ABSTRACT: The present study aimed to investigate the hospital-acquired Extended Spectrum β-Lactamase production (ESBL) producing Escherichia coli, to identify the significant ESBL peptide spectra after exposure to ceftazidime and amoxicillin/clavulanic acid, and to identify the resistant peptide spectra after exposure to ciprofloxacin. ESBL production and the minimal inhibitory concentration of ciprofloxacin were investigated using the Epsilometer strip test. Their colonies were collected to analyze the peptide spectra by VITEK^® Mass Spectrometry. The significant difference in spectrum intensity for suspected ESBL peptide spectra, and DNA starvation/stationary phase protection (Dps) -like peptide spectra were also determined. Among 228 isolates, the high ESBL-producing E. coli which exhibited high ciprofloxacin resistance was demonstrated as 22.63%. The significant reduction of spectrum intensity after exposure to ceftazidime and amoxicillin/clavulanic acid was only detected at spectrum 9714 Da in the high ESBL-producing group (P ≤0.001). Among four selected high ESBL producers, only one significant spectrum located at 9063 Da was detected in the high ciprofloxacin-resistant group (P ≤0.001) after being exposed to ceftazidime and ciprofloxacin. The highest percentage increase of spectrum 9063 Da was 69.42%. Multidrug resistance was demonstrated in the tested E. coli isolates, especially β-lactam resistance which may be caused by their ESBL production. It may be said that the peptide spectrum located at 9714, and 9063 Da were suspected as the ESBL indicative peptide spectrum in the high ESBL producers, and as the Dps indicative peptide spectrum in the high ciprofloxacin-resistant isolates. 

Keywords: Peptide spectra analysis, DNA starvation/stationary phase protection like protein, Hospital-acquired infection, Extended spectrum β-lactamase, Escherichia coli, Matrix-Assisted Laser Desorption Ionization-Time Of Flight

Funding: This work was supported by the Faculty of Medicine, Chiang Mai University, grant No. 4-2565.

Citation: Daoaroonkiet, P., Kwangsukstith, S., Srichai, S., Jaidee, S., Emthanom, M., Sujitpiriyakit, P., Sastraruji, T., Sookkhee, S., 2023. Peptide spectra analysis of DNA starvation/stationary phase protection-like protein found in hospital-acquired ESBL producing Escherichia coli by MALDI-TOF technique. Nat. Life Sci. Commun. 22(2): e2023032.

INTRODUCTION

With the spread of antibiotic resistance, rapid diagnosis to indicate the mechanism of antibiotic resistance has been important for eradicating the causative pathogens. The most common pathogen is Escherichia coli, especially Extended-Spectrum β-Lactamase producing E. coli (ESBL-EC). It is the major cause of urinary tract infection (UTI), affecting the urinary bladder, urethra, and kidneys (Flores-Mireles et al., 2015). Although this infection could be treated with the drugs of choice, aminoglycoside, and polymyxin, this pathogen still produces Extended-Spectrum β-Lactamase (ESBL) and/or other mechanisms and transfers the ESBL and/or other antibiotic-resistance genes to other bacteria. In the past, UTIs were easily cured by antibiotics. However, inappropriate use of antibiotics has led to the worldwide emergence of antibiotic-resistant strains of E. coli (Cantón et al., 2011), resulting in an increased mortality rate due to difficulties in treating UTIs. ESBL produced by E. coli can degrade the β-lactam rings which is the component of penicillin, cephalosporin, and monobactam groups. Therefore, fluoroquinolone is frequently used to treat ESBL-EC infection instead (Wiener et al., 2015). Nevertheless, it was found that the rate of fluoroquinolone resistance has significantly increased among ESBL-EC but not in non-ESBL-EC (Hirakata et al., 2005; National Antimicrobial Resistance Surveillance Center, 2019; Talan et al., 2016). There are several mechanisms of fluoroquinolone resistance, such as mutation of genes on target enzyme, efflux upregulation, decreased porin protein, plasmid-mediated quinolone resistance (PMQR) via qnr gene (Redgrave et al., 2014), and DNA starvation/stationary phase protection (Dps) protein expression (Alhajouj et al., 2020; Pinto et al., 2010).

After proteomically identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in the previous study (Sookkhee et al., 2017), a 19-kDa protein resembling the Dps-like protein produced by E. coli O157:H7 and E. coli B7A was detected in high ciprofloxacin-resistant strains of ESBL-EC that were exposed to various concentrations of ciprofloxacin. This protein may be responsible for protecting E. coli when exposed to stressors such as ciprofloxacin, nutritional stress, ultraviolet radiation, gamma radiation, or thermal stress (Wolf et al., 1999). It protects DNA from stressors by promoting DNA condensation and reducing the formation of reactive oxygen species (Karas et al., 2015). Lately, a new technique, Matrix-Assisted Laser Desorption Ionization-Time Of Flight (MALDI-TOF), is performed to identify the pathogen species, and identify antibiotic-resistant proteins or peptides to be less time-consuming, less expensive, and more precise compared to the conventional method. VITEK^® Mass Spectrometry (VITEK^® MS) apparatus, which uses the MALDI-TOF technique, was recently carried out for routine in vitro diagnosis (IVD) in hospitals in Thailand. Moreover, VITEK^® MS has been applied to analyze the proteins in specific peptide spectra of antibiotic resistance (Clark et al., 2013). However, there are no reports on this technique being used to analyze the peptide spectra in ESBL-EC after previous exposure to ciprofloxacin. In the present study, the objectives were to investigate the significant peptide spectra after exposure to ceftazidime (CAZ) compared with amoxicillin/clavulanic acid (AMC), and with ciprofloxacin (CIP) according to ESBL production and minimal inhibitory concentration of ciprofloxacin (MICCIP) for suspected ESBL indicative peptide spectrum, and as the Dps indicative peptide spectrum.

