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Background Recent years, multi drug resistant pathogens and their pathogenicity were increased worldwide due to unauthorized consumption of antibiotics. In addition, correlation between multi drug resistant bacteria and biofilm formation is heightened due to the production of more virulence behavior. There is no better identification methods are available for detection of biofilm producing gram negative bacteria. Materials and methods In this research work, multi drug resistant strains of Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) were identified based on the specific antibiotics and third generation cephalosporin discs by disc diffusion assay. Subsequently, biofilm forming ability of selected pathogens were identified tissue culture plate and tube test. Based on the multi-drug resistant ability and biofilm production, the molecular identification of P. aeruginosa and K. pneumoniae were confirmed by PCR using universal primers. Results and conclusions No zone of inhibition present around the discs of muller hinton agar plates were confirm, selected P. aeruginosa and K. pneumoniae strains were multi drug resistant pathogens. Performed third generation cephalosporin antibiotics were also highly sensitive to selected pathogens of P. aeruginosa and K. pneumoniae. Further, biofilm forming ability of selected P. aeruginosa and K. pneumoniae was confirmed by tissue culture plate and tube methods. Finally, molecular identification of P. aeruginosa and K. pneumoniae was named as P. aeruginosa and K. pneumoniae. Our result was conclude, selected P. aeruginosa and K. pneumoniae as biofilm producing pathogens and also highly resistant to current antibiotics.

Based on the excellent nutrient level, the current study was focused on isolation and anti-bacterial activity of the actinomycetes from marine mangrove soil samples. As result, 10 different strains of actinomycetes strains were identified on actinomycetes isolation agar plates. The identified strains were shown with white, clear, uncontaminated well matured spore producing ability. Based on the initial observation, the isolated colonies were actinomycetes. The partially extracted crude compound shown excellent anti-bacterial activity against P. aeruginosa and K. pneumoniae with 15 mm and 13 mm zone of inhibitions were observed at 500 μL concentrations. The minimum inhibition concentration result was also confirmed the 500 μL concentration against both the tested concentration with high inhibition rate. Then, the intracellular damages, decreased cell growth of the crude actinomycetes extract treated bacterial strains were clearly observed by confocal laser scanning electron microscope. The extracellular damages of bacterial cell wall and shape of the both the pathogens were clearly shown by scanning electron microscope. Therefore, all the results were clearly supported to the partially extracted crude compound and it has excellent anti-bacterial activity against tested multi drug resistant bacteria.

The multi-drug resistant effect of the Gram negative bacteria K. pneumoniae was identified by disc diffusion method using specific UTI panel discs of Kleb 1 HX077 and Kleb 2 HX090 HEXA. Among the multi-drug resistant bacteria, the carbapenem resistant (CR) effect of the K. pneumoniae was screened by specific carbapenem detection antibiotics of HEXA HX066 and HX0103 HEXA by disc diffusion method. In addition, the effective antibiotics were further performed against K. pneumoniae by minimum inhibition concentration method. Further, the carbapenemase genes of VIM 1 and IMP 1 were detected from the isolated strains by multiplex PCR method. Furthermore, the biofilm forming ability of selected carbapenem resistant K. pneumoniae was initially identified by tissue culture plate method and confirmed by exopolysaccharide arrest ability of congo red agar assay. Finally, our result was proved that the identified K. pneumoniae is carbapenemase producing strain, and its virulence was extended with strong biofilm formation.

Urinary tract infections are second most important diseases worldwide due to the increased amount of antibiotic resistant microbes. Among the Gram negative bacteria, P. mirabilis is the dominant biofilm producer in urinary tract infections next to E. coli. Biofilm is a process that produced self-matrix of more virulence pathogens on colloidal surfaces. Based on the above fact, this study was concentrated to inhibit the P. mirabilis biofilm formation by various in-vitro experiments. In the current study, the anti-biofilm effect of essential oils was recovered from the medicinal plant of Solanum nigrum, and confirmed the available essential oils by liquid chromatography-mass spectroscopy analysis. The excellent anti-microbial activity and minimum biofilm inhibition concentration of the essential oils against P. mirabilis was indicated at 200 µg/mL. The absence of viability and altered exopolysaccharide structure of treated cells were showed by biofilm metabolic assay and phenol–sulphuric acid method. The fluorescence differentiation of P. mirabilis treated cells was showed with more damages by confocal laser scanning electron microscope. Further, more morphological changes of essential oils treated cells were differentiated from normal cells by scanning electron microscope. Altogether, the results were reported that the S. nigrum essential oils have anti-biofilm ability.

