Molecular Characterization and Epidemiology of Antibiotic Resistance Genes of β-Lactamase Producing Bacterial Pathogens Causing Septicemia from Tertiary Care Hospitals

Septicemia is a systematic inflammatory response and can be a consequence of abdominal, urinary tract and lung infections. Keeping in view the importance of Gram-negative bacteria as one of the leading causes of septicemia, the current study was designed with the aim to determine the antibiotic susceptibility pattern, the molecular basis for antibiotic resistance and the mutations in selected genes of bacterial isolates. In this study, clinical samples (n = 3389) were collected from potentially infected male (n = 1898) and female (n = 1491) patients. A total of 443 (13.07%) patients were found to be positive for bacterial growth, of whom 181 (40.8%) were Gram-positive and 262 (59.1%) were Gram-negative. The infected patients included 238 males, who made up 12.5% of the total number tested, and 205 females, who made up 13.7%. The identification of bacterial isolates revealed that 184 patients (41.5%) were infected with Escherichia coli and 78 (17.6%) with Pseudomonas aeruginosa. The clinical isolates were identified using Gram staining biochemical tests and were confirmed using polymerase chain reaction (PCR), with specific primers for E. coli (USP) and P. aeruginosa (oprL). Most of the isolates were resistant to aztreonam (ATM), cefotaxime (CTX), ampicillin (AMP) and trimethoprim/sulfamethoxazole (SXT), and were sensitive to tigecycline (TGC), meropenem (MEM) and imipenem (IPM), as revealed by high minimum inhibitory concentration (MIC) values. Among the antibiotic-resistant bacteria, 126 (28.4%) samples were positive for ESBL, 105 (23.7%) for AmpC β-lactamases and 45 (10.1%) for MBL. The sequencing and mutational analysis of antibiotic resistance genes revealed mutations in TEM, SHV and AAC genes. We conclude that antibiotic resistance is increasing; this requires the attention of health authorities and clinicians for proper management of the disease burden.


Introduction
Blood is a connective tissue which forms about 8% of total body weight; 5-7 L of blood is present in an average human body. The main components of blood are plasma (liquid portions 55%) and cells (45%): white blood cells, platelets and leucocytes [1,2]. Blood functions as the transportation medium for nutrients and aids in the excretion of waste materials by specialized organs. In vertebrates, blood is important for the maintenance of the body's temperature [3].
Blood is a sterile medium, but its contamination with pathogens or toxins leads to blood stream infections (BSIs), which are some of the leading causes of mortality and Antibiotics 2023, 12, 617 2 of 11 morbidity around the globe. BSIs are associated with fatal health conditions, which require admission to intensive care units [4]. In the United States, BSIs have been correlated with various risk factors, including exposure to microorganisms and the use of central venous catheters [5]. Causative agents for septicemia vary from region to region; it can be caused by both Gram-positive and Gram-negative bacteria, the most common of these being E. coli, P. aeruginosa, Staphylococcus aureus, Klebsiella pneumonia and Salmonella typhi [6]. Among these, the Gram-negative bacteria most associated with septicemia is E. coli [7][8][9].
The most common classes of antibiotics used to treat BSIs are penicillin, cephalosporins, aminoglycosides, glycopeptides, lincosamides, tetracyclines, fluoroquinolones and carbapenems. Due to overuse and misuse of antibiotics, bacteria have developed resistance to them, resulting in global health hazards [10,11]. Drug resistance in E. coli and other Gram-negative bacteria continues to rise, resulting in the emergence of multidrug-resistant strains. Treating the infections caused by these pathogens is a challenging issue [12]. An estimated 700,000 patients die globally each year due to high antibiotic resistance, and this number continues to rise [13]. A study in 2017 reported a total of 48.9 million cases of morbidity and 11 million of mortality worldwide, which constitutes a total of 20% mortality. Of the total, 85% of the cases of sepsis, including those of sepsis associated with death, were reported in low-middle income countries, and in Pakistan 60% of sepsis cases were fatal because of the infection being caused by multidrug-resistant strains and the misuse of antibiotics [14,15]. A study on neonatal sepsis in Sub-Saharan Africa revealed that E. coli accounted for 10% of the reported cases and was mostly resistant to aminoglycosides and β-lactams [16].
Determining the common pathogens and the antimicrobial susceptibility pattern causing septicemia is essential in order to select appropriate antibiotic therapies to decrease mortality and morbidity [17]. Keeping in view the importance of Gram-negative bacteria as one of the leading causes of septicemia, the current study was designed with the aim to determine the antibiotic susceptibility pattern, the molecular basis for antibiotic resistance and the mutations in selected genes of the bacterial isolates in Peshawar, Khyber-Pakhtunkhwa, Pakistan.

