Cloning, sequencing, and characterizing of soil antibiotic active-producing Streptomyces species-specific DNA markers.

Association of the antibiotic activity of the soil Streptomyces isolates to their genetic profiles analyzed through RAPD-PCR fingerprints prompted us here in this study to use the most common bands as specific markers to identify homologous proteins within these isolates by cloning, sequencing, and characterizing these markers. Six out of twelve DNA bands ranged between 600 and 1350 bp previously obtained by RAPD-PCR analysis were purified out of the RAPD gels, and then cloned into pGEM-T Easy vector system. Success of the cloning process was confirmed by digesting purified plasmids with EcoRI. The clones namely No. 54, 55, 20, 56, 57, and 58 were sequenced using the DNA BigDye Terminator Sequencing System utilizing the M13 primer. Results indicated that the size of the inserted sequences is 599, 566, 522, 870, 857, and 254 bp, in clones No. 54. 55, 20, 56, 57, and 58, respectively. Homologous proteins of the six cloned sequences generated by DNA blast software indicated that the highest score of protein homology was scored for clone No. 54 with 87 % homology to putative secreted pectate lyase [Streptomyces coelicolor A3(2)]. The other clones showed less homology with 77 % homology for the clones No. 55 and 56, 73 % homology for the clone No. 20, and 55 % homology for the clones No. 57 and 58. The association of homologous proteins to the reported RAPD pattern is confirmed here for the first time, and the resulting DNA cloned fragments deserve further molecular analysis.

Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii isolated from three major hospitals in Jordan.

BACKGROUND: In the last decade, incidences of carbapenem-resistant Acinetobacter baumannii have been increasingly reported worldwide. Consequently, A. baumannii was included in the World Health Organization's new list of critical pathogens, for which new drugs are desperately needed. The objective of this research was to study the molecular epidemiology and antimicrobial susceptibility of clinical carbapenem-resistant A. baumannii isolated from Jordanian hospitals. METHODS: A total of 78 A. baumannii and 8 Acinetobacter spp. isolates were collected from three major hospitals in Jordan during 2018. Disc diffusion and microdilution methods were used to test their susceptibility against 19 antimicrobial agents. Multilocus sequence typing (MLST) was performed using the Pasteur scheme, followed by eBURST analysis for all isolates. PCR was used to detect ß-lactam resistance genes, blaOXA-23-like , blaOXA-51-like , and blaNDM-1 . RESULTS: Of the 86 tested isolates, 78 (90.6%) exhibited resistance to carbapenems, whereas no resistance was recorded to tigecycline or polymyxins. Based on the resistance profiles, 10.4% and 84.8% of isolates were classified into multidrug resistant (MDR) or extensively drug resistant (XDR), respectively. The most prevalent carbapenems resistance genes amongst isolates were blaOXA-51-Like (89.5%), followed by blaOXA-23-Like (88.3%) and blaNDM-1 (10.4%). MLST revealed the presence of 19 sequence types (STs), belonging to eight different international complexes. The most commonly detected clonal complex (CC) was CC2, representing 64% of all typed isolates. CONCLUSIONS: This is the first study to report the clonal diversity of A. baumannii isolates in Jordan. A high incidence of carbapenem resistance was detected in the isolates investigated. In addition, our findings provided evidence for the widespread of blaOXA-23-like harbouring carbapenem-resistant A. baumannii and belonging to CC2. The number of XDR isolates identified in this study is alarming. Thus, periodic surveillance and molecular epidemiological studies of resistance factors are important to improve treatment outcomes and prevent the spread of A. baumannii infections.

Genomic detection of waterborne enteric viruses as water quality indicators in Al-Zarqa River, Jordan.

