Перспективе за откривање нових антимикробних агенаса за борбу против мулти-резистентних патогена

Nastanak i širenje antibiotske rezistencije predstavlja sve veći problem u lečenju infektivnih bolesti, posebno kod osetljivih osoba. Problem u lečenju već sada odnosi preko milion ljudskih života godišnje, a očekuje se da će taj broj rasti i 2050 dostići 10 miliona, tako da rešenje ovog problema zahteva angažovanost nauke, medicine i celokupnog društva. Takođe ovde treba dodati i štete koje ovaj problem nanosi u veterinarskoj medicini i poljoprivredi. S obzirom da je problem nastao usled prekomerne upotrebe i zloupotrebe antibiotika jedno od rešenja jeste njihova ispravna upotreba. Pored toga postoji potreba za otkrivanjem novih antimikrobnih agenasa koji bi se koristili kao dopuna ili zamena za antibiotike kako se civilizacija ne bi vratila u preantibiotsko doba. Perspektive za otkrivanje novih potentnih agenasa poput antibiotika su male tako da su istraživanja fokusirana na veći broj alternativa kao što su: antimikrobni peptidi (u kliničkoj upotrebi se već nalazi nekoliko peptidnih antibiotika kao što su bacitracin i kolistin), različiti produkti biljaka, hemijski sintetisana antimikrobna jedinjenja, specifična antitela, vakcine, probiotici, bakteriofagi i drugi molekuli. U rešavanje ovog globalnog problema naučnici su uključili i veštačku inteligenciju (mašinsko učenje) kako bi na što brži način iskoristili dosadašnja saznanja za dizajniranje novih jedinjenja i himernih molekula koja bi bila dostojna zamena kliničkim antibioticima, što je već rezultovalo u otkrivanju novog veoma potentnog antibakterijskog jedinjenja, halicina. Svesnost o ozbiljnosti problema u kome se ceo svet nalazi se mora podići na viši nivo i to kroz angažovanje svih relevantnih subjekata, a posebno nauke, medicine, službi javnog informisanja i vlada svih država sveta.Настанак и ширење антибиотске резистенције представља све већи проблем у лечењу инфективних болести, посебно код осетљивих особа. Проблем у лечењу већ сада односи преко милион људских живота годишње, а очекује се да ће тај број расти и 2050 достићи 10 милиона, тако да решење овог проблема захтева ангажованост науке, медицине и целокупног друштва. Такође овде треба додати и штете које овај проблем наноси у ветеринарској медицини и пољопривреди. С обзиром да је проблем настао услед прекомерне употребе и злоупотребе антибиотика једно од решења јесте њихова исправна употреба. Поред тога постоји потреба за откривањем нових антимикробних агенаса који би се користили као допуна или замена за антибиотике како се цивилизација не би вратила у преантибиотско доба. Перспективе за откривање нових потентних агенаса попут антибиотика су мале тако да су истраживања фокусирана на већи број алтернатива као што су: антимикробни пептиди (у клиничкој употреби се већ налази неколико пептидних антибиотика као што су бацитрацин и колистин), различити продукти биљака, хемијски синтетисана антимикробна једињења, специфична антитела, вакцине, пробиотици, бактериофаги и други молекули. У решавање овог глобалног проблема научници су укључили и вештачку интелигенцију (машинско учење) како би на што бржи начин искористили досадашња сазнања за дизајнирање нових једињења и химерних молекула која би била достојна замена клиничким антибиотицима, што је већ резултовало у откривању новог веома потентног антибактеријског једињења, халицина. Свесност о озбиљности проблема у коме се цео свет налази се мора подићи на виши ниво и то кроз ангажовање свих релевантних субјеката, а посебно науке, медицине, служби јавног информисања и влада свих држава света.Knjiga sažetaka: Treći Kongres biologa Srbije, Zlatibor, Srbija 21 - 25. 9. 2022

Analysis of large conjugative plasmid of Lactococcus lactis subsp. lactis biovar. diacetylactis S50

