Neopterin has been extensively used as a clinical marker of immune activation during inflammation in a wide range of conditions and stresses. However, the analysis of neopterin alone neglects the cellular reactions that generate it in response to interferon-γ. Neopterin is the oxidation product of 7,8-dihydroneopterin, which is a potent antioxidant generated by interferon-γ-activated macrophages. 7,8-Dihydroneopterin can protect macrophage cells from a range of oxidants through a scavenging reaction that generates either neopterin or dihydroxanthopterin, depending on the oxidant. Therefore, plasma and urinary neopterin levels are dependent on both macrophage activation to generate 7,8-dihydroneopterin and subsequent oxidation to neopterin. This relationship is clearly shown in studies of exercise and impact-induced injury during intense contact sport. Here, we argue that neopterin and total neopterin, which is the combined value of 7,8-dihydroneopterin and neopterin, could provide a more comprehensive analysis of clinical inflammation than neopterin alone. The level of clinical inflammation occurring as a result of physical trauma, cardiovascular disease, cancer, bacterial, parasitic infections, and viral infections is often assessed by measuring the concentration of plasma and urinary neopterin ,2,3,4,5.The convenience of measuring neopterin ignores the basic biology that macrophages do not enzymatically synthesize neopterin. Neopterin is an oxidized form of 7,8-dihydroneopterin, a product of γ-interferon-mediated upregulation of GTP cyclohydrolase I (GTPCH1) . TherefoInterferons α and γ, lipopolysaccharide, and possibly phorbol ester all upregulate the activity of monocyte-derived macrophage GTPCH1 . GTPCH1 In turn, 7,8-dihydroneopterin and neopterin are readily transported in either direction across the cell membrane. In cell culture, the addition of 7,8-dihydroneopterin to an incubation media results in the relatively rapid accumulation of 7,8-dihydroneopterin within the cells .7,8-Dihydroneopterin is a potent radical scavenging and chain-breaking antioxidant, which can out compete α-tocopherol for the lipid peroxyl radical during low-density lipoprotein (LDL) oxidation, even though it is water soluble . Peroxyl7,8-Dihydroneopterin, but not neopterin or xanthopterin, downregulates the level of CD36, which is the primary scavenger receptor responsible for the uptake of oxidized LDL and foam cell formation in atherosclerosis ,39. OxLDNeopterin has been reported to activate inducible nitric oxide synthase (iNOS) in rat smooth muscle cells , but supThe evidence suggests that measuring either neopterin or 7,8-dihydroneopterin alone cannot provide a complete picture of oxidative stress or immune activation. Rather, the combination of both biomarkers elucidates a more robust mechanism of the inflammatory process, with an analysis of neopterin being a measure of the oxidative status within the cells, and 7,8-dihydroneopterin being a measure of interferon-mediated cellular activation. Moreover, measuring the ratio between 7,8-dihydroneopterin and neopterin may elucidate differences in overall oxidative stress versus immune activation in different disease models and physical trauma.Neopterin was first isolated from human urine in 1967  and usedAlthough ELISA offers high throughput analysis within hospitals, small-scale clinical and research laboratory analysis have used HPLC. Since neopterin is highly fluorescent, nanomolar concentrations can be reliably detected after separation from other compounds. C18 has been popular, but we have found isocratic analysis on strong cation exchange (SCX) columns at pH 2.5 that were excellent for urine analysis ,35. HoweAlthough an abundance of detection methodologies is promising, sample preparation still remains an important aspect. Since urine contains very low levels of proteins, and HPLC columns have become relatively inexpensive compared to sample clean-up costs, an HPLC analysis of urine usually involves the direct injection of diluted urine into the HPLC . In compIn spite of 7,8-dihydroneopterin’s biosynthetic heritage providing potential for an excellent marker of immune activation, it has been widely neglected in clinical or experimental research. 7,8-Dihydroneopterin’s low fluorescence and nM concentration in plasma make it relatively difficult to detect. 7,8-Dihydroneopterin can be easily detected at μM concentration by its absorbance at 254 nm, but in clinical samples, it is usually at the nM level. There is also no ELISA kit that is commercially available for 7,8-dihydroneopterin, kits are only available for neopterin. 