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An Assay for Isolation of Zinc(II)-Containing Bacterial Proteins Using Microfluidic Coupled with Paramagnetic Particles

Staphylococcus aureus has become a looming threat due to its increasingly multiple drug resistance to many antibiotics. In 1947, four years after mass production of penicillin began S. aureus penicillin resistant strains were identified [1]. In 1959, methicillin, a new antibiotic, was found to be effective against penicillin-resistant staphylococci. The bacteria eventually became resistant to methicillin which consequently led to the discovery of the first methicillin resistant strain of S. aureus (MRSA) in 1961. Currently, approximately 65% of staphylococcal strains have been identified with MRSA phenotype (resistance to beta-lactam antibiotics and the cephalosporins) [2]. Emergence and the increase number of antibiotic resistance bacteria have resulted in exploring alternatives to antibiotics such as heavy metals complexes [3-7], heavy metal nanomaterials and nanoparticles [8].

Heavy metals, namely zinc based complexes seem to be promising antibacterial agents; however, bacteria pathogens are able to develop or acquire resistance to heavy metals. In 1991, loss of methicillin resistance due to elimination of resistance to heavy metals and tetracycline was reported [9]. Studies found that exposure to nutraceutics, namely zinc derivatives, echinacea and garlic products exhibited resistance to antibiotics in S. aureus [10]. These investigations suggested a strong association between methicillin resistance and zinc [11]. Moodley et al. demonstrated that feed supplemented with tetracycline or zinc increased the number of MRSA in the nasal cavity of pigs [12]. It was discovered that gene czrC encoding zinc and cadmium resistance in MRSA was related to methicillin resistance [13]. This gene is widespread both in humans and animals and is predicted to encode transporters, metal-carrying proteins. Zinc-dependent endopeptidases, , specifically resistance factor HmrA from MRSA, have been found to belong to a group of proteins characterized with zinc and antibiotic resistance [14]. HmrA, an antibiotic resistance factor of methicillin-resistant Staphylococcus aureus, is common among bacterial species. Two zinc-dependent metalloproteases, ZmpA and ZmpB, have been shown to influence the resistance of B. cenocepacia to host antimicrobial peptides [15]. In addition, it was discovered that a fusion protein with a penicillin-binding site and zinc-binding metalloproteinase domain is involved in signal transferring in staphylococcal resistance to beta-lactam antibiotics [16]. Associations between types and levels of heavy metals and specific patterns of antibiotics resistance share cross resistance. Several mechanisms underlie this process and is most important due to their non-specific properties [17]. With the exception of the previous mentioned associations with multiple antibiotic resistance, the following zinc bound proteins possess staphylococcal pathogenic properties: superantigens [18, 19], lysostafin [20] and other extracellular proteases [21], enterotoxins [22], adhesion factors [23], transporter proteins [24], biofilm formation [25], transcription factors, and DNA-binding proteins [26, 27].

Particle-based proteins extraction is a novel and simple process employing paramagnetic beads coupled with specific antibody, ligand protein or low-molecular mass ligand. There are several protocols for antibody immobilization, proteins, organelles or whole cells. Similar to other immunochemical assays, beads-based protocols maximize optimization and give reliable results [28, 29] as bead-based sensors for pesticides [30], toxins [31], viruses [32] and other pathogens [33, 34], biomarkers [35, 36], detection or separation of rare cells [37-39] or cells sorting and counting [40]. Heavy metal immunoassays utilized anti-heavy-metal antibodies that monitored binding [41] environment pollutants, Pb(II) [42], Hg(II) [43], As(IV, V), Cd(II) [44], Cu(II) [45], U(VI) [46], and rare heavy metals Ru(II) [47] and In(III) [48]. Uranyl-specific antibodies suitable for cellular imaging were developed [49] based on these studies. Antibodies against Zn(II) were successfully prepared in previous investigations [50].

Magnetic bead-based analytical procedures can be miniaturized, is broadly applicable and widely applied in lab-on-a-chip and robotic sample handling systems [51]. They provide high sensitivity and specificity. The objective of the study was to explore automatic immune-paramagnetic-bead-based methods for cell isolation and techniques for specific protein group separations. Zn(II) binding proteins were extracted from Staphylococcus aureus via specific anti-Zn(II) antibody with minimal handling. The comprehensive isolation scheme is outlined in Fig. 1.

Výzkum byl podpořen díky projektu CEITEC CZ.1.05/1.1.00/02.0068

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