In-vitro high-throughput library screening-Kinetics and molecular docking studies of potent inhibitors of α-glucosidase.

High throughput screening of synthetic compounds against vital enzymes is the way forward for the determination of potent enzyme inhibitors. In-vitro high throughput library screening of 258 synthetic compounds (comp. 1-258), was performed against α-glucosidase. The active compounds out of this library were investigated for their mode of inhibition and binding affinities towards α-glucosidase through kinetics as well as molecular docking studies. Out of all the compounds selected for this study, 63 compounds were found active within the IC50 range of 3.2 µM to 50.0 µM. The most potent inhibitor of α-glucosidase out of this library was the derivative of an oxadiazole (comp. 25). It showed the IC50 value of 3.23 ± 0.8 µM. Other highly active compounds were the derivatives of ethyl-thio benzimidazolyl acetohydrazide with IC50 values of 6.1 ± 0.5 µM (comp. 228), 6.84 ± 1.3 µM (comp. 212), 7.34 ± 0.3 µM (comp. 230) and 8.93 ± 1.0 µM (comp. 210). For comparison, the standard (acarbose) showed IC50 = 378.2 ± 0.12 µM. Kinetic studies of oxadiazole (comp. 25) and ethylthio benzimidazolyl acetohydrazide (comp. 228) derivatives indicated that Vmax and Km, both change with changing concentrations of inhibitors which suggests an un-competitive mode of inhibition. Molecular docking studies of these derivatives with the active site of α-glucosidase (PDB ID:1XSK), revealed that these compounds mostly interact with acidic or basic amino acid residues through conventional hydrogen bonds along with other hydrophobic interactions. The binding energy values of compounds 25, 228, and 212 were -5.6, -8.7 and -5.4 kcal.mol-1 whereas RMSD values were 0.6, 2.0, and 1.7 Å, respectively. For comparison, the co-crystallized ligand showed a binding energy value of -6.6 kcal.mol-1 along with an RMSD value of 1.1 Å. Our study predicted several series of compounds as active inhibitors of α-glucosidase including some highly potent inhibitors.

Effect of varying thickness properties of the slow release fertilizer films on morphology, biodegradability, urea release, soil health, and plant growth.

Green biomass is a renewable and biodegradable material that has the potential use to trap urea to develop a high-efficiency urea fertilizer for crops' better performance. Current work examined the morphology, chemical composition, biodegradability, urea release, soil health, and plant growth effects of the SRF films subjected to changes in the thickness of 0.27, 0.54, and 1.03 mm. The morphology was examined by Scanning Electron Microscopy, chemical composition was analyzed by Infrared Spectroscopy, and biodegradability was assessed through evolved CO2 and CH4 quantified through Gas Chromatography. The chloroform fumigation technique was used for microbial growth assessment in the soil. The soil pH and redox potential were also measured using a specific probe. CHNS analyzer was used to calculate the total carbon and total nitrogen of the soil. A plant growth experiment was conducted on the Wheat plant (Triticum sativum). The thinner the films, the more they supported the growth and penetration of the soil's microorganisms mainly the species of fungus possibly due to the presence of lignin in films. The fingerprint regions of the infrared spectrum of SRF films showed all films in soil changed in their chemical composition due to biodegradation but the increase in the thickness possibly provides resistance to the films' losses. The higher thickness of the film delayed the rate and time for biodegradation and the release of methane gas in the soil. The 1.03 mm film (47% in 56 days) and 0.54 mm film (35% in 91 days) showed the slowest biodegradability as compared to the 0.27 mm film with the highest losses (60% in 35 days). The slow urea release is more affected by the increase in thickness. The Korsymer Pappas model with release exponent value of < 0.5 explained the release from the SRF films followed the quasi-fickian diffusion and also reduced the diffusion coefficient for urea. An increase in the pH and decrease in the redox potential of the soil is correlated with higher total organic content and total nitrogen in the soil in response to amending SRF films with variable thickness. Growth of the wheat plant showed the highest average plant length, leaf area index and grain per plant in response to the increase in the film's thickness. This work developed an important knowledge to enhance the efficiency of film encapsulated urea that can better slow the urea release if the thickness is optimized.

Hybrid molecularly imprinted polymers for targeted separation and enrichment of 10-hydroxycamptothecin in Camptotheca acuminata Decne.

