Fluoroquinolone resistance and mutational profile of gyrA in pulmonary MDR tuberculosis patients.

BACKGROUND: Fluoroquinolones (FQs) are potential drugs that inhibit DNA synthesis and are used in the treatment of multidrug-resistant tuberculosis (TB) and short-term anti-TB regimens. In recent years, a high proportion of FQ resistance has been observed in Mycobacterium tuberculosis isolates. The development of FQ resistance in multidrug-resistant TB negatively impacts patient treatment outcome and is a serious threat to control of TB. METHODS: The study included a total of 562 samples from patients with pulmonary TB that had been on anti-tuberculosis therapy. MTBDRsl assays were performed for the molecular detection of mutations. Sequence analysis was performed for the characterization and mutational profiling of FQ-resistant isolates. RESULTS: FQ resistance was observed in 104 samples (18.5%), most of which were previously treated and treatment failure cases. A total of 102 isolates had mutations in DNA gyrase subunit A (gyrA), while mutations in gyrB were observed in only two isolates. Mutational analysis revealed that the mutations mostly alter codons 94 (replacing aspartic acid with glycine, D94G) and 90 (replacing alanine with valine, A90V). In MDR and treatment failure cases, resistance to FQs was most commonly associated with the D94G mutation. In contract, a high proportion of A90V mutations were observed in isolates that were newly diagnosed. CONCLUSION: The findings suggest that genotypic assays for FQ resistance should be carried out at the time of initial diagnosis, before starting treatment, in order to rule out mutations that impact the potential use of FQs in treatment and to control drug resistance.

Sero-epidemiology and Risk Factor Analysis of Measles Among Children in Pakistan.

Comparative cross sectional study was conducted on blood samples (n = 231) collected from children of 1 to 10 years of age in Punjab Pakistan through convenient sampling method. Indirect haemagglutination assay (IHA) was standardized and used for serodiagnosis and evaluation of humoral immunity against measles. Associated risk factors including age, gender, locale, and vaccination status were analyzed. Geometric mean titre (GMT) of vaccinated individuals was significantly higher (p < 0.001) than that of non-vaccinated individuals showing that IHA titre of vaccinated individuals was a measure of humoral immune response; whereas, in case of non-vaccinated individuals an indicative of exposure to the measles infection.

Co-application of copper nanoparticles and metal tolerant Bacillus sp. for improving growth of spinach plants in chromium contaminated soil.

Chromium (Cr) is classified as a toxic metal as it exerts harmful effects on plants and human life. Bacterial-assisted nano-phytoremediation is an emerging and environment friendly technique that can be used for the detoxification of such pollutants. In current study, pot experiment was conducted in which spinach plants were grown in soil containing chromium (0, 5, 10, 20 mgkg-1) and treated with selected strain of Bacillus sp. and Cu-O nanoparticle (CuONPs). Data related to plant's growth, physiological parameters, and biochemical tests was collected and analyzed using an appropriate statistical test. It was observed that under chromium stress, all plant's growth parameters were significantly enhanced in response to co-application of CuONPs and Bacillus sp. Similarly, higher levels of catalase, superoxide dismutase, malondialdehyde, and hydrogen peroxide were also observed. However, contents of anthocyanin, carotenoid, total chlorophyll, chlorophyll a & b, were lowered under chromium stress, which were raised in response to the combined application of CuONPs and Bacillus sp. Moreover, this co-application has significant positive effect on total soluble protein, free amino acid, and total phenolics. From this study, it was evident that combined application of Bacillus sp. and CuONP alleviated metal-induced toxicity in spinach plants. The findings from current study may provide new insights for agronomic research for the utilization of bacterial-assisted nano-phytoremediation of contaminated sites.

Biofabrication of ZnO nanoparticles using Acacia arabica leaf extract and their antibiofilm and antioxidant potential against foodborne pathogens.

