Domenico Iannelli

Experimental Phage Therapy against Staphylococcus aureus in Mice

ABSTRACT The present study describes a bacteriophage (MSa) active against Staphylococcus aureus, including methicillin-resistant staphylococcal strains. When inoculated into mice simultaneously with S. aureus A170 (108 CFU/mouse), phage (109 PFU) rescued 97% of the mice; when applied to nonlethal (5 × 106 CFU/mouse) 10-day infections, the phage also fully cleared the bacteria. The phage MSa, 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.

Bacteriophage Therapy of Salmonella enterica: A Fresh Appraisal of Bacteriophage Therapy

BACKGROUND The most serious criticisms leveled at bacteriophage therapy are as follows: phages induce neutralizing antibodies, phages are active only when administered shortly after bacterial infection, and phage-resistant bacteria emerge rapidly in the course of therapy. METHODS Phages lytic for several Salmonella enterica serovars were isolated by means of standard protocols from feces of patients with gastroenteritis. Growth of S. enterica serovar Paratyphi B (Salp572(phi1S)) in the presence of phage phi1 (selected from among 8 phages for its larger host range) provided a phage phi1-resistant bacterial strain (Salp572(phi1R)). The properties of the Salp572(phi1S) and Salp572(phi1R) strains and of phage phi1 were studied in a mouse model of experimental infection. RESULTS Phages induced nonneutralizing antibodies and were active 2 weeks after experimental infection of mice; phage-resistant bacteria were avirulent and short lived in vivo. More importantly, phage-resistant bacteria were excellent vaccines, protecting against lethal doses of heterologous S. enterica serovars. CONCLUSIONS Phage therapy effectiveness has not yet been properly assessed.

Genetic Resistance to Brucella abortus in the Water Buffalo (Bubalus bubalis)

ABSTRACT 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; χ2, 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.

Bacteriophage-Resistant Staphylococcus aureus Mutant Confers Broad Immunity against Staphylococcal Infection in Mice

In the presence of a bacteriophage (a bacteria-attacking virus) resistance is clearly beneficial to the bacteria. As expected in such conditions, resistant bacteria emerge rapidly. However, in the absence of the phage, resistant bacteria often display reduced fitness, compared to their sensitive counterparts. The present study explored the fitness cost associated with phage-resistance as an opportunity to isolate an attenuated strain of S. aureus. The phage-resistant strain A172 was isolated from the phage-sensitive strain A170 in the presence of the MSa phage. Acquisition of phage-resistance altered several properties of A172, causing reduced growth rate, under-expression of numerous genes and production of capsular polysaccharide. In vivo, A172 modulated the transcription of the TNF-α, IFN-γ and Il-1β genes and, given intramuscularly, protected mice from a lethal dose of A170 (18/20). The heat-killed vaccine also afforded protection from heterologous methicillin-resistant S. aureus (MRSA) (8/10 mice) or vancomycin-intermediate S. aureus (VISA) (9/10 mice). The same vaccine was also effective when administered as an aerosol. Anti-A172 mouse antibodies, in the dose of 10 µl/mouse, protected the animals (10/10, in two independent experiments) from a lethal dose of A170. Consisting predominantly of the sugars glucose and galactose, the capsular polysaccharide of A172, given in the dose of 25 µg/mouse, also protected the mice (20/20) from a lethal dose of A170. The above results demonstrate that selection for phage-resistance can facilitate bacterial vaccine preparation.

Protective Effect of the Nramp1 BB Genotype against Brucella abortus in the Water Buffalo (Bubalus bubalis)

ABSTRACT 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.

In Vitro Activity against Staphylococcus aureus of a Novel Antimicrobial Agent, PRF-119, a Recombinant Chimeric Bacteriophage Endolysin

ABSTRACT Antistaphylococcal activity of the novel chimeric endolysin PRF-119 was evaluated with the microdilution method. The MIC50 and MIC90 of 398 methicillin-susceptible Staphylococcus aureus isolates were 0.098 μg/ml and 0.391 μg/ml, respectively (range, 0.024 to 0.780 μg/ml). Both the MIC50 and MIC90 values of 776 methicillin-resistant S. aureus isolates were 0.391 μg/ml (range, 0.024 to 1.563 μg/ml). All 192 clinical isolates of coagulase-negative staphylococci exhibited MIC values of >50 μg/ml. In conclusion, PRF-119 exhibited very good activity specifically against S. aureus.

