Genomic characteristics of cfr and fexA carrying Staphylococcus aureus isolated from pig carcasses in Korea.

The emergence of transferable linezolid resistance genes poses significant challenges to public health, as it does not only confer linezolid resistance but also reduces susceptibility to florfenicol, which is widely used in the veterinary field. This study evaluated the genetic characteristics of linezolid-resistant Staphylococcus aureus strains isolated from pig carcasses and further clarified potential resistance and virulence mechanisms in a newly identified sequence type. Of more than 2500 strains isolated in a prior study, 15 isolated from pig carcasses exhibited linezolid resistance (minimum inhibitory concentration ≥ 8 mg/L). The strains were characterized in detail by genomic analysis. Linezolid-resistant S. aureus strains exhibited a high degree of genetic lineage diversity, with one strain (LNZ_R_SAU_64) belonging to ST8004, which has not been reported previously. The 15 strains carried a total of 21 antibiotic resistance genes, and five carried mecA associated with methicillin resistance. All strains harbored cfr and fexA, which mediate resistance to linezolid, phenicol, and other antibiotics. Moreover, the strains carried enterotoxin gene clusters, including the hemolysin, leukotoxin, and protease genes, which are associated with humans or livestock. Some genes were predicted to be carried in plasmids or flanked by ISSau9 and the transposon Tn554, thus being transmittable between staphylococci. Strains carrying the plasmid replicon repUS5 displayed high sequence similarity (99%) to the previously reported strain pSA737 in human clinical samples in the United States. The results illustrate the need for continuous monitoring of the prevalence and transmission of linezolid-resistant S. aureus isolated from animals and their products.

Prevalence and Characteristics of Phenicol-Oxazolidinone Resistance Genes in Enterococcus Faecalis and Enterococcus Faecium Isolated from Food-Producing Animals and Meat in Korea.

The use of phenicol antibiotics in animals has increased. In recent years, it has been reported that the transferable gene mediates phenicol-oxazolidinone resistance. This study analyzed the prevalence and characteristics of phenicol-oxazolidinone resistance genes in Enterococcus faecalis and Enterococcus faecium isolated from food-producing animals and meat in Korea in 2018. Furthermore, for the first time, we reported the genome sequence of E. faecalis strain, which possesses the phenicol-oxazolidinone resistance gene on both the chromosome and plasmid. Among the 327 isolates, optrA, poxtA, and fexA genes were found in 15 (4.6%), 8 (2.5%), and 17 isolates (5.2%), respectively. Twenty E. faecalis strains carrying resistance genes belonged to eight sequence types (STs), and transferability was found in 17 isolates. The genome sequences revealed that resistant genes were present in the chromosome or plasmid, or both. In strains EFS17 and EFS108, optrA was located downstream of the ermA and ant(9)-1 genes. The strains EFS36 and EFS108 harboring poxtA-encoding plasmid cocarried fexA and cfr(D). These islands also contained IS1216E or the transposon Tn554, enabling the horizontal transfer of the phenicol-oxazolidinone resistance with other antimicrobial-resistant genes. Our results suggest that it is necessary to promote the prudent use of antibiotics through continuous monitoring and reevaluation.

Emergence of fexA in Mediating Resistance to Florfenicols in Campylobacter.

Florfenicol belongs to a class of phenicol antimicrobials widely used as feed additives and for the treatment of respiratory infections. In recent years, increasing resistance to florfenicol has been reported in Campylobacter spp., the leading foodborne enteric pathogens causing diarrheal diseases worldwide. Here, we reported the identification of fexA, a novel mobile florfenicol resistance gene in Campylobacter Of the 100 Campylobacter jejuni strains isolated from poultry in Zhejiang, China, 9 were shown to be fexA positive, and their whole-genome sequences were further determined by integration of Illumina short-read and MinION long-read sequencing. The fexA gene was found in the plasmid of one strain and chromosomes of eight strains, and its location was verified by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting. Based on comparative analysis, the fexA gene was located within a region with the tet(L)-fexA-catA-tet(O) gene arrangement, demonstrated to be successfully transferable among C. jejuni strains. Functional cloning indicated that acquisition of the single fexA gene significantly increased resistance to florfenicol, whereas its inactivation resulted in increased susceptibility to florfenicol in Campylobacter Taken together, these results indicated that the emerging fexA resistance is horizontally transferable, which might greatly facilitate the adaptation of Campylobacter in food production environments where florfenicols are frequently used.

