Emergence of a multidrug-resistant Salmonella enterica serovar Amager lineage carrying the blaCTX-M-65-positive pESI megaplasmid

This study characterizes a multidrug-resistant *Salmonella* Amager strain isolated from a Chilean river that carries the blaCTX-M-65-positive pESI megaplasmid, highlighting the emergence of a globally significant lineage linked to human infections in the US and UK and underscoring the critical role of environmental surveillance in detecting such threats.

The Gist

🦠 The Big Picture: A "Super-Invader" in the Water

Imagine the world of bacteria as a crowded city. Most bacteria are just regular citizens, but some are troublemakers that can make people sick. One of the most notorious troublemakers is a germ called Salmonella. Usually, if you eat food contaminated with it, you get a bad stomach ache, but your body can fight it off. However, doctors have a special "heavy artillery" of antibiotics (like strong weapons) to treat severe cases.

Recently, scientists discovered a new, dangerous version of this germ. It's not just a regular Salmonella; it's a multidrug-resistant "Super-Salmonella" that has stolen a powerful shield, making it immune to almost all our best medicines.

🔍 The Discovery: Finding the "Smoking Gun" in a River

In October 2023, scientists in Chile were acting like environmental detectives. They were sampling water from the Mapocho River (a major river in Santiago) to see what kind of bacteria were living there.

They found a specific strain called Salmonella Amager. At first glance, it looked like a normal river bacterium. But when they tested it in the lab, they realized it was a "Super-Salmonella."

• The Problem: It was resistant to fluoroquinolones (a common antibiotic) and beta-lactams (the heavy artillery used for serious infections).
• The Analogy: Imagine a burglar who doesn't just pick the lock; they have a master key that opens every door in the city, including the bank vault. This bacterium has a master key that unlocks resistance to our best drugs.

🧬 The Secret Weapon: The "pESI" Mega-Backpack

How did this river bacterium get so tough? The scientists used advanced technology (like a high-powered microscope for DNA) to look inside the germ. They found the culprit: a massive piece of genetic equipment called a megaplasmid.

• The Analogy: Think of a bacterium as a small car. Usually, it carries a small glovebox with a few tools. But this Salmonella Amager was carrying a giant, 300,000-mile-long backpack (the pESI megaplasmid) strapped to its back.
• What's in the backpack? This backpack was stolen from a different, very famous criminal gang called Salmonella Infantis. Inside this backpack were instructions (genes) for:
  • The "Shield" (blaCTX-M-65): A gene that acts like a chemical shield, destroying antibiotics before they can kill the bacteria.
  • The "Lockpick" (qnrB19): A gene that stops fluoroquinolones from working.
  • Other tools: Genes that help it survive in harsh environments and resist other drugs like tetracycline and chloramphenicol.

The scientists found that this backpack was 99.98% identical to the one carried by the Salmonella Infantis gang. It's like finding a burglar wearing a stolen uniform and carrying a stolen toolbox that belongs to a different, more famous gang.

🌍 The Global Connection: It's Not Just in Chile

The most alarming part of the story is that this isn't just a local river problem. The scientists looked at a global database of bacteria (like a massive international wanted poster) and found that this exact same lineage (family tree) of Salmonella Amager was causing infections in humans in the United States and the United Kingdom.

• The Timeline: By analyzing the DNA like a family tree, they figured out this "Super-Salmonella" lineage likely emerged around 2010. Since then, it has been spreading quietly, hopping from bacteria to bacteria, and from animals to humans.
• The Spread: It seems the Salmonella Infantis bacteria (the original owner of the backpack) passed this giant backpack to the Salmonella Amager bacteria. Once the Amager bacteria got the backpack, it became a super-germ and started traveling the world, causing human infections.

🚨 Why Should We Care?

This paper is a wake-up call for three main reasons:

1. The "First-Line" Defense is Broken: The antibiotics doctors use first to treat severe Salmonella infections (like cephalosporins) no longer work on this strain. If a person gets infected, they might need much stronger, more toxic, or more expensive drugs, or in worst-case scenarios, have no treatment options.
2. The Environment is a Warning System: This dangerous germ was found in a river before it was widely reported in human hospitals. This proves that if we watch our rivers, soil, and water, we can spot these "Super-Bugs" early, acting as an early warning system to protect human health.
3. Bacteria Share "Loot": Bacteria are like thieves that can swap backpacks. A harmless river bacterium can steal a "super-villain" backpack from a known pathogen and become a threat itself.

💡 The Takeaway

This study tells us that a dangerous, drug-resistant version of Salmonella has emerged in Chile, carrying a massive genetic "backpack" stolen from another bacteria. It is now spreading globally, causing infections in people in the US and UK.

The key lesson? We need to keep an eye on our environment. Just like a smoke detector in a house, monitoring our rivers and water sources can alert us to new, dangerous germs before they become a full-blown crisis in our hospitals.

Technical Summary

1. Problem Statement

Non-typhoidal Salmonella is a leading cause of foodborne illness globally. While typically self-limiting, it causes severe invasive infections in vulnerable populations, requiring first-line treatments like third-generation cephalosporins and fluoroquinolones. The emergence of multidrug-resistant (MDR) strains, particularly those producing Extended-Spectrum $\beta$-Lactamases (ESBLs), poses a critical public health threat.

