Recent studies on advance spectroscopic techniques for the identification of microorganisms: A review

The continuous development of resistance to antibiotic drugs by microorganisms causes high mortality and morbidity. Pathogens with distinct features and biochemical abilities make them destructive to human health. Therefore, early identification of the pathogen is of substantial importance for quick ailments and healthcare outcomes. Several phenotype methods are used for the identification and resistance determination but most of the conventional procedures are time-consuming, costly, and give qualitative results. Recently, great focus has been made on the utilization of advanced techniques for microbial identification. This review is focused on the research studies performed in the last five years for the identification of microorganisms particularly, bacteria using advanced spectroscopic techniques including mass spectrometry (MS), infrared (IR) spectroscopy, Raman spectroscopy (RS), and nuclear magnetic resonance (NMR) spectroscopy. Among all the techniques, MS techniques, particularly MALDI-TOF/MS have been widely utilized for microbial identification. A total of 44 bacteria i.e., 6 Staphylococcus spp., 3 Enterococcus spp., 6 Bacillus spp., 4 Streptococcus spp., 6 Salmonella spp., and one from each genus including Escherichia, Acinetobacter, Pseudomonas, Proteus, Clostridioides, Candida, Brucella, Burkholderia, Francisella, Yersinia, Moraxella, Vibrio, Shigella, Serratia, Citrobacter, and Haemophilus (spp.) were discussed in the review for their identification using the above-mentioned techniques. Among all the identified microorganisms, 21% of studies have been conducted for the identification of E. coli, 14% for S. aureus followed by 37% for other microorganisms.     

Free flow and capillary isoelectric focusing of bacteria from the tomatoes plant tissues

The means of the preconcentration and preseparation of selected species or pathovars of bacteria directly from the plant tissue suspension by free flow isoelectric focusing are introduced here. After the focusing, the resulting fraction of microorganisms, native or dynamically modified by the non-ionogenic tenside on the basis of pyrenebutanoate, was separated by capillary isoelectric focusing and/or cultivated and positively identified by gas chromatographic analysis of fatty acid methyl esters. Simultaneously, capillary isoelectric focusing with UV and fluorometric detection was used for the rapid estimation of unknown isoelectric points of the examined plant pathogenic species of genus Clavibacter, Xanthomonas and Pseudomonas prior to the preconcentration and preseparation. The microorganisms were of different origin, native and/or dynamically modified by the non-ionogenic tenside.     

Cebulantin,a Cryptic Lanthipeptide Antibiotic Uncovered Using Bioactivity‐Coupled HiTES

The majority of natural product biosynthetic gene clusters in bacteria are silent under standard laboratory growth conditions, making it challenging to uncover any antibiotics that they may encode. Herein, bioactivity assays are combined with high‐throughput elicitor screening (HiTES) to access cryptic, bioactive metabolites. Application of this strategy in Saccharopolyspora cebuensis, with inhibition of Escherichia coli growth as a read‐out, led to the identification of a novel lanthipeptide, cebulantin. Extensive NMR spectroscopic analysis allowed the elucidation of the structure of cebulantin. Subsequent bioactivity assays revealed it to be an antibiotic selective for Gram‐negative bacteria, especially against Vibrio species. This approach, referred to as bioactivity‐HiTES, has the potential to uncover cryptic metabolites with desired biological activities that are hidden in microbial genomes.     

Cebulantin,a Cryptic Lanthipeptide Antibiotic Uncovered Using Bioactivity‐Coupled HiTES

The majority of natural product biosynthetic gene clusters in bacteria are silent under standard laboratory growth conditions, making it challenging to uncover any antibiotics that they may encode. Herein, bioactivity assays are combined with high‐throughput elicitor screening (HiTES) to access cryptic, bioactive metabolites. Application of this strategy in Saccharopolyspora cebuensis, with inhibition of Escherichia coli growth as a read‐out, led to the identification of a novel lanthipeptide, cebulantin. Extensive NMR spectroscopic analysis allowed the elucidation of the structure of cebulantin. Subsequent bioactivity assays revealed it to be an antibiotic selective for Gram‐negative bacteria, especially against Vibrio species. This approach, referred to as bioactivity‐HiTES, has the potential to uncover cryptic metabolites with desired biological activities that are hidden in microbial genomes.     

