A scheme for identification of nonfermentative gram-negative bacteria.

A scheme was designed to speed the identification of 50 nonfermentative gram-negative bacteria to genus, species, and biotype using a minimal numer of biochemical tests. When the scheme was evaluated in our laboratory, one technologist identified 93.6% of 188 known isolates (46 species). Later another technologist, given 161 of the same isolates (46 species) as unknowns, identified 91.9%. The isolates that were misidentified in both cases were atypical organisms with aberrant biochemical reactions, plus 1 typical organism with a delayed oxidation of glucose.     

Identification of an epidemic strain ofAcinetobacter baumanii using electrophoretic typing methods

Nosocomial infections due toAcinetobacter baumannii dramatically increased in a Lebanese medical center following an outbreak of hostilities in Lebanon in 1984. The incidence of infection caused by this organism has remained high in this institution, thus requiring the implementation of a strain typing system to aid in infection control. Three methods were investigated for their utility in differentiating among a representative group of 36 nosocomialAcinetobacter baumannii isolates obtained over a 10 month period from specimens of hospitalized patients. Isolates were typed by antibiogram analyses, plasmid fingerprinting, and total cell protein profiles. Only three distinct total cell protein profiles were detected, with one pattern accounting for 26 (72.2%) of the isolates. However, eight different plasmid profiles were observed, with 20 (55.5%) isolates having the same profile. Eleven distinct antibiograms were seen with the most prevalent pattern occuring in 21 isolates. Twenty of the 21 (95%) isolates with the common antibiogram also had the same plasmid profile and total protein profile (44.4% of total isolates). The combination of these three typing methods was useful in tracing the spread of these organisms in the medical center. The data obtained suggest the distribution of a common strain among at least six wards of this hospital.     

RAPD typing in microbiology—a technical review

Many biochemical and molecular techniques can be used for distinguishing isolates of a given bacterial species. Traditional typing techniques based on phenotypic characteristics such as serotyping are being increasingly challenged by the use of DNA-based methods. The introduction of the polymerase chain reaction (PCR) has led to typing techniques based on DNA amplification. Randomly amplified polymorphic DNA (RAPD) typing (also known as arbitrarily primed-polymerase chain reaction, APPCR) is one such technique which is being used increasingly to type micro-organisms, especially during clinical outbreaks. The applications and potential problems and solutions of RAPD typing are discussed and the role of such techniques among established typing methods is addressed.     

青岛地区医院内感染耐甲氧西林金黄色葡萄球菌分子分型研究

目的 探讨青岛地区医院内感染的耐甲氧西林金黄色葡萄球菌(MRSA)分子流行病学特征及脉冲场凝胶电泳(PFGE)型别与菌株表型、一般临床资料间的关系.方法 收集2003-2007年间青岛地区主要医院内感染MRSA 360株,Sma Ⅰ酶切菌株染色体DNA后,进行PFGE电泳,用Bionumericus 2.0软件对电泳图谱进行比较和聚类分析,绘制进化树.同时对患者的性别、年龄、菌株来源等进行多变量统计分析.应用纸片扩散法测定分离菌株的药物敏感谱,并与PFGE型别进行比较分析.PCR扩增不同PFGE型别MRSA代表株25株分离株的7个管家基因进行序列测定和多位点测序分型分析(MLST).结果 所有菌株经PFGE电泳后共分为5型(M0～M4型),其中M1型为优势菌型,M2型次之,M4型相对少见,M0为独特型,明显不同于其他已知PFGE型别.统计学分析发现5种PFGE型别在患者性别、年龄分布上的差异无统计学意义,但在菌株分离部位、来源有统计学的差异:M2型多分离自伤口感染,而M3型菌株多来自ICU病房,5种PFGE型在不同医院间及医院内的分布存在差异.M1与M2两型构成各医院分离菌株的主要型别.抗生素敏感性测定中未发现万古霉素耐药菌株,亦未发现某种PFGE型别与某种特定抗生紊抗性之间的直接相关性.MIST分型发现优势型M1与M3共属于国内常见ST239型,M2型则归类于ST5,M4型属于ST240,独特型中的2种PFGE谱型则分属于ST45及ST398.结论 ST239菌株为青岛地区医院内感染MRSA优势菌株;医院内MRSA的PFGE分型与菌株来源明显相关,与患者年龄、性别无关,MRSA感染普遍存在于各年龄人群中.     

