Presencia de genes fimH y afa en aislamientos urinarios de Escherichia coli productora de betalactamasas de espectro extendido en Lima, Perú / Presence of fimH and afa genes in urinary isolates of extended-spectrum beta-lactamases producing Escherichia coli in Lima, Peru

RESUMEN Con el objetivo de determinar la presencia de los genes fimH y afa en aislamientos urinarios de Escherichia coli productoras de betalactamasas de espectro extendido (BLEE), se realizó un estudio descriptivo, con aislamientos del cepario del proyecto TO-06/09 del Instituto Nacional de Salud del Niño en Lima, Perú. Se incluyeron 75 aislamientos urinarios de Escherichia coli. La identificación de genes se realizó por reacción en cadena de la polimerasa. De los 75 aislamientos, 74 (98,7%) fueron positivos para el gen fimH y 6 (8,0%) fueron positivos para el gen afa. Se evidenció la presencia de los factores de virulencia producidos por los genes fimH y afa en aislamientos urinarios de Escherichia coli productoras de BLEE.

ABSTRACT Descriptive study conducted in order to determine the presence of the fimH and afa genes in urinary isolates of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli. Isolates from project TO-06/09 of the Instituto Nacional de Salud del Niño in Lima, Peru were used. A total of 75 urinary isolates of Escherichia coli were included. Gene identification was performed by polymerase chain reaction. From the 75 isolates, 74 (98.7%) were positive for the fimH gene and 6 (8.0%) were positive for the afa gene. Virulence factors produced by the fimH and afa genes were evident in urinary isolates of ESBL producing Escherichia coli.

Betaine supplementation improved L-threonine fermentation of Escherichia coli THRD by upregulating zwf (glucose-6-phosphate dehydrogenase) expression

BACKGROUND: The supplementation of betaine, an osmoprotective compatible solute, in the cultivation media has been widely used to protect bacterial cells. To explore the effects of betaine addition on industrial fermentation, Escherichia coli THRD, an L-threonine producer, was used to examine the production of L-threonine with betaine supplementation and the underlying mechanism through which betaine functions was investigated. RESULTS: Betaine supplementation in the medium of E. coli THRD significantly improved L-threonine fermentation parameters. The transcription of zwf and corresponding enzyme activity of glucose-6-phosphate dehydrogenase were significantly promoted by betaine addition, which contributed to an enhanced expression of zwf that provided more nicotinamide adenine dinucleotide phosphate (NADPH) for L-threonine synthesis. In addition, as a result of the betaine addition, the betaine-stimulated expression of enhanced green fluorescent protein (eGFP) under the zwf promoter within a plasmid-based cassette proved to be a transcription-level response of zwf. Finally, the promoter of the phosphoenolpyruvate carboxylase gene ppc in THRD was replaced with that of zwf, while L-threonine fermentation of the new strain was promoted by betaine addition. Conclusions: We reveal a novel mode of betaine that facilitates the microbial production of useful compounds. Betaine supplementation upregulates the expression of zwf and increases the NADPH synthesis, which may be beneficial for the cell growth and thereby promote the production of L-threonine. This finding might be useful for the production of NADPH-dependent amino acids and derivatives in E. coli THRD or other E. coli strains.

Carbapenem Resistance in Gram-negative Bacteria in South-western Nigeria: The Role of Extended-spectrum lactamase CTX-M-15 / Resistencia al carbapenem en las bacterias gramnegativas en Nigeria Suroccidental: papel de la betalactamasa de espectro extendido tipo CTX-M-15

