Abstract
Besides its live-saving properties, antibiotic treatment affects the commensal microbiota facilitating colonization with potentially harmful microorganisms. Here we tested how commonly applied antibiotics induced gut microbiota changes and predisposed to intestinal carriage of multi-drug resistant Pseudomonas aeruginosa (MDR Psae) upon exposure. Therefore, mice received either vancomycin, ciprofloxacin, ampicillin plus sulbactam (A/S) or no antibiotics via the drinking water and were perorally challenged with a clinical MDR Psae isolate after antibiotic withdrawal. Whereas 100% of A/S and 55% of ciprofloxacin pretreated mice harbored Psae in their feces seven days post-challenge, intestinal Psae carriage rates were 20.0% and 26.3% in vancomycin pretreated and untreated mice, respectively. Microbiota analyses revealed that immediately before MDR Psae challenge, A/S pretreated mice displayed the lowest total bacterial, lactobacilli and Clostridium leptum fecal loads compared to other cohorts. Seven days following Psae exposure, however, higher numbers of apoptotic colonic epithelial cells were observed in A/S pretreated versus untreated mice that were accompanied by more enhanced innate and adaptive immune cell responses and nitric oxide secretion in colonic and ileal biopsies in the former versus the latter. In conclusion, distinct gut microbiota shifts following A/S pretreatment facilitate pronounced intestinal MDR Psae colonization and pro-inflammatory immune responses upon oral exposure.
Introduction
Strictly aerobic Gram-negative Pseudomonas aeruginosa (Psae) bacteria are typically found in aqueous conditions both, on surfaces and in soil. Their inherent antibiotic resistance and adaptability help them survive in a variety of environments, including medical institutions such as hospitals [1]. While bacterial motility and surface adhesion are facilitated by a single flagellum and numerous cell surface pili [2], Psae virulence is further influenced by alginate secretion, biofilm formation, quorum-sensing, and an intricate secretion system [1, 2]. In health-care environments, sinks and water bottles, but also respiratory equipment are common ambient reservoirs of Psae [3, 4]. The opportunistic pathogenic bacteria constitute a major source of nosocomial infections, particularly in critical care units, and can be responsible for ventilator-associated pneumonia, urinary tract infections, superinfections of burn wounds, and bloodstream infections, that are associated with fatality rates of over 30% [5]. Acquired extended-spectrum β-lactamases, carbapenemases, and 16S rRNA methylases have contributed to the emergence of multi-drug resistant (MDR) Psae strains, which have put immunocompromised individuals, patients suffering from cystic fibrosis and other chronic pulmonary morbidities, and intensive care unit patients at higher risk for a longer hospital stay and death [5–7]. As a result, the World Health Organization (WHO) has highlighted the urgent need for new treatment options and classified MDR Gram-negative bacteria, including Psae, as a major threat to human health [8]. Usually, Psae infection occurs after colonization, which can occur either endogenously originating from the patient's natural microbiota or exogenously if obtained from cross-contamination from other patients or the hospital environment, for instance [9]. Notably, it has been demonstrated that intestinal Psae colonization of individuals before intensive care unit admission is linked to a nearly 15-fold higher risk of future infection as compared to non-colonized subjects [10]. The invasion and establishment of opportunistic pathogenic bacteria including Psae in mice and humans are facilitated by antimicrobial drugs that disrupt the complex and diverse commensal gut microbiota [11–14], which physiologically exerts colonization resistance to the host [15, 16]. Besides antibiotics also proton pump inhibitors, steroids, and opioids have been shown to induce dysbiosis associated with abrogation of colonization resistance [17]. Notably, antibiotic pretreatment was shown to be predictors for high colonization pressure of patients with MDR Psae causing subsequent blood stream infections [18]. This prompted us to test in our actual study to what extent pretreatment with distinct antibiotic compounds and different antimicrobial spectra including vancomycin (Van), ciprofloxacin (Cip), and ampicillin plus sulbactam (A/S) that are commonly applied antibiotics for treatment of human infections compromised the fecal microbiota composition and in turn, predisposed mice to intestinal carriage of MDR Psae upon oral exposure.
