In contrast to planktonic cells, bacteria imbedded biofilms are notoriously refractory to treatment by antibiotics or bacteriophage (phage) used alone. Given that the mechanisms of killing differ ...profoundly between drugs and phages, an obvious question is whether killing is improved by combining antibiotic and phage therapy. However, this question has only recently begun to be explored. Here, in vitro biofilm populations of Pseudomonas aeruginosa PA14 were treated singly and with combinations of two phages and bactericidal antibiotics of five classes. By themselves, phages and drugs commonly had only modest effects in killing the bacteria. However some phage-drug combinations reduced bacterial densities to well below that of the best single treatment; in some cases, bacterial densities were reduced even below the level expected if both agents killed independently of each other (synergy). Furthermore, there was a profound order effect in some cases: treatment with phages before drugs achieved maximum killing. Combined treatment was particularly effective in killing in Pseudomonas biofilms grown on layers of cultured epithelial cells. Phages were also capable of limiting the extent to which minority populations of bacteria resistant to the treating antibiotic ascend. The potential of combined antibiotic and phage treatment of biofilm infections is discussed as a realistic way to evaluate and establish the use of bacteriophage for the treatment of humans.
The canonical view of phage - bacterial interactions in dense, liquid cultures is that the phage will eliminate most of the sensitive cells; genetic resistance will then ascend to restore high ...bacterial densities. Yet there are various mechanisms by which bacteria may remain sensitive to phages but still attain high densities in their presence - because bacteria enter a transient state of reduced adsorption. Importantly, these mechanisms may be cryptic and inapparent prior to the addition of phage yet result in a rapid rebound of bacterial density after phage are introduced. We describe mathematical models of these processes and suggest how different types of this 'phenotypic' resistance may be elucidated. We offer preliminary in vitro studies of a previously characterized E. coli model system and Campylobacter jejuni illustrating apparent phenotypic resistance. As phenotypic resistance may be specific to the receptors used by phages, awareness of its mechanisms may identify ways of improving the choice of phages for therapy. Phenotypic resistance can also explain several enigmas in the ecology of phage-bacterial dynamics. Phenotypic resistance does not preclude the evolution of genetic resistance and may often be an intermediate step to genetic resistance.
The maximum exponential growth rate, the Malthusian parameter (MP), is commonly used as a measure of fitness in experimental studies of adaptive evolution and of the effects of antibiotic resistance ...and other genes on the fitness of planktonic microbes. Thanks to automated, multi-well optical density plate readers and computers, with little hands-on effort investigators can readily obtain hundreds of estimates of MPs in less than a day. Here we compare estimates of the relative fitness of antibiotic susceptible and resistant strains of E. coli, Pseudomonas aeruginosa and Staphylococcus aureus based on MP data obtained with automated multi-well plate readers with the results from pairwise competition experiments. This leads us to question the reliability of estimates of MP obtained with these high throughput devices and the utility of these estimates of the maximum growth rates to detect fitness differences.
A total of 20 different strains were isolated, purified and screened for polyhydroxyalkanoate (PHA) production. PHA-producing strains were screened by Nile blue staining and confirmed by Sudan Black ...B staining. Strain 1.1 was selected for further analysis due to its high PHA production ability. PHA production was optimized and time profiling was calculated. PHA production on various different cheap carbon sources, i.e., sugar industry waste (fermented mash, molasses, spent wash) and corn oil, was compared. Cell dry weight and PHA content (%) were calculated and compared. The 12.53 g/L is the CDW of bacterial strain when grown in medium containing corn oil. It was found that corn oil at 12.53 g/L medium can serve as a carbon source for bacterial growth, allowing cells to accumulate PHA up to 35.63 %. The
Pha
C gene was amplified to confirm the genetic basis for the production of PHAs. Moreover, 16S rRNA gene sequence analysis showed that strain 1.1 belongs to
Pseudomonas
species.
