on 26-Sep-2025 (Fri)

Genetic variability may occur by a variety of mechanisms. Point mutations may occur at a nucleotide base pair (bp), which is referred to as microevolutionary change

Point mutations at crucial locations on “old” β-lactamase genes (e.g., genes for Temoneira-1 [TEM-1], sulfhydryl variable-1 [SHV-1]) are primarily responsible for the remarkable array of newly recognized extended-spectrum β-lactamases (ESBLs)

A second level of genomic variability in bacteria is referred to as a macroevolutionary change and results in whole-scale rearrangements of multiple nucleotide sequences as a single event. These rearrangements may include inversions, duplications, insertions, deletions, or the transposition of segments of DNA from one location of a bacterial chromosome or plasmid to another location in the genome. These large-scale alterations of the bacterial genome are frequently generated by specialized genetic elements such as integrons, transposons, or insertion sequences, which have the capacity to move and insert independently throughout the bacterial genome.

A third level of genetic variability in bacteria is created by the acquisition of large segments of foreign DNA carried by resistance (R) plasmids, bacteriophages, naked sequences of DNA, or specialized transposable genetic elements known as integrative and conjugative elements (ICE) from other bacteria.2

Inheritance of foreign DNA further contributes to bacterial genetic variability and its capacity to respond to selection pressures imposed by antimicrobial agents.3 These mechanisms endow bacteria with the seemingly unlimited capacity to develop resistance to any antimicrobial agent (Fig. 18.1).

When an antibiotic-resistance gene evolves, this gene can spread between bacteria by transformation, transduction, conjugation, or transposition.

Antibiotic-resistance genes existed well before the introduction of antimicrobials for treatment for human infections and can be found within bacterial genomes preserved in Arctic region permafrost samples untouched by human hands for over 30,000 years.11

synthesized by antibiotic-producing bacteria when they are about to enter the late static growth phase and are about to become dormant or sporulate. Antibiotic-producing bacteria are resistant to the antibiotics they produce. Therefore, antibiotics left in the microenvironment by dormant bacteria are avoided as potentially toxic to competitors yet serve as a ready source of carbon (food) for the next generation of the antibiotic-producing bacterial strain once the growth phase begins again

Aquatic environ- ments are particularly rich with bacterial populations replete with antibiotic-resistance genes.12

Although some antibiotic-resistance genes place a metabolic “burden” on bacteria, many microorganisms evolved strategies to limit this cost by repressing gene expression when not needed or by phase variation. This allows favorable, but sometimes “costly,” antibiotic-resistance genes to be held in reserve in the absence of antibiotic selection pressure yet express their resistance potential on reexposure to antibiotics.15

This is accomplished through at least two mechanisms: (1) species- and strain-specific DNA modifying enzymes (e.g., methylation of selected sequences of host DNA into specific patterns) and restriction enzymes that survey cellular host DNA and degrade foreign DNA that lacks appropriate DNA modification sequences; and (2) a type of adaptive defense system against foreign DNA known as CRISPR (clustered regularly interspaced short palindromic repeats).16

CRISPRs are detectable in nearly 50% of all bacterial genomes, and this genetic element protects their genomes from attack by foreign DNA during transformation, phage invasion, or plasmid insertion.

Recent evidence in the enterococci indicates that deletion of CRISPR elements is inversely related to multiple antibiotic-resistance development

Maintaining the fidelity of the host genome, while permitting limited variation by microevolutionary and macroevolutionary changes, allows pathogens to strike a balance between genomic stability and plasticity in rapidly changing microenvironments.

(A) Genetic exchange may occur by transforma- tion (naked DNA transfer for dying bacteria to a competent recipient). This generally results in transfer of homologous genes located on the chromosome by recombination enzymes (RecA).

(B) Transduction also may transfer antibiotic-resistance genes (usually from small plasmids) by imprecise packaging of nucleic acids by transducing bacteriophages.

(C) Conjugation is an efficient method of gene transfer, requiring physical contact between donor and recipient. Self-transferable plasmids mediate direct contact by forming a mating bridge between cells. Smaller nonconjugative plasmids might be mobilized in this mating process and be transported into the recipient.

