Introduction
The wise stewardship of antimicrobial use begins with an understanding of the impact of the drug on microbial populations. Microbiomes are the innocent bystanders of antimicrobial therapy; the potential impact antimicrobials may have on the host is among the reasons to de-escalate antimicrobial therapy. The impact of antimicrobials, and other drugs, is an adverse event that is insidious in its presentation. This manuscript will focus on the impact of drugs, especially antimicrobials, on host microbiota.
Microbiota/Microbiome
Defined: Microbiota are ecological communities of commensal, symbiotic and pathogenic microorganisms. The populations, or taxa, may be defined based on a particular site (ocean, atmospheric, skin, gastrointestinal tract), habitat or period (perhaps including age). Microbiome is used to refer to the population or the collective genome of that population. The two terms are often used interchangeably.1 In general, acquisition of the microbiome occurs after birth through contact with maternal and environmental microbes,2 thus helping to shape the immune and nutritional statuses3. Although the most commonly recognized microbiome occurs in the gastrointestinal tract, other microbiomes exist in the body. Depending on the species, microbiomes have been described for the skin,4 reproductive tract,5 and (using culture-independent methods) organs previously thought to be sterile such as the lung,6 urinary tract,7 and even the eye8 and brain.
The Human Microbiome Project (https://hmpdacc.org/) of the National Institutes of Health was established in 2008 and provides a good foundation for persons interested in exploring the scope of research regarding interactions between microbiomes and the human body, and the subsequent impact on both health and disease. Among the pertinent observations is that the number of microorganisms making up the microbiota is estimated to outnumber human somatic and germ cells by a factor of 10. The number of genes in the human gut microbiome is 150 times more than the human genome. Although bacteria are the most commonly acknowledged organisms, the importance of others increasingly is being recognized (e.g., fungi [mycobiome], archaea, viruses). Regardless of the location in the body, the microbiome is diverse and dynamic, being influenced by many factors, including host genotype, lifestyle, environment (including other household members, which also include pets9) and disease. Using the gut microbiome as an example, multiple factors influence the composition, including age, genetics, diet, and drugs (as reviewed by10).
Role of the Microbiota in Health
Gut microbiome: Description of the gut microbiome is based largely on molecular (16S ribosomal subunits) rather than culture techniques.2 The population (at least in the large intestine) reflects approximately 99% oxygen intolerant/obligate anaerobes. Although the microbiome is markedly variable, in the human gastrointestinal tract, Bacteroidetes, Firmicutes (together, representing about 90% of all bacterial species), Actinobacteria, Proteobacteria and Cerrucomicrobia are the primary phyla represented.11 Using the gut as an example,2,12 direct effects of the microbiota include protection against pathogens by competing for nutrients, influencing the local environment (altering local pH and O2), and producing compounds (biocidins, antimicrobial peptides) that inhibit microbes or alter their virulence. Indirect effects of commensals include providing signals that influence maturation and organization of mucosal-associated lymphoid tissue (Th17 and Tregs; IgA production); induction of mucin production by goblet cells and antimicrobials by Paneth cells. Information regarding specificity of immune responses for certain bacteria (e.g., Clostridium and T cell differentiation) is emerging; vaccine efficacy in particular is emerging as a therapeutic intervention that may be impacted.3 The commensal microbiome can confer immune-mediated resistance against pathogens in the gut, but also other organs: the gut microbiota appears to be particularly important to mucosal tolerance in the lung.6
Other microbiomes have been described. The importance of the skin microbiome to healthy skin has been understood: dysbiosis of the skin can be transferred from one animal to another.4 Reductions in Streptococcus or Propionibacterium have been associated with disease, and Acinetobacter may protect against skin allergies.13 The urinary tract, including urine, is not sterile. Whereas culture and susceptibility (C&S) is the gold standard for verifying the presence of prototypic rapidly growing aerobic uropathogens, the development of molecular techniques has allowed demonstration of the presence and emerging role of more slowly growing organisms in the urinary microbiome. Enhanced C&S techniques now allow identification of bacteria in otherwise “sterile” urine. The role these microbiomes have in complicating interpretation of culture and susceptibility testing remains to be addressed. Lactobacillus appears to be an important member of the urinary microbiome. Lactobacillus also appears to be a dominant species in vaginal microbiomes, with those producing lactic acid potentially being particularly helpful in preventing infection. Other lactobacilli that interface with anaerobic organisms, on the other hand, may increase the risk of bacterial vaginosis. Pregnancy decreases diversity, perhaps decreasing the risk of ascending infection.5
Organisms found in the lung microbiome of healthy humans include Pseudomonas, Streptococcus, Prevotella, Fusobacterium and Veillonella.6 The microbiota in the unhealthy lung is profoundly complex, with up to 60 species, for example, found in patients with cystic fibrosis. Bronchial hypersensitivity of asthma and chronic obstructive pulmonary disease also is associated with over 100 taxa, but particularly Proteobacteria. Even the brain may have a microbiome, as is being demonstrated again as a result of advances in sequencing technologies.