Al-Zarqa River is the second main tributary to River Jordan after the Yarmouk River. The river flow has been modified by discharge of industrial wastewater and treated domestic water. Concerns about the occurrence of waterborne pathogenic viruses in the surface waters of Al-Zarqa River prompted the analysis of the surface water quality with respect to the presence of enteric viruses. Viruses were concentrated from a total of 33 different water environmental samples including raw sewage, effluent samples and river water collected from and around the river over a period of 11 months. Calculated recovery yields for these concentration methods ranged between 2 and 8%. Polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), nested RT-PCR and southern blotting hybridization analysis were used for the detection of hepatitis A virus, norovirus, astrovirus and human adenovirus 40/41, with the later one being detected in 21 (64%) of the samples that also showed previous positive presence for enteroviruses. To our knowledge, this is the first molecular biology report in Jordan describing the circulation of adenoviruses, which were detected more frequently than enteroviruses in sewage and water samples, and therefore, they can be used as an index for the presence of human pathogenic viruses in water environment.

Phenotypic and molecular analysis of dominant occurring antibiotic active-producing Streptomyces soil flora in Northern Jordan.

This investigation aimed to determine the relatedness of dominant occurring soil Streptomyces spp. in Northern Jordan based on their RAPD-PCR fingerprints, and to compare RAPD technique with the conventional phenotypic characterization of Streptomyces isolates. Fifty-eight white and gray color-bearing aerial mycelia antibiotic active-producing Streptomyces soil isolates along with three reference strains were genetically analyzed by RAPD-PCR. Polymorphisms between the isolates showed 1 to 10 bands per isolate and ranged from 200 to 3200 bp in size. Results revealed one common band of ~600 bp shared by ~85% of the isolates, and the observation of bands specific to some reference strains and some soil isolates. When RAPD patterns were analyzed with the UPGMA, results revealed clustering the tested isolates into two equal main super clusters (50% each). Super cluster I appeared to be homogenous and include the three reference strains. However, super cluster II was heterogeneous and but not including any of the reference strains. The association of the antibiotic activity of the dominant white and gray aerial mycelium-bearing Streptomyces isolates to RAPD clustering is reported for the first time, and the RAPD-PCR fingerprints generated here deserve to be cloned, characterized and sequenced in future as Streptomyces species-specific DNA markers. The more random primers used in the analysis may add to RAPD technique a cost-effective, fast, precise result, and less labor work solution for analyzing the similarities and differences among the Streptomyces isolates.

Phenolic Composition and Antimicrobial Activity of Different Emirati Date (Phoenix dactylifera L.) Pits: A Comparative Study.

The biochemical composition, secondary metabolites (phenolic compounds, flavonoids) and antimicrobial potential of different varieties of Emirati date (Phoenix dactylifera L.) pits were investigated. Total phenolic acids (TPC) and total flavonoid contents (TFC) of the different date pits were measured using a Folin-Ciocalteau reagent. Different organic solvents [(n-hexane; H2O: EtOH (1:1); ethyl acetate; acetone: Water (1:1); and methanol: Chloroform (1:1)] were compared to evaluate the phytotoxicity of Ajwa, Fard, Khalas, Khodari, Abu Maan, Lulu, and Mabroom date pits. The antimicrobial activity of the date pit extracts were evaluated by means of agar-well diffusion assay on Staphylococcus aureus (ATCC 29123), Escherichia coli (ATCC 25922) and Candida albicans (ATCC 66027). Minimum inhibitory concentrations (MICs) were measured following clinical laboratory standardization institute (CLSI) protocol. The biochemical analyses of date pits indicate that TPC were ranged from 7.80 mg of equivalent gallic acid/100 g dry weight in Ajwa to 4.65 mg in Mabroom. The TFC were ranged between 1.6-4.54 mg of equivalent catechin/100 g dry weight. Ajwa pit extract showed good quality traits (higher protein, lower ash content, and intermediate dietary fiber). The results indicate that the ethyl acetate extract of Khalas and Khodari inhibited S. aureus with an inhibition zone diameter of 20 mm and MIC of 10 mg/mL. Abu Mann pit extract inhibited the S. aureus and also decreased the population of E. coli. The diameter of inhibition zone was 15, 16, and 18 mm after treatment with Ajwa extracts, while the MICs were 7.5 and 5 mg/mL. The MeOH: CFM extract of Abu Mann and Ajwa was more potent against E. coli bacteria than any other extract. This work demonstrates that the Emirati date pits extract has antimicrobial (antibacterial, antifungal) potential and can be used as phytotoxic natural compounds.

