Antimicrobial Drug Resistance Trends of Bacteria Causing Bloodstream Infections in a Diagnostic Centre in Lahore

Bacteraemia due to multidrug-resistant (MDR) bacteria, particularly those producing carbapenemase or extended-spectrum beta-lactamase (ESBL), causes a signi�cant threat to patients and associated morbidity and mortality. The global rise in the incidence of bacteremia necessitates the rapid and accurate identi�cation of pathogens to ensure effective patient health management. Objective: To investigate antimicrobial drug resistance trends among bacteria causing bloodstream infections from a diagnostic centre in Lahore. Methods: This research was conducted at the Institute of Microbiology and Molecular Genetics, University of the Punjab, Lahore and Citilab and Research Centre, Lahore, from January 2020 to December 2022. A total of 2919 blood samples were cultured to screen the bacteremia patients. Following standard protocols, four hundred twenty isolates proceeded for gram-staining, biochemical characterization, and antimicrobial susceptibility testing (AST). The AST results of each strain calculated multiple antibiotic resistance (MAR). Results: Of 420 bacterial isolates, Gram-negative and Gram-positive isolates accounted for 48.57% and 51.43%, respectively. The predominant pathogens were Staphylococcus epidermidis (48.10%) and Salmonella typhi (27.14%), with other signi�cant pathogens including Klebsiella spp., Pseudomonas spp., Enterobacter , Acinetobacter spp., Escherichia coli , Staphylococcus aureus , Enterococcus spp., Citrobacter , Morganella morganii , and Proteus mirabilis . AST revealed high resistance to Cephalosporins, Nitrofurantoin, Fosfomycin, and Quinolones. In contrast, Carbapenems demonstrated notable sensitivity. Salmonella typhi and Staphylococcus epidermidis exhibit the highest MAR values. Conclusions: The study highlights the prevalence of multidrug resistance bacteremia-causing pathogens, with a concerning trend towards decreasing antibiotic e�cacy.

pneumoniae, Pseudomonas aeruginosa, or Salmonella spp. have gained prominence in the context of communityacquired bacteraemia [3]. The existence of bacteria in the bloodstream, known as bacteraemia, presents an immediate concern for public health, capable of triggering severe illnesses and in icting signi cant economic tolls on the global economy annually. Bacteraemia can lead to clinical sepsis, characterised by life-threatening organ dysfunction [5]. Diagnosis is accomplished by examining the patient's clinical symptoms and conducting laboratory tests [1]. Bacteraemia is linked to fever and chills but can also manifest without symptoms [5]. The clinical diagnosis of bacteraemia is con rmed through microbiological examination of blood samples. Blood cultures remain the de nitive test for identifying individuals with bacteraemia. Isolating the microorganism from the bloodstream validates the diagnosis, enabling medical practitioners to pinpoint the infection's origin and subsequently prescribe suitable antimicrobial treatments [6]. The rise of antimicrobial resistance (AMR) in most bacterial pathogens presents a substantial global public health challenge. AMR remains an escalating concern within healthcare facilities, and it is linked to the rapid dissemination of multidrug resistant clones from hospital-acquired contexts to infections acquired within the community [7]. The "ESKAPE" pathogens can evade the bactericidal effects of antibiotics. This group includes E: Enterococcus faecium, S: Staphylococcus aureus, K: Klebsiella pneumoniae, A: Acinetobacter baumannii, P: Pseudomonas aeruginosa, and E: Enterobacter spp. These pathogens can acquire and propagate antibiotic resistance, contributing to over half of all infections associated with healthcare settings, and are closely linked to high rates of antimicrobial resistance. They pose a signi cant risk to patients, particularly those developing bloodstream infections, as limited treatment options and effective management strategies are available [7]. Bacterial infections with MDR have been linked to increased occurrences of complications, mortality, and recurrence. The constrained progress in creating new antibiotics has resulted in the challenging treatment of MDR infections [8]. The improper utilisation of antibiotics is associated with the emergence and dissemination of antibiotic-resistant bacteria. Interventions focusing on the prudent use of antimicrobials have been implemented to target antibiotic prescription practices within primary care environments [9]. By identifying the most prevalent drugresistant pathogens and delineating the key drivers of resistance, the current study aims to evaluate the bacterial pathogens involved in BSIs and their antimicrobial susceptibility patterns in patients and to inform evidencebased interventions, guide antimicrobial stewardship efforts, and optimize therapeutic strategies for

