Phenotypic and Molecular Characterization of CTX-M Type B-Lactamases in Gram Negative Bacterial Strains Isolated from Hospitals, Lahore, Pakistan.

One of the principal mechanisms that contribute resistance to antibiotics is the production of extended spectrum beta lactamase (ESBL) in Gram negative bacteria. In the present study, molecular methods were used to evaluate the prevalence of the extended-spectrum beta-lactamase (ESBL)-encoding CTX-M gene among Gram negative bacterial strains. In total, 148 clinical samples were collected from different tertiary care hospitals of Lahore, Pakistan. Disc synergy diffusion method was used to detect the presence of ESBL production. Moreover, antibiotic resistance patterns and molecular detection of bla CTX-M ESBLs, were also studied. The pathogens isolated from the 148 samples included Escherichia coli (43%) followed by Klebsiella sp. (28%), Proteus sp. (18%) and Pseudomonas sp. (11%). In all 148 strains, 95 (64%) were ESBL producers while 53 (36%) were non ESBL producers. The strains which were phenotypically ESBL producers, bla CTX-M were found in 46% E. coli strains, while 50% Klebsiella sp. were harboring the gene. A high resistance rate was observed against cephalosporins (cefopodoxime 67%, cefoperazone 73%, cephalexin 63% sparaxin 61%). Lower resistance was observed against meropenem among all isolated bacterial strains. Genotypic detection of bla CTX-M genes by PCR revealed 46% of E. coli and 50% of Klebsiella strains harbored bla CTX-M gene. The present study showed that ESBLs producers were resistant to commonly used antibiotics. Similarly, bla CTX-M ESBL production is more prevalent in our clinical isolates.

Prevalence of Complications Associated with Diabetes among Pakistani Patients: A Questionnaire-based Survey.

BACKGROUND: The rate of mortality is increasing in diabetic patients due to diabetes-associated complications. The common complications include neuropathy, nephropathy, retinopathy, foot ulcer, slow wound healing, kidney dysfunction, amputation, dysfunction of organs, frequent infections, sepsis, skin diseases, hearing impairment, cardiovascular disorders, etc. These complications can be diagnosed following some common symptoms such as increased thirst, frequent urination, extreme hunger, unexplained weight loss, fatigue, irritability, blurred vision, slow-healing sores, etc. This survey was designed to study the prevalence of various complications in a group of diabetic patients so that effective treatment options could be developed against the most prevalent complications. METHODS: This cross-sectional study was conducted in 2019 in a tertiary care hospital of Karachi after the approval of the ethical committee of the hospital as well as in the University of Karachi. To perform this study, a questionnaire was designed comprised of different questions related to diabetic complications. The consent form was attached to each questionnaire in which the patient agreed to participate voluntarily in this survey. The diabetic patients who visited the General Physician OPD were the subjects of this survey. All designed questions included in the questionnaire were asked either directly from the patients or their attendants. RESULTS: A total of 160 diabetic subjects were part of the study range between the ages of 11 to 90 years. Out of 160 patients, 52 were males, and 108 were females. Among all subjects, 124 (78 %) patients were type 2 while 57 (36 %) were type 1 diabetic patients. 117 (73 %) showed confusion of mind, 104 (65 %) complained of blood pressure, 105 (66 %) had hypertension, 106 (66 %) had eye damage (retinopathy), 96 (60 %) were facing trouble focusing vision, and 70 (44 %) were experiencing seizures, 63 (39 %) patients had laser treatment, 68 (43 %) showed wounds on foot and slow wounds healing, 49 (31 %) were having kidney damage (nephropathy), 79 (49 %) had pain in legs or knee, 35 (22 %) and 26 (16 %) complained of heart problems and liver damage respectively. Some patients were found to deal with more hunger, i.e., 99 (62 %) patients, 118 (74 %) were experiencing frequent urine desire, 138 (86 %) showed fatigue, 123 (77 %) complained of thirst, 35 (22 %) had nausea, 30 (19 %) had a frequent cold, 36 (23 %) had skin problems, 17 (11 %) patients showed frequent vomiting, 19 (12 %), 13 (8 %) and 16 (10 %) were experiencing acne formation, stroke and nerve damage (neuropathy) respectively. CONCLUSION: All age groups showed diabetes-associated complications and different abnormal body conditions. However, the age groups ranging from thirty to eighty years showed more complications. The most prevalent complications reported were retinopathy, nephropathy, diabetic wounds on the foot, slow wound healing, seizures, hypertension, neuropathy, skin infections, cardiovascular disorders, liver damage, and stroke in both types of diabetic patients. Our survey may aid in pointing out the most prevalent diabetic complications prevailing in our population so that effective treatment options could be developed to reduce these life-threatening complications.

