Control of the Spread of Resistance

Control of the Spread of Resistance

The 60-year period during which antibiotics have been available has seen dramatic changes in the disease burden caused by infections. Outcomes from infections such as pneumococcal pneumonia, tuberculosis, and streptococcal puerperal sepsis, that used to cause considerable morbidity and mortality, are now frequently benign, at least in developed countries. We can also prevent much infection by using antibiotics during high-risk procedures, notably in the peri-operative period. The immense social, economic, and health benefits that are due to antibiotic use are, however, increasingly overshadowed by the issue of resistance. Indeed, the emergence and spread of multiresistant strains (sometimes referred to emotively as ‘superbugs’) have raised the spectrum of untreatable infection. The reality is that such instances remain extremely rare. However, resistance does limit antibiotic choice available to prescribers, sometimes meaning that less effective, more toxic or more expensive drugs have to be used. For example, the antibiotics needed to treat multiresistant forms of tuberculosis are over 100 times more expensive than the first-line drugs used to treat disease caused by fully susceptible strains. Such excess costs mean that some infections can no longer be treated in poor communities where resistance to first-line drugs is widespread. Furthermore, significant slowing in the development of genuinely new antibiotics (i.e. those with novel modes of action to which cross-resistance to older agents does not occur) has increased the potential for this threat to become a reality that once again compromises patient outcome.

In 1945 during his Nobel Prize acceptance speech Sir Alexander Fleming said ‘It is not difficult to make microbes resistant to penicillin in the laboratory by exposing them to concentrations not sufficient to kill them, and the same thing has occasionally happened in the body.’ This warning was evident less than a decade after the introduction of penicillin, when a particular penicillin-resistant Staphylococcus aureus strain started to cause outbreaks of postoperative and perinatal infection in hospitals across the world. Poor hospital cleaning, increasing dependence on antibiotics and changing healthcare practices were blamed. Unfortunately, these issues are again topical, with frequent media headlines about ‘superbugs’ and their spread.

Compared with most other drugs of similar potency, antibiotics are remarkably safe, and they are also remarkably effective. This has inevitably led to liberal, even lavish use, and concern has frequently been expressed that excessive and inappropriate use of these agents is the chief cause of the widespread emergence of resistant organisms. Misuse of most drugs tends to have consequences only for the individual patient. Unfortunately, inappropriate antibiotic use can have adverse consequences for both the individual and for wider populations. Below Figure shows the disturbing relationship between the prescribing of penicillin-like antibiotics in multiple populations and the respective prevalence of pneumococcal strains with reduced susceptibility (or frank resistance) to penicillin. Of course, many of the antibiotics prescribed would not have been specifically for pneumococcal infection. This is, therefore, evidence of the selective pressure for resistance emergence in (respiratory tract) flora and subsequent spread of bacteria within populations. Such effects have been referred to as the collateral damage associated with antibiotic therapy.

Availability of antibiotics

Most developed countries have tightly regulated systems for the control of the manufacture, importation, distribution, sale, supply and description of medicinal products, including antibiotics, for human and veterinary use. In the global market for medicines, licensing authorities will increasingly be required to ensure that there is a consistency of approach to medicines availability. Currently there are many examples of inconsistencies in the availability and recommendations for use of antibiotics throughout both the developed and developing world. The availability of antibiotics, notably newer more expensive agents is an issue in poorer countries. Pharmaceutical companies have a part to play in helping to ensure that antimicrobial agents, including critical antimalarial, antituberculosis, and antiretroviral drugs are priced and advertised appropriately in these markets.

While the sale and distribution of antibiotics are fairly tightly controlled in rich, developed countries, the marketing of these agents is much less restricted in the poorer, and numerically much larger, developing world. Paradoxically, the use of antibiotics in the developing countries needs to be extended, not restricted, if standards of health are to be brought up to those of the developed world. A key issue here is unregulated ‘over the counter’ availability of antibiotics. Controversy persists about striking a balance between making effective medicines available in a timely fashion to those who need them, against the potential detrimental effects of uncontrolled or indiscriminate use. This argument is most pertinent in the case of antimicrobial drugs, as there is no other example in therapeutics in which local misuse of an efficacious agent can lead to a general diminution in its effectiveness. It was no great surprise that chloramphenicol-resistant typhoid bacilli first emerged in South America and penicillin-resistant gonococci in south-east Asia, where unrestricted availability of antibiotics is commonplace. In some countries antibiotics can still be purchased easily as single tablets resulting in inappropriate use, suboptimal dosing and the consequent encouragement of resistance.

Correlation between antimicrobial use (outpatient prescribing of penicillins) and resistance (prevalence of penicillin-non-susceptible Str. pneumoniae) in 19 countries in Europe. Reprinted from: Goossens H, Ferech M, Vander Stichele R, Elseviers M. Outpatient antibiotic use in Europe and association with resistance: a cross-national database study. Lancet 2005; 365: 579-587 with permission from Elsevier.

In the UK fluconazole and aciclovir have been available for over-the-counter purchase without the need for a prescription for more than a decade. There is no convincing evidence that this availability additional to prescribed courses has increased the emergence of resistance to these agents in the target pathogens—Candida albicans and herpes simplex virus—for which they are commonly used. This may, however, reflect inherent properties of these drugs uncommonly to select for resistant variants. There is pressure to extend the availability of over-the-counter antibiotics to include drugs such as trimethoprim for use in urinary tract infections. It will be important to monitor any such changes closely to determine the benefits and drawbacks of any such deregulation of antibiotics. A related issue is the extension of capacity to prescribe antibiotics (and other drugs) to other healthcare professionals, including pharmacists and nurses. Such extended roles clearly need to be underpinned by appropriate training and education, and the availability of carefully constructed guidelines (see below).

