Integrons and multidrug resistance across phylogenetic groups of clinical isolates of Escherichia coli.

Objective: This study was aimed to investigate the multidrug resistance patterns in clinical isolates of Escherichia coli and their correlation with integrons and phylogenetic groupings. Methods: A total of 37 clinical E. coli isolates were evaluated for drug resistance patterns by disk diffusion method. Phylogenetic groupings and the presence of integrons among E. coli were determined by multiplex PCR assays. Results: Multidrug resistance was identified in 84% of the clinical isolates of E. coli with higher resistance found against cephalosporins (94.6%) and fluoroquinolones (83.8%), while lower resistance was observed against polymyxins (24.3%) and carbapenems (29.7%). Metallo-ß-lactamases were found in all carbapenem resistant isolates. The phylogenetic group B2 was the most dominant (40.5%), followed by groups A (35.1%), D (13.5%) and B1 (10.8%). Integrons were detected in 25 (67.6%) isolates and intI1, intI2, and intI3 genes were found in 62.2%, 18.9% and 10.8% of isolates respectively. Conclusion: Our results show that phylogenetic classification of E. coli is not relevant with antimicrobial resistance. However, there was strong association between the integron classes and resistance against ß-lactam and fluoroquinolones antimicrobials. Additionally, this study highlighted that the presence of integrons plays a crucial role in the development of multidrug resistance in clinical isolates of E. coli. Most significantly, this is the first report of detection of three classes of integron among clinical isolates of E. coli in Pakistan.

Incorporating dietary fiber from fruit and vegetable waste in meat products: a systematic approach for sustainable meat processing and improving the functional, nutritional and health attributes.

Background: Every year, the food business produces a sizeable amount of waste, including the portions of fruits and vegetables that are inedible, and those that have reached a stage where they are no longer suitable for human consumption. These by-products comprise of components such as natural antioxidants (polyphenols, carotenoid etc.), dietary fiber, and other trace elements, which can provide functionality to food. Due to changing lifestyles, there is an increased demand for ready-to-eat products like sausages, salami, and meat patties. In this line, meat products like buffalo meat sausages and patties are also gaining the interest of consumers because of their rich taste. Meat, however, has a high percentage of fat and is totally deprived of dietary fiber, which poses severe health problems like cardiovascular (CV) and gastrointestinal diseases. The health-conscious consumer is becoming increasingly aware of the importance of balancing flavor and nutrition. Therefore, to overcome this problem, several fruit and vegetable wastes from their respective industries can be successfully incorporated into meat products that provide dietary fiber and play the role of natural antioxidants; this will slow down lipid oxidation and increase the shelf-life of meat products. Methodology: Extensive literature searches have been performed using various scientific search engines. We collected relevant and informative data from subject-specific and recent literature on sustainable food processing of wasted food products. We also looked into the various applications of waste fruit and vegetable products, including cereals, when they are incorporated into meat and meat products. All relevant searches meeting the criteria were included in this review, and exclusion criteria were also set. Results: The pomace and peels of fruits like grapes, pomegranates, cauliflower, sweet lime, and other citrus are some of the most commonly used fruit and vegetable by-products. These vegetable by-products help inhibit oxidation (of both lipids and proteins) and the growth of pathogenic and spoilage bacteria, all without altering the consumer's acceptability of the product on a sensory level. When included in meat products, these by-products have the potential to improve the overall product quality and lengthen its shelf-life under certain circumstances. Conclusion: Cost-effective and easily accessible by-products from the fruit and vegetable processing industries can be used in meat products to enhance their quality features (physicochemical, microbial, sensory, and textural aspects) and health benefits. Additionally, this will provides environmental food sustainability by lowering waste disposal and improving the food's functional efficacy.

Incorporation of betel leaf extract provides oxidative stability and improves phytochemical, textural, sensory and antimicrobial activities of buffalo meat sausages.

