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Infections caused by resistant microorganisms are a complex global public health challenge, and the way to combat the increase of resistance is the development of more modern and faster techniques for resistance detection. This study aimed to evaluate the transport of inactivated bacteria impregnated in a filter paper disk to detect carbapenem resistance genes by multiplex real-time PCR (qPCR) using high-resolution melting (HRM). A total of 88 isolates of 10 different species of Enterobacterales harboring well-characterized carbapenem resistance genes were evaluated. A full 10-µL loop of fresh growth of bacteria were impregnated in a filter paper disk, which was left at room temperature for 2 days in order to simulate the time spent in transportation. Bacterial inactivation was performed with 70% ethanol at 15 min. Afterwards, the DNA was extracted from the paper disks for further analysis by qPCR HRM. The time of 15 min in 70% ethanol was enough to inactivate all the isolates tested. It was possible to correctly identify the presence of the carbapenem resistance gene by HRM qPCR in 87 isolates (98.87%) that were transported in the filter paper disks. Our results indicated that it is possible to use filter paper to transport inactivated bacteria and to identify carbapenem resistance genes by qPCR HRM. This alternative tends to facilitate the access to this technology by many laboratories which do not have the qPCR equipment.

Hydrogen sulfide (H2S) is known to participate in bacteria-induced inflammatory response in periodontal diseases. Therefore, it is necessary to quantify H2S produced by oral bacteria for diagnosis and treatment of oral diseases including halitosis and periodontal disease. In this study, we introduce a paper-based colorimetric assay for detecting bacterial H2S utilizing silver/Nafion/polyvinylpyrrolidone membrane and a 96-well microplate. This H2S-sensing paper showed a good sensitivity (8.27 blue channel intensity/μM H2S, R2 = 0.9996), which was higher than that of lead acetate paper (6.05 blue channel intensity/μM H2S, R2 = 0.9959). We analyzed the difference in H2S concentration released from four kinds of oral bacteria (Eikenella corrodens, Streptococcus sobrinus, Streptococcus mutans, and Lactobacillus casei). Finally, the H2S level in Eikenella corrodens while varying the concentration of cysteine and treatment time was quantified. This paper-based colorimetric assay can be utilized as a simple and effective tool for in vitro screening of H2S-producing ability of many bacteria as well as salivary H2S analysis.

Aim of this research was to treat the organics enriched Paper and Pulp Industry (PPI) effluents using multi-metal tolerant predominant indigenous bacterial species. In addition, assessing the potential of treated bacterial biomass as a single cell protein (SCP). The multi-metal tolerant Streptomyces tuirus OS1 was enumerated from the Paper and Pulp Industry (PPI) effluents was identified through standard molecular characterization. S. tuirus OS1 proficiently ameliorated organic contaminants in PPI effluent in the in study at 35 °C, 45 °C, and 25 °C. Fortunately, the S. tuirus OS1 considerably increased the dissolved oxygen level in treated PPI effluent in 30 days of bioremediation process. Interestingly, at 35 °C of bioremediation process the S. tuirus OS1 demonstrated increased dried biomass (7.1 g L

The increasing prevalence of antibiotic-resistant bacteria needs rapid identification and efficient destruction routes. This study proposes testing paper derived from electrospun fibrous mats and aggregation-induced emission (AIE) probes for trace sensing and simultaneous destruction of antibiotic-resistant E. coli. Aptamers are conjugated on fibers for selective capture of E. coli, and the capture capability can be regenerated via rinsing with salt solution. Hydroxyl tetraphenylethene (TPE) is linked with two cephalosporin molecules to construct TPE-Cep probes, and the fluorescence emission is turned on specifically in the presence of β-lactamase, which is a critical marker for screening resistant bacteria. Fibrous mats are lit up only in the presence of antibiotic-resistant bacteria, and the fluorescence intensity changes could be statistically fitted into an equation for quantitative analysis. Fibrous strips display apparent color changes from blue to green for a visual readout of bacterial levels, and the limit of detection (LOD) is much lower than those of previous paper substrates. In addition, the TPE-Cep probes could produce reactive oxygen species (ROS) under room light illumination to kill the captured bacteria. Thus, the integration of aptamer-grafted electrospun fibers and functional AIE probes provides potential for selective capture, trace imaging and photodynamic destruction of antibiotic-resistant bacteria.

This study aimed to evaluate the filter paper as a means to transport inactivated Gram-negative non-fermentative (GNNF) bacteria and Haemophilus spp. for analysis using MALDI-TOF MS. A total of 133 isolates were evaluated and the analysis of each isolate was performed directly from original bacterial colony and in filter paper after the processing. To evaluate the agreement between the identification performed directly from the colony and after impregnation in filter paper, we assign the scores: >2·3 as excellent (E); 2·0 to 2·3 as very good (VG); 1·7–1·99 as good (G)

Infections caused by bacteria that produce β-lactamases (BLs) are a major problem in hospital settings. The phenotypic detection of these bacterial strains requires culturing samples prior to analysis. This procedure may take up to 72 h, and therefore it cannot be used to guide the administration of the first antibiotic regimen. Here, we propose a multisensor for identifying pathogens bearing different types of β-lactamases above the infectious dose threshold within 90 min that does not require culturing samples. Instead, bacterial cells are preconcentrated in the cellulose scaffold of a paper-based multisensor. Then, 12 assays are performed in parallel to identify whether the pathogens produce carbapenemases and/or cephalosporinases, including metallo-β-lactamases, extended-spectrum β-lactamases (ESBLs), and AmpC enzymes. The multisensor generates an array of colored spots that can be quantified with image processing software and whose interpretation leads to the detection of the different enzymes depending on their specificity toward the hydrolysis of certain antibiotics, and/or their pattern of inhibition or cofactor activation. The test was validated for the diagnosis of urinary tract infections. The inexpensive paper platform along with the uncomplicated colorimetric readout makes the proposed prototypes promising for developing fully automated platforms for streamlined clinical diagnosis.

Environmentally recalcitrant pollutants (chromium and sulphate) from mixed effluents of tannery and paper mills were treated simultaneously using highly metal-resistant dissimilatory sulphate-reducing bacteria. The remedial efficiency of the bacteria appeared zero when the bacterial pure culture was employed without the addition of any growth substrate from external source, while the bacterial sulphate reduction and consequent metal removal appeared maximum when sodium lactate was added as growth substrate in another set of experiments. In this set of experiments, almost 100% precipitation of the chromium (700 ppm) occurred in the first 7 days of anaerobic incubation. Sulphate was also removed totally (1225 ppm) in the first 7 days of incubation. pH of the media remained in the neutral to slightly basic range throughout the experimental trial. The achievement of maximum remedial efficiency also depicts the ultimate preference of lactate as carbon source by sulphate-reducing bacteria. The outcomes of the present investigation can be fruitful for designing efficient, environment-friendly and economical bioremedial strategies.

