The role of oxygen limitation in protecting Pseudomonas aeruginosa strains growing in biofilms from killing by antibiotics was investigated in vitro. Bacteria in mature (48-h-old) colony biofilms were poorly killed when they were exposed to tobramycin, ciprofloxacin, carbenicillin, ceftazidime, chloramphenicol, or tetracycline for 12 h. It was shown with oxygen microelectrodes that these biofilms contain large anoxic regions. Oxygen penetrated about 50 μm into the biofilms, which averaged 210 μm thick. The region of active protein synthesis was visualized by using an inducible green fluorescent protein. This zone was also limited to a narrow band , approximately 30 μm wide, adjacent to the air interface of the biofilm. The bacteria in mature biofilms exhibited a specific growth rate of only 0.02 h ⁻¹ . These results show that 48-h-old colony biofilms are physiologically heterogeneous and that most of the cells in the biofilm occupy an oxygen-limited, stationary-phase state. In contrast, bacteria in 4-h-old colony biofilms were still growing, active, and susceptible to antibiotics when they were challenged in air. When 4-h-old colony biofilms were challenged under anaerobic conditions, the level of killing by antibiotics was reduced compared to that for the controls grown aerobically. Oxygen limitation could explain 70% or more of the protection afforded to 48-h-old colony biofilms for all antibiotics tested. Nitrate amendment stimulated the growth of untreated control P. aeruginosa isolates grown under anaerobic conditions but decreased the susceptibilities of the organisms to antibiotics. Local oxygen limitation and the presence of nitrate may contribute to the reduced susceptibilities of P. aeruginosa biofilms causing infections in vivo.

The present study describes a bacteriophage (M ^Sa ) active against Staphylococcus aureus , including methicillin-resistant staphylococcal strains. When inoculated into mice simultaneously with S. aureus A170 (10 ⁸ CFU/mouse), phage (10 ⁹ PFU) rescued 97% of the mice; when applied to nonlethal (5 × 10 ⁶ CFU/mouse) 10-day infections, the phage also fully cleared the bacteria. The phage M ^Sa , delivered inside macrophages by S. aureus , kills the intracellular staphylococci in vivo and in vitro. The phage can also prevent abscess formation and reduce the bacterial load and weight of abscesses. These results suggest a potential use of the phage for the control of both local and systemic human S. aureus infections.

Microbial contamination in food processing plants can play a fundamental role in food quality and safety. The aims of this study were to learn more about the possible influence of the meat processing environment on initial fresh meat contamination and to investigate the differences between small-scale retail distribution (SD) and large-scale retail distribution (LD) facilities. Samples were collected from butcheries (n= 20), including LD (n= 10) and SD (n= 10) facilities, over two sampling campaigns. Samples included fresh beef and pork cuts and swab samples from the knife, the chopping board, and the butcher's hand. The microbiota of both meat samples and environmental swabs were very complex, including more than 800 operational taxonomic units (OTUs) collapsed at the species level. The 16S rRNA sequencing analysis showed that core microbiota were shared by 80% of the samples and includedPseudomonasspp.,Streptococcusspp.,Brochothrixspp.,Psychrobacterspp., andAcinetobacterspp. Hierarchical clustering of the samples based on the microbiota showed a certain separation between meat and environmental samples, with higher levels ofProteobacteriain meat. In particular, levels ofPseudomonasand severalEnterobacteriaceaemembers were significantly higher in meat samples, whileBrochothrix,Staphylococcus, lactic acid bacteria, andPsychrobacterprevailed in environmental swab samples. Consistent clustering was also observed when metabolic activities were considered by predictive metagenomic analysis of the samples. An increase in carbohydrate metabolism was predicted for the environmental swabs and was consistently linked toFirmicutes, while increases in pathways related to amino acid and lipid metabolism were predicted for the meat samples and were positively correlated withProteobacteria. Our results highlighted the importance of the processing environment in contributing to the initial microbial levels of meat and clearly showed that the type of retail facility (LD or SD) did not apparently affect the contamination.IMPORTANCEThe study provides an in-depth description of the microbiota of meat and meat processing environments. It highlights the importance of the environment as a contamination source of spoilage bacteria, and it shows that the size of the retail facility does not affect the level and type of contamination.

Arginine enhanced the killing of Pseudomonas aeruginosa by ciprofloxacin and tobramycin under anaerobic, but not aerobic, growth conditions. Arginine or nitrate also enhanced the killing by these antibiotics in mature biofilms, reducing viable cell counts by a factor of 10 to 100 beyond that achieved by antibiotics alone.

The development of a simple and low cost electrochemical impedance immunosensor based on screen printed gold electrode for rapid detection of Escherichia coli in water is reported. The immunosensor is fabricated by immobilizing anti-E. coli antibodies onto a gold surface in a covalent way by the photochemical immobilization technique, a simple procedure able to bind antibodies upright onto gold surfaces. Impedance spectra are recorded in 0.01 M phosphate buffer solution (PBS) containing 10 mM Fe(CN)₆³⁻/Fe(CN)₆⁴⁻ as redox probe. The Nyquist plots can be modelled with a modified Randles circuit, identifying the charge transfer resistance R_ct as the relevant parameter after the immobilization of antibodies, the blocking with BSA and the binding of E. coli. The introduction of a standard amplification procedure leads to a significant enhancement of the impedance increase, which allows one to measure E. coli in drinking water with a limit of detection of 3 × 10¹ CFU mL⁻¹ while preserving the rapidity of the method that requires only 1 h to provide a “yes/no” response. Additionally, by applying the Langmuir adsorption model, we are able to describe the change of R_ct in terms of the “effective” electrode, which is modified by the detection of the analyte whose microscopic conducting properties can be quantified.

Brucellosis is a costly disease of water buffaloes (Bubalus bubalis). Latent infections and prolonged incubation of the pathogen limit the efficacy of programs based on the eradication of infected animals. We exploited genetic selection for disease resistance as an approach to the control of water buffalo brucellosis. We tested 231 water buffalo cows for the presence of anti-Brucella abortusantibodies (by the agglutination and complement fixation tests) and theNramp1genotype (by PCR-denaturing gradient gel electrophoresis). When the 231 animals (58 cases and 173 controls) were divided into infected (seropositive) and noninfected (seronegative) groups and theNramp1genotypes were compared, the seropositive subjects were 52 out of 167 (31%) in theNramp1A⁺(Nramp1AAorNramp1AB) group and 6 out of 64 (9.4%) in theNramp1A⁻(Nramp1BB) group (odds ratio, 4.37; 95% confidence limits, 1.87 to 10.19; χ², 11.65 for 1 degree of freedom). Monocytes fromNramp1BBsubjects displayed significantly (P< 0.01) higher levels ofNramp1mRNA thanNramp1AAsubjects and also a significantly (P< 0.01) higher ability in controlling the intracellular replication of severalBrucellaspecies in vitro. Thus, selection for theNramp1BBgenotype can become a valuable tool for the control of water buffalo brucellosis in the areas where the disease is endemic.