Specific Polymerase Chain Reaction Primers Research Articles

Solid waste dumpsite leachate and contiguous surface water contain multidrug-resistant ESBL-producing Escherichia coli carrying Extended Spectrum β-Lactamase (ESBL) genes

Dumpsites generate leachates containing bacteria that may carry antibiotic resistance genes, such as extended spectrum β-lactamase (ESBL). However, the contribution of dumpsite leachates in the environmental spread of ESBL genes has not been investigated in greater detail. This study aimed to quantify the impact of Ajakanga dumpsite leachate on the spread of ESBL genes through surface water. The susceptibility of Escherichia coli isolated from dumpsite leachate and the accompanying surface water to selected antibiotics was assessed by the standardized disc diffusion method. The isolates were evaluated for phenotypic ESBL production using the double disc synergy test (DDST). The detection of ESBL genes in the isolates was carried out using a primer-specific polymerase chain reaction (PCR). Escherichia coli isolates from leachate (n = 26/32) and surface water (n = 9/12) expressed ESBL phenotype. The ESBL-producing isolates showed the highest level of resistance to the 3rd generation cephalosporin antibiotics: cefotaxime (100%), cefpodoxime (97%), ceftazidime (97%), with low resistance observed to imipenem (6%) and azithromycin (3%). All the isolates were multidrug-resistant, showing resistance to three or more classes of antibiotics. All the ESBL-producing E. coli obtained carried blaCTX−M, 21/35 (60%) carried blaTEM while none of the isolates bore blaSHV. This study found that ESBL-producing Escherichia coli from dumpsite leachate and nearby surface water had identical resistance signatures indicating the relatedness of the isolates, and that dumpsite leachate could contribute to the transfer of ESBL-producing bacteria and their genes to receiving surface water. This study has necessitated the need for a review of the guidelines and operational procedures of dumpsites to forestall a potential public health challenge.

Common RBC antigens in O type Tunisian blood donors and their importance in alloimmunization.

The presence of some red blood cell (RBC) antigens may affect the preference for using type O blood in emergency situations because they may induce complex or multiple alloimmunization in special circumstances. A subgroup of 77 type O blood Tunisian donors were genotyped for 19 common blood alleles using the single specific primer-polymerase chain reaction method. The statistical analysis was done using HaploView software. The study showed the dominance of the alleles RH*5, KEL*2, FY*2, and CO*1 and the absence of the homozygous state of the KEL*1 and CO*2 alleles. Furthermore, a complete linkage disequilibrium between the RH*2/RH*4 and RH*3/RH*5 loci and the FY*Null/FY*Exp and FY*A/FY*B loci was detected. Additionally, it seems that sensitization to MNS:3, FY:1, and RH:3 may constitute a potential factor for alloimmunization after transfusion with O blood type units: the probabilities of simple alloimmunizations are 24.5 per 100, 18.5 per 100, and 18 per 100, respectively. Multiple alloimmunization against RH:1;KEL:1 or RH:1;KEL:1;RH:3 phenotypes may occur, with probabilities of 7 per 1000 and 2 per 1000, respectively. Some O-type RBC units may contain blood with very immunogenic phenotypes, the use of which in an emergency requires great caution because it can be a step towards subsequent alloimmunization.

Iraqi patients with a single-nucleotide polymorphism of interleukin-10 -1082G/A and interleukin-6 -174G/C susceptibility to asthma

The effects of genetic variations in the IL-10 -1082G/A gene and IL-6 -174G/C gene, as well as the genotypes and alleles linked to the prevalence of asthma disease, were investigated using a molecular and immunological study. Between October 2018 and the end of July 2020, 40 healthy individuals (20 females and 20 males) served as a control group for the study, which involved 50 asthmatic patients (31 females and 19 males) at the Allergy Centre, Al-Anbar Teaching Hospital, in Al-Anbar City. The study used the Single Specific Primer-Polymerase Chain Reaction (SSP-PCR) technique to show that the single nucleotide polymorphisms IL-10 -1082G/A and IL-6 -174G/C had a considerably high prevalence rate (P<0.05) among asthma case and that there was an association between the polymorphism and the asthma risk. The findings indicate that asthma patients had considerably higher (P<0.05) IL-10 A alleles and heterozygous GA genotypes (1082G/A) compared to the control group. Genetic variations affecting IL-10 production and the genotypes affecting IL-10 serum levels are associated with the occurrence of asthma and are attributed to the IL-10 -1082G/A promoter gene polymorphism. There was a strong correlation between cytokine levels, of disease development, and the genotypes of the AA and AG genes, indicating that IL-10 -1082A/G predisposition to asthma may be influenced by the gene promoter polymorphism. Asthma development and immunological markers (IL-10) are substantially correlated. One theory links allergic rhinitis to both the development of asthma and its risk. Inducing long-term immunological and clinical tolerance in patients was a good use of HDM immunotherapy. The current study's findings indicate a substantial difference between the asthma patients and the control group in terms of gene type and allele frequency of the IL-6 -174G/C polymorphism. The patients exhibited a higher prevalence of the G allele and the GG homozygous genotype than the control group. Therefore, it was shown that those with GG genotypes had a 2-fold increased probability of having asthma, indicating that patients were more prone to the condition.