The molecularly imprinted polymer was synthesized using 3-aminopropylthiosilane-methacrylic acid monomer (APTES-MAA) as the functional monomer and 10-hydroxycamptothecin (HCPT) as the template, based on computer simulation. The hybrid molecularly imprinted polymers (HMIPs) were characterized using Fourier transform infrared spectroscopy, thermogravimetric analysis, particle size measurement, scanning electron microscopy and energy dispersive X-ray spectroscopy. It has been shown that HMIPs are irregularly shaped and porous, with particle sizes ranging mainly from 130 to 211 nm. At 298 K, the HMIPs exhibit a maximum adsorption capacity of 8.35 mg·g-1 for HCPT and demonstrate good adsorption specificity (α = 5.38). The pseudo-second-order reaction mechanism suggests that the equilibrium adsorption capacity of HCPT on HMIPs is 8.11 mg·g-1. Finally, HCPT was successfully separated and enriched from the extract of Camptotheca acuminata Decne. seeds using HMIPs.

First identification of potato tuber rot caused by Penicillium solitum, its silver nanoparticles synthesis, characterization and use against harmful pathogens.

Potato is one of the highly consumed vegetable crop grown in different regions across Pakistan that is affected by fungal diseases. The current research was conducted to identify fungal pathogen causing mold-like disease of potato in Khyber Pakhtunkhwa (KP), Pakistan. For molecular identification and characterization of the fungal disease; potato tuber samples were collected followed by culturing on potato dextrose agar (PDA). Based on morphological features, the pathogen was identified as a Penicillium species. This result was obtained in 45 different isolates from potato tubers. Molecular identification was done using ß-tubulin primers and ITS5 sequencing of 13 different isolates that releveled 98% homology with BLAST (GenBank accession no. KX958076) as Penicillium solitum (GenBank accession nos. ON307317; ON307475 and ON310801). Phylogenetic tree was constructed that showed Penicillium solitum prevalence along with Penicillium polonicum and Penicillium citrinum on potato tubers. Based on this, Penicillium solitum based silver nanoparticles (Ag NPs) were synthesized and characterized using UV-visible spectroscopy, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), energy dispersive X-ray (EDX) and field emission scanning electron microscopy (FE SEM). UV-analysis showed a characteristic peak at 410 nm confirming synthesis of Penicillium solitum based Ag NPs. This was further confirmed by XRD followed by EDX and SEM that showed face cubic crystal structure with Ag as major constituent of 18 nm formed spherical Ag NPs. FTIR showed band stretching of O-H, N-O and C-H of biological origin. Similarly, Penicillium solitum based Ag NPs presented strong anti-bacterial and anti-fungal activity at 0.5 level of significance LSD. According to our knowledge, this is the first report of Penicillium solitum identification in Pakistan, its Ag NPs synthesis and characterization to be used against pathogens of agricultural significance.

Process optimization, antioxidant, antibacterial, and drug adjuvant properties of bioactive keratin microparticles derived from porcupine (Hystrix indica) quills.

A structural protein called keratin is often employed in the medical industry to create medication carriers. Process improvement, antioxidant, antibacterial, and adjuvant drug studies of synthetic bioactive keratin microparticles made from lipids and keratin derived from porcupine (Hystrix indica) quills are the main objectives of this study. After coating the keratin microparticles with lipids which were obtained from the same porcupine quills, the bioactive keratin microparticles were produced. The response surface technique was applied to optimize the conditions for extraction of the keratin protein and sizing of the keratin microparticles. An infrared spectroscopy was used to analyze the chemical shifts in compositions of keratin microparticles while the optical microscopy was used to measure the size of the keratin microparticles. The results of this work revealed that a yield 27.36 to 42.25% of the keratin protein could be obtained from porcupine quills. The keratin microparticles were sized between 60.65 and 118.87 µm. Through response surface optimization, mercaptoethanol and urea were shown to be the main variables which positively affected the yield and the size of the keratin protein. The lipid stacking on the keratin microparticles' surface was confirmed by infrared spectroscopy. The 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonate) assay confirmed the keratin microparticle's antioxidant activity of 29.83%. Compared to lipid alone, the antibacterial properties of the keratin microparticles against Escherichia coli-a gram-negative-and Staphylococcus aureus-a gram-positive-bacteria enhanced by up to 55% following the coating of the microparticles with the lipids. The pharmacological action against these bacterial species was further improved by the lipid-loaded erythromycin that was carried on the surface of keratin microparticles. This work has demonstrated the design and uses of the keratin microparticles obtained from porcupine quills for clinical applications.

