Bacteriophage ZCKP1: a potential treatment for Klebsiella pneumoniae isolated from diabetic foot patients

The recorded growth in infection by multidrug resistant bacteria necessitates prompt efforts towards developing alternatives to antibiotics, such as bacteriophage therapy. Immuno-compromised patients with diabetes mellitus are particularly prone to foot infections by multidrug resistant Klebsiella pneumoniae, which may be compounded by chronic osteomyelitis. Bacteriophage ZCKP1, isolated from freshwater in Giza, Egypt, was tested in vitro to evaluate its lytic activity against a multidrug resistant K. pneumoniae KP/01, isolated from foot wound of a diabetic patient in Egypt. Characterization of ZCKP1 phage indicated that it belonged to the Myoviridae family of bacteriophages with a ds-DNA genome size of 150.9 kb. Bacteriophage ZCKP1 lysed a range of osteomyelitis pathogenic agents including Klebsiella spp., Proteus spp. and E. coli isolates. The bacteriophage reduced the bacterial counts of host bacteria by ≥2 log10 CFU/ml at 25°C, and demonstrated the ability to reduce bacterial counts and biofilm biomass (> 50%) when applied at high multiplicity of infection (50 PFU/CFU). These characteristics make ZCKP1 phage of potential therapeutic value to treat K. pneumoniae and associated bacteria present in diabetic foot patients

High-throughput sequencing reveals genetic determinants associated with antibiotic resistance in Campylobacter spp. from farm-to-fork

[EN]Campylobacter species are one of the most common causative agents of gastroenteritis worldwide. Resistance against quinolone and macrolide antimicrobials, the most commonly used therapeutic options, poses a serious risk for campylobacteriosis treatment. Owing to whole genome sequencing advancements for rapid detection of antimicrobial resistance mechanisms, phenotypic and genotypic resistance trends along the “farm-to-fork” continuum can be determined. Here, we examined the resistance trends in 111 Campylobacter isolates (90 C. jejuni and 21 C. coli) recovered from clinical samples, commercial broiler carcasses and dairy products in Cairo, Egypt. Multidrug resistance (MDR) was observed in 10% of the isolates, mostly from C. coli. The prevalence of MDR was the highest in isolates collected from broiler carcasses (13.3%), followed by clinical isolates (10.5%), and finally isolates from dairy products (4%). The highest proportion of antimicrobial resistance in both species was against quinolones (ciprofloxacin and/or nalidixic acid) (68.4%), followed by tetracycline (51.3%), then erythromycin (12.6%) and aminoglycosides (streptomycin and/or gentamicin) (5.4%). Similar resistance rates were observed for quinolones, tetracycline, and erythromycin among isolates recovered from broiler carcasses and clinical samples highlighting the contribution of food of animal sources to human illness. Significant associations between phenotypic resistance and putative gene mutations was observed, with a high prevalence of the gyrA T86I substitution among quinolone resistant isolates, tet(O), tet (W), and tet(32) among tetracycline resistant isolates, and 23S rRNA A2075G and A2074T mutations among erythromycin resistant isolates. Emergence of resistance was attributed to the dissemination of resistance genes among various lineages, with the dominance of distinctive clones. For example, sub-lineages of CC828 in C. coli and CC21 in C. jejuni and the genetically related clonal complexes ‘CC206 and CC48’ and ‘CC464, CC353, CC354, CC574’, respectively, propagated across different niches sharing semi-homogenous resistance patterns.SIThis work was partially funded by the Zewail City internal research fund (agreement number ZC 004-2019) and joint ASRT-BA research grant (project number 1110) awarded to Dr. Mohamed Elhadidy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Integration of Epidemiological Evidence in a Decision Support Model for the Control of Campylobacter in Poultry Production

