Molecular and bacteriological investigations for the co-existence CRISPR/Cas system and ß-lactamases of types extended-spectrum and carbapenemases in Multidrug, extensive drug and Pandrug-Resistant Klebsiella pneumoniae.

The recent approach towards combating the antimicrobial resistance has led to the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated sequence to overcome the challenges of antimicrobial resistance. Thus, this study aimed to detect the underlying resistance mechanisms such as ESBLs and carbapenemases and whether there is a correlation between multidrug, extensive drug and pan drug resistance and the occurrence of CRISPR loci. A total of one hundred study isolates were subjected to antimicrobial susceptibility testing using the AST card of the Vitek technique to detect resistance patterns involving ESBLs and carbapenemase (CRE). An investigation of the genes encoding CRISPR/Cas systems using PCR was achieved. Out of 81 (81.0%) resistant Klebsiella pneumoniae isolates, 71 (71%) and 21 (21.0%) produced ESBLs and carbapenemases, respectively. Also, 53 (53.0%), 19 (19.0%) and 9 (9.0%) were MDR, XDR, and PDR respectively. It was noted that Cas1, Cas3, CRISPR1, CRISPR2 and CRISPR3 were positive in 38 (38.0%) of the isolates, while CRISPR1 for incomplete CRISPR1-Cas systems alone was detected in 78 (78.0%). Further, the number of intact CRISPR1, intact CRISPR2 and intact CRISPR3 types were 7 (27.0%), 34 (34%) and 18 (18.0%) respectively. It is concluded that antibiotic resistance levels were inversely correlated with the existence of CRISPR/Cas systems. The absence of the CRISPR/Cas system increases the prevalence of MDR, XDR and PDR in ESBL and carbapenem-producing Klebsiella pneumoniae. With the increase in the degree of antibiotic resistance (MDR, XDR to PDR), the occurrence ratio of the (CRISPR)/CRISPR-associated sequence decreased.

Molecular cytogenetic analysis of multi-miscarriage products of conception in clinical cases from Al-Anbar Governorate, west of Iraq.

Most clinical miscarriages often occur throughout the first trimester of pregnancy, with fetal chromosomal abnormalities being identified as the primary reason for such occurrences. The objective is to analyze the fetal chromosomal aberrations in the product of conception among Iraqi patients suffering from recurrent miscarriages. The cross-sectional study was performed on 60 cases of products of conception in women suffering from multiple miscarriages, obtained from Department of Obstetrics and Gynecology is located in Ramadi Teaching Hospital for Child and Maternity, as well as other Private Clinics in the Ramadi City. Long-term culture of conventional cytogenetic analysis using the G-banding technique was employed to determine the chromosomal disorder of fetal tissue part or villus samples. Fetal chromosomal abnormalities were detected in 86.7 %. Numerical chromosomal abnormalities were revealed in 98.1 %, while structural abnormalities were detected in 1.9 %. Additionally, the commonest gestation loss occurs in parents under 35 years in the first trimester (92.3 %). Trisomy 21 was the most frequent (46.2 %) in gestational loss. Fetal chromosomal abnormalities have been linked with gestational loss in Iraqi couples. Therefore, it is recommended that cytogenetic analysis should be performed to identify the genetic cause of recurrent miscarriage. This is important for providing appropriate genetic counseling and educating couples about the risk of future pregnancies.

lncRNA-microRNA axis in cancer drug resistance: particular focus on signaling pathways.

Cancer drug resistance remains a formidable challenge in modern oncology, necessitating innovative therapeutic strategies. The convergence of intricate regulatory networks involving long non-coding RNAs, microRNAs, and pivotal signaling pathways has emerged as a crucial determinant of drug resistance. This review underscores the multifaceted roles of lncRNAs and miRNAs in orchestrating gene expression and cellular processes, mainly focusing on their interactions with specific signaling pathways. Dysregulation of these networks leads to the acquisition of drug resistance, dampening the efficacy of conventional treatments. The review highlights the potential therapeutic avenues unlocked by targeting these non-coding RNAs. Developing specific inhibitors or mimics for lncRNAs and miRNAs, alone or in combination with conventional chemotherapy, emerges as a promising strategy. In addition, epigenetic modulators, immunotherapies, and personalized medicine present exciting prospects in tackling drug resistance. While substantial progress has been made, challenges, including target validation and safety assessment, remain. The review emphasizes the need for continued research to overcome these hurdles and underscores the transformative potential of lncRNA-miRNA interplay in revolutionizing cancer therapy.