ABSTRACT Objective: To determine the role of extended-spectrum β-lactamases in carbapenem-resistant Gram-negative bacteria from south-western Nigeria. Methods: Twenty-seven carbapenem-resistant isolates that were found to be non-carbapenemase producers (15 Escherichia coli, 9 Klebsiella pneumoniae and 3 Pseudomonas aeruginosa) were further studied. These isolates were subjected to analysis including phenotypic and genotypic detection of various β-lactamases, efflux activity, outer membrane protein, plasmids replicon typing, detection of transferable genes and resistances and typing using random amplified polymorphic DNA tests. Results: No isolates demonstrated de-repression of efflux, but all showed either complete loss or reduced production of outer membrane proteins. Transconjugants from these strains contained various genes including plasmid-mediated quinolone resistance and extended-spectrum beta-lactamases. All the transconjugants carried the blaCTX-M-15 gene. The transconjugants had varying minimum inhibitory concentrations of carbapenems ranging from 0.03 μg/ml to 8 μg/ml. Varying resistances to other antimicrobial agents were also transferred with the plasmids. The donor isolates were not clonally related by molecular typing. Conclusion: Resistance to carbapenem antibiotics in this sample was not mediated only by carbapenemases but also by production of extended-spectrum β-lactamases (largely CTX-M-15), accompanied by protein loss. This was an important mechanism underpinning carbapenem resistance in these clinical isolates of various species.

RESUMEN Objetivo: Determinar el papel de las betalactamasas de espectro extendido en la resistencia al carbapenem en las bacterias gramnegativas en Nigeria. Métodos: Veintisiete aislados resistentes al carbapenem que fueron hallados productores de no carbapenemasas (15 Escherichia coli, 9 Klebsiella pneumoniae, y 3 Pseudomonas aeruginosa) fueron estudiados con mayor profundidad. Estos aislados fueron sometidos a análisis incluyendo la detección fenotípica y genotípica de varias betalactamasas, la actividad de eflujo, las porinas de la membrana externa, la tipificación del replicón plasmídico, la detección de genes transferibles y resistencias y tipificación usando pruebas de ADN polimórficas amplificadas aleatorias. Resultados: Ninguno de los aislamientos mostró desrepresión de eflujo, pero todos demostraron la pérdida completa o la producción reducida de porinas externas de la membrana. Los transconjugantes de estas cepas contenían varios genes incluyendo resistencia a la quinolona mediada por plásmidos y betalactamasas de espectro extendido. Todos los transconjugantes portaban el gen blaCTX-M-15. Los transconjugantes tenían diversas concentraciones inhibitorias mínimas de carbapenemas que oscilaban entre 0.03 μg/ml y 8 μg/ml. Varias resistencias a otros agentes antimicrobianos fueron también transferidas con los plásmidos. Los aislamientos del donante no estuvieron relacionados clonalmente por tipificación molecular. Conclusión: La resistencia al antibiótico carbapenem en esta muestra no fue mediada solamente por las carbapenemasas, sino también por la producción de betalactamasas de espectro extendido (en gran parte CTX-M-15), acompañado por pérdida de porina. Éste era un mecanismo importante que sustentaba la resistencia al carbapenem en estos aislados clínicos de varias especies.

Study of pandrug and heavy metal resistance among E. coli from anthropogenically influenced Delhi stretch of river Yamuna

Abstract Escalating burden of antibiotic resistance that has reached new heights present a grave concern to mankind. As the problem is no longer confined to clinics, we hereby report identification of a pandrug resistant Escherichia coli isolate from heavily polluted Delhi stretch of river Yamuna, India. E. coli MRC11 was found sensitive only to tobramycin against 21 antibiotics tested, with minimum inhibitory concentration values >256 µg/mL for amoxicillin, carbenicillin, aztreonam, ceftazidime and cefotaxime. Addition of certain heavy metals at higher concentrations were ineffective in increasing susceptibility of E. coli MRC11 to antibiotics. Withstanding sub-optimal concentration of cefotaxime (10 µg/mL) and mercuric chloride (2 µg/mL), and also resistance to their combinatorial use, indicates better adaptability in heavily polluted environment through clustering and expression of resistance genes. Interestingly, E. coli MRC11 harbours two different variants of blaTEM (blaTEM-116 and blaTEM-1 with and without extended-spectrum activity, respectively), in addition to mer operon (merB, merP and merT) genes. Studies employing conjugation, confirmed localization of blaTEM-116, merP and merT genes on the conjugative plasmid. Understanding potentialities of such isolates will help in determining risk factors attributing pandrug resistance and strengthening strategic development of new and effective antimicrobial agents.