Despite the green energy generation with low cost compared to conventional fuel cells, microbial fuel cells (MFCs) still suffer with anode related constraints including laborious pretreatment and modification process of conventional electrodes, limited bacterial loading capacity, and inferior extracellular electron transfer efficiency. Accordingly, this investigation explores the waste tissue paper derived three dimensional (3D) carbon aerogel (CA) integrated with cerium dioxide (CeO2) nanotubes decorated nitrogen-doped reduced graphene oxide nanosheets (NRGO) as a competent anode to address these technical complements. The direct growth of NRGO and CeO2 over CA in the form of freestanding and binder-free NRGO/CeO2(1:2)/CA alleviates the significant constrains of conventional anode fabrication. The 3D hierarchical architectures of CA with open porous structure provide easy access of bacteria, thus increases the bacterial colonies per unit volume. Furthermore, the hydrogen bonding between the interfacial oxygen atoms of CeO2 and lysine residues of the cytochrome c in bacteria yields excellent extracellular electron transfer efficiency. The electrostatic interaction between the NRGO and bacteria cells improves the bacterial adhesion and biofilm formation, leading to the compact biofilm formation for the improved direct electron transference. With the profits of above, the MFC with NRGO/CeO2(1:2)/CA demonstrates a maximum power output and good lifespan performances. The present exploration facts thus access advanced avenues to converting waste matters of tissue paper, human urine, and wastewater into profitable constituents for the development of efficient and durable power producing systems.

In the wood-free paper industry, whitewater is usually a mixture of additives for paper production. We are currently lacking an efficient, cost-effective purification technology for their removal. In closed whitewater cycles the additives accumulate, causing adverse production problems, such as the formation of slime and pitch. The aim of our study was to find an effective bio-based strategy for whitewater treatment using a selection of indigenous bacterial isolates. We first obtained a large collection of bacterial isolates and then tested them individually by simple plate and spectrophotometric methods for their ability to degrade the papermaking additives, i.e., carbohydrates, resin acids, alkyl ketene dimers, polyvinyl alcohol, latex, and azo and fluorescent dyes. We examined correlation between carbon source use, genera, and inoculum source of isolates using two multivariate methods: principal component analysis and FreeViz projection. Of the 318 bacterial isolates, we selected a consortium of four strains (Xanthomonadales bacterium sp. CST37-CF, Sphingomonas sp. BLA14-CF, Cellulosimicrobium sp. AKD4-BF and Aeromonas sp. RES19-BTP) that degrade the entire spectrum of tested additives by means of dissolved organic carbon measurements. A proof-of-concept study on a pilot scale was then performed by immobilizing the artificial consortium of the four strains and inserting them into a 33-liter, tubular flow-through reactor with a retention time of < 15 h. The consortium caused an 88% reduction in the COD of the whitewater, even after 21 days., GREENER H2020

Paper has become the basic elixir in everyone's activities and usage of paper has increased day by day, the waste generated by paper is also enormous. The primary source of paper is wood (tree) yet, waste paper is environmentally good and biodegradable; however, it is the primary source of deforestation. Current research aims to find an alternate way to recycle paper in the biological approach. Hence in our work, twelve cellulose-producing bacteria were isolated, out of which one bacterial strain proved to be the best. Cellulase enzyme was extracted and purified, and used for enzymatic de-inking of photocopy papers. The optimal conditions for cellulase synthesis were at 60 °C, glucose as the only carbon source, and potassium nitrate as the nitrogen source. The enzyme demonstrated excellent de-inking at a lower pulp consistency of 3% with a 20% enzyme dose. The cellulose and hemicellulose levels decreased, which can be attributed to fiber breaking. Further, the changes in the functional groups identified by Fourier-transform infrared spectroscopy analysis and the changes in the surface morphology of the pulp fibers were obtained using scanning electron microscope analysis.

Infections caused by foodborne microorganisms are a great threat to the global environment and public healthcare today. Thus, rapid, portable and sensitive assays that can realize the identification of foodborne bacteria are highly desired. In this study, a smart fluorescent and colorimetric dual-readout sensing system has been established for simple and rapid E. coli determination by utilizing the Cu2+-triggered oxidation of o-phenylenediamine (OPD). Initially, Cu2+ can oxidize OPD to OPDox, resulting in an orange-yellow fluorescence and visible pale-yellow color. However, E. coli can effectively reduce Cu2+ into Cu+, inhibiting the Cu2+-triggered oxidation of OPD to OPDox. Consequently, the introduction of E. coli can turn off both the fluorescence and the UV-vis absorbance signals of the OPD-Cu2+ system, illustrating an original mechanism for fluorescent and colorimetric dual-channel detection of E. coli. Moreover, a filter paper-based visual sensor was built and coupled with OPD-Cu2+ solution under the assistance of a UV lamp. The as-prepared sensor can detect E. coli quantitatively with the help of a typical smartphone color-scanning application (APP). Thus, this study offers a valid dual-mode assay for sensitive and on-site visible detection of E. coli, guaranteeing the reliability of the results and is more attractive for practical use. Graphical Abstract Schematic illustration of the smartphone-integrated sensing system for fluorescent and colorimetric dual-channel detection of E. coli based on the Cu2+-OPD system.

Rapid and sensitive detection of enteropathogenic Escherichia coli (EPEC) in fluids with complex background is an important task for safety quality control in the field of medicine, environment, and food. In this study, a gold foil paper–based aptasensor was developed for the detection of enteropathogenic EPEC O26:K60 with surface-enhanced Raman spectroscopy (SERS) and magnetic separation technology mediated by Fe3O4@Au composite. The gold foil paper was firstly modified with thiolated capture probe and SERS tag. The thiolated aptamer probe for EPEC was immobilized onto a Fe3O4@Au composite. In the presence of EPEC, highly specific recognition between the aptamer probe and EPEC made the Fe3O4@Au composite partially dissociated from the gold foil paper. This led to a decreased Raman intensity response, which showed an obvious negative linear correlation with increasing concentration of EPEC over a wide concentration range from 10 to 107 CFU/mL under an excitation wavelength of 633 nm. The detection limit was about 2.86 CFU/mL in a buffer solution and a licorice extractum and the detection time was only 2.5 h. The results demonstrate that the gold foil paper–based aptasensor can be an excellent biosensing platform that offers a reliable, rapid, and sensitive alternative for EPEC detection.

