Antimicrobials in practice part 3: treating resistant infections (Proceedings)

Treatment failures can occur due to the presence of resistant bacteria, such as methicillin resistant Staphylococcus aureus/pseudintermedius, extended-spectrum beta-lactamase producing Enterobacteriaceae, or vancomycin resistant enterococci.  Additionally, treatment failure can occur due to a failure to culture and identify the bacteria responsible for infection.  A frequent cause of this is anaerobic infections, where the cultures must be handled under special conditions.  The emergence of antimicrobial resistance among anaerobic isolates is also increasing, particularly for such bacteria as Bacteroides fragilis.  Infections caused by these types of bacteria can be extremely difficult and frustrating to treat.  The following are some options for treatment in companion animals.

Types of resistant bacteria

Methicillin-resistant Staphylococci

Some strains of S. aureus have developed resistance to the anti-staphylococcal penicillins, such as methicillin. Because of this, they are termed methicillin-resistant S. aureus, or MRSA.  MRSA has become a serious cause of nosocomial infections in human and veterinary medicine worldwide. The resistance in MRSA is mediated via the mecA gene which encodes for penicillin binding protein 2a (PBP2a) that has a very low affinity for beta-lactam antibiotics. The incidence of MRSA infections in dogs and cats has increased in recent years, and the majority are associated with post-operative infections, open wounds, and surgical implants. In horses, isolation rates have been reported to be as high as 5.3% in tertiary care facilities.  Resistance patterns and genetic screening of MRSA from dogs and humans are virtually identical, therefore cross-contamination from human to animal and animal to human is likely to occur.  Other methicillin resistant staphylococci have been reported to occur in animals, including S. intermedius and S. pseudintermedius.  A recent phylogenetic study has shown that S. pseudintermedius, not S. intermedius, is the common cause of canine pyoderma, whereas S. intermedius is the species associated with pigeons.  Accordingly, the canine pathogen is likely to be reclassified as S. pseudintermedius.

Extended spectrum beta-lactamase bacteria

Gram-negative bacteria commonly produce beta-lactamases that inactivate penicillins, but not the extended spectrum cephalosporins.  In the last few decades, bacteria (mainly E. coli and Klebsiella sp) have been discovered that produce what are known as extended-spectrum beta-lactamases (ESBL).  These enzymes hydrolyze the extended-spectrum cephalosporins that have an oxyimino side chain.  These cephalosporins include cefotaxime, ceftriaxone and ceftazidime.  The expression of ESBLs is plasmid mediated, and these plasmids often encode for other genes that infer resistance to antimicrobials of other classes (ie aminoglycosides).

Enterococci

Enterococcal organisms, particularly E. faecalis and E. faecium, are opportunistic pathogens that are considered commensal organisms in the GI tract. They a not typically virulent, but are often multi-drug resistant. They most commonly infect the urinary tract in small animals and are frequently cultured in combination with E.coli.  If they are cultured in combination, we recommend treating the other bacterium, and then reculturing the urine. In many instances, clearing the primary organism will allow the immune system to clear the enterococcal infection.  If this is the only bacterium cultured and the animal is showing clinical signs, treatment options are often limited. They are typically sensitive to vancomycin, however vancomycin-resistant enterococci (VRE) are increasing. Acquired vancomycin resistance is due to the acquisition of van genes (van A, vanB, vanD, vanE, vanG) that result in the production of peptidoglycan precursors with reduced affinity for glycopeptides antibiotics.

Antimicrobials for treatment of resistant bacterial infections

Due to the emergence of many resistant bacteria in human medicine, newer antibiotics are being developed to combat these infections. Currently, their use is not recommended in veterinary medicine without documented sensitivity testing.  Under no circumstances should these antibiotics be used as a first line treatment for disease.

Carbapenems

The carbapenems are beta-lactam antibiotics and include the drugs imipenem (in combination with cilastatin), meropenem and ertapenem.  These drugs have a very broad spectrum of activity that includes bacteria that are resistant to most other drugs.  The only notable exceptions include MRSA and resistant Enterococcus faecium.  The high activity of these drugs is due to the ability to withstand the beta-lactamases (including ESBL) and the ability to penetrate porin channels that usually exclude other drugs.  They are more rapidly bactericidal and less likely to cause endotoxin release following administration to septic animals.  Of the drugs in this class, meropenem may be the best choice for use in animals because it is easily administered SC and is less likely than imipenem to be nephrotoxic.  The recommended dose for Enterobactericeae and other sensitive organisms is 8.5 mg/kg SC every 12 hr, or 24 mg/kg IV every 12 hr. For infections caused by Pseudomonas aeruginosa, or other similar organisms that may have MIC values as high as 1.0 μg/mL, the recommended dose is 12 mg/kg q8h, SC, or 25 mg/kg q8h, IV.

