Restriction Enzymes XbaI Research Articles

Epidemiology and molecular characterization of carbapenem-resistant Klebsiella pneumoniae isolated from neonatal intensive care units in General Hospital of Ningxia Medical University, China, 2017–2021

ObjectivesThis study aimed to retrospectively investigate the epidemiology and molecular characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates from neonatal intensive care units (NICU) between 2017 and 2021.MethodsThe antibacterial susceptibility of all strains was assessed using the VITEK 2 compact system. The presence of antibiotic resistance, virulence genes, sequence types (STs), capsular (K) types, and the wzi genes was determined through polymerase chain reaction (PCR). Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI. Additionally, the virulence potential of peg344-positive strains was evaluated using the string test and mouse intraperitoneal infection models. Whole-genome sequencing was conducted on the DNB system and PacBio platforms.ResultsA total of 46 CRKP isolates were collected during the study period. Out of these, 93.47% (43/46) were identified as CRKP strains belonging to the ST76-K10 type carrying blaNDM-5. It was observed that CRKP infection resulted in more severe clinical symptoms compared to CRKP colonization. Among the CRKP strains, a hypervirulent CRKP strain called KP-63, belonging to the ST23 type, was identified. This strain exhibited high mortality in the mouse infection model and was found to possess virulence genes. Genomic alignment analysis revealed a significant similarity between the virulence plasmid from KP-63 strain (pKP-63) and pK2044 from the hypervirulent K. pneumoniae strain NTUH-2044.ConclusionsThere has been a potential dissemination of ST76-K10 type CRKP carrying blaNDM-5 in the NICU at Ningxia Hospital. Neonatal CRKP infection has been found to cause more severe clinical symptoms than colonization. Furthermore, we have discovered a CR-hvKP strain of ST23 with serotype K1, which exhibits a significant resemblance in its virulent plasmid to pK2044. Therefore, it is crucial to enforce effective measures to restrict the spread and hinder the evolution of CRKP within the hospital.

Construction, Identification, and Expression of Lactococcus lactis-Based Recombinant Vaccine for Echinococcus granulosus

To construct Lactococcus lactis (LL)-based recombinant LL-Eg95 (rLL-Eg95) vaccine for Echinococcus granulosus (Eg) and to examine its expression efficiency. Eg95 gene was obtained by PCR from the template of pCD-Eg95. Then, pMG36e was inserted in the Eg95 gene after double cleaving with restriction endonucleases XbaⅠ and HindⅢ to construct recombinant plasmid pMG36e-Eg95, which was transformed into E.coli BL2 (DE3) competent cells. The recombinant plasmid was extracted and identified by double restriction endonuclease digestion and was then electroporated into LL MG1363 to construct rLL-Eg95 vaccine. Then, the plamid was extracted and identified by PCR. Examination of the recombinant plasmid by double restriction endonuclease digestion showed that the segment was of the expected length. PCR showed that 471 base pairs of Eg95 gene were amplified when the plasmid extracted from roxithromycin-resistant recombinant LL was used as the template. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the relative molecular mass of the Eg95 protein expressed was approximately 16.5×103 and that the amount of the expressed protein was 17% of the total bacterial proteins. Western blot findings suggested that the expressed protein could be recognized by mice serum infected with hydatid cyst. The rLL-Eg95 vaccine was successfully constructed, expressing Eg95 protein that has specific antigenicity.

A CRISPR-based approach using dead Cas9-sgRNA to detect SARS-CoV-2.

Rapid, highly specific, and robust diagnostic kits to detect viruses and pathogens are needed to control disease spread and transmission globally. Of the many different methods proposed to diagnose COVID-19 infection, CRISPR-based detection of nucleic acids tests are among the most prominent. Here, we describe a new way of using CRISPR/Cas systems as a rapid and highly specific tool to detect the SARS-CoV-2 virus using the in vitro dCas9-sgRNA-based technique. As a proof of concept, we used a synthetic DNA of the M gene, one of the SARS-CoV-2 virus genes, and demonstrated that we can specifically inactivate unique restriction enzyme sites on this gene using CRISPR/Cas multiplexing of dCas9-sgRNA-BbsI and dCas9-sgRNA-XbaI. These complexes recognize and bind to the target sequence spanning the BbsI and XbaI restriction enzyme sites, respectively, and protect the M gene from digestion by BbsI and/or XbaI. We further demonstrated that this approach can be used to detect the M gene when expressed in human cells and from individuals infected with SARS-CoV-2. We refer to this approach as dead Cas9 Protects Restriction Enzyme Sites, and believe that it has the potential to be applied as a diagnostic tool for many DNA/RNA pathogens.