Materials and methods
Mice, antibiotic pretreatment regimens
Conventional C57BL/6j wildtype mice were bred and housed in the Forschungsinstitute für Experimentelle Medizin, Charité – Universitätsmedizin Berlin, Germany under specific pathogen free (SPF) conditions. Mice were kept in cages that were equipped with filter tops within an experimental semi-barrier under standard conditions (i.e., 22–24 °C room temperature, 55 ± 15% humidity, 12 h light/12 h dark cycle) and had ad libitum access to autoclaved water and standard chow (food pellets: ssniff R/M-H, V1534-300, Sniff, Soest, Germany). Eight-week-old female mice were transferred to sterile cages (maximum of 3–4 animals per cage) and received either ampicillin plus sulbactam (A/S; 2 g L−1; Dr. Friedrich Eberth Arzneimittel, Ursensollen, Germany), vancomycin (Van; 500 mg L−1; Hikma Pharmaceuticals, London, UK) or ciprofloxacin (Cip; 200 mg L−1; Bayer Vital, Leverkusen, Germany) via the drinking water (ad libitum) for 19 days (i.e., from day −21 until day −2), whereas a control group remained untreated. To assure antibiotic washout, the antimicrobial compounds were withdrawn two days prior bacterial challenge (i.e, day −2) and replaced by sterile tap water.
Culture-independent analysis of fecal microbiota composition
DNA was extracted from fecal samples, quantitated applying the Quant-iT PicoGreen reagent (Invitrogen, Paisley, UK) and adjusted to 1 ng per µL. as reported earlier [19]. Then, total eubacterial loads as well as the main bacterial groups abundant in the murine commensal gut microbiota including enterobacteria, enterococci, lactobacilli, bifidobacteria, Bacteroides/Prevotella species, Clostridium coccoides group, Clostridium leptum group and Mouse Intestinal Bacteroides were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genera- or group-specific 16S rRNA gene primers (Tib MolBiol, Berlin, Germany) and numbers of 16S rRNA gene copies per ng DNA of each sample determined as described previously [19–21].
MDR P. aeruginosa infection and quantitative assessment of fecal bacterial loads
The MDR Psae isolate was initially cultured from respiratory material of a patient suffering from nosocomial pneumonia and kindly provided by Prof. Dr. Bastian Opitz (Charité – University Medicine, Berlin, Germany). Notably, the bacterial strain displayed antimicrobial sensitivity to fosfomycin and colistin only [22]. Prior infection, the Psae strain was grown on cetrimid agar (Oxoid, Wesel, Germany) for 48 h in an aerobic atmosphere at 37 °C. On days 0 and 1, mice were perorally challenged with 109 colony-forming units (CFU) of the MDR Psae strain by gavage in a total volume of 0.3 mL phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) as reported earlier [22]. For quantitative assessment of fecal Psae loads over time post-challenge, fecal samples were homogenized in sterile PBS, serial dilutions streaked onto cetrimid agar and onto columbia agar supplemented with 5% sheep blood (both from Oxoid, Wesel, Germany) and incubated in an aerobic atmosphere at 37 °C for 48 h as described previously [22]. Fecal weights were determined by the difference of the sample weights before and after preservation. The detection limit of viable bacteria was 100 CFU per g.
Sampling procedures
Mice were sacrificed 7 days post MDR Psae challenge by CO2 asphyxiation. The large intestinal lengths were measured with a ruler (in cm). Tissue samples from mesenteric lymph nodes (MLN), colon, and ileum were removed under sterile conditions. Intestinal samples were collected in parallel from each mouse for microbiological, immunohistochemical, and immunological analyses.
Immunohistochemistry
Five µm thin paraffin sections of colonic ex vivo biopsies were used for in situ immunohistochemical analysis as reported previously [23, 24]. In brief, primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), F4/80 (#14-4801, clone BM8, eBioscience, San Diego, CA, USA, 1:50), CD3 (No. N1580, Dako, Glostrup, Denmark, 1:10), FOXP3 (clone FJK-165, no. 14-5773, eBioscience, 1:100), and B220 (No. 14-0452-81, eBioscience, San Diego, CA, USA, 1:200) were used to assess apoptotic cells, macrophages/monocytes, T lymphocytes, regulatory T cells, and B lymphocytes, respectively. The average numbers of positively stained cells within at least six high power fields (HPF, 0.287 mm2; 400× magnification) were determined by an independent investigator.