Multi-activity tetracoordinated pallado-oxadiazole thiones as anti-inflammatory, anti-Alzheimer, and anti-microbial agents: Structure, stability and bioactivity comparison with pallado-hydrazides.

Herein, we report a comparative study based on structure, thermal and solution stability, and biopotency against lipoxygenase (LOX), butyrylcholinesterase (BChE) and microbes for Pd(II) compounds of N,O,S bearing 5-(C5H4XR)-1,3,4-oxadiazole-2-thiones (L') of type [PdL'Cl2] (P'n) and N,O bearing respective hydrazides (L) of type trans-[PdL2Cl2] (Pn) {X = C, R = 4-I, 2-Br, 4-NO2, 3-NO2, 2-Cl, 3-Cl (n = 1-6, serially); X = N (n = 7)}. Spectral techniques (IR, EI-MS, NMR) and physicochemical evaluations successfully characterized the new compounds. The L' behaved as bidentate S-N donors bonded through exocyclic sulfur and N-3' nitrogen, while L acted as amino N donors. UV-vis (solution speciation) and thermal degradation profiles consistently confirmed the greater stability for P'n than Pn compounds. These compounds manifested varying degree in vitro potential to inhibit LOX, BChE and several bacteria and fungi, affected mainly by Pd(II) presence, M-L binding mode, nature and position of R, or halo groups electronegativity. Molecular docking with human 5-LOX and BChE further validated the respective experimental inhibition findings and explored several putative mechanistic interactions (H-bonding, π-stacking, π-alkyl, π-S, etc.) at the enzyme active sites. Pn generally offered superior antimicrobial and anti-LOX (anti-inflammatory) potential than respective P'n compounds, with P3/P'5, P(2,3,7)/P'3, and P6 being comparable, better and equivalent to ampicillin, nystatin and baicalein, the reference antibacterial, antifungal and anti-LOX drugs, respectively. Contrarily, the anti-BChE activity of P'n was found better than Pn compounds, showing P'2/P1 as the most promising anti-Alzheimer drug candidates. This study bares important structural and mechanistic aspects in optimizing antimicrobial, anti-inflammatory and anti-Alzheimer activities, highlighting some potential future pallado-drug candidates.

Impact of Interplay between Obese Gut Microbiota and Diet in Developing Obesity in Synthetic Community Mice.

This study aims at investigating the effects of cultured gut microbiota (GM) of obese human coupled high fat diet (HFD) or chow diet (CD) in development of obesity in mice. 20 stool samples were collected from obese patients and isolated bacteria were identified morphologically and biochemically. Identified isolates were mixed in equal proportions to synthesize obese GM. In vivo study was performed using obese GM combined with HFD/CD using mouse model for three months. Albino mice were treated with ampicillin from one week prior to birth until weaning of the pups at seven weeks of age and then inoculated with obese GM. Sixteen mice were divided into four groups: i.e. group 1 (G1) mice fed with CD, group 2 (G2) mice with HFD, group 3 (G3) mice with GM + HFD and group 4 (G4) mice with GM + CD. Mice from groups 3-4 were considered synthetic community (SC) mice due to transfer of synthesize human GM. 16S rRNA sequencing identified five abundant bacteria as Pseudomonas aeruginosa, Staphylococcus sp., Escherichia coli, Morganella morganii, and Klebsiella oxytoca (accession numbers: MZ150742-MZ150746). In vivo study indicated that GM combination with either HFD/CD caused significantly increased body weight in SC mice (BMI; Kg/m2) compared to HFD or CD fed mice groups. One way ANOVA revealed highly significant increase (p ≤ 0.001) in levels of total cholesterol (TC), triglycerides and low density lipoprotein (LDL) in GM coupled diet groups (G3-G4; SC mice) compared to significant increase in HFD group (G2) versus CD group (G1). Our study is first of its kind to report significant effects of using purified strains as obese GM plus diet (HFD/CD) in inducing obesity in SC mice and elevated serum liver parameters as metabolic indicators, hence providing strong evidence about significance of modified GM combination with HFD in developing obesity in SC mice.