Background Worldwide, multi-drug resistant Klebsiella pneumoniae (K. pneumonia) and their virulence’s were contributed more in the multi-drug resistant effect. According to the World Health organization report, it is an emerging thread in developing countries and also comes under first ever critical list. In this context, the current study was concentrated on detection of extended spectrum beta lactamase (ESBL) producing strain and their antimicrobial susceptibility study of K. pneumoniae. Materials and methods Firstly, the multi-drug resistant effect of the K. pneumoniae was identified from specific CLSI guidelines recommended antibiotics by disc diffusion method. Consecutively, the primary ESBL identification test was performed using ceftazidime and cefotaxime, followed by double disc combination and phenotypic confirmation tests using ceftazidime/clavulanic acid and cefotaxime/clavulanic acid. Finally, the minimum inhibition concentration of some important sensitive antibiotics were performed against selected K. pneumoniae was confirmed by micro broth dilution method. Results and conclusions The current result was most favorable to selected K. pneumoniae with more multi drug resistant characteristic nature. All the performed antibiotics were almost more sensitive to selected K. pneumoniae. The effective antibiotics of piperacillin was also exhibited more resistant effect against tested bacteria and it cleaved the bacterial enzyme clearly. The present result of primary ESBL identification test result was exhibited with ≤22 mm and ≤27 mm against ceftazidime and cefotaxime were observed respectively. Followed result of double disc combination and phenotypic confirmation experiments results were clearly stated that the selected K. pneumoniae was ESBL producer. The ceftazidime, cefotaxime and ceftazidime/clavulanic acid and cefotaxime/clavulanic acid were exhibited with merged zones and ≥5 mm zones around the combination disc when compared with disc alone were observed. All the ESBL detection test results were clearly indicated that the selected K. pneumoniae strain was ESBL producer.

Background Recent years, the treatment of multi-drug resistant bacteria and their effect are very difficult due to the virulence factors modification. Based on the world wide thread, we have tried to identify the ESBLs producing bacteria from urinary tract infection patients. In result, the highly antibiotic resistant effect of Pseudomonas aeruginosa and Klebsiella pneumoniae were identified. Methods Initially, Hexa disc diffusion method was performed to detect the multi-drug resistant bacteria using respective antibiotics of HX066, HX033 and HX077, HX012 discs. Consecutively, the ESBL producing ability of confirmed multi drug resistant bacteria was performed to detect their ESBL producing ability using specific extended spectrum beta lactamase (ESBLs) detection discs of Hexa G-minus 24. Furthermore, the ESBL producing ability of the bacteria was confirmed by ESBLs detection Ezy MIC™ E-test stripe method. Results and conclusions In result, 10, 5 and 4 mm and 10, 14 and 8 mm zone of inhibition against imipenem (IPM), Ticarcillin/Clavulanic acid (TCC), Cefoperazone (CPZ) and Ampicillin (AMP), Norfloxacin (NX), Nalidixic acid (NA) antibiotics for P. aeruginosa and 16, 22 and 10, 18 mm zone of inhibition against ceftazidime (CAZ), methicillin (MET), ampicillin amoxyclav (AMC), co-trimoxazole (COT) of the HX077 HX012 for K. pneumoniae were observed. Based on the Clinical & Laboratory Standards Institute (CLSI) guidelines, both the bacteria were more resistant to tested antibiotics and it could be developed more resistant against all the tested antibiotics. In addition, the phenotypic detection of ESBL production effect was also performed against both the selected uropathogens, and the results were shown ≥22 mm, ≥27 zone of inhibition against all the tested antibiotics. Further, the genetic identification of multi plux PCR result was shown TEM, SHV and CTX-m genes were present in both the selected uropathogens. Finally, our results were correlated each other and concluded that the selected uropathogens were multi drug resistant effect and also ESBLs producer.

The present study was designed to identify the potential bioactive compound from endophytic actinomycetes (EA) Nocardiopsis sp. GRG 2 (KT 235641) against selected extended spectrum beta lactamase (ESBL) producing Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae). Initially, the multi drug resistance (MDR) effect of selected uropathogens was confirmed by respective UTI panel of Hexa antibiotics disc methods. The zone of inhibition ≤22 mm for ceftazidime, ≤ 27 mm for cefotaxime and ≤8 mm zone of MIC stripe against both the uropathogens of phenotypic methods confirmed, the selected strains were ESBL producer. Among the various EA extracts, GRG 2 extract showed excellent antibacterial activity against both ESBL producing P. aeruginosa and K. pneumonia by agar well diffution method. The molecular identification of selected GRG 2 strain was named as Nocardiopsis sp. GRG 2 (KT235641). The antibacterial metabolites present in the TLC elution was exhibited at 274 nm by UV visible spectrometer. The partial purification of preparative HPLC fraction 3 showed 14, 16 mm against P. aeruginosa and K. pneumoniae, respectively. Based on the antibacterial effect, the FT-IR, GC-MS and LC-MS analysis of fraction 3 was confirmed as 1, 4-diaza-2, 5-dioxo-3-isobutyl bicyclo[4.3.0]nonane (DDIBN). Further, the dose dependent inhibition of DDIBN against both ESBL producing pathogens was observed at 75 μg/mL by minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC). The increased cell death and disrupted cell membrane integrity were observed at MIC of DDIBN by confocal laser scanning electron microscope (CLSM) and scanning electron microscope (SEM). The results were proved that the DDIBN has potential antibacterial metabolites against ESBL producing pathogens and it can be applied for various other biomedical fields.

