Abstract
The complex bacterial populations that constitute the gut microbiota can harbor antibiotic-resistance genes (ARGs), including those encoding for β-lactamase enzymes (BLA), which degrade commonly prescribed antibiotics such as ampicillin. While it is known that ARGs can be transferred between bacterial species, with dramatic public health implications, whether expression of such genes by harmless commensal bacterial species shields antibiotic-sensitive pathogens in trans by destroying antibiotics in the intestinal lumen is unknown. To address this question, we colonized GF mice with a model intestinal commensal strain of E. coli that produces either functional or defective BLA. Mice were subsequently infected with Listeria monocytogenes or Clostridioides difficile followed by treatment with oral ampicillin. Production of functional BLA by commensal E. coli markedly reduced clearance of these pathogens and enhanced systemic dissemination during ampicillin treatment. Pathogen resistance was independent of ARG acquisition via horizontal gene transfer but instead relied on antibiotic degradation in the intestinal lumen by BLA. We conclude that commensal bacteria that have acquired ARGs can mediate shielding of pathogens from the bactericidal effects of antibiotics.
Importance
The wide use of antibiotics in human populations and in livestock has led to increasing prevalence of pathogenic and commensal bacterial species that harbor antibiotic resistance genes (ARGs), such as those encoding for ampicillin-degrading β-lactamases. We investigated whether harmless autochthonous bacteria might degrade orally administered antibiotics, thereby impairing their ability to combat intestinal pathogens. Here we report that antibiotic degradation by a resident intestinal strain of E. coli reduces the effectiveness of oral ampicillin against two intestinal pathogens, L. monocytogenes and C. difficile, resulting in increased intestinal and systemic bacterial burden. We demonstrate that expression of ARGs by non-pathogenic members of the gut microbiota shields antibiotic-sensitive pathogens and enhances their expansion and dissemination.
Observation
Antibiotic administration has markedly reduced the morbidity and mortality associated with bacterial infections in the pre-antibiotic era. Increasing antibiotic-resistance in pathogenic microbes, mediated in part by acquired genes that encode antibiotic-degrading enzymes, represents a major threat to human health (1).
The gut microbiota contains trillions of commensal bacteria that can also harborantibiotic resistance genes (ARGs) (2). Notably, antibiotic exposure can increase ARG generepresentation and expression by the gut microbiota (3). Horizontal ARG transfer represents a mechanism by which drug-sensitive microbes can acquire resistance, e.g. by acquisition of genes encoding antibiotic-degrading hydrolases (4, 5). Thus, it is possible that commensal bacterial species transfer ARGs to intestinal pathogens upon antibiotic exposure in the gut lumen. However, another possibility is that production of antibiotic-degrading enzymes by the resident microbiota protects otherwise drug-sensitive pathogens in trans, thereby facilitating their replication and spread in the host.
To test this hypothesis in a controlled system, we reconstituted germ-free (GF) mice with an E. coli strain, utilized here as a model commensal, that expresses either a WT form of β-lactamase (TEM-1) or an inactive point mutant (hereby referred to as WT BLA or mut BLA, respectively) (Figure 1A) (6). This approach yielded cohorts of mice that, with the exception of one codon, harbor identical genomes, thus excluding differences in microbiota functions (e.g., immune activation, colonization resistance, etc.) that are not related to the β-lactam degradation.
Although WT BLA and mut BLA E. coli reached identical luminal bacterial densities in reconstituted mice, a colorimetric assay confirmed that only the intestinal content of mice reconstituted with WT BLA E. coli retained the capacity to hydrolyze β-lactams (Figure 1 B, C). One week after reconstitution, mice were orally infected with the foodborne pathogen Listeria monocytogenes 10403s (Lm). Lm is highly sensitive to β-lactam antibiotics and can expand in the gut lumen of mice that lack colonization resistance (7). Mice were then administered ampicillin on day +1 and +2 post Lm infection and sacrificed on day +3. As expected, Lm reached identical densities in the intestines of WT BLA or mut BLA E. coli reconstituted mice on day +1, indicating that the 2 E. coli strains did not differ in their inability to provide colonization resistance against Lm. However, we found significantly higher Lm burden in multiple organs in mice harboring WT BLA E. coli on day +3, consistent with the notion that β-lactamase-dependent ampicillin degradation shielded Lm from the therapeutic antibiotic’s action. To exclude the possibility that Lm might have acquired resistance to ampicillin via horizontal gene transfer, we inoculated single Lm colonies recovered from the cecal content of WT BLA E. coli-reconstituted, Lm-infected mice, into liquid medium either in the presence or absence of ampicillin. Notably, none of the inoculated Lm colonies grew in the presence of ampicillin, in contrast to WT BLA-expressing E. coli colonies recovered from the same mice (Figure 1F). Furthermore, none of the Lm colonies tested positive for the presence of the β-lactamase gene, which was uniformly detected in colonies of WT BLA E. coli by PCR (Figure 1G).