Managing food contamination from bacteria has been an ongoing issue in the public health and industrial fields. Enzymatic substrates possessing optical properties, e.g. fluorescence or color manifestation, are widely exploited in pathogenic/non-pathogenic bacteria culture methods. Recently, various chromogenic substrates have been utilized in the development of point-of-care diagnostic tools. Herein, four types of chromogenic substrates were exploited to develop paper-based sensors for major foodborne pathogens. We designed a compact sized three-dimensional paper device with a simple user interface. By inserting functional layers in the middle of multilayers, pre-lysis and pH regulation steps were excluded and the analysis time was subsequently reduced, while only one sample droplet was needed for the whole analysis process. After the enzymatic reactions had proceeded, target-specific colors appeared. When it was combined with enrichment, 101 cfu mL-1 of pathogens were successfully detected in 4-8 hours, while those in milk samples were readily sensed in 12 hours. The proposed bacteria sensor exhibited great advantages of low cost, portability and simple operation, while showing a respectable limit-of-detection as low as 101 cfu mL-1 and below. Significantly, we emphasize that it takes fewer steps than existing methods and provides a reduced analysis time owing to the layer functionalization.

Rapid and reliable identification of bacteria is an important issue in food, medical, forensic, and environmental sciences; however, conventional procedures are time-consuming and often require extensive financial and human resources. Herein, we present a label-free method for bacterial discrimination using surface-enhanced Raman spectroscopy (SERS) and partial least squares discriminant analysis (PLS-DA). Filter paper decorated with gold nanoparticles was fabricated by the dip-coating method and it was utilized as a flexible and highly efficient SERS substrate. Suspensions of bacterial samples from three genera and six species were directly deposited on the filter paper-based SERS substrates before measurements. PLS-DA was successfully employed as a multivariate supervised model to classify and identify bacteria with efficiency, sensitivity, and specificity rates of 100% for all test samples. Variable importance in projection was associated with the presence/absence of some purine metabolites, whereas confidence intervals for each sample in the PLS-DA model were calculated using a resampling bootstrap procedure. Additionally, a potential new species of bacteria was analyzed by the proposed method and the result was in agreement with that obtained via 16S rRNA gene sequence analysis, thereby indicating that the SERS/PLS-DA approach has the potential to be a valuable tool for the discovery of novel bacteria. Graphical abstract This paper describes the discrimination of bacteria at the genus and species levels, after minimal sample preparation, using paper-based SERS substrates and PLS-DA with uncertainty estimation.

Bacterial infections are the leading cause of morbidity and mortality in the world, particularly due to a delay in treatment and misidentification of the bacterial species causing the infection. Therefore, rapid and accurate identification of these pathogens has been of prime importance. The conventional diagnostic techniques include microbiological, biochemical, and genetic analysis, which are time-consuming, require large sample volumes, expensive equipment, and reagents, as well as trained personnel. In response, we have now developed a paper-based ratiometric fluorescent sensor array. Environment-sensitive fluorescent dyes (3-hydroxyflavone derivatives) pre-adsorbed on paper microzone plates fabricated using photolithography, upon interaction with bacterial cell envelopes, generate unique fluorescence response patterns. The stability and reproducibility of the sensor array response were thoroughly investigated, and the analysis procedure was refined for optimal performance. Using neural networks for response pattern analysis, the sensor was able to identify 16 bacterial species and recognize their Gram status with an accuracy rate greater than 90%. The paper-based sensor was stable for up to six months after fabrication and required 30 times lower dye and sample volumes as compared to the analogous solution-based sensor. Therefore, this approach opens avenues to a state-of-the-art diagnostic tool that can be potentially translated into clinical application in low-resource environments.

Thermophilic methane fermentation was a valid approach for handling the stillage eluted from ethanol fermentation of waste paper and kitchen waste. The wide organic loading rate (OLR) range (2-14 g VTS/(L d)) for stable performance and relatively high energy recovery efficiency (79.0%) were achieved, and OLR of 8 g VTS/(L d) was optimum for achievement of highest biogas evolution and VTS removal efficiency. Microbial community analysis revealed that hydrolysis of cellulose was the critical step for methane production from the stillage.

β-Lactam antibiotics are widely used for the treatment of bacterial infections; however, an increase in the number of antibiotic-resistant bacteria, which produce β-lactamases that inactivate β-lactam antibiotics, can lead to serious untreatable diseases. Thus, proper prescribing of antibiotics and the development of new antimicrobial agents with strong resistance to hydrolysis by β-lactamases can be a solution to reduce antibiotic resistance and treat various bacterial infections. Accordingly, simple and rapid methods for the screening of antibiotics that show β-lactamase resistance are required. Therefore, we designed a multi-channel microfluidic paper-based analytical device (μPAD) for colorimetric screening of β-lactam antibiotics for β-lactamase resistance; the μPAD, created by modifying paper with self-assembling organosilane and a 4-(2-pyridylazo)resorcinol (PAR)–Hg2+ complex, can screen β-lactam antibiotics for hydrolytic resistance to multiple β-lactamases at once. To demonstrate its suitability for this task, we confirmed the resistance of already studied β-lactam antibiotics—penicillin G, cloxacillin, and ceftazidime—toward different classes of β-lactamases (penicillinase, TEM-1, and CTX-M-1) using the μPAD. Based on ligand exchange between hydrolyzed β-lactam antibiotics and PAR in the PAR–Hg2+ complex, colorimetric detection of the relative susceptibilities of β-lactam antibiotics to a β-lactamase is possible even with the naked eye in only 10 minutes. The portable and equipment-free μPAD allows for simple multi-screening of diverse classes of β-lactam antibiotics for β-lactamase resistance.