Emergence of multidrug resistant pathogens is increasing globally at an alarming rate with a need to discover novel and effective methods to cope infections due to these pathogens. Green nanoparticles have gained attention to be used as efficient therapeutic agents because of their safety and reliability. In the present study, we prepared zinc oxide nanoparticles (ZnO NPs) from aqueous leaf extract of Acacia arabica. The nanoparticles produced were characterized through UV-Visible spectroscopy, scanning electron microscopy, and X-ray diffraction. In vitro antibacterial susceptibility testing against foodborne pathogens was done by agar well diffusion, growth kinetics and broth microdilution assays. Effect of ZnO NPs on biofilm formation (both qualitatively and quantitatively) and exopolysaccharide (EPS) production was also determined. Antioxidant potential of green synthesized nanoparticles was detected by DPPH radical scavenging assay. The cytotoxicity studies of nanoparticles were also performed against HeLa cell lines. The results revealed that diameter of zones of inhibition against foodborne pathogens was found to be 16-30 nm, whereas the values of MIC and MBC ranged between 31.25-62.5 µg/ml. Growth kinetics revealed nanoparticles bactericidal potential after 3 hours incubation at 2 × MIC for E. coli while for S. aureus and S. enterica reached after 2 hours of incubation at 2 × MIC, 4 × MIC, and 8 × MIC. 32.5-71.0% inhibition was observed for biofilm formation. Almost 50.6-65.1% (wet weight) and 44.6-57.8% (dry weight) of EPS production was decreased after treatment with sub-inhibitory concentrations of nanoparticles. Radical scavenging potential of nanoparticles increased in a dose dependent manner and value ranged from 19.25 to 73.15%. Whereas cytotoxicity studies revealed non-toxic nature of nanoparticles at the concentrations tested. The present study suggests that green synthesized ZnO NPs can substitute chemical drugs against antibiotic resistant foodborne pathogens.

Development, Biological Characterization, and Immunological Evaluation of Virosome Vaccine against Newcastle Disease in Pakistan.

Newcastle disease (ND) is a highly fatal, infectious, viral disease, and despite immunization with live and inactivated vaccines, the disease is still endemic, causing heavy morbidity and mortality leading to huge economic losses to the poultry industry in Pakistan. Therefore, the present study was aimed for the first time in the country at using novel virosomal technology to develop the ND vaccine using an indigenous highly virulent strain of the virus. ND virosome was prepared using Triton X-100, and SM2 Bio-Beads were used to remove the detergent and reconstitute the viral membrane into virosome. Confirmation was done by transmission electron microscopy and protein analysis by SDS-PAGE. In vitro cell adhesion property was observed by incorporating green fluorescent protein (GFP), producing plasmid into virosome and in vitro cell culture assay. Sterility, safety, and stability of the vaccine were tested before in vivo evaluation of immunogenicity and challenge protection study in commercial broiler. The virosome vaccine was administered (30 µg/bird) at days 7 and 14 through the intranasal route in comparison with commercially available live and inactivated ND vaccines. Results revealed significantly high (p < 0.05) and clinically protective hemagglutination inhibition (HI) antibody titers at 7, 14, 21, and 28 days postimmunization with the virosome vaccine in comparison to the negative control. The GMTs were comparable to live and inactivated vaccines with nonsignificant (p > 0.05) differences throughout the experiment. Antibody levels increased in all vaccinated groups gradually from the 7th day and were maximum at 28th-day postvaccination. In the virosome-administered group, GMT was 83.18 and 77.62 at 21st and 28th-days postvaccination, respectively. Challenge revealed 100%, 90%, and 80% protection in virosome, live, and inactivated vaccinated groups, respectively. Under given experimental conditions, we can conclude that ND virosome vaccine prepared from the indigenous virus was found to be safe and immunogenic.

The prospects of antimicrobial coated medical implants.

The implants are increasingly being a part of modern medicine in various surgical procedures for functional or cosmetic purposes. The progressive use of implants is associated with increased infectious complications and prevention of such infections always remains precedence in the clinical settings. The preventive approaches include the systemic administration of antimicrobial agents before and after the surgical procedures as well as the local application of antibiotics. The relevant literature and existing clinical practices have highlighted the role of antimicrobial coating approaches in the prevention of implants associated infections, although the applications of these strategies are not yet standardized, and the clinical efficacy is not much clear. The adequate data from the randomized control trials is challenging because of the unavailability of a large sample size although it is compulsory in this context to assess the clinical efficacy of preemptive practices. This review compares the efficacy of preventive approaches and the prospects of antimicrobial-coated implants in preventing implant-related infections.

Extensively Drug-resistant Acinetobacter baumannii Belonging to International Clone II from A Pet Cat with Urinary Tract Infection; The First Report from Pakistan.