人-猪链球菌感染性综合征研究

目的 描述人－猪链球菌感染性综合征的临床表现,探讨病原特征,以及感染来源。方法 观察２５例原因不明的“急性感染性中毒性出血性休克综合征”,用十几种培养基进行病原分离、菌种鉴定、药敏试验和动物试验,用ＰＣＲ指纹图技术分析人源与猪源株的同源性,用流行病学方法调查感染来源等。结果 猪链球菌感染性综合征临床表现主要有：链球菌中毒性休克综合征和链球菌脑膜炎型综合征。从患的血液和脑脊液中各分离到３株链球菌,     

Polymerase chain reaction identification of coagulase-negative Staphylococci and of strain diversity and spread of Staphylococcus epidermidis in a major medical center in Lebanon.

A 2-step polymerase chain reaction (PCR) assay and random amplification of polymorphic DNA (RAPD) analysis, respectively, were assessed to identify coagulase-negative staphylococci organisms to the species level and to determine the strain diversity and spread of Staphylococcus epidermidis, the most frequently isolated species, in a medical center in Beirut, Lebanon. Our data indicated that PCR was faster and was more efficient in identifying S. epidermidis isolates than is conventional biochemical testing. RAPD analysis have shown that S. epidermidis strains were scattered across the different clinical services, demonstrating various clusters of infection in the medical center.     

Evaluation of the pathogenicity of African Horsesickness (AHS) isolates in vaccinated animals

BACKGROUND: The polyvalent African Horsesickness (AHS) attenuated live vaccine (ALV) produced by Onderstepoort Biological Products (OBP) Ltd., South Africa, has been associated with some safety concerns and alleged cases of vaccine failure or vaccine-induced disease. The risk of reassortment and reversion to virulence is a common concern associated with the use of ALVs, and a phenomenon reported for viruses with segmented RNA genomes. The purpose of this study was to determine whether or not reassortment of AHS vaccine strains could result in reassortants and reversion to virulence and therefore cause AHS in susceptible horses. METHODS: Clinical or field isolates of AHS were obtained from horses with AHS symptoms or disease post vaccination. AHS-na?ve horses were inoculated with these isolates and monitored for clinical reactions. Laboratory tests were performed at intervals to determine immune responses and viraemia. Viral RNA extraction and complete genome amplification of monovalent AHS-ALV vaccine strains and isolates collected post-vaccination was conducted. cDNA of the genome segments were run on PAGE to determine mobility patterns and genome segments 2, 3, 4, 5 and 6 sequenced for phylogenetic analysis. RESULTS: No clinical symptoms typical of AHS were observed in inoculated horses and all showed a good immune response. A comparison of mobility patterns of the amplified cDNA genome on PAGE allowed the identification and differentiation of reassortants, which were confirmed by sequence and phylogenetic analysis of the nucleotide sequences. CONCLUSION: This study, however, showed no indications that vaccine reassortants were pathogenic or lethal after inoculation in susceptible horses. Assumptions of virulence or reversion to virulence of vaccine reassortants post-vaccination in horses could not be substantiated.     

Prevalence of Pseudomonas aeruginosa pyocin and antibiotic biotypes in four Tehran hospitals

