药物的耐药性.结果 30株MRSA中,SCCmecⅡ型6株(20.0%);SCCmecⅢ型15株(50.0%);SCCmecⅣa型1株和SCCmecⅤ型2株.6株MecA基因阳性菌株未能分型.PVL总阳性率36.7%(11/30),其中SCCmecⅡ1株、SCCmecⅢ5株、SCCmecⅣa 1株,SCCmecⅤ2株、未分型2株.SCCmecⅡ型和SCCmecⅢ型菌株除对β内酰胺类抗生素耐药以外,对其他抗菌药物呈现多重耐药.SCCmecaⅣa和SCCmecⅤ型的菌株除对β内酰胺类抗生素耐药以外,对其他类抗菌药物均敏感.结论 我院临床分离MRSA的SCCmec型别主要以SCCmecⅢ和SCCmecⅡ为主;PVL基因的阳性率较高;携带SCCmecⅡ和SCCmecⅢ的MRSA对抗菌药物呈现多重耐药现象.     

Nationwide study of the prevalence, characteristics, and molecular epidemiology of extended-spectrum-beta-lactamase-producing Enterobacteriaceae in France

Among 10,872 isolates of Enterobacteriaceae from a nationwide study of 88 French hospitals in 2005, 169 (1.7%) expressed an extended-spectrum beta-lactamase. The most prevalent species were Escherichia coli (48.5%), Enterobacter aerogenes (23.7%), and Klebsiella pneumoniae (14.8%). Molecular analysis underlined the polyclonal spread of CTX-M-expressing E. coli, primarily isolates of the CTX-M-1 subgroup.     

Multicentre surveillance of the prevalence and molecular epidemiology of macrolide resistance among pharyngeal isolates of group A streptococci in the USA

OBJECTIVES: Rates of macrolide resistance in group A streptococci (GAS) were reported to be low in the US in the 1990s. However, we documented an unexpectedly high rate of macrolide resistance among GAS in Pittsburgh, PA, in 2001 and 2002. In an effort to define the current prevalence of macrolide-resistant GAS in the US, a multicentre surveillance project was initiated. METHODS: Between October 2002 and May 2003, 50 pharyngeal GAS isolates per month were requested from each of the nine participating sites representing a wide geographical distribution. Standard susceptibility testing was performed and the macrolide resistance phenotype was assessed using double-disc diffusion testing. Monthly and annual rates of macrolide resistance were calculated for each site. An adjusted overall rate of macrolide resistance was determined to account for differences in the numbers of GAS isolates sent from each centre. RESULTS: Overall, 171 of the 2797 collected isolates of GAS (6.1%) were resistant to erythromycin. The adjusted overall resistance rate was 5.2%. Rates of macrolide resistance varied by site (range 3.0-8.7%) and also by month (<2% to >10%). The M phenotype of macrolide resistance accounted for >60% of all macrolide-resistant isolates recovered in this study. CONCLUSIONS: These data suggest an increasing prevalence and broad geographical distribution of macrolide-resistant GAS in the US, indicating the need for ongoing local and national longitudinal surveillance to define the extent of this problem.     

Antimicrobial susceptibility and molecular epidemiology of beta-lactamase-producing, aminoglycoside-resistant isolates of Enterococcus faecalis.

beta-Lactamase-producing (BL+), aminoglycoside-resistant (AR) Enterococcus faecalis is endemic in our hospital, having caused widespread colonization and infection. Suitable therapy for infections caused by these organisms has been problematic. We compared the antimicrobial and bactericidal activities, by broth macrodilution and time-kill methods, of several antibiotics, alone and in combination, against BL+, AR isolates of E. faecalis and determined the transmissibility of antibiotic resistance markers. Ampicillin-sulbactam, imipenem, daptomycin, and ciprofloxacin were the most active antibiotics with MICs for 90% of isolates tested of 2, 1, 2, and 1 microgram/ml, respectively, against inocula of 10(3) and 10(5) CFU/ml. Little inoculum effect was noted with imipenem, vancomycin, daptomycin, or ciprofloxacin, while the addition of sulbactam to ampicillin partially inhibited the effect of the increased inoculum. Penicillin-sulbactam and ampicillin-sulbactam combinations in a 2:1 ratio were most frequently bactericidal (greater than or equal to 3-log10-unit decrease in bacterial titers at 24 h for 13 of 20 isolates), followed by daptomycin (8 of 20 isolates) and ciprofloxacin (2 of 20 isolates). Bactericidal activity was not demonstrated for imipenem or teicoplanin. beta-Lactamase production and aminoglycoside resistance were associated with a 60- to 65-MDa plasmid which was easily transferred to a plasmid-free E. faecalis recipient. The 840-bp beta-lactamase gene probe hybridized to purified plasmid DNA from BL+ donor isolates of E. faecalis and transconjugants but not from BL- isolates. Ampicillin-sulbactam and daptomycin (an investigational antibiotic) seem to be reasonable choices for the empiric therapy of presumed enterococcal infections in hospitals in which BL+, AR E. faecalis strains are isolated. Their use should ideally be supported by tests for bactericidal activity.     