Tối ưu quy trình phân lập hệ gen virus BK từ mẫu máu, nước tiểu của bệnh nhân sau ghép thận

BK polyomavirus (BKV) is a common opportunistic pathogen in the community, infecting humans in their early life stages, often remaining in a latent state. BK virus-associated nephropathy occurs in 2 to 10% of kidney recipients, and up to 40 to 90% of these patients have permanent loss of graft function due to delay in diagnosis. In the present study, we have successfully optimized the component and thermal cycling for the long-PCR assay to amplify the BKV genome, including middle-concentration samples such as plasma (concentration about 105 copy/ml). We have successfully amplified the BKV genome in 41/50 samples, including 04 plasma and 39 urine samples. The optimized amplification protocol of full-length BKV genome includes the following steps: 1) Extraction of DNA-BKV from plasma and urine samples and quantification of BKV-DNA concentrations to ensure that about 105 copies/ml (plasma) and 107 copies/ml (urine); 2) Each full-length genome reaction in the volume of 50 μl containing 1X Phusion HF master mix (Thermo Scientific, USA); 0.5 μM of wBK_a(F+R) primers, 3-5 μl of genomic DNA. Cycling conditions were 98°C for 30 s, followed by 35 cycles of 98°C for 10 s; 58°C for 20 s; 72°C for 2 min, and 72ºC for 6 min and a 4oC hold; 3) All 5.1 kb purified PCR were verified and confirmed by restriction enzymes XbaI, EcoRI, and direct sequencing.

Implementation of immuno-chemoinformatics approaches to construct multi-epitope for vaccine development against Omicron and Delta SARS-CoV-2 variants

BackgroundThe new coronavirus is still a life-threatening menace, because of its changing nature and capacity to produce many mutations to bypass the immune system. The vaccination is the first effective weapon against COVID-19. AimThe study's goal was to design a multi-epitope peptide vaccine (MEPV) for a mix of Omicron and Delta Coronavirus strains using immuno-chemoinformatics tools. MethodsTo create the vaccine epitopes, seven proteins from the Omicron and Delta coronavirus strains were selected (ORF1a, ORF3a, surface protein, membrane protein, ORF7a, ORF8, and nucleocapsid protein). Antigenicity, toxicity, and allergenicity of the epitopes were evaluated. ResultsThe designed vaccine is made up of 534 amino acids that are homogeneous, antigenic, and non-toxic. Sticky restriction enzymes (XhoI and XbaI) were used to incorporate the MEPV into the pmirGLO luciferase vector. SnapGene server was used to create primers for PCR testing. Developing the MEPV is a terrific cost-effective strategy. The created MEPV's physiochemical properties have been determined to be basic, hydrophobic, and stableImmunogenicity and immune response profiles of the developed vaccine candidate were better assessed using in silico immunological simulations. ConclusionsWe advocate moving the built vaccine to the biological validation step, where it may test our findings using appropriate model organisms.

Implementation of immuno-chemoinformatics approaches to construct multi-epitope for vaccine development against Omicron and Delta SARS-CoV-2 variants

