Emerging Antibiotic Resistance in Mycoplasma Microorganisms, Designing Effective and Novel Drugs / Therapeutic Targets: Current Knowledge and Futuristic Prospects

Citation: Mohd. Iqbal Yatoo, Oveas Raffiq Parray, Riyaz Ahmed Bhat, Muheet, Arumugam Gopalakrishnan, Archana Saxena, Sandip Chakraborty, Ruchi Tiwari, Sandip Kumar Khurana, Shoor Vir Singh and Kuldeep Dhama, Emerging Antibiotic Resistance in Mycoplasma Microorganisms, Designing Effective and Novel Drugs / Therapeutic Targets: Current Knowledge and Futuristic Prospects, J Pure Appl Microbiol., 2019; 13(1):27-44 doi: 10.22207/JPAM.13.1.03


INTRODUCTION
Mycoplasmoses, infections caused by mycoplasma (smallest prokaryotes lacking cell wall) microorganisms have become common in both livestock and humans in recent past which might be due to emergence of these pathogens and secondly due to resistance to antibiotics 1,2,3,4,5 .
For farmers it has vast economic constraints. Mycoplasmoses has gained importance both due to occurrence of frequent outbreaks, inability to diagnose and difficulty to treat 4,5,6,7,8 . Utilization of inappropriate doses of mycoplasma non specific antibiotics is the routine practice in both medical and veterinary practice 4,9,10 . Continuous use of antibiotics against other microbial pathogens also flares up mycoplasma growth and hence frequent emergence of outbreaks 9,10,11,12 . Lack of prophylactic measures adds to the problem 4,8 . Fastidious nature and special requirements of costly media for mycoplasma along with lack of laboratory facilities renders diagnosis difficult hence non utilization of mycoplasma specific therapies or control measures for mycoplasma infections usually get unnoticed or ignored or remain undiagnosed 4,8,13,14 . Prolonged usage of antibiotics against microbial infections in inappropriate doses leads to development of antibiotic resistance which is recent concern of importance 15,16 .
Even though Mycoplasma lack cell wall but many other cellular systems are similar to bacteria. They are cellular parasites and derive their requirements from host 17,18 and have limited synthetic ability due to lack of machinery and hence parasitise host cells in vivo and require specialized media in vitro 17,19,20,21 . These wall less organisms contain capsule, lipoprotein plasma membrane, genetic material (DNA), ribosomes, soluble proteins, metabolites and enzymes 18,20 . Of these capsule or plasma membrane is believed to be of importance for antigenicity. Despite such limited capability and specific requirements genitalium), infections of the genitourinary tract, infertility, septic abortion and amnionitis (M. pneumonia, M. hominis), disorders of the hematopoietic, cardiovascular, central nervous, musculoskeletal, cutaneous and gastrointestinal systems (M. pneumonia, M. hominis) 24,25,37,38,39 .
Among the mycoplasmal diseases of livestock, respiratory tract infection, mastitis, arthritis, and septicemia are considered to cause heavy economic losses and are often reported from countries of Africa and Asia, including India 4,35,36,40,41, . These usually terminate in chronic complications, leading to huge economic losses in the form of diminished production, treatment cost, high mortality and decreased export, and are causing threat to production, creating carriers and constant risk for disease outbreak. Due to lack of vaccines, preventive measures are non specific and usually rarely adopted against mycoplasma. Diagnostic facilities are not well equipped for culture, isolation or detection of mycoplasma. Moreover there is also difficulty in controlling the diseases because of lack in consistency of expression of disease 30 . Hence the only possible option and current practice of dealing with mycoplasma is utilizing antibiotics for therapeutic purposes. They are usually used for treating mycoplasma affected animals and now a days few have been used for prophylactic purposes for preventing mycoplasma infections in risk herds e.g. oxytetracycline. Treatment of affected flocks with broad acting antibiotics is practiced to control the spread of the infection. These treatments and the antibiotics used however are having fewer efficacies, thus there is a doubt on the efficacy of antibiotic therapy 42 .A very significant instance is the disease contagious caprinepleuropneumonia (CCPP) in India wherein antibiotic therapy (usual) is proven to be a failure ultimately resulting in quick disease spread 43 . Since Mollicutes lack cell wall, hence the cell wall acting antibiotics, such as betalactam antibiotics, glycopeptides, and fosfomycin, do not affect them and also the biological characteristic features of mycoplasmas results in the ineffectiveness of a number of other antimicrobials (sulfonamides, trimethoprim, rifampin, polymyxin, nalidixic acid, linezolid, and some others) 44 . In addition to this use of inappropriate doses of antibiotics and use at large scale in field conditions endangers to the Journal of Pure and Applied Microbiology risk of antibiotic resistance.
