Mobile genetic elements associated with carbapenemase genes in South American Enterobacterales

ABSTRACT Introduction: Carbapenem resistance in members of order Enterobacterales is a growing public health problem causing high mortality in developing and industrialized countries. Its emergence and rapid propagation worldwide was due to both intercontinental spread of pandemic strains and horizontal dissemination via mobile genetic elements (MGE) such as plasmids and transposons. Objective: To describe MGE carrying carbapenem resistance genes in Enterobacterales which have been reported in South America. Search strategy and selection criteria: A search of the literature in English or Spanish published until 2019 in PubMed, Google Scholar, LILACS and SciELO databases was performed for studies of MGE in Enterobacterales reported in South American countries. Results: Seven South American countries reported MGE related to carbapenemases. Carbapenemase-producing Klebsiella pneumoniae belonging to clonal complex 258 were the most prevalent pathogens reported; others carbapenemase-producing Enterobacterales such as Escherichia coli, Serratia marcescens, and Providencia rettgeri also have been reported. The MGE implicated in the spread of the most prevalent carbapenemase genes are Tn4401 and non-Tn4401 elements for bla KPC and ISAba125 for bla NDM, located in different plasmid incompatibility groups, i.e. L/M, A/C, FII and bacterial clones. Conclusion: This review indicates that, like in other parts of the world, the most commonly reported carbapenemases in Enterobacterales from South America are being disseminated through clones, plasmids, and transposons which have been previously reported in other parts of the world.

Genotipificación de aislamientos del complejo Mycobacterium tuberculosis mediante MIRU-VNTR, Cali, Colombia, 2013-2015 / MIRU-VNTR genotyping of Mycobacterium tuberculosis in a population of patients in Cali, Colombia, 2013-2015

Resumen Introducción. La tuberculosis continúa siendo uno de los problemas de salud más importantes a nivel mundial y, con la infección por el virus de la inmunodeficiencia humana (HIV), constituye la principal causa de muerte por infecciones. En el 2016, se notificaron 6,3 millones de casos nuevos de la enfermedad. Objetivo. Describir los patrones genéticos determinados mediante la genotipificación del número variable de repeticiones en tándem de unidades repetitivas interespaciadas de micobacterias (Mycobacterial Interspersed Repetitive Units - Variable Number of Tandem Repeats, MIRU-VNTR) en la población de estudio y compararlos con los hallados en otros estudios locales e internacionales. Materiales y métodos. Mediante MIRU-VNTR, entre el 2013 y el 2015 se hizo la genotipificación de 105 muestras de ADN extraídas del esputo o de aislamientos en cultivo de M. tuberculosis provenientes de pacientes residentes en Cali con diagnóstico de tuberculosis pulmonar. La amplificación de 24 loci MIRU-VNTR se hizo por medio de la reacción en cadena de la polimerasa (PCR). Los amplicones resultantes se visualizaron por electroforesis en geles de agarosa (2 %) teñidos con SYBR Safe™. La asignación de los alelos se hizo con un análisis gráfico con el programa GelAnalyzer 2010. Los resultados obtenidos se analizaron con el algoritmo UPGMA y se compararon con las bases de datos internacionales MIRU-VNTRplus y SITVITWEB. Resultados. Se genotipificaron por completo 62 de las muestras y se obtuvieron 58 perfiles diferentes de MIRU-VNTR. Al comparar con las bases de datos internacionales, su distribución por linajes fue la siguiente: 54,8 % para el LAM, 25,8 % para el Haarlem, 14,5 % para el S, 3,2 % para el Beijing y 1,6 % para el Cameroon. Los patrones MIRU-VNTR correspondieron a 20 tipos internacionales de MIRU (MIRU International Types, MIT) diferentes, y los más frecuentes fueron el MIT 190 y el MIT 110, con 22,6 y 6,5 %, respectivamente. Conclusión. Estos resultados confirmaron hallazgos previos sobre el predominio de los linajes LAM y Haarlem en la ciudad y la presencia de los MIT encontrados en otra ciudad de Colombia.