MATERIALS AND METHODS

Bacterial Populations

The sample size was calculated with G*Power program version 3.1.9.4 by analysis of variance (F-tests) with a priori power analysis (McCrum-Gardner, 2010). The significance level (α) was determined at 0.05. The calculated sample size was at least 66 isolates. Two hundred and twenty-eight non-duplicated E. coli isolates were randomly collected from the positive urine-cultured MacConkey plates at the Diagnostic Laboratory Unit, Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand in 2019-2020. These collected isolates were grown on MacConkey’s agar and incubated at 37°C for 18-24 hours. The colonies that appeared as lactose fermenters were further biochemically identified. E. coli would exhibit A/AG (Glucose, lactose, and/or sucrose fermenter with gas production) in a Triple Sugar Iron tube. Gram stain, motility test, catalase test, indole production test, urease test, and utilization of citrate were also conducted. The colonies that were confirmed as E. coli were inoculated in Tryptic Soy Broth supplemented with 20% glycerol and were kept in a -80°C freezer at the Department of Microbiology, Faculty of Medicine, Chiang Mai University until further use. E. coli strains ATCC25922 and ATCC8739 were used as the standard control strains.

Antibiotic Susceptibility

The Kirby-Bauer assay of agar disk diffusion technique was performed to determine the antibiotic susceptibility of each E. coli isolate according to CLSI guideline 2020 (Clinical Laboratory Standards Institute, 2020). Briefly, a single colony of each isolate was inoculated in Müeller Hinton Broth (MHB), adjusting the turbidity to McFarland standards No. 0.5. It was later swabbed on Müeller Hinton Agar (MHA) and antibiotic disks were placed then incubated at 37°C for 18-24 hours. Diameter of the inhibition zone of each isolate to thirteen antibiotics including amoxicillin/clavulanic acid (AMC, 20/10 µg/disk), ampicillin (AMP, 10 µg/disk), cefoxitin (FOX, 30 µg/disk), ceftazidime (CAZ, 30 µg/disk), cefotaxime (CTX, 30 µg/disk), piperacillin/ tazobactam (PTZ, 100/10 µg/disk), imipenem (IPM, 10 µg/disk), meropenem (MEM, 10 µg/disk), doripenem (DOR, 10 µg/disk), kanamycin (K, 30 µg/disk), gentamicin (CN, 10 µg/disk), ciprofloxacin (CIP, 5 µg/disk), and tetracycline (TE, 30 µg/disk) was measured and interpreted according to the CLSI guideline 2020. Among 13 antibiotics, the multidrug-resistant isolates were categorized that exhibited the numbers of antibiotic susceptibility less than seven drugs and then assumed to be called resistant group while the isolates were also categorized and called the susceptible or intermediate resistant groups if they exhibited the numbers of antibiotic susceptibility more than ten, or eight-nine drugs, respectively.

Detections of ESBL Production

According to the recommendation by CLSI guideline 2020 as described above, ESBL production can be detected by using Double Disk Synergy Test (DDST). A single colony of E. coli from the resistant group was done with Kirby-Bauer assay as described above before placing the AMC disk at 30 mm apart from CTX and CAZ disks and then incubated at 37°C for 18-24 hours. The inhibition zone and the keyhole phenomenon that appeared between AMC with CTX or/and with CAZ were observed. ESBL production was screened according to their CTX and CAZ susceptibilities. The keyhole phenomenon was also focused on ESBL production.

Subpopulations of ESBL-EC were categorized by using the Epsilometer test (E-test). A colony was inoculated in MHB, then the turbidity was adjusted to McFarland standards No. 0.5 and swabbed on MHA. The E-test strip containing the gradient concentration of CAZ was placed at one end and ceftazidime/clavulanic acid (CAZ/Clav) at the other end. They were incubated and MIC levels of both sides were obtained. MIC ratio was then calculated between CAZ and CAZ/Clav. If the MIC ratio was lower than eight-fold, the result was interpreted as non-ESBL production (ESBL^N). If the MIC ratio was greater or equal to eight-fold, it was assumed to be categorized as low ESBL production (ESBL^L), while MIC ratio was greater or equal to 250-fold, it was assumed to be categorized as high ESBL production (ESBL^H), according to our laboratory criteria which were modified from Division of Central Laboratory, Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand.

Ciprofloxacin Susceptibility

According to our laboratory criteria which were modified from the Division of Central Laboratory, Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand, MIC_CIP levels of each ESBL-EC isolate was also determined by using E-test as described above with CIP E-strip. Results were interpreted into four groups namely the high CIP resistant (CIP^HR), low CIP resistant (CIP^LR), CIP intermediate resistant (CIP^INTR), and CIP susceptible (CIP^SUS) groups. If the MIC level is > 8 µg/ml, it was classified as CIP^HR isolate, while MIC levels ≥ 4 µg/ml and ≤ 8 µg/ml were classified as CIP^LR isolate. CIP^INTR and CIP^SUS isolates had MIC levels of 1 - 4 µg/ml and ≤ 1 µg/ml, respectively.