BackgroundThe new coronavirus is still a life-threatening menace, because of its changing nature and capacity to produce many mutations to bypass the immune system. The vaccination is the first effective weapon against COVID-19. AimThe study's goal was to design a multi-epitope peptide vaccine (MEPV) for a mix of Omicron and Delta Coronavirus strains using immuno-chemoinformatics tools. MethodsTo create the vaccine epitopes, seven proteins from the Omicron and Delta coronavirus strains were selected (ORF1a, ORF3a, surface protein, membrane protein, ORF7a, ORF8, and nucleocapsid protein). Antigenicity, toxicity, and allergenicity of the epitopes were evaluated. ResultsThe designed vaccine is made up of 534 amino acids that are homogeneous, antigenic, and non-toxic. Sticky restriction enzymes (XhoI and XbaI) were used to incorporate the MEPV into the pmirGLO luciferase vector. SnapGene server was used to create primers for PCR testing. Developing the MEPV is a terrific cost-effective strategy. The created MEPV's physiochemical properties have been determined to be basic, hydrophobic, and stableImmunogenicity and immune response profiles of the developed vaccine candidate were better assessed using in silico immunological simulations. ConclusionsWe advocate moving the built vaccine to the biological validation step, where it may test our findings using appropriate model organisms.

Molecular subtyping of Salmonella spp. strains in provincial abattoirs with no hazard analysis critical control point from Buenos Aires, Argentina

We subtyped 32 Salmonella enterica strains isolated from carcasses (n=10), the environment (n=14), head meat (n=1) and viscera washing and chilling water (n=7) in provincial abattoirs with no Hazard Analysis Critical Control Point (HACCP) system from Buenos Aires, Argentina, before and after implementing improvement actions. Pulsed-field gel electrophoresis (PFGE) was carried out using the XbaI restriction enzyme. Strains belonged to six serovars, from which 10 restriction patterns were obtained (five unique patterns and five clusters). We found different clones of S. enterica serovars in the same abattoir by XbaI-PFGE. In addition to promoting good hygiene practices, the implementation of an HACCP plan is necessary to meet the zero-tolerance criteria for Salmonella on beef.

Pathogenic detection and molecular tracing of a bacillary dysentery outbreak by Shigella sonnei in Huainan city

Objective: To understand the virulence gene and drug resistance profile of Shigella sonnei outbreak in Huainan city, and conduct pathogenic traceability analysis. Methods: Water samples and feces related to an infectious diarrhea outbreak in Huainan city in August 2020 were collected for multiple pathogen detection. Virulence gene, drug sensitivity, pulse-field gel electrophoresis and whole genome sequencing of Shigella isolates were analyzed respectively. Results: 38 strains of Shigella sonnei were detected in 56 samples of mucilage feces with a positive rate 67.86%, and all serotypes were Shigella sonnei Phase I. Three strains of Shigella sonnei were detected by fluorescence PCR in the Gram-negative (GN) bacterial enrichment solution of terminal water and well water. Virulence genes were ipaH positive (38), ipaH/ial (31) and ipaH/ial/sen positive (1), respectively. The drug resistance spectrum showed that 9 of 14 antibiotics were 100% resistant, and only imipenem, chloramphenicol, ceftazidime and ciprofloxacin were effective drugs. XbaⅠ restriction enzyme map type of 36 isolates was completely consistent, and the ST type analysis of 3 strains was ST152. Whole genome sequencing and analysis verified that the outbreak was caused by a single clonal group of strains, and revealed that the isolates of the outbreak were clustered into a large cluster with 3 Chinese strains and 1 Korean strain in the database, far away from the strains of other countries. Conclusion: The outbreak is caused by a single clone of Shigella sonnei, which are low virulence strains and have multiple drug resistance.

Molecular characterisation of an outbreak of NDM-7-producing Klebsiella pneumoniae reveals ST11 clone expansion combined with interclonal plasmid dissemination

The aim of this study was to characterise a hospital outbreak of NDM-7-producing Klebsiella pneumoniae associated with the successful multidrug-resistant (MDR) high-risk clone ST11 between 2017 and 2019 in southern Spain. A total of 46 NDM-7-producing isolates were recovered during the outbreak, including 16 from clinical samples, 27 from surveillance samples and 3 from environmental samples. All isolates were MDR, including carbapenem-resistant. Pulsed-field gel electrophoresis using XbaI restriction enzyme (XbaI-PFGE) showed three pulsotypes belonging to three different clones by multilocus sequence typing (MLST): ST307 (1 isolate); ST152 (1 isolate); and ST11 (44 isolates). Representative isolates were selected for characterisation of blaNDM-7-carrying plasmids using PCR-based replicon typing and whole-genome sequencing analysis. IncX3 plasmids containing NDM-7 were identified in the three clones. The blaNDM-7-carrying plasmids from the ST307 and ST11 clones were identical and were very similar to the IncX3 NDM-7 plasmid previously described. The NDM-7 carbapenemase was introduced into the hospital by means of the ST307 clone, while the ST11 high-risk clone was responsible for NDM-7 dissemination. It is essential to develop and implement strategies to control the introduction and spread of successful MDR clones in hospitals that include active surveillance programmes to detect colonised patients.

