Cloning and high-level expression of Thermus thermophilus RecA in E. coli: purification and novel use in HBV diagnostics

ABSTRACT We studied the role of Thermus thermophilus Recombinase A (RecA) in enhancing the PCR signals of DNA viruses such as Hepatitis B virus (HBV). The RecA gene of a thermophilic eubacterial strain, T. thermophilus, was cloned and hyperexpressed in Escherichia coli. The recombinant RecA protein was purified using a single heat treatment step without the use of any chromatography steps, and the purified protein (>95%) was found to be active. The purified RecA could enhance the polymerase chain reaction (PCR) signals of HBV and improve the detection limit of the HBV diagnosis by real time PCR. The yield of recombinant RecA was ∼35 mg/L, the highest yield reported for a recombinant RecA to date. RecA can be successfully employed to enhance detection sensitivity for the diagnosis of DNA viruses such as HBV, and this methodology could be particularly useful for clinical samples with HBV viral loads of less than 10 IU/mL, which is interesting and novel.

Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α

A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.

Structural studies on Mycobacterium tuberculosis RecA: Molecular plasticity and interspecies variability.

Structures of crystals of Mycobacterium tuberculosis RecA, grown and analysed under different conditions, provide insights into hitherto underappreciated details of molecular structure and plasticity. In particular, they yield information on the invariant and variable features of the geometry of the P-loop, whose binding to ATP is central for all the biochemical activities of RecA. The strengths of interaction of the ligands with the P-loop reveal significant differences. This in turn affects the magnitude of the motion of the ‘switch’ residue, Gln195 in M. tuberculosis RecA, which triggers the transmission of ATP-mediated allosteric information to the DNA binding region. M. tuberculosis RecA is substantially rigid compared with its counterparts from M. smegmatis and E. coli, which exhibit concerted internal molecular mobility. The interspecies variability in the plasticity of the two mycobacterial proteins is particularly surprising as they have similar sequence and 3D structure. Details of the interactions of ligands with the protein, characterized in the structures reported here, could be useful for design of inhibitors against M. tuberculosis RecA.

Caracterização de um programa de triagem auditiva neonatal / Description of the neonatal hearing screening program / Caracterización de un programa para triagem auditiva neonatal

Objetivo: Caracterizar o programa de Triagem Auditiva Neonatal (TAN) em uma população de neonatos. Métodos: Estudo retrospectivo realizado com um total de 993 bebês. Resultados: Dos 993 bebês que realizaram a TAN, 492 (49,55%) eram meninos e 501 (50,45%), meninas. Em relação aos fatores de risco, 37 (3,73%) apresentaram hiperbilirrubinemia; 24 (2,42%), medicação ototóxica; 20 (2,02%), ventilação mecânica; 17 (1,71%), infecções; 13(1,31%), malformações craniofaciais; cinco (0,50%), síndromes congênitas; dois (0,20%), doenças neurodegenerativas, e 38 (3,83%) permaneceram em unidade de terapia intensiva por mais de cinco dias. A idade mínima, ao realizar a TAN, foi de cinco horas de vida e a máxima, de dois meses. Um segundo teste foi agendado em 15 dias para 35,75% dos bebês que falharam na TAN. No segundo teste, a idade média foi de 34,82 dias, sendo a mínima de dois dias, a máxima de nove meses e o índice de falha de 7,49% para a orelha direita e, de 6,89% para a orelha esquerda. Quarenta e cinco bebês não compareceram ao segundo teste e 8,68% foram encaminhados para diagnóstico. Houve relação estatisticamente significativa, entre maior ocorrência de falha para neonatos e menor tempo de vida. O número de bebês que realizaram a TAN aumentou desde o início do programa. Conclusão: Foi possível caracterizar o programa de TAN no hospital.

