JPMA. The Journal of the Pakistan Medical Association, 2018
Multidrug resistance (MDR) in gram-negative pathogens is the emerging threat to clinicians. The c... more Multidrug resistance (MDR) in gram-negative pathogens is the emerging threat to clinicians. The current study was designed to determine the prevalence and pattern of multidrug resistance in gram-negative clinical isolates. It was conducted at the COMSATS Institute of Information Technology, Islamabad, Pakistan, from June to October 2014. Of the 8, 300 samples collected, 729(8.8%) clinically important gram-negative pathogens were retrieved. These pathogens were subjected to phenotypic and biochemical detection and were further processed for multidrug resistance pattern. It was observed that gram-negative pathogens were simultaneously resistant to many antibiotics. The prevalence of extended spectrum b-lactamase phenomenon was 220(100%) in Klebsiella pneumoniae, 195(75%) in Escherichia coli. Resistance to carbapenem was 174(79%) in Klebsiella pneumoniae and 14(5.4%) in Escherichia coli. Resistance against fluoroquinolones also displayed an escalating trend. The current study found tha...
Horizontal gene transfer plays a potent role in the evolution of prokaryotes. A rigorous sequence... more Horizontal gene transfer plays a potent role in the evolution of prokaryotes. A rigorous sequence and phylogenetic analysis was carried out using the robust ClustalW, motifs/domains finding suites and neighbor-joining based ProtDist and BioNJ. This paper reports a few cases of horizontal gene transfer events between archaea and bacteria. Some of these events have been found to be unique to the bacterial pathogenic members and have not been observed in respective non-pathogenic counterparts. Two cases have been shown to exhibit particular importance. The first one is a Cps4I gene that codes for capsule polysaccharide biosynthesis protein in Streptococcus pneumoniae. The other gene has been detected in Streptococcus agalactiae that codes for N-acetyl neuramic acid synthetase, which is involved in the synthesis of N-acetyl neuramic acid or sialic acid. We believe that these genes, having been retained in the genome through selective advantage, have key functions in the organism’s biolo...
Neuropeptide S (NPS) is a naturally occurring appetite stimulant, associated with anxiety, stress... more Neuropeptide S (NPS) is a naturally occurring appetite stimulant, associated with anxiety, stress, and excitement regulation. Neuropeptide S serves as a hypothalamic energy regulator that enhances food intake with a reduced level of satiety. NPS activates fat angiogenesis and the proliferation of new adipocytes in obesity. NPS has an established role in energy regulation by many pre-clinical investigations; however we have limited data available to support this notion in humans. We found significant association of Neuropeptide S receptor (NPSR1) Asn107Ile (rs324981, A>T) polymorphism with obese male participants. The current investigation carried out genotype screening of NPSR1 allele to assess the spectrum of the Asn107Ile polymorphism in obese and healthy Pakistani individuals. We revealed a significant (p = 0.04) difference between AA vs TT + AT genotype distribution of NPSR1 (SNP rs324981,) between obese and healthy individuals (p = 0.04). In this genotype analysis of (SNP rs...
Purpose To identify the genetic defects underlying retinitis pigmentosa (RP) in Pakistani familie... more Purpose To identify the genetic defects underlying retinitis pigmentosa (RP) in Pakistani families. Methods Genome-wide high-density single-nucleotide-polymorphism microarray analysis was performed using the DNA of nine affected individuals from two large families with multiple consanguineous marriages. Data were analyzed to identify homozygous regions that are shared by affected sibs in each family. Sanger sequencing was performed for genes previously implicated in autosomal recessive RP and allied retinal dystrophies that resided in the identified homozygous regions. Probands from both families underwent fundus examination and electroretinogram measurements. Results The tubby-like protein 1 gene (TULP1) was present in the largest homozygous region in both families. Sequence analysis identified a previously reported mutation (c.1138A>G; p.Thr380Ala) in one family and a novel pathogenic variant (c.1445G>A; p.Arg482Gln) in the other family. Both variants were found to be presen...
Many of the virulence determinants secreted from pathogenic Gram-negative bacteria are autotransp... more Many of the virulence determinants secreted from pathogenic Gram-negative bacteria are autotransporter proteins, which are usually either exported to the bacterial cell surface or secreted into the external environment. There appears to be dearth of information regarding the exact mechanism of their processing into the surface-exposed passenger domains and C-terminal, βdomains. The C-terminal porin domains mediate the final step of autotransporter secretion by threading of the passenger domain through the outer membrane (OM). The native structure is formed only after this final secretion step, which does not require any energy involvement. Despite sequence divergence and functional diversity among autotransporter passenger domains almost all of them are predicted to form parallel s-helices, indicating this structural topology may be important for secretion. The possible involvement of periplasmic environment in regulating the export of pertactin, an autotransporter passenger domain ...
