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1.  Comparative growth analysis of capsulated (Vi+) and acapsulated (Vi-) Salmonella typhi isolates in human blood 
EXCLI Journal  2015;14:213-219.
Salmonella enterica serovar Typhi (S. Typhi) is a human restricted pathogen. It biosynthesizes a virulence capsular polysaccharide named as Vi antigen. S. Typhi regulates expression of genes involved in the biosynthesis of Vi antigen in response to osmolarity. Beside Vi-positive isolates, Vi-negative (acapsulated) isolates are also pathogenic. However, Vi-positive isolates are more prevalent. The present study was planned to investigate comparative growth of Vi-positive and Vi-negative S. Typhi isolates in an ex vivo human whole blood model. Four isolates of each type were tested for growth in human whole blood and in an enrichment medium (Tryptic soy broth-TSB) as a control. It was found that capsulated (Vi-positive) strains formed smooth circular colonies and grew with shorter lag and generation time than Vi-negative isolates. Overall growth pattern of S. Typhi isolates both in vitro and ex vivo conditions showed that Vi-positive isolates grew at a faster rate. Especially in human blood, the lag time of acapsulated isolates was almost doubled as compared to capsulated S. Typhi isolates. It was also observed that Vi-negative isolates reduced in number up to 81 % during the first 12 hours of incubation in human whole blood. Interestingly, both types of isolates had similar growth curve in TSB indicating that Vi capsule is dispensable for bacterial growth in vitro. This study shows for the first time that absence of capsular antigen retards the growth of Vi-negative isolates on initial contact with human blood, but with passage of time they adjust themselves according to the new environment.
PMCID: PMC4553862  PMID: 26417360
Salmonella Typhi; Vi+; Vi-; growth; human blood
4.  A SET-domain-independent role of WRAD complex in cell-cycle regulatory function of mixed lineage leukemia 
Nucleic Acids Research  2014;42(12):7611-7624.
MLL, the trithorax ortholog, is a well-characterized histone 3 lysine 4 methyltransferase that is crucial for proper regulation of the Hox genes during embryonic development. Chromosomal translocations, disrupting the Mll gene, lead to aggressive leukemia with poor prognosis. However, the functions of MLL in cellular processes like cell-cycle regulation are not well studied. Here we show that the MLL has a regulatory role during multiple phases of the cell cycle. RNAi-mediated knockdown reveals that MLL regulates S-phase progression and, proper segregation and cytokinesis during M phase. Using deletions and mutations, we narrow the cell-cycle regulatory role to the C subunit of MLL. Our analysis reveals that the transactivation domain and not the SET domain is important for the S-phase function of MLL. Surprisingly, disruption of MLL–WRAD interaction is sufficient to disrupt proper mitotic progression. These mitotic functions of WRAD are independent of SET domain of MLL and, therefore, define a new role of WRAD in subset of MLL functions. Finally, we address the overlapping and unique roles of the different SET family members in the cell cycle.
PMCID: PMC4081079  PMID: 24880690
8.  A novel multiplex PCR for detection of Pseudomonas aeruginosa: A major cause of wound infections 
Background and Objective: Wound infections are often difficult to treat due to various bacterial pathogens. Pseudomonas aeruginosa is one of the common invaders of open wounds. Precise diagnosis of this etiological agent in wound infections is of critical importance particularly in treatment of problematic cases. The existing diagnostic methods have certain limitations particularly related to specificity. Our objective was to to establish a comprehensive and reliable multiplex PCR to confirm diagnosis of P. aeruginosa.
Methods: A multiplex PCR test was developed for rapid and comprehensive identification of P. aeruginosa. Four highly specific genes were targeted simultaneously for detection of genus, species and exotoxin production (16S rDNA, gyrB, oprL and ETA) in P. aeruginosa; additionally one internal control gene (invA) of Salmonella was used. The specificity of the multiplex PCR was confirmed using internal and negative controls. Amplified fragments were confirmed by restriction analysis and DNA sequencing.
Results: The developed method was applied on 40 morphologically suspected P. aeruginosa isolates (from 200 pus samples) and 18 isolates were confirmed as P. aeruginosa. In comparison, only 12 could be identified biochemically.
Conclusions: Combination of the four reported genes in multiplex PCR provided more confident and comprehensive detection of P. aeruginosa which is applicable for screening of wound infections and assisting treatment strategy.