The impact of circulating 25-hydroxyvitamin D and vitamin D receptor variation on leukemia-lymphoma outcome: Molecular and cytogenetic study.

Vitamin D (VD) potentially has a crucial function in the development of cancerous cells. This study aims to detect the role of vitamin D concentration and its receptor polymorphisms as possible prognostic biomarkers in patients with leukemia/lymphoma and further will attempt to detect the presence of the Philadelphia chromosome abnormality in chronic myeloid leukemia (CML). Seventy-five patients, in addition to 50 healthy individuals were included. Three single nucleotide polymorphisms of the vitamin D receptor (FokI, Tru91, and ApaI) were identified via Polymerase Chain Reaction- Fragment Length Polymorphism (PCR-RFLP). Sanger sequencing and karyotyping for all patients has been undertaken. Out of 75 patients, 69 (92.0%) were vitamin D deficient. The homozygous genotype TT of FokI is the most commonly found in non-Hodgkin's lymphoma, while the heterozygous CT is observed markedly in CML, chronic lymphoid leukemia, and Hodgkin's lymphoma. The AC and CC genotypes of ApaI are more frequent in patients with CML, while the AC genotype is the most common in HL. In Tru9I, the GG genotype has a wider distribution in individuals diagnosed with leukemia. The PCR-RFLP and Sanger sequencing techniques together confirmed significant genotype respectively. The Philadelphia chromosome, t (9;22) was found in five (17%) cases with CML. There is a marked relationship between FokI, ApaI, and Tru91 polymorphisms and the chance of developing leukemia. In lymphoma, a significant connection between the polymorphisms of FokI and ApaI is frequently detected. Cytogenetic and molecular testing are essential for detection of CML and monitoring therapy response.

Study of vacuolating cytotoxin A (vacA) genotypes of ulcerogenic and non-ulcerogenic strains of Helicobacter pylori and its association with gastric disease.

Globally, Helicobacter pylori (H. pylori), a stomach pathogen, is present in around 50 % of the population. This bacterial infection produces persistent inflammation, which significantly raises the risk of duodenal, gastric ulcer, and stomach cancer. The goal of this study is to identify the vacA genotypes in H. pylori and analyze how they relate to medical conditions brought on by the bacteria and clarithromycin resistance. PCR was used to describe 115 endoscopic stomach samples from infected patients and identify vacA gene. Of the 115 research participants, H. pylori was found in 81 (70.4 %) of them. Of the isolated cultures, only 38 (69.1 %) were resistant to clarithromycin. VacA was discovered in 55 (67.9 %) of the samples that had H. pylori in them. Patients with gastritis were more likely to have s2m2 strains of infection (66.7 %), while those with gastric and duodenal ulcers were more likely to have s1m1 strains (64.7 %). VacA-positive H. pylori strains (60 % n = 33) were more resistant to clarithromycin versus (19.2 % n = 5) for vacA-negative bacteria. Clarithromycin resistance was significantly linked to vacA s2m2 in H. pylori isolates (75.9 %). According to the study's results, the vacA variants s1m1 and s2m2 have a strong connection with the emergence of H. pylori infections that cause peptic ulcer disease in the population of Iraq. Genetic testing is essential in predicting both the course of treatment and the outcome of H. pylori disease.

Inverse Association between the Existence of CRISPR/Cas Systems with Antibiotic Resistance, Extended Spectrum ß-Lactamase and Carbapenemase Production in Multidrug, Extensive Drug and Pandrug-Resistant Klebsiella pneumoniae.