(D) Transposons are specialized sequences of DNA that possess their own recombination enzymes (transposases), allowing transposition (“hopping”) from one location to another, independent of the recombination enzymes of the host (RecA independent). They may transpose to nonho- mologous sequences of DNA and spread antibiotic-resistance genes to multiple plasmids or genomic locations throughout the host. Some transposons possess the ability to move directly from a donor to a recipient, independent of other gene transfer events (conjugative transposons or integrative and conjugative elements)

Plasmids are autonomously replicating genetic elements that generally consist of covalently closed, circular, double-stranded DNA molecules ranging from less than 10 kilobase pairs (kbp) to more than 400 kb. They are extremely common in bacteria.14

Although multiple copies of a specific plasmid, or multiple different plasmids, or both may be found in a single bacterial cell, closely related plasmids often cannot coexist in the same cell. This observation led to a classification scheme of plasmids based on incompat- ibility (Inc) groups.1,4

Plasmids may determine a wide range of functions besides antibiotic resistance, including virulence and metabolic capacities. All plasmids possess an origin of replication for DNA polymerase to bind and replicate plasmid DNA. Plasmids must also retain a set of genes that facilitate their stable maintenance in host bacteria

Mercury released from dental fillings may increase the number of antibiotic-resistant bacteria in the mouth.19

Transposons can translocate as a unit from one area of the bacterial chromosome to another or between the chromosome and plasmid or bacteriophage DNA

Transposable genetic elements possess a specialized system of recombination that is independent of the generalized recom- bination system that classically permits recombination of largely homologous sequences of DNA by crossover events (the recA system of bacteria). The recA-independent recombination system (“transposase”) of transposable elements usually occurs in a random fashion between nonhomologous sequences of DNA and results in whole-scale modifica- tions of large sequences of DNA as a single event (Fig. 18.2).2,5

There are two types of transposable genetic elements, called trans- posons and insertion sequences, which have similar characteristics.

Transposons differ from insertion sequences in that they encode functional genes that mediate a recognizable phenotypic characteristic, such as an antibiotic- resistance marker.

These inverted- repeat DNA termini are essential to the transposition process. Trans- posons (Tn) and insertion sequences (IS) are incapable of autonomous self-replication and must exist on a replicon, such as the chromosome, bacteriophage, or plasmid, to be replicated and maintained in a bacterial population

Some transposons have the capability to move from one bacterium to another without being fixed within a plasmid or bacte- riophage. These elements are referred to as conjugative transposons, or integrative and conjugative elements (ICE)

An important variant of transposition is “one-ended” transposition, wherein only one end of the transposon is responsible for asymmetrical replication. This type of element is highly efficient in mobilizing resistance genes adjacent to its insertion site. These elements play a prominent role, along with ICE and integrons, in the evolution of large regions of the chromosome where multiple resistance genes accumulate into one set of resistance gene cassettes known as resistance genomic islands.2

These islands serve as convenient depots for insertions of new DNA without the risk of insertion into a critical metabolic or structural gene of the bacterial genome

The “fitness cost” of acquiring new resistance genes is minimized by deposition of genes in genomic resistance islands.2 However, the metabolic cost of retaining all these accessory, antibiotic-resistance genes by host bacteria remains. The extra metabolic burden is worth it only in the presence of repeated environmental antibiotic exposures, as might exist in hospitals and special care areas, such as the critical care unit, where antibiotic selection pressures favor multidrug-resistant organisms

Genetic analysis of sequences of DNA adjacent to antibiotic- resistance genes has revealed that unique integration units often exist near promoter sites.32 These integration elements, called integrons,33 function as recombina- tion “hot spots” for site-specific recombination events between largely nonhomologous sequences of DNA. Integrons facilitate the lateral transfer and integration of antibiotic-resistance genes from mobile gene cassettes.

Integrons do not transpose independently as a specific unit structure from one sequence of DNA to another. This capability of autonomous movement of large sequences of DNA is reserved primar- ily for transposons, insertion sequence elements, and bacteriophages. Integrons can become flanked, however, by transposable elements and become mobilized as an integrated structure into an existing transposon

Five classes of integrons that encode antibiotic-resistance genes have been recognized, with type 1 integrons being the most common in pathogenic microorganisms.36

The principal role of integrons is to provide a convenient insertion site for antibiotic-resistance genes from foreign DNA sources

Integrons also serve as expression cassettes for antibiotic-resistance genes in that an efficient promoter site is provided in close proximity to the 5′ end of the newly inserted DNA sequence. The frequency of transcription of integrated cassettes of antibiotic- resistance genes depends on the proximity of the gene to the promoter at the 5′ upstream end of the integron

The level of expression of a resistance gene diminishes as the distance between the promoter and the specific antibiotic-resistance gene cassette increases.31,32

At least eight distinctive mechanisms of antibiotic resistance have been described in bacteria (Tab le 1 8.1 ).