PMCID: PMC3817763  PMID: 24353667
Multiplex PCR; wound infections; P. aeruginosa
9.  Rapid emergence of ESBL producers in E. coli causing urinary and wound infections in Pakistan 
Objectives: Production of extended spectrum beta -lactamases (ESBLs) by clinical isolates of pathogenic E. coli is a very serious therapeutic threat. This study was aimed to investigate the prevalence of ESBLs and associated drug resistance in E. coli isolates from urine and pus, and to report the drift from 2005 to 2009-10.
Methodology: Among 173 E. coli isolates, 82 were phenotypically detected as ESBL producers by standard cefotaxime / clavulanic acid and ceftazidime / clavulanic acid disc diffusion tests. Antimicrobial resistance of all ESBL producers was assessed by disc diffusion method. Presence of CTX-M, TEM, SHV and OXA groups was investigated by PCR.
Results: The prevalence of ESBL producing E. coli increased significantly from 33.7% in 2005 to 60.0% in 2009-10 (urine: 31.8% to 62.9%; pus: 41.1% to 55.5%). Resistance to cefotaxime, ceftazidime, ciprofloxacin, gentamicin, nalidixic acid, ticarcillin-clavulanic acid, and trimethoprim-sulfamethoxazole was above 85% in both sets of isolates. Imipenem and Fosfomycin resistance was non-existent in 2005 but ranged from 3-15% in 2009-10. Remarkable increase from 9.5% to 64.7% in urinary tract isolates and from 0 to 55% in pus isolates was observed in colistin sulphate resistance. The dissemination of genes encoding ESBLs was: CTX-M 3.5%; TEM 10.7%; both CTX-M and TEM 3.5% in 2005, and CTX-M 42.5%; TEM 48.1%; both CTX-M and TEM 29.6% in 2009-10.
Conclusions: Our results showed very rapid emergence of multidrug resistant ESBL producing E. coli in Pakistan posing a very serious threat in the treatment of nosocomial and community acquired infections.
PMCID: PMC3809246  PMID: 24353573
Pathogenic E. coli; ESBL production; Drug resistance; Genotypic characterization
11.  Virulence profile of different phylogenetic groups of locally isolated community acquired uropathogenic E. coli from Faisalabad region of Pakistan 
Uropathogenic E.coli (UPEC) are among major pathogens causing urinary tract infections. Virulence factors are mainly responsible for the severity of these emerging infections. This study was planned to investigate the distribution of virulence genes and cytotoxic effects of UPEC isolates with reference to phylogenetic groups (B2, B1, D and A) to understand the presence and impact of virulence factors in the severity of infection in Faisalabad region of Pakistan.
In this study phylogenetic analysis, virulence gene identification and cytotoxicity of 59 uropathogenic E.coli isolates obtained from non-hospitalized patients was studied.
Among 59 isolates, phylogenetic group B2 (50%) was most dominant followed by groups A, B1 (19% each) and D (12%). Isolates present in group D showed highest presence of virulence genes. The prevalence hlyA (37%) was highest followed by sfaDE (27%), papC (24%), cnf1 (20%), eaeA (19%) and afaBC3 (14%). Highly hemolytic and highly verotoxic isolates mainly belonged to group D and B2. We also found two isolates with simultaneous presence of three fimbrial adhesin genes present on pap, afa, and sfa operons. This has not been reported before and underlines the dynamic nature of these UPEC isolates.
It was concluded that in local UPEC isolates from non-hospitalized patients, group B2 was more prevalent. However, group D isolates were most versatile as all were equipped with virulence genes and showed highest level of cytotoxicity.
PMCID: PMC3475034  PMID: 22867028
Uropathogenic E.coli; Phylogenicity; Virulence genes
12.  Multiple drug resistance patterns in various phylogenetic groups of uropathogenic E.coli isolated from Faisalabad region of Pakistan 
Brazilian Journal of Microbiology  2011;42(4):1278-1283.
The objective of this work was the phylogenetic characterization of local clinical isolates of uropathogenic E. coli with respect to drug resistance. A total of 59 uropathogenic E. coli responsible for community acquired urinary tract infections were included in this study. A triplex PCR was employed to segregate each isolate into four different phylogenetic groups (A, B1, B2 and D). Drug resistance was evaluated by disc diffusion method. The drugs used were ampicillin, aztreonam, cefixime, cefoperazone, ceftriaxone, cephradine among β-lactam group; amikacin, gentamicin, and streptomycin among aminoglycosides; nalidixic acid and ciprofloxacin from quinolones; trimethoprim-sulfomethoxazole, and tetracycline. Among 59 uropathogenic E. coli isolates majority belonged to phylogenetic group B2 (50%) where as 19% each belonged to groups A and B1, and 12% to group D. All the isolates were multiple drug resistant (MDR). Most effective drugs against Group A, B1, and B2 were gentamicin, amikacin and cefixime; ceftriaxone and quinolones; and ceftriaxone and amikacin, respectively. Group D isolates were found to be highly resistant to all drugs. Our results have shown emergence of MDR isolates among uropathogenic E. coli with dominance of phylogenetic group B2. However, it was found that group D isolates were though less frequent, more drug resistant as compared with group B2. Groups A and B1 were relatively uncommon. Amikacin, ceftriaxone and gentamicin were the most effective drugs in general.