Dogs and cats: Information regarding microbiomes of dogs and cats is limited to the gastrointestinal microbiome. The gut microbiome has been described for dogs14-16 and cats.17 The canine and human microbiomes are similar (compared to pig or mice), including response to diets,18 and the relationship between human and animal microbiomes2,9 has been described. Sharing of microbes has been demonstrated between pets, their people, and other persons.19 Response of the canine microbiome to pre-, pro- and synbiotics has been described.20
Role of the Microbiota in Disease
It is beyond the scope of this paper to delineate the evidence supporting the role of the microbiome in diseases, but a synopsis is provided here. The breadth of diseases now being considered impacted by an unhealthy microbiome is broad, including gastrointestinal diseases (including inflammatory bowel diseases and cirrhosis), immune-mediated diseases, including allergies, skin diseases (e.g., atopy), cardiovascular diseases, metabolic diseases such as diabetes and obesity, to neurologic diseases including behavioral disorders, epilepsy, and substance abuse, and cancer. The term “dysbiosis” is emerging as a common terminology to refer to the role of an unbalanced microbiome. The imbalance can reflect the loss of attributes of the microbiome. Humans: Dysbiosis of the gut microbiome has been associated with inflammatory bowel diseases, with microbes being essential to the inflammatory component. Diversity decreases, particularly Firmicutes and Bacteroides and Saccharomyces. Selected organisms increase in population (Basidiomycota, Ascomycota, and Candida albicans.10,21 Populations of adherent, invasive bacteria increase, although the role of cause and effect is not clear.21 Although the prevalence of small intestinal bacterial overgrowth has been demonstrated to be greater in human patients with IBD compared to controls, a cause-effect relationship remains controversial.22 Inflammation may favor the growth of Enterobacteriaceae with these populations decreasing as inflammation decreases. Fusobacterium is another inflammatory/invasive organism that may be associated with IBD. The relationship between the GI microbiota and the brain is well recognized with bidirectional signals occurring through at least 3 interactive pathways involving the endocrine, nervous, and immune systems.23 The autonomic nervous system has the capability of altering the microbiota by virtue of altered motility, acid, bicarbonate, and mucus secretion, permeability, and mucosal immune response. Alterations have been implicated in a variety of diseases, including irritable bowel syndrome, constipation, metabolic disorders such as obesity, and psychiatric/neurologic disorders including depression, anxiety, autism spectrum disorder, and others. Diseases of the cardiovascular system, including type 2 diabetes mellitus, kidney disease (including exacerbation of uremia) also have been linked to dysbiosis of the gut, primarily by virtue of changes in small molecule metabolites such as trimethylamine-N-oxide and short chain fatty acids.5
Dysbiosis of the urinary tract microbial composition and diversity is likely related to the development of UTI, with a decrease in diversity being problematic.24 Native bacteria might prevent overgrowth of uropathogens (competing for nutrition/attachment sites), causing disruption of biofilm, production of antimicrobial peptides, immunomodulation, and regulation of gene expression. Diseases of the urinary system that may reflect dysbiosis in humans may include interstitial cystitis, urgency urinary incontinence, and prostatitis (for which Burkholderia was more commonly present).
The role of the microbiome in disease has been reviewed in dogs15,16 and others. Dysbiosis of the microbiome has been reviewed in dogs and cats,25 as well as its specific role in the presence of inflammation for both cats26 and dogs15,26,27. Other specific reports include dysbiosis, or at least altered microbiomes and its association with gastric dilatation-volvulus in high risk dogs,28 conspecific aggression29.
The role of antimicrobials in treating presumed dysbiosis in the gastrointestinal tract has been described after dietary manipulation,30 treatment with synbiotics,31,32 and fecal transplantation33.