Abstract Introduction: Tuberculosis continues to be one of the main public health problems in the world. Together with the HIV infection, it is one of the main causes of death due to infections worldwide. In 2016, 6.3 million new cases of the disease were reported. Objective: To describe the genetic patterns determined by genotyping using variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) in the study population and compare them with other studies carried out in Cali, Colombia, and the world. Materials and methods: We genotyped a total of 105 DNA samples extracted from sputum or culture isolates of the Mycobacterium tuberculosis complex, which were obtained from pulmonary tuberculosis diagnosed patients over the period 2013-2015, in Cali. We performed PCR amplification of 24 loci by MIRU-VNTR on the DNA extracted from the samples. The amplicons were visualized in agarose gel electrophoresis (2%) with SYBR Safe™ staining. Then, the alleles were designated by graphical analysis using the GelAnalyzer 2010 software. These results were analyzed using the UPGMA logarithm and compared with the registers from the MIRU-VNTR plus and SITVITWEB databases. Results: We genotyped 62 of the samples completely and we obtained 58 different MIRU-VNTR profiles. By comparing with the international databases, we determined the following distributions per lineage: LAM, 54.8%; Haarlem,25.8%; S, 14.5%; Beijing, 3.2%, and Cameroon, 1.6%. The MIRU-VNTR patterns corresponded to 17 different MITs; the most frequent were MIT 190 and MIT 110, with 22.6% and 6.5%, respectively. Conclusions: These results demonstrated previous observations about the predominance of the LAM and Haarlem lineages in the city, and the presence of the MITs found in another city of Colombia.

Methicillin-resistant Staphylococcus aureus isolated from an intensive care unit in Minas Gerais, Brazil, over a six-year period

ABSTRACT To characterize methicillin-resistant Staphylococcus aureus isolates from an intensive care unit of a tertiary-care teaching hospital, between 2005 and 2010. A total of 45 isolates were recovered from patients admitted to the intensive care unit in the study period. Resistance rates higher than 80% were found for clindamycin (100%), erythromycin (100%), levofloxacin (100%), azithromycin (97.7%), rifampin (88.8%), and gentamycin (86.6%). The SCCmec typing revealed that the isolates harbored the types III (66.7%), II (17.8%), IV (4.4%), and I (2.2%). Four (8.9%) isolates carried non-typeable cassettes. Most (66.7%) of the isolates were related to the Brazilian endemic clone from CC8/SCCmec III, which was prevalent (89.3%) between 2005 and 2007, while the USA100/CC5/SCCmec II lineage emerged in 2007 and was more frequent in the last few years. The study showed high rates of antimicrobial resistance among methicillin-resistant S. aureus isolates and the replacement of Brazilian clone, a well-established hospital lineage, by the USA100 in the late 2000s, at the intensive care unit under study.

Molecular diversity analysis of Tetradium ruticarpum (WuZhuYu) in China based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers / 中国天然药物

"Wu zhu yu", which is obtained from the dried unripe fruits of Tetradium ruticarpum (A. Jussieu) T. G. Hartley, has been used as a traditional Chinese medicine for treatment of headaches, abdominal colic, and hypertension for thousands of years. The present study was designed to assess the molecular genetic diversity among 25 collected accessions of T. ruticarpum (Wu zhu yu in Chinese) from different areas of China, based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers. Thirteen ISSR primers generated 151 amplification bands, of which 130 were polymorphic. Out of 165 bands that were amplified using 10 iPBS primers, 152 were polymorphic. The iPBS markers displayed a higher proportion of polymorphic loci (PPL = 92.5%) than the ISSR markers (PPL = 84.9%). The results showed that T. ruticarpum possessed high loci polymorphism and genetic differentiation occurred in this plant. The combined data of iPBS and ISSR markers scored on 25 accessions produced five clusters that approximately matched the geographic distribution of the species. The results indicated that both iPBS and ISSR markers were reliable and effective tools for analyzing the genetic diversity in T. ruticarpum.

Molecular diversity analysis of Tetradium ruticarpum (WuZhuYu) in China based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers / 中国天然药物

"Wu zhu yu", which is obtained from the dried unripe fruits of Tetradium ruticarpum (A. Jussieu) T. G. Hartley, has been used as a traditional Chinese medicine for treatment of headaches, abdominal colic, and hypertension for thousands of years. The present study was designed to assess the molecular genetic diversity among 25 collected accessions of T. ruticarpum (Wu zhu yu in Chinese) from different areas of China, based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers. Thirteen ISSR primers generated 151 amplification bands, of which 130 were polymorphic. Out of 165 bands that were amplified using 10 iPBS primers, 152 were polymorphic. The iPBS markers displayed a higher proportion of polymorphic loci (PPL = 92.5%) than the ISSR markers (PPL = 84.9%). The results showed that T. ruticarpum possessed high loci polymorphism and genetic differentiation occurred in this plant. The combined data of iPBS and ISSR markers scored on 25 accessions produced five clusters that approximately matched the geographic distribution of the species. The results indicated that both iPBS and ISSR markers were reliable and effective tools for analyzing the genetic diversity in T. ruticarpum.

