Abstract Extended-spectrum β-lactamase-producing Escherichia coli (ESBL-E) are becoming increasingly widespread in Vietnam. Antibiotics are detected in many Vietnamese foods; however, the effect of ESBL-E and antibiotic consumption on intestinal bacteria has not been studied sufficiently. Here, we investigated the effect of oral administration of ESBL-E (TB19) and cefotaxime on luminescence-emitting cefotaxime-sensitive E. coli (X14). Mice were given water containing TB19 and then received three injections of 1.0 × 108 CFU of X14 harboring a luciferase gene. The mice were administered 100 μg of cefotaxime and luminescent bacteria were monitored over 24 h, following which luminescent bacteria were isolated from mouse feces. Luminescence continued to be detected in mice administered TB19 24 h after cefotaxime ingestion. Fecal analysis revealed two types of luminescent colonies: cefoxitin-resistant E. coli (X14-R) and Pseudomonas aeruginosa. Pulse-field gel electrophoresis confirmed that X14-R was a clonal strain of X14, suggesting that X14 survived using ESBLs originating from TB19 and acquired cefoxitin resistance due to cefotaxime consumption. Moreover, in vitro analysis of X14 indicated that expression of the ampC gene was upregulated by cefotaxime. Overall, ESBL-E and cefotaxime promoted the expansion of cefoxitin-resistant E. coli in the absence of plasmid-mediated gene transfer. AmpC β-lactamase-producing Escherichia coli, extended-spectrum β-lactamase-producing Escherichia coli, luminescent Escherichia coli, cefotaxime INTRODUCTION Antimicrobial-resistant bacteria are associated with persistent diseases and their global prevalence is continuously growing, warranting investigation (Morrissey et al.2013). Extended-spectrum β-lactamases (ESBLs) are enzymes produced by Enterobacteriaceae, including Escherichia coli and Klebsiella pneumoniae. ESBLs block the activity of β-lactam antibiotics, such as penicillin and cephalosporin (Falagas and Karageorgopoulos 2009). Recent studies demonstrated a high prevalence of ESBL-producing bacteria in various communities in Asian countries (Tian et al.2008; Sasaki et al.2010; Hsueh et al.2011; Luvsansharav et al.2011). In a previous study, we investigated food contamination with ESBL-producing E. coli (ESBL-E) in a Vietnamese market. The study revealed that ESBL-E are more predominant in chicken than in beef, pork or shrimp (Lee et al.2015). Moreover, analysis revealed that chicken harbors high concentrations of antibiotic residues (Yamaguchi et al.2015). These findings may be explained by the lack of antibiotic usage regulation at meat production sites (Carrique-Mas et al. 2013). Additionally, the prevalence of ESBL-E carriers among healthy individuals is higher in Hanoi, Vietnam, than in Japan (Nakayama et al.2015). Generally, the expansion of ESBL-E is related to plasmid-meditated horizontal gene transfer. Indeed, Hoang et al. (2017) reported the presence of 104–139 kbp plasmids encoding blaCTX-M-55 in ESBL-E in pork, market workers and patients with urinary tract infections. In vivo imaging systems are important tools for analyzing bacterial behavior in the host (Lau et al.2015). Mouse models have been used to study the pathogenesis of infectious diarrhea caused by E. coli, and in vivo imaging was used to evaluate E. coli behavior in the mouse gut (Rhee et al.2011). Moreover, antibiotic treatments including the effect of systemic and topical antibiotics on bacterial infections have also been examined using in vivo imaging systems (Andrue et al.2013; Guo et al.2013; Hertlein et al.2013; Niska et al., 2013; Bernut et al.2014). This method was proven useful in monitoring the bacterial burden, representing an alternative method of evaluating preclinical in vivo efficacies of systemic and topical antimicrobial agents. Accordingly, in vivo imaging systems are increasingly employed in various biomedical fields. Although the presence of ESBL-E in humans is thought to be associated with consumption of food contaminated with ESBL-E, no