Human leukocyte antigen HLA-B*57:01 status in HIV-1 patients developing hypersensitivity reactions in Benin: a pilot study

BackgroundAntiretroviral drugs in people living with HIV-1 (PLHIV-1) often trigger side effects which may lead to discontinuation or failure of treatment. Human Leukocyte Antigen B*57:01 (HLA-B*57:01) allele is known to predict hypersensitivity reactions to Abacavir. Very few data are available on the prevalence of HLA-B*57:01 allele in PLHIV-1 in African countries. This study aimed to screen for HLA-B*57:01 allele in PLHIV-1 in Benin.MethodsThis pilot study was carried out on one hundred ten PLHIV-1 enrolled in two health facilities in Benin. Socio-demographic and clinical data were collected. Biological data were determined and HLA-B*57:01 allele was genotyped, using Single Specific Primer-Polymerase Chain Reaction in blood samples.Results70% of participants were female. PLHIV-1 were under TDF + 3TC + DTG (47.2%) or TDF + 3TC + EFV (57.3%). Their median age was 41 [36-48.75] years and the average CD4 + T cell count was 249 [130-381.25] cells/µl. The average viral load in treatment failure PLHIV-1 was 4.7 [3.9–5.2] Log10. At the inclusion date, twenty-nine (26.4%) PLHIV-1 under TDF + 3TC + EFV have developed hypersensitivity reactions. None of 110 patients had shown HLA-B*5701 allele.ConclusionOur study revealed that HLA-B*57:01 allele was very rare in PLHIV-1 in Benin, suggesting that its screening before starting the Abacavir regimen did not seem necessary.

Identification of genetically plastic forms among Belarusian ancient flax (Linum usitatissimum convar. elongatum Vav. et Ell.) varieties using the Linum Insertion Sequence LIS-1

The Linum Insertion Sequence 1 (LIS-1) occurs in the genetically plastic flax genotypes in response to the lack or excess of mineral and water nutrition, but also naturally, and can be transmitted to the progeny. We have analyzed 21 ancient Belarusian varieties of flax Linum usitatissimum convar. elongatum Vav. et Ell. The LIS-1 presence or absence was checked for individual plants in at minimum two generations with primer-specific polymerase chain reaction (PCR) and agarose gel electrophoresis. The studied flax varieties formed four groups: non-responsive varieties (LIS-1 was not found, group NR); responsive, which formed and completely lost the insertion (group R0); responsive, which formed and retained LIS-1 (group R1); and responsive unstable (group R2). A statistically significant difference was found in ‘plant height’ (p < 0.05), ‘technical length of the stem’ (p < 0.05) between R0 and NR, and R2 and NR LIS-1 groups. The machine learning algorithm random forest classifier was used to predict the presence, absence or heterozygosity of LIS-1 in flax plants based on their growth and reproductive characteristics. As a result, the accuracy of the prediction was 98% on test data. In terms of sources for the selection of fibre flax varieties adaptive to environmental challenges, the most promising group consists of responsive varieties that have formed LIS-1 insertion (R0, R1 and R2 groups).

Evaluation of the genetic diversity of KIR genes in patients with endometrial cancer