Nitric oxide measurement
Ex vivo biopsies (approximately 1 cm2) derived from colon and ileum (both cut longitudinally and washed in PBS), as well as MLN (3 nodes) were placed in 24-flat-bottom well culture plates (Thermo Fisher Scientific, Waltham, MA, USA) containing 500 μL serum-free RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with penicillin (100 U mL−1, Biochrom, Berlin, Germany) and streptomycin (100 μg mL−1; Biochrom, Berlin, Germany). After 18 h at 37 °C, culture supernatants were tested for nitric oxide secretion by the Griess method as stated earlier [25].
Statistical analysis
Significance levels and medians were calculated using GraphPad Prism (version 9; San Diego, CA, USA). Normal distribution of the data was determined with the Anderson-Darling test. For multiple comparisons, the one-sided ANOVA with Tukey post-hoc test (for normally distributed data) and the Kruskal-Wallis test with Dunn's post-hoc test (for not normally distributed data) were applied. Two-sided probability (p) values ≤ 0.05 were considered significant. Data were pooled from four independent experiments.
Results
Fecal microbiota composition before differential antibiotic pretreatment of mice
In our present study we addressed to what extent distinct gut microbiota changes in consequence of defined antibiotic treatment regimens commonly applied in human medicine predisposed the vertebrate host to intestinal carriage of MDR Psae following oral challenge. Therefore, mice received either Van, Cip, A/S or no antibiotic compound for a period of 19 days via the drinking water (i.e., from day −21 until day −2). Our comprehensive survey of the fecal gut microbiota composition immediately before initiation of antibiotic pretreatment on day −21 applying culture-independent 16S rRNA-based analyses revealed comparable total bacterial loads as well as similar fecal numbers of aerobic, facultative anaerobic and obligate anaerobic bacterial genera and species such as enterobacteria (including Escherichia coli), enterococci, lactobacilli, bifidobacteria, Bacteroides/Prevotella species, C. coccoides and leptum groups and Mouse Intestinal Bacteroides in respective treatment and control cohorts (Fig. 1).
Fecal microbiota composition of mice upon initiation of antibiotic treatment.
Fresh fecal samples were taken immediately before initiation of treatment with different antibiotic compounds (white circles), namely, vancomycin (Van), ciprofloxacin (Cip), and ampicillin plus sulbactam (A/S) on day (d) −21, and the fecal commensal microbiota composition quantitatively assessed by culture-independent 16S rRNA methodology (see methods). (A) Total eubacterial loads, (B) enterobacteria, (C) enterococci, (D) lactobacilli, (E) bifidobacteria, (F) Bacteroides/Prevotella species (spp.), (G) Clostridium coccoides group, (H) Clostridium leptum group, and (I) Mouse Intestinal Bacteroides were expressed as gene copies per ng DNA. A group without antibiotic application served as control cohort (w/o, grey symbols). Medians (black bars) and numbers of analyzed mice (in parentheses) are indicated. Data shown were pooled from four independent experiments
Citation: European Journal of Microbiology and Immunology 2025; 10.1556/1886.2025.00015
Fecal microbiota composition after completion of antibiotic regimens and before MDR Psae infection
We next asked to what extent respective antibiotic pretreatment had affected the fecal gut microbiota composition predisposing the mice for subsequent intestinal MDR Psae carriage upon oral exposure. To address this, we surveyed the fecal microbiota composition immediately before Psae gavage on day 0. Our quantitative 16S rRNA based molecular analyses revealed that in the A/S cohort the total eubacterial loads as well as the numbers of lactobacilli and C. leptum group were lower when compared to all other cohorts (P < 0.01–0.001; Fig. 2A, D and H). Furthermore, fecal enterobacterial loads were lower in both, A/S and Cip pretreated mice when compared to vancomycin treated and the untreated groups (P < 0.01–0.001; Fig. 2B). In addition, the fecal numbers of the obligate anaerobic Bacteroides/Prevotella species, C. coccoides group, and Mouse Intestinal Bacteroides were lower in the A/S and Van pretreated cohorts if compared to the Cip and untreated groups on day 0 (P < 0.01–0.001; Fig. 2F, G and I). Hence, differential antibiotic pretreatment is accompanied by distinct changes in the intestinal microbiota composition.
