Purification and characterization of α-galactosidase isolated from Klebsiella pneumoniae in the human oral cavity.

The enzyme α-Galactosidase (α-D-galactoside galactohydrolase [EC 3.2.1.22]) is an exoglycosidase that hydrolyzes the terminal α-galactosyl moieties of glycolipids and glycoproteins. It is ubiquitous in nature and possesses extensive applications in the food, pharma, and biotechnology industries. The present study aimed to purify α-galactosidase from Klebsiella pneumoniae, a bacterium isolated from the human oral cavity. The purification steps involved ammonium sulfate precipitation (70 %), dialysis, ion exchange chromatography using a DEAE-cellulose column, and affinity monolith chromatography. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to determine the molecular weight of the purified enzyme. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were determined by using p-nitrophenyl-α-D-galactopyranoside as substrate. The results showed that the purification fold, specific activity, and yield were 126.52, 138.58 units/mg, and 21.5 %, respectively. The SDS-PAGE showed that the molecular weight of the purified enzyme was 75 kDa. The optimum pH and temperature of the purified α-galactosidase were detected at pH 6.0 and 50 °C, respectively. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were 4.6 mM and 769.23 U/ml, respectively. α-galactosidase from Klebsiella pneumoniae was purified and characterized. (SDS-PAGE) analysis showed that the purified enzyme appeared as single band with a molecular weight of 75 kDa.

Occurrence of some common carbapenemase genes in carbapenem-resistant Klebsiella pneumoniae isolates collected from clinical samples in Tabriz, northwestern Iran.

OBJECTIVES: This study aimed to evaluate the antibiotic resistance patterns and prevalence of carbapenemase genes in Klebsiella pneumoniae isolates in different clinical samples from Tabriz city, northwestern Iran. RESULTS: This cross-sectional study was conducted in the Department of Microbiology, Islamic Azad University, Ahar Branch, Iran, in 2020. K. pneumoniae isolates were collected from different clinical samples, including blood, wounds, sputum, and urine. The isolates were identified using a series of standard bacteriological tests. Antibiotic resistance was determined by the disc diffusion method. The presence of blaVIM, blaNDM, blaKPC, blaOXA, and blaIMP genes were screened by polymerase chain reaction (PCR). A total of 100 non-duplicated K. pneumoniae isolates were collected from 57 urine samples, 27 blood samples, 13 wound samples, and 3 sputum samples. Overall, 70.0% of the samples were from inpatients, while 30.0% were from outpatients. The most resistance rate was related to ampicillin (94.0%), while the lowest resistance rate was related to imipenem (18.0%) and meropenem (20.0%). Overall, 25.0% of the isolates were carbapenem-resistant, of which 13.0% were resistant to both imipenem and meropenem. The PCR showed the total prevalence of 23.0% for carbapenemase genes, including 18.0% for blaKPC, 3.0% for blaVIM, 1.0% for blaIMP, and 1.0% for blaOXA gene. The blaNDM gene was not detected in any isolate. The prevalence of carbapenemase-producing K. pneumoniae isolates was relatively lower in northwestern Iran than in other regions of the country. However, special attention should be paid to the proper use of antibiotics, particularly carbapenems, to prevent further spread of antibiotic resistance and its related genes.

Immunological Study of IFN-γ, ICAM-4, and Vitamin D3 Markers among Gastrointestinal Tumor Patients in Babylon Province, Iraq.

OBJECTIVE: The current study was conducted to investigate the roles of ICAM-4, IFN-γ, and vitamin D3 markers among benign and malignant gastrointestinal stromal tumors (GISTs). METHODOLOGY: Eighty-eight participants, admitted to the Babylon GIT Center, Merjan Medical City, Iraq from April to December 2020, were recruited for the study. Blood samples were collected from the participants, who were divided into four groups: malignant GIT tumor (N = 42), benign GIT tumor (N = 29), irritable bowel disease as a positive control (N = 10), and healthy individuals as a negative control (N = 7). Serum ICAM-4, IFN-γ, and vitamin D3 levels were determined using the blood samples. RESULTS: The younger males were more affected by malignant GIT tumors at a mean age of 53.39 years than benign GIT tumors, IBD, and healthy individuals. There is also an increase in ICAM-4, IFN-γ, and a decrease in vitamin D3 levels compared to healthy individuals. The vitamin D3 level decreased progressively with age and rose in ICAM-4 with a decrease in vitamin D3 level in patients, increasing the probability of infection with GIT tumor. ICAM-4 levels may grow and increase as interferon levels rise. CONCLUSION: The younger males are more prone to malignant GIT and the serum levels of ICAM-4, vitamin D3, and IFN-γ are high in malignant patients compared with benign GIT tumors and lower than the control.

Synergistic effects of silybin and curcumin on virulence and carbapenemase genes expression in multidrug resistant Klebsiella oxytoca.

