Specific growth rates and td of
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He has more than 96 SCI publications, he acted as an academic editor, reviewer, and he holds several registered patents.",coeditorOneBiosketch:"Researcher in enteric health, most notably probiotics and their relationship to nutrition and disease protection in poultry as well as the design of avian enteric inflammation models for the study of the impact of diet and microbiome on growth and development.",coeditorTwoBiosketch:"My research focuses mainly on apicomplexan parasites, such as Toxoplasma Cryptosporidium, Eimeria, and minor on nematodes. Prof.Alali has more than 30 publications and he acts as a reviewer in many journals.",coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"209746",title:"Dr.",name:"Saeed",middleName:null,surname:"El-Ashram",slug:"saeed-el-ashram",fullName:"Saeed El-Ashram",profilePictureURL:"https://mts.intechopen.com/storage/users/209746/images/system/209746.png",biography:"Dr. Saeed El-Ashram is a professor at Foshan University, China, and Kafrelsheikh University, Egypt, and a research professor at Zhaoqing Dahuanong Biology Medicine Co., Ltd., China. Dr. El-Ashram\\'s research focuses on parasitic diseases. He has more than 100 journal publications to his credit. He is currently an academic editor and reviewer and holds several registered patents. The primary focus of his research is to understand how the animal immune system recognizes and responds to parasitic infections with and/or without a microbial community. Some are the causative agents of significant diseases in humans, such as toxoplasmosis, cryptosporidiosis, alveolar echinococcosis, and fascioliasis. Others are a substantial financial burden to food producers because of the effects these parasites have on domestic animals, for example, coccidiosis and cryptosporidiosis (livestock and poultry).",institutionString:"Foshan University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Foshan University",institutionURL:null,country:{name:"China"}}}],coeditorOne:{id:"73465",title:"Dr.",name:"Guillermo",middleName:null,surname:"Téllez",slug:"guillermo-tellez",fullName:"Guillermo Téllez",profilePictureURL:"https://mts.intechopen.com/storage/users/73465/images/system/73465.jpg",biography:"Guillermo Tellez-Isaias received his DVM and MS in Veterinary Sciences from the National Autonomous University of Mexico (UNAM), and his Ph.D. from Texas A&M University. He worked as a professor at UNAM for sixteen years, eight as head of the Avian Medicine Department, College of Veterinary Medicine. Dr. Tellez was president of the National Poultry Science Association of Mexico and is a member of the Mexican Veterinary Academy and the Mexican National Research System. Currently, he works as a research professor at the Center of Excellence in Poultry Science, University of Arkansas. His research is focused on poultry gastrointestinal models to evaluate the beneficial effects of functional foods to enhance intestinal health and disease resistance.",institutionString:"University of Arkansas at Fayetteville",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"8",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"University of Arkansas at Fayetteville",institutionURL:null,country:{name:"United States of America"}}},coeditorTwo:{id:"437285",title:"Dr.",name:"Firas",middleName:null,surname:"Alali",slug:"firas-alali",fullName:"Firas Alali",profilePictureURL:"https://mts.intechopen.com/storage/users/437285/images/17927_n.jpg",biography:"Academic reviewer for many journals.\r\nAssociate Professor at University of Kerbala, Iraq. Firas Alali works at the Department of Veterinary Parasitology of Veterinary Medicine college, Kerbala University. Firas does research in Parasitology, Entomology, and Vector-Borne Diseases including zoonoses.",institutionString:"University of Kerbala",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:null},coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"13",title:"Immunology and Microbiology",slug:"immunology-and-microbiology"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"453623",firstName:"Silvia",lastName:"Sabo",middleName:null,title:"Mrs.",imageUrl:"https://mts.intechopen.com/storage/users/453623/images/20396_n.jpg",email:"silvia@intechopen.com",biography:null}},relatedBooks:[{type:"book",id:"1591",title:"Infrared Spectroscopy",subtitle:"Materials Science, Engineering and Technology",isOpenForSubmission:!1,hash:"99b4b7b71a8caeb693ed762b40b017f4",slug:"infrared-spectroscopy-materials-science-engineering-and-technology",bookSignature:"Theophile Theophanides",coverURL:"https://cdn.intechopen.com/books/images_new/1591.jpg",editedByType:"Edited by",editors:[{id:"37194",title:"Dr.",name:"Theophile",surname:"Theophanides",slug:"theophile-theophanides",fullName:"Theophile Theophanides"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3161",title:"Frontiers in Guided Wave Optics and Optoelectronics",subtitle:null,isOpenForSubmission:!1,hash:"deb44e9c99f82bbce1083abea743146c",slug:"frontiers-in-guided-wave-optics-and-optoelectronics",bookSignature:"Bishnu Pal",coverURL:"https://cdn.intechopen.com/books/images_new/3161.jpg",editedByType:"Edited by",editors:[{id:"4782",title:"Prof.",name:"Bishnu",surname:"Pal",slug:"bishnu-pal",fullName:"Bishnu Pal"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"371",title:"Abiotic Stress in Plants",subtitle:"Mechanisms and Adaptations",isOpenForSubmission:!1,hash:"588466f487e307619849d72389178a74",slug:"abiotic-stress-in-plants-mechanisms-and-adaptations",bookSignature:"Arun Shanker and B. 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This fact concerns also the short ripened ewes’ lump cheese traditionally produced immediately after milking in Slovakian upland cottages. The cheese is curdled with rennet, fermented by native lactic acid bacteria and briefly ripened for 7 to 10 d. Then it is usually sent to a cheese factory for production of the soft Slovakian „Bryndza” cheese [2].
This chapter deals with the behaviour of coagulase-positive staphylococci as their populations belong to the ubiquitous microflora of ewes’ milk.
For growth it requires B vitamins (thiamine and nicotic acid), inorganic salts and amino acids as a nitrogen source, especially arginine, cystein, proline and valine. Glutamic acid, leucine and tyrosine are not required for growth, but they are essential for enterotoxin production. Deprivation of any amino acid is much less responsive in SEA production than for SEB or SEC production. Arginine seems to be essential for enterotoxin B production [5,7,10].
Most strains hydrolyse native animal proteins (casein, gelatine, fibrin), lipids, phospholipoproteins and Tween. They also coagulate animal plasma with the assistance of a coagulase and the clumping factor. Besides that, the typical enzymatic activity of
The role of enzymes like coagulase, catalase, hyaluronidase, lipase, heat-resistant nuclease, staphylokinase and β-galactosidase is to disrupt cell structure, degrade cell lipids and hyaluronic acid, and to convert fibrinogen to fibrin. All those mechanisms promote
Toxins (leukocidins, haemolysins and epidermolytic toxin) possess haemolytic, cytotoxic, dermonecrotic and lethal activity. They are able to paralyse smooth and skeletal muscles, damage blood vessels, cause extensive lesions on the skin and reveal a moist glistering surface (called also Ritter’s disease) and finally have a toxic effect on the central nervous system [5,11,14].
In addition to surface factors, enzymes and cytotoxins, strains of
From the food point of view, the production of one or more staphylococcal enterotoxins (SEs) is crucial, because they are causative agents of staphylococcal food poisoning (SFP) outbreaks in human.
Staphylococcal enterotoxins are heat-stable exoproteins consisting from 236 to 296 aminoacids with a molecular mass of 25-35 kDa. Upon hydrolysis, 18 amino acids are present, mostly aspartic acid, glutamic acid, lysine and tyrosine. For the majority of these, an isolectric point of pH 5.7-8.6 is considered. There are five different types of classical enterotoxins (SEA-SEE) which are distinct in antigen reaction. Recently, new types of enterotoxins and enterotoxin-like types (SEG-SEV) have been described in
The SFP is characterized as a relatively mild intoxication which occurs after ingestion of at least 20 ng of staphylococcal enterotoxins presented in the food. Although the numbers of outbreaks caused by bacterial toxins are generally underestimated, official EU data [18] reported 558 outbreaks in 2009 from which almost 53% were caused by
Enterotoxins are produced in a narrower range of temperature than the growth is noticed. In general, enterotoxins production is expected in a temperature range of 10-46 °C, with the optimum temperature for production in the range 40-45 °C [6,8,10,20]. Enterotoxins are heat-stable in milk. Their resistance to heating is represented by D-values at 121°C and 100°C ranging from 9.9-11.4 to 70.0 minutes, respectively [21,22]. Their heat-resistance decreases following SECSEBSEA and is also significantly reduced in acidic conditions [10]. It should also be noticed that 99.6% of cells are destroyed by the pasteurisation of milk at 72 °C for 15s, and at 72 °C for 35s all cells are killed. Enterotoxins can resist both the process of milk pasteurisation or sterilisation of canned foods [6,7].
