Comparación de tres técnicas para subtipificación molecular de Listeria monocytogenes / Comparison of three molecular subtyping techniques for Listeria monocytogenes

Abstract Listeria monocytogenes is a foodborne pathogen. The recent alert for L. monocytogenes in vegetables from Argentina warns about the importance of reinforcing its isolation, characterization and subtyping in food, clinical and environmental samples. The aim of the present study was to compare the discriminatory power of enterobacterial repetitive interpower; genic consensus polymerase chain reaction (ERIC-PCR), automated ribotyping and pulsed-field gel electrophoresis (PFGE) to subtype strains of L. monocytogenes isolated from Argentine meat and environmental samples. Simpson's Diversity Index (DI) was calculated on the basis of based on the dendrograms obtained in the by cluster analysis, showing the following discriminatory power: ApaI-PFGE (0.980), AscI-PFGE (0.966), ribotyping (0.912), ERIC-PCR (0.886). The ID values between ApaI- and AscI-PFGE and between ribotyping and ERIC-PCR were not significantly different. Of the three techniques evaluated, PFGE showed the highest discriminatory power. However, the subtyping techniques should be accompanied by effective food monitoring strategies and reliable clinical and epidemiological studies.

Resumen Listeria monocytogenes es un patógeno alimentario. La reciente alerta por la presencia de L. monocytogenes en vegetales en Argentina advierte sobre la importancia de reforzar el aislamiento, la caracterización y la subtipificación de esta bacteria en muestras clínicas de alimentos y ambientales. El objetivo del presente estudio fue comparar el poder discriminatorio de enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), la ribotipificación automatizada y la pulsed-field gel electrophoresis (PFGE) para subtipificar cepas de L. monocytogenes aisladas de carne y de muestras ambientales en Argentina. El índice de diversidad (ID) de Simpson, calculado a partir de los dendrogramas obtenidos en el análisis de agrupamiento, mostró los siguientes resultados: Apal-PFGE (0,980), AscI-PFGE (0,966), riboti-pado (0,912), ERIC-PCR (0,886). Los valores obtenidos no fueron significativamente diferentes al comparar entre Apal- y AscI-PFGE, ni entre ribotipadoy ERIC-PCR. De las técnicas evaluadas, la PFGE presentó el mayor poder discriminatorio. Sin embargo, las técnicas de subtipificación deberían acompañarse de estrategias de control de los alimentos efectivas y de estudios clínicos y epidemiológicos confiables.

Standardization and application on ribotyping library of Clostridioides difficile in China / 中华流行病学杂志

Objective@#To establish a standard operation procedure (SOP) for ribosome genotyping (ribotyping) on Clostridioides (C.) difficile, supplement and verify ribotyping typing library, so as to improve the comparability of data between different laboratories and to develop surveillance network of C. difficil in China.@*Methods@#Molecular typing of 54 reference strains from the United States and Europe of C. difficile were performed by using the SOP referencing correspondence from abroad and from our laboratory with a BioNumerics 7.6 software to estimate the reference library of types of C. difficile. Identification of 374 clinical and animal isolates of C. difficile from 13 cities in China between 2010 and 2018, to supplement the library information. Kappa test was used to evaluate the consistency.@*Results@#Results of capillary electrophoresis of reference strains appeared clear and stable, which guaranteed the clustering results being fast and accurate. Results from the supplementary typing showed that there were 84 types of isolates, of which 25 RT types were consistent with reference strains from abroad, while 58 RT types were different from referenced types. In the 40 referenced types, 15 RT types were not found in this study. In the consistency evaluation, the Kappa value was 0.891 and (P<0.01), showing the two Molecular typing as consistent and with close resemblance.@*Conclusions@#The result of capillary electrophoresis by applying SOP for ribotyping on C. difficile base on QIAxcel capillary electrophoresis system, appeared clear and stable. The standardized library seemed more easily used for comparability and data sharing between the laboratories.