7,8-Dihydroneopterin is also both heat and UV light-sensitive in comparison to neopterin .The usual approach to measure 7,8-dihydroneopterin is oxidation to neopterin using an acidic iodine solution ,13,58 orAnother reason that neopterin has been favored over total neopterin analysis is because 7,8-dihydroneopterin can be labile. In air-saturated solution at 25 °C, 7% of the 7,8-dihydroneopterin was lost over 4 h . This raThe two-step process to measure 7,8-dihydroneopterin can be avoided using HPLC coupled with mass spectrometry (LC-MS or LC-tandem mass spectrometry (LC-MS/MS)) ,59. The While neopterin has been measured repeatedly as an assessment of macrophage activation and infiltration for several decades ,61,62,63Recently, neopterin and 7,8-dihydroneopterin have been used as indicators of immune system activation in sport and exercise medicine, and are gaining momentum over other traditional inflammatory markers . Their bNeopterin has been routinely measured in several high-intensity exercise studies. Neopterin has been shown to rise in response to high-intensity cycling , ultra-eTypically, exercise studies have only measured neopterin ,7. WhilsRecent studies examining impact-induced trauma during exercise have begun to elucidate the importance of measuring neopterin in addition to 7,8-dihydroneopterin. The combined analysis provides a comprehensive overview of exercise-induced changes in oxidative status and immune system activation ,71,72. FTo complicate the exercise-related research on neopterin, recent evidence ,71,72 haCollectively, sport and exercise research that have measured neopterin and 7,8-dihydroneopterin have provided evidence that the change in one does not necessarily result in a change in the other. To critically and correctly evaluate a clinical outcome, treatment efficacy, or the oxidative status/immune system activation of an individual, neopterin and 7,8-dihydroneopterin should be simultaneously measured. There is a clear need to bring the analysis of neopterin and 7,8-dihydroneopterin from sport and exercise medicine back into the classical clinical environment. The measurement of neopterin and total neopterin would provide a clearer and more sensitive measure of patient oxidative stress and inflammation during clinical events.


Zephyranthes concolor (Lindl.) Benth. & Hook. f. (Amaryllidaceae), an endemic species to Mexico, were found to contain the alkaloids chlidanthine, galanthamine, galanthamine N-oxide, lycorine, galwesine, and epinorgalanthamine. Since currently only partial and low resolution 1H-NMR data for chlidanthine acetate are available, and none for chlidanthine, its 1D and 2D high resolution 1H- and 13C-NMR spectra were recorded. Unambiguous assignations were achieved with HMBC, and HSQC experiments, and its structure was corroborated by X-ray diffraction. Minimum energy conformation for structures of chlidanthine, and its positional isomer galanthamine, were calculated by molecular modelling. Galanthamine is a well known acetylcholinesterase inhibitor; therefore, the isolated alkaloids were tested for this activity. Chlidanthine and galanthamine N-oxide inhibited electric eel acetylcholinesterase , indicating they are about five times less potent than galanthamine, while galwesine was inactive at 10−3 M. Inhibitory activity of HIV-1 replication, and cytotoxicity of the isolated alkaloids were evaluated in human MT-4 cells; however, the alkaloids showed poor activity as compared with standard anti-HIV drugs, but most of them were not cytotoxic.The bulbs and aerial parts of  Zephyranthes Herb.  is composed of approximately 60 species distributed in the American continent. A number of species are cultivated due to their gorgeous flowers, and are known by plant breeders as “rain lilies”, owing this name to their tendency to flower after a rain period D20 = −129° . IR  νmax cm−1 2923, 2853, 1604, 1460, 1254, 1088. EI-MS m/z (%): 287(100) M+ (C17H21NO), 272(9) M+−Me, 256(65) M+−OMe, 244(10), 230(6), 225(14), 212(29), 202(45) (C17H12NO2), 115(42), 77(39). For 1H- and 13C-NMR spectral data see Galanthamine (2). The 1H- and 13C-NMR spectra data were as reported by Carrol et al.  (DNTB) were obtained from Sigma Aldrich Chemie . Fifty microliters of AChE solution (500 U/mL) in phosphate buffer , and 50 μL of the compound dissolved in the same buffer were added to each of the wells. The plates were incubated for 30 min at room temperature before addition of 100 μL of the substrate solution . Absorbance was recorded in a Labsystems Microplate Reader  at 405 nm after 6 min. Enzyme activity was calculated as percentage compared to an assay using a buffer without the tested compound. The AChE inhibitory data were analysed with the Prism software package .The assay for measuring acetylcholinesterase (AChE) inhibitory activity was performed in 96 well microtitre plates as previously described ,15. EC50Anti-HIV activity and cytotoxicity were evaluated in human MT-4 cells based on viability of cells that have been infected or not infected with HIV-1 following exposure to various concentrations of the test compound according to the procedure previously described ,36. AfteZephyranthes concolor contain the alkaloids chlidanthine, galanthamine, galanthamine N-oxide, lycorine, galwesine, and epinorgalanthamine. The structure of chlidanthine was determined by 1D and 2D high resolution 1H-, 13C-NMR spectra, and X-ray diffraction. Minimum energy conformation for structures of galanthamine, and its structural isomer chlidanthine were calculated by molecular modelling, suggesting structure solvatation plays a role in the conformation distributions. Chlidanthine and galanthamine N-oxide inhibited electric eel acetylcholinesterase, but were about five times less potent than galanthamine, while galwesine was inactive at 10−3 M. Inhibitory activity of HIV-1 replication, and cytotoxicity of the isolated alkaloids were evaluated in human MT-4 cells; however, the alkaloids showed poor activity as compared with standard anti-HIV drugs, although most of them were not cytotoxic.The bulbs and aerial parts of


Phyllanthus (L.) is one of the most important groups of plants belonging to the Phyllantaceae family. Phyllanthus urinaria (L.) is an annual perennial herbal species found in tropical Asia, America, China, and the Indian Ocean islands. P. urinaria is used in folk medicine as a cure to treat jaundice, diabetes, malaria, and liver diseases. This review provides traditional knowledge, phytochemistry, and biological activities of P. urinaria. The literature reviewed for this article was obtained from the Web of Science, SciFinder, PubMed, ScienceDirect, and Google Scholar journal papers published prior to December 2017. Phytochemical investigations reveal that the plant is a rich source of lignans, tannins, flavonoids, phenolics, terpenoids, and other secondary metabolites. Pharmacological activities include anticancer, hepatoprotective, antidiabetic, antimicrobial, and cardioprotective effects. Thus, this present review summarizes the phytochemical constituents and their biological activities including biological studies on various crude extracts and fractions both in vitro and in vivo, and on clinical trial information about P. urinaria. This review compiles 93 naturally occurring compounds from P. urinaria along with their structures and pharmacological activities. The review is expected to stimulate further research on P. urinaria, and its pharmacological potential to yield novel therapeutic agents.The genus  Traditional or indigenous medicine denotes medical practices developed by local ethnic people using natural herbs. Different world locations have their own history of traditional medicine. For example, Ayurveda medicine originated from Southeast Asia, Unani medicine originated from Arab countries in the Middle East, and acupuncture and traditional Chinese medicine (TCM) originated from China  belongs to a family of flowering plants Phyllanthaceae and consists of more than 1000 species widely distributed in various parts of the world . Previous reviews broadly highlight the biological activities of Phyllanthus species, mostly from P. amurus Schum. & Thonn., P. emblica L. or P. niruri L. , decoction of the whole plant of P. urinaria (Chinese name: Yexiazhu) can clear heat-toxin and remove dampness so it is employed to treat jaundice, enteritis, diarrhea, and dropsy  by reduced blood vessel density, matrix induced tube formation, and cell migration  through extracellular signal-related kinase (ERK) and hypoxia pathways  cancer cell line through MAPKs, Myc/Max, NFκB, and hypoxia pathways  cells through decreased expression of matrix MMP-2 and MMP-9, as well as transcription of MMP-2 mRNA, suggesting suppression of the function of MMPs by extracts [ethanol/water (1:1)] obtained from P. urinaria leaves  have significant growth inhibitory potential against various types of cancer cells nt Table . ReporteP. urinaria extracts in various cancer cell lines in vitro. An aqueous P. urinaria whole plant extract has anti-angiogenesis and reduced tumor growth in Lewis lung carcinoma in vivo -2 infection in vitro  DNA synthesis in HBV wild type and LMV-resistant-infected HepG2 cells via the COX-2 and IL-6 signaling