Quick and In Situ Detection of Different Polar Allelochemicals in Taxus Soil by Microdialysis Combined with UPLC-MS/MS.

The action of allelopathy need that allelochemicals exist in the soil and reach a certain concentration. Also, the detection of allelochemicals in the soil is one of the most important research topics in the process of exploring allelopathy. To solve the problem of the simultaneous detection of allelochemicals with low concentrations and different polarities, a novel strategy for the quick detection of the allelochemicals in Taxus soil by microdialysis combined with UPLC-MS/MS on the basis of in situ detection without destroying the original structure of soil was developed for the first time in the work. The dialysis conditions were optimized by the Box-Behnken design (BBD): 70% methanol, 3 µL/min flow rate, and 3 cm long membrane tube. A reliable UPLC-MS/MS program was systematically optimized for the simultaneous detection of nine allelochemicals with different polarities. The results proved the differences in the contents and distributions of nine allelochemicals in three different Taxus soils.

Clinical Prevalence, Antibiogram Profiling and Gompertz Growth Kinetics of Resistant Staphylococcus epidermidis Treated with Nanoparticles of Rosin Extracted from Pinus roxburghii.

The rise of methicillin-resistant Staphylococcus epidermidis (MRSE) makes it difficult to treat infections that increase morbidity and mortality rates in various parts of the world. The study's objectives include identifying the clinical prevalence, antibiogram profile, and Gompertz growth kinetics of MRSE treated with synthetically created nanoparticles of rosin obtained from Pinus roxburghii. A total of 64 of 200 clinical isolates of S. epidermidis (32% of the total) displayed sensitivity (40.62%) and resistance (59.37%) to seven different antibiotic classes. The most sensitive patterns of antibiotic resistance were seen in 20 (78.95%) and 24 (94.74%) isolates of MRSE against piperacillin/tazobactam and cephradine, respectively. Fosfomycine was found to be the most effective antibiotic against MRSE in 34 (89.47%) isolates, followed by amoxicillin. Successfully produced, described, and used against MRSE were rosin maleic anhydride nanoparticles with a size range of 250 nm to 350 nm. Five different concentrations of 25, 50, 75, 100, and 150 mg mL-1 rosin maleic anhydride nanoparticles were investigated to treat MRSE resistance. According to Gompertz growth kinetics, the maximal growth response was 32.54% higher and the lag phase was also 10.26% longer compared to the control when the amount of rosin maleic anhydride nanoparticles was increased in the MRSE. Following the application of rosin maleic anhydride nanoparticles, the growth period is extended from 6 to 8 h. A potential mechanism for cell disintegration and distortion is put forth. This investigation came to the conclusion that rosin maleic anhydride nanoparticles better interfere with the surface of MRSE and demonstrated a preferred bacteriostatic action.

Overexpression of antisense phosphatase 2C affords cold resistance in hybrid Populus davidiana × Populus bolleana.

BACKGROUND: Overexpression of the abiotic and biotic stress-resistance genes of the plant signaling pathway is well known for its significant role in the regulation of plant growth and enhancement of the productivity of agricultural land under changing climatic conditions. OBJECTIVES: This research aimed to clone Populus davidiana × Populus bolleana PP2C (PdPP2C) gene and analyze its structure and function, and downregulate PdPP2C by overexpression of its antisense PdPP2C (AS-PdPP2C) gene for enhancing cold resistance in transgenic lines of hybrid P. davidiana × P. bolleana. METHODS: PdPP2C was cloned and transformed to identify its function, and its antisense was overexpressed via downregulation to increase the cold resistance in transgenic lines of hybrid P. davidiana × P. bolleana. RESULTS: Antisense inhibition of protein phosphatase 2C accelerates the cold acclimation of Poplar (P. davidiana × P. bolleana) gene in terms of antifreeze. CONCLUSION: PdPP2C was expressed in the roots, stems, and leaves of P. davidiana × P. bolleana, and the expression was higher in the leaves. The expression of PdPP2C was also significantly downregulated at low-temperature (0 °C and 4 °C) stress. The relative conductivity and malondialdehyde content of non-transgenic lines were higher than those of AS-PdPP2C lines after 2 days of cold treatment at - 1 °C. The leaves of the transgenic lines were not wilted and showed no chlorosis compared with those of the non-transgenic lines. The AS-PdPP2C transgenic lines also showed higher freezing resistance than the non-transgenic lines. AS-PdPP2C participated in the regulation of freezing resistance.