Antimicrobial resistance, with the production of extended-spectrum ß-lactamases (ESBL) and carbapenemases, is common in the opportunistic pathogen, Klebsiella pneumoniae. This organism has a genome that can contain clustered regularly interspaced short palindromic repeats (CRISPRs), which operate as a defense mechanism against external invaders such as plasmids and viruses. This study aims to determine the association of the CRISPR/Cas systems with antibiotic resistance in K. pneumoniae isolates from Iraqi patients. A total of 100 K. pneumoniae isolates were collected and characterized according to their susceptibility to different antimicrobial agents. The CRISPR/Cas systems were detected via PCR. The phenotypic detection of ESBLs and carbapenemases was performed. The production of ESBL was detected in 71% of the isolates. Carbapenem-resistance was detected in 15% of the isolates, while only 14% were susceptible to all antimicrobial agents. Furthermore, the bacteria were classified into multidrug (77%), extensively drug-resistant (11.0%) and pandrug-resistant (4.0%). There was an inverse association between the presence of the CRISPR/Cas systems and antibiotic resistance, as resistance was higher in the absence of the CRISPR/Cas system. Multidrug resistance in ESBL-producing and carbapenem-resistant K. pneumoniae occurred more frequently in strains negative for the CRISPR/Cas system. Thus, we conclude that genes for exogenous antibiotic resistance can be acquired in the absence of the CRISPR/Cas modules that can protect the bacteria against acquiring foreign DNA.

Cytogenetic screening of chromosomal abnormalities and genetic analysis of FSH receptor Ala307Thr and Ser680Asn genes in amenorrheic patients.

Objective: Amenorrhea is a rare reproductive medical condition defined by the absence of menstruation during puberty or later life. This study aims to establish the frequency and pattern of chromosomal abnormalities (CA) in both primary amenorrhea (PA) and secondary amenorrhea (SA), and further to detect the genetic changes in exon 10 at nucleotide positions 919 and 2039 of the genotypes Thr307Ala, and Asn680Ser, respectively. Design settings and patients: This cross-sectional study was conducted on a sample of seventy amenorrhoeic women according to the Helsinki declaration rules of medical ethics, as divided into 40 (57.14%) with PA and 30 (42.86%) with SA, and 30 healthy women with normal menstruation as the control. The chromosomal karyotyping was performed according to the ISCN, 2020. PCR products were submitted to RFLP and Sanger sequencing for women with normal karyotype and high FSH serum levels. Results: The classical Turner Syndrome was the most common CA in PA, followed by isochromosome X [46, Xi(X)(q10)], mosaicism of Turner and isochromosome X [45, X /46, Xi(X)(q10)], sex reversal (46, XY) and (46, XX,-3,+der3,-19,del 19 p). Abnormal SA cases were characterized by mosaicism Turner syndrome (45,X/46,XX) and (46,XX,-3,+der3,X,+derX). The homozygous genotypes AA and GG of Ala307Thr (rs6165) in the FSHR gene are most common in PA, while the homozygous genotype AA is more common in SA. GG and AG genotypes of Ser680Asn (rs6166) are more frequent in Iraqi patients with PA and SA compared to the healthy control women. Both PCR-RFLP and Sanger sequencing indicated a marked matching between genotypes. Conclusions: The study emphasizes the need for cytogenetic analysis to determine the genetic basis of PA and SA. Further, genotyping for women with normal karyotype and high FSH serum concentrations via PCR-RFLP should be considered for the precise diagnosis and development of appropriate management of and counselling for these patients.

Antibiotic resistance: The challenges and some emerging strategies for tackling a global menace.