Analysis of toxin and multilocus sequence typing of Clostridium difficile strains isolated from China-Japan Friendship Hospital / 中华微生物学和免疫学杂志

Objective To analyze the characteristics of toxin, the PCR-ribotyping(RT) and the multilocus sequence typing(MLST) of Clostridium difficile strains isolated from China-Japan Friendship Hospital in order to provide a basis for monitoring the outbreak of nosocomial Clostridium difficile infection.Methods A total of 321 samples were collected from the patients with suspected Clostridium difficile infection(CDI) in China-Japan Friendship Hospital(CJFH) during 2012 to 2013.All Clostridium difficile strains were isolated and identified by the standard phenotypic culture method.Cytotoxicity test was performed to detect toxin B.Toxin genes (tcdA and tcdB) and binary toxin genes (cdtA and cdtB) harbored by those strains were analyzed.RT and MLST were used for homologous analysis.Clinical data of the patients were collected to analyze the isolation rate of Clostridium difficile in different populations.Results Forty-eight strains of Clostridium difficile were isolated from 46 patients with diarrhea and three of them were isolated from the same patient.The incidence of CDI among all patients, outpatients and inpatients were 14.3%(46/321), 12.8%(5/39) and 14.5%(41/282), respectively.Toxin B was detected in all of the strains as indicated by the cytotoxicity test.Strains of sequence type 1(ST1) showed the strongest cytotoxicity of all the isolated Clostridium difficile strains.Ten out of the 48 strains (20.8%) were tcdA(-)/tcdB(+) strains, which belonged to either ST37 or ST81.The results of RT and MLST were consistent in assigning the strains into nine types, in which the predominant type was ST1/RT027 accounting for 27.1% (13/48).All of the ST1/RT027 strains presented a toxin gene profile of tcdA(+)/tcdB(+) and cdtA(+)/cdtB(+).Most of the ST1/RT027 strains were isolated from the Traditional Chinese Medicine Department of Respiratory, where smallnosocomial outbreaks of ST1/RT027 strain infection might happen.Conclusion CDI diagnosed in CJFH mainly belongs to nosocomial infection.Most of the isolated strains harbor tcdA(+)/tcdB(+) genes.Surveillance for the outbreaks of CDI caused by ST1/RT027 strains over producing toxins A and B should be strengthened in hospitals.

Establishment of digital polymerase chain reaction-ribotyping and database for Clostridium difficile genotyping / 中华传染病杂志

Objective To develop a digital polymerase chain reaction (PCR) ribotyping method and database for Clostridium difficile genotyping.Methods Sequencer based fluorescence capillary gel electrophoresis was used,instead of agarose gel electrophoresis,to establish the digital PCR-ribotyping of Clostridium difficile.Forty Clostridium difficile reference strains,consisting of 10 PCR-ribotypes (RT),were genotyped by the new digital PCR-ribotyping method to set-up the database.Results The sequencer based fluorescence capillary gel electrophoresis correctly detected PCR-ribotyping products of the 40 reference strains,and showed as digital figure;significant differences of these digital figures were found between the 10 RT.High similar digital figures were shown in twenty-one RT027 strains,three RT002 strains and two RT014 strains.However,seven RT001 strains were typed as four subtypes,and two RT014 strains as two subtypes,respectively.Conclusion A digital PCR-ribotyping,and a reference database consisting of 10 RT are successfully established.

25S rDNA genotype and antifungal susceptibility of vaginal Candida albicans / 中华妇产科杂志