Due to the presence of various organic contaminants, improper disposal of pulp-paper wastewater poses harm to the environment and human health. In this work, pulp-paper sludge (PPS) after secondary treatment were collected from M/s Century Pulp-paper Mills in India, the chemical nature of the organic pollutants was determined after solvent extraction. All the isolates were able to produce lipase (6.34–3.93 U ml− 1) which could account for the different fatty acids detected in the PPS. The dominant strains were in the classes of α and γ Proteobacteria followed by Firmicutes. The Shannon-Weiner diversity indexes for phylotype richness for the culturable and non-culturable bacterial community were 2.01 and 3.01, respectively, indicating the non- culturable bacterial strains has higher species richness and diversity compared to the culturable bacterial strains. However, the culturable strains had higher species evenness (0.94 vs 0.90). Results suggested only a few isolated strains were resistant to the POPs in the PPS, whereas non-cultural bacteria survived by entering viable but non-cultural state. The isolated strains (Brevundimonas diminuta, Aeromonas punctata, Enterobacter hormaechei, Citrobacter braakii, Bacillus pumilus and Brevundimonas terrae) are known for their multidrug resistance but their tolerance to POPs have not previously been reported and deserved further investigation. The findings of this research established the presence of POPs which influence the microbial population. Tertiary treatment is recommended prior to the safe disposal of pulp paper mill waste into the environment.

To recover the biogas from sewage sludge and paper waste (PW), the methanogenic performance of thermophilic anaerobic co-digestion of sewage sludge with PW was assessed by a continuous experiment. The effects on the biogas production and microbial community were investigated by changing the PW ratio from 0 to 66.7%. The optimal performance was obtained at the ratio of sewage sludge: PW = 4:6 (total solids), where the COD removal efficiency and biogas production increased from 58.34±5.90% to 72.92±0.08% and 438±53 to 594±72 mL/g-VSadded, respectively. By investigating the trend of carbohydrate and protein degradation rates, the competition between carbohydrate and protein degradation was quantified. The critical PW addition ratio was about (63.64%), where the protein degradation rate decreased to zero with increasing PW addition. Meanwhile, the microbial analysis showed that cellulolytic bacteria outcompeted proteolytic bacteria and to be the predominant group after PW addition.

Suaeda maritima is a halophyte that has been evolutionary adapted to cope with saline and heavy metal conditions. The aim of the present study was to examine the biochemical response of Suaeda maritima against phytoextracting heavy metals and salts from paper mill effluent. Suaeda maritima seedlings have been grown for 120 days with an irrigation solution of 250 ml of 75% raw paper mill effluent after four drenching. Analysis of biochemical parameters revealed that maximum synthesis of chlorophyll, protein, phenol, proline and glycinebetaine may be involved in their ability to cope with heavy metal and salt stress. The present study confirms that the accumulation and increase of biochemical constituents can be considered as an efficient ROS scavenger and also maintain cellular homeostasis and metabolic functions in photosynthetically active leaves in Suaeda maritima, during phytoextraction studies. Keywords: phytoextraction, paper mill effluent, Suaeda maritima, biochemical, heavy metal and salts

Designing novel isolation methods could enhance the diversification of the available bacterial strains to biotechnology. In this study, the new ex situ and in situ cultivation methods are introduced for the isolation of actinobacteria. In the ex situ experiments, the soil suspension was spread on the isolation media located above some ordinary papers in immediate contact with the slurry of soil substrate and incubated for 16 weeks. The paper was wholly immersed in the cave soil for in situ cultivations, and the containers were buried under layers of soil in Hampoeil cave for 10 weeks. Fruit wrapping kraft coated paper, with 68.8% recovery of isolates, was a better choice in isolation of actinobacteria than other studied filter paper. Based on the molecular identification results, 19% of the isolates obtained from the in situ cultivation method had less than 98.5% similarity to known taxa of actinobacteria and potentially may represent new species. In contrast, in the standard cultivation method, 1.3% of the isolates had less than 98.5% similarity 16Sr RNA gene. This data shows that the introduced cultivation method is a promising technique for isolating less culturable or new actinobacteria.

This study reports the synthesis of bacterial cellulose (BC) hydrogel sheets and their utilization as a support for silver‑nickel oxide nanocomposites (Ag/NiO). A two-step facile hydrothermal method was employed for the preparation of Ag/NiO, followed by impregnation into BC hydrogel sheets. A 20% Ag/NiO composition was revealed by dry weight analysis. The stability of nanocomposites-Hydrogel was confirmed by Ag

Purpose To study the metagenomics of the microbes isolated from the canaliculus of patients with infective canaliculitis. Methods A prospective study was performed on five consecutive canalicular samples obtained for the metagenomic analysis from the patients with infective canaliculitis who underwent non-incisional canalicular curettage at a tertiary care Dacryology service. The canalicular concretions were collected intraoperatively soon after a canalicular curettage and immediately transported on ice to the laboratory. Following DNA extraction and library preparation, a whole shotgun metagenome sequencing was performed on the Illumina™ platform. The downstream processing and bioinformatics of the samples were performed using multiple software packaged in SqueezeMeta™ pipeline or MG-RAST™ pipeline. Results The taxonomic hit distribution across the samples showed that bacteria were the most common isolates (mean—80.5%), followed by viruses (mean—0.74%), and archaea (0.01%). The five major phyla identified across the samples of infective canaliculitis were, Fusobacteria, Bacteroidetes, Proteobacteria, Actinobacteria, and Firmicutes. The prevalent organisms include Fusobacterium nucelatum, Fusobacterium periodonticum, Parvimonas micra, Prevotella oris, Selonomonas noxia, Pseudopropionobacterium propoinicum, Campylobacter showae, and Streptococcus anginosus, amongst few others. Actinomycetes israelii was noted in all the samples, though it was not the most abundant. The microbial gene mapping and protein prediction demonstrated proteins with known functions to range from 69.91% to 87.09% across the samples. The functional subsystem profiling demonstrated genes associated with carbohydrate, amino acid, and co-enzyme transport and metabolism, cell wall or cell membrane biogenesis, energy production and conversion, transcription, translation, and cellular communications. Conclusion This is the first whole metagenome sequencing of infective canaliculitis. Infected canaliculi harbor diverse microbial communities, including bacteria, viruses, and archaea. Functional analysis has provided newer insights into the ecosystem dynamics and strategies of microbial communities.