Inappropriate antibiotic use

Attention has repeatedly been drawn to the worldwide public health problem of the spread and persistence of drug-resistant organisms, and there have been frequent calls for regulation to curb the unnecessary use and misuse of antimicrobial drugs in some countries. The following practices have been clearly identified as contributing to the present situation:

• Inappropriate prescribing of antibiotics; e.g. for ailments for which they are ineffective, such as for sore throats where 80% of infective cases are caused by viruses.
• Incorrect dose or duration of use; e.g. in uncomplicated urinary tract infection more than 3 days of antibiotic treatment does not increase the chance of success, but does increase the risk of selection of resistance bacteria in the gut flora and adverse drug effects.
• Excessive use of antibiotic prophylaxis; e.g. for most types of surgery there is no value in giving more than one dose of antibiotic(s). Excess antibiotic doses may encourage resistance emergence or side effects including antibiotic-associated diarrhoea.
• Antibiotic use without prescription; e.g. the uncontrolled availability of antibiotics ‘over the counter’, which can result in unnecessary use or intermittent, suboptimal dosing. The increasing availability of antibiotics through the internet may exacerbate this risk. In some countries, poorly formulated or manufactured, counterfeited or expired antibiotics are sold and used for self-medication or prophylaxis.
• Animal/agricultural use of antibiotics; e.g. using clinically useful antibiotics as growth promoters in animal feeds and on agricultural crops (see below).

Antibiotics use in animals

More than half of all antibiotics produced worldwide are used in animals, primarily as part of the food production chain. Two aspects of this use are particularly concerning. First, there is a large overlap between the types of antibiotics given to animals and those used to treat infection in man. Secondly, large quantities of antibiotics are used not to treat overt infection but instead as animal growth promoters to increase weight gain and therefore market value of animals. Combining these two issues, it is not surprising therefore that there is mounting evidence of resistant bacteria developing in animals and either infecting human beings or acting as a source of resistance genes for human pathogens. For example, avoparcin use in animals is linked to the development of resistance to glycopeptides in animal strains of enterococci and possibly also in human strains. Avoparcin was banned as a growth promoter in Denmark in 1995, at which point about 80% of Danish broiler chickens were colonized with vancomycin-resistant enterococci; the current prevalence is less than 5%. Similarly, fluoroquinolone use in animals has been clearly associated with the increase in prevalence of fluoroquinolone resistance in salmonella and campylobacter strains that infect man. Notably, a multiresistant Salmonella enterica serotype Typhimurium strain (DT104) has spread in animals, foods and, subsequently, in human beings.

All use of antimicrobial agents for growth promotion is now banned in the European Union. There is some concern that the therapeutic use of antibiotics in animals may increase as use of antibiotic growth promoters is curtailed, but this is unlikely to have the same negative consequences as seen with unrestricted use of antibiotics in animals.

Antibiotic prescribing in the community and hospital

The European Union, the US Food and Drug Administration and the World Health Organization have initiated national and regional campaigns aimed at professionals and the public to reduce the unnecessary prescribing of antibiotics. Efforts have been concentrated on prescribing in the community, not least because this accounts for 80% of all human use of antimicrobial drugs. Principles such as not prescribing antibiotics for viral sore throats, or simple coughs and colds, and avoiding the use of new and more expensive antibiotics (e.g. quinolones and cephalosporins) when standard and less expensive antibiotics remain effective have been emphasized. Prescribing of antibiotics started to fall in England in 1995-1996. The decrease subsequently stabilized, with a slight rise in 2003-2004.

It is not clear whether this decrease in prescribing has been driven by reduced incidence of infections (such as respiratory tract infections), reduced consultation rates, or because general practitioners are following prescribing guidance for infections more closely. However, prescribing of paediatric antibiotic preparations fell by almost 50%—a much greater reduction than that seen for the whole population. This suggests that public (parental) expectation, and hence pressure, for the doctor to prescribe an antibiotic following a consultation may be decreasing.

Trends in prescribing of antibacterial drugs in general practice in England. Reproduced from: National Health Service Business Services Authority prescription pricing division website.

It is estimated that up to 50% of antibiotic usage in hospitals is inappropriate. Interventions to improve antibiotic prescribing for hospital inpatients can be successful, and importantly may reduce antimicrobial resistance or hospital-acquired infections, such as Clostridium difficile infection. A key issue is choosing the most appropriate control methods for a given setting and ensuring that they are sustainable. The scope of measures that can be used to reduce inappropriate antibiotic prescribing is too large to consider in detail here, but can generally be grouped into educational or restrictive approaches or a combination of the two.

Appropriate antibiotic use

The World Health Organization advocates the following 12 key interventions to promote more rational use of medicines in general; all are applicable to antibiotic use:

1. Establishment of a multidisciplinary national body to coordinate policies on medicine use.
2. Use of clinical guidelines.
3. Development and use of national essential medicines list.
4. Establishment of drug and therapeutics committees in districts and hospitals.
5. Inclusion of problem-based pharmacotherapy training in undergraduate curricula.
6. Continuing in-service medical education as a licensure requirement.
7. Supervision, audit, and feedback.
8. Use of independent information on medicines.
9. Public education about medicines.
10. Avoidance of perverse financial incentives.
11. Use of appropriate and enforced regulation.
12. Sufficient government expenditure to ensure availability of medicines and staff.

