Fabrizio Pantanella

Metallo-β-Lactamase Producers in Environmental Microbiota: New Molecular Class B Enzyme in Janthinobacterium lividum

ABSTRACT Eleven environmental samples from different sources were screened for the presence of metallo-β-lactamase-producing bacteria by using a selective enrichment medium containing a carbapenem antibiotic and subsequently testing each isolate for production of EDTA-inhibitable carbapenemase activity. A total of 15 metallo-β-lactamase-producing isolates, including 10 Stenotrophomonas maltophiliaisolates, 3 Chryseobacterium spp., one Aeromonas hydrophila isolate, and one Janthinobacterium lividumisolate (a species in which production of metallo-β-lactamase activity was not previously reported), were obtained from 8 samples. In the J. lividum isolate, named JAC1, production of metallo-β-lactamase activity was elicited upon exposure to β-lactams. Screening of a JAC1 genomic library for clones showing a reduced imipenem susceptibility led to the isolation of a metallo-β-lactamase determinant encoding a new member (named THIN-B) of the highly divergent subclass B3 lineage of metallo-β-lactamases. THIN-B is most closely related (35.6% identical residues) to the L1 enzyme of S. maltophilia and more distantly related to the FEZ-1 enzyme of Legionella gormanii (27.8% identity) and to the GOB-1 enzyme of Chryseobacterium meningosepticum(24.2% identity). Sequences related toblaTHIN-B, and inducible production of metallo-β-lactamase activity, were also detected in the J. lividum type strain DSM1522. Expression of theblaTHIN-B gene in Escherichia coliresulted in decreased susceptibility to several β-lactams, including penicillins, cephalosporins (including cephamycins and oxyimino cephalosporins), and carbapenems, revealing a broad substrate specificity of the enzyme. The results of this study indicated that metallo-β-lactamase-producing bacteria are widespread in the environment and identified a new molecular class B enzyme in the environmental species J. lividum.

CAU-1, a subclass B3 metallo-beta-lactamase of low substrate affinity encoded by an ortholog present in the Caulobacter crescentus chromosome

ABSTRACT The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-β-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned in Escherichia coli from C. crescentus type strain DSM4727. Expression studies confirmed the metallo-β-lactamase activity of its product, CAU-1. The enzyme produced in E. coli was purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (>9), and unlike the L1 enzyme of Stenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various β-lactams were poor overall (Km values were always >100 μM and often >400 μM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. In C. crescentus, the CAU-1 enzyme is produced independently of β-lactam exposure and, interestingly, the blaCAU determinant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a metallo-β-lactamase in a member of the α subdivision of the class Proteobacteria.

FoodBioTimerAssay: a new microbiological biosensor for detection of Escherichia coli food contamination

Background : Prevention of foodborne diseases is a fundamental goal for public health and industries engaged in food preparation and distribution. The correct procedure to ensure an effective prevention of foodborne diseases consists essentially in microbiological monitoring and enumeration of indicator microorganisms of faecal contamination at critical control points along the food producing procedures. Here, we propose a new microbiological biosensor, called FoodBioTimerAssay (FBTA), for rapid and reliable detection of Escherichia coli as indicator of faecal contamination in food and surface samples. Methods : A total of 122 samples were analysed using both experimental FBTA and Reference method. FBTA employs FBTM medium and counts bacteria through microbial metabolism measure: the time required for colour switch (red-to-yellow) of FBTM, due to E. coli metabolism, is correlated to initial bacterial concentration. Results : FBTA results showed an overall agreement percentage with Reference method equal to 97.54%. Discrepancies concerned three samples (1 food and 2 surface samples). Moreover, the time required to perform FBTA method was 3-fold shorter than Reference one. Conclusions : FBTA method may be considered a useful tool for detection of E. coli contamination in food and surface samples. Therefore, FBTA method may be successfully employed in risk analysis of foodborne diseases.

Influence of sub-inhibitory antibiotics and flow condition on Staphylococcus aureus ATCC 6538 biofilm development and biofilm growth rate: BioTimer assay as a study model