pathways -induced cytotoxicity. An ethanolic extract of P. urinaria whole plant attenuates the acetaminophen-induced hepatotoxicity and inhibition of the cytochrome P450 CYP2E1 enzyme in mice , contains corilagin, flavonoids and polysaccharides; it attenuates steatohepatitis in cultured hepatocytes in vitro and in methionine-and-choline-deficient diet–fed mice in vivo , induction of fatty acid oxidation (upregulation of CYP4a10 and suppression of C/EBPβ), and antioxidant properties (reduced CYP2e1 expression) -radical assay with an SC50 (50%-scavenging concentrations) value of 14.3 mg/mL). The presence of flavonoids, tannins and phenolic compounds in P. urinaria suggest that they contribute the observed antioxidant activity. P. urinaria extracts have anti-nociceptive effects in mice , coxsackie virus A16, and CA16  and HSV-2 with selective index (SI) value >33.6 [(SI = 50% cytotoxic concentration (CC50)/ half inhibitory concentration (IC50)]; the P. urinaria extract may act against the early infection stage and the replication stage in cells in vitro  by 24%, after three h.  indicates that the methanolic extract of P. urinaria is as active as the dichloromethane extract  strains of P. falciparum with an IC50 = 4.1 μg/mL  diseases, and patients often combine herbal products with CV medications. Extracts of ts Table . The ethP. urinaria whole plant stimulate antiarthritic activity in vitro anthracene (DMBA)-induced skin papillomagenesis in vivo , urinatetralin (7), dextrobursehernin (11), and urinaligran (12) , (-)-syringaresinol (18) and (-)-episyringaresinol (19) , is traditionally applied in the treatment of many liver diseases and has antioxidant, anti-inflammatory, immunomodulatory, and hepatoprotective activities (Table 1) attenuates the CCl4 and galactosamine induced cytotoxicity in rat hepatocytes  has cytotoxic effects in CHO and J774 cell lines with IC50 values of 8.07 and 6.00 μM, respectively  and heliobuphthalmin lactone (10) against CHO and J774 cell lines with IC50 values ranging from 15.82–41.30 μM  has anti-proliferative properties in Hep2, EL-1 monocytes, HeLa and MCP7 cells, and induces apoptosis through inhibited telomerase activity and activation of c-myc and caspase 3 and 8  , corilagin (30), isostrictinin (31) chebulagic acid (32), phyllanthusiin C (33), phyllanthusiin B (34) and phyllanthusiin U (35) are identified  are identified as markers of P. urinaria , and human enterovirus 71 (EV71)-induced cytotoxicity in Vero cells with IC50 = 5.6 and 32.33 μg/mL, respectively  and 30 isolated from the acetone extract of P. urinaria act differently in suppressing HSV infection. The isolate 30 did not affect HSV-1 or HSV-2 infection, but compound 38 prevented HSV-2 infection with no effect on HSV-1 replication  has anti-inflammatory activity in cystic fibrosis bronchial IB3-1 cells involving inhibition of NF-κB/DNA interactions, IL-8 gene expression, and MCP-1 and RANTES secretion  which contain catechin and epicatechin oligomers. Tannins interact with one or more protein molecules to form water insoluble complexes. Tannins have various beneficial biological activities including anticancer, cardio-protective, antimicrobial, antioxidant and free radical scavenging activities  and quercetin 3-O-α-L-rhamnopyranoside (49) have been isolated  was isolated from the methanolic extract of P. urinaria  and are found in most fruits and vegetables. Flavonoids have various pharmacological activities including anticancer, anti-inflammatory, antioxidant, anti-diabetic, and antiviral activites through various cell-signaling pathways , methylgallate (58), and methyl brevifolincarboxylate (55) , and (E)-3--2-propenoic acid (68) were isolated from the n-butanol fraction from methanolic extract obtained from whole plants of P. urinaria , brevifolin carboxylic acid (54), and ellagic acid (66) were identified as markers of P. urinaria , 54, 56, 66, and Phyllanthusiin E (70) are also identified  was isolated from ethanolic extract obtained from whole plants of P. urinaria  was isolated and has antinociceptive activity in vivo  has no noticeable effect on HBV replication and its polymerase activity or on HBsAg secretion. However, it potently inhibits HBeAg secretion in HepG2 2.2.15 cells with an IC50 of 0.07 μg/mL , brevifolin (53) and its derivatives , gallic acid (56), and its derivatives (57–60) and ellagic acid (66).Phenolic compounds are the major group of phytochemicals that include at least one aromatic ring, with one or more hydroxyl groups attached. Phytochemical investigation of ethanolic extract from whole plants of 72–74), two diterpenoids (75 and 76), two