Camponotus modoc, seek honeydew excretions from Cinara splendens aphids based solely on the presence of sugar constituents (H1), prefer sugar solutions containing aphid-specific sugars (H2) and preferentially seek sugar solutions with higher sugar content (H3). We further tested the hypothesis (H4) that workers of both Ca. modoc and European fire ants, Myrmica rubra, selectively consume particular mono-, di- and trisaccharides. In choice bioassays with entire ant colonies, sugar constituents in honeydew (but not aphid-specific sugar) as well as sugar concentration affected foraging decisions by Ca. modoc. Both Ca. modoc and M. rubra foragers preferred fructose to other monosaccharides  and sucrose to other disaccharides . Conversely, when offered a choice between the aphid-specific trisaccharides raffinose and melezitose, Ca. modoc and M. rubra favoured raffinose and melezitose, respectively. Testing the favourite mono-, di- and trisaccharide head-to-head, both ant species favoured sucrose. While both sugar type and sugar concentration are the ultimate cause for consumption by foraging ants, strong recruitment of nest-mates to superior sources is probably the major proximate cause.Ants select sustained carbohydrate resources, such as aphid honeydew, based on many factors including sugar type, volume and concentration. We tested the hypotheses (H1–H3) that western carpenter ants,  ForagAphid honeydew is consumed by many ant species  and ofteLasius niger, heavily tend those aphid species that produce large amounts of aphid sugars, and preferentially feed on aphid sugars, particularly melezitose .Ca. modoc colony a choice between the complete aqueous SB (1 ml) and the partial aqueous SB (1 ml).In each of five replicates, we offered a . 2.5.3Ca. modoc colonies aqueous solutions of fructose  with increasing fructose content .To test H3, we offered each of four . 2.5.4Ca. modoc colonies and six M. rubra colonies choices between aqueous solutions [5% (w/v)] of (i) single monosaccharides  , (ii) single disaccharides  , (iii) single trisaccharides [D-(+)-raffinose pentahydrate or D-(+)-melezitose]  and (iv) the preferred monosaccharide [D-(-)-fructose], disaccharide [D-(+)-sucrose], and trisaccharide   (see Results). All bioassays with M. rubra , but not with Ca. modoc , included plain water (1 ml) as an additional test stimulus.To test H4, we offered six . 2.6M. rubra did not consume certain sugar solutions, some consumption data became less than 0 following weight loss subtraction due to passive water evaporation measured in evaporation controls (see above). To improve model fit, we excluded from analyses those sugar solutions which (in one-sample T-tests) had mean ‘consumption’ values (in grams) significantly less than 0. Following this procedure, sugar solutions with remaining less than 0 consumption values were assigned ‘0' values (less than 0 consumption is not possible), and 0-value data together with all other data were entered into the statistical model. We used a likelihood ratio test to compare the effect of sugar treatment on the mean proportion of sugar solutions consumed by colonies. We compared the pairwise differences in the estimated marginal mean proportion of sugar solution consumed between treatments with a Tukey's honest significant difference (HSD) test using the emmeans package which is appropriate for linear-mixed effects models  and R-studio (v. 1.1.456) . To calcpackage; ). For ex. 3. 3.1Ci. splendens honeydew  revealed the presence of two monosaccharides , three disaccharides  and one trisaccharide [D-(+)-raffinose pentahydrate (50 µg)] ] . Erlose . 3.2Ci. splendens and a blend of select synthetic honeydew sugars tested at equal concentration prompted similar consumption rates by Ca. modoc , indicating that honeydew constituents (e.g. amino acids) other than these select sugars did not modulate foraging responses (but see , . 3.5. 3.5.1Ca. modoc  and by M. rubra . However, we did not detect differences in consumption by colonies of Ca. modoc in post hoc pairwise comparisons between any of the sugar solutions . Numerically, fructose had higher consumption rates than the other monosaccharides, but this difference could not be shown statistically due to the limited sample size. Colonies of M. rubra consumed more of the fructose solution than of the glucose or xylose solution, with the xylose solution and plain water prompting equally low consumption Solutions of single monosaccharides differed in their ability to prompt consumption by . 3.5.2Ca. modoc  and M. rubra . Colonies of Ca. modoc consumed more of the sucrose solution than of maltose, melibiose or trehalose solutions , with the latter three solutions prompting similarly low and equal consumptions . As consumptions of melibiose solutions, trehalose solutions and of water by M. rubra colonies differed significantly from zero , we compared proportional consumption only between maltose and sucrose solutions, with the latter being preferred .Solutions of single disaccharides differed in their ability to prompt consumption by . 3.5.3Ca. modoc  and M. rubra . Colonies of Ca. modoc consumed more of the raffinose solution than of the melezitose solution . By contrast, colonies of M. rubra consumed more of the melezitose solution than of the raffinose solution  and more of the melezitose or raffinose solution than of a plain water control .Solutions of single trisaccharides differed in their ability to prompt consumption by . 3.5.4Ca. modoc and M. rubra in preceding experiments 4–6, prompted similar consumption by Ca. modoc  but dissimilar consumption by M. rubra . Colonies of M. rubra consumed more of the sucrose than of the fructose solution , as much fructose as melezitose solution , and as much sucrose as melezitose solution . Any sugar solution prompted more consumption than plain water .When concurrently offered, single-sugar solutions of the mono-, di- or trisaccharide preferentially consumed by . 4Ca. modoc sought honeydew based solely on the presence of sugar constituents (a) and preferentially consumed sugar solutions with higher sugar content . Below, we shall elaborate on our results.As predicted, tituents a and pre content . Also as figures –8. Unexpa. modoc b. Below,Ca. modoc workers of Ci. splendens honeydew . Similarly, many other ant species preferred common sugars to aphid-derived sugars or had no preference [L. niger and the red imported fire ant, Solenopsis invicta, preferred aphid-derived sugars  to the common sugar sucrose [L. niger recognize sugar-valuable aphid colonies based on their cuticular hydrocarbon profile [Aphis fabae, irrespective of low or high melezitose content in honeydew secretions [The effect of aphid signature sugars on foraging responses by ants is not consistent among the ant species studied thus far. For example, the presence and absence of raffinose in sugar blends had no effect on foraging responses by ur study b. Similaeference –38,72, w sucrose ,73,74. C sucrose , it seem sucrose –38,72. H profile  and theycretions ,76. Decicretions –79, the cretions ,29,36,80cretions ,82.Ca. modoc and M. rubra clearly distinguished between different types of sugar. When offered a choice between separate solutions of monosaccharides , both Ca. modoc and M. rubra preferentially consumed fructose solutions . Their selection of a specific disaccharide was equally consistent. When offered a choice between separate solutions of maltose, melibiose, trehalose or sucrose, workers of both Ca. modoc and M. rubra preferentially consumed the sucrose solution  their favourite sugars is probably linked to both its nutritional value and digestibility by these ants. Enzymes such as invertase that are capable of breaking sucrose down to its glucose and fructose constituents occur commonly in ants [Camponotus species [M. rubra [M. rubra workers [M. rubra strongly discriminated against (b), are not readily metabolized by ants [Apis mellifera capensis [Ca. modoc and M. rubra discriminated against the disaccharides maltose and trehalose is somewhat surprising because M. rubra has the enzymatic ability of maltose breakdown [Formica polytena [M. rubra [Ca. modoc and M. rubra (figures M. rubra, a lethal bait containing their favourite sugar sucrose will probably improve bait uptake, transport to the nest and trophallaxis with nest-mates, thereby expediting the demise of nests.The top choice of sucrose by  in ants –87. They species ,86 and aM. rubra . As fruc workers . Convers against b, are no by ants ,89 and rcapensis . Our finreakdown , and trereakdown . Howeverpolytena . β-GalacM. rubra , whereasM. rubra —have notM. rubra ,94. Conc figures –8 were dPreferential consumption of certain sugar solutions figures –8 is theCa. modoc, resulted in almost linearly increasing consumption rates (Camponotus mus, the Argentine ant, Linepithema humile, and the ponerine ant, Odontomachus chelifer [Ca. modoc nest-mates may have been recruited to high-dose fructose solutions, if foraging ants—on their return trip to the nest—deposited trail pheromone, and if recruited nest-mates reinforced the trail with their own pheromone deposits. For example, having fed on more concentrated sugar solutions, more worker ants of Camponotus rufipes pheromone-marked foraging trails [L. niger workers pheromone-marked trails after feeding on sucrose droplets that were greater than their crop volume [Sugar concentration affecting consumption was clearly visible in our dose-response experiment . Selection rates . Howeverchelifer –97. Antschelifer ,99. Moreg trails . Similarp volume , with fep volume .L. niger returning to the nest marked trails most intensely when they had fed on the aphid-derived sugars melezitose and raffinose, and on the common sugar sucrose [Ca. modoc and M. rubra, revealing that sugar type and sugar consumption by ants are strongly linked (figures Sugar type, in addition to volume and concentration of sugar resources, also modulates the ants' trail marking propensity. For example, foragers of  sucrose . In our  figures –8.Ca. modoc seek Ci. splendens honeydew for its sugar constituents rather than other macro- or micronutrients, but their foraging decisions were not guided by aphid-specific sugars. Sucrose was a top-choice sugar for both Ca. modoc and M. rubra foragers probably due to its digestibility and nutritional value. While both sugar type and sugar concentration are ultimate causes for uptake of sugar sources by foraging ants, strong recruitment of nest-mates to superior sugar sources is probably the major proximate cause.In conclusion, workers of Click here for additional data file.