Endometrial cancer (EC) constitutes more than half of all genital cancers in women, with an increasing incidence in different countries. Natural killer cells (NK cells) are kinds of innate immune cells that are controlled by sets of receptors, such as killer cell Ig-like receptors (KIRs), which can inhibit or activate NK cells. In this study, we evaluated the diversity and genetic association of KIRs in confirmed cases of endometrial cancer compared to healthy controls. A total of 151 women with EC and 167 age/race-matched healthy controls were analyzed for KIR genes. Demographic and histopathologic data were gathered in questionnaires, and 16 KIR genes along with two variants of KIR2DS4 (KIR2DS4fl and KIR2DS4del), were genotyped by usingsequence specific primers-polymerase chain reaction (SSP-PCR) method. A comparison between cases and controls revealed that although there were not any significant differences in A haplotype associated genes and also the variants of KIR2DS4 (p >0.05), B haplotype associated genes such as KIR2DS2 and KIR2DL2 decreased significantly in EC patients in comparison with healthy controls (p=0.03 and p=0.01, respectively). Furthermore, we found that EC mostly developed in cases with the AA genotype; however, the carriers of Bx and C4T4 genotypes were less frequent in patients with EC. Our results revealed that KIR2DS2 and KIR2DL2, along with Bx and C4T4 genotypes, have a protective impact against developing endometrial cancer in Iranians.

Association between HLA-B*27:04 and genetic susceptibility to ankylosing spondylitis in Hunan Province.

Human leukocyte antigen (HLA) B27 is a susceptibility allele of ankylosing spondylitis (AS), and HLA-B27 antigen typing is an important indicator for clinical diagnosis of AS, but current typing methods such as sequence specific primer polymerase chain reaction (PCR-SSP) still possess limitation. Therefore, this study aims to analyze the correlation between B27 subtypes and susceptibility to AS in Hunan Province by applying high-resolution polymerase chain reaction-sequence-based typing (PCR-SBT). Peripheral blood of 116 patients with suspected AS (suspected AS group) and 121 healthy volunteers (control group) admitted to the Second Xiangya Hospital from January 2020 to December 2020 were collected for HLA-B genotyping by PCR-SBT. Among the patients in the suspected AS group, 23 patients were finally diagnosed with AS (confirmed AS group), and the remaining 93 undiagnosed patients served as the non-confirmed AS group. PCR-SBT and PCR-SSP were used to detect HLA-B27 typing in 116 patients with suspected AS, and the results of the 2 methods were compared. The HLA-B27 allele frequency in the suspected AS group was significantly higher than that in the control group [11.63% vs 2.48%; P<0.001, odds ratio (OR)=5.18, 95% confidence interval (CI) 2.097 to 12.795]. B*27:04, B*27:05, B*27:06, and B*27:07 were detected in the suspected AS group and the control group. The frequency of the B*27:04 allele in the suspected AS group was significantly higher than that in the control group (9.48% vs 1.24%; P<0.001, OR=8.346, 95% CI 2.463 to 28.282). The positive rate of B27 in the suspected AS group and the confirmed AS group (B27+/+ and B27+/-) was significantly higher than that in the control group (χ2=16.579, P<0.001; χ2=94.582, P<0.001, respectively). Among the confirmed AS group, 21 were HLA-B27 carriers, and the B27 positive rate in the confirmed AS group was 91.3%. PCR-SBT could achieve high resolution typing of the HLA-B gene locus, with higher sensitivity, specificity, positive predictive value, negative predictive value, and accuracy than PCR-SSP. PCR-SBT typing analysis shows a strong correlation between HLA-B * 27:04 and AS in Hunan province. The PCR-SBT method can be used as the preferred option for the auxiliary diagnosis of clinical AS.

The Interleukin 6 Protein Level as well as a Genetic Variants, (rs1800795, rs1800797) Are Associated with Adverse Cardiovascular Outcomes within 10-Years Follow-Up.

Worldwide, cardiovascular disease (CVD) is the leading cause of premature death. The proinflammatory cytokine interleukin 6 (IL-6) is a essential marker of innate immunity that is considered to play an important proatherogenic role for cardiovascular disease. The aim of this study (substudy of ClinTrials.gov identifier: NCT01045070) was to evaluate IL-6 protein level and genetic variants (rs1800795, rs1800797) with respect to CV outcome (combined endpoint: myocardial infarction, stroke/transient ischemic attack, cardiac death, death according to stroke) among patients CVD within 10-years follow-up. Overall 1002 in-patients with CVD were included. IL-6 protein level was determined by electrochemiluminescence immunoassay (fasting, between 7 and 8 a.m.). Genetic analyses were carried out by single specific primer-polymerase chain reaction. In survival analyses, IL-6 protein levels of ≥6.4 pg/mL (log-rank test: p = 0.034; cox regression: p = 0.032, hazard ratio = 1.29) and CC genotype of rs1800795 (log-rank test: p < 0.001, cox regression: p < 0.001, hazard ratio = 1.72) and AA genotype of rs180797 (log-rank test: p = 0.002, cox regression: p < 0.001, hazard ratio = 1.62) were associated with a poorer CV prognosis considering combined CV endpoint. This study was the first to investigate both elevated IL-6 levels and genetic variants for their prognostic value for adverse CV outcomes in CVD patients within the 10-year follow-up period.