Old leaves accumulate more heavy metals than other parts of the desert shrub Calotropis procera at a traffic-polluted site as assessed by two analytical techniques.

Calotropis procera is a perennial big shrub that has the potential to accumulate high concentrations of heavy metals. Metal sequestration in old organs has been considered as a mechanism for plant survival in polluted soils. The aim of the present study was to assess the role of the old leaves as a sink for HMs accumulation in C. procera. Two instruments were used: atomic absorption spectroscopy (AAS) and X-ray fluorescence (XRF) microscopy. Soil and plant samples were collected from around one of the worst congested traffic areas in the United Arab Emirates (UAE). Samples from roots, stem, and green and old leaves were prepared and analyzed by both instruments. Calotropis procera was able to concentrate Fe, Mn, Sr, and Zn in the roots, but their translocation to stem and green leaves was low. Old leaves had greater ability to accumulate significantly higher concentrations of different metals, especially Fe and Sr, than other parts of the plants, indicating that C. procera uses these metabolically less-active leaves as sinks for heavy metals. Fe and Sr attained higher bioconcentration and accumulation values, compared to Zn and Mn. There were significant positive correlations between XRF and AAS for all elements in the different organs.

Potential Biotechnological Strategies for the Cleanup of Heavy Metals and Metalloids.

Global mechanization, urbanization, and various natural processes have led to the increased release of toxic compounds into the biosphere. These hazardous toxic pollutants include a variety of organic and inorganic compounds, which pose a serious threat to the ecosystem. The contamination of soil and water are the major environmental concerns in the present scenario. This leads to a greater need for remediation of contaminated soils and water with suitable approaches and mechanisms. The conventional remediation of contaminated sites commonly involves the physical removal of contaminants, and their disposition. Physical remediation strategies are expensive, non-specific and often make the soil unsuitable for agriculture and other uses by disturbing the microenvironment. Owing to these concerns, there has been increased interest in eco-friendly and sustainable approaches such as bioremediation, phytoremediation and rhizoremediation for the cleanup of contaminated sites. This review lays particular emphasis on biotechnological approaches and strategies for heavy metal and metalloid containment removal from the environment, highlighting the advances and implications of bioremediation and phytoremediation as well as their utilization in cleaning-up toxic pollutants from contaminated environments.

Complement components as potential therapeutic targets for asthma treatment.

Asthma is the most common respiratory disorder, and is characterized by distal airway inflammation and hyperresponsiveness. This disease challenges human health because of its increasing prevalence, severity, morbidity, and the lack of a proper and complete cure. Asthma is characterized by T(H)2-skewed inflammation with elevated pulmonary levels of IL-4, IL-5, and IL-13 levels. Although there are early forays into targeting T(H)2 immunity, less-specific corticosteroid therapy remains the immunomodulator of choice. Innate immune injury mediated by complement components also act as potent mediators of the allergic inflammatory responses and offer a new and exciting possibility for asthma immunotherapy. The complement cascade consists of a number of plasma- and membrane-bound proteins, and the cleavage products of these proteins (C3 and C5) regulate the magnitude of adaptive immune responses. Complement protein are responsible for many pathophysiological features of asthma, including inflammatory cell infiltration, mucus secretion, increases in vascular permeability, and smooth muscle cell contraction. This review highlights the complement-mediated injury during asthma inflammation, and how blockade of active complement mediators may have therapeutic application.

Isolation of Cronobacter spp. (formerly Enterobacter sakazakii) from infant food, herbs and environmental samples and the subsequent identification and confirmation of the isolates using biochemical, chromogenic assays, PCR and 16S rRNA sequencing.

