The microbial composition in coastal water of the Port Dickson beach in Negeri Sembilan, Malaysia was analyzed using several microbial indicators for the purpose of selecting the best indicator for marine water pollution. The indicators studied were total coliform (TC), fecal coliform (FC), fecal streptococci (FS) and coliphage. Five locations were selected along the Port Dickson beaches and samplings were carried out in 1998 and 2001. The results showed an increase in the number of total coliform (TC), fecal coliform (FC) and fecal streptococci (FS) between these two sampling by 98.12%, 86.12% and 99%, respectively. The numbers of TC, FC and FS exceeded the recommended limit for recreational seawater based on U.S. EPA 1986 standard. There was a positive correlation between TC, FC and FS and negative to coliphages.
Bacteriophage EC1-UPM is an N4-like bacteriophage which specifically infects Escherichia coli O78:K80, an avian pathogenic strain that causes colibacillosis in poultry. The complete genome sequence of bacteriophage EC1-UPM was analysed and compared with other closely related N4-like phage groups to assess their genetic similarities and differences.
Male-specific RNA coliphages (FRNA) have been recommended as indicators of fecal contamination and of the virological quality of water. In this study, 16 river water and 183 animal fecal samples were examined for the presence of FRNA coliphages by a plaque assay using Salmonella typhimurium WG49 and WG25 to differentiate between male-specific and somatic phages, a RNase spot test to differentiate between DNA and RNA phages and a reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific identification of FRNA phages. The overall recovery rate for F-specific coliphages was 8.0%. (4.4% from animal fecal matter and 50% from river water samples). Plaque counts were generally low (< 6 x 10(2) pfu per g feces or ml water), with FRNA (6.5%) and Male-specific DNA coliphages (FDNA) (7.0%) phages occurring at almost equal frequencies. The RT-PCR was positive in all FRNA plaques and was able to identify FRNA phages in mixed populations of FRNA, FDNA and somatic phages.
Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.
Disease inflicted by avian pathogenic Escherichia coli (APEC) causes economic losses and burden to the poultry industry worldwide. In this study, the efficacy of chitosan nanoparticles loaded ΦKAZ14 (C-ΦKAZ14 NPs) as an oral biological therapy for Colibacillosis was evaluated. C-ΦKAZ14 NPs containing 10(7) PFU/ml of ΦKAZ14 (Myoviridae; T4-like coliphage) bacteriophage were used to treat experimentally APEC-infected COBB 500 broiler chicks. C-ΦKAZ14 NPs and ΦKAZ14 bacteriophage were administered orally in a single dose. The clinical symptoms, mortality, and pathology in the infected birds were recorded and compared with those of control birds that did not receive C-ΦKAZ14 NPs or naked ΦKAZ14 bacteriophage. The results showed that C-ΦKAZ14 NP intervention decreased mortality from 58.33 to 16.7% with an increase in the protection rate from 42.00 to 83.33%. The bacterial colonization of the intestines of infected birds was significantly higher in the untreated control than in the C-ΦKAZ14 NP-treated group (2.30×10(9) ± 0.02 and 0.79×10(3) ± 0.10 CFU/mL, respectively) (P ≤ 0.05). Similarly, a significant difference in the fecal shedding of Escherichia coli was observed on d 7 post challenge between the untreated control and the C-ΦKAZ14 NP-treated group (2.35×10(9) ± 0.05 and 1.58×10(3) ± 0.06 CFU/mL, respectively) (P ≤ 0.05). Similar trends were observed from d 14 until d 21 when the experiment was terminated. Treatment with C-ΦKAZ14 NPs improved the body weights of the infected chicks. A difference in body weight on d 7 post challenge was observed between the untreated control and the C-ΦKAZ14 NP-treated group (140 ± 20 g and 160 ± 20 g, respectively). The increase was significant (P ≤ 0.05) on d 21 between the 2 groups (240 ± 30 g and 600 ± 80 g, respectively). Consequently, the clinical signs and symptoms were ameliorated upon treatment with C-ΦKAZ14 NPs compared with infected untreated birds. In all, based on the results, it can be concluded that the encapsulation of bacteriophage could enhance bacteriophage therapy and is a valuable approach for controlling APEC infections in poultry.
The use of chitosan as a delivery carrier has attracted much attention in recent years. In this study, chitosan nanoparticles (CS-NP) and chitosan-ΦKAZ14 bacteriophage-loaded nanoparticles (C-ΦKAZ14 NP) were prepared by a simple coercavation method and characterized. The objective was to achieve an effective protection of bacteriophage from gastric acids and enzymes in the chicken gastrointestinal tract. The average particle sizes for CS-NP and C-ΦKAZ14 NP were 188 ± 7.4 and 176 ± 3.2 nm, respectively. The zeta potentials for CS-NP and C-ΦKAZ14 NP were 50 and 60 mV, respectively. Differential scanning calorimetry (DSC) of C-ΦKAZ14 NP gave an onset temperature of -17.17 °C with a peak at 17.32 °C and final end set of 17.41 °C, while blank chitosan NP had an onset of -20.00 °C with a peak at -19.78 °C and final end set at -20.47. FT-IR spectroscopy data of both CS-NP and C-ΦKAZ14 NP were the same. Chitosan nanoparticles showed considerable protection of ΦKAZ14 bacteriophage against degradation by enzymes as evidenced in gel electrophoresis, whereby ΦKAZ14 bacteriophage encapsulated in chitosan nanoparticles were protected whereas the naked ΦKAZ14 bacteriophage were degraded. C-ΦKAZ14 NP was non-toxic as shown by a chorioallantoic membrane (CAM) toxicity assay. It was concluded that chitosan nanoparticles could be a potent carrier of ΦKAZ14 bacteriophage for oral therapy against colibacillosis in poultry.
Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes (mprA and lrhA) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production.IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival and persistence during infection. The K1 serotype is one example of a polysaccharide capsule type and is strongly associated with UPEC strains that cause UTIs, bloodstream infections, and meningitis. The number of UTIs caused by antibiotic-resistant UPEC is steadily increasing, highlighting the need to better understand factors (e.g., the capsule) that contribute to UPEC pathogenesis. This study describes the original and novel application of lytic capsule-specific phage killing, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing to define the entire complement of genes required for capsule production in UPEC. Our comprehensive approach uncovered new genes involved in the regulation of this key virulence determinant.
To explore new approaches of phage-based bio-process of specifically pathogenic Escherichia coli bacteria in food products within a short period. One hundred and forty highly lytic designed coliphages were used. Escherichia coli naturally contaminated and Enterohemorrhagic Escherichia coli experimentally inoculated samples of lettuce, cabbage, meat, and egg were used. In addition, experimentally produced biofilms of E. coli were tested. A phage concentration of 10(3) PFU/ml was used for food products immersion, and for spraying of food products, 10(5) PFU/ml of a phage cocktail was used by applying a 20-s optimal dipping time in a phage cocktail. Food samples were cut into pieces and were either sprayed with or held in a bag immersed in lambda buffer containing a cocktail of 140 phages. Phage bio-processing was successful in eliminating completely E. coli in all processed samples after 48 h storage at 4°C. Partial elimination of E. coli was observed in earlier storage periods (7 and 18 h) at 24° and 37°C. Moreover, E. coli biofilms were reduced >3 log cycles upon using the current phage bio-processing. The use of a phage cocktail of 140 highly lytic designed phages proved highly effective in suppressing E. coli contaminating food products. Proper decontamination/prevention methods of pathogenic E. coli achieved in this study can replace the current chemically less effective decontamination methods.