Resistance to β-lactam antibiotics occurs primarily through production of β-lactamases, enzymes that inactivate these antibiotics by splitting the amide bond of the β-lactam ring. They have likely coevolved with bacteria as mechanisms of resistance against natural antibiotics over time.39,40 The selective pressure exerted by the widespread use of anti- microbial therapy in modern medicine may have accelerated their development and spread.41–43

The first β-lactamase was described as a “penicillinase” capable of hydrolyzing penicillin in E. coli in 1940, about the same time as the first clinical use of penicillin was reported in the literature.41 The next years witnessed the rapid spread of plasmid-encoded penicillin resistance among the majority of S. aureus clinical isolates.42

Among gram-negative organisms, the rise in ampicillin resistance in the 1960s was ascribed to the emergence of TEM-1, a plasmid-encoded β-lactamase named after a Greek patient, Temoniera, in whom the first isolate was recovered.43

Similarly, both chromo- somally encoded and plasmid-mediated SHV-type β-lactamases, with a molecular structure related to TEM enzymes, became widely prevalent among E. coli and K. pneumoniae isolates.

The pharmaceutical industry’s development of third-generation cepha- losporins, initially stable to the action of TEM- and SHV-type

soon followed by the emergence and global spread of ESBL, capable of hydrolyzing monobactam and broad-spectrum cephalosporins.3,43–45

TEM-1 is the most common β-lactamase in gram-negative bacteria, and it can hydrolyze penicillins and narrow- spectrum cephalosporins in Enterobacteriaceae, N. gonorrhoeae, and H. influenzae.3

The extended-spectrum of activity of TEM-derived ESBLs occurs through changes in a single or a few amino acids that change the configuration of the enzyme at its active site, making it more accessible to the bulky R1 oxymino side chains of third-generation cephalosporins (cefotaxime, cefpodoxime, ceftazidime, ceftriaxone), and monobactam aztreonam

The first TEM-derived ESBL, TEM-3, was reported in 1988.48 There are now more than 220 TEM-derived ESBLs (see Lahey Clinic [Burlington, MA] website, www.lahey.org/Studies/temtable.asp).

These enzymes are found primarily in E. coli and K. pneumoniae isolates but also in other Enterobacteriaceae, such as Enterobacter aerogenes, Morganella morganii, Proteus spp., and Salmonella spp.

The majority of TEM-derived ESBLs remain susceptible to inhibition by clavulanic acid, although inhibitor-resistant TEM variants have also been described.49

The SHV-1 β-lactamase has a biochemi- cal structure similar to that of TEM-1 (68% of amino acids are shared48–50), and its ESBL derivatives are also produced by point mutations (one or more amino-acid substitutions) at its active site.

SHV-type β-lactamases are found primarily in K. pneumoniae strains.

Cefotaxime-M (CTX-M) β-lactamases are not evolutionarily related to SHV and TEM families because they are thought to have been acquired by plasmids from the chromosomal ampicillin C (AmpC) enzymes of Kluyvera spp., environmental gram-negative rods of low pathogenic potential.51

In general, the CTX-M family hydrolyzes cefotaxime and ceftriaxone better than ceftazidime, and they are inhibited more by tazobactam than by clavulanic acid,50 although point mutations leading to increased activity against ceftazidime can occur.

CTX-M enzymes have disseminated rapidly and are now among the most prevalent ESBLs worldwide.

