Investigation of the effect of β-lactam antibiotics and serum on growth and gene expression in Escherichia coli strain JJ 1886

* Correspondence: elif.bozcal@istanbul.edu.tr

phase of E. coli, and genetic location of the bla CTX-M gene (11). Similarly, upregulation of the bla CTX-M- 15 gene has been reported in Shigella sonneii in the presence of CAZ (12). Therefore, it can be inferred that genes encoding CTX-M-type β-lactamases can be upregulated or downregulated in the presence of β-lactam antibiotics. Moreover, the control of β-lactamase gene expression is regulated by a histidine kinase/response regulator pair in Vibrio parahaemolyticus (4). In addition, AmpE is a putative signaling protein in β-lactamase regulation in E. coli, and AmpE might help reveal the effect of β-lactam on peptidoglycan biosynthesis (13).
On the occasions that the CTX-M-15-type β-lactamase-producing E. coli ST131 pandemic strain has been found in the blood causing septicemia and bacteremia, it has been resistant to the bactericidal effects of serum (21). Many virulence factors are involved in E. coli resistance to the bactericidal activity of serum. To illustrate, the proteins associated with lipopolysaccharide (LPS) biosynthesis and the K capsule have significant contributions to serum survival, as does the outer membrane protein (OmpA) (21,22). Moreover, serum survival factors, including Trat, Iss, and phage proteins encoded by plasmids, are serum survival factors in E. coli (23). Other factors or proteins that may be involved in serum resistance have been reported, including NlpI lipoprotein, murein lipoprotein (Lpp), and the phosphate transport system (24)(25)(26).
Given that the E. coli ST131 strain exhibits resistance to many antibiotics and serum, it is important to study the inhibitory and bactericidal effect of antibiotics and serum on this strain. Here we investigate the effect of β-lactam antibiotics plus human serum on E. coli strain JJ1886 (025b:H4-ST131) growth and the gene expression of β-lactamase and serum resistance-associated genes.

Bacterial strains and normal human serum
The CTX-M-15-type β-lactamase-producing E. coli epidemic strain JJ1886 was used as the test microorganism. E. coli strain JJ1886 was kindly provided by Professor James R Johnson (University of Minnesota, VA Medical Center, Minneapolis, MN, USA). E. coli ATCC 25922 strain was used as a quality control strain for minimum inhibitory concentration (MIC) testing analysis. Normal human serum (NHS) was commercially purchased (Panbiotech, Germany). To inactivate the complement system of NHS, serum was incubated at 56 °C for 30 min, resulting in heatinactivated serum (HIS) (27). NHS and HIS were stored at -80 °C until further analysis.

Dimethyl sulfoxide stocks of bacterial strains
Dimethyl sulfoxide (DMSO) stocks of E. coli strain JJ1886 and E. coli ATCC 25922 were prepared as follows: bacterial cultures were incubated overnight, diluted to 1/100 in Luria Bertani (LB) broth, and grown until the logarithmic growth phase at 37 °C (OD 600 : 0.8). Next, DMSO was added to the bacterial culture, giving a final concentration of 8%. Bacterial cultures were dispensed in volumes of 120 µL and stored at -80 °C (28).

Nitrocefin test
Nitrocefin is a chromogenic cephalosporin. Through the procedure provided by the manufacturer (Bioanalyse), we phenotypically tested whether E. coli strain JJ1886 is a β-lactamase producer. E. coli ATCC 25922 was used as a quality control strain. Nitrocefin disks change from yellow to red within 5 min (as quickly as 15 s) when the β-lactam antibiotic is hydrolyzed by bacterial induction of the β-lactamase enzyme.

Time-dependent effect of NHS
E. coli strain JJ1886 from DMSO stock was inoculated into LB broth and incubated for 24 h at 37 °C. The concentration of 24-h bacterial culture was adjusted in PBS to the 0.5 McFarland standard (1.5 × 10 8 CFU/mL). The final bacterial concentration was diluted to 5 × 10 6 CFU/mL. Next, LB broth, NHS (1:2 v/v), HIS (1:2 v/v), and E. coli strain JJ1886 were added to the wells of a 96-well plate. The bacteria were treated with NHS, HIS, and LB by incubation at 37 °C without shaking. Samples were taken at time intervals of 0, 1, 2, and 3 h (31). Serial dilution was performed in a sterile 1X PBS buffer and plated on Luria agar (LA) plates by drop test, as previously described (32). The time-dependent effect of serum on bacterial growth was calculated as log 10 CFU/mL after incubation at 37° C for 24 h (n = 3).