L. infantum and L. donovani—and cutaneous leishmaniasis—caused by many species, including L. major, L. tropica and L. braziliensis. As for certain other trypanosomatids, sexual reproduction has been confirmed in these parasites, and formation of hybrids can contribute to virulence, drug resistance or adaptation to the host immune system. In the present work, the capability of intraclonal and interspecies genetic exchange has been investigated using three parental strains: L. donovani, L. tropica and L. major, which have been engineered to express different fluorescent proteins and antibiotic resistance markers in order to facilitate the phenotypic selection of hybrid parasites after mating events. Stationary and exponential-phase promastigotes of each species were used, in in vitro experiments, some of them containing LULO cells . Several intraclonal hybrids were obtained with L. tropica as crossing progenitor, but not with L. donovani or L. major. In interspecies crossings, three L. donovani x L. major hybrids and two L. donovani x L. tropica hybrids were isolated, thereby demonstrating the feasibility to obtain in vitro hybrids of parental lines causing different tropism of leishmaniasis. Ploidy analysis revealed an increase in DNA content in all hybrids compared to the parental strains, and nuclear analysis showed that interspecies hybrids are complete hybrids, i.e. each of them showing at least one chromosomal set from each parental. Regarding kDNA inheritance, discrepancies were observed between maxi and minicircle heritage. Finally, phenotypic studies showed either intermediate phenotypes in terms of growth profiles, or a decreased in vitro infection capacity compared to the parental cells. To the best of our knowledge, this is the first time that in vitro interspecies outcrossing has been demonstrated between Leishmania species with different tropism, thus contributing to shed light on the mechanisms underlying sexual reproduction in these parasites.Diseases caused by trypanosomatids are serious public health concerns in low-income endemic countries. Leishmaniasis is presented in two main clinical forms, visceral leishmaniasis—caused by  L. infantum and L. donovani) and a cutaneous form . Although it is now known that sexual reproduction occurs in these parasites, more studies are necessary to characterize the ability of Leishmania to generate hybrids, which may represent an important mechanism for virulence, drug resistance or adaptation to the host immune system. Therefore, several experiments were conducted to generate either intraclonal or interspecies hybrids in vitro. Results demonstrated that hybrids can be formed even with outcrosses between parasites causing visceral and cutaneous forms of the disease. Characterization of hybrids in terms of ploidy, kDNA content, growth rate and infection capacity provide important information about sexual reproduction in these parasites.Neglected Tropical Diseases (NTDs) represent a serious threat to humans, especially for those living in poor or developing countries. Leishmanianiosis is considered a zoonotic NTD transmitted by the bite of female phlebotomine sandflies, and is manifested mainly as a visceral form (caused by  Trypanosoma brucei (responsible for sleeping sickness in Africa), Trypanosoma cruzi (responsible for Chagas disease in South America) and Leishmania spp. are single-celled protists causing some of the most neglected and deadly transmissible diseases produced by eukaryotes in low-income countries. Leishmania is a digenetic parasite transmitted to humans by species of sandflies of the genus Phlebotomus and Lutzomyia, which maintain the zoonotic cycle [Leishmania causes different disorders: i) cutaneous leishmaniasis, produced among other species by L. major and L. tropica in the Old World and by L. braziliensis in the New World, is the less severe form and may lead to the formation of disfiguring sores and scars in exposed parts of the body [L. donovani and L. infantum in the Old World, and by L. infantum chagasi in the New World, and can derive into a rare skin form of the disease called post-kala azar dermal leishmaniasis when the antimony therapy fails [Major pathogenic trypanosomatids, ic cycle ,2. In ththe body ; ii) mucthe body ; iii) vipy fails ,6.The mode of reproduction of these species is no longer just a basic biology issue, but a source of information that can provide explanations to important clues regarding parasite virulence, drug resistance or adaptation to the host immune system. Nowadays, it is unquestionable that sexual reproduction and related processes such as gene recombination and chromosome segregation, takes place in trypanosomatids. As for other eukaryotes, meiosis-specific genes for the formation of gametes by reductive division during meiosis as well as their cytoplasmic fusion (syngamy) during mating, have been described in trypanosomatids –9. The dT. brucei are capable of generating viable hybrids by genetic exchange [Since the late 80s, there is confirmed evidence that, under controlled laboratory conditions, different strains of exchange . It was exchange ,17. The exchange , or the exchange . Finallyexchange –22.Leishmania spp. are more recent and confirm those obtained with other trypanosomatids. The use of antibiotic resistance markers served to detect the first interclonal genetic exchange event between different strains of L. major in the presence of either the natural vector Phlebotomus duboscqi, or the permissive Lutzomyia longipalpis sandfly. Similar to T. brucei, a percentage of the hybrid generation had an unexpectedly higher ploidy than that of the parental strains [L. infantum (self-mating or selfing) [L. infantum and L. major, whose filial hybrid generation retained part of the characteristics of each parent [The results obtained with  strains ,24. Furtselfing) . Moreoveh parent .Leishmania had only been demonstrated in the midgut of the permissive Lu. Longipalpis or non-permissive Ph. papatasi and Ph. duboscqi sand flies fed with infected blood. However, a recent work demonstrated that these events could also take place between promastigotes in the absence of the insect vector in axenic conditions [Until 2020, genetic exchange events in nditions , and mignditions . These nT. brucei showed that unlike minicircles [Other important results obtained from genetic exchange processes were related to the inheritance of extrachromosomal kinetoplast DNA (kDNA). kDNA is an intricate mesh of both maxicircles  and minicircles (very numerous and responsible for the editing of parasite mRNAs) . Geneticicircles , the DNAicircles .Leishmania we have proceeded to perform interspecies and intraclonal hybridization studies between lines of L. donovani, L. tropica and L. major. These experiments have been conducted in the absence of the vector, but assessing the effect of an embryonic cell line from the permissive vector Lu. longipalpis (LULO cells) as feed layer for hybridization experiments. The inheritance of the hybrids obtained from each of the parental strains, their ploidy, kDNA composition, viability and infectivity in macrophages have been analysed to contribute to decipher the mechanism behind reproduction in Leishmania.Encouraged by the importance of genetic exchange in Experiments in this study involving animals have been carried out in accordance with Spanish and European Union legislation . The protocols used in this study have been approved by the Ethics Committee of the University of León (Spain), Project license number OEBA-ULE-007-2019.Leishmania strains. Mice were infected intraperitoneally with 5x108 metacyclic promastigotes of different Leishmania strains (see below) and after 6 weeks, animals were euthanized. Livers, spleens and lymph nodes were aseptically processed in order to recover the parasites as previously described [Six-to-eight week old female Balb/c mice, purchased from Janvier Laboratories  were used to maintain the escribed ,33.L. donovani MHOM/ET/67/HU3, also known as LV9 or L-82 , L. major LV39c5 (RHO/SU/59/P) , L. tropica MRAT/IQ/72/ADHANIS1  and hybrid parasites were cultured at 26°C in M199 medium (Sigma), supplemented with 25 mM HEPES pH 6.9, 10 mM glutamine, 7.6 mM hemin, 0.1 mM adenosine, 0.01 mM folic acid, 1x RPMI 1640 vitamin mix (Sigma), 10% (v/v) heat-inactivated fetal bovine serum (FBS), and antibiotic mixture (200 U/mL penicillin and 200 μg/mL streptomycin). Stationary-phase parasites were harvested after 3 days in this phase, while exponential-phase promastigotes were collected 3 days after starting the culture. Both exponential and stationary phase promastigotes were confirmed microscopically before performing the experiments.Parental strains, 6 promastigotes/mL in complete M199 medium and parasite numbers were determined daily on a Z1 Beckman Coulter.To prepare growth curves, parental and hybrid lines were seeded at a density of 10Lutzomyia longipalpis [4 LULO cells in 8-well Ibidi chambers and adding promastigotes 24 hours later at a 1:5 ratio. Interaction was visualized by confocal microscopy. For this purpose, parasites were stained with Hoechst 33342 (Fisher Scientific) during 30 minutes at 37°C and images were acquired on a Zeiss LSM800 confocal microscope.LULO cells were kindly provided by Dr. Felio Jesús Bello . LULO is an adherent embryonic cell line derived from the permissive sandfly gipalpis . LULO cein vitro crossing experiments, one of the transgenic lines was generated previously in our lab, namely L. major mCh HYG (expressing the gene encoding mCherry (mCh) and the hygromicin resistance cassette (HYG)). In addition to this transgenic line, some new fluorescent cell lines were generated after electroporation of the corresponding plasmids (see below) and further selection of the parasites on semisolid medium containing specific antibiotic markers (see below) [To perform e below) .L. donovani mCh HYG and L. tropica mCh HYG were obtained after electroporation of these strains with pLEXSY-mCh-HYG construct [L. tropica with pLEXSY-iRFP-PAC [L. tropica iRFP PAC, which