Staphylococcus biofilm exhibits high antibiotic resistance and therapeutic doses of antibiotics are often sub-inhibitory. Whereas data are available on the effect of sub-inhibitory antibiotics on matrix formation, little is known on their influence on biofilm population. Here, using BioTimer Assay (BTA), a method developed to quantify biofilm population, the influence of sub-inhibitory gentamicin, ofloxacin and azithromycin on Staphylococcus aureus ATCC 6538 biofilm population in flow with respect to static condition was assessed. Antibiotics and flow condition increased biofilm population even if at different extent, depending on the antibiotic molecule. The greatest bacterial population was found in biofilm developed under flow condition in the presence of azithromycin. A significant increase in biofilm matrix was recorded for biofilm developed in the presence of antibiotics in flow with respect to static condition. The growth rates (GRs) of 24-h biofilm developed under the influence of antibiotics and flow condition were also evaluated using BTA and a specific mathematical model. Antibiotics and flow condition affected the GRs of 24-h biofilm even if at different extent. The lowest GR value was recorded for biofilm developed under flow condition in the presence of ofloxacin. Although further studies are needed, our data indicate that antibiotics and flow condition influenced biofilm development by increasing both bacterial population and matrix formation and affected the GRs of the developed biofilm. To the best of our knowledge, BTA is unique in allowing the calculation of the GRs of biofilm and it may be considered to be a useful study model to evaluate the activity of antibiofilm molecules.

A New Approach to Use of Bacteriolytic Enzymes as a Tool for Species Identification: Selection of Species-Specific Indicator Strains with Bacteriolytic Activity towards Enterococcus Strains

We describe the bacteriolytic activity of 377 group D Enterococcus isolates expressed towards 25 Enterococcus strains belonging to different species and Micrococcus luteus ATCC 4698. Of the 26 indicator strains used to reveal bacteriolytic activity, 5 were lysed by all of the strains of some species and were not lysed by all of the strains of other species. The use of these indicator strains allowed us to devise a new method to differentiate group D Enterococcus strains, based on qualitative analysis (lysis or no lysis of the indicator strains) of bacteriolytic activity. The bacteriolytic patterns obtained fell into six bacteriolytic groups corresponding (98% agreement) to species or groups of enterococci as determined by a comparison with data from a phenetic similarity study.

A New Biosensor to Enumerate Bacteria in Planktonic and Biofilm Lifestyle

Remarkable interest for human health concerns the microbiological risk assessment due infections by bacteria possessing the ability to adhere to host cell or abiotic surfaces as well as to live in aggregated and biofilm lifestyle. Biofilm is multicellular bacterial community held together by a self-produced extracellular matrix in response to several factors. These factors may include recognition of specific or non-specific attachment sites on cell surfaces, electrostatic interactions on abiotic surfaces, nutritional cues, or in some cases, exposure to stress conditions in the environment as well as into the host. Biofilm lifestyle is comparatively more common than the planktonic one and it has been shown that biofilm plays a crucial role in human health (Brady et al., 2008; Bryers, 2008). As matter of fact, the eradication of bacterial biofilm by administration of antibiotics often fails due to the high drug resistance of bacteria in this lifestyle. Since 2008, European Centre for Disease Prevention and Control reports epidemiological data on the increasing of antimicrobial resistance constituting an important concern in public health hazard. For this reason, rapid assays to determine biofilm susceptibility to antibacterial drugs can significantly improve the outcome of infected patients by enabling a fast selection of efficient antibiotic treatments, thus decreasing the period and the related costs of hospitalization, as well as the incidence of morbidity and mortality ( Gfeller et al., 2005). Therefore, a fundamental prerequisite in studying, counteracting and eradicating biofilm is the possibility to quantify the actual number of bacteria involved. Bacterial counts have deep implications in microbiological diagnosis and therapeutic treatments (Bryers, 2008), in water and food quality analysis (Ramalho et al., 2001; Lee et al., 2007; Rueckert et al., 2005), in environmental applications and consumers’ safety. The standard method used to evaluate the number of bacteria, based on determination of Colony Forming Units (CFUs) (ISO method), can be considered fully appropriate only when bacteria are in planktonic lifestyle but it is unreliable to count bacteria in aggregated, adherent and biofilm lifestyle (Berlutti et al., 2003, Pantanella, 2008; Berlutti, 2008 a; Frioni,

Rebuilding the Gut Microbiota Ecosystem

A microbial ecosystem in which bacteria no longer live in a mutualistic association is called dysbiotic. Gut microbiota dysbiosis is a condition related with the pathogenesis of intestinal illnesses (irritable bowel syndrome, celiac disease, and inflammatory bowel disease) and extra-intestinal illnesses (obesity, metabolic disorder, cardiovascular syndrome, allergy, and asthma). Dysbiosis status has been related to various important pathologies, and many therapeutic strategies aimed at restoring the balance of the intestinal ecosystem have been implemented. These strategies include the administration of probiotics, prebiotics, and synbiotics; phage therapy; fecal transplantation; bacterial consortium transplantation; and a still poorly investigated approach based on predatory bacteria. This review discusses the various aspects of these strategies to counteract intestinal dysbiosis.