Over a 15‐month period, 340 strains of Pseudomonas aeruginosa were isolated from clinical specimens, i.e. sputum, wound swabs, pus, burns, urine, stool, etc., and the environment, i.e. sink, floor, bed sheets, etc., in four Tehran hospitals. Identification of Pseudomonas aeruginosa was carried out by standard methods. Pyocin typing and subtyping were done using the spotting method of Govan, and using a set of 13 indicator strains. Results of the study showed that the dominant pyocin types were PT10, PT4 and PT3, with frequencies of 30.6%, 21.7% and 8.8%, respectively. The pyocins had a wide spectrum of activity, in contrast to most other bacteriocins known. The dominant pyocin subtypes observed in this study were b, d, a, and c, with frequencies of 18.4%, 14.9%, 12.7%, 9.2%, respectively. Approximately 95.9% of the isolates were typable. 98.8% of the Pseudomonas aeruginosa isolates were resistant to 1 or more antibiotics tested. Antibiotic sensitivity tests were performed using the disc diffusion method, employing Mueller‐Hinton Agar. The following antibiotics were tested: Aztreonam (ATM), Ceftazidime (CAZ), Ceftriaxone (CRO), Imipenem (IMP), Cefoperazone (CFP), Amikacin (AN), Sisomicin (SIS), Polymyxin B (PB), Gentamicin (GM), Tobramycin (NN), Netilmicin (NET), Tetracycline (TE) and Carbenicillin (CB). The highest resistance was observed against tetracycline (98.2%), while no resistance was detected against polymyxin B. Among betalactams, ceftazidime and among aminoglycosides, amikacin showed the greatest activities. Distribution of these isolates from the hospital environments was alarming. The results of these findings were reported to the hospital authorities and a number of preventive and control measurements, including more effective methods of disinfection for all instruments that come into contact with patients, were suggested to reduce the spread of the bacterium.     

2008-2009年北京市沙门菌流行特征和分子分型

目的 分析2008-2009年北京市沙门菌流行特征及脉冲场凝胶电泳(PFGE)分子分型. 方法 对2008-2009年通过WHO全球沙门菌监测系统及北京市肠道门诊监测系统分离到的137株沙门菌进行生化鉴定、血清分型和相关流行病学分析;利用PFGE进行分子分型. 结果 北京市2008-2009年沙门菌的流行具有明显季节性,6-9月份高发,共分离菌株84株,占64.1%(84/131);患者年龄多为18～40岁,占46.1%(58/128);男性80例,女性51例,男女比例为1.57:1.137株沙门菌分属于20种血清型,其中肠炎沙门菌和鼠伤寒沙门菌为优势菌型,分别占46.7%(64/137)和17.5%(24/137).共分71种PFGE带型,其中肠炎沙门菌和鼠伤寒沙门菌都有16种PFGE型别.肠炎沙门菌的4种PFGE型别(JEGX01.CN0001、JEGX01.CN0003、JFGX01.CN0002、JEGX01.CN0019)和鼠伤寒沙门菌的JPXX01.CN0001为优势分子型别. 结论 2008-2009年北京市沙门菌的流行具有性别、年龄和季节性分布特征;PFGE分子型别较多,且存在差异明显的多个克隆系.

Abstract：

Objective To study the epidemiological characteristics and molecular phenotypes of Salmonella by pulsed-field gel electrophoresis (PFGE) in Beijing from 2008 to 2009. Methods A total of one hundred thirty-seven isolates recovered from the WHO Global Samonella Surveillance system and entero clinic surveillance system were identified by biochemical tests and serotyping.The related epidemiological informations were also analyzed.The isolates were further typed by PFGE. Results The prevalence of Salmonella from 2008 to 2009 showed obvious seasonal character.High incidence occurred from June to September,and 64.1% (84/131) isolates were recovered in this period.Patients of 18-40 year-old were 46.1% (58/128) and 80 patients were male and 40 patients were female with the ratio of 1.57:1.These 137 Salmonella isolates belonged to 20 serotypes,including Enteritidis (46.7%,64/137) and Typhimurium (17.5%,24/137) as the dominant serotype.In total,71 PFGE profiles were identified.Four PFGE patterns of S.Enteritidis isolates (JEGX01.CN0001,JEGX01.CN0003,JEGXO1.CN0002,JEGX01.CN0019) and S.Typhimurium pattern of JPXX01.CN0001 were dominant patterns. Conclusion The prevalence of Salmonella from 2008 to 2009 showed distribution characteristics of sex,age and seasons.The numerous PFGE patterns of Salmonella showed diversity of these isolates and different clones existed in Beijing.     

Characterization of Mycobacterium orygis as M. tuberculosis complex subspecies

The oryx bacilli are Mycobacterium tuberculosis complex organisms for which phylogenetic position and host range are unsettled. We characterized 22 isolates by molecular methods and propose elevation to subspecies status as M. orygis. M. orygis is a causative agent of tuberculosis in animals and humans from Africa and South Asia.     