Expression profiles of sporulation genes in multidrug-resistant Bacillus species isolated from intensive care units of Ghanaian hospital.

Sporulating bacteria such as Bacillus spp. have contributed to severity of opportunistic hospital acquired infections, including postoperative wounds and respiratory tract infections. This study determines the expression profiles of sporulation markers in multidrug-resistant Bacillus spp. isolated from Ghanaian hospital environments. Antimicrobial resistance (AMR) profiles of the bacteria were determined with disk diffusion and broth microdilution. Primer-specific polymerase chain reaction (PCR) amplification was used to profile the sporulation markers, and quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used for the expression of the sporulation markers at different antibiotic concentrations. The strains are multidrug resistant (70-100%) to at least two of the eight classes of the antibiotics tested including cephalosporins, penicillin, aminoglycosides, and glycopeptide. The strains showed different resistance patterns to all the tested antibiotics, which might indicate diverse resistance mechanisms. Common (spoVK spoVE, spoJ, and sigF) and not commonly (sigJ, soJ, yrbC, and yjcE) reported sporulation markers were detected in the strains. The study showed an association of the sporulation markers with AMR as indicated by their expression profiles.

Study of frequency and inheritance model of ACE1 I/D and ACE2 rs2285666 polymorphisms in COVID-19 patients with varying severity of lung involvement and its effect on serum cytokines levels.

The angiotensin-converting enzyme (ACE) has been shown to play a role as a receptor for the COVID-19 virus. This virus usually gets into cells and infects them by attaching to their glycoprotein receptors, which are found on the ACE2 receptor. The aim of this study was to evaluate the frequency and inheritance of ACE1 I/D and ACE2 rs2285666 polymorphisms in COVID-19 patients with varying severity of lung involvement and its effect on serum cytokines levels of interleukin (IL)-1 and IL-6 and laboratory parameters. One hundred eighty-fiveCOVID-19 patients were grouped according to the severity of lung involvement. (I/D) polymorphism of the ACE1 gene and rs2285666 polymorphism of the ACE2 gene were determined by single specific primer-polymerase chain reactionand restriction fragment length reaction-polymerase chain reactionmethods, respectively. Serum levels of IL-1 and IL-6 were also measured by the enzyme linked immunosorbent assay technique. No statistically significant association of ACE2 rs2285666 polymorphism genotypes and ACE1 I/D with the severity of lung involvement was noted. However, there was a statistically significant association between I/D ACE1 polymorphism genotypes and IL-6, white blood cells (WBC), and neutrophil-to-lymphocyte ratio (NLR) levels. Also, there was no statistically significant association between rs2285666 polymorphism genotypes and patients' blood oxygen saturation level, IL-6, IL-1β, lactate dehydrogenaseactivity, WBC count, and NLR. In patients with COVID-19, the rs2285666 polymorphism of the ACE2 gene and the I/D polymorphism of the ACE1 gene were not significantly associated with the severity of COVID-19 disease and serum IL-6 and IL-1 cytokine levels.

Relationship between KIR genotypes and HLA-ligands with SARS-CoV-2 infection in the Saudi population

AimTo ascertain whether killer cell immunoglobulin-like receptors (KIR) genes polymorphisms and HLA-I ligands are associated with COVID-19 in Saudi Arabia. MethodsEighty-seven COVID-19 patients who tested positive for SARS-CoV-2 and onehundred and fourteen healthy controls were enrolled in this study for genotyping of the 16 KIR genes, HLA-C1 and -C2 allotypes and HLA-G 14-bp indels polymorphisms using the sequence specific primer polymerase chain reaction (SSP-PCR) method. KIR genotype frequency differences and combination KIR-HLA-C ligand were tested for significance. ResultsFramework genes KIR2DL4, KIR3DL2, KIR3DL3, and KIR3DP2 were present in all individuals. The frequencies of KIR2DL2 and KIR2D4 were higher in COVID-19 positive patients than in healthy individuals. The frequencies of the combination KIR2DL2-HLA-C2 was also significantly higher in patients affected by COVID-19 compared with healthy controls. ConclusionIt was found that the inhibitory KIR2DL2 gene in isolation or combined with its HLA-C2 ligand could be associated with susceptibility to COVID-19 in the Saudi population.

Molecular identification of diarrheagenic Escherichia coli pathotypes and their antibiotic resistance patterns among diarrheic children and in contact calves in Bahir Dar city, Northwest Ethiopia.