Recent reports of community-acquired bloodstream infections with multire- sistant, CTX-M E. coli isolates from Spain and Israel have raised significant public health concerns.52,53 The ST131 (O25:H4) clone associated with the CTX-M-15 enzymes has emerged as an important multidrug-resistant pathogen and may have been responsible for the majority of infections with multidrug-resistant E. coli infections in Europe and the United States since 2007.54

Oxacillin (OXA)-type β-lactamases are also plasmid derived and hydrolyze oxacillin and its derivatives very effectively; they are poorly inhibited by clavulanic acid.42,47 OXA-derived ESBLs have been described mainly in P. a e r u g i n o s a , in which they confer high-level resistance to oxymino-β-lactams.47

AmpC β-lactamases are primarily chromosomal enzymes that confer resistance to penicillins, narrow- spectrum cephalosporins, oxymino-β-lactams, and cephamycins and are not susceptible to β-lactamase inhibitors such as clavulanic acid (molecular class C, functional group 1).55

Cefepime and aztreonam are usually poor substrates, although modulation by point mutations at the R2 loop of the active site have been responsible for variants with increased ability to hydrolyze cefepime.

AmpC production in gram-negative bacilli is normally repressed. However, a transient increase in production (10- to 100-fold) can occur in the presence of β-lactam antibiotics in the fol- lowing species that possess inducible AmpC enzymes: Enterobacter, Citrobacter freundii, Serratia, M. morganii, Providencia, and P. aerugi- nosa.55

AmpC β-lactamase production returns to low levels again after antibiotic exposure is discontinued, unless spontaneous mutations occur in the ampD locus of the gene, leading to permanent hyperproduction (derepression) in these species. Third-generation cephalosporin use in Enterobacter spp. infections can therefore select for the overgrowth of these stably derepressed mutants, leading to the emergence of antibiotic resistance during treatment.56

More than 20 plasmid-mediated AmpC enzymes, derived from chromosomally encoded genes in Enterobacteriaceae or Aeromonas spp., have been described in E. coli, K. pneumoniae, Salmonella enterica, and Proteus mirabilis. β-Lactam resistance attributable to this system appears to be increasing and confers a resistance phenotype similar to that of Enterobacter spp.44

Carbapenemases confer the largest antibiotic- resistance spectrum because they can hydrolyze not only carbapenems but also broad-spectrum penicillins, oxymino-cephalosporins, and cephamycins.

The K. pneumoniae carbapenemase (KPC) enzymes are currently the most important class A serine carbapenemases.

Since initially reported from K. pneumoniae isolates in several northeastern US outbreaks,57 KPCs have been found worldwide in multiple other gram-negative species, such as E. coli, Citrobacter, Enterobacter, Salmo- nella, Serratia, and P. aeruginosa.6

Class B MBLs use a Zn2+ cation for hydrolysis of the β-lactam ring; are susceptible to ion chelators, such as ethylenediaminetetraacetic acid (EDTA); and are resistant to clavulanic acid, tazobactam, and sulbactam. They confer resistance to all β-lactam antibiotics except monobactams.

Chromosomally encoded MBLs are primarily found in environmental isolates of Aeromonas, Chryseobacterium, and Stenotrophomonas spp. and are of usually low pathogenic potential.58 Most clinically important MBLs belong to five different families (imipenem [IMP], Verona integron-encoded metallo-β-lactamase [VIM], Sao Paulo metallo-β- lactamase [SPM], German imipenemase [GIM], and Seoul imipenemase [SIM]), typically transmitted by mobile gene elements inserted into integrons and spread through P. aeruginosa, Acinetobacter, other gram- negative nonfermenters, and enteric bacterial pathogens

The New Delhi metallo-β-lactamase–1 (NDM-1) has received the most attention recently. Originally described in a K. pneumoniae isolate from India in 2008, NDM-1 enzymes have since been reported in the United States, the United Kingdom, and a number of other countries, primarily in connection with travel to India or Pakistan.59,60 These enzymes confer resistance to all β-lactams except aztreonam. However, most MBLs reside on mobile gene cassettes inserted into integrons that harbor additional antibiotic-resistance genes to other antimicrobial classes; this multidrug resistance can be transferred to other species via transposons and plasmids, severely limiting therapeutic options in serious infections.61

Recent meta-genomic analyses have indicated that the majority of MBLs probably originate from Shewanella spp. and that the genes for many more, as yet uncharacterized, metallo-enzymes are present in environmental and commensal bacterial species.62

Finally, class D carbapenemases have been described among four subfamilies of OXA-type β-lactamases (OXA-23, OXA-24, OXA-58, and OXA-146), primarily in A. baumannii. The intrinsically weaker carbapenemase activity is augmented by coupling β-lactamase production with an additional resistance mechanism, such as decreased membrane permeability or increased active efflux.63