Mycoplasmas have devised a number of resistance mechanisms for combating the antibiotics. Due to lack of cell wall mycoplasma have intrinsic resistance to antibiotic classes beta-lactams and to all antimicrobials which target the cell wall 45 . Target alterations and efflux mechanisms are novel acquired resistance mechanisms of mycoplasma developed through genetic mutation or transfer of new genes 44 . Target alterations, in ribosome and in topoisomerase II genes have been found as resistance mechanisms to macrolide and tetracycline and fluoro-quinolone class of antibiotics, respectively 44, 45 . However other mechanisms of antibiotic resistance in mycoplasma need to be explored 46 .
As there is rise of antibiotic resistance against mycoplasma, ineffectiveness of commonly available and used antibiotics adds to problem hence there is dire need for exploring novel strategies and novel targets for antimycoplasma therapy 47,48,49 . These include the follow ups for antibiotic sensitivity testing, employing alternativeantibiotics, and alternate therapies. Novel targets are based on proteomics 46,50 , genomics, enzymes 52 or metabolism 53 , besides there are numerous other novel targets which are under investigation 44,47,48,49 and further exploration can help in identifying specific and effective therapeutic and drug targets in future. All these measures will help in preventing antibiotic resistance in mycoplasma, emergence of resistant species, exploring novel targets, safe and effective therapy of mycoplasma diseases in future.
The present review highlights the situation of emerging antibiotic resistance in Mycoplasma microorganisms, and designing effective and novel drugs / therapeutic targets to counter these pathogens for safeguarding health of animals and humans.

Antimycoplasma therapy
Commonly used therapeutics in antimycoplasma therapy include antibiotics and rarely anti-inflammatory, antipyretic, analgesic and antiallergic agents. The antibiotics used against mycoplasma are usually of class tetracycline, flouroquonolones, macrolides, aminoglycosides, and cephalosporins. Among these oxytetracycline is commonly used followed by enrofloxacin, tylosin 5,8,54 , streptomycin, sometimes danofloxacin, tiamulin, and azithromycin 55,56 . In humans, commonly used antibiotics for mycoplasma belong to tetracyclines, fluoroquinolones, and macrolides besides commonly used ones include lincosamides, streptogramines, and ketolides 57 . The antimycoplasmal activity of triclosan against species of the entity (Mycoplasma) which are both sensitive as well as resistant to fluroquinolones has been well documented 58 . Spectinomycin therapy has been documented against urethritis caused by Mycoplasma genitalium (macrolide resistant) in human 59 . A list of antibiotics used against various species of mycoplasma is given in Table 1. Prolonged use of these antibiotics in inappropriate doses in common herds may lead to adverse effects including abortion and teratogenicity (tetracyclines), tooth decay and bone weakness (flouroquonolones), ototoxicity (amino-glycosides) besides predisposing to menace of antibiotic resistance 5,8 .
N o v e l , s p e c i f i c a n d e f f e c t i v e antimycoplasma antibiotics need to be explored for better therapeutic results. In vitro testing 54 , alternating antibiotics 81,84,85 , long acting antibiotics 86 , novel macrolide, tetracyclines, aminoglycosides or quinolones 81,84 , pleuro-mutilin antibiotics like valnemulin and tiamulin 87,88 and advanced antimicrobials 89,90 are the future prospects with possible promising results for making antibiotic therapy of mycoplasma safe and effective and combating antibiotic resistance 44 .