Peptide Preparation of MALDI-TOF Technique

 Kirby-Bauer assay was performed to expose ESBL-EC to antibiotics as described above. CIP, AMC, and CAZ disks were placed on MHA which was swabbed with each culture at McFarland standards No. 0.5. Then the plate was incubated at 37°C for 18-24 hours. Each exposed area as shown in Figure 1 was collected for further peptide preparation on VITEK^® MS-DS Target Slide (Biomerieux, Marcy l’Etoile, France). The culture of E. coli ATCC8739, carried out as the standard control, was spotted on the calibrator well on this slide. Next, 1 µL of alpha-cyano-4-hydroxycinnanic acid (CHCA) matrix (Biomerieux, Marcy l’Etoile, France) was dropped into each well of VITEK^® MS-DS Target Slide. Once all wells were dried, the slides were placed into the VITEK^® MS apparatus (Biomerieux, Marcy l’Etoile, France). The MALDI-TOF technique was performed by using a laser frequency of 50 Hz, laser wavelength of 337 nm, ion source voltage of 20 kV, and lens voltage of 6 kV. The mass and spectra intensities of each tested well were exhibited. Spectra analysis was further carried out to determine the significant peptide.

Figure 1. The positions of the collected spots of each culture with the exposure areas: 1) CIP-exposed area, 2) CAZ-exposed area, and 3) CAZ and AMC-exposed area.

Spectra Analysis

Peptide spectra from all tested wells of each ESBL-EC isolate were analyzed. The normality of the data was initially analyzed by the Shapiro-Wilk test. As shown in Figure 1, the significant difference in spectra intensity between the CAZ-exposed area (Area 3) and CAZ and AMC-exposed area (Area 2) was calculated by using the paired t-test. This significant difference indicated the presence of ESBL production. In the present study, it was called the ESBL peptide spectra. According to our previous report, the presence of Dps-like protein was revealed in the culture of CIP^HR isolates namely 8/14, 21/7, 23/9, 24/5, and 26/40. In contrast, the absence of Dps protein was found in the culture of CIP^SUS isolates namely 23/5, 23/48, and ATCC25922. In the present study, the peptide spectra of these isolates, CIP^HR and CIP^SUS isolates were also tested. Dps indicative peptide spectra of these isolates were determined. The significant difference in spectra intensity compared between the CIP-exposed and non-CIP-exposed areas (Areas 1 and 2 shown in Figure 1) were analyzed by using paired t-test. Peptide spectra that demonstrated the significant difference only in CIP^HR E. coli were suspected as Dps peptide spectra.

Dps Peptide Spectra Analysis

To determine the presences of Dps peptide spectra in three groups of ESBL production, and three groups of CIP resistance, the significant difference in Dps spectra intensity compared between the CIP-exposed and non-CIP-exposed (or CAZ-exposed) areas (Areas 1 and 2 shown in Figure.1) were analyzed by using paired t-test among the ESBL^H, ESBL^L, and ESBL^N groups, and CIP^HR, CIP^L, and CIP^INTR+SUS groups. The prevalence of Dps peptide spectra exhibited in CIP^HR and CIP^LR groups was also determined.

RESULTS

Antibiotic susceptibility

Among 228 E. coli isolates, 51 (22.37%), 40 (17.54%), and 137 (60.09%) isolates were categorized into three groups including susceptible (S), intermediate resistant (I), and multidrug-resistant (R), respectively, according to their susceptibility toward 13 antibiotics. Apart from nine antibiotics, all isolates were susceptible to four antibiotics namely MEM, K, CN, and DOR. Only the isolates resistant to at least six antibiotics, considered to multidrug-resistant, and categorized in the multidrug-resistant group were selected to further determine ESBL production. According to Table 1, the most common antibiogram (12.28%) were resistant to 6 of 13 antibiotics, except PTZ, IPM, MEM, CN, K, CIP, and DOR. Due to not being ESBL substrates, therefore, these antibiotics were not affected by ESBL. CIP is a fluoroquinolone. MEM and IPM are carbapenems. CN and K are aminoglycosides. PTZ is piperacillin combined with tazobactam.

Among 13 antibiotics, AMP and CTX were the most resisted drugs with percentages of 84.21% and 83.77%, 100% and 100%, and 100% and 96.61% among total, ESBL^H, and ESBL^L isolate, respectively. TE was also found as the third most resisted drug with resistance percentages of 75.00%, 97.01%, and 62.71% among total, ESBL^H, and ESBL^L isolates, respectively. The results are demonstrated in Figure 2. While none of the isolates were susceptible to four agents including MEM, CN, K, and DOR.

Table 1. Antibiograms of all tested isolates toward 13 antibiotics. Number of drugs susceptibility, groups, and % prevalence arranged descendingly according to % prevalence.