Contribution of AcrAB efflux pump to ciprofloxacin resistance in Klebsiella pneumoniae isolated from burn patients

Resistance to fluoroquinolones has been recently increased among bacterial strains isolated from outpatients. Multidrug-resistant K. pneumoniae is one of the major organisms isolated from burn patients and the AcrAB efflux pump is the principal pump contributing to the intrinsic resistance in K. pneumoniae against multiple antimicrobial agents including ciprofloxacin and other fluoroquinolones. Fifty-two K. pneumoniae isolated from burn patients in Shahid Motahari hospital and confirmed by conventional biochemical tests. Antimicrobial susceptibility testing was done according to CLSI 2011 guidelines, to determine the antimicrobial resistance pattern of isolates. AcrA gene was detected among ciprofloxacin-resistant isolates by PCR assay. MICs to ciprofloxacin were measured with and without carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Forty out of the 52 K. pneumoniae isolated from burn patients in Shahid Motahari hospital were resistant to ciprofloxacin according to breakpoint of CLSI guideline. PCR assay for acrA gene demonstrated that all ciprofloxacin-resistant isolates harbored acrA gene coding the membrane fusion protein AcrA and is a part of AcrAB efflux system. Among these isolates, 19 strains (47.5%) showed 2 to 32 fold reduction in MICs after using CCCP as an efflux pump inhibitor. The other 21 strains (52.5%) showed no disparity in MICs before and after using CCCP. In conclusion, the AcrAB efflux system is one of the principal mechanisms contribute in ciprofloxacin resistance among K. pneumoniae isolates but there are some other mechanisms interfere with ciprofloxacin resistance such as mutation in target proteins of DNA gyrase of topoisomerase IV enzymes.     

北京市40株肠炎沙门菌多重耐药和分子分型研究

目的分析2009-2010年北京市肠炎沙门菌多重耐药和脉冲场凝胶电泳（pulsed-field gel electropho-resis,PFGE）分子分型。方法对2009-2010年北京市肠道门诊监测系统分离到的40株肠炎沙门菌进行生化鉴定、血清分型并运用纸片法进行药敏检测,并采用PFGE进行分子分型。结果发现40株肠炎沙门菌中,32株多重耐药菌株,其中4～5种抗生素耐药27株（84.38%）,10种抗生素耐药1株（3.13%）;可分为14个PFGE带型,其中4个PFGE型的菌株数超过1株。结论北京市肠炎沙门菌分离株多重耐药性比较严重,PFGE分子型别较多,且存在差异明显的多个克隆系,但同一PFGE型菌株的多重耐药谱较为接近。     

Investigation of a pseudo-outbreak of 'Pseudomonas thomasii' in a special-care baby unit by numerical analysis of SDS-PAGE protein patterns

Forty-two cultures of pseudomonas comprising 28 clinical isolates from a pseudo-outbreak on a Special-Care Baby Unit and 14 reference strains, including 9 type strains, of various Pseudomonas species, were characterized by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins. The protein patterns were highly reproducible and were used as the basis for a numerical analysis which divided the strains into 9 phenons. Two of the 28 clinical isolates were identified by biochemical tests as P. pickettii and their identification was confirmed by SDS-PAGE as they fell in the same phenon as the type strain of the species. The remaining 26 isolates, which could not be identified on phenotypic tests, fell in the same phenon as three reference strains of 'P. thomasii'. The protein patterns provided the first clear evidence that P. pickettii and 'P. thomasii' were separate taxa and that the 'outbreak' was polymicrobial in origin, in line with the probable aqueous source of contamination. We conclude that high-resolution SDS-PAGE of proteins provides an effective method of identifying and differentiating pseudomonads, especially where this cannot be done adequately using conventional biochemical tests.     