Antimycoplasma antibiotic resistance
Antimycoplasma antibiotic resistance is the current issue of emergence and requires timely intervention for proper resolution. Since decades mycoplasmosis is being treated mainly with antimicrobials with varying degree of success, as vaccination is either lacking or has not been effective 49,91 . Antimicrobials like tetracyclines, marcolides,aminoglycosides, fluoroquinolones, and cephalosporins are common agents used in animals and humans against mycoplasmosis 34,57,92,93,94 . Other antibiotics used are lincosamides, streptogramines, and ketolides 57 . Since mycoplasmas are among the fastest evolving bacteria, with high mutation rates hence resistance to antibiotics has developed 44 . Also due to indiscriminate use of antimicrobials that too in inappropriate doses drug resistance has developed 91, 95 . It has been also observed that in Mycoplamas there is development of intrinsic resistance to β-lactams and other antimicrobials targeting the bacterial cell wall 96 .
It is interesting to note that the antibiotics to which the entities are usually sensitive may prove to be ineffective due to development of resistance 97 . Recently a number of studies have been carried out for evaluating antibiotic sensitivity of mycoplasma species against antimycoplasma antibiotics 98,99,100,101,102 .
Antibiotic resistance in microbes is an emergent issue, which poses a serious concern throughout the world 3,104,105 . In the initial stages of discovery, most pathogenic organisms were susceptible to antibiotics and hence were cured successfully but with the passage of time resistance developed and effectiveness of antibiotics decreased 106 . In the present times commonly tetracyclines, macrolides, flouroquinolones and aminoglycosides are being used against mycoplasma. Generally, marcolides such as tylosin is considered as the drug of choice and is extensively used in practice for the treatment of caprine, bovine and avian mycoplasmosis. Enrofloxacin was considered as the most efficacious among the antimicrobial agents against mycoplasma infection 93,107,108 . In many European countries, antimicrobial resistance of Mycoplasma spp. has been reported against tylosin, oxytetracycline and spectinomycin 109 . Resistance to tetracyclines, quinolones, and macrolides in mycoplasma develops in a similar manner as observed in other bacteria. However in some mycoplasmas such mechanisms are yet to be known 57 indicating unexplored mechanisms of resistance or adaptations in mycoplasma 44 . Resistance to oxytetracycline is suspected in Of the various antimicrobials used against mycoplasma, tetracyclines are the most commonly and widely in humans and farm animals 57,110,111 . Their mechanisms of action involves inhibition of protein synthesis by affecting 30S subunit of ribosome through blocking the attachment of charged aminoacyl-tRNA to the A site on the 30S subunit ribosome 112 . Thus, it prevents introduction of new amino acids to the nascent peptide chain 113 . Because of prolonged and continuous use of tetracyclines against microbial infections the problem of antibiotic resistance has developed. The different mechanisms involved in resistance are active cellular efflux, expression of protective proteins, antibiotic degradation with enzymes, interfering drug entry, and target modification 112,114 . These are considered to be the main mechanisms of tetracyclines resistance in classic bacteria. The development of tetracycline resistance in mycoplasmas in some cases is associated with the acquisition of tet(M) determinants located at the Tn916 transposon 115 . The transposon encodes the TetM protein, protecting ribosomes from the effects of tetracyclines. This protein is homologous to the eF-Tu and eF-G elongation factors. It can cause conformational changes in the 30S ribosomal subunit, preventing it from binding to tetracyclines. TetM determinant when present is found to be associated with a greater degree of tetracycline resistance. This is actually responsible for mycoplasmal cross resistance to other members of tetracycline group of antibiotics 44,57 . Also, resistance mechanisms in mycoplasma are also believed to be due to mutations in the tetracycline-binding unit of 16S rRNA 116,117 . Preliminary studies suggest that Mccp may have developed resistance to oxytetracycle 5,8 .
Macrolides are recommended as drug of choice against mycoplasma and are also being applied in few areas. The mechanism of action of macrolides is by inhibition of bacterial protein biosynthesis, by preventing peptidyltransferase from adding the growing peptide attached to tRNA to the next amino acid 118 . Resistance to macrolides is noted in both classical bacteria and mycoplasma (e.g. Mycoplasma pneumoniae) 119,120,121 . Target modification, alteration in drug efflux, and degradation of antibiotics by enzyme are the three mechanisms of macrolide resistance 122,123 . Development of resistance to macrolides in mycoplasma is on rise and is linked to inhibition of efflux of antibiotic and changes in structure of 50S ribosomal sub unit 57 . The alteration in the central loop of domain V of 23S rRNA can also cause resistance in mycoplasmas 57 . Mutation can also lead to resistance to macrolides in mycoplasma 44 . It has been observed that due to point mutations at various sites in the 23S rRNA domain V there may be development of resistance to macrolides 124,125,126,127 . It is to be noted that resistance of greater intensity to macrolides (14-as well as 15-membered) is associated with mutation A2058G 128 . In vivo mutation has been reported in certain instances in the L4 as well as L22 proteins (ribosomal) 12,120,129 . Rise of new mutants can also infer resistance 98 .
