INTRODUCTION

Worldwide, multidrug-resistant (MDR) bacteria have become a global problem due to the treatment challenges and a great issue to public health [1, 2]. They are estimated to cause more than 700,000 deaths worldwide every year and may exceed 10 million deaths by 2050 and lead to a high economic loss [3]. Infections by MDR bacteria can result in failure of treatment, in addition to the high cost of medical treatment and hospitalization stay [4]. Generally, antimicrobial resistance mechanisms are categorized into four types: (1) limitation of drug uptake; (2) modification of a drug target; (3) inactivation of a drug; (4) activation of drug efflux. Worldwide, the production of extended-spectrum β-lactamases (ESBLs) by Enterobacteria is a real threat to public health [5, 6]. There are three major families of ESBLs, namely; CTX-M, SHV, and TEM, besides a large variety of other types of enzymes. CTX-M enzymes are the most prevalent ESBLs worldwide [7]. ESBL-producing G- bacteria are resistant to cephalosporins, penicillins, monobactams, and even carbapenems [8]. These organisms are sometimes resistant to fluoroquinolones and aminoglycosides. Metallo-β-lactamases (MBLs) or (carbapenemase producers) are known as class B, they have a broad spectrum and can resist all beta-lactam antibiotics except monobactams. They are neither inhibited by tazobactam, sulbactam, clavulanate, nor metal chelators (EDTA) [9, 10]. The spread of the VIM- and IMP-type MBL in Gram-negative bacteria, has been noticed since 1990 [11, 12]. The spread of MBL genes induces the emergence of multidrug-resistant bacteria. Aminoglycosides and cephalosporins (third generation) are generally used in the treatment of infections caused by G- pathogens. These antibiotics become less effective due to the synthesis of β-lactamase by these bacteria. Carbapenems are the current choice to treat infections caused by ESBL producers; however, resistance to carbapenem by the production of carbapenemases in G- bacteria is fast running out and may leave treatment options with potentially toxic drugs such as polymyxin and colistin. Now the MBL poses a therapeutic challenge worldwide [13] because these enzymes are capable of hydrolyzing penicillins in addition to the 3^rd- and 4^th-generation cephalosporins (carbapenems) [14]. The coexistence of different classes of β-lactamases in gram-negative bacteria has been reported widely, thus rising diagnostic and treatment challenges [15]. It is an indication of an increase in the resistance mechanisms, thus rendering the antibacterial drug ineffective. The study aimed to evaluate the coexistence of ESBLs and carbapenemase-producing genes in confirmed ESBLs-producing Gram-negative bacteria.

MATERIALS AND METHODS

Identification of Samples

220 G- bacteria stocks were previously collected from 5 tertiary hospitals during pilgrimage seasons in Makkah, KSA between 2018 and 2020, and kept at -70°C. The isolates were identified with a VITEK-2 Compact System using AST-GNI cards (Biomerieux) and biochemical tests including motility, urease, citrate utilization, Indole, Kligler iron agar (KIA), and oxidase tests.

Susceptibility Tests

The susceptibility of bacteria to antibiotics was done using the VITEK 2 system (AST no. 12 card) and the disc-diffusion method following the standards of the Clinical and Laboratory Standards Institute [16]. The antibiotics included; cefepime (30µg), ceftriaxone, cefotaxime, ceftazidime, amikacin, gentamicin, amoxicillin/clavulanic acid, ciprofloxacin, imipenem, ertapenem, and meropenem (Oxoid, UK).

Screening for ESBLs -Producing Strains

ESBL production was confirmed by Double-Disc Synergy Test. An inoculum Standard of (0.5 McFarland) of each isolate was prepared and then inoculated on MHA plates. ceftriaxone (30µg) and Ceftazidime (30µg) discs were then applied with a distance of 1.5cm away from amoxicillin-clavulanic acid (20µg/10µg) disc and then incubated at 37 °C for 18h. Positive results were confirmed when the zone of inhibition was extended towards amoxicillin/clavulanic acid disc >5mm.

EDTA-Disc Synergy Test

MBL production by bacterial isolates was tested using EDTA-Disc Synergy Test. The plates were swabbed with 0.5-McFarland isolates at turbidity standards. After brief drying, two 10µg meropenem discs were placed 20mm apart, and 10µl EDTA (0.5M) was added to one disc. Zone enhancement in the area between 2 discs indicated a positive result.