Sensibilidad a fosfomicina en Escherichia coli productoras de betalactamasas de espectro extendido / Sensitivity to Fosfomycin in Extended-spectrum Betalactamase-producing Escherichia coli

RESUMEN Las infecciones urinarias son causadas mayormente por Escherichia coli (E. coli), el uso indiscriminado de antibióticos ha originado un aumento de infecciones por cepas productoras de betalactamasas de espectro extendido (BLEE). Con el objetivo de determinar la sensibilidad a fosfomicina se realizó un estudio en cepas de E. coli productoras de BLEE aisladas de urocultivos provenientes de un hospital de Perú. Se recolectaron 266 cepas de E. coli identificadas por métodos convencionales como productoras de BLEE. Se determinó la sensibilidad de fosfomicina por concentración inhibitoria mínima mediante el método de dilución en agar y por el método de disco difusión. Se encontró 192 (72,2 %) cepas de E. coli productora de BLEE sensibles a fosfomicina. Se concluye que la fosfomicina presenta actividad antimicrobiana frente a cepas de E. coli productoras de BLEE, y podría ser considerada una buena opción terapéutica frente a cepas resistentes.

ABSTRACT Urinary infections are caused mainly by Escherichia coli (E. coli); indiscriminate use of antibiotics has caused an increase in infections due to extended-spectrum beta-lactamase (ESBL)-producing strains. Aiming to determine the sensitivity to fosfomycin, a study was conducted in ESBL-producing E. coli strains isolated from urine cultures at a hospital in Peru. Two hundred and sixty-six (266) strains of E. coli were collected, which were determined by conventional methods to be ESBL- producing. Sensitivity to fosfomycin was determined through minimum inhibitory concentration with the agar dilution method and the diffusion disc method. One hundred and ninety-two (192) (72.2%) strains of ESBL-producing E. coli strains sensitive to Fosfomycin were found. It, therefore, follows that fosfomycin exhibits antimicrobial activity against ESBL-producing E. coli strains and that it could be considered a good treatment option for resistant strains.

Clonal relation and antimicrobial resistance pattern of extended-spectrum ß-lactamase- and AmpC ß-lactamase-producing Enterobacter spp. isolated from different clinical samples in Tehran, Iran

Abstract INTRODUCTION: Here, we determined the genes encoding antibiotic resistance enzymes and virulence factors and evaluated the genetic relationship between Enterobacter spp. isolated from different clinical samples. METHODS: A total of 57 clinical isolates of Enterobacter spp. were tested for the production of extended-spectrum β-lactamases (ESBLs), carbapenemase, and AmpC using phenotypic and genotypic methods. RESULTS: The most common ESBLs and AmpC β-lactamases were bla TEM (63.3%) and bla EBC (57.7%), respectively. The most prevalent virulence gene was rpos (87.7%). The random amplified polymorphic DNA (RAPD) patterns of strains were genetically unrelated. CONCLUSIONS: RAPD polymerase chain reaction analysis revealed high genetic diversity among isolates.

Clonal relationships, antimicrobial susceptibilities, and molecular characterization of extended-spectrum beta-lactamase-producing Escherichia coli isolates from urinary tract infections and fecal samples in Southeast Iran

