Phenotypic Characterization and Antibiotic Resistance Patterns of Extended-Spectrum β-Lactamase- and AmpC β-Lactamase-Producing Gram-Negative Bacteria in a Referral Hospital, Saudi Arabia

Background Emergence of pathogenic bacteria carrying β-lactamase-resistant determinants has become a major health problem in the hospital setting. The study aimed to determine antibiotic-resistant patterns and frequency of extended-spectrum β-lactamase- (ESBL-) producing Gram-negative bacteria (GNB) and AmpC β-lactamase-producing GNB. Methodology A prospective cross-sectional study was conducted during a period from September 2017 to August 2018 at King Abdullah Hospital, Bisha Province, Saudi Arabia. GNB (n = 311) were recovered from patients' clinical specimens including sputum, urine, wound pus, blood, tracheal aspirates and high vaginal swabs, umbilical discharge, eye discharge, and cerebrospinal fluids. Isolates were identified by the Phoenix identification system. Antimicrobial susceptibility was tested by the Kirby–Bauer disk procedure. Phenotypic characterization of ESBLs and AmpC β-lactamases was performed utilizing the double-disk synergy test and inhibitor-based method, respectively. Associations with outcome measures were determined by simple descriptive statistics and a chi-square test. Results Out of 311 GNB isolates, the frequency of ESBL and AmpC β-lactamase producers was 84 (27%) and 101 (32.5%), respectively. Klebsiella pneumoniae and Escherichia coli were common ESBL producers. AmpC β-lactamases predominate among Acinetobacter spp. and Pseudomonas aeruginosa. Coproduction of ESBLs and AmpC β-lactamases was found in 36 (11.6%) isolates, with very close relative frequencies among K. pneumoniae, Acinetobacter spp., and P. aeruginosa. β-Lactamase producers were predominantly found in the surgical department (56.5%) and ICUs (44.2%). ESBL producers revealed high resistance for cefuroxime (96.4%), cefotaxime (92.9%), and trimethoprim/sulfamethoxazole (90.5%). The resistance rates were significantly higher among ESBL producers than nonproducers for cephalosporins (p < 0.001), amoxicillin/clavulanate (p < 0.001), piperacillin/tazobactam (p = 0.010), nitrofurantoin (p = 0.027), aztreonam (p < 0.001), ciprofloxacin (p = 0.002), and trimethoprim/sulfamethoxazole (p < 0.001). Significantly higher (p < 0.05) resistance rates were observed among AmpC β-lactamase producers than nonproducers for all tested antibiotics. Conclusions This finding showed a high prevalence of ESBL- and AmpC β-lactamase-producing GNB in our hospital. Quality control practice and routine detection of β-lactamase producers before deciding on antibiotic therapy are advocated.


Introduction
e controlling of infectious diseases caused by pathogenic bacteria has become challenged in the last years due to the extension of bacterial resistance to several antibiotics [1]. Infections caused by bacteria carrying resistant determinants have been associated with increased rates of mortality, hospital stay, therapeutic failure, and health costs [2,3]. Antibacterial agents of the β-lactam group are frequently prescribed medications for the treatment of infections caused by Gram-negative bacteria (GNB) [4]. Members of GNB can hydrolyze many β-lactam antibiotics through the production of one or both of extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases [3,4]. e production of ESBLs and AmpC β-lactamases mediated by both chromosomal and plasmid genes can transfer horizontally between GNB members [5,6]. Bacterial strains carrying such enzymes are capable of being resistant to a wide variety of antibiotics, including β-lactam drugs [3]. AmpC β-lactamases are clinically significant cephalosporinases encoded on chromosomes of Gram-negative rods which mediate resistance to cefoxitin, cephalothin, cefazolin, most of the penicillins, and β-lactamase inhibitor [7].
Studies of the antimicrobial susceptibility of GNB revealed an increased resistance due to hyperproduction of ESBL and AmpC enzymes over time [1,8,9].
is phenomenon was observed commonly in E. coli, K. pneumoniae, and other GNB as well [9]. Increasing antibiotic resistance due to the hyperproduction of AmpC enzymes among Enterobacter and Citrobacter has been reported in Europe [9]. One study in the United States reported a high incidence of ESBL-and AmpC-resistant genes among E. coli and Klebsiella spp. [10]. In countries of the Gulf Cooperation Council, the high prevalence of ESBL-producing GNB associated with nosocomial infections has been well established [11]. Although considerable studies in Saudi Arabia have been focused on epidemiology and resistant traits of ESBL-producing microorganisms [1,12,13], such data are still limited about AmpC β-lactamase producers [14]. However, the mechanisms that underpin antibiotic resistance are not thoroughly investigated in certain areas [14,15]. Routine phenotypic detection of β-lactamases carrying resistant strains would be a useful guide for antibiotic therapy and minimizes spreading of these bacteria in hospital settings [4,5].
is study, therefore, set out to determine resistance patterns and the frequency of ESBLand AmpC β-lactamase-producing GNB from patients at King Abdullah Hospital, Bisha Province, southwest of Saudi Arabia.

