RESISTANCE TO TETRACYCLINE IN ESCHERICHIA COLI AND STAPHYLOCOCCUS AUREUS : BRIEF OVERVIEW ON MECHANISMS OF RESISTANCE AND EPIDEMIOLOGY

In this work we briefl y present the mechanisms of resistance to te-tracyclines. Tetracycline’s were introduced in clinical practice in 1948, and are used for the therapy in human and veterinary medicine or as growth promoters in livestock industry. Th ere are three major mechanisms of resistance to tetracyclines. Gram negative bacteria utilize effl ux pump system of proteinaceous transporters in eliminating the drug from the cell. Th is mechanism of resistance is encoded by tet genes that belong to the group 1. Gram positive bacteria promote resistance to tetracyclines by producing soluble cytoplasmatic ribosomal protection proteins and the most frequent once are TetM and TetO proteins. Enzymatic inactivation is not widespread mechanism and the responsible gene is termed tetX . Epidemiological importance of tetracyclines is well documented in number of research papers. We described few works showing that tetracycline’s are provoking resistance to other classes of antibiotics or vice versa. Th is phenomenon is probably due to the fact that resistance determinants are oft en situated on mobile genetic elements. Withdrawal of the therapy does not exclude resistance in short time frame due to the various environmental factors and animal feeding habits. Most oft en resistance to tetracycline is reported in Escherichia coli isolates from pigs, chickens and turkeys. Th e TetM and TetK proteins are most oft en found in methicillin resistant Staphylococcus aureus .


INTRODUCTION
Th e tetracyclines are one of the oldest antibiotics that are used in livestock industry.Th ey have been discovered in 1948, and have been used under generic name chlortetracycline and oxytetracyline.Over the years tetracycline antibiotics have been improved and marketed under various trade names.Th e range of activities for these antibiotics is very broad encompassing therapy of infections caused by Gram-negative and Gram positive bacteria but also chlamydiae, mycoplasmas, rickettsiae and protozoan parasites.Importantly, they are also used as growth promoters of farm animals worldwide.It is not surprising that resistance to these antibiotics has spread in various bacterial communities.Th eir mode of action requires inhibition of bacterial protein synthesis by preventing association of the aminoacyl t-RNA to the ribosome A site (Chopra and Roberts, 2001).
Resistance to tetracycline has been developed utilizing various mechanisms but the most prominent is the effl ux pump driven by the proton motive force.Such effl ux proteins belong to the major facilitator superfamily (MSF) of transporters.Bacteria can produce ribosomal protection proteins and over two decades ago, enzymatic inactivation of tetracycline has been discovered as well (Speer et al., 1991).Detection of the respective genes encoding resistance to tetracycline raises a possibility to analyze epidemiological aspects of the resistance and to discover mobile genetic elements that may have contributed in dissemination of the tetracycline resistance genes (tet) in nature.Indeed the tet resistant genes are found on mobile genetic elements the transposones.Th is is the case with the tet(A) gene that was found on transposone Tn1721 while the tetM gene was detected on Tn916 (Frech and Schwarz, 1999;Chopra and Roberts, 2001).Even though Tn1721 is non-conjugative transposone it is inserted in conjugative plasmids and this is facilitating dissemination of the tet genes in the environment.On the other hand the Tn916 transposone is conjugative so it could be self transferable.

Effl ux pump
Th e effl ux proteins belong to a large group of transmembrane proteins encoded by tet genes.Based on sequence amino acid identity tet genes are placed in six groups (Chopra and Roberts, 2001).Th e tet genes belonging to the group 1, are most abundant and represented by tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(I), tet(J), tet(K), tet(L), tet(V), tet(Y), tet(Z), tet(30), tet(31), tcr3, otr(B), tetP(A).Th e amino acid identity between these proteins is 41 to 78% and for the repressor protein it is 37 to 88%.Th e TetZ protein is found also in Gram positive bacteria, but all other proteins from the group 1 belong to Gram-negative bacteria (Chopra, 2002).Th e structure of Tet proteins has been studied and it is evident that they consist of 12 predicted transmembrane α-helices with nonconserved central loops connecting α-helicis 6 and 7. Th e effl ux proteins are inserted in the lipid bilayer and their hydrophilic amino acid loops are protruding into the periplasm and cytoplasm.Effl ux proteins utilize the energy by exchanging a proton for a tetracycline cation complex against a concentration gradient (Chopra and Roberts, 2001).Namely, upon entry to the bacteria cell by diff usion process tetracycline are releasing proton (H+) and become increasingly receptive for divalent cations (the Me+).Th e monovalent cationic reactive complex strives to binds to repressor -operator complex due to its own instability.Th is process disables the repressor binding to DNA by triggering conformational changes in the repressor protein.Even very small amount of tetracycline's are suffi cient for separation of the TetR from the DNA.Th is mechanism leads to the constitutive expression of the tet genes encoding effl ux proteins.In the absence of antibiotic, the repressor protein TetR negatively regulates tet gene expression by binding to the operator (TetO).It has been noted that tetracycline-metal complex present one of the most sensitive eff ector-inducible systems of transcriptional regulators.(Hillen and Barrens, 1994).In Gram negative bacteria two or more effl ux genes may be commonly responsible for resistance to tetracycline while in Gram positive bacteria, the effl ux mechanism is oft en coupled with the ribosomal protection mechanism which gives opportunity for development of new generation of drugs aiming at both resistance targets (Chopra, 2002).