M E T H O D S
bacteraemia patients in Lahore.
This study was based on an analysis of blood culture data to evaluate the rate of bacteria causing bloodstream infections. A total of 2919 patients visited the diagnostic Centre in Lahore, for blood culture tests. All data recorded on blood cultures submitted for detection of blood infection with antibiotic sensitivity testing (AST) was collected from the Microbiology Department of Citilab and Research Centre, Lahore. Demographic information of patients of blood sample were obtained. All samples were taken in blood culture bottles containing Tryptic Soy Broth with SPS and incubated at 37°C overnight. Culture broth with positive signs of bacterial growth was streaked on Blood agar, Chocolate agar and MacConkey's agar plates and incubated for 24-48 hours at 37°C. The puri ed colonies were selected to identify based on colony morphology and gram staining. According to the microbiological manual, the biochemical characterization was carried out by multiple tests, including triple sugar iron, citrate utilization, methyl red, oxidase, motility, Voges Proskauer, and indole. Antibiotic susceptibility testing of strains was performed on Muller Hinton agar (MHA) using Kirby Bauer's method. Around 30 antibiotic discs were tested for antimicrobial activity against the organisms under study. The plates were prepared by pouring MHA to a depth of 3 mm and then swabbing them with the test organisms. The paper discs were placed on the surface of agar plates aseptically and at well-spaced intervals of >30mm. For gram negative isolates, antibiotics discs . After incubation, the plates were observed for the zone of inhibition around the disc. For 18 hours, the bacterial test plates were incubated at 37°C. Finally, the inhibition zone diameters were measured in millimeters. A relative antibiotic resistance pro le among different clinical pathogens is determined using the Multiple Antibiotic Resistance Index. The MAR index of any isolate is calculated as a ratio of 'a' to 'b', where 'a' is the number of antibiotics for which resistance has been determined, and 'b' is the number of antibiotics used on each isolate. For each isolate, the MAR Index was calculated.
A total of 420 (14.38%) bacteria were isolated from 2919 blood cultures received in the Microbiology section of the Lab. The most affected age group was 1-20 years. 120 (40.13%), followed by 21-40 years 93 (31.10%), 41-60 years 45 (15.05%) >1 year 23 (7.69%) and 61-80 years 17 (5.68%). Blood infections were more predominant in females 180 (53.0%) than in males 159 (46%) ( Table 1).     Bacteremia was more common in women than men, with 52.8% and 47.2%, respectively. In contrast to our results, male was more likely to be affected in a retrospective study  . Another study highlighted the rapid increase in c a r b a p e n e m r e s i s t a n c e r a te s to a n t i b i o t i c s i n Acinetobacter species and a noticeable rise in third and four th-generation cephalosporins resistance in Salmonella typhi in the last ve years. Whereas S. aureus with decreasing resistance trends was observed between 2011-2015. Two studies in Pakistan reported the emergence of MDR isolates in Salmonella typhi and XDR was 76% (182 isolates) and 48% (115 isolates) during previous outbreaks [23,24]. Extensive use of beta-lactam drugs, aminoglycosides and other antibiotics is one of the major factors that lead to the emergence of extended-spectrum beta-lactamase Enterobacteriaceae (ESBL), carbapenemresistant Enterobacteriaceae (CRE), gentamicin-resistant Gram-negatives, methicillin-resistant S. aureus (MRSA), and vancomycin-resistant enterococci (VRE) [25]. Moreover, AMR trends differ from country to country due to different antimicrobial usage, healthcare facilities and policies for infection management [26]. Nowadays, the antibiotic use rate in Pakistan is higher than in other Asian countries [7]. These elevated levels of consumption of antibiotics are the major reason behind AMR rise [27]. Regular updating protocols to use antibiotics and antimicrobial resistance pro les of prevalent bacteria are essential to control drug-resistant bacteraemia.

C O N C L U S I O N S
Bacteraemia mainly occurs in females. The infected individuals were mostly between 1-20 and 21-40 years old. The leading gram-positive and gram-negative bacteria were Staphylococcus epidermidis and Salmonella typhi. Antibiotic susceptibility tests showed 100% resistant antibiotics, including cephalosporins, nitrofurantoin, fosfomycin, and quinolones. Carbapenems were among the most sensitive antibiotics. Ampicillin, cefepime and sulphamethoxazole had the highest antimicrobial activity. S. typhi had the highest MAR value in the range 0.71-0.8 and S. epidermidis in the range 0.51-0.6. Pathogens are becoming resistant to previously used antibiotics, and the trend is shifting towards multidrug resistance.