Among gram-positive bacteria, staphylococci are the major pathogens that produce β-lactamase. Staphylococcal β-lactamases preferentially hydrolyze penicillins. Most are inducible and are excreted extracellularly.3

The β-lactamases produced by Bacteroides fragilis are predominantly cephalosporinases, some of which have been found to hydrolyze cefoxitin and imipenem and may be transferable.71

The efficiency of the β-lactamase in hydrolyzing an antibiotic depends on (1) its rate of hydrolysis and (2) its affinity for the antibiotic. Other variables are (3) the amount of β-lactamase produced by the bacterial cell, (4) the susceptibility of the target protein (penicillin-binding protein [PBP]) to the antibiotic, and (5) the rate of diffusion of the antibiotic into the periplasm of the cell.

Another model is exemplified by gram-negative bacteria, which (1) produce a β-lactamase that remains trapped in the periplasmic space and (2) have no barrier to antibiotic penetration. An example is H. influenzae strains that produce the TEM-1 β-lactamase.72 In this model and the first one discussed, a marked inoculum effect occurs in that the MIC for a large inoculum (106 colony-forming units [CFUs] per milliliter) may be 1000-fold greater than that for a small inoculum (102 CFUs/mL). Lysis of the organism by ampicillin releases the trapped β-lactamase into the microenvironment, providing partial protection to adjacent bacteria residing in the same location. If a high inoculum exists before ampicillin exposure occurs, the release of all the periplasmic β-lactamase enzymes in a confined space might be sufficient to protect some of the remaining viable bacteria of the original population of microorganisms.

When Enterobacter strains are exposed to two β-lactam antibiotics, one of which is a potent inducer (e.g., cefamandole), antagonism between the two antibiotics may result.70

Among aerobic bacteria, aminoglycoside resistance is most commonly due to enzymatic inactivation through aminoglycoside-modifying enzymes.

Aminoglycoside phos- photransferase (APH)(3′) and APH(3″) are distributed widely among gram-positive and gram-negative species worldwide and have led to decreased use of kanamycin and streptomycin.

The two most interesting developments in aminoglycoside-modifying enzymes have been the discovery of bifunctional enzymes. The first example is the AAC(6′) APH(2″) enzyme, which has two functioning active sites, one for acetylation and the other for phosphorylation of aminoglycosides. This bifunctional enzyme probably arose from a fusion event of the genes for these two enzymes. This enzyme is now widespread in staphylococci and enterococci, frequently residing on a common transposon Tn4001 found on the chromosome and on transferable plasmids.

Recently, another major discovery with regard to aminoglycoside- modifying enzymes involves evidence of variant enzymes that can modify the structure of an entirely different class of antimicrobial agent. The first bifunctional enzyme that can modify aminoglycosides and a fluo- roquinolone (ciprofloxacin) was described in 2006.80

AAC(6′)-Ib-cr, not only acetylates kanamycin, gentamicin, and tobramycin, but also acetylates the piperazinyl side group of cip- rofloxacin. This acetylated quinolone is fourfold less active than the parent compound and may lead to clinically significant resistance in enteric bacteria, particularly in strains that possess other mechanisms for diminished quinolone activity.

The minimum requirements for growth are a source of carbon and nitrogen, an energy source, water, and various ions.

Iron is so important that many bacteria secrete special proteins (siderophores) to concen- trate iron from dilute solutions, and our bodies will sequester iron to reduce its availability as a means of protection

Oxygen (O2 gas), although essential for the human host, is actually a poison for many bacteria. Some organisms (e.g., Clostridium perfringens, which causes gas gangrene) cannot grow in the presence of oxygen

Aerobic bacteria produce superoxide dismutase and catalase enzymes, which can detoxify hydrogen peroxide and superoxide radicals that are the toxic byproducts of aerobic metabolism

Bacteria that can rely entirely on inorganic chemicals for their energy and source of carbon (carbon dioxide [CO2]) are referred to as autotrophs (lithotrophs), whereas many bacteria and animal cells that require organic carbon sources are known as het- erotrophs (organotrophs)

The metabolism of normal flora bacteria is optimized for the pH, ion concentration, and types of food present in their environment within the body.