Diarrheagenic Escherichia coli strains are an essential cause of diarrheal infection in younger children and animals. The study was focused on understanding the associated characteristics of various DEC strains among children and calves, establishing the possible zoonotic transmission, and determining their antibiotic resistance patterns. Samples from 144 acute diarrheic children and 50 diarrheic calves were collected and processed using traditional culture methods. The molecular identification of pathotypes was completed using primer-specific polymerase chain reaction (PCR) targeting ten virulence genes (stx1, stx2, eae, aatA, lt, st, ial, hlyA bfpA, and daaE) related to six DEC pathotypes (EPEC, ETEC, EHEC, EAEC EIEC, and DAEC). The antimicrobial susceptibility testing was carried out using the Kirby-Bauer disk diffusion method. Colonies from 74 study subjects (54 diarrheic children and 20 diarrheic calves) were positive for E. coli isolates. Subsequent PCR detection discovered that 77% of children and 85% of calves’ isolates were positive for one or more virulence genes typical of particular strains. Among those ETEC [(18%), (26%)] is being the maximum predominant, and [(15%), (15%)] were positive for STEC, [(13%), (8%)] for atypical EPEC, [(6%), (7%)] for EHEC, [(6%), (5%)] for EAEC, and [(6%), (4%)] for EIEC strains in children’s and calves, respectively. Of the identified E. coli isolates, about 29% were found to be hybrid isolates. ETEC (66.7%) and STEC (58.9%) strains showed a better detection rate in contact children with diarrheic calves than children with no contacts. Most antibiotic resistances were obtained towards amoxicillin (64.9%), gentamycin (56.8%), and ampicillin (54.1%). Up to sixty-five percent of isolates were resistant to a minimum of three categories of antibiotics. This is the primary report on the wide occurrence of the six-diarrheagenic Escherichia coli strains, and ETEC was found to be the predominant pathotype among children and contact calves in Ethiopia.

Relationship Between Angiotensin II Type 1 Receptor Antibody Positivity and Cytokine Gene Polymorphism in Renal Transplant Patients When Organ Rejection Occurs.

Kidney transplant remains the gold standard for the treatment of end-stage renal disease. Relationships between the presence of non-HLA antibodies, antibodies to AT1R, and cytokine gene polymorphisms with rejection have recently been shown. We sought to determine whether the presence of antibodies to AT1R and cytokine gene polymorphisms affected the development of rejection in pediatric and adult patients, whether a relationship is present between cytokine polymorphism and level of antibodies to AT1R, and whether their presence can be a biomarker pretransplant. Our study included 100 pediatric and adult kidney transplant patients plus 50 healthy controls. Levels of AT1R antibodies (by enzyme-linked immunosorbent assay) and gene polymorphisms of the cytokines transforming growth factor β, tumor necrosis factor α, interleukins 6 and 10, and interferon gamma cytokines (by sequence- specific primer-polymerase chain reaction) were studied retrospectively and evaluated with the SPSS statistical program. We found no statistically significant relationship between levels of antibodies to AT1R and gene polymorphisms among the studied cytokines in patients with rejection compared with the healthy controls and patients with uneventful courses posttransplant. However, higher levels of antibodies to AT1R were observed in pediatric compared with adult transplant recipients (P < .001). A statistically significant relationship was also observed between transforming growth factor β1 C/C G/C low-release and interleukin 6 G/C high-release gene polymorphism and levels of antibodies to AT1R (P < .001). Because we observed that some gene polymorphisms among the studied cytokines may affect AT1R antibody levels, future studies are needed to understand the mechanism of the relationship. In addition, studies with larger groups are required to sufficiently confirm that higher antibody levels are present in pediatric versus adult patients.

Cellular immune response of Staphylococcus aureus enterotoxin B in Balb/c mice through intranasal infection.