Fecal microbiota composition in mice after antibiotic pretreatment and immediately before P. aeruginosa challenge
Two days after cessation of antibiotic pretreatment with either vancomycin (Van), ciprofloxacin (Cip) or ampicillin plus sulbactam (A/S) and hence, immediately before oral multi-drug resistant P. aeruginosa infection on day 0 (d0), the fecal commensal microbiota composition was quantitatively assessed by culture-independent 16S rRNA-based qRT-PCR. (A) Total eubacterial loads, (B) enterobacteria, (C) enterococci, (D) lactobacilli, (E) bifidobacteria, (F) Bacteroides/Prevotella species (spp.), (G) Clostridium coccoides group, (H) Clostridium leptum group, and (I) Mouse Intestinal Bacteroides were expressed as gene copies per ng DNA. A group without antibiotic application served as control cohort (w/o, grey symbols). Medians (black bars), significance levels (P values) determined by the Kruskal-Wallis test with Dunn's post-hoc test and numbers of analyzed mice (in parentheses) are indicated. Data shown were pooled from four independent experiments
Citation: European Journal of Microbiology and Immunology 2025; 10.1556/1886.2025.00015
Fecal Psae loads over time following infection of mice that had been subjected to differential antibiotic pretreatment
Two and three days after cessation of the respective antibiotic pretreatment regimen (i.e., on days 0 and 1), mice were perorally challenged with 109 viable bacterial cells of a MDR Psae patient isolate by gavage and the fecal bacterial loads were assessed over time post-challenge (Figs 3 and 4). Our cultural analyses revealed that 24 h after the latest Psae application (i.e., on day 2), more than 50% of the untreated mice with a complex gut microbiota had already expelled the facultative pathogenic bacteria with median bacterial loads below the detection limit (Figs 3A and 4A). All mice from the A/S group, however, harbored Psae with median loads of approximately 107 CFU per gram feces (P < 0.01 versus the without antibiotics cohort (w/o); Figs 3A and 4D). In the Van and Cip groups, 80% and 60% of mice displayed MDR Psae in their intestines on day 2 p.i., respectively (Figs 3A, 4B and C). From day 3 until day 7 post challenge, fecal MDR Psae loads were higher in A/S pretreated mice as compared to those with Van or Cip or without any antibiotic pretreatment (P < 0.05–0.001; Fig. 3B–F). At the end of the observation period, 100% of the A/S and 55% of the Cip pretreated mice were intestinal MDR Psae carriers, whereas only 20.0% and 26.3% of the Van pretreated animals and the control cohort without antibiotic challenge harbored MDR Psae in their feces, respectively (Figs 3F and 4). Hence, differential antibiotic pretreatment predisposes mice for intestinal carriage of MDR Psae upon peroral challenge with the facultative pathogenic bacteria.
Fecal P. aeruginosa loads following oral bacterial challenge of mice that had been subjected to differential antibiotic pretreatment.
Two and three days after cessation of antibiotic pretreatment with either vancomycin (Van), ciprofloxacin (Cip) or ampicillin plus sulbactam (A/S), mice were perorally challenged with multi-drug resistant P. aeruginosa on day 0 (d0) and d1. The intestinal P. aeruginosa loads were determined in fecal samples taken at defined time points post-infection (A–F) by culture and expressed as colony-forming units per gram feces (CFU/g). A group without antibiotic application served as control cohort (w/o, grey symbols). Medians (black bars), numbers of P. aeruginosa positive out of the total number of analyzed mice (in parentheses), and significance levels (P values) determined by the Kruskal-Wallis test with Dunn's post-hoc test are indicated. Data shown were pooled from four independent experiments
Citation: European Journal of Microbiology and Immunology 2025; 10.1556/1886.2025.00015
Time course of fecal P. aeruginosa loads following oral bacterial challenge of mice that had been subjected to antibiotic pretreatment.