In addition to these measures, good antimicrobial prescribing needs to be informed by timely and accurate information on the likely infecting pathogens. Delays in diagnosis occur through poor or non-existing sampling techniques, delay in transport, slow and laborious laboratory techniques, and unsatisfactory reporting methods. A major problem in dealing with patients in whom an infection is suspected is distinguishing between infection and colonization. Patients with an undiagnosed fever may well be colonized with potentially pathogenic micro-organisms, but may not be infected. The distinction is not always obvious, and under these circumstances it is understandable for a clinician to prescribe antibiotics. It is not rational, however, to treat patients merely because they have a raised temperature. Good practice dictates that all relevant samples for culture should ideally be collected before treatment, unless this requirement could compromise outcome (for example, in patients with suspected meningitis where prompt antibiotic therapy may be life saving). The initial choice of antimicrobial therapy will depend on the most likely infecting organism, the severity of the illness, and the type of patient. If the identity of the organism is known then treatment can be specific and a single, narrow-spectrum antibiotic used. If the infecting organism can be targeted then broad-spectrum antibiotics do not need to be used, thus leaving much of the body's normal flora undisturbed.

Initial (often empirical) antibiotic therapy is based on good surveillance and prompt guidance informed by accessible policies (see Chapter 18) or from infection specialists. Crucially, antibiotic prescriptions should be reviewed regularly to determine whether the drug or route of administration is still appropriate. Oral antibiotics tend to be considerably cheaper than intravenous alternatives and of course do not require an access device that itself may be a source of infection. For these reasons, intravenous antibiotics should be reviewed after 48-72 h and switched to an ‘equivalent’ oral formulation, provided oral absorption is satisfactory and the oral antibiotic has the requisite pharmacokinetic characteristics. New microbiological or other information (e.g. fever defervescence for at least 24 h, marked clinical improvement; low C-reactive protein) should prompt a review of therapy and consideration of whether a switch to oral antibiotic(s), a narrow spectrum intravenous alternative, or cessation of antibiotics (no infection present) is appropriate. Laboratory reports should contain information on a restricted number of antibiotic susceptibilities.

Antibiotic policies and resistance surveillance

Even in relatively straightforward clinical situations there are often several equally effective agents that might be used. Choice may then be determined by a locally agreed set of guidelines for the rational use of antibiotics. The antibiotic formulary is a locally agreed list of available antibiotics, usually including some degree of restriction on particular agents. Guidance on the most appropriate use of antibiotics should not be too restrictive, should reflect local needs, and should be formulated with the agreement of the local users. Advice should of course facilitate the most effective treatment for the individual patient, but should take into account the potential consequences for the wider population.

The best antibiotic policies are grounded in good microbiology laboratory surveillance, which is required to detect important change in bacterial resistance. Clinicians need to be aware of the local and changing patterns of infection and antibiotic resistance in their locality. Information about new agents, together with an assessment of their likely place in therapy, should be available. There are a number of caveats to pathogen and antibiotic surveillance data in general. Bias inherent in the way samples or pathogens are collected is a common problem. For example, uncomplicated urinary tract and respiratory tract infections are usually treated empirically and indeed without samples being submitted. General practitioners tend to reserve the submission of urine or sputum samples for those cases that have complicated courses or where recurrence of symptoms occurs. Thus, antibiotic treatment policies based entirely on the results of such samples and pathogens will tend to be skewed towards more antibiotic-resistant pathogens, and in turn may recommend unnecessarily broad spectrum or newer antibiotics. Such issues can be overcome by using sentinel (sometimes also called spotter) practices that submit samples from patients with ‘normal’ infections, usually for set periods of the year.

Antibiotic rotation

The selective pressure that results from relying on one or a few antibiotics has led some to explore whether antibiotic rotation (also called antibiotic cycling), particularly in the intensive care unit, can reduce or delay the emergence of resistance. However, this theory has several important unanswered issues: how often should antibiotics be rotated? Is the optimum period of usage the same for all antimicrobial drugs? Which antibiotics and classes should be rotated and in what order? What are the practicalities of ensuring compliance with a rotational policy? Current consensus is that routine antibiotic rotation should not be implemented. Indeed, several studies have been unable to demonstrate a reduction in the prevalence of resistance while antibiotic rotation was being used, and some have found that resistance actually increased during some parts of the cycle. Ironically, diverse antimicrobial prescribing may be associated with reduced emergence of resistance. This should not be interpreted as an argument for entirely unrestricted prescribing, as this is likely to be associated with suboptimal therapy for some patients.

Monitoring antibiotic policies

Blind faith in a restrictive antibiotic policy is not the answer to control of antibiotic usage since bacterial resistance patterns change over time owing to selective pressure. Periodic antibiotic audit should be mandatory in all areas where prescribing occurs. This should not be viewed as a policing exercise, so implying a threat to the clinician's freedom to prescribe, but instead should serve as a need to justify selection of antimicrobial agents in the light of critical analysis. Monitoring antibiotic usage should provide ward, unit, and hospital-wide information on prescribing patterns. This should prove useful for trend analysis and allow discrepancies to be identified. Such information lends itself to detailed scrutiny to differentiate between rational, questionable, and irrational antibiotic usage. Clinical efficacy and adverse events can be evaluated. Correlations between antibiotic usage and antimicrobial resistance should be sought, and changes can be made. A ‘defined daily dose’ for each antibiotic can be used as a standard unit of measurement, and can be useful to identify qualitative as well as quantitative variability in prescribing.