The food packaging industry generally uses papers and paperboards (PPBs) especially for disposable products. According to the Framework Regulation (EC) No. 1935/2004 of the European Union, no transfer of contamination should occur from food packaging material to the food items. The aim of this study was to determine the presence, numbers, source and different kinds of bacteria present in food packaging PPBs with various contents of pulp fiber. The samples were randomly collected from popular confectioners and fast food restaurants in Saharanpur, India. The results indicated the presence of bacteria in all the samples, ranging from 1.3 × 102 to 6.1 × 103 cfu/g. Most of the samples contained bacteria in more than the permitted concentration of 2.5 × 102 cfu/g as set by Food and Drug Administration (FDA). The detected bacteria were from genera Bacillacea, Staphylococcus and Pseudomonas. According to the FDA declaration, pathogenic bacteria such as B. cereus and S. aureus have been associated with food borne diseases (FBD). Some contaminants in food packaging PPBs were found to be B. subtilis and P. aeroginosa, which produce enzymes like peroxidases and lipoxygenases that are odor generating enzymes.

The pulp and paper industry is a major source of lignocellulose-containing streams. The components of lignocellulose material are lignin, hemicellulose, and cellulose that may be hydrolyzed into their smaller components and used as feedstocks for valorization efforts. Much of this material is contained in underutilized streams and waste products, such as black liquor, pulp and paper sludge, and wastewater. Bacterial fermentation strategies have suitable potential to upgrade lignocellulosic biomass contained in these streams to value-added chemicals. Bacterial conversion allows for a sustainable and economically feasible approach to valorizing these streams, which can bolster and expand applications of the pulp and paper industry. This review discusses the composition of pulp and paper streams, bacterial isolates from process streams that can be used for lignocellulose biotransformations, and technological approaches for improving valorization efforts. KEY POINTS: • Reviews the conversion of pulp and paper industry waste by bacterial isolates. • Metabolic pathways for the breakdown of lignocellulose components. • Methods for isolating bacteria, determining value-added products, and increasing product yields.

Lysine crotonylation is a newly discovered and reversible posttranslational modification involved in various biological processes, especially metabolism regulation. A total of 5159 lysine crotonylation sites in 2272 protein groups were identified. Twenty-seven motifs were found to be the preferred amino acid sequences for crotonylation sites. Functional annotation analyses revealed that most crotonylated proteins play important roles in metabolic processes and photosynthesis. Bioinformatics analysis suggested that lysine crotonylation preferentially targets a variety of important biological processes, including ribosome, glyoxylate and dicarboxylate metabolism, carbon fixation in photosynthetic organisms, proteasome and the TCA cycle, indicating lysine crotonylation is involved in the common mechanism of metabolic regulation. A protein interaction network analysis revealed that diverse interactions are modulated by protein crotonylation. These results suggest that lysine crotonylation is involved in a variety of biological processes. HSP70 is a crucial protein involved in protecting plant cells and tissues from thermal or abiotic stress responses, and HSP70 protein was found to be crotonylated in paper mulberry. This systematic analysis provides the first comprehensive analysis of lysine crotonylation in paper mulberry and provides important resources for further study on the regulatory mechanism and function of the lysine crotonylated proteome.

This work aimed to study the profiling and efficiency of microbial communities and their abundance in the pulp and paper industry wastewater, which contained toxic metals, high biological oxygen demands, chemical oxygen demand, and ions contents. Sequence alignment of the 16S rRNA V3-V4 variable region zone with the Illumina MiSeq framework revealed 25356 operating taxonomical units (OTUs) derived from the wastewater sample. The major phyla identified in wastewater were Proteobacteria, Bacteroidetes, Firmicutes, Chloroflexi, Actinobacteria, Spirochetes, Patesibacteria, Acidobacteria, and others including unknown microbes. The study showed the function of microbial communities essential for the oxidation and detoxifying of complex contaminants and design of effective remediation techniques for the re-use of polluted wastewater. Findings demonstrated that the ability of different classes of microbes to adapt and survive in metal-polluted wastewater irrespective of their relative distribution, as well as further attention can be provided to its use in the bioremediation process.

Nowadays, thanks to nanotechnological progress, which itself guides us more and more closely toward not only the efficient design of innovative nanomaterials or nanostructures, but to the improvement of their functionality, we benefit from an important asset in the battle against pathogenic illnesses. Herein, we report a versatile biocompatible plasmonic nanoplatform based on a Whatman paper incorporating positively-charged gold nanospherical particles via the immersion approach. The morphological characterization of the as-engineered-plasmonic paper was examined by SEM (scanning electron microscopy) and HRTEM (high-resolution transmission electron microscopy) investigations, while its surface chemical modification with a synthetic polypeptide, specifically RRWHRWWRR-NH2 (P2), was proved by monitoring the plasmonic response of loaded gold nanospheres and the emission signal of P2 via fluorescence spectroscopy. The as-functionalized plasmonic paper is non-cytotoxic towards BJ fibroblast human cells at bactericidal concentrations. Finally, the antimicrobial activity of the P2-functionalized plasmonic paper on both planktonic bacteria and biofilms was tested against two reference strains: Gram-positive Bacteria, i.e., Staphylococcus aureus and the Gram-negative Bacteria, i.e., Escherichia coli, determining microbial inhibition of up to 100% for planktonic bacteria. In line with the above presented nanoplatform&rsquo, s proper design, followed by their functionalization with active antimicrobial peptides, new roads can be open for determining antibiotic-free treatments against different relevant pathogens.

Phenolic compounds such as catechol and resorcinol are toxic and persistent pollutants in the aqueous environment. Detection procedures such as chromatographic and spectrophotometric methods are time-consuming and require sophisticated instruments with skilled manpower. Development of a simple, cost effective, portable and disposable paper based biosensor could be a better alternative to the conventional methods. The present study attempted to develop a paper based biosensor by immobilizing horseradish peroxidase enzyme to detect catechol and resorcinol in aqueous samples. Horseradish peroxidase catalyzes the oxidation of phenolic compounds to semiquinones, which on reaction with a chromogen, 3-methyl 2-benzothiazolinone hydrazine (MBTH) gives faint pink to red color depending on the compound and its concentration in the sample is the basis for biosensing application. Different methods of enzyme immobilization on filter paper like physical adsorption, covalent coupling, and polysaccharide entrapment were executed. The performance of the various enzyme immobilization methods was evaluated by analyzing the developed color intensity using ImageJ software. Entrapment technique is the most effective method of immobilizing enzyme on the filter paper that produces the highest color intensity with better stability. The visible limit of detection (LoD) was observed as 0.45 mM (50 mg/L) for catechol and 0.09 mM (10 mg/L) for resorcinol in aqueous samples.