Impact of Drugs on the Microbiota
Antimicrobials
The established gastrointestinal microbiota becomes resistant to environmental disturbances including dietary changes and short-term antimicrobial use (as reviewed by10). Antibiotics have always played a role in microbial sensing and signaling. Their ancient presence is, not surprisingly, accompanied by antimicrobial resistance, which is thus equally ancient, and crucial to the healthy microbiome. Patterns of resistance, however, are influenced by exposure to drugs, including antimicrobials. The term resistomes refers to the collections of antimicrobial resistance genes within a microbiome; they can persist for more than a year post antimicrobial exposure.
Humans
A number of studies have reviewed the impact of antimicrobials on the gut microbiome. Bhalodi34 reviewed the impact of antimicrobials used to treat sepsis on patient fecal microbiome. Populations that declined in response to beta lactam and fluoroquinolone therapy included Enterobacteriaceae, bifidobacteria, and lactobacilli, while enterococci increased. Fluoroquinolone therapy among the observations was the large percent (71%) of ICU patients that receive antimicrobials (including when used for non-antimicrobial effects, such as prokinetics [macrolides], anti-inflammatory or others. Interestingly, while the short-term use of antimicrobials does not appear to lead to antimicrobial resistance, discontinuation of antibiotics was accompanied by recolonization with multidrug-resistant bugs (perhaps supporting a role of appropriate probiotics?). Willman35 quantified changes in the gut microbiome in patient with leukemias receiving prophylactic antimicrobials (either ciprofloxacin or sulfamethoxazole/trimethoprim). Both significantly impacted the microbiome with the spread of resistance genes more efficient in the latter group.
Companion Animals
A number of studies have demonstrated potentially detrimental effects in the microbiome of the gastrointestinal tract in dogs. Imipenem, metronidazole, and clindamycin, each of which has an excellent (and for metronidazole, exclusive) anaerobic bacterial spectrum, have been demonstrated, at subminimum inhibitory concentrations, to inhibit biofilm formation by Bacteroides fragilis (in vitro), whereas enrofloxacin increased formation.36
Diversity
Among the drugs commonly used to treat diarrhea in dogs are metronidazole and tylosin. Because the former is used in humans, more information is available regarding the impact of this drug in human microbiota (see above).
1. The impact of metronidazole has also been described in dogs. The impact of metronidazole (12.5 mg/kg q 12 h) on fecal microbiota on 16S rRNA was compared to that of prednisolone (1 mg/kg/day) (RCCT?) in dogs treated for 14 days with measurements pre and 14, 28 and 42 days.37 At all time points, Firmicutes was by far the most predominant phylum identified in both groups and was not substantially altered. However, metronidazole significantly impacted bacterial composition (decreased diversity) with decreases in Bacteroidaceae, Clostridiaceae, Fusobacteriaceae (possibly detrimental?) and others. Increases occurred in Bifidobacteriaceae (possibly beneficial?), Enterobacteriaceae, Enterococcaceae and Streptococcaceae, with these populations close to baseline by day 42. A significant effect could not be detected for prednisolone. No treated dog exhibited signs of dysbiosis.
2. Using a DB-RCCT design, oral metronidazole (10 to 15 mg/kg q 12 h; n=14) was compared to placebo (cellulose; n=17) for treatment of acute diarrhea in dogs.38 Although the time for resolution of diarrhea was significantly less in treated dogs (2.1±1.6 days vs. 3.6±2.1 days), the clinical relevance of this difference is not clear. However, seven control dogs had diarrhea >4 and two of these >7days, both of which resolved within 1.1 and 4.9 days. At seven days, 3/13 treated dogs remained positive for C. perfringens vs. 11/14 untreated dogs, although the relevance of this finding is not clear (27/31 dogs were positive prior to therapy). (Does this suggest that metronidazole might be withheld until 4 days? Do we need a clinical trial with a reasonable non-antimicrobial alternative, such as bismuth subsalicylate or kaolin pectin?).
3. Using a DB-RCCT, the effect of oral metronidazole (9–31 mg/kg q 12 h) was compared to a commercial probiotic (containing Bifidobacterium, Lactobacillus species) or placebo (sucrose) (n=20 per group). A commercial “diarrhea panel” was performed, and selected drugs were allowed as per clinician discretion. Investigators were unable to demonstrate a benefit of either treatment, probably reflecting an issue with study sample size.
Amoxicillin: Amoxicillin/clavulanic (8.75 mg/kg SC or IV q 8 h) alone (n=20) or in combination with metronidazole (10 mg/kg IV plus saline placebo q 12 h; n=14) was compared in dogs with acute hemorrhagic diarrhea. Based on duration of hospitalization and a “canine hemorrhagic gastroenteritis score,” no significant difference between the treatment groups could be demonstrated. The authors acknowledged the need to study whether or not any antimicrobials are necessary.