Bioremediation of Hydrocarbons Contaminating Sewage Effluent Using Man-made Biofilms: Effects of Some Variables

Biofilm samples were established on glass slides by submerging them in oil-free and oil-containing sewage effluent for a month. In batch cultures, such biofilms were effective in removing crude oil, pure n-hexadecane, and pure phenanthrene contaminating sewage effluent. The amounts of the removed hydrocarbons increased with increasing biofilm surface area exposed to the effluent. On the other hand, addition of the reducing agent thioglycollate dramatically inhibited the hydrocarbon bioremediation potential of the biofilms. The same biofilm samples removed contaminating hydrocarbons effectively in three successive batch bioremediation cycles but started to become less effective in the cycles thereafter, apparently due to mechanical biofilm loss during successive transfers. As major hydrocarbonoclastic bacteria, the biofilms harbored species belonging to the genera Pseudomonas, Microvirga, Zavarzinia, Mycobacterium, Microbacterium, Stenotrophomonas, Gordonia, Bosea, Sphingobium, Brachybacterium, and others. The nitrogen fixer Azospirillum brasilense and the microalga Ochromonas distigma were also present; they seemed to enrich the biofilms, with nitrogenous compounds and molecular oxygen, respectively, which are known to enhance microbiological hydrocarbon degradation. It was concluded that man-made biofilms based upon sewage microflora are promising tools for bioremediation of hydrocarbons contaminating sewage effluent.     

A Rapid and Sensitive Aptamer‐Based Electrochemical Biosensor for Direct Detection of Escherichia Coli O111

A sensitive and specific electrochemical biosensor based on target‐induced aptamer displacement was developed for direct detection of Escherichia coli O111. The aptamer for Escherichia coli O111 was immobilized on a gold electrode by hybridization with the capture probe anchored on the electrode surface through Au‐thiol binding. In the presence of Escherichia coli O111, the aptamer was dissociated from the capture probe‐aptamer duplex due to the stronger interaction between the aptamer and the Escherichia coli O111. The consequent single‐strand capture probe could be hybridized with biotinylated detection probe and tagged with streptavidin‐alkaline phosphatase, producing sensitive enzyme‐catalyzed electrochemical response to Escherichia coli O111. The designed biosensor showed weak electrochemical signal to Salmonella typhimurium, Staphylococcus aureus and common non‐pathogenic Escherichia coli, indicating high specificity for Escherichia coli O111. Under the optimal conditions, the proposed strategy could directly detect Escherichia coli O111 with the detection limit of 112 CFU mL^?1 in phosphate buffer saline and 305 CFU mL^?1 in milk within 3.5 h, demonstrated the sensitive and accurate quantification of target pathogenic bacteria. The designed biosensor could become a powerful tool for pathogenic microorganisms screening in clinical diagnostics, food safety, biothreat detection and environmental monitoring.     

Microbial synthesis and characterization of physiochemical properties of polyhydroxyalkanoates (PHAs) produced by bacteria isolated from activated sludge obtained from the municipal wastewater works in Hong Kong

The first objective of this study was the measurement of physical properties of P(3HB-co-3HV) copolymers with different (hydroxybutyrate) HB to (hydroxyvalerate) HV ratios produced by Bacillus cereus (TRY2) isolated from activated sludge. The 3HV PHBV copolymers were 0.05, 22.6, 39.2, 54.1, and 69.1 mol%, respectively. The second objective was to study possible wastewater treatment and production of PHAs at the same time by B. cereus (TRY2) and Pseudomonas spp. (TOB17) (both were isolated from activated sludge), recombinant Bacillus DH5α, and a combination of the above three bacteria. The results were satisfactory; the maximum COD and TOC of the sewage sludge reduced were 53.5% and 67.5%, respectively.     

