Seasonal prevalence of ESBL-producing pseudomonas aeruginosa in bottled mineral water: A hidden public health threat undetectable by fecal indicators – A study from Tehran, Iran

ABSTRACT

Overall Description: A conceptual flowchart divided into three main vertical sections: Sampling, Laboratory Analysis, and Policy Recommendations, illustrating that bottled mineral water in Tehran may harbor antibiotic-resistant Pseudomonas aeruginosa undetectable by standard fecal indicator tests. Section 1: Sampling States "100 samples, Tehran retail, Apr–Sep 2019". Section 2: Laboratory Analysis Highlights "Seasonal contamination" occurring exclusively during warm months (May, July, September). It shows a green checkmark next to "Pseudomonas aeruginosa" and indicates negative results for "Coliforms & E.coli". Below this, it lists the Resistance Profile (n=5): Resistant to SXT, CFM, CFLEX; Susceptible to GM, AK, IMI, CO, CRO. It also notes the presence of ESBL Genes: blaTEM-1 (100%) and blaCTX-M (40%). Section 3: Policy Recommendation Features a "CALL TO ACTION" banner to "Update Global Bottled Water Standards". It highlights "THE BLIND SPOT" with three bullet points: Fecal indicators do NOT detect this threat (All Negative), ESBL genes present (blaCTX-M 40%), and Risk in WarmMonths (Secondary contamination). Finally, it lists "RECOMMENDED ACTIONS": Add P. aeruginosa to mandatory screening (ISO 16266), Require Temperature Labelingon packaging, and Shift surveillance from Factory to Retail outlets.

Bottled mineral water is widely promoted as a ‘safe’ and ‘pure’ alternative to tap water, especially in urban settings such as Tehran. However, this perception may be dangerously inaccurate, as conventional safety assessments rely solely on fecal indicator bacteria – such as coliforms and Escherichia coli – which fail to detect non-fecal opportunistic pathogens like Pseudomonas aeruginosa. In a six-month study (spring to autumn 2019), we randomly collected 100 bottled mineral water samples from retail outlets across Tehran and analyzed them for P. aeruginosa, antimicrobial resistance profiles, and extended-spectrum beta-lactamase (ESBL)-encoding genes (bla TEM-1, bla CTX-M, bla SHV), alongside standard fecal indicators. P. aeruginosa was isolated from 5% of samples (5/100), exclusively during warm months (May, July, September; p = 0.032), suggesting temperature-dependent secondary contamination. Critically, all contaminated samples tested negative for fecal indicators, revealing a major blind spot in current regulatory frameworks. All isolates were susceptible to gentamicin, amikacin, imipenem, colistin, and ceftriaxone, but showed 100% resistance to trimethoprim–sulfamethoxazole. Genotypically, all harbored blaTEM-1, 40% carried bla CTX-M (indicative of recent human or hospital-associated origins), and none carried bla SHV. These findings demonstrate that bottled water passing standard fecal indicator tests can still harbor ESBL-producing P. aeruginosa, likely due to post-bottling contamination during warm-season storage or transport. As the first comprehensive report from the Middle East to simultaneously assess fecal indicators and ESBL genes in bottled water, this study calls for urgent updates to international safety standards – including Codex Stan 227-2001 and U.S. FDA regulations (21 CFR Part 165)—to include mandatory screening for opportunistic pathogens, temperature labeling on packaging, and enhanced retail surveillance, particularly in warm-climate regions. This is not only a microbiological concern but a pressing public health policy imperative.