Characterization of BoLA class II DQA and DQB by PCR-RFLP, cloning, and sequencing reveals sequence diversity in crossbred cattle

The BoLA class II DQA and DQB genes in crossbred cattle were studied using PCR-RFLP, cloning, and sequencing techniques. Seventy-two crossbred cattle (Vrindavani) were used in the current study. HaeIII and XbaI restriction enzymes digested DQA exon 2-3, revealing seven (HaeIII-A-G) and three (XbaI A-C) motifs, respectively. The BoLA-DQB gene was analyzed using PCR-RFLP with PstI and TaqI restriction enzymes, yielding five restriction motifs for each restriction enzyme (PstI-A-E and TaqI-A-E). In crossbred cattle, addition, deletion, and substitutions were observed in distinct sequences, resulting in variations in overall gene length. Changes in nucleotides at positions 64–80, 110–200, and 207–264 were largely responsible for polymorphism in DQA exon 2. The phylogenetic analysis predicted a high degree of nucleotide and amino acid changes in DQA exon 2-3 and DQB exon 2. DQA genes had a nucleotide dissimilarity of 0.3–25.4 percent, while DQB genes had a nucleotide dissimilarity of 1.5–14.3 percent. We cloned and sequenced 20 genotypes based on PCR-RFLP of the DQA and DQB genes. The current study observed variation in the DQA and DQB genes and will serve as a foundation for future research on the BoLA DQA and DQB genes.

First identification of telomeric DNA sequences in Trichomonas vaginalis

Trichomoniasis is the most common nonviral sexually transmitted disease; it is caused by Trichomonas vaginalis and seriously threatens human reproductive health. Telomeres are specialised DNA–protein complexes at the ends of chromosomes that have a protective function. The aim of the present study was to identify and characterise the telomeric DNA of T. vaginalis—which has not been previously reported—by multiple molecular methods including sequencing, the Bal nuclease (BAL) 31 nuclease assay, fluorescence in situ hybridisation (FISH), and Southern blotting. We found numerous repeated units of TTTTAGGG in T. vaginalis genomic DNA digested with S1 nuclease in combination with XbaI restriction enzyme. The (TTTTAGGG)n tandem repeats were also highly sensitive to BAL 31 exonuclease digestion. We confirmed that the (TTTTAGGG)n repeats were located at the end of T. vaginalis chromosomes by FISH. Restriction enzyme digestion combined with Southern blotting using a digoxigenin-labelled (TTTTAGGG)5 probe showed that the T. vaginalis telomeric DNA length varied from 1.0 to 1.5 kb. This is the first report on the telomeric DNA sequence of T. vaginalis which includes the length and distribution on chromosomes; our findings lay a foundation for further study on telomere maintenance mechanisms in T. vaginalis.

Determining the DNA Fingerprinting Profiles of Salmonella Isolates from Raw Poultry Meats and Human Clinical Samples from the Same Geographic Area Using Pulsed-Field Gel Electrophoresis