Study Design and Setting.
A prospective cross-sectional study was conducted between September 2017 and August 2018 at King Abdullah Hospital, Bisha Province, Saudi Arabia.
is hospital is a referral hospital with 365 beds distributed into different specialized units [15]. Various clinical specimens were collected from patients of all age groups and submitted to the hospital microbiology laboratory for routine microbiological investigations. e specimens were sputum, urine, wound pus, blood, tracheal aspirates and high vaginal swabs, umbilical discharge, eye discharge, and cerebrospinal fluids. Ethical approval was obtained from the research and ethical committee, College of Medicine, University of Bisha.

Identification of Pathogens.
Isolation and identification of GNB were carried out based on cultural characteristics, Gram stains, oxidase test, and conventional biochemical tests following standard assay [16]. en, full identification of isolate was performed using Phoenix system identification method (Becton, Dickinson, USA). e Phoenix panels were inoculated according to the manufacturer's instructions. Depending on the site of infections and types of specimens, significant growth of each pathogen was identified and processed for antimicrobial susceptibility testing. Every single significant growth of GNB was included in this study. Clinical samples with missed patient personal information and/or yielded more than two isolates were being excluded from the study.

Double-Disk Synergy Test (DDST).
e DDST was performed to detect ESBL producers as described by Jarlier et al. [18]. e test was performed immediately along with susceptibility testing of each isolate. A susceptibility disk containing amoxicillin/clavulanate (20/10 μg) was placed in the center of the plate. ree disks of cephalosporin agents, namely, ceftazidime (30 μg), cefotaxime (30 μg), and cefepime (30 μg), were located 30 mm apart (center to center) from the amoxicillin/clavulanate disk. All cultured plates were aerobically incubated overnight at 37°C. A visible distortion or extension of the edge of the inhibition zone of cephalosporin towards amoxicillin/clavulanate was interpreted as positive for the production of ESBLs. Strains of E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were served as a negative and positive control, respectively.

Inhibitor-Based Method.
AmpC β-lactamase production was detected by an inhibitor-based method on disk containing boronic acid as previously described [19]. Isolates showed inhibition with zone diameters less than 18 mm for cefoxitin disk (30 μg), which was processed for confirmation of AmpC production. A 0.5 McFarland suspension of tested isolates was inoculated evenly on a Mueller-Hinton agar plate (Oxoid, England). Two disks of cefoxitin (30 μg) with and without boronic acid (400 μg) were placed onto the surface of the plate at a distance of 30 mm. After overnight incubation at 37°C aerobically, a zone of 5 mm or greater around the disk of cefoxitin containing boronic acid compared to the cefoxitin disk was considered for AmpC β-lactamase production.

Statistical Analysis.
Data management and analysis were performed using the Statistical Package for Social Sciences (SPSS; Version 16.0). Simple descriptive statistics were presented to analyze the outcome data. A chi-square test was used to compare between the resistant patterns of β-lactamase-and non-β-lactamase-producing isolates. All values less than 0.05 were considered as statistically significant.