Tylosin is considered as the drug of choice and is widely used in practice for the treatment of caprine, bovine and avian mycoplasmosis 130 . But 131 Shahet al. has reported that tylosin showed resistance in clinical cases of CCPP and attributed it to indiscriminate use of the antimicrobial drug.
Fluoroquinolones though constitute the most popular group of drugs in human medicine against mycoplasma 57,132,133 but are occasionally used in animal mycoplasma diseases 134,135 . These antimicrobials act by interfering DNA synthesis targeting DNA gyrase and topoisomerase IV 136 . The resistance mechanisms involve either alterations in the drug target or alterations in permeability 135,134,136,137 . Quinolone-modifying or degrading enzymes have not been identified yet for resistance mechanism in bacteria but in some fungi 138 . There may be development of mutations in the genes coding for particular enzyme significantly and that depends on the antibiotic. For instance resistance to ofloxacin, trova-floxacin and ciprofloxacin has been observed in Mycoplasma hominis. This is due to topo-isomerase IV gene mutation. Due to mutation of the gene coding for DNA gyrase, sparfloxacin resistance is observed 44, 139 .
Main mechanisms of fluoroquinolone resistance in bacteria include target modifications due to mutations in the quinolone resistancedetermining region (QRDR) of the target genes gyrA (DNA gyrase subunit A), gyrB (DNA gyrase subunit B), parC (topoisomerase IVsubunit A), parE (topoisomerase IV subunit B), reduced drug assimilation by inhibiting influx and increasing efflux and acquiring resistance through genes 140 . In case of mycoplasmas, development of drug resistance is most probably associated with mutations in the QRDR region of the target genes (DNA gyrase and topoisomerase IV) 57 . Fluoroquinolones show cross-resistance among members of this group which is related to mutations and their location 57,137,141 .
Aminoglycosides are the other mainly used antibiotic class against mycoplasma. They inhibit protein synthesis by interfering with 30S subunit of ribosome and contain an aminomodified glycoside (sugar) 142 . Due to prolonged and large scale use, emergence of resistant strains decreased the therapeutic potential of aminoglycosides 143 . Resistance mechanisms include alteration of the ribosomal binding sites in case of streptomycin, reduced uptake of drug and expression of drug inactivating enzymes 144,145,146 .
In case of mycoplasma species, mutational resistance has been recorded to aminoglycosides 147 . In case of Gram-negative and Gram-positive bacteria three types of antibiotic modifying enzymes are found viz., phosphotransferases, acetyltransferases and nucleotransferases 148 but none of these enzymes have been revealed in mycoplasma. Experimentally, one transposon encoding for gentamicin resistance was introduced by transformation into mycoplasma strains, which expressed them 149,150 .
The other resistance mechanisms to these classes of drugs which have been noted in other classical microbes are yet to be deciphered in mycoplasma. The resistance mechanisms to other occasionally used antibiotics are also under investigation. Further resistance mechanisms in various species and subspecies of mycoplasma causing severe diseases in humans and animals are also not known. Proper evaluation of antibiotic resistance mechanisms in mycoplasma will help in devising better future therapeutics.

Combating antibiotic resistance
The regulation of antimycoplasma antibiotic therapy is an important aspect of prevention of resistance to antibiotics besides making antibiotic therapy effective. Antibiotic usage needs to be routinely checked in livestock rearing areas. Farmers should be made aware about disadvantages and side effects of prolonged usage of single type of antibiotics in animals. Proper dose of antibiotic is essential as subnormal doses can lead to ineffectiveness and hence resistance. Alternating two or more antibiotics periodically can help in preventing antibiotic resistance. Though novel, specific and effective antimycoplasma antibiotics are need of the hour but better is to develop prophylactic measures as need for the use of antibiotics should not arise. Better managemental practices along with prophylactics can help in preventing mycoplasma diseases and hence limiting use of antibiotics.