The fexA gene in Campylobacter: whether the spread has occurred among various hosts in eastern China.

OBJECTIVES: The emergence of the florfenicol resistance gene fexA in Campylobacter poses a serious threat to public health, but the extent of the spread of fexA in Campylobacter from various hosts has not been well understood. This study aimed to investigate the fexA in Campylobacter isolates from different hosts. METHODS: PCR was used to identify fexA-positive Campylobacter from different hosts during 2008-2019 in China, and the fexA-positive isolates were characterized by susceptibility tests, whole-genome sequencing, and natural transformation. RESULTS: A total of 69 (2.54%, 69/2721) fexA-positive Campylobacter were identified, and the fexA-positive isolates increased remarkably (0.42%-16.90%) since it was first detected in 2010. By source, the 69 isolates were obtained from chickens (3.57%, 57/1595), geese (3.43%, 7/204), ducks (1.02%, 2/197), and environments (2.86%, 3/105); the fexA-positive isolates were not isolated in humans and pigs. In addition to fexA, these isolates also carried other antimicrobial resistance genes and exhibited multidrug resistance. Whole-genome sequencing analysis showed the fexA gene can disseminate clonally or horizontally via either multidrug resistance genomic islands or insertion sequences among the Campylobacter. The genetic structure IS1216-∆ISEfa11-hp-fexA-NAD(P)H-∆ISEfa11-IS1216 was conserved and widespread in the Campylobacter of various origins, and the IS1216 can form fexA-carrying circular intermediates, emphasizing that IS1216 plays an important role in the spread of fexA in Campylobacter. CONCLUSIONS: This study indicates the wide spread of fexA-positive Campylobacter in poultry and environments. Because multidrug resistance genomic islands and IS1216 can facilitate the transmission of fexA, systematic surveillance should be implemented to prevent the spread of fexA to humans.

Molecular characterization of florfenicol and oxazolidinone resistance in Enterococcus isolates from animals in China.

Florfenicol is widely used for the treatment of bacterial infections in domestic animals. The aim of this study was to analyze the molecular mechanisms of florfenicol and oxazolidinone resistance in Enterococcus isolates from anal feces of domestic animals. The minimum inhibitory concentration (MIC) levels were determined by the agar dilution method. Polymerase chain reaction (PCR) was performed to analyze the distribution of the resistance genes. Whole-genome sequencing and comparative plasmid analysis was conducted to analyze the resistance gene environment. A total of 351 non-duplicated enteric strains were obtained. Among these isolates, 22 Enterococcus isolates, including 19 Enterococcus. faecium and 3 Enterococcus. faecalis, were further studied. 31 florfenicol resistance genes (13 fexA, 3 fexB, 12 optrA, and 3 poxtA genes) were identified in 15 of the 19 E. faecium isolates, and no florfenicol or oxazolidinone resistance genes were identified in 3 E. faecalis isolates. Whole-genome sequencing of E. faecium P47, which had all four florfenicol and oxazolidinone resistance genes and high MIC levels for both florfenicol (256 mg/L) and linezolid (8 mg/L), revealed that it contained a chromosome and 3 plasmids (pP47-27, pP47-61, and pP47-180). The four florfenicol and oxazolidinone resistance genes were all related to the insertion sequences IS1216 and located on two smaller plasmids. The genes fexB and poxtA encoded in pP47-27, while fexA and optrA encoded in the conjugative plasmid pP47-61. Comparative analysis of homologous plasmids revealed that the sequences with high identities were plasmid sequences from various Enterococcus species except for the Tn6349 sequence from a Staphylococcus aureus chromosome (MH746818.1). The current study revealed that florfenicol and oxazolidinone resistance genes (fexA, fexB, poxtA, and optrA) were widely distributed in Enterococcus isolates from animal in China. The mobile genetic elements, including the insertion sequences and conjugative plasmid, played an important role in the horizontal transfer of florfenicol and oxazolidinone resistance.

Prevalence of the phenicol resistance gene fexA in Campylobacter isolated from the poultry supply chain.