Foods of animal origin, such as poultry, eggs, and pork, are recognized sources of Salmonella infections, but determination of the proportion of foodborne infections associated with various food sources has been challenging. In the present study, 141 Salmonella isolates recovered from 1,322 poultry product samples purchased over a 1-year period from retail stores across Seattle, WA were subtyped by pulsed-field gel electrophoresis (PFGE) using restriction enzyme XbaI. The objectives of the study were (i) to analyze the longitudinal distribution of Salmonella PFGE profiles throughout the sampling period and their clonality within and between poultry processing establishments, (ii) to determine the association between PFGE profiles of Salmonella isolates from locally distributed poultry products and those of clinical isolates submitted to the Washington State Department of Health (WA-DOH) laboratories, and (iii) to compare the PFGE profiles of Salmonella isolates from the National Antimicrobial Resistance Monitoring System (NARMS) retail meats program. During the 1-year sampling period, multiple indistinguishable PFGE patterns were found across multiple poultry processing establishments. Twelve of the 30 unique PFGE profiles of Salmonella isolates from locally purchased poultry products were indistinguishable from the PFGE profiles of clinical Salmonella isolates submitted to the WA-DOH. When the PFGE profiles from the poultry samples were compared with those found in the NARMS database, eight indistinguishable PFGE matches were found with isolates recovered from chicken breasts, ground turkey, and ground beef from multiple states. Although this study revealed some association between PFGE profiles from raw poultry products and those of clinical isolates from the same geographical area, these results do not prove that all of those clinical isolates were from infections acquired through consumption or handling of poultry.

Identification of the Pseudomonas fluorescens group as being responsible for blue pigment on fresh cheese

New cases of blue cheese discoloration has led to recent research to identify the causal agent and factors that favor blue pigment appearing. Nonetheless, very few reports have described the source of contamination and the measurements to eradicate the microbiological source on cheese farms by determining the relation between blue discoloration on fresh cheese and the Pseudomonas fluorescens group. Thus, 60 samples from a cheese farm (cheese, equipment surfaces, tap water, and raw and pasteurized milk) were analyzed by phenotypical, MALDI-TOF, 16S rRNA sequencing and pulsed-field gel electrophoresis tests to determine the causal agent. The results obtained by pulsed-field gel electrophoresis with restriction enzymes XbaI and SpeI confirmed tap water as the initial contaminated source. The above-mentioned result was essential to avoid Pseudomonas contamination due to the most residual microorganisms being inactivated through a new disinfection program.

An outbreak of food poisoning due to Escherichia coli serotype O7:H4 carrying astA for enteroaggregative E. coli heat-stable enterotoxin1 (EAST1).

In June 2020, a large-scale food poisoning outbreak involving about 3000 elementary and junior high school students occurred in Yashio, Saitama, Japan. A school lunch was the only food stuff ingested by all of the patients. Escherichia coli serotype O7:H4 carrying the astA gene for enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1) was detected in faecal specimens from the patients, and sample inspection revealed its presence in a seaweed salad and red seaweed (Gigartina tenella) as one of the raw materials. Analysis of the antibiotic sensitivity of the isolates revealed resistance to ampicillin and cefotaxime. All isolates were confirmed to be of the same origin by pulsed-field gel electrophoresis after digestion with the restriction enzyme XbaI, and single nucleotide polymorphism analysis using whole genome sequencing. To our knowledge, this is the first report of a large-scale food poisoning caused by E. coli O7:H4, which lacks well-characterized virulence genes other than astA.

Heterogeneity of Salmonella isolates obtained from various sources in Russia 2010–2019

Aim: the goal of the study was to evaluate the heterogeneity of the Salmonella enterica subsp. enterica strains isolated from clinical specimens and various environmental sources in the Russian Federation during the period 20112019.
 Materials and methods. The data of 3076 non-typhoid isolates of Salmonella obtained from sporadic and outbreak cases of salmonellosis (n = 2518), food and water samples (n = 558) were used. These isolates were serotyped according to the KaufmanWhite scheme and genotyped by Pulsed-Field Gel Electrophoresis (PFGE) using XbaI and BlnI restriction endonucleases according to a standard PFGE-protocol developed by PulseNet International Network.
 Results. The studied Salmonella isolates were differentiated into 73 serotypes and 601 PFGE types. A comparative analysis of isolates from various sources made it possible to identify subtypes that differed significantly in their prevalence in humans and potential transmission factors (sources). A significant proportion of chicken, turkey, and pork meat samples contained PFGE-subtypes which did not occur in clinical samples. Regional differences in the heterogeneity of the Salmonella spp. were also identified.
 Conclusions. Genetic heterogeneity of the Salmonella population from humans and other sources shows significant variability of virulent properties and indicates the necessity of differentiated assessment of their epidemiological potential.