Abstract INTRODUCTION: Multidrug-resistant (MDR) Escherichia coli, a species that is a leading cause of urinary tract infections (UTIs) and is a major global public health concern. This study was designed to detect the differences in antibiotic resistance patterns, the production and type of extended spectrum β-lactamases (ESBLs), and the clonal relationships among E. coli isolates from UTIs and fecal samples. METHODS: Antibacterial resistance was determined by the disk diffusion method. ESBL, carbapenemase, and AmpC-producing isolates were detected phenotypically. Then, the ESBL genes were sequenced to detect the type. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) was performed on the ESBL-positive isolates. RESULTS: The most common effective antibacterial agents were colistin, imipenem, and amikacin. Among the isolates, 204 (56.6%) were MDR. Of the 163 ESBL-positive isolates, 11 (6.7%) produced AmpC, and the frequencies of beta-lactamase-positive genes were as follows: bla CTX-Mgroup1, 76%; bla TEM1, 74.8%; bla SHV12, 1.2%; and bla OXA1, 12.88%. ERIC PCR showed a diverse pattern, suggesting that clonal spread of E. coli in this area is uncommon, and that most of the infecting strains are endogenous. CONCLUSIONS: The high rates of antibacterial-resistant and MDR isolates are quite important since these strains can act as source of resistant bacteria that can be spread in the community. Controlling antibiotic use, against inappropriate use and abuse, in the community and continuous surveillance of emerging resistance traits are critical to controlling the spread of resistance.

Caracterización molecular y patrón de susceptibilidad antimicrobiana de Escherichia coli productora de β -lactamasas de espectro extendido en infección del tracto urinario adquirida en la comunidad / Molecular characterization and antimicrobial susceptibility pattern of extended-spectrum β -lactamase-producing Escherichia coli as cause of community acquired urinary tract infection

Resumen Introducción Las infecciones del tracto urinario adquiridas en la comunidad (ITUac) causadas por cepas de Escherichia coli productoras de β-lactamasas de espectro extendido (BLEE), principalmente por cepas que contienen el gen blaCTX-M-15, es un fenómeno creciente a nivel mundial. Objetivo Determinar el patrón de susceptibilidad a antimicrobianos de cepas de E. coli productoras de BLEE causantes de ITUac y conocer su patrón molecular. Materiales y Métodos Se realizó un estudio descriptivo en Oaxaca, México, donde se incluyeron 288 cepas de E. coli aisladas de pacientes adultos con posible ITUac. Para obtener los patrones de susceptibilidad antimicrobiana se siguieron los criterios del CLSI y para obtener el análisis molecular se utilizó la técnica de RPC. Resultados Del total de cepas de E. coli aisladas, 31,3% fueron productoras de BLEE, presentando una menor susceptibilidad a antimicrobianos que las cepas no productoras de estas enzimas. El 95,6% de las cepas BLEE estudiadas fueron portadoras del gen blaCTX-M. Conclusiones Un tercio de las ITUac causadas por E. coli en nuestra población fueron causadas por cepas BLEE, mostrando un alto nivel de resistencia a los antimicrobianos comúnmente utilizados en su tratamiento y disminuyendo las opciones terapéuticas para tratamientos empíricos en esta población.

Background Community acquired urinary tract infections (CaUTI) caused by strains of extended-spectrum β-lactamases (ESBL) - producing Escherichia coli, mainly by strains carrying the blaCTX-M-15 gene, is a growing phenomenon worldwide. Aim To determine the antibiotic susceptibility pattern of ESBL-producing E. coli as cause of CaUTI and to identify their molecular pattern. Methods A descriptive study was performed in the city of Oaxaca, Mexico, from where 288 strains of CaUTI-producing strains of E. coli in adults with possible UTI were isolated. The CLSI criteria was followed to determine the antimicrobial susceptibility patterns, and their molecular characterization was performed by using PCR. Results 31.3% of E. coli strains isolated in our population were ESBL producers, which presented higher levels of antibiotic resistance than those of non-producers of these enzymes. 95.6% of the studied strains were carriers of the blaCTX-M gene. Conclusions One-third of the Ca-UTI caused by E. coli in our population are caused by ESBL-producing strains, which present high levels of resistance to the antibiotics widely used in our community. This situation considerably decreases the number of antibiotics available for an empiric treatment against these infections.