Antibiotic sensitivity testing (AST) is one of the most commonly used and convenient method for evaluating sensitivity of mycoplasma to antibiotics 99,100 . It can predict decrease of sensitivity and rise of resistance 99 . Regulation of antibiotic application in mycoplasmosis based on AST will be helpful in preventing antibiotic resistance. The antibiotics showing better sensitivity can be employed in mycoplasmosis. Recently a number of studies have been carried out for evaluating antibiotic sensitivity of mycoplasma species against antimycoplasma antibiotics 99,100,101,102 . The antibiotics under focus included enrofloxacin, marbofloxacin, spiramycin, tulathromycin, erythromycin, tylosin, florfenicol, oxytetracycline, doxycycline, tilmicosin, lincomycin, tiamulin, gamithromycin, tildipirosin and valnemulin. These studies predict decrease of sensitivity and rise of resistance to antibiotics. Novel methods of AST are being employed for sensitivity testing 151 .
In vitro testing will help in determining sensitivity and standardizing appropriate dose, long acting antibiotics will help in minimizing frequent use of antibiotics, novel members of commonly used classes of antibiotics, and advanced antimicrobials like CRISPER designed antibiotics will be resistance free. Antibiotic alternatives are also being evaluated for effective therapy and prevention of rise of antibiotic resistance 152 . The use of antibiotic alternatives along with immunomodulators (systemic) viz., corticosteroids or immunoglobulins intra-venously are found to be efficacious to treat infections due to macrolide-resistant Mycoplasma pneumoniae (MRMP) 153,154,155 . Identifying the novel drug targets for designing effective anti-mycoplasma antibiotics has future prospects for exploration.

Novel targets for antimycoplasma therapy
The rise of resistance against commonly available and used antimycoplasma antibiotics ensues exploration of novel targets of mycoplasma for therapeutic purposes. This can also be helpful in designing effective prophylactic measures. As the mycoplasma consists of cell membranes and genetic material hence molecular structures of these organelles can be targeted besides the mechanisms in which these are involved like adhesion, invasion, pathogenesis, transcription and translation (DNA and protein synthesis) 48,49 . Capsular lipopoly-saccharides (LPS) of mycoplasma have been investigated 156 . The main components of mycoplasma cell membranes are lipoproteins and immediate targets can be lipids or proteins of membrane 157 . Proper exploration of biological membranes of mycoplasma for various molecular components can enable better evaluation of molecular targets. Such studies are lacking. Other possible targets are enzymes, proteins, metabolites, metabolic or cycle pathways 47,48,49 . Besides therapeutics based on these various targets,having different mechanisms of action can also be helpful in preventing antibiotic resistance as mutations of molecular targets can be overpowered. Exploration of novel antigenic structures can be exploited for raising antibodies or serum for therapeutic use or developing vaccines for prophylactic use. For all these novel approaches, exploration of molecular structures of mycoplasma is must. Common structures of mycoplasma include DNA, ribosome, membrane lipoproteins, soluble proteins, enzymes and metabolites. Fig. 1 depicts the common structural components of mycoplasma cell. Among these plasma membrane 157 , proteins 174 , toxic molecules, metabolic pathways, and attachment and motility 49 , peptidoglycan synthesis or ribosomal activity 48 , glycolipids 175 , genomics, proteomics, and metabolic pathways 47 are being evaluated as novel targets of antimycoplasma therapy.
Some of the novel therapeutics approved and/or under experimental trials that target these pathways, enzymes or proteins have been highlighted previously. They target glycolysis/ gluconeogenesis, pentose phosphate pathway, fructose and mannose metabolism (phosphoglycolohydroxamic acid), cysteine and methionine metabolism, biosynthesis of amino acids (adenine), Recently glycolytic enzymes e.g. pyruvate dehydrogenases A to C (PdhA-C), glyceraldehyde-3-phosphate dehydrogenase (GapA), lactate It has also been observed that for the purpose of designing therapeutics in order to treat infections caused by Mycoplasma pneumonia, community-acquired respiratory distress syndrome (CARDS) toxin acts as a target (promising) as well as a significant candidate. The NLRP3 inflammasome is activated by the toxin intracellularly which leads to vigorous inflammation in association with infection due to M. pneumoniae 49,176 . CARDS is a significant virulent factor generating antibody titers at high level 177,178,179 .