BACKGROUND: Cronobacter spp. (formerly Enterobacter sakazakii), are a group of Gram-negative pathogens that have been implicated as causative agents of meningitis and necrotizing enterocolitis in infants. The pathogens are linked to infant formula; however, they have also been isolated from a wide range of foods and environmental samples. RESULTS: In this study, 233 samples of food, infant formula and environment were screened for the presence of Cronobacter spp. in an attempt to find its source. Twenty nine strains were isolated from samples of spices, herbs, infant foods, and dust obtained from household vacuum cleaners. Among the 76 samples of infant food, infant formula, milk powder and non-milk dairy products tested, only one sample of infant food contained Cronobacter spp. (1.4%). The other Cronobacter spp. isolates recovered include two from household vacuum dust, and 26 from 67 samples of herbs and spices. Among the food categories analyzed, herbs and spices harbored the highest number of isolates, indicating plants as a possible reservoir of this pathogen. Initial screening with API 20E test strips yielded 42 presumptive isolates. Further characterization using 3 chromogenic media (alpha-MUG, DFI and EsPM) and 8 sets of PCR primers detecting ITS (internal transcribed spacer sequences), 16S rRNA, zpx, gluA, gluB, OmpA genes followed by nucleotide sequencing of some PCR amplicons did not confirm the identity of all the isolates as none of the methods proved to be free of both false positives or false negatives. The final confirmation step was done by 16S rRNA sequence analysis identifying only 29 of the 42 isolates as Cronobacter spp. CONCLUSION: Our studies showed that Cronobacter spp. are highly diverse and share many phenotypic traits with other Enterobacteriaceae members highlighting the need to use several methods to confirm the identity of this pathogen. None of the biochemical, chromogenic or PCR primers proved to be a reliable method for confirmation of the identity of the isolates as all of them gave either false positives or false negatives or both. It is therefore concluded that 16S rRNA sequencing is pivotal to confirm the identity of the isolates.

Influence of culture conditions of Streptomyces sp. (strain S242) on chitinase production.

The purpose of this study was to determine the influence of growth conditions and medium composition on the production ofchitinase by Streptomyces sp. (strain S242). Production of chitinase by strain S242 was detected on colloidal chitin agar (CCA) medium after 8 days of incubation at 28 degrees C resulting in a clear zone 10 mm around the colony. Chitinase activity was assayed as the amount of N-acetylglucosamine released in micromol/ml/min using the dinitrosalicylic acid assay method. The crude enzyme had maximum activity (0.162 U ml/l) after 4 days of incubation at pH 7 and 30 degrees C when the broth medium was supplemented with 1.6% of colloidal chitin. However, enzyme activity was strongly decreased at 40 degrees C and extreme acidic and alkaline pH values. SDS-PAGE and zymogram analysis revealed six distinctive bands that range from 39 to 97 kDa with chitinolytic activity. The findings of this investigation create a possibility for the use of the organism in the commercial production of chitinase. In addition, it can be a source of DNA for cloning the chitinase gene(s) to generate phytopathogen resistant transgenic plants.

Microbial populations of crude oil spill polluted soils at the Jordan-Iraq desert (the Badia region) / População microbiana de solos contaminados pelo derramamento de petróleo no deserto Jordão-Iraque (região de Badia)

Microbial populations' inhabitants in crude petroleum contaminated soils were analyzed in relation with the soil characteristics. A noticeable greater decline of bacterial counts and diversity but a prevalence of the genus Pseudomonas over the other identified genera in the fresh contaminated soils as compared to the old ones was observed.

Analisou-se a população microbiana de solos contaminados pelo derramamento de petróleo em função das características do solo. Uma diminuição notável foi observada nas contagens e diversidade bacterianas, mas observou-se a prevalência de Pseudomonas em relação aos demais gêneros identificados em solos recentemente contaminados, quando comparado com solos com contaminação antiga.

Usefulness of strb1 and 16S rDNA-targeted PCR for detection of Streptomyces spp. in environmental samples.

