Influence of the subinhibitory concentration of antibiotics to biofilm production and adherence to matrix proteins of invasiveand noninvasive isolates of group a beta-hemolytic streptococcus

Infekcije izazvane bakterijama koje produkuju biofilm su jedan od najaktuelnijih problema u bakteriologiji. Biofilm je način odbrane bakterija od djelovanja antibiotika i imunskog sistema domaćina. Terapija infekcija izazvanih biofilmom je uglavnom neuspješna, jer su za eradikaciju biofilma potrebne visoke koncentracije antibiotika koje bi bile izuzetno toksične po domaćina. Zbog neodgovarajuće i neuspješne terapije, bilo bi veoma značajno otkriti faktore i uslove sredine koji dovode do nastanka biofilma ili ga čak i indukuju. Streptococcus pyogenes ili streptokok grupe A (GAS) je jedan od najučestalijih humanih patogena, koji izaziva širok spektar oboljenja, koje se na osnovu patogenetskih mehanizama nastanka mogu podijeliti na piogene infekcije, toksemične infekcije i poststreptokokne sekvele. Ovako širok spektar oboljenja GAS može da zahvali mnogobrojnim faktorima virulencije. Sposobnost formiranja biofilma kao dodatnog faktora virulencije je još uvijek nedovoljno ispitana za GAS, iako je biofilm kao značajan faktor virulencije potvrđen kod mnogobrojnih bakterija. Početni i najvažniji korak u formiranju biofilma je adherencija za supstrat, bilo da su u pitanju adherencija za ćelije domaćina ili za vještačke materijale. Streptococcus pyogenes raspolaže sa znatnim brojem adhezina koji mu omogućavaju adherenciju za različite humane ćelije. Najveći značaj adherencije je u pripajanju za ćelije domaćina, agregaciji blisko postavljenih bakterija, stvaranju signala za prelazak u kolektivni način života, pri kojem bakterije dobijaju zaštitni matriksni omotač, koji u planktonskom načinu života ne postoji, a koji je odgovoran za većinu odbrambenih mehanizama kojima bakterije u biofilmu izbjegavaju eradikaciju sa mjesta infekcije. Smatra se da produkcija biofilma omogućava in vitro osjetljivim sojevima rezistenciju na antibiotike i dovodi do terapijskog neuspjeha. Uočeno je da su bolji produktori neinvazivni sojevi različitih vrsta streptokoka u odnosu na invazivne sojeve, kao i da su GAS sojevi osjetljivi na eritromicin bolji produktori u odnosu na eritromicin rezistentne sojeve. Takođe, pretpostavlja se da streptokokno kliconoštvo najvjerovatnije nastaje prodorom bakterija u epitelne ćelije ili stvaranjem biofilma u kriptama tonzila...Biofilms have been increasingly recognized as being important in human disease. Problems associated with human biofilm infections result from two distinct characteristics of all biofilms. First, biofilms are highly resistant to immune killing and clearance, and to treatment with antimicrobial agents. Second, protected biofilms might be capable of shedding individual bacteria and sloughed pieces of biofilm into surrounding tissues and the circulatory system. Such shed cells might be responsible for acute illness, which might recur despite vigorous antimicrobial treatments. So, it is important to establish factor that induce biofilm production. Group A Streptococcus (GAS) is a beta-hemolytic, Gram- positive human pathogen capable of causing a wide variety of human disease ranging from noninvasive diseases, such as pharyngitis and impetigo, to more severe, invasive infections, including necrotizing fasciitis, sepsis, and toxic shock-like syndrome. A large number of secreted or cell-attached virulence factors expressed by this microorganism have been investigated so far. Recent observations suggesting that biofilm may also have a role in S. pyogenes infections have been reported. First and most important step in biofilm production is adherence to biotic and abiotic surfaces. After successful adherence, bacteria aggregate and get quorum signal for biofilm formation, sessile cells secreted exopolysaccharide matrix that protect bacteria as shield from surroundings effects. The nature of biofilm structure and the physiological attributes of biofilm organisms confer an inherent resistance to antimicrobial agents. Although no strain of GAS that is resistant to penicillin in vitro has been identified in clinical laboratories using current susceptibility methods, treatment failure rates of up to one-third have been observed in clinical practice. Ogava et al. proposed that biofilm formation or internalization into epithelial cells enable Streptococcus pyogenes to establish pharyngeal carriage in patients with pharyngitis. However, little is known about whether the ability to form biofilm is a characteristic of all group A streptococcal strains and is there relationship between biofilm formation and clinical source of isolates..

