Anti-microbial resistance (AMR) origin, development 

and dissemination, How to avoid encountering

       Totally Resistant to Treatment of Microbial Infection

Anti-microbial resistance (AMR) is the ability of microorganisms, especially bacteria,  and p to survive and even propagate despite the exposure to antimicrobial drugs meant to kill or inhibit them. AMR can originate from different sources, such as mutations in the microorganisms’ genes or the acquisition of resistance genes from other organisms through horizontal gene transfer (HGT). HGT is the movement of genetic material between different organisms without sexual reproduction. HGT can be mediated by mobile genetic elements (MGEs) such as plasmids, transposons, and integrons12. MGEs can capture resistance genes from chromosomes and transfer them to other bacteria via plasmids or phages23. Some resistance genes have been traced back to antimicrobial-producing organisms, such as the erm genes that confer resistance to macrolides3. AMR can develop and spread locally and globally through various routes, such as human and animal waste, food production and consumption, travel and trade, and environmental contamination34. AMR poses a serious threat to public health and animal welfare, as it reduces the effectiveness of available treatments and increases the risk of infections and mortality.

 

The public and farming Community and entrepreneurs should be made aware of how Microbial Resistance - originates, develop, and disseminate to avoid Human Body encountering Totally Resistant Treatment of Microbial Infections

The field of Global Medicine and Health Affairs recently encountered a worsening experience and emanated a grief concern for the future of Antibiotic Medication - as a treatment for Bacterial Infections of any type - especially of non-contagious diseases (only for the time being) - such as wounds of any origin. The bad experience is that in a hospital in the United States, a patient was found with a Bacterial Infection, that is resistant to all the modern-day antibiotics and antibacterial - and hence heavily depended on wound irrigation of stronger and yet milder disinfectants acceptable to the body tissues exposed in the wound – A practice holds resemblance to the era before the inventing the antibiotic for the therapy and medications a many centuries older practice at the inception history of humanitarian medical care and attention.

The concern emanated here is not the treatment of the wound infected with Bacteria that is totally resistant to all the available antibiotics, but if such bacteria becomes an internal pathogen (causing the disease within the body) – or rather an internal pathogen attains the resistant to all known antibiotics - then it is troublesome, and if not the appropriate antibiotic is found in time, will lead to disastrous consequences not just only for individual cases - it will become total mayhem of the entire medical care system, including the veterinary field because the Bacterial Resistance can spread rapidly Bacteria to Bacteria in no time and a resistant bacteria can be contracted by a normal individual introducing it a fresh nonresistant colony of commensals and saprophytes of that animal/individual. Already present-day practitioners of all respects are having enough trouble with Resistant Bacterial infections for antibiotics and anti-bacterial, and bacteria developing resistance during the cause of treatment - particularly after selecting an antibiotic as suitable, through Antibiotic Sensitive Test (ABST).

The Origin of Antibiotic and Anti-Microbial Resistance by Microbes

A few decades ago, there were no proper regulatory mechanisms or authority on the use of antibiotics, even in the countries invented, involved in mass-scale production of these drugs - at that time; even on deriving newer and newer antibiotics and whenever producing its analogue synthetically= (Antibacterial)   is celebrated as of a breakthrough for future course, not realizing what is stored in by the targeted Microorganisms for the new antibiotic. A monster of propaganda by the promoters of research – a monstrous act not knowing the resistance developing before the promotion – but it is in fact a constraint in the industry of antibiotics produced from another live microbe or fungi.

The most important thing we all must remember is, no antibiotic can arrest or kill all the bacteria that cause diseases (pathogens) = each antibiotic can act only against a certain group of pathogens, for example, the earliest antibiotic Penicillin – and its modern-day analogues can act only, if these drugs penetrate the cell wall of the bacteria, and it is most effective only at the multiplication stages of bacterial infection. If a bacteria can shunt the penetration of the antibiotic into its wall or inside of its cytoplasm – that is called natural resistance, and if the microstructure of the bacteria prevents the antibiotic from acting against these organisms – it is called natural inert resistance, and the researchers do work only on these aspects – to produce an improvised antibiotic or similar analog antibacterial, never they can study about the acquiring resistance before at least partially release it to the field of practice, despite the fact such action itself responsible for spread of fresh mode of AMR.

