Nanobody-based immunosensor for the detection of H. pylori in saliva.

Helicobacter pylori (H. pylori) infection is highly prevalent worldwide, affecting more than 43% of world population. The infection can be transmitted through different routes, like oral-oral, fecal-oral, and gastric-oral. Electrochemical sensors play a crucial role in the early detection of various substances, including biomolecules. In this study, the development of nanobody (Nb)-based immunosensor for the detection of H. pylori antigens in saliva samples was investigated. The D2_Nb was isolated and characterized using Western blot and ELISA and employed in the fabrication of the immunosensor. The sensor was prepared using gold screen-printed electrodes, with the immobilization of Nb achieved through chemical linkage using cysteamine-glutaraldehyde. The surface of the electrode was characterized using EIS, FTIR and SEM. Initially, the Nb-based immunosensor's performance was evaluated through cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV). The sensor exhibited excellent linearity with an R2 value of 0.96. However, further assessment with the DPV technique revealed both a low limit of detection (5.9 ng/mL, <1 cfu/mL) and high selectivity when exposed to a mixture of similar antigens. Moreover, the immunosensor demonstrated robust recovery rates (96.2%-103.4%) when spiked into artificial saliva and maintained its functionality when stored at room temperature for 24 days.

Novel spa type and antimicrobial resistance gene profiles of Staphylococcus aureus strains isolated from bovine mastitis in Jordan.

Staphylococcu aureus is the most prevalent microorganism associated with mastitis in cattle. This study was designed to determine the spa types of Staph. aureus and to assess the resistance genes profile of isolated strains in dairy farms in Jordan. In total, 747 milk samples of cattle suffering from subclinical mastitis were collected from 37 dairy farms and tested for Staph. aureus. To detect antimicrobial resistance genes, all 219 strains of Staph. aureus were tested. Furthermore, 21 isolates of Staph. aureus were typed using spa typing. As a result, different proportions of resistance genes were found for Staph. aureus. High resistance genes were in tetK 100%, blaZ 99%, and tetM 97%. Moderate resistance genes were in aac(6')/aph(2'' 52%, ant(4')-Ia 48%, and ermC 41%. Low resistance genes were in ermA is 24%, aph(3')-III is 15%, and mecA is 15%. The spa typing of 21 isolates revealed six spa types, of which five were previously known. For the first time, a novel spa type (t17158) was identified as the main cause of mastitis in dairy cows in Jordan. The identification of resistance genes and spa types is helpful in determining the most effective treatments for cows and plays a significant role in reducing the transmission of pathogens.

First molecular characterization of capsule expression and antibiotic susceptibility profile of Staphylococcus aureus isolates from bovine mastitis in Jordan.

Background and Aim: Bovine mastitis has long been considered the most important cause of economic losses in the dairy industry. Staphylococcus aureus is the most frequently isolated pathogen from bovine mastitis cases worldwide. Capsular polysaccharides (CPs) of serotype 5 (CP5) or serotype 8 (CP8) are the most prevalent capsule genotypes related to infections associated with S. aureus in humans. However, a variety of CPs has been reported in ruminants and other hosts. Information regarding the relationship between genotypic and phenotypic capsule variation and bovine mastitis in Jordan is scarce. Thus, we aimed to determine the prevalence of S. aureus capsule genotypes CP5 and CP8 in milk from bovine mastitis cases and the antimicrobial susceptibility profile of the recovered isolates in 27 dairy farms in Jordan. Materials and Methods: Staphylococcus aureus strains were isolated from bovine mastitis cases in two districts of Jordan. All S. aureus isolates were initially identified using conventional biochemical and microbiological methods. Subsequently, confirmation of the identity of S. aureus was performed by polymerase chain reaction (PCR) targeting nuc gene. Capsule polysaccharide typing was performed by PCR specific for CP5 and CP8. In addition, we assessed the antibiotic susceptibility profile of S. aureus isolates against commonly used antimicrobials by the disk diffusion method according to Clinical and Laboratory Standards Institute guidelines. Results: We collected 148 clinical isolates of S. aureus from bovine mastitis cases in the Zarqa (67.6%, n = 100) and Irbid (32.4%, n = 48) districts. Most isolates possessed capsule genotypes (91.3%), predominantly CP8 (88.6%). Only 8.7% of the isolates were nontypeable by PCR. In addition, we found statistically significant differences between the geographical region and the status of methicillin-resistant capsule genotypes (p < 0.05). The rates of resistance to ß-lactam, macrolide, and fluoroquinolone antibiotics were very low, but resistance to tetracyclines was considerably high (22.3%). Significantly, mastitis isolates from Irbid showed a higher rate of resistance to ciprofloxacin (8.3% vs. 0%), while isolates from Zarqa showed a significantly higher rate of resistance to gentamicin (12.0% vs. 6.2%). Conclusion: We established associations between capsule genotypes and antimicrobial resistance and the pathogenic behavior of S. aureus isolated from bovine mastitis cases. Further studies are necessary to fully elucidate the role and mechanisms of capsular expression in the epidemiological and molecular variability of S. aureus in bovine mastitis.