Novel drugs/therapies against mycoplasma infections
Considering the rise of resistance against main antimycoplasma antibiotics like tetracyclines, macrolides or fluoroquinolones, novel drugs or therapies are being explored. Josamycin, pristinamycin, marbofloxacin, solithromycin, lefamulin, sitafloxacin, zoliflodacin, tigecycline, spectinomycin and moxifloxacin are newer antimycoplasma antibiotics that have shown promising results in antimycoplasma therapy 181,182 . Herbal medicines 182,183 , traditional medicine 184 , or their combination 185 have been elucidated for mycoplasma infections. Novel technologies for safeguarding health of humans and animals are being evaluated 186 . Phage therapy 187,188 has proven effective in some antimycoplasma therapies. Immunotherapy 189 , cytokine therapy 190,191 and immunomodulation 192 have potent biomedical applications and are being applied in mycoplasma infections also 189,190 . Immuno-modulators which help in boosting immunity 193 can be helpful in mycoplasma therapy. Nano therapeutics 194 involving nanomedicines 195 are advanced therapeutics in biomedicine and pharmacotherapy and can help in effective targeting and accurate drug delivery 196 . These novel and emerging therapies can overcome resistance problems in mycoplasma infections. Still many aspects of these drugs or therapies are yet to be explored however they hold a great promise for future of antimycoplasma therapies.

CONCLUSION AND FUTURE PROSPECTS
Antibiotics are frequently being used as antimicrobial agents against mycoplasma and as first line of treatment for the therapeutic management of mycoplasmoses infection. Commonly macrolides, tetracyclines, fluoroquinolones, and aminoglycosides are the main classes of antibiotics being employed worldover. Among these, oxytetracycline has been the most commonly used antibiotic for a prolonged period followed by enrofloxacin, tylosin, streptomycin and rarely danofloxacin, lincomycin, spiramycin, erythromycin, gamithromycin, azithromycin, clarithromycin, gentamicin, doxycycline, tulathromycin have been employed. Due to emergence of drug resistant strains of Mycoplasmathe effectiveness of these antibiotics isdramaticallyreduced. Frequent prolonged usage of single type of antibiotics and inappropriate doses can lead to menace of antibiotic resistance. Different microbial mechanisms have been developed by mycoplasma for resistance to antibiotics. This usually include acquisition of proteins affecting ribosomal subunits, inhibition of antibiotic efflux, structural changes in the ribosomal subunit, target mutations, expression or production of enzymes. Still the exploration of several novel mechanisms to counter Mycoplasma infections isunder evaluation. Besides alternating anti-biotics, antimycoplasma antibiotic sensitivity Journal of Pure and Applied Microbiology evaluation should be undertaken on routine basis for checking the emerging drug resistance. Evaluation of specific doses and novel mycoplasma specific class of antibiotics need to be explored. Exploring novel therapeutic and drug targets in mycoplasma for effective antibiotic therapy is the recent area of research with promising results. The rise of antibiotic resistance and ineffectiveness of conventional antibiotic therapy in mycoplasmosis has led to devising strategies and exploring possibilities for combating antimycoplasma resistance and treating mycoplasmosis effectively. It cannot be denied that changes in genomic, transcriptomic, secretomic as well as proteomic profiles of the microbes (including Mycoplasma) have influenced the phenomenon of antimicrobial resistance significantly. Regulating antibiotic use, alternating therapies along with appropriate doses of antibiotics, antibiotic sensitivity testing and exploring novel antimycoplasma antibiotics could solve the problem. Novel therapeutic targets are being explored in mycoplasma for antibiotic action which can help in safe and effective treatment of mycoplasmosis. These targets may be toxins, toxic metabolites, enzymes, proteins, metabolic pathways, metabolites, cell division, nucleotides, or cellular structural parts of mycoplasma. However to be successful, these measures need to be explored after proper experimental evaluation followed by application in clinical cases. Future of antibiotic therapy in mycoplasmosis relies solely on devising safe and effective antibiotics with mycoplasma specific targets or mechanism of action and less prone to development of resistance.

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