Participation of the arcRACME protein in self-activation of the arc operon located in the arginine catabolism mobile element in pandemic clone USA300

ABSTRACT Staphylococcus aureus pandemic clone USA300 has, in addition to its constitutive arginine catabolism (arc) gene cluster, an arginine catabolism mobile element (ACME) carrying another such cluster, which gives this clone advantages in colonisation and infection. Gene arcR, which encodes an oxygen-sensitive transcriptional regulator, is inside ACME and downstream of the constitutive arc gene cluster, and this situation may have an impact on its activation. Different relative expression behaviours are proven here for arcRACME and the arcACME operon compared to the constitutive ones. We also show that the artificially expressed recombinant ArcRACME protein binds to the promoter region of the arcACME operon; this mechanism can be related to a positive feedback model, which may be responsible for increased anaerobic survival of the USA300 clone during infection-related processes.

Detection of Integrons and Staphylococcal Cassette Chromosome mec Types in Clinical Methicillin-resistant Coagulase Negative Staphylococci Strains

OBJECTIVES: Integrons are thought to play an important role in the spread of antibiotic resistance. This study investigates class 1 and 2 integron-positive methicillin-resistant coagulase-negative staphylococci strains isolated in Iran and characterizes their patterns of antimicrobial resistance. METHODS: Hundred clinical isolates of coagulase-negative staphylococci were characterized for integron content and staphylococcal cassette chromosome mec (SCCmec) type. RESULTS: Sixteen isolates carried class 1 (intI1) integrons and four isolates carried class 2 (intI2) integrons. One resistance gene array was identified among the class 1 integrons (aadA1 cassette). The distribution of SCCmec types in 50 methicillin-resistant coagulase-negative staphylococci strains showed that SCCmec types III and V dominated among the tested strains. CONCLUSION: This is the first report of methicillin-resistant coagulase-negative staphylococci strains that carry two mobile genetic elements, including class 1 and 2 integrons and SCCmec, in Iran.

Correlations between major risk factors and closely related Mycobacterium tuberculosis isolates grouped by three current enotyping procedures: a population-based study in northeast Mexico

The characteristics of tuberculosis (TB) patients related to a chain of recent TB transmissions were investigated. Mycobacterium tuberculosis (MTB) isolates (120) were genotyped using the restriction fragment length polymorphism-IS6110 (R), spacer oligotyping (S) and mycobacterial interspersed repetitive units-variable number of tandem repeats (M) methods. The MTB isolates were clustered and the clusters were grouped according to the similarities of their genotypes. Spearman’s rank correlation coefficients between the groups of MTB isolates with similar genotypes and those patient characteristics indicating a risk for a pulmonary TB (PTB) chain transmission were ana- lysed. The isolates showing similar genotypes were distributed as follows: SMR (5%), SM (12.5%), SR (1.67%), MR (0%), S (46.67%), M (5%) and R (0%). The remaining 35 cases were orphans. SMR exhibited a significant correlation (p < 0.05) with visits to clinics, municipalities and comorbidities (primarily diabetes mellitus). S correlated with drug consumption and M with comorbidities. SMR is needed to identify a social network in metropolitan areas for PTB transmission and S and M are able to detect risk factors as secondary components of a transmission chain of TB.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

Genome-Wide Identification and Classification of MicroRNAs Derived from Repetitive Elements

MicroRNAs (miRNAs) are known for their role in mRNA silencing via interference pathways. Repetitive elements (REs) share several characteristics with endogenous precursor miRNAs. In this study, 406 previously identified and 1,494 novel RE-derived miRNAs were sorted from the GENCODE v.19 database using the RepeatMasker program. They were divided into six major types, based on their genomic structure. More novel RE-derived miRNAs were confirmed than identified as RE-derived miRNAs. In conclusion, many miRNAs have not yet been identified, most of which are derived from REs.