The control of human Campylobacteriosis is a priority in public health agendas all over the world. Poultry is considered a significant risk factor for human infections with Campylobacter and risk assessment models indicate that the successful implementation of Campylobacter control strategies in poultry will translate on a reduction of human Campylobacteriosis cases. Efficient control strategies implemented during primary production will reduce the risk of Campylobacter introduction in chicken houses and/or decrease Campylobacter concentration in infected chickens and their products. Consequently, poultry producers need to make difficult decisions under conditions of uncertainty regarding the implementation of Campylobacter control strategies. This manuscript presents the development of probabilistic graphical models to support decision making in order to control Campylobacter in poultry. The decision support systems are constructed as probabilistic graphical models (PGMs) which integrate knowledge and use Bayesian methods to deal with uncertainty. This paper presents a specific model designed to integrate epidemiological knowledge from the United Kingdom (UK model) in order to assist poultry managers in specific decisions related to vaccination of commercial broilers for the control of Campylobacter. Epidemiological considerations and other crucial aspects including challenges associated with the quantitative part of the models are discussed in this manuscript. The outcome of the PGMs will depend on the qualitative and quantitative data included in the models. Results from the UK model and sensitivity analyses indicated that the financial variables (cost/reward functions) and the effectiveness of the control strategies considered in the UK model were driving the results. In fact, there were no or only small financial gains when using a hypothetical vaccine B (able to decrease Campylobacter numbers from two to six logs in 20% of the chickens with a cost of 0.025 £/chicken) and reward system 1 (based on similar gross profits in relation to Campylobacter levels) under the specific assumptions considered in the UK model. In contrast, significant reductions in expected Campylobacter numbers and substantial associated expected financial gains were obtained from this model when considering the reward system 2 (based on quite different gross profits in relation to Campylobacter levels) and the use of a hypothetical cost-effective vaccine C (able to reduce the level of Campylobacter from two to six logs in 90% of the chickens with a cost of 0.03 £/chicken). The flexibility of probabilistic graphical models allows for the inclusion of more than one Campylobacter vaccination strategy and more than one reward system and consequently, diverse potential solutions for the control of Campylobacter may be considered. Cost-effective Campylobacter control strategies that can significantly reduce the probability of Campylobacter introduction into a flock and/or the numbers of Campylobacter in already infected chickens, and translate to an attractive cost-reward balance will be preferred by poultry producers

Remote sensing techniques and geochemical constraints on the formation of the Wadi El-Hima mineralized granites, Egypt: new insights into the genesis and accumulation of garnets

The Wadi El-Hima Neoproterozoic I- and A-type granites in the Southern Eastern Desert of Egypt are rich in garnets (up to 30 vol%) and are cut by NW–SE strike-slip faults, as confirmed from structure lineament extraction maps. These mineralized granites and garnet mineralization zones can be successfully discriminated using remote sensing techniques. Spectral angle mapper and matched filtering techniques are highly effective for mapping garnet-rich zones and show that the highest garnet concentrations occur along the intrusive contact zone of NW–SE striking faults. El-Hima granites have high SiO2 (73.5–75.1 wt%), Al2O3 (13.4–15.3 wt%) and total alkali (6.7–8.7 wt%) contents, suggesting that they were sourced from peraluminous (A/CNK > 1) parental magmas. Garnet-bearing trondhjemites are metasomatic in origin and formed after I-type tonalite-granodiorites, which originated in a volcanic arc tectonic setting. Garnet-rich syenogranites and alkali-feldspar granites are both post-collisional A-type granites: the syenogranites formed from peraluminous magmas generated by partial melting of lower crustal tonalite and metasedimentary protoliths during lithospheric delamination, and the alkali-feldspar granites crystallized from highly fractionated, felsic and alkali-rich peraluminous magmas in the upper crust. Garnets in El-Hima mineralized granites occur in three forms: (1) subhedral disseminated crystals, (2) vein-type crystals, and (3) aggregated subhedral crystals, reflecting different mechanisms of accumulation. All are dominantly almandine in composition (Alm76Sps10 Prp7Grs6Adr1) and have high average concentrations of heavy rare earth elements (HREE) (ΣHREE = 1636 ppm), Y = (3394 ppm), Zn (325 ppm), Li (39.17 ppm) and Ga (34.94 ppm). Garnet REE patterns show strong negative Eu anomalies with HREE enriched relative to LREE, indicating a magmatic origin. These magmatic garnets are late-stage crystallization products of Al-rich hydrous magmas, and formed at low temperature (680–730 °C) and pressure (2.1–2.93 kbar) conditions in the upper continental crust. Peculiar garnet concentrations in syenogranites near and along contact zones with alkali feldspar granites are related to peraluminous parent hydrous magma compositions. These garnets formed by in situ crystallization from A-type granite melts, alongside accumulation of residual garnets left behind after partial melting of the host garnet-rich granites along the intrusive contact. Magmatic-fluid flow along the NW–SE striking fault of Najd system enhanced garnet accumulation in melts, which formed clots and veins of garnet