Identification of Bacterial Isolates
All the isolates were identified by being cultured on MacConkey and blood agar media, followed by Gram staining (pink color colonies under microscope), API strips (as per API codes and reading scales) and on the molecular level by USP for E. coli and oprL for P. aeruginosa (Figure 1).

Identification of Bacterial Isolates
All the isolates were identified by being cultured on MacConkey and blood agar media, followed by Gram staining (pink color colonies under microscope), API strips (as per API codes and reading scales) and on the molecular level by USP for E. coli and oprL for P. aeruginosa (Figure 1).

Antibiotic Susceptibility Pattern of Clinical Isolates
The resulting antibiotic sensitivity patterns of identified E. coli and P. aeruginosa revealed resistance to AMP, SXT and CIP, and sensitivity to MEM, IPM and TOB (Table 2).

Antibiotic Susceptibility Pattern of Clinical Isolates
The resulting antibiotic sensitivity patterns of identified E. coli and P. aeruginosa revealed resistance to AMP, SXT and CIP, and sensitivity to MEM, IPM and TOB (Table 2).

Determination of Minimum Inhibitory Concentration
The potency of the antibiotics depends on their minimum inhibitory concentration (MIC) values. The higher the MIC value, the less potent the antibiotic, and vice versa. The ESBLs, MBLs and AmpC β-lactamases producing E. coli and P. aeruginosa isolates were highly resistant to CTX and CAZ with high MIC values as well as non-β-lactam drugs. SXT, CIP, DO, CN and AK were susceptible to MEM and to TGC with low MIC values (Tables 3  and 4).

Discussion
Antibiotic resistance is a major health threat and is responsible for high morbidity and mortality around the globe. Gram-negative bacteria have developed ways to combat the available antibiotics, making bacterial infections hard to treat. In the current study, the results confirmed this phenomenon, which is affecting community health and the economy. In the current study, 41.5% prevalence of E. coli was reported, which is similar to other findings [14]. The positivity ratios of infection of E coli were 64.7% in female patients and 35.3% in male patients. In our study, 27.1% of E. coli isolates were reported in the age group of 41-60 years; this may be due to weakened immune systems or to frequent exposure. This was followed by 26.01% in 21-40 years, which is in contrast to the reported literature [15]. The E. coli isolates of this study showed resistance to AMP, CTX, CAZ, CIP and LEV and sensitivity towards SCF, CO, MEM, TGC, AK, FOS and TZP, in agreement with the literature [16]. The prevalence rate of P. aeruginosa in the current study is 17.6%, 23.1% of this was in female patients and 76.9% in male patients, as supported by the reported study [17]. A 2016 study conducted in Pakistan found P. aeruginosa in 13% of septicemia patients, 55.8% males and 44.2% females. The prevalence of P. aeruginosa in our study at 17.6% implies that it has increased in the last few years. This directly indicates an increase in antibiotic resistance in Pakistan [18]. In the current study, ESBL genes in E. coli isolates were screened, in which CTX-M was detected in 70%, TEM in 63.7% and SHV in 35%. Another reported study had lower prevalence of CTX-M (57.7%), TEM (20.3%) and SHV (15.4%) [19]. Similar to that study, in the current study, 36.6% of the MBL targeted genes were detected, in which NDM1 gene prevalence was almost 30%. An Indian study reported the same results of MBL Ec with 28% prevalence of NDM-1 [20]. In this study, AmpC β-lactamase was found in 85.0%, CIT gene in 16.4% and DHA gene in 11.9% of the total clinical isolates, which supported earlier reported studies [21,22]. The mutations in the selected gene may offer a molecular explanation for the antibiotic resistance in the isolates of the current study.