PCR Testing

DNA of each isolate was extracted using the boiling method with modification and used for PCR [17]. Multiplex PCR was carried out to detect β-lactamase genes blaCTX-M, blaTEM, blaSHV, blaIMP, and blaVIM-1. Table 1 shows the primers used for PCR. Each reaction had a volume of 25μL, including 10μL master mix (Promega, USA), 1.0μL of each primer, 4μL DNA template, and 10μL purified distilled water. The multiplex PCR reaction was initiated with a pre-denaturation step at 94°C for 5min, followed by 35 amplification cycles (denature at 94 °C (30s), annealing at 55°C (30s), and extension at 72 °C (1min)), then finalized with an extension at 72°C for 10min. The PCR products were detected by 1% agarose gel electrophoresis.

Ethics Statement

The study was approved by the institutional review board of the faculty of medicine at Umm Alquraa University. Consent was obtained from each participant.

Statistical Analysis

Statistical analysis of data was performed using SPSS software (v.25, IBM, US). Different variables were compared using the Chi-Square test, and a p-value <0.05 was considered statistically significant.

RESULTS AND DISCUSSION

In this study, 35.4% (n=78) of Gram-negative bacteria were found to be phenotypic ESBL producers (Table 2), out of which 53.8% (n=42) were K. pneumonia, 20.5% (n=16) E. coli, 10.3% (n=8) Ps. aeruginosa, 9.0% (n=7) Enterobacter spp., 3.8% (n=3) P. mirabilis, and 2.6%(n=2) were A. baumannii. Of these 78 isolates, 32.1% (n=25) were found to be MBLs, 15.3% (n=12) belonged to K. pneumonia, 7.7% (n=6) belonged to E. coli, 6.4% (n=5) belonged to Ps. aeruginosa and 2.6% (n=2) belonged to Enterobacter spp (Table 2). The study showed that rates of resistance against cephalosporins; cefotaxime, ceftazidime, ceftriaxone, and cefepime were 100%, 98.7%, 96.2%, and 66.7%, respectively. Resistance rates against carbapenems; ertapenem, meropenem, and imipenem, were 38.5%, 32.1%, and 23.1% respectively. Resistance rates against amikacin, gentamicin, ciprofloxacin, and amoxicillin/clavulanic acid antibiotics were 64.1%, 61.5%, 52.5%, and 50.0%, respectively (Table 3). PCR confirmed the presence of ≥1 ESBL genes in 61 isolates (78.2%) as shown in Table 4 and Figure 1. The study found that 57 (73.1%) of the isolates were harboring at least one of the tested MBL genes. About 8 (10.3%) isolates carried only one gene, namely SHV (5.1%), TEM (2.6%), and IMP (2.6%) while 55 (70.5%) of the isolates carried more than one gene (Table 3). Neither CTX-M nor VIM gene was found alone. Among the studied isolates, 31 (39.7%) carried two genes, 8 (10.8%) strains carried three genes, and 16 (20.5%) strains carried 4 genes (Table 4). CTX-M was the highest detected gene (70.5%) followed by IMP (61.5%), TEM (27%), VIM (21.8%), and SHV (21.8%) (Table 5 and Figure 2). The coexistence of two genes (CTX-M+IMP) mostly was seen in 26 (33.4%) isolates. In addition, the coexistence of three genes (TEM+ CTX-M+ IMP) was mostly seen in 6 (7.7%) while the coexistence of four genes (CTX-M+ TEM+ SHV+ IMP) was mostly seen in 6 (7.7%) isolates as shown in Table 4 and Figure 1. The results showed that 17 K. pneumoniae isolates carried (CTX-M+ IMP) genes, 4 isolates harbored (TEM+ CTX-M+ IMP), and 4 isolates harbored (CTX-M+ TEM+ SHV+ VIM) genes simultaneously. Moreover, the simultaneous presence of (CTX-M+ IMP) and (CTX-M+ TEM+ SHV+ IMP) genes was observed in 6 and 2 strains of E. coli, respectively. Also the presence of (CTX-M+ IMP), (TEM+ CTX-M+ IMP), (IPM+ CTX-M+ VIM+TEM), (IMP+CTX-M+ SHV+ VIM), (CTX-M+ TEM+ SHV+ IMP) genes was observed in 5 separate Ps. aeruginosa isolates (Table 4). Significant association was detected between CTX-M and IMP gene carriers and between SHV and VIM gene carriers (P < 0.05) (Table 5).