OBJECTIVE: Silybin and curcumin have potential antimicrobial effects. This study aimed to evaluate the synergistic antimicrobial effects of silybin and curcumin on virulence and carbapenemase genes expression among multidrug-resistant (MDR) Klebsiella oxytoca. RESULTS: A total of 70 MDR K. oxytoca (carrying blaIMP and blaOXA-48-like genes) were included. The antibiotic susceptibility and biofilm production of isolates were determined. The silybin and curcumin at concentrations 10-500 mg/mL alone and in combination were exposed to bacterial isolates in Mueller Hinton broth medium for 24 h. The expression of blaIMP, blaOXA-48-like, mrkA, pilQ, matB and fimA genes was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). The mean minimum inhibitory concentration (MIC) of curcumin and silybin were 250 mg/mL and 500 mg/mL, respectively. The anti-virulent effect of 100 mg/mL of silybin and curcumin was shown by significant reduction in the expression of fimA (2.1-fold, P < 0.0001) and mrkA (2.1 fold, P < 0.0001) genes. Moreover, these compounds significantly decreased the expression of blaIMP1 (3.2-fold, P < 0.0001) gene. Notably, there was no significant effect on pilQ, matB and blaOXA-48-like genes. The results showed that silybin and curcumin can be candidate as natural way for control the MDR virulent strains of K. oxytoca.

Extended-spectrum Beta-lactamases Encoding Genes among Salmonella Enterica serovar Typhi Isolates in Patients with Typhoid Fever from four Academic Medical Centers Lagos, Nigeria.

BACKGROUND: There is scarce information about the occurrence of extended-spectrum ß-lactamases (ESBLs) in Salmonella enterica serovar Typhi (S. Typhi) from patients with typhoid fever. OBJECTIVE: To study the antimicrobial resistance and ESBL encoding genes among S. Typhi isolates in aforesaid patients from Lagos, Nigeria. METHODS: S. Typhi isolates were collected from blood samples of typhoid fever patients from 4 academic medical centers in Lagos, Nigeria. The identification of isolates and their antibiotic susceptibility testing were performed by standard bacteriological techniques and disc diffusion method, respectively. The production of ESBLs was investigated using combination disk test (CDT) and polymerase chain reaction (PCR). RESULTS: A total of 27 S. Typhi isolates was collected. All isolates were susceptible to imipenem and nitrofurantoin. Fifteen (55.6%) isolates were multidrug-resistant (MDR). The CDT test showed 11 (40.7%) ESBL producer isolates. However, the PCR revealed a higher occurrence rate for ESBL producers (66.7%, n = 18/27). The ESBL genes were as follows: blaCTX-M (37.0%, n = 10/27), blaSHV (18.5%, n = 5/27), and blaTEM (44.4%, n = 12/27). All ESBL positive S. Typhi isolates were MDR. CONCLUSIONS: This study showed the emergence of ESBL-harboring S. Typhi in patients with typhoid fever from Nigeria.

Extended-spectrum Beta-lactamases Encoding Genes among Salmonella Enterica serovar Typhi Isolates in Patients with Typhoid Fever from four Academic Medical Centers in Lagos, Nigeria

ABSTRACT Background: There is scarce information about the occurrence of extended-spectrum β-lactamases (ESBLs) in Salmonella enterica serovar Typhi (S. Typhi) from patients with typhoid fever. Objective: To study the antimicrobial resistance and ESBL encoding genes among S. Typhi isolates in aforesaid patients from Lagos, Nigeria. Methods: S. Typhi isolates were collected from blood samples of typhoid fever patients from 4 academic medical centers in Lagos, Nigeria. The identification of isolates and their antibiotic susceptibility testing were performed by standard bacteriological techniques and disc diffusion method, respectively. The production of ESBLs was investigated using combination disk test (CDT) and polymerase chain reaction (PCR). Results: A total of 27 S. Typhi isolates was collected. All isolates were susceptible to imipenem and nitrofurantoin. Fifteen (55.6%) isolates were multidrug-resistant (MDR). The CDT test showed 11 (40.7%) ESBL producer isolates. However, the PCR revealed a higher occurrence rate for ESBL producers (66.7%, n = 18/27). The ESBL genes were as follows: blaCTX-M (37.0%, n = 10/27), blaSHV (18.5%, n = 5/27), and blaTEM (44.4%, n = 12/27). All ESBL positive S. Typhi isolates were MDR. Conclusions: This study showed the emergence of ESBL-harboring S. Typhi in patients with typhoid fever from Nigeria.

Metallo-ß-lactamase and AmpC genes in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa isolates from abattoir and poultry origin in Nigeria.