Molecular analysis for the OprD gene among Pseudomonas aueroginosa clinical isolates obtained from hospitals in Jordan.

INTRODUCTION: Pseudomonas aeruginosa has increasingly been associated with the emergence of antibiotic resistance. Antibiotic resistance among P. aeruginosa isolates is an ambiguous and complicated mechanism utilizing several enzymes and structural proteins. This study was conducted to investigate the prevalence of mutations in the chromosomal OprD gene that show resistance to carbapenems among clinical isolates of P. aeruginosa. METHODOLOGY: Sixty-three clinical isolates of P. aeruginosa resistant to meropenem were collected from public hospitals in Irbid city, north of Jordan. Analysis of antimicrobial susceptibility was carried out and their susceptibility was categorized. Molecular analysis of mutations in the OprD gene was performed using restriction fragment length polymorphism (RFLP) and DNA sequencing. RESULTS: Molecular analysis of P. aeruginosa isolates showed 52% of the common molecular modifications among the collected isolates. These alterations could be associated and affect meropenem-susceptibility rather than imipenem. The most frequent molecular changes among the resistant isolates were the F170L substitution mutation. This was detected in 22 (35%) of the isolates with an unusual insertion sequence (IS) of 100 bp within the 590 bp DNA segment downstream of the restriction site. The divergent sequence of 10 amino acids 372(VDSSSSYAGL)383 was detected in 7 (11%) of the isolates. CONCLUSIONS: A significant alteration in the OprD gene in P. aeruginosa clinical isolates was found. Alterations in the OprD gene could be linked to protein permeability of the outer membrane of P. aeruginosa associated with meropenem resistance. Further investigations with a larger number of bacterial isolates are needed to validate the proposed association.

Potential antimicrobial effect of plant essential oils and virulence genes expression in methicillin-resistant Staphylococcus aureus isolates.

AIM: This study aimed to investigate the antibacterial efficacy of eight commercially available essential oil (EO) blends and characterize the effect on the expression of some virulence genes against methicillin-resistant Staphylococcus aureus (MRSA). MATERIALS AND METHODS: In vitro evaluation of the antimicrobial effects of oils against MRSA was performed using the disk diffusion method and by measuring the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The EOs (A-F) were contained (ß-pinene, carvacrol, carvone, dimethyl trisulfide, linalool, limonene, menthol, monoterpene hydrocarbons, and thymol) in different amounts. In addition, a real-time polymerase chain reaction was also used to determine the gene expression of the virulence genes (intercellular adhesion cluster [ica]-9, ica-15, and RNA III) against MRSA (ATCC 43300) after treatment with selected oils. RESULTS: Among the eight EOs evaluated, EO (D), (E), and (A) showed, in general, the greatest antimicrobial activity against MRSA. EO at 1/3 MIC has effectively down-regulated ica-9 and ica-15 of MRSA by 17.83 and 4.94 folds, respectively. Meanwhile, EO (A) has effectively down-regulated RNAIII by 3.74 folds. Our results indicated that some of the EOs exhibit promising antimicrobial effects against MRSA isolates. Moreover, the results of the analyzed virulence genes related to the pathogenicity of MRSA were down-regulated at the sub-MIC concentrations of EOs, indicated that EOs could be successfully used to suppress the virulence factors and, consequently, decreased the pathogenicity of MRSA. CONCLUSION: These encouraging results indicate that some of the EOs used in this study can be utilized as a natural antibiotic for the treatment of MRSA disease.

Antimicrobial resistance and putative virulence genes of Pseudomonas aeruginosa isolates from patients with respiratory tract infection.

