Identification and Characterization of Klebsiella pneumoniae from Farmed American Bullfrogs (Rana catesbeiana)

Klebsiella pneumoniae is recognized as the most common multidrug-resistant bacterial pathogen in humans, and little is known about its pathogenicity in aquatic animals. Recently, K. pneumoniae was found to cause substantial mortality and morbidity in American farm frogs. ABSTRACT Klebsiella pneumoniae is a major cause of nosocomial infection and is considered a clinically important bacterium with antibiotic-resistant strains. There are few reports of K. pneumoniae infections in cultured aquatic animals, and no natural infection has been reported in amphibians. From September to October 2021, a high-mortality disease outbreak occurred in a pond-raised American bullfrog farm in Guangzhou, China. The infected bullfrogs were characterized by multiple organ congestive enlargement and inflammation. A pathogenic bacterium was isolated from the viscera of infected bullfrogs and confirmed to be K. pneumoniae by morphological, biochemical, and phylogenetic analyses. Infection experiments confirmed the virulence of the pathogenic strain against bullfrogs and tadpoles. A histopathological examination showed that the strain was harmful to multiple organs. Antibiotic resistance experiments indicated the isolate was a carbapenemase-producing multidrug-resistant K. pneumoniae (MDR-KP) strain. This study is the first report of K. pneumoniae infected American bullfrogs (Rana catesbeiana) and amphibians. These results will shed light on the pathogenicity of K. pneumoniae and help prevent and control K. pneumoniae infections in bullfrogs. IMPORTANCE Klebsiella pneumoniae is recognized as the most common multidrug-resistant bacterial pathogen in humans, and little is known about its pathogenicity in aquatic animals. Recently, K. pneumoniae was found to cause substantial mortality and morbidity in American farm frogs. This was the first report of K. pneumoniae infecting amphibians. In this study, we analyzed the biochemical, growth, and phylogenetic characteristics of the K. pneumoniae strain and described the symptoms and pathological features of infected bullfrogs and tadpoles; this will provide useful data for the prevention and control of infectious diseases, which has been suggested to decrease economic losses in bullfrog farming and reduce the potential threat to public health posed by K. pneumoniae.

[1]  Yongtao Liu,et al.  Vibrio cholerae was found in cultured bullfrog , 2022, Epidemiology and Infection.

[2]  P. C. Vasconcelos,et al.  CTX-M-15-producing Klebsiella pneumoniae ST273 associated with nasal infection in a domestic cat. , 2022, Journal of global antimicrobial resistance.

[3]  C. Struve,et al.  Genetics and pathology associated with Klebsiella pneumoniae and Klebsiella spp. isolates from North American Pacific coastal marine mammals. , 2021, Veterinary microbiology.

[4]  L. Cerdeira,et al.  Multidrug-resistant Klebsiella pneumoniae: a retrospective study in Manaus, Brazil , 2021, Archives of Microbiology.

[5]  Stephen C. Watts,et al.  A genomic surveillance framework and genotyping tool for Klebsiella pneumoniae and its related species complex , 2021, Nature Communications.

[6]  Jia Hua Cheng,et al.  Virulence profiles of Klebsiella pneumoniae isolated from 2 large dairy farms in China. , 2021, Journal of dairy science.

[7]  Jiabin Li,et al.  Deoxycholic Acid and Lithocholic Acid Alleviate Liver Injury and Inflammation in Mice with Klebsiella pneumoniae-Induced Liver Abscess and Bacteremia , 2021, Journal of inflammation research.

[8]  Chen Yuhua,et al.  Klebsiella pneumoniae: A pathogenic bacteria transmitted through Hirudo nipponia that may cause illness in humans. , 2020 .

[9]  Yongtao Liu,et al.  Klebsiella pneumonia: A pathogenic bacteria transmitted through Hirudo nipponia that may cause illness in humans. , 2020, Transboundary and emerging diseases.

[10]  A. Earl,et al.  Adaptive evolution of virulence and persistence in carbapenem-resistant Klebsiella pneumoniae , 2020, Nature Medicine.

[11]  K. Holt,et al.  Population genomics of Klebsiella pneumoniae , 2020, Nature Reviews Microbiology.

[12]  J. Bengoechea,et al.  Klebsiella pneumoniae infection biology: living to counteract host defences , 2018, FEMS microbiology reviews.

[13]  L. Cerdeira,et al.  Multidrug-resistant CTX-M-15-producing Klebsiella pneumoniae ST231 associated with infection and persistent colonization of dog. , 2018, Diagnostic microbiology and infectious disease.

[14]  L. Guelfand,et al.  Comparison of the identification results of Candida species obtained by BD Phoenix™ and Maldi-TOF (Bruker Microflex LT Biotyper 3.1). , 2018, Revista Argentina de microbiologia.

[15]  C. Shi,et al.  Aeromonas shuberti as a cause of multi-organ necrosis in internal organs of Nile tilapia, Oreochromis niloticus. , 2018, Journal of fish diseases.

[16]  M. Shi,et al.  The evolutionary history of vertebrate RNA viruses , 2018, Nature.

[17]  R. Martin,et al.  Colonization, Infection, and the Accessory Genome of Klebsiella pneumoniae , 2018, Front. Cell. Infect. Microbiol..