Objective To investigate 25S rDNA genotype distribution of vaginal Candida albicans and its relationship with antifungal susceptibility. Methods A total of 954 isolates of Candida albicans were subjected to genotype analysis according to the presence and the size of transposable groupⅠintron in 25S rDNA with PCR; 156 isolates were enrolled to detect their antifungal susceptibility of 5 antifungal agents with the M27-A3 broth microdilution method. Results Totally 876 isolates were determined to be intronless genotype A (91.8%,876/954); 58 (6.1%, 58/954) and 20 isolates (2.1%, 20/954) were intron-containing genotype B and genotype C, respectively. The minimal inhibitory concentrations (MIC) of itraconazole and fluconazole against the intron-containing group were significantly higher than those of the intronless group (0.25 versus 0.125μg/ml, P<0.05;0.25 versus 0.125μg/ml, P<0.01), whereas the MIC of nystatin against the intron-containing group was lower than that of the intronless group (4 versus 8μg/ml, P<0.01). The resistance rate of Candida albicans to itraconazole of the intron-containing group was significantly higher than that of the intronless group [24% (19/78) versus 3% (2/78), P<0.01]. Cross-resistance analysis revealed 5 of the 21 (24%) itraconazole resistant isolates were cross-resistant to fluconazole ,and 5 of the 6 (5/6) fluconazole-resistant strains were cross-resistant to itraconazole. Besides, all the five strains simultaneously resistant to itraconazole and fluconazole belonged to the intron-containing group. Conclusion The presence of the transposable group Ⅰ intron in 25S rDNA of vaginal Candida albicans might be important in affecting itraconazole, fluconazole and nystatin susceptibility.

Detection of genotype and toxin associated gene of hypervirulent Clostridium difficile clinical isolates / 国际检验医学杂志

Objective To investigate the genotype and variance of toxin associated genes of moxifloxacin‐resistant Clostridium difficile clinical isolates in Sydney .Methods Twenty‐two moxifloxacin‐resistant Clostridium difficile clinical isolates were collected from Sydney ,which were genotyped by using sequencer capillary gel electrophoresis based PCR‐ribotyping ,and toxin A and B cod‐ing gene tcdA and tcdB ,and binary toxin coding gene cdtA and cdtB were detected by using PCR method .Toxin regulator gene tc‐dC was analyzed by using PCR‐sequencing ,and was aligned with reference sequence of VPI 10463 (Genbank accession number :X92982) ,and the tcdC sequence types of all 22 isolates were identified by using blast tool in NCBI .Results Twenty‐one isolates were genotyped as hypervirulent PCR‐ribotypes 027 (RT027) ,and one isolate as RT078 ;all 22 isolates contained tcdA and tcdB for toxin A and B and cdtA and cdtB for binary toxin (tcdA+ tcdB+ cdtA+ cdtB+ ) .The tcdC sequence types of the 21 RT027 i‐solates belong to sc1 ,and that of the one RT078 isolate belongs to WA39 .Compared with tcdC reference sequence of VPI 10463 ,a consecutive 18 bp deletion (nt341 to 379) and one nucleotide deletion at position 117 were found in the 21 RT027 isolates ,and a consecutive 39 bp deletion (nt330 to 368) and one nucleotide mutation at position 184(C> T) were found in the one RT078 isolate . Conclusion Clostridium difficile hypervirulent RT027 was the common moxifloxacin resistant genotype ;Clostridium difficile hy‐pervirulent RT027 and RT078 clinical isolates contained genes for toxin A and B and binary toxin ,and contained gene sequence mu‐tation in toxin regulator gene tcdC .

Application of FTIR in specified microorganisms source tracking for pharmaceutical products / 中国药学杂志

OBJECTIVE: To preliminarily explore the application of FTIR in microbial source tracking for specified microorganisms in pharmaceutical products. METHODS: The repeatability, intermediate precision, and accuracy of FTIR were investigate in selected conditions. RESULTS: Under standard operation and culture conditions, the repeatability, intermediate precision, and accuracy of FTIR met the basic requirements for microbial source tracking as judged by a threshold value of similarity index of greater than 0.95. The tracking results of 12 bacterial strains were almost the same as RiboPrinter analysis. CONCLUSION: FTIR is economic and fast. It is advantageous for promotion of specified microorganisms source tracking in enterprises.