reports to date have described this phenomenon. Nevertheless, we confirmed the presence of antibiotic-contaminated foods in Vietnam (Lee et al.2015). In the current study, we aimed to clarify the effect of orally administered ESBL-E on luminescent antibiotic-sensitive E. coli in the mouse gut following cefotaxime consumption. METHODS Bacterial strains Escherichia coli TB1 and TB19 were isolated from backyard chicken farms in Thai Binh, Vietnam. The TB1 strain is susceptible to cefotaxime and the TB19 strain possesses ESBL (Table 1). The luminescent E. coli Xen14 strain (X14) was obtained from Perkin Elmer (Waltham, MA, USA). X14 chromosomes and plasmids were confirmed to possess the luxCDABE operon. All E. coli strains were cultured in Luria-Bertani (LB) medium (Sigma-Aldrich, St. Louis, MO, USA). Table 1. Antimicrobial susceptibilities of bacterial strains. Antimicrobial susceptibility test Disk diffusion test Minimum inhibitory concentration (μg/mL) Aminoglycoside Quinolone β-lactam β-lactam Strain Laboratory name Lumine- scence Type of antibiotic resistance TC CP ST SM KM GM NA CPFX ABPC CTX CAZ CFX MEM ABPC CTX CAZ CFX MEM Bacteria administered to mice TB1 No – R R S R R S S S R S S S S 256 0.094 0.125 2 0.032 (R) (S) (S) (S) (S) E. coli TB19 No ESBL R S R R S S S S R R S S S >256 32 0.75 1.5 0.032 (R) (R) (S) (S) (S) X14 Yes – S S S R R S S S I S S S S 24 0.25 0.38 8 0.047 (I) (S) (S) (S) (S) Luminescent bacteria isolated from feces L1 Yes – – – – – – S – S – – S – S – – 1 >256 0.25 (S) – (S) P. aeruginosa L2 Yes – – – – – – S – S – – S – S – – 1 >256 0.19 (S) – (S) L3 Yes – – – – – – S – S – – S – S – – 0.75 >256 0.125 (S) – (S) X14-R1 Yes AmpC S S S R R S S S R I S R S >256 2 4 64 0.064 E. coli (R) (I) (S) (R) (S) X14-R2 Yes AmpC S S S R R S S S R I S R S 128 2 4 192 0.047 (R) (I) (S) (R) (S) Antimicrobial susceptibility test Disk diffusion test Minimum inhibitory concentration (μg/mL) Aminoglycoside Quinolone β-lactam β-lactam Strain Laboratory name Lumine- scence Type of antibiotic resistance TC CP ST SM KM GM NA CPFX ABPC CTX CAZ CFX MEM ABPC CTX CAZ CFX MEM Bacteria administered to mice TB1 No – R R S R R S S S R S S S S 256 0.094 0.125 2 0.032 (R) (S) (S) (S) (S) E. coli TB19 No ESBL R S R R S S S S R R S S S >256 32 0.75 1.5 0.032 (R) (R) (S) (S) (S) X14 Yes – S S S R R S S S I S S S S 24 0.25 0.38 8 0.047 (I) (S) (S) (S) (S) Luminescent bacteria isolated from feces L1 Yes – – – – – – S – S – – S – S – – 1 >256 0.25 (S) – (S) P. aeruginosa L2 Yes – – – – – – S – S – – S – S – – 1 >256 0.19 (S) – (S) L3 Yes – – – – – – S – S – – S – S – – 0.75 >256 0.125 (S) – (S) X14-R1 Yes AmpC S S S R R S S S R I S R S >256 2 4 64 0.064 E. coli (R) (I) (S) (R) (S) X14-R2 Yes AmpC S S S R R S S S R I S R S 128 2 4 192 0.047 (R) (I) (S) (R) (S) ABPC: ampicillin, CTX: cefotaxime, CAZ: ceftazidime, CFX: cefoxitin, MEM: meropenem, SM: streptomycin, KM: kanamycin, GM: gentamicin, CPFX: ciprofloxacin NA: nalidixic acid, TC: tetracycline, CP: chloramphenicol, ST: sulfamethoxazole/trimethoprim S: susceptible, I: intermediate, R: resistant View Large Confirmation of bacterial luminescence After subculturing X14 for 17 h, 5 μL of the culture was added to 5 mL of LB medium and incubated at 37°C for 24 h. Then, 2 mL of the X14 broth was used to measure luminescence by an IVIS Lumina II imaging system (Perkin Elmer). For all in vitro experiments, the IVIS system was set up for 1-s exposure and the region of interest was adjusted to 25% of the threshold to quantify luminescence. Serial dilutions of subcultured X14 broth were used to determine both luminescence and bacterial concentrations. For bacterial concentration analysis, bacterial broth was diluted in sterile phosphate-buffered saline and then 100 μL of diluted bacterial broth was spread on LB agar. The plates were incubated at 37°C for 24 h and then colony forming units (CFUs) were counted. The length of time for which E. coli emitted luminescence in vitro was investigated as follows: after subculturing, 5 μL of X14 broth was