Cancer is characterized by uncontrolled proliferation of cells that can invade local and distant tissues, leading to metastasis and disruption of normal physiological processes (1,2,3). Tumors may infiltrate the bone marrow, impair hematopoiesis, and cause immunosuppression, particularly affecting neutrophils and lymphocytes, which are key components in defending against bacterial, viral, and fungal pathogens (4). Antineoplastic treatments, including chemotherapy, radiotherapy, and immunotherapy, improve survival and increase cancer-free intervals but also exacerbate immunocompromised states by disrupting innate and adaptive immune responses (5). Consequently, oncology patients are highly susceptible to infections, which may involve the urinary, respiratory, and gastrointestinal systems, contributing significantly to morbidity and mortality (2). The prevalence and severity of infections in cancer patients are influenced by malignancy type, prior therapies, long-term indwelling catheters, and comorbidities (6). Neutropenia, a frequent consequence of chemotherapy, is associated with an elevated risk of bloodstream infections (BSIs) and opportunistic infections, with infection-related mortality ranging from 24% in developed countries to 33% in underprivileged regions (4). Epidemiological trends show a shift in microbial prevalence: Gram-negative bacteria, particularly Escherichia coli, predominate in adult oncology patients, whereas Gram-positive pathogens are more common in pediatric populations (7). Fungal infections, including Candida and Aspergillus species, further complicate clinical outcomes due to invasive potential and recurrent hospital admissions (8).

Extended-spectrum β-lactamase (ESBL) production by E. coli is a major driver of antimicrobial resistance (AMR), with ESBL enzymes hydrolyzing β-lactam antibiotics such as penicillin’s and cephalosporins, thus limiting therapeutic options (9). The biochemical properties of β-lactamase, including substrate affinity (Km) and maximal hydrolysis rate (Vmax), impact drug efficacy and pharmacological management (10). Understanding these mechanisms is essential for guiding empiric therapy, particularly in immunocompromised populations where infections may be systemic and rapidly progressive (11). Global AMR data indicate that frequent and inappropriate antibiotic use has contributed to a dramatic increase in drug-resistant infections (12). For urinary tract infections (UTIs), particularly those caused by E. coli, inappropriate antibiotic prescriptions have led to resistance against β-lactams and fluoroquinolones, which complicate treatment decisions and elevate mortality risk (13). The growing incidence of multidrug-resistant pathogens emphasizes the need for timely antimicrobial susceptibility testing (AST) to guide treatment selection (14). AST involves rapid microbiological diagnostic investigations to identify pathogens and determine their resistance profiles. Cultures from blood, urine, and sputum, including enrichment and plate cultivation, are used to generate reliable susceptibility data, enabling early targeted therapy and reducing the risk of adverse outcomes (15,16). Despite its importance, limited data are available integrating clinical, microbiological, biochemical, and pharmacological perspectives in oncology patients, especially in developing countries like Pakistan. This study addresses these gaps by evaluating urinary, respiratory, and gastrointestinal infections in immunocompromised cancer patients, assessing the prevalence of ESBL-producing E. coli, and correlating enzymatic activity with antimicrobial susceptibility patterns. The findings aim to support evidence-based antimicrobial practices and improve clinical outcomes for high-risk oncology populations.

A cross-sectional study was conducted at a tertiary care public hospital in Karachi, Pakistan, from April 2021 to May 2023. A total of 170 patients were recruited from the department of oncology with documented or clinically suspected urinary, respiratory, or gastrointestinal infections after their informed consent. Inclusion criteria include all those patients who are undergoing chemotherapy or radiotherapy, while exclusion criteria include prior organ transplantation or chronic immunosuppressive therapy unrelated to malignancy. Hence, structured questionnaires were used to record demographic details, medical history, type of cancer, and clinical characteristics. Urine, sputum, and stool samples were collected under aseptic conditions. Urine samples underwent physical examination, microscopic analysis, and bacteriological culture using MacConkey agar and/or cysteine lactose electrolyte-deficient (CLED) agar.

Sputum samples were analyzed for macroscopic appearance and biochemical composition, while stool specimens were evaluated for bacteria, protozoa, and helminths, with Cryptosporidium. Colony morphology, Gram staining, and standard biochemical tests were done for microbial identification. ESBL production in E. coli was confirmed using nitrocefin hydrolysis assays. Biochemical parameters such as β-lactamase activity (U/mL), maximum hydrolysis rate (Vmax), and substrate affinity (Km), were quantified spectrophotometrically. Antimicrobial susceptibility testing (AST) of urinary E. coli isolates was performed using the Kirby-Bauer disk diffusion method according to CLSI guidelines. Antibiotic tests included β-lactams, fluoroquinolones, aminoglycosides, nitrofurantoin, co-trimoxazole, and carbapenems. Data was analyzed using SPSS version 24. Normality was assessed via the Shapiro-Wilk test. Continuous variables were expressed as mean ± standard deviation (SD), while categorical variables were expressed as frequencies and percentages.