Two and three days after cessation of antibiotic treatment with either vancomycin (B), ciprofloxacin (C) or ampicillin plus sulbactam (D), mice were perorally challenged with multi-drug resistant P. aeruginosa on day 0 (d0) and d1. A group without antibiotic application served as control cohort (grey symbols, A). The intestinal colonization densities were assessed in fecal samples at defined time points post-infection by culture and expressed as colony-forming units per gram feces (CFU/g). Medians (black bars), numbers of P. aeruginosa positive out of the total number of analyzed mice (in parentheses), and significance levels (P values) determined by the Kruskal-Wallis test with Dunn's post-hoc test are indicated. Data were pooled from four independent experiments
Citation: European Journal of Microbiology and Immunology 2025; 10.1556/1886.2025.00015
Macroscopic and microscopic sequelae of MDR Psae infection in mice that had been subjected to a differential antibiotic pretreatment
Next, we tested whether intestinal MDR Psae carriage in antibiotic pretreated mice was associated with macroscopic and/or microscopic signs of intestinal inflammation. Since inflammatory conditions within the intestinal tract have been shown to be associated with shortening of the affected intestines [19, 26], we measured the colonic lengths upon necropsy, but did not find any differences between the experimental groups (n.s.; Fig. 5A). Furthermore, we stained colonic paraffin sections with an antibody against cleaved caspase3 constituting a marker for cell apoptosis. On day 7 post MDR Psae exposure, median apoptotic colonic epithelial cells numbers were twice as high in A/S pretreated as compared to untreated control mice (P < 0.01; Fig. 5B). Hence, intestinal MDR Psae carriage in A/S pretreated mice is associated with apoptotic colonic epithelial cell responses.
Macroscopic and microscopic inflammatory sequelae upon P. aeruginosa challenge of mice that had been subjected to differential antibiotic pretreatment.
Two and three days after cessation of antibiotic pretreatment with either vancomycin (Van), ciprofloxacin (Cip) or ampicillin plus sulbactam (A/S), mice were perorally challenged with multi-drug resistant P. aeruginosa on day 0 (d0) and d1. Upon sacrifize on d7 post-challenge, (A) the colonic lengths were measured (in cm) and the average number of (B) apoptotic (cleaved caspase-3+, Casp3+) epithelial cells in six high power fields (HPF) were determined in colonic paraffin sections applying in situ immunohistochemistry. A group without antibiotic application served as control cohort (w/o, grey symbols). Medians (black bars), numbers of analyzed mice (in parentheses), and significance levels (P values) assessed by the Kruskal- Wallis test with Dunn's post-hoc test are indicated. Data shown were pooled from four independent experiments
Citation: European Journal of Microbiology and Immunology 2025; 10.1556/1886.2025.00015
Colonic immune cell responses upon MDR Psae infection of mice that had been subjected to a differential antibiotic treatment
Next, we addressed whether distinct intestinal immune cell responses could be observed following oral MDR Psae challenge of mice after differential antibiotic pretreatment. Therefore, we stained large intestinal paraffin sections with antibodies directed against specific innate and adaptive immune cell populations (Fig. 6). Our quantitative immunohistochemical analyses revealed that on day 7 after oral MDR Psae exposure numbers of F4/80+ cells indicative for macrophages and monocytes were higher in the colonic mucosa and lamina propria of A/S and Van pretreated mice as compared to untreated counterparts (P < 0.01 and P < 0.05, respectively; Fig. 6A). Whereas colonic CD3+ T lymphocyte and FOXP3+ regulatory T cell counts did not differ among respective cohorts (n.s.; Fig. 6B and C), B220+ B lymphocytes were higher in the large intestinal mucosa and lamina propria of all pretreated groups versus the untreated cohort on day 7 post bacterial challenge (P < 0.001; Fig. 6D). Hence, both, innate and adaptive immune cell responses could be observed in the large intestines upon MDR Psae challenge in mice that had been subjected to differential antibiotic pretreatment.