added to 5 mL of LB medium, incubated at 37°C and then cell luminescence was determined using the IVIS imaging system after 24, 48, 72 and 96 h. To investigate the effect of cefotaxime on X14, the bacteria were subcultured for 17 h and then 1.0 × 108 CFU of X14 was added to LB medium containing 0, 5, 10, 50 or 75 μg/mL cefotaxime. The cultures were incubated at 37°C for 6 h, and then cell luminescence was evaluated using the IVIS imaging system (see below). Animal experiments Twelve-week-old specific-pathogen-free C57BL/6 mice (Japan Clea, Tokyo, Japan) were acclimatized to standard laboratory conditions and provided free access to rodent chow and water for 1 week. The experimental schedule is outlined in Fig. 2A. Three different types of drinking water (200 mL) were prepared (bacteria-free water, given to four mice; water containing 1.0 × 106 CFU of TB1, given to three mice; and water containing TB19, given to four mice). The mice had free access to the prepared water for 3 days, after which all mice were administered 500 μL (1.0 × 109 CFU) of luminescent E. coli per day for 3 days using an oral gavage (Fuchigami, Kyoto, Japan). Then, 3 h after the final luminescent E. coli administration, mice were given 100 μg of cefotaxime in 500 μL of sterile phosphate-buffered saline. Bacterial luminescence was detected using the IVIS imaging system at 0, 6 and 24 h after cefotaxime consumption. Mouse feces were sampled 24 h after cefotaxime consumption. Sterile phosphate-buffered saline (500 μL) was added to the sampled feces and then homogenized. Homogenized feces samples were serially diluted and spread on CHROMagar ECC (CHROMagar, Paris, France) plates containing 1 μg/mL cefotaxime and incubated at 37°C for 24 h. Luminescence of the resultant colonies was determined by the IVIS imaging system. All animal experiments in the current study were performed according to the guidelines of the Animal Care Committee of Osaka University. In vivo imaging using the IVIS system To monitor luminescent E. coli in vivo, the IVIS Lumina II imaging system was used. Mice were injected intraperitoneally with 40 μL of anesthetic solution prepared by mixing 10 mL of ketamine (Daiichi-Sankyo, Tokyo, Japan) with 2.2 mL of 2% xylazine (Bayer, Leverkusen, Germany) to immobilize the animals for imaging. The IVIS imaging system was set up for a 3-min exposure, and the region of interest was adjusted to a threshold of 25% for luminescence quantification. Identification of bacteria from fecal colonies Fecal colonies were examined for luminescence emission, and luminescent colonies were selected for analysis. Selected colonies were then cultured at 37°C for 24 h, and DNA was extracted using the boiling method (Nakayama et al.2015). Next, 1 ng/μL of extracted DNA was subjected to polymerase chain reaction (PCR) to amplify the 1500-bp fragment of the 16S rRNA gene (35 cycles of 95°C for 30 s, 53°C for 30 s and 72°C for 90 s) using a thermal cycler (Takara Bio, Shiga, Japan). A pair of 16S universal rRNA primers (forward, 5΄-agagtttgatcctggctcag-3΄; reverse, 5΄-tacggttaccttgttacgactt-3΄) (Nakayama and Oishi 2013) was ordered from Gene Design (Osaka, Japan). The amplified gene fragment was purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany), and sequenced at Macrogen Japan (Kyoto, Japan). Sequencing results were analyzed by Lebibi (https://umr5558-bibiserv.univ-lyon1.fr/lebibi/lebibi.cgi). Pulse-field gel electrophoresis Pulse-field gel electrophoresis (PFGE) was used to determine if a clonal strain of E. coli was isolated from feces, as previously described (Hoang et al.2017) and based on the manufacturer's protocol (Bio-Rad, Hercules, CA, USA). After bacterial subculture, E. coli colonies were sampled and cells were washed with distilled water. To prepare plugs, cells suspended in 150 μL of distilled water were mixed with 150 μL of 1% Seakem Gold agarose (Lonza Inc., Allendale, USA). The plugs were soaked in 1 μg/mL protease K solution (Roche, Basel, Switzerland), incubated at 50°C for 17 h, treated