Surveillance methodology for Vibrio cholerae in environmental samples

The purpose of this study was to examine the prevalence of Vibrio cholerae in environmental water samples by using a series of biochemical tests. A total of 223 V. cholerae-like bacteria were isolated from TCBS agar after spreading the alkaline peptone water enriched sewer (n = 21) and water (n = 16) samples. All oxidase positive isolates were subjected to confirmation for V. cholerae by seven other biochemical tests and polymerase chain reaction. Only 74.2% isolates were found to be V. cholerae by PCR using primers against an outer membrane protein (ompW) gene, out of which only 2 isolates were positive for cholera toxin (ctxAB) gene. Among the various biochemical tests studied, arginine hydrolysis, arabinose fermentation and string test showed 92 - 100% sensitivity and 42 - 67% specificity. Eight isolates including the toxigenic ones, showed agglutination with V. cholerae O1 antiserum. The present study showed that no biochemical test is 100% specific for V. cholerae. However, a few tests, if performed in a sequence after growing the alkaline peptone water enriched samples onto TCBS media can be used for screening of V. cholerae from the environmental samples. This study also showed that most of the environmental isolates are non-O1/non-O139 and the chances of presence of toxigenic V. cholerae are very rare in the environment.     

广州市两例B群流脑病例分离株的MLST分型研究

目的对从广州市报告的两例B群流行性脑脊髓膜炎（流脑）病例中分离的4株脑膜炎奈瑟菌（Neisseria meningitidis,Nm）进行多位点序列分型（multi—locus sequence typing,MLST）研究。方法分纯并提取菌株DNA、运用MI。ST分析对7个管家基因进行扩增并测序,确定菌株的序列型,对其相关性进行分析。结果两例病例分离株7个管家基因位点序列均不相同,第一例病例分离株的序列型为ST-7型,属高致病性ST-5complex／subgroupIII克隆系;第二例病例分离株的序列型为新发现的ST一9804型。结论广州两例B群流脑病例虽然发生时间相近,但分属不同的序列型,无同源性。MLST分型技术对研究不同流脑病例间的流行病学关系有重要意义。     

深圳市龙岗区近年伤寒沙门菌耐药性及脉冲场电泳分子分型研究

目的了解深圳市龙岗区近年伤寒沙门菌的耐药性和同源性。方法收集深圳市龙岗区2004—2007年肠道传染病和食物中毒样品中的伤寒沙门菌并用生化和血清学方法进行鉴定,采用改良K-B法进行药敏试验,伤寒沙门菌基因组经限制性内切酶XbaI酶切后,采用脉冲场电泳（PFGE）获得电泳图谱,再利用BioNumerics软件对电泳图谱进行同源性分析。结果共分离到17株伤寒沙门菌,所有菌株均对青霉素和苯唑西林耐药,对第3代头孢菌素、氨基糖苷类、氟喹诺酮类、氯霉素和复方新诺明敏感。64.7%的菌株对3种或以上抗生素中度耐药或耐药。BioNumerics分析结果显示,共有14个不同的PFGE带型出现,除2株以外其余15株分布于3个相似性在85%以上的簇内。结论深圳市龙岗区伤寒沙门菌的耐药已经成为一种非常普遍的现象,需加强监测。氟喹诺酮类和第3代头孢菌素类抗生素在临床上治疗伤寒沙门菌感染仍然可以取得满意效果。深圳市龙岗区可能存在3个伤寒沙门菌的流行克隆,其传播可能是该区伤寒沙门菌发生的主要原因。     

Investigation of classical epidemiological links between patients harbouring identical, non-predominant meticillin-resistant Staphylococcus aureus genotypes and lessons for epidemiological tracking