Reduction of nitric oxide by denitrifying bacteria

Two heterotrophic denitrifying bacteria,Paracoccus denitrificans andPseudomonas denitrificans, have been shown to utilize nitric oxide (NO) as a terminal electron acceptor and succinate, yeast extract, and heat/alkali pretreated municipal sewage sludge as carbon and energy sources. Complete removal of NO (0.50%) from a feed gas sparged into the cultures was observed. It is suggested that reduction of NO may be a common feature of denitrifying bacteria and that a microbial process to dispose of NO_x may be economically viable.     

Synthesis,characterization and antimicrobial activity of 3,5-di-tert-butylsalicylaldehyde-S-methylthiosemicarbazones and their Ni(II) complexes

Ni(II) complexes were prepared by the reactions of 3,5-di-tert-butylsalicylaldehyde-S-methylisothiosemicarbazone (L) with salicylaldehyde or 2-hydroxy-1-naphthaldehyde in the presence of NiCl₂·6H₂O. The complexes and starting material L were characterized by physic-chemical analysis and spectroscopic techniques such as ¹HNMR, ¹³CNMR, IR and UV–VIS. Antimicrobial activity studies of L and the two complexes standards strains of bacteria (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Bacillus cereus, Enterococcus faecalis, Streptococcus pneumoniae, Listeria monocytogenes, Escherichia coli, Salmonella typhi and Candida albicans) and 22 clinically isolated microorganisms, including multidrug resistant pathogenic microorganisms, were carried out. The free thiosemicarbazone L showed a significant inhibition of the growth all of Gram-positive bacteria tested.     

Construction of Recycling Photocatalytic Gels for the Disinfection of Pathogens and Degradation of Organic Pollutants

Bismuth oxybromide (BiOBr) nanosheets are exciting photocatalysts for microbial disinfection and organic dye degradation. However, it remains a great challenge to easily recycle these nanomaterials and improve their photocatalytic ability. Herein, we constructed a novel photocatalytic BiOBr@PAG gel containing BiOBr nanosheets and polyacrylamide gel (PAG), based on peroxydisulfate-induced polymerization reaction. The photocatalytic gel had equally distribution of BiOBr nanosheets on the surface, and could be easily recycled from water. More strikingly, the gel could also rapidly kill all tested pathogenic bacteria (i. e., Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) under irradiation. Its disinfection activity is attributed to remarkable intracellular ROS production and oxidative cell damage. Furthermore, the gel had higher photocatalytic activity than BiOBr nanosheets alone during degradation of organic dyes. This study developed a novel strategy for preparation of easy-recycling and high-efficiency photocatalytic systems for practical application in environmental treatment and medicinal disinfection.     

Biocide Use in the Antimicrobial Era: A Review

Biocides are widely used in healthcare and industry to control infections and microbial contamination. Ineffectual disinfection of surfaces and inappropriate use of biocides can result in the survival of microorganisms such as bacteria and viruses on inanimate surfaces, often contributing to the transmission of infectious agents. Biocidal disinfectants employ varying modes of action to kill microorganisms, ranging from oxidization to solubilizing lipids. This review considers the main biocides used within healthcare and industry environments and highlights their modes of action, efficacy and relevance to disinfection of pathogenic bacteria. This information is vital for rational use and development of biocides in an era where microorganisms are becoming resistant to chemical antimicrobial agents.     

Antimicrobial Activity of Broth Fermented with Kefir Grains

Kefir grains originate from the Caucasus region and are used for preparing beverages using sugar solution, milk, and fruit juice. As long as they are formed by a microbial consortium useful in the intestine, the produced drinks can be called probiotics. The aim of this study was to determine the antimicrobial activity during kefir fermentation in sugar broth. Fermentations with three kinds of carbohydrates (molasses, demerara sugar, and brown sugar) as carbon source were carried out. Brown sugar promoted the greatest antimicrobial activities, producing inhibition halos corresponding to 35, 14, 12, 14, and 14 mm for Candida albicans, Salmonella typhi, Shigella sonnei, Staphylococcus aureus, and Escherichia coli, respectively. Different carbon source concentrations and the time of fermentation influenced the size of the inhibition halos of the pathogenic microorganisms.     