Colonic immune cell responses upon P. aeruginosa challenge of mice that had been subjected to differential antibiotic pretreatment.
Two and three days after cessation of antibiotic pretreatment with either vancomycin (Van), ciprofloxacin (Cip) or ampicillin plus sulbactam (A/S), mice were perorally challenged with multi-drug resistant P. aeruginosa infection on day 0 (d0) and d1. Upon sacrifize on d7 post-challenge, the average number of distinct immune cell populations such as (A) macrophages and monocytes (F4/80+), (B) T lymphocytes (CD3+), (C) regulatory T cells (FOXP3+), and (D) B lymphocytes (B220+) in six high power fields (HPF) were determined in colonic paraffin sections applying in situ immunohistochemistry. A group without antibiotic application served as control cohort (w/o, grey symbols). Medians (black bars), numbers of analyzed mice (in parentheses), and significance levels (P values) assessed by the Kruskal-Wallis test with Dunn's post-hoc test or by the one-sided ANOVA test with Tukey post-hoc test are indicated. Data shown were pooled from four independent experiments
Citation: European Journal of Microbiology and Immunology 2025; 10.1556/1886.2025.00015
Intestinal nitric oxide secretion upon MDR Psae infection of mice that had been subjected to a differential antibiotic treatment
Then, we tested whether the intestinal immune responses following MDR Psae challenge in antibiotic pretreated mice were accompanied by enhanced pro-inflammatory mediator secretion in the intestinal tract. Therefore, we measured nitric oxide in ex vivo biopsies derived from different intestinal compartments on day 7 post MDR Psae exposure and detected higher nitric oxide concentrations in the colon (P < 0.01–0.001; Fig. 7A) and ileum (P < 0.05–0.01; Fig. 7B), but not the MLN (n.s.; Fig. 7C) derived from the A/S cohort as compared to the remaining experimental groups. Hence, A/S pretreated mice carrying MDR Psae exhibited enhanced small and large intestinal nitric oxide secretion.
Intestinal nitric oxide secretion upon P. aeruginosa challenge of mice that had been subjected to differential antibiotic pretreatment.
Two and three days after cessation of antibiotic pretreatment with either vancomycin (Van), ciprofloxacin (Cip) or ampicillin plus sulbactam (A/S), mice were perorally challenged with multi-drug resistant P. aeruginosa infection on day 0 (d0) and d1. Upon sacrifize on d7 post-infection, nitric oxide secretion was assessed in ex vivo biopsies taken from the (A) colon, (B) ileum, and (C) mesenteric lymph nodes (MLN). A group without antibiotic application served as control cohort (w/o, grey symbols). Medians (black bars), numbers of analyzed mice (in parentheses), and significance levels (P values) assessed by the Kruskal-Wallis test with Dunn's post-hoc test are indicated. Data shown were pooled from four independent experiments
Citation: European Journal of Microbiology and Immunology 2025; 10.1556/1886.2025.00015
Discussion
The complex gut microbiota provides a physiological shield protecting the vertebrate host against invasion and establishment of (opportunistic) enteropathogenic bacteria [15]. In our actual study we tested the effects of pretreatment with distinct antibiotic compounds and different antimicrobial spectra on inducing gut microbiota dysbiosis that predisposed mice to intestinal carriage of MDR Psae upon oral exposure. As expected, an intact gut microbiota protected untreated control mice from stable intestinal Psae colonization. On the contrary, as early as 3 days following oral Psae challenge, mice from the A/S group displayed much higher fecal bacterial carriage rates if compared to the remaining pretreated or nontreated cohorts. In fact, at the end of the observation period (i.e., day 7 post-challenge) MDR Psae could be isolated from the feces of all A/S pretreated mice, whereas the fecal bacterial carriage rates were much lower in the other pretreated or untreated cohorts ranging from 20% to 55%. When assessing the fecal microbiota composition after the 3-week-course of antibiotic pretreatment and immediately before oral Psae challenge it turned out that A/S application resulted in most prominent gut microbiota shifts towards decreased aerobic as well as anaerobic bacterial numbers if compared to the other cohorts. The significant reduction of distinct bacterial groups provided vacant ecological niches and nutrients facilitating stable colonization of the Psae bacteria in the gut lumen. Particularly A/S lowered fecal lactobacilli and virtually eradicated the C. leptum group. Our results are supported by our previous study showing that colonization resistance against the enteropathogen Campylobacter jejuni was abolished upon A/S pretreatment [27]. The analyses of both, the C. jejuni colonization dynamics and microbiota