Adherence and Biofilm Production of Streptococcus pyogenes

Streptococcus pyogenes (group A streptococcus – GAS) can cause numerous human infections, varying from mild skin infections to life‐threatening, e.g. necrotizing fasciitis. Adherence and biofilm production are important in streptoccocal pathogenesis. GAS adhesins are numerous and diverse, with the ability to bind to several different receptors at the same time, which leads to difficulties in their precise identification and classification. Biofilm production is one of the most probable explanation for therapeutic failure in the treatment of GAS infections. Most researchers agreed that biofilm formation is a trait of individual strains rather than a general serotype attribute. The aim of our study is to investigate differences in adherence to laminin and biofilm production between invasive and non‐invasive isolates (NI) of GAS. In this study the correlation between adherence to laminin and invasiveness in GAS isolates is noticed. The strains isolated from GAS carriers and highly invasive (HI) GAS strains have excellent capacity for binding to laminin. When testing biofilm production, there was noticeable positive correlation between adherence and biofilm production among non‐invasive isolates. Non‐invasive isolates were stable biofilm productors. There was no correlation between adherence and biofilm production among invasive isolates. Invasive isolates were also unstable biofilm productors

Distribution of macrolide-resistant genes among isolates of macrolideresistant Streptococcus pyogenes and Streptococcus pneumoniae in Serbia

Macrolide resistance in Streptococcus pneumoniae and in group A streptococci (GAS) is a significant problem worldwide. In Serbia, data on the mechanisms of resistance and the corresponding resistance genes in streptococci are largely lacking. Therefore, we analyzed the distribution of macrolide resistance phenotypes and genotypes in 44 macrolideresistant GAS (MRGAS) and 50 macrolide-resistant S. pneumoniae (MRSP) isolates collected in the same period. The double disk diffusion test and PCR were used to analyze resistance phenotypes and resistance genes, respectively. Among MRSP, the MLSB phenotype dominated, whereas the M phenotype was the most prevalent among MRGAS isolates. Consequently, in MRSP, the ermB gene was the most common (n=40, 80%), followed by the mefA gene (n=7,14%). In MRGAS strains, mefA dominated (n=27,61%), followed by ermA (n=15, 33%) and ermB (n=3, 7%). In 3 MRSP isolates no resistance genes were detected, while one MRGAS strain with iMLSB phenotype harbored both ermA and mefA genes