Background and Aim:Staphylococcus aureus produces various superantigen exotoxins, including staphylococcal enterotoxin B (SEB). It causes fatal anaphylactic reactions and toxic shock. This study aimed to evaluate the reaction of leukocytes and histopathological changes in the respiratory organs of Balb/c mice after intranasal infection with enterotoxigenic S. aureus (SEB).Materials and Methods:The presence of the seb gene in S. aureus was established in this study using polymerase chain reaction-specific primer. Two groups of 8-week-old male Balb-c mice consist of six mice in each group. The treated group was infected with 50 μL and 100 μL of SEB intranasal on days 1 and 14, respectively. NaCl was administered in the second group and was considered as a control group. Blood samples were collected through the retro-orbital plexus on days 1, 4, 7, 14, and 22 after infections. Total cell counts were analyzed with an independent sample t-test and compared using the statistical package for the social sciences (SPSS) version 16.0 (IBM Corp., NY, USA). The infected tissues of the respiratory organ were observed descriptively and compared to the control group.Results:The seb gene with a molecular size of 478 bp, indicating the SEB strain, is present in S. aureus used in this study. Intranasal administration of SEB showed increased leukocytes, lymphocytes, monocytes, and eosinophils on day 22 post-infection. Significant leukocytosis was seen on days 6 and 14; lymphocytosis on days 1, 4, 6, and 16; and eosinophilia on days 6, 14, and 22 compared with the control group (p > 0.05). In contrast, the neutrophil decreased after an increase of immature band cells compared to the control group, indicating a severe acute infection with SEB. The lungs and trachea of the test group had an inflammatory cell accumulation in the respiratory organ.Conclusion:Intranasal route infection of S. aureus containing seb gene significantly induced the cellular immune response and caused pathological changes in the respiratory tissues of the Balb/c mice model. The hematological changes were aligned with marked pathological changes in the respiratory tract. Balb/c mice could be an excellent experimental model to study toxic and anaphylactic shock against SEB to define the future therapeutic agents.

THE IMPACT OF VERY LATE ANTIGEN 4 POLYMORPHISM ON DRUG RESPONSIVENESS IN PATIENTS WITH MULTIPLE SCLEROSIS INITIATION

Background: Very late antigen 4 (VLA4) integrin facilitates the immune cells migration to central nervous system (CNS) through blood brain barrier (BBB), so the polymorphism in this gene may be considered as genetic risk factor for multiple sclerosis (MS) occurrence. It may interact with the responsiveness level of Natalizumab. Objective: To show if VLA4 single nucleotide gene polymorphism (SNP) (C-269-A) considered as genetic predisposition factor for MS and if have a role in Natalizumab (Tysabri) drug non-responsiveness. Methods: Sixty-six (66) person with MS and 60 healthy persons involved in this study, their ages were range from 14 to 67 years. They attended to seek treatment in the MS outpatient’s clinic, at Baghdad Teaching Hospital, Medical City Complex from December 2018 to March 2020. Patient were divided into two group; resistant group (34) and response group (32). The VLA-4 SNP polymorphism investigated by sequence specific primer polymerase chain reaction (SSP-PCR) technique. Results: The VLA4 gene SSP-PCR genotyping revealed no significant differences between patients and control group also between responder patients and non-responder to Natalizumab. Conclusion: VLA-4 polymorphisms at the level of SNP at positions 269 (C/A) have no role in MS susceptibility or Natalizumab responsiveness. Keywords: MS, Natalizumab, VLA-4 Citation: Khaliel AH, Abbas AA, Hatem AO, Abdulamir AS. The impact of Very Late Antigen 4 polymorphism on drug responsiveness in patients with multiple sclerosis initiation. Iraqi JMS. 2022; 20(1): 83-89. doi: 10.22578/IJMS.20.1.11

First Report of Burkholderia cepacia Causing Finger-Tip Rot on Banana Fruit in the Guangxi Province of China.