Efficient discrimination by MIRU-VNTRs of Mycobacterium tuberculosis clinical isolates belonging to the predominant SIT11/EAI3-IND ancestral genotypic lineage in Kerala, India

The present study evaluated the ability of MIRU-VNTRs to discriminate Mycobacterium tuberculosis [MTB] clinical isolates belonging to the SIT11/EAI3-IND ancestral genotypic lineage, which is highly prevalent in Kerala, India. Starting from 168 MTB clinical isolates, spoligotyping [discriminatory index of 0.9113] differentiated the strains into 68 distinct patterns, the biggest cluster being SIT11/48 SIT11 [n = 48]. The present study shows that 12-loci MIRUs and 3 ETRs allowed an efficient discrimination of these isolates [discriminatory indexes of 0.7819 and 0.5523, respectively]

ermA, ermC , tetM and tetK are essential for erythromycin and tetracycline resistance among methicillin-resistant Staphylococcus aureus strains isolated from a tertiary hospital in Malaysia.

The objective of this study was to determine the expression and transferability of tetracycline and erythromycin resistance among 188 MRSA strains from a Malaysian tertiary hospital. The minimum inhibitory concentrations (MICs) for oxacillin, erythromycin, tetracycline and ciprofloxacin ranged from 4 to 512 μg/ml, 0.25 to 256 μg/ml, 0.5 to 256 μg/ml and 0.5 to 512 μg/ml, respectively. Tetracycline-resistant strains showed co-resistance towards ciprofloxacin and erythromycin. There was a significant increase (P<0.05) of high-level tetracycline (≥MIC 256 μg/ml) and erythromycin (≥MIC 128 μg/ml) resistant strains in between the years 2003 and 2008. All erythromycin-resistant strains harboured ermA or ermC gene and all tetracycline-resistant strains harboured tetM or tetK gene. The blaZ was detected in all MRSA strains, whereas ermA, tetM, ermC, tetK and msrA genes were detected in 157 (84%), 92 (49%), 40 (21%), 39 (21%) and 4 (2%) MRSA strains, respectively. The blaZ, tetM, ermC and tetK genes were plasmid-encoded, with ermC gene being easily transmissible. Tn5801-like transposon was present in 78 tetM-positive strains. ermA and tetM genes were the most prevalent erythromycin and tetracycline resistance determinants, respectively, in MRSA strains. The association of resistance genes with mobile genetic elements possibly enhances the spread of resistant traits in MRSA.

SXT constin among Vibrio cholerae isolates from a tertiary care hospital.

Background & objectives: The SXT element, also known as ‘constin’ (conjugable, self transmissible, integrating element) is an integrating conjugative element (ICE) in Vibrio cholerae discovered in the chromosome of epidemic V. cholerae O139 strain MO10 (SXTMO10) which arose in late 1992 in Chennai, India. SXT related ICEs have become widespread and currently, most if not all Asian V. cholerae clinical isolates contain SXT related ICEs. The present study attempts to determine the presence of SXT Int gene in V. cholerae recovered between 2005 to 2007 in a tertiary care hospital, demonstrate its conjugal nature and also detect co-presence and co-transfer of plasmids in representative isolates. Methods: This prospective study was done on 116 V. cholerae isolates [114- O1 (107 ogawa and 7 inaba) and 2 - Non O1 Non O139 V. cholerae] from watery stools between 2005 to 2007 recovered from equal number of patients. PCR was carried out using SXT Int specific primers that produced a 592 bp internal fragment of SXT element, and rifampicin resistant strain of E.coli K-12 was used as recipient in conjugation experiments to study transfer of SXT, as also co-transfer of resistance to tetracycline, erythromycin, and nalidixic acid. Antibiotic susceptibility was performed against various antibiotics. Results: Of the 116 isolates, 110 (94.8%) were positive for SXT element by PCR. It was demonstrated in 94.7 per cent of the O1, and 100 per cent of non O1 non O139 V. cholerae. All 2005 isolates, 25 per cent of 2006 isolates and 96.6 per cent of 2007 isolates were positive for SXT. Thirty two drug resistance patterns were observed and the 2007 isolates showed resistance to as many as eight antibiotics. The resistance of SXT positive isolates was higher than those of SXT negative and the typical drug resistance pattern corresponding to SXTET and SXTMO10 was shown by only one V. cholerae O1 isolate. Successful conjugal transfer of SXT was seen in 31 (88.6%) of the 35 isolates studied without any co-transfer while, presence of plasmids was observed in two of the 31 donor V. cholerae studied. Interpretation & Conclusions: The demonstration of SXT element and its successful horizontal transfer in V. cholerae isolates studied emphasizes the need for its detection to monitor antibiotic resistance and dissemination in V. cholerae.