Multirresistencia y factores asociados a la presencia de betalactamasas de espectro extendido en cepas de Escherichia coli provenientes de urocultivos / Multiresistance and factors associated with the presence of extended-spectrum beta-lactamases in Escherichia coli strains isolated from urine culture

RESUMEN El tratamiento empírico para la infección urinaria se ve complicado frente a la presencia de multirresistencia y de betalactamasas de espectro extendido (BLEE). El objetivo del estudio fue describir los patrones de resistencia antibiótica de cepas de Escherichia coli aisladas en urocultivos y los factores clínico-epidemiológicos asociados a la presencia de BLEE en un grupo pediátrico y adulto. Se recolectaron durante 14 meses, 353 cepas provenientes de Emergencia y Hospitalización del Hospital Cayetano Heredia, 45,9% fueron cepas multirresistentes. La incidencia de BLEE en población pediátrica fue 16,3% vs. 31,1% en la adulta, el 63,6% provenía de pacientes ambulatorios. La presencia de BLEE se asoció con encontrarse hospitalizado en pediatría, así cómo al uso de pañal y vejiga neurogénica en adultos. Estos factores deben considerarse al momento de elegir un tratamiento antibiótico. Asimismo, es necesario implementar programas de reporte epidemiológico y modelos de prevención de factores de riesgo.

ABSTRACT The empirical treatment of urinary infections is complicated by the presence of multiresistance and resistance to extendedspectrum beta-lactamases (ESBLs). The objective of this study was to describe the patterns of antibiotic resistance of Escherichia coli strains isolated from urine cultures and the clinical-epidemiological factors associated with the presence of ESBLs in a pediatric and an adult group. A total of 353 strains were collected from the Emergency and Hospitalization Sector of the Cayetano Heredia Hospital over 14 months, and 45.9% of the isolated strains were multiresistant. The rate of resistance to ESBLs in the pediatric and adult population was 16.3% and 31.1%, respectively, and 63.6% of the resistant strains were isolated from outpatients. The presence of ESBLs was associated with hospitalization in pediatrics, use of diapers, and the presence of neurogenic bladder in adults. These factors should be considered in selection of an antibiotic treatment. Moreover, epidemiological reporting programs and models should be implemented for reduction of risk factors.

Meningitis neonatal precoz causada por transmisión vertical de Escherichia coli productora de beta-lactamasa de espectro extendido en parto prematuro con rotura prematura de membranas / Early neonatal meningitis caused by vertical transmission of extended-spectrum beta-lactamase-producing Escherichia coli in premature labor with premature rupture of membranes

Se comunica el caso de un recién nacido producto de un parto prematuro con rotura prematura de membranas, que desarrolló precozmente meningitis neonatal por Escherichia coli productora de beta-lactamasa de espectro extendido. Los cultivos en líquido céfalo raquídeo y sangre neonatal fueron tempranamente positivos para esta bacteria. No obstante no aislarse este microorganismo en la madre, los hallazgos de la biopsia placentaria y la precocidad de la infección neonatal son determinantes en señalar que se trató de infección intraamniótica con transmisión vertical al neonato. La meningitis neonatal fue tratada con meropenem y el niño se dio de alta en buenas condiciones después de 41 días de hospitalización. Las guías perinatales actuales, preconizan el tamizaje de muestras vaginales para la prevención del parto prematuro y de los resultados adversos asociados a infección bacteriana ascendente durante el embarazo.

We report the case of a newborn resultant of premature delivery with premature rupture of membranes, which developed early-onset neonatal meningitis caused by transmission of Escherichia coli producer of betalactamasa of spectrum extended. Cultures in cerebrospinal fluid and neonatal blood were early positive for this bacterium. Although this microorganism is not isolated in the mother, the findings of the placenta biopsy and the precocity of the neonatal infection are determinant in indicating that it was an intraamniotic infection with vertical transmission to the neonate. Neonatal meningitis was treated with meropenem and the child was discharged in good condition after 41 days of hospitalization. The current perinatal guidelines support the screening of vaginal samples for the prevention of preterm birth and the adverse outcomes associated with ascending bacterial infection during pregnancy.