The carbapenem-resistant Acinetobacter baumannii (CRAB) has got global attention as a notorious nosocomial pathogen. This study describes a case of urinary tract infection in a 2-years old pet female cat infected with A. baumannii. The susceptibility profiling, screening for the resistance determinants, and the multilocus sequence typing was performed. The A. baumannii isolate was found to harbor the blaOXA23-like gene and corresponded to International clone II that has been widely reported to be involved in human infections. The study proposes that the pets may contribute towards the spread of clinically relevant antimicrobial-resistant pathogens.The carbapenem-resistant Acinetobacter baumannii (CRAB) has got global attention as a notorious nosocomial pathogen. This study describes a case of urinary tract infection in a 2-years old pet female cat infected with A. baumannii. The susceptibility profiling, screening for the resistance determinants, and the multilocus sequence typing was performed. The A. baumannii isolate was found to harbor the blaOXA23-like gene and corresponded to International clone II that has been widely reported to be involved in human infections. The study proposes that the pets may contribute towards the spread of clinically relevant antimicrobial-resistant pathogens.

Neurological and cognitive significance of probiotics: a holy grail deciding individual personality.

The role of the human microbiome in the brain and behavioral development is an area of increasing attention. Recent investigations have found that diverse mechanisms and signals including the immune, endocrine and neural associations are responsible for the communication between gut microbiota and the brain. The studies have suggested that alteration of intestinal microbiota using probiotic formulations may offer a significant role in the maturation and organization of the brain and can shape the brain and behavior as well as mood and cognition in human subjects. The understanding of the possible impact of gut microflora on neurological function is a promising phenomenon that can surely transform the neurosciences and may decipher the novel etiologies for neurodegenerative and psychiatric disorders.

Extended-spectrum beta-lactamases producing extensively drug-resistant Salmonella Typhi in Punjab, Pakistan.

INTRODUCTION: The multidrug-resistant (MDR) Salmonella enterica serovar Typhi isolates have been increasingly reported from the Asian and African countries. The emergence of isolates with decreased susceptibility to fluoroquinolones and cephalosporins has worsened the situation. Recently, an outbreak from Sindh, Pakistan was reported caused by extensively drug-resistant (XDR) S. Typhi strains. METHODOLOGY: In the present study, a total of 82 cases of typhoid have been investigated during 2018 from the febrile children referred to a tertiary care hospital in the population-wise largest province (Punjab) of Pakistan. S. Typhi was identified by standard microbiological techniques and isolates were characterized for antimicrobial resistance profiling and minimum inhibitory concentrations were determined. The presence of various ESBL genes in S. Typhi was confirmed by the PCR. RESULTS: Out of the 82 isolates tested, 35 (43%) were found to be XDR; resistant to the first-line drugs. The resistance to third-generation cephalosporins was mainly mediated by extended-spectrum beta-lactamases i.e. blaTEM and blaCTX-M genes. CONCLUSIONS: The higher prevalence of ESBL producing Salmonella typhi clinical strains raises the concern about transmission prevention and infection management in the community as well as clinical settings. Moreover, the study highlights the problem concerning the declining antibiotic arsenal for the therapeutic management of typhoid fever and the emergence and spread of XDR strains in Pakistan.

Emergence of Resistance to Fluoroquinolones and Third-Generation Cephalosporins in Salmonella Typhi in Lahore, Pakistan.

Extensively drug-resistant (XDR) Salmonella Typhi has been reported in Sindh province of Pakistan since 2016. The potential for further spread is of serious concern as remaining treatment options are severely limited. We report the phenotypic and genotypic characterization of 27 XDR S. Typhi isolated from patients attending Jinnah Hospital, Lahore, Pakistan. Isolates were identified by biochemical profiling; antimicrobial susceptibility was determined by a modified Kirby-Bauer method. These findings were confirmed using Illumina whole genome nucleotide sequence data. All sequences were compared to the outbreak strain from Southern Pakistan and typed using the S. Typhi genotyping scheme. All isolates were confirmed by a sequence analysis to harbor an IncY plasmid and the CTX-M-15 ceftriaxone resistance determinant. All isolates were of the same genotypic background as the outbreak strain from Sindh province. We report the first emergence of XDR S. Typhi in Punjab province of Pakistan confirmed by whole genome sequencing.

Quorum quenching: role of nanoparticles as signal jammers in Gram-negative bacteria.

Quorum sensing (QS) is a cell density dependent regulatory process that uses signaling molecules to manage the expression of virulence genes and biofilm formation. The study of QS inhibitors has emerged as one of the most fascinating areas of research to discover novel antimicrobial agents. Compounds that block QS have become candidates as unusual antimicrobial agents, as they are leading players in the regulation of virulence of drug-resistant pathogens. Metal and metal oxide nanoparticles offer novel alternatives to combat antibiotic resistance in Gram-negative bacteria aiming their capacity as QS inhibitors. This review provides an insight into the quorum quenching potential of metal and metal oxide nanoparticles by targeting QS regulated virulence of Gram-negative bacteria.