What we are discussing here is only about Acquiring Resistance of Microbes against Antibiotics, as it is more threatening and difficult to handle during treatments.

Why do Microbes Acquire resistance? It is simply surviving mechanism for Microbes, as for any other living organism, whatever material or element, microbes realize as harmful to their survival – they will try to neutralize it in the most effective way and spread it from the bacterium to the bacterium in a same rapid manner.  Eradication of disease-causing Pathogens shall not kill the commensals and benefitting organisms, within the body such an eradication would signal the onset of deficiency of certain vitamins, leading to another disease condition.  

Until the late 1980s in Sri Lanka, the ordinary groceries located near the extensive cultivation areas stored Tetracycline capsules as part of their sale grocery – and the farmers returning home would ask for “Gaha Karal” or “Laksha-Paha Karal” in Sinhala or in Tamil “Iyanthu Ladcham kulusai” and the boutique owner will dispense one or two Tetracycline capsules, as requested by the tired customer, which is most often less than three. They use this purchase for themselves or for their farming oriented animals. This is the reason for me to write this in simplest terms.

Therefore Tetracycline a broadband antibiotic (with action against a large number of bacteria and some other microorganisms) is exposed to all the bacteria in the gut – Pathogens, commensals, and benefitting organisms, and the time gap was more than enough for all kinds of bacteria to elicit a war against their killer antibiotics – through establishing Resistance Against antibiotics, and the bacteria are in possession of an abundance of DNA resources, outside of its central nuclear DNA, to mediate the acquiring resistance genetically, bacterium to bacterium in in-enumerative speed within a part of a second.

In the modern day though there are many regulatory mechanisms are in place all over the world misuse of Antibiotics continues by many means; I mentioned about groceries around the extensive farming areas selling Tetracycline – and the farmers consume for themselves for aberration and cut wounds, as well use it to drench it orally to farm animals found slightly ill – It is a crime against humanity and animal welfare, because all the farm animals are totally depended on bacteria, Fungi and Protozoa of their large four compartmented stomach for their digestion – and drenching Tetracycline for slightest illness would disturb the digestion further, by reducing bacteria and Protozoa off the maintained balance of Rumino-Reticulo stomach   Micro-flora and orally feeding all the farm animals would culminate in undesired consequences. In these animals development of resistance is only of secondary importance, in contrast to the mono-gastric animals – human, dogs and cats as explained in the previous episode oral or systemic treatment with antibiotics have to be guided by well versed respective field specialist.

Never give oral antibiotics to any mature ruminants of the wild or Farm,

Never take antibiotics on your own, doesn't matter however lower dosage,

Which is too dangerous than taking higher dose,

 But higher dose you may risk your life!

Still staying on the old practices of yesteryears to focus on the origin of Bacterial Resistance, field of Veterinary Medicine, and farming productions have paid heavily, in origin and development of Bacterial Resistance – Disease outbreaks threatening the total capital pumped into Poultry farming for egg and Meat productions demanded inclusion of antibiotics Penicillin and Tetracycline and  antibacterial such as sulfonamides and furazolidones into their feeds as low as 0.1% to 1.0% per ton of feeds as preventive measures against major outbreak of various diseases and the farming entrepreneurs were happy that the preventive measures very successful. This led to antibiotic residuals reaching the gut of those who consuming egg and meat, exposing those individuals’ commensal microbes to the antibiotics, inducting the bacteria to elicit an effective measures against their potential threat. Further the farming entrepreneurs found that the same antibiotic incorporated in the feed in trace amounts – interacting with feed components acting as growth promoting factor; this made the farming entrepreneurs and feed mixing mills become obsessive of mixing antibiotics or antibacterial in trace amounts and it spread to all meet production farming of poultry, swine, lamb and beef and all went cut lose for the hell transpired now. This in spite of few sulfonamides and furazolidones causing stunned growth as well. The same approach in the dairy industry made disastrous impact by interfering in dairy by products such as curd, yogurts and particularly various kinds of Cheeses, particularly of much smaller cottage dairy industry, rather than the technically advanced industrial dairying, since all these byproducts require microbial fermentation for maturing as a product.  