Biodegradation of Polyhydroxyalkanoate Films in Natural Environments

Summary: Biodegradation of film specimens from polyhydroxyalkanoates (PHAs) of two types – poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) – was analysed in different environments: tropical sea waters of the South China Sea (Nha Trang, Vietnam) and soils in the environs of Hanoi (Vietnam), Nha Trang (Vietnam) and Krasnoyarsk (Siberia, Russia). In seawater, the mass loss of the specimens of both types was almost equal. However, in tropical soils, PHB degraded quicker than PHBV. In the Siberian soil, the degradation rate of the PHBV was generally higher than that of PHBV. Analysis of molecular mass of PHA specimens showed its decreasing during biodegradation. In the tropical sea conditions, PHA degrading microorganisms were represented by bacteria of Enterobacter, Bacillus and Gracilibacillus genera. Among PHA degrading bacteria, Burkholderia, Alcaligenes, Bacillus, Mycobacterium and Streptomyces genera were identified in Vietnamese soils, and Variovorax, Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus and Xanthomonas genera in Siberian soils. Micromycetes of Gongronella, Paecilomyces, Penicillium and Trichoderma genera exhibited PHA degrading activity in Vietnamese soils, and Paecilomyces, Penicillium, Acremonium, Verticillium and Zygosporium genera – in Siberian soils.     

Interaction of bacteria and ion‐exchange particles and its potential in separation for matrix‐assisted laser desorption/ionization mass spectrometric identification of bacteria in water

Identification of microbial contaminants in drinking water is a challenge to matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) due to low levels of microorganisms in fresh water. To avoid the time‐consuming culture step of obtaining enough microbial cells for subsequent MALDI‐MS analysis, a combination of membrane filtration and nanoparticles‐ or microparticles‐based magnetic separation is a fast and efficient approach. In this work, the interaction of bacteria and fluidMAG‐PAA, a cation‐exchange superparamagnetic nanomaterial, was investigated by MALDI‐MS analysis and transmission electron microscopy. FluidMAG‐PAA selectively captured cells of Salmonella, Bacillus, Enterococcus and Staphylococcus aureus. This capture was attributed to the aggregation of negatively charged nanoparticles on bacterial cell regional surfaces that bear positive charges. Three types of non‐porous silica‐encapsulated anion‐exchange magnetic microparticles (SiMAG‐Q, SiMAG‐PEI, SiMAG‐DEAE) were capable of concentrating a variety of bacteria, and were compared with silica‐free, smaller fluidMAG particles. Salmonella, Escherichia coli, Enterococcus and other bacteria spiked in aqueous solutions, tap water and reservoir water were separated and concentrated by membrane filtration and magnetic separation based on these ion‐exchange magnetic materials, and then characterized by whole cell MALDI‐MS. By comparing with the mass spectra of the isolates and pure cells, bacteria in fresh water can be rapidly detected at 1 × 10³ colony‐forming units (cfu)/mL. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolic profiling of meat: assessment of pork hygiene and contamination with Salmonella typhimurium

Spoilage in meat is the result of the action of microorganisms and results in changes of meat and microbial metabolism. This process may include pathogenic food poisoning bacteria such as Salmonella typhimurium, and it is important that these are differentiated from the natural spoilage process caused by non-pathogenic microorganisms. In this study we investigated the application of metabolic profiling using gas chromatography-mass spectrometry, to assess the microbial contamination of pork. Metabolite profiles were generated from microorganisms, originating from the natural spoilage process and from the artificial contamination with S. typhimurium. In an initial experiment, we investigated changes in the metabolic profiles over a 72 hour time course at 25 °C and established time points indicative of the spoilage process. A further experiment was performed to provide in-depth analysis of the metabolites characteristic of contamination by S. typhimurium. We applied a three-way PARAllel FACtor analysis 2 (PARAFAC2) multivariate algorithm to model the metabolic profiles. In addition, two univariate statistical tests, two-sample Wilcoxon signed rank test and Friedman test, were employed to identify metabolites which showed significant difference between natural spoiled and S. typhimurium contaminated samples. Consistent results from the two independent experiments were obtained showing the discrimination of the metabolic profiles of the natural spoiled pork chops and those contaminated with S. typhimurium. The analysis identified 17 metabolites of significant interest (including various types of amino acid and fatty acid) in the discrimination of pork contaminated with the pathogenic microorganism.     