Lactococcus lactis subsp. lactis biovar. diacetylactis S50 je prirodni izolat iz maslacne maje. Soj S50 sintetise bakteriocin uskog spektra delovanja i proteinazu PI tipa. Soj S50 poseduje tri vidijiva plazmida koji se mogu izolovati (pS50-7, pS50-10a i pS50-10b). Ciscenjem plazmida iz soja S50 istovremenim tretmanom subletalnom temperaturom i novobiocinom (10u:g/m!) dobijen je Bac- i Prt- derivat (S50-1). Analizom genoma soja S50 i derivata S50-1 na elektroforezi u pulsirajucem polju (PFGE) nakon digestije restrikcionim enzimima ustanovijeno je da se geni za sintezu proteinaze PI tipa i bakteriocina S50 nalaze na plazmidu velicine oko 290kb koji je oznacen kao plazmid pS50-290. Na osnovu razdvajanja secene DNK na PFGE i hibridizacije sa probama za proteinazni (Q; i Qo) i bakteriocinski gen (LenA) odredjena je restrikciona mapa plazmida pS50-290 i odredjen je polozaj gena na njemu. Plazmid pS50-290 pokazuje visok stepen retardacije na PFGE sto moze biti posledica vezivanja proteina za njega. Soj S50 je ukrstan sa vecim brojem recipijentnih sojeva (MG7284, 1L1403, VEL1122 i ocisceni derivati soja S50) u kojima su dobijani Bact konjugnti sa priblizno istom frekvencom. Na osnovu toga se moze tvrditi da je plazmid pS50-290 autokonjugabilan ili Tra+ plazmid, odnosno da na sebi poseduje sve neophodne gene za sopstveni transfer. Pored sinteze bakteriocina ustanovljeno je da svi dobijeni konjuganti sintetisu i proteinazu PI tipa istih karakteristika kao i soj S50. Analizom plazmidnog sastava konjuganata ustanovijeno je da i drugi plazmidi soja S50 (pS50-7 i pS50-10b) poseduju sposobnost konjugacionog transfera, najverovatnije zato sto poseduju gen za Mob protein i oriT sekvencu. Restrikcionom analizom plazmida izolovanih iz konjuganata, rekonjuganata i derivata S50- 20 (digestijia Smal restrikcionim enzimom i razdvajanje dobijenih fragmenata na PFGE) ustanovijeno je da je u njima plazmid pS50-290 (oznacen kao pS50-290A) skracen i da poseduje samo jedno Smal restrikciono mesto za razliku od plazmida pS50-290 izolovanog iz sojaS50 koji poseduje dva. Fragment koji se deletira je velicine nekoliko kilobaza i pokazuje homologiju sa ostalim plazmidima soja S50 (pS50-7, pS50-10a i pS50-10b). Hibridizacionim ekspeimentima u kojima je plazmid pS50-7 koriscen kao proba pokazano je da svi plazmidi soja S50 poseduju homologe sekvence cije prisustvo govori o zajednickom poreklu ovih plazmida. U procesu izolacije spektinomicin-rezistentnih derivata soja S50 izolovani su mutanti koji pokazuju visok nivo rezistencije na spektinomicin i koji ujedno poseduju inverziju u hromozomu velicine izmedju 180 i 790kb. Istu inverziju je posedovao i mutant derivata S50-20-62 rezistentan na visoku koncentraciju spektinomicina.Lactococcus lactis subsp. lactis biovar. diacetylactis S50 was isolated from butter starter culture. Strain S50 produced bacteriocin which has narrow antibacterial spectrum, In addition, strain S50 synthesized extracellular cell wall-associated proteinase of Pl-type. The strain S50 has three plasmids detectable by conventional procedures for isolation of plasmids (pS50-7, pS50-10a and pS50-10b). Curing experiments of strain S50 with novobiocin (10u1g/ml) and sublethal temperature (41°C) resulted in obtaining a Prt. Bac- derivative (S50-1). The analysis of original strain S50 and Prt-. Bac- derivative S50-1 by restriction enzymes on PFGE revealed that prt and bac genes are located on the large plasmid (approximately 290 kb) named pS50-290. Restriction map of plasmid pS50-290 was done by concomitant using PFGE and hybridization experiments with prt (Q; and Qg2) and bac (LenA) probes. The plasmid pS50-290 shows significant retardation on PFGE. DNA sequence which is target for binding protein(s) is tocated between Smal restriction site (at position 80 kb) and Ncol (148 kb) on restriction map. The plasmid pS50-290 is self-transmissible (Tra+) plasmid. Strain S50 was used as a donor in conjugation experiments for transfer of plasmid pS50-290 to the other lactococcal strains (MG7284, 1L1403 and VEL1122). All conjugations gave high number of conjugants. The obtained transconjugants (frequency of conjugation were about 10-7) produced bacteriocin S50 and proteinase of PI type. These data show that ability for conjugal transfer depends only on genetic elements located on pS50-290 plasmid. d Conjugants MG10 and SIL102, besides plasmid pS50-290 acquired smaller plasmids of strain S50 (MG10 has plasmid pS50-7 and SVEL102 pS50-10b). Transfer of plasmids pS50-7 and pS50-10b (they are non-self-transmissible plasmids) appeared to be dependent on cotransfer of the conjugative plasmid pS50-290. The analysis of conjugants and derivative S50-20 (derivative of the strain S50 cured of plasmids pS50-7 and pS50-10b) by restriction enzyme Smal and PFGE showed that the plasmid pSS50-290 (designated pS50-290A) present in them has only one Smal site. Hybridization experiments (labelled pS50-7 was used as a probe) revealed that plasmids pS50- 290, pS50-10a and pS50-10b contain homologous sequences to the particular region of plasmid pS50-7. Plasmid pS50-290A which is present in conjugants and derivative S50-20 hasn't shown homology because it has deleted that region. During the isolation of spectinomycin-resistant mutants of the derivative S50-1 as well as S50-20-62 to Sbe used as a recipients in conjugation crosses an inversion within chromosomal DNA has occurred in them