The next stage on from monitoring antibiotic usage is antibiotic audit, thereby closing the audit loop (See The Table Below).

Control of the transmission of antibiotic-resistant bacteria

It is essential that an active infection control programme is also in place, so that patients harbouring multiresistant bacteria are appropriately nursed, managed, and treated. While a full account of the optimal infection control procedure to minimize the risk of pathogen transmission is not appropriate here, some important principles are worth emphasizing. Isolation of patients and ensuring scrupulous hand hygiene, such as with alcohol-based hand rubs, can reduce the risk of transmission and the spread of pathogens. Much has been written about hospital cleanliness and the risk of hospital infection, notably the spread of antibiotic-resistant bacteria such as methicillin-resistant Staph. aureus (MRSA), but the lack of data to substantiate a link between these is stark. The great majority of infections acquired during healthcare arise because of poor hand hygiene. Compliance with hand hygiene policies should therefore be monitored. Although the need to isolate a patient may conflict with other pressures on healthcare delivery, this should not prevent infection control teams implementing this fundamental way of minimizing pathogen dissemination risk where appropriate. Patients may be isolated in single rooms or cohort-isolated in groups of beds or on dedicated units.

Audit of antibiotic prescribing

Much infection control practice is based on empiricism, and policies are frequently based on experience rather than controlled trial data. This does not mean that such policies are optional! There is ample evidence that when infection control measures are strictly enforced, the incidence of infection with resistant organisms can be reduced. Effective ways of preventing cross-infection with and spread of antibiotic-resistant pathogens still need to be defined and refined. Crucially, these approaches may need to differ depending on whether a particular antibiotic-resistant pathogen has already become established (endemic) or is rare (sporadic). A good analogy is plugging the holes in a leaking dyke: eventually more than fingers are needed to sustain the barrier. Controversy still exists about the true control benefit of screening for specific potential pathogens such as MRSA. The role and benefit of new rapid screening methods, usually based on DNA detection, remain to be determined. Alternative approaches include targeted prophylaxis against such pathogens in patients undergoing high-risk procedures such as surgery.

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Genetics of Resistance

Genetics of Resistance

All the properties of a microbial cell, including those of medical importance such as antibiotic resistance and virulence determinants, are determined ultimately by the microbial genome, which in turn comprises the three sources of genetic information in the cell: the chromosome, plasmids, and bacteriophages. Resistance of bacteria to antibiotics may be either intrinsic or acquired.

Intrinsic resistance is the ‘natural’ resistance possessed by a bacterial species and is usually specified by chromosomal genes. An example of a bacterial species with a high degree of intrinsic resistance is Pseudomonas aeruginosa. By contrast, acquired resistance occurs in formerly susceptible cells, either following alterations to the existing genome or by transfer of genetic information between cells. Thus, a basic knowledge of microbial genetics is essential to understand the development and spread of resistance to antimicrobial drugs.

The heritable information that specifies a bacterial cell, and passes to daughter cells at cell division, is carried in bacteria, as in all living cells, as an ordered sequence of nucleotide pairs along molecules of DNA. The process of transcription of this information into messenger RNA, and its subsequent translation into functioning proteins by ribosomes, is also similar in bacteria and in other cells.

The bacterial chromosome

The main source of genetic information in a bacterial cell is the chromosome. Each bacterial cell has a single chromosome, which, in the vast majority of cases, is known to form a single closed circular DNA molecule. In Escherichia coli, the organism studied most intensively, this single DNA molecule comprises about 4 × 10³ kb (kilobases) and is about 1.4mm in length. Considering the average cell is about 1-3 µm in length, only by ‘super-coiling’ of DNA can the chromosome fit inside the bacterium. Enzymes known as DNA gyrases control the process of super-coiling DNA. Conversely, DNA uncoiling, which is necessary for messenger RNA production or chromosome replication, is controlled by DNA topoisomerases.

The chromosome is found in the cytoplasm of the cell, not separated from it by a nuclear membrane. Transcription of DNA and translation of the resulting messenger RNA can therefore proceed simultaneously. Most bacterial chromosomes contain sufficient DNA to encode for 1000-3000 different genes. Not all of these genes need to be expressed at any one time, and indeed it would be wasteful for the cell to do so. Gene regulation is therefore necessary, and this can occur at either the transcriptional or translational level.

Chromosomal mutations to antibiotic resistance

Mutations result from rare mistakes in the DNA replication process and occur at the rate of between 10^-4 and 10^-10 per cell division. They usually involve deletion, substitution, or addition of one or only a few base pairs, which cause an alteration in the amino acid composition of a specific protein. Such mistakes are random and spontaneous. They occur continuously in cell genes and are independent of the presence or absence of a particular antibiotic. The vast majority of mutations are repaired by the cell without any noticeable effect. In the presence of an antibiotic some of these occasional spontaneous antibiotic-resistant mutants that are present in a large susceptible population of bacteria may be selected. In such a situation, the susceptible cells will be killed or inhibited by the antibiotic, whereas the resistant mutants will survive and proliferate to become the predominant type. Most chromosomal resistance mutations result in alterations to permeability or specific antibiotic target sites, but some result in enhanced production of an inactivating enzyme or bypass mechanism. The latter types are mutations at the transcriptional or translational level in gene regulatory mechanisms.