Florfenicol, an animal-specific broad-spectrum antibiotic, has been widely used in livestock and poultry breeding, which leads to the high antimicrobial resistance (AMR) of Campylobacter in food animals. Recently, a new florfenicol resistance gene, fexA, often located on various multidrug resistance genomic islands (MDRGIs) and confers resistance to various antimicrobial agents, was characterized in Campylobacter. However, the prevalence and genetic environments of fexA and its associated MDRGIs in Campylobacter in the poultry supply chain need further characterization. Here, a total of 111 (15.48 %) Campylobacter isolates (63 C. jejuni, 40 C. coli, 8 C. lari) were obtained from 717 samples from farms, slaughterhouses, and supermarkets. Both phenotypic and genotypic analyses indicated that the AMR of C. coli was significantly higher than that of C. jejuni. PCR amplification and whole genome sequencing showed that the fexA gene was present in 26 out of 35 florfenicol-resistant Campylobacter isolates. This gene was located in the tet(L)-fexA-tet(O) MDRGI. The fexA-harboring isolates detected in the above sources could be clustered into the same branch, indicating that they may have the same ancestor. In addition, the erm(B) gene was identified in 17 Campylobacter isolates, and the A2075G point mutation in the 23S rRNA gene occurred in 26 isolates, emphasizing the high resistance of Campylobacter to macrolides. In summary, these results indicate that fexA within the MDRGI of Campylobacter can be transmitted through bacteria in the animal-based food supply chain, and it is necessary to strengthen the monitoring of the prevalence and spread of fexA in foodborne Campylobacter spp.

Coexistence of optrA and fexA in Campylobacter.

Previous studies indicated that Campylobacter has developed several mechanisms that confer resistance to florfenicol, which is used in food animal production. This study describes the coexistence of optrA and fexA in Campylobacter jejuni and Campylobacter coli isolates from pigs and poultry. Moreover, whole-genome sequencing data showed that the two genes are located in various multidrug resistance genomic islands within different regions of the Campylobacter genomes. The emergence of optrA and fexA may support the spread of florfenicol-resistant Campylobacter strains of animal origin.IMPORTANCE Florfenicol is widely used for the treatment of respiratory infections and as a feed additive in food animal production. As a foodborne pathogen, Campylobacter is constantly exposed to florfenicol, and resistance to this antimicrobial agent has increased in recent years. Previous studies indicated that Campylobacter has developed several mechanisms that confer resistance to florfenicol. This study describes for the first time the coexistence of the florfenicol exporter FexA and the ribosomal protective protein OptrA in Campylobacter jejuni isolated from pigs. The two genes were located in various multidrug resistance genomic islands within different regions of the Campylobacter genomes. Although phenicols are not commonly used for the treatment of Campylobacter infections, the extensive use of florfenicol in food animals may play a role in the coselection of multidrug resistance genomic island (MDRGI)-carrying Campylobacter isolates which also exhibited resistance to critically important antimicrobial agents (macrolides, aminoglycosides, and tetracyclines) commonly used for the treatment of human campylobacteriosis.

Molecular characteristics of optrA-carrying Enterococcus faecalis from chicken meat in South Korea.

The purpose of this study was to identify the genetic environment of optrA gene in linezolid (LZD)-resistant Enterococcus faecalis from chicken meat and to describe the probable mechanism of dissemination of the optrA gene through plasmid or chromosomal integration. Whole genome sequencing and analysis revealed that all 3 E. faecalis isolates confirmed as LZD- and chloramphenicol-resistant carried fexA adjacent to the optrA gene as well as a variety of resistance genes for macrolides, tetracyclines, and aminoglycosides, simultaneously. But, the other genes conferring LZD resistance, cfr and poxtA, were not detected in those strains. Two isolates harboring the optrA gene in their chromosomal DNA showed >99% similarity in arrangement to the transposon Tn6674 and the transposase genes, tnpA, tnpB, and tnpC and were located in the first open reading frame for transposase. One isolate harboring an optrA-carrying plasmid also showed >99% similarity with the previously reported pE439 plasmid but had 2 amino acid changes (Thr96Lys and Tyr160Asp) and a higher minimum inhibitory concentration against LZD of 16 mg/L than that of pE439 (8 mg/L). Mobile genetic elements such as transposons or plasmids flanking the optrA gene conduct a crucial role in the dissemination of antimicrobial resistance genes. Further investigations are required to identify the way by which optrA is integrated into chromosomal DNA and plasmids.