Today everything banned, no more feed premix of antibiotics; authorities were excessively late, and Bacteria multiplying over supersonic speed picking up at least one factor of resistance at each multiplication – the hell have been disastrously established. Today in place is, even antibiotic treated for a known disease be given a time length before slaughtering, known as Antibiotic Withdrawal Time (AWT); for each antibiotic or antibacterial AWT varies; antibiotic to antibiotic of same group as well. Even milk of a cow treated for mastitis, until minimum of five days gone after the last local infiltration not accepted for collection, systemic treated cows with tetracycline have 7 days Antibiotic  Withdrawal Time.

How Bacteria Effect the Resistance (Development of Anti-Microbial Resistance AMR)

The first antibiotic to be invented is Penicillin, it is the first antibiotic the encountered the resistance during treatment as well, and therefore had undergone extensive research for improvisation derivatives, culminated in producing numerous of penicillins from live microorganisms and synthetic analogues; at least 15 Penicillin group of drugs, including Amoxicillin to Imepenem and Ticarcillin are in active use in the Veterinary Medicine and very similar to penicillins are Cephalosporins and Cephamycins are also proliferating very fast in practice. Actually any antibiotic found with ß lactam ring within their structure has been placed within the Penicillin group and all these ß lactam (beta lactam drugs) has one only mode of action against the bacteria   - arrest the bacterial multiplication by interfering the cell wall synthesis through stopping the interlinks between Peptidoglycan strands of the cell wall – by antibiotic binding to trans peptidase enzyme responsible for bridging the strands. Even though all the ß lactam drugs have one and only mode of action against the bacteria, it can exert resistance in many facets – in various biochemical forms of challenges.  Hence ß lactam antibiotics has undergone extensive studies for resistance as well – explaining its’ mode of resistances – can be a model for basic biochemical explanations given for resistance to all the antibiotics by the Bacteria with slight deviations to the particular antibiotic’s mode action against the bacteria.

When it comes to explaining Penicillin group activity against bacteria, we cannot avoid the explaining  the Staining Property of the Bacteria – The first and the oldest method of staining the invented by the Danish scientist Hans Christian Gram (1853–1938) still worth fully warranted by medical practice of any type including Veterinary and Dental sector. The Bacteria with thicker mesh of cell wall would always accept the Gram Staining and would appear dark blue to dark purplish particles under the Microscope – all the organisms that accept the Gram Staining are popularly known as Gram Positive (+ve) organisms. All the organisms with thinner and membranous cell wall will not accept the Gram Staining and are known as Gram  Negative(-ve)  organisms and these organisms would appear Light pink to pink particles under microscope

A colony of mixed Gram Positive and Gram negative organisms under Microscope.

Most of the Gram Positive (+ve) organisms will allow penetration by the Penicillin or ß lactam drugs and are somewhat susceptible to the antibiotics. But Gram Negative organisms would sieve and shunt the penetration by the ß lactam drugs in varying decrees based on the antibiotics and individual bacteria. The bacterium Haemophilus influenza a Gram Negative (-ve) bacteria readily allows penetration by the ß lactam antibiotics but still resistant to all those antibiotics (see below), whereas Escherichia coli a common Gram-ve bacteria shows greater obstacles for ß lactam antibiotics and becomes responsible further spread of acquired resistance whereas the species Pseudomonas show varying permeability, still permits with greater difficulty in higher doses. Therefore barrier mode of resistance though naturally mediated, through chromosomes, it can be acquired by the bacteria through plasmids (extra chromosomal) as well.  Therefore any bacteria can impose permeability barrier to any antibiotic, as mode of resistance either by acquiring it or by chromosomally mediated.