BACKGROUND: Antibiotic resistance is currently the most serious global threat to the effective treatment of bacterial infections. Antibiotic resistance has been established to adversely affect both clinical and therapeutic outcomes, with consequences ranging from treatment failures and the need for expensive and safer alternative drugs to the cost of higher rates of morbidity and mortality, longer hospitalization, and high-healthcare costs. The search for new antibiotics and other antimicrobials continues to be a pressing need in humanity's battle against bacterial infections. Antibiotic resistance appears inevitable, and there is a continuous lack of interest in investing in new antibiotic research by pharmaceutical industries. This review summarized some new strategies for tackling antibiotic resistance in bacteria. METHODS: To provide an overview of the recent research, we look at some new strategies for preventing resistance and/or reviving bacteria's susceptibility to already existing antibiotics. RESULTS: Substantial pieces of evidence suggest that antimicrobials interact with host immunity, leading to potent indirect effects that improve antibacterial activities and may result in more swift and complete bactericidal effects. A new class of antibiotics referred to as immuno-antibiotics and the targeting of some biochemical resistance pathway components including inhibition of SOS response and hydrogen sulfide as biochemical underlying networks of bacteria can be considered as new emerging strategies to combat antibiotic resistance in bacteria. CONCLUSION: This review highlighted and discussed immuno-antibiotics and inhibition of SOS response and hydrogen sulfide as biochemical underlying networks of bacteria as new weapons against antibiotic resistance in bacteria.

Prevalence of CRISPR-Cas Systems and Their Possible Association with Antibiotic Resistance in Enterococcus faecalis and Enterococcus faecium Collected from Hospital Wastewater.

Purpose: This study aimed to evaluate the presence of CRISPR-Cas system genes and their possible association with antibiotic resistance patterns of Enterococcus faecalis and Enterococcus faecium species isolated from hospital wastewater (HWW) samples of several hospitals. Methods: HWW samples (200 mL) were collected from wastewater discharged from different hospitals from October 2020 to March 2021. The isolation and identification of enterococci species were performed by standard bacteriology tests and polymerase chain reaction (PCR). Antibiotic resistance was determined using the disc diffusion. The presence of various CRISPR-Cas systems was investigated by PCR. The association of the occurrence of CRISPR-Cas systems with antibiotic resistance was analyzed with appropriate statistical tests. Results: In total, 85 different enterococci species were isolated and identified using phenotypic methods. The results of PCR confirmed the prevalence of 50 (58.8%) E. faecalis and 35 (41.2%) E. faecium, respectively. In total, 54 (63.5%) of 85 isolates showed the presence of CRISPR-Cas loci. The incidence of CRISPR-Cas was more common in E. faecalis. CRISPR1, CRISPR2, and CRISPR3 were present in 35 (41.2%), 47 (55.3%), and 30 (35.3%) enterococci isolates, respectively. The CRISPR-Cas positive isolates showed significant lower resistance rates against vancomycin, ampicillin, chloramphenicol, erythromycin, rifampin, teicoplanin, tetracycline, imipenem, tigecycline, and trimethoprim-sulfamethoxazole in comparison with CRISPR-Cas negative isolates. The results showed that the presence of CRISPR-Cas genes was lower in multidrug-resistant (MDR) isolates (53.1%, n = 26/49) compared to the non-MDR enterococci isolates (77.8%, n = 28/36) (P = 0.023). Conclusion: This study revealed the higher prevalence of E. faecalis than E. faecium in HWWs. Also, the lack of CRISPR-Cas genes was associated with more antibiotic resistance rates and multidrug resistance in E. faecalis and E. faecium isolates with HWW origin.

Cross-sectional study of chromosomal aberrations and immunologic factors in Iraqi couples with recurrent pregnancy loss.

BACKGROUND: Parental chromosomal aberrations are important causes of recurrent pregnancy loss (RPL). Some immunological factors such as antiphospholipid antibodies and interleukin-6 (IL-6) also contribute to this complication. The aim of this study was to determine the frequency of chromosomal abnormalities and to evaluate some of the immunological factors in couples with RPL from different cities in Iraq. METHODS: This study was conducted on 25 couples (50 individuals) who had more than two first trimester abortions in the past and 25 healthy females as controls. Karyotyping was performed on peripheral blood of all participants. Anticardiolipin (IgG and IgM), antiphosopholipid (IgG and IgM), lupus anticoagulant, and IL-6 were assayed. Data were analyzed using appropriate statistical tests. RESULTS: Chromosomal abnormalities were found in 28.0% (n = 7/25) of RPL couples. Of these five (10.0%) were female and two (4.0%) were male. The types of structural abnormalities were as follows: 45, XX, t(21; 21); 45, XX, rob (14, 15); 46, XX, add (21) (p13); 46 XY, add (21)(p13); 46, XX, 21ps+; 46, XY, per inv (9) (p11q12) and 45, XX, t(13q, 13q). No chromosomal abnormalities were found in the control group. Also, no significant differences were found in the immunological parameters of the couples with RPL and the control group. CONCLUSION: In this study, karyotyping revealed a high number of chromosomal abnormalities associated with the RPL in Iraqi couples. Since identification of genetic causes of miscarriage is important for genetic counseling and educating couples about the risk of future pregnancies, it is recommended that conventional karyotyping be investigated in patients with RPL.