Antimicrobial susceptibilities and molecular epidemiology of clinical isolates of Clostridium difficile in taiwan

The antimicrobial susceptibility and virulence factors of Clostridium difficile clinical isolates in Taiwan have not previously been reported. One hundred and thirteen isolates were collected from two major teaching hospitals in Taiwan from 2001 to 2009. Molecular typing was performed by an automated repetitive extragenic palindromic sequence-based PCR (rep-PCR) method (DiversiLab; Bacterial Barcodes, Inc., Athens, GA) and PCR ribotyping. Detection of tcdA, tcdB, cdtA, and cdtB genes was performed using a multiplex PCR assay, and gyrA and gyrB genes of moxifloxacin-nonsusceptible isolates were sequenced. All isolates were susceptible to vancomycin and metronidazole. Ninety-five (84%) isolates were susceptible to moxifloxacin, and the MIC(90) for nemonoxacin was 4 μg/ml. Tigecycline showed favorable antibacterial activity (MIC(90) of 0.06 μg/ml). Thirteen rep-PCR types were identified as a predominant rep-PCR type (type A; non-North American pulsed-field gel electrophoresis type 1 [NAP1], -NAP7, or -NAP8) accounting for 52.2% (59 isolates). Nine of 18 moxifloxacin-nonsusceptible isolates belonged to the rep-PCR type A. The rep-PCR type A and C isolates were distinct from NAP1 (ribotype 027) and NAP8 (ribotype 078) as determined by PCR ribotyping. Seventy-four (65%) isolates harbored tcdA and tcdB, and 15 (13%) harbored cdtAB encoding binary toxin. Eleven isolates had a gene deletion in tcdC, including a 39-bp deletion (9 isolates) and an 18-bp deletion (2). In conclusion, dissemination of a predominant C. difficile clone in southern and northern Taiwan was noted. However, no NAP1 (ribotype 027) isolate could be discovered in this study.     

Implementation of a T4 extraction control for molecular assays of cerebrospinal fluid and stool specimens

The use of appropriate extraction and amplification controls for acellular specimens is not standardized in the clinical laboratory community. Extraction controls and checks for inhibitors of amplification in cellular specimens are most often accomplished by amplification of an internal human genomic target. This approach is not feasible for acellular specimens, which may contain little or no amplifiable genomic material. Other specimen types, such as stool, frequently contain amplification inhibitors. Failure to test for these inhibitors can result in the reporting of false-negative results. The goal of this study was to evaluate the use of a T4 bacteriophage as an extraction and amplification control for acellular specimens. The T4 bacteriophage assay was evaluated for use as a control in 290 specimens, including cerebrospinal fluid, serum, and filtered stool. Extraction procedures on two automated instruments were assessed, including the Roche MagNAPure Compact (Roche Diagnostics, Indianapolis, IN) and the QIAGEN BioRobot M48 (QIAGEN, Valencia, CA), along with the manual QIAGEN extraction method. The T4 bacteriophage can be extracted reliably and reproducibly from cerebral spinal fluid, serum, and filtered stool and, therefore, is useful as both an extraction control and inhibitor check for these specimen sources.     

20株亚胺培南耐药肺炎克雷伯菌耐药机制及分子流行病学研究

目的对20株亚胺培南耐药肺炎克雷伯菌耐药机制和分子流行病学进行研究。方法改良Hodge试验检测碳青霉烯酶表型;PCR法检测碳青霉烯酶、Amp C酶和超广谱β-内酰胺酶(ESBLs)耐药基因及整合子,并进行测序及BLAST比对确定基因型;脉冲场凝胶电泳(PFGE)和多位点序列分型(MLST)分析菌株同源性和遗传相关性;并进行药敏试验结果分析。结果20株菌均携带KPC-2、TEM-1、SHV和CTX-M型基因,未检测到IMP、VIM、OXA和NDM碳青霉烯酶基因和amp C酶基因;20株菌均有I类整合子,其中18株的整合子整合有耐药基因盒,主要整合基因是aad A2。20株菌中19株被PFGE分型为13个型别,其中P1型和P7型各有3株,P2和P 10型各有2株;20株菌中17株为ST11型,其余3株分别为ST290、ST147和ST967型;20株菌呈多重耐药或泛耐药。结论携带KPC-2是20株肺炎克雷伯菌耐亚胺培南的主要原因,多重耐药或泛耐药与其他β-内酰胺酶和整合子有关;院内存在克隆株流行;检出ST290和ST967型别产KPC肺炎克雷伯菌。     