Xylella fastidiosa comparative genomic database is an information resource to explore the annotation, genomic features, and biology of different strains

The Xylella fastidiosa comparative genomic database is a scientific resource with the aim to provide a user-friendly interface for accessing high-quality manually curated genomic annotation and comparative sequence analysis, as well as for identifying and mapping prophage-like elements, a marked feature of Xylella genomes. Here we describe a database and tools for exploring the biology of this important plant pathogen. The hallmarks of this database are the high quality genomic annotation, the functional and comparative genomic analysis and the identification and mapping of prophage-like elements. It is available from web site http://www.xylella.lncc.br.

Mycobacterial Interspersed Repetitive Unit typing in Mycobacterium tuberculosis isolates from Sichuan Province in China.

Background & objectives: Emergence and spread of drug resistant Mycobacterium tuberculosis is a serious threat to tuberculosis (TB) control programme. Therefore, the objective of this study was to genotype drug-resistant M. tuberculosis strains isolated from patients in Sichuan, China, using Mycobacterial Interspersed Repetitive Units (MIRU) for epidemiological analysis. Methods: Drug-resistance testing of M. tuberculosis isolates from pulmonary TB patients was confirmed by proportion method. Twelve MIRU loci were analyzed on 80 drug-resistant and 9 susceptible isolates by polymerase chain reaction and agarose gel electrophoresis. Hunter-Gaston discriminatory index (HGI) values were determined for each 12 MIRU loci for the evaluation of their discrimination power. Results: Among 12 MIRU loci examined, polymorphic bands could be generated on 11 loci. Sixty five isolates had distinct MIRU patterns, while other 24 belonged to 8 clusters and resistant to at least one anti-TB drug tested. The association between the MIRU patterns and the mutation patterns of drug-resistance relevant target genes was not significant among the drug-resistant isolates. Interpretation & conclusions: The results showed that with a satisfactory discrimination power exhibited, the 12 loci based MIRU typing could be a valuable tool for epidemiological studies in M. tuberculosis isolates from Sichuan.

Exploring the Antarctic soil metagenome as a source of novel cold-adapted enzymes and genetic mobile elements / Explorando el metagenoma del suelo antártico como fuente de nuevas enzimas adaptadas al frío y elementos génicos móviles

Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications, and for the detection of genetic elements associated with gene mobilization, respectively. Fourteen lipase/esterase-, 14 amylase-, 3 protease-, and 11 cellulase-producing clones were detected by activity-driven screening, with apparent maximum activities around 35 °C for both amylolytic and lipolytic enzymes, and 35-55 °C for cellulases, as observed for other cold-adapted enzymes. However, the behavior of at least one of the studied cellulases is more compatible to that observed for mesophilic enzymes. These enzymes are usually still active at temperatures above 60 °C, probably resulting in a psychrotolerant behavior in Antarctic soils. Metagenomics allows to access novel genes encoding for enzymatic and biophysic properties from almost every environment with potential benefits for biotechnological and industrial applications. Only intI- and tnp-like genes were detected by PCR, encoding for proteins with 58-86 %, and 58-73 % amino acid identity with known entries, respectively. Two clones, BAC 27A-9 and BAC 14A-5, seem to present unique syntenic organizations, suggesting the occurrence of gene rearrangements that were probably due to evolutionary divergences within the genus or facilitated by the association with transposable elements. The evidence for genetic elements related to recruitment and mobilization of genes (transposons/integrons) in an extreme environment like Antarctica reinforces the hypothesis of the origin of some of the genes disseminated by mobile elements among "human-associated" microorganisms.