Characterization and molecular subtyping of Shiga toxin–producing Escherichia coli strains in provincial abattoirs from the Province of Buenos Aires, Argentina, during 2016-2018

We characterized Shiga toxin–producing Escherichia coli (STEC) O157 (n = 20) and non-O157 (n = 68) isolated from carcasses (n = 54), the environment (n = 20), head meat (n = 3) and viscera washing and chilling water (n = 11) in provincial abattoirs before and after implementing improvement actions. The strains were tested for eae, saa, ehxA and fliCH7 genes. Variants stx1 and stx2 were also determined. Pulsed-field gel electrophoresis (PFGE) was carried out with restriction enzymes XbaI and BlnI. All twenty O157 STEC strains [H7; H21; HNM] carried genes rfbO157 and ehxA; 90.0 % were positive for eae and 15.0 % were negative for fliCH7 and positive for saa. Results of PFGE showed 17 XbaI patterns, of which 14 were unique and three formed clusters. From the 68 non-O157 STEC strains, 66.2 %, 55.9 % and 2.9 % were positive for ehxA, saa and eae genes, respectively. Fifty-three XbaI patterns were obtained (49 unique and four forming clusters). Cross-contamination between products and between the environment and products was confirmed in all abattoirs. While the proposed improvements reduced the risk of contamination, Good Hygiene Practices and Good Manufacturing Practices should be implemented in provincial abattoirs, stressing the importance of having a uniform national food safety standard.

A Closed-Tube Loop-Mediated Isothermal Amplification Assay for the Visual Detection of Staphylococcus aureus.

Food-borne diseases induced by Staphylococcus aureus contamination seriously affect human health and food safety. Therefore, a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus was developed in this study. Firstly, two pairs of outer and inner primers were designed targeting on a conserved fragment of gyrB gene in different S. aureus strains. Secondly, the weakly buffered gyrB-LAMP assays were optimized under various pH values and other conditions, followed by the visual evaluation of five pH-sensitive indicators, and the cresol-red was chosen as the best dye for the best visual performance. Thirdly, the cresol-red-based LAMP assay showed good sensitivity with the detection limit of 5.4copies/μL for purified DNAs, and good specificity with no cross-reaction with other related species. The specificity of the amplified products was further confirmed by XbaI restriction enzyme digestion analysis. Finally, the cresol-red-based LAMP assay was validated by the clinical-dried fish samples inoculated with known numbers of S. aureus and further validated by 20 blind samples. To our knowledge, this is the first report of a closed-tube LAMP assay based on pH-sensitive indicators for the visual detection of the food-borne S. aureus by the gyrB gene.

Distribution of polymorphism rs693 of ApoB gene in a sample of Colombian Caribbeans.

Introduction:Several studies have reported that the single nucleotide polymorphism rs693 of Apo lipoprotein B gene is associated with high levels of plasma lipids and high body mass index, which are risk factors for cardiovascular diseases. The distribution of this single nucleotide polymorphism and its association with the phenotype depend on the genetic background of each population. Objective:To evaluate the distribution of single nucleotide polymorphism rs693 and its association with lipid profile and body mass index in a sample of Colombian Caribbeans.Methods: 108 non-related adult subjects of both gender were included in this study. Body mass index and lipid profile that included total cholesterol, triglycerides, Low Density Lipoprotein and High Density Lipoprotein were determined. The single nucleotide polymorphism rs693 was determined by Polymerase Chain Reaction/Restriction Fragment Length Polymorphism from genomic DNA followed by digestion with the restriction enzyme XbaI. The chi-square test was used to analyze the genotype distribution of rs693 and the genotype-phenotype association was evaluated through different inheritance model. Results:The genotype frequencies for single nucleotide polymorphism rs693 were CC (45.0%), TT (16.5%) and CT (38.5%). The allele frequencies were C (64.0%) and T (36.0%). The single nucleotide polymorphism was in Hardy-Weinberg equilibrium in the studied sample. No association of the single nucleotide polymorphism rs693 with lipid profile nor the body mass index was found (p >0.05).Conclusion:There is no significant association between single nucleotide polymorphism rs693 and body mass index nor lipid profile, in a sample of Colombian Caribbeans.