AggLb Is the Largest Cell-Aggregation Factor from Lactobacillus paracasei Subsp paracasei BGNJ1-64, Functions in Collagen Adhesion, and Pathogen Exclusion In Vitro

Eleven Lactobacillus strains with strong aggregation abilities were selected from a laboratory collection. In two of the strains, genes associated with aggregation capability were plasmid located and found to strongly correlate with collagen binding. The gene encoding the auto-aggregation-promoting protein (AggLb) of Lactobacillus paracasei subsp. paracasei BGNJ1-64 was cloned using a novel, wide-range-host shuttle cloning vector, pAZILSJ. The clone pALb35, containing a 11377-bp DNA fragment, was selected from the SacI plasmid library for its ability to provide carriers with the aggregation phenotype. The complete fragment was sequenced and four potential ORFs were detected, including the aggLb gene and three surrounding transposase genes. AggLb is the largest known cell-surface protein in lactobacilli, consisting of 2998 aa (318,611 Da). AggLb belongs to the collagen-binding superfamily and its C-terminal region contains 20 successive repeats that are identical even at the nucleotide level. Deletion of aggLb causes a loss of the capacity to form cell aggregates, whereas overexpression increases cellular aggregation, hydrophobicity and collagen-binding potential. PCR screening performed with three sets of primers based on the aggLb gene of BGNJ1-64 enabled detection of the same type of aggLb gene in five of eleven selected aggregation-positive Lactobacillus strains. Heterologous expression of aggLb confirmed the crucial role of the AggLb protein in cell aggregation and specific collagen binding, indicating that AggLb has a useful probiotic function in effective colonization of host tissue and prevention of pathogen colonization

The acetyl xylan esterase of Bacillus pumilus belongs to a family of esterases with broad substrate specificity