Ribosomal protection proteins
Ribosomal protection proteins (RPP) are soluble cytoplasmic proteins and they are protecting ribosome from the tetracycline including doxycycline and monocycline.Th e best studied mechanisms of the RPP are encoded by tet(M) and tet(O) genes.TetM proteins induce release of tetracycline from the ribosome by using the energy from GTP hydrolysis.Th e analysis of the upstream and downstream sequence of the structural gene and GC content (<40%) of the tetM gene supports the postulate that tet(M) gene may have originated from Gram positive bacteria and indicate that these genes are been transferred to Gram negative microorganisms (Chopra and Roberts, 2001).TetO proteins are changing the architecture of the ribosome inducing alterations on tetracycline binding site.Th ese alterations are transient but mechanisms involved to prevent rebinding of the tetracycline to the ribosome are presumptive.Two interesting postulates, yet to be experimentally proven, relay on a possibility that Tet proteins promote aa-tRNA binding to the A site or that Tet(O) acts successively before aa-tRNA binding.Even though the conformational changes at the primary binding site of the tetracycline have been recognized, it is to be answered how ribosome's returns and sustains elongation cycles in the presence of tetracycline antibiotics (Connell et al., 2003).
Th e tetM gene was most frequently found in methicillin resistant Staphylococcus aureus (MRSA) from 24 clinical isolates, the tetK gene was represented by 21 isolate and the tetKM genotype was found in 21 isolates (Trzcinski et al., 2000).In their research, for the fi rst time, a single tetK gene in heterogeneous MRSA isolates from Poland was discovered.It was established that all isolates possessing tetM gene are resistant to all tetracycline's while tetK gene does not induce resistance to monocycline.A high level of resistance to tetracycline's (MIC > 128 mg/L) was obtained when tetKM genes were present in Staphylococcus aureus isolates comparing to isolates with single determinants.It was elucidated that preincubation with subinhibitory concentration of tetracyclines or monocyclines (except for tetK positive isolates where preincubation with monocycline only slightly increased MIC) induced higher MICs in MRSA isolates hence involving improved screening method (the preincubation with antibiotics before MIC analysis).
Th e abundance of tet(M) genes in the collection of clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) was documented in the research of Schmitz et al. (2001).Th e tetM gene was detected in 76% of MRSA isolates while tetK gene was present in 73% of the isolates.Both genes the tetM and tetK were present in half of the MRSA isolates.Th ey have revealed that in methicilin susceptible/tetracycline resistant S. aureus (MSSA) the tet(K) gene was the most frequently found (in 96% of isolates), while tetM gene was detected in 10% of isolates.In MSSA isolates combination of tet(M) and tet(K) genes were represented at 6%.

Enzymatic inactivation and other mechanisms of resistance to tetracyclines
TetX protein is responsible for the enzymatic inactivation of tetracyclines.Th e tet(X) gene encoding the relevant protein was found on transposon of the strict anaerobes of the genus Bacterodies.Less research was directed to reveal the actual presence of tet(X) genes in nature and because of that the Bacteroides group is the only, yet to be known, species having tetX genes.In their natural host, the tetX gene is probably not functional since it requires oxygen and NADPH to chemically modify tetracycline.Th ere are some unknown mechanisms conferring low level of resistance to tetracycline's encoded by tet(U) gene.Th e sequence of the tet(U) gene is however not similar to other sequences obtained for Tet determinants and it is diffi cult to reveal their mechanisms of resistance.Th e otr(C) gene was found in Streptomycetes, but the whole sequence was not obtained and the resistance mechanisms are not yet explained (Chopra and Roberts, 2001).