To confirm that the increased Lm burden observed above was due to antibiotic degradation by resident E. coli, we collected the cecal contents of mice reconstituted with either WT BLA or mut BLA E. coli and treated with ampicillin in the drinking water for two consecutive days to allow for luminal accumulation of the antibiotic. Inoculation of Lm into serial dilutions of the cecal content supernatants revealed that the cecal contents from mice reconstituted with mut BLA E. coli had a higher inhibitory capacity compared to cecal contents recovered from mice reconstituted with WT BLA E. coli (Figure 1G and Supplementary Figure 1). Since the presence of active β-lactamase was the only bona fide difference between the cecal contents of the two cohorts of mice, we conclude that the microbiota-encoded enzymatic activity curtailed the efficacy of ampicillin treatment against Lm.
To expand our observations beyond the Listeria model, and to assess whether commensal-mediated antibiotic degradation may represent a mechanism that is relevant to other infectious agents, we adapted our experimental strategy to an established C. difficile infection model (8) (Figure 2A), an important intestinal pathogen that is also sensitive to ampicillin (Supplementary Figure 2). Of note, this model allowed us to investigate the relevance of our findings in a setting where expansion of an antibiotic-resistant microbe takes place following antibiotic-mediated depletion of the intestinal microbiota, a common occurrence in hospitalized patients (9). Similar to the results obtained with Lm, we observed indistinguishable levels of expansion for both E. coli and C. difficile on day +1 after reconstitution or infection, respectively, in all groups of mice (Figure 2B, C). In agreement with our previous findings, the C. difficile burden was significantly reduced by ampicillin treatment in mice reconstituted with mut BLA E. coli, but not in mice reconstituted with WT BLA E. coli (Figure 2D). Direct comparison of the ampicillin-treated mice confirmed a significantly higher burden in mice whose intestinal flora had the capacity to hydrolyze β-lactams (Figure 2D).
These findings suggest that ARGs expressed by commensal bacteria can shape the chemical niche of the intestine and confer an apparent antibiotic resistant phenotype to pathogens in trans, without direct acquisition of ARGs by the pathogenic microbe. We refer to this activity as commensal-mediated pathogen shielding. Using two different infection models, we show that production of β-lactamases, a prototypical antibiotic resistance factor, by resident intestinal microbes can significantly reduce the effectiveness of ampicillin treatment, thereby generating a safe environment in which otherwise sensitive pathogens are shielded from this drug. Importantly, previous studies in healthy volunteers demonstrated that upon treatment with cephalosporins, subjects harboring BLA-producing commensal strains, unlike BLA-negative subjects, had undetectable concentrations of the drug in the feces and maintained a rich microbiota, providing evidence that BLA concentrations sufficient to inactivate antibiotics are commonly achieved in humans (10, 11).
Whether or not ARGs enrichment within the gut microbiota is detrimental to host health is a complex question, and the answer is likely to be context-dependent.
For instance, oral administration of recombinant beta lactamase or BLA-producing bacteria was shown to preserve the integrity of the microbiota following parenteral administration of beta-lactam antibiotics in animal models, without affecting drug concentration in the serum (12-17). These approaches were shown to be advantageous in that they preserved colonization resistance against pathogens (12-17).