Note:  AMP, Ampicillin; FOX, Cefoxitin; CTX, Cefotaxime; CAZ, Ceftazidime; AMC, Amoxicillin/ clavulanic acid; PTZ, Piperacillin/tazobactam; IPM, Imipenem; MEM, Meropenem; CN, Gentamicin; K, Kanamycin; CIP, Ciprofloxacin; DOR, Doripenem; TE, Tetracycline; R, Resistant; S, Susceptible; R, Multidrug-Resistant group, numbers of drug susceptibility ≤7; I, Intermediate resistant group, numbers of drug susceptibility 8-9; S, Susceptible group, numbers of drug susceptibility ≥10.

Detections of ESBL Production

According to DDST, CAZ and CTX resistances, and keyhole phenomenon were detected in 159 out of 177 isolates (89.83%) in the resistant and intermediate-resistant groups. The keyhole phenomenon could be observed as the clear zone between AMC and CAZ or AMC and CTX disks. It could be interpreted that these isolates were ESBL producers. Positive results of the ESBL screening test were detected in all resistant isolates (n=137). These isolates were selected for further determination of ESBL peptide spectra.

According to the ESBL confirmation test using an E-strip test with CAZ and CAZ/clavulanic acid at opposite ends, 67 (48.91%), 59 (43.07%), and 11 (8.03%) isolates were classified as ESBL^H, ESBL^L, and ESBL^N E. coli with MIC_CAZ/MIC_CAZ/Clav ≥ 250, 8 to 250, and ≤ 8, respectively.

Figure 2. Percentages of resistant isolates toward 13 antibiotics in the order of total isolates (orange bar), ESBL^H (yellow bar) and ESBL^L (green bar) ESBL-producing groups.

Ciprofloxacin Susceptibility

All ESBL^H and ESBL^L E. coli according to the result of the ESBL E-strip test were then carried out to determine their CIP susceptibility using the E-strip test. Fifty-four ESBL-producing isolates with MIC_CIP ≥ 16 µg/mL were grouped into CIP^HR ESBL-producing isolates. Fifty-seven and seven isolates were classified into CIP^LR and CIP^INTR groups, respectively. Only 19 isolates (13.87%) exhibited as the CIP^SUS isolates. This classification was also shown in Supplemented Table 1. According to their ESBL production and CIP susceptibility, each isolate was separately plotted and categorized into nine groups as shown in Figure 3.

Figure 3. Distribution of 137 resistant isolates according to their MIC_CAZ/MIC_CAZ/Clav and MIC_CIP by categorizing them into nine groups. Each dot represents isolates that had the same values of MIC_CAZ/MIC_CAZ/Clav and MIC_CIP. Area 1, ESBL^N, CIP^HR; Area 2, ESBL^L, CIP^HR; Area 3, ESBL^H, CIP^HR; Area 4, ESBL^N, CIP^LR; Area 5, ESBL^L, CIP^LR; Area 6, ESBL^H, CIPL^R; Area 7, ESBL^N, CIP^INTR+SUS; Area 8, ESBL^L, CIP^INTR+SUS; Area 9, ESBL , CIP^INTR+SUS.

Figure. 3 showed the distribution of nine groups of the tested isolates according to their ESBL production and CIP resistant level, MIC_CAZ/MIC_CAZ/Clav, and MIC_CIP, respectively. From this figure, the ESBL^H and CIP^HR groups, Area 3, could be suggested that the ESBL-tolerant β-lactams such as MEM and DOR, and other groups of agents such as CN should be used to treat the infection of these isolates. Details of this figure exhibit six isolates (4.38%) were ESBL^N, and CIP^HR (Area 1). Seventeen isolates (12.41%) were ESBL^L, and CIP^HR (Area 2). Thirty-one isolates (22.63%) were ESBL^H, and CIP^HR (Area 3). Three isolates (2.19%) were ESBL^N, and CIP^LR (Area 4). Thirty-one isolates (22.63%) were ESBL^L, and CIP^LR (Area 5). Twenty-three isolates (16.79%) were ESBL^H, and CIP^LR (Area 6). Two isolates (1.46%) were ESBL^N, and CIP^INTR+SUS (Area 7). Eleven isolates (8.03%) were ESBL^L, and CIP^INTR+SUS (Area 8). Thirteen isolates (9.49%) were ESBL^H, and CIP^INTR+SUS (Area 9). Their ESBL production and CIP susceptibility were categorized as shown in Supplemented Table 2.

Spectra Analysis

Out of 83 isolates of E. coli that were analyzed using the MALDI-TOF technique by VITEK^® MS, 30, 25, and 28 isolates were ESBL^H, ESBL^L, ESBL^N, and 27, 12, and 44 were CIP^HR, CIP^LR, and CIP^INTR+SUS, respectively.

Considering the manner of ESBL indicative peptide spectra, the spectrum intensities of these spectra should be significantly decreased after comparing the CAZ and AMC exposed areas. Among 199 peptide spectra that were visible after being analyzed by VITEK^® MS, nine peptide spectra of ESBL^H isolates were selected according to the ESBL manner.

Figure 4. Spectrum intensities after exposure to AMC (blue bar) and CAZ (orange bar) of the ESBL^H (A) and ESBL^L (B) groups. The positive standard error of each spectrum is also shown. *, P ≤ 0.001.