OBJECTIVE To determine Candida colonisation/infection in renal transplant patients and to determi... more OBJECTIVE To determine Candida colonisation/infection in renal transplant patients and to determine the resistance pattern against antifungal drugs. METHODS This prospective, observational study was conducted at Al-Sayyed Hospital, Rawalpindi, Pakistan, from January to October 2014, in collaboration with the Microbiology and Public Health Laboratory's, Islamabad campus..The clinical specimens investigated included respiratory tract secretions, blood, urine, high vaginal swab, skin scrapings, and plastic devices samples. RESULTS Of the 7,850 samples, 164(2.08%) were positive for Candida. Candida albicans were most prevalent as they were found in 114(69%) samples. Besides, 56(34%) of the positive samples were resistant to one or more antifungal agents. Highest resistance was obtained against fluconazole. We found only 5(3.04%) positive samples of Candida glabrata; of them, 3(60%)were resistant. In case of Candida spp, 27(48%) resistance was observed. In Candida albicans, 23(41%) o...
The spectrum of emerging new diseases as well as re-emerging old diseases is broadening as infect... more The spectrum of emerging new diseases as well as re-emerging old diseases is broadening as infectious agents evolve, adapt, and spread at enormous speeds in response to changing ecosystems. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recent phenomenon and may take a while to understand its transmission routes from less traveled territories, ranging from fomite exposure routes to wastewater transmission. The critical challenge is how to negotiate with such catastrophic pandemics in high-income countries (HICs ~20% of the global population) and low-and middle-income countries (LMICs ~ 80% of the global population) with a total global population size of approximately eight billion, where practical mass testing and tracing is only a remote possibility, particularly in low-and middle-income countries (LMICs). Keeping in mind the population distribution disparities of high-income countries (HICs) and LMICs and urbanisation trends over recent years, traditional wastew...
BackgroundAcute diarrhea is a leading cause of morbidity and mortality in children particularly i... more BackgroundAcute diarrhea is a leading cause of morbidity and mortality in children particularly in developing countries of Asia and Africa. The present study was conducted to detect the two most important pathogens, rotavirus and Campylobacter Jejuni in children suffering with diarrhea in Rawalpindi and Islamabad, Pakistan in 2014. The clinical and epidemiological aspects of the disease were also investigated.MethodsA total of 500 stool samples were collected from children presented with clinical signs and symptoms of acute diarrhea. The samples were initially screened for the presence of rotavirus A (RVA) via ELISA (Enzyme-linked immunosorbent assay) and RT-PCR (Reverse Transcriptase PCR) and then were analysed for C. jejuni by using species specific PCR assay.ResultsThe detection rate of RVA was 26.4% (132/500) while, Campylobacter was detected in 52% (260/500) of samples with C. jejuni accounted for 48.2% (241/500) of all study cases. Co-infection of C. jejuni with RVA was identified in 21.8% of all cases. Children with RVA and C. jejuni co-infection showed a higher probability (p = 0.01) to be dehydrated. A significant association (p = 0.02) was found between C. jejuni positive status and fever in children. The median age of children with both RVA and C. jejuni infection was 6–11 months. The RVA detection rate was high in winter months of the year while, C. jejuni infections were documented high in summer over 1 year study period.ConclusionsThe overall results have demonstrated the high prevalence of C. jejuni in Rawalpindi, Islamabad, Pakistan in 2014. The results of present study will not only help to calculate disease burden caused by C. jejuni and rotavirus but also will provide critical information to health authorities in planning public health care strategies against these pathogens.
Type 2 diabetes mellitus (T2DM) accounts for 90% of diabetes cases worldwide. The majority of T2D... more Type 2 diabetes mellitus (T2DM) accounts for 90% of diabetes cases worldwide. The majority of T2DM patients are obese. Dysbiosis in the gut microflora is strongly associated with the pathogenesis of obesity and T2DM; however, the microbiome of obese-T2DM individuals in the Pakistani population remains unexplored. The gut microbiota signature of 60 Pakistani adults was studied using 16S rRNA sequencing targeting V 3-V 4 hypervariable regions. The sequence analysis revealed that bacteria from Firmicutes were predominant along with those from Clostridia and Negativicutes, whereas bacteria from Verrucomicrobia, Bacteroidetes, Proteobacteria, and Elusimicrobia were less abundant among the obese T2DM patients. These data distinctively vary from those in reports on the Indian population. The difference in gut microbiota could presumably be related to the distinct lifestyle and eastern dietary habits (high carbohydrate and fat intake, low fiber intake) and unregulated antibiotic consumption. This is the first study carried out to understand the gut microbiome and its correlation with individual life style of obese T2DM patients in the Pakistani population.