The Bacillus pumilus gene encoding acetyl xylan esterase tare) was identified and characterized. The axe gene was expressed and the recombinant enzyme produced in Escherichia coli was purified and characterized. The recombinant enzyme displayed similar properties to the acetyl xylan esterase (AXE) from B. pumilus. The AXE primary structure was 76% identical to the cephalosporin C deacetylase of B. subtilis, and 40% to two recently identified AXEs from Thermoanaerobacterium and Thermotoga maritima. These four proteins are of similar sire and represent a new family of esterases having a broad substrate specificity. The recombinant AXE was demonstrated to have activity on several acetylated substrates, including on cephalosporin C

Identification and molecular characterization of Chryseobacterium vrystaatense ST1 isolated from oligomineral water of southeast Serbia

The isolation and molecular characterization of bacterial strains isolated from water sources in the Vlasina Mountain in southeast Serbia, confirmed the presence of a new species Chryseobacterium vrystaatense ST1. This Gram- negative species showed an extremely low level of biochemical reactivity in biochemical tests. The gene for 16S rRNA was amplified by PCR using universal primers and sequenced. Comparison of 16S rRNA gene sequence and phenotypic features indicated that the isolate ST belonged to Chryseobacterium vrystaatense. A BLAST search of sequenced 1088 nucleotides of the 16S rRNA gene with all sequences deposited in the NCBI collection showed the highest similarity (98%) with the strain Chryseobacterium vrystaatense sp. nov., designated as strain R-23533. The very high homology of these two strains allowed classification of our strain at the species level, but some differences indicate, and indirectly confirm, that the isolate ST is an authentic representative. On the basis of these results, we could conclude that Chryseobacterium vrystaatense ST was for first time isolated in Serbia, which is particularly important when one bears in mind that there are only three sequences of this species deposited in the NCBI collection

Molecular characterization of semi-hard homemade cheese microflora

U poslednjoj deceniji zabeležen je nagli razvoj molekularnih tehnika baziranih na 16S i 23S rRNK, koje se koriste u izučavanju biodiverziteta mikroorganizama. U ovom radu ispitivana je mikroflora polutvrdog sira pripremljenog u domaćinstvu. Zbog visokog sadržaja masti u ovom siru razvili smo novu tehniku za izolaciju totalne DNK iz sira (metod je baziran na bead beating-u). Brza izolacija DNK iz mikroflore sira omogućila nam je molekularnu identifikaciju BMK (BAKTERIJE MLEČNE KISELINE) na osnovu umnožavanja gena za 16S rRNK PCR metodom. Za umnožavanje su korišćeni prajmeri specifični za gene 16S rRNK lakto-bacila, ali su uslovi PCR reakcije bili takvi da su omogućavali i umnožavanje gena 16S rRNK laktokoka. Rezultati RFLP analize pokazali su da mikrofloru sira pripremljenog u domaćinstvu čine predominantno laktokoke.This decade has shown an impressive development in the application of molecular techniques based on 16S and 23S rRNA genes to study the microbial diversity in various ecosystems. Microflora of semi-hard homemade cheese was examined in this work. We developed a novel technique for DNA extraction (a bead beating based method) due to high fat content of this cheese. Rapid extraction of DNA from cheese microflora enabled a molecular identification of the LAB (Lactic Acid Bacteria) strains based on PCR amplification of 16S RNA coding sequences. The specific primers for 76S RNA gene of lactobacilli were used for amplification. The PCR reaction was performed at lower temperature, where the specificity of the annealing reaction was reduced and lactococcal sequences of 16S RNA genes were also amplified. The results of RFLP analysis revealed that the microflora of Doboj homemade cheese encompases mostly lactococci

Overexpression of sgm 5’ UTR mRNA reduces gentamicin resistance in both Escherichia coli and Micromonospora melanosporea cells