Microbial attack on lignocellulose in the rumen

The microbial population of the rumen consists of many types and species of anaerobic and facultatively anaerobic microorganisms, often at high population densities, living in symbiosis with the animal. The animal is unable to synthesize enzymes capable of digesting the major structural components of plant cell walls (fiber) and relies on the rumen microbes to ferment this material with the generation of volatile fatty acids and microbial cells that the animal can utilize. The crude fiber fraction of ruminant forages consists mainly of cellulose, hemicellulose, and lignin. The rumen contents are rich in organic matter, which has a pH of 5.5–6.9, is maintained at about 39‡C, and has a low redox potential with little free oxygen in the rumen liquor. When plant material enters the rumen, it is invaded by many species of microorganisms, some of which digest certain structural components of the plant cell walls. Measurable digestion of plant cell walls occurs 4–5 h after ingestion by the animal. The rate and extent of digestion is affected by many factors, including the nature and degree of adaptation of the microbial population to the diet, the species of plant and its lignin content, the form in which it is presented to the animal, the amount consumed, and the rate of passage through the rumen. Because the rumen contents are anoxic, there is little degradation of lignin. Some solubilization occurs and lignin-hemicellulose complexes are found in rumen liquor, probably released by cellulolytic and hemicellulolytic microorganisms. One species of bacterium isolated from the rumen preferentially attacked highly lignified cell walls and grew on phenolic acids both aerobically and anaerobically (Akin, 1980), but the abundance and distribution of the species is not known. Phenolic acids are also released and further metabolized in the rumen, and methoxyal groups removed from lignin. Since lignin is covalently bound to cellulose and hemicellulose in the plant cell wall, it probably protects these polysaccharides from microbial attack both physically and chemically. Delignification increases the digestibility of cell wall polysaccharides several-fold. Cellulose may also be shielded from microbial attack by encrusting xylan or xyloglucans (Albersheim, 1975). When plant tissues enter the rumen, they are invaded by both cellulolytic and noncellulolytic microorganisms. Initial invasion is by ciliate protozoa and motile bacteria, followed by phycomycete zoospores and, later, nonmotile bacteria, including cellulolytic species. Some species of ciliate protozoa (e.g.,Epidinium ecaudatum) immediately commence to digest the plant cell walls, but cellulolysis by phycomycete fungi and bacteria is delayed because of preferential metabolism of soluble carbohydrates and the lag before significant attachment of cellulolytic bacteria has occurred. In the predominant cellulolytic rumen bacteria,Ruminococcus albus, R. flavefasciens, andBacteroides succinogenes (Forsberg et al., 1981), adhesion is a prerequisite to cellulolysis because the cellulase enzymes are bound to the cell surface, but some strains ofB. succinogenes also release vesicle-bound enzymes. Cellulases in some species of protozoa (e.g.,Epidinium ecaudatum,Eudiplodinium maggii, andEremoplastron bovis) are now considered to be of protozoon origin (Coleman et al., 1976), but ingested bacteria and adsorbed enzymes may contribute to protozoon cellulolysis. All species of rumen phycomycete fungi examined (Orpin, 1981) have been shown to be cellulolytic and grow on plant cell walls. The cellulase produced by one strain ofNeocallimastix frontalis was associated in part with membranous vesicles released into the medium. Digestion of plant cell walls by the phycomycete fungi is accompanied by the loss of up to 20% of the lignin as a ligninhemicellulose complex. The enzymology of cellulolysis in the rumen is not completely known. It is clear that more than one enzyme is responsible for complete hydrolyses of cellulose to glucose or cellobiose, and endo-1,4-Β-glucanases and Β-1,4-glucosidases have been found in several species of rumen microorganisms. There is also evidence for a ‘swelling factor’ being necessary prior to cellulolysis by some rumen species. All of the cellulolytic rumen microorganisms can also attack, to some degree, the hemicelluloses of the plant cell walls. In some species, cellulase and xylanase activity is associated with the same enzyme complex (e.g.,Bacteroides succinogenes). Other bacteria may attack hemicelluloses, but not cellulose (e.g., some strains ofButyrivibrio fibrisolvens), and others (e.g.,Lachnospira multiparus) digest pectin which allows plant cells to separate and make more cell wall components available to microbial attack. All these organisms act in a consortium resulting in extensive digestion of the plant cell walls.     