Intestinal nematodes in dogs and cats in Banja Luka, Republika Srpska

Pojedine intestinalne nematode pasa i mačaka pokazuju zoonozni potencijal, pa samim tim imaju javno-zdrastveni značaj. Na teritoriji grada Banja Luke je pregledano 149 uzoraka (131 od pasa i 18 od mačaka), s tim da je 40 uzoraka fecesa pasa sakupljeno sa dvije javne površine u gradu. Parazitološkim pregledom (flotacija sa zasićenim rastvorom cink sulfata i determinacija adulta i larvi parazita u fecesu) dobijeni su sledeći rezultati: Toxocara canis (9,16%), Ancylostomatidae (5,34%), Trichuris vulpis (7,63%), Toxascaris leonina (1,52%), Toxocara cati (33,3%), Trichuris spp. (5,5%) i Hydatigera taeniformis (5,5%). Na osnovu dobijenih rezultata možemo zaključiti da su intestinalne nematode u daleko većem stepenu raširene nego što se pretpostavljalo i da zbog toga predstavljaju zdrastveni problem kod pasa i mačaka na teritoriji Banja Luke.Some intestinal nematodes of dogs and cats show zoonotic potential, and therefore they are of public-health significance. In the territory of the city of Banja Luka, 149 samples were examined (131 of dogs and 18 cats), with 40 samples of dog feces collected from two public areas in the city. Parasitological examination (saturated zinc sulfate floatation and determination of larvae and adult parasites in feces) resulted in the following data: Toxocara canis (9.16%), Ancylostomatidae (5.34%), Trichuris vulpis (7.63%), Toxascaris leonina (1.52%), Toxocara cati (33.3%), Trichuris spp. (5.5%) and Hydatigera taeniaeformis (5.5%). On the basis of the obtained results we can conclude that the intestinal nematodes are more widespread than is generally assumed and therefore represent a health problem for dogs and cats in the territory of Banja Luka

INTESTINAL NEMATODES IN DOGS AND CATS IN BANJA LUKA, REPUBLIKA SRPSKA

Some intestinal nematodes of dogs and cats show zoonotic potential, and therefore theyare of public-health significance. In the territory of the city of Banja Luka, 149 samples wereexamined (131 of dogs and 18 cats), with 40 samples of dog feces collected from two public areasin the city. Parasitological examination (saturated zinc sulfate floatation and determination of larvaeand adult parasites in feces) resulted in the following data: Toxocara canis (9.16%),Ancylostomatidae (5.34%), Trichuris vulpis (7.63%), Toxascaris leonina (1.52%), Toxocara cati(33.3%), Trichuris spp. (5.5%) and Hydatigera taeniaeformis (5.5%). On the basis of the obtainedresults we can conclude that the intestinal nematodes are more widespread than is generallyassumed and therefore represent a health problem for dogs and cats in the territory of Banja Luka

Influence of subinhibitory antibiotic concentration on Streptococcus pyogenes adherence and biofilm production

In this study, the focus was on the effects of sub-MICs of the antibiotics on adherence, hydrophobicity, and biofilm formation by two groups of Streptococcus pyogenes strains, which were responsible for different clinical cases. The aim of this study was to explore the effects of sub-MICs of penicillin, ceftriaxone, erythromycin, and clindamycin on adherence, surface hydrophobicity, and biofilm biomass in two selected collections of group A streptococcus (GAS): strains isolated from carriers (CA) and strains isolated from patients with tonsillopharyngitis (TPh). Isolates were tested for hydrophobicity to xylene, adherence, and biofilm production in uncoated microtiter plates before and after treatment with 1/2 and 1/4 MICs of antibiotics. Penicillin reduced adherence and biofilm production in TPh strains, whereas ceftriaxone diminished adherence and biofilm formation in CA group. On the contrary, clindamycin enhanced adherence and biofilm production in both groups of strains. Erythromycin did not significantly alter adherence, but triggered biofilm production in both groups of isolates. Hydrophobicity of both groups of strains was significantly reduced after exposure to all antibiotics. Beta-lactams displayed anti-biofilm activity; penicillin diminished both adherence and biofilm production in TPh strains, whereas ceftriaxone reduced it in strains isolated from CA

Efficacy of biocides with different chemical compounds on the eradication of Salmonella spp. biofilm