Co-production of hydrogen and ethanol by Escherichia coli SS1 and its recombinant

Background: The development of a potential single culture that can co-produce hydrogen and ethanol is beneficial for industrial application. Strain improvement via molecular approach was proposed on hydrogen and ethanol co-producing bacterium, Escherichia coli SS1. Thus, the effect of additional copy of native hydrogenase gene hybC on hydrogen and ethanol co-production by E. coli SS1 was investigated. Results: Both E. coli SS1 and the recombinant hybC were subjected to fermentation using 10 g/L of glycerol at initial pH 7.5. Recombinant hybC had about 2-fold higher cell growth, 5.2-fold higher glycerol consumption rate and 3-fold higher ethanol productivity in comparison to wild-type SS1. Nevertheless, wild-type SS1 reported hydrogen yield of 0.57 mol/mol glycerol and ethanol yield of 0.88 mol/mol glycerol, which were 4- and 1.4-fold higher in comparison to recombinant hybC. Glucose fermentation was also conducted for comparison study. The performance of wild-type SS1 and recombinant hybC showed relatively similar results during glucose fermentation. Additional copy of hybC gene could manipulate the glycerol metabolic pathway of E. coli SS1 under slightly alkaline condition. Conclusions: HybC could improve glycerol consumption rate and ethanol productivity of E. coli despite lower hydrogen and ethanol yields. Higher glycerol consumption rate of recombinant hybC could be an advantage for bioconversion of glycerol into biofuels. This study could serve as a useful guidance for dissecting the role of hydrogenase in glycerol metabolism and future development of effective strain for biofuels production.

Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli

Background: Pullulanase production in both wild-type strains and recombinantly engineered strains remains low. The Shine-Dalgarno (SD) sequence and stem-loop structure in the 5' or 3' untranslated region (UTR) are well-known determinants of mRNA stability. This study investigated the effect of mRNA stability on pullulanase heterologous expression. Results: We constructed four DNA fragments, pulA, SD-pulA, pulA-3t, and SD-pulA-3t, which were cloned into the expression vector pHT43 to generate four pullulanase expression plasmids. The DNA fragment pulA was the coding sequence (CDS) of pulA in Klebsiella variicola Z-13. SD-pulA was constructed by the addition of the 5' SD sequence at the 5' UTR of pulA. pulA-3t was constructed by the addition of a 3' stem-loop structure at the 3' UTR of pulA. SD-pulA-3t was constructed by the addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of pulA. The four vectors were transformed into Escherichia coli BL21(DE3). The pulA mRNA transcription of the transformant harboring pHT43-SD-pulA-3t was 338.6%, 34.9%, and 79.9% higher than that of the other three transformants, whereas the fermentation enzyme activities in culture broth and intracellularly were 107.0 and 584.1 times, 1.2 and 2.0 times, and 62.0 and 531.5 times the amount of the other three transformants (pulA, SD-pulA, and pulA-3 t), respectively. Conclusion: The addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of the pulA gene is an effective approach to increase pulA gene expression and fermentation enzyme activity.

Perfil de sensibilidad antimicrobiana de microorganismos causantes de infecciones urinarias adquiridas en la comunidad en pacientes con diabetes mellitus en Colombia / Antimicrobial susceptibility profile in urinary pathogens causing community-acquired infections in diabetic patients in Colombia

