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Antimicrobial Resistance

Genome-wide screening of candidate genes associated with the vancomycin nonsusceptibility phenotype in Staphylococcus aureus

S. aureus is a major human pathogen associated with both nosocomial and community-acquired infections. Vancomycin is mainly used for the treatment of patients with S. aureus infections; however, decreased susceptibility to vancomycin is increasingly common. Therefore, the identification of novel markers for the development of diagnostic tools for vancomycin-intermediate S. aureus (VISA) is important. We applied a genome-wide association approach to discriminate between VISA and vancomycin-susceptible S. aureus (VSSA) using 79 whole-genome sequences (24 clinical isolates and 55 publicly available genome sequences). We identified 46 single nucleotide polymorphisms (SNPs) associated with the VISA phenotype. Among them, 27 showed nonsynonymous amino acid substitutions and 10 showed no amino acid change (Figure 20).
 Flow diagram for genotype-phenotype association analysis (left). Overview of the mutations associated with the VISA phenotype (right).
Figure 20. Flow diagram for genotype-phenotype association analysis (left). Overview of the mutations associated with the VISA phenotype (right).

Development of diagnostic tools for carbapenemase-producing Gram-negative pathogens

Carbapenem resistance in Gram-negative pathogens is an emerging problem worldwide. Rapid detection and identification of carbapenemases are important for epidemiological investigations and infection control measures. To develop a multiplex real-time PCR assay for detection of the most common genetic types of carbapenemases (KPC, NDM, OXA-48, IMP, VIM, and GES), 296 clinical isolates were tested. These isolates included 14 species of Gram-negative bacteria, with 204 carbapenemase-producing and 92 non-carbapenemase-producing strains. The 204 carbapenemase-producing Gram-negative bacterial strains included KPC type (69), NDM type (32), OXA-48 type (25), IMP type (25), VIM type (27), GES type (9), and co-producing (17). Six types of carbapenemase genes were detected by two sets of triplex real-time PCR. Set 1 detected KPC, NDM, and OXA-48, and Set 2 identified IMP, VIM, and GES. The sensitivities and specificities were 100% and 99.3%, respectively, for all carbapenemase genes, and there was almost perfect agreement (Cohen Ҡ) between conventional with multiplex real-time PCR (0.98, 95% confidence interval [CI], 0.81-1) (Figure 21).
Determination of the sensitivity of the multiplex real-time PCR: 10-fold serial dilution of gDNA (100-0.0001 ng/mL)

Figure 21. Determination of the sensitivity of the multiplex real-time PCR: 10-fold serial dilution of gDNA (100-0.0001 ng/mL).


One Health Approach to AMR

The necessity of the One Health Approach to AMR and the start of a One Health-based research project


Antimicrobial-resistant pathogens can trigger an outbreak and be transmitted through diverse pathways such as agriculture, food-producing animals, fisheries, food, and the environment as well as human to human, for which comprehensive measures across all government departments are imperative (Figure 22). International organizations (WHO, OIE, and FAO) are also participating in concerted efforts to combat AMR, adopting the ‘One Health’ approach which recognizes the interconnectedness of the health of humans, animals, plants, and the environment. In 2015, WHO proposed a global action plan that strongly urged countries to establish national plans and to participate in global collaboration, and a number of countries (e.g. the UK in 2015, the US in 2015, and Japan in 2016) have published national action plans to address AMR.
Outbreak of antimicrobial-resistant bacteria and transmission pathways.

Figure 22. Outbreak of antimicrobial-resistant bacteria and transmission pathways.

In August 2016, the Korean government established the National Action Plan on AMR including R&D support to strengthen interministerial cooperation in the One Health approach. In 2017, the Korea Centers for Disease Control & Prevention (KCDC) first launched a One Health-based research project including investigation, studies on transmission pathways, mechanism of AMR, and building of an integrated system for AMR in human-animal-environment interfaces (Table 3). The target pathogens included MRSA, vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-resistant Enterococci (VRE), carbapenem-resistant Enterobacteriaceae (CRE), multidrug-resistant Acinetobacter baumannii, and multidrug-resistant Pseudomonas aeruginosa, Escherichia coli, Salmonella spp., and Campylobacter spp.