Chromosomal mutations to antibiotic resistance can be divided into single-step and multi-step types.

Single large-step mutations

With these mutations, a single mutational change results in a large increase in the minimum inhibitory concentration of a particular antibiotic. Single-step mutations may lead to treatment failure when these drugs are used alone. In some Gram-negative bacilli, mutations in the genetic regulatory system for the normally low-level chromosomal β-lactamase may result in a vast overproduction (sometimes referred to as ‘derepression’) of this enzyme with resulting slow hydrolysis of compounds such as cefotaxime and ceftazidime that are considered under normal circumstances to be β-lactamase stable.

Multistep (stepwise) mutations

These are sequential mutations that result in cumulative gradual stepwise increases in the minimum inhibitory concentration of a particular antibiotic. They are clinically quite common, especially in situations where only low concentrations of antibiotic can be delivered to the site of an infection.

Plasmids

The bacterial chromosome carries all the genes necessary for the survival and replication of the bacterial cell under most circumstances. Many, perhaps all, bacteria also carry additional molecules of DNA (usually between 2 and 200 kb in size) known as plasmids, which are separate from, and normally replicate independently of, the bacterial chromosome. Plasmids can carry genes that confer a wide range of properties on the cells that carry them. In general, these are properties that are not essential for the survival of the cell under normal circumstances, but which offer the cells a survival advantage in unusual or adverse conditions. Examples of such properties are:

• Fertility: the ability to conjugate with and transfer genetic information into other bacteria
• Resistance to antibiotics: antibiotic resistance encountered clinically is often associated with plasmids
• Ability to produce bacteriocins: proteins inhibitory to other bacteria that may be ecological competitors
• Exotoxin production
• Immunity to some bacteriophages
• Ability to use unusual sugars and other substrates as foods.

Plasmids differ in size, DNA base composition, the DNA fragments that can be recognized after treatment with restriction endonucleases (‘plasmid fingerprints’), and in their incompatibility behaviour. Compatible plasmids can coexist in the same host cell, while incompatible plasmids cannot, and so tend to be unstable and displace one another. There are at least 20 incompatibility (Inc) groups within the plasmids found in enteric Gram-negative bacilli, and similar incompatibility schemes are used to subdivide staphylococcal plasmids and those found in Pseudomonas spp.

Bacteriophages

The third possible source of genetic information in a bacterial cell is a bacteriophage. Bacteriophages (phages) are viruses that infect bacteria.
Most phages will attack only a relatively small number of strains of related bacteria—they have a narrow and specific host range. Phages can be divided into two main types:

• Virulent phages inevitably destroy by lysis any bacteria that they infect, with the release of numerous new phage particles from each lysed cell.
• Temperate (lysogenic) phages may either lyse or lysogenize infected bacterial cells. In the state of lysogeny, the phage nucleic acid is replicated in a stable and dormant fashion within the infected cell, often following insertion into the host cell chromosome. Such a dormant phage is known as a prophage. However, while in the prophage state, some prophage genes may be expressed and may confer additional properties on the cell. Once in every few thousand cell divisions, a prophage becomes released from the dormant state and enters the lytic cycle, with subsequent destruction of its host cell and release of new phage particles into the surrounding medium.

The possibility of using naturally occurring phages for the treatment of some infections (phage therapy) has been suggested, partly in response to the threat posed by antibiotic resistance pathogens.

Transfer of genetic information

There are three ways in which genetic information can be transferred from one bacterial cell into another: transformation, transduction, and conjugation.

• Transformation involves lysis of a bacterial cell and the release of naked DNA into the surrounding medium. Under certain circumstances, intact bacterial cells in the vicinity can acquire some of this DNA. This process has been much studied in the laboratory, but there are few convincing demonstrations of its occurrence in vivo. The process depends crucially on the ability of the recipient cells to be competent for uptake of free DNA.
• Transduction involves the accidental incorporation of bacterial DNA, either from the chromosome or a plasmid, into a bacteriophage particle during the phage lytic cycle. The phage particle then acts as a vector and transfers the bacterial DNA to the next cell that it infects.
• Conjugation involves physical contact between two bacterial cells. The cells adhere to one another and DNA passes unidirectionally from one cell, termed the donor, into the other, the recipient. Ability to conjugate depends on carriage of an appropriate plasmid or transposon by the host cell.

These transfer mechanisms means that bacteria do not have to rely solely on a process of mutation and selection for their evolution. They can, therefore, acquire and express blocks of genetic information that have evolved elsewhere. A bacterial cell can, for example, acquire by conjugation a plasmid that carries genes conferring resistance to several different antibiotics. As a result, within a very short time following the receipt of such a plasmid by a susceptible cell, the bacteria in a given niche may change from being predominantly susceptible to being resistant to multiple drugs. Of course, the ability to transfer genes in this way does not eliminate the need for these to evolve; however, once they have evolved, it ensures their eventual widespread dissemination under appropriate selection pressures.