Campylobacter jejuni is the one of the leading cause of bacterial food borne gastroenteritis. Pgl... more Campylobacter jejuni is the one of the leading cause of bacterial food borne gastroenteritis. PglB, a glycosyltransferase, plays a crucial role of mediating glycosylation of numerous periplasmic proteins. It catalyzes N-glycosylation at the sequon D/E-X1-N-X2-S/T in its substrate proteins. Here we report that the PglB itself is a glycoprotein which self-glycosylates at N534 site in its DYNQS sequon by its own catalytic WWDYG motif. Site-directed mutagenesis, lectin Immunoblot, and mobility shift assays confirmed that the DYNQS is an N-glycosylation motif. PglB's N-glycosylation motif is structurally and functionally similar to its widely studied glycosylation substrate, the OMPH1. Its DYNQS motif forms a solvent-exposed crest. This motif is close to a cluster of polar and hydrophilic residues, which form a loop flanked by two α helices. This arrangement extremely apposite for auto-glycosylation at N534. This self-glycosylation ability of PglB could mediate C. jejuni's ability to colonize the intestinal epithelium. Further this capability may also bear significance for the development of novel conjugated vaccines and diagnostic tests.
Vibrio cholerae O1 infections mainly are responsible for significant mortality and morbidity amon... more Vibrio cholerae O1 infections mainly are responsible for significant mortality and morbidity amongst children, however, non-O1/non-O139 V. cholerae have also been reported to cause mild to severe infections because of their virulence potential. The pathogenic mechanisms of non-O1, non-O139 isolates are not as clearly understood as for that of O1 and O139 isolates. Type three secretion system (TTSS) is also considered one of the important virulent factors and during the current study, we investigated the role of TTSS in association with non-O1/non-O139 clinical isolates. We report that the presence of TTSS in non-O1/non-O139 V. cholerae clinical isolate (D13) from a child confers more virulence compared to the one lacking it (D15) in another clinical case during the small cholera epidemic. Moreover, the antibiotic susceptibility profiles of D13 and D15 indicate that they are multiple drug resistance (MDR) isolates. The sequence analysis for TTSS cluster was carried out for D13 and compared with the TTSS positive reference Vibrio parahaemolyticus RIMD2210633 and V. cholerae AM19226 non-O1/non-O139. Furthermore, the pathogenic potential of D13 & D15 was also explored in simple and economical invertebrate host model, Galleria mellonella and the results revealed that TTSS+ve isolate (D13) was more virulent compared to TTSS-ve isolate (D15). We suggest that this distinct genetic difference, seen in natural variants D13 and D15, is also reflected by the clinical picture of the former in contributing towards the severity of disease symptoms and this finding was further validated by assessing virulence potential of both isolates using inexpensive G. mellonella infection model.
OBJECTIVES Campylobacter jejuni has been classified as a member of priority pathogens group due t... more OBJECTIVES Campylobacter jejuni has been classified as a member of priority pathogens group due to the rapid emergence of multidrug resistant isolates. In the present study we planned to determine the prevalence, antibiotic resistance patterns and source tracking of clinical C. jejuni isolates from pediatric diarrheal patients in Pakistan. METHODS A total of 150 stool samples from children were processed for the presence of Campylobacter jejuni using culture, biochemical tests and by species specific PCR. Antibiotic susceptibility of the isolates was determined by disc diffusion method and metallo-beta-lactamase (MBL) producers were detected using gene specific PCR. Source tracking was done using source predictive PCR. RESULTS Campylobacter jejuni was present in 54.6% of the processed samples. More than 80% of the isolated strains were resistant to 7 out of 12 antibiotics tested. High level of susceptibility was observed against imipenem (12.2%) and tigecycline (9.7%). Six isolates (7.3%) were metallo-beta-lactamase producers and were positive for at least one of the five metallo-beta-lactamase genes. Source tracking showed that 57.3% of the isolates belonged to livestock associated cluster (C1 to C6) and 42.8% were assigned to non-livestock /environmental clusters (C7-C9). Isolates belonging to livestock cluster had high Multiple Antibiotic Resistance (MAR) index (p value<0.001) as compared to non-livestock. CONCLUSION High prevalence of multidrug resistant C. jejuni among pediatric diarrheal patients was observed. Moreover, association of these isolates to livestock clades suggest transmission to human population via food chain and presence of imipenem resistant MBL producing C. jejuni can lead to serious public health concerns.