[18]  M. Ouellette,et al.  Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. , 2017, The Lancet. Infectious diseases.

[19]  K. Holt,et al.  Population genomics of hypervirulent Klebsiella pneumoniae clonal-group 23 reveals early emergence and rapid global dissemination , 2017, bioRxiv.

[20]  Junfa Yuan,et al.  Pathogenic Elizabethkingia miricola Infection in Cultured Black-Spotted Frogs, China, 2016 , 2017, Emerging infectious diseases.

[21]  H. Ingmer,et al.  Transfer of Antibiotic Resistance in Staphylococcus aureus. , 2017, Trends in microbiology.

[22]  O. Călinescu,et al.  Competition is the basis of the transport mechanism of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae , 2017, PloS one.

[23]  Peng Li,et al.  Apparent digestibility coefficients and amino acid availability of common protein ingredients in the diets of bullfrog, Rana (Lithobates) catesbeiana , 2015 .

[24]  T. Russo,et al.  Hypervirulent Klebsiella pneumoniae , 2014, Open forum infectious diseases.

[25]  K. Poole Bacterial stress responses as determinants of antimicrobial resistance. , 2012, The Journal of antimicrobial chemotherapy.

[26]  M. Araújo,et al.  Additive threats from pathogens, climate and land-use change for global amphibian diversity , 2011, Nature.

[27]  F. Gulland,et al.  Pleuritis and suppurative pneumonia associated with a hypermucoviscosity phenotype of Klebsiella pneumoniae in California sea lions (Zalophus californianus). , 2010, Veterinary microbiology.

[28]  B. Bruun,et al.  Genetic heterogeneity of Flavobacterium meningosepticum demonstrated by DNA-DNA hybridization. , 2009, Acta pathologica, microbiologica, et immunologica Scandinavica. Section B, Microbiology.

[29]  D. Hillis,et al.  Taxonomic Freedom and the Role of Official Lists of Species Names , 2009 .

[30]  J. W. Snyder,et al.  Direct Comparison of the BD Phoenix System with the MicroScan WalkAway System for Identification and Antimicrobial Susceptibility Testing of Enterobacteriaceae and Nonfermentative Gram-Negative Organisms , 2008, Journal of Clinical Microbiology.

[31]  S. Abbott,et al.  Multisystemic Abscesses in African Green Monkeys (Chlorocebus aethiops) with Invasive Klebsiella pneumoniae—Identification of the Hypermucoviscosity Phenotype , 2008, Veterinary pathology.

[32]  S. Abbott,et al.  16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls , 2007, Journal of Clinical Microbiology.

[33]  Malik Peiris,et al.  Aetiology: Koch's postulates fulfilled for SARS virus , 2003, Nature.

[34]  Debra L Miller,et al.  Bacterial Pathogens Isolated from Cultured Bullfrogs (Rana Castesbeiana) , 2002, Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

[35]  John W. Beaber,et al.  Genomic and Functional Analyses of SXT, an Integrating Antibiotic Resistance Gene Transfer Element Derived from Vibrio cholerae , 2002, Journal of bacteriology.

[36]  Catherine Dauga,et al.  Evolution of the gyrB gene and the molecular phylogeny of Enterobacteriaceae: a model molecule for molecular systematic studies. , 2002, International journal of systematic and evolutionary microbiology.

[37]  Z. Q. Li,et al.  Characterization of an iridovirus from the cultured pig frog Rana grylio with lethal syndrome. , 2001, Diseases of aquatic organisms.

[38]  S. Yamanaka,et al.  Isolation of myxobacteria from the marine environment. , 1998, FEMS microbiology letters.

[39]  D E Green,et al.  Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[40]  H. Korkeala,et al.  Effect of Sodium Nitrite and Sodium Chloride on Growth of Lactic Acid Bacteria , 1992, Acta Veterinaria Scandinavica.

[41]  A. Bauer,et al.  Antibiotic susceptibility testing by a standardized single disk method. , 1966, American journal of clinical pathology.

[42]  Dr. C. Friedlaender Ueber die Schizomyceten bei der acuten fibrösen Pneumonie , 1882, Archiv für pathologische Anatomie und Physiologie und für klinische Medicin.

[43]  B. Das,et al.  Isolation, identification and characterization of Klebsiella pneumoniae from infected farmed Indian Major Carp Labeo rohita (Hamilton 1822) in West Bengal, India , 2018 .

[44]  Seung-Ho Heo,et al.  Rapid identification of Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae using triplex polymerase chain reaction in rodents. , 2013, Experimental animals.

[45]  Didier Raoult,et al.  The rpoB gene as a tool for clinical microbiologists. , 2009, Trends in microbiology.

[46]  M. Rouf,et al.  Pathogenicity ofAeromonas hydrophila in red leg disease in frogs , 2007, Current Microbiology.

[47]  M. Ferraro Performance standards for antimicrobial susceptibility testing , 2001 .

[48]  Kanako Watanabe,et al.  ICB database: the gyrB database for identification and classification of bacteria , 2001, Nucleic Acids Res..

[49]  Griffith,et al.  General Zoology Or Systematic Natural History , 1999 .