In this study, we revealed rapid detection of streptomycin-producing Streptomyces spp. by extraction of total soil DNA from 14 soil samples using a modified lysis method followed by PCR amplification ofa genus-specific sequence in the Streptomyces' 16S rDNA gene. DNA band of the expected size (438 bp) was seen with all the samples. Additionally, specific amplification of the streptomycin-coding gene (strb1) directly from soil revealed the presence of a single DNA band of 940 bp. These results indicate that PCR-amplification of Streptomyces specific genes could be used for direct detection of streptomycin-producing Streptomyces species from soil.

Susceptibility of local Agrobacterium tumefaciens strains to streptomycetes isolates from Jordan soils.

Five strains of Agrobacterium tumefaciens were isolated and characterized from 12 different plant tumors. The susceptibility of these phytopathogens to antibiotics and to soil Streptomyces isolates was tested. Among the 90 Streptomyces isolates, only 12 were able to inhibit the growth of at least one A. tumefaciens strain. Four strains of A. tumefaciens were susceptible to streptomycin and cefotaxime. In addition, Streptomyces 404 strain was able to inhibit the growth of four strains of the Agrobacterium pathogens with an inhibition zone diameter ranged between 10 and 16 mm. The strong inhibitory effects of Streptomyces 404 strain on A. tumefaciens suggest the use of this strain as a promising agent to control crown gall disease.

Screening for soil streptomycetes from North Jordan that can produce herbicidal compounds.

A total of 231 different soil Streptomyces isolates were recovered from 16 different locations in North Jordan. They were assessed for their phytotoxic activity on seeds of cucumber (Cucumis sativus L.) and ryegrass (Lolium perenne L.) placed adjacent to a 2 cm wide Streptomyces culture strips grown at 28C degrees for 3 weeks on starch casein nitrate (SCN) agar. Phytotoxicity was ascertained on the basis of suppressed seed germination, discoloration of the root tip, reduced root and the shoot growth and eventual death of the root. Twenty one of the isolates exhibited adverse effect against growth of germinated cucumber seeds, germination and growth of ryegrass seeds. Using filter paper bioassay method, culture filtrate from the SCN broth of the isolate R9; identified as Streptomyces aburaviensis, significantly inhibited seed germination, radicle and shoot growth ofryegrass, reduced radicle and shoot growth of cucumber and suppressed the shoot growth of milk thistle (Silybum marianum L.). Also, culture filtrate from the glucose-peptone-molasses (GPM) broth diluted (1:1) with sterilized distilled water caused complete inhibition of seed germination of redroot pigweed (Amaranthus retroflexus L.). Dichloromethane extracted fraction of S. aburaviensis (strain R9) culture filtrate from GPM broth completely inhibited seed germination of ryegrass when applied at doses of 3 and 5 mg of dry weight, and the seedling growth of cucumber and milk thistle was severely reduced by the same doses.

Microbial populations of crude oil spill polluted soils at the Jordan-Iraq desert (the Badia region).

Microbial populations' inhabitants in crude petroleum contaminated soils were analyzed in relation with the soil characteristics. A noticeable greater decline of bacterial counts and diversity but a prevalence of the genus Pseudomonas over the other identified genera in the fresh contaminated soils as compared to the old ones was observed.

Selection of bacteria and plant seeds for potential use in the remediation of diesel contaminated soils.

Enumeration and recovery of the dominant bacteria from a chronically fuel contaminated soil has been investigated. Bacterial counts from these polluted soils ranged between 0.70x10(8) and 28.20x10(8) CFU/g soil. Three different types of bacterial colonies have been recovered on the agar plates. Biochemical examination of the recovered bacteria revealed that they mainly belonged to the genus Pseudomonas, Micrococcus and Bacillus. Turbidity, cell biomass (dry weight basis), and physical appearance determined the growth of these bacteria on diesel. A noticeable decline in alfalfa (Medicago sativa) seeds germination of 15-30% was shown at 500 mg/kg diesel or higher. Under these contaminated conditions, fescue grass (Cyndon dactylon) exhibited a higher viability than alfalfa indicating that C. dactylon seeds are relatively tolerant to diesel and can possibly be used in phytoremediation of diesel contaminated soils. Results of diesel phyotoxicity to seed germination of these two plants were based on filter paper media and therefore; should be considered as first indication only. Extrapolation of such results to actual soil conditions should be cautiously approached taking into account diesel sorption on soil and mechanisms of its bioavailability.