Time-dependent effect of NHS and β-lactam antibiotics
Measurement of the time-dependent effect of NHS and β-lactam antibiotics including CTX, CAZ, and CAR was performed as mentioned in Section 2.5. Briefly, the concentrations of CTX, CAZ, and CAR were diluted in LB broth in 96-well plates as follows: 512-0.125 µg/mL, 5120-2.5 µg/mL, and 20-0.625 mg/mL, respectively. After that, LB broth, NHS (1:2 v/v), HIS (1:2 v/v), and E. coli strain JJ1886 were added to each well containing CTX, CAZ, or CAR. Samples were withdrawn at time intervals of 0, 1, 2, 3, and 24 h from the well equal to the MIC value for each antibiotic. Time-dependent effects of NHS and β-lactam antibiotics was calculated as log 10 CFU/mL after 24 h of incubation at 37 °C (n = 3).

Standard PCR
In order to determine the level of expression of serum resistance and β-lactamase-associated genes in the presence of β-lactam antibiotics and NHS, the genes encoding β-lactamase (bla CTX-M ), β-lactamase regulator (ampE), lipoprotein NlpI (nlpI), murein lipoprotein (lpp), lipopolysaccharide core heptose (I) kinase (rfaP), LPS biosynthesis protein (wbbJ), capsule biosynthesis (kpsS), peptidoglycan glycosyltransferase (ftsI), and phosphate transport system regulator (phoU) were confirmed by standard PCR as follows: isolation and purification of bacterial genomic DNA were performed using the GeneALL Genomic DNA Purification Kit (Korea). PCR was carried out in a final volume of 25 µL containing 1.25 U of Taq DNA polymerase (Thermo Fisher Scientific) in 1X PCR buffer, 0.2 µM of each dNTP, 2 mM MgCl 2 , and 1 µM of each primer ( Table 1). The program of PCR included initial denaturation at 95 °C for 2 min, followed by 30 cycles of 95° C for 5 s and 55.5 °C for 1 min, with a final extension step at 72 °C for 2 min. The amplicon size was determined by 1.0% agarose gel electrophoresis ( Table  1). The primers for each gene were designed according to the genome-sequenced E. coli strain JJ1886 (NCBI Reference Sequence: NC_022648; the reference sequence was derived from CP006784).

Total RNA isolation and cDNA synthesis
For the examination of the expression of serum resistance and β-lactamase-associated genes, total RNA isolation of E. coli strain JJ1886 was performed using the Hybrid-R Total RNA Isolation Kit (Korea). Briefly, the concentration of 24-h bacterial culture was adjusted to OD 600 of 0.05 with fresh LB medium. Next, bacterial culture was immediately added to the LB medium (control) and LB medium consisting of NHS (v/v 1:2), HIS (v/v 1:2) NHS + CTX, NHS + CAZ, NHS + CAR, HIS + CTX, HIS + CAZ, HIS + CAR, CTX, CAZ, and CAR. Final antibiotic concentrations were calculated according to the MIC values and incubated at 37 °C for 24 h with shaking (50 rpm). The bacterial cultures (24 h) of 12 different conditions were used for total RNA isolation. Isolated mRNA was stored at -80 °C until cDNA synthesis analysis.
cDNA synthesis analysis was carried out, followed by RNA isolation from each condition (LB mediumcontrol; LB medium consisting of NHS [v/v 1:2], HIS [v/v 1:2], NHS + CTX, NHS + CAZ, NHS + CAR, HIS + CTX, HIS + CAZ, HIS + CAR, CTX, CAZ, and CAR). cDNA synthesis was performed using a Biospeedy cDNA synthesis kit (Turkey) as follows: 12 µL of mRNA was mixed with 4 µL of oligo-dT and incubated for 10 min at 70 °C. cDNA synthesis was carried out in a final volume of 40 µL including 8 µL of 5X reaction buffer, 2 µL of dNTP mix, 2 µL of reverse transcriptase, and 12 µL of RNAse and nuclease-free water. The reaction mixture was incubated at 37 °C for 60 min. The obtained cDNA was stored at -20 °C.

Statistics
The GraphPad Prism 5.0 (San Diego, CA, USA) program was used for all statistical analyses. Two-way ANOVA was carried out. Bonferroni posttests were applied. P-values of less than 0.0001 were considered significant.