All cells require a constant supply of energy to survive. This energy is derived from the controlled breakdown of various organic substrates (carbohydrates, lipids, and proteins). This process of substrate breakdown and conversion into usable energy is known as catabolism. The energy produced may then be used in the synthesis of cellular constituents (cell walls, proteins, fatty acids, nucleic acids), a process known as anabolism. Together these two processes, which are inter- related and tightly integrated, are referred to as intermedi- ary metabolism.

The metabolites are converted via one or more pathways to one common univer- sal intermediate, pyruvic acid. From pyruvic acid, the carbons may be channeled toward energy production or the synthesis of new carbohydrates, amino acids, lipids, and nucleic acids

high-energy phosphate bond in adenosine triphosphate (ATP) or guanosine triphosphate (GTP), whereas electro- chemical energy is stored by reduction (adding electrons to) of nicotinamide adenine dinucleotide (NAD) to NADH or flavin adenine dinucleotide (FAD) to FADH2. The NADH can be converted by a series of oxidation-reduction reactions into chemical (pH) and electrical potential gradients (Eh) across the cytoplasmic membrane.

Bacteria can produce energy from glucose by—in order of increasing efficiency—fermentation, anaerobic respiration (both of which occur in the absence of oxygen), or aerobic respiration.

In yeast, fermentative metabolism results in the conversion of pyruvate to ethanol plus CO2. Alcoholic fermentation is uncommon in bacteria, which most commonly use the one- step conversion of pyruvic acid to lactic acid. This process is responsible for making milk into yogurt and cabbage into sauerkraut.

Other bacteria use more complex fermentative pathways, producing various acids, alcohols, and often gases (many of which have vile odors). These products lend flavors to various cheeses and wines and odors to wound and other infections

The TCA cycle allows the organism to generate substan- tially more energy per mole of glucose than is possible from glycolysis alone.

Whereas fermentation produces only 2 ATP molecules per glucose, aerobic metabolism with electron transport and a complete TCA cycle can generate as much as 19 times more energy (38 ATP molecules) from the same starting material (and it is much less smelly)

In addition to efficient generation of ATP from glucose (and other carbohydrates), the TCA cycle provides a means by which carbons derived from lipids (in the form of acetyl CoA) may be shunted toward either energy production or the generation of biosynthetic precursors.

The last two functions make the TCA cycle a so-called amphibolic cycle (i.e., it may function to break down and synthesize molecules)

Bacteria usually have only one copy of their chromosomes (they are therefore haploid), whereas eukaryotes usually have two distinct copies of each chromosome (they are therefore diploid).

With only one chromosome, alteration of a bacterial gene (mutation) will have a more obvious effect on the cell. In addition, the structure of the bacterial chromosome is main- tained by polyamines, such as spermine and spermidine, rather than by histones

In addition to protein-structural genes (cistrons, which are coding genes), the bacterial chromosome contains genes for ribosomal and transfer ribonucleic acid (RNA). Bacterial genes are often grouped into operons or islands (e.g., pathogenicity islands) that share function or to coor- dinate their control. Operons with many structural genes are polycistronic

Bacteria may also contain extrachromosomal genetic elements such as plasmids or bacteriophages (bacterial viruses). These elements are independent of the bacterial chromosome and in most cases can be transmitted from one cell to another

RNA synthesis is performed by a DNA-dependent RNA poly- merase. The process begins when the sigma factor recog- nizes a particular sequence of nucleotides in the DNA

Promoter sequences occur just before the start of the DNA that actually encodes a protein

Sigma factors bind to these promoters to provide a docking site for the RNA polymerase. Some bacteria encode several sigma factors to coordinate transcription of a group of genes under special conditions such as heat shock, starvation, special nitrogen metabolism, or sporulation

Once the polymerase has bound to the appropriate site on the DNA, RNA synthesis proceeds with the sequential addition of ribonucleotides complementary to the sequence in the DNA. Once an entire gene or group of genes (operon) has been transcribed, the RNA polymerase dissociates from the DNA, a process mediated by signals within the DNA

Each tRNA molecule con- tains a three-nucleotide sequence complementary to one of the codon sequences. This tRNA sequence is known as the anticodon; it allows base pairing and binds to the codon sequence on the mRNA. Attached to the opposite end of the tRNA is the amino acid that corresponds to the particular codon-anticodon pair