Maria Cristina Thaller

Tracking acquired antibiotic resistance in commensal bacteria of Galápagos land iguanas: no man, no resistance.

Background Antibiotic resistance, evolving and spreading among bacterial pathogens, poses a serious threat to human health. Antibiotic use for clinical, veterinary and agricultural practices provides the major selective pressure for emergence and persistence of acquired resistance determinants. However, resistance has also been found in the absence of antibiotic exposure, such as in bacteria from wildlife, raising a question about the mechanisms of emergence and persistence of resistant strains under similar conditions, and the implications for resistance control strategies. Since previous studies yielded some contrasting results, possibly due to differences in the ecological landscapes of the studied wildlife, we further investigated this issue in wildlife from a remote setting of the Galapagos archipelago. Methodology/Principal Findings Screening for acquired antibiotic resistance was carried out in commensal enterobacteria from Conolophus pallidus, the terrestrial iguana of Isla Santa Fe, where: i) the abiotic conditions ensure to microbes good survival possibilities in the environment; ii) the animal density and their habits favour microbial circulation between individuals; and iii) there is no history of antibiotic exposure and the impact of humans and introduced animal species is minimal except for restricted areas. Results revealed that acquired antibiotic resistance traits were exceedingly rare among bacteria, occurring only as non-dominant strains from an area of minor human impact. Conclusions/Significance Where both the exposure to antibiotics and the anthropic pressure are minimal, acquired antibiotic resistance traits are not normally found in bacteria from wildlife, even if the ecological landscape is highly favourable to bacterial circulation among animals. Monitoring antibiotic resistance in wildlife from remote areas could also be a useful tool to evaluate the impact of anthropic pressure.

Proteolytic enzymes: a new treatment strategy for prosthetic infections?

Among the different mechanisms of bacterial resistance to antimicrobial agents that have been studied, biofilm formation is one of the most widespread. This mechanism is frequently the cause of failure in the treatment of prosthetic device infections, and several attempts have been made to develop molecules and protocols that are able to inhibit biofilm-embedded bacteria. We present data suggesting the possibility that proteolytic enzymes could significantly enhance the activities of antibiotics against biofilms. Antibiotic susceptibility tests on both planktonic and sessile cultures, studies on the dynamics of colonization of 10 biofilm-forming isolates, and then bioluminescence and scanning electron microscopy under seven different experimental conditions showed that serratiopeptidase greatly enhances the activity of ofloxacin on sessile cultures and can inhibit biofilm formation. Images

Enterococcus flavescens sp. nov., a New Species of Enterococci of Clinical Origin

Four yellow-pigmented group D enterococci of uncertain taxonomic position were isolated from several humans with severe infections. The results of DNA composition, DNA-DNA hybridization, fatty acid content, and biochemical property studies demonstrated that these organisms were slightly related to other previously described yellow-pigmented enterococcal species and constitute a new species, for which we propose the name Enterococcus flavescens. The type strain of E. flavescens is strain CCM 4239 [corrected].

Diagnosis of vascular graft infections with antibodies against staphylococcal slime antigens

Late-onset infections of synthetic vascular grafts (LO-SVGIs) are generally caused by staphylococci that produce a slime polysaccharide and grow as a biofilm on the graft surface. We developed an ELISA to detect serum antibodies against staphylococcal slime polysaccharide antigens (SSPA). Patients with an ongoing staphylococcal LO-SVGI had greater titres of IgM antibodies against SSPA than did patients in other groups. Antibody titres of 0.40 ELISA units (EU) or more, or 0.35 EU or more detected 97% and 100% of staphylococcal LO-SVGIs, respectively, 0% and 2% titre/unit false-positive results. Our findings suggest that such an ELISA represents a sensitive, specific, and non-invasive diagnostic test for staphylococcal LO-SVGIs.

Biochemical Characterization of the THIN-B Metallo-β-Lactamase of Janthinobacterium lividum

ABSTRACT The THIN-B metallo-β-lactamase, a subclass B3 enzyme produced by the environmental species Janthinobacterium lividum, was overproduced in Escherichia coli by means of a T7-based expression system. The enzyme was purified (>95%) by two ion-exchange chromatography steps and subjected to biochemical analysis. The native THIN-B enzyme is a monomeric protein of 31 kDa. It exhibits the highest catalytic efficiencies with carbapenem substrates and cephalosporins, except for cephaloridine, which acts as a poor inactivator. Individual rate constants for inactivation by chelators were measured, suggesting that inactivation occurred by a mechanism involving formation of a ternary complex.