Recently it has been recognized that bacteriophages, the natural predators of bacteria can be used efficiently in modern biotechnology. They have been proposed as alternatives to antibiotics for many ...antibiotic resistant bacterial strains. Phages can be used as biocontrol agents in agriculture and petroleum industry. Moreover phages are used as vehicles for vaccines both DNA and protein, for the detection of pathogenic bacterial strain, as display system for many proteins and antibodies. Bacteriophages are diverse group of viruses which are easily manipulated and therefore they have potential uses in biotechnology, research, and therapeutics. The aim of this review article is to enable the wide range of researchers, scientists, and biotechnologist who are putting phages into practice, to accelerate the progress and development in the field of biotechnology.
Aeromonas punctata is the causative agent of septicemia, diarrhea, wound infections, meningitis, peritonitis, and infections of the joints, bones and eyes. Bacteriophages are often considered ...alternative agents for controlling bacterial infection and contamination. In this study, we described the isolation and preliminary characterization of bacteriophage IHQ1 (family Myoviridae) active against the Gram-negative bacterial strain A. punctata. This virulent bacteriophage was isolated from stream water sample. Genome analysis indicated that phage IHQ1 was a double-stranded DNA virus with an approximate genome size of 25-28 kb. The initial characterization of this newly isolated phage showed that it has a narrow host range and infects only A. punctata as it failed to infect seven other clinically isolated pathogenic strains, i.e., methicillin-resistant Staphylococcus aureus 6403, MRS A 17644, Acinetobacter 33408, Acinetobacter 1172, Pseudomonas aeruginosa 22250, P. aeruginosa 11219, and Escherichia coli. Proteomic pattern of phage IHQ1, generated by SDS-PAGE using purified phage particles, showed three major and three minor protein bands with molecular weights ranging from 25 to 70 kDa. The adsorption rate of phage IHQ1 to the host bacterium was also determined, which was significantly enhanced by the addition of 10 mM CaCl₂. From the single-step growth experiment, it was inferred that the latent time period of phage IHQ1 was 24 min and a burst size of 626 phages per cell. Moreover, the pH and thermal stability of phage IHQ1 were also investigated. The maximum stability of the phage was observed at optimal pH 7.0, and it was totally unstable at extreme acidic pH 3; however, it was comparatively stable at alkaline pH 11.0. At 37°C the phage showed maximum number of plaques, and the viability was almost 100%. The existence of Aeromonas bacteriophage is very promising for the eradication of this opportunistic pathogen and also for future applications such as the design of new detection and phage typing (diagnosis) methods. The specificity of the bacteriophage for A. punctata makes it an attractive candidate for phage therapy of A. punctata infections.
Shigella dysenteriae is a normal inhabitant of the human gastrointestinal tract, but sometimes it causes severe infection known as shigellosis (bacillary dysentery). Bacteriophages are considered ...very safe and effective agents for controlling bacterial infections and contaminations. In this study, we describe the isolation and characterization of bacteriophage WZ1, isolated from waste water which inhibits the growth of S. dysenteriae. Phage WZ1 showed maximum stability at 37 °C and was stable up to 65 °C but was totally inactive at 70 °C. The pH stability increased from low to high and was totally inactive at pH 3 while maximum stability was observed at optimal pH 7. Phage WZ1 adsorption rate to the host bacterium was significantly enhanced by the addition of CaCl2. It has a latent time and burst time of 24 min and about 430 virions/cell, respectively. Transmission electron microscopy of phage WZ1 revealed a head width of 10 ± 0.5 nm and length of 10 ± 0.2 nm with a contractile tail of 128 ± 25 nm long and 21 ± 0.5 nm wide and belongs to family Myoviridae of order Caudovirales. Twelve structural proteins ranging from 22 to 150 kDa were detected by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS‐PAGE). The genome was found to be double stranded DNA with an approximate size of 38 kb. It has a very good reduction potential for S. dysenteriae by lowering abruptly the optical density of the planktonic S. dysenteriae culture. Phage WZ1 is a very promising candidate for phage therapy and other applications such as phage typing.