sesquiterpenes (77 and 78), two monoterpenes (79 and 80), and four sterols (81–84) have been isolated from the extracts of P. urinaria  that has anti-HCV activity through inactivation of virus particles, revocation of HCV attachment and reduced viral fusion  has anti-inflammatory, anti-nociceptive, antimicrobial, and anti-apoptotic activities  and its derivatives have therapeutic potential against various types of cancers in vitro and in vivo  are compounds derived from one or more five-carbon isoprene units. These compounds represent the most diverse class of beneficial phytochemicals with anticancer, anti-cardiovascular, anti-Alzheimers, and anti-malarial activities. The terpenoids such as taxol, artemisinin, and ginkgolides have therapeutic effects on a variety of diseases -cucurbic acid (85), (+)-methyl cucurbate (86), methyl -2-(5′-hydroxy-pent-2′-enyl)-3-oxocyclopentaneacetate (87), and 5-hydroxymethyl-2-furaldehyde (93)  that stimulates leaf closing of P. urinaria in the daytime, without affecting other nyctinastic plants  through an improved immune system, reversion of liver fibrosis, blockage of the induced hepatocarcinoma cell cycle and inhibition of angiogenesis. The HBV-DNA levels decrease ≥2 log in 22.2% (10/45) of patients in the treatment group compared with the control group at 5.0% (2/40). The number of antibodies that test positive in the treated group is lower (1.08 ± 1.01) after the treatment period of 24 months compared with the control group (2.11 ± 1.12) , 2 g (0.33 ± 1.08), or 3 g (0.85 ± 1.30) compared to a placebo (0.28 ± 0.85). Also there was no difference in the HBeAg conversion and ALT normalization of treated compared to control groups  and non-alcoholic fatty liver disease (NAFLD). Histologically, there is a minor reduction in steatosis and hepatocyte ballooning in the treated group, however, it is not significant. Perhaps, P. urinaria might not be a suitable agent to treat NASH . There is no difference in toxicological measurements between treated and control groups; in both groups some subjects experienced mild negative effects.Little data is available regarding the toxicity profiles of n et al.  demonstrP. urinaria. There is evidence that the crude extracts and pure compounds found within P. urinaria have anticancer, hepatoprotective, antimicrobial, antidiabetic, and cardioprotective activities through various signaling pathways. Although the chemical structure and its biological potential of some of the constituents are known, generally, the mechanisms of action need to be investigated for further development into therapeutics.This review summarizes information regarding the traditional uses of phytochemicals, pharmacological activities of crude extracts as well as pure compounds, analysis of active compounds, and clinical trials related to P. urinaria and to identify the bioactive molecules responsible for the pharmacological activities. If possible, specific targets  need to be identified. The reported clinical data for P. urinaria against HBV is limited and consequently limits the use of herbal medicines to treat chronic liver disease. The compounds brevifolin and its derivatives, corilagin, ellagic acid, gallic acid, geraniin, loliolide, phyllanthin may be drug candidates for treating liver diseases because of their potent antiviral activites including anti-hepatitis activity. The high concentration of these compounds in P. urinaria suggests their use and indicates that studies are needed to assess the absorption, distribution, metabolism, and excretion of candidate compounds. Mechanism of action studies on the liver protecting effect of P. urinaria preparations and purified compounds when combined with conventional medicines, are also expected to lead the way in the discovery of new agents with improved pharmacological properties.Systematic efficacy studies are necessary to examine standardized extracts of P. urinaria preparations and toxicological studies. Toxicological studies need to address the mycotoxin, heavy metal, and pesticide concentrations as well as the general toxicity of P. urinaria extracts and purified compounds. Attempts need to be made to gain regulatory approval of P. urinaria preparations as nutraceuticals or medicinal drugs.The herbal medicines cultivated in different geographical regions differ in their composition as well as their therapeutic effects demanding quality control of MG wrote the manuscript. S-TD edited the manuscript. Both authors have seen and agreed on the finally submitted version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SC and handling Editor declared their shared affiliation.