Resumen Introducción. La infección de las vías urinarias es la más frecuente en pacientes diabéticos, y es un factor determinante de la morbilidad y la mortalidad en este grupo de pacientes. El aumento de la resistencia de los microorganismos adquiridos en la comunidad a los antibióticos comúnmente utilizados para combatirla es alarmante. Objetivo. Determinar el perfil de sensibilidad a los antibióticos de los microorganismos responsables de infecciones urinarias adquiridas en la comunidad en pacientes diabéticos atendidos en algunos hospitales de Colombia. Materiales y métodos. Se hizo un estudio descriptivo de un subgrupo de pacientes diabéticos en el marco de una investigación en adultos con infección de origen comunitario de las vías urinarias. Durante un año, se recolectaron aislamientos de Escherichia coli, Klebsiella spp. y Proteus mirabilis en nueve hospitales de Colombia y se determinó su perfil de sensibilidad mediante métodos microbiológicos y moleculares, para establecer la presencia de betalactamasas de espectro extendido del tipo AmpC y de carbapenemasas del tipo KPC. Resultados. Se recolectaron 68 aislamientos (58 de E. coli, nueve de Klebsiella spp. y uno de P. mirabilis). Cuatro (6,9 %) de los aislamientos de E. coli expresaron dichas betalactamasas, en dos (3,4 %) de ellos, pertenecientes al grupo filogenético B2 y al clon ST131, se detectaron las betalactamasas TEM-1 y CTM-X-15. En otros cuatro (6,9 %) aislamientos de E. coli se encontró el fenotipo AmpC, y en tres de ellos se produjeron las betalactamasas TEM-1 y CMY-2. Un aislamiento de K. pneumoniae expresó la carbapenemasa KPC-3. Conclusión. Se confirmó la presencia de cepas productoras de betalactamasas de espectro extendido y carbapenemasas en microorganismos responsables de infección urinaria adquirida en la comunidad en pacientes diabéticos.

Abstract Introduction: Urinary tract infection is the most common pathology in diabetic patients, and an important determinant of morbidity and mortality among them. The increasing resistance of uropathogens acquired in the community to commonly used antibiotics is alarming. Objective: To identify the profile of antibiotic susceptibility of uropathogens responsible for community-acquired infections among diabetic patients in hospitals in Colombia. Materials and methods: We conducted a descriptive study in a subgroup of diabetic patients in the framework of a larger study in adults with urinary tract infection acquired in the community. Over one year, we collected Escherichia coli, Klebsiella spp. and Proteus mirabilis isolates from nine hospitals in Colombia. Their susceptibility profile was determined using microbiological and molecular methods to establish the presence of extended-spectrum AmpC betalactamases and KPC carbapenemases. Results: We collected 68 isolates (58 E. coli, nineKlebsiella spp. and oneProteus mirabilis). Four (6.9%) of the E. coli isolates expressed extended spectrum betalactamases,two (3.4%) of thembelonged to the phylogenetic group B2 andto ST131 clone and expressed the TEM-1 and CTM-X-15 betalactamases. The AmpC phenotype was found in four(6.9%) of the E. coli isolates, three of which producedTEM-1 and CMY-2 betalactamases. One K. pneumoniaeisolate expressed the KPC-3 carbapenemase. Conclusion: The presence of extended spectrum betalactamases and carbapenemases in uropathogens responsible for community-acquired infection was confirmed in diabetic patients.

Improvement of hydrogen yield of ethanol-producing Escherichia coli recombinants in acidic conditions