Discussion
β-Lactamase production among pathogenic bacteria is becoming important resistance mechanisms in hospitals worldwide [3,4]. ere are no current data about the existences of ESBL-and AmpC β-lactamase-producing GNB in King Abdullah Hospital, Bisha, Saudi Arabia. In the present study, β-lactamase-producing bacteria were commonly found in ICU and surgical departments. is finding is similar to that reported in the eastern region of Saudi Arabia [20] and many parts of the world, such as in Algeria [21] and Nigeria [22]. It has been suggested that the higher use of invasive devices and the selective pressure of newer β-lactams for patients at ICU and surgery unit result in the emergence of such pathogens [20]. Noteworthy, GNB collected from the hospital units tend to be β-lactamase producers.
is might be due to clonal spreading and transmission of β-lactamase genes between GNB in the hospital. However, this hypothesis could be to understand by analyzing the clonal similarity of bacterial isolates collected from different hospital wards using specific molecular markers.
In the present study, the prevalence of ESBL-producing GNB was 27%, which is almost similar to that found in the eastern region of the country (30.6%) [12]. However, our result was lower than 72% reported at a tertiary care hospital in Riyadh capital [1]. High prevalence of ESBL-producing bacteria has been observed in many African and Asian countries, such as 47.6% in Algeria [21], 60% in Pakistan [8], and 65% in Nigeria [22]. By contrast, the lowest proportions of ESBL-producing Enterobacteriaceae have been reported in Europe, such as below 1% in Sweden [23] and 5% in Netherlands [24]. ese reports coupled with the current findings indicated the global dissemination of β-lactamaseproducing microorganisms, notably in developing countries including Saudi Arabia. is could be attributed to lack of antibiotic policy, poor hygiene conditions in developing countries [25]. Moreover, increasing global trade and international travel were found to be significant risk factors for emerging these resistant bacteria [5]. Indeed, Saudi Arabia has become a significant place for spreading of ESBL microorganisms due to the hosting of mass gathering during Haj and Umrah and population flow from many parts of the world [26]. erefore, local and national surveillance coupled with the international effort to compact spreading of β-lactamase-producing microorganisms is needed.
In the present study, K. pneumoniae and E. coli were the major ESBL producers although diverse GNB expressed ESBL production. is finding is consistent with previous studies in Saudi Arabia [12,20,25]. Likewise, studies in African countries found that K. pneumoniae and E. coli were the leading ESBL producers in the hospital settings [4,27].    AmpC β-lactamase-producing bacteria may cause nosocomial outbreaks in hospital settings and leading to affect therapeutic choices [28]. e present study showed a higher prevalence of AmpC β-lactamase producers (32.5%) compared to a recent study conducted in Saudi Arabia (5.5%) [14]. However, our finding is in agreement with a report from India, where AmpC phenotype was recorded in 36.5% of Enterobacteriaceae in a multicenter study [7]. e highest frequency of AmpC β-lactamases in this study was found among Acinetobacter spp. (73.5%). As well, high incidence of AmpC enzymes in Acinetobacter spp. has been reported in China (72%) [29] and India (60%) [30].
ese results with our current findings indicated that regular carrying out of infection control procedures could play an important role to reduce spreading of AmpC β-lactamase-producing organisms. However, screening of cefoxitin resistance during routine sensitivity tests can aid in early detection of AmpC β-lactamase producers and setting of effective antibiotic therapy [7,28].
ese elevated rates were also reported in Riyadh capital by Marie et al. (2013) [1]. Furthermore, AmpC β-lactamase-producing GNB demonstrated high rate of resistance to meropenem (49.5%) and imipenem (57.2%). Such figures are of great concern since carbapenems are considered the drugs of choice for therapy of serious ESBLand AmpC β-lactamase-associated infections in the country [20,33]. e possible explanation for increasing resistance rates might attribute to antibiotic misuse in general and frequent prescription of carbapenems in our hospital. erefore, phenotypic detection of β-lactamase-producing GNB should be carried routinely before antibiotic therapy. However, several phenotypic tests are available, cheap, and easier to conduct routinely in concurrent with routine susceptibility testing of GNB in the hospital laboratory.
In the present study, coproduction of ESBLs and AmpC β-lactamases was found among 11.6% of the isolates. is proportion is relatively lower than 14.3% reported among P. aeruginosa in Pakistan [34]. Likewise, several studies identified the production of ESBLs and AmpC β-lactamases together by GNB [3,35].
Bacterial strains producing both ESBLs and AmpC β-lactamases are often more resistant to β-lactam, β-lactamase inhibitor combinations, carbapenems, and several antibiotic groups [26]. In this study, GNB carrying ESBLs and AmpC β-lactamases revealed high resistant rates to most of the antibiotics. A strong association between plasmid AmpC β-lactamases with ESBL, plasmid-mediated quinolone resistance, and aminoglycoside-modifying enzymes has been well documented in the literature [14,20]. However, the genotypic study of different families of β-lactamaseencoded resistant genes might be essential to develop a full picture of β-lactamases resistance mechanisms in GNB.

Conclusion
In conclusion, the present study reported high prevalence of ESBL-and AmpC β-lactamase-producing GNB, mainly among K. pneumoniae, E. coli, and Acinetobacter spp. in King Abdullah Hospital. Coexistence of ESBLs and AmpC β-lactamases was found among 11.6% of the isolates, making the infection caused by those bacteria more challenging to treat. Escalating levels of antibiotic resistance among ESBL and AmpC β-lactamase producers were observed, leaving limited therapeutic options. e presences of ESBLs and AmpC β-lactamases were fundamental mechanisms of increasing resistance rates among Gram-negative pathogens in our hospital, although other resistant determinants have not investigated yet. ese findings impose that regular carrying out of infection control procedures could play an important role to reduce spreading of ESBL-and AmpC β-lactamaseproducing isolates. Routine detection of ESBL-and AmpC β-lactamase-producing isolates before deciding on antibiotic therapy is advocated.

Data Availability
All the data supporting our findings were incorporated within the article. Raw data can be presented by the principal investigator upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
MEI designed and conducted the study, collected and analyzed and interpreted the data, and wrote initial draft. MA designed and conducted the study. AMA revised the study for important intellectual contents. BKE interpreted the data and revised the manuscript for important intellectual content. All authors have investigated the final draft and are accountable for the content and similarity index of the manuscript.