On the other hand, early work (reviewed in (18)) revealed that beta-lactamase-producing, non-pathogenic bacteria, can hinder the efficacy of penicillins in vitro and in vivo, using models of subcutaneous and tonsil infection. Clinical data also suggested that the presence of one beta-lactamase-producing bacterial strain at the site of infection could enhance persistence of a pathogen upon antibiotic treatment (19). In these settings, members of the Bacteroides genus, among the most highly represented genera in the human intestine(20), were also identified as BLA-carriers.
Consistent with these observations, our laboratory recently showed that a few bacterial strains, out of the dozens composing the microbiota of a mouse colony treated with ampicillin for over 8 years, had the capacity to hydrolyze ampicillin, while the other bacterial strains, in isolation, remained sensitive to ampicillin and thus were protected in trans by a minor subset of the microbiota (21)(see Figure 4A in (22)).
In conclusion, we propose that commensal-mediated pathogen shielding can impair the effectiveness of some antibiotic treatments during infection. While pharmacokinetic studies have generally focused on antibiotic absorption, distribution, enzymatic modification, protein binding and biliary/renal clearance, the role of microbiota-mediated antibiotic degradation in the gut lumen and its potential for dramatically impacting responses to antibiotic treatment has received less attention. Our findings extend the recently uncovered broad capacity of the gut microbiota to metabolize drugs, affecting their efficacy (23, 24). Within this model, antibiotics represent an additional class of xenobiotics that commensals can metabolize.
Our study suggests that presence or absence of commensal bacterial strains that inactivate beta-lactam antibiotics is likely to impact clinical responses to antibiotic treatment, possibly contributing to inter-individual variability in therapy outcomes. Furthermore, the occurrence of pathogen shielding might be a relevant element to consider in the engineering of probiotic bacterial strains to be employed in clinical practice.
Methods
Mouse Husbandry
All experiments using wild-type mice were performed with C57BL/6J female mice that were 6–8 weeks old; mice were purchased from Jackson Laboratories. Germ-free (GF) mice were bred in-house in germ-free isolators. Following reconstitution mice were housed in sterile, autoclaved cages with irradiated food and acidified, autoclaved water. All animals were maintained in a specific-pathogen-free facility at Memorial Sloan Kettering Cancer Center Animal Resource Center. Experiments were performed in compliance with Memorial Sloan-Kettering Cancer Center institutional guidelines and approved by the institution’s Institutional Animal Care and Use Committee.
Generation of E. coli strains
Plasmids encoding for WT TEM-1 βlactamase (pDIMC8-TEM1) or mutated TEM-1 βlactamase (pDIMC8-TEM1 W208G) were extracted from the RH06 and RH09 E. coli strains, published elsewhere (6), gel-purified and utilized for transformation of Stellar competent cells (Takara Bio) according to manufacturer’s instructions. The resulting strains were utilized for experiments throughout this study. Of note, the plasmids conferred resistance to chloramphenicol, and while expression of the TEM-1 gene was placed under the regulation of a tac promoter, we did not induced it by IPTG treatment, but rather exclusively relied on leaky transcription of the gene, to produce more physiologically-relevant conditions.
Antibiotic treatment, reconstitution and infections
GF mice were gavaged with either of two strains of E. coli, encoding for a functional or a point-mutated version of TEM-1 β-lactamase, respectively. 1 week post reconstitution mice were gavaged with 109 CFUs of L. monocytogenes (Lm) strain 10403s and administered 1 mg of ampicillin (Fisher) by oral gavage daily for 2 consecutive days. Animals were euthanized at day 3 post infection. Reconstitution of WT mice with E. coli strains for in vitro experiments involving dilution of cecal content, mice were treated for 3 days with metronidazole and vancomycin in drinking water (0.5 g/l), left on regular water for 1 day, and then gavaged with the appropriate E. coli strain. 1 week post reconstitution mice were treated with ampicillin in drinking water (0.5 g/l) for two days prior to being euthanized.
For C. difficile infection experiments, WT C57Bl/6 mice were administered a combination of metronidazole, neomycin and vancomycin (0.25 g/l each) in drinking water for 3 days, and 24h post antibiotic regimen cessation were injected i.p. with clindamycin (200 μg). On the following day mice were reconstituted with either WT or mut BLA E. coli (5×104 CFUs) and 200-500 spores of C. difficile strain VPI10463 (ATCC #43255).