Background: An effective single culture with high glycerol consumption and hydrogen and ethanol coproduction yield is still in demand. A locally isolated glycerol-consuming Escherichia coli SS1 was found to produce lower hydrogen levels under optimized ethanol production conditions. Molecular approach was proposed to improve the hydrogen yield of E. coli SS1 while maintaining the ethanol yield, particularly in acidic conditions. Therefore, the effect of an additional copy of the native hydrogenase gene hycE and recombinant clostridial hydrogenase gene hydA on hydrogen production by E. coli SS1 at low pH was investigated. Results: Recombinant E. coli with an additional copy of hycE or clostridial hydA was used for fermentation using 10 g/L (108.7 mmol/L) of glycerol with an initial pH of 5.8. The recombinant E. coli with hycE and recombinant E. coli with hydA showed 41% and 20% higher hydrogen yield than wild-type SS1 (0.46 ± 0.01 mol/mol glycerol), respectively. The ethanol yield of recombinant E. coli with hycE (0.50 ± 0.02 mol/mol glycerol) was approximately 30% lower than that of wild-type SS1, whereas the ethanol yield of recombinant E. coli with hydA (0.68 ± 0.09 mol/mol glycerol) was comparable to that of wild-type SS1. Conclusions: Insertion of either hycE or hydA can improve the hydrogen yield with an initial pH of 5.8. The recombinant E. coli with hydA could retain ethanol yield despite high hydrogen production, suggesting that clostridial hydA has an advantage over the hycE gene in hydrogen and ethanol coproduction under acidic conditions. This study could serve as a useful guidance for the future development of an effective strain coproducing hydrogen and ethanol.

Efficient expression and characterization of a cold-active endo-1, 4-β -glucanase from Citrobacter farmeri by co-expression of Myxococcus xanthus protein S

Background: Cold-active endo-1, 4-β-glucanase (EglC) can decrease energy costs and prevent product denaturation in biotechnological processes. However, the nature EglC from C. farmeri A1 showed very low activity (800 U/L). In an attempt to increase its expression level, C. farmeri EglC was expressed in Escherichia coli as an N-terminal fusion to protein S (ProS) from Myxococcus xanthus. Results: A novel expression vector, pET(ProS-EglC), was successfully constructed for the expression of C. farmeri EglC in E. coli. SDS-PAGE showed that the recombinant protein (ProS-EglC) was approximately 60 kDa. The activity of ProS-EglC was 12,400 U/L, which was considerably higher than that of the nature EglC (800 U/L). ProS-EglC was active at pH 6.5-pH 8.0, with optimum activity at pH 7.0. The recombinant protein was stable at pH 3.5-pH 6.5 for 30 min. The optimal temperature for activity of ProS-EglC was 30°C-40°C. It showed greater than 50% of maximum activity even at 5°C, indicating that the ProS-EglC is a cold-active enzyme. Its activity was increased by Co2+ and Fe2+, but decreased by Cd2+, Zn2+, Li+, methanol, Triton-X-100, acetonitrile, Tween 80, and SDS. Conclusions: The ProS-EglC is promising in application of various biotechnological processes because of its cold-active characterizations. This study also suggests a useful strategy for the expression of foreign proteins in E. coli using a ProS tag.

CTX-M extended-spectrum ß-lactamase-producing Klebsiella spp, Salmonella spp, Shigella spp and Escherichia coli isolates in Iranian hospitals

Abstract This study was conducted in Iran in order to assess the distribution of CTX-M type ESBLs producing Enterobacteriaceae. From January 2012 to December 2013, totally 198 E. coli, 139 Klebsiella spp, 54 Salmonella spp and 52 Shigella spp from seven hospitals of six provinces in Iran were screened for resistance to extended-spectrum cephalosporins. After identification and susceptibility testing, isolates presenting multiple-drug resistance (MDR) were evaluated for ESBL production by the disk combination method and by Etest using (cefotaxime and cefotaxime plus clavulanic acid). All isolates were also screened for bla CTX-M using conventional PCR. A total of 42.92%, 33.81%, 14.81% and 7.69% of the E. coli, Klebsiella spp, Salmonella spp and Shigella spp isolates were MDR, respectively. The presence of CTX-M enzyme among ESBL-producing isolates was 85.18%, 77.7%, 50%, and 66.7%, in E. coli, Klebsiella spp, Salmonella spp and Shigella spp respectively. The overall presence of CTX-M genes in Enterobacteriaceae was 15.4% and among the resistant isolates was 47.6%. This study indicated that resistance to β-lactams mediated by CTX-M enzymes in Iran had similar pattern as in other parts of the world. In order to control the spread of resistance, comprehensive studies and programs are needed.

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.