Production of 2-methylisoborneol by Streptomyces violaceusniger and its transformation by selected species of Pseudomonas.

The effect of glucose, glycerol and yeast-extract (YE) on 2-methylisoborneol (MIB) production by Streptomyces violaceusniger (C4-S strain) was determined. Glycerol as a sole carbon source promoted MIB production was maximum concentration (40.65 microg/l) after 10 days incubation, while glucose favored biomass production. Increasing YE concentration from 0.1 to 1% (W/V) repressed MIB production with 62 and 83% reduction after 10 and 14 days, respectively. Transformation of this odorous compound was investigated using seven different Pseudomonas species. Action of the different Pseudomonas spp. on the C4-S extract was followed at 1 hour, 2 hours, 6 hours and 12 hours. Pseudomonas spp. 1, 2, 3 and 5 indicated a positive reaction; however, Pseudomonas spp. 4, 6 and 7 showed no effect. These data suggest that 2-methylisoborneol can be transformed by Pseudomonas spp. and the test adopted by this study can be applied for screening organisms for their ability to transform MIB.

Evaluation of combined 16S rDNA and strb1 gene targeted PCR to identify and detect streptomycin-producing Streptomyces.

In this study, two designed primers were evaluated to identify soil Streptomyces and to detect streptomycin production by strb1 targeted PCR. Potential Streptomyces-specific signatures were identified in their 16S rDNA sequences in regions located around nucleotide positions 576 and 995. Primer pair RI7/RI8 derived from these regions was investigated for its specificity in detecting and identifying Streptomyces isolates by PCR assays using DNA from pure cultures. The constructed primer pair showed high specificity in detecting and identifying Streptomyces type strains as well as soil isolates. Streptomycin-producers were detected by PCR assays through the selective amplification of streptomycin biosynthetic gene (strb1). Results suggest that PCR assay facilitates the differential identification of Streptomyces-specific antibiotic producers and a resident population of Streptomyces in Jordan with the capacity of streptomycin-production is present.

Diagnosis of Mycobacterium tuberculosis in cases of pulmonary infections from Jordanian hospitals.

The aim of this study is to use the diagnostic utility of smear technique for the detection of Mycobacterium tuberculosis in clinical specimens obtained from patients suspected of having pulmonary tuberculosis. A total of 305 respiratory specimens (broncoalveolar lavage, sputum and pleural fluid) were collected from 298 patients having pulmonary infections as bronchitis, tuberculosis, pneumonia and other respiratory diseases. Results revealed that 25 specimens collected from 22 patients were positive (8.2%) by acid fast (AF) smear. Data indicated that the specificity and positive predictive value of the conventional smear assay were 100%, however, the sensitivity of the smear examination was 73.5%. Conventional smear technique actually detected M. tuberculosis in respiratory specimens and it could be applied for early and specific diagnosis of tuberculosis in such patients.

Genotypic and phenotypic characteristics of antibiotic-producing soil Streptomyces investigated by RAPD-PCR.

Random amplified polymorphic DNA (RAPD) analysis has been used to determine the relatedness of 73 antibiotic-producing soil Streptomyces isolates that were recovered from different soil habitats in Jordan based on their RAPD-PCR fingerprints. Genetic polymorphisms between these isolates showed three common bands of 2777, 800 and 250 bp shared by approximately (95%) of them. Some specific bands were also observed. Further analysis of RAPD patterns with the UPGMA resulted in clustering the tested isolates into two main super clusters. Super cluster I was more homogenous than super cluster II and contained all the reference strains. However, super cluster II consists of unrelated isolates within five small groups. As RAPD fingerprints of the tested isolates linked to their phenotypes, differentiation between isolates with different cultural properties was observed.