CFUs enumeration and Selective plating
L. monoctogenes was identified through plating of serial dilutions of homogenized organs (prepared as described elsewhere (7)) or fecal material (resuspended 100 mg/ml in PBS) ontoBHI plates supplemented with streptomycin (100 μg/ml) and nalidixic acid (50 μg/ml).
E. coli CFUs were enumerated following plating of serial dilution of fecal material onto LB plates supplemented with chloramphenicol (50 μg/ml). E. coli CFU numbers obtained from plating of ex-GF mice at day of infection onto LB plates (not supplemented with antibiotics) yielded identical numbers, indicating that plasmids carrying CM resistance cassette as well as the WT/mut BLA gene were maintained even in the absence of any selective pressure.
For detection of C. difficile, fecal pellets or cecal content were resuspended in deoxygenated phosphate-buffed saline (PBS), and ten-fold dilutions were plated on BHI agar supplemented with yeast extract, taurocholate, L-cysteine, cycloserine and cefoxitin at 37°C in an anaerobic chamber (Coylabs) overnight.
Lm culture in cecal content
Cecal contents were recovered from E. coli reconstituted WT or GF animals, resuspended in PBS at 300 mg/ml (WT) and spun down at 3000 rpm for 10’. Serial 1:2 dilutions of the resulting supernatant were generated using PBS and 100 μl of each dilution were plated in replicate in flat bottom 96 well plates. An equal volume of BHI medium supplemented with streptomycin (200 μg/ml) and nalidixic acid (100 μg/ml) acid (to prevent growth of residual E. coli) containing 100-1000 CFUs Lm 10403s, was added on top. Lm for this assay was prepared by re-inoculating an overnight culture in liquid BHI at 37°C on shaker, until logarithmic phase of growth was reached (OD=0.1-0.4). After an overnight incubation at 37°C, the plate was assayed by OD 600 reading and individual dilutions plated onto BHI-Strep-NA plates for assessment of Lm growth. Normalized inhibition index was calculated as 1/first dilution allowing for Lm growth, with the initial dilution being 1:2 to take into account the addition of a volume of BHI equivalent to that of the medium. For example, if the first dilution where Lm was detected was 1:16, the resulting inhibition index would be 16. Within each experiment samples were then normalized to the baseline, obtained by averaging the values obtained in the control group, represented by mice reconstituted with mut BLA E. coli.
PCR
PCR for was carried out using the following primers: β-lactamase (fw: 5’-GCTATGTGGCGCGGTATTAT-3’; rev: 5’-AAGTAAGTTGGCCGCAGTGT-3’, product: 191 bp); p60 (fw: 5’-GCGCAACAAACTGAAGCAAAGGATGC-3’; rev: 5’- CTCGCGTTACCAGGCAAATAGATGGACG-3’, product: 1300 BP), using the SapphireAMp Fast PCR master mix (Takara Bio) and the following conditions: 94°C × 1’, 30 × (98°C ×; 5’’, 58°C × 5’’, 72°C × 15’’).
Competing financial interests
E.G.P. has received speaker honoraria from Bristol Myers Squibb, Celgene, Seres Therapeutics, MedImmune, Novartis and Ferring Pharmaceuticals and is an inventor on patent application # WPO2015179437A1, entitled “Methods and compositions for reducing Clostridium difficile infection” and #WO2017091753A1, entitled “Methods and compositions for reducing vancomycin-resistant enterococci infection or colonization” and holds patents that receive royalties from Seres Therapeutics, Inc.
Acknowledgments
We thank Ying Taur and Peter McKenney for critical discussion of the manuscript. RH06 and RH09 E. coli strains carrying the pDIMC8-TEM1 and pDIMC8-TEM1 W208G plasmids were a kind gift of Prof. Marc Ostermeier (Johns Hopkins University). This work was supported by NIH grant P30 CA008748 (to MSKCC), Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease Awards (T.M.H.).
S.B. was supported by an Early Postdoc Mobility Fellowship from the Swiss National Science Foundation (P2EZP3_159083) and an Irvington Fellowship from the Cancer Research Institute (49679).