They are shown in Figure 4A. A spectrum located at 9714 Da exhibited a significant difference in the spectrum intensities between these exposures as shown by the P-value of ≤ 0.001. It could be possible that the significant decrease in spectrum intensity after exposure to AMC may be caused by the destructive action of clavulanic acid toward the ESBL enzyme. This decreased manner of this spectrum after being exposed to AMC may be suggested as the characteristics of ESBL enzyme in the ESBL^H isolates. From Figure 4B, ten peptide spectra of ESBL^L isolates are shown. Their decreased intensities were also detected after comparing the exposure with CAZ and AMC. However, there were no peptide spectra that have a significant difference in spectra intensities.

As shown in Figure 5, only one spectrum at 9714 Da was suspected to be ESBL indicative peptide spectrum. It was lower spectrum intensity after being exposed to AMC than CAZ. Reductions of spectrum intensity between AMC-exposed and CAZ-exposed points of isolate A2C7, U438, B2D2, and U1050 were 48.14% (Δ=61440.73; 66184.62 when exposed to AMC, and 127625.4 when exposed to CAZ), 71.31% (Δ=82683.37; 33265.55 when exposed to AMC, and 115948.9 when exposed to CAZ), 38.94% (Δ=56450.8; 88536.22 when exposed to AMC, and 144987 when exposed to CAZ), and 80.58 (Δ=90575.18; 21828.19 when exposed to AMC, and 112403.4 when exposed to CAZ), respectively. The manner of the reduction of spectrum intensity at 9714 Da in four ESBL^H isolates was implied as to the specific inhibition of clavulanic acid towards β-lactamase, suggesting that the manner of this spectrum could be applied to identify the ESBL^H production in E. coli. Spectrum intensities of high, low, and non-ESBL producing groups were demonstrated in Supplemented Table 3.

Figure 5. Comparison of the % reduction of spectrum intensity at 9714 Da between the exposure with CAZ and AMC. The suspected ESBL enzyme are shown in the red boxes and labeled with black arrows. These CIP^HR isolates were A2C7 (A), U438 (B), B2D2 (C), and U1050 (D).

Dps Peptide Spectra Analysis

To analyze the peptide spectra indicating Dps among the CIP^HR and CIP^SUS ESBL-EC, CIP^HR isolates A2D7, AA3, B2A6, T101, and CIP^SUS ESBL-EC, isolates A2F2, B1B1, B2F2 were renamed from isolates 8/14, 21/7, 26/40, 23/9, and 23/48, 24/5, 23/4, respectively as shown in the previous study (Sookkhee et al., 2017). From the former results, Dps protein was demonstrated in all CIP^HR isolates but not shown in all CIP^SUS isolates. With VITEK^®-MS, Dps suspected protein is shown in the form of peptide spectra and should be also shown in the CIP^HR isolates. After being exposed to the sub-MIC of CIP, the stress proteins involved with the increased CIP concentration were expected to be demonstrated in the CIP^HR isolates. It was found that there were some peptide spectra exhibited only in the CIP^HR group but not in the CIP^SUS group. Among the CIP^HR group, the increase in spectrum intensities was significantly detected after compared between the exposure to CIP and CAZ.

Figure 6 shows the spectra intensities of six significant peptide spectra, namely 4531, 4777, 8350, 9063, 9551, and 10477 Da, selected based on the presence or absence of these spectra in four representatives of CIP^HR and three representatives of CIP^SUS ESBL-EC previously reported by Sookkhee et al (Sookkhee et al., 2017). Four significant spectra located at 4531, 8350, 9063, and 10477 were detected only in CIP^HR isolates and absent in CIP^SUS isolates, suggesting that they may play a role in the resistance to CIP. These selected spectra may be suspected as Dps-like protein as described above. In contrast, spectra 4777 and 9551 were expressed only in CIP^SUS isolates and absent in CIP^HR isolates. This suggested that these peptide spectra could be used to distinguish between CIP^HR and CIP^SUS ESBL-EC. Further statistical analysis of the spectra 4531, 8350, 9063, and 10477 should be performed for all isolates to determine if any or all of them are expressed among the CIP^HR group in a statistically significant amount for the likeliest spectrum of Dps. It may be supposed that only one spectrum at 9063 Da (in red box) may be considered specific to the Dps-like protein. However, we mentioned ESBL-EC isolates for investigating the significant difference. Spectrum intensities of high, low, and non-ESBL producing groups were demonstrated in Supplemented Table 4.

Among CIP^HR representative isolates as shown in Figure 6, the spectrum intensity of 4531 Da after being exposed to CIP was 106160.6, 96764.72, 90151.11, and 23079.16 for A2D7, AA3, B2A6, and T101, respectively. The intensity of 8350 Da was 49509.08, 39086.93, and 57215.58 for A2D7, AA3, and B2A6, respectively. The intensity of 10477 Da was 38647.53, 70789.87, and 38347.45, by A2D7, B2A6, and T101, respectively. Only one spectrum at 9063 Da was suspected to be Dps indicative peptide spectrum. The spectrum intensity increased after being exposed to CIP than CAZ. Their intensities were 257205.3, 199994.9, 239033.8, and 95899.94, by A2D7, AA3, B2A6, and T101, respectively. While the CIP^SUS isolates, the intensity of 4777 Da were 29781.71, 31740.7, and 75675.06 for A2F2, B1B1, and B2F2, respectively. The intensity of 9551 Da was 23373.05 and 28276.13 for B1B1 and B2F2, respectively. The increase of spectrum intensity at 9714 Da in 4 CIP^HR representative isolates was implied as to the specific induction of ciprofloxacin towards Dps-like protein, suggesting that this spectrum may be applied to investigate one of ciprofloxacin resistance in E. coli.