Glycopeptides

Often, the only effective drug against MRSA and Enterococcus species will be a glycopeptide. Of the glycopeptides, vancomycin is the only one used in veterinary medicine. Vancomycin has been given as an IV infusion administered over 30 to 60 minutes. It is not absorbed orally and is too painful when injected IM. The dose to maintain concentrations within the therapeutic range, and avoid toxicity is 15 mg/kg, q6h, IV. For successful therapy of serious infections, an aminoglycoside such as gentamicin or amikacin should be administered with vancomycin.

Oxazolidinones

Linezolid is the first in the class of oxazolidinones to be used in medicine. It works by binding to the bacterial 23S ribosomal RNA of the 50S subunit and prevents the formation of a functional 70S initiation complex. It is currently being used in people to treat vancomycin resistant gram-positive infections caused by enterococci and streptococci, as well as MRSA. Linezolid is absorbed orally and has been used successfully in dogs and cats at NCSU for the treatment of MRSA at a dose of 10 mg/kg PO or IV q8-12h. Linezolid is available in 400 and 600 mg tablets ($53 per tablet!), oral suspension, and injection.

Glycylcyclines

Tigecycline is the first in a new class of glycylcyclines with activity against susceptible or multidrug-resistant staphylococci, enterococci or streptococci as well as most Enterobacteriaceae and anaerobic pathogens. It has recently been approved for use in humans for the treatment of skin, soft-tissue and intra-abdominal infections.  Its use in veterinary medicine has not been reported.

Streptogramins

Quinupristin and dalfopristin are straptogramin antibiotics that are used in human medicine for cases of MRSA and VRE. They work in concert at the level of the bacterial ribosome to inhibit protein synthesis.  Dalfopristin works in the early phases of protein synthesis, while quinupristin works on the later phases. Veterinary use has not been reported.

Daptomycin

Daptomycin represents a class of antibiotics known as the peptolides.  It is active against a wide variety of bacteria, including gram-positives, gram-negatives, MRSA and VRE. The mechanism of action involves the disruption of amino acid transport by the cell membrane and alterations of the cytoplasmic membrane potential. The bactericidal activity of daptomycin is concentration dependent and is influenced by pH and ionized calcium concentrations.

Anaerobic infections

Anaerobic infections in animals are most commonly caused by Clostridium, Actinomyces, Bacteroides, Fusobacterium and Peptostreptococcus spp.  These bacteria are classified as obligate anaerobes, and have varying degrees of tolerance to oxygen.  They are commensal organisms found in the gastrointestinal tract, particularly in the large intestines, and other mucosal surfaces such as the oropharynx and vagina.  Infections generally follow contamination from these sites and include pyometra, pyothorax, osteomyelitis, fasciitis, peritonitis and bite wound abscesses.  The pathology associated with anaerobic infections is due to the elaboration of enzymes and toxins from the bacteria that result in tissue damage and necrosis, setting up further proliferation of the bacteria by enhancing the anaerobic environment.  Furthermore, anaerobic infections are often polymicrobial, with a symbiotic relationship developing.

Clinicians may become suspicious of an anaerobic bacterial infection if a putrid odor and tissue gases are present.  Other distinguishing clinical signs include black wound exudates, necrotic tissue and bone sequestration.  Infections in closed body cavities such as the thorax and abdomen have a high likelihood of anaerobic infections and should be treated as such.  Pyogenic infections that do not culture positive for bacteria using routine methods, and infections that do not respond to antibiotic therapy with aminoglycosides or sulfonamides should also be treated as anaerobic.  Gram stains and anaerobic cultures can be used to definitively diagnose the infection.

For treatment of anaerobic infections, surgical excision and lavage of devitalized tissue should be the primary step, whenever possible.  Medical therapy accompanies surgery, or is used as the primary therapy in cases where surgery is not possible.  Therapy is often prolonged and can be difficult, due to the lack of blood flow to the necrotic tissue.  Additionally, there are high concentrations of bacteria in areas such as abscesses, and these bacteria may produce toxins that inactivate the antibiotics.  Culture and sensitivity results are rarely available for anaerobic infections, therefore therapy is often empiric and it is essential to know the spectrum of activity as well as the ability of the drug to penetrate into the tissues when choosing an antibiotic.  Rational choices include beta-lactams, nitroimidazoles, lincosamides and chloramphenicol.  The development of antibiotic resistance among anaerobic bacteria also appears to be increasing, particularly among Bacteroides and Clostridium spp.