Molecular mechanisms of antibiotic co-resistance among carbapenem resistant Acinetobacter baumannii.

INTRODUCTION: The spread of carbapenem-resistant Acinetobacter baumannii (CRAB) is difficult to control especially in the hospitals due to the successful mobilization and evolution of the genetic elements harboring the resistant determinants. The study was conducted to examine the distribution of aminoglycosides, tetracycline, and sulfonamide-resistant determinants among CRAB isolates that carry the blaOXA-23 gene. METHODOLOGY: For a total of 160 CRAB strains isolated at tertiary care hospitals of Pakistan that mainly carried blaOXA-23 gene were included in the study to evaluate the assortment of antibiotic resistance genes. RESULTS: The susceptibility rates of CRAB for other than beta-lactam drugs were 2.5% for both ciprofloxacin and aminoglycosides and 18% and 25% for sulfonamides and tetracyclines, respectively. Polymyxin B (MIC90, 1 g/mL) Colistin (MIC90, 1 g/mL) and Tigecycline (MIC90, 2 g/mL) were most active against these extensively drug-resistant CRAB isolates. The isolates were found to possess various genes mainly the tetB and sul2 for tetracycline and sulfonamide but the genes conferring resistance to aminoglycosides were varied with various combinations. CONCLUSION: Despite the CRAB clones containing blaOXA-23 have been previously reported in Pakistani hospitals, the screening of genetic determinants responsible for other antimicrobial agents is crucial for developing an effective surveillance and mitigation system for infection management.

In vitro and in vivo activity of Manuka honey against NDM-1-producing Klebsiella pneumoniae ST11.

AIM: To determine the therapeutic potential of Manuka honey against New Delhi metallo-ß-lactamase-1-producing Klebsiella pneumoniae ST11 in vitro and in vivo. MATERIALS & METHODS: Carbapenamases and metallo-ß-lactamases-producing K. pneumoniae ST11 isolated from blood culture was confirmed by VITEK-2® system, matrix-assisted laser desorption ionization-time of flight and multilocus sequence typing, followed by determination of minimum inhibitory concentration (µg/ml) using VITEK-2 system. Genetic analysis of bla NDM-1 was done by PCR, pulsed-field gel electrophoresis and DNA hybridization. In vitro and in vivo efficacy of Manuka honey was performed by microbroth dilution assay and BALB/c mice model respectively. RESULTS: K. pneumoniae ST11 displayed resistance to commonly used antibiotics. bla NDM-1 was located on 150 and 270kb plasmids. Minimum inhibitory concentration and minimum bactericidal concentration of Manuka honey was 30% (v/v) and substantial reduction of bacterial mean log value (>1 log) was observed in mice. Histological analysis of mice liver and kidneys demonstrated mild to moderate inflammation. CONCLUSION: Manuka honey can be used as an alternate therapeutic approach for management of New Delhi metallo-ß-lactamase-producing pathogens.

Nanoantibiotics: Future nanotechnologies to combat antibiotic resistance.

The discovery of antibiotics was hailed as a historic breakthrough for the human race in the fight against bacterial and malignant infections. However, in a very short time, owing to their acute and aggressive nature, bacteria have developed resistance against antibiotics and other chemotherapeutics agents. Potentially, this situation could again result in bacterial infection outbreaks. Metal and metal oxide nanoparticles have been proven as better alternatives; the combination of antibiotics and metal oxide nanoparticles was shown to decrease the toxicity and enhance the antibacterial, antiviral, and anticancer efficacy of the agents. This review provides a detailed view about the role of metal and metal oxide nanoparticles in the treatment of infections in conjunction with antibiotics, their modes of action, and synergism. In addition, the problems of multidrug resistance are addressed and will allow the development of a comprehensive, reliable, and rational treatment plan. It is expected that this comprehensive review will lead to new research opportunities, which should be helpful for future applications in biomedical science.

Isolation, Characterization, and Antibacterial Activity of Bacteriophages Against Methicillin-Resistant Staphylococcus aureus in Pakistan.