Figure 6. Comparison of 4 CIP^HR isolates and 3 CIP^S isolates when exposed to CIP.  The % reduction of spectrum intensity at 9063 Da between the exposure with CIP and CAZ. The suspected Dps-like protein are shown in the red boxes and labeled with black solid arrows. Different spectra which were absent in the CIP^HR isolates (A-D) can be detected in the CIP^SUS isolates (E-G) and present as the black dotted arrows. These isolates were A2D7 (A), AA3 (B), B2A6 (C), T101 (D), A2F2 (E), B1B1 (F), and B2F2 (G).

Figure 7. Spectrum intensities after exposure to CIP (blue bar) and CAZ (orange bar) of the CIP^HR (A), CIP^LR (B), and CIP^INTR+SUS groups (C). *, P ≤ 0.05; **,P-value ≤ 0.001.

Figure 7 shows the spectrum intensities of four peptide spectra including 4531, 8350, 9063, and 10477 Da after being exposed to CIP and CAZ. From Figure 7A, the CIP^HR groups, the increase of spectra intensity at 9063 Da was significantly different between the exposure of CIP compared to CAZ (P ≤ 0.001). As well as Figure 7B, the increase of spectra intensity at 9063 Da in the CIP^LR groups was significantly different (P ≤ 0.05). In contrast to the spectra intensity of peptide spectrum 8350 Da, the spectrum intensity after exposure to CIP was significantly lower than the intensity after exposure to CAZ. This significant difference was also demonstrated in the CIP^INTR+SUS groups. It may be suggested that the presence of spectrum 9063 Da after exposure to CIP could be used as a criterion to identify CIP^HR in ESBL-EC. From the results of the previous study as described above, the peptide spectrum at 9063 Da may be suspected to be Dps protein.

From the results of Figure 8, it may be suggested that only one spectrum at 9063 Da was suspected to be Dps protein. The spectrum intensity was higher after being exposed to CIP than CAZ. Reductions of spectrum intensity between CIP-exposed and CAZ-exposed points of isolate B1B6, B2A6, B2C2, and U1362 were 41.19% (Δ=84807.05; 205911.70 when exposed to CIP, and 121104.70 when exposed to CAZ), 40.56% (Δ=96948.31; 239033.80 when exposed to CIP, and 142085.50 when exposed to CAZ), 69.42% (Δ=110974.70; 159852.20 when exposed to CIP, and 48877.51 when exposed to CAZ), and 69.21% (Δ=112628.50; 162735.20 when exposed to CIP, and 50106.65 when exposed to CAZ), respectively.

Figure 8. Comparison of the % reduction of spectrum intensity at 9063 Da between the exposure with CIP and CAZ. The suspected Dps-like protein are shown in the red boxes and labelled with black arrows. These CIP^HR isolates were B1B6 (A), B2A6 (B), B2C2 (C), and U1362 (D).

DISCUSSION

β-lactams, aminoglycosides, fluoroquinolones, and tetracyclines are recommended to treat Gram-negative bacterial infections including E. coli. One of the E. coli infections in the hospital is the hospital-acquired UTI that demonstrated the occurrence of antibiotic resistance including resistance from β-lactamase production and fluoroquinolone resistance. The antibiotic susceptibility and the resistance of β-lactamase-producing strains in hospital-acquired UTIs require rapid and accurate identification for the reassessment of existing treatment protocols.

According to a previous study, ESBL-producing isolates were most resistant to ampicillin followed by ceftriaxone, cefpodoxime, cefotaxime, aztreonam, cefepime and ceftazidime (Maheshwari et al., 2016). This finding was similar to our study, which found that AMP, CTX, and TE were three of the most resisted antibiotics, while none of the isolates were resistant to MEM, CN, K, or DOR. It can be confirmed that the representative antibiotics in the aminoglycoside group, K and CN, were appropriate to be prescribed as the drug of choice for UTI due to the antibiotic resistance rates of these drugs were still low as also previously reported (Soleimani et al., 2014)  