Regarding pH,
Fast acidification down to values unacceptable for growth is the most efficient way of
In general, a tolerance of
Complete inhibition of
Similarly to temperature effect on enterotoxins production, the pH range allowing production of enterotoxins is also more limited than those for growth. The practical limit in acidic foods is pH 5.0, with an optimum of 7.0. The SEA is produced under a wider range of pH than SEB or SEC [6,20].
A characteristic feature that distinguishes
The ability of
With respect to enterotoxins production requirements, values of water activity for their production are mostly in the same range as for the growth of the producer. In food with decreased water activity and at aerobic conditions, the enterotoxins can be produced even if the value is from 0.86 to 0.89 aw. The production of SEB appears to be more sensitive to reduced water activity than SEA production, whereas SEA is produced up to aw 0.87-0.89, SEB is produced only in the narrow range of water activity values 0.99-0.97 [10,28].
In generally,
The majority of disinfectants routinely used in the food industry (halogens, quarternary ammonium salts) will be effective when applied correctly. After inappropriate sanitation however, the cells can recover and become more resistant [8].
Pathogenic
The perspective targets for drugs in
Staphylococci compete poorly with indigenous bacteria and are inhibited by the antagonistic activities of other organisms. Therefore the presence of
Media for isolation and determination of
In the first group are media such as tryptone soya broth, brain heart infusion broth, mannitol-salt agar, salt meat broth. They use sodium chloride as the selective agent and metabolizable substrates such as mannitol, blood or milk as diagnostic agents are incorporated. However, higher concentrations of salt and the lack of resuscitators in the media may inhibit injured or stressed cells (false negative results). Moreover, other microorganisms are salt-tolerant or can metabolize substrates, so the media are not specific enough.
In the second group are media which contain combinations of selective and diagnostic agents. The list of selective agents which includes sodium azide, sodium chloride, lithium chloride, potassium tellurite, glycine and antibiotics (polymyxin or sulphametathine) is not large but provides many combinations. Media like tellurite-polymyxin agar, KRANEP agar, Giolitti-Cantoni broth, Baird-Parker agar and its modifications, and some other media are found in this group. The mode of diagnostic action is fermentation of mannitol, egg yolk reaction – clear zones around colonies, black colonies (reduction of tellurite to tellurium) and pigment production [5]. The problems of this media are that some animal strains of
To correct the discrepancies of media in the previous groups, the addition of plasma (from rabbit, pig or rat) with bovine fibrinogen instead of egg yolk is used. These media allow the detection of coagulase directly on the plate due to the formation of fibrin zones around the colonies. Such media include Baird-Parker agar with plasma and Rabbit-plasma fibrinogen (RPF) medium. Because of the cost and variable performance of commercially available plasmas, they are not used in routine examinations.
Nowadays, there is also the possibility to use chromogenic media for detection of
The first step in the identification of suspected colonies is the Gram-staining, microscopic examination of the morphology, catalase test and also β-haemolysis surrounding colonies on the sheep-blood agar [16,17,33-37].
One of the preferred examinations is the coagulase test, either as a tube format for the presence of unbounded extracellular coagulase or as a slide coagulase test for the presence of a clumping factor - cell wall associated enzyme. There are commercially available rapid and convenient tests, and also laboratory procedures are permissible to detect the presence of coagulase. It should also be noted that the production of coagulase is not a property of only
From among biochemical tests, the API-Staph system and the VITEK Gram-positive Identification Card are widely used. They are based on the reaction of microorganism with a set of specific substrates. There is also the possibility of fluorescence microscopy detection without previous growth of culture on selective media by the use of the VIT-Staphylococcus system. This is based on the penetration of a specific gene probe into the bacteria cell, marking the individual signature of the gene sequence with the dye and illuminating them. Subsequently, the samples are examined under fluorescence microscopy. Bacteria belonging to the genus
However, the most reliable way to identify a suspicious colony as
From the human health point of view, methods for the detection of staphylococcal enterotoxins are required. Firstly, the presence of genes encoding enterotoxins (
The natural ecological niches of
In primary production and the dairy environment, except for milk producing animals, human beings and operational environment belong among the main sources of product contamination. One third of people are the asymptomatic carriers of
According to references [34,45,46], the frequency of
In Slovakia, a similar incidence (4-9%) of
The lack of proper hygienic measures during food processing would also increase the counts of
In the study performed by Kousta et al. [51], 96% of both unpasteurized and pasteurized milk cheeses met the EU regulations for
The correlation between the presence of a respective gene and real enterotoxin production is about 70-80%, which might be explained by the incomplete expression of the enterotoxigenic genes. This is influenced by environmental conditions, such as temperature, pH and water activity which are important both for the growth and production of enterotoxins [20,42,52,54]. For this reason, it is necessary to know cardinal values of intrinsic and extrinsic factors preventing the growth of
The growth of various
Growth of
T [°C] | ||||
[h-1] | td [h] | [h-1] | td [h] | |
7 | 0.006 | 120.3 | ||
8 | 0.026 | 27.0 | ||
10 | 0.055 | 12.7 | ||
12 | 0.103 | 6.8 | 0.082 | 8.5 |
15 | 0.148 | 4.7 | 0.145 | 4.8 |
18 | 0.313 | 2.2 | 0.264 | 2.6 |
21 | 0.545 | 1.3 | 0.484 | 1.4 |
25 | 0.711 | 1.0 | ||
30 | 1.215 | 0.6 | ||
35 | 1.664 | 0.4 | ||
39 | 1.931 | 0.4 | ||
43 | 1.903 | 0.4 | ||
46 | 0.562 | 1.2 |
Specific growth rates and td of
Despite the slow growth of the 2064 strain, the temperature of 7 °C can be considered as the minimal temperature for growth of
In order to know the variability of growth rates as calculated from the growth curves, we performed static cultivations of the 28 confirmed
Comparing the determined parameters with values generated by the Combase Predictor (
Taking into account that 12-37% of the bound of reliability during cultivation experiments is tolerable; these findings demonstrate that the duration of the lag phase and the growth rate of
0.163 | 13.8 | 2.93 | 8.17 | 4.3 | |
0.025 | 3.2 | 0.59 | 0.29 | 0.6 | |
15.3 | 23.0 | 20.0 | 3.6 | 14.0 | |
0.104 | 4.4 | 0.78 | 7.19 | 2.2 | |
0.318 | 21.4 | 3.72 | 9.02 | 6.6 | |
0.159 | 14.3 | 3.13 | 8.19 | 4.4 |
Growth parameters of
Within quantitative predictive microbiology the secondary models are used to characterise the influence of intrinsic or extrinsic food factors on specific growth parameters. Among the temperature models, the Ratkowsky-type and cardinal temperature models are appreciated by users despite the basically empirical nature of the relationships [66].
The specific growth rates of three
Based on the testing of goodness of fit, the per cent of variance (%V) confirmed high correlation coefficients
Comparison of the Ratkowsky model applied to the strains of
The effect of temperature in the whole range from 7 °C to 51 °C on the ability of
According to the recommendation of Ratkowsky [68], maximal temperature for
From the food practice point of view, the model of Gibson et al. [69] is useful for the prediction of the time (
In the case of initial
Plots of the square root of specific growth rates (
As temperature was the only modifying environmental factor, lag phase was described by means of the model developed by Davey et al. [71] according to the following equation (
In dairy practice, the initial numbers of
Growth dynamics of
Growth and fermentative metabolism of lactic acid bacteria, as a permanent component of raw milk microflora, are offered by a wide variety of fermented dairy products. Besides the most effective inhibitive activity against pathogen and spoilage microorganisms, which includes production of organic acids and subsequent pH decrease, they produce bacteriocins, H2O2, and aromatic compounds and act as a strong competitor for nutritional factors (nicotineamide, biotine or niacine) [23,72,73].