Persistence and safety assessment of novel probiotic strain Lactobacillus plantarum 1 strain Lp86 and Lp36 in Salmonella typhi infected mice.

The species of Lactic acid bacteria are known to confer beneficial effects on the host by inhabiting in their gastrointestinal tract (GIT). They succeed in surviving the harsh conditions of the GIT by exhibiting strong tolerance against gastric acids, digestive enzymes and bile simultaneously antagonizing the pathogens by production of antimicrobials. This study has been conducted to elaborate these probiotic characteristics in vivo for which mice were intragastrically given a probiotic approved dose of 1011cfu/ml for 4 days to assess the persistence of two probiotic candidates Lactobacillus plantarum Lp36 and Lactobacillus plantarum Lp86. The fecal count of the test probiotic candidates were seen to persist well in the GIT for 15 days with a count ranging between 104-108cfu/ mg of feces (p>0.01). The safety assessment of L. plantarum Lp36 in healthy and S. typhi in infected mice showed an increase in cell count from (day zero of inoculation) 106cfu/100mg of feces to108cfu/100mg (p>0.01) which was maintained till day six, suggesting the persistence in the GIT. The sections of the mice intestinal lining under scanning electron microscope revealed the adherence of Lp36 and Lp86 to the intestinal epithelia. The mice did not show any adverse effect on its health. These findings make our strains promising probiotic candidates to be used to promote health benefits after further assessments.

Optimization of physicochemical parameters for maximum amylase production by indigenously isolated Bacillus cereus AS2 strain.

Amylases are enzymes that catalyze the hydrolysis of starch into highly valuable products of economic significance. Amylases are used extensively in various industrial sectors. Microbial sources particularly Bacillus species are well known for the cost effective commercial production of amylase enzyme. Present study focuses on the enhancement of amylase enzyme production from an indigenously isolated Bacillus cereus AS2 strain via one variable at a time (OVAT) optimization of different physical and chemical factors. Purposely, eight parameters possibly affecting the amylase production including temperature, pH, incubation time, inoculum size, substrate concentration, metal ions, carbon and nitrogen sources were investigated. According to the results, amylase production was significantly boosted at maximum when the Bacillus cereus AS2 was grown at 45°C on pH 7.0 for 72 hours in the medium supplemented with 4% starch and 0.5% glycine. Among the different metal ions tested, CaCl2 (0.05%) was found significant to accelerate extracellular amylase production.

Hypoxic Preconditioning Improves the Therapeutic Potential of Aging Bone Marrow Mesenchymal Stem Cells in Streptozotocin-Induced Type-1 Diabetic Mice.

Insulin replacement is the current therapeutic option for type-1 diabetes. However, exogenous insulin cannot precisely represent the normal pattern of insulin secretion. Another therapeutic strategy is transplantation of pancreatic islets, but this is limited by immune rejection, intrinsic complications, and lack of donor availability. Stem cell therapy that results in the regeneration of insulin-producing cells represents an attractive choice. However, with advancing age, stem cells also undergo senescence, which leads to changes in the function of various cellular processes that result in a decrease in the regeneration potential of these aging stem cells. In this study, the effect of young and aging mesenchymal stem cells (MSCs) on the regeneration of pancreatic beta cells in streptozotocin (STZ)-induced type-1 diabetic mice was observed after hypoxic preconditioning. Hypoxia was chemically induced by 2, 4-dinitrophenol (DNP). Plasma insulin and glucose levels were measured at various time intervals, and pancreatic sections were analyzed histochemically. The effect of DNP was also analyzed on apoptosis of MSCs by flow cytometry and on gene expression of certain growth factors by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). We observed that hypoxic preconditioning caused changes in the gene expression levels of growth factors in both young and aging MSCs. Young MSCs showed significant regeneration potential compared with the aging cells in vivo. However, hypoxic preconditioning was able to improve the regeneration potential of aging MSCs. It is concluded from the present study that the regeneration potential of aging MSCs into pancreatic ß-cells can be enhanced by hypoxic preconditioning, which causes changes in the gene expression of certain growth factors.