Салмонеле увијек представљају интересантну микробиолошку тематику, јер спадају међу најучесталије зоонотске бактеријске патогене који се лако и једноставно преносе са животиња на људе преко термички недовољно обрађених намирница. Иако је током COVID-19 пандемије дошло до смањења учесталости хуманих салмонелоза за чак 22% на глобалном нивоу, салмонеле и даље представљају другу најчешћу цријевну бактеријску зоонозу са годишњом инциденцом од 13,3 случајева на 100 000 становника. На подручју Републике Српске салмонелозе су на првом мјесту кад су у питању цријевне инфекције, са значајним падом учесталости са 14, 5 промила на свега 4 промила због епидемиолошких мјера и препорука током пандемије. С обзиром да је проглашен крај пандемије и да више не постоје епидемиолошке мјере, очекивано је да ће се и учесталост салмонелозних инфекција вратити на претходни ниво. Салмонелозе се најчешће повезују са недовољно термички обрађеним пилећим месом, као и са преливима, кремама и сосевима у чији састав улазе термички необрађена јаја, односно жуманца, али треба нагласити и значај свињског меса и производа од свињског меса, као и свјеже и сухо воће, поготово поријеклом из егзотичних земаља. Захваљујући развоју науке и технологије, схватили смо да бактерије могу преживљавати у два облика, као појединачни, планктонски облици, и удружене, у форми биофилма. Бактеријски биофилм је вид заједничког суживота и начин преживљавања бактерија у неповољним условима. Унутар биофилма се налазе бактеријски сојеви који се метаболички и генетски разликују у односу на планктонске сојеве од којих су потекли. Метаболизам бактерија унутар биофилма је значајно успорен у односу на планктонске форме, због ограничене количине доступних храњивих материја. Такође, и на генетском нивоу је уочена потпуно другачија експресија гена у односу на планктонске сојеве. Додатну заштиту бактеријама унутар биофилма пружа омотач од полисахарида, који бактерије самe стварају, али тек након што добију сигнал да пређу у форму биофилма. Биофилм се ствара у неколико корака, који подразумијевају прво адхеренцију (приљубљивање) појединачних облика, затим њихово размножавање на датом мјесту, након чега добијају сигнал за прелазак у форму биофилма и продукцију заштитног полисахаридног омотача. Крајњи исход је формирање зрелог биофилма. Биофилм представља заједнички начин егзистенције свих врста микроорганизма у природи, како на живим бићима, тако и на неживим објектима. У том контексту је веома важно утврдити способност продукције биофилма код салмонела које се изолују из намирница животињског поријекла, поготово на различитим површинама (метал, стакло, пластика), са којима могу доћи у дотицај у оквиру прехрамбене индустрије и угоститељства. Наш циљ је да утврдимо који серотипови се најчешће детектују и да анализирамо евентуално присутну разлику у продукцији биофилма код различитих серотипова. Ерадикација, тј. уклањање бактеријског биофилма представља прави изазов због егзополисахаридног омотача које синтетишу бактерије унутар биофилма. Овај полисахаридни омотач им омогућава да се снажно залијепе за површину, а даје им и отпорност на велики број различитих хемијских супстанци, тако да једини и најсигурнији начин уклањања биофилма представља физичко одстрањивање са супстрата. Тамо гдје није могуће на такав начин уклонити биофилм, примјењују се биоциди (дезинфицијенси) који имају мању или већу ефикасност у ерадикацији зрелог биофилма. Додатни проблем представља чињеница да неки биоциди могу оштетити површину на којој се налази биофилм, што представља велики проблем у иднустрији или угоститељству због оштећења машина или апарата који се користе у наведеним гранама. Због тога је веома важно утврдити ефикасност биоцида различитог хемијског састава на ерадикацију зрелог биофилма. Подаци из доступне литературе говоре да су препарати хлора имају бољу ефикасност у односу на алкохол и водоник пероксид, па је и наш циљ да се у блиској будућности спроведу слична испитивања и код нас. Резултати будућих истраживања би имали и практични значај, јер би се могли користити у виду препорука за практичну примјену у зеинфекцији машина у прехрамбеној индустрији, као и за дезинфекцију површина у угоститељству, здравству и сточарству.Salmonella spp. are always an interesting microbiological topic, because they are among the most common zoonotic bacterial pathogens which can be easily transmitted from animals to humans through thermally