Identifying and quantifying ESKAPEE pathogens in and around sinks in high burden hospitals

A prospective study in two high-burden hospitals in La Paz, Bolivia, utilized culture and 16S rDNA sequencing to reveal that sinks and their surrounding environments are heavily contaminated with viable ESKAPEE pathogens, particularly *Klebsiella* and *Enterobacter* species, with contamination levels decreasing as distance from the drain increases.

The Gist

Imagine a hospital as a bustling city where patients are the residents and doctors are the guardians. In this city, there are invisible invaders called ESKAPEE pathogens. Think of these as a "rogue gang" of super-bacteria (including Staph, Klebsiella, Pseudomonas, and others) that are tough to kill with normal medicine and love to cause infections in sick people.

This study is like a detective investigation into where this gang is hiding. The detectives (the researchers) suspected that the sinks and drains in two busy hospitals in La Paz, Bolivia, were the gang's secret headquarters.

Here is the story of what they found, explained simply:

1. The Suspect: The Sink Drain

You know how a sink drain has a little U-shaped pipe (called a P-trap) where water sits? The researchers realized this isn't just a place for water; it's a bacterial hotel.

• The Mechanism: When you turn on the faucet, the water swirls and agitates the water in that pipe. It's like shaking a soda can. This action sprays tiny, invisible droplets of water (aerosols) into the air, carrying the bacteria with them.
• The Result: These droplets land on the sink, the counter, the faucet handles, and even float into the air, potentially landing on a patient's bed or being breathed in.

2. The Investigation: Two Methods

The team went to two different hospitals (let's call them Hospital A and Hospital B) and played two different games to catch the bacteria:

• Game 1: The Petri Dish (Culture): They swabbed surfaces and air, then put the samples on special jelly plates (like a garden) to see if the bacteria would grow into visible colonies. This tells us if the bacteria are alive and active.
• Game 2: The DNA Scanner (16S Sequencing): They took a snapshot of the genetic code of everything in the sample. This is like taking a photo of every person in a crowd to see who is there, even if they are sleeping or hiding.

3. The Findings: The Gang is Everywhere!

The results were startling. The sinks were heavily infested.

• The Hotspot: The inside of the sink basin (the bowl part) was the most contaminated area. It was like the gang's main clubhouse.
• The Spread: As you moved away from the sink (to the faucet handle, then the counter, then the air), the number of bacteria dropped, but they were still there.
• The Air: Even the air in the room wasn't safe; about 74% of air samples had these bacteria.
• The Leaders:
  • S. aureus (Staph) was the most common guest (found in over half the samples).
  • Klebsiella/Enterobacter were the heaviest hitters (found in the highest numbers).

4. The Differences Between the Hospitals

The two hospitals had different "neighborhood vibes":

• Hospital A (The Crowded One): This hospital was smaller and more crowded. The bacteria here were very distinct. The sinks looked like a different world compared to the rest of the room.
• Hospital B (The Spacious One): This hospital was larger. Here, the bacteria on the sink were more similar to the bacteria on the high-touch surfaces (like door handles).
• Why the difference? The researchers guessed it might be because Hospital B has a dedicated cleaning crew, while in Hospital A, the nurses are responsible for cleaning. Also, Hospital A had porcelain sinks, while Hospital B had stainless steel. Just like some clothes hold dirt better than others, different sink materials might hold bacteria differently.

5. The Big Picture: Why This Matters

In low-income countries like Bolivia, these infections are a huge problem, but it's hard to know exactly where they come from. This study proves that sinks are a major source.

• The Analogy: Imagine trying to keep a house clean, but the faucet itself is constantly spraying dirty water onto the clean dishes. No matter how much you wipe the counter, if the faucet is spraying germs, the house stays dirty.
• The Problem: Current cleaning methods (like wiping with a rag) aren't enough to stop the bacteria from growing back in the drain pipes. It's like trying to mop up a leak without fixing the pipe.

The Conclusion

The researchers are sounding the alarm: Hospital sinks in these settings are breeding grounds for dangerous, drug-resistant bacteria.

They aren't just saying "wash your hands." They are saying we need new, smarter ways to stop the bacteria from growing in the drains and spraying into the air. Until we fix the "plumbing problem," patients in these hospitals will remain at high risk of catching infections from the very sinks meant to help them stay clean.

In short: The sink drain is the villain, the bacteria are the villains' army, and we need a better plan to defeat them before they infect the patients.

Technical Summary

1. Problem Statement

Hospital-acquired infections (HAIs) driven by ESKAPEE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli) are a critical global health threat, particularly in Low- and Middle-Income Countries (LMICs) like Bolivia. These multidrug-resistant organisms often originate in premise plumbing, specifically sink drains and P-traps. When faucets are used, water agitation aerosolizes bacteria from the drain, leading to surface contamination and potential patient inhalation. Despite the known risks, there is a lack of surveillance data regarding the prevalence and concentration of these pathogens in Bolivian healthcare facilities, where diagnostic capacity is limited.