The antioxidant effect of betel leaf extract (BLE) on lipid and protein oxidation, microbial count and physicochemical attributes was investigated in meat sausages during refrigerated storage at 4 ± 1 °C. Buffalo meat sausages were developed after incorporating 0, 250, 500 and 750 mg kg-1 of BLE (BLE0, BLE1, BLE2 and BLE3) respectively. The sausages showed no changes in proximate composition due to BLE inclusion, but there was an improvement in microbial quality, color score, textural properties and lipid and protein oxidative stability. Further, higher sensory scores were observed for the BLE-incorporated samples. The images from scanning electron microscopy (SEM) revealed a reduction in surface roughness and unevenness showing microstructure modification in BLE treated sausages compared to the control sausages. Hence, to improve the storage stability and impede the rate of lipid oxidation in sausages, BLE incorporation proved to be an effective strategy.

First Detection of Extensively Drug-Resistant Salmonella Typhi Isolates Harboring VIM and GES Genes for Carbapenem Resistance from Faisalabad, Pakistan.

Rapid emergence of resistance in Salmonella enterica serovar Typhi (Salmonella Typhi) against most of the available therapeutic options for typhoid has rendered its treatment more difficult. This study sought to determine the current scenario of antimicrobial resistance in local isolates of Faisalabad following several treatment failure reports. Out of 300 clinical specimens collected in 2018, 45 isolates were identified as Salmonella Typhi. To assess changes, we compared their antibiogram profile with 31 Salmonella Typhi strains isolated in 2005. The isolates collected during 2018 showed a significant rise in antimicrobial drug resistance as compared with isolates revived from the cultures of 2005, including 15 multidrug-resistant (MDR), 20 extensively drug-resistant, and 14 pan drug-resistant isolates compared with only 8 MDRs from 2005. Notably, in 2018 isolates, resistance to azithromycin was seen in 75% of the isolates. Extended-spectrum beta-lactamase production was detected in 47% of Salmonella Typhi isolates and 18% isolates showed resistance against carbapenems. The sequences of two carbapenemase genes, VIM and GES, found in Salmonella Typhi were submitted in NCBI. The carbapenem resistance is rare in Enterobacteriaceae and probably first time reported in Salmonella Typhi. H58 haplotype was identified in the 2018 Salmonella Typhi isolates and PCR-restriction fragment length polymorphism method identified 16.7% of H58 strains that belonged to lineage I, 19.4% of H58 strains that belonged to lineage II, and the remaining 63.9% that belonged to the node. The regional difference in the antimicrobial resistance trend needs effective epidemiological studies.

Level of biofilm production by Staphylococcus aureus isolates is critical for resistance against most but not all antimicrobial drugs.

Background and Objective: Staphylococcal biofilms cause a wide range of acute and chronic infections, both in hospital and community settings across the world. This study explores biofilm forming propensity among Staphylococcus aureus clinical isolates from Faisalabad, Pakistan and their association with antimicrobial drug resistance. Methods: The study was conducted during July to December 2020. The biofilm forming ability of S. aureus isolates was assessed by crystal violet staining in 96 well plates. Antimicrobial susceptibility was determined by disk diffusion method against ten antimicrobials representing whole spectrum of antimicrobial drugs. Results: All the isolates (n=22) produced biofilm; 14 (63.6%) were strong, and 8 (36.4%) moderate biofilm producers. Comparative data were obtained for moderate and strong biofilm producers. Increased biofilm production did not affect azithromycin, clindamycin and mupirocin. However, stronger biofilm production significantly increased resistant isolates in case of augmentin (23.2%), cefoxitin (17.9%), levofloxacin (26.8%), tetracycline (23.2%), vancomycin (14.3%) and trimethoprim (21.4%). Conclusions: Our findings indicate that the ability to produce large amount of biofilm is an important factor, and S. aureus isolates with this ability, do not require acquisition of drug resistance genes from other bacteria. Our study also provides a guideline for selection of antimicrobials which are not adversely affected by level of biofilm production by various strains of S. aureus.

Immunological characterization of chitosan adjuvanted outer membrane proteins of Salmonella enterica serovar Typhi as multi-epitope typhoid vaccine candidate.