16S rRNK metilaze su eksprimirane u većini bakterija koje proizvode antibiotike da bi se zaštitile od dejstva antibiotika putem metilacije 16S rRNK na pozicijama koje su bitne za njihovo dejstvo. Gen sgm koji je odgovoran za rezistenciju na sisomicin i gentamicin u soju Micromonospora zionensis, metilu je G1405 u okviru A mesta 16S rRNA gde se nalazi i CCGCCC heksanukleotid. Isti heksanukleotid se nalazi i 14 nukleotida ispred mesta vezivanja ribozoma na sgm informacionoj RNK. Predloženi model translacione regulacije sgm gena pretpostavlja da se Sgm protein vezuje za ovaj motiv kako na 16S rRNK, tako i na 5’ netranslirajućem regionu (UTR) sopstvene informacione RNK. 5’ UTR sekvenca je overeksprimirana na sgm informacionoj RNK sa skraćenim 3’ krajem i testiran je efekat na gentamicinsku rezistenciju u ćelijama E. coli i Micromonospora melanosporea. Overekspresija ove regulatorne sekvence dovodi do smanjenja rezistencije u oba testirana soja najverovatnije zbog titracije Sgm molekula od strane 5’ UTR-a.The 16S rRNA methylases are expressed by most of the antibiotic producing bacteria in order to protect themselves against antibiotics by methylation of 16S rRNA at positions which are crucial for their action. The sgm sisomicin-gentamicin resistance gene from Micromonospora zionensis methylates G1405 positioned in the A site of 16S rRNA, which includes a CCGCCC hexanucleotide. The same hexanucleotide is also present 14 nucleotides in front of the ribosome binding site of sgm mRNA. The model proposed for translational regulation of sgm assumes that Sgm binds to this motif, both on 16S rRNA and on the 5’ untranslated region (UTR) of its own mRNA. The 5’ UTR mRNA sequence was overexpressed on 3’-truncated sgm mRNA, and the effect on gentamicin resistance conferred by Sgm was tested in Escherichia coli and in Micromonospora melanosporea. Overexpression of the sgm mRNA regulatory region decreases the resistance to gentamicin in both E. coli and M. melanosporea. This effect is likely to be due to titration of Sgm molecules by the overexpressed 5’ UTR

Dynamics of sodium dodecyl sulfate utilization andantibiotic susceptibility of strain Pseudomonas sp. ATCC19151

Poznato je da soj Pseudomonas sp. ATCC19151 poseduje gen koji kodira Potencijalnu alkilsulfatazu. U ovom radu analizirana je sposobnost rasta ovog soja u minimalnom medijum usa različitim koncentracijama natrijum dodecilsulfata (0.5, 0.75 i 1 %) kao jedinim izvorom ugljenika. Pokazano je da Pseudomonas sp. ATCC19151 ispoljava najbolji rast uminimalnom medijumusa 0.5 % natrijum dodecil sulfata, te je stoga ova koncentracija uzeta kao optimalna za testiranje dinamike korišćenja natrijum dodecil sulfata tokom različitih faza rasta. Dinamika korišćenja natrijum dodecil sulfata podudarala se sa rastom kulture. Pored toga u cilju detaljnije karakterizacije soja, analizirana je i osetljivost Pseudomonas sp. ATCC19151 na antibiotike. Pokazano je da je analizirani soj rezistentan na šest (ampicilin, tetraciklin, hloramfenikol, tobramicin, nalidiksičnukiselinui gentamicin) od devet analiziranih antibiotika.Pseudomonas sp. ATCC19151 harbors a gene encoding a putative alkylsulfatase (sdsA). Here we report a growth ability of this strain in minimal media containing 0.5, 0.75, and 1% sodium dodecyl sulfate as the sole carbon source. The most prominent growth was detected for the minimal medium with 0.5% SDS, so this concentration of SDS was used to monitor Pseudomonas sp. ATCC19151 SDS biodegradation dynamics. Bacterial growth coincided with the disappearance of SDS. Antibiotic susceptibility was tested as well. Pseudomonas sp. ATCC19151 was resistant to six out of nine tested antibiotics, including ampicillin, tetracycline, chloramphenicol, tobramycin, nalidixic acid, and gentamycin