Evolution of new resistance gene combinations

The distinction between chromosomal and plasmid genes is not absolute. Where appropriate regions of DNA homology exist, classic (‘normal’ or ‘homologous’) recombination can occur, both between different plasmids and between plasmids and the chromosome. Although this process can lead to the formation of new antibiotic resistance gene combinations, it is relatively uncommon in bacteria because there are few regions of sequence homology between the bacterial chromosome and plasmids that can be exploited for this purpose. Homologous recombination is used by researchers to create ‘knockout’ cells in which the function of a specific gene is disrupted. A more important mechanism by which antibiotic resistance genes can pass naturally from one bacterial replicon to another is the ‘illegitimate’ recombination process known as transposition.

Transposons

Transposition depends on the existence of specific genetic elements termed transposons. These elements are discrete sequences of DNA capable of translocation (transposition) from one replicon (plasmid or chromosome) to another. Unlike classic (‘normal’) recombination, transposons do not share extensive regions of homology with the replicon into which they insert. In many cases, transposons consist of individual resistance genes, or groups of genes, bounded by DNA sequences called either direct or inverted repeats, i.e. a sequence of bases at one end of the transposon that also appears, either in direct or reverse order, at the other end. These repeats may be relatively short, often of the order of 40 base pairs, but longer examples have been identified. It is likely that these DNA sequences provide highly specific recognition sites for certain enzymes (transposases) that catalyse the movement of transposons from one replicon to another, without the need for extensive regions of sequence homology. Depending upon the transposon involved, insertion may occur at only a few or at many different sites on the host replicon. Transposons may carry genes conferring resistance to many different antibiotics, as well as other metabolic properties, and their existence helps to explain how a single antibiotic resistance gene can become disseminated over a wide range of unrelated replicons.

Isolated DNA sequences analogous to the terminal sequences of transposons can also move from one replicon to another, or be inserted in any region of any DNA molecule. Such insertion sequences appear to contain only genes that are related to insertion functions; however, in principle at least, two similar insertion sequences could bracket any assemblage of genes and convert it into a transposon. Thus, theoretically, all replicons are accessible to transposition and all genes are potentially transposable. This theory is of crucial evolutionary importance since it explains how genes of appropriate function can accumulate on a single replicon under the impact of selection pressure. Transposons and insertion sequences therefore play a vital part in plasmid evolution.

Integrons

Transposons may contain combinations of genes conferring resistance to various different antibiotics. An important question concerns the mechanism by which new combinations of antibiotic resistance genes are formed. It is now apparent that special molecular structures, termed integrons, may enable the formation of new combinations of resistance genes within a bacterial cell, either on a plasmid or within a transposon, in response to selection pressures.

Integrons appear to consist of two conserved segments of DNA located either side of inserted antibiotic resistance genes. Individual resistance genes seem to be capable of insertion or removal as ‘cassettes’ between these conserved structures. The cassettes can be found inserted in different orders and combinations. Integrons also act as an expression vector for ‘foreign’ antibiotic resistance genes by supplying a promoter for transcription of cassettes derived originally from completely unrelated organisms. Integrons lack many of the features associated with transposons, including direct or inverted repeats and functions required for transposition. They do, however, possess site-specific integration functions, notably a special enzyme termed an integrase. The precise role of integrons in the evolution and spread of antibiotic resistance genes remains to be determined, but they have been found, together with their associated antibiotic resistance gene cassettes, in many different Gram-negative bacteria. At least three potential mechanisms of spread exist:

• the potential mobility of an integron itself by site-specific insertion;
• spread following insertion of an integron into a transposon;
• horizontal transfer of integrons on plasmids.

Whatever the mechanism, unrelated clinical isolates from different worldwide locations have been shown to carry the same integron structures, and it seems that these structures may play a key role in the formation and dissemination of new combinations of antibiotic resistance genes.

The process of evolution and spread of antibiotic resistance genes continues. The origin of resistance genes carried by integrons, transposons, or plasmids, or even the origin of these elements themselves, is generally not known, but it has been possible to observe a steady increase in the numbers of resistant bacterial strains following the introduction of successive chemotherapeutic agents into clinical use. There are many examples and the evolutionary process is a continuous event. The qnrA genes that encode plasmid mediated quinolone resistance are embedded in complex integrons. Similar genes have been identified in the water-borne species Shewanella algae, so emphasizing the potential for spread of resistance mechanisms from environmental bacteria.

Genotypic resistance

To summarize the earlier discussion, genes conferring resistance to antibiotics are often found inserted into integrons, and may be part of the bacterial chromosome or may be carried on plasmids, transposons, or as part of a phage genome. The distribution of these genes between the chromosome and other elements reflects to some extent the biochemical mechanisms involved. For example, resistance that results from mutational alteration of an existing target protein will normally be chromosomal in location and will not be integron-associated, whereas resistance genes for entirely new enzymes, such as the aminoglycoside-modifying enzymes, novel β-lactamases, or trimethoprim-resistant dihydrofolate reductases, are commonly carried on plasmids and transposons as part of integrons. This reflects the fact that the evolution of any new enzyme is likely to be a very long process; the occurrence of the genes for such enzymes on plasmids, transposons, and integrons enables spread of these genes between different strains, species, and genera rather than requiring evolution of the genes afresh by each bacterial strain for itself. The discovery of a variant gene encoding an aminoglycoside modifying (acetyltransferase) enzyme that can mediate quinolone resistance has highlighted the plasticity of resistance mechanisms. In this case, the new mechanism is all the more startling given that antimicrobial-modifying enzymes have traditionally been antibiotic class specific.