BACKGROUND: Outer membrane proteins (OMPs) of Gram-negative bacteria have been known as potential vaccine targets due to their antigenic properties and host specificity. Here, we focused on the exploration of the immunogenic potential and protective efficacy of total OMPs of Salmonella enterica serovar Typhi due to their multi epitope properties, adjuvanted with nanoporous chitosan particles (NPCPs). The study was designed to extrapolate an effective, low cost prophylactic approach for typhoid fever being getting uncontrolled in Pakistan due to emergence of extensively drug resistant (XDR) strains. METHODS & RESULTS: The OMPs of two S. Typhi variants (with and without Vi capsule) alone and with nanoporous chitosan particles as adjuvant were comparatively analyzed for immunogenic potential in mice. Adaptive immunity was evaluated by ELISA and relative quantification of cytokine gene expression (IL4, IL6, IL9, IL17, IL10, TNF, INF and PPIA as house keeping gene) using RT-qPCR. Statistical analysis was done using Welch's test. The protection was recorded by challenging the immunized mice with 50% ×LD50 of S. Typhi. The Vi + ve-OMPs of S. Typhi showed the most promising results by ELISA and significantly high expression of IL-6, IL-10 and IL-17 and 92.5% protective efficacy with no detectable side effects. CONCLUSION: We can conclude that the OMPs of Vi + ve S. Typhi are the most promising candidates for future typhoid vaccines because of cost effective preparation without expensive purification steps and multi-epitope properties. Chitosan adjuvant may have applications for oral protein based vaccines but found less effective in injectable preparations.

Mutational Diversity in the Quinolone Resistance-Determining Regions of Type-II Topoisomerases of Salmonella Serovars.

Quinolone resistance in bacterial pathogens has primarily been associated with mutations in the quinolone resistance-determining regions (QRDRs) of bacterial type-II topoisomerases, which are DNA gyrase and topoisomerase IV. Depending on the position and type of the mutation (s) in the QRDRs, bacteria either become partially or completely resistant to quinolone. QRDR mutations have been identified and characterized in Salmonella enterica isolates from around the globe, particularly during the last decade, and efforts have been made to understand the propensity of different serovars to carry such mutations. Because there is currently no thorough analysis of the available literature on QRDR mutations in different Salmonella serovars, this review aims to provide a comprehensive picture of the mutational diversity in QRDRs of Salmonella serovars, summarizing the literature related to both typhoidal and non-typhoidal Salmonella serovars with a special emphasis on recent findings. This review will also discuss plasmid-mediated quinolone-resistance determinants with respect to their additive or synergistic contributions with QRDR mutations in imparting elevated quinolone resistance. Finally, the review will assess the contribution of membrane transporter-mediated quinolone efflux to quinolone resistance in strains carrying QRDR mutations. This information should be helpful to guide the routine surveillance of foodborne Salmonella serovars, especially with respect to their spread across countries, as well as to improve laboratory diagnosis of quinolone-resistant Salmonella strains.

Characterization of the multidrug efflux transporter styMdtM from Salmonella enterica serovar Typhi.

Salmonellae are foodborne pathogens and the major cause of gastroenteritis in humans. Salmonellae express multidrug efflux transporters that play a key role in their drug resistance, which is becoming an increasing problem for therapeutic intervention. Despite their biomedical importance, the mechanisms underlying substrate transport by multidrug efflux transporters remain poorly understood. Here, we describe the first characterization of a multidrug transporter belonging to the major facilitator superfamily from the genus Salmonella. We show that several clinical Salmonella Typhi (S. Typhi) isolates constitutively express the styMdtM (STY4874) gene, which encodes a known multidrug-resistance (MDR) transporter. Guided by the structure of the Escherichia coli (E. coli) homolog, we studied two residues critical for substrate transport, Asp25 and Arg111. Mutation of Asp25 to glutamate did not affect the transport function of styMdtM, whereas mutation to alanine reduced its transport activity, suggesting that a negative charge at this position is critical for substrate translocation across the membrane. Substrate-affinity measurements by intrinsic fluorescence spectroscopy showed that the Asp25Ala mutant retained its capacity to bind substrate, albeit at a lower level. Mutation of Arg111 to alanine resulted in a decrease in secondary structure content of the transporter, and mutation to lysine completely destabilized the structure of the transporter. A homology model of styMdtM suggests that Arg111 is important for stabilizing the transmembrane domain by mediating necessary interactions between neighboring helices. Together, our studies provide new structural and mechanistic insights into the Salmonella MDR transporter styMdtM.