Characterisation of aerotolerant forms of a robust chicken colonizing Campylobacter coli

Campylobacter contaminated poultry meat is a major source of human foodborne illness. Campylobacter coli strain OR12 is a robust colonizer of chickens that was previously shown to outcompete and displace other Campylobacter strains from the chicken’s gastrointestinal tract. This strain is capable of aerobic growth on blood agar. Serial aerobic passage increased this aerotolerance as assessed by quantitative assays for growth and survival on solid media. Aerotolerance was also associated with increased peroxide stress resistance. Aerobic passage did not alter cellular morphology or motility or hinder the microaerobic growth rate. Colonization of broiler chickens by aerotolerant C. coli OR12 was significantly lower than the wild-type strain at 3 days after challenge but not by 7 days, suggesting adaptation had occurred. Bacteria recovered from chickens had retained their aerotolerance, indicating this trait is stable. Whole genome sequencing enabled comparison with the wild-type sequence. Twenty-three point mutations were present, none of which were in genes known to affect oxidative stress resistance. Insertions or deletions caused frame shifts in several genes including, phosphoglycerate kinase and the b subunit of pyruvate carboxylase that suggest modification of central and carbohydrate metabolism in response to aerobic growth. Other genes affected include those encoding putative carbonic anhydrase, motility accessory factor, filamentous haemagglutinin, and aminoacyl dipeptidase proteins. Aerotolerance has the potential to affect environmental success and survival. Increased environmental survival outside of the host intestinal tract may allow opportunities for transmission between hosts. Resistance to oxidative stress may equate to increased virulence by virtue of reduced susceptibility to oxidative free radicals produced by host immune responses. Finally, resistance to ambient atmospheric oxygen may allow increased survival on chicken skin, and therefore constitutes an increased risk to public health

Bacteriophage ZCSE2 is a Potent Antimicrobial against Salmonella enterica Serovars: Ultrastructure, genomics and efficacy

© 2020 by the authors. Developing novel antimicrobials capable of controlling multidrug-resistant bacterial pathogens is essential to restrict the use of antibiotics. Bacteriophages (phages) constitute a major resource that can be harnessed as an alternative to traditional antimicrobial therapies. Phage ZCSE2 was isolated among several others from raw sewage but was distinguished by broad-spectrum activity against Salmonella serovars considered pathogenic to humans and animals. Lytic profiles of ZCSE2 against a panel of Salmonella were determined together with low temperature activity and pH stability. The morphological features of the phage and host infection processes were characterized using a combination of transmission electron and atomic force microscopies. Whole genome sequencing of ZCSE2 produced a complete DNA sequence of 53,965 bp. No known virulence genes were identified in the sequence data, making ZCSE2 a good candidate for phage-mediated biological control purposes. ZCSE2 was further tested against S. Enteritidis in liquid culture and was observed to reduce the target bacterium to below the limits of detection from initial concentrations of 107–108 Colony Forming Units (CFU)/mL. With a broad host-range against pathogenic Salmonella serovars, phage ZCSE2 constitutes a potential tool against a major cause of human and animal disease

Characterization and comprehensive genome analysis of novel bacteriophage, vB_Kpn_ZCKp20p, with lytic and anti-biofilm potential against clinical multidrug-resistant Klebsiella pneumoniae

IntroductionThe rise of infections by antibiotic-resistant bacterial pathogens is alarming. Among these, Klebsiella pneumoniae is a leading cause of death by hospital-acquired infections, and its multidrug-resistant strains are flagged as a global threat to human health, which necessitates finding novel antibiotics or alternative therapies. One promising therapeutic alternative is the use of virulent bacteriophages, which specifically target bacteria and coevolve with them to overcome potential resistance. Here, we aimed to discover specific bacteriophages with therapeutic potential against multiresistant K. pneumoniae clinical isolates.Methods and ResultsOut of six bacteriophages that we isolated from urban and medical sewage, phage vB_Kpn_ZCKp20p had the broadest host range and was thus characterized in detail. Transmission electron microscopy suggests vB_Kpn_ZCKp20p to be a tailed phage of the siphoviral morphotype. In vitro evaluation indicated a high lytic efficiency (30 min latent period and burst size of ∼100 PFU/cell), and extended stability at temperatures up to 70°C and a wide range of (2-12) pH. Additionally, phage vB_Kpn_ZCKp20p possesses antibiofilm activity that was evaluated by the crystal violet assay and was not cytotoxic to human skin fibroblasts. The whole genome was sequenced and annotated, uncovering one tRNA gene and 33 genes encoding proteins with assigned functions out of 85 predicted genes. Furthermore, comparative genomics and phylogenetic analysis suggest that vB_Kpn_ZCKp20p most likely represents a new species, but belongs to the same genus as Klebsiella phages ZCKP8 and 6691. Comprehensive genomic and bioinformatics analyses substantiate the safety of the phage and its strictly lytic lifestyle.ConclusionPhage vB_Kpn_ZCKp20p is a novel phage with potential to be used against biofilm-forming K. pneumoniae and could be a promising source for antibacterial and antibiofilm products, which will be individually studied experimentally in future studies