Chromosomal and plasmid-mediated types of resistance may be equally important in the antibiotic management of an individual patient. However, the plasmid-encoded variety has achieved greater notoriety because of the spectacular fashion in which bacteria may acquire resistance to a number of unrelated agents by a single genetic event. Furthermore, the potential for spread of plasmid borne resistance to other species or genera highlights the importance of control of pathogens that are antibiotic resistant by virtue of such plasmid genes. Certainly, it has been plasmid-encoded resistance that has caused most problems in the highly selective environment of the hospital. Nevertheless, mutational resistance involving the bacterial chromosome is also a common cause of treatment failure with some compounds. Antibacterial agents for which resistance is not known to be encoded on plasmids (e.g. rifampicin) generally suffer from mutational resistance problems instead.

Phenotypic resistance

So far as is known, phenotypic resistance to antibacterial agents is rare, although it is not always possible to be sure that phenotypic changes brought about in the microenvironment of a lesion do not contribute to insusceptibility of bacteria in the infected host. In the laboratory, phenotypic resistance can sometimes be induced; for example, varying the conditions of growth of Ps. aeruginosa can alter the outer envelope, and this affects susceptibility to polymyxins.

Another example is the failure of penicillins and cephalosporins to kill ‘persisters’ (those cells in a bacterial population that survive exposure to concentrations of β-lactam agents lethal to the rest of the culture). This does not result from a genetic event since the resistance is not heritable, and it is probable that the ‘resistant’ bacteria are caught in a particular metabolic state at the time of first encounter with the drug.

A peculiar form of phenotypic resistance is observed with mecillinam, a β-lactam antibiotic which, unusually, does not affect bacterial cell division. Mecillinam induces surface changes in susceptible Gram-negative bacilli which generally lead to cell death by osmotic rupture. However, those cells in the population that happen to have low internal osmolality survive, and, as mecillinam lacks the ability to prevent growth and division, such bacteria continue to grow in a morphologically altered form. On withdrawal of the drug, the bacteria resume their normal shape and, in due course, revert to the same mixed susceptibility as the original parent culture.

The influence of antibiotic selection pressure

Antibiotic resistance genes, and the genetic elements that carry them, existed before the introduction of antibiotics into human medicine. However, it is clear that the emergence and survival of predominantly resistant bacterial populations is due to the selective pressure associated with the widespread use of antibiotics. Resistant cells survive in a given niche at the expense of susceptible cells of the same or other species. In some cases, however, there is a fitness cost to resistant bacterial cells that may mean that they are less able to compete once the selective pressure imparted by the antibiotic is removed. In such cases, any antibiotic susceptible progeny cells that remain may be counterselected in preference to these unfit mutants. Individual cells may lose their plasmids and chromosomal mutations may revert to being antibiotic susceptible

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Pathogenic Action

Bacteria can damage cells at all around the invasions site directly; most of this damage occurs as a result of natural bacterial life processes such as movement, metabolism and reproduction.

For example simply by penetrating the plasma membrane of a host cell or by secreting enzymes a bacterium can damage the cell; more over when too many bacteria infect the cell either of by invasion or reproduction within the cell the host cell may be lysed.

Compared with direct actions; the production of toxins is more efficient pathogenic mechanism; released toxins can travel via the bloodstream and damaged tissues far from the original invasion site; most toxins cause damaged by damaging host cell membranes or by inhibiting protein synthesis within the cell; there are two types of bacterial toxins exotoxin and endotoxin.

Exotoxins in general are produced by gram positive species; exotoxins may be released into the body of a host by bacteria that have invaded and colonized in it; alternatively a host may eat a contaminated piece of rot beef that already contains the exotoxins.

exotoxins travel through the bloodstream and active at specific sites to produce characteristic diseases; for example the exotoxins produced by Corynebacterium diphtheriae causes  diphtheria a disease that affects the respiratory membranes; the exotoxins produced by Clostridium tetani causes tetanus a disease that affects muscle contraction.

In contrast Endotoxins are substances that generally produced by gram negative bacteria; in particular lipopolysaccharides are released from the cell walls of gram negative bacteria; unlike exotoxins which produce specific diseases at specific sites in the body lipopolysaccharides produce the same general effects throughout the body; fever, weakness, aching regardless of the bacteria that produce them; typhoid fever and meningitis are among the disease caused by endotoxins.

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Cellular Structure of Bacteria

The basic components of bacterial cells are:-

• Cell wall
• Plasma membrane
• Cytoplasm
• Chromosome
• Ribosome

The outer most structure of bacteria is the cell wall a semi rigid envelope that maintains the integrity of the cell in the same way that the skin maintains the integrity of the human body.

The cell wall helps protect the cell against environmental changes for example heat, cold and drugs that would otherwise damage or destroy it.

It allows most molecules to pass through it. This layer is composed of molecules called Peptidoglycans; this is often called Peptidoglycans layer.

Just inside the cell wall is a second less rigid envelope the plasma membrane sometimes called the inner membrane or cytoplasmic membrane which encloses the cell contents; in bacterial cells the plasma membrane has two primary functions:-

First it serves as a selective barrier to molecules that penetrating the cell wall; allowing some - such as water or oxygen - to flow easily into the cell enteria and restricting the passage of another such as proteins.

Second it contains enzymes; proteins that cause chemical reactions to occur as vital to the life functions of the cell.