Molecular screening approach to identify protozoan and trichostrongylid parasites infecting one-humped camels (Camelus dromedarius)

Little is known about the diversity of many parasites infecting camels, with most relying on morphological parameters. DNA extracted from different tissues (n = 90) and fecal samples (n = 101) from dromedary camels (Camelus dromedarius) in Egypt were screened for multiple parasites using different molecular markers. Screening of tissue samples (heart) for Toxoplasma gondii and Sarcocystis spp. was performed using B1 and 18S rRNA gene markers, respectively. T. gondii was further genotyped using multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP). Sarcocystis was analyzed using PCR-RFLP characterization (XbaI and MboI restriction enzymes). A taxonomically challenging but important group of nematodes (Trichostrongylidae family) were screened using the ITS-2 ribosomal DNA (rDNA) species-specific markers. Furthermore, nested PCR was used for the detection of Cryptosporidium spp. (SSU rRNA gene) and positive samples were genotyped after RFLP (SspI and VspI) and sequencing. Cryptosporidium parvum isolates were subtyped by sequence analysis of the 60-kDa glycoprotein gene. This study revealed that many parasites infect the investigated camels, including T. gondii (1.1%), Sarcocystis spp. (64.4%), Cryptosporidium spp. (5.9%) and Trichostrongylidae nematodes (22.7%). The species contribution for nematodes was as follows: Haemonchus spp. (95.6%), Trichostrongylus axei (26%), Trichostrongylus colubriformis (65.2%) and Cooperia oncophora (60.8%). Mn-PCR-RFLP typing for Toxoplasma was only successful for three markers: 5′-SAG2 (type II), 3′-SAG2 (type II) and alt. SAG2 (type II). PCR-RFLP using XbaI showed possible mixed Sarcocystis infection. Moreover, the Cryptosporidium genotypes detected were C. parvum (IIdA19G1 and IIaA15G1R1), Cryptosporidium rat genotype IV and a novel genotype (camel genotype). This approach revealed the unique Cryptosporidium genotypes infecting the investigated camels, and the high genetic diversity of the investigated parasites.

Isolation and In Vitro Evaluation of Bacteriophage against Methicillin-Resistant Staphylococcus aureus (MRSA) from Burn wounds

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a major threat to human health. It is considered to be a Multi-Drug Resistant (MDR) pathogen capable of causing a variety of diseases. MRSA is also one of the most important nosocomial pathogens in burn infection. As a treatment strategy against MRSA infections phage therapy has the potential of becoming alternative remedy. Thus, the aim of present study was to isolate and characterize lytic bacteriophage from hospital sewage effective against burn wounds infecting MRSA isolates. Materials and methods: Staphylococcus aureus strains were isolated from hospitalized burnt patients. Strains were confirmed as MRSA by Kirby-Bauer disk diffusion tests to penicillin, methicillin, and oxacillin and also PCR assay for mecA gene. Phage was isolated from hospital sewage sample and tittered by Double Layer Agar (DLA) method. Spot test was used for host range determination. The latent period and burst size were estimated from one step growth curve. Phage morphology was observed by electron microscopy. The nature of the nucleic acid of isolated bacteriophage was confirmed by using Rnase A, Dnase I and 6 restriction enzymes. Results: The titer, latent period and burst size of the isolated phage was determined to be 1×109 PFU/ml, 20 min and 190 PFU per infected cell, respectively. It displayed a wide host range for MRSA bacteria by spot test (27 isolate from 30).Electron microscopy observation demonstrated that the phage belonged myoviridea family. Digestion profiles of Rnase A, Dnase I and XbaI restriction enzyme on 1% agarose gel Electrophoresis, show that the genome of isolated phage was a double-stranded DNA with a size of <∼43 kbp. Conclusion: The isolated phage was active against a wide range of MRSA strains recovered from burnt patients. Therefore it can be considered as a suitable candidate for use in prophylaxis and treatment of these clinical infections and for the preparation of anti-staphylococcal cocktails.