2. Methodology

The study employed a prospective observational design conducted between May and August 2025 in two hospitals in La Paz, Bolivia (Hospital A: smaller, higher density; Hospital B: larger, more space).

• Sampling Strategy:
  • Total Samples: 272 samples collected (233 surface swabs, 39 air samples).
  • Surface Categories:
    1. Basin interior (inside the sink).
    2. Sink surfaces (handles, edges).
    3. Near-sink surfaces (<1m from basin).
    4. High-touch surfaces (>1m from basin).
  • Air Sampling: Collected using an AirPrep Cub Sampler (200 L/min for 30 mins).
• Culture-Based Analysis:
  • Samples were plated on selective chromogenic media (CHROMagar Orientation, ESBL, Acinetobacter, and Pseudomonas) to identify and quantify viable ESKAPEE pathogens as Colony Forming Units (CFU).
  • Incubation was performed at 37°C (or 30°C for Pseudomonas) for 24 hours.
  • Results were normalized to 100 cm² for surfaces and 6000 L for air.
• Molecular Analysis (16S rDNA Sequencing):
  • DNA was extracted from eluates preserved in DNA/RNA Shield.
  • Sequencing: Full-length 16S rRNA genes were amplified and sequenced using PacBio Revio (long-read sequencing).
  • Bioinformatics: Data processing involved DADA2 and QIIME2 pipelines for denoising, ASV inference, and taxonomy assignment using the GTDB database.
  • Statistical Analysis: Beta-diversity was assessed via Bray-Curtis dissimilarity and PCoA; significance was tested using PERMANOVA. Differential abundance was analyzed using Wilcoxon rank-sum tests with Benjamini-Hochberg correction.

3. Key Contributions

• Dual-Method Approach: The study integrates traditional culture methods with long-read 16S sequencing to provide a comprehensive view of both viable pathogens and total microbial community composition.
• LMIC Context: It provides rare, high-resolution data on environmental pathogen loads in Bolivian hospitals, a region with limited AMR surveillance.
• Granular Spatial Mapping: It quantifies the "distance decay" effect of pathogen concentration from the sink drain to surrounding surfaces and air.
• Comparative Analysis: It contrasts two distinct hospital environments (different cleaning protocols, sink materials, and patient densities) to identify factors influencing microbial ecology.

4. Key Results

• High Prevalence:
  • 74.7% of surface swabs and 74.4% of air samples tested positive for at least one viable, presumptive ESKAPEE pathogen.
  • Hospital A showed higher culture positivity (85%) compared to Hospital B (66.9%).
• Spatial Distribution:
  • Sink basins were the most contaminated locations (Mean: 31 CFU/100 cm²; 95% CI: 16–46).
  • Contamination levels decreased with distance from the drain.
  • Air samples had the lowest concentration (Mean: 1 CFU/100 cm²) but remained highly positive for presence.
• Pathogen Specifics:
  • Most Frequent: S. aureus was detected in 54.4% of all samples.
  • Highest Concentration: Klebsiella/Enterobacter spp. showed the highest mean concentration (100 CFU/100 cm²).
  • Hospital Differences: Hospital A basins had higher Pseudomonas abundance, while Hospital B showed higher Streptococcus and Staphylococcus abundance.
• Microbial Community Structure:
  • PERMANOVA analysis confirmed that sample location significantly explained microbial variation (21.9% in Hospital A vs. 11.9% in Hospital B).
  • Methodological Discrepancy: While culture data showed high S. aureus presence, 16S sequencing showed low relative abundance of Staphylococcus spp. Conversely, Klebsiella abundance was consistent across both methods. This highlights the limitations of 16S sequencing in detecting specific viable pathogens compared to culture.

5. Significance and Implications

• Transmission Risk: The study confirms that sinks in these high-burden hospitals act as significant reservoirs for ESKAPEE pathogens, posing a direct risk for aerosolization and surface transmission to patients and staff.
• Operational Insights: Differences between hospitals suggest that cleaning protocols (e.g., dedicated cleaning teams in Hospital B vs. nurse-led cleaning in Hospital A) and infrastructure (porcelain vs. stainless steel sinks) significantly impact pathogen load and community composition.
• Public Health Action: The findings underscore the urgent need for targeted interventions in LMICs. While standard cleaning is insufficient to prevent recolonization, the study suggests a need for novel mitigation strategies (e.g., point-of-use filtration, bacteriophage treatment, or material changes) to break the sink-to-patient transmission cycle.
• Limitations: The study notes that culture methods may overestimate pathogen presence due to non-pathogenic genus members, and 16S sequencing may underestimate viable counts. Additionally, air sampling viability may have been compromised by the collection method.

In conclusion, this research provides critical evidence that hospital sinks in Bolivia are heavily colonized by multidrug-resistant ESKAPEE pathogens, necessitating immediate and sustained environmental control measures to reduce HAI transmission.