The aim of this study is to identify potential antibacterial compounds of endophytic actinomycetes (EA) from marine algae Caulerpa racemosa for inhibit the multi drug resistant (MDR) Gram negative bacteria (GNB) including Eschericia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Proteus mirabilis (P. mirabilis), Klebsiella pnumonia (K. pnumoniae) and Enterobacter sp., which were confirmed by UTI panel antibiotic discs. Among the 100 EA, five strains were showed better antogonistic activity against all the selected uropathogens. In the ethyl acetate extract, the NMS 5 strains of EA showed excellent anti-bacterial activity against all the MDR uropathogen. Further, the NMS 5 extract was partially purified from thin layar chramatogaraphy (TLC) and spot 3 of TLC crude compound showed 14, 12 mm zone of inhibition against both the uropathogens. In addition, the minimum inhibition concentration (MIC) of crude exhibited the inhibition percentage was 87 and 84 at 120 µg/mL concentration. The MIC was confirmed by minimum bactericidal concentration (MBC) and it was indicated same as MIC. The treated NMS 5 crude compound of P. aeruginosa and K. pneumonia cells were showed more dead cells, intracellular damage and morphological damage with disaggregated cells by confocal laser scanning electron microscope (CLSM) and scanning electron microscope (SEM). Hence, the present study was revealed that the algae derived EA have the potential anti-bacterial metabolites for inhibition of MDR bacteria and it can be applied for various other infections after further purification.

The aim of the current study is to identify bioactive compound from marine endophytic actinomycetes (MEA) isolated from Gulf of Mannar region, Southeast coast of India. Among the isolated actinomycetes, strain GRG 4 exhibited excellent ability to inhibit isolated colistin resistant (CR) Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae), which is a emerging threat to the world. The strain was identified as Streptomyces coeruleorubidus GRG 4 (KY457708), based on morphological, biochemical, phenotypic and genotypic characters. The bioactive metabolites present in the methanolic extract were partially purified by TLC and preparative HPLC. The active HPLC fraction 2 showed 15, 20 mm zone of inhibition against both CR P. aeruginosa and K. pneumoniae respectively. Analytical HPLC and FT-IR results of fraction 2 showed with carbonyl group. Both GC-MS and LC-MS results confirmed that the fraction 2 contained chemical constituents of Bis (2-Ethylhexyl) Phthalate (BEP). The compromised structure with loosely integrated and ruptured cell wall of BEP treated CR bacteria were observed by confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) at 75 μg/mL of minimum inhibitory concentration (MIC) dose. Further, cytotoxic effect of BEP against A549 human lung cancer cells revealed complete inhibition by cell proliferation and apoptosis was observed at 100 μg/mL in 24 h treatment. In addition, irreversible ROS dependent oxidative damage was clearly observed at the IC50 concentration of BEP. The toxicity of BEP was also studied against Vibrio fischeri (V. fischeri) and found to be highly toxic after 15 and 30 min of treatment. Based on the results it could be concluded that the identified compound BEP is a potent inhibitor for CR bacteria and A549 lung cancer cells.

Based on the untreated biofilm formation in urinary tract infections (UTIs), current study was focused on the inhibition of Pseudomonas aeruginosa (P. aeruginosa) biofilm formation using Nerium oleander (N. oleander) essential oils. Initially, the LC-MS analysis of antibacterial potential of hydro distillated N. oleander essential oils was detected by LC-MS analysis. Initially, the antibacterial nature of essential oils was detected by agar well diffusion method against biofilm forming P. aeruginosa. Then, the possible anti-bacterial fractions of the essential oils were purified by preparative HPLC, and confirmed by analytical HPLC peaks. Possible anti-bacterial components of the fractions were merged together and confirmed by agar well diffusion method. Consecutively, the and minimum biofilm inhibition concentration experiment was proved that the concentration of 200 µg/mL of purified essential oils was very effective against P. aeruginosa biofilm formation. The decrease survival rate of the MTT assay result was also supported the minimum biofilm inhibition concentration result. Finally, all the results were more evident; the HPLC purified N. oleander essential oils as a promising anti-biofilm factor against P. aeruginosa biofilm formation.