High frequency of simultaneous presence of ESBL and carbapenemase producers among nosocomial coliform isolates in Faisalabad, Pakistan.

OBJECTIVES: The objective of the current study was to find prevalence of relevant ESBL and carbapenemase producing genes in nosocomial E. coli and K. pneumoniae isolates and to check phenotypic susceptibility of all ESBL positive isolates to carbapenems. METHODS: Forty ESBL producing clinical isolates of Escherichia coli (n=33) and Klebsiella pneumoniae (n=7) were examined for the presence of ß-lactamase genes (CTX-M, CTX-M-1, 2, 3, 4 and TEM). Carbapenem resistance was checked phenotypically and by presence of blaNDM-1 gene. RESULTS: Nine (27%) were positive for CTX-M genes, and 10 (30%) for TEM among E. coli isolates. Importantly, six isolates showed co-existence of CTX-M and TEM genes. In K. pneumoniae, two (28%) isolates were positive for CTX-M and one (14%) for TEM genes. Eight (24%) E. coli and one (14%) K. pneumoniae isolates were positive for CTX-M-1. Respective figures for CTX-M-4 were three (10%) and one (14%). CTX-M-2 and CTX-M-3 groups were not represented. Twenty (50%) isolates were resistant to both imipenem and meropenem out of which only four isolates expressed blaNDM-1 gene. CONCLUSIONS: The significant presence of both ESBL and carbapenemase producers and co-existence of ESBL and carbapenemases in the same isolates is worrisome.

Selective laser melting processed Ti6Al4V lattices with graded porosities for dental applications.

Dental implants need to support good osseointegration into the surrounding bone for full functionality. Interconnected porous structures have a lower stiffness and larger surface area compared with bulk structures, and therefore are likely to enable better bone-implant fixation. In addition, grading of the porosity may enable large pores for ingrowth on the periphery of an implant and a denser core to maintain mechanical properties. However, given the small diameter of dental implants it is very challenging to achieve gradations in porosity. This paper investigates the use of Selective Laser Melting (SLM) to produce a range of titanium structures with regular and graded porosity using various CAD models. This includes a novel 'Spider Web' design and lattices built on a diamond unit cell. Well-formed interconnecting porous structures were successfully developed in a one-step process. Mechanical testing indicated that the compression stiffness of the samples was within the range for cancellous bone tissue. Characterization by scanning electron microscopy (SEM) and X-ray micro-computed tomography (µCT) indicated the designed porosities were well-replicated. The structures supported bone cell growth and deposition of bone extracellular matrix.

Virulotyping of Salmonella enterica serovar Typhi isolates from Pakistan: Absence of complete SPI-10 in Vi negative isolates.

The pathogenesis of Salmonella enterica serovar Typhi (S. Typhi), the cause of typhoid fever in humans, is mainly attributed to the acquisition of horizontally acquired DNA elements. Salmonella pathogenicity islands (SPIs) are indubitably the most important form of horizontally acquired DNA with respect to pathogenesis of this bacterium. The insertion or deletion of any of these transferrable SPIs may have impact on the virulence potential of S. Typhi. In this study, the virulence potential and genetic relatedness of 35 S. Typhi isolates, collected from 2004 to 2013 was determined by identification of SPI and non-SPI virulence factors through a combination of techniques including virulotyping, Whole Genome Sequencing (WGS), and Variable Number of Tandem Repeats (VNTR) profiling. In order to determine the virulence potential of local S. Typhi isolates, 56 virulence related genes were studied by PCR. These genes are located in the core as well as accessory genome (SPIs and plasmid). Major variations among studied virulence determinants were found in case of SPI-7 and SPI-10 associated genes. On the basis of presence of virulence related genes, the studied S. Typhi isolates from Pakistan were clustered into two virulotypes Vi-positive and Vi-negative. Interestingly, SPI-7 and SPI-10 were collectively absent or present in Vi-negative and Vi-positive strains, respectively. Two Vi-negative and 11 Vi-positive S. Typhi strains were also analyzed by whole genome sequencing (WGS) and their results supported the PCR results. Genetic diversity was tested by VNTR-based molecular typing. All 35 isolates were clustered into five groups. Overall, all Vi-negative isolates were placed in a single group (T5) whereas Vi-positive isolates were grouped into four types. Vi-negative and Vi-positive isolates were mutually exclusive. This is the first report on the comparative distribution of SPI and non-SPI related virulence genes in Vi-negative and Vi-positive S. Typhi isolates with an important finding that SPI-10 is absent in all Vi-negative isolates.