with pefabloc (Sigma-Aldrich) and then incubated at 4°C for 20 min. The supernatant was discarded and removed, and samples were digested with Xba1 (Nippon Gene, Tokyo, Japan) at 37°C for 2 h. Electrophoresis was performed using a CHEF-DRIII system (Bio-Rad) with 1% Seakem Gold agarose. Electrophoresis conditions were as follows: initial time, 2.2 s; final time, 54.2 s; voltage, 6 V/cm; run time, 19 h. After electrophoresis, gels were stained with GelRed nucleic acid stain (Biotium, Hayward, USA) and imaged using a FluorChem Q system (Cell Bioscience, Santa Clara, USA). Antimicrobial susceptibility testing Six antibiotic groups, namely β-lactams (ampicillin, cefotaxime, ceftazidime, cefoxitin and meropenem), quinolones (nalidixic acid and ciprofloxacin), aminoglycosides (kanamycin, streptomycin and gentamicin), tetracycline, chloramphenicol and folic acid inhibitors (trimethoprim-sulfamethoxazole), were used to evaluate antimicrobial susceptibility of E. coli. Gentamicin, ciprofloxacin, ceftazidime and meropenem were also used to test antimicrobial susceptibility of Pseudomonas aeruginosa. Antibiotic susceptibility was determined by the disk diffusion method using antibiotic disks (Becton Dickinson, Franklin Lakes, NJ, USA) according to the standard procedure of the Clinical and Laboratory Standards Institute (CLSI; Wayne, PA, USA). Susceptibility test results were interpreted using the CLSI document, M100-S24. PCR amplification of luxCDABE and blaCTX-M-9 The presence of luxCDABE and blaCTX-M-9 genes was evaluated in luminescent colonies as follows: DNA was extracted from bacteria after culturing for 17 h as described above for bacterial identification. Then, 1 ng/μL of extracted DNA was used to amplify luxCDABE and blaCTX-M-9 genes by PCR in a thermal cycler (luxCDABE, 35 cycles of 95°C for 30 s, 53°C for 30 s and 72°C for 30 s; blaCTX-M-9, 25 cycles of 95°C for 30 s, 60°C for 90 s and 72°C for 90 s). Primers were designed by Gene Design (luxCDABE forward, 5΄-cgataattgggtcaagcaag-3΄ and reverse, 5΄-aaattggggaggttggtatg-3΄; blaCTX-M-9 forward, 5΄-gtgcaacggatgatgttcgc-3΄ and reverse, 5΄-gaagcgtctcatcgccgatc-3΄). RNA extraction and real-time reverse transcription-PCR To quantify gene expression, real-time reverse transcription (RT)-PCR was performed. Bacteria were subcultured and 2 μL of the X14 culture was added to 2 mL of LB medium containing 0, 0.2 or 2.0 μg/mL cefotaxime. mRNA expression of X14-R ampC was also investigated by adding 2 μL X14-R to 2 mL LB medium without cefotaxime. Cells were then cultured for 17 h, and RNA extraction was performed as described previously (Nakayama, Nomura and Matsumura 2007). After subculturing, 5 μL of bacterial cells was added to 5 mL of LB medium and cultured for 17 h. Cultured E. coli cells were washed with sterile phosphate-buffered saline, centrifuged at 4°C for 10 min and then the bacterial pellet was mixed with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), glass beads and chloroform. After centrifugation, the upper phase was sequentially extracted with isopropanol and 70% ethanol. Extracted RNA was subjected to RT for cDNA synthesis using a Roche cDNA synthesis kit (Roche, Basel, Switzerland). Real-time PCR was then performed (40 cycles of 94°C for 20 s, 53°C for 20 s and 72°C for 30 s) using a 7900HT fast real-time PCR system (Applied Biosystems, Paisley, UK). Three sets of primers from Gene Design were used to evaluate the expression of three genes, ampC (forward, 5΄-gcatcgcacaattaccccgc-3΄; reverse, 5΄-aatagcgtcgccaccaagca-3΄), luxCDABE (described above) and E. coli 16S-23S rRNA (forward, 5-΄caattttcgtgtccccttcg-3΄; reverse, 5΄-gttaatgatagtgtgtcgaaac-3΄; Nakayama and Oishi 2013). Data were analyzed using the ΔΔCt method. Relative intensity was calculated as the target gene signal intensity divided by the 16S-23S rRNA gene signal intensity (ampC or luxCDABE/E. coli 16S-23S rRNA). Statistical analysis Significance of the results was established with the Pearson correlation test (Fig. 1A) or Student's t-test (Figs 1B