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 m into the biofilms, which averaged 210 m 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 m 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.

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.

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 included Pseudomonas spp., Streptococcus spp., Brochothrix spp., Psychrobacter spp., and Acinetobacter spp. Hierarchical clustering of the samples based on the microbiota showed a certain separation between meat and environmental samples, with higher levels of Proteobacteria in meat. In particular, levels of Pseudomonas and several Enterobacteriaceae members were significantly higher in meat samples, while Brochothrix, Staphylococcus, lactic acid bacteria, and Psychrobacter prevailed 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 to Firmicutes, while increases in pathways related to amino acid and lipid metabolism were predicted for the meat samples and were positively correlated with Proteobacteria. 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. Meat is a complex niche with chemical and physical properties that allow the colonization and development of a variety of microorganisms, especially bacteria (1, 2). Several factors can influence the occurrence of microbes in meat. After slaughtering, meat can be contaminated by microorganisms from water, air, and soil, as well as from the workers and equipment involved in the processing. In the later processing steps of the fresh meat chain (i.e., handling, cutting, and storage), abiotic factors such as temperature, gaseous atmosphere, pH, and NaCl levels select for certain bacteria, allowing colonization of the meat surface by different spoilage-related species and...

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 abortus antibodies (by the agglutination and complement fixation tests) and the Nramp1 genotype (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 the Nramp1 genotypes were compared, the seropositive subjects were 52 out of 167 (31%) in the Nramp1A ؉ (Nramp1AA or Nramp1AB) group and 6 out of 64 (9.4%) in the Nramp1A ؊ (Nramp1BB) group (odds ratio, 4.37; 95% confidence limits, 1.87 to 10.19; 2 , 11.65 for 1 degree of freedom). Monocytes from Nramp1BB subjects displayed significantly (P < 0.01) higher levels of Nramp1 mRNA than Nramp1AA subjects and also a significantly (P < 0.01) higher ability in controlling the intracellular replication of several Brucella species in vitro. Thus, selection for the Nramp1BB genotype can become a valuable tool for the control of water buffalo brucellosis in the areas where the disease is endemic.

The development of fast and ultrasensitive methods to detect bacterial pathogens at low concentrations is of high relevance for human and animal health care and diagnostics. In this context, surface-enhanced Raman scattering (SERS) offers the promise of a simplified, rapid, and high-sensitive detection of biomolecular interactions with several advantages over previous assay methodologies. In this work, we have conceived reproducible SERS nanosensors based on tailored multilayer octupolar nanostructures which can combine high enhancement factor and remarkable molecular selectivity. We show that coating novel multilayer octupolar metastructures with proper self-assembled monolayer (SAM) and immobilized phages can provide label-free analysis of pathogenic bacteria via SERS leading to a giant increase in SERS enhancement. The strong relative intensity changes of about 2100% at the maximum scattered SERS wavelength, induced by the Brucella bacterium captured, demonstrate the performance advantages of the bacteriophage sensing scheme. We performed measurements at the single-cell level thus allowing fast identification in less than an hour without any demanding sample preparation process. Our results based on designing well-controlled octupolar coupling platforms open up new opportunities toward the use of bacteriophages as recognition elements for the creation of SERS-based multifunctional biochips for rapid culture and label-free detection of bacteria.

We tested 413 water buffalo cows (142 cases and 271 controls) for the presence of anti-Brucella abortus antibodies (by the skin test, the agglutination test, and the complement fixation test) and the Nramp1 genotype (by capillary electrophoresis). Four alleles (Nramp1A, -B, -C, and -D) were detected in the 3 untranslated region of the Nramp1 gene. The BB genotype was represented among only controls, providing evidence that this genotype confers resistance to Brucella abortus. The monocytes from the BB (resistant) subjects displayed a higher basal level of Nramp1 mRNA and a lower number of viable intracellular bacteria than did the monocytes from AA (susceptible) subjects. The higher basal level of the antibacterial protein Nramp1 most probably provides the BB animals with the possibility of controlling bacteria immediately after their entry inside the cell.

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 Rct 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 Rct in terms of the “effective” electrode, which is modified by the detection of the analyte whose microscopic conducting properties can be quantified.