The multidrug resistant Gram negative bacteria (MDRGNB) is an emerging burden and now represents a daily challenge for the management of antimicrobial therapy in healthcare settings. The present study was aimed to detect the prevalence of TEM and CTX-M type genes from GNB on urinary tract infection (UTIs). The ciprofloxacin resistant uropathogens were detected by HEXA UTI 5 disc diffusion method. The phenotypic detection of uropathogens producing extended spectrum beta lactamases (ESBLs) was confirmed by double disc combination test (DDCT) and phenotype confirmation test (PCT). The prevalence of TEM and CTX-M genes of uropathogens was identified by multiplex PCR analysis. The in vitro antimicrobial susceptibility of E. coli producing ESBL (26), 21 isolates of P. mirabilis, 17 P. aeruginosa, 14 K. pneumoniae and 6 Enterobacter sp. were detected. Based on the extension of the cephalosporin zone edge towards augmentin disc in the DDST method proved 84% of the isolates were ESBL positive. Similar results were obtained in phenotypic confirmatory test (PCT) by the increases of ≥5 mm zone of inhibition in the combination disc when compared with ceftazidime disc alone. The prevalence of TEM and CTX-M genes were determined from multidrug resistance uropathogens (MDU) respectively as 83%, 75%, 71%, 63%, 60%, 55%, 54%, 50%. The most prevalent (TEM + CTX-M) genes were also detected in ciprofloxacin resistant strains P. mirabilis BDUMS1 (KY617768) and E. coli BDUMS3 (KY617770). Due to the increase of ESBL genes in uropathogens, sustained supervision for using favorable antibiotics and decreasing the infection is essential.

Urinary tract infections (UTIs) are the most disregarded diseases in both developing and developed countries and accountable for one fourth of the health care related infections. Even though the women and children are the most susceptible reservoir, the infections are at a significant amount in men of all ages. In UTI, the multidrug resistant bacteria (MDRB) is an emerging burden and now represents a daily challenge for the management of antimicrobial therapy in healthcare settings. A total of 100 uropathogens of Gram negative bacteria (GNB) were used in the current study to detect the ciprofloxacin resistant effect due to extended spectrum beta lactameses production (ESBLs). The phenotypic identification of ESBLs producing uropathogens from UTIs were detected by primary ESBL identification test (PMIT) and double disc combination method (DDCM). In addition, the ESBL production was further confirmed by MIC stripe method. Further, the prevalence of TEM, SHV, OXA and CTX-M type genes of isolated GNB and ciprofloxacin resistant strains was detected by multiplex PCR method. Among the 100 uropathogens, 84% was found to produce ESBLs. Out of 84, 60 strains were identified as ciprofloxacin resistant by Hexa discs and they developed resistance against all antibiotics. The PMIT and DDCM proved the result including Escherichia coli (26), 21 isolates of Proteusmirabilis, 17 of Pseudomonas aeruginosa, 14 of Klebsiellapneumoniae and 6 of Acinetobacter sp. In particular, the high number of CTX-M and TEM genes were frequently detected from collected uropathogens and all the TEM, SHV, OXA, and CTX-M genes were identified from ciprofloxacin resistant strains only. Due to the increase of multiple ESBL genes in uropathogens, sustained supervision for using favorable antibiotics and the decreasing the infection is essential. Keywords: Antimicrobial resistance, Extended spectrum beta lactamase (ESBL), Urinary tract infections (UTIs), Gram negative bacteria (GNB)

Our study is to evaluate the potential bioactive compound of Nocardiopsis sp. GRG1 (KT235640) and its antibacterial activity against multi drug resistant strains (MDRS) on urinary tract infections (UTIs). Two brown algae samples were collected and were subjected to isolation of endophytic actinomycetes. 100 strains of actinomycetes were isolated from algal samples based on observation of morphology and physiological characters. 40 strains were active in antagonistic activity against various clinical pathogens. Among the strains, 10 showed better antimicrobial activity against MDRS on UTIs. The secondary metabolite of Nocardiopsis sp. GRG1 (KT235640) has showed tremendous antibacterial activity against UTI pathogens compared to other strains. Influence of various growth parameters were used for synthesis of secondary metabolites, such as optimum pH 7, incubation time 5–7 days, temperature (30 °C), salinity (5%), fructose and mannitol as the suitable carbon and nitrogen sources. At 100 μg/ml concentration MIC of Nocardiopsis sp. GRG1 (KT235640) showed highest percentage of inhibition against Proteus mirabilis (85%), and E.coli, Staphylococcus auerues, Psuedomonas aeroginasa, Enterobactor sp and Coagulinase negative staphylococci 78–85% respectively.