A partir de muestras de suelo antártico se obtuvo la metagenoteca PP1. Esta fue sometida a análisis funcionales y genotípicos para el aislamiento de nuevas enzimas adaptadas al frío con potenciales aplicaciones, y para la detección de elementos génicos asociados a la movilización de genes, respectivamente. Por tamizaje fenotípico se detectaron 14, 14, 3 y 11 clones productores de lipasas/esterasas, proteasas, amilasas y celulasas, respectivamente, con actividades máximas aparentes de 35 °C para las amilasas y lipasas, y de 35-55 °C para las celulasas, tal como se observó para otras enzimas adaptadas al frío. Sin embargo, una celulasa parece ser compatible con enzimas mesófilas, las que usualmente se mantienen activas hasta por sobre 60 °C. Este hecho probablemente esté asociado a un comportamiento psicrotolerante en los suelos antárticos. La metagenómica permite acceder a una nueva miríada de productos metabólicos con potenciales beneficios para aplicaciones biotecnológicas e industriales. Se detectaron los genes tipo intI y tnp por PCR, y sus productos génicos deducidos tuvieron identidades del 58 al 86 % y del 58 al 73 % con secuencias conocidas, respectivamente. Dos clones, BAC 27A-9 y BAC 14A-5, parecen presentar organizaciones sintéticas únicas, lo cual sugiere la existencia de rearreglos génicos probablemente debidos a divergencias evolutivas dentro del género o facilitados por la asociación de elementos de transposición. La evidencia de elementos génicos relacionados con el reclutamiento y la movilización de genes en ambientes extremos como la Antártida refuerza la hipótesis sobre el origen de algunos genes diseminados por elementos móviles entre los microorganismos asociados al ser humano.

Avaliação dos locos CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) em estudos epidemiológicos de cepas de Yersinia pestis / Evaluation of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci in epidmiologic study of strains of Yersinia pestis

Yersinia pestis é o agente causador da peste, doença primária de roedores, transmitida por pulgas infectadas, podendo infectar o homem e outros mamíferos. A maioria dos estudos de genotipagem realizada em cepas brasileiras de Y. pestis demonstrou baixo poder discriminatório, revelando padrões genotípicos semelhantes para cepas com diferentes características epidemiológicas. A análise do clustered regularly interspaced short palindromic repeats (CRISPR) vem sendo utilizada para genotipagem e estudos filogenéticos em diferentes gêneros bacterianos, inclusive de Y. pestis. Neste estudo foi realizada a genotipagem de cepas de Y. pestis da coleção de cultura do Serviço Nacional de Referência em Peste do Centro de Pesquisas Aggeu Magalhães - CPqAM/FIOCRUZ, isoladas nos três focos de peste do Estado de Pernambuco, pela análise dos locos CRISPR. Para as amplificações das 51 amostras, foram utilizados primers dirigidos às sequências CRISPR descritas na literatura (YPa, YPb e YPc). Os amplicons obtidos, após PCR, foram sequenciados, a fim de analisar a estrutura de cada loco CRISPR. Dois locos se apresentaram polimórficos: YPa, com alelos de 150pb a 570pb e YPb, com alelos de 330pb a 331pb. O loco YPc se mostrou monomórfico com alelos de 208pb. Os padrões CRISPR obtidos para cepas de Y. pestis de focos de outros países foram os mesmos observados nas cepas brasileiras. Diferentes arranjos dos espaçadores (YPa, YPb e YPc) foram observados e possibilitou que as cepas fossem agrupadas em 12 perfis genotípicos (PG1-PG12). Dois perfis foram distribuídos nos três focos estudados: PG1 perfil mais frequente (38 cepas) e PG3 (seis cepas). Os demais perfis foram específicos de uma determinada região de isolamento: PG2 para o foco de Triunfo e PG4-PG7 para o foco de Araripe. Os perfis PG8 a PG12 representaram o restante das cepas de focos de outros países. As mesmas cepas foram analisadas, anteriormente, através de onze locos VNTR (MLVA), e foram agrupadas em 35 perfis genotípicos

A menor diversidade observada no CRISPR ocorre, provavelmente, devido à região estar envolvida na codificação gênica e com maior pressão seletiva, portanto, com menor risco de sofrer mutação decorrente de estocagem. A análise dos locos CRISPR, complementar ao uso do MLVA, será útil na identificação e rastreamento de novas cepas, contribuindo para o estabelecimento de medidas de controle adequadas nas áreas de foco para prevenção da peste e na investigação de novos casos que possam surgir no Brasil

Comparison of the genetic convergence degree of Mycobacterium tuberculosis [TB] strains isolated from patients infected with TB by MIRU-VNTR technique