Significance of Vi Negative Isolates of Salmonella Enterica Serovar Typhi.

Typhoid is a major global disease. The causative agent, Salmonella enterica serovar Typhi (S. Typhi) has a capsular antigen called Vi antigen which is traditionally considered to be the main cause of virulence. All the current vaccines are based on Vi antigen. However, the realization of the fact that there are S. Typhi strains which lack Vi antigen but still exist naturally and can cause disease has stirred great scientific interest. It is also interesting to note that their relative prevalence is affected by climatic conditions. Now it is established that Vi positive and Vi negative S. Typhi have different modes of pathogenesis; and as recent studies suggest, different structure of polysaccharide antigens. This means that current vaccines are not effective against a significant number of S. Typhi strains which not only affect the success of vaccination programs but also help in rapid emergence of Vi negative S. Typhi due to natural selection. The focus should be on vaccines based on antigens which are universally present in all S. Typhi. One such candidate is O-specific polysaccharides (OSPs). Successful attempts have been made to prepare conjugate vaccines based on OSPs.

Rapid screening of pyogenic Staphylococcus aureus for confirmation of genus and species, methicillin resistance and virulence factors by using two novel multiplex PCR.

OBJECTIVES: Emergence of methicillin resistant Staphylococcus aureus (MRSA) is a major medical problem of current era. These bacteria are resistant to most drugs and rapid diagnosis can provide a clear guideline to clinicians. They possess specific virulence factors and relevant information can be very useful. We designed this study to develop multiplex PCRs to provide rapid information. METHODS: We studied 60 Staphylococcus aureus isolates and detected methicillin resistance by cefoxitin sensitivity and targeting of mecA gene. After initial studies with uniplex PCRs we optimized two multiplex PCRs with highly reproducible results. The first multiplex PCR was developed to confirm genus, species and methicillin resistance simultaneously, and the second multiplex PCR was for screening of virulence factors. RESULTS: We found 38.33% isolates as methicillin resistant. α -toxin, the major cytotoxic factor, was detected in 40% whereas ß-hemolysin was found in 25% cases. Panton Valentine leucocidin was detected in 8.33% and toxic shock syndrome toxin in5% cases. The results of uniplex and multiplex PCRs were highly compatible. CONCLUSIONS: These two multiplex PCRs when run simultaneously can provide vital information about methicillin resistance and virulence status of the isolate within a few hours as compared to several days needed by routine procedures.

Detection of high levels of resistance to linezolid and vancomycin in Staphylococcus aureus.

Both methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) are rapidly overcoming the current array of drugs. One hundred and fifty isolates from a hospital were studied for resistance towards linezolid and vancomycin. Fifty-four (36.0 %) isolates were MRSA. Both MRSA and MSSA showed high resistance towards linezolid when using the disc diffusion method, with the figures being 48.1 and 29.2 %, respectively. The figures for the E-test were 46.3 and 27.0 %, respectively. The vancomycin resistance was remarkable in MRSA (14.8 %), but relatively low in MSSA (3.1 %). The E-test results were 13.0 and 4.16 %, respectively. The cfr gene was detected in 78 % of linezolid-resistant isolates and the vanA operon was detected in 74 % of vancomycin-resistant isolates. This level of resistance against linezolid and vancomycin is unprecedented. These results are alarming and highlight the threat of non-treatable S. aureus strains.

Oral Administration of Ethanolamine Glycerophospholipid Containing a High Level of Plasmalogen Improves Memory Impairment in Amyloid ß-Infused Rats.