shifts identified lactobacilli and C. leptum as key bacterial groups that were associated with the colonization resistance against the enteropathogens further supporting our actual data. However, the gut microbial composition was less affected by Van and Cip pretreatment, which could account for the preserved colonization resistance seen in these groups as shown in our very recent [27] and actual study. Notably, mice from the Van or Cip cohorts retained distinct bacterial groups such as lactobacilli that have been shown to exert effective defense mechanisms including competition for adhesion sites and production of antimicrobial and immune-modulatory metabolites which in turn, protected from infections by enteropathogens such as C. jejuni, Salmonella spp., E. coli, and Listeria monocytogenes [28–34]. The importance of lactobacilli in mediating colonization resistance is further underscored by a survey showing that lactobacilli were more abundant in the intestinal tract of hospitalized patients who were not colonized by MDR (facultative) pathogenic bacteria including Psae if compared to MDR bacterial carriers [35]. In support, several studies provided evidence that viable bacteria, but also defined probiotic metabolites and bacteriocins derived from distinct Lactobacillus strains (including Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus acidophilus, and Lactobacillus gasseri) were able to inhibit growth and biofilm formation by Psae including MDR strains [36–40] and could additionally down-regulate expression of major virulence gene such as efflux pumps [39]. Remarkably, purified L-glutaminase derived from L. gasseri inhibited not only MDR Psae growth but also bacterial biofilm formation in an in vivo vaginosis model [40].
Interestingly, Pettigrew and colleagues provided evidence that intestinal abundance of defined bacteria taxa such as Clostridiales were negatively associated with MDR Psae carriage indicative for a potentially protective role of clostridia from MDR bacterial acquisition [17]. These data support our actual observation that A/S pretreated mice in which not only commensal lactobacilli but also C. leptum were virtually depleted were highly susceptible to stable intestinal MDR Psae colonization. Given these results, one might hypothesize that application of viable probiotic bacterial species either alone or in combination or even transfer of a complex microbiota rich in potentially probiotic bacteria might be reasonable approaches to repress MDR Psae from their ecological niches and be a therapeutic measure in the combat on MDR Psae carriage. In order to address the latter hypothesis, we previously subjected secondary abiotic mice generated by broad-spectrum antibiosis that were carrying more than 108 viable of Psae per gram feces with a complex murine fecal microbiota perorally [41] that is characterized not only by a pronounced microbial richness (i.e., high diversity), but also by high abundances of potentially probiotic bacterial genera and families including lactobacilli, clostridia, bifidobacteria, and Mouse Intestinal Bacteroides among others [14, 28, 42–44]. Our study revealed that upon murine fecal microbiota transplantation the intestinal MDR Psae loads could be lowered by approximately 4 orders of magnitude [41]. It is tempting to speculate that the concerted action of yet to be defined murine commensals including the afore-mentioned bacterial taxa such as lactobacilli and clostridia and derived antimicrobial molecules including bacteriocins and short chain fatty acids [45, 46] might have contributed to the Psae-lowering and in single cases, even clearing effects [41]. Remarkably, a clinical trial revealed that application of a probiotic mixture consisting of lactobacilli, bifidobacteria, and Saccharomyces boulardii to hospitalized patients who had to be treated with amoxicillin plus clavulanate resulted in decreased intestinal colonization with Psae [47].
Even though Van does not directly tackle obligate anaerobic bacteria, we found that, when compared to untreated counterpart mice, both anaerobic Gram-negative (Bacteroides/Prevotella spp. and Mouse Intestinal Bacteroides) and Gram-positive (C. coccoides and C. leptum) groups declined two days after antibiotic pretreatment had ended (i.e., day 0 and just before MDR Psae challenge). The decreased levels of Bacteroides/Prevotella spp. upon Van treatment observed in our actual and recent study [27] are further supported by a report from Russel and colleagues [48]. Notably, at the end of the Van pretreatment, we found a brief blossoming of enterobacteria in the feces, which is consistent with earlier findings that enterobacterial proliferation occurs during Van-induced dysbiosis which is most likely due to changes within the intraluminal milieu [49, 50].
