Researchers at The University of Texas Medical Branch have collaborated with a team of scientists from Emory University’s Rollins School of Public Health, the FDA, the University of Maryland, the Maryland VA, and Emory University School of Medicine to examine the common waterborne bacterium Aeromonas hydrophila, which has increasingly been implicated in serious human infections.
Credit: CDC Dr. W.A.Clark
By correlating clinical and experimental findings with genome sequencing data, the scientists have found key factors that distinguish bacteria that can cause necrotizing skin infections (“flesh-eating bacteria”) from other bacteria commonly found in freshwater sources. Together, the group employed whole-genome sequencing of clinical Aeromonas strains, followed by corresponding laboratory analysis, to identify disease-causing characteristics.
A paper entitled “Characterization of Aeromonas hydrophila Wound Pathotypes by Comparative Genomic and Functional Analyses of Virulence Genes” documenting the study is published in the April 23, 2013 edition of the Open Access Journal of Clinical Microbiology (mBio) as a follow-up to a case report titled, “Aminoglycoside-Resistant Aeromonas hydrophila as Part of a Polymicrobial Infection following a Traumatic Fall into Freshwater,” published in mBio’s March 2011 edition.
For the new study, scientists sequenced the entire genomes of bacteria isolated from an individual infected after falling into Georgia’s Chattahoochee River — their goal to determine key differences between the bacteria that caused severe disease and bacteria of the same species that are harmless. Findings indicated a number of genes unique to each strain, but one particular strain, identified as E1, contained certain genes that may explain why it was so aggressive and persistent in human infection.
“We did a comprehensive analysis of which genes were in each strain and found a number of genes in the E1 strain that could enhance the ability of this bacterium to cause disease,” explains report co-author Joshua Shak, an MD/PhD student at Emory’s Rollins School of Public Health. “We then performed a variety of laboratory experiments to ensure that what we were seeing at the genetic level was also present at the phenotypic, or behavioral, level.”
Researchers at the University of Texas Medical Branch analyzed a number of behaviors in the laboratory such as bacterial motility, or the ability to move using whip-like appendages called flagella. Results indicated that the E1 strain was a more dominant swimmer (moving through a semi-solid media) and swarmer (spreading across the surface of media). Based on the findings, the researchers concluded that the E1 strain was more pathogenic on three different levels:
• Clinical: This bacterial strain caused an aggressive, rapidly advancing infection in a human.
• Genomic: Examination of the entire genetic sequence revealed genes encoding for toxins, capsules that surround and protect the bacteria, and motility-enabling flagella.
• Experimental: When tested in the lab, the E1 strain displayed a superior ability to break open red blood cells, secrete toxins, survive in human blood, move rapidly, and kill mice.
“These findings are fascinating because, although A. hydrophila is common in freshwater and usually harmless, a small subset is more prone to causing disease,” Shak commented to HealthNewsDigest.com “Furthermore, our findings highlight the diagnostic potential of whole-genome sequencing. With future advances in rapid sequencing and genome analysis, we will be able to provide timely information on the identity and the disease-causing potential of clinically-isolated microscopic organisms.”
A nasty little critter first discovered in 1962, Aeromonas hydrophila (A. hydrophila) is a heterotrophic, Gram-negative, rod-shaped species of bacterium mainly found in warm climate areas in either fresh or brackish water.
According to a Public Health Agency of Canada Infectious Substances abstract, infection with Aeromonas hydrophila can result in gastrointestinal or non-gastrointestinal complications, with symptoms of gastrointestinal infection ranging from watery diarrhea to dysenteric or bloody diarrhea. Chronic infection is also possible. Non-gastrointestinal complications that may arise subsequent to A. hydrophila infection include hemolytic syndrome and kidney disease, cellulitis, wound and soft-tissue infection, meningitis, bacteremia and septicemia, ocular infections, pneumonia and respiratory tract infections, urinary tract infection in neonates, osteomyelitis, peritonitis and acute cholecystitis. Severe infection can result from non resolved intermittent diarrhea, which can occur months after the initial infection. A. hydrophila can also cause disease in aquatic animals, and is present in aquatic ecosystems in most parts of the world. Although the role of Aeromonas hydrophila as a causative agent of human disease is reportedly controversial, but some estimates say it may cause 13 % of gastroenteritis cases in United States, although some jurisdictions may not identify these organisms as pathogens.