insufficiently processed foods. Although the incidence of human salmonellosis has decreased by 22% globally during the COVID-19 pandemic, these bacteria still occupy the second place among the most common intestinal bacterial zoonosis with an annual incidence of 13.3 cases per 100,000 population globally. In the Republic of Srpska, salmonellosis is the most common intestinal infection, with a significant drop in frequency from 14.5 per thousand to only 4 per thousand due to epidemiological measures and recommendations during the pandemic. Given that the end of the COVID-19 pandemic has been declared and that there are no longer any epidemiological measures, it is expected that the frequency of salmonellosis will be as high as before the pandemic. Salmonellosis is the most often associated with improperly thermal processing of chicken meat, as well as with dressings, creams and sauces that include non-thermally processed eggs or egg yolks, but the importance of pork meat and pork meat products, as well as fresh and dried fruit, should also be emphasized. With the development of science and technology, we realized that bacteria can survive in two forms, as individual, planktonic forms, as it is well known, and also, as we recently have discovered, associated in the form of biofilms. Bacterial biofilm is a form of coexistence and a mode for bacterial survival in unfavorable conditions. Inside of the biofilms, bacterial strains differ metabolically and genetically from the planktonic strains from which they originated. The metabolism of bacteria within the biofilm is significantly slowed down compared to free-living planktonic forms, due to the limited amount of available nutrients. Also, at the genetic level, completely different levels of gene expression was observed compared to planktonic strains. Additional protection for the bacteria inside the biofilm is provided by a exopolysaccharide coat, produced by bacteria, after they give to each other a signal to switch from planktonic to the biofilm form. A biofilm is created in several steps, which include the adherence tu substratum of individual forms as the first step in this process, then bacterial replication, after which they receive a signal for transitioning in the biofilm mode and the production of a protective exopolysaccharide coat. The end result of this complex process is the formation of a mature bacterial biofilm. Biofilm represents a survival mode of all types of microorganisms in nature, both on living creatures and on nonhost objects. In this context, it is very important to determine the ability of Salmonella spp. isolated from foods of animal origin to produce biofilms on different surfaces (metal, glass, plastic), with which they may come into contact during food processing in food industry and catering. Our goal is to determine which serotypes are most often detected and to analyze if there are any differences in biofilm production potential among different bacterial serotypes. Eradication of the bacterial biofilm is a real challenge due to the exopolysaccharide coat synthesized by the bacteria placed inside of the biofilm community. This coat allows them to have strong fixation to the nonhost surface, and also gives them impenetrable resistance and protection against different chemical substances. Sometimes, the only way for biofilm removal is physical elimination from the substrate. In the cases where it is not possible for physical biofilm displacement, biocides (disinfectants) are applied, which have a different potential for the eradication of the mature biofilm. An additional problem is the fact that some biocides can damage the nonhost surface and this represents a huge problem in the food industry or catering due to damage of the machines or devices. Therefore, it is very important to determine the effectiveness of biocides of different chemical composition on the eradication of mature biofilm. Available data show that chlorine preparations are more effective than alcohol and hydrogen peroxide, so our goal is to conduct similar tests in our country. The results of future research would also have practical significance, as they could be used in the form of recommendations for practical application in the disinfection of machines in the food industry, as well as for the disinfection of surfaces in catering, healthcare and animal husbandry.Zbornik radova i kratkih sadržaj