A/S pretreated mice did not only display highest intestinal Psae burdens, but also bacteria-induced apoptosis of colonic epithelial cells that were accompanied by pronounced innate (i.e., macrophages and monocytes) and adaptive (B lymphocytes) immune cell responses and enhanced colonic and ileal nitric oxide secretion if compared to control mice with low Psae carrier rates. Interestingly, previous studies showed that Pseudomonas species were able to produce nitric oxide themselves depending on changes of the growth conditions (e.g., shift from aerobic to anaerobic milieu) and the pH values in their habitat [51]. Conversely, Psae could also use host nitric oxide to evade killing by neutrophilic granulocytes in the lung, thereby promoting pathogenic colonization [52]. Our actual data are further supported by our previous study showing enhanced apoptotic cell responses in colonic epithelia and pro-inflammatory mediator secretion in intestinal and even systemic compartments following Psae challenge of mice harboring a complex human versus murine gut microbiota [22].
Conclusion and future perspectives
In summary, distinct gut microbiota shifts upon pretreatment with antibiotic compounds differentially predisposed mice to intestinal carriage of MDR Psae upon oral exposure. A/S induced dysbiosis with virtually depleted lactobacilli and C. leptum resulted not only in high intestinal Psae loads in all challenged mice but was also accompanied by enhanced distinct pro-inflammatory immune responses and colonic epithelial cell apoptosis. Future studies should include metabolomic and metagenomic analyses to fully describe the changes in the commensal gut community's structure as well as functional (i.e., pathophysiological) implications brought on by antibiotics as well as to further dissect which metabolic pathways and metabolites are affected by antibiotic-induced dysbiosis and how they impact the gastrointestinal colonization by distinct MDR (facultative) pathogenic bacteria. Furthermore, long-term examinations of these alterations may shed light onto the relationship between immunological reprogramming and microbial recovery, thereby identifying possible targets for interventive measures.
Ethics statement
All animal experiments were conducted according to the European Guidelines for animal welfare (2010/63/EU) with approval of the local commission for animal experiments (Landesamt für Gesundheit und Soziales, Berlin). Animal welfare was monitored daily by assessment of clinical conditions of mice.
Conflict of interest
SB and MMH are members of the Editorial Board of the journal, therefore they did not take part in the review process in any capacity and the submission was handled by a different member of the editorial board. The submission was subject to the same process as any other manuscript and editorial board membership had no influence on editorial consideration and the final decision.
Author's contributions
MMH: Designed and performed experiments, analyzed data, wrote paper
SM: Analyzed data, co-edited paper
NWS: Analyzed data, co-edited paper
ABL: performed experiments;
IP: performed experiments;
GR: performed experiments
SB: Provided advice in design and performance of experiments, co-edited paper.
Funding
This work was supported by grants from the German Research Foundation (DFG; SFB633, TP A7 and B6) and also funded by the European Union's Horizon 2020 research and innovation programme (under the Marie Sklodowska-Curie grant agreement No. 956279; COL_RES project). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
List of abbreviations
| A/S | ampicillin plus sulbactam |
| Casp3 | caspase-3 |
| CFU | colony-forming units |
| Cip | ciprofloxacin |
| d | day |
| HPF | high power fields |
| MDR | multi-drug resistant |
| MLN | mesenteric lymph nodes |
| PBS | phosphate buffered saline |
| Psae | Pseudomonas aeruginosa |
| qRT-PCR | quantitative real-time polymerase chain reaction |
| spp | species |
| Van | vancomycin |
Acknowledgments
We thank Ulrike Escher, Ulrike Fiebiger, Eliane von Klitzing, Michaela Wattrodt, and André Fischer for excellent technical assistance and the staff of the animal research facility of the Charité – University Medicine Berlin for animal breeding are caretaking.
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