The production of paper-based bacterial cellulose-chitosan (BC-Ch) nanocomposites was accomplished following two different approaches. In the first, BC paper sheets were produced and then immersed in an aqueous solution of chitosan (BC-ChI); in the second, BC pulp was impregnated with chitosan prior to the production of paper sheets (BC-ChM). BC-Ch nanocomposites were investigated in terms of physical characteristics, antimicrobial and antioxidant properties, and the ability to inhibit the formation of biofilms on their surface. The two types of BC-Ch nanocomposites maintained the hydrophobic character, the air barrier properties, and the high crystallinity of the BC paper. However, BC-ChI showed a surface with a denser fiber network and with smaller pores than those of BC-ChM. Only 5% of the chitosan leached from the BC-Ch nanocomposites after 96 h of incubation in an aqueous medium, indicating that it was well retained by the BC paper matrix. BC-Ch nanocomposites displayed antimicrobial activity, inhibiting growth of and having a killing effect against bacteria Staphylococcus aureus and Pseudomonas aeruginosa and yeast Candida albicans. Moreover, BC-Ch papers showed activity against the formation of a biofilm on their surface. The incorporation of chitosan increased the antioxidant activity of the BC paper. Paper-based BC-Ch nanocomposites combined the physical properties of BC paper and the antimicrobial, antibiofilm, and antioxidant activities of chitosan.

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A bacterial “systematic evolution of ligands by exponential enrichment” protocol was developed herein to identify nucleic acid aptamers capable of binding molecules from three common nosocomial and antibiotic-resistant bacteria: Acinetobacter baumannii, Escherichia coli, and multidrug-resistant Staphylococcus aureus. This high-affinity and high-specificity process featured three selection stages for screening of bacteria-specific aptamers, and the aptamers identified were integrated into a microfluidic system. The biotin-labeled aptamers were first bound to nitrocellulose membranes housed within the chip and then incubated with bacteria; a tetramethyl benzidine (TMB)-streptavidin (blue) color reaction was next exploited upon binding of secondary aptamers to primary ones, thereby permitting bacterial detection. This new dual-aptamer microfluidic chip possesses many advantages over its traditional-scale counterparts, such as faster detection times (35 min), smaller size (7.0 cm × 5.0 cm × 1.2 cm), higher specificity, and the capability to detect multiple pathogens simultaneously; it may therefore be promising for point-of-care bacterial diagnostics.

Cellulose nanofibril (CNF) paper has various applications due to its unique advantages. Herein, we present the intrinsic mechanical properties of CNF papers, along with the preparation and properties of nanoparticle-reinforced CNF composite papers. The literature on CNF papers reveals a strong correlation between the intrafibrillar network structure and the resulting mechanical properties. This correlation is found to hold for all primary factors affecting mechanical properties, indicating that the performance of CNF materials depends directly on and can be tailored by controlling the intrafibrillar network of the system. The parameters that influence the mechanical properties of CNF papers were critically reviewed. Moreover, the effect on the mechanical properties by adding nanofillers to CNF papers to produce multifunctional composite products was discussed. We concluded this article with future perspectives and possible developments in CNFs and their bionanocomposite papers.

This paper addresses two key challenges toward an integrated forward error-correcting biosensor based on our previously reported self-assembled quick-response (QR) code. The first challenge involves the choice of the paper substrate for printing and self-assembling the QR code. We have compared four different substrates that includes regular printing paper, Whatman filter paper, nitrocellulose membrane and lab synthesized bacterial cellulose. We report that out of the four substrates bacterial cellulose outperforms the others in terms of probe (gold nanorods) and ink retention capability. The second challenge involves remote activation of the analyte sampling and the QR code self-assembly process. In this paper, we use light as a trigger signal and a graphite layer as a light-absorbing material. The resulting change in temperature due to infrared absorption leads to a temperature gradient that then exerts a diffusive force driving the analyte toward the regions of self-assembly. The working principle has been verified in this paper using assembled biosensor prototypes where we demonstrate higher sample flow rate due to light induced thermal gradients.

Development of a foam-formed cellulose filter paper with high wet strength was carried out for application as a drinking water filter. The wet strength and antimicrobial activity of cellulose foam paper against several bacteria species (Bacillus subtilis MTCC 441 (Gram +ve), B. cereus NCDC 240 (Gram +ve), Pseudomonas aeruginosa NCDC 105 (Gram -ve), Klebsiella pneumonia NCDC 138 (Gram -ve), and Escherichia coli MTCC 40 (Gram -ve)) were investigated. The morphology and structure of the cellulose foam paper were characterized using scanning electron microscopy (SEM). The results of our study confirmed that glutaraldehyde solution or 1,2,3,4-butanetetracarboxylic acid (BTCA) added to cellulose foam paper pretreated with cationic polyacrylamide (C-PAM) provided very high and stable wet strength performance together with excellent antimicrobial properties.

A bacterium isolated from effluent of thermal paper industry and identified as Pseudomonas aeruginosa (PAb1) based on 16SrRNA gene sequence analysis which could grow on basal mineral salt medium upon bisphenol A, which functions as an exclusive carbon source. Physicochemical variables of thermal paper industry effluent noted were significantly greater than the typical limit due to pollution of the acquiring water systems. The mathematic kinetic models like Monod, Moser and Tesier models were applied for batch fermentation of bisphenol A degradation in basal salt medium and the half saturation coefficient (KS) and the regression coefficient R2 using Monad, Moser and Tesier kinetic models registered as 9.947 g/L, 12.46 g/L and 14.14 g/L and 0.91, 0.94 and 0.84 respectively. Besides, the utmost specific growth rate μmax was witnessed as 0.841 h−1 for the P. aeruginosa (PAb1) regarding BPA degradation. Metabolic intermediates like phenol, acetophenone, and hydroquinone and p-hydroxybenzoic acid were also determined through the degradation process by GC-MS. The metabolic pathway of BPA degradation by the bacterial isolates was also designed in today's analysis. A probabilistic statistical model originated using Box-Behnken response surface methodology and process variables were optimized by nonlinear optimization.