Purpose: To describe the Neonatal Hearing Screening Program (NHSP) in a population of newborns. Method: Retrospective study totalizing 993 babies. Results: Out of the 993 babies that undergone the NHS, 492 (49.55%) were boys and 501 (50.45%), girls. In relation to risk factors, 37 (3.73%) had hyperbilirubinemia; 24 (2.42%), ototoxic medication; 20 (2.02%), mechanical ventilation; 17 (1.71%), infections; 13 (1.31%), craniofacial malformations; five (0.50%), congenital syndromes; two (0.20%), neurodegenerative diseases, and 38 (3.83%) remained in the neonatal intensive care unit for more than five days. When submitted to the NHS, the babies were between five hours old and two months old. A second test was scheduled 15 days later for the 35.75% babies who had failed in the first NHS. In the second test, the average age was 34.82 days old, the minimum, two days old and the maximum, nine months old. The failure rate was 7.49% for the right ear and 6.89% for the left ear. Forty-five babies did not attend the second test and 8.68% were referred for diagnosis. There was a statistically significant relationship between a higher occurrence of failure for neonates with shorter time of life. The number of babies who has undergone the NHS has increased since the beginning of the program. Conclusion: It was possible to characterize the program of NHS in this hospital.

Objetivo: Caracterizar el programa para Triagem Auditiva Neonatal (TAN) en una población de neonatos. Método: Estudio retrospectivo realizado con un total de 993 bebés. Resultados: De los 993 niños que realizaron la TAN, 492 (49,55%) eran varones y 501 (50,45%) niñas. En cuanto a los factores de riesgo, 37(3,73%) presentaron hiperbilirrubinemia, 24 (2,42%), medicación ototóxica, 20 (2,02 %), ventilación mecánica, 17 (1,71%), infecciones; 13 (1,31 %), malformaciones craneofaciales, 5 (0,50%) , síndromes congénitos, 2 (0,20 %), enfermedades neurodegenerativas, y 38 (3,83 %) permanecieron en unidad de cuidados intensivos durante más de 5 días. La edad mínima al realizar la TAN fue de 5 horas de vida y la máxima, de 2 meses. Una segunda prueba fue programada en 15 días para 35,75% de los bebés que fallaron en la TAN. En la segunda prueba, la edad pormedio fue de 34,82 días, siendo la mínima de 2 días, la máxima de 9 meses y la tasa de fracaso de 7.49% para el oído derecho y de 6,89% para el oído izquierdo. Cuarenta y cinco bebés no asistieron a la segunda prueba y el 8,68 % fueron remitidos para el diagnóstico. Se encontró una correlación estadísticamente significativa entre mayor incidencia de fallas para los recién nacidos y menor tiempo de vida. El número de bebés que fueron sometidos TAN aumentó desde que comenzó el programa. Conclusión: Es posible caracterizar el programa de TAN en el hospital.

A simple negative selection method to identify adenovirus recombinants using colony PCR

Background: The AdEasy system is a fast-track system for generating recombinant adenoviruses using the efficient homologous recombination machinery between shuttle and adenovirus backbone plasmids in Escherichia coli BJ5183 cells. The key step is homologous recombination in BJ5183 cells, which is driven by RecA activity. However, culture time is stringently limited to reduce the damage to recombinant plasmids by RecA activity. Therefore, rapid identification of recombinant adenoviruses within the limited time-period is critical. Results: We developed a simple negative selection method to identify recombinant adenoviruses using colony PCR, which improves the efficiency of adenovirus recombination screening and packaging. Conclusions: The negative selection method to identify AdEasy adenovirus recombinants by colony PCR can identify the recombined colony within a short time-period, and maximally avoid damage to the recombinant plasmid by limiting recombination time, resulting in improved adenovirus packaging.