Prevalence, characteristics, and molecular epidemiology of macrolide and fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae at five tertiary-care hospitals in Korea

The genes erm(B), mef(A), and both erm(B) and mef(A) were identified in 42.6, 10.1, and 47.3%, respectively, of the erythromycin-resistant Streptococcus pneumoniae isolates. Of the strains, 3.8% were nonsusceptible to levofloxacin and had 1 to 6 amino acid changes in the quinolone resistance-determining region, including a new mutation, Asn94Ser, in the product of parC. Levofloxacin with reserpine was highly specific for efflux screening.     

Assessment of the prevalence and diversity of emergent campylobacteria in human stool samples using a combination of traditional and molecular methods

This study aims to assess the diversity of campylobacteria (Campylobacter and Arcobacter) in human fecal samples from patients with diarrhea (n = 140) and asymptomatic controls (n = 116) in Chile, using a combination of traditional culture and molecular methods. The culture methods detected campylobacteria in 10.7% of the patients with diarrhea and in 1.7% of the controls. In contrast, the molecular methods detected campylobacteria more often than the traditional culture, with a prevalence of 25.7% and 5.2%, respectively. The traditional methods only recovered the species Campylobacter jejuni, Campylobacter coli, and Arcobacter butzleri, whereas the molecular methods additionally detected the emergent species Campylobacter concisus and Campylobacter ureolyticus.     

Evaluation of assay systems for the detection of rotavirus in stool specimens

We tested 41 rotavirus positive and 42 negative specimens as determined by electron microscopy. The assays systems used were an indirect NIH-ELISA, Meritec-Rotavirus, Virogen Rotatest, and Rotazyme II. Meritec and Virogen (latex agglutination assays) were the most sensitive tests, detecting 95% of the positive specimens. The NIH-ELISA detected 81% and Rotazyme detected 63%. Rotazyme was the most specific (100%), followed by the NIH-ELISA (95%) and the two latex agglutination systems (91%). To determine the level of rotavirus detection, we tested three systems against serial two-fold dilutions of ten positive stools. The NIH-ELISA detected rotavirus in an average dilution of 1:723. Rotazyme detected rotavirus in an average stool dilution of 1:366, and Meritec showed an average of 1:164. Rotavirus strains representing serotypes 1-4 were also tested. Meritec was able to detect all four serotypes. Virogen did not react with serotype 2 strains. The NIH-ELISA and rotazyme were unable to detect serotype 3. These data suggest that some latex agglutination assays may be a useful alternative to ELISAs in the clinical laboratory.     

Detection of enterotoxigenic Escherichia coli in stool specimens by polymerase chain reaction

A polymerase chain reaction (PCR) protocol for rapid (7 h) detection of enterotoxigenic Escherichia coli (ETEC) is described. This protocol has been validated on 57 stool samples from young children by comparing it with the colony hybridization technique. A good agreement was found between the two methods with Cohen’s kappa statistics of 0.87 and 0.79 for the detection of the heat-stable toxin (ST) and heat-labile toxin (LT), respectively. Of 26 samples positive for LT and 15 samples positive for ST by colony hybridization, 21 (81%) and 15 (100%) were also found to be positive for LT and ST by PCR, respectively. Only one sample identified as LT-negative by colony hybridization was found to be positive by PCR. However, 3 of 42 samples of ST-negative by colony hybridization were detected as positive by PCR. A reconstruction experiment revealed that PCR could detect LT-producing and ST-producing ETEC at minimal concentrations of 2.5 × 10³ cfu and 2.5 × 10² cfu per gram of feces, respectively. These data indicate the possible use of this method for rapid identification of ETEC-associated diarrhea in clinical and epidemiological settings.     