Abstract The scientific and industrial interest in antimicrobial nano-carriers has significantly ... more Abstract The scientific and industrial interest in antimicrobial nano-carriers has significantly increased in recent years due to post-processing contamination. Liposomes are considered as promising food-grade carrier systems for bioactive-agents. However, due to relatively low stability of liposomes, surface modification or coating is required to improve its efficiency. The purpose of this study was to fabricate chitosan (CA) stabilized nano-liposomes (chitosomes) to enhance the controlled release and antimicrobial effect of nisin-Z against multidrug resistant (MDR) foodborne pathogens. Chitosome characteristics including encapsulation efficiency (EE %), SEM, thermal stability (TGA, DSC), FTIR analysis, antimicrobial potential and in vitro controlled release were investigated. Overall, chitosomes (CS) were thermally more stable, showed higher EE% (86 ± 1%) as compared to liposomes (56 ± 2%) and controlled S. aureus, E. faecalis and L. monocytogenes growth better than free or liposomal-nisin. SEM confirmed the size of liposomes from 54 to 108 nm, which increased after coating with chitosan. FTIR analysis revealed the interaction between CA and liposomes by merger of amide I and II peaks. Thus, CA-stabilized liposomes not only improve physico-chemical attributes while ensuring sustained release of nisin but also provide a potential approach to control bacterial contamination in food products for enhanced food safety.
Infectious diarrhea caused by the food borne pathogen, Campylobacter jejuni, is a major threat to... more Infectious diarrhea caused by the food borne pathogen, Campylobacter jejuni, is a major threat to public health worldwide leads high incidence of child mortality each year. In the present study, hydrothermal synthesis of Silver-Graphene-TiO2 nanocomposites along with TiO2, TiO2-Graphene and TiO2-silver nanocomposites was done and the samples were characterized using X-ray diffraction (XRD), tunneling electron microscopy (TEM) and UV-Vis Spectroscopy. Effect of silver and graphene addition on the broad spectrum antibacterial ability of TiO2 was studied under visible light. Moreover, the effects on bacterial survival, membrane integrity, cellular motiltiy and biofilm formation of C. jejuni were also evaluated. A synergetic effect of silver and graphene on Silver-Graphene-TiO2 nanocomposites was observed as indicated by its increased visible light sensitivity and enhanced antibacterial activity under visible light compared to its parent derivatives. Silver-Graphene-TiO2 composites effectively reduced growth and caused leakage of protein and DNA from C. jejuni cell. Atomic Force Microscopy was used to confirm bacterial cell damage. Besides, it also reduced motillity, hydrophobicity and autoaggregation of C. jejuni and showed excellent inhibition of biofilm formation. Furthermore, no significant cytotoxicity of synthesized nanoparticles was observed in human cell lines. We propose that Silver-Graphene-TiO2 composites can be used as effective antimicrobial agents to control the spread of C. jejuni by preventing both bacterial growth and biofilm formation.
The Type VI Secretion System (T6SS) provides enhanced virulence to Campylobacter jejuni and has b... more The Type VI Secretion System (T6SS) provides enhanced virulence to Campylobacter jejuni and has been associated with a high incidence of bloody diarrhea. The hemolysin-coregulated protein (Hcp)-the hallmark of the T6SS-can act as a structural and effector protein. Unlike other T6SS-harboring bacteria, which possess multiple Hcp proteins each performing different functions, C. jejuni possesses only one Hcp protein, and its structural and functional role has not been elucidated previously. Here, we report the structure and functional studies of Hcp from C. jejuni. We found similarities between the hexameric ring structure of Hcp-Cj and that of Hcp3 from Pseudomonas aeruginosa. Through functional studies, we showed two roles for Hcp-Cj that is, in cytotoxicity toward HepG2 cells and in biofilm formation in C. jejuni. In structure-based mutational analyses, we showed that an Arg-to-Ala mutation at position 30 within the extended loop results in a significant decrease in cytotoxicity, suggesting a role for this loop in binding to the host cell. However, this mutation does not affect its biofilm formation function. Collectively, this study supports the dual role of Hcp-Cj as a structural and effector protein in C. jejuni.
Study design: We determined the G type, the gene 4 genotype, the electropherotype, and the genomi... more Study design: We determined the G type, the gene 4 genotype, the electropherotype, and the genomic RNA constellation (genogroup) of a group A rotavirus strain isolated from an adult with severe diarrhea. This patient, the first documented case of rotavirus diarrhea in adult in ...
Background and Objective: Vibrio cholerae continues to emerge as a dangerous pathogen because of ... more Background and Objective: Vibrio cholerae continues to emerge as a dangerous pathogen because of increasing resistance to a number of antibiotics. This paper provides a solution to emerging antibiotic resistance by introducing novel proteins as vaccine candidates against cholera. Materials and Methods: Vibrio cholerae genome versatility is a hurdle for developing a vaccine to combat diarrhoeal infection, so its core gene information was used to determine a potential vaccine candidate. Whole genome sequence data of more than 100 Vibrio cholerae strains were used simultaneously to get core genome information. The VacSol pipeline based on reverse vaccinology was selected to address the problem of safe, cheap, temperature-stable, and effective vaccine candidates which can be used for vaccine development against Vibrio cholerae. VacSol screens vaccine candidates using integrated, well-known, and robust algorithms/tools for proteome analysis. The proteomes of the pathogens were initially ...