Ethanolamine plasmalogen (PlsEtn), a major phospholipid in neuronal membranes [60-90 mol% of ethanolamine glycerophospholipid (EtnGpl)], is specifically decreased in brains from patients with Alzheimer's disease (AD). The present study investigated how PlsEtn administration affects cognitive deficits and lipid composition in an animal model of AD. AD model rats were infused with amyloid-ß (Aß) into the cerebral ventricle and divided into three groups. Control, Egg, and Ascidian groups were then orally administered vehicle, egg yolk EtnGpl (260 µmol as EtnGpl/kg BW/day; 10 µmol as PlsEtn/kg BW/day), or ascidian viscera EtnGpl (260 µmol as EtnGpl/kg BW/day; 209 µmol as PlsEtn/kg BW/day), respectively. After 4 weeks of dosing, Aß-infused rats were tested for learning ability in an 8-arm radial maze. The administration of ascidian viscera EtnGpl improved both reference and working memory-related learning abilities. In lipid analysis, the Ascidian group showed higher levels of PlsEtn species in the plasma, erythrocytes, and liver when compared to other groups. In addition, although there were no differences at levels of total plasmalogen including choline plasmalogen, the Ascidian group had significantly higher levels of 18:0ol/22:6-PlsEtn in the cerebral cortex. These levels of 18:0ol/22:6-PlsEtn in the cerebral cortex were correlated with working memory-related learning ability. Moreover, 18:0ol/22:6-PlsEtn levels in the cerebral cortex showed positive correlations with those in the erythrocytes and liver. In summary, dietary PlsEtn, especially that with 22:6n-3 (docosahexaenoic acid, DHA), may ameliorate learning deficiencies in AD by altering lipid composition in the brain.

Purification and antigenic detection of O-specific polysaccharides of Salmonella enterica serovar Paratyphi A isolate from Pakistan: an emerging threat.

BACKGROUND: Paratyphoid fever caused by Salmonella enterica serovar Paratyphi A is becoming a serious health problem in Asian countries particularly Pakistan, China and India and situation is aggravated by current unavailability of a licensed vaccine. This study was designed to purify the O-specific polysaccharides (OSP) produced by an isolate of Salmonella Paratyphi A from Pakistan and detect antigenicity of extracted lipopolysaccharide (LPS) and purified OSP pioneerly in South Asian region as candidate for conjugate vaccine preparation. RESULTS: S. Paratyphi A isolates were identified through PCR using primers of fliC-a gene (329 bp) and confirmed via nested PCR using fliC-nested primers (289 bp). Yield of the LPS of S. Paratyphi A isolate was 40 mg/L of the bacterial culture using hot phenol method. The purified LPS revealed the characteristic ladder like pattern of S. Paratyphi A LPS on SDS-PAGE with silver staining. Purified OSP obtained by acid hydrolysis yielded 23 mg/L of culture broth and was not detected by silver staining. Antigenic interaction of the purified LPS and OSP with hyper immune mice sera was confirmed by single precipitin line evaluated through immunodiffusion assay. The antigenicity was found well intact. CONCLUSIONS: The purified antigenic OSP from S. Paratyphi A may have the potential to be coupled with a carrier protein to develop low cost conjugate vaccine candidates against S. Paratyphi A in paratyphoid endemic regions.

Molecular detection of antimicrobial resistance in local isolates of Staphylococcus epidermidis from urinary tract infections in Faisalabad region of Pakistan.