Detection of clarithromycin resistance and 23SrRNA point mutations in clinical isolates of Helicobacter pylori isolates: Phenotypic and molecular methods.

BACKGROUND AND OBJECTIVES: Peptic ulcer disease, chronic gastritis, and stomach cancer are all caused by H. pylori. The most notable drug for the treatment is the antibiotic clarithromycin, which is currently the drug of choice. H. pylori clarithromycin resistance has been associated with point mutations in 23srRNA, the most prominent of which are A2143 and A2144G. In H. pylori bacteria, methylase synthesis, macrolide-inactivating enzyme activity, and active efflux have all been found to be resistance mechanisms. The goal of the study is to determine how resistant H. pylori is to clarithromycin and what the minimum inhibitory concentration is for various antimicrobials. Furthermore, gastro-endoscopy will be performed on Iraqi patients to detect the presence of A2143G and A2144G point mutations in Helicobacter pylori infections, as diagnosed from the pyloric region and other anatomical regions. METHODS: One hundred fifteen samples were collected from patients strongly suspected of H. pylori infection presented for upper gastrointestinal endoscopy at Ramadi Teaching Hospitals and Private Clinics for the period from January 2020 until February 2021. Specimens were cultured on brain heart infusion agar containing various antibiotics and were incubated at 37 °C under microaerophilic conditions. For identification of H. pylori, isolates of the biochemical tests and RT-PCR assay were applied. The Epsilometer test was used in the antibiotic susceptibility testing as dependent on the CLSI standard. The Restriction Fragment Length Polymorphism technique was used to determine point mutations. RESULTS: In total, 55 (47.8%) Helicobacter pylori isolates were cultured from the 115 biopsy specimens, among which 16 (29.1%), 38 (69.1%), 20 (36.4%), and 40 (72.7%) revealed some degree of resistance to levofloxacin, clarithromycin, ciprofloxacin, and metronidazole, respectively. The frequency of A2144G and A2143 point mutations were 23 (60.5%) and 19 (50%), respectively. CONCLUSIONS: According to our results, Helicobacter pylori showed high resistance to clarithromycin. Our results demonstrate the requirement for antibiotic susceptibility testing and molecular methods in selecting drug regimens.

Detection of Helicobacter Pylori infection by invasive and non-invasive techniques in patients with gastrointestinal diseases from Iraq: A validation study.

There is still no agreement on the gold standard technique for diagnosing of H. Pylori in Iraq, as well as a paucity of data on the validity of different diagnostic techniques. This study aimed to investigate the prevalence of this bacterium with different methods and compare them to the quantitative polymerase chain reaction (qPCR) as a golden standard technique among Iraqi patients. In total, 115 Iraqi patients strongly suspected of H. pylori infection were enrolled in the current study. Invasive techniques including rapid urease testing (RUT) and gastric tissue culture in addition to non-invasive techniques including 14C-Urea breath test (14C-UBT), stool antigen test (SAT), CagA-IgG serology, and qPCR were performed to confirm the H. pylori infection. The qPCR was used as the gold standard to estimate the sensitivity, specificity, positive and negative predictive values for the studied diagnostic tests. Overall, the prevalence rate of H. pylori in Iraqi patients was ranged from 47.8 to 70.4% using different methods. The positive results for each test were as follows: qPCR 81, (70.4%) UBT 79 (68.7%), SAT 77 (67%), RUT 76 (66.1%), Cag-IgG 61 (53%), and culture 55 (47.8%). The 14C-UBT showed the highest overall performance with 97.5% sensitivity, 97% specificity, and total accuracy of 97.3% followed by SAT, RUT, Cag-IgG, and culture method. Based on the accuracy of the studied methods for H. pylori detection, they can be arranged in order as follows: qPCR > UBT > SAT > RUT> CagA IgG > culture. Since a universal gold standard assay for the diagnosis of H. pylori has not yet been established in Iraq, the UBT may be recommended as first choice due to its higher performance compared to other methods.