BACKGROUND: In recent years, antibiotic resistance has been indicated as a paramount threat to public health. The use of bacteriophages appears to be a safer alternative for the control of bacterial infections. OBJECTIVES: The present study aims to explore sewage water for the presence of indigenous bacteriophages, and to investigate their antibacterial potential against Methicillin-resistant Staphylococcus aureus (MRSA). METHODS: Bacterial isolates were first collected and identified from pus samples taken from the surgical and burn units using standard microbiological procedures. A cefoxitin disk screen test was then used and interpreted according to the clinical laboratory standards institute (CLSI) guidelines for the detection of MRSA. The sewage samples were processed and the phages enriched using S. aureus as a host organism. Turbid and clear plaques of different sizes were isolated using an overlay method, purified, and then enumerated by means of a dilution method. RESULTS: The phages exhibited good lytic activity against MRSA when tested in-vitro, and the highest activity was attained within three to six hours of phage infection. The isolated phage pq/48 was also found efficient in decreasing the bacterial count during an in-vivo trial in rabbits. A protein analysis using SDS-PAGE revealed 10 proteins of between 20 kDa and 155 kDa in size. CONCLUSIONS: The overall results indicated that bacteriophages isolated from sewage exhibited excellent lytic activity against MRSA strains. In conclusion, bacteriophages can be further characterized and appear to be a promising candidate for phage therapy against MRSA in the future.

Evaluation of Risk Factors for Peste des Petits Ruminants Virus in Sheep and Goats at the Wildlife-Livestock Interface in Punjab Province, Pakistan.

Peste des petits ruminants virus (PPRV) is causing infectious disease with high morbidity and mortality rate in domestic and wild small ruminants of Pakistan with valuable economical losses. The present study was carried out to investigate risk factors of PPRV in domestic small ruminants which were present in the vicinity of wildlife parks. A total of 265 sera samples (27 wild ruminants and 238 domesticated small ruminants) from apparently healthy animals from two different wildlife parks were collected and analysed for PPRV antibodies. Also, 20 nasal swabs from domestic small ruminants showing respiratory signs were collected to check for presence of PPRV antigen. Competitive ELISA revealed highest proportions of anti-PPRV antibodies in domestic small ruminants around the Wildlife Park at Lahore (35%) as compared to Faisalabad (13%), with no existence of PPRV antibodies in tested serum of wild ruminants at these parks. Higher seropositivity was observed in females (25.6%) than in males (5.1%) and in goats (34.5%) compared to sheep (11.2%). The results of N-gene based RT-PCR highlight the absence of PPRV due to lack of current PPR outbreak in the region during study period. Even though grazing was not a significant risk factor, there is still a possibility of wildlife-livestock interactions for feed and water reservoirs, resulting in spillover of PPR to wildlife. Keeping in view the high seropositivity and risk of PPR, vaccination should be adopted to avoid circulation of PPRV among wild and domestic small ruminants (sheep and goats).

Occurrence and antibacterial susceptibility pattern of bacterial pathogens isolated from diarrheal patients in Pakistan.

OBJECTIVE: To determine the occurrence of bacterial pathogens responsible for diarrhea and to engender information regarding the effectiveness of commonly used antibiotic against diarrhea. METHODS: This cross-sectional study was conducted between April and July 2014. Samples were collected from the Divisional Headquarter and Allied Hospital, Faisalabad, Pakistan. The differential and selective media were used to isolate bacterial pathogens, which were identified through cultural characteristics, microscopy, and biochemical tests. Disc diffusion assay was carried out using Muller Hinton agar medium, and minimum inhibitory concentration was determined using broth dilution method against isolated pathogens. RESULTS: One hundred and forty-one (100%) samples were positive for some bacteria. Frequency of occurrence was Bacillus cereus (B. cereus) (66%), Escherichia coli (E.coli) (48.5%), Salmonella typhi (S. Typhi) (27.7%), Pseudomonas aeruginosa (P. aeruginosa) (8.5%), and Staphylococcus aureus (S. aureus) (4.3%). Single pathogen was detected in 20 (14.2%) samples whereas combinations were found in 121 (85.8%) samples. Bacillus cereus and E.coli were the most frequently detected pathogens followed by the S. Typhi, P. aeruginosa, and Staph. aureus. The percentage occurrence of isolated pathogens was 31% in B. cereus, 31% in E. coli, 18% in S. Typhi, 5% in P. aeruginosa, and 3% in Staph. aureus. CONCLUSION: Pseudomonas aeruginosa showed resistance against Amoxicillin and Cefotaxime, whereas S. aureus was found resistant against Cefotaxime. Statistical analysis using one way Analysis of Variance revealed that Ofloxacin and Gentamicin had significant (p less than 0.05) differences against all isolates as compared with other antibiotics used in this study.