The occurrence and dissemination of methicillin and vancomycin-resistant Staphylococcus in samples from patients and health professionals of a university hospital in Recife, State of Pernambuco, Brazil

Introduction Methicillin-resistant Staphylococcus aureus (MRSA) strains have been responsible for many nosocomial outbreaks. Within hospitals, colonized employees often act as reservoirs for the spread of this organism. This study collected clinical samples of 91 patients admitted to the intensive care unit (ICU), hemodialysis/nephrology service and surgical clinic, and biological samples from the nasal cavities of 120 professionals working in those environments, of a University Hospital in Recife, in the State of Pernambuco, Brazil. The main objective of this study was to determine the occurrence and dissemination of methicillin- and vancomycin-resistant Staphylococcus spp. Methods The isolates obtained were tested for susceptibility to oxacillin and vancomycin and detection of the mecA gene. In addition, the isolates were evaluated for the presence of clones by ribotyping-polymerase chain reaction (PCR). Results MRSA occurrence, as detected by the presence of the mecA gene, was more prevalent among nursing technicians; 48.1% (13/27) and 40.7% (11/27) of the isolates were from health professionals of the surgical clinic. In patients, the most frequent occurrence of mecA-positive isolates was among the samples from catheter tips (33.3%; 3/9), obtained mostly from the hemodialysis/nephrology service. Eight vancomycin-resistant strains were found among the MRSA isolates through vancomycin screening. Based on the amplification patterns, 17 ribotypes were identified, with some distributed between patients and professionals. Conclusions Despite the great diversity of clones, which makes it difficult to trace the source of the infection, knowledge of the molecular and phenotypic profiles of Staphylococcus samples can contribute towards guiding therapeutic approaches in the treatment and control of nosocomial infections. .

The etiology, molecular epidemiology and clinical characteristics of Clostridium difficile infections in Xiangya Hospital / 中华检验医学杂志

Objective A preliminary study on the etiology , the gene typing , the PCR-ribotyping and the clinical features of Clostridium difficile from clinical isolates at Xiangya Hospital could improve the isolation rate and provide the basis for effectively prevention of C.difficile.Methods A prospective observational study was performed.A total of 452 stool samples were collected during June to December 2012 at Xiangya Hospital.All stools were anaerobic cultured by selective medium and identified by API 20A for C.difficile.The positive isolates were detected the toxin genes ( tcdA, tcdB, cdtA, cdtB ) and ribotyping (16S-23S internal spacer region ) by PCR.The clinical data of all patients were collected and analyzed through Logistic regression to discover the risk factors for the development of C.difficile infection ( CDI ) . Results The rate of CDI occurrence was 13.94%(63/452), among them, 42.86%(36/63) were A-B+strains and only 14.29%(9/63) were obtained from community acquired-CDI.No binary toxin was detected in any of the isolates.Eleven different PCR ribotypes were identified , the dominant ribotype CD017 accounted for 22.22%(14/63).Logistic regression analysis showed that the risk factors for CDI included age>55(P=0.016;OR=4.45;95%CI:1.33-14.91), diarrhea frequency(P=0.007, OR=0.03;95%CI:0.002 -0.38 ) and the duration of diarrhea ( P =0.015; OR =7.86; 95%CI: 1.50 -41.16 ) . Conclusions C.difficile is the main pathogens of diarrhea patients and is mainly from hospital infections with higher detection rate of A -B+ in Xiangya Hospital.Ribotyping exist comparative advantages type CD017.No evidence suggests outbreak of C.difficile infection.

Molecular epidemiology of Vibrio cholerae causing outbreaks & sporadic cholera in northern India.