Relação entre o peso corporal da adolescente grávida e as medidas antropométricas do recém-nascido / Relación entre el peso corporal de la adolescente embarazada y las medidas antropométricas del recién nacido / Relation between the body weight of pregnancy adolescent and the anthropometric measures of the newborn

O estudo teve como objetivo de relacionar o peso da adolescente grávida com as medidas antropométricas do recém-nascido (peso, comprimento, perímetro cefálico). Trabalhou-se na abordagem quantitativa, retrospectiva, com amostra de 585 prontuários. Para determinar o peso da adolescente grávida foi utilizada a Curva de Rosso. As análises foram realizadas no Statistical Package for the Social Science (SPSS), para as associações bivariadas foram utilizados os coeficientes gama e d de Sommer (significativo p≤0,05). As adolescentes grávidas no final da gestação foram classificadas como peso normal (31,4%), baixo peso (46%), sobrepeso (11.3%) e obesidade (11,3%). O peso da adolescente grávida apresentou associação estatisticamente significativa com o peso recém-nascido (d=0,208), comprimento (d=0,11) e perímetro cefálico (d=0,11). Concluiu-se que há indicativos de que o peso materno pode ter uma relação direta com a antropometria do recém-nascido. Nesse sentido, sugere-se que outros estudos sejam realizados, considerando esta possibilidade, de modo a embasarem o estímulo à adoção de estilos de vida e hábitos alimentares saudáveis no acompanhamento da gestante adolescente.

The study aimed to relate the weight of the pregnant adolescent with the anthropometric measures (weight, stature, cephalic perimeter) of the newborn. The study was performed by quantitative and retrospective approach, collecting data of 585 records, the Rosso’s curve was used in order to determine the weight of pregnant adolescent, the analyses were performed using Statistical Package for the Social Science (SPSS), the bivariate association was described by gama and Sommer d coeficient (statistically significant when p≤0.05). Pregnant teens in late pregnancy were classified as normal weight (36.4%), low weight (46%), overweight (11.3%) and obese (11.3%). The association between weight of the pregnant adolescent and weight of the newborn was statistically significant (d=0.208), stature (d=0.11) and cephalic perimeter (d=0.11). In conclusion, there are indications that maternal weight may have a direct relationship with the anthropometry of the newborn. In this sense, it is suggested that further studies are conducted, considering this possibility, in the way of supporting a stimulus to the adoption of healthy lifestyles and eating habits in monitoring the pregnant adolescent.

El objetivo fue relacionar el peso de la adolescente embarazada con medidas antropométricas del recién nacido (peso, estatura, perímetro cefálico). El trabajo fue cuantitativo, retrospectivo, la muestra fue de 585 historias médicas. Para determinar el peso de la adolescente embarazada, se usó la Curva de Rosso, los análisis fueron realizados en el programa Statistical Package for the Social Science (SPSS), para asociaciones bivariadas se utilizaron los coeficientes de gama y d de Sommer (significativo p≤0,05). Las adolescentes embarazadas en el final del embarazo fueron clasificadas como peso normal (31,4%), bajo peso (46%), sobrepeso (11,3%) y obesidad (11,3%). El peso materno tuvo asociación significativa con el peso del recién nacido (d=0,208), estatura (d=0,11) y perímetro cefálico (d=0,11). Se concluye que hay indicativos que el peso materno puede tener una relación directa con la antropometría del recién nacido. En ese sentido se sugiere que otros estudios sean realizados, considerando esta posibilidad, de modo de fundamentar el estímulo a la adopción de estilos de vida y hábitos alimenticios saludables en el seguimiento de la adolescente embarazada.

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. seropedicaeRecA protein (RecA Hs) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

A rapid and reliable species-specific identification of clinical and environmental isolates of Vibrio cholerae using a three-test procedure and recA polymerase chain reaction.