In recent decades, epidemiology has significantly been considered in hygienic studies and disease control, and has made a way into all the programs and hygiene policies. By examining the convergence of harmful lineage genetic patterns, the common infectious resources among the patients can be inferred. The purpose of this study was to compare the Mycobacterium tuberculosis genetic patterns convergence isolated from patients infected with Mycobacterium Tuberculosis by MIRU-VNTR technique. After isolation the samples from Lowenstein Jensen culture environment and taking segregate tests and drug susceptibility, the DNA was extracted using CTAB/NaCl technique. The genetic patterns of lineages were calculated according to 12 loci format with MIRU-VNTR technique. Demographic and molecular information of patients was used for epidemiological purposes. After performing drug sensitivity test, 65/140 [64/4%] samples fall into MDR, 29 [20/7%] samples in non MDR category, and the rest of them were among drug - sensitive lineages. Lineage genetic pattern analysis indicated that 49 [35%] of samples related to Delhi/CAS, 28 [20%] to Uganda I, 16 [11/4%] to New I, 1 [0.7%] to EAI, 3[2/1%] to Haarlem, and 5[3/5%] to H37RV families. The genetic pattern convergence comparison exhibited that the most common and variant genetic patterns was seen in Tehran province which were mostly connected to south [from the South of Tehran to Azadi Square] and to the border cities neighboring Afghanistan, Iraq, Turkmenistan and cities with extreme percentage of immigration, all of which signified shared polluted resources among patients

Selection and genotyping of lactobacillus with potential preventive effect by repetitive element sequence-based PCR analysis / 生物工程学报

We selected and characterized isolates of Lactobacillus crispatus (L. crispatus) for potential preventing infections of the female reproductive tract. We cultured vaginal swabs from healthy volunteers on de Man, Rogosa and Sharpe (MRS) agar and identified the isolates at the species level by 16S rRNA sequence and genotyped the isolates of Lactobacillus by PCR amplification of repetitive bacterial DNA elements (rep-PCR). Furthermore, 10 L. crispatus strains were assessed for hydrogen peroxide (H2O2) and acid production. Overall 65 isolates were confirmed to be Lactobacillus by sequence analogy, among them 19 were L. crispatus, 17 were Lactobacillus jensenii and 12 were Lactobacillus fermentum. rep-PCR produced specie and strain-specific genomic fingerprints for the Lactobacillus isolates. The selected 10 L. crispatus isolates produced highly acidic environment after growth in MRS. The isolates T22-3 and T29-5 demonstrated high production of H2O2. This study indicated that there are individual differences with vaginal Lactobacillus colonization, and strain diversity within vaginal L. crispatus isolates, T22-3 and T29-5 might be candidates for restoring urogenital health environment in females.

Application of Single-nucleotide Polymorphism and Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats Analyses to Clinical Mycobacterium tuberculosis Isolates from Korea / 대한진단검사의학회지

BACKGROUND: Single-nucleotide polymorphism (SNP) analysis is a powerful strategy for large-scale molecular population studies examining phylogenetic relationships among bacterial strains. Mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) can be easily digitized to share data among laboratories. This study applied SNP and MIRU-VNTR analyses for molecular strain typing of Mycobacterium tuberculosis isolates collected throughout Korea. METHODS: We studied 102 clinical M. tuberculosis isolates, including 6 paired strains, collected from 11 university hospitals in Korea in 2008 and 2009. SNPs were detected using hairpin primer assays, and then, MIRU-VNTR analysis was performed. RESULTS: Thirty-five SNPs contained polymorphisms that helped differentiate the 96 tested isolates. The isolates were classified into 15 clusters. The Beijing family strains were distributed within closely related clusters in the SNP dendrogram. For MIRU-VNTR analysis, the 96 isolates were divided into 12 groups. The discriminatory index in 8 of these groups (MIRU-10, -23, -26, and -31; ETR-A, -B, -C, and -F) was high (Hunter-Gaston diversity index > 0.6). Unlike the SNP method, MIRU-VNTR analysis did not identify any notable localizations of Beijing or non-Beijing family isolates in specific clusters. CONCLUSIONS: SNP and MIRU-VNTR analyses are surrogate molecular strain-typing methods for M. tuberculosis in Korea where Beijing family isolates are predominant.