The present study was, therefore, designed to investigate the prevalence and epidemiology of NDM-1 producers in Quetta, Pakistan.Extensive drug resistant Gram-negative bacilli, harboring New Delhi metallo-β-lactamase-1 (NDM-1bla gene was recognized by Polymerase Chain Reaction (PCR).This study was carried out in Microbiology Laboratory, Bolan Medical Complex Hospital Quetta, Biotechnology laboratory, BUITEMS Quetta and Hi-tech laboratory, CASVAB, University of Balochistan, Quetta, from March to June 2018, during the hot season. Biochemical and molecular approaches were applied for the identification of bacterial isolates. Minimum Inhibitory Concentrations (MICs) were determined using E-test method. Carbapenemase activity was ascertained by Modified Hodge Test (MHT) and the presence of blaNDM-1 harboring isolates of three different species namely Morganella morganii (n=2) Enterobacter cloacae (n=2) and Citrobacter freundii (n=1), from 300 pus samples. These isolates were found extensive drug resistant (XDR). Strikingly, two isolates of M. morganii were displaying resistance against 23 antibiotics of sulphonamides, aminoglycosides, polypeptide, monobactams, tetracyclines, quinolones, macrolides, cephalosporins, phosphonic acid and β-lactams groups, suggesting Pan Drug Resistance (PDR).We isolated five M. morganii producing NDM-1 in the province of Balochistan, Pakistan. The presence of blaNDM-1 in different bacterial species and their extensive rather pan drug resistance pattern poses a momentous clinical threat.This is the first report on emergence of PDR strain of  M. morganii and E. cloacae which are members of Enterobacteriaceae family play a significant role in causing nosocomial infections like, wounds, skin, soft tissue, urinary tract, respiratory tract, and biliary tract, mostly in immunocompromised patients. NDM-1 was reported from different hospitals in the UK in Morganella morganii, Citrobacter freundii, Enterobacter cloacae, Providencia, Klebsiellapneumonia and Escherichia coli.6Carbapenem group of antibiotics is one of the most effective drugs that has been used as a last remedy for the treatment of infections caused by Multi Drug Resistant (MDR) Gram negative bacilli.blaNDM-1 has been reported.Enterobacteriaceae, raised with the ability of rapid clonal distribution due to its presence on mobile elements such as plasmids.blaNDM-1from different parts of Pakistan.United Kingdom, Canada, France, China, Japan, Oman, Iraq, Bangladesh and United States are one of the few other countries from where Three hundred patients of both genders, adults and children from surgical wards, burn wards, and burn intensive care units (ICU’s) of three tertiary care hospitals  in Quetta were selected in this study. Detailed history was taken from each patient regarding, wound, time and duration of pus discharge, previous antibiotic therapy, pre-existing clinical complications and the areas/climate from where they belonged to. Those patients were preferred for the study whose pus was still oozing from their wounds after several antibiotic therapies. Antibiotic therapy of the patients was discontinued for 72 hours before sample collection.This study was carried out in Microbiology Laboratory, Bolan Medical Complex Hospital Quetta, Biotechnology laboratory, BUITEMS Quetta and Hi-tech laboratory, CASVAB, University of Balochistan, Quetta, from March to June 2018, during the hot season.Ethical approval for the present study was taken from the Institutional Bioethical Committee Bolan Medical Complex Hospital, PMRC R.No G 263 Quetta Balochistan.Pus samples in sterile 5cc B.D syringes (USA) and with sterile cotton swabs (prepared in lab) from surgical/burn wounds of the patients were taken aseptically. The surface area of the wound was cleaned with sterilized cotton. Efforts were made to collect fresh pus from inside the wound after applying slight pressure. Samples were labeled and transported to the Microbiology laboratory immediately. All the samples were inoculated within one hour of collection.http://www.biomerieux-usa.com/clinical/api). Thermo Scientific Genomic Purification