According to molecular epidemiology theory, two isolates belong to the same chain of transmission if they are similar according to a highly discriminatory molecular typing method. This has been demonstrated in outbreaks, but is rarely studied in endemic situations. Person-to-person transmission cannot be established when isolates of meticillin-resistant Staphylococcus aureus (MRSA) belong to endemically predominant genotypes. By contrast, isolates of infrequent genotypes might be more suitable for epidemiological tracking. The objective of the present study was to determine, in newly identified patients harbouring non-predominant MRSA genotypes, whether putative epidemiological links inferred from molecular typing could replace classical epidemiology in the context of a regional surveillance programme. MRSA genotypes were defined using double-locus sequence typing (DLST) combining clfB and spa genes. A total of 1,268 non-repetitive MRSA isolates recovered between 2005 and 2006 in Western Switzerland were typed: 897 isolates (71%) belonged to four predominant genotypes, 231 (18%) to 55 non-predominant genotypes, and 140 (11%) were unique. Obvious epidemiological links were found in only 106/231 (46%) patients carrying isolates with non-predominant genotypes suggesting that molecular surveillance identified twice as many clusters as those that may have been suspected with classical epidemiological links. However, not all of these molecular clusters represented person-to-person transmission. Thus, molecular typing cannot replace classical epidemiology but is complementary. A prospective surveillance of MRSA genotypes could help to target epidemiological tracking in order to recognise new risk factors in hospital and community settings, or emergence of new epidemic clones.     

2010年医院感染细菌的耐药性分析

目的 了解医院2010年1-12月全年临床分离菌株对各类抗菌药物的耐药性,以指导临床合理使用抗菌药物.方法 使用纸片扩散法进行抗菌药物监测,结果参照CLSI 2005年版进行判断.结果 临床分离1073株细菌中,革兰阳性菌占31.3％,革兰阴性菌占68.7％;耐甲氧西林金黄色葡萄球菌(MRSA)及耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)分别检出54.1％及65.3％,产ESBLs大肠埃希菌及肺炎克雷伯菌检出率分别为42.7％和28.9％.结论 该资料对医院细菌性感染的治疗和抗菌药物的合理选用具有重要的参考价值.     

乳酸杆菌及嗜热链球菌脉冲场凝胶电泳分子分型方法建立及应用

目的 建立乳酸杆菌及嗜热链球菌的PFGE分子分型方法,并对北京市售酸奶中分离的乳酸杆菌及嗜热链球菌进行分子分型.方法 选取ApaⅠ、NotⅠ、sfiⅠ、XbaⅠ和SmaⅠ共5种PFGE分析中常用的限制性内切酶,对从北京市售酸奶中分离到的52株乳酸杆菌、嗜热链球菌以及相应的标准菌株进行酶切,优化PFGE限制性内切酶种类及电泳条件,并用优化出的实验条件对菌株进行分子分型,同时进行聚类分析,与生化鉴定及16s rRNA基因鉴定结果进行对比分析.结果 限制性内切酶NotⅠ对保加利亚乳酸杆菌、发酵乳酸杆菌和德氏乳酸杆菌的酶切效果较好,而限制性内切酶Apa Ⅰ对嗜热链球菌、嗜酸乳酸杆菌及干酪乳酸杆菌的酶切效果较好.24株保加利亚乳酸杆菌被分为8个PFGE型,15株嗜热链球菌被分为8个PFGE型,7株嗜酸乳酸杆菌被分为3个PFGE型,2株德氏乳酸杆菌分属于2个不同的PFGE型.结论 建立的PFGE方法分析结果与生化鉴定及16s rRNA基因鉴定结果高度符合,所建方法适用于乳酸杆菌及嗜热链球菌的分子分型.     

人-猪土生克雷伯菌感染综合征研究

目的描述人-猪土生克雷伯菌感染综合征临床表现,探讨病原特征以及感染来源.方法观察8例不明原因急性感染中毒休克综合征患者,用多种培养基进行病原菌分离培养,菌种鉴定,动物实验,DNAG C%mol含量测定,药物敏感性试验等方法,确定8例患者引起感染的病原体;用流行病学方法调查感染来源.结果人-猪土生克雷伯菌感染综合征临床表现:发热、呕吐、心慌、呼吸困难、腹痛、腹泻、昏迷、休克、皮肤呈玫瑰红瘀点、瘀斑,主要分布在四肢、头面部及胸部;从患者血液,死猪肉中分离5株细菌,其形态学、生理特征、毒力试验、细菌DNAG C%mol含量测定、药物敏感性试验结果均为一致,证实为土生克雷伯菌肺炎亚种;动物实验表明:此菌株引起动物的症状和人的临床表现一致.结论8例不明原因急性感染中毒休克综合征病原体是土生克雷伯菌肺炎亚种;经流行病学调查表明:此疾病感染来源是病、死猪,病牛.