Lack of safe drinking water is a major concern, mainly in developing countries. Consequently, there is a significant need for affordable technologies to provide clean drinking water. In this paper, two approaches for drinking water disinfection using nanocellulose and the antimicrobial compound phenyl bis-diphenylphosphinato bismuth (III) complex are studied. The first approach investigates the deactivation of bacteria when contaminated water percolates through a filter paper with a microfibrillated cellulose (MFC) coating containing Bi-complex. Antibacterial activity was studied by filtering bacterial broth with a Gram-positive pathogen Staphylococcus aureus as a test strain and measuring live bacteria in the filtrate. The filter composites did not show any sign of instantaneous killing of bacteria, irrespective of the Bi-complex loading or base paper substrate. However, the effluent demonstrated drastic reduction in bacterial population after resting at 37 °C. Bacteria were completely eradicated 24 h after passing through 5 wt% and 10 wt% Bi-coated filters. If also active against locally important drinking water contaminants, this approach could be used for purification of storage water. The second approach uses a Bi-complex loaded nanofibrillated cellulose (NFC) membrane. Herein, complete and immediate exclusion of bacteria was achieved in the NFC membrane when bacterial broth was passed through it at 1 bar. The 2.5 wt% Bi-loaded membrane could significantly minimise the risk of biofilm formation, even after 4 days of contact with bacterial broth. The Bi-complex concentration in the filtrate (130.6 ± 1.9 ppb) from the 2.5 wt% Bi-loaded microfilter was shown to be non-toxic to murine fibroblast cells. This study outlines the proof of concept of two sustainable prototypes for stored or immediate drinking water purification, but the approach would need to be extended to a broader range of relevant contaminating pathogens of drinking water to fully understand its utility.

Bacteria detection and toxicity measurement are essential in many aspects. Becoming increasingly popular in recent years, paper-based analytical devices (PADs) have proven to be cost-effective, portable and eco-friendly with quantitative diagnostic results. In this work, by a straightforward soaking-drying method, a resazurin-deposited PAD has been developed for rapid bacteria detection and biotoxicity measurement. The colorimetric response on the PAD was generated from metabolic reduction of resazurin by Enterococcus faecalis, a facultative anaerobic bacterial strain. After recording and quantifying the colorimetric response with Hue value by a smartphone, the bioassay on PAD enables the detection of resazurin reduction kinetics difference among bacteria at various densities in 10 min. Thereby, the bioassay on PAD was applied to study the toxicity of two chlorophenols, i.e. pentachlorophenol (PCP) and 4-chlorophenol (4-CP), to E. faecalis. Compared to growth-based inhibition test, which takes 5 h, this assay shows higher efficiency, i.e. in 30 min, the biotoxicity difference between PCP and 4-CP can be identified.

Aims This study was designed to analyse the prevalence of class 1 and class 2 integron integrase genes among antibiotic-resistant coliform bacteria isolated from paper currency circulating in Pakistan. Methods and results A total of 500 individual currency notes were collected from different food vending sites at Lahore, Pakistan. Bacterial population were identified by biochemical and PCR techniques. Antimicrobial susceptibility testing was performed by disc diffusion assay. The highest bacterial population on currency was found from street vendors and butcher shops. Escherichia coli was found to be the most prevalent coliform bacteria followed by Klebsiella sp. and Enterobacter sp. PCR amplification of antimicrobial resistance gene showed the presence of ampC, blaTEM , blaNDM-1 , qnrA, tet(A) and tet(B) genes among coliform isolates. A total of 47 integron integrase bearing strains of coliform bacteria were analysed. Sequence analysis showed the presence of dfrA1-aadA1, dfrA1, dfrA5, dfrA7, aadA1, aadA4 cassette arrays in class 1 integron and dfrA1-sat2-aadA1 in class 2 integrase genes. Conclusion Circulating currency was heavily contaminated with antimicrobial-resistant coliform bacteria bearing class 1 and class 2 integron integrase genes. Significance and impact of the study This study describes a potential threat of severe bacterial infections due to improper hand hygiene and community sanitation when dealing with the currency notes.

Three of the primary bottlenecks, which should be consider for practical, point-of-need use of microbial fuel cell (MFC) analytical devices were surpassed in this work: i) the use of a diffusive barrier, hence, an electrogenic biofilm; ii) longer enrichment/stabilization times to produce a biofilm, made in a laboratory environment, over the electrode; and iii) difficulty comparing results obtained from MFCs based on electrogenic biofilms with standardized bioassays, a setback to be adopted as a new method. Here we show an easy way to determine water toxicity employing planktonic bacteria as biorecognition agents. The paper-based MFC contain an electron carrier (or mediator) to facilitate charge transfer from bacteria to the anode. In this way, there is no need to use biofilms. As far as we know this is the first paper-based MFC containing P. putida KT2440, a well characterized non-pathogenic bacteria previously used in standardized water toxicity bioassays. Results were obtained in 80 min and an effective concentration 50 of 9.02 mg L−1, calculated for Zn2+ (a reference toxic agent), was successfully compared with previously published and ISO standardized bioassays, showing a promising future for this technology. The practical design and cost (less than one U.S. dollar) of the paper-based MFC toxicity test presented will open new market possibilities for rapid and easy-to-use MFC analytical devices.

Pulp and paper industry is coming under one of the most water polluting industries, and generated wastewater is highly toxic in nature. The paper mill requires huge quantity (~50–60 m3 of water to produce one ton of paper) of water, and accordingly huge quantity of chemical contaminated wastewater is discharged. The paper mill effluents have identified 240–250 chemicals in different stages of paper making. Various chemical constituents such as high chemical oxygen demand, biochemical oxygen demand, AOX, chlorinated compounds, color, suspended materials, lignin and their derivatives are released in the wastewater. The present review study is focused on the paper mill processes, wastewater generation and its effective treatment by microorganisms. The biological treatment has been identified as cost-effective and eco-friendly methods for the degradation of xenobiotic compounds for paper mill wastewater. Various studies have been performed so far to investigate the complex nature of wastewater by the application of bacteria, fungi and their enzymes at industrial scale. Therefore, the article discussed the importance of biological method as an effective technique for the degradation of paper mill wastewater.

In the present work, nanoparticles of copper and silver synthesized via pulsed laser ablation of the respective targets in distilled water are applied to cellulose filter paper to check their effectiveness in the annihilation of bacteria from contaminated water. The treatment of the filter paper with the nanoparticles is found to be an excellent way to get rid of two common bacteria, Staphylococcus aureus and Escherichia coli, from contaminated water. The spread plate method on agar, employed to test the antibacterial efficacy of the nanoparticle-treated papers, clearly shows the absence of bacterial growth upon coming into contact with the nanoparticles in the filter paper. These results were further substantiated by the growth kinetic study of the bacteria that exhibited slow growth of the bacteria that were exposed to the nanoparticles. The morphology of the bacteria that came into contact with the nanoparticles is found to be adversely affected by the nanoparticles. Both copper and silver nanoparticles show a similar extent of antibacterial activity.