Aeromonas spp. are commonly found in ground water; drinking water at treatment plants, distribution systems, and reservoirs; and in both clean and polluted lakes and rivers. Water is the main reservoir, with the bacteria found in about 1% – 27 % of drinking water, but it can also be found in mud, soil, fresh produce, meat (beef, poultry, pork, fish, shellfish, and shrimp) and dairy products. Hosts include humans, animals, birds, fish, and cold-blooded marine and freshwater reptiles. The infectious dose for humans and animals is greater than 10 10 organisms. Infection is typically spread via fecal-oral transmission during direct ingestion or drinking of contaminated water or foods, but can also be transmitted by eating contaminated meat, dairy, shrimp, or fish. The pathogen can be transferred from human-to-human by contact with infected wounds, feces or blood, and can also be transferred during sports; especially when played in muddy environments involving transfer of infected soil.
Agents used to combat Aeromonas hydrophila infections include fluoroquinolones (ciproflaxin) , aminoglycosides (except streptomycin), tetracycline, chloramphenicol, carbapenems, polymyxin, streptomycin, gentamicin), and trimethoprim-sulfamethoxazole, however almost all Aeromonas spp. are resistant to penicillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, and cephalothin. Susceptibility has been shown for the disinfectants 1% sodium hypochlorite, 2% glutaraldehyde, 70% ethanol, iodines, phenolics, and formaldehyde. A. hydrophila is also sensitive to silver in water and free chlorine, and exposure to temperatures of 62 oC or greater for more that a few minutes is also lethal to A. hydrophila. The bacteria can be inactivated by moist heat (121°C for 15 min – 30 min) and dry heat (160-170°C for 1-2 hours). The bacteria can also be inactivated by ultrasonic waves delivered under high pressure (200 KPa) and elevated temperatures (40 °C) . Simultaneous sonication at temperature higher than 70°C and 0.5 MPa is also effective.
The most common A. hydrophilia infection vector in humans is through an open wound in contaminated water. Mild symptoms of an infection include fever and chills. If the infection becomes septic, symptoms can include abdominal pain, nausea, vomiting, and diarrhea. Three different types of wounds that can result from aeromonas hydrophila infection in humans: cellulitis, myonecrosis, and ecthyma gangrenosum, with cellulitis, an inflammation of the subcutaneous tissue being the most common type. The other two, myonecrosis and ecthyma gangrenosum, are less common but have worse results.
The 2013 paper “Characterization of Aeromonas hydrophila Wound Pathotypes by Comparative Genomic and Functional Analyses of Virulence Genes,” (doi: 10.1128/ mBio.00064-1 23 April 2013 mBio vol. 4 no. 2 e00064-13) abstract notes that Aeromonas hydrophila has increasingly been implicated as a virulent and antibiotic-resistant etiologic agent in various human diseases.To better understand the differences between pathogenic and environmental strains of A. hydrophila, the research team conducted comparative genomic and functional analyses of virulence-associated genes of these two wound isolates (E1 and E2), the environmental type strain A. hydrophila ATCC 7966T, and four other isolates belonging to A. aquariorum, A. veronii, A. salmonicida, and A. caviae. Full-genome sequencing of strains E1 and E2 revealed extensive differences between the two and strain ATCC 7966T.
The more persistent wound infection strain, E1, harbored coding sequences for a cytotoxic enterotoxin (Act), a type 3 secretion system (T3SS), flagella, hemolysins, and a homolog of exotoxin A found in Pseudomonas aeruginosa. Corresponding phenotypic analyses with A. hydrophila ATCC 7966T and SSU as reference strains demonstrated the functionality of these virulence genes, with strain E1 displaying enhanced swimming and swarming motility, lateral flagella on electron microscopy, the presence of T3SS effector AexU, and enhanced lethality in a mouse model of Aeromonas infection. By combining sequence-based analysis and functional assays, the research team characterized an A. hydrophila pathotype, exemplified by strain E1, that exhibited increased virulence in a mouse model of infection, likely because of encapsulation, enhanced motility, toxin secretion, and cellular toxicity.
The scientists observe that Aeromonas hydrophila has increasingly been implicated in serious human infections, and that while many determinants of virulence have been identified in Aeromonas, rapid identification of pathogenic versus nonpathogenic strains remains a challenge for this genus, as it is for other opportunistic pathogens. The paper demonstrates that by using whole-genome sequencing of clinical Aeromonas strains, followed by corresponding virulence assays, that comparative genomics can be used to identify a virulent subtype of A. hydrophila that is aggressive during human infection and more lethal in a mouse model of infection. This aggressive pathotype contained genes for toxin production, toxin secretion, and bacterial motility that likely enabled its pathogenicity. The study’s results highlight the potential of whole-genome sequencing to transform microbial diagnostics; with further advances in rapid sequencing and annotation, genomic analysis will be able to provide timely information on the identities and virulence potential of clinically isolated microorganisms.