Purpose: Vibrio cholerae, the cause of cholera, is one of the leading causes of morbidity and mortality in many developing countries. Most laboratories initially rely on biochemical tests for a presumptive identification of these strains, followed by a polymerase chain reaction (PCR)-based method to confirm their identification. The aim of this study is to establish a rapid and reliable identification scheme for V. cholerae using a minimal, but highly specific number of biochemical tests and a PCR assay. Materials and Methods: We developed a species-specific PCR to identify V. cholerae, using a housekeeping gene recA, and used that to evaluate the sensitivity and specificity of 12 biochemical tests commonly used for screening and / or presumptive identification of V. cholerae in the clinical and environmental samples. Results: Here we introduced a combination of three biochemical tests, namely, sucrose fermentation, oxidase test, and growth in trypton broth containing 0% NaCl, as also the PCR of the recA gene, for rapid identification of V. cholerae isolates, with 100% sensitivity and specificity. The established method accurately identified a collection of 47 V. cholerae strains isolated from the clinical cases (n = 26) and surface waters (n = 21), while none of the 32 control strains belonging to different species were positive in this assay. Conclusion: The triple-test procedure introduced here is a simple and useful assay which can be adopted in cholera surveillance programs for efficient monitoring of V. cholerae in surface water and fecal samples.

Evaluación de los sistemas comerciales automatizados VITEK 2 y API 20NE para la identificación de organismos del complejo Burkholderia cepacia aislados de muestras clínicas / Evaluation of commercial systems VITEK 2 and API 20NE for identification of Burkholderia cepacia complex bacteria from clinical samples

Las especies del complejo Burkholderia cepacia (CBC) son capaces de causar infecciones crónicas del tracto respiratorio en pacientes con fibrosis quística y en otros individuos inmunocomprometidos. La mayoría de estas especies exhiben alta resistencia a la terapia antibiótica, lo que genera la necesidad de una detección rápida y precisa para poder implementar estrategias de control adecuadas. En este trabajo se utilizó la técnica de reacción en cadena de la polimerasa (PCR) para amplificar el gen recA (PCR-recA), con el fin de identificar microorganismos pertenecientes al CBC. Con este método molecular como referencia, se evaluó la sensibilidad (S) y la especificidad (E) de dos sistemas de identificación comerciales automatizados, VITEK 2 y API 20NE (bioMérieux®), así como también el valor de las pruebas bioquímicas manuales más representativas para la identificación de estos microorganismos. El método VITEK 2 presentó una S del 71,1 % y una E del 100 %; para el método API 20NE, estos valores fueron 69,7 % y 90,2 %, respectivamente. En cuanto a las pruebas fenotípicas manuales, los resultados obtenidos fueron más heterogéneos, lo que posiblemente se deba a que estas bacterias podrían sufrir presión selectiva para sobrevivir en pacientes crónicos y perder factores fenotípicos característicos. La técnica de PCR-recA resultó de fácil implementación, por lo que cabe considerar a esta técnica de identificación como una opción viable, aun en laboratorios de diagnóstico clínico de mediana complejidad.

Species belonging to the Burkholderia cepacia complex (BCC) are capable of causing chronic respiratory tract infections in patients suffering from cystic fibrosis as wel as in immunocompromised individuals. Most of these species are highly resistant to antibiotic therapy, generating the need for their rapid and accurate detection for the proper treatment and clinical management of these patients. In this wok, the polymerase chain reaction (PCR) technique based on the amplification of the recA gene (PCR-recA) was applied for an accurate identification of bacteria belonging to the BCC. Sensitivity (S) and specificity (E) of two biochemically-based commercial automated systems, API 20NE and VITEK 2 (bioMérieux®), and of the most representative biochemical manual tests for the identification of the Burkholderia cepacia complex were herein evaluated. The commercial systems VITEK 2 and API 20NE showed the following sensitivity and specificity vaues for identification to the species level, S: 71.1 %, E: 100 %, S: 69.7 %, E: 90.2 %, respectively. More complex results were observed for phenotypic manual tests, since BCC bacteria can undergo selective pressure to survive in chronic patients causing the loss of their typical phenotypic characteristics. The PCR-recA technique was easy to implement even in medium-complexity clinical diagnostic laboratories.