constitutively expresses the iRFP gene. Finally, electroporation of promastigotes of L. donovani, L. major and L. tropica with the construct pLEXSY-CTN-PAC (see below) and selection of transfectants in medium with puromycin (200 μg/mL) gave rise to L. donovani CTN PAC, L. major CTN PAC and L. tropica CTN PAC, respectively, which produce the citrine (CTN) protein. For the construct of pLEXSY-CTN-PAC  and confocal microscopy. For this purpose, 2x104 exponential phase promastigotes were collected, washed twice in 1xPBS and placed in 8-well Ibidi chambers previously treated with poly-L-lysine (Sigma). Parasites were stained with Hoechst 33342 (Fisher Scientific) during 30 minutes at 37°C and images were acquired on a Zeiss LSM800 confocal microscope.The correct integration of both the reporter and antibiotic resistance cassettes in the In vitro hybrids were generated by mixing equal numbers (2.5x106) of parental promastigotes , either on empty 96 well plates or on 96 well plates previously seeded with confluent LULO cells. Several days after mixing parental cell lines  parasites were transferred into 48-well plates including fresh complete M199 medium containing the selection antibiotics , in order to select the double resistant hybrids generated due to genetic exchange.6 promastigotes were collected, washed twice in 1xPBS and fixed by adding 70% ethanol dropwise in vortex. Samples were kept at -20°C during at least 24 hours and up to 1 week until analysis. For this, fixed cells were washed again in 1xPBS twice, stained with propidium iodide (40 μg/mL) and treated with 200 μg/mL RNase (Sigma) at 37°C for 30 minutes. Analysis was carried out on MACSQuant Analyzer 10 flow cytometry equipment. Data were analysed with FlowJo v10 software. L. major Friedlin (largely diploid) and L. braziliensis Mb2904 (approximately 3n) were used as ploidy-content reference cell lines in order to compare them with hybrids and parental strains.Ploidy of hybrids and parental strains was determined through the analysis of their total DNA content. Briefly, 1x10Leishmania strains was performed by sequencing ITS1 [The characterization of parental NA genes), using tL. donovani mCH HYG x L. major CTN PAC and L. donovani mCh HYG x L. tropica iRFP PAC) and in their parental strains was carried out by SNP-CAPs using the SNP2CAPS tool described by Thiel et al., (2004) [Leishmania strain can be found in Analysis of specific loci placed on different chromosomes and kinetoplastid DNA in the interspecific hybrids  . DetaileL. donovani x L. major hybrids) and 1:1, 1:3 and 3:1 ratios (for the L. donovani x L. tropica hybrids). Then, several loci were analysed by SNP-CAPs as indicated above. Subsequently, the ratio obtained between the largest band digested with the restriction enzyme belonging to one parental allele, and the uncut band corresponding to the allele of the other parental, was calculated using the Gene Tools 4.3.9 (Syngene) software. The ratio obtained in simulated 3n and 4n was compared to that obtained by the hybrids, thus allowing us to classify the hybrids according to one of the control ratios by statistical analysis using k-means analysis in SPSS COR statistical software.In order to determine the origin of the extra chromosome sets in the different hybrids, we based on the experiment carried out by Romano et al in 2014 . Simulatin vitro was tested in RAW murine macrophages. For this purpose, 5x104 RAW cells were seeded in 8-well Ibidi chambers and infected in a proportion of 1:10 with promastigotes. After 2 hours, the co-culture was washed three times in PBS. Three days after the infection, samples were stained with Hoechst 33342 (Fisher Scientific) during 30 minutes at 37°C, and images were acquired on a Zeiss LSM800 confocal microscope.The ability of intraclonal and interspecies hybrids to infect in vitro infective competence of hybrids vs. parents was determined using bone marrow-derived macrophages (BMMs) obtained from Balb/c mice. Briefly, after euthanasia, the animals were dissected and the femur and tibia were cleaned and separated in sterile conditions using scissors and forceps. The heads of the bones were cut off, and 5 mL of 1xHBSS  were injected through the bone channel with a 25G syringe. The undifferentiated cells were collected [The ollected  and frozollected . BrieflyL. major mCh HYG, which had been already generated in our laboratory and was able to stably express the gene encoding mCh and the hygromycin resistance cassette [In order to ease the phenotypic analysis of the parental and hybrid strains, in addition to cassette , we prepL. tropica mCh HYG and L. tropica CTN PAC were mixed in the presence of a feed layer of cells isolated from Lu. longipalpis embryos (LULO cells) (Stationary-phase promastigotes of O cells) , and theL. tropica strain before mating and in three hybrids. These results clearly show that unlike the parental strains, the hybrids contain genes from both parents either for antibiotic resistance or for fluorescence emission, thus confirming that genetic exchange events occurred during the outcrossing.These hybrids have yellow color  , are resLeishmania). A recent paper, however, described the mating of two different strains of L. tropica promastigotes in axenic conditions [in vitro using a feed layer of LULO cells, which were used to mimic the conditions within the insect midgut. L. tropica was initially chosen as model microorganism due to the fact that this species has a greater mating success in flies [Typically, the outcrossing experiments carried out in trypanosomatids have been performed in the midgut of the vector’s digestive tract between promastigotes –26,44,45nditions . The yieLeishmania species, namely L. major (L. major mCh HYG x L. major CTN PAC) and L. donovani (L. donovani mCh HYG x L. donovani CTN PAC). Based on our previous results with L. tropica, we used stationary-phase promastigotes and LULO cells, and the addition of selection antibiotics was carried out on day 3 after mixing. Unfortunately, no hybrids were obtained under these conditions. Therefore, we decided to modify the promastigote phase of growth and use exponential-phase promastigotes in addition to stationary-phase promastigotes in the absence of LULO cells in an attempt to increase the probability of success, since as previously reported, mating-competent forms of Leishmania can also occur in axenic conditions [Leishmania species. These results confirm the better capacity of L. tropica to generate mating-competent forms compared to L. major [L. donovani as well.We also attempted to generate intraclonal hybrids for other nditions . DespiteL. major  and, as L. donovani x L. major, L. donovani x L. tropica and L. major x L. tropica, using either exponential or stationary-phase promastigotes and in the presence and absence of LULO cells. Two different experiments were performed and addition of antibiotics was carried out on day 3 after mixing. As shown in L. donovani mCh HYG x L. major CTN PAC, which have yellow color    or the presence of an insect cellular feed layer, which confirms that LULO cells are not strictly required for in vitro formation of hybrids.Interspecies hybrid formation was also attempted between L. donovani mCh HYG x L. major CTN PAC and L. donovani mCh HYG x L. tropica iRFP PAC hybrids showed a growth profile in between their parental strains; the highest parasite number being provided by the parental L. donovani mCh HYG and the lowest parasite number being provided by the other parental strains (i.e. L. major CTN PAC and L. tropica iRFP PAC) (RFP PAC) . Inheritin vitro interspecies outcrossing in Leishmania, and in this specific case, between species with different tropism; i.e. visceral (L. donovani) and cutaneous (L. tropica and L. major).To the best of our knowledge, this is the first report describing Once the hybrids were identified phenotypically, namely by their dual antibiotic resistance and mixed color detected by confocal microscopy, the ploidy of both the parental and filial generations was determined by flow cytometry in order to assess whether reductive or additive chromosome processes might have occurred after mating.L. tropica mCh HYG x L. tropica CTN PAC crossing . As indicated by flow cytometry, the ploidy of the parental L. tropica strain was 3n, whereas ploidy of the filial strains after in vitro mating was approximately 4n. Regarding interspecies hybrids, flow cytometry results indicated that the three hybrids obtained from L. donovani x L. major (both 2n) were 3n . As far as we know, intraclonal or interespecies hybrids from 3n parentals have never been described before, either in sandflies or in vitro.These results indicate that either intraclonal or interspecies crossings give rise to additive chromosome events in the hybrid progeny. This phenomenon has been previously observed by other authors, who have obtained in their crosses a mixture of hybrids with variable ploidy, with the presence of 2n, 3n and even 4n hybrids from 2n parents, regardless of whether the hybrids were obtained in the sandfly or in vitro –27. In oLeishmania chromosomes as indicated in L. donovani mCh HYG x L. major CTN PAC and L. donovani mCh HYG x L. tropica iRFP PAC  in the corresponding hybrid clones. Results indicated that the hybrids obtained from L. donovani x L. major crosses, inherited the extra trisomic chromosomes 15, 22 and 35 from L. donovani  (L. donovani x L. tropica hybrids are grouped within the category 1:3 (L. donovani:L. tropica) , or L. tropica in the case of L. donovani x L. tropica hybrids (4n).The origin of the extra chromosome sets in the different hybrids was determined as indicated in the Materials and methods section. . major) , whereastropica) , thus coLeishmania species [The differences in ratios for each locus may reflect mosaic aneuploidy in the progeny. This process has been demonstrated in several  species , and has species .Cyb, ND5, ND7 and 12sRNA) were analysed. Inheritance of Cyb and ND5 was assessed by SNP-CAPs   in minicircle kDNA in nia spp.  Click here for additional data file.