Results
E. coli strain JJ1886 was determined to be a β-lactam producer using the nitrocefin phenotypic test. By MIC testing, E. coli strain JJ1886 was shown to be resistant to CTX, CAZ, and CAR, with MICs of 1280 µg/mL, 16 µg/ mL, and 20 mg/mL, respectively. Because E. coli strain JJ1886 was resistant to CAR concentrations between 512 and 0.125 µg/mL, the MIC was detected at a higher concentration (20 mg/mL) for this antibiotic.
During the analysis of the time-dependent effect of NHS on the growth of E. coli strain JJ1886 during the exponential growth phase, the bacterium was treated with serum at 37 °C. We found that the growth rate decreased between the 0-h and 1-h time intervals. However, the bacterial cell number increased after 2 h of treatment with NHS, likely due to its resistance to NHS ( Figure  1A). For the quality control strain (E. coli ATCC 25922), a rapid decrease in cell number was reported in the first hour of the treatment with NHS ( Figure 1B). In addition, we found that HIS-treated E. coli strain JJ1886 and E. coli ATCC 25922 showed a significant increase in cell numbers (7-8 log 10 CFU/mL) after 2 h of treatment at 37 °C. E. coli strain JJ1886 was treated with NHS or HIS plus CTX (1280 µg/mL), CAZ (16 µg/mL), and CAR (20 mg/ mL). During the treatment with CAZ (16 µg/mL), in the exponential growth phase, E. coli strain JJ1886 showed resistance to NHS in the first hour. However, compared with HIS + CAZ in LB medium, the number of cells decreased after 2 h. During the 3-to 24-h time interval, NHS + CAZ showed bactericidal activity against E. coli strain JJ1886. There was a decrease in cell count after 3 h when HIS and CAZ were present in growth media. However, when only CAZ was present, bacterial growth was inhibited until the third hour of treatment with the serum, but the number of cells doubled after 24 h of culturing compared to the beginning of treatment (0 h) (Figure 2).
Similar results were obtained with CTX when E. coli strain JJ1886 was treated with 1280 µg/mL CTX, NHS, and HIS. However, it was observed that the number of cells decreased after 3 h of treatment with NHS + CTX. We found that NHS + CTX had a bactericidal effect at 24 h. When both HIS and CTX were in the growth medium, the cell number did not change until the 3-h time point, but the cell number increased swiftly at 24 h. However, when only CTX was present in the medium, bacterial growth was inhibited until the third hour of treatment, but cell number increased at 24 h ( Figure 3). A decrease in cell number was observed after 1 h of treatment of E. coli strain JJ1886 with 20 mg/mL CAR, NHS, and HIS. However, when NHS and CAR were used together in the growth medium, E. coli O25b:H4 showed resistance at the second hour and the cell number increased at 24 h ( Figure 4).
The quantitative expression of serum resistance and β-lactam-associated genes of E. coli strain JJ1886 was investigated by treatment with CAZ, CTX, CAR, NHS, and HIS (LB as a control; NHS, HIS, NHS + CTX, NHS + CAZ, NHS + CAR, HIS + CTX, HIS + CAZ, HIS + CAR, CTX, CAZ, and CAR). Serum resistance and β-lactamase-related genes are shown in Table 1. All relative gene expression was evaluated for downregulation or upregulation according to the control value of 1. A minimum 0.5-fold change was used as the cut-off for downregulation and a minimum 2-fold relative increase for upregulation (Table 2; Figure 5). There was no significant relative gene expression change for CTX-M-15, ampE, ftsI, nlpI, lpp, rfaP, wbbJ, kpsS, or phoU. Thus, the coexistence of serum and antibiotics in the growth medium did not alter the relative expression of the indicated genes. However, a significant downregulation was observed for the nlpI gene following treatment with CAR ( Figure 5; Table 2).