JPMA. The Journal of the Pakistan Medical Association, 2018
Multidrug resistance (MDR) in gram-negative pathogens is the emerging threat to clinicians. The c... more Multidrug resistance (MDR) in gram-negative pathogens is the emerging threat to clinicians. The current study was designed to determine the prevalence and pattern of multidrug resistance in gram-negative clinical isolates. It was conducted at the COMSATS Institute of Information Technology, Islamabad, Pakistan, from June to October 2014. Of the 8, 300 samples collected, 729(8.8%) clinically important gram-negative pathogens were retrieved. These pathogens were subjected to phenotypic and biochemical detection and were further processed for multidrug resistance pattern. It was observed that gram-negative pathogens were simultaneously resistant to many antibiotics. The prevalence of extended spectrum b-lactamase phenomenon was 220(100%) in Klebsiella pneumoniae, 195(75%) in Escherichia coli. Resistance to carbapenem was 174(79%) in Klebsiella pneumoniae and 14(5.4%) in Escherichia coli. Resistance against fluoroquinolones also displayed an escalating trend. The current study found tha...
Horizontal gene transfer plays a potent role in the evolution of prokaryotes. A rigorous sequence... more Horizontal gene transfer plays a potent role in the evolution of prokaryotes. A rigorous sequence and phylogenetic analysis was carried out using the robust ClustalW, motifs/domains finding suites and neighbor-joining based ProtDist and BioNJ. This paper reports a few cases of horizontal gene transfer events between archaea and bacteria. Some of these events have been found to be unique to the bacterial pathogenic members and have not been observed in respective non-pathogenic counterparts. Two cases have been shown to exhibit particular importance. The first one is a Cps4I gene that codes for capsule polysaccharide biosynthesis protein in Streptococcus pneumoniae. The other gene has been detected in Streptococcus agalactiae that codes for N-acetyl neuramic acid synthetase, which is involved in the synthesis of N-acetyl neuramic acid or sialic acid. We believe that these genes, having been retained in the genome through selective advantage, have key functions in the organism’s biolo...
Neuropeptide S (NPS) is a naturally occurring appetite stimulant, associated with anxiety, stress... more Neuropeptide S (NPS) is a naturally occurring appetite stimulant, associated with anxiety, stress, and excitement regulation. Neuropeptide S serves as a hypothalamic energy regulator that enhances food intake with a reduced level of satiety. NPS activates fat angiogenesis and the proliferation of new adipocytes in obesity. NPS has an established role in energy regulation by many pre-clinical investigations; however we have limited data available to support this notion in humans. We found significant association of Neuropeptide S receptor (NPSR1) Asn107Ile (rs324981, A>T) polymorphism with obese male participants. The current investigation carried out genotype screening of NPSR1 allele to assess the spectrum of the Asn107Ile polymorphism in obese and healthy Pakistani individuals. We revealed a significant (p = 0.04) difference between AA vs TT + AT genotype distribution of NPSR1 (SNP rs324981,) between obese and healthy individuals (p = 0.04). In this genotype analysis of (SNP rs...
Purpose To identify the genetic defects underlying retinitis pigmentosa (RP) in Pakistani familie... more Purpose To identify the genetic defects underlying retinitis pigmentosa (RP) in Pakistani families. Methods Genome-wide high-density single-nucleotide-polymorphism microarray analysis was performed using the DNA of nine affected individuals from two large families with multiple consanguineous marriages. Data were analyzed to identify homozygous regions that are shared by affected sibs in each family. Sanger sequencing was performed for genes previously implicated in autosomal recessive RP and allied retinal dystrophies that resided in the identified homozygous regions. Probands from both families underwent fundus examination and electroretinogram measurements. Results The tubby-like protein 1 gene (TULP1) was present in the largest homozygous region in both families. Sequence analysis identified a previously reported mutation (c.1138A>G; p.Thr380Ala) in one family and a novel pathogenic variant (c.1445G>A; p.Arg482Gln) in the other family. Both variants were found to be presen...
Many of the virulence determinants secreted from pathogenic Gram-negative bacteria are autotransp... more Many of the virulence determinants secreted from pathogenic Gram-negative bacteria are autotransporter proteins, which are usually either exported to the bacterial cell surface or secreted into the external environment. There appears to be dearth of information regarding the exact mechanism of their processing into the surface-exposed passenger domains and C-terminal, βdomains. The C-terminal porin domains mediate the final step of autotransporter secretion by threading of the passenger domain through the outer membrane (OM). The native structure is formed only after this final secretion step, which does not require any energy involvement. Despite sequence divergence and functional diversity among autotransporter passenger domains almost all of them are predicted to form parallel s-helices, indicating this structural topology may be important for secretion. The possible involvement of periplasmic environment in regulating the export of pertactin, an autotransporter passenger domain ...