BACKGROUND: Gram-negative bacteria (GNB) including Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae represent the most relevant reservoir of resistance genes such as metallo-ß-lactamase (MBL) and AmpC genes that give them the undue advantage to resist antimicrobial onslaught. This study aimed to investigate the occurrence of MBL (blaIMP-1, blaIMP-2, blaVIM-1, blaVIM-2) and AmpC (blaFOX, blaDHA, blaCMY, blaACC) resistance genes in aforementioned GNB collected from abattoir and poultry sources in Nigeria. RESULTS: In total, 370 isolates were collected from abattoir tables (n = 130), anal region of cows (n = 120), and the cloacae of poultry birds (n = 120). The test isolates showed high rate of resistance to cephalosporins and carbapenems. The MBLs were phenotypically detected in 22 E. coli, 22 P. aeruginosa, and 18 K. pneumoniae isolates using combined disc test (CDT). However, only 11 E. coli, 24 P. aeruginosa, and 18 Klebsiella pneumoniae isolates were phenotypically confirmed to be AmpC producers using cefoxitin-cloxacillin double disk synergy test (CC-DDST). MBL encoding genes (particularly the blaIMP-1 genes and blaIMP-2 genes) were detected by polymerase chain reaction (PCR) in 12 (54.6%) E. coli, 15 (83.3%) K. pneumoniae, and 16 (72.7%) P. aeruginosa isolates. AmpC genes (particularly the blaCMY genes and blaFOX genes) were found in a total of 5 (29.4%) E. coli isolates, 5 (27.8%) isolates of K. pneumoniae, and 10 (41.7%) isolates of P. aeruginosa. CONCLUSIONS: Our study showed the circulation of MBL and AmpC genes in GNB from abattoir and poultry origin in Nigeria. Adoption of regular control policies is necessary to reduce the spread of these species as soon as possible, especially in poultry and slaughterhouses.

Study of the D-dimer, C-reactive protein, and autoantibodies markers among HBV infected patients in Babylon province, Iraq.

BACKGROUND: Hepatitis B can be defined as one of the dangerous diseases caused by the hepatitis B virus (HBV), which infects the liver and causes liver failure, cirrhosis, and death. AIMS: This study aimed to evaluate the D-dimer, C-reactive protein (CRP), and autoantibodies markers among HBV-infected patients in Babylon province, Iraq, compared to a healthy control group. METHODS: In this cross-sectional study, all patients referred to GIT and liver centers in Merjan Medical City, Babylon, Iraq from January 2016 to January 2018 were screened for HBV infection by quantitative real-time polymerase chain reaction (qPCR). Antinuclear antibody (ANA), dsDNA, D-dimer, and CRP markers were examined using fluorescence technique in randomly selected patients and some healthy individuals as control group. Statistical analysis was performed using SPSS program. RESULTS: In this study, 424 HBV patients from different areas of Babylon province, Iraq were enrolled. A total of 40 patients and 15 healthy participants were selected for evaluation of D-dimer, CRP, ANA, and dsDNA levels. The results of the distribution of HBV-infected patient by gender per area revealed that males accounted for a higher percentage than females in all parts of Babylon provinces. Also, a highly significant increase in serum levels was observed in the patients compared to the control subjects for all the studied parameters D-dimer, ANA, dsDNA, and CRP. Overall, 5.5% of HBV patients (3/40) had a positive ANA titer. None of the HBV patients had a positive dsDNA titer. CONCLUSION: This study showed that, HBV-infected patients had elevated levels of D-dimer and CRP compared to the control group. Also, the seropositivity titers of ANA and anti-dsDNA autoantibodies were low in Iraqi HBV patients.

Evaluation of the phytoconstituents of Auricularia auricula-judae mushroom and antimicrobial activity of its protein extract.

INTRODUCTION: The growing resistance to antibiotics and the complexity of defeating multi-drug resistant bacteria have led to an increase in the search for novel and effective antimicrobials from various plants. This study aimed to determine the bioactive contents of Auricularia auricular-judae mushroom and evaluate the antimicrobial potential of its protein extract against some selected human bacterial and fungal pathogens which could serve as a lead to the discovery of new antimicrobial agents. METHODS: The constituents of the A. auricular-judae were evaluated by standard phytochemical analysis methods. The agar well diffusion, micro-broth dilution, and time-kill kinetic assays were used to determine the antimicrobial activity of the extracts against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, yeast (Candida albicans), and dermatophytic pathogens. RESULTS: The preliminary phytochemical analysis of the extracts revealed the presence of carbohydrate (43.15 %; 38.30 %) proteins (23.75 %; 23.75 %), flavonoids (1.20 %; 0.80 %), alkaloids (0.60 %; 1.00 %), saponin (6.00 %; 2.40 %), tannin (1.65 %; 1.57 %), cyanide (0.24 %; 0.40 %), ash (12.40 %; 10.40 %), moisture (6.00 %;6.00 %), lipids(6.00 %;6.00 %), and fiber (8.70 %; 6.45 %) for the Tris buffer and warm aqueous extracts, respectively. The Tris and warm aqueous protein extracts showed antimicrobial effects toward all the human bacterial pathogens and two fungal isolates. CONCLUSIONS: This study revealed the potential ability of A. auricula-judae for use as a herbal antimicrobial in the treatment of human bacterial and fungal pathogens.