Mechanisms of resistance in anaerobes

Numerous mechanisms of resistance have been determined in various anaerobic bacteria, and many of them are common among multiple isolates.  The horizontal transmission of antibiotic resistance genes within and from anaerobic bacteria is an important factor to consider.  Bacterial conjugation is the most common method of gene transfer, and can occur via transposon, plasmid and chromosomal transfer. Some of the genes involved in anaerobic resistance include erm (erythromycin ribosomal methylase), nim (nitroimidazole resistance genes), and ccrA and cfiA (class B beta-lactamase) genes.

Antimicrobials for treatment of anaerobic infections

Beta-lactams

The penicillins have historically been the drugs used most frequently in anaerobic infections.  The mechanism of action of the penicillins against anaerobic bacteria is identical to their action against aerobic bacteria.  This involves inhibiting cell wall synthesis.  They are bactericidal and time-dependent.  Toxicity is minimal and numerous inexpensive drugs are available.  Penicillin G remains the drug of choice for clostridial and peptostreptococcal infections.  It also has good activity against most Actinomyces and Fusobacterium isolates.  Bacteroides species are sometimes sensitive, with the exception of B. fragilis.  The aminopenicillins (ampicillin and amoxicillin) have a similar spectrum of activity to penicillin G.  Amoxicillin typically achieves higher serum concentrations following parenteral and oral administration, therefore it is preferred over ampicillin.  Additionally, when combined with clavulanic acid, amoxicillin has much higher activity against Bacteroides species due to the inhibition of beta-lactamase by the clavulanic acid.  The antistaphylococcal penicillins (oxacillin and cloxacillin) are less active against anaerobes, and therefore not recommended.  Extended spectrum penicillins (including ticarcillin and piperacillin) offer no advantage in treatment to penicillin G, except in cases of B. fragilis infections. 

The cephalosporins have a similar activity against anaerobic bacteria as the penicillins do, with the notable exception of Clostridium sp, which have limited susceptibility to cephalosporins.  In general, the first-generation cephalosporins (cephalexin, cephalothin, cephapirin, cefazolin and cefadroxil) are more active than other second or third generation cephalosporins.  However, Bacteroides species are often resistant.  The second generation cephalosporins, cefoxitin and cefotetan, are considered the antibiotic of choice for B. fragilis infections.  Since they are also fairly broad spectrum, with activity against both gram-positive and gram-negative infections, they can be used successfully as a monotherapy for mixed bacterial infections.  Cefotaxime, a third-generation cephalosporin is also useful against most anaerobes, however it is more expensive than cefoxitin. The carbapenems are highly active against gram-positive, gram-negative and anaerobic bacteria, more so than the other beta-lactam antibiotics.  They are relatively non-toxic, however their expense precludes the use of these antibiotics in most cases.

Resistance of anaerobic bacteria to beta-lactam antibiotics can occur in a variety of ways.  The majority of clinically isolated B. fragilis are beta-lactamase producers. Most of the enzymes are chromosomally mediated cephalosporinases, with activities against many narrow- and broad-spectrum penicillins and cephalosporins. These enzymes are inhibited by beta-lactamase inhibitors and were assigned to group 2e and molecular class A, being encoded by chromosomal or rarely plasmid cepA gene. So, the preferred beta-lactams for treatment of B. fragilis-associated infections include cefoxitin, beta-lactam/beta-lactamase inhibitor combinations and carbapenems. However, the production of a metallo-β-lactamase by B. fragilis strains has compromised the clinical use of cefoxitin and more recently of carbapenems. This enzyme, encoded by the chromosomal or plasmid ccrA and cfiA genes, and belonging to molecular class B and functional group 3, hydrolyses a broad spectrum of β-lactams, including cephamycins and carbapenems.  Other mechanisms of resistance to β-lactams include low affinity penicillin binding proteins (altered PBP2 or PBP 1) and reduced cell wall permeability.

Nitroimidazoles

Of the nitroimidazole antibiotics, metronidazole is most commonly used in veterinary medicine. Other drugs include ronidazole and tinidazole.  They have activity against protozoa and anaerobic bacteria, but no activity against other bacteria.  The mechanism of action involves the reduction of the nitro group on the antibiotic by nitroreductases produced by susceptible bacteria.  This results in the formation of highly reactive intermediates that disrupt bacterial DNA.  These antibiotics are only active in anaerobic conditions because oxygen will compete with the antibiotic for electrons necessary in the nitroreductase reaction.  Metronidazole is well absorbed following oral administration and has excellent distribution into tissues, including the CNS and abscesses.  It has quick onset of action and is rapidly bactericidal.  Metronidazole has excellent activity against B. fragilis, but is poor against Actinomyces and Proprionobacterium. 