INTRODUCTION: Pseudomonas aeruginosa is a common agent causing community acquired and nosocomial respiratory tract infections, with particularly life-threatening manifestations in patients who are immunocompromised of who have cystic fibrosis. This study investigated the occurrence of extended-spectrum ß-lactamases (ESBLs) and metallo ß-lactamase (MBL) in association with important putative virulence genes and genotypes variation among P. aeruginosa isolates from respiratory tract infection of Jordanian patients. METHODS: Over a period of 8-month, a total of 284 respiratory tract samples were obtained from patients diagnosed with respiratory tract infection while attending the Pulmonary Clinic/Intensive Care Unit, Jordan University Hospital (JUH). At the time of sampling most were inpatients (86.9%). Samples were cultured specifically for P. aeruginosa. RESULTS: A total of 61/284 (21.5%) P. aeruginosa isolates were recovered from respiratory samples of patients. The percentage of MDR P. aeruginosa isolates was 52.5%, and all isolates were susceptible to colistin with lower rates of susceptibility to other tested antibiotics. Positive genes of blaCTX-M, blaVEB, blaTEM, blaGES and blaSHV were detected in 68.9%, 18.9%, 18.9%, 15.6% and 12.5% of isolates, respectively. Genotyping revealed no significant genetic relationship among MDR P. aeruginosa isolates from hospitalized patients as judged by the constructed dendrogram and the presence of 14 genotypic groups. The percentages of the virulence genes algD, lasB, toxA, exoS, and exoU among P. aeruginosa isolates were 98%, 98%, 80%, 33% and 33%, respectively, and 87% of isolates produced pyocyanin. CONCLUSION: The present study demonstrates high occurrence of MDR P. aeruginosa isolates carrying blaCTX-M genes. No specific associations were found between antibiotic resistance, virulence genes and genotypes among MDR isolates.

Major characteristics of Staphylococcus aureus colonizing Jordanian infants.

BACKGROUND: Colonization of infants with methicillin-resistant Staphylococcus aureus (MRSA) carries specific toxin genes. In particular, Panton-Valentine leukocidin (PVL) are a risk factor for subsequent infection during hospitalization. This prospective study investigated important epidemiological characteristics of Staphylococcus aureus colonizing the nares and intestines of Jordanian infants. METHODS: A total of 860 nasal and stool specimens were obtained from each of the 430 infants admitted to the neonatal intensive care unit or referred to outpatient clinics of Jordan University Hospital. All specimens were cultured to recover S. aureus, all isolates were tested for antimicrobial susceptibility and the MRSA strains for presence of specific toxin genes and SCCmec using polymerase chain reaction. RESULTS: Eighty of the 430 (18.6%) infants were colonized with S. aureus, of these, 27 (6.3%) harbored the organism in both the nose and intestine. The frequency of S. aureus nasal and intestinal carriage in outpatient infants compared to inpatients admitted to the neonatal intensive care unit was significantly higher (27.3% vs 2.8%) and (17.1% vs 2.3%), respectively. MRSA accounted for 57/107 (53.3%) of all isolates, and of these 16/57 (28%) were PVL-positive and carried SCCmec type IV, except one, which was type III. All nasal and intestinal MRSA carried at least one toxin gene (tst, eta, seb), but few carried two toxin genes. CONCLUSION: This study demonstrates that S. aureus strains are more frequently colonizing Jordanian outpatient infants than inpatients and all MRSA strains carried 1-3 clinically important staphylococcal toxin genes. Further studies are needed to investigate the role of these toxins in hospitalized infants.

Identification of differentially expressed small non-protein-coding RNAs in Staphylococcus aureus displaying both the normal and the small-colony variant phenotype.

The emerging interest in RNA research is due to the discovery that bacterial non-protein-coding RNAs (npcRNAs; often referred to as "non-coding RNAs") are central regulatory molecules. While single npcRNAs have been described in Staphylococcus aureus, mostly based on computational-based approaches, experimental data on npcRNAs and their impact on the formation of different phenotypes of S. aureus are missing. Consequently, two specialized cDNA libraries were constructed from total RNA collected from different growth phases of an isogenic clinical strain pair of S. aureus displaying both the normal and the small-colony variant phenotype. Overall, 142 candidates for novel npcRNAs were identified and their expression analyzed by Northern blot assays. Of these, the presence of 18 novel npcRNAs in S. aureus was experimentally confirmed. In fact, growth phase-specific regulation was detected for almost all of the novel npcRNAs, with different npcRNA expression patterns detectable for both phenotypes. Of particular interest, S. aureus phenotype-specific expression of four novel npcRNAs was documented. Thus, the presence of differentially expressed npcRNAs in S. aureus may help to understand the phenotypic variation and its associated pathogenicity.