Recent advances in microchip electrophoresis for analysis of pathogenic bacteria and viruses

Life-threatening diseases, such as hepatitis B, pneumonia, tuberculosis, and COVID-19, are widespread due to pathogenic bacteria and viruses. Therefore, the development of highly sensitive, rapid, portable, cost-effective, and selective methods for the analysis of such microorganisms is a great challenge. Microchip electrophoresis (ME) has been widely used in recent years for the analysis of bacterial and viral pathogens in biological and environmental samples owing to its portability, simplicity, cost-effectiveness, and rapid analysis. However, microbial enrichment and purification are critical steps for accurate and sensitive analysis of pathogenic bacteria and viruses in complex matrices. Therefore, we first discussed the advances in the sample preparation technologies associated with the accurate analysis of such microorganisms, especially the on-chip microfluidic-based sample preparations such as dielectrophoresis and microfluidic membrane filtration. Thereafter, we focused on the recent advances in the lab-on-a-chip electrophoretic analysis of pathogenic bacteria and viruses in different complex matrices. As the microbial analysis is mainly based on the analysis of nucleic acid of the microorganism, the integration of nucleic acid-based amplification techniques such as polymerase chain reaction (PCR), quantitative PCR, and multiplex PCR with ME will result in an accurate and sensitive analysis of microbial pathogens. Such analyses are very important for the point-of-care diagnosis of various infectious diseases.     

Microbiological control and antibacterial action of a propolis-containing mouthwash and control of dental plaque in humans

Propolis is a bee product with several biological properties. This study aimed at investigating a propolis-containing mouthwash, its organoleptic properties, microbial contamination and its antibacterial action in vitro. This mouthwash was assessed in vivo to control dental plaque in humans. The presence of microorganisms was analyzed and the minimum inhibitory concentration against Streptococcus mutans was determined. A comparative study was done in vivo using propolis, chlorhexidine, and propolis plus chlorhexidine in lower concentrations for 14 days. Dental plaque was analyzed by the Patient Hygiene Performance (PHP) index. The odontological product was yellow, cloudy, free of microbial contamination, and exerted an inhibitory action in vitro. Individuals who used a propolis-containing mouthwash for 14 consecutive days in combination or not to chlorhexidine showed a similar PHP index to chlorhexidine alone. The product exerted an antibacterial action in vitro and in vivo, exhibiting a positive action in the control of dental plaque.     

Prevalence of Antimicrobial Resistant Bacteria from Conjunctival Flora in an Eye Infection Prone Breed (Saint Bernard)

The conjunctival bacterial resident and opportunistic flora of dogs may represent a major source of dissemination of pathogens throughout the environment or to other animals and humans. Nevertheless, contamination with bacteria from external sources is common. In this context, the study of the antimicrobial resistance (AMR) pattern may represent an indicator of multidrug resistant (MDR) strains exchange. The present study was focused on a single predisposed breed—Saint Bernard. The evaluated animals were healthy, but about half had a history of ocular disease/treatment. The swabs collected from conjunctival sacs were evaluated by conventional microbiological cultivation and antimicrobial susceptibility testing (AST). The most prevalent Gram-positive was Staphylococcus spp.; regardless of the history, while Gram-negative was Pseudomonas spp.; exclusively from dogs with a history of ocular disease/treatment. Other identified genera were represented by Bacillus, Streptococcus, Trueperella, Aeromonas and Neisseria. The obtained results suggest a possible association between the presence of mixed flora and a history of ocular disease/treatment. A high AMR was generally observed (90%) in all isolates, especially for kanamycin, doxycycline, chloramphenicol and penicillin. MDR was recorded in Staphylococcus spp. and Pseudomonas spp. This result together with a well-known zoonotic potential may suggest an exchange of these strains within animal human populations and the environment.