Presence of tet resistance genes in humans and animals
Resistance to tetracycline is widely distributed and occurs because of the application of the antibiotics in human and veterinary medicine.Th is type of resistance is oft en found in multi drug resistant isolates and oft en is the most prevalent resistance in commensal and clinical isolates (Miles et al., 2006).It is not surprising that single resistance to TET is rare among family of Enterobacteriaceae (Dolejska et al., 2009).Th e clinical impact on resistance to tetracycline was estimated during the tigecycline phase 3 clinical trials.Patients all over the world were enrolled in the clinical trial.Th e incidence of resistance to tetracycline in E. coli was 39%.Multiple effl ux pump determinants were represented by 33% of isolates.It was concluded that resistance rate to tetracycline depend on its presence in the environment, excessive use of biocides and genetic transfer of resistance elements among bacterial species (Tuckman et al., 2007).Cross selection of resistance to tetracycline occurs in patients treated with other antibiotics, but it is important to note that resistance to tetracycline in E. coli from stool specimens of infants that have not received any antibiotic was similar to the E. coli from children that were treated with β-lactam antibiotics.Colonization capacity of the TET r versus susceptible strains of E. coli in infants was similar showing that resistance traits was not related to treatment with antibiotics.Rather it was infl uenced by the presence of virulence determinant, presumably the P fi mbriae and aerobactin.Hence the colonizing capacity and invasiveness of the bacteria really on virulence factors in infantile intestinal microbiota (Karami et al., 2006).A longitudinal study on antimicrobial drug resistance in E.coli isolates from humans and food animals in the USA has revealed that the most prevalent was resistance to tetracycline.Also this type of resistance was commonly found with the resistance to streptomycin, sulfonamide, ampicillin and chloramphenicol (Tadesse et al., 2012).Th e withdrawal of antibiotics especially those that have been used as growth promoters, has led to a moderate decrease in resistance rate.So was the case when application of antimicrobial agents was terminated for subtherapeutic use in specifi c pathogen free Yorkshire herd of pigs, held at the Kentucky Agricultural Experiment Station Research farm in the USA.Decrease of resistance to tetracycline in lactose positive enteric bacteria was 82 to 24%, but this decrease did not show steady trends aft er 126 months of withdrawal, since resistance to tetracycline remains at the level of 40% (Langlois et al., 1983).Resistance to tetracycline's was the most commonly found by Miles et al. (2006) in E. coli isolates from humans (in 43.8% of isolates) and poultry (in 82.4% of isolates) in Jamaica.Th e cross resistance of tetracycline with kanamycin and nalidixic acid was limited to avian isolates, while isolates from humans were frequently cross resistant to the aminoglycosides, ampicillin and quinolones.Th e tetB and tetD genes, encoding the active effl ux proteins, were detected on transferable plasmids in E. coli.Authors have discussed that E. coli from humans and poultry in Jamaica may not have a common source because they have diff erent resistance patterns.Th e levels of resistance to tetracycline's was tested on 1263 isolates of E. coli from humans, domestic and wild animals in a research work of Bryan et al. (2004).Th e highest resistance was found in chickens, turkeys and pigs while other species like goats, horses, ducks, geese and deer have shown low level of resistance.Th e multiplex PCR assay aimed to detect tet resistant genes was performed in isolates having MIC to TET ≥ 93 mg/L.Th e obtained MIC represented high resistance to tetracyclines and presumably off er a possibility of resistance gene detection.In total 325 strains were analyzed by PCR and 97% have been found to contain one or more tet resistance genes.Th e distribution of genes was within the following order: tetB gene was detected most abundantly i.e. it was found in 63% of isolates, tetA gene was detected in 35% of isolates followed with the various frequencies of tetC, tetD and tetM gene detection.Th e tetM gene was found in E. coli from pigs and chickens only.Tetracycline genes in pair were found in 30% of isolates originated from turkey, pigs and horses while 4.5% of isolates from pigs have carried three tet resistant genes.Th e presence of several genes comparing to one tet resistant determinant was not correlated to the MIC values.
Even though it is a well established opinion that treatment of humans and animals with antibiotics select for antimicrobial resistance in pathogenic and commensal bacteria, there is a plausible lines of evidence that even in circumstances when antibiotics are not provided on farms or in cases when long withdrawal period of antibiotic use was established, resistance to tetracycline's and/or ampicillin is still documented.Th is is explained by the interchange of mobile genetic elements among bacteria that present the normal fl ora of the gastrointestinal tract of animals, age of animals and the diet provided.Another important fact is that resistance to some antibiotics coselects resistance to other and this is usually related to transferable nature of resistance mechanisms common in some bacterial species such as E. coli (Mirzaagha et al., 2011).

CONCLUDING REMARKS
Th e application of antibiotics for the therapy and for growth promotion in livestock industry is the main reason for resistance development in commensal and pathogenic bacteria.In human medicine problems occur if antibiotic treatment is required in life threatening diseases caused by multiresistant bacteria that are diffi cult to treat with standard antibiotics.Th e prominent examples now days are resistance of Staphylococcus aureus to vancomycin, but also resistance to metallo-β-lactamases of Acinetobacter baumannii, Pseudomonas aeruginosa as well as Enterobacter spp.and Klebsiella spp.from the hospital environment.In developed countries patients infected with such microorganism are isolated in special units and this practice has shown to be adequate to restrict spreading of multiple resistant bacteria in the hospital settings (Levy and Marshall, 2004).Because of the broad antimicrobial activity of tetracycline's it is important to continue developing new generation of antibiotics, such as glycylcyclines, competent to effl ux and ribosomal protection mechanisms, for treatment of infections caused by Gram positive and Gram negative bacteria (Chopra, 2002).