Background & objectives: Several outbreaks of cholera have been reported in Chandigarh region during a span of seven years from 2002-2008. The genetic characteristics of Vibrio cholerae isolates obtained during these outbreaks have not been adequately studied. The aim of this study was to do molecular typing of V. cholerae isolated from the sporadic and outbreak cases by pulsed-field gel electrophoresis (PFGE), Rep-PCR and ribotyping. Methods: Fifty representative isolates of V. cholerae from outbreak as well as sporadic cases were subjected to molecular typing by PFGE, 173 isolates (163 clinical and 10 environmental) were typed by rep-PCR and ribotyping. Ribotyping was done by determination of rRNA restriction pattern of BglI restriction digestion and hybridization with 7.2 kb rRNA probe of pKK3535 plasmid using DIG DNA labelling and detection kit. Universal VC1 primer was used for rep-PCR. Results: PFGE generated 15 pulsotypes, of which four matched the published pulsotypes and there were 11 new pulsotypes. PFGE was the most discriminatory method that could differentiate between isolates belonging to single ribotype. Pulsotype P1 corresponding to known pulsotype H1 was the major pulsotype till 2003. Pulsotype P3 corresponding to known pulsotype L emerged in 2004. The 2007 outbreaks in Punjab and Haryana were caused by P5 though P1 and P3 were isolated from the sporadic cases from the same region. The 2008 outbreak was caused by pulsotypes P6 and P7. Ribotype IV was the most predominant followed by RIII. This ribotype was not isolated after 2003 and ribotype IV became the most predominant 2004 onwards. Of the two unknown ribotypes (UNI and UN2), UNI was more common (27 isolates). Rep-PCR was the least discriminatory and divided all clinical isolates into four major profiles. The dendrogram analysis of PFGE revealed similarity of some clinical isolates with environmental isolates indicating the genetic relatedness. Interpretation & conclusion: Our findings showed that Rep-PCR was least discriminatory method. Ribotyping was a reliable and reproducible method. Ribotype IV was predominant ribotype followed by RIII. A total of 15 pulsotypes were generated and 11 of these were not reported earlier. Genetic relatedness was shown by clinical and environmental isolates which needs to be confirmed in future studies.

Automated ribotyping of Salmonella and Staphylococcus aureus in food poisoning of Guangdong province / 中华流行病学杂志

Objective To understand the genetic polymorphism of Salmonella and Staphylococcus aureus in Guangdong province, as well as to explore methods for identifying and tracing the source of these two foodbome pathogens. Methods Using the automated ribotyping system, two foodbome pathogens were tested with either EcoR Ⅰ or Pvu Ⅱ restriction enzymes. BioNumerics software was then applied for image analysis, database establishment and other corresponding analysis. Results Digestion of 32 Salmonella isolates with Pvu Ⅱ yielded 19 different ribotypes,and digestion of 14 Salmonella isolates with EcoR Ⅰ yielded 2 different ribotypes. Staphyloccus aureus isolates showed greater genetic diversity, whereas EcoR Ⅰ digestion of 49 different isolates yielded 31 different ribotypes. Conclusion Unique Salmonella and Staphylococcus aureus isolates could be identified through ribotyping. Although Salmonella serotyping and ribotyping were not strongly correlated, the combination of both restriction enzymes could be used to more effectively identify the genetic relationship among different strains as well as the source of food poisoning. Thus, not only could the genetic relationships amongst the different strains be inferred through ribotyping skills, the source of food poisoning and mode of transmission could also be determined under the use of this method.

Comparative study on the phenotypic characteristics and molecular typing of foodborne Vibrio parahaemolyticus in Guangdong province / 中华流行病学杂志

Objective To understand the phenotypic characteristics of foodbome Vibrio parahaemolyticus in Guangdong province through carrying out a comprehensive comparison including pulse field gel electrophoresis,ribotyping and serotyping.Methods 74 different Vibrio parahaemolyticus strains isolated from seafood and cases due to food poisoning in Guangdong province were under serotyping and susceptibility testing,in addition to the testing of direct heat hemolysin(tdh)and the heat hemolysin-related hemolysin hormone(trh)via PCR.Ribosomal genotyping(ribotyping)with EcoR Ⅰ restriction enzyme was utilized on 74 different Vibrio parahaemolyticus isolates,whereas pulsed-field gel electrophoresis(PFGE)with the Not Ⅰ restriction enzyme was used on 74 different Vibrio parahaemolyticus isolates.BioNumerics software was used to compare the isolates from different sources,times and places in order to elicit the correlation between different strains.Results Although Vibrio parahaemolyticus was 100.00％ sensitive to chloramphenicol,it still presented different levels of resistance against 13 other antibiotics.Among the 74 different strains of Vibrio parahaemolyticus under testing,24.32％ showed positive for the tdh virulence gene,whereas 4.05％ positive for trh.74 different Vibrio parahaemolyticus strains were found to belong to 26 serotypes,where the O5:K17 and O2:K28 serotypes were dominant in those isolates that causing seafood-poisoning.The O3:K6 serotype was found to be the dominant of those isolates that causing food-poisoning.Based on ribosomal genotyping,the 74 Vibrio parahaemolyticus isolates were divided into 62 different ribotypes,whereas the 74 strains of Vibrio parahaemolyticus were divided into 67 different PFGE types,thus exhibiting considerable genetic diversities of the strains.Conclusion Majority of the isolates causing food-poisoning carried tdh virulence gene.PFGE was shown to have the highest resolution,followed by ribotyping with serotyping being the lowest,where the combination of the three could improve the resolution.

