Detection of Linezolid-Resistant Enterococcus faecalis and Enterococcus faecium Isolates from the Layer Operation System in Korea.

Linezolid (LNZ) is one of the most important antimicrobial agents against infections caused by gram-positive bacteria, including enterococci. In a layer operation system, antimicrobial resistance can be transferred to commercial layers via the fecal-oral route. This study investigated the presence and distribution of LNZ-resistant Enterococcus faecalis and Enterococcus faecium in a layer operation system. Among 117 E. faecalis and 154 E. faecium, 10 (8.5%) E. faecalis and 5 (3.2%) E. faecium isolates showed resistance to LNZ and chloramphenicol, and they exhibited multidrug resistance against 5 or more classes of antimicrobial agents. Among the resistant isolates, 9 (90.0%) and 2 (20.0%) E. faecalis harbored optrA and cfr genes, respectively. The optrA and fexA genes were not detected in five LNZ-resistant E. faecium. None of the 15 LNZ-resistant isolates harbored the fexA gene, and no mutations were observed in the genes encoding domain V of 23S ribosomal RNA (rRNA) and ribosomal proteins L3 (rplC) and L4 (rplD). Transferability was identified in three of the nine optrA-positive LNZ-resistant isolates. The tetM, tetL, and ermB genes were cotransferred with the optrA gene in all optrA-positive transconjugants. The results indicate that optrA is well-distributed in E. faecalis, implying a greater level of transferability. Thus, enhanced surveillance efforts are needed to monitor the emergence and spread of optrA in enterococci in layer operation system.

Impacts and characteristics of antimicrobial resistance of Escherichia coli isolates by administration of third-generation cephalosporins in layer hatcheries.

We investigated the characteristics and persistence of Escherichia coli resistant to third-generation cephalosporins (3GCs) by early administration of ceftiofur or gentamicin and to analyze the impact of 3GC use in hatcheries. We studied 10 ceftiofur-treated flocks (CTFs) and 10 gentamicin-treated flocks (GTFs) of layers. Fecal samples were collected at 1, 2, 4, 8, 18, and 30 weeks of age for all flocks. Among the 446 E. coli isolates, 58 (29.0 %) of 200 isolates in CTFs were identified as 3GC-resistant E. coli and 28 (11.4 %) of 246 isolates in GTFs were identified as 3GC-resistant E. coli. The presence of 3GC-resistant E. coli isolates at 1, 2, and 4 weeks was significantly higher in CTFs than in GTFs (p < 0.05). Moreover, the rate of resistance to 3GCs gradually decreased from 83.3 % at 1 week of age to 4.4 % at 30 weeks of age in CTFs. Of the 86 3GC-resistant E. coli isolates, 32 isolates had ß-lactamase-encoding gene: blaCTX-M-14 (ten isolates), blaCTX-M-15 (three isolates), blaCMY-2 (five isolates), and blaTEM-1 (twenty-five isolates) genes. Plasmid replicon typing revealed that blaCTX-M-14, blaCTX-M-15, blaCMY-2, and blaTEM-1 were located on F, F and FIB, I1 and K, and I1 and FII, respectively. Furthermore, 18 isolates carried class 1 integrons, with four different gene cassettes. These results revealed that ceftiofur used in hatcheries can lead to an increase in the number of 3GC-resistant E. coli with many characteristics. A voluntary ban must be imposed on the use of 3GCs for 1-day-old chicks in poultry industry.

Molecular characterization of antimicrobial-resistant Enterococcus faecalis and Enterococcus faecium isolated from layer parent stock.

Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) are ubiquitous intestinal bacteria in humans and animals that can easily acquire antimicrobial resistance, which allows them to have roles as antimicrobial resistance indicators. In addition, layer parent stock produces thousands of eggs for the production of commercial laying hens and can transfer a variety of viral and bacterial agents to chicks. The objective of this study was to determine the prevalence and characteristics of antimicrobial-resistant E. faecalis and E. faecium isolated in the layer parent stock level of the egg-layer operating system in South Korea. A total of 129 E. faecalis and 166 E. faecium isolates from 74 flocks of 30 layer parent stock were tested for resistance in this study. The prevalence of doxycycline- (51.9%), erythromycin- (53.5%), high-level gentamicin- (13.2%), high-level kanamycin- (31.0%), high-level streptomycin- (30.2%), and tetracycline- (64.3%) resistant E. faecalis isolates were higher than those for E. faecium isolates (P < 0.05). The ermB gene was detected in 66 (95.7%) erythromycin-resistant E. faecalis isolates, which was higher than that of 32 (71.7%) erythromycin-resistant E. faecium isolates. Twenty-one high-level gentamicin-resistant Enterococcus spp. (17 E. faecalis and 4 E. faecium) carried at least one aminoglycoside-modifying enzyme gene, aac(6')Ie-aph(2')-Ia or ant(6)-Ia. Fourteen isolates that harbored both aac(6')Ie-aph(2')-Ia and ant(6)-Ia exhibited pattern A with IS256 at both ends. Ten high-level ciprofloxacin-resistant Enterococcus spp. (8 E. faecalis and 2 E. faecium) showed amino acid changes from serine to isoleucine at codons 83 in gyrA, and 80 in parC. Also, the virulence genes ace, asa1, efaA, and gelE were detected in this study. To the best of our knowledge, this is the first study to examine the prevalence and characteristics of antimicrobial-resistant E. faecalis and E. faecium isolates in the layer parent stock. Our findings support the need for a surveillance program to monitor the emergence of antimicrobial-resistant E. faecalis and E. faecium in layer operating system.

Emergence of CMY-2-Producing Escherichia coli in Korean Layer Parent Stock.

The use of antimicrobials in food animals is the major determinant for the propagation of resistant bacteria in the animal reservoir. Especially, parent stock (PS) produces thousands of eggs for commercial laying hens and can transfer a variety of viral and bacterial agents to chicks. The objective of this study was to investigate the presence and distribution of third-generation cephalosporin-resistant and extended-spectrum ß-lactamase (ESBL)- and plasmid-mediated AmpC (pAmpC)-producing Escherichia coli in the PS level of the layer operation system in Korea. A total of 591 E. coli isolates were tested in this study and resistance to the first-generation cephalosporins ranged from 60.0% to 71.1%, whereas the resistance to the second- and third-generation cephalosporins ranged from 18.3% to 28.9% and from 8.3% to 14.5%, respectively. Of the 65 third-generation cephalosporin-resistant E. coli isolates, 51 (78.5%) showed multidrug resistance and 18 (27.7%) exhibited phenotypic and genotypic characteristics of ESBL/pAmpC-producing E. coli. With regard to ESBL/pAmpC gene prevalence, only the CMY-2 gene was identified in all 18 ESBL/pAmpC-producing E. coli and two isolates possessed both the CMY-2 and TEM-1 genes. Pulsed-field gel electrophoresis (PFGE) analysis of the 18 CMY-2-producing E. coli isolates revealed four PFGE patterns from the same PS farm and flock and accorded both CMY-2 genes and antimicrobial resistance pattern. To the best of our knowledge, this is the first study to investigate the prevalence and characteristics of third-generation cephalosporin-resistant and CMY-2-producing E. coli isolated at the layer PS level; our findings support the critical need for a comprehensive surveillance program to monitor the emergence and dissemination of third-generation cephalosporin resistance.

Molecular characteristics of extended-spectrum and plasmid-mediated AmpC ß-lactamase-producing Escherichia coli isolated from commercial layer in Korea.

