The efficacy of isolated bacteriophages from pig farms against ESBL/AmpC- producing Escherichia coli from pig and Turkey farms

Extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases are plasmid (but also chromosomally) encoded enzymes found in Enterobacteriaceae, determining resistance to a variety of important antibiotics including penicillins, cephalosporins, and monobactams. In recent decades, the prevalence of ESBL /AmpC-producing bacteria has increased rapidly across the world. Here, we evaluate the potential use of bacteriophages in terms of a reduction of antibiotic-resistant bacteria in healthy animals. The aim of our studies was to isolate bacteriophages capable of destroying ESBL/AmpC-producing Escherichia coli isolated from livestock habitats. The efficacy of isolated phages against ESBL/AmpC E. coli strains varies, but creation of a phage cocktail with broad activity spectrum is possible. This may indicate that the role of phages may not be limited to phage therapy, but bacterial viruses may also be applied against spread of bacteria with antibiotic resistance genes in the environment. We also addressed the hypothesis, that phages, effective for therapeutic purposes may be isolated from distant places and even from different environments other than the actual location of the targeted bacteria. This may be beneficial for practical purposes, as the construction of effective phage preparations does not require access to disease outbreaks

POTENTIAL APPLICATION OF LYOPHILIZATION IN COMMERCIAL USE OF BACTERIOPHAGE PREPARATIONS IN VETERINARY MEDICINE

Microbial contamination in livestock, resulting in foodborne illnesses, poses a serious problem for veterinary. Rapidly growing number of antibiotic-resistant bacterial strains triggers increased interest in phage therapy, which has already been tested against zoonotic pathogens in animals. The aim of the study was to investigate the potential application of lyophilization process in commercial processing of phages. We lyophilized three phages active against common animal pathogens E. coli, Salmonella and Enterococcus, in the presence of three different cryoprotectants. The activity of phages was determined after 10 days, 1 month, 3 months, 6 months and 1 year storage at room temperature. All conditions of storage were established to consider practical aspects of phages processing. Skim milk appeared to be the most effective cryoprotectant, however the obtained results varied for different phages, suggesting that efficiency of this process was phage-dependent. This in turn, may be problematic during optimization of phage lyophilization for commercial processing. Nevertheless, further commercialization of phage preparations seems to be unavoidable and the development of new methods for phage processing and storage is required. Key words: bacteriophages; lyophilization; freeze-drying; long-term preservation of bacteriophages   MOŽNOSTI UPORABE LIOFILIZACIJE V KOMERCIALNE NAMENE ZA PRIPRAVO BAKTERIOFAGOV V VETERINARSKI MEDICINI Mikrobiološka kontaminacija hrane v živinoreji, ki povzroča bolezni, predstavlja resen problem v veterinarski medicini. Hitro naraščajoče število bakterijskih sevov, odpornih proti antibiotikom vzbuja vedno večje zanimanje za uporabo t.i. fagoterapije, zdravljenja z bakterijskimi virusi bakteriofagi, ki je bila že preizkušena kot metoda za preprečevanje okužb pri živalih. Cilj raziskave je bil proučiti potencialno uporabo postopka liofilizacije za komercialno obdelavo fagov. Liofilizirali smo tri fage, aktivne proti skupnim živalskim patogenom Escherichia coli, Salmonella sp. in Enterococcus faecalis v prisotnosti treh različnih krioprotektantov. Dejavnost fagov je bila določena po 10 dneh, 1 mesecu, 3 mesecih, 6 mesecih in 1 letu skladiščenja pri sobni temperaturi. Vzpostavljeni so bili vsi pogoji shranjevanja, z namenom upoštevanja praktičnih vidikov obdelave fagov. Posneto mleko je bilo najučinkovitejši krioprotektant, vendar so se dobljeni rezultati razlikovali pri različnih fagih, kar kaže na to, da je učinkovitost postopka odvisna od vrste faga. To bi lahko predstavljalo težavo pri optimizaciji liofilizacije fagov v komercialne namene. Kljub temu je nadaljnja komercialna priprava preparatov s fagi neizogibna in bo potrebno razvijati nove metode za obdelavo in shranjevanje fagov. Ključne besede: bakteriofagi; liofilizacija; sušenje z zamrzovanjem; dolgoročno ohranjanje bakteriofago

Characteristics of Environmental <i>Klebsiella pneumoniae</i> and <i>Klebsiella oxytoca</i> Bacteriophages and Their Therapeutic Applications

In recent years, multidrug-resistant (MDR) strains of Klebsiella pneumoniae have spread globally, being responsible for the occurrence and severity of nosocomial infections. The NDM-1-kp, VIM-1 carbapenemase-producing isolates as well as extended-spectrum beta lactamase-producing (ESBL) isolates along with Klebsiella oxytoca strains have become emerging pathogens. Due to the growing problem of antibiotic resistance, bacteriophage therapy may be a potential alternative to combat such multidrug-resistant Klebsiella strains. Here, we present the results of a long-term study on the isolation and biology of bacteriophages active against K. pneumoniae, as well as K. oxytoca strains. We evaluated biological properties, morphology, host specificity, lytic spectrum and sensitivity of these phages to chemical agents along with their life cycle parameters such as adsorption, latent period, and burst size. Phages designated by us, vB_KpnM-52N (Kpn52N) and VB_KpnM-53N (Kpn53N), demonstrated relatively broad lytic spectra among tested Klebsiella strains, high burst size, adsorption rates and stability, which makes them promising candidates for therapeutic purposes. We also examined selected Klebsiella phages from our historical collection. Notably, one phage isolated nearly 60 years ago was successfully used in purulent cerebrospinal meningitis in a new-born and has maintained lytic activity to this day. Genomic sequences of selected phages were determined and analyzed. The phages of the sequenced genomes belong to the Slopekvirus and Jiaodavirus genus, a group of phages related to T4 at the family level. They share several features of T4 making them suitable for antibacterial therapies: the obligatorily lytic lifestyle, a lack of homologs of known virulence or antibiotic resistance genes, and a battery of enzymes degrading host DNA at infection