Secondly the enzyme trans-peptidase enzyme responsible for bridging the strands between Peptidoglycan strands of the bacterial cell wall acts as Penicillin Binding Protein as well so that to initiate the cell wall weakening – This double action enzyme may lose the affinity for antibiotic by acquired alteration in the target enzyme or Antibiotic Binding Protein - for ß lactam antibiotics both are one and same (PBP) whereas for other antibiotics it may be two different proteins.

Thirdly and more importantly any bacteria can synthesize an enzyme to inactivate the antibiotics so that to survive the antibacterial action – a classical example is demonstrated by some Gram Positive (+ve) and Gram Negative (-ve) organisms inactivate the ß lactam drugs by producing ß lactamase enzyme that cleave the ß lactam ring and there are six types ß lactamase enzyme that are somewhat Bacterial specific as well. The earliest identified one was just known as Penicillinase, but among the modern day ß lactamase enzymes some act exclusively on Penicillins only and some others are only against cephalosporins, all the other ß lactamase enzymes readily hydrolyses both penicillins and cephalosporins.

The type and concentrations of ß lactamase secreted are also bacterial species specific,

Gram +ve bacteria secreting ß lactamase is generally excreted as external enzyme to the environment they live, in larger quantity, and active only against penicillins not against  cephalosporins, mediated only through inducible, single determinant plasmid – only through transduction (see below) – primarily not a chromosomal constitutive – unable to initiate self-transmission of resistance. Staphylococcal strains are very fast to develop these type of resistance.  In contrast

Gram-ve bacteria secrets ß lactamase within the periplasmic space of the bacteria, in very small quantity, primarily constitutive, multiple determinant – heterogeneous mediated, less often inducible, able to initiate self-transmission by conjugation of two bacteria, and active against both penicillins and cephalosporins (inactivate both group of antibiotic).

 Gram –ve bacteria, capable of developing resistance, through ß lactamase secretion as of above include Escherichia, Haemophilus, Klebsiella, Pasteurella, Proteus, Pseudomonas and Salmonella spp; it is also noteworthy some of the strains of above bacteria may take longer time  to develop resistance despite being heterogenic origin.

Other form of resistances to ß lactam drugs would be shown through bacterial phenotypic changes, incomplete cell wall (Spheroplasts) or absence of cell wall Protoplasts as found in Renal Medulla, becoming Quiescent and Tolerance are not mediated genetically – just environmental responses to survive the antibacterial effects.

General methods for Resistance of Microorganisms to Antibacterial agents

Microbiologic resistance implies an increase in the Minimum Inhibitory Concentration (MIC) range to the levels too high to be reached at standard therapeutic dose rates, and the general term used to describe is unexpected lack of response to treatment in a clinical case.

Resistance can be classified by the method of acquisition such resistance by the Bacteria.

1.      Selection of Resistant Clones

2.      Chromosomal Mutation

3.      Phage transduction and

4.      R factor acquisition by conjugation

Generalized Biochemical methods of Resistances – means of Bacteria protecting themselves.

1.      Increased production of inactivating enzymes; may be inducible or constitutive e.g. Penicillins, Cephalosporins, aminoglycosides group, chloramphenicol

2.      Defective production of autolytic enzymes – environmental response e.g.  Penicillins, Cephalosporins

3.      Alterations in the specific configuration of the target sites e.g. oxacillin, cloxacillin macrolides, lincomycin,  streptomycin,

4.      Decreased enzyme affinity e.g.  trimethoprim,

5.      Induction of membrane transport system to remove the antibacterial e.g. tetracyclines

6.      Inhibition or change in membrane transport system to prevent entry of the antibacterial e.g. aminoglycosides

7.      Utilization of (host) alternative pathways e.g. sulfonamides, trimethoprim,

8.      Increased synthesis of key metabolic intermediate e.g.  Para-amino benzoic acid(PABA)  in sulfonamide resistance

9.      Development of impermeable cell walls with extremely narrow poring e.g.  Pseudomonas aeruginosa to most of the antibiotics

In all of these cases a modification protein synthesis and enzyme activity is necessary to confer resistance; thus this adaptation is genetically determined.  