A specific concern is the pESI megaplasmid (approx. 300 kbp), originally identified in Salmonella Infantis. This plasmid carries multiple virulence and resistance factors, including the ESBL gene blaCTX-M-65. While pESI has been documented in other serovars (e.g., Agona, Muenchen), its spread to Salmonella Amager, a serovar not previously associated with this specific high-risk lineage, and its concurrent acquisition of fluoroquinolone resistance determinants, represents an emerging global threat that limits treatment options.

2. Methodology

The study employed a combination of phenotypic characterization, hybrid genome sequencing, and large-scale genomic epidemiology:

• Isolation and Phenotyping:
  • A strain, Salmonella Amager 19-MAP-20-4, was isolated from the Mapocho River (Chile) in October 2023 using modified Moore swabs and standard enrichment protocols.
  • Antimicrobial Susceptibility Testing (AST): Performed via broth microdilution (Sensititre plates) against 34 antibiotics to determine Minimum Inhibitory Concentrations (MICs).
• Genomic Sequencing and Assembly:
  • Hybrid Sequencing: Utilized Illumina NovaSeq X (short reads) and Oxford Nanopore (long reads) to generate a high-quality hybrid assembly.
  • Assembly: De novo assembly was performed using Flye, polished with Pilon, and circularized using Circlator.
• Bioinformatic Analysis:
  • Resistance Gene Detection: Used ABRicate and AMRFinderPlus to identify AMR genes (e.g., blaCTX-M-65, qnrB19) and mutations.
  • Plasmid Analysis: MOB-suite was used for plasmid typing and reconstruction. The pESI-like megaplasmid was identified by screening for $\ge$220 genes matching the reference pN55391.
  • Phylogenetics:
    • Host Genome: A time-measured Maximum Clade Credibility (MCC) tree was constructed for 61 S. Amager isolates from the HC50_1458 cluster using core-SNP alignment (Snippy, Gubbins) and BEAST v1.10.4.
    • Plasmid Phylogeny: 1,548 pESI-like plasmids carrying blaCTX-M-65 were analyzed. Representative plasmids were dereplicated and aligned to the pN55391 reference to infer evolutionary relationships using IQ-TREE.

3. Key Results

• Characterization of the Chilean Isolate (19-MAP-20-4):
  • Phenotype: The strain exhibited MDR resistance to $\beta$-lactams (ampicillin, cefotaxime, ceftriaxone), chloramphenicol, tetracycline, and fluoroquinolones (ciprofloxacin). The MIC for cefotaxime/clavulanic acid was 64-fold lower than cefotaxime alone, confirming ESBL production.
  • Genotype:
    • Megaplasmid: A 311,303 bp plasmid was recovered, sharing 99.98% sequence identity with the S. Infantis pESI-like megaplasmid (pN55391). It carried blaCTX-M-65, aadA1, aph(4)-Ia, aac(3)-IVa, floR, sul1, and tet(A).
    • Secondary Plasmid: A 2.7 kbp plasmid carrying the qnrB19 gene (conferring fluoroquinolone resistance) was identified.
• Global Population Structure:
  • The Chilean isolate belongs to the HC20_35565 cluster (linked by $\le$20 cgMLST alleles).
  • This cluster includes human clinical isolates from the United States (n=7) and United Kingdom (n=1) collected between 2022 and 2024, indicating international spread.
• Temporal Evolution:
  • Phylogenetic dating suggests the emergence of this pESI-carrying S. Amager lineage occurred around 2010.5 (95% HPD: 2004.4–2014.8).
  • The lineage has since disseminated across environmental and human niches globally.
• Plasmid Origin:
  • Plasmid phylogeny shows that the pESI-like elements in S. Amager cluster tightly within S. Infantis-dominated subclusters, strongly suggesting a recent horizontal transfer event from S. Infantis to S. Amager.

4. Key Contributions

• First Report of pESI in S. Amager: This study documents the first emergence of a Salmonella Amager lineage carrying the pESI megaplasmid, expanding the known host range of this high-risk plasmid.
• Convergence of Resistance Mechanisms: It highlights the co-occurrence of the pESI megaplasmid (providing ESBL and other resistances) with a separate qnrB19-carrying plasmid, creating a "super-resistant" phenotype that compromises both cephalosporin and fluoroquinolone therapies.
• Environmental Surveillance Validation: The isolation of this strain from river water in Chile, prior to or concurrent with human cases in the US and UK, demonstrates the utility of environmental surveillance in detecting emergent pathogens before they cause widespread human outbreaks.

5. Significance

The findings underscore a critical shift in the epidemiology of Salmonella. The pESI megaplasmid, previously associated primarily with S. Infantis, is now a mobile genetic element capable of driving the emergence of MDR lineages in diverse serovars like S. Amager. The global dissemination of the blaCTX-M-65-positive pESI lineage across continents (Chile, USA, UK) and its presence in both environmental and clinical samples signals a high risk of further spread.

This study calls for:

1. Enhanced Surveillance: Integrating environmental monitoring with clinical data to anticipate outbreaks.
2. One Health Approach: Recognizing that water systems act as reservoirs for the exchange of resistance plasmids between animal, environmental, and human hosts.
3. Clinical Awareness: Healthcare providers and surveillance systems must be alerted to the potential for ESBL-producing, fluoroquinolone-resistant Salmonella in serovars beyond Infantis, necessitating alternative treatment strategies for severe infections.