Reduced susceptibility to 5-nitroimidazole drugs is generally associated with the presence of a nitroimidazole reductase encoded by a nim gene. This enzyme converts 4- or 5-nitroimidazole to 4- or 5-aminoimidazole, thus avoiding the formation of the toxic nitroso radicals that are essential for antimicrobial activity. Currently, at least 8 nim genes have been described in Bacteroides and Prevotella spp. 

Lincosamides

Clindamycin is a lincosamide antibiotic that has bacteriostatic activity against gram-positive aerobic bacteria and most anaerobes.  It works through inhibition of peptide synthesis by binding to the 50s ribosome subunit.  It is well absorbed orally, and drug concentrations are often higher in the tissues than in the plasma.  The one exception to this is the central nervous system, as concentrations in the CSF are generally low.  Clindamycin has been considered the gold standard for treatment of anaerobic infections in humans as well as small animals.  It has reliable activity against most Bacteroides sp and has been proven to be effective in cases of pyothorax and lung abscesses.  Clindamycin reaches higher concentrations in abscesses than either penicillin or chloramphenicol, and it accumulates within leukocytes by an energy-dependent process, making it highly effective in cases of intracellular bacteria.  Lincomycin, another lincosamide antibiotic, is not as effective as clindamycin against anaerobic bacteria.

Resistance to clindamycin among certain anaerobic bacteria has been steadily increasing over the last few decades, particularly among Bacteroides and Clostridium spp.  In humans, Bacteroides resistance has been reported to be as high as 44%, while Cl. difficile resistance can be as high as 67%.  In small animals, up to 17% of Bacteroides sp and 20% of Clostridium sp are reported to be resistant. Resistance is mediated by various erm genes which have been demonstrated to be expressed on Bacteroides (particularly B. fragilis), Cl. difficile, Cl. perfringens, Prevotella, Porphyromonas, Peptostreptococcus and Eubacterium spp. 

Chloramphenicol

Chloramphenicol is a naturally occurring antibiotic that works through a similar mechanism to clindamycin.  It binds to the bacterial ribosomal 50s subunit, inactivating peptidyl transferase enzymes, and preventing peptide bond formation.  At physiologic concentrations, it is bacteriostatic.  Following absorption, it is widely distributed to most tissues in the body, including the eye, the CSF and the prostate.  Chloramphenicol is metabolized by hepatic glucuronyl transferase, an enzyme which is deficient in cats and neonates.  Chloramphenicol has high in vitro activity against anaerobic bacteria and is an excellent choice for empirical therapy.  It also has a broad spectrum of activity against aerobic bacteria, making it good for mixed population infections. 

Resistance to chloramphenicol in Bacteroides species (in particular, B. fragilis) and Cl. perfringens, arises due to the enzymatic inactivation of the drug by acetylation. When resistance occurs in other genera, it is typically plasmid determined; it is also plasmid associated in resistant strains of Cl. perfringens and B. ochraceus.

Vancomycin

Vancomycin has activity against some Gram-positive anaerobic bacteria, but is not effective against Gram-negative anaerobic bacteria.  It is used in human medicine for the treatment of Cl. difficile diarrhea. Clostridial resistance to vancomycin is extremely rare.  The van genes reported to occur in various enterococci have not been found in clostridial species.  

Drugs that do not have activity against anaerobic bacteria

Aminoglycoside antibiotics are not effective against anaerobes because drug uptake across the bacterial cell membrane requires enzymes lacking in anaerobic bacteria and the drug uptake is an oxygen-dependent process.  Fluoroquinolones used in veterinary medicine have a decreased activity under anaerobic conditions due to an inability to penetrate bacteria, similar to the aminoglycosides.  Ciprofloxacin is bactericidal against normally susceptibile bacteria, like E. coli and S. aureus, however it becomes bacteriostatic under anaerobic conditions.  Newer fluoroquinolone antibiotics (ie trovafloxacin) have structural changes that increase their activity against anaerobes.  Anaerobic bacteria can also develop DNA gyrase mutations that create fluoroquinolone resistance, similar to aerobic bacteria. Tetracycline antibiotics exhibit widespread resistance to anaerobes, as does erythromycin.  Sulfonamides and trimethoprim-sulfonamide combinations show some in vitro activity against anaerobes, however in vivo, efficacy is poor.  This may be due to the production of enzymes, such as para-aminobenzoic acid, a product of cellular breakdown that inhibits the activity of sulfonamides.