Aislamiento y caracterización molecular, ribotipificación, de cepas nativas de Bacillus thuringiensis aisladas en suelos y larvas muertas de Hylesia metabus en la región nororiental de Venezuela / Isolation and molecular characterization, ribotyping, of Bacillus thuringiensis native strains isolated from soils and Hylesia metabus death larvae at northeast region of Venezuela

Bacillus thuringiensis es una bacteria esporogénica grampositiva del grupo B. cereus, caracterizada por la producción de δ-endotoxinas codificadas por una familia de genes cry, mayoritariamente plasmídicos con actividad patógena específica contra larvas de insectos. Esta propiedad ha sido usada en la formulación de productos comerciales para control de insectos plaga como Hylesia metabus “palometa peluda” que afecta a los pobladores de la región nororiental de Venezuela. En el Centro Venezolano de Colecciones de Microorganismos se ha iniciado la construcción de una colección de cepas nativas de B. thuringiensis, a partir de muestras de suelos y larvas muertas de H. metabus colectadas en zonas afectadas, que pudiera ser utilizada en la elaboración local de bioinsecticidas para control de ésta y otras plagas. El empleo de condiciones selectivas de crecimiento, pruebas bioquímicas complementarias y presencia de inclusiones parasporales, seguido de la caracterización molecular empleando la técnica de ribotipificación automatizada, permitió la identificación de 11 cepas nativas de B. thuringiensis. La variedad de ribotipos encontrados refleja la heterogeneidad genética de B. thuringiensis en la muestra examinada.

Bacillus thuringiensis is a Gram positive spore-forming bacteria of the B. cereus group, that produces δ-endotoxins, encoded mainly by cry genes, with pathogenic specific activity against some insect larvae. This property has been used in products applied to the control of Hylesia metabus, a pest known as “palometa peluda” that affects living population in the northeast region of Venezuela. In the Venezuelan Center for Culture Collections we have initiated the construction of a collection of B. thuringiensis native strains isolated from soils and H. metabus death larvae that could be applied in pest control. The use of selective growth conditions, biochemical tests and detection of parasporal inclusions, followed by ribotyping methods, allowed the identification of 11 B. thuringiensis native strains. Variety of ribotypes shows the genetic heterogeneity of B. thuringiensis identified.

Antimicrobial resistance and PCR-ribotyping of Shigella responsible for foodborne outbreaks occurred in southern Brazil

Little information about Shigella responsible for foodborne shigellosis is available in Brazil. The present study aimed to investigate the antimicrobial resistance and PCR-ribotyping patterns of Shigella isolates responsible for foodborne outbreaks occurred in Rio Grande do Sul State (RS), Southern Brazil in the period between 2003 and 2007. Shigella strains (n=152) were isolated from foods and fecal samples of victims of shigellosis outbreaks investigated by the Surveillance Service. Identification of the strains at specie level indicated that 71.1 percent of them were S. flexneri, 21.5 percent S. sonnei, and 0.7 percent S. dysenteriae. Ten strains (6.7 percent) were identified only as Shigella spp. An increasing occurrence of S. sonnei was observed after 2004. Most of the strains were resistant to streptomycin (88.6 percent), followed by ampicillin (84.6 percent), and sulfamethoxazole/trimethoprim (80.5 percent). Resistant strains belonged to 73 patterns, and pattern A (resistance to ampicillin, sulfamethoxazole/trimethoprim, tetracycline, streptomycin, chloramphenicol, and intermediate resistance to kanamycin) grouped the largest number of isolates (n=36). PCR-ribotyping identified three banding patterns (SH1, SH2, and SH3). SH1 grouped all S. flexneri and SH2 grouped all S. sonnei. The S. dysenteriae strain belonged to group SH3. According to the results, several Shigella isolates shared the same PCR-rybotyping banding pattern and the same resistance profile, suggesting that closely related strains were responsible for the outbreaks. However, other molecular typing methods need to be applied to confirm the clonal relationship of these isolates.