Treatment of wounds, especially chronic ones, is a major challenge in healthcare, with serious clinical and economic burdens. Multiple treatment approaches, including the usage of silver and iodine, have dramatically improved wound healing and reduced the incidence of infection. However, once infected by drug-resistant bacteria, treatment of wounds becomes a serious complication, with limited availability of effective antibiotic drugs, leading to high morbidity and mortality. Therefore, alternative therapeutic agents are required to address this gap in wound management. The introduction of manuka honey as a therapeutic agent against infected wounds was the result of extensive research about its activity against both planktonic and biofilm bacterial growth. Likewise, several types of Saudi honey  showed promising in vitro and in vivo antimicrobial activity against wound pathogens. This short review summarizes literature that investigated the activity of common types of Saudi honey in relation to wound infections and explores their clinical utility. Wounds can be defined as breakage or injury of the  layers of the skin, leading to loss of its integrity and function . GeneralWound infectionsBacterial colonization of wounds can sometimes be observed without causing an infection . This isTreating chronic wounds is a major challenge in healthcare globally . There iAntimicrobial resistanceSince the discovery of penicillin by Alexander Fleming in 1928, the ongoing use and misuse of antibiotics have led to the emergence of antimicrobial resistance (AMR), where antimicrobial compounds have become less effective against resistant bacteria in the clinical setting . The emeHoney as a wound remedyHoney, as a natural product, has been used by different cultures as a wound remedy over many centuries ,23, owinRecently, a type of honey from New Zealand, Manuka honey, was optimized for use as medical-grade honey for wound dressing . This waA recent in vivo study has shown an interesting positive impact upon the healing process of chronic wounds when treated with medical-grade honey (MGH) while the absence of any antibiotic drugs in almost all patients (eight of nine cases) . This isSaudi honeyS. aureus, S. mutans, K. pneumonia, and E. coli [S. mutans followed E. coli, S. aureus, and K. pneumoniae based on the optical density of tested bacteria before and after exposure to the honey as follows: 1.809 to 0.359, 1.528 to 0.411, 1.721 to 0.497 and 1.746 to 0.511, respectively [Nigella sativa types of honey (50% w/v) showed an in vitro bacteriostatic effect against wound isolates of imipenem-resistant and sensitive strains of P. aeruginosa, while the same concentration of Manuka honey was shown to have bactericidal activity against the same bacteria [Bacillus cereus and S. aureus exhibited increased in vitro sensitivity to Sidr honey (33% w/v), evidenced by the large zone of inhibition  compared to E.coli and Salmonella enteritidis  [Enterococcus faecalis and S. aureus) at lower the concentration of Manuka honey was observed while its absent/limited effect on Gram-negative bacteria (P. aeruginosa and E. coli) [Saudi honey, e.g., Sidr (Ziziphus spina-christi) honey, showed promising in vitro results against pathogenic drug-resistant bacteria, such as  E. coli . The mosectively . Multiplectively . In essebacteria . The actbacteria . Althougctively) . This isE. coli) .E. coli (zone of inhibition 30 mm) compared to P. aeruginosa (zone of inhibition 18mm) and A. baumannii (zone of inhibition 19 mm)  and Gram-negative  bacteria [Sidr honey from the Najran region has high in vitro antimicrobial activity (40% w/v) against bacteria . Therefobacteria . Thymus vulgaris) honey is another type of Saudi honey, with specific medicinal properties, including promising in vitro activity against pathogenic bacteria [S. aureus and B. cereus) than Gram-negative  strains (Table 2 CFU) following day 17 of application, compared to bacterial load (9 x 104 CFU) recorded after using dressing made of povidone-iodine and saline [Talh  showed promising broad-spectrum bacteriostatic activity, which seemed to be more potent against Gram-positive bacteria. There remains a need for focused studies to evaluate the potential prophylactic use of Saudi honey against antibiotic-resistant and biofilm-forming bacteria in wounds. Further, systems biology studies to elucidate the impact of Saudi honey on planktonic and biofilm growth of various wound bacteria at the cellular and genomic levels are required. Such data may guide the selection of optimized Saudi honey with higher activity against pathogenic bacteria, with a role as medical-grade honey for treating wound infections.


Sarcocystis are protozoan parasites that infect mammals, birds, and reptiles. Sarcocystis spp. have an obligatory two-host prey-predator life cycle. Sarcocysts form in the muscles and central nervous system of the intermediate host, while oocysts and sporocysts develop in the small intestine of the definitive host. There is a lack of studies on omnivorous birds of family Corvidae as potential definitive hosts of Sarcocystis spp. Until now, only S. ovalis has been confirmed to be transmitted via corvids. In the current study, 91 small intestine samples from six corvid species from Lithuania were examined for the presence of Sarcocystis spp. that use birds, carnivorous mammals, and cervids as intermediate hosts. Oocysts of Sarcocystis spp. were observed in 43 samples (47.3%) using a light microscope. Based on molecular methods, 11 Sarcocystis spp.,  were identified. These results indicate that corvids may transmit some species of Sarcocystis that use birds and mammals as intermediate hosts.Members of the genus Sarcocystis spp., but research about this is lacking. In the present study, intestinal samples from 91 corvids collected in Lithuania were molecularly tested by species-specific PCR targeting the ITS1 and cox1 genes and subsequently sequenced for the presence of Sarcocystis spp. Under a light microscope, oocysts of Sarcocystis spp. were observed in 43 samples (47.3%), while molecular methods, detected Sarcocystis spp. in 77 birds (84.6%). Eleven Sarcocystis spp.  were identified in the intestinal samples from six corvid species from Lithuania. Infections with multiple Sarcocystis spp. were detected in 79.2% of the infected corvid birds. Three of the identified Sarcocystis spp. use corvids as intermediate hosts (IH); therefore, corvids may serve as IH and DH of the same Sarcocystis species. Based on molecular results and on corvid diet, omnivorous corvids may play an important role in transmitting Sarcocystis spp.Members of the family Corvidae are ecologically flexible omnivorous birds, particularly adaptive to urban habitats, and living in proximity to humans; these birds may serve as definitive hosts (DH) for  Sarcocystis (Apicomplexa: Sarcocystidae) are parasitic protozoa widespread in reptiles, birds, and mammals. They are characterised by an obligatory prey-predator two-host life cycle [Sarcocystis spp., and species cannot be distinguished according to the morphology of parasite sexual stages observed in the DH [Representatives of the genus fe cycle . Asexualfe cycle . The sarn the DH .Sarcocystis spp. worldwide [Sarcocystis spp. that mainly use birds and small mammals  as IH [Sarcocystis spp. is unclear; however, based on phylogenetic studies, omnivorous birds are likely to be involved in transmitting several Sarcocystis spp. that use carnivores and ungulates as IH [Birds have been shown to be DH of at least 17 orldwide ,5,6,7,8..) as IH ,10. The es as IH ,12,13. Corvus corax) and hooded crows (C. cornix) [Sarcocystis spp. [Sarcocystis species, S. ovalis, is confirmed to be transmitted by corvids [Alces alces), red deer (Cervus elaphus) and sika deer (C. nippon) as IH [Pica pica) [C. macrorhynchos) as DH [Corvidae is a family of ecologically flexible omnivorous birds, particularly adaptive to urban habitats close to humans ,15,16,17 cornix) ,18. Basetis spp. , but thetis spp. ,20. Curr corvids . This spn) as IH ,23,24 anca pica)  and JapaSarcocystis spp. in intestinal samples from six corvid species from Lithuania. The aim of the current study was to molecularly identify Coloeus monedula), 5 rooks (Corvus frugilegus), 4 common magpies and 3 Eurasian jays (Garrulus glandarius) were examined for Sarcocystis spp. Oocysts/sporocysts of Sarcocystis spp. were isolated from the intestinal mucosa of each bird using previously described methodology [A total of 91 birds from the Corvidae family were collected between 2015 and 2021. All birds were found dead  and obtained from the Kaunas T. Ivanauskas Zoology Museum, the Lithuanian national authority responsible for monitoring dead or wounded wild birds. Bird samples were kept frozen at −20 °C until a microscopic examination had been conducted. Intestinal samples of 33 hooded crows, 25 common ravens, 21 western jackdaws  are found in the muscles of birds as IH; two species,  use carnivorous mammals as IH; and four remaining species  employ cervids as IH [Sixteen ithuania . Ten of ds as IH ,12,26,27Sarcocystis spp. that use birds and carnivores as IH [cox1) gene of Sarcocystis spp. that use cervids as IH [Approximately 200 µL of intestinal sediment was taken from each sample and prepared for DNA extraction using GeneJET Genomic DNA Purification Kit . Nested PCR (nPCR) was used to amplify DNA fragments of all parasite species in the study. The external primers SU1F/5.8SR2 amplified the internal transcribed spacer 1 (ITS1) region in es as IH . The SF1ds as IH . Internads as IH , with mods as IH . Nucleotds as IH . The seqSarcocystis spp. detection rates in the analysed host species, it was estimated the overall frequency of positive parasite cases, which was calculated as the ratio of positive cases from the host species sample size multiplied by the 16 Sarcocystis species tested. The unconditional exact test, which is more sensitive in detecting differences, especially in small sample sizes, evaluated differences in the prevalence of the detected Sarcocystis spp. and in the frequency of positive parasite cases [The statistical analyses were performed with Quantitative Parasitology 3.0 software . The Stete cases .Sarcocystis spp. oocysts in 47.3% (43/91) of birds examined (n = 69) were seen under a light microscope in intestinal mucosa, while free sporocysts were not detected. The molecular method detected Sarcocystis spp. 1.8 times more often , which was significantly higher (p < 0.001) than the microscopic method. In four cases (two hooded crows and two western jackdaws), Sarcocystis spp. were observed microscopically, but species-specific nPCR did not detect amplified DNA fragments of parasites. Molecular examination revealed high