Zoonotic bacterial intestinal infections in republic of srpska and their significance within the one health principle

Koncept jednog zdravlja se zasniva na činjenici postoji veza između zdravlja ljudi, životinja i životne sredine u pogledu zoonoznih patogena, antimikrobne rezistencije i bezbjednosti hrane. Povećanje brojnosti ljudske populacije na Zemlji utiče na zdravlje ne samo naše vrste, već i na zdravlje životinja i životne sredine, kao i na bezbjednost hrane. Povećana potrošnja mesa i mesnih produkata, globalizacija u snabdijevanju hranom i stalne migracije ljudi i životinja igraju ključnu ulogu u problemima kojima se bavi koncept jednog zdravlja. Značajan porast broja tovnih životinja, selekcija određenih vrsta i nove prakse uzgoja životinja u cilju povećanja proizvodnje uzrokuju nedostatak genetske raznovrsnosti i sve više monotipski karakter životinjskih krda i jata. Ovo može dovesti do povećane osjetljivost životinja na nove patogene i epidemijsko širenje novih bolesti. Zbog brzog transporta ljudi i životinja s jednog na drugi kraj planete, ljudi koji imaju kontakt sa životinjama u udaljenim dijelovima zemalja u razvoju mogu da se ukrcaju u avion i sljedećeg dana doputuju u industrijalizovanu zemlju, te tako prenesu zoonotske patogene. Realno, hiljade ljudi svakog dana prelazi međunarodne granice dovodeći do toga da zoonotske bolesti imaju veći potencijal za izazivanje epidemija i pandemija.The concept of One Health proposes that there is a connection between human, animal, and environmental health with respect to zoonotic pathogens, antibiotic resistance and foodsafety. The earth’s increasing human population impacts the health not only of our own species, but that of food and companion animals and of our environment. Increased meat consumption, globalization of the food supply, and migration of humans and animals all play a pivotal role in how we address current and future challenges. The sheer growth in numbers of food animals, use of intensive genetic selection, and enhanced breeding practices to increase production is causing a lack of genetic diversity and an increasingly monotypic character of animal herds and flock. These practices may increase the susceptibility of animals to novel disease pathogens and outbreaks. Because of the ease of global travel, humans who have contact with animals in a remote area of a developing country can board a plane and travel to an industrialized country the following day. In fact, thousands of people cross international borders each day. Thus zoonotic diseases have greater potential than ever before to initiate global outbreaks in humans

Prevalence and antimicrobial susceptibility of extended-spectrum beta-lactamase producing urinary isolates of Escherichia coli in outpatients

Introduction. In Gram-negative bacteria, the production of beta-lactamases is the most important mechanism of resistance to beta-lactam antibiotics. In the Banja Luka region, there were no extensive researches on the prevalence and antimicrobial resistance of the extended-spectrum beta-lactamase (ESBL) producing Escherichia coli (E. coli) isolates. Objective. The aim of the present study was to determine the presence of ESBL producing E. coli isolates as the cause of the urinary tract infections in outpatients, the distribution of these ESBL isolates according to age and gender of patients and their susceptibility to antimicrobials. Methods. Urine specimens obtained from outpatients were cultured on chromogenic CPS-ID3 media. All plates showing significant (>105 cfu/ml) growth of E. coli in pure culture were further processed. Antimicrobial susceptibility testing was performed on VITEK TWO Compact using AST-GN27 cards for testing Gram negative bacteria and detection of ESBL producers. Results. Out of 2,195 isolates, 177 (8.1%) were ESBL producers. Ninety-two isolates were obtained from female patients (5% of E. coli isolated from women) and 85 isolates from male patients (23% of E. coli isolated from men). High percentage of ESBL isolates was detected in the infant age group under one year (36.7%) and in the age group over 60 years (28.8%). All ESBL isolates were susceptible to imipenem and resistant to ampicillin, piperacillin, cefazolin, cefotaxime, ceftazidime and cefepime. There was a significant resistance to amikacin (79.1%), gentamicin (76.8%), amoxicillin/clavulanate (54.8%) and trimethoprim/sulphamethoxazole (45.8%). Resistance to nutrofurantoin was 13.6%. Conclusion. This study has demonstrated the presence of ESBL producing E. coli urinary isolates in outpatients, and their extensive susceptibility to imipenem and nitrofurantoin