Tylosin (20 to 22 mg/kg q 24 h x 14 days) also demonstrated an impact on canine microbiome diversity in healthy dogs (n=5) using a jejunal fistula and 16S rRNA.39 Fusobacteria, Bacteroidales and Moraxella tended to decrease during therapy while Enterococcus, Pasteurella, Escherichia increased. Changes were evident through 28 days (2 weeks after withdrawal), the last sampling time. Spirochaetes, Streptomycetaceae and Prevotellaceae were considered to have failed to recover by day 28. Clinical sequela of these changes were not evident.
Diversity and Resistance
That routine use of amoxicillin is demonstrated with emergent resistance has been demonstrated in dogs. Using standard C&S techniques, our lab has demonstrated that 5 days of amoxicillin therapy in healthy dogs (10 mg/kg PO q 12 h) renders essentially all remaining E. coli resistant to amoxicillin but resistance patterns return close to baseline by 3 weeks post discontinuation of therapy.40 Although high level (MIC>256 mcg/ml), resistance was primarily only toward amoxicillin. Likewise, fecal E. coli in healthy dogs receiving enrofloxacin (5 mg/kg a 24) for 5 days developed high level (MIC 90>64 mcg/ml) resistance, but resistance was MDR (susceptibility only to aminoglycosides) due to efflux pump expression and persisted (no change in proportion) at least 3 weeks after the final dose. In a more comprehensive study, based on fecal samples from dogs (n=127) pre, immediately after, and 1 and 3 months post treatment, dogs receiving amoxicillin-clavulanic acid (n=28), cephalexin (n=32), cefovecin (n=24), clindamycin (n=29) or a fluoroquinolone (n=14) was associated with emergent resistance.41 Likewise, the impact of routine therapy with the same drugs on mucosal staphylococcal isolates was reported with the proportion of coagulase negative versus positive and antimicrobial-resistant isolates changing.42 Cefovecin similarly impact the fecal microbiota in healthy dogs,43 initially (day 3) causing a decrease in total number of E. coli, but eventually (day 7 through 28, study end) a higher number of cefovecin-resistant E. coli. Enterococci also increased, as did beta-lactam resistance.
An example of an undesirable sequela of antimicrobial therapy, including non-judicious, is represented by Campylobacter gastroenteritis in humans. Campylobacter is a leading cause of human bacterial gastroenteritis. Outbreaks in humans have been associated with transmission from pet-store-purchased puppies.44 Campylobacter (including ciprofloxacin-resistant) shedding also has been demonstrated in shelter puppies.45 Increasing outbreaks led to investigation by the Center for Disease Control who subsequently reported on an outbreak of multidrug-resistant Campylobacter jejuni linked to puppy exposure (Montgomery MP MMR 2019). In one month, 118 cases were reported, 29 of which were pet store employees across 18 states (at least 107 hospitalized). Puppies were infected before reaching the pet stores. Subsequent study of records of puppies purchased from pet stores (n=20 stores) found that 95% of 142 puppies had received antibiotics, 55% for prophylaxis versus 38% for prophylaxis and treatment and 1% for treatment only, for a median total of 15 days (2–67 range). Four antibiotics accounted for 81% of those used: metronidazole (63%), sulfadimethoxine (without trimethoprim; 54%), doxycycline (27.5%) and azithromycin (13%).
Non-Antimicrobial Drugs
The impact of drugs on microbiota is not limited to antimicrobials. While they may present more harm to the microbe compared to antimicrobial drugs, microbes are likely to perceive many compounds that “don’t belong” as a potential threat and thus mount resistance.46 For example, due to induction of efflux pumps, fluoxetine induces multidrug resistance in E. coli.47 As such, the more comorbidities a patient has, including the more drugs it is receiving, the more important it is to reassess assumptions regarding antimicrobial susceptibilities.48
Conclusions
These combined studies demonstrate that multiple organs, even those previously thought sterile, have microbiomes; that routine and even non-routine antimicrobial (and non-antimicrobial) therapy results in substantive changes in microbiomes, with changes in both number, diversity, and susceptibility patterns and that the time necessary for the microbiome to return to normal is variable. Further studies are necessary to support approach to antimicrobial de-escalation, such as identifying which diseases are associated with bacterial infection, which organisms (if any) are the cause of diseases, and the impact of dose and duration, thus providing support for de-escalation of antimicrobial therapy by narrowing the spectrum, the need for antimicrobial therapy in select diseases.
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