Banana (Musa acuminata) is one of the most popular and widely consumed fruit crops in the world. During late October to early November 2020, a banana finger-tip rot disease was observed in the banana (cultivar 'Brazil', AAA group) orchard of about 12 hectares located in Zhongcun, Zhangmu Town, Fumian District, Yulin City, Guangxi province, China. The disease incidence was about 0.5% at the surveyed field. Infected fingers and their tips were usually normal in the appearances and then turned to brown to black discoloration in the central fruit pulp adjacent to the fingertips (Fig. 1A). In severe infection, diseased fingers showed brown to black discoloration in both the central and the periphery fruit pulp, and along the longitudinal axis throughout the fruit (Fig. 1B-C). The symptomatic banana fingers were surface-disinfected with 1% sodium hypochlorite for 30 sec, 75% ethanol for 30 sec then rinsed three times with sterile distilled water. The flesh tissues were ground in a sterile mortar and soaked in 1 ml of sterile distilled water for 30 min. A 50 μl of tissue suspensions was streaked onto Luria-Bertani (LB) medium. Single colonies were picked and re-streaked onto new LB medium. The cultures were incubated at 37°C for 24 h. Two representative strains, GX and GX2, were obtained from symptomatic pulps and used in the following studies. To molecularly identify the bacterial species, we performed a polymerase chain reaction (PCR) using 16S rRNA and recA primers (Turner et al. 1999; Lee and Chan 2007) and amplified 1,442 bp and 1,019 bp sequences, respectively. The amplified sequences were deposited in GenBank under the accession numbers MZ267253 and MZ961355 for the 16S rRNA and MZ287336 and MZ983484 for the recA genes. BLASTn searches shared more than 99% similarity with the reference sequences of B. cepacia strains (MK680073.1 and KC261418.1 for 16S rRNA; AY598028.1 and KF812859.1 for recA). Phylogenetic trees were constructed using the 16 rRNA and recA sequences and showed that the representative strains, GX and GX2, strongly clustered with B. cepacia type strains (Fig. 2). To further determine the genomovars of strain GX, we used specific PCR primers to the B. cepacia epidemic strain marker (BCESM), type III secretion gene cluster (bcscV) and cable pilin subunit gene (cblA) (Lee and Chan 2007; Ansari et al. 2019). The presence of bcscV and BCESM were confirmed by PCR, while cblA was not observed in the strains GX and GX2, suggesting that the isolated strains belong to B. cepacia genomovar III and are slightly different from the Iranian and Taiwan strains of B. cepacia (Lee and Chan 2007; Ansari et al. 2019). Pathogenicity test was conducted on banana fingers (cultivar 'Zhongjiao No.3') at the immature and full ripe stages. A final suspensions of 106 CFU/ml, was injected into the banana fingers (100 μl per finger) through the center of the stigma (Lee and Chan 2007; Ansari et al. 2019). The fingers inoculated with sterile water were used as negative control. To maintain humidity, the treated fingertips were wrapped with Parafilm. For each treatment, ten independent replicates were conducted. At 10 days post-inoculation (dpi), the pulp of immature bananas exhibited reddish brown decaying tissue, which symptoms were similar to those observed in the field (Fig. 1D). Moreover, the pulp tissues of ripe bananas showed a dark brown discoloration in the tip at 5 dpi, whereas the controls remained symptomless (Fig. 1E). The same bacterium was re-isolated from diseased tissues and its identification confirmed by 16S rRNA, thus fulfilling the Koch's postulates. This disease was first described in Honduras in Latin America, and then reported in Taiwan province of China, and Iran (Buddenhagen 1968; Lee et al. 2003; Ansari et al. 2019). To our knowledge, this is the first report of banana finger-tip rot caused by B. cepacia in the Guangxi province, China. It is necessary to determine the distribution of B. cepacia and to prevent its spread in Guangxi province of China.

Biocontrol of Wheat Crown Rot Using Bacillus halotolerans QTH8.

Fusarium pseudograminearum causes crown rot in wheat. This study aimed to assess the effects of the bacterial strain QTH8 isolated from Cotinus coggygria rhizosphere soil against F. pseudograminearum. Bacterial strain QTH8 was identified as Bacillus halotolerans in accordance with the phenotypic traits and the phylogenetic analysis of 16S rDNA and gyrB gene sequence. Culture filtrates of bacterial strain QTH8 inhibited the mycelial growth of F. pseudograminearum and resulted in mycelial malformation such as tumor formation, protoplast condensation, and mycelial fracture. In addition, bacterial strain QTH8 also inhibited the mycelial growth of Hainesia lythri, Pestalotiopsis sp., Botrytis cinerea, Curvularia lunata, Phyllosticta theaefolia, Fusarium graminearum, Phytophthora nicotianae, and Sclerotinia sclerotiorum. The active compounds produced by bacterial strain QTH8 were resistant to pH, ultraviolet irradiation, and low temperature, and were relatively sensitive to high temperature. After 4 h exposure, culture filtrates of bacterial strain QTH8—when applied at 5%, 10%, 15%, 20%, 25%, and 30%—significantly reduced conidial germination of F. pseudograminearum. The coleoptile infection assay proved that bacterial strain QTH8 reduced the disease index of wheat crown rot. In vivo application of QTH8 to wheat seedlings decreased the disease index of wheat crown rot and increased root length, plant height, and fresh weight. Iturin, surfactin, and fengycin were detected in the culture extract of bacterial strain QTH8 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Bacterial strain QTH8 was identified for the presence of the ituC, bacA, bmyB, spaS, srfAB, fend, and srfAA genes using the specific polymerase chain reaction primers. B. halotolerans QTH8 has a vital potential for the sustainable biocontrol of wheat crown rot.

Validation of PCR-based Markers Associated with Sex Determination in Date Palm (Phoenix dactylifera L.)