Table 3. One Health-based Research Project, 2017
One Health-based Research Project, 2017

This project is expected to be extended with the participation of related ministries with the construction of an interministerial framework for AMR and the strategic investment in R&D such as the development of diagnostic tools and control technologies for AMR. The One Health approach to AMR may produce meaningful data to enable analysis of AMR occurrence and transmission, and the establishment of AMR prevention and control programs.


AMR of E. coli and K. pneumoniae isolated from companion animal hospitals

E. coli and K. pneumoniae have become important pathogens in nosocomial infections. To establish the prevalence of AMR among E. coli and K. pneumoniae in companion animals, humans, and the environment, 3,137 samples were collected from companion animals, their owners, animal hospital staffs, and hospital environments from June to December 2017.
A total of 238 E. coli strains were isolated; these isolates were most prevalent (n = 195, 22.6%) in companion animals, followed by owners (n = 35, 4.1%) and hospital environments (n = 8, 0.9%). The resistance rates to cefotaxime, ceftazidime, and imipenem from companion animals, humans, and hospital settings isolates were 72.3% (172/238), 42.9% (102/238), and 1.7% (4/238), respectively (Figure 23). Imipenem-resistant E. coli were isolated from four hospitalized dogs in the same animal hospital and contained carbapenemase-producing E. coli carrying blaNDM-5.
Antimicrobial resistance (AMR) rates (%) of E. coli collected from companion animals, their owners, animal hospital workers, and hospital settings.
Figure 23. Antimicrobial resistance (AMR) rates (%) of E. coli collected from companion animals, their owners, animal hospital workers, and hospital settings.

 A total of 45 K. pneumoniae strains were isolated and were most prevalent (n = 32, 3.7%) in companion animals, followed by hospital environments (n = 10, 1.2%) and owners (n = 3, 0.3%). Resistance to cefotaxime and ceftazidime from companion animals and hospital settings, except for humans, accounted for 82.2% (37/45) and 73.3% (33/45) of isolates, respectively. Ten extended-spectrum beta-lactamases (ESBL) subtypes were identified from both companion animals and hospital settings. blaCTX-M15 was most dominant (n = 29, 52.7%), followed by blaDHA-1 (n = 13, 23.6%), blaDHA-1-like (n = 10, 18.1%), blaTEM-1 (n = 7, 12.7%), blaSHV-12 (n = 4, 7.3%), blaCTX-M14, blaCMY-2-like (n = 3, 5.5%), blaSHV-26 (n = 2, 3.6%), blaSHV-28, and blaDHA-9 (n = 1, 1.8%).

AMR of livestock-associated S. aureus from pig farms

 The emergence and spread of livestock-associated methicillin-resistant S. aureus (LA-MRSA) in food-producing animals have raised public health concerns, particularly regarding the possible transmission to humans. This investigation assessed the prevalence and genotypes of MRSA and methicillin-sensitive S. aureus (MSSA) strains isolated from pig farms and associated environments. A total of 1,027 samples were collected from pigs (n = 760), farm workers (n = 153), and farm environments (n = 114) from June to December 2017 and a total of 85 S. aureus (47 MRSA and 38 MSSA strains, respectively) were isolated.
Multilocus sequence typing (MLST) analysis revealed that 64% of the MRSA isolates were ST398 and 13% were ST541 (Figure 24). Similarly, the majority of MLST types among the MSSA isolates were ST398 (39%) and ST5 (37%). ST398-SCCmecV-agrI (55%), ST541-SCCmecV-argI (13%), and ST2084-SCCmecIV-agrI (11%) were the predominant MRSA clones in pig farms. Almost all ST398-MRSA and ST398-MSSA isolates were resistant to tetracycline (100% and 96%, respectively). The ST398-MRSA/MSSA strains tended to have higher levels of multiple antibiotic resistance compared to that in the non-ST398-MRSA strains.

AMR and multilocus sequence type (MLST) rates (%) of methicillin-resistant S. aureus (MRSA) isolates collected from pigs, farm workers, and associated environments.

Figure 24. AMR and multilocus sequence type (MLST) rates (%) of methicillin-resistant S. aureus (MRSA) isolates collected from pigs, farm workers, and associated environments.

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