and C, 2C and 4B and C) with using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Figure 1. View largeDownload slide Optimization of the detection of luminescent E. coli. (A) Correlationship between E. coli numbers and luminescence intensity. Data were analyzed by Pearson correlation tests. (B) Escherichia coli continues to emit luminescence. (C) Effect of cefotaxime concentration on bacterial luminescence. Data were analyzed by Student's t-test. * P < 0.01. Figure 1. View largeDownload slide Optimization of the detection of luminescent E. coli. (A) Correlationship between E. coli numbers and luminescence intensity. Data were analyzed by Pearson correlation tests. (B) Escherichia coli continues to emit luminescence. (C) Effect of cefotaxime concentration on bacterial luminescence. Data were analyzed by Student's t-test. * P < 0.01. Figure 2. View largeDownload slide Detection of bacterial luminescence over a 24-h period following oral administration of ESBL-E and cefotaxime ingestion. (A) Experimental schedule. (B) Imaging of the luminescence of TB19 24 h after cefotaxime consumption. (C) Effect of TB19 consumption and TB1 consumption on bacterial luminescence. Data were analyzed by Student's t-test. * P < 0.05. Figure 2. View largeDownload slide Detection of bacterial luminescence over a 24-h period following oral administration of ESBL-E and cefotaxime ingestion. (A) Experimental schedule. (B) Imaging of the luminescence of TB19 24 h after cefotaxime consumption. (C) Effect of TB19 consumption and TB1 consumption on bacterial luminescence. Data were analyzed by Student's t-test. * P < 0.05. RESULTS Detection of luminescent bacteria Luminescent X14 cells were visualized using the IVIS imaging system, and the minimum concentration of bacteria detected was 1.0 × 106 CFU/mL. A clear relationship was observed between luminescence and bacterial concentration (P = 0.0007; Fig. 1A). Evaluation of luminescent signal duration revealed that high-intensity luminescence was detectable for 2 days and gradually decreased thereafter (Fig. 1B). The effect of several concentrations of cefotaxime on X14 luminescence was investigated. As shown in Fig. 1C, luminescence of X14 was affected by the presence of cefotaxime at concentrations exceeding 50 μg/mL. In vivo imaging of mice administered luminescent bacteria Mice were orally administered luminescent bacteria and evaluated by in vivo imaging. Six hours after cefotaxime treatment, luminescent signals were detected in all mice in the abdomen. Notably, 24 h after cefotaxime consumption, luminescence was detected in all mice administered TB19 but not in mice administered TB1 (Fig. 2B). Moreover, quantification of luminescent intensity based on relative light units (RLUs) revealed that TB19 resulted in significantly higher RLUs than did TB1 24 h after cefotaxime consumption (Fig. 2C). Identification of bacteria in fecal luminescent colonies After cefotaxime consumption (24 h), mouse feces were collected and two types of luminescent colonies were detected on CHROMagar ECC plates containing 1 μg/mL cefotaxime. Luminescent colonies were identified as E. coli (X14-R1 and X14-R2, obtained from two mice) and P. aeruginosa (L1, L2 and L3, obtained from three mice) by 16S rRNA sequencing (Table 1). All isolated bacteria harbored the luxCDABE gene (Fig. 3A) but not the blaCTX-M-9 gene. PFGE analysis of E. coli, X14 and luminescent isolates from the mouse feces revealed that the only difference between X14 and X14-R was the size of the second largest PFGE band (Fig. 3B). Figure 3. View largeDownload slide Confirmation that luminescent bacteria harbor the luxCDABE gene and identification of E. coli by molecular techniques. (A) Detection of the luxCDBAE genes by PCR, and (B) identification of the isolated cells from luminescent fecal colonies using PFGE. The white arrow indicates the second largest PFGE band in X14-R. Figure 3. View largeDownload slide Confirmation that luminescent bacteria harbor the luxCDABE gene and identification of E. coli by