Sarcocystis spp. rates in examined corvid species, varying from 66.7% (in western jackdaw and Eurasian jay) to 100% (in common raven and common magpie). Comparing Sarcocystis spp. infection prevalence obtained in three corvid species with adequate sample size to draw reliable conclusions (N > 20), molecular analysis detected significantly lower prevalence (p = 0.0014) in western jackdaw  than in common raven .Microscopic examination detected, examined . Oocystscox1 sequences generated in the current study revealed the presence of 11 out of 16 examined Sarcocystis spp.   and S. lutrae (0–4.6%). The GenBank accession numbers of analysed sequences of Sarcocystis spp. listed in All amplified samples were sequenced and identified as belonging to the species for which the species-specific primers were designed. Comparing ITS1 or iformis) . Sequenc species . DetecteSarcocystis spp. identified in this study were detected in hooded crow samples , S. halieti (n = 48) and S. kutkienae (n = 45), were detected more often than other examined species (Sarcocystis cornixi (n = 16), S. lari (n = 13) and S. turdusi (n = 13) were detected slightly less, and other Sarcocystis spp. were identified in no more than in five birds. Sarcocystis spp. that use carnivores and cervids as IH were detected only in hooded crow and common raven; however, the number of investigated individuals of other host species was lower, except for western jackdaw (n = 21). When comparing the frequency of positive parasite cases (p = 0.0054) and common raven  had significantly higher infection rates than western jackdaw .All 11  samples . Nine Sa species . Sarcocyte cases  between Sarcocystis spp. Multiple species of Sarcocystis were common in all corvid host species  of examined corvid birds were positive for at least one  species . InfectiSarcocystis spp. in IH, since parasite species cannot be differentiated by the stages found in DH (oocyst or sporocyst) [Sarcocystis spp. from intestinal and faecal samples of predators or omnivorous animals [Sarcocystis spp. DNA from intestinal and/or faecal samples does not conclusively prove the role of tested animal as DH of these parasites. The detected DNA may also belong to Sarcocystis spp. that had been present in the carrion the bird has been feeding on without infecting analysed animal species. Thus, life cycle experiments are necessary to confirm the results obtained [Sarcocystis spp. were identified in mucosal scrapings of corvids based on species-specific nPCR targeting ITS1 or cox1 and subsequent sequencing (Sarcocystis spp. detection rate was significantly higher (p < 0.001) by molecular methods (84.6%) than by microscopic examination (47.3%)  . Whereas animals ,25,35,36obtained . In the quencing . The ove (47.3%) . The stuSarcocystis have a diheteroxenous life cycle, meaning different animal species serve as IH and DH [Sarcocystis spp. that use mice, rats and lizards as IH  have been shown to have both diheteroxenous and dihomoxenous life cycles, allowing transmission via cannibalism [Sarcocystis spp. identified in the present study,  [Sarcocystis cornixi and S. kutkienae form sarcocysts in muscles of corvids, while S. halieti is multi-host adapted, employing birds of several different orders as IH [S. cornixi and S. halieti [S. kutkienae [Sarcocystis spp. is intriguing, and more research is needed to reveal this interesting phenomenon. Our data indicate that corvids can feed on carrion of other birds of the Corvidae family, including their conspecifics. For example, groups of hooded crows were observed feeding on carcasses of hooded crows and rooks (presumably killed by poisoning) in a waste disposal area of Klaipeda, Lithuania in January 2016. In several cases, common ravens were recorded feeding on carrion of rooks and western jackdaws killed by cars in Lithuania.Representatives of the genus H and DH . Howevernibalism ,40,41,42 cornixi , S. kutkutkienae  and S. hed data) . Sarcocyrs as IH ,46. Bird halieti , and basutkienae . As mentSarcocystis spp.  were identified in the intestinal samples of corvids  from Germany [S. halieti was one of the most commonly detected species; it was confirmed in hooded crows (n = 20), in common ravens (n = 18), in western jackdaws (n = 7) and in common magpies (n =3)  . Thus, cSarcocystis spp. were identified in all six studied bird species . Hooded crow and common raven had significantly higher frequency of positive Sarcocystis spp. cases than western jackdaw. This observation agrees with diet studies of corvids, since common raven and hooded crow are the main scavengers among corvids in Lithuania [Sarcocystis spp.In the current study, ithuania ,51. Carcithuania . Common ithuania ,51. WestS. calchasi, S. corvusi, S. fulicae, S. hardangeri, and S. frondea were not detected in corvids in this study. Pathogenic S. calchasi infects birds of several orders and has been detected in Germany [Accipiter hawks [S. calchasi has not been confirmed in Lithuania yet, may be explained by the possibility that corvids are not the DH of this parasite, or this species is absent or rare in Lithuania. In 2013, S. corvusi was described in the leg muscles of two migrating jackdaws from Lithuania [S. fulicae were identified solely in muscles of Eurasian coot (Fulica atra) [Sarcocystis hardangeri was identified only in cervids from specific regions of Norway and Iceland [S. frondea was found only in introduced sika deer [S. frondea was not observed in examined corvids due to this species’ low prevalence in Lithuania.Oocysts or sporocysts of  Germany ,54, USA  Germany ,56, and  Germany . This sper hawks ,54 prevaer hawks ,52; howeithuania , and aftca atra) , a waterca atra) ,51,52. S Iceland ,59, so, ika deer . TherefoSarcocystis species [Sarcocystis spp. infections in 79.2% of the infected corvid birds. The mixed infections in intestinal or faecal samples of naturally infected wild animals cause issues with identifying Sarcocystis spp. In this work, species-specific PCR was applied to detect Sarcocystis spp. Despite the high Sarcocystis spp. detection rate (86.0%), identification of species is sure to be limited. For instance, four samples were microscopically positive for Sarcocystis spp., but molecular analysis did not identify the examined species. These results indicate the presence of other Sarcocystis spp. not tested in the intestinal samples of corvids. Other authors have applied methods apart from species-specific PCR, such as cloning, and metabarcoding, to detect Sarcocystis spp. from samples containing oocysts or sporocysts [Sarcocystis spp. in certain hosts [Sarcocystis spp. in intestines and faecal samples of omnivorous animals and predators should be further developed.One DH can harbour multiple  species ,8,10,25.orocysts ,61,62,63in hosts . In summcox1 and subsequent sequencing 11 Sarcocystis spp. that employ birds , cervids  and carnivorous mammals (S. arctica and S. lutrae) as their IH were identified in the intestinal samples of six corvid species. Ten of these Sarcocystis species were confirmed in corvid intestines for the first time. Therefore, the present study’s results indicate that widespread omnivorous corvids, which live close to humans, could be involved in transmitting these Sarcocystis spp., including potentially pathogenic S. halieti. However, the question remains whether corvids are DH of these identified Sarcocystis spp. or the detected DNA was a residue of food particles present in the intestine of tested birds.Based on species-specific nPCR targeting ITS1 or Sarcocystis spp. that were not identified by molecular methods. Several other studies also reported that molecular methods were not efficient in revealing the full diversity of Sarcocystis spp. in the intestinal and faecal samples of predators or omnivorous animals. Thus, more sensitive Sarcocystis spp. identification techniques from naturally infected DH must be developed.In four cases, light microscopy detected oocysts of


Escherichia coli infection therapy. Traditional methods for detecting Escherichia coli (E. coli), such as serological experiments, immunoassays, polymerase chain reaction, and isothermal microcalorimetry have some drawbacks. As a result, detecting E. coli in a timely, cost-effective, and sensitive manner is critical for various areas of human safety and health. Intelligent devices based on nanotechnology are paving the way for fast and early detection of E. coli at the point of care. Due to their specific optical, magnetic, and electrical capabilities, nanostructures can play an important role in bacterial sensors. Another one of the applications involved use of nanomaterials in fighting microbial infections, including E. coli mediated infections. Various types of nanomaterials, either used directly as an antibacterial agent such as metallic nanoparticles (NPs) , or as a nanocarrier to deliver and target the antibiotic to the E. coli and its infected area. Among different types, polymeric NPs, lipidic nanocarriers, metallic nanocarriers, nanomicelles, nanoemulsion/ nanosuspension, dendrimers, graphene, etc. proved to be effective vehicles to deliver the drug in a controlled fashion at the targeted site with lower off-site drug leakage and side effects.The exploration of multiplexed bacterial virulence factors is a major problem in the early stages of  Escherichia coli (E. coli) is a gram-negative bacteria and causative agent of many infectious diseases in humans. Many bacterial infections such as urinary tract infections, bloodstream infections, pneumonia, surgical site infections [E. coli [fections ,2,3, bacfections ,5, and n[E. coli .The Gram-negative bacteria are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane (OM) ,8. The OE. coli infections. However, huge numbers of drug-resistance strains have appeared due to antibiotics misuse in the last 50 years [E. coli therapy have been revealed to stimulate the Shiga toxin release from E. coli [E. coli [E. coli infections using antibodies [E. coli has been used to robust the immune responses in humans. However, the short duration of the vaccine producing immunity against bacterial infections is a major drawback for treating E. coli [The use of antibiotics is an efficient, prevailing, and the utmost method for treating 50 years ,14. Furt50 years . Numerou E. coli . In addi[E. coli . Still, tibodies . Vaccine E. coli . Despite E. coli .E. coli infections for several years, including enzyme-linked immune sorbent assay (ELISA) and polymerase chain reaction (PCR) [E. coli producing bacterial infections [Conventional methods have been used for the diagnosis of on (PCR) . The nonon (PCR) . Meanwhion (PCR) . PCR-bason (PCR) . A multifections . ELISA ifections . Neverthfections ,28. To tfections . Recentlfections ,31,32. Tfections ,34,35,36fections ,38. Zincfections . The catfections ,41.E. coli infections [Nanotechnology-based approaches such as gold NPs, silver NPs, magnetic NPs, and quantum dots (QDs) reveal selective target-binding characteristics ,55,56,57fections ,61,62,63fections . The entfections ,66,67.E. coli have significant disadvantages that restrict their implementation in real-world situations [Specific and accurate identification of bacteria is a critical component of early illness diagnosis and surveillance, and it has the ability to avert outbreaks and the spread of devastating epidemics. It is also vital to quickly confirm the presence of these bacteria at low concentrations to prevent these diseases. As previously stated in the introduction, traditional approaches for detecting tuations ,69,70.E. coli in wastewater, food, and the human body. Different nanomaterials used in the detection of E. coli are summarized in Early diagnosis of specific species of bacteria has numerous medical, environmental, and food safety uses . NPs havE. coli recognition. In comparison to native Au, gold NPs have distinct physical, chemical, and electrical properties [E. coli O157:H7 bacteria by using gold NP labelling, antibody affinity binding, and inductively coupled plasma mass spectrometry (ICPMS) [E. coli O157:H7 cells in 1 mL of sample or 500 CFU mL−1 using the signal amplification ability of Au NPs and the perfect sensitivity of ICPMS. The assay had good specificity for E. coli O157:H7 in tests with non-pathogenic E. coli . Each experiment took 40 min to complete. Demonstration of this assay for E. coli O157:H7 suggests its potential for detecting a variety of bacterial pathogens.Due to its versatility in dimension and arrangement, such as circular, diamond, crystalline, triangular, and spiral geometries, gold NPs have been extensively used in operties . In this (ICPMS) . The tecE. coli O157:H7 DNA identification using nanogold modification and mass amplification [E. coli O157:H7 eaeA gene connected to the NP-modified electrode surface through the Au-SH binding. The outer nanogold was used as a mass amplifier to boost the signal, and the biotin-avidin system was used to bond with the target DNA. This biosensor identified target DNA corresponding to 2 × 103 CFU mL−1E. coli O157:H7, indicating that developing an appropriate and sensitive QCM biosensor for harmful bacteria diagnosis based on specific DNA analysis is feasible. In order to attract more attention, this DNA biosensor should be combined with some micro- and nano-fabrication techniques to realize more promising and practical applications, and more attention should be focused on the further improvement of the sensitivity and the shortening of analysis time.In another study, Wang et al. reported a quartz crystal microbalance (QCM) biosensor for fication . 1,6-HexE. coli, the human infections Pseudomonas aeruginosa and Vibrio cholerae, and two strains of the plant pathogen Xanthomonas campestris were all detected. There was no cross-reactivity among the Gram-negative bacterial species examined in this assay, which detected about 100 cells. The assay takes less than 60 min to complete and is resistant to various media, including seawater and human serum. This strategy combines highly evolved biological materials with the optical properties of gold NPs to achieve the simple, sensitive, and specific detection of bacterial species. A schematic representation for chimeric phages and processes of pathogen detection is given in Based on the interaction between bacterial cells and the viruses that infect them (phages), Peng et al. provided a straightforward technique for identifying a range of bacterial species . As phag2–PANI nanocomposite, Raj et al. created a label-free and extremely sensitive biosensor based on electrochemical measurement [−1, and identified E. coli in less than 30 min. In addition, the proposed biosensor showed a high linear detection range and practical use in urine samples, and electrode regeneration experiments. The suggested electrochemical biosensor for E. coli is shown in Using the Au@MoSsurement . The Au@E. coli [E. coli  and Pseudomonas aeruginosa (P. aeruginosa), was statistically significant. This means this new PEPTIR-1.0-based biosensor can be used in the rapid, sensitive, and selective detection of E. coli in aqueous matrices. Pao et al. investigated carbohydrate-protein interactions and developed a label-free nanosensor to recognize E. coli using surface-modified Au NPs and solid-liquid contact electrification [E. coli in a wide range of concentrations of 2 × 104–2 × 107 CFU mL−1, with a LOD of 4 × 103 CFU mL−1. The current work highlights the bright prospect of TENS as a new prototype of sensing technology for the label-free and rapid analysis of carbohydrate-protein interactions, as well as other pathogenic microorganisms. Ropero-Vega et al. described a bioinformatic architecture of a peptide based on TIR protein, an Intimin membrane protein receptor found in E. coli . This pe[E. coli . The biofication . The desE. coli were described by Hwang et al. [−1 than silver or silver mono-metallic nanohole arrays, which is around 40% higher. Furthermore, the -NHA provided exceptionally durable material stability against corrosion and oxidation over a one-month observation period. The ultra-sensitive -NHA enables label-free identification of E. coli at concentrations ranging from 103 to 108 CFU mL−1, with a LOD of 59 CFU mL−1. This alloy plasmonic material provides a new outlook for widely applicable biosensing and bio-medical applications.Plasmonic nanosensors use the electromagnetic field localization of noble metal NPs to identify biological substances with great sensitivity. Due to their unusual optical features, nanocomposites and nanoalloys of two plasmonic metals have received much attention in recent years . For exag et al. . CompareE. coli [E. coli, glassy carbon electrodes treated with Chi-silver NPs increased its signal. Identification was achieved over a wide concentration range of 0.001–100 ng mL−1 and 10–107 CFU mL−1 (many orders of magnitude). At very low concentrations, the nanosensors could reliably detect LPS and E. coli. Chi-AgNPs have potential as low-cost, sensitive nanobiosensors for Gram-negative bacteria, due to strong electrostatic interaction with LPS present in their outer membranes.In another study, for the construction of a new biosensor, a polymer-metal method was also used. Imran et al. developed a sensitive electrochemical nanobiosensor using positively charged chitosan stabilized silver NPs (Chi-silver NPs) for the identification of negatively charged lipopolysaccharide (LPS) or E. coli . In the E. coli [5–1.0×108 CFU mL−1. At pH 7.0, 25 °C, and a 20-min incubation duration, the optimal fluorescence intensity for identifying E. coli was observed. E. coli has a LOD of 4.6 × 104 CFU mL−1 under optimal conditions. The quenching was discussed to be a static quenching procedure, which was proved by the quenching efficiency of QDs, which decreased as the temperature increased.As a result of their high light emission capabilities and flexibility to be modified with a range of functional groups to identify diverse analytes, QDs have recently demonstrated to be invaluable tools for biosensing applications. To take advantage of this, Wu et al. developed a new fluorescent probe based on ZnTe QDs modified with mannose (MAN) for detecting E. coli . AccordiE. coli O157:H7 antibody for specific recognition of E. coli. The detection range of the nanosensor was 10–108 CFU mL−1 and LOD of 3 CFU mL−1 . Interestingly, the sensitivity of CdS@ZIF-8 for the detection of E. coli was 16 times greater than CdS QDs.Zhong et al. also synthesized a zeolitic imidazolate framework-8 (ZIF-8), loaded with CdS QDs (core-shell CdS@ZIF-8) for the identification of O157:H7 and then used as a signal enhancer tag . In the E. coli [E. coli cells, was conjugated to CGQDs. The findings could be useful in instances where rapid, consistent detection of bacteria in liquids, such as drinking water, is required, especially considering the low range of E. coli concentrations (103 to 106 CFU mL−1) within which two biosensing tests have been proven to perform together. These findings have the potential to be used in situations where rapid, reliable detection of bacteria in liquids, such as drinking water, is required. A schematic representation for a fluorescent-assisted nanosensor array, based on graphene QDs attached cecropin P1 for identifying pathogens is given in Bruce et al. used a biosensing test that emphasized monitoring changes in fluorescence intensity to examine the application of conjugated carboxylated graphene CGQDs to identify E. coli . CecropiE. coli [E. coli detection capability. The proposed electrochemical biosensor showed good selectivity, detecting E. coli O157:H7 in water samples with LOD of 1.3 × 1018 M.Carbon nanomaterials have been used to make electrochemical biosensors in recent years due to their unique mix of intrinsic features, such as high conductance, durability, and bioactivity, making them a suitable candidate for bio-sensing material . For exaE. coli . The eleS. aureus and Staphylococcus epidermidis) isolated from various human illnesses may be differentiated. This type of multiplexing with NIR fluorescent nanosensors enables remote detection and differentiation of important pathogens and the potential for smart surfaces. A schematic diagram for identifying pathogens by this method is given in Nibler et al. developed a set of near-infrared (NIR) fluorescence nanosensors based on single-walled carbon nanotubes (SWCNTs) and utilized them for remote fingerprinting of clinically important bacteria . Based oE. coli O157:H7, using a nanostructured platform made of boron-carbon nanorods coated with nickel NPs (BC-Ni) [E. coli O157:H7 preferentially in water, juice, and faeces samples with a LOD of 10 CFU mL−1 and a dynamic detection range of 100 to 105 CFU mL−1.Kaur et al. reported the invention of a label-free impedimetric aptasensor for specific and accurate identification of  (BC-Ni) . The eleE. coli K12 and S. aureus at the same time [E. coli K12 and S. aureus were used to functionalize each junction. Compared to the stationary sensor, the constructed junction sensor linked with the fluidic channel showed improved electric signal outputs for identifying E. coli K12. In the sensing range of 102–105 CFU mL−1, a linear regression was seen for both the E. coli and S. aureus functionalized array sensors. Within 2 min, multiplexed identification of bacteria at sensing levels as low as 102 CFU mL−1 was obtained for E. coli K12 and S. aureus.Lee et al. developed a continuous flow multijunction biosensor for detecting ame time . Gold-plE. coli  [E. coli antibodies before being tested as an E. coli biosensor. The biosensor can identify analytes with concentrations ranging from 1.3 × 102 to 1.3 × 108 CFU mL−1, with a detection limit of 3 CFU mL−1, and has a detection time of 5 min and a total analysis time of 20–25 min.Metal-organic frameworks (MOFs) are chemical types that combine metal ions or clusters with organic ligands to produce one, two, or three-dimensional architectures. They are a type of coordination polymer that has the unique property of being porous. MOFs are considered a suitable medium for the construction of biosensors for various analytes found in the environment . In thisic acid) . Tb-BTC E. coli bacteria was reported by Gupta et al. [3(BTC)2  was combined with polyaniline (PANI) to create a MOF-based electrochemically active platform. Cu3(BTC)2-PANI thin films were bio-interfaced with anti-E. coli antibodies and used as a biosensing electrode on an indium-tin-oxide (ITO) substrate. The sensor described above, which used the electrochemical impedance spectroscopy (EIS) signal measuring methodology, had great sensitivity for detecting very low E. coli (2 CFU mL−1) concentrations in a fast reaction time (2 min) and was also sensitive in the presence of other non-specific bacteria. This new MOF/PANI based detection platform for E. coli has shown improved performance compared to many of the previously reported electrochemical biosensors.The design of a Cu-MOF based electrochemical biosensor for the very selective detection of a et al. . Cu3, the adoption of SNP-RB as a sensing platform lowered response time dramatically, while also providing good analytical performance in terms of selectivity and sensitivity.To examine and unravel biological processes and related mechanisms, accurate, low-toxicity, and real-time biochemical tests are necessary. Sensors and probes made of silicon nanomaterials have the ability to meet the above-mentioned criteria . To testiosensor . The preE. coli bacteria in environmental water samples. The aminated DNA probe was mounted on 3-aminopropyltriethoxysilane-functionalized hollow silica microspheres (HSMs) and placed onto a screen-printed electrode (SPE) carbon paste with integrated gold NPs [E. coli DNA sensor had a dynamic linear response range of 1 × 10−10 M to 1 × 10−5 M (R2 = 0.982), with a LOD of 1.95 × 10−15 M. The designed DNA biosensor had a sensibility that was equivalent to non-complementary and single-base mismatched DNA. At 4 °C and pH 7, the DNA biosensor showed a steady response after 21 days of storage. Over three regeneration and rehybridization cycles, the DNA biosensor response was regenerated. The schematic procedure for label-free detection of E. coli by DNA attached-hollow silica NPs was depicted in Yuhana et al. developed a new label-free electrochemical DNA biosensor for detecting gold NPs . The bioE. coli O157:H7 [E. coli antibody-coated magnetic nanobeads were combined with an E. coli sample and used to extract and concentrate the bacteria. The substance was immersed in a redox probe solution and placed on an interdigitated electrode that had been screen-printed. The impedance was examined after a magnetic field concentrated the cells on the electrode’s surface. Without pre-enrichment, the impedance immunosensor could identify E. coli O157:H7 at a concentration of 1400 bacterial cells in a volume of 25 L in less than 1 h. Between 104 and 107 CFU mL−1, a linear relationship between bacteria concentration and impedance value was observed. Despite the presence of a redox probe during impedance measurement, analysis of the equivalent circuit model revealed that the impedance shift was predominantly caused by two factors: double-layer capacitance and resistance owing to electrode surface roughness. The magnetic field and impedance were simulated using COMSOL Multiphysics software. The architecture illustration of antibody-covered nanobeads and the low concentration detection of E. coli using a magnetic field procedure is given in Magnetic NPs have been widely utilized in biosensing for pathogen identification, mainly for the immunomagnetic separation of bacteria from a sample, or as labels to improve the biosensor’s responsiveness ,104. Wan O157:H7 . Anti-E.E. coli O157:H7 in ground beef specimens [E. coli antibodies were immobilized onto streptavidin-coated magnetic NPs, which were utilized to extract and concentrate E. coli O157:H7 from ground beef samples. In the presence of 0.1 M mannitol solution, the magnitude of impedance and phase angle were evaluated over a frequency range of 10 Hz–1 MHz. The biosensor’s lowest LOD for E. coli O157:H7 in pure culture and ground beef samples were 7.4 × 104 and 8.0 × 105 CFU mL−1, respectively. By concentrating bacterial cells linked to MNAC in the active layer of IDAM above the surface of electrodes with the help of a magnetic field, the sensitivities of the impedance biosensor were increased by 35%. According to equivalent circuit analysis, the impedance shift caused by the presence of E. coli O157:H7 on the surface of IDAM was caused by bulk resistance and double-layer capacitance. This impedance biosensor did not use surface immobilization methods, redox probes, or sample incubation. From sampling to testing, the entire detection time was 35 min.Varshney et al. developed and tested an impedance biosensor based on an interdigitated array microelectrode (IDAM) combined with magnetic NP–antibody conjugates (MNAC) for the quick and precise identification of pecimens . Biotin-3O4 (10 nm) and shell of Au. Then, core-shell multifunctional immunomagnetic NPs was modified with anti-E. coli O157:H7 antibodies for simultaneous identification and isolation of O157:H7. The nanosensor could detect bacteria in the lettuce matrix with a LOD of 1 log CFU mL−1 and no-enrichment procedure. The method, which requires no pre-enrichment, provides an alternative to conventional microbiological detection methods and can be used as a rapid screening tool for many food samples.In an interesting paper, Lee et al. combined two features of localized surface plasmon resonance and target separation of immunomagnetic NPs for the low-concentration detection of O157:H7 in lettuce . ImmunomE. coli in a liquid medium [E. coli were fictionalized on the membrane’s back surface. The immobilization of antibodies was studied as a function of antibody suspension concentration, pH, and incubation period in order to maximize bacterial immunocapture. Detection tests in various conditions for bacterial suspensions ranging from 103–108 CFU mL−1 were used to assess the biosensor’s performance. For suspensions ranging from 103–107 CFU mL−1, a linear relationship between the frequency response and the logarithm of E. coli concentration was observed, with the biosensor’s LOD estimated to be 103 CFU mL−1. The biosensor capability for the desired operation in complicated biological media is demonstrated by the 5-fold regeneration and high selectivity towards E. coli identified at 104 CFU mL−1 in a suspension coloured with Bacillus subtilis at 106 CFU mL−1. The schematic construction of a ZnO/GaAs BAW nanosensor for identification of E. coli is depicted in Pathogenic bacteria must be detected in various disciplines, including food safety, environmental water analysis, and clinical diagnostics. Despite the fact that fast and specific procedures based on the quick and simple binding of recognition elements and targets have been developed, the sensitive identification of bacterial pathogens has been limited due to their low targets. As ZnO NPs have a large binding capacity, they can provide additional reactive sites to bind with bacterial targets, improving detection sensitivity significantly . Chawichd medium . The bioE. coli O157:H7 DNA was constructed by attachment of ssDNA on the surface of LAPS. The developed nanosensor was added to a solution of E. coli ssDNA molecules until the hybridization procedure of target DNA and probe was completed. The findings show that different signal variations can be observed and recorded in order to identify E. coli ssDNA. The target ssDNA had a LOD of 1.0 × 102 CFU mL−1 for E. coli O157:H7 detection in solution. All the results demonstrate that this DNA biosensor, based on the electrostatic detection of ssDNA, provides a novel approach for the sensitive and effective detection of bacterial DNA, which has promising prospects and potential applications in the quality control of food and water. A schematic diagram of the nanosensor arrangement built up from ZnO nanorods arrays, probe ssDNA, and silica layer for detecting E. coli is shown in Tian et al. developed a label-free nanosensor based on the light-addressable potentiometric sensor (LAPS) and ssDNA-ZnO nanorod arrays (NRAs) . The recE. coli infection.In the 21st century, NPs have emerged as the most innovative system for drug delivery and therapeutic purposes. Different nanomaterials were used to prepare NPs according to their unique characteristics, such as controlled drug release, site-specific targeting, lower side-effects and toxicities, and higher therapeutic efficiency. Meanwhile, bacterial infections are suitably treated with these NPs with promising outcomes. E. coli bacteria and to retard bacterial “planktonic” and “biofilm” growth [co-glycolic acid (PLGA), polylactic acid (PLA), polyethyleneimine (PEI), polycaprolactone (PCL), etc.) are utilized to construct polymeric nanocarriers [Polymeric nanocarriers have a leading biomedical role in drug delivery and therapeutics. Various polymeric NPs were devised to target ” growth . Polymer” growth ,107. SevE.coli [In a recent attempt, nettle essential oil was encapsulated inside chitosan NPs to assess its antibacterial activity . The nanE.coli . E. coli bacteria and retarded invasion of gut epithelial cells by the E. coli in a dose-dependent manner [Similarly, cranberry proanthocyanidin chitosan nanocomposites were prepared without using any cross-linker. Instead, ratio adjustment of the proanthocyanin and chitosan modulate hydrogen binding were used . The nant manner .E. coli bacterial membranes and improved antibiofilm properties as compared to plain chitosan NPs. The modified chitosan NPs are of special interest in combating E. coli associated multidrug resistance. The anti-bacterial activity was assessed through antibiofilm assay, time-kill activity, and polystyrene adherence. Findings indicated greater inhibition and biofilm disruption of E. coli and other gram-positive bacteria, thus emphasizing its role against multidrug-resistant, biofilm-forming bacteria in resistant infections. [Further, the intrinsic anti-bacterial property of chitosan was enhanced through its mannosylation via reductive amination reaction, and NPs were