In the poultry industry, commercial layer farms play an important role in meeting the protein demand through the supply of eggs. However, the risk of contamination by ß-lactamase-producing Escherichia coli in eggs laid by commercial chickens is significant. In this study, we investigated the rate of extended-spectrum ß-lactamase (ESBL) and plasmid-mediated AmpC (pAmpC) ß-lactamase-producing E. coli isolated from layer hens and characterized their molecular background. Among the 92 cefotaxime-resistant E. coli isolates, 66 (71.7%) were identified as multidrug resistant and 29 showed phenotypic and genotypic characteristics of ß-lactamase-producing E. coli. The ESBL/pAmpC genes blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, and blaCMY-2 were detected in 1, 6, 5, and 4 isolates, respectively. The non-ESBL/pAmpC gene blaTEM-1 was found in 16 isolates. Three isolates harbored both blaTEM-1 and blaCTX-M-14 genes. A total of 12 isolates also carried class 1 integrons, with 3 different gene cassette arrangements found in 8 of these isolates. A pulsed-field gel electrophoresis (PFGE) analysis of the 29 ß-lactamase-producing E. coli isolates revealed that 4 PFGE patterns were consistent with the ß-lactamase gene and layer farm origin, and showed a similar antibiotic resistance pattern. Our results suggest that comprehensive surveillance and more prudent use of third-generation cephalosporins in commercial layer farms is necessary to prevent the dissemination of ESBL/pAmpC-producing E. coli.

Comparative genetic characterization of third-generation cephalosporin-resistant Escherichia coli isolated from a layer operation system in Korea.

The layer operation system has a pyramidal structure in which the grandparent stock (GPS) is at the top, followed by the parent stock (PS), and commercial chickens (CC) that produce eggs at the bottom of the pyramid. Unfortunately, this vertical integration of the layer industry allows the transfer of Escherichia coli(E. coli) to the next step. The objective of this study was to genetically characterize and investigate the prevalence of third-generation cephalosporin-resistant E. coli at all levels of the layer operation system in Korea. A total of 561 E. coli samples were tested in this study, and antimicrobial resistance to third-generation cephalosporin, cefotaxime, and ceftazidime gradually increased from GPS to CC. Fifty-one (9.1%) isolates produced ß-lactamase genes: GPS harbored blaTEM-1 (1 isolate) or blaCMY-2 (2 isolates) genes; PS harbored blaTEM-1 (7 isolates) or blaCMY-2 (6 isolates) genes; CC harbored blaCTX-M-1 (1 isolate), blaCTX-M-14 (1 isolate), blaTEM-1 (13 isolates), blaCTX-M-14+TEM-1 (7 isolates), or blaCMY-2 (4 isolates) genes; and eggs harbored blaCTX-M-1 (2 isolates), blaCTX-M-14 (1 isolate), blaTEM-1 (3 isolates), or blaCMY-2 (3 isolates) genes. Pulsed-field gel electrophoresis (PFGE) analysis was performed on the 51 ß-lactamase-producing E. coli isolates. In PFGE, E. coli included 7 PFGE patterns showing the same production stage and exhibited both ß-lactamase genes and the antimicrobial resistance pattern. Our findings suggest that there is a critical need for comprehensive surveillance of third-generation cephalosporin-resistant E. coli at all levels of the layer-production pyramid and provide important considerations for the control of infection in large-scale layer operations in Korea.

Characterization of avian paramyxovirus type 1 from migratory wild birds in chickens.

Newcastle disease virus (NDV) is one of the most important infectious agents in the poultry industry, and vaccines against it have been widely used for prevention and control. Live vaccines, which can replicate in the respiratory and digestive systems, have been especially needed in areas with outbreaks of viscerotropic velogenic Newcastle disease. Towards the goal of searching for a new live vaccine candidate, avian paramyxovirus type 1 (APMV-1) was isolated from the faeces of wild birds. Three APMV-1 strains thus isolated were characterized in terms of phylogeny, pathogenicity, immunogenicity and tissue tropism, and on the basis of these analyses were classified as lentogenic genotype I NDV. CBU2179, one of the three APMV-1 strains, was selected and was evaluated in terms of its efficacy and safety in specific pathogen-free chickens and commercial broilers. The manufactured trial vaccine from this strain, also called CBU2179, induced similar immune responses to those of VG/GA and B1 commercial vaccines, and provided 100% protection against challenge from viscerotropic velogenic NDV, KJW/49 strain (the official challenge strain in Korea). Also, the CBU2179 virus was re-isolated and persisted as long as or longer than other vaccine strains in both the respiratory and alimentary tracts. Therefore, the CBU2179 strain may represent a good candidate for a live Newcastle disease vaccine to protect chickens against viscerotropic velogenic NDV.