Analysis of drought and salt-alkali tolerance in tobacco by overexpressing WRKY39 gene from Populus trichocarpa.

WRKY is one of the largest families of transcription factors in plants. It not only regulates plant growth and development but also participates in the regulation of plant defense against biological and abiotic stresses. In this study, research was aimed to overexpress WRKY39 gene of P. trichocarpa (PtWRKY39) and to identify its important role played in drought and saline-alkali tolerance in tobacco model plant. Under the control of CaMV35S promoter, the overexpression of PtWRKY39 gene was increased to more than 10 times in T3 generation of transgenic tobacco plant. The drought resistance and saline-alkali tolerance were evidenced in overexpressed PtWRKY39 transgenic lines at germination/seedling stage. The overall germination rate, fresh weight, and chlorophyll contents of overexpressed lines were significantly higher while the level of malondialdehyde was significantly lower in PtWRKY39 transgenic lines than that of wild type (WT) lines. The content of H2O2 in leaves was detected by the 3, 3-Diaminobenzidine method showed that the overexpression of PtWRKY39 gene could reduce the accumulation of ROS (mainly H2O2) and enhance salt-alkali tolerance. Phenotypic analysis at 7-leaf pot transgenic seedlings stage treated with the saline-alkali soil extract and salt NaCl under root irrigation stress, revealed growth of the transgenic line was significantly higher than that of WT. This work concludes that overexpression of PtWRKY39 gene can improve the regulation of drought resistance and saline-alkali tolerance of transgenic plants during seed germination and vegetative growth.

CO1-Based DNA barcoding for assessing diversity of Pteropus giganteus from the state of Azad Jammu Kashmir, Pakistan.

The flying fox (Pteropus giganteus) also familiar with the name of the greater Indian fruit Bat belongs to the order Chiroptera and family Pteropodidae. Current research emphasis on the DNA barcoding of P. giganteus in Azad Jammu Kashmir. Bat sequences were amplified and PCR products were sequenced and examined by bioinformatics software. Congeneric and conspecific, nucleotide composition and K2P nucleotide deviation, haplotype diversity and the number of haplotypes were estimated. The analysis showed that all of the five studied samples of P. giganteus had low G contents (G 19.8%) than C (27.8%), A (25.1%) and T (27.3%) contents. The calculated haplotype diversity was 0.60% and the mean intraspecific K2P distance was 0.001% having a high number of transitional substitutions. The study suggested that P. giganteus (R=0.00) do not deviate from the neutral evolution. It was determined from the conclusion that this mtDNA gene is a better marker for identification of Bat species than nuclear genes due to its distinctive characteristics and may serve as a landmark for the identification of interconnected species at the molecular level and in the determination of population genetics.

WITHDRAWN: Sustainable liquid membrane separation using interfacial engineering of deep eutectic solvent and cellulose acetate.

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CO1-Based DNA barcoding for assessing diversity of Pteropus giganteus from the state of Azad Jammu Kashmir, Pakistan / Código de barras de DNA à base de CO1 para avaliar a diversidade de Pteropus giganteus do estado de Azad Jammu e Caxemira, Paquistão

Abstract The flying fox (Pteropus giganteus) also familiar with the name of the greater Indian fruit Bat belongs to the order Chiroptera and family Pteropodidae. Current research emphasis on the DNA barcoding of P. giganteus in Azad Jammu Kashmir. Bat sequences were amplified and PCR products were sequenced and examined by bioinformatics software. Congeneric and conspecific, nucleotide composition and K2P nucleotide deviation, haplotype diversity and the number of haplotypes were estimated. The analysis showed that all of the five studied samples of P. giganteus had low G contents (G 19.8%) than C (27.8%), A (25.1%) and T (27.3%) contents. The calculated haplotype diversity was 0.60% and the mean intraspecific K2P distance was 0.001% having a high number of transitional substitutions. The study suggested that P. giganteus (R=0.00) do not deviate from the neutral evolution. It was determined from the conclusion that this mtDNA gene is a better marker for identification of Bat species than nuclear genes due to its distinctive characteristics and may serve as a landmark for the identification of interconnected species at the molecular level and in the determination of population genetics.