Bacteria can be divided into two main groups based on differences in the cell walls structure; this difference was first noticed as differences in staining with a dye called gram’s stain.

Gram positive organisms have the structure defined so far.

But Gram negative bacteria have an additional membrane outside the peptidoglycans layer called the outer membrane; this which contains openings called channels some them formed by protein called Porins which allows nutrients, waste products, fluids to flow into and out of the cell.

Some of these channels are non-specific they allow any molecule up to certain size to flow through them; other channels are specific; only certain particular molecules can pass through them while prohibiting the entry of other molecules including antibiotic drugs into the cell; the cell wall channels protect the cell contents and enhance the likelihood of the cells of survival.

Cytoplasm is a viscid, thick, sticky substance that serves as the matrix fully interior contents of the cell; cytoplasm is made up primarily of water accounting for eighty percent of the total content; it also contains enzymes and nutrients such as carbohydrates, lipids, fats and other molecules.

In most bacteria the genetic material is contained in one single circular chromosome and contains all hereditary information required for bacteria; the chromosome consists of genes which made up of deoxyribonucleic acid (DNA)

DNA is a material that is composed of long and twisted strands of compounds called nucleotides arranged in a double helix pattern; DNA Carries the codes of reproducing specific proteins which determine characteristics or functions of the organism; in the higher organisms such as humans and plants chromosomes are enclosed in the cell nucleus; therefore these organisms are called eukaryotes; in contrast bacterial cells are prokaryotes since they lack of cell nucleus in addition to the existence of a single chromosome.

In addition to single chromosome; many bacterial cells it contains circular molecules of DNA called plasmid; it may carry genes the codes helps for resistance to antibiotics and for production of toxins both of which are important to bacterial survival; however the DNA contained in the plasmid is not for reproduction or other of basic cell function.

There are more than one type of ribonucleic acid RNA; ribosomal RNA and other proteins complex to form the ribosome; ribosomes are structured to serve as protein factories for the cell; because the cell cannot survive without proteins; ribosomes are vital to cell life like DNA; RNA is a component of all living cells and is composed of nucleotides and arranged in long strands that unlike the double stranded DNA; RNA is a single strand of nucleic acid and the primary function of RNA is the protein synthesis; the genetic code contained in the DNA is transcribed into messenger RNA which then travels to the ribosomes;  the genetic code is then translated into proteins.

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Pathogenic Sub-Type

In a human body normal flora a normally harmless bacteria that lives primarily on the skin and in the eyes, mouth, upper respiratory system, the gastrointestinal tract mainly in the large intestine (colon) and urogenital tract; however under certain conditions some of the normal flora may be become pathogenic then by causing opportunistic infections

The co-existence of bacteria and the human hosts is called symbiosis; normal flora existing one of three symbiotic relationships with the human hosts.

In commensalism the bacteria benefit from the relationship and the human host is neither harm nor benefit it; examples of commensals include the corynebacterium species that inhabit the tissues surrounding the eye and mycobacterium species that normally lives in the ear and external genitals; these bacteria use normal body secretions and died cells as nutrients and cause no harm to the host.

In mutualism both the bacteria and the host benefit; examples of mutualistic bacteria include the E. coli species that inhabit the large intestine; these bacteria live on the nutrients that pass through intestine and in return they produce essential vitamins use such as K and B for the human host.

In parasitism the bacteria benefit at the expense of the host; parasites in the human body are pathogenic bacteria that responsible for many diseases; the important bacterial sub-types that frequently cause diseases in humans are:-

• Cocci and Bacilli.
• Mycoplasmas.
• Mycobacteria.
• Spirochetes.
• Rickettsias.
• Nocardia Forms.

Cocci are round bacteria and the bacilli are rod shaped; pathogenic cocci and bacilli include gram positive, gram negative, aerobic or anaerobic sub-types.

Staphylococcus aureus which are gram positive aerobe cocci cause skin and lung infections; Pseudomonas aeruginosa which are gram negative aerobic bacilli cause numerous respiratory and urinary tract infections.

In contrast Mycoplasmas are very small bacteria that lake cell walls as a result they are irregularly shaped, they atypically facultative anaerobe; mycoplasmas such as Mycoplasma pneumoniae are primarily associated with respiratory infections.

Mycobacteria are a group of aerobic non-motile, non-endospore-forming rods; mycobacterium tuberculosis is responsible for tuberculosis and Mycobacterium leprae causes leprosy; the Mycobacterium avium, Mycobacterium intracellulare complex a group of microorganisms that rarely infect humans but it has now classified as one of the leading opportunistic infections associated with AIDS.

Spirochetes are motile bacteria with coiled shapes since they lack flagella they moved by rotating in a corkscrew fashion spirochetes can be an aerobic or anaerobic; the pathogenic species of spirochetes cause syphilis and Lyme disease.

Rickettsias are gram negative and non motile and can be either the rods or cocci; they also parasites since they most inhabit another organism to survive a and cannot reproduce outside a host cell; rickettsias are almost always transmitted to humans via insect and tick bite; vehicles diseases such as epidemic typhus and Rocky Mountains spotted fever

Chlamydia trachomatis are coccoid rickettsia is that can cause blindness and two types of sexually transmitted diseases (STDs) one of which Non-Gonococcal Urethritis is probably the most common STD in the U.S.

Finally Nocardia forms are aerobic rods; Nocardia asteroides can cause chronic tuberculosis like respiratory infection.

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