Post-operative endophthalmitis: Antibiogram & genetic relatedness between Pseudomonas aeruginosa isolates from patients & phacoemulsifiers.

Background & objectives: Though not frequently but there are reports showing phacoemulsifiers as a potent source of infection in post-operative cases of endophthalmitis. This study was carried out to find antibiogram and genetic relatedness between Pseudomonas aeruginosa isolates from a post-cataract surgery endophthalmitis outbreak (3 patients) and internal tubings of 5 phacoemulsifiers. Methods: In vitro antimicrobial sensitivity patterns of the 8 bacterial isolates were observed. Genetic analysis of the bacterial isolates was done using random amplification of polymorphic DNA (RAPD) assay and PCR ribotyping. The resulting DNA band patterns were examined visually and by computer assisted analysis using unweighted pair group method. Results: The three P. aeruginosa patient isolates were found to be different from the five phacoemulsifier isolates in sensitivity towards 3 antibiotics and by genetic analysis (33 and 44% homology by RAPD assay and PCR ribotyping). Two of the patient isolates shared 100 per cent genetic homology by RAPD assay and another pair shared 100 per cent homology by PCR ribotyping. The five isolates from phacoemulsifiers did not share significant genetic homology. There was significant genetic variation between bacterial isolates from patients and phaco emulsifiers. Interpretation & conclusion: Though the three P. aeruginosa isolates obtained from the patients were phenotypically similar and genetically close, they differed from the phaco-machine isolates both genetically, and in their antibiogram profile. However, the five phacoemulsifier isolates were genetically diverse though they shared the same antibiogram profile. Therefore the Ringer’s lactate from phacomachines could not be conclusively proven to be the source of infection.

Investigation of Toxin Gene Diversity, Molecular Epidemiology, and Antimicrobial Resistance of Clostridium difficile Isolated from 12 Hospitals in South Korea / 대한진단검사의학회지

BACKGROUND: Clostridium difficile is a major cause of antibiotic-associated diarrhea. The objective of this study was to characterize clinical isolates of C. difficile obtained from various regions in Korea with regard to their toxin status, molecular type, and antimicrobial susceptibility. METHODS: We analyzed a total of 408 C. difficile isolates obtained between 2006 and 2008 from 408 patients with diarrhea in 12 South Korean teaching hospitals. C. difficile toxin genes tcdA, tcdB, cdtA, and cdtB were detected by PCR. Molecular genotyping was performed by PCR ribotyping. Antimicrobial susceptibilities of the 120 C. difficile isolates were assessed by agar dilution methods. RESULTS: Among 337 toxigenic isolates, 105 were toxin A-negative and toxin B-positive (A-B+) and 29 were binary toxin-producing strains. PCR ribotyping showed 50 different ribotype patterns. The 5 most frequently occurring ribotypes comprised 62.0% of all identified ribotypes. No isolate was susceptible to cefoxitin, and all except 1 were susceptible to piperacillin and piperacillin-tazobactam. The resistance rates of isolates to imipenem, cefotetan, moxifloxacin, ampicillin, and clindamycin were 25%, 34%, 42%, 51%, and 60%, respectively. The isolates showed no resistance to metronidazole or vancomycin. CONCLUSIONS: This is the first nationwide study on the toxin status, including PCR ribotyping and antimicrobial resistance, of C. difficile isolates in Korea. The prevalence of A-B+ strains was 25.7%, much higher than that reported from other countries. Binary toxin-producing strains accounted for 7.1% of all strains, which was not rare in Korea. The most prevalent ribotype was ribotype 017, and all A-B+ strains showed this pattern. We did not isolate strains with decreased susceptibility to metronidazole or vancomycin.