Emergence of cfr-Mediated Linezolid Resistance in Staphylococcus aureus Isolated from Pig Carcasses.

Altogether, 2547 Staphylococcus aureus isolated from cattle (n = 382), pig (n = 1077), and chicken carcasses (n = 1088) during 2010-2017 were investigated for linezolid resistance and were further characterized using molecular methods. We identified linezolid resistance in only 2.3% of pig carcass isolates. The linezolid-resistant (LR) isolates presented resistance to multiple antimicrobials, including chloramphenicol, clindamycin, and tiamulin. Molecular investigation exhibited no mutations in the 23S ribosomal RNA. Nevertheless, we found mutations in ribosomal proteins rplC (G121A) and rplD (C353T) in one and seven LR strains, respectively. All the LR isolates carried the multi-resistance gene cfr, and six of them co-carried the mecA gene. Additionally, all the LR isolates co-carried the phenicol exporter gene, fexA, and presented a high level of chloramphenicol resistance. LR S. aureus isolates represented 10 genotypes, including major genotypes ST433-t318, ST541-t034, ST5-t002, and ST9-t337. Staphylococcal enterotoxin and leukotoxin-encoding genes, alone or in combination, were detected in 68% of LR isolates. Isolates from different farms presented identical or different pulsed-field gel electrophoresis patterns. Collectively, toxigenic and LR S. aureus strains pose a crisis for public health. This study is the first to describe the mechanism of linezolid resistance in S. aureus isolated from food animal products in Korea.

Live Feeds Used in the Larval Culture of Red Cusk Eel, Genypterus chilensis, Carry High Levels of Antimicrobial-Resistant Bacteria and Antibiotic-Resistance Genes (ARGs).

The culture of red cusk eel Genypterus chilensis is currently considered a priority for Chilean aquaculture but low larval survival rates have prompted the need for the continuous use of antibacterials. The main aim of this study was to evaluate the role of live feed as a source of antibacterial-resistant bacteria in a commercial culture of G. chilensis. Samples of rotifer and Artemia cultures used as live feed were collected during the larval growth period and culturable bacterial counts were performed using a spread plate method. Rotifer and Artemia cultures exhibited high levels of resistant bacteria (8.03 × 104 to 1.79 × 107 CFU/g and 1.47 × 106 to 3.50 × 108 CFU/g, respectively). Sixty-five florfenicol-resistant isolates were identified as Vibrio (81.5%) and Pseudoalteromonas (15.4%) using 16S rRNA gene sequence analysis. A high incidence of resistance to streptomycin (93.8%), oxytetracycline (89.2%), co-trimoxazole (84.6%), and kanamycin (73.8%) was exhibited by resistant isolates. A high proportion of isolates (76.9%) carried the florfenicol-resistance encoding genes floR and fexA, as well as plasmid DNA (75.0%). The high prevalence of multiresistant bacteria in live feed increases the incidence of the resistant microbiota in reared fish larvae, thus proper monitoring and management strategies for live feed cultures appear to be a priority for preventing future therapy failures in fish larval cultures.

Detection of oxazolidinone and phenicol resistant enterococcal isolates from duck feces and carcasses.