Identification,clinical aspects,susceptibility pattern,and molecular epidemiology of beta-haemolytic group G Streptococcus anginosus group isolates from central Taiwan

No literature is available on the prevalence and clinical aspects of beta-haemolytic group G Streptococcus anginosus group in central Taiwan. In this study, we used 16S rRNA gene sequencing and 16S-23S rDNA intergenic spacer sequencing (where necessary) as the gold standard for molecular identification. Twenty-seven S. anginosus group isolates were identified from 273 beta-haemolytic GGS isolates collected from patients in central Taiwan between February 2007 and August 2011. Of the 27 isolates, 22 were S. anginosus and 5 were Streptococcus constellatus. The 3 commercial methods, Rapid ID 32 Strep, API 20 Strep, and Vitek 2 GP card, identified 77.8%, 40.7%, and 37.0% of S. anginosus group isolates, respectively, with acceptable %ID or probability level. All the S. constellatus isolates possessed the lmb gene (encoding laminin-binding protein); however, none of the S. anginosus isolates possessed this gene. All the 27 isolates were susceptible to penicillin. Five S. anginosus group isolates (18.5%) were resistant to erythromycin. The resistance genes, ermB and mefA, were detected in 3 (2 S. anginosus and 1 S. constellatus) and 2 (2 S. anginosus) isolates, respectively. Pulsed field gel electrophoresis showed that most S. anginosus group isolates were genetically diverse. This is the first study to evaluate 3 commercial methods for the identification of beta-haemolytic group G S. anginosus group species, and only the Rapid ID 32 Strep system showed considerable ability. The clinical aspects, susceptibility pattern, and molecular epidemiology of beta-haemolytic group G S. anginosus group isolates from central Taiwan were also first presented.     

上海市浦东新区副溶血弧菌病原学与分子流行病学特征分析

目的 了解上海市浦东新区副溶血弧菌病原学与分子流行病学特征。 方法 运用玻片凝集法对505株副溶血弧菌菌株进行血清学分型,运用K-B纸片法对菌株进行12种抗生素药敏试验,同时对菌株基因组DNA经限制性内切酶NotⅠ酶切后进行脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)分子分型,利用BioNumerics Version 6.64 分析软件对图谱进行聚类分析,初步建立PFGE分子分型数据库。 结果 505株菌株中有117株血清型未能分型,O3:K6为患者分离株中的优势血清型,其中69.14%(56/81)的食物中毒分离株和61.87%(185/299)的散发病例分离株血清型为O3:K6型;监测食品分离株血清型分布呈现多样性,无优势菌株。99.41%(502/505)菌株对氨苄西林耐药。505株菌株共获得221个不同的PFGE带型,有26株PFGE未能分型。监测食品分离株的PFGE呈现遗传多样性,无优势带型,并且与散发及食物中毒患者分离株的PFGE带型不同。食物中毒与散发病例分离株的优势带型相同。耐2种或以上抗生素的菌株与其他菌株的PFGE型不相同,相同PFGE带型内的菌株血清型相同,不同时间、不同腹泻监测点之间存在完全相同PFGE条带。 结论 浦东新区未出现多重耐药菌株,存在多起疑似聚集性病例事件,但与监测食品分离株的遗传谱系关系较远。     

南通地区2014年肠道病毒71型的分子流行病学研究

目的分析2014年江苏南通地区肠道病毒71型(EV71)的分子流行病学特征。方法采集52例手足口病患儿咽拭子标本,提取病毒核酸,采用实时荧光定量PCR(RT-PCR)扩增病毒的VP1序列。获得的序列与来自其他地区的序列进行比对分析,构建系统发育树。结果 52份临床标本中分离到12株EV71 VP1序列,12株VP1序列的核苷酸、氨基酸同源性为93.4%~99.1%,进化树分析显示12株VP1序列全部属于C4基因型的C4a亚型。结论 2014年南通地区分离的EV71流行病毒株与近年来中国其他地区的流行株有相同的起源,均属于EV71C4基因的C4a亚型。     

Detection of verotoxin-producing Escherichia coli in stool specimens by the polymerase chain reaction

In this retrospective study a total of 404 stools kept at −70°C were tested for the presence of verotoxin-producing Escherichia coli (VTEC) by the polymerase chain reaction (PCR). Thirteen positive samples from 11 patients were identified by PCR which correlated with previous isolation of E. coli O157:H7. There was no failure to detect VTEC by PCR but PCR did not identify further VTEC isolates. We concluded that (a) the occurrence of VTEC other than serotype O157:H7 is rare in our demographic area, (b) PCR is effective in the identification of E. coli O157:H7, and (c) PCR has the additional advantage over conventional culture methods of identifying VTEC, including sorbitol-fermenting serotypes, which might have been detected if we had extended our sample size.