Resumo A raposa-voadora (Pteropus giganteus), também conhecida como morcego indiano, pertence à ordem dos Chiroptera e à família Pteropodidae. A presente pesquisa dá ênfase ao código de barras de DNA de P. giganteus em Azad Jammu e Caxemira. Sequências genéticas dos morcegos foram amplificadas, e os produtos de PCR foram sequenciados e examinados por software de bioinformática. De espécies congenérica e coespecífica, foram estimados composição nucleotídica e desvio de nucleotídeos K2P, diversidade de haplótipos e número de haplótipos. A análise mostrou que todas as cinco amostras estudadas de P. giganteus apresentaram baixos teores de G (19,8%) em comparação com C (27,8%), A (25,1%) e T (27,3%). A diversidade de haplótipos calculada foi de 0,60%, e a distância média intraespecífica de K2P foi de 0,001%, com um elevado número de substituições transicionais. O estudo sugeriu que P. giganteus (R = 0,00) não se desviou da evolução neutra. É possível concluir que o gene mtDNA é um marcador favorável para identificação de espécies de morcegos do que genes nucleares por causa de suas características distintivas e pode servir como um marco para a identificação de espécies interconectadas em nível molecular e para a determinação genética de populações.

CO1-Based DNA barcoding for assessing diversity of Pteropus giganteus from the state of Azad Jammu Kashmir, Pakistan

Abstract The flying fox (Pteropus giganteus) also familiar with the name of the greater Indian fruit Bat belongs to the order Chiroptera and family Pteropodidae. Current research emphasis on the DNA barcoding of P. giganteus in Azad Jammu Kashmir. Bat sequences were amplified and PCR products were sequenced and examined by bioinformatics software. Congeneric and conspecific, nucleotide composition and K2P nucleotide deviation, haplotype diversity and the number of haplotypes were estimated. The analysis showed that all of the five studied samples of P. giganteus had low G contents (G 19.8%) than C (27.8%), A (25.1%) and T (27.3%) contents. The calculated haplotype diversity was 0.60% and the mean intraspecific K2P distance was 0.001% having a high number of transitional substitutions. The study suggested that P. giganteus (R=0.00) do not deviate from the neutral evolution. It was determined from the conclusion that this mtDNA gene is a better marker for identification of Bat species than nuclear genes due to its distinctive characteristics and may serve as a landmark for the identification of interconnected species at the molecular level and in the determination of population genetics.

Resumo A raposa-voadora (Pteropus giganteus), também conhecida como morcego indiano, pertence à ordem dos Chiroptera e à família Pteropodidae. A presente pesquisa dá ênfase ao código de barras de DNA de P. giganteus em Azad Jammu e Caxemira. Sequências genéticas dos morcegos foram amplificadas, e os produtos de PCR foram sequenciados e examinados por software de bioinformática. De espécies congenérica e coespecífica, foram estimados composição nucleotídica e desvio de nucleotídeos K2P, diversidade de haplótipos e número de haplótipos. A análise mostrou que todas as cinco amostras estudadas de P. giganteus apresentaram baixos teores de G (19,8%) em comparação com C (27,8%), A (25,1%) e T (27,3%). A diversidade de haplótipos calculada foi de 0,60%, e a distância média intraespecífica de K2P foi de 0,001%, com um elevado número de substituições transicionais. O estudo sugeriu que P. giganteus (R = 0,00) não se desviou da evolução neutra. É possível concluir que o gene mtDNA é um marcador favorável para identificação de espécies de morcegos do que genes nucleares por causa de suas características distintivas e pode servir como um marco para a identificação de espécies interconectadas em nível molecular e para a determinação genética de populações.