Kit, Lithuania, was used for DNA extraction, according to the manufacturer’s instructions. Internal fragment of 1500 bp of 16S rDNA gene was amplified using universal primers, 27F-5’- AGA GTT TGA TCC TGG CTC AG -3’ and RD1-5’- AAG GAG GTG ATC CAG CC -3’ with Initial denaturation 95°C for 2 min, followed by 35 cycles of 30 sec. at 95°C, 30 sec. at 55°C and 2 min at 72°C. Final extension was set at 72°C for 10 min.11Conventional microbiological procedures were adopted for bacterial isolation from pus samples. Each sample was streaked simultaneously on MacConkey and Blood agar plates  followed by incubation aerobically at 37°C for 24 hours.10 Plates were observed for bacterial colonies and the isolated colonies were further triple cloned. Isolates were identified by analytical profile index, API 20E system  according to the manufacturer’s instructions  guidelines.Klebsiella pneumoniae BBA 1706 (negative control), Klebsiella pneumoniae BBA 1705 (positive control) and Escherichia coli 25922, were used in the assay.Phenotypic detection of carbapenemase enzyme activity of bacterial isolates was achieved by Modified Hodge Test (MHT), as described by (CLSI).blaNDM-1 gene was amplified with the following set of primers; blaNDM-1 F-5’- GGG CAG TCG CTT CCA ACG GT-3’ and blaNDM-1 R-5’- GTA GTG CTC AGT GTC GGC AT -3’. Conditions for PCR were set to; initial denaturation at 95°C for 5 min, followed by 30 Cycles of 95°C for 40 sec, 58°C for 30 sec, 72°C for 30 sec with final extension 72°C for 5 min.15Plasmid DNA was extracted for selected phenotypically carbapenems resistant isolates using GeneJET Plasmid Miniprep Kit by Thermo Fisher Scientific Lithuania, according to the manufacturer’s instructions. The blaNDM-1 genes of the representative samples was carried out commercially through Macrogen, South Korea. Deduced sequences were aligned using Basic Local Alignment Search Tool (BLAST). NCBI at (https://blast.ncbi.nlm.nih.gov/Blast.cgi).Sequencing of the PCR product of 16S rDNA and Staphylococcus aureus (n=56) and Klebsiella pneumoniae (n=39) as predominant, while Acinetobacter baumannii (n=6) and Morganella morganii (n=2) were the least dominant  showed no bacterial growth whereas, 198 (66%) exhibited bacterial growth on MacConkey and blood agar plates after aerobic incubation at 37°C for 24 hrs. Based on cultural, biochemical and morphological characterization, and using API system, 10 different bacterial types were tentatively identified, which included dominant .Morganella morganii, (n=2), Enterobacter cloacae (n=2) and Citrobacter freundii (n=1). These five isolates were tested against twenty-three available antibiotics from, aminoglycosides, tetracyclines, quinolones, β-lactams, monobactams, sulphonamides, macrolides, cephalosporins, phosphonic acid and polypeptide classes of antimicrobials, showed increased resistance. Strikingly two isolates of M. morganii  exhibited an extreme level of resistance against β-lactam and non β-lactam classes of antimicrobials, whereas out of 23 antibiotics Enterobacter cloacae (Ec-93 and Ec-188) were sensitive to tigecycline and Citrobacter freundii (Cf-276) to polymyxin-B, tigecycline and colistin only. The resistance pattern of these isolates indicated that these are XDR and/or PDR , Enterobacter cloacae (n=2) and Citrobacter freundii (n=1), were subjected to MHT for phenotypic detection of carbapenemase enzyme activity as described by CLSI.blaNDM-1 producers, four gave positive clover-leaf like indentation whereas, isolate Ec-93 was negative. The sensitivity of the test was 80 %.All five isolates, blaNDM-1 producers were successfully amplified for 16S rDNA gene. The amplified products (1500 bp) of the representative isolates were sequenced. The obtained sequences were BLAST to ascertain the molecular based identification and confirmation of the isolates. The retrieved sequences, as a result of BLAST, showed 100 % similarity to the previously reported genes. https://blast.ncbi.nlm.nih.gov/Blast.cgiAll five suspected blaNDM-1 gene. An amplicon size of 475 bp confirmed the existence of blaNDM-1 gene in the tested isolatesblaNDM-1 gene. Representative PCR