Molecular identification of Burkholderia cepacia complex and species distribution among cystic fibrosis patients seen at the reference center in southern Brazil / Identificação molecular do complexo Burkholderia cepacia e distribuição por espécie entre pacientes com fibrose cística em um centro de referência no sul do Brasil

Background: Burkholderia complex (Bcc) infections in cystic fibrosis (CF) patients are associated with decline in lung function and reduced survival. The potential transmissibility of Bcc among CF patients has been reported, indicating that strict segregation of CF patients with Bcc is crucial. Aims: To standardize the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) assay in order to identify Bcc species and to establish the prevalence of Bcc species and their susceptibility profile among CF patients seen at the Hospital de Clínicas de Porto Alegre (HCPA). Methods: The classification of the clinical isolates recovered from respiratory tract specimens of CF patients as Bcc was achieved using the API-20NE® phenotypic commercial system. The identification of the Bcc species was performed using PCR-RFLP. The antimicrobial disc diffusion susceptibility testing was performed according to the CLSI (2006). Results: API-20NE® was able to identify Bcc isolates (244 specimens), such as B. cepacia, indicating that it was not able to distinguish among the Bcc species. The PCR-RFLP molecular method discriminated the eight reference Bcc species, thus validating the method for clinical isolates. Bcc prevalence determined by PCR-RFLP was 10.6% (26/244). The molecular analysis identified B. cenocepacia in 53.8% (14/26) of infected patients, B. multivorans in 15.4% (4/26), and B. vietnamiensis and B. ambifaria in 7.7% (2/26). The antibiotic resistance profile was variable among Bcc species. Conclusions: The PCR-RFLP method was validated for the identification of Bcc species. B. cenocepacia proved to be the most prevalent species among the CF patients seen at the HCPA.

Introdução: Infecções por bactérias do complexo Burkholderia cepacia (CBC) em pacientes com fibrose cística (FC) estão associadas a declínio da função pulmonar e diminuição da sobrevida. O potencial de transmissibilidade de CBC entre pacientes com FC é uma realidade, tornando-se importante a estrita segregação dos pacientes infectados.Objetivos: Padronizar a técnica de PCR-RFLP (reação em cadeia da polimerase seguida de clivagem com enzimas derestrição) para diferenciação das espécies de CBC e estabelecer a prevalência dessas espécies e seus perfis de sensibilidade em pacientes com FC atendidos no Hospital de Clínicas de Porto Alegre (HCPA). Métodos: A identificação dos isolados clínicos do trato respiratório de pacientes com FC como CBC foi feita pelo sistema deidentificação fenotípica comercial API-20NE®. A diferenciação das espécies de CBC foi realizada por PCR-RFLP, e o teste de suscetibilidade aos antimicrobianos por disco-difusão foi realizado de acordo com o CLSI (2006).Resultados: O sistema API-20NE® identificou todos os isolados do CBC (244 amostras) como B. cepacia, indicando claramente que não distingue as espécies do complexo. O método molecular de PCR-RFLP discriminou as oito espécies de referência de CBC, validando o método para isolados clínicos. A prevalência de CBC por PCR-RFLP foi de 10,6% (26/244).A análise molecular apontou B. cenocepacia colonizando em 53,8% (14/26) dos pacientes infectados, B. multivorans em 15,4% (4/26) e B. vietnamiensis e B. ambifaria em 7,7% (2/26). O perfil de resistência entre as espécies de CBC para os antibióticos testados foi variado. Conclusão: Foi validada a aplicação do método molecular PCR-RFLP para identificar espécies de CBC, e B. cenocepaciafoi a espécie mais prevalente entre os pacientes fibrocísticos atendidos no HCPA.

A description of genes of Corynebacterium pseudotuberculosis useful in diagnostics and vaccine applications

Corynebacterium pseudotuberculosis, a Gram-positive intracellular pathogen, is the etiological agent of caseous lymphadenitis or CLA. This bacterium infects goats and sheep and causes great economic losses worldwide annually, mainly for goat producers. Despite its importance, CLA is still poorly characterized. However, with advances in the genomic field, many C. pseudotuberculosis genes have already been characterized, mainly those related to virulence such as phospholipase D. Here, we examined the use of the several available genes of C. pseudotuberculosis and reviewed their applications in vaccine construction, more efficient diagnostics for CLA, and control of this disease, among other applications.