If there is slow and insufficient acid production in the growth environment, no inhibitive effect is observed. This was the case of
Thus, it is interesting to select an appropriate starter culture of LAB which is able to efficiently inhibit
The requirements assigned to a starter culture of LAB include fast growth and survival in dairy environment, rapid production of lactic acid resulting in pH value diminution and no production of toxic or other technologically and sensorially unacceptable metabolites.
The effectiveness of a starter culture of LAB is related to the rate at which it can produce sufficient amounts of lactic acid, predominantly in the first six hours of fermentation. It is connected with the phenomena of the pH lag phase. It is obvious in Fig. 5 that the higher the incubation temperature, the more intensive the metabolism of LAB, and the sooner a pH decrease will occur. The intensity of pH drop is determined by the initial counts of the starter culture, as was confirmed in our co-culture experiments with
Period without any pH change (lagpH) found during static cultivation of the starters Fresco 1010 (Christian Hansen, Hørsholm, Denmark) and
It was also observed that during co-cultivation of Fresco culture with
The ratio between the initial inoculum of
However, our data did not showed a direct relation between the inhibition of
Besides initial concentration of LAB, the applied temperature also has a strong effect on the microbial growth dynamics. With an increasing of the incubation temperature, the duration of pH and microbial lag phase is shortened. On the other hand, the higher the temperature, the higher the growth rates.
The combined effect of temperature and the initial Fresco culture is depicted in Fig. 7. From it, one is able to calculate the necessary addition of Fresco culture and thermal mode during milk or young cheese fermentation to ensure a minimal increase in the numbers of
Alomar et al. [76] also found that
Although acidification plays an important role in
Dependence of specific growth rate (
Dependence of increase in
Original ewes’ lump cheese is an artisanal full-fat, soft rennet cheese from raw ewes’ milk manufactured on the farm level in Slovakian mountain areas according to the technological steps described and pictured in Fig. 8 and 9, respectively. After two weeks of ripening at temperatures from 18 °C to 21 °C, it is used for industrial production of the popular Slovakian “Bryndza” cheese [2]. Fermentation of the lump cheese relies on native mesophilic lactic acid bacteria (LAB) such as
Flow diagram of artisanal production of ewes’ lump cheese
Generally, cheeses are considered as one of the safest foods currently consumed. However, pathogenic bacteria which can be transmitted by dairy products cannot be underestimated. Historically, there have been several outbreaks related to the consumption of cheeses. The predominantly responsible organisms
Photo-documentation illustrating the artisanal manufacture of ewes’ lump cheese on the farm level in Slovakian mountain areas (Author: Ľ. Valík)
Despite the raw milk origin and substantial proportion of raw milk cheeses containing enterotoxigenic
According to our investigations of eight products manufactured under upland farm conditions, the acidification of the curd started after a 10-20 h period and went on intensively for 20 h. Thus, a level of acidity equivalent to pH of 5.2-4.9 was usually reached in young cheese after 30-40 h. Such a fairly long time permits to the growth not only LAB but also of undesirable bacteria, including
The first 24 h of the process of making raw milk cheese appeared to be critical for
In order to prevent
As seen in Fig. 10a and 10b, a pH of 5.0, unacceptable for growth of
Growth dynamics and pH value changes during fermentation of ewes’ lump cheese at 18 °C without starter culture (10a) and with addition of 1% Fresco culture (10b); ■ presumptive LAB on M17 agar, presumptive LAB on MRS agar, counts of
In order to keep the numbers of
This assumption was also confirmed by some other authors. Olarte et al. [82] observed differences in
A rapid decrease in pH values from 6.7 to an average value of 5.24 was observed in 24 h old raw cows’ milk cheeses [58]. From an initial average density of 1.89 log
In raw milk cheeses collected by Jakobsen et al. [80], a significant decline in pH values was observed after 5-6 h of fermentation and pH lower than 5.5 was achieved in all samples after 24 h. The highest
In raw goats’ milk cheeses, the initial log counts of
On the other hand, in raw milk Mexican cheese Fresco, pH decline from pH 6.7 to value 5.6 was achieved only after 10 days of ripening at 4 °C. Counts of
Based on these results and observations from literature, it is strongly recommended, to use the starter culture at least in artisanal cheese production. Rapid fermentation process prevents against the growth of
The variation in the responses of
Even if pasteurization kills
The inhibitory potential of LAB on
Artisanal raw milk cheese production poses a few critical factors limiting its safety. With reference to the growth of
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein modules are found to be a part of adaptive antivirus defense systems in archaea and bacteria and mediate immunity by a three-stage process called adaption, processing of the primary transcript, and interference. These systems incorporate fragments of foreign DNA (known as spacers) into CRISPR cassettes, then transcribe the CRISPR arrays including the spacers, and process them to make a guide crRNA or the clustered regularly interspaced short palindromic repeats ribonucleic acid (CRISP RNA) which is employed to specifically target and cleave the genome of the cognate virus or plasmid. Earlier classic methods such as zinc finger motif, meganucleases, and transcription activator-like effector nucleases were deployed for genome editing but due to its prerequisite for different fusion proteins, the technology raised hurdles in its applicability. The characteristic feature of single guide RNA of CRISPR to regulate Cas protein to target specific gene sequence is highly advantageous to overcome the barriers posing from classic methods. Proteins cas1 and cas2 genes are found to be the core and active part of the information processing subsystems of the three distinct types of CRISPR/Cas systems [1]. Due to the current problems with the vast diversity and complexity of the architecture of CRISPR/Cas systems, the classification is still challenging. Based on the presence of three signature genes, the classification is as follows:
Typical type I loci contain the signature cas3 gene, which codes for helicase and DNase activities within a single large protein. The detailed sequence and structural comparisons have led to the recognition of many of these proteins in the RAMP superfamily including Cas5 and Cas6 families. Type I systems are currently divided into six subtypes, I-A to I-F, each of which has its own signature gene and distinct features of operon organization [2, 3].
These contain cas9 as a signature gene encoding for a multidomain protein that combines all the functions of effector complexes and the target DNA cleavage and is essential for the maturation of the crRNA. These systems use cellular (not encoded within the CRISPR/Cas loci) RNase III and tracrRNA for the processing of pre-crRNA. Type II CRISPR-Cas systems are currently classified into three subtypes such as II-A, II-B, and II-C. Type II-A encompasses an additional signature gene csn2. Protein csn2 is found to be engaged in spacer integration. Type II-B systems belong to the Cas family of proteins with 5′-single-stranded DNA exonuclease activity. The recently proposed type II-C CRISPR-Cas systems possess only three protein-coding genes (cas1, cas2, and cas9) and are common in sequenced bacterial genomes (Figure 1) [2, 3].
Simplified model of the immunity mechanisms of class 1 and class 2 CRISPR-Cas systems. The CRISPR-Cas systems are composed of a
All type III systems possess the signature gene cas10 which encodes a multidomain protein containing a palm domain similar to that in cyclases and polymerases of the PolB family (Table 1) [2, 3].
Classification | Type I | Type II | Type III | Type V |
---|---|---|---|---|
Signature protein | Cas 3 | Cas 9 | Csm (III-A) or Cmr (III-B) | Cas 12a |
Effector | Cascade | CrRNA | sgRNA | CrRNA |
Cleavage product | SSBs | DSBs | SSBs | DSBs |
Different types of CRISPR/Cas based on signature protein, effector, and cleavage product [3].
Initially, CRISPR-Cas 9 was found to nick the DNA along with the guide RNA Cas 12a belonging to class II Type VA system, derived from
Type II is further divided into six types based on their structure and function. The Cas12a protein contains a RuvC endonuclease domain, which sequentially cleaves the non-targeting strand and the targeting strand to form DSBs (double-stranded base pairs). Compared to the CRISPR/Cas9 system, this system has several remarkable differences, including the signature protein, PAM sequence, and cleavage product [6]. CRISPR/Cas12a based sensing methods focus on fluorescence readout with reduced transduction efficiency as studies report a direct correlation between the catalysis systems with recognition elements (i.e., aptamers), thus greatly improving the working efficiency of the detection platform. Cas 13 consists of four subtypes and is involved in RNA interference activities. Off-target editing is critical to Cas 13 and requires significant attention in retrieving obstacles for protein analysis [7].