Staphylococci are one of the foremost causes of urinary tract infections (UTIs) in humans. The emergence of multiple drug resistance (MDR) among Staphylococci poses serious challenges in antimicrobial therapy for UTIs. Most work has been done on S. aureus while coagulase negative Staphylococci (mainly S. epidermidis) are often neglected. This study was conducted to establish a baseline profile of drug resistance in local S. epidermidis isolates from UTIs. Eighty urine samples were collected from suspected UTIs cases and screened for S. epidermidis. Twenty isolates were suspected as S. epidermidis based on colony morphology and Gram staining. Molecular detection by polymerase chain reaction (PCR) confirmed 13 isolates as S. epidermidis. Using disc diffusion method, phenotypic drug resistance of the isolates was observed towards erythromycin (100 %), gentamycin, azithromycin and tetracycline (92.3 %), ampicillin and oxytetracyclin (84.6 %), amikacin and srteptomycin (76.9 %), methicillin (69.2 %), cephradine, cefaclor and cefazolin (53.8 %) and vancomycin (15.3 %). Eighteen most commonly reported genes responsible for conferring resistance towards these drugs were targeted by PCR: among these tetM gene was found most prevalent (46.1 %) followed by tetK (30.7 %), aac(6')/aph(2") (30.7 %), aacA-aphD (23 %), ermA (23 %), blaZ (23 %), mecA (23 %) blaTEM-1 (23 %), MeccA (23 %) and mecA (15.3 %). No gene fragment for vancomycin resistance was detected. The salient finding was that all S. epidermidis isolates were multiple drugs resistant as they showed resistance against at least three structurally different antimicrobial agents. It is concluded that in addition to the mostly used antimicrobial agent vancomycin, the cephalosporins including cephradine, cefaclor and cefazolin are also the drugs of choice against UTIs caused by S. epidermidis.

Simplest identification, O-specific polysaccharide purification and antigenic evaluation of Salmonella enterica serovar Typhi Vi negative isolate.

Currently licensed typhoid vaccines are based on Vi capsular polysaccharides. Recent molecular reports from typhoid endemic countries state that Salmonella enterica serovar Typhi (S. Typhi) Vi negative strains occur naturally and cause typhoid fever which is indistinguishable from disease caused by Vi positive strains. Vaccine based on Vi polysaccharide may not protect patients if the invading S. Typhi are negative for Vi. The lipopolysaccharide (LPS) is an essential component of S. Typhi outer membrane in which O-specific polysaccharide (OSP) is a protective antigen and universal candidate for vaccine development. In this study, S. Typhi Vi negative isolates were discriminated from Vi positive isolates through a duplex PCR using primers of fliC-d (599bp) and tviA (495bp) genes. The LPS of S. Typhi Vi negative isolates was extracted by hot phenol method and OSP was purified by core hydrolysis. The yield of extracted LPS was 91 mg/L and that of purified OSP was 49.14 mg/L of culture broth. LPS showed ladder like appearance by zinc imidazole staining following SDS-PAGE. Whole cell challenged mice sera were used for in vitro antigenicity evaluation of the purified LPS and OSP. The antigenicity was found adequate by immunodiffusion assay. To our knowledge, this is the first report of purification and antigenic evaluation of LPS of a Vi negative S. Typhi isolate. The purified OSP from S. Typhi Vi negative isolate may be coupled with a carrier protein to produce universal low cost conjugate vaccine candidates for use in typhoid endemic regions.

Comparative growth analysis of capsulated (Vi+) and acapsulated (Vi-) Salmonella typhi isolates in human blood.

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.

Metal nanoparticle assisted polymerase chain reaction for strain typing of Salmonella Typhi.

Salmonella enterica serotype Typhi (S. Typhi) is the causative agent of typhoid fever and remains a major health threat in most of the developing countries. The prompt diagnosis of typhoid directly from the patient's blood requires high level of sensitivity and specificity. Some of us were the first to report PCR based diagnosis of typhoid. This approach has since then been reported by many scientists using different genomic targets. Since the number of bacteria circulating in the blood of a patient can be as low as 0.3 cfu ml(-1), there is always a room for improvement in diagnostic PCR. In the present study, the role of different types of nanoparticles was investigated to improve the existing PCR based methods for diagnosis and strain typing of S. Typhi (targeting Variable Number of Tandem Repeats [VNTR]) by using optimized PCR systems. Three different types of nanoparticles were used i.e., citrate stabilized gold nanoparticles, rhamnolipid stabilized gold and silver nanoparticles, and magnetic iron oxide nanoparticles. The non-specific amplification was significantly reduced in VNTR typing when gold and silver nanoparticles were used in an appropriate concentration. More importantly, the addition of nanoparticles decreased the non-specificity to a significant level in the case of multiplex PCR thus further validating the reliability of PCR for the diagnosis of typhoid.