Analysis of Bacteriolytic Activity Patterns, a Novel Approach to the Taxonomy of Enterococci

The bacteriolytic activities of different group D streptococcal species on various media and substrates were studied. Our results showed that all of the enterococcal species which we tested had bacteriolytic activity on at least one of the media used, while the group D nonenterococcal species had no such activity. In addition, using culture media containing different additives and different pH values, we defined seven major groups of bacteriolytic activity (lyogroups), each of which overlapped with one species (four lyogroups), two species (two lyogroups), or four species (one lyogroup). The detection of enterococcal lyogroups proved to be as reliable for species identification as the conventional methods presently in use.

Isolation and partial characterization of bacteriophages infecting Pseudomonas syringae pv. actinidiae, causal agent of kiwifruit bacterial canker

The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker of kiwifruit. In the last years, it has caused severe economic losses to Actinidia spp. cultivations, mainly in Italy and New Zealand. Conventional strategies adopted did not provide adequate control of infection. Phage therapy may be a realistic and safe answer to the urgent need for novel antibacterial agents aiming to control this bacterial pathogen. In this study, we described the isolation and characterization of two bacteriophages able to specifically infect Psa. φPSA1, a member of the Siphoviridae family, is a temperate phage with a narrow host range, a long latency, and a burst size of 178; φPSA2 is a lytic phage of Podoviridae family with a broader host range, a short latency, a burst size of 92 and a higher bactericidal activity as determined by the TOD value. The genomic sequence of φPSA1 has a length of 51,090 bp and a low sequence homology with the other siphophages, whereas φPSA2 has a length of 40 472 bp with a 98% homology with Pseudomonas putida bacteriophage gh‐1. Of the two phages examined, φPSA2 may be considered as a candidate for phage therapy of kiwifruit disease, while φPSA1 seems specific toward the recent outbreaks isolates and could be useful for Psa typing.

Effect of slime production on the antibiotic susceptibility of isolates from prosthetic infections.

The antibacterial activity of 6 antibiotics towards 10 gram-positive and 6 gram-negative glycocalyx-producing strains, has been evaluated by employing a method which partially simulates the in vivo colonization of prosthetic devices. The results showed that routine antibiotic sensitivity tests are not predictive about the response of the glycocalyx-embedded bacteria, and that prophylaxis may be useful with ofloxacin and clindamycin, before placing a prosthetic device. Once bacterial colonization had already occurred, however, none of the tested antibiotics was able to eradicate the sessile bacterial form. The minimum bactericidal concentration (MBC) values, indeed, were much higher than those determined on the planktonic form, and were much higher than serum and tissue levels that can be reached in vivo.

Coping with antibiotic resistance: contributions from genomics

Antibiotic resistance is a public health issue of global dimensions with a significant impact on morbidity, mortality and healthcare-associated costs. The problem has recently been worsened by the steady increase in multiresistant strains and by the restriction of antibiotic discovery and development programs. Recent advances in the field of bacterial genomics will further current knowledge on antibiotic resistance and help to tackle the problem. Bacterial genomics and transcriptomics can inform our understanding of resistance mechanisms, and comparative genomic analysis can provide relevant information on the evolution of resistant strains and on resistance genes and cognate genetic elements. Moreover, bacterial genomics, including functional and structural genomics, is also proving to be instrumental in the identification of new targets, which is a crucial step in new antibiotic discovery programs.

Bacterial acid phosphatase gene fusions useful as targets for cloning-dependent insertional inactivation

The Morganella morganii phoC gene, encoding a class A acid phosphatase, was used to generate gene fusions with modified amino‐terminal moieties of the Escherichia coli lacZ gene carrying a multiple‐cloning site flanked by phage‐specific promoters and recognition sites for universal sequencing primers. The corresponding hybrid proteins retained a PhoC‐like enzymatic activity which is easily detectable by a plate histochemical assay, rendering similar gene fusions potentially useful as targets for cloning‐dependent insertional inactivation. Cloning experiments performed in plasmids carrying similar lacZ‐phoC fusions confirmed their usefulness as cloning vectors for direct screening of recombinants. As compared to conventional lacZ α‐complementation‐based vectors, which can only be used in E. coli hosts carrying specific lacZ mutations, the lacZ‐phoC fusion‐based vectors can be used in combination with any E. coli host and require a less expensive histochemical assay for screening of recombinants, while retaining all the advantageous features that made the former so popular as general purpose cloning vehicles.