Pormohammad et al. reported the prevalence of multidrug resistance (MDR) in human, environmental, and animal E. coli isolates at 22%, 31.3%, and 5.7%, respectively (Pormohammad et al., 2019). Similarly, urine specimens in our study showed a high prevalence of the representative β-lactam and CIP resistance (n of R groups, %), thus, MDR was mentioned. CIP was one of the antibiotics used to treat E. coli.  It acts by binding to bacterial DNA gyrase and/or topoisomerase IV (Hamed et al., 2018). However, the emergence of CIP resistance in ESBL-EC, particularly in UTI, has been increasing over the years as well, with MIC assessments indicating a prevalence of 43% in Thailand (Polwichai et al., 2009). Several studies reported on how E. coli, especially ESBL-EC, employed various mechanisms to resist CIP and/or other fluoroquinolones. The mechanisms of resistance include the mutation of the gyrA gene which is encoded to bacterial DNA gyrase (Weigel et al., 1998), the mutation of ParC subunits which are encoded to bacterial topoisomerase IV (Gonzalez et al., 1998), the upregulation of efflux pump (Giraud et al., 2000), the downregulation of porin protein (Coldham et al., 2006), and the use of plasmid-mediated quinolone resistance (Hamed et al., 2018). In addition, another mechanism that helps to protect bacterial cell from oxidative stress during the stationary phase while exposed to antibiotics is the production of DNA Starvation/Stationary Phase Protection Protein (Dps) (Ceci et al., 2004)., which acts as the stress protein. This stress protein was highlighted to be investigated in our laboratory. Dps-like protein was also reported in the previous study of our colleague (Sookkhee et al., 2017)as the demonstrated protein at 19 kDa found in the isolates of CIP^HR and ESBL^H E. coli after being exposed to sub-MIC of CIP. It possessed a high identity to Dps protein as described previously (Choi et al., 2000; Nair et al., 2004). However, this characteristic of Dps analyzed from the proteomic analysis did not provide the specific spectrum of the Dps-like protein after being analyzed by VITEK^® MS. The present study would like to investigate the specific spectrum of this stress protein which is analyzed by VITEK^® MS. The most common resistant mechanism of the resistance of E. coli is ESBL production, resulting in resistance to penicillins, cephalosporins, and monobactams. However, a higher prevalence of CIP resistance was statistically significant in ESBL-producing clinical isolates compared to non-ESBL-producing clinical isolates (Maheshwari et al., 2016). A study by Abera et al. (Abera et al., 2016) reported that ESBL-producing Enterobacteriaceae showed higher levels of resistance against chloramphenicol, cotrimoxazole and CIP than non-ESBL producers (P = 0.001). This outcome suggested that there is a possible relationship between the production of ESBL and CIP resistance. The co-existence of the two resistance mechanisms could be explained by them being transferred on the same plasmid that carried genes encoding ESBL enzymes and other resistant proteins of different antibiotics, especially CIP-resistant markers (Maheshwari et al., 2016). The higher resistance to ciprofloxacin in ESBL-producing isolates could be confirmed by ESBL-encoding genes and CIP-resistant genes, plasmid-mediated quinolone resistance (PMQR) gene, being in the same plasmid (Azargun et al., 2018). Farajzadeh Sheikh et al. reported a prevalence of qnr genes which is one of the PMQR genes as 68.29% and 95.23% of qnr S and qnr S-B genes among 58 ESBL-producing E. coli isolates, respectively (FarajzadehSheikh et al., 2019). These findings indicate a possible association between ESBL production and the presence of qnr genes. The rise of plasmids co-harboring multiple drug-resistance genes could lead to a higher prevalence of UTI (Xiong et al., 2008). A significant relationship between the presence of PMQR genes consisted of qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB, and aac genes in E. coli that caused nosocomial UTI and ESBL production was also confirmed (Zaki et al., 2021). Several reports described that the close relationship between the resistance to third-generation cephalosporins and CIP can be found in other Enterobacteriaceae, since both E. coli and K. pneumoniae possess this characteristic (Paterson et al., 2000). Possibly, the relationship between resistance to third-generation cephalosporins and quinolones may also be explained by other mechanisms such as the active efflux, outer membrane protein alterations, and the presence of Dps, requiring further study. Although several studies have confirmed the co-existence of PMQR genes and other antibiotic resistances Dps-encoding gene has not yet been determined to be at plasmid or chromosome. In the present study, we mentioned the relation between the presence of Dps-like protein and high production of ESBL, hence, the localization of the Dps-encoding gene would be an interesting aspect to be further studied. In our previous study, Dps-like protein was found in high ESBL-producing isolates which also exhibited CIP^HR resistance (Sookkhee et al., 2017). Hence, we hypothesized that the presence of Dps-like protein could be detected in the resistant E. coli after being analyzed by VITEK^® MS. To investigate other proteins which contributed to the resistance, the lack of porin protein and encoded genes of our ESBL^H isolates in this study may be further studied on cephalosporin, carbapenem and CIP resistances as same as the study on carbapenem resistance that described the isolates with loss of porin genes may be suspected to also resist carbapenem (Doumith et al., 2009).

To detect antimicrobial resistance, the five main approaches are molecular genetics analysis, biochemical methods, rapid immunoassays, electrochemical assay, and mass spectrometry. All the approaches yield high sensitivity and specificity with more than 90% in both values. However, the difference in turnaround time (TAT) is an important factor for consideration. MALDI-TOF MS, a mass spectrometry technology, not only yielded in as little TAT as 30 minutes (Oviaño et al., 2019), but also has a low cost per test (Tran et al., 2015). Apart from the identification of antimicrobial resistance, species identification and strain typing are also possible for MALDI-TOF MS. MALDI-TOF MS is also appropriate for routine laboratory In Vitro Diagnostics (IVD), as the conventional proteomic analysis is expensive and time-consuming while MALDI-TOF technique via VITEK^® MS apparatus is more effective and cost-saving (Jang et al., 2018). It has become more acceptable and widespread in clinical laboratory diagnosis. This method has high sensitivity and efficiency, with the ability to analyze molecules in attomolar (10^-18 mmol/liter) to femtomolar (10^-15 mmol/liter) concentrations. Unlike other mass spectrometry techniques, MALDI-TOF MS uses soft ionization to ionize large molecules which are then detected and analyzed to generate a spectrum of the components based on mass-to-charge ratios. This enables MALDI-TOF MS to analyze biomolecules without fragmentation. Concerning the identification of antimicrobial resistance, MALDI-TOF MS is performed in three aspects, which are measuring antibiotics modifications caused by bacterial enzymes, analyzing the antibiotic resistance-related spectra patterns, and measuring the bacterial growth after being exposed to antibiotics. Through these methods, many antimicrobial-resistant proteins could be detected including β-lactamases, which have the most advanced in terms of detection by measuring antibiotics modifications (Hrabák et al., 2013). For the analysis of the peptide spectra approach of β-lactamases, the peptide spectrum located at 11109 Da was identified as the carbapenemase in KPC-producing Klebsiella pneumoniae (Lau et al., 2014). This spectrum was also detected in 88.2% of KPC-producing Klebsiella pneumoniae (Gaibani et al., 2016). Our experiment was the first study to use the MALDI-TOF MS technique via VITEK^® MS to investigate the specific peptide spectra of ESBL and Dps-like protein in ESBL-EC after exposure to CIP to indicate the mechanism of resistance and result in the appropriate use of antibiotics.

