Molecular docking and in vitro antibacterial activity of chiral phthalimide on ESBL producing gram negative bacteria.

Antibiotic resistance is tricky enemy that challenges our healthcare system. It is a stealthy, adaptive and ever evolving opponent, which can take years to develop but can spread like wildfire. In this study, derivatives of chiral phthalimides were developed with this aim to control the growth of resistant strains of Klebsiella pneumonia, Escherichia coli and Pseudomonas aeruginosa by targeting their resistance causing proteins and explore their binding interaction focal points through computational docking. Total 8 novel chiral phthalimides were synthesized and its antibiogram analysis was done on Muller-Hinton Agar by disc diffusion method. Cytotoxicity studies were made to check efficacy of tested compounds on human RBCs and monitor release of hemoglobin absorbance at 540nm. By using in silico molecular approach, crystal structure of target protein was retrieved from Protein Data Bank and docked through Autodock vina and PyRx. The obtained results revealed that seven out of eight compounds have active inhibitory effects against virulent strains. Minimum Inhibitory Concentration (MIC) was measured for most potent compounds i.e., 2-(1,3-dioxoisoindolin-2-yl)-3-(4-hydroxyphenyl) propanoic acid (compound 7) and 3-(1,3-dioxoisoindolin-2-yl) propanoic acid (compound 8). Docking studies displayed a report of highest affinity binding points i.e., amino acids LYS315, ALA318, TYR150, THR262, HIS314 and ARG148 for compound 7 while ALA 318, LYS 315, ARG14 and ILE291 for compound 8.

Investigations on Acinetophage, QAB 3.4, Targeting Extensively Drug-Resistant Acinetobacter baumannii Isolates.

PURPOSE: Drug resistance against antimicrobials is on the rise at alarmingly high rates. Acinetobacter baumannii is one of the six ESKAPE pathogens which are a significant "one health" issue. Clinical isolates of A. baumannii exhibit MDR phenotype mostly and infrequently the XDR and PDR phenotype. As a result, these infections have one of the highest mortality rates in hospitals. Alternative therapies are urgently needed. METHODS: Various phages were enriched against XDR clinical strain of A. baumannii. A potent phage, QAB 3.4, was further tested against 100 clinical strains. Because of its broad lytic activity, it was further tested for stability, resistance development and as an infection control agent. RESULTS: Phage QAB 3.4 showed broad lytic activity against 100 MDR and XDR clinical isolates representing a wide diversity of infection sites. Assays conducted to document the phage's stability, and ability of clinical isolates to develop resistance against it, showed promising outcomes for its potential use in clinical applications. Phage QAB 3.4 was able to eradicate A. baumannii from pre-inoculated solid surfaces. It provides a proof of concept that phages can be used as environmentally friendly infection control agents. CONCLUSION: We propose the phage QAB 3.4 is a promising candidate for further pre-clinical and clinical studies to test its biosafety and efficacy.

Correlation between antibiotic resistance and phylogenetic types among multidrug-resistant Escherichia coli isolated from urinary tract infections.

OBJECTIVES: Emergence of multidrug resistance has reduced the choice of antimicrobial regimens for UTIs. To understand the association of phenotype and genotype among uropathogens. MATERIALS AND METHODS: Six hundred and twenty-eight (628) urine samples were collected and analyzed. Antibiotic sensitivity pattern was determined by the Kirby-Bauer Disc Diffusion Method and minimum inhibitory concentration (MIC) was tested by the E test. Fluoroquinolone resistant mutations in QRDR of gyrA and ParC, phylogenetic groups, and PAIusp subtype were detected by PCR. RESULTS: Most prevalent uropathogens were Escherichia coli (53.2%) followed by Klebsiella pneumoniae (21%). Multidrug- resistance was observed in > 50% cases for third-generation cephalosporins and ciprofloxacin and lowest in meropenem. E. coli (66.2%) and K. pneumonia (64.4%) were extended-spectrum ß-lactamases (ESBLs) producers. MIC to trimethoprim-sulfamethoxazole was highest in E. coli (>1024 µg/ml). In 80 (24%) of the 334 E. coli isolates analyzed in detail, 54 fluoroquinolones (FQ) resistant isolates carried mutations (S83L, D87N, S80I, E84V) in QRDR of gyrA and ParC. Out of 54 FQ-resistant isolates, 43 (79.6%) isolates belonged to the phylogenetic group B2, and 11(20.4%) belonged to group D. Isolates belonged to group B2, 38 (88.4%) of the 43 isolates carried PAIusp subtype IIa and high frequency of mutation E84V in ParC was detected in 37 (97.4%). Other mutations, such as S80I, S83L in gyrA and D87N in ParC were found in all resistant isolates. CONCLUSION: Correlations between phenotype and genotype provided a basis to understand the resistance development in uropathogens, and PAIusp subtyping indicated that E. coli belonged to the B2 group.

Drug resistance profile of Mycobacterium tuberculosis and predictors associated with the development of drug resistance.