Role Played by Human Mannose-Binding Lectin Polymorphisms in Pulmonary Tuberculosis

BACKGROUND Mannose-binding lectin (MBL) activates the complement system in an antibody-independent manner, enhances complement-mediated phagocytosis, and plays a major role in the regulation of inflammatory cytokine release by monocytes. METHODS Case patients (277 patients with pulmonary tuberculosis) and control subjects (288 household contacts) were tested by polymerase chain reaction (PCR) for polymorphisms at the promoter and the exon 1 regions of the MBL gene. Diagnosis of pulmonary tuberculosis, based on findings from chest radiography and sputum smear examination, was confirmed by PCR and bacteriological tests. RESULTS HYA/HYA subjects were protected against tuberculosis (odds ratio [OR], 0.09 [95% confidence interval {CI}], 0.023-0.408; P < 1 X 10 (-6)). LYB/LYD subjects were susceptible to disease (OR, 49 [95% CI, 2.9-812.5]; P < 1 X 10(-6)). CONCLUSIONS This study supports the conclusion that MBL can protect or predispose the host to tuberculosis, depending on the hosts haplotype pair.

Synergistic antibacterial and anti-inflammatory activity of temporin A and modified temporin B in vivo.

Temporins are antimicrobial peptides secreted by the granular glands of the European red frog (Rana temporaria). They are 10–14 amino acid long polypeptides active prevalently against gram positive bacteria. This study shows that a synthetic temporin B analogue (TB-YK), acquires the capacity to act in synergism with temporin A and to exert antimicrobial and anti-inflammatory activity in vivo against gram positive and gram negative bacteria. Administration of 3.4 mg/Kg of temporin A (TA)+1.6 mg/Kg TB-YK, given to individual mice concurrently with a lethal dose of bacteria (gram positive or negative), rescued 100% of the animals. More importantly, the same doses of temporins, administered one week after experimental infection with a sub lethal dose of bacteria, sterilized 100% of the animals within 3–6 days. Also, it is described an animal model based on the use of sub lethal doses of bacteria, which closely mimics bacterial infection in humans. The model offers the possibility to test in a preclinical setting the true potential of TA and TB-YK in combination as antimicrobial and anti-inflammatory agents.

Mannose-binding lectin haplotypes influence Brucella abortus infection in the water buffalo (Bubalus bubalis)

A case-control study established that the haplotype pair HYA/HYA at the MBL (mannose binding lectin) locus of water buffalo is associated with resistance to Brucella abortus infection (P < 10−7) and the haplotype pairs LYD/LYD with susceptibility to the same pathogen (P < 10−7). The subjects included in the present study were tested twice—at a 1-month interval—for the presence of anti-B. abortus antibodies in the serum by agglutination, complement fixation and flow cytometry. Cases (335 subjects) included animals consistently positive to all these tests; controls (335 subjects) comprised animals exposed yet negative by the same tests. The serum from genetically resistant subjects displayed in vitro significantly higher antibacterial activity compared to the serum from genetically susceptible subjects, lending biological significance to the results from the association study. Inhibition of the antibacterial activity following heat treatment of the serum, addition of specific MBL inhibitors (EDTA, mannose, N-acetyl-d-glucosamine) or anti-human MBL antiserum provide convincing evidence that the antibacterial activity present in the serum results from the interaction between MBL and B. abortus. A replication study (comprising 100 cases and 100 controls) confirmed the results from the original study.

Simultaneous detection of cucumber mosaic virus, tomato mosaic virus and potato virus Y by flow cytometry.

The simultaneous detection is described of cucumber mosaic virus (CMV), potato virus Y (PVY) and tomato mosaic virus (ToMV) by flow cytometry. Extracts from leaves of healthy and CMV or PVY infected plants were incubated with latex particles, each with a diameter of 3 microm. Extracts from ToMV infected or uninfected plants, however, were incubated with particles, each with a diameter of 6 microm. Beads were washed and incubated in succession with primary and secondary antibodies, the latter labeled with phycoerythrin (PE) or fluorescein (FITC). CMV and PVY were distinguished on the basis of the fluorescence emitted by FITC and PE; ToMV was distinguished from CMV and PVY on the basis of the different diameter (6 microm) of the particles on which it was adsorbed. The three viruses were detected also by another approach. Latex particles with a diameter of 3, 6 and 10 microm were separately sensitized with antibodies specific for CMV, PVY and ToMV. An equal number of sensitized particles was mixed and incubated with the plant extracts containing the three viruses and then with anti-CMV, anti-PVY and anti-ToMV antibodies labeled with FITC. The study describes also a virus purification method based on the use of antibody coated latex particles. The method is simple technically and applicable to the purification of large as well as minute amounts of different viruses (CMV, PVY and ToMV).