molecular techniques. (A) Detection of the luxCDBAE genes by PCR, and (B) identification of the isolated cells from luminescent fecal colonies using PFGE. The white arrow indicates the second largest PFGE band in X14-R. Antibiotic susceptibility testing of fecal isolates Evaluation of antibiotic susceptibility and minimum inhibitory concentrations of luminescent E. coli indicated cefoxitin, streptomycin and kanamycin resistance. Luminescent P. aeruginosa showed high cefoxitin resistance (Table 1). Effect of cefotaxime on luxCDABE and ampC gene expression To determine the effects of cefotaxime on luxCDABE and ampC gene expression, in vitro imaging and quantitative RT-PCR were performed. In vitro imaging revealed that bacteria growing in the presence of cefotaxime exhibited increased luminescence than bacteria growing in the absence of the antibiotic in both liquid (Fig. 4A and B) and agar media (data not shown). Expression levels of ampC and luxCDABE evaluated by real-time PCR (Fig. 4C) revealed that cefotaxime promoted bacterial luminescence and AmpC β-lactamase gene expression. X14-R ampC expression was also determined, showing that in the absence of cefotaxime, ampC expression was higher than in X14. Figure 4. View largeDownload slide Low-concentration cefotaxime affects luminescent E. coli X14 in a dose-dependent manner in vitro. (A) Imaging analysis of the effect of cefotaxime on bacterial luminescence. (B) Data from (A) analyzed by IVIS. Quantification of the RLUs is shown. (C) Quantitative RT-PCR analysis of the effect of cefotaxime on ampC and luxCDABE expression. The ampC expression was also investigated in X14-R in the absence of cefotaxime. Data were analyzed by Student's t-test. Figure 4. View largeDownload slide Low-concentration cefotaxime affects luminescent E. coli X14 in a dose-dependent manner in vitro. (A) Imaging analysis of the effect of cefotaxime on bacterial luminescence. (B) Data from (A) analyzed by IVIS. Quantification of the RLUs is shown. (C) Quantitative RT-PCR analysis of the effect of cefotaxime on ampC and luxCDABE expression. The ampC expression was also investigated in X14-R in the absence of cefotaxime. Data were analyzed by Student's t-test. DISCUSSION Inappropriate use of antibiotics has led to the expansion of antimicrobial-resistant bacteria. One mechanism of expansion is horizontal gene transfer mediated by mobile genetic elements, including plasmids (Abd El-Aziz and Gharib 2015; Yu et al.2016). ESBL-related genes, such as blaCTX-M, are mainly encoded by plasmids and can also be transferred to other E. coli or Enterobacteriaceae via plasmids (Sandegren et al.2012; De-Toro et al.2013); however, not all plasmids can be transferred to E. coli or Enterobacteriaceae. ESBL-E TB19 harbors an ESBL-related blaCTX-M-9 gene-encoding plasmid that can be transferred to other E. coli or Enterobacteriaceae. Thus, we hypothesized that a plasmid encoding blaCTX-M-9 might also be transferred to X14 or mouse intestinal bacteria. However, after mice were administered cefotaxime, isolated fecal luminescent bacteria were not positive for the blaCTX-M-9 gene, suggesting that blaCTX-M-9-encoding plasmids derived from TB19 were not suitable for transfer to X14 or mouse intestinal bacteria. Interestingly, mice orally administered TB19 exhibited abdominal luminescence 24 h after cefotaxime ingestion, while luminescence in other mouse groups disappeared over time. This suggests that TB19 released ESBL enzymes that helped X14 survive in the gut during the initial phase after cefotaxime consumption and subsequently allowed cefotaxime to impart X14 cefotaxime resistance (generating X14-R). In addition, luminescence intensity (6 h after cefotaxime ingestion) of mice that were only administered X14 was similar to that of mice that were administered TB19 and X14. On the other hand, the intensity of luminescence in mice administered TB1 and X14 was lower than that of the other two groups. These findings suggest that oral administration of TB1 and X14 did not allow