Phoenix dactylifera (Date palm) is one of the essential commercial and economical fruit crops which mainly grows in hot arid areas. A good source of food, shelter, and energy, date palm is widely known due to its unique characteristics. The dioecious nature of date palm made it challenging to prognosticate its sex until they flower almost 5 to 8 years after plantation. Researchers have developed many strategies to determine sex, including morphological study, biochemical studies, molecular-based studies as sequence characterized amplified regions (SCAR), randomly amplified polymorphic DNA (RAPD) marker, and polymerase chain reaction (PCR) assays, but still more endeavours are integral. To foretell sex at the seedling stage, specific PCR primers were employed in this study. These primers were designed on sex-linked regions that determine their gender. Date palm DNA was extracted through the CTAB method. The DNA was then amplified through PCR using sex-linked specific primers. PCR products have run on gel electrophoresis using 1.2% agarose to score the bands for sex determination. Four primers were employed in this study to determine sex. Two primers were SCAR markers, and one was a sex-linked tetra primer. Tetra-primer amplified the double band fragment of 430 and 320bp respectively in male plants, while a band of 430bp was amplified in female plants. The other primer named ALAMER1 was amplified by the single band fragment of 186bp in males with no band in females. The fourth primer, named as SCARdp, amplified the band fragment of 354bp fragment in males. These SCAR markers were established to be male-specific. Sex-linked markers provide a platform for robust, efficient, and accurate determination of sex in date palms.

Collagen-VI Specific Primer Design Identification in Rats (Rattus norvegicus) Pancreas

Type 2 diabetes mellitus, the most common diabetes type characterized by hyperglycemia, is caused by abnormal secretion and activity of pancreatic insulin enzymes. The extracellular matrix (ECM) plays a vital role in keeping β pancreatic cells intact and undissociated. The ECM in the pancreas can play a role in influencing insulin function and production. The most abundant ECM in the pancreas is collagen type VI. Collagen type VI has an essential role in the survival of pancreatic islet cells, including pancreatic β cells. Nowadays, Polymerase Chain Reaction (PCR) technology is widely utilized for molecular biology analysis. One of the most critical factors for successful PCR is designing the correct specific PCR primers. The objective of this study was to design a specific primer for collagen VI in the pancreas of Rattus norvegicus. The primer was designed and analyzed using MEGA.11, primer three-plus, and primer-BLAST. Five primer pairs were analyzed based on the characteristics of primer length, product amplicon length, Tm value, GC percentage, and secondary structure. Primer pair 3 (F:5’-TGTTTGGCTTTGTCGCGGGC-3’ and R:5’-TTGTTGCTGCCGACACTGGC-3’); Col6a2 (F:5’-TGTGGTCAACAGGCTGGGCG-3’ and R:5’-TCTGGCGCCGGCTCTCTTTG-3’) were considered as the best primer for the Collagen VI expression detection from the pancreas of Rattus norvegicus, which produce amplicon about 250pb and 245pb, respectively.

The Role of HLA-DRB1 Alleles in Pulmonary Cystic Fibrosis.

Cystic fibrosis (CF) is the most common lethal autosomal recessive disease in white Caucasians. It affects many organs including the lung, pancreas, and liver. Whilst CF is a monogenic disease, several studies revealed a complex relationship between genotype and clinical phenotype of diseases. We examined the expression of human leukocyte antigen (HLA) class II alleles among Iranian CF patients with disease-related microbial infection. This study was conducted on 50 hospitalized CF patients (27 males, 23 females aged 15.5±6.5 years), and 50 healthy age- and gender-matched control subjects. 5ml whole blood was harvested and after isolation of genomic DNA, HLA-DRB1 subtypes were determined by single specific primer polymerase chain reaction methods. HLA-DRB1*10 was less frequent and HLA-DRB1*04 and HLA-DRB1*11 was the most frequent allele in CF patients, but none reached significance. HLA-DRB1*04 allele was frequently seen among16 CF patients with high serum IgE levels (430.25±219.7 IU/mL) and 27 CF patients that were positive for Pseudomonas aeruginosa colonization. A total of 31 CF patients had candida Albicans colonization in whom HLA-DRB1*11 was mostly seen. A total of 3 CF patients had allergic bronchopulmonary aspergillosis and two were diabetic. The DR4 and DR11 serotypes that recognize the HLA-DRB1*04 and HLA-DRB1*11 gene products respectively are not significantly enriched in the Iranian CF population. Further research should be conducted on DR4 and DR11 in CF patients to understand their possible role in infection and IgE expression.