CRISPR/Cas systems generally play a role as RNA-guided endonucleases (crRNA). The crRNA guides cas proteins to specific DNA sequences whereby the hybridization leads to cas protein activation which later results in cleavage of DNA sequence [8]. Figure 2 shows the components of Cas9, Cas12a, Cas12f, and Cas13a.
Basic components of CRISPR/Cas9, Cas12a, Cas12f, and Cas13a pink triangle indicates cis-cleavage site [
Cas9 is an endonuclease and the single-guide RNA (sgRNA) of CRISPR-Cas9 systems contains a hairpin-rich region that binds to Cas9 and a 20-nucleotide “spacer” region that binds with the complementary “protospacer” region in the target strand of a dsDNA duplex. Binding between the sgRNA and the DNA target brings Cas9 into close proximity to the target (Figure 2). The His-Asn-His (HNH) domain of Cas9 cleaves the strand that is complementary to sgRNA (target strand) and the RuvC domain of Cas9 cleaves the other strand of the dsDNA (non-target strand). Single-guide RNA (sgRNA) (Figure 3) [13].
Combining functional nucleic acids and molecular translators with CRISPR/Cas technology for detection of non-nucleic acids such as proteins. Adapted from Ref. [
Recent findings indicate that the cas12a proteins have both trans and cis cleavage activities on ssDNA regardless of the sequence. Notably, Cas12a is the first Cas protein to be identified whose ternary complex has been shown to have trans-ss DNA cleavage ability. Research shows that Cas12a may have acquired single-stranded DNA ability through evolution due to the abundance of viruses in the environment. Thus, gaining a significant role as a powerful and dominating weapon to eliminate invasion by foreign ss DNA. The well-characterized variants of cas12, cas12a, and cas12f, formerly known as cas14 lack the HNH domain but nevertheless, achieved the PAM dependant cleavage with RuvC domain alone. Recent findings have reported Cas13a also called C2Ca, an RNA-guided and RNA-targeting CRISPR effector from the class 2 type VI CRISPR system, was found to have the trans-cleavage activity on RNA. Additionally, the RuvC catalytic pocket of both C2c1 and Cas12a was responsible for the cleavage of both strands of targeted dsDNA [9].
Electrochemical biosensors register physical-chemical and biological change and possess high throughput of the biological recognition process. Depending on the type of biological recognition, sensors are classified into biocatalytic devices and affinity sensors. Biocatalytic sensors integrate enzymes and whole cells as recognition elements leading to exquisite specificity and a significant rise in the rate of reaction whereas affinity sensors make use of extreme selectivity and specificity for acquiring higher sensitivity. The electrochemical transducer responds to the binding event and converts the electrical response to an output that can be amplified, stored, and displayed [14]. Due to its signal-off architecture, these electrochemical sensors provide limited sensitivity and productivity. To overcome these limitations the CRISPR/Cas12a based electron sensing biosensors have been developed for non-nucleic acid targets. CRISPR/Cas12a-based immobilization-free electrochemical biosensors can detect small molecules and proteins by adjusting regions for target recognition in RCA components [15]. Transcription factors (TFs) assay seems to be path-breaking as it is found to be involved directly in many diseases including cancers. CRISPR/Cas 12a based biosensors for the detection of transcription factors have been developed. The biosensing mechanism is based on the interaction of TF’s with double-stranded DNA activator eliminating Cas12a/crRNA from contacting and interacting with the 14 activators, thus inhibiting Cas12a activation. As a consequence of this strategy, the DNase activity of Cas12a was controlled and several TFs with well-defined binding sites could be quantified at the picomolar level with high precision [16, 17].
Recent findings report that the implementation of various nucleic acid amplification strategies led to improvements in analytical specificity and sensitivity and the development of point of care (POC) diagnostics. For example, the best-studied reaction is the amplification employing the Cas9 nickase (Cas9nAR) which when combined with polymerase and primers may substantially duplicate double-stranded DNA (dsDNA) without requiring heat cycling as does the polymerase chain reaction (PCR) [18].
In contrast to PCR, isothermal proximity assay seems to be an effective protein quantification assay for disease biomarkers and point of care diagnostics. Recent advances in the CRISPR/Cas technique specifically combining recombinase polymerase activity (RPA) and ssDNAse activity have led to the discovery of a series of isothermal assays for protein quantification. iPCCA relies on proximity binding for target recognition due to which it holds the potential for detecting non-nucleic acid targets such as proteins. However, isothermal amplification does not necessitate the use of advanced and sophisticated thermal cyclers and hence is more commonly used in biosensing [9].
The most widely used bioassay, ELISA (enzyme-linked immunosorbent assay) has revolutionized the ability to detect a wide variety of antigens. Complex chemical structure and restricted catalytic efficiency of HRP has a direct correlation with poor sensitivity in picomolar and nanomolar concentration. However, conventional ELISA is still not sensitive enough to detect ultralow concentrations of biomarkers for the early diagnosis of cancer, cardiovascular risk, neurological disorders, and infectious disease. CRISPR/Cas 13a based signal amplification strategy also called CLISA has been used to develop a 10 fold high-sensitive method for detecting low abundance. Recently, CRISPR/Cas13a has been recently demonstrated to have RNA-directed RNA cleavage ability. This RNA-guided trans-endonuclease activity is highly specific, being activated only when the target RNA has the perfect complementary sequence to the crRNA and is highly efficient. This potent signal amplification ability of CRISPR/Cas13a enables the development of direct RNA assays with a sensitivity down to the femtomolar level [19, 20].
Aptamer, a highly selective recognition element has been combined with various analytical techniques to increase the sensitivity of protein assay. Amongst these, an electrochemical technique using specific aptamers as recognition elements exhibits great promise in detecting protein duo to its attractive merits, such as high selectivity and sensitivity, the potential for miniaturization, and ease of integration with additional components [21]. Recent findings have demonstrated that the electrochemical aptasensor has been effectively used for the detection of thrombin in femtomolar concentration. It has been reported that once CRISPR RNA (crRNA)-directed Cas12a binds to a specific target DNA, the conserved RuvC nuclease domain in Cas12a will non-specifically cut single-stranded DNA (ssDNA). A homogeneous electrochemical aptasensor has been reported for sensitive and specific detection of thrombin by utilizing binding-induced DNA strand displacement strategy as the transduction element of thrombin and rolling circle amplification-regulated CRISPR/Cas12a for signal amplification. Importantly, this homogeneous electrochemical aptasensor can detect the femtomolar range of thrombin, and exhibited good specificity relative to other interfering blood-relevant proteins. The BIDSD-RCA-CRISPR/Cas12a is implemented in three steps, but this electrochemical aptasensor dispenses with the need for probe surface-immobilization procedure, simplifying the preparation process, and reducing the operating cost of the analysis. The strategy further could be applied to detect another disease-related protein biomarker in early diagnosis in the future [22].
The struggle for life between bacteria and their infecting viruses (phages) has led to the development of numerous bacterial defense mechanisms and their phage-encoded opponents. Recently, anti-CRISPR proteins have been identified, which inhibits the CRISPR/Cas system. The mechanism by which anti-CRISPR proteins inhibit CRISPR/Cas provides an extensive set of valuable tools to both understand and manipulate CRISPR [21]. Several findings report that the growing number of anti-CRISPR families has a significant impact on CRISPR/Cas function and has been a driving force in the evolution of CRISPR-Cas. These Anti-CRISPR systems rely heavily on Aca proteins due to their extensive interaction with anti-CRISPRs and the presence of Aca genes has the potential to act as anti-anti CRISPR playing a vital role in CRISPR-based antibacterial technologies [22, 23]. Anti-CRISPR ranges from 50 to 150 amino acids with no sequence similarity. Recent finding demonstrated that phage carries atleast one anti-CRISPR gene to avoid elimination by competent hosts. The unique mechanism of anti-CRISPR results in sequence-specific transcriptional repression system. Type II anti-CRISPRs have more evident biotechnological uses, given the widespread usage of CRISPR-Cas9 genome editing tools. Their application could be critical for gene drive and gene therapy technologies [24].