Citrobacter freundii is a worldwide emerging nosocomial pathogen with escalating incidence of multidrug resistance. Citrobacter freundii exists in natural environment, especially in health care ...settings and is difficult to eradicate. Phage therapy is considered as an alternative way of controlling bacterial infections and contaminations. In this study, we have described isolation and characterization of a virulent bacteriophage LK1 capable of specifically infecting Citrobacter freundii. A virulent bacteriophage LK1, specific for Citrobacter freundii was isolated from sewage water sample. TEM showed that phage Lk1 has an icosahedral head 70 nm in diameter and short tail of 17 nm, and can be classified as a member of the Podoviridae family. Restriction analysis indicated that phage LK1 was a dsDNA virus with an approximate genome size of 20–23 kb. Proteomic pattern generated by SDS PAGE using purified LK1 phage particles, revealed three major and six minor protein bands with molecular weight ranging from 25 to 80 kDa. Adsorption rate of LK1 relative to the host bacterium was also determined which showed significant improvement in adsorption with the addition of CaCl2. In a single step growth experiment, LK1 exhibited a latent period of 24 min and burst size of 801 particle/cell. Moreover, pH and thermal stability of phage LK1 demonstrated a pH range of 5.0–6.0 and phage viability decreased to 0% at 65 °C. When LK1 was used to infect six other clinically isolated pathogenic strains, it showed relatively narrow host range. LK1 was capable of eliciting efficient lysis of Citrobacter freundii, revealing its potential as a non‐toxic sanitizer for controlling Citrobacter freundii infection and contamination in both hospital and other public environments.
Diabetic foot infections (DFIs) constitute a major complication of diabetes mellitus. DFIs contribute to the development of gangrene and non-traumatic lower extremity amputations with a lifetime risk ...of up to 25%. The aim of the present study was to identify the presence of neuropathy and determine the ulcer grade, microbial profile and phenotypic and genotypic prevalence of the methicillin-resistance gene mecA and extended spectrum β-lactamase (ESBL)-encoding genes in bacterial isolates of DFI in patients registered at the Pakistan Institute of Medical Sciences (Islamabad, Pakistan). The results indicated that 46/50 patients (92%), exhibited sensory neuropathy. The most common isolate was Staphylococcus aureus (25%), followed by Pseudomonas aeruginosa (P. aeruginosa; 18.18%), Escherichia coli (16.16%), Streptococcus species (spp.) (15.15%), Proteus spp. (15.15%), Enterococcus spp. (9%) and Klebsiella pneumoniae (K. pneumoniae; 3%). The prevalence of the mecA gene was found to be 88% phenotypically and 84% genotypically. K. pneumoniae was shown to have the highest percentage of ESBL producers with a prevalence of 66.7% by double disk synergy test, and 100% by the cefotaxime + clavulanic acid/ceftazidime + clavulanic acid combination disk test. P. aeruginosa and K. pneumoniae had the highest (100%) proportion of metallo β-lactamase producers as identified by the EDTA combination disk test. The overall prevalence of β-lactamase (bla)-CTX-M, bla-CTX-M-15, bla-TEM, bla-OXA and bla-SHV genes was found to be 76.9, 76.9, 75.0, 57.7 and 84.6%, respectively, in gram-negative DFI isolates. The prevalence of mecA and ESBL-related genes was found to be alarmingly high in DFIs, since these genes are a major cause of antibiotic treatment failure.
Background: In Pakistan, most patients with Hepatitis B and C have history of facial and armpit shaving from barbers. Objective: Evaluate the awareness and risk factors associated with barbers in ...transmission of Hepatitis B and C. Subjects and methods: A cross sectional survey was conducted in Rawalpindi and Islamabad, the twin capital cities of Pakistan between January and July 2009. Five hundred and eight barbershops were surveyed. Results: Out of 508 barber shops, 99.8% and 98.2% were washing their razor with water and water plus antiseptic solution, respectively, while 99.8% were using new blades. Only 39.6% knew that hepatitis B virus (HBV) and hepatitis C virus (HCV) were viral diseases, 26.6% knew that it can lead to cancer, 90.7% thought that hepatitis could be transferred by blade sharing, 47.8% knew that a vaccine for HBV was available, and 43.0% had education beyond the primary level. None of the barbers used a new or washed apron/towel on every customer. Conclusion: In Pakistan, a small number of barbers are shaving clients with an old style razor with a permanent blade. There is moderate awareness about the various modes of transmission of hepatitis among the barbers, and most of them don’t know about vaccination. A predominant number of them are considering interferon treatment as a vaccine for hepatitis B and C.
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