amplified products were sequenced. The retrieved sequences, as a result of BLAST, showed 99% similarity to the previously reported blaNDM-1 genes. https://blast.ncbi.nlm.nih.gov/Blast.cgiPhenotypically five XDR and/or PDR isolates were amplified by PCR for blaNDM-1), which was initially reported in a Swedish patient of Indian origin in 2008,17New Delhi Metallo- β-lactamase-1 (Enterobacteriaceae (CRE) of three different spp. from three hundred pus samples. blaNDM-1gene was detected in Morganella morganii, (n=2), Enterobacter cloacae (n=2) and Citrobacter freundii (n=1). Of all Enterobacteriaceae in our study, NDM-1 was present in 4.27 % as opposed to 2.7% in Kuwait, 1.2% reported in Pakistan, India and the United Kingdom and 1.1% reported in Vietnam.In the present study we isolated five extensive and/or pan drug resistant NDM-1 producers belonging to carbapenem resistant Morganella morganii to tigecycline and colistin as well.Multi drug resistance was observed in NDM-1 and NDM-5 variants that exhibited resistance to ciprofloxacin, amikacin and aztreonam, and pan drug resistance in other variants like NDM-4, NDM-7 and NDM-9 that showed resistance to nearly all antimicrobials used in clinical practice excluding tigecycline and colistin.Morganella morganii, (67%), Enterobacter cloacae (18%) and Citrobacter freundii (11%) to the carbapenems was quite variable in our case.In an epidemiological report the occurrence of carbapenem resistance in the Asian countries from 2002-2012 was 2.4% to meropenem and 1.9% to imipenem,Enterobacter cloacae (CRE) was substantial against meropenem, ciprofloxacin, gentamycin, imipenem, tobramycin, levofloxacin and cefepime. Remarkably 65.9% were kept in the category of Multi Drug Resistant (MDR), use of their resistance against three or more antibiotic groups.E. cloacae) were sensitive to tigecycline but resistant to polymyxin-B and colistin, showing probable genetic diversity of the isolates.In Southwest China resistance pattern of Morganella morganii indeed show resistance to polymyxin B and E, when isolated from blood and urine specimens was efficiently treated by combination therapy of fosfomycin in double dose and meropenem. Citrobacter freundii WCHCF65 isolated from hospital sewage showed resistance to meropenem, ciprofloxacin, imipenem and ceftazidime and was sensitive to tigecycline, colistin and amikacin,The presence of NDM-1 in diverse microbial species necessitates precise and early detection particularly in view of the limited treatment options available. Addressing the issue, our investigations clearly suggest PCR assays more sensitive (100% sensitivity). than Modified Hodge test (80% sensitivity) in case of NDM-1 carbapenemase producers.Morganella morganii in Pakistan. The present study not only revealed the presence of three different bacterial species harboring blaNDM-1 in the population of Balochistan, the largest province of Pakistan, but also profiled their extensive and pan drug resistance pattern against a range of antibiotics including carbapenems.To the best of our knowledge, this is the maiden report on NDM producers in Balochistan, and pan drug resistant blaNDM-1, showing resistance to a range of antibiotics including carbapenems. Importantly, two isolates of Morganella morganii displayed resistance against all 23 antibiotics used in the study. The spread of blaNDM-1, extensive and pan drug resistance, lack of new antimicrobials particularly in carbapenem resistant Enterobacteriaceae (CRE) is an alarming clinical threat.Our study which intended to interpret the first detection, prevalence and epidemiology of NDM-1producers in clinical samples from Quetta, Pakistan, indicated five isolates harboring MD, NA, KMB and DS designed study outset and experiments, wrote the manuscript and contributed in biological, molecular experiments and sequencing data analysis.SA, AA, DG and MD contributed in sample collection, data acquisition, interpretation and manuscript writing.All authors collectively contributed to the critical review, significant rational amendment in the final manuscript and approval of the article to be published.