Genetics of Bacterial Dissemination of Resistance   

We all know that bacteria have two types of genetic structures that can confer resistance, Chromosomes and Plasmids both of which are of double stranded DNA, and at times both are associated with the bacterial inner cell membrane which would facilitate any transfer. Unlike Chromosomal DNA, Plasmids are not essential for live of bacteria, but do carry genetic determinants that confer resistance as well as for virulence (the measure of vigor in pathogenicity) of bacteria. Bacteria may carry many Plasmids, but all are of one type only, this is known as “Incompatibility” conformity.  

Plasmids are smaller unit than central chromosome, but play a very active and complex role in the disseminations of Bacterial Resistance than the chromosome. Plasmids may contain 20-500 genes that can carry resistance to 3 to 6 different antibacterial commonly – but up to 9 antibacterial have been recorded – as well as to specific virulence factors. To this effect many plasmids have been isolated, characterized and identified. Plasmid-mediated resistance are known as R-factor or Acquired resistance.

Three possible mechanisms by which plasmids may migrate from one bacteria to another are Transformation, Transduction and Conjugation.

1.      In Transformation, naked DNA pass from the donor to the recipient through medium they grow in and this process is confined only to limited range of bacteria.

2.      In Transduction the transfer is mediated by a bacteriophage that makes use its specialized molecular equipment adapted for the insertion of DNA into the recipient bacteria, commonly it is the phage DNA that is transferred; in certain cases some DNA from the episome (additional genetic element inside bacteriophages that can replicate independently) in the bacterial cell replaces the proper phage DNA - nucleic acid sequence - assumes as a special inserter.  Phage mediated Transduction take place generally in Gram-ve species, but in some Gram+ve bacteria as well including Staphylococcus aureus.  

3.      In Conjugation the DNA passes from the donor cell to recipient via a bridge formed during direct cell to cell contact. This is the most sophisticated and thus an important process gene transfer under natural conditions. The sophistication that ensures the resistant gene is transmitted, by the facet it requires a surface appendage known as sex pilus to form the bridge. This pilus is coded for by a resistance transfer factor on the plasmid and is called a conjugative sequence = and the opposite is referred to as non-conjugative plasmid without a resistance transfer factor.  Thus many types of bacteria can act as recipients and resistance can pass through freely from saprophytic (commonly) in the gut of the animals to pathogenic bacteria.

Though in general conjugation transfer occurs more frequently in between Gram-ve organisms Pasteurella and Pseudomonas very poor performers of conjugation; this type of transfer of gene material rarely occurs in between two Gram+ve organisms. Conjugation allows the passage of a number of distinct genes at one time. Thus resistance to several antibiotics – all mediated by different biochemical means may be acquired in a single step. The great efficiency of the Conjugation process makes the probability of gene transfer to a super-infecting pathogen high.  

Another mechanism involved gene transfer within a bacterium, but rather indirectly bacteria to bacteria, capability of genetic sequences to migrate from Plasmids to Chromosome and back from Chromosome to Plasmids – and the element showing this phenomenon is called Transposons and number of Transposons responsible for the transfer of R-factor resistance have been isolated, characterized and identified.

Chromosomal Acquiring of Resistance

This type resistance to antibacterial agents depends heavily on a mutation in the bacterial genes that leads to resistance to particular antimicrobial agent. Here the antibacterial drug act only as selective agents that allow the resistant mutants to emerge either by a single step or sequential mutations and hence their genesis is independent of the presence of the agent. Mutated bacteria are often metabolically deranged and are at selective growth disadvantage; but for Quinolones and related fluoroquinolones bacteria have developed resistance rapidly – by chromosomal mutations.