PMCID: PMC3768734  PMID: 24031752
uropathogenic E. coli; phylogenetic analysis; drug resistance
13.  Microsatellite isolation and marker development in carrot - genomic distribution, linkage mapping, genetic diversity analysis and marker transferability across Apiaceae 
BMC Genomics  2011;12:386.
The Apiaceae family includes several vegetable and spice crop species among which carrot is the most economically important member, with ~21 million tons produced yearly worldwide. Despite its importance, molecular resources in this species are relatively underdeveloped. The availability of informative, polymorphic, and robust PCR-based markers, such as microsatellites (or SSRs), will facilitate genetics and breeding of carrot and other Apiaceae, including integration of linkage maps, tagging of phenotypic traits and assisting positional gene cloning. Thus, with the purpose of isolating carrot microsatellites, two different strategies were used; a hybridization-based library enrichment for SSRs, and bioinformatic mining of SSRs in BAC-end sequence and EST sequence databases. This work reports on the development of 300 carrot SSR markers and their characterization at various levels.
Evaluation of microsatellites isolated from both DNA sources in subsets of 7 carrot F2 mapping populations revealed that SSRs from the hybridization-based method were longer, had more repeat units and were more polymorphic than SSRs isolated by sequence search. Overall, 196 SSRs (65.1%) were polymorphic in at least one mapping population, and the percentage of polymophic SSRs across F2 populations ranged from 17.8 to 24.7. Polymorphic markers in one family were evaluated in the entire F2, allowing the genetic mapping of 55 SSRs (38 codominant) onto the carrot reference map. The SSR loci were distributed throughout all 9 carrot linkage groups (LGs), with 2 to 9 SSRs/LG. In addition, SSR evaluations in carrot-related taxa indicated that a significant fraction of the carrot SSRs transfer successfully across Apiaceae, with heterologous amplification success rate decreasing with the target-species evolutionary distance from carrot. SSR diversity evaluated in a collection of 65 D. carota accessions revealed a high level of polymorphism for these selected loci, with an average of 19 alleles/locus and 0.84 expected heterozygosity.
The addition of 55 SSRs to the carrot map, together with marker characterizations in six other mapping populations, will facilitate future comparative mapping studies and integration of carrot maps. The markers developed herein will be a valuable resource for assisting breeding, genetic, diversity, and genomic studies of carrot and other Apiaceae.
PMCID: PMC3162538  PMID: 21806822
14.  Detection of Vi-Negative Salmonella enterica Serovar Typhi in the Peripheral Blood of Patients with Typhoid Fever in the Faisalabad Region of Pakistan 
Journal of Clinical Microbiology  2005;43(9):4418-4425.
The synthesis and transportation proteins of the Vi capsular polysaccharide of Salmonella enterica serovar Typhi (serovar Typhi) are encoded by the viaB operon, which resides on a 134-kb pathogenicity island known as SPI-7. In recent years, Vi-negative strains of serovar Typhi have been reported in regions where typhoid fever is endemic. However, because Vi negativity can arise during in vitro passage, the clinical significance of Vi-negative serovar Typhi is not clear. To investigate the loss of Vi expression at the genetic level, 60 stored strains of serovar Typhi from the Faisalabad region of Pakistan were analyzed by PCR for the presence of SPI-7 and two genes essential for Vi production: tviA and tviB. Nine of the sixty strains analyzed (15%) tested negative for both tviA and tviB; only two of these strains lacked SPI-7. In order to investigate whether this phenomenon occurred in vivo, blood samples from patients with the clinical symptoms of typhoid fever were also investigated. Of 48 blood samples tested, 42 tested positive by fliC PCR for serovar Typhi; 4 of these were negative for tviA and tviB. Three of these samples tested positive for SPI-7. These results demonstrate that viaB-negative, SPI-7-positive serovar Typhi is naturally occurring and can be detected by PCR in the peripheral blood of typhoid patients in this region. The method described here can be used to monitor the incidence of Vi-negative serovar Typhi in regions where the Vi vaccine is used.
PMCID: PMC1234127  PMID: 16145086

Results 1-14 (14)