Ribotyping, biotyping and capsular typing of Haemophilus influenzae strains isolated from patients in Campinas, southeast Brazil

Forty-five Haemophilus influenzae strains isolated from patients were characterized based on biochemical characteristics. Their capsular types were determined by polymerase chain reaction (PCR); they were compared, using two molecular methods [ribotyping with a specific DNA probe amplified from the 16S rDNA region from H. influenzae and through restriction fragment length polymorphism (RLFP) of an amplified 16S DNA region]. The strains were better discriminated by the ribotyping technique that used the 16S probe and by the combination of both techniques. Biotypes I and IV were the most common, followed by biotypes VI, VIII and III. Biotypes II and VII were not found. Most of the capsular samples were nontypable (89 percent), with capsular types a and b found in 2 and 9 percent of the samples, respectively. We concluded that there is a very close genetic identity among pathogenic and non-pathogenic strains.

Application of PCR ribotyping and tDNA-PCR for Klebsiella pneumoniae identification

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90,6 percent of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.

Ribotyping and virulence markers of Yersinia pseudotuberculosis strains isolated from animals in Brazil

Ribotyping and virulence markers has been used to investigate 68 Yersinia pseudotuberculosis strains of serogroups O:1a and O:3. The strains were isolated from clinical material obtained from healthy and sick animals in the Southern region of Brazil. Ribotypes were identified by double digestion of extracted DNA with the restriction endonucleases SmaI and PstI, separation by electrophoresis and hybridization with a digoxigenin-labeled cDNA probe. The presence of the chromosomal virulence marker genes inv, irp1, irp2, psn, ybtE, ybtP-ybtQ, and ybtX-ybtS, of the IS100 insertion sequence, and of the plasmid gene lcrF was detected by polymerase chain reaction. The strains were grouped into four distinct ribotypes, all of them comprising several strains. Ribotypes 1 and 4 presented distinct profiles, with 57.3 percent genetic similarity, ribotypes 2 and 3 presented 52.5 percent genetic similarity, and genetic similarity was 45 percent between these two groups (1/4 and 2/3). All strains possessed the inv, irp1, and irp2 genes. Additionally, strains of serogroup O:1a carried psn, ybtE, ybtP-ybtQ, ybtX-ybtS, and IS100. As expected lcrF was only detected in strains harboring the virulence plasmid. These data demonstrate the presence of Y. pseudotuberculosis strains harboring genotypic virulence markers in the livestock from Southern Brazil and that the dissemination of these bacteria may occur between herds.

Molecular Analysis of Clostridium difficile Isolates by Arbitrarily Primed-Polymerase Chain Reaction and Polymerase Chain Reaction-Ribotyping / 감염

BACKGROUND: Clostridium difficile is known as the major cause of nosocomially acquired diarrhea. Various phenotypic and genotypic methods have been used to subtype C. difficile strains. The purpose of the present study is to evaluate several typing methods which can be used as tools for subtyping C. difficile isolates for epidemiological studies. METHODS: In two Korean tertiary care hospitals, a total of 81 C. difficile isolates were collected from symptomatic, hospitalized patients in 1998. All isolates were examined for the release of toxin A and toxin B by PCR assay and cell culture assay. Also arbitrarily primed-PCR and PCR-ribotyping profiles were determined for the typing of C. difficile strains on a genetic level. RESULTS: The toxin B gene was detected in 65.4% (54/81) of isolates by both PCR assay and cell cultureassay. Nine types were identified with T-7 primer, and 13 types were identified with PG-05 primer in AP- PCR. Sixteen types were identified in PCR-ribotyping. When two typing methods were compared, reproducibility by PCR-ribotyping was 100%, while it was only 83% and 33% AP-PCR with primer T-7, and PG-05, respectively. The discrimination index was 0.88 for PCR-ribotyping, 0.82 for AP-PCR with primer T-7 and 0.81 with primer PG-05. CONCLUSION: These data suggest that PCR-ribotyping provides a reproducible, discriminatory, and simple alternative to conventional molecular approaches for typing strains of C. difficile.