OBJECTIVE To determine Candida colonisation/infection in renal transplant patients and to determi... more OBJECTIVE To determine Candida colonisation/infection in renal transplant patients and to determine the resistance pattern against antifungal drugs. METHODS This prospective, observational study was conducted at Al-Sayyed Hospital, Rawalpindi, Pakistan, from January to October 2014, in collaboration with the Microbiology and Public Health Laboratory's, Islamabad campus..The clinical specimens investigated included respiratory tract secretions, blood, urine, high vaginal swab, skin scrapings, and plastic devices samples. RESULTS Of the 7,850 samples, 164(2.08%) were positive for Candida. Candida albicans were most prevalent as they were found in 114(69%) samples. Besides, 56(34%) of the positive samples were resistant to one or more antifungal agents. Highest resistance was obtained against fluconazole. We found only 5(3.04%) positive samples of Candida glabrata; of them, 3(60%)were resistant. In case of Candida spp, 27(48%) resistance was observed. In Candida albicans, 23(41%) o...
The spectrum of emerging new diseases as well as re-emerging old diseases is broadening as infect... more The spectrum of emerging new diseases as well as re-emerging old diseases is broadening as infectious agents evolve, adapt, and spread at enormous speeds in response to changing ecosystems. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recent phenomenon and may take a while to understand its transmission routes from less traveled territories, ranging from fomite exposure routes to wastewater transmission. The critical challenge is how to negotiate with such catastrophic pandemics in high-income countries (HICs ~20% of the global population) and low-and middle-income countries (LMICs ~ 80% of the global population) with a total global population size of approximately eight billion, where practical mass testing and tracing is only a remote possibility, particularly in low-and middle-income countries (LMICs). Keeping in mind the population distribution disparities of high-income countries (HICs) and LMICs and urbanisation trends over recent years, traditional wastew...
BackgroundAcute diarrhea is a leading cause of morbidity and mortality in children particularly i... more BackgroundAcute diarrhea is a leading cause of morbidity and mortality in children particularly in developing countries of Asia and Africa. The present study was conducted to detect the two most important pathogens, rotavirus and Campylobacter Jejuni in children suffering with diarrhea in Rawalpindi and Islamabad, Pakistan in 2014. The clinical and epidemiological aspects of the disease were also investigated.MethodsA total of 500 stool samples were collected from children presented with clinical signs and symptoms of acute diarrhea. The samples were initially screened for the presence of rotavirus A (RVA) via ELISA (Enzyme-linked immunosorbent assay) and RT-PCR (Reverse Transcriptase PCR) and then were analysed for C. jejuni by using species specific PCR assay.ResultsThe detection rate of RVA was 26.4% (132/500) while, Campylobacter was detected in 52% (260/500) of samples with C. jejuni accounted for 48.2% (241/500) of all study cases. Co-infection of C. jejuni with RVA was identified in 21.8% of all cases. Children with RVA and C. jejuni co-infection showed a higher probability (p = 0.01) to be dehydrated. A significant association (p = 0.02) was found between C. jejuni positive status and fever in children. The median age of children with both RVA and C. jejuni infection was 6–11 months. The RVA detection rate was high in winter months of the year while, C. jejuni infections were documented high in summer over 1 year study period.ConclusionsThe overall results have demonstrated the high prevalence of C. jejuni in Rawalpindi, Islamabad, Pakistan in 2014. The results of present study will not only help to calculate disease burden caused by C. jejuni and rotavirus but also will provide critical information to health authorities in planning public health care strategies against these pathogens.
Type 2 diabetes mellitus (T2DM) accounts for 90% of diabetes cases worldwide. The majority of T2D... more Type 2 diabetes mellitus (T2DM) accounts for 90% of diabetes cases worldwide. The majority of T2DM patients are obese. Dysbiosis in the gut microflora is strongly associated with the pathogenesis of obesity and T2DM; however, the microbiome of obese-T2DM individuals in the Pakistani population remains unexplored. The gut microbiota signature of 60 Pakistani adults was studied using 16S rRNA sequencing targeting V 3-V 4 hypervariable regions. The sequence analysis revealed that bacteria from Firmicutes were predominant along with those from Clostridia and Negativicutes, whereas bacteria from Verrucomicrobia, Bacteroidetes, Proteobacteria, and Elusimicrobia were less abundant among the obese T2DM patients. These data distinctively vary from those in reports on the Indian population. The difference in gut microbiota could presumably be related to the distinct lifestyle and eastern dietary habits (high carbohydrate and fat intake, low fiber intake) and unregulated antibiotic consumption. This is the first study carried out to understand the gut microbiome and its correlation with individual life style of obese T2DM patients in the Pakistani population.