CRISPR/Cas based technology has a lot of potential as a tool for treating a range of medical conditions that have a genetic component, including cancer, hepatitis B, or even high cholesterol [25, 26, 27]. It is likely to be many years before CRISPR/Cas technology is used routinely in humans. CRISPR/Cas technology emerged as a versatile technology with wide application in the genome sequence editing, molecular studies of various gene functions, protein detection, gene therapy, and in the biomedical science as a diagnostic technology for detection of covid 19 like viral, bacterial, and various genetic disease [28]. Cancer is one of the fatal diseases that has severely threatened human life and caused a tremendous burden for society [29]. Early diagnosis of cancer is of great benefit to treat patients in early stages which leads to improve the survival rate of cancer patients. In body fluids detection of cancer related biomarkers is a critical kind of noninvasive technique for cancer diagnosis. Nevertheless, existing techniques of cancer biomarker detection always depends on a large-scale instruments and required sophisticated operation [30]. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR/Cas) based in vitro diagnosis can simplify the detection procedures and improve sensitivity and specificity, with great promise as the next-generation molecular diagnosis [31]. In the future, genome-wide screening for various genetic disorders, and cancer subtypes should be conducted to identify specific genetic and epigenetic targets for CRISPR technology to be most effective. The functionality of the identified mutations and their related signaling pathways need to be thoroughly analyzed before they are manipulated for therapy with CRISPR technology [32]. More in vivo research on Cas9 epigenetic regulation is needed to better understand its impact on cancer epigenetics. The use of synthetic biology for Cas9 modulation can be further extended to create real-time predictive algorithms for specific metastatic pathways that update as epigenetic regulation progress and the cancer advances so that treatment can always be precisely one step ahead of cancer. Ongoing research has the potential to optimize and advance CRISPR technology, culminating in the clinical realization of its full potential for breast cancer diagnosis, modeling, and treatment [29, 33, 34]. In the future, CRISPR/Cas technology will be used as a unique promising technology to study the various genes for their function, for identification of mutations and their correction, this technology will be used in tumor angiogenesis research for cancer treatment [35], CRISPR technology also used for modification of genetic sequence to develop various organisms for the benefit of human and environmental protection. Much research is still focusing on its use in animal models or isolated human cells, with the aim to eventually use the technology to routinely treat diseases in humans (Figure 4).
Applications of CRISPR/Cas system in detection of various molecules [
From many years scientists have learned about genetics and gene function by studying the effects of alteration in DNA sequence. Artificially by making a change in a gene, either in a cell line or a whole organism, it is possible to study the effect of that change to understand what the function of that gene is. For a long period geneticists used chemicals or radiation to create mutations but this approach is not precise and specific and due to its randomness for several years scientists have been using ‘gene targeting’ to introduce changes in specific places in the genome, by deletion or insertion either whole genes or single bases. Conventional gene targeting has been very valuable for studying genes and genetics, however it takes a long time to create a mutation and is fairly expensive But the CRISPR/Cas9 system based technology currently stands out as the fastest, cheapest and most reliable system for ‘editing’ genes. In the last decade CRISPR/Cas is a genome editing technology that is creating a an atmosphere of excitement in the science world because of its faster, cheaper, promising, precise, sensitive and efficient and more accurate nature than previous conventional techniques of genome engineering and it has a wide range of potential applications. CRISPR/Cas technology have made it possible to edit the genomes of most cell types precisely and efficiently hence (CRISPR)/Cas9 system is a novel, versatile and easy-to-use tool to edit genomes irrespective of their complexity, with multiple and applications in almost all branches of life science, biomedicine and facilitating the elucidation of target gene function in biology and diseases. CRISPR/Cas technology able to detect various targets starting from nucleic acids to proteins. Incorporating CRISPR/Cas systems with numerous nucleic acid amplification strategies allows the generation of amplified detection signals, enrichment of low-abundance molecular targets, enhancements in analytical specificity and sensitivity, and development of point-of-care diagnostic techniques. It is concluded that the CRISPR/Cas systems in association with functional nucleic acids (FNAs) and molecular translators permits the detection of non-nucleic acid targets, like proteins, metal ions, and tiny molecules. Productive integrations of CRISPR technology with nucleic acid amplification techniques lead to sensitive and fast detection of Protein.
The authors would like to thank Dr. B.A. Mehere, Principal and Head of the Department of Biochemistry and Biotechnology, Dr. Ambedkar College, Deekshabhoomi, Nagpur, India, for providing research space and facility.
The authors declare no conflict of interest.
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His studies in robotics lead him not only to a PhD degree but also inspired him to co-found and build the International Journal of Advanced Robotic Systems - world's first Open Access journal in the field of robotics.",institutionString:null,institution:{name:"TU Wien",country:{name:"Austria"}}},{id:"441",title:"Ph.D.",name:"Jaekyu",middleName:null,surname:"Park",slug:"jaekyu-park",fullName:"Jaekyu Park",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/441/images/1881_n.jpg",biography:null,institutionString:null,institution:{name:"LG Corporation (South Korea)",country:{name:"Korea, South"}}},{id:"465",title:"Dr",name:"Christian",middleName:null,surname:"Martens",slug:"christian-martens",fullName:"Christian Martens",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"479",title:"Dr.",name:"Valentina",middleName:null,surname:"Colla",slug:"valentina-colla",fullName:"Valentina Colla",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/479/images/358_n.jpg",biography:null,institutionString:null,institution:{name:"Sant'Anna School of Advanced Studies",country:{name:"Italy"}}},{id:"494",title:"PhD",name:"Loris",middleName:null,surname:"Nanni",slug:"loris-nanni",fullName:"Loris Nanni",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/494/images/system/494.jpg",biography:"Loris Nanni received his Master Degree cum laude on June-2002 from the University of Bologna, and the April 26th 2006 he received his Ph.D. in Computer Engineering at DEIS, University of Bologna. On September, 29th 2006 he has won a post PhD fellowship from the university of Bologna (from October 2006 to October 2008), at the competitive examination he was ranked first in the industrial engineering area. He extensively served as referee for several international journals. He is author/coauthor of more than 100 research papers. He has been involved in some projects supported by MURST and European Community. His research interests include pattern recognition, bioinformatics, and biometric systems (fingerprint classification and recognition, signature verification, face recognition).",institutionString:null,institution:null},{id:"496",title:"Dr.",name:"Carlos",middleName:null,surname:"Leon",slug:"carlos-leon",fullName:"Carlos Leon",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Seville",country:{name:"Spain"}}},{id:"512",title:"Dr.",name:"Dayang",middleName:null,surname:"Jawawi",slug:"dayang-jawawi",fullName:"Dayang Jawawi",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Technology Malaysia",country:{name:"Malaysia"}}},{id:"528",title:"Dr.",name:"Kresimir",middleName:null,surname:"Delac",slug:"kresimir-delac",fullName:"Kresimir Delac",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/528/images/system/528.jpg",biography:"K. Delac received his B.Sc.E.E. degree in 2003 and is currentlypursuing a Ph.D. degree at the University of Zagreb, Faculty of Electrical Engineering andComputing. His current research interests are digital image analysis, pattern recognition andbiometrics.",institutionString:null,institution:{name:"University of Zagreb",country:{name:"Croatia"}}},{id:"557",title:"Dr.",name:"Andon",middleName:"Venelinov",surname:"Topalov",slug:"andon-topalov",fullName:"Andon Topalov",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/557/images/1927_n.jpg",biography:"Dr. Andon V. Topalov received the MSc degree in Control Engineering from the Faculty of Information Systems, Technologies, and Automation at Moscow State University of Civil Engineering (MGGU) in 1979. He then received his PhD degree in Control Engineering from the Department of Automation and Remote Control at Moscow State Mining University (MGSU), Moscow, in 1984. From 1985 to 1986, he was a Research Fellow in the Research Institute for Electronic Equipment, ZZU AD, Plovdiv, Bulgaria. In 1986, he joined the Department of Control Systems, Technical University of Sofia at the Plovdiv campus, where he is presently a Full Professor. He has held long-term visiting Professor/Scholar positions at various institutions in South Korea, Turkey, Mexico, Greece, Belgium, UK, and Germany. And he has coauthored one book and authored or coauthored more than 80 research papers in conference proceedings and journals. His current research interests are in the fields of intelligent control and robotics.",institutionString:null,institution:{name:"Technical University of Sofia",country:{name:"Bulgaria"}}},{id:"585",title:"Prof.",name:"Munir",middleName:null,surname:"Merdan",slug:"munir-merdan",fullName:"Munir Merdan",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/585/images/system/585.jpg",biography:"Munir Merdan received the M.Sc. degree in mechanical engineering from the Technical University of Sarajevo, Bosnia and Herzegovina, in 2001, and the Ph.D. degree in electrical engineering from the Vienna University of Technology, Vienna, Austria, in 2009.Since 2005, he has been at the Automation and Control Institute, Vienna University of Technology, where he is currently a Senior Researcher. His research interests include the application of agent technology for achieving agile control in the manufacturing environment.",institutionString:null,institution:null},{id:"605",title:"Prof",name:"Dil",middleName:null,surname:"Hussain",slug:"dil-hussain",fullName:"Dil Hussain",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/605/images/system/605.jpg",biography:"Dr. Dil Muhammad Akbar Hussain is a professor of Electronics Engineering & Computer Science at the Department of Energy Technology, Aalborg University Denmark. Professor Akbar has a Master degree in Digital Electronics from Govt. College University, Lahore Pakistan and a P-hD degree in Control Engineering from the School of Engineering and Applied Sciences, University of Sussex United Kingdom. Aalborg University has Two Satellite Campuses, one in Copenhagen (Aalborg University Copenhagen) and the other in Esbjerg (Aalborg University Esbjerg).\n· He is a member of prestigious IEEE (Institute of Electrical and Electronics Engineers), and IAENG (International Association of Engineers) organizations. \n· He is the chief Editor of the Journal of Software Engineering.\n· He is the member of the Editorial Board of International Journal of Computer Science and Software Technology (IJCSST) and International Journal of Computer Engineering and Information Technology. \n· He is also the Editor of Communication in Computer and Information Science CCIS-20 by Springer.\n· Reviewer For Many Conferences\nHe is the lead person in making collaboration agreements between Aalborg University and many universities of Pakistan, for which the MOU’s (Memorandum of Understanding) have been signed.\nProfessor Akbar is working in Academia since 1990, he started his career as a Lab demonstrator/TA at the University of Sussex. After finishing his P. hD degree in 1992, he served in the Industry as a Scientific Officer and continued his academic career as a visiting scholar for a number of educational institutions. In 1996 he joined National University of Science & Technology Pakistan (NUST) as an Associate Professor; NUST is one of the top few universities in Pakistan. In 1999 he joined an International Company Lineo Inc, Canada as Manager Compiler Group, where he headed the group for developing Compiler Tool Chain and Porting of Operating Systems for the BLACKfin processor. The processor development was a joint venture by Intel and Analog Devices. In 2002 Lineo Inc., was taken over by another company, so he joined Aalborg University Denmark as an Assistant Professor.\nProfessor Akbar has truly a multi-disciplined career and he continued his legacy and making progress in many areas of his interests both in teaching and research. 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He was head of this department from 1993 to 2003. His specializations are medicine, gastroenterology, clinical pharmacology, clinical nutrition, and dietetics. His research fields are biochemical pharmacological examinations in the human gastrointestinal (GI) mucosa, mechanisms of retinoids, drugs, capsaicin-sensitive afferent nerves, and innovative pharmacological, pharmaceutical, and nutritional (dietary) research in humans. He has published about 360 peer-reviewed papers, 197 book chapters, 692 abstracts, 19 monographs, and has edited 37 books. He has given about 1120 regular and review lectures. He has organized thirty-eight national and international congresses and symposia. He is the founder of the International Conference on Ulcer Research (ICUR); International Union of Pharmacology, Gastrointestinal Section (IUPHAR-GI); Brain-Gut Society symposiums, and gastrointestinal cytoprotective symposiums. He received the Andre Robert Award from IUPHAR-GI in 2014. 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The preliminary objectives of the study are to understand and develop the evidence-based tools and interventions for the control and prevention of malaria in different sites of the INDIA. Alongside, with the help of next-generation genomics study, the team has studied the antimalarial drug resistance in India. Further, he has extended his research in the development of Humanized mice for the study of liver-stage malaria and identification of molecular marker(s) for the Artemisinin resistance. At present, his research focuses on understanding the role of B cells in the activation of CD8+ T cells in malaria. Received the CSIR-SRF (Senior Research Fellow) award-2018, FIMSA (Federation of Immunological Societies of Asia-Oceania) Travel Bursary award to attend the IUIS-IIS-FIMSA Immunology course-2019',institutionString:"Nirma University",institution:{name:"Nirma University",country:{name:"India"}}},{id:"334383",title:"Ph.D.",name:"Simone",middleName:"Ulrich",surname:"Ulrich Picoli",slug:"simone-ulrich-picoli",fullName:"Simone Ulrich Picoli",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/334383/images/15919_n.jpg",biography:"Graduated in Pharmacy from Universidade Luterana do Brasil (1999), Master in Agricultural and Environmental Microbiology from Federal University of Rio Grande do Sul (2002), Specialization in Clinical Microbiology from Universidade de São Paulo, USP (2007) and PhD in Sciences in Gastroenterology and Hepatology (2012). She is currently an Adjunct Professor at Feevale University in Medicine and Biomedicine courses and a permanent professor of the Academic Master\\'s Degree in Virology. She has experience in the field of Microbiology, with an emphasis on Bacteriology, working mainly on the following topics: bacteriophages, bacterial resistance, clinical microbiology and food microbiology.",institutionString:null,institution:{name:"Universidade Feevale",country:{name:"Brazil"}}},{id:"229220",title:"Dr.",name:"Amjad",middleName:"Islam",surname:"Aqib",slug:"amjad-aqib",fullName:"Amjad Aqib",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229220/images/system/229220.png",biography:"Dr. Amjad Islam Aqib obtained a DVM and MSc (Hons) from University of Agriculture Faisalabad (UAF), Pakistan, and a PhD from the University of Veterinary and Animal Sciences Lahore, Pakistan. Dr. Aqib joined the Department of Clinical Medicine and Surgery at UAF for one year as an assistant professor where he developed a research laboratory designated for pathogenic bacteria. Since 2018, he has been Assistant Professor/Officer in-charge, Department of Medicine, Manager Research Operations and Development-ORIC, and President One Health Club at Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan. He has nearly 100 publications to his credit. His research interests include epidemiological patterns and molecular analysis of antimicrobial resistance and modulation and vaccine development against animal pathogens of public health concern.",institutionString:"Cholistan University of Veterinary and Animal Sciences",institution:null},{id:"62900",title:"Prof.",name:"Fethi",middleName:null,surname:"Derbel",slug:"fethi-derbel",fullName:"Fethi Derbel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62900/images/system/62900.jpeg",biography:"Professor Fethi Derbel was born in 1960 in Tunisia. He received his medical degree from the Sousse Faculty of Medicine at Sousse, University of Sousse, Tunisia. He completed his surgical residency in General Surgery at the University Hospital Farhat Hached of Sousse and was a member of the Unit of Liver Transplantation in the University of Rennes, France. He then worked in the Department of Surgery at the Sahloul University Hospital in Sousse. Professor Derbel is presently working at the Clinique les Oliviers, Sousse, Tunisia. His hospital activities are mostly concerned with laparoscopic, colorectal, pancreatic, hepatobiliary, and gastric surgery. He is also very interested in hernia surgery and performs ventral hernia repairs and inguinal hernia repairs. He has been a member of the GREPA and Tunisian Hernia Society (THS). During his residency, he managed patients suffering from diabetic foot, and he was very interested in this pathology. For this reason, he decided to coordinate a book project dealing with the diabetic foot. Professor Derbel has published many articles in journals and collaborates intensively with IntechOpen Access Publisher as an editor.",institutionString:"Clinique les Oliviers",institution:null},{id:"300144",title:"Dr.",name:"Meriem",middleName:null,surname:"Braiki",slug:"meriem-braiki",fullName:"Meriem Braiki",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/300144/images/system/300144.jpg",biography:"Dr. Meriem Braiki is a specialist in pediatric surgeon from Tunisia. She was born in 1985. She received her medical degree from the University of Medicine at Sousse, Tunisia. She achieved her surgical residency training periods in Pediatric Surgery departments at University Hospitals in Monastir, Tunis and France.