Analysis via VITEK^® MS in the present study found that some specific peptide spectra were demonstrated in significantly different levels between CAZ-exposed and CAZ and AMC-exposed areas to determine the indicative peptide spectra of ESBL, and CIP-exposed, and non-CIP exposed areas of ESBL^H isolates. Statistical analysis showed that the peptide spectrum at 9714 Da was more significantly different, and this is supposedly correlated to the production of ESBL, and the peptide spectrum at 9063 Da was significantly different among CIP^HR, CIP^LR, CIP^INTR and CIP^SUS. This finding may suggest that the peptide spectrum at 9063 Da was supposed to be the suspected Dps-like protein as reported in our previous study. The further proteomic study is required to precisely confirm if this spectrum plays a role as a Dps-like protein. Although the detection of antimicrobial resistance using MALDI-TOF MS is carried out with software, VITEK^® MS still lacks the software to automatically detect antimicrobial-resistant protein. Hence, if we can identify the peptide spectra which are specifically indicated for the ESBL^H and Dps-like protein, it would be beneficial for the development of rapid and simple antimicrobial resistance detection by VITEK^® MS in the future.

CONCLUSION

Fluoroquinolones, frequently used to treat ESBL-EC infection, are becoming more ineffective in recent years due to the emergence of multidrug-resistant strains. Although the detection of antimicrobial resistance using VITEK^® MS was carried out with software, VITEK^® MS cannot still automatically detect antimicrobial-resistant proteins. Identifying spectra corresponding to resistant proteins, including Dps-like protein, would be beneficial for the development of precise, accurate, and rapid antimicrobial resistance detection by VITEK^® MS. In this study, the peptide spectra of hospital-acquired ESBL-EC, which were multidrug-resistant, were analyzed upon the MALDI-TOF MS technique of the VITEK^® MS apparatus. Results showed the peptide spectrum located at 9714 Da was supposed to be ESBL-indicative peptide spectra, and the peptide spectrum located at 9063 Da was separately supposed to be a Dps-like protein as previously reported by our proteomic analysis in ESBL-EC. Further proteomic analysis can also be done to confirm this finding. This finding could potentially help expedite the routine laboratory diagnosis using VITEK^® MS in hospitals and allow for early detection of fluoroquinolone-resistance and ESBL^H.

ACKNOWLEDGMENTS

The authors would like to thank the Department of Microbiology, Chiang Mai University for providing microbiological support. We are grateful to Maharaj Nakorn Chiang Mai Hospital for providing the clinical isolates. We are also grateful to all technicians at the Diagnostic Laboratory Unit, Maharaj Nakorn Chiang Mai Hospital, Faculty of Medicine for their guidance on performing the VITEK^® MS apparatus. We would also like to thank the Institutional Biosafety Committee (IBC) for approving our work done on Biosafety Level 2(CMUIBC02018/2564).

AUTHOR INFORMATION

PD participated in the proposal sending, analyzing data, and proofing the manuscript. SK participated in the proposal sending and participated in the microbiologic experiment. SSr participated in the microbiologic experiment and drafted the manuscript. SJ analyzed data and designed the figure. ME participated in the microbiologic experiment and drafted the manuscript. PS collected the bacteria and performed the VITEK^® MS experiment. TS performed the statistic analysis. SSo carried out the research funding, designed the research methods, collected the bacteria, participated in the microbiologic experiment, and drafted the manuscript. All authors have read and approved the final manuscript.

CONFLICT OF INTEREST

The authors have no competing interests.

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Table 1. Data of ESBL screening result, the presence of keyhole phenomenon, the ratio of MIC_CAZ/MIC_CAZ/Clav, and the ESBL confirmation results of resistant and intermediate resistant isolates.

Table 2. ESBL production after exposed to CAZ, and CIP susceptibility of 137 resistant E. coli. ESBL production after exposed to CAZ is categorized by the MIC ratio between CAZ, and CAZ /Clav. CIP susceptibility is categorized by the MIC of CIP.

Table 3. Mean, SE, and P-values of spectra intensities after compared between the AMC and CAZ exposure in high, low, and non-ESBL producing groups.

Table 4. Mean, SE, and P-value of spectra intensities after compared between the CIP and CAZ exposure in high, low, intermediate resistant, and CIP susceptible groups.