These drugs are most preferred in practice for their rapid action on the target bacterial DNA – and instantly kill the bacteria and for the delay in appearing viable resistant mutants. All quinolone drugs act on the enzyme DNA Gyrase of the Gram negative bacteria and on Topoisomerase IV of Gram positive bacteria, - these enzymes nicks the Bacterial DNA in certain points that facilitates the protective super coiling of DNA; when these enzymes inactivated by quinolones, protective super coiling fails and the Bacterial DNA as well the bacteria carrying it are readily disintegrated.  In these resistance develop through chromosomal mutation and rapid selection of mutants that are capable of preventing the drug influx and inducting efflux by the alterations in the quinolone target enzymes. Additional mutations in the next most susceptible target, as well as in genes controlling drug accumulation, augment resistance further, so that the most-resistant isolates have mutations in several genes. Resistance to quinolones can also be mediated by plasmids that code for the synthesis the Qnr protein, which protects the Microbes from inhibition by the Quinolone family antibiotics.  

The Clinical Relevance of Resistance for Antibacterial

Acquired – ß lactamase induced resistance is wide spread in veterinary isolates; (50 – 60) % of the Staphylococcus spp strains and 70% of the E. coli strains resistant to Penicillin G. and 40% of later isolates from farm animals resistant to Ampicillin. Hemorrhagic septicemia caused by very low efficient conjugative transmission of resistance Pasteurella multicida – readily responsive to treatments by sulfonamides/ trimethoprim 80:20 intravenous injection even on very late stages of disease or in the midst of outbreaks. Use of tetracycline is wide spread in veterinary medicine, since it is very effective against Intra-Cellular infections of both protozoa and Bacteria in cattle. Thus more exposed to plasmid mediated resistance conferring by diminishing the uptake and influx and also efflux of active ingredient by the bacteria. Another modern drug in extensive use is quinolones for its rapid effectiveness against wide range of intracellular infections such as normally naturally resistant Brucella, Mycoplasma and Chlamydia spp had rendered these groups of drugs to be potent and rapid chromosomal resistant mutant selectors. Still if quinolones shows larger or equal diameter in an ABST, than any other antibiotic disc inserted, it should be the drug of choice – for its rapidity in disintegrating the microbes through direct DNA action.

During intestinal infection many pathogenic bacteria can act as recipients and R-factor resistance can pass through freely from saprophytic bacteria commonly found in the gut of the animals that act as a reservoir of R-factor resistance.  Use of low level of antibiotics as in animal feeds and improper dosing had led to high incidence R-factor resistance in a given population. Further indiscriminate use of antibiotics which may totally eliminate the effectiveness of many antibiotics.

Guidelines to minimize the emergence of Bacterial Resistance.

1.      A broad-spectrum antibacterial should not be used, if a narrow-spectrum agent is effective against the causative agents. This warrants the selection of narrow spectrum but extra quick antibiotic. A proper ABST interpretation shall guide the practitioner.

2.      Information regarding endemic infections and sensitivity pattern should be obtained through ABST and considered when choosing an antibiotic.

3.      Appropriate dose rates should always be followed

4.      When a combination regimen is used, to prevent the development of resistant strains, individual agents should be used at full dosage.

5.      Antibacterial for topical application should selected from agents that are un-common for resistance.

6.      For prophylaxis an antibacterial agent that prevents colonization of a specific organism or eradicates it shortly after it has become established should be used.

7.      In consistent with reasonable practice – every effort should be made to use antibiotics only when the medical indications clear for such use and to avoid newer agents when already available agents are effective.

As such in an outbreak of endemic or non-endemic situation vaccination program shall be coupled with vigorously treating current ill animals with most effective antibiotic meticulously selected against isolated organism is important, so that to minimize the exposing the population to the antibiotic repeatedly.  

The latest drugs to develop resistance is Colistin or Polymyxin E of same family of Polymyxin B, that are capable the solubilizing the membrane of Gram-ve organisms even in isotonic environment. The first Colistin-resistance gene that is carried in a plasmid that can be transferred between bacterial strains has been described in May 2016. Use of Colistin to treat Acinetobacter baumannii infections has led to the development of resistant bacterial strains. These resistant strains have also been shown to develop resistance to antimicrobial compounds, including LL-37 and lysozyme, produced as part of the human host's immune system. A tragedy in forecast.

Use of Colistin is higher in the Mediterranean and South-East Asia (Korea and Singapore), where Colistin resistance rates are continually increasing.