\r\nShe is currently working at the Pediatric surgery department, Sidi Bouzid Hospital, Tunisia. Her hospital activities are mostly concerned with laparoscopic, parietal, urological and digestive surgery. She has published several articles in diffrent journals.",institutionString:"Sidi Bouzid Regional Hospital",institution:null},{id:"229481",title:"Dr.",name:"Erika M.",middleName:"Martins",surname:"de Carvalho",slug:"erika-m.-de-carvalho",fullName:"Erika M. de Carvalho",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229481/images/6397_n.jpg",biography:null,institutionString:null,institution:{name:"Oswaldo Cruz Foundation",country:{name:"Brazil"}}},{id:"186537",title:"Prof.",name:"Tonay",middleName:null,surname:"Inceboz",slug:"tonay-inceboz",fullName:"Tonay Inceboz",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/186537/images/system/186537.jfif",biography:"I was graduated from Ege University of Medical Faculty (Turkey) in 1988 and completed his Med. PhD degree in Medical Parasitology at the same university. I became an Associate Professor in 2008 and Professor in 2014. I am currently working as a Professor at the Department of Medical Parasitology at Dokuz Eylul University, Izmir, Turkey.\n\nI have given many lectures, presentations in different academic meetings. I have more than 60 articles in peer-reviewed journals, 18 book chapters, 1 book editorship.\n\nMy research interests are Echinococcus granulosus, Echinococcus multilocularis (diagnosis, life cycle, in vitro and in vivo cultivation), and Trichomonas vaginalis (diagnosis, PCR, and in vitro cultivation).",institutionString:"Dokuz Eylül University",institution:{name:"Dokuz Eylül University",country:{name:"Turkey"}}},{id:"71812",title:"Prof.",name:"Hanem Fathy",middleName:"Fathy",surname:"Khater",slug:"hanem-fathy-khater",fullName:"Hanem Fathy Khater",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/71812/images/1167_n.jpg",biography:"Prof. Khater is a Professor of Parasitology at Benha University, Egypt. She studied for her doctoral degree, at the Department of Entomology, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, USA. She has completed her Ph.D. degrees in Parasitology in Egypt, from where she got the award for “the best scientific Ph.D. dissertation”. She worked at the School of Biological Sciences, Bristol, England, the UK in controlling insects of medical and veterinary importance as a grant from Newton Mosharafa, the British Council. Her research is focused on searching of pesticides against mosquitoes, house flies, lice, green bottle fly, camel nasal botfly, soft and hard ticks, mites, and the diamondback moth as well as control of several parasites using safe and natural materials to avoid drug resistances and environmental contamination.",institutionString:null,institution:{name:"Banha University",country:{name:"Egypt"}}},{id:"99780",title:"Prof.",name:"Omolade",middleName:"Olayinka",surname:"Okwa",slug:"omolade-okwa",fullName:"Omolade Okwa",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/99780/images/system/99780.jpg",biography:"Omolade Olayinka Okwa is presently a Professor of Parasitology at Lagos State University, Nigeria. She has a PhD in Parasitology (1997), an MSc in Cellular Parasitology (1992), and a BSc (Hons) Zoology (1990) all from the University of Ibadan, Nigeria. She teaches parasitology at the undergraduate and postgraduate levels. She was a recipient of a Commonwealth fellowship supported by British Council tenable at the Centre for Entomology and Parasitology (CAEP), Keele University, United Kingdom between 2004 and 2005. She was awarded an Honorary Visiting Research Fellow at the same university from 2005 to 2007. \nShe has been an external examiner to the Department of Veterinary Microbiology and Parasitology, University of Ibadan, MSc programme between 2010 and 2012. She is a member of the Nigerian Society of Experimental Biology (NISEB), Parasitology and Public Health Society of Nigeria (PPSN), Science Association of Nigeria (SAN), Zoological Society of Nigeria (ZSN), and is Vice Chairperson of the Organisation of Women in Science (OWSG), LASU chapter. She served as Head of Department of Zoology and Environmental Biology, Lagos State University from 2007 to 2010 and 2014 to 2016. She is a reviewer for several local and international journals such as Unilag Journal of Science, Libyan Journal of Medicine, Journal of Medicine and Medical Sciences, and Annual Research and Review in Science. \nShe has authored 45 scientific research publications in local and international journals, 8 scientific reviews, 4 books, and 3 book chapters, which includes the books “Malaria Parasites” and “Malaria” which are IntechOpen access publications.",institutionString:"Lagos State University",institution:{name:"Lagos State University",country:{name:"Nigeria"}}},{id:"273100",title:"Dr.",name:"Vijay",middleName:null,surname:"Gayam",slug:"vijay-gayam",fullName:"Vijay Gayam",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/273100/images/system/273100.jpeg",biography:"Dr. Vijay Bhaskar Reddy Gayam is currently practicing as an internist at Interfaith Medical Center in Brooklyn, New York, USA. He is also a Clinical Assistant Professor at the SUNY Downstate University Hospital and Adjunct Professor of Medicine at the American University of Antigua. He is a holder of an M.B.B.S. degree bestowed to him by Osmania Medical College and received his M.D. at Interfaith Medical Center. His career goals thus far have heavily focused on direct patient care, medical education, and clinical research. He currently serves in two leadership capacities; Assistant Program Director of Medicine at Interfaith Medical Center and as a Councilor for the American\r\nFederation for Medical Research. As a true academician and researcher, he has more than 50 papers indexed in international peer-reviewed journals. He has also presented numerous papers in multiple national and international scientific conferences. His areas of research interest include general internal medicine, gastroenterology and hepatology. He serves as an editor, editorial board member and reviewer for multiple international journals. His research on Hepatitis C has been very successful and has led to multiple research awards, including the 'Equity in Prevention and Treatment Award” from the New York Department of Health Viral Hepatitis Symposium (2018) and the 'Presidential Poster Award” awarded to him by the American College of Gastroenterology (2018). He was also awarded 'Outstanding Clinician in General Medicine” by Venus International Foundation for his extensive research expertise and services, perform over and above the standard expected in the advancement of healthcare, patient safety and quality of care.",institutionString:"Interfaith Medical Center",institution:{name:"Interfaith Medical Center",country:{name:"United States of America"}}},{id:"93517",title:"Dr.",name:"Clement",middleName:"Adebajo",surname:"Meseko",slug:"clement-meseko",fullName:"Clement Meseko",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/93517/images/system/93517.jpg",biography:"Dr. Clement Meseko obtained DVM and PhD degree in Veterinary Medicine and Virology respectively. He has worked for over 20 years in both private and public sectors including the academia, contributing to knowledge and control of infectious disease. Through the application of epidemiological skill, classical and molecular virological skills, he investigates viruses of economic and public health importance for the mitigation of the negative impact on people, animal and the environment in the context of Onehealth. \r\nDr. Meseko’s field experience on animal and zoonotic diseases and pathogen dynamics at the human-animal interface over the years shaped his carrier in research and scientific inquiries. He has been part of the investigation of Highly Pathogenic Avian Influenza incursions in sub Saharan Africa and monitors swine Influenza (Pandemic influenza Virus) agro-ecology and potential for interspecies transmission. He has authored and reviewed a number of journal articles and book chapters.",institutionString:"National Veterinary Research Institute",institution:{name:"National Veterinary Research Institute",country:{name:"Nigeria"}}},{id:"158026",title:"Prof.",name:"Shailendra K.",middleName:null,surname:"Saxena",slug:"shailendra-k.