A total of 170 patients were recruited from the department of oncology. The mean age was 36.8 ± 12.9 years, and the mean weight was 66.8 ± 14.2 kg. Most patients were male (55.9%, 95/170) compared to females (44.1%, 75/170). Solid tumors predominated (73.5%, 125/170) over hematologic malignancies (26.5%, 45/170). The most frequent cancers were associated with the oral cavities (30%), breast (22%), and colon (10%), followed by ovarian, rectal, esophagus, stomach, lung, gallbladder, liver, cervix, urinary bladder, and pancreas cancers. Chemotherapy exposure was reported in 88.2% of patients, radiotherapy in 47.1%, and 65.9% had a history of prior antibiotic use, as shown in Table 1.

Table 1: Demographic and Clinical Characteristics

The frequency and type of infections varied across cancer types. Gastrointestinal infections were most common in the oral cavity (12.4%), breast (10.5%), and ovarian cancers (4.1%), whereas respiratory tract infections were more frequent in the lung (3.5%) and rectal cancers (2.9%). Urinary tract infections were most frequently reported in oral cavities (15.8%) and breast cancer (8.8%), as shown in Table 2.

Table 2: Distribution of Infections by Cancer Type

Escherichia coli was the most frequent pathogen in UTIs (78.2%), Staphylococcus aureus in RTIs (50.4%), and Salmonella spp. and Shigella spp. in GI infections (15.6% each). Other urinary isolates included K. pneumoniae (16.5%) and Enterococcus faecalis (2.7%), as shown in Table 3.

Table 3: Pathogens Isolated in Different Infection Types

As shown in Table 4, the mean β-lactamase activity was 12.5 ± 3.2 U/mL, significantly higher than the reference range (<2 U/mL). The Km (substrate affinity) was within normal limits (1.8 ± 0.4 mM), whereas Vmax was elevated (24.6 ± 4.1 µmol/min). The hydrolysis rate was markedly increased at 78.3 ± 5.2%, confirming high ESBL activity.

Table 4: Biochemical Profile of ESBL Activity

As shown in Table 5, antibiotic susceptibility testing revealed high resistance to amoxicillin (100%), co-trimoxazole (85%), and ciprofloxacin (72%). Imipenem (98% sensitivity) and amikacin (85% sensitivity) were most effective.

Table 5: Antimicrobial Susceptibility of ESBL-Producing E. coli

As shown in Table 6, Candida was observed in UTIs (5.8%) and RTIs (11.7%), whereas Cryptosporidium was detected only in GI infections (5.8%). Bacterial co-infections were also reported in all types of infection.

Table 6: Fungal, Parasitic, and Bacterial Co-Infections

ESBL-producing E. coli represents a critical threat for oncological patients, predominantly causing urinary infections (17). This study demonstrates a high prevalence (78.2%) in urinary isolates, consistent with global trends linking neutropenia and prior antibiotic exposure to multidrug resistance. The predominance of S. aureus in respiratory infections and Salmonella/Shigella in gastrointestinal infections highlights systemic colonization under immunocompromised conditions (18). Biochemical analyses revealed markedly elevated β-lactamase activity and hydrolysis rates, confirming enzymatic mechanisms as the key drivers of resistance (19). Pharmacologically, this renders penicillin’s and cephalosporins ineffective, necessitating carbapenem use. Aminoglycosides and nitrofurantoin offer alternatives but require careful dosing and monitoring (20).

Correlation between urinary, respiratory, and gastrointestinal infections indicate widespread multidrug resistance, likely influenced by systemic antibiotic exposure. Infection control and programs are vital to prevent cross-site colonization and mitigate resistance escalation (21). Strengths of this study include integrated clinical, microbiological, biochemical, and pharmacological analysis, providing mechanistic insights for therapy optimization. Limitations include single-center design, limited follow-up, and lack of molecular characterization of ESBL genes, which may affect generalizability. Multicenter studies with longitudinal data and genotypic analysis are recommended. The findings of the study underscore the importance of combining biochemical profiling with AST to guide precision therapy in immuno-compromised cancer patients. Understanding enzyme kinetics can improve treatment efficacy, reduce failures, and inform rational antimicrobial practices.

ESBL-mediated resistance in urinary Escherichia coli isolates among immunocompromised cancer patients is driven by elevated β-lactamase activity and enhanced enzymatic hydrolysis, resulting in multidrug-resistant infections across urinary, respiratory, and gastrointestinal sites. Carbapenems remain the most effective therapeutic option, while aminoglycosides and nitrofurantoin provide alternative strategies under careful monitoring. Integrating biochemical profiling with microbiological and pharmacological analyses is essential for informed antimicrobial selection, personalized patient care, and robust programs. Continuous surveillance, evaluation of enzyme kinetics, and targeted interventions are crucial to mitigate the growing threat of ESBL-mediated antimicrobial resistance in high-risk oncology populations

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