Campylobacter jejuni is the one of the leading cause of bacterial food borne gastroenteritis. Pgl... more Campylobacter jejuni is the one of the leading cause of bacterial food borne gastroenteritis. PglB, a glycosyltransferase, plays a crucial role of mediating glycosylation of numerous periplasmic proteins. It catalyzes N-glycosylation at the sequon D/E-X1-N-X2-S/T in its substrate proteins. Here we report that the PglB itself is a glycoprotein which self-glycosylates at N534 site in its DYNQS sequon by its own catalytic WWDYG motif. Site-directed mutagenesis, lectin Immunoblot, and mobility shift assays confirmed that the DYNQS is an N-glycosylation motif. PglB's N-glycosylation motif is structurally and functionally similar to its widely studied glycosylation substrate, the OMPH1. Its DYNQS motif forms a solvent-exposed crest. This motif is close to a cluster of polar and hydrophilic residues, which form a loop flanked by two α helices. This arrangement extremely apposite for auto-glycosylation at N534. This self-glycosylation ability of PglB could mediate C. jejuni's ability to colonize the intestinal epithelium. Further this capability may also bear significance for the development of novel conjugated vaccines and diagnostic tests.
Vibrio cholerae O1 infections mainly are responsible for significant mortality and morbidity amon... more Vibrio cholerae O1 infections mainly are responsible for significant mortality and morbidity amongst children, however, non-O1/non-O139 V. cholerae have also been reported to cause mild to severe infections because of their virulence potential. The pathogenic mechanisms of non-O1, non-O139 isolates are not as clearly understood as for that of O1 and O139 isolates. Type three secretion system (TTSS) is also considered one of the important virulent factors and during the current study, we investigated the role of TTSS in association with non-O1/non-O139 clinical isolates. We report that the presence of TTSS in non-O1/non-O139 V. cholerae clinical isolate (D13) from a child confers more virulence compared to the one lacking it (D15) in another clinical case during the small cholera epidemic. Moreover, the antibiotic susceptibility profiles of D13 and D15 indicate that they are multiple drug resistance (MDR) isolates. The sequence analysis for TTSS cluster was carried out for D13 and compared with the TTSS positive reference Vibrio parahaemolyticus RIMD2210633 and V. cholerae AM19226 non-O1/non-O139. Furthermore, the pathogenic potential of D13 & D15 was also explored in simple and economical invertebrate host model, Galleria mellonella and the results revealed that TTSS+ve isolate (D13) was more virulent compared to TTSS-ve isolate (D15). We suggest that this distinct genetic difference, seen in natural variants D13 and D15, is also reflected by the clinical picture of the former in contributing towards the severity of disease symptoms and this finding was further validated by assessing virulence potential of both isolates using inexpensive G. mellonella infection model.
OBJECTIVES Campylobacter jejuni has been classified as a member of priority pathogens group due t... more OBJECTIVES Campylobacter jejuni has been classified as a member of priority pathogens group due to the rapid emergence of multidrug resistant isolates. In the present study we planned to determine the prevalence, antibiotic resistance patterns and source tracking of clinical C. jejuni isolates from pediatric diarrheal patients in Pakistan. METHODS A total of 150 stool samples from children were processed for the presence of Campylobacter jejuni using culture, biochemical tests and by species specific PCR. Antibiotic susceptibility of the isolates was determined by disc diffusion method and metallo-beta-lactamase (MBL) producers were detected using gene specific PCR. Source tracking was done using source predictive PCR. RESULTS Campylobacter jejuni was present in 54.6% of the processed samples. More than 80% of the isolated strains were resistant to 7 out of 12 antibiotics tested. High level of susceptibility was observed against imipenem (12.2%) and tigecycline (9.7%). Six isolates (7.3%) were metallo-beta-lactamase producers and were positive for at least one of the five metallo-beta-lactamase genes. Source tracking showed that 57.3% of the isolates belonged to livestock associated cluster (C1 to C6) and 42.8% were assigned to non-livestock /environmental clusters (C7-C9). Isolates belonging to livestock cluster had high Multiple Antibiotic Resistance (MAR) index (p value<0.001) as compared to non-livestock. CONCLUSION High prevalence of multidrug resistant C. jejuni among pediatric diarrheal patients was observed. Moreover, association of these isolates to livestock clades suggest transmission to human population via food chain and presence of imipenem resistant MBL producing C. jejuni can lead to serious public health concerns.