Conclusions
The findings of this study have several key implications for health policymakers, clinicians and researchers. These findings highlight the need to update infection-prevention measures to be better able to manage the diseases caused by E. coli and P. aeruginosa. The increase in antibiotic resistance is an alarming situation, and necessitates a rationalization of the treatment strategy to control BSIs. The unavailability of newer drugs, and the constant increase in antibiotic resistance have led to the use of limited drugs such as colistin by physicians. This has resulted in a condition called pan-drug resistance, necessitating the discovery of new antimicrobial drugs.

Materials and Methods
The study was conducted in the Khyber Teaching Hospital Peshawar, Hayatabad Medical Complex Peshawar and the Center of Biotechnology and Microbiology, University of Peshawar, using standard microbiological procedures. A total of 3389 blood samples were collected from suspected septicemic patients in EDTA tubes aseptically, from both sexes and from various age groups, and were processed by automated blood culture systems. An overview of the whole methodology is represented in Scheme 1. Informed consent was obtained from all patients on a prescribed proforma before taking blood samples.
Medical Complex Peshawar and the Center of Biotechnology and Microbiology, University of Peshawar, using standard microbiological procedures. A total of 3389 blood samples were collected from suspected septicemic patients in EDTA tubes aseptically, from both sexes and from various age groups, and were processed by automated blood culture systems. An overview of the whole methodology is represented in Scheme 1. Informed consent was obtained from all patients on a prescribed proforma before taking blood samples.

Scheme 1.
Flowchart of the methodology used in the study.

Scheme 1.
Flowchart of the methodology used in the study.

Molecular-Level Identification
For molecular-level identification of the bacterial isolates (USP for E. coli and oprL for P. aeruginosa) and detection of antibiotic-resistant genes ( Table 9), DNA of the bacterial isolates was extracted using Thermo Scientific GeneJET Genomic DNA purification kits as per the manufacturer's protocol. The extracted DNA was confirmed by gel electrophoresis (1% agarose gel in 1X triacetate EDTA buffer) and visualized by a gel documentation system.

Antimicrobial Susceptibility Testing
The Kirby-Bauer disk diffusion method [21] was used to determine the antibiotic sensitivity pattern of the identified bacterial isolates against selected antibiotic disks (Table 10) as per Clinical Laboratory and Standard Institute (CLSI) guidelines. The pure cultures of the bacterial isolates (0.5 McFarland standard) were inoculated on sterile Muller-Hinton agar (MHA) media, and the antibiotic disks were applied, followed by 24 h of incubation at 37 • C. The zones of inhibition were measured and were evaluated as resistant (R), intermediate (I) and sensitive (S), as per CLSI guidelines [19]. Table 10. Antibiotic disks along with the concentration used in the study.

S. No
Name of Antibiotic

Minimum Inhibitory Concentration
The minimum inhibitory concentrations (MICs) of the selected antibiotics were determined using MIC strips (Table 11). The strips were placed along with inoculation of the pure isolates on sterilized MHA media, followed by overnight incubation at 37 • C [24].

Detection of Antibiotic-Resistant Genes by Polymerase Chain Reaction
The selected antibiotic resistance genes, as per antibiotic resistance pattern, were amplified by polymerase chain reaction (PCR) using specific primers ( Table 9). The PCR contained 12.5 µL of Taq Master Mix (Thermo Fisher Scientific TM, , Waltham, MA, USA), 11.5 µL nuclease-free water, 0.5 µL of forward and reverse primers (oligo nucleotide Microgen, Seoul, Korea) each and 2 µL of DNA sample. Under optimized conditions