prepared through ionic gelation . Mannoseections. .co-β-amino ester) nanocarriers for targeting E. coli bacteria in urinary tract infections [E. coli and efficiently treated a mouse cystitis model under photodynamic therapy [Apart from natural polymers, charged synthetic polymers were also used against bacterial infections. In one such attempt, a photosensitizer, Chlorin e6 (Ce6) was encapsulated inside charge-conversion polymeric (poly(PEG-fections . The Ce6 therapy .E. coli (MIC: 12.5 μg/mL) and Leishmania , proving the efficacy of the nanoformulation [Further, dual antibacterial and antiparasitic multifunctional PLGA NPs were synthesized with coloaded caffeic acid phenethyl and juglone drugs . The commulation .E. coli and S. aureus. The formed NPs endured antibacterial properties to the dyes, inks, and different coatings. It was found that the NPs have long-term anti-bacterial properties with 99% efficacy at a concentration of 40 µg/mL [Another innovation is developing a novel antibacterial polymer, cationic acrylate copolymeric polyvidone with double active centres and then complexed with iodine . Further40 µg/mL .E. coli. The total amount of chlorhexidine loaded on the coated catheter was around 4.55 mg. The NP-coated catheter was immersed in artificial urine containing microorganisms. Drug release was exceeded up to 2 weeks, with satisfactory inhibition of bacterial growth and proliferation into the catheter up to 14 days [PCL is another polymer with wide applications in nanomedicine. In a study, chlorhexidine-loaded PCL nanospheres were prepared and coated on the urinary catheters for anti-bacterial action against uropathogens, including  14 days . This inLipids are a basic component of the cell membrane, hence, lipid-based nanovesicles mimic natural components and easily transfer the drug molecule inside the cell. This property allows lipid-based nanovesicles to specifically target bacterial cells.E. coli [E. coli even in a dose that was half of the free drug dose [Nanostructured lipidic carriers (NLCs) are lipidic carriers made up of lipids, surfactants, and co-surfactants, with a higher capacity to load drugs. For instance, ceftriaxone-loaded NLCs were prepared using the double emulsion solvent evaporation method for bactericidal action against E. coli . Ceftriarug dose .E. coli O157:H7 strains, thus proving their role in delivering lipophilic drugs for biomedical applications and prospective role in treating E. coli infections [Solid lipidic nanocarriers (SLNs) are another versatile lipidic nanocarrier with long-term stability. A novel class of SLNs was developed using propylene glycol monopalmitate and glyceryl monostearate lipidic mixtures. Carvacrol was loaded as a drug with >98% encapsulation efficiency. Carvacrol-loaded SLNs demonstrated higher anti-bacterial effects against fections .E. coli. BSP16Lys-liposome have reduced about 1.6-log colony-forming units (CFU)/mL of the viable E. coli, demonstrating the potential of these liposomes against resistant E. coli infections [E. coli led infections. Furthermore, antimicrobial lipid, dioctadecyldimethyammonium bromide (DODAB), was used to form lipidic bi-layered systems and entrap antimicrobial peptide, gramicidin [E. coli with no toxicity against eukaryotic cells [Liposomes are a bi-layered vesicular system with biocompatible, biodegradable, and non-toxic nature, and can encapsulate all types of drugs from hydrophilic to hydrophobic. In a recent attempt, endolysin BSP16Lys containing cationic DPPC liposomes were prepared to act against antibiotic-resistant gram-negative bacteria . Endolysfections . Likewisamicidin . Gramidiic cells . E. coli [For enhanced coverage against bacterial infections, a combination of different types of nanocarriers or materials is employed. For instance, ceftriaxone-loaded polymer-lipid hybrid NPs were prepared using the self-assembly method . ChitosaE. coli .E. coli infection and biofilm formation on catheters [E. coli biofilms were grown on the catheters and treated with chitosan-NLCs. The biofilms were collected after 24 h of treatment with chitosan-NLCs. Results indicated that the chitosan-NLCs hybrid system considerably inhibited the viability of biofilms at all ages and could control the growth of both young and mature E. coli biofilms on the catheters [Similarly, chitosan-coated nanostructured lipidic carriers (NLCs) were developed to combat atheters . E. coliatheters .Different metallic nanocarriers, such as iron (Fe), silver (Ag), gold, zinc (Zn), etc., have shown promising roles as antibacterial and antibiotic drug delivery carriers.E. coli infection [+ conductance with lower flux with detrimental antibiotic action against antibiotic-resistant strains of E. coli, i.e., ampicillin-resistant E. coli DH5α-Puc 18 and kanamycin-resistant E. coli Parg-25. Thus, iron-oxide-oleic acid NPs have the potential to be used as a strong anti-bacterial agent against antibiotic-resistant organisms in the future [Iron oxide NPs are of particular importance in metallic NPs. For instance, iron oxide NPs with biocompatible oleic acid coating were synthesized in the size of 10.64 nm to combat nfection . The syne future .E. coli with a minimum inhibitory concentration (MIC) of 2.8 µg/mL. The MIC is about 100 times lesser than the human toxic, confirming the low toxicity potential. The only dose above 100 µg/mL for the 2 weeks produced damage to the kidney and the liver. Thus, it demonstrated that nano-magnetite itself could be used to treat infections with a wide therapeutic window [Magnetite is one of the forms of iron oxide. Magnetite NPs were prepared by the green, surfactant-free electrochemical method . In thisc window .Beta vulgaris, Brassica oleracea, Cinnamomum verum, and Cinnamomum tamala. In-vitro characterization demonstrated suitable size and morphology. Moreover, ZnO NPs proved their anti-bacterial activity against E. coli and S. aureus [Zinc is an important micro-ingredient for normal body function with natural anti-bacterial activity. In a study, zinc oxide (ZnO) NPs were synthesized through green, eco-friendly, and economical methods using 4 plant extracts: . aureus .E. coli and demonstrated a 100% inhibition ratio with two stepped inhibition and sterilization process against E. coli. The whole inhibition-sterilization process was analysed through growth curves, FTIR, SEM, and MDA concentrations [Ag is a well-known antimicrobial agent. In the recent past, various studies reported the efficacy of Ag NPs ,122. In trations .12O19/xCoFe2O4 were fabricated using the sol–gel method [E. coli was found promising and thus, proving their efficacy as an antimicrobial agent [Magnetic nanocomposites consisting of BaFel method . In-vitral agent .E. coli and S. aureus. Suitable-sized niosomes are formed from appropriate ratios of span 80, drug, and cholesterol. Prepared niosomes have good stability and good antibacterial activity [Niosomes are a vesicular system prepared from non-ionic surfactant and a stabilizer. Due to their peculiar nature, niosomes have a wide window to encapsulate various types of drug moieties. In a recent study, simvastatin-loaded niosomal gel was prepared for the activity against activity .E. coli, S. aureus, Bacillus subtilis, etc. [Nanoemulsion is a versatile liquid system containing two immiscible liquids with the help of an emulsifier. Liquid dosage forms have importance in killing microorganisms. In an effort, ε-polylysine and D-limonene nanoemulsion was formulated using Tween-80 as a surfactant. Both components have shown greater synergistic effect against bacteria, including is, etc. . E. coli O157:H7 and S. aureus in the tryptic soy broth and milk than free thyme oil. Thus, the nanoemulsion can be used as a preservative and treat infections [Likewise, thyme oil and sodium caseinate-based neutral self-assembled nanoemulsion was prepared for antimicrobial action . Encapsufections .E. coli infection. Trp-terminated dendrimers have demonstrated splendid activity against extended-spectrum beta-lactamases (ESBL)-producing and multidrug resistance (MDR) isolates of E. coli. The peptide-based dendrimers were found to be stable in plasma over a longer period with minimal hemolysis and low genotoxicity [E. coli inhibition is compiled in Dendrimers are highly branched, symmetric, nanosized molecules used for drug delivery, diagnostics, and therapeutic purposes. Researchers developed bioinspired peptide Trp-terminated dendrimers as an effective treatment of toxicity . A summaSalmonella enterica, Escherichia coli, and Listeria monocytogenes, were specifically quantified in apple juice with limits of detection of about 10 CFU/mL [2O2 solution. Then, the virus concentration is detected in correlation with the change in beads motion. The developed NBC system could detect ZIKV in samples with virus concentrations as low as 1 particle/μL. The NBC platform technology has the potential to be used in the development of point-of-care diagnostics for pathogen detection and disease management in developed and developing countries [Pathogens, and all diseases associated with them, are a significant concern worldwide . Diagnos0 CFU/mL . In anotountries . Of courountries ,134,135.The safety assessment of medical devices containing or deriving from nanotechnology is carried out by the US-FDA’s Centre for Devices and Radiological Health (CDRH), housing a Nanotechnology Regulatory Science Research Programme that is based on three pillars: physicochemical characterization methods, in vitro and in vivo models, and  risk assessment ,137. TheE. coli infected area and inhibiting E. coli at a much lower dose. Moreover, some of the nanomaterials have antibacterial action and produced synergistic action along with antibiotics to combat E. coli.NPs have been shown to have considerable potential in the development of bacterial sensing devices. Bacterial sensing can be conducted in a lab setting by evaluating physiological fluids, or in vivo by tracking bacteria in real-time within the body. In a label-free methodology, NPs can act as both a detection element and a reporter when the surface is coupled with a specific recognition element. Integrating artificial intelligence, a sub-type of machine learning with nanotechnology, is paving the way for advancements in microbe detection systems and is working closely toward the human dream of having a sensor that fully fits all of the aforementioned criteria. Even though several biosensors for detecting food-borne diseases have been created, researchers continue to face difficulties in fabricating biosensors for the accurate and reliable assessment of microorganisms in real food samples. In short, a variety of nanocarriers have shown promising efficiency in delivering antibiotic to the