-saxena",fullName:"Shailendra K. Saxena",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRET3QAO/Profile_Picture_2022-05-10T10:10:26.jpeg",biography:"Professor Dr. Shailendra K. Saxena is a vice dean and professor at King George's Medical University, Lucknow, India. His research interests involve understanding the molecular mechanisms of host defense during human viral infections and developing new predictive, preventive, and therapeutic strategies for them using Japanese encephalitis virus (JEV), HIV, and emerging viruses as a model via stem cell and cell culture technologies. His research work has been published in various high-impact factor journals (Science, PNAS, Nature Medicine) with a high number of citations. He has received many awards and honors in India and abroad including various Young Scientist Awards, BBSRC India Partnering Award, and Dr. JC Bose National Award of Department of Biotechnology, Min. of Science and Technology, Govt. of India. Dr. Saxena is a fellow of various international societies/academies including the Royal College of Pathologists, United Kingdom; Royal Society of Medicine, London; Royal Society of Biology, United Kingdom; Royal Society of Chemistry, London; and Academy of Translational Medicine Professionals, Austria. He was named a Global Leader in Science by The Scientist. He is also an international opinion leader/expert in vaccination for Japanese encephalitis by IPIC (UK).",institutionString:"King George's Medical University",institution:{name:"King George's Medical University",country:{name:"India"}}},{id:"94928",title:"Dr.",name:"Takuo",middleName:null,surname:"Mizukami",slug:"takuo-mizukami",fullName:"Takuo Mizukami",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/94928/images/6402_n.jpg",biography:null,institutionString:null,institution:{name:"National Institute of Infectious Diseases",country:{name:"Japan"}}},{id:"233433",title:"Dr.",name:"Yulia",middleName:null,surname:"Desheva",slug:"yulia-desheva",fullName:"Yulia Desheva",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/233433/images/system/233433.png",biography:"Dr. Yulia Desheva is a leading researcher at the Institute of Experimental Medicine, St. Petersburg, Russia. She is a professor in the Stomatology Faculty, St. Petersburg State University. She has expertise in the development and evaluation of a wide range of live mucosal vaccines against influenza and bacterial complications. Her research interests include immunity against influenza and COVID-19 and the development of immunization schemes for high-risk individuals.",institutionString:'Federal State Budgetary Scientific Institution "Institute of Experimental Medicine"',institution:null},{id:"238958",title:"Mr.",name:"Atamjit",middleName:null,surname:"Singh",slug:"atamjit-singh",fullName:"Atamjit Singh",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/238958/images/6575_n.jpg",biography:null,institutionString:null,institution:null},{id:"333753",title:"Dr.",name:"Rais",middleName:null,surname:"Ahmed",slug:"rais-ahmed",fullName:"Rais Ahmed",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/333753/images/20168_n.jpg",biography:null,institutionString:null,institution:null},{id:"252058",title:"M.Sc.",name:"Juan",middleName:null,surname:"Sulca",slug:"juan-sulca",fullName:"Juan Sulca",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/252058/images/12834_n.jpg",biography:null,institutionString:null,institution:null},{id:"191392",title:"Dr.",name:"Marimuthu",middleName:null,surname:"Govindarajan",slug:"marimuthu-govindarajan",fullName:"Marimuthu Govindarajan",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/191392/images/5828_n.jpg",biography:"Dr. M. Govindarajan completed his BSc degree in Zoology at Government Arts College (Autonomous), Kumbakonam, and MSc, MPhil, and PhD degrees at Annamalai University, Annamalai Nagar, Tamil Nadu, India. He is serving as an assistant professor at the Department of Zoology, Annamalai University. His research interests include isolation, identification, and characterization of biologically active molecules from plants and microbes. He has identified more than 20 pure compounds with high mosquitocidal activity and also conducted high-quality research on photochemistry and nanosynthesis. He has published more than 150 studies in journals with impact factor and 2 books in Lambert Academic Publishing, Germany. He serves as an editorial board member in various national and international scientific journals.",institutionString:null,institution:null},{id:"274660",title:"Dr.",name:"Damodar",middleName:null,surname:"Paudel",slug:"damodar-paudel",fullName:"Damodar Paudel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/274660/images/8176_n.jpg",biography:"I am DrDamodar Paudel,currently working as consultant Physician in Nepal police Hospital.",institutionString:null,institution:null},{id:"241562",title:"Dr.",name:"Melvin",middleName:null,surname:"Sanicas",slug:"melvin-sanicas",fullName:"Melvin Sanicas",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/241562/images/6699_n.jpg",biography:null,institutionString:null,institution:null},{id:"337446",title:"Dr.",name:"Maria",middleName:null,surname:"Zavala-Colon",slug:"maria-zavala-colon",fullName:"Maria Zavala-Colon",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Puerto Rico, Medical Sciences Campus",country:{name:"United States of America"}}},{id:"338856",title:"Mrs.",name:"Nur Alvira",middleName:null,surname:"Pascawati",slug:"nur-alvira-pascawati",fullName:"Nur Alvira Pascawati",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Universitas Respati Yogyakarta",country:{name:"Indonesia"}}},{id:"441116",title:"Dr.",name:"Jovanka M.",middleName:null,surname:"Voyich",slug:"jovanka-m.-voyich",fullName:"Jovanka M. Voyich",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Montana State University",country:{name:"United States of America"}}},{id:"330412",title:"Dr.",name:"Muhammad",middleName:null,surname:"Farhab",slug:"muhammad-farhab",fullName:"Muhammad Farhab",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Agriculture Faisalabad",country:{name:"Pakistan"}}},{id:"349495",title:"Dr.",name:"Muhammad",middleName:null,surname:"Ijaz",slug:"muhammad-ijaz",fullName:"Muhammad Ijaz",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Veterinary and Animal Sciences",country:{name:"Pakistan"}}}]}},subseries:{item:{id:"93",type:"subseries",title:"Inclusivity and Social Equity",keywords:"Social contract, SDG, Human rights, Inclusiveness, Equity, Democracy, Personal learning, Collaboration, Glocalization",scope:"\r\n\tThe environment is subject to severe anthropic effects. Among them are those associated with pollution, resource extraction and overexploitation, loss of biodiversity, soil degradation, disorderly land occupation and planning, and many others. These anthropic effects could potentially be caused by any inadequate management of the environment. However, ecosystems have a resilience that makes them react to disturbances which mitigate the negative effects. It is critical to understand how ecosystems, natural and anthropized, including urban environments, respond to actions that have a negative influence and how they are managed. It is also important to establish when the limits marked by the resilience and the breaking point are achieved and when no return is possible. The main focus for the chapters is to cover the subjects such as understanding how the environment resilience works, the mechanisms involved, and how to manage them in order to improve our interactions with the environment and promote the use of adequate management practices such as those outlined in the United Nations’ Sustainable Development Goals.
",coverUrl:"https://cdn.intechopen.com/series_topics/covers/39.jpg",keywords:"Anthropic effects, Overexploitation, Biodiversity loss, Degradation, Inadequate Management, SDGs adequate practices"},{id:"38",title:"Pollution",scope:"\r\n\tPollution is caused by a wide variety of human activities and occurs in diverse forms, for example biological, chemical, et cetera. In recent years, significant efforts have been made to ensure that the environment is clean, that rigorous rules are implemented, and old laws are updated to reduce the risks towards humans and ecosystems. However, rapid industrialization and the need for more cultivable sources or habitable lands, for an increasing population, as well as fewer alternatives for waste disposal, make the pollution control tasks more challenging. Therefore, this topic will focus on assessing and managing environmental pollution. It will cover various subjects, including risk assessment due to the pollution of ecosystems, transport and fate of pollutants, restoration or remediation of polluted matrices, and efforts towards sustainable solutions to minimize environmental pollution.
",coverUrl:"https://cdn.intechopen.com/series_topics/covers/38.jpg",keywords:"Human activity, Pollutants, Reduced risks, Population growth, Waste disposal, Remediation, Clean environment"},{id:"41",title:"Water Science",scope:"