SEN virus genotype H distribution in ß-thalassemic patients and in healthy donors in Iraq: Molecular and physiological study.

The SEN virus (SENV) has been linked to transfusion-associated non-A-E hepatitis; however, information regarding SENV infections in patients with thalassemia, particularly in those with hepatitis virus co-infections, remains limited. This study investigated the frequency of SENV (genotypes D and H) infections in Iraqi patients with thalassemic patients infected and not infected with hepatitis C virus. The study involved 150 ß-thalassemia patients (75 with HCV infections and 75 without) and 75 healthy blood donors. Patient levels of vitamins C and E, liver function markers, and glutathione peroxidase (GPX) were determined. Recovered viral nucleic acids were amplified using the conventional polymerase chain reaction (SENV DNA) or the real-time polymerase chain reaction (HCV RNA) techniques. Only 10% of healthy donors had evidence of SENV infection. Among patients with thalassemia, 80% and 77% of patients with and without concurrent HCV infections, respectively, had SENV infections. DNA sequencing analyses were performed on blood samples obtained from 29 patients. Patients with thalassemia, particularly those with SENV infections, had higher levels of several enzymatic liver function markers and total serum bilirubin (P < 0.05) than did healthy blood donors. Among the examined liver function markers, only gamma-glutamyl transferase demonstrated significantly higher levels in HCV-negative patients infected with SENV-H than in those infected with SENV-D (P = 0.01). There were significantly lower vitamin C, vitamin E, and glutathione peroxidase levels in patients than in healthy donors (P < 0.05), but only glutathione peroxidase levels were significantly lower in HCV-negative thalassemia patients infected with SENV than in those without SENV infections (P = 0.04). The SENV-H genotype sequences were similar to the global standard genes in GenBank. These results broaden our understanding the nature of the SENV-H genotype and the differential role of SENV-H infections, compared to SENV-D infections, in patients with thalassemia, in Iraq.

Isolation and identification of potential probiotic Lactobacillus species from feces of infants in southwest Iran.

OBJECTIVES: To evaluate the potential probiotic properties of Lactobacillus strains isolated from feces of infants and also to determine their antimicrobial activity against some enteropathogenic bacteria. METHODS: The Fecal samples were prepared from 120 infants aged less than 24 months. In total, 105 Lactobacillus strains were identified by phenotypic tests. Thirty isolates were randomly selected to study their potential probiotic properties. These isolates were examined for resistance to acid (pH: 2.5, 2 h) and bile (oxgall 0.3%, 8 h), adhesion to HT-29 cells, antibiotic susceptibility, and antimicrobial activities. RESULTS: On basis of 16S rRNA sequencing, 30 isolates identified as Lactobacillus fermentum (n = 11; 36.7%), Lactobacillus plantarum (n = 9; 30%), Lactobacillus rhamnosus (n = 6; 20%), and Lactobacillus paracasei (n = 4; 13.3%). All tested strains survived at acid and bile conditions. Six Lactobacillus strains revealed high adherence to HT-29 cells. Three strains including the L. fermentum (N2, N7), and the L. plantarum (N20) showed good probiotic potential and inhibited the growth of Yersinia enterocolitica ATCC 23715, Shigella flexneri ATCC 12022, Salmonella enterica ATCC 9270, and enteropathogenic Escherichia coli (EPEC) ATCC 43887. The antibiotic resistance test showed that all the isolates were susceptible to tetracycline, and chloramphenicol. CONCLUSIONS: Lactobacillus strains like L. fermentum (N2, N7), and the L. plantarum (N20), could be potential probiotic, but further in vitro and in vivo studies on these probiotic strains are still required.