OBJECTIVES: Drug-resistant tuberculosis (DR-TB) is a major challenge to national TB control programmes in developing countries. In the Pakistan province of Punjab, the extent and development of DR-TB is not well known. The current study was therefore conducted to assess the incidence and predictors of DR-TB in Punjab Province. METHODS: Drug susceptibility testing was performed for 863 confirmed culture-positive Mycobacterium tuberculosis isolates using the proportion method. Patients were enrolled in the Programmatic Management of Drug-Resistant TB Unit of Gulab Devi Chest Hospital (Lahore, Pakistan) from August 2011 to September 2013. Data analysis was performed using IBS SPSS Statistics v.20. Multivariate logistic regression analysis was performed to assess risk factors for DR-TB. RESULTS: The rate of resistance to at least one drug (i.e. DR-TB) was 35.0% (302/863) and the rate of multidrug-resistant TB (MDR-TB) was 30.0% (259/863). Among DR-TB cases, the number of females was relatively higher (167/302; 55.3%) compared with males. The majority of DR-TB patients resided in a rural area (229/302; 75.8%). Significant predictors of DR-TB were age 18-45 years, previous TB treatment, rural residence, being a housewife, being married, duration of sickness >1year and unemployment. CONCLUSION: The problem of DR-TB in Pakistan is significant. The strongest risk factors were young age and previous anti-TB treatment. Being married, being a housewife, rural residence and unemployment were also risk factors, culminating in an urgent need for effective control, early diagnosis and treatment policies for DR-TB.

Determination of antimicrobial effect of protamine by transmission electron microscopy and SDS PAGE on Pseudomonas aeruginosa isolates from diabetic foot infection.

OBJECTIVES: Diabetic foot infection is one of the major complications of diabetes leading to lower limb amputations. Isolation and identification of bacteria causing diabetic foot infection, determination of antibiotic resistance, antimicrobial potential of protamine by electron microscopy and SDS-PAGE analysis, arethe aims of this study. MATERIALS AND METHODS: 285 pus samples from diabetic foot infection patients were collected from different hospitals of Karachi and Capital Health Hospital, Halifax, Canada. Clinical history of each patient was recorded. Bacterial isolates were cultured on appropriate media; identification was done by morphology, cultural and biochemical tests. Effect of protamine against multi drug resistant strains of Pseudomona aeruginosa was checked by minimum inhibitory concentration in 96 well micro-titer plates. The isolates were grown in bactericidal concentration of protamine on plates to isolate mutants. Effect of protamine on protein expression was checked by SDS- PAGE and ultra-structural morphological changes by transmission electron microscopy. RESULTS: Results indicated prevalence of foot infection as 92% in diabetic patients. Major bacterial isolates were Staphylococcus aureus 65 (23%), P. aeruginosa 80 (28.1%), Klebsiella spp. 37 (13%), Proteus mirabilis 79 (27.7%), and Escherichia coli 24 (12%). These isolates were highly resistant to different antibiotics. MIC value of protamine was 500 µg/ml against P. aeruginosa. SDS-PAGE analysis revealed that protamine can suppress expression of various virulence proteins and electron micrographs indicated condensation of cytoplasm and accumulation of protamine in cytoplasm without damaging the cell membrane. CONCLUSION: P. aeruginosa and S. aureus were the major isolates expressing multi-drug resistance and protamine sulfate represented good antimicrobial potential.

Can phylogenetic type predict resistance development?

OBJECTIVES: To determine whether phylogenetic type is associated with the development of multidrug resistance to antibiotics. METHODS: Urinary tract infection (UTI) isolates from three hospitals in Pakistan were collected over a period of 10 months, and analysed in terms of causative bacterial species and drug susceptibility. RESULTS: Multidrug resistance was widespread and resistance frequencies were >50% for several of the most commonly used antibiotics, including ciprofloxacin and third-generation cephalosporins for Escherichia coli isolates. The great majority of E. coli isolates remained susceptible to meropenem and fosfomycin. Sixty E. coli isolates were analysed in detail to determine correlations between resistance phenotypes and genotypes, mutation rates and phylogenetic group. Most isolates had elevated mutation rates, suggesting this was being selected. The majority of ciprofloxacin-resistant isolates carried a specific set of mutations in the quinolone resistance-determining region of gyrA and parC (S83L, D87N, S80I and E84V). In addition, 67% of the ciprofloxacin-resistant E. coli isolates carried one or more horizontally transmissible determinants of resistance to ciprofloxacin, including aac(6')-Ib-cr, qepA, qnrA and qnrB. There was a significant correlation between resistance to third-generation cephalosporins, being an extended-spectrum ß-lactamase producer, being resistant to ciprofloxacin and belonging to phylogenetic group B2. CONCLUSIONS: The data suggest that features of the bacterial genotype might facilitate the development of multidrug resistance in particular lineages. Better understanding of the mechanistic basis for correlations between drug resistance and genotype could potentially be exploited to develop molecular tools for the prediction of resistance development.