Summary Extensive research has been conducted on the development of three groups of naturally occurring antimicrobials as novel alternatives to antibiotics: bacteriophages (phages), bacterial cell wall hydrolases (BCWH), and antimicrobial peptides (AMP). Phage therapies are highly efficient, highly specific, and relatively cost‐effective. However, precautions have to be taken in the selection of phage candidates for therapeutic applications as some phages may encode toxins and others may, when integrated into host bacterial genome and converted to prophages in a lysogenic cycle, lead to bacterial immunity and altered virulence. BCWH are divided into three groups: lysozymes, autolysins, and virolysins. Among them, virolysins are the most promising candidates as they are highly specific and have the capability to rapidly lyse antibiotic‐resistant bacteria on a generally species‐specific basis. Finally, AMP are a family of natural proteins produced by eukaryotic and prokaryotic organisms or encoded by phages. AMP are of vast diversity in term of size, structure, mode of action, and specificity and have a high potential for clinical therapeutic applications.

A novel method is presented for the specific and direct detection of bacteria using bacteriophages as recognition receptors immobilized covalently onto functionalized screen-printed carbon electrode (SPE) microarrays. The SPE networks were functionalized through electrochemical oxidation in acidic media of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) by applying a potential of +2.2 V to the working electrode. Immobilization of T4 bacteriophage onto the SPEs was achieved via EDC by formation of amide bonds between the protein coating of the phage and the electrochemically generated carboxylic groups at the carbon surface. The surface functionalization with EDC, and the binding of phages, was verified by time-of-flight secondary ion mass spectrometry. The immobilized T4 phages were then used to specifically detect E. coli bacteria. The presence of surface-bound bacteria was verified by scanning electron and fluorescence microscopies. Impedance measurements (Nyquist plots) show shifts of the order of 10(4) Omega due to the binding of E. coli bacteria to the T4 phages. No significant change in impedance was observed for control experiments using immobilized T4 phage in the presence of Salmonella. Impedance variations as a function of incubation time show a maximum shift after 20 min, indicating onset of lysis, as also confirmed by fluorescence microscopy. Concentration-response curves yield a detection limit of 10(4) cfu/mL for 50-microL samples.

The human immunodeficiency virus type 1 (HIV-1) vpu gene product is a class I integral membrane phosphoprotein that is capable of oligomerization. Two distinct biological activities have been attributed to Vpu: induction of CD4 degradation in the endoplasmic reticulum and enhancement of viral particle release from the plasma membrane of infected cells. These two biological activities were shown to involve two separable structural domains: the N-terminal transmembrane (TM) domain and the C-terminal cytoplasmic domain. The TM domain mediates enhancement of viral particle release, whereas phosphorylation of the cytoplasmic domain is essential for Vpu-induced CD4 degradation. In this study, we performed a mutational analysis of the TM domain of Vpu to delineate amino acids that are important in the process of viral particle release or in Vpu-induced CD4 degradation. Substitution of conserved amino acids from the N-terminal, middle, or C-terminal parts of the native VpuTM domain generated proteins that integrated normally into canine pancreatic microsomal membranes, exhibited subcellular localization similar to those of wild-type Vpu, but partially lost their ability to enhance viral particle release, strongly suggesting that the VpuTM domain contains determinants responsible for Vpu-mediated enhancement of viral particle release. Interestingly, the C-terminal TM mutant VpuIVW, in contrast to the other mutants, also lost its ability to bind and consequently degrade the CD4 molecule, indicating that the alteration of the C-terminal part of the TM did interfere with this function of Vpu. Taken together, our study supports the notion that both structural elements of Vpu (TM and cytoplasmic) contribute to the biological activities of Vpu.

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The HIV-1-specific vpu gene encodes an integral membrane phosphoprotein which affects three aspects of the HIV-1 infectious cycle: it enhances virion release from infected cells; it causes degradation of the CD4 protein in the endoplasmic reticulum; and it delays syncytia formation in HIV-1-infected CD4+ T-cells. Although little is known about how Vpu mediates these effects, it has been proposed to function as a nonspecific cation channel. In this report, voltage clamp measurements of Xenopus oocytes show that Vpu expression is not associated with increased transmembrane currents. Instead, Vpu expression diminishes membrane conductance. Injection of 4.6 ng of Vpu mRNA into these cells reduces endogenous potassium conductance by 50%. Only Vpu mutants which retain the ability to degrade CD4 can diminish K+ conductance. Inhibition by Vpu is not unique to K+ channels as it is also observed on several coexpressed membrane proteins but not on a coexpressed cytoplasmic protein. These results indicate that the CD4 degradative capability of Vpu and the Vpu-mediated modulation of membrane protein expression are mechanistically coupled and that Vpu may contribute to HIV pathogenesis by altering plasma membrane protein expression at the cell surface.

A phage replication-competitive enzyme-linked immunosorbent assay (PR-cELISA) was developed for the detection of multiple antibiotic-resistant Salmonella Typhimurium DT104. In the PR-cELISA procedure, a phage, BP1, was inoculated into a log-phase bacterial culture at a ratio of 1:100. After a 3-h incubation of the mixture, BP1 replication was measured by cELISA based on the competitive binding between BP1 and biotinylated BP1 to Salmonella Typhimurium smooth lipopolysaccharide. Among the 84 Salmonella strains and 9 non-Salmonella strains that were tested by PR-cELISA, BP1 detected 39 of 40 Salmonella Typhimurium strains, 2 of 10 Salmonella non-Typhimurium somatic group B strains, and 5 of 18 Salmonella somatic group D1 strains. With the addition of chloramphenicol to the culture medium, PR-cELISA detected all 27 multiple antibiotic-resistant Salmonella Typhimurium DT104 and none of the other Salmonella strains or non-Salmonella strains tested. The results demonstrated that PR-cELISA has potential applications for the detection of multiple antibiotic-resistant Salmonella Typhimurium DT104.

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The growing interest in applications of bacteriophages creates a need for improvements in the production processes. Continuous monitoring of the phage production is an essential aspect of any control strategy and, at present, there is no completely satisfactory option. The approach presented here uses IR-spectrometry to continuously measure the rate of respiration (CO(2) released) of Escherichia coli infected by phage T4 at various multiplicities of infection (MOI). Within the trends in these data, or in other aspects of the rate of respiration, it was possible to reliably and reproducibly identify five features that reflected specific events in the infection process. These included two events in the host cell apparent growth rate and events in the magnitude of the host cell density, in the measurement of OD(600) or in the specific rate of respiration. All of these correlations were within 95% confidence showing that they are suitable for the monitoring and control of E. coli populations infected by phage T4. This method is reliable, cheap, and can be operated in-line and in real time.