In the present work, we report the synthesis of chitosan:starch‑silver nanoparticle (Cht:St-AgNPs) coated papers for antimicrobial packaging applications. The starch-assisted synthesized St-AgNPs are spherical in shape with an average particle size of 7 nm. Chitosan was mixed into the synthesized St-AgNPs solution with different ratios of 9:1, 8:2, 7:3, and 5:5 by weight. Further, the influence of different ratios of Cht:St-AgNPs on the various paper properties such as mechanical properties, water and oil resistance, and antimicrobial activities was investigated. It was observed that the properties of the coated papers were strongly dependent on the composition of Cht:St-AgNPs. The Cht:St-AgNPs-coated paper prepared with the ratio of 9:1 showed excellent mechanical properties and good resistance properties against water and oil. The Cht:St-AgNPs coated papers showed a remarkable enhancement in mechanical strength, oil and water resistance, and antibacterial and antifungal activity, which can make them a potential candidate for functional antimicrobial packaging applications.

Salmonella causes over a million foodborne illnesses per year in the United States resulting in more hospitalizations and deaths than any other foodborne bacterial pathogen. To help prevent outbreaks, a rapid, portable, sensitive, and reliable method for onsite detection of bacteria that can be used in different sample matrices would be beneficial. Herein, we present a colorimetric paper-based analytical device (PAD) combined with immunomagnetic separation (IMS) for detecting Salmonella typhimurium. IMS anti-Salmonella coated magnetic beads were applied to capture and separate bacteria from the sample matrix and preconcentrate it into small volumes before testing on paper. To directly detect S. typhimurium after IMS, a sandwich immunoassay was implemented into the procedure with β-galactosidase (β-gal) as the detection enzyme. Using the antibody/enzyme complex, we performed a colorimetric assay with chlorophenol red-β-d-galactopyranoside (CPRG) for bacteria quantification. The method was confirmed to be highly specific to S. typhimurium without interference from other pathogenic bacteria like Escherichia coli. Using this system, the limit of detection of S. typhimurium was found to be 10

Switching between metabolically active and dormant states provides bacteria with protection from environmental stresses and allows rapid growth under favorable conditions. This rapid growth can be detrimental to the environment, e.g., pathogens in recreational lakes, or to industrial processes, e.g., fermentation, making it useful to quickly determine when the ratio of dormant to metabolically active bacteria changes. While a rapid increase in metabolically active bacteria can cause complications, a high number of dormant bacteria can also be problematic, since they can be more virulent and antibiotic-resistant. To determine the metabolic state of Escherichia coli and Salmonella Typhimurium, we developed two paper-based colorimetric assays. The color changes were based on oxidoreductases reducing tetrazolium salts to formazans, and alkaline phosphatases cleaving phosphates from nitrophenyl phosphate salt. Specifically, we added iodophenyl-nitrophenyl-phenyl tetrazolium salt (INT) and methylphenazinium methyl sulfate to metabolically active bacteria on paper and INT and para-nitrophenyl phosphate salt to dormant bacteria on paper. The color changed in less than 60 min and was generally visible at 103 CFU and quantifiable at 106 CFU. The color changes occurred in both bacteria, since oxidoreductases and alkaline phosphatases are common bacterial enzymes. On one hand, this feature makes the assays suitable to a wide range of applications, on the other, it requires specific capture, if only one type of bacterium is of interest. We captured Salmonella or E. coli with immobilized P22 or T4 bacteriophages on the paper, before detecting them at levels of 102 or 104 CFU, respectively. Determining the ratio of the metabolic state of bacteria or a specific bacterium at low cost and in a short time, makes this methodology useful in environmental, industrial and health care settings.

Water quality monitoring is becoming an essential part of our lives as increasing human activities continue to spill unknown and unexpected contaminants into our water systems. To ensure the provision of safe and clean water to the public and the ecosystem, the development of rapid and sensitive in situ early warning systems for water toxicity monitoring is crucial. In this work, an entirely paper-based microbial fuel cell sensor utilizing freeze-dried bacteria is demonstrated as a portable and disposable water toxicity sensor. The bacterial cells were preinoculated on the anode reservoir of the device, and they were freeze-dried, making their on-site and on-demand applications possible. Upon rehydration of the bacteria with the water samples, current readings were obtained, and inhibition ratios (IRs) were calculated for different concentrations of formaldehyde as a model toxin. For 0.001, 0.01, and 0.02% of formaldehyde, IRs of 7.88, 16.08, and 23.14% were obtained, respectively. These IRs showed a very good linearity with the formaldehyde concentrations at R2 = 0.995. Additionally, the shelf life of the freeze-dried microbial fuel cell sensor was investigated. Even after 14 days of storage in the desiccator, at 4, and at −20 °C, the performance outputs compared to the new device were all at 96%.

Reduced graphene oxide (RGO) multilayer sheets were synthesized using waste paper pulp as carbon source. Formation of RGO was confirmed by Raman spectroscopy. RGO was mixed with cement at different concentrations (0.001, 0.01, 0.05 and 0.1% w/w) to obtain graphene cement composites (GCC). RGO is supposed to impart hydrophobicity to cement; hence, the capillary test was conducted. Hydrophobic property of GCC was found to be dependent on time, concentration and temperature. Antimicrobial property of GCC was checked which included antibacterial and antifungal studies. GCC showed better antibacterial performance (90%) against gram positive bacteria (Bacillus subtilis) than the gram-negative bacteria (Escherichia coli, 85%). Gram negative bacteria possess structurally more complex cell membranes than gram positive bacteria; hence, it was more resistant to GCC. Our finding supports the concept of involvement of membrane bound macromolecules in antibacterial performance of RGO. GCC was also found to be effective (88% inhibition) against fungal strain (Aspergillus niger).

The health and well-being of cattle is a significant concern for global agricultural output. In dairy production within low and middle income countries (LMICs), there is a significant biosensing challenge in detecting sexually transmitted infection (STI) pathogens during animal husbandry, due in part to difficulties associated with the limited infrastructure for veterinary medicine. Here we demonstrate low-cost, multiplexed and sample-to-answer paper-origami tests for the detection of three bovine infectious reproductive diseases in semen samples, collected at a test site in rural India. Pathogen DNA from one viral pathogen, Bovine Herpes virus-1 (BoHV-1) and two bacteria (Brucella and Leptospira) was extracted, amplified (using loop-mediated isothermal amplification, LAMP) and detected fluorescently, enabling