The heavy use or abuse of antimicrobials in food animals has caused an increase in antimicrobial resistance in enterococci of animal origin, which could get transmitted to those of human origin via the food chain. Since duck meat consumption has been on the rise in Korea, we conducted this study to provide information about the antimicrobial resistance of the enterococci obtained from healthy ducks and their carcasses. A total of 82 Enterococcus faecium and 174 E. faecalis isolated from duck fecal and carcass samples were investigated for antimicrobial resistance to 16 agents, using broth dilution method, and were further characterized using molecular methods. Most of E. faecium (84.1%) and E. faecalis (87.9%) isolates were resistant to one or more antimicrobials. Multi-drug resistant (MDR) isolates were observed in both E. faecium (40.2%) and E. faecalis (33.9%) with high frequencies. High rate of resistance was observed for tetracycline, ciprofloxacin, chloramphenicol, and erythromycin in both E. faecium and E. faecalis. Resistance to gentamicin, vancomycin, and daptomycin, in both E. faecium and E. faecalis, was, if at all, very rare. However, linezolid resistance was observed in nine E. faecium (11.0%) and one E. faecalis (0.6%). All, but one, Linezolid resistant (LR) isolates were also resistant to chloramphenicol and florfenicol. The novel transferable oxazolidinone and phenicol resistant gene, optrA, was found in six E. faecium isolates. All of them co-carried phenicol exporter gene fexA. None of the LR isolates had mutation in the 23S ribosomal RNA and in the ribosomal protein L3. Six LR E. faecium isolates had Asn130Lys mutation in the ribosomal protein L4, of which five also carried optrA gene. None of the isolates carried the multi-resistance gene cfr. Transfer of oxazolidinone and phenicol resistance was observed in five among the 10 LR isolates; two of them had optrA and fexA genes. Multi-drug resistant Enterococcus that also carried the resistance gene to a last-resort antimicrobial is a major concern for public health. Thus, to prevent the introduction of last-resort antimicrobial resistance into food chain, continuous surveillance of antimicrobial resistance in duck is imperative.

The cytochrome d oxidase complex regulated by fexA is an Achilles' heel in the in vivo survival of vibrio vulnificus.

Vibrio vulnificus is a halophilic estuarine bacterium causing severe opportunistic infections. To successfully establish an infection, V. vulnificus must adapt to redox fluctuations in vivo. In the present study, we show that deletion of V. vulnificus fexA gene caused hypersensitivity to acid and reactive oxygen species. The ΔfexA mutant exhibited severe in vivo survival defects. For deeper understanding the role of fexA gene on the successful V. vulnificus infection, we analyzed differentially expressed genes in ΔfexA mutant in comparison with wild type under aerobic, anaerobic or in vivo culture conditions by genome-scale DNA microarray analyses. Twenty-two genes were downregulated in the ΔfexA mutant under all three culture conditions. Among them, cydAB appeared to dominantly contribute to the defective phenotypes of the ΔfexA mutant. The fexA deletion induced compensatory point mutations in the cydAB promoter region over subcultures, suggesting essentiality. Those point mutations (PcydSMs) restored bacterial growth, motility, cytotoxicity ATP production and mouse lethality in the ΔfexA mutant. These results indicate that the cydAB operon, being regulated by FexA, plays a crucial role in V. vulnificus survival under redox-fluctuating in vivo conditions. The FexA-CydAB axis should serve an Achilles heel in the development of therapeutic regimens against V. vulnificus infection.

Detection of novel oxazolidinone and phenicol resistance gene optrA in enterococcal isolates from food animals and animal carcasses.

Altogether 7720 Enterococcus faecalis and 3939 E. faecium isolated from food animals and animal carcasses during 2003-2014 in Korea were investigated to determine if linezolid-resistant (LR) enterococci (≥8µg/ml) are present. Overall, 12 E. faecalis and 27 E. faecium recovered from chickens (n=32), pigs (n=6), and cattle (n=1) were resistant to linezolid and were further characterized using molecular methods Most LR isolates were also resistant to chloramphenicol (97.44%) and florfenicol (92.31%). Molecular analysis showed no mutations in the 23S ribosomal RNA and in the ribosomal protein L3. The optrA gene was found in 89.74% of the LR enterococci, including 12 E. faecalis and 23 E. faecium isolates. Among them, 30 optrA-positive isolates co-carried phenicol exporter gene fexA. Seven LR E. faecium isolates had Asn130Lys mutations in the ribosomal protein L4, of which six also carried optrA gene. None of the isolates carried the mutliresistance gene cfr. Transfer of optrA gene was observed in 16 of the 35 optrA-positive isolates by conjugation. Pulsed-field gel electrophoresis demonstrated that the vast majority of Enterococcus strains carrying optrA gene were genetically heterogeneous. Multi-locus sequence typing revealed eight novel Sequence types among E. faecalis and E. faecium strains. To our knowledge, this is the first report of optrA gene in isolates from cattle and animal carcasses. This is also the first report of optrA gene in Korea. Active surveillance of optrA in enterococci is urgently warranted.