for sufficient X14 growth during the early phase after cefotaxime consumption. According to previous studies, cefoxitin resistance can arise due to genetic mutations (Mulvey et al.2005; Berrazeg et al.2015) or transfer of gene-harboring plasmids (Mulvey et al.2005; Abd El-Aziz and Gharib). In the current study, we demonstrated a single band difference between X14 and X14-R using PFGE, suggesting that X14-R originated from X14 and that the X14 chromosome may have mutated in response to cefotaxime ingestion by the host mouse. ampC promoter and attenuator region mutations are the main mechanisms behind AmpC overproduction (Lim and Nikaido 2010; Berrazeg et al.2015); AmpC overproduction is usually associated with a reduced susceptibility to third-generation cephalosporins (Mulvey et al.2005). Moreover, a consensus –35 box (TTGACA) is the most critical factor affecting the ampC promoter (Tracz et al.2007). In the current study, we did not observe any sequence differences in ampC promoter or attenuator regions of X14 and X14-R cells (data not shown). According to antibiotic susceptibility tests, X14 was originally resistant to ampicillin. The β-lactam-susceptible strain harbored a T at position –32 of the ampC promoter –35 box, while both X14 and X14-R had an A at this position; therefore, the original X14 strain possessed a mutated ampC promoter and showed ampicillin resistance. Furthermore, in vitro analysis revealed that both luxCDABE and ampC genes were upregulated by cefotaxime consumption, although we were unable to identify the chromosome sequence mutated in response to cefotaxime ingestion by the host. Results suggest that cefotaxime administration promoted luminescence expression and antibiotic resistance. AcrAB-TolC is a well-known multidrug efflux transporter that extrudes a diverse range of β-lactams from the cell (Lim and Nikaido 2010). In addition, resistance to β-lactams most commonly involves alterations of penicillin-binding proteins or expression of β-lactamases (Everaert and Coenye 2016). We did not evaluate efflux transporters or alterations in penicillin-binding proteins in the current study; therefore, it is possible that these changes also occurred in the isolated X14-R cells. In mice orally administered TB19, luminescent P. aeruginosa was also detected. According to Zhu et al. (2013), P. aeruginosa easily becomes resistant to β-lactam antibiotics and almost all P. aeruginosa cells are naturally resistant to cefoxitin due to AmpC β-lactamase production (Wolter et al.2009). Notably, we observed no fecal bacteria growth on agar containing 1 μg/mL cefotaxime before cefotaxime ingestion, suggesting that P. aeruginosa was not resistant to originally cefotaxime at that time. However, after cefotaxime ingestion, ESBL was produced by TB19 and P. aeruginosa acquired cefoxitin resistance. Moreover, isolated P. aeruginosa cells also harbored the luxCDABE genes obtained from X14, a strain which harbors plasmid luxCDABE. In the current study, P. aeruginosa did not possess luxCDABE genes. Although we could not clarify how lux CDABE was transferred to P. aeruginosa, it is possible that it occurred through plasmid conjugation, as shown in previous studies (Winson et al.1998; Kassem et al.2010; Danino et al.2013). In conclusion, cefotaxime susceptibility of E. coli was rescued by ESBL-E in the presence of cefotaxime in high concentrations. Furthermore, cefotaxime consumption by the animal host induced cefoxitin resistance in cefotaxime-susceptible E. coli. Our study revealed that the presence of ESBL—even in the absence of plasmid-mediated genetic transfer—may lead to the expansion of antimicrobial-resistant bacteria. Acknowledgements We thank the research staff of the Laboratory of Clinical Research on Infectious Disease, RIMD, Osaka University who helped us with the IVIS imaging system. 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FEMS Microbiology Letters – Oxford University Press
Published: Mar 1, 2018
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