Bacteriophages to control multi-drug resistant enterococcus faecalis infection of dental root canals

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. Phage therapy is an alternative treatment to antibiotics that can overcome multi-drug resistant bacteria. In this study, we aimed to isolate and characterize lytic bacteriophages targeted against Enterococcus faecalis isolated from root canal infections obtained from clinics at the Faculty of Dentistry, Ismalia, Egypt. Bacteriophage, vB_ZEFP, was isolated from concentrated wastewater collected from hospital sewage. Morphological and genomic analysis revealed that the phage belongs to the Podoviridae family with a linear double-stranded DNA genome, consisting of 18,454, with a G + C content of 32.8%. Host range analysis revealed the phage could infect 10 of 13 E. faecalis isolates exhibiting a range of antibiotic resistances recovered from infected root canals with efficiency of plating values above 0.5. One-step growth curves of this phage showed that it has a burst size of 110 PFU per infected cell, with a latent period of 10 min. The lytic activity of this phage against E. faecalis biofilms showed that the phage was able to control the growth of E. faecalis in vitro. Phage vB_ZEFP could also prevent ex-vivo E. faecalis root canal infection. These results suggest that phage vB_ZEFP has potential for application in phage therapy and specifically in the prevention of infection after root canal treatment

Encapsulation of E. coli phage ZCEC5 in chitosan-alginate beads as a delivery system in phage therapy

© 2019, The Author(s). Bacteriophages can be used successfully to treat pathogenic bacteria in the food chain including zoonotic pathogens that colonize the intestines of farm animals. However, harsh gastric conditions of low pH and digestive enzyme activities affect phage viability, and accordingly reduce their effectiveness. We report the development of a natural protective barrier suitable for oral administration to farm animals that confers acid stability before functional release of bead-encapsulated phages. Escherichia coli bacteriophage ZSEC5 is rendered inactive at pH 2.0 but encapsulation in chitosan–alginate bead with a honey and gelatin matrix limited titer reductions to 1log10PFUmL−1. The encapsulated phage titers were stable upon storage in water but achieved near complete release over 4–5h in a simulated intestinal solution (0.1% bile salt, 0.4% pancreatin, 50mM KH2PO4 pH 7.5) at 37°C. Exposure of E. coli O157:H7 to the bead-encapsulated phage preparations produced a delayed response, reaching a maximal reductions of 4.2 to 4.8log10CFUmL−1 after 10h at 37°C under simulated intestinal conditions compared to a maximal reduction of 5.1log10CFUmL−1 at 3h for free phage applied at MOI = 1. Bead-encapsulation is a promising reliable and cost-effective method for the functional delivery of bacteriophage targeting intestinal bacteria of farm animals

Phage-Encoded Endolysins

Due to the global emergence of antibiotic resistance, there has been an increase in research surrounding endolysins as an alternative therapeutic. Endolysins are phage-encoded enzymes, utilized by mature phage virions to hydrolyze the cell wall from within. There is significant evidence that proves the ability of endolysins to degrade the peptidoglycan externally without the assistance of phage. Thus, their incorporation in therapeutic strategies has opened new options for therapeutic application against bacterial infections in the human and veterinary sectors, as well as within the agricultural and biotechnology sectors. While endolysins show promising results within the laboratory, it is important to document their resistance, safety, and immunogenicity for in-vivo application. This review aims to provide new insights into the synergy between endolysins and antibiotics, as well as the formulation of endolysins. Thus, it provides crucial information for clinical trials involving endolysins