Caracterización genotípica de Staphylococcus aureus resistente a la meticilina prevalente en hospitales de Pakistán / Genotypic characterization of prevalent methicillin resistant Staphylococcus aureus in Pakistani community hospitals / Caracterização de Staphylococcus aureus resistente à meticilina prevalecente em hospitais da comunidade no Paquistão

El gen de la proteína A se usó como marcador genético para la caracterización de aislados de Staphylococcus aureus resistentes a la meticilina (SAMR). De un total de 130 aislados de Staphylococcus aureus, 90 fueron identificados como SAMR y 81 de éstos se pudieron caracterizar por tipificación spa. Todos estos aislados fueron obtenidos de cinco Hospitales Nacionales de la Comunidad. Se utilizaron dos juegos diferentes de cebadores para amplificar la región-X del gen de la proteína A en las cepas de SAMR. Un conjunto de cebadores, spa-F/spa-R ha identificado tres tipos de repeticiones diferentes, a saber, 7 repeticiones (spa 2), 8 repeticiones (spa 3) y 10 repeticiones (spa 4) y otro conjunto de cebadores, spa-1113F/spa-1514R ha identificado 4 tipos de repeticiones diferentes, a saber, 6 repeticiones (spa 1), 15 repeticiones (spa 6), y 17 repeticiones (spa 7) y 19 repeticiones (spa 8). Se identificó la repetición 11 (spa 5) con ambos conjuntos de cebadores. Los tipos de SAMR esporádicos que portaban las repeticiones 6, 7, 10, 17 y 19 fueron poco prevalentes mientras que los SAMR epidémicos con 8, 11, y 15 repeticiones fueron más prevalentes y se los consideró involucrados en la transmisión entre los pacientes dentro de los diferentes hospitales. Este trabajo concluye que la técnica spa es lo suficientemente eficiente como para diferenciar las cepas epidémicas, esporádicas y aquéllas que se transforman lentamente de esporádicas a epidémicas.

Protein A gene was used as a genetic marker for the characterization of Pakistani methicillin resistant Staphylococcus aureus (MRSA) isolates. Out of a total of 130 Staphylococcus aureus isolates, 90 were identified as MRSA and of these 90 MRSA, 81 MRSA isolates were characterized by spa typing. All of these isolates were collected from five National Community Hospitals. Two different sets of primers were used to amplify the X-region of Protein A gene in MRSA strains. One set of primers i.e, spa-F/spa-R identified three types of different repeats viz., 7 repeats (spa 2), 8 repeats (spa 3) and 10 repeats (spa 4) and another set of primers i.e. spa-1113F/spa-1514R identified 4 types of different repeats viz., 6 repeats (spa 1), 15 repeats (spa 6), and 17 repeats (spa 7) and 19 repeats (spa 8). Repeat 11 (spa 5) was identified with both sets of primers. Sporadic MRSA types carrying 6, 7, 10, 17 and 19 repeats were less prevalent, while the epidemic MRSA with 8, 11 and 15 repeats were more prevalent and considered to be involved in transmission among the patients within different hospitals. Research work concludes that spa technique is efficient enough to differentiate spa strains carrying variations in general and those slowly transforming from sporadic to epidemic outbreak in particular.

O gene da proteína A foi usado como marcador genético para a caracterização de isolados de Staphylo­coccus aureus resistentes à meticilina (SAMR). De um total de 130 isolados de Staphylococcus aureus, 90 foram identificados como SAMR e 81 destes puderam se caracterizar por tipificação spa. Todos estes isolados foram obtidos de cinco Hospitais Nacionais da Comunidade. Utilizaram-se dois jogos diferentes de cevadores para amplificar a região-X do gene da proteína A nas cepas de SAMRUn conjunto de ce­vadores, spa-F/spa-R tem identificado três tipos de repetições diferentes, a saber, 7 repetições (spa 2), 8 repetições (spa 3) e 10 repetições (spa 4) e outro conjunto de cevadores, spa-1113F/spa-1514R tem identificado 4 tipos de repetições diferentes, a saber, 6 repetições (spa 1), 15 repetições (spa 6), e 17 repetições (spa 7) e 19 repetições (spa 8). Foi identificada a repetição 11 (spa 5) com ambos os conjuntos de cevadores. Os tipos de SAMR esporádicos que tinham as repetições 6, 7, 10, 17 e 19 foram pouco prevalecentes enquanto que os SAMR epidêmicos com 8, 11, e 15 repetições foram mais prevalecentes e são considerados envolvidos na transmissão entre os pacientes dentro dos diferentes hospitais. Este trabalho conclui que a técnica spa é o suficientemente eficiente como para diferenciar as cepas epidêmicas, esporádicas e aquelas que se transformam lentamente de esporádicas em epidêmicas.