Abstract The scientific and industrial interest in antimicrobial nano-carriers has significantly ... more Abstract The scientific and industrial interest in antimicrobial nano-carriers has significantly increased in recent years due to post-processing contamination. Liposomes are considered as promising food-grade carrier systems for bioactive-agents. However, due to relatively low stability of liposomes, surface modification or coating is required to improve its efficiency. The purpose of this study was to fabricate chitosan (CA) stabilized nano-liposomes (chitosomes) to enhance the controlled release and antimicrobial effect of nisin-Z against multidrug resistant (MDR) foodborne pathogens. Chitosome characteristics including encapsulation efficiency (EE %), SEM, thermal stability (TGA, DSC), FTIR analysis, antimicrobial potential and in vitro controlled release were investigated. Overall, chitosomes (CS) were thermally more stable, showed higher EE% (86 ± 1%) as compared to liposomes (56 ± 2%) and controlled S. aureus, E. faecalis and L. monocytogenes growth better than free or liposomal-nisin. SEM confirmed the size of liposomes from 54 to 108 nm, which increased after coating with chitosan. FTIR analysis revealed the interaction between CA and liposomes by merger of amide I and II peaks. Thus, CA-stabilized liposomes not only improve physico-chemical attributes while ensuring sustained release of nisin but also provide a potential approach to control bacterial contamination in food products for enhanced food safety.
Infectious diarrhea caused by the food borne pathogen, Campylobacter jejuni, is a major threat to... more Infectious diarrhea caused by the food borne pathogen, Campylobacter jejuni, is a major threat to public health worldwide leads high incidence of child mortality each year. In the present study, hydrothermal synthesis of Silver-Graphene-TiO2 nanocomposites along with TiO2, TiO2-Graphene and TiO2-silver nanocomposites was done and the samples were characterized using X-ray diffraction (XRD), tunneling electron microscopy (TEM) and UV-Vis Spectroscopy. Effect of silver and graphene addition on the broad spectrum antibacterial ability of TiO2 was studied under visible light. Moreover, the effects on bacterial survival, membrane integrity, cellular motiltiy and biofilm formation of C. jejuni were also evaluated. A synergetic effect of silver and graphene on Silver-Graphene-TiO2 nanocomposites was observed as indicated by its increased visible light sensitivity and enhanced antibacterial activity under visible light compared to its parent derivatives. Silver-Graphene-TiO2 composites effectively reduced growth and caused leakage of protein and DNA from C. jejuni cell. Atomic Force Microscopy was used to confirm bacterial cell damage. Besides, it also reduced motillity, hydrophobicity and autoaggregation of C. jejuni and showed excellent inhibition of biofilm formation. Furthermore, no significant cytotoxicity of synthesized nanoparticles was observed in human cell lines. We propose that Silver-Graphene-TiO2 composites can be used as effective antimicrobial agents to control the spread of C. jejuni by preventing both bacterial growth and biofilm formation.
The Type VI Secretion System (T6SS) provides enhanced virulence to Campylobacter jejuni and has b... more The Type VI Secretion System (T6SS) provides enhanced virulence to Campylobacter jejuni and has been associated with a high incidence of bloody diarrhea. The hemolysin-coregulated protein (Hcp)-the hallmark of the T6SS-can act as a structural and effector protein. Unlike other T6SS-harboring bacteria, which possess multiple Hcp proteins each performing different functions, C. jejuni possesses only one Hcp protein, and its structural and functional role has not been elucidated previously. Here, we report the structure and functional studies of Hcp from C. jejuni. We found similarities between the hexameric ring structure of Hcp-Cj and that of Hcp3 from Pseudomonas aeruginosa. Through functional studies, we showed two roles for Hcp-Cj that is, in cytotoxicity toward HepG2 cells and in biofilm formation in C. jejuni. In structure-based mutational analyses, we showed that an Arg-to-Ala mutation at position 30 within the extended loop results in a significant decrease in cytotoxicity, suggesting a role for this loop in binding to the host cell. However, this mutation does not affect its biofilm formation function. Collectively, this study supports the dual role of Hcp-Cj as a structural and effector protein in C. jejuni.
Study design: We determined the G type, the gene 4 genotype, the electropherotype, and the genomi... more Study design: We determined the G type, the gene 4 genotype, the electropherotype, and the genomic RNA constellation (genogroup) of a group A rotavirus strain isolated from an adult with severe diarrhea. This patient, the first documented case of rotavirus diarrhea in adult in ...
Background and Objective: Vibrio cholerae continues to emerge as a dangerous pathogen because of ... more Background and Objective: Vibrio cholerae continues to emerge as a dangerous pathogen because of increasing resistance to a number of antibiotics. This paper provides a solution to emerging antibiotic resistance by introducing novel proteins as vaccine candidates against cholera. Materials and Methods: Vibrio cholerae genome versatility is a hurdle for developing a vaccine to combat diarrhoeal infection, so its core gene information was used to determine a potential vaccine candidate. Whole genome sequence data of more than 100 Vibrio cholerae strains were used simultaneously to get core genome information. The VacSol pipeline based on reverse vaccinology was selected to address the problem of safe, cheap, temperature-stable, and effective vaccine candidates which can be used for vaccine development against Vibrio cholerae. VacSol screens vaccine candidates using integrated, well-known, and robust algorithms/tools for proteome analysis. The proteomes of the pathogens were initially ...
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Papers by Habib Bokhari