Copper oxide nanoparticles (CuO NPs) were synthesized biologically using leaf extract of Camilla japonica. The typical UV–visible spectral peak of CuO NPs was observed at a wavelength of ∼290 nm, which confirmed their successful synthesis. From scanning electron microscope (SEM) and transmission electron microscope (TEM) analyses, the synthesized CuO NPs were found to possess spherical shape. Energy dispersive X-ray analyzer (EDX) results revealed that the CuO NPs are almost pure with atomic percentages of 50.92 for Cu and 49.08 for O. Fourier transform infrared (FTIR) confirmed the presence of an absorption peak located at a wavenumber position of ∼480 cm−1 typical for highly pure CuO NPs. TEM images displayed that the particles are relatively uniform in size ∼15–25 nm. The P. aeruginosa and K. pneumonia showed complete resistance against Hexa 077 antibiotic discs. The result of ≤22 ceftazidime and ≤27 cefotaxime confirmed that both the uropathogens were ESBL producers. The ≥8 mm of the MIC stripe further confirmed that both the uropathogens were ESBL producers. Furthermore, the antibacterial activity of CuO NPs against selected ESBL producing P. aeruginosa and K. pneumoniae at minimum inhibition concentration (MIC) of 100 μg/mL. The decreased cell viability and damaged membrane construction of both the uropathogens were observed by confocal laser scanning microscope (CLSM) using AO/EB stains at desired MIC dose. The morphological damage of the bacterial cells was demonstrated by SEM analysis. Hence, based on the above in vitro findings, the results suggested that the CuO NPs are efficient antibacterial compounds against ESBL producing bacteria, and that the plant leaf mediated CuO NPs can be considered as novel and promising material to act against various infectious bacteria.

Urinary tract infections (UTIs) are diverse public health complication and caused by range of pathogens, however mostly Gram negative bacteria cause significant life threatening risks to different populations. The prevalence rate and antimicrobial resistance among the Gram negative uropathogens alarmed significantly heighten the economic burden of these infections. In this study, we investigated the antibiofilm efficiency of Pyrrolo [1,2-a] pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) extracted from endophytic actinomycetes Nocardiopsis sp. GRG 1 (KT235640) against P. mirabilis and E. coli. The extracted compound was characterized through TLC, HPLC, GC-MS, LC-MS and confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM). The compound, Pyrrolo [1,2-a] pyrazine-1, 4-dione, hexahydro-3-(2-methylpropyl) inhibits both bacterial biofilm formation as well as reduces the viability of preformed biofilms. Furthermore, CLSM image shows cell shrinkage, disorganized cell membrane and loss of viability. The SEM result also confirms the cell wall degradation in treated cells of the bacteria. Hence, the Pyrrolo [1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) is active against P. mirabilis and E. coli.

The accelerative outgrowth of extended spectrum β-lactamases (ESBLs) producing Escherichia coli (E. coli) and Proteus mirabilis (P. mirabilis) was mainly due to incessant relentless influence of antibiotics thereby increasing incidence and death rate which was obvious from the survey of ESBLs producing bacteria related health problem. In the present paper, we synthesized and characterized zinc oxide nanoparticles (ZnO NPs) employing using Camellia japonica leaf extract, bactericidal action of these NPs against extended spectrum β lactamases (ESBLs) positive E. coli and P. mirabilis clinical strains owing the minimal inhibitory concentration (MIC) percentage 83, 81% at 100 μg/mL concentration and minimum bactericidal concentration (MBC) final inhibiting concentration at 150 μg/mL. Moreover, confocal laser scanning microscopy (CLSM) and scanning electron microscope (SEM) results evident for loss of viability, cell shrinkage, disarrangement of cell membrane, and cell wall lysis activity of ZnO NPs against ESBLs positive E. coli BDUMS3 (KY617770) and P. mirabilis BDUMS1 (KY617768) strains. From the results, it was observed that the biologically synthesized ZnO NPs has stronger antibacterial effect against ESBLs producing bacterial strains. Nevertheless, current date there is no reports of antibacterial activity of metal oxide (ZnO) NPs against ESBL producing gram negative bacteria. Consequently, this finding is the first report in this respect and it shows band gap energy and ROS accumulation to damage the cell wall and inhibit the growth of ESBL producing gram negative strains.