Discussion
Certain E. coli strains present in the bloodstream may lead to serious infections, such as sepsis and urinary tract infections. Surviving in the bloodstream and overcoming host defense mechanisms are necessary virulence traits for E. coli. These virulence traits involve iron acquisition systems and serum resistance-associated factors (23). Previous studies have shown that serum survival factors allow pathogenic bacteria to survive in serum. These factors (16μ g/mL)   include outer membrane proteins, capsule, O-antigens of the LPS, periplasmic protease, murein lipoprotein, and the phosphate transport system (21,24,26,33). Many pathogenic E. coli pathotypes are known to be serumresistant, including CFTO73, RS218, 536, CP9, and EC958 (23). Among these, EC958 is a O25b:H4-ST131 strain disseminated globally and a known producer of the CTX-M-15-type ESBL (34). Similarly, the E. coli strain JJ1886 is an epidemic clone (O25b:H4-ST131) that is resistant to numerous β-lactams, including cephalosporins (20). In this study, we report that E. coli strain JJ1886 is resistant to serum and β-lactams, including CAZ, CTX, and CAR. However, CAZ and CTX together with NHS had bactericidal activity against E. coli strain JJ1886 at the tested time points.
A previous investigation reported that the E. coli CFTO73 strain survived when treated with serum during the first hour of culture. However, cell number decreased after the first hour of NHS treatment. These findings show that the E. coli CFTO73 strain can tolerate serum during the lag phase of growth (28). In our study, we investigated the time-dependent effect of NHS on growth E. coli strain JJ1886 in the exponential growth phase when treated with NHS at 37 °C. We found that the cell number decreased between the 0-and 1-h intervals. However, the cell number increased after 2 h of treatment. It can be speculated that the susceptibility and tolerance of different E. coli strains vary when E. coli strains are treated with NHS.
CAZ is a broad-spectrum cephalosporin used against gram-negative bacteria (35). In our study, only CAZ (16  (36). In other words, β-lactam antibiotics inhibit the formation of peptidoglycan synthesis during bacterial growth and cause cell lysis, thus affecting the growth rate and tolerance of the bacterium. Therefore, the inhibitory effect of β-lactam antibiotics can be timedependent for E. coli strain JJ1886 cells until a certain time interval. Afterwards, E. coli strain JJ1886 cells proliferated during the 24-h treatment with CAZ, CTX, and CAR. Thus, it can be concluded that E. coli JJ1886 cells could tolerate the inhibitory effect of CAZ, CTX, and CAR when exposed to β-lactam antibiotics over a longer period. Another reason why E. coli JJ1886 cells regrew at the end of the 24-h culture period is the inoculum effect of β-lactamase-producing strains when compared to nonβ-lactamase-producing strains (37). It has been suggested that the starting inoculum (1 × 10 6 to 5 × 10 6 CFU/mL) could lead to regrowth of bacteria. When E. coli JJ1886 cells were incubated with β-lactam antibiotics (CAZ and CTX) together with serum, the cell number decreased, and eventually a bactericidal effect of NHS and β-lactam antibiotics (CAZ and CTX) was detected (Figures 2 and 3). However, when NHS and CAR were added together to growth media, cell number increased at the 3-and 24-h time points ( Figure  4). A previous investigation of the bactericidal effect of antibiotics in human serum demonstrated that the complement-mediated effect makes bacterial cells more susceptible to antibiotics (38). However, here we found that, even though CAR + NHS were present in growth media, E. coli strain JJ1886 was resistant (Figure 4). Based on this finding, it can be inferred that E. coli strain JJ1886 is tolerant to both CAR and NHS.
We investigated the relative gene expression of serum resistance and β-lactamase-associated genes in the presence of NHS, HIS, CAZ, CTX, and CAR. A significant downregulation was observed for the nlpI gene, which encodes lipoprotein NlpI, when E. coli strain JJ1886 was treated with CAR only ( Figure 5; Table 2). Lipoprotein NlpI is an outer membrane lipoprotein involved in cell division. It has been reported that NlpI together with periplasmic protease (Prc) controls peptidoglycan synthesis by changing levels of MepS, which is an endopeptidase that breaks the peptide crosslink of the peptidoglycan component. In other words, NlpI binds to the Prc and MepS in order to degrade MepS via Prc (39). NlpI also contributes to the deposition of the complement regulator C4bp of E. coli in order to escape killing by serum (24). In addition to being associated with NlpI, Prc (also known as Tsp) contributes to pathogenicity by improving serum survival. Moreover, Prc processes the carboxy C-terminal region of the periplasmic protein and penicillin-binding protein 3, which is responsible for mediating peptidoglycan synthesis (33). In our study, growth analysis showed that E. coli strain JJ1886 was resistant to CAR and NHS during the 24-h incubation, and the nlpI gene encoding lipoprotein NlpI was downregulated in the presence of CAR. CAR is a carboxypenicillin, and E. coli strains that have surface β-lactamase are resistant to CAR (40). The mode of action of CAR is to inhibit cell wall synthesis during peptidoglycan cross-linking; this could affect the cell wall and periplasm membrane (41). Downregulation of the nlpI gene might be related to the peptidoglycan biosynthesis process. However, the exact reason why a downregulation was observed in the nlpI gene is unclear. To develop a more accurate conclusion requires further studies on the nlpI gene in E. coli strain JJ1886.
Taken together, our data show that during the exponential growth phase, E. coli strain JJ1886 is resistant to NHS. When the bacterium was treated with NHS together with β-lactam antibiotics, including CAZ and CTX, we detected a bactericidal effect. However, E. coli strain JJ1886 cells were resistant to NHS in combination with CAR. Our findings may show that these cells can tolerate the inhibitory effect of CAR and NHS. Moreover, there was no significant change in gene expression for encoding class A extended-spectrum beta-lactamase CTX-M-15, β-lactamase regulator, penicillin-binding protein 3, and serum survival-related factors, except for the nlpI gene encoding lipoprotein NlpI.