Linezolid Resistance Genes Research Articles

Detection of poxtA- and optrA-carrying E. faecium isolates in air samples of a Spanish swine farm

ObjectiveTwo linezolid-resistant Enterococcus faecium isolates, C10004 and C10009, were recovered from air samples of a Spanish swine farm and comprehensively characterized. MethodsDetection of linezolid resistance mechanisms (mutations and acquisition of resistance genes) was performed by PCR/sequencing. Isolates were characterized by multilocus sequence typing (MLST), antimicrobial susceptibility testing, detection of antimicrobial resistance and virulence genes, and analysis of the genetic environment of the linezolid resistance genes. The characterization of isolate C10009 was performed by Whole-Genome-Sequencing and of isolate C10004 by PCR and amplicon sequencing, where applicable. Conjugation experiments to assess the transferability of the optrA and poxtA genes implicated in linezolid resistance were performed. ResultsThe linezolid-resistant E. faecium isolates C10004 and C10009, assigned to ST128 and ST437, respectively, harbored the optrA and poxtA genes. Neither mutations in the 23S rRNA nor in the genes for the ribosomal proteins L3, L4 and L22 were detected. C10004 and C10009 carried fourteen and thirteen antimicrobial resistance genes, respectively. The sequence alignment indicated that the genetic environment of the poxtA gene was identical in both isolates, with a downstream-located fexB gene. The poxtA gene was transferred by conjugation together with the fexB gene, and also with tet(M) and tet(L) in the case of isolate C10004. The optrA gene could not be transferred. ConclusionsThis is the first report of the poxtA gene in Spain. The presence of poxtA- and optrA-carrying E. faecium isolates in air samples represents a public health concern, indicating an involvement of swine farms in the spread of linezolid-resistant bacteria.

Dispersal of linezolid-resistant enterococci carrying poxtA or optrA in retail meat and food-producing animals from Tunisia.

The epidemiology of Enterococcus resistant to priority antibiotics including linezolid has mainly been investigated in developed countries and especially in hospitals. We aimed to evaluate the contribution of different non-human reservoirs for the burden of MDR enterococci in Tunisia, where scarce data are available. Samples (n = 287) were collected from urban wastewater (n = 57), retail meat (n = 29; poultry/bovine/ovine), milk (n = 89; bovine/ovine), farm animal faeces (n = 80; poultry/bovine/ovine) and pets (n = 32; rabbit/dogs/cats/birds) in different Tunisian regions (2014-17). They were plated onto Slanetz-Bartley agar after pre-enrichment without antibiotics. Standard methods were used for bacterial identification and characterization of antibiotic resistance and virulence genes (PCR), antibiotic susceptibility testing (disc diffusion/broth microdilution; EUCAST/CLSI) and clonality (SmaI-PFGE/MLST). All samples carried Enterococcus (n = 377 isolates) resistant to antibiotics considered to be critical or highly important by WHO. Even without antibiotic selection, 38% of Enterococcus faecalis (Efs) and 22% of Enterococcus faecium (Efm) were identified as MDR. Linezolid-resistant isolates (5%; MIC = 8 mg/L) comprised six poxtA-carrying Efm (cow milk), seven optrA-carrying Efs (chicken faeces/meat) and five Efm lacking cfr/optrA/poxtA (poultry/bovine/ovine/wastewater). Clinically relevant Efm clones (clade A1) were identified in animal/meat sources. Ampicillin resistance (1%) was confined to ST18/ST78-like MDR Efm clones from bovine meat/milk samples carrying relevant virulence markers (e.g. ptsD/IS16). This study provides evidence of the contribution of livestock and foodstuffs to the dispersal of acquired linezolid resistance genes including poxtA and optrA. We report the first poxtA-carrying Efm in Tunisia, and for the first time in bovine samples, stressing the urgent need for alternative measures to counteract the spread of linezolid-resistant enterococci globally.

Whole-genome sequencing of Enterococcus hirae CQP3-9, a strain carrying the phenicol-oxazolidinone-tetracycline resistance gene poxtA of swine origin in China.

The aim of this study was to characterise the whole genome sequence of linezolid-intermediate Enterococcus hirae strain CQP3-9 isolated from a large-scale swine farm in Sichuan Province, China, in August 2018. An Illumina MiSeq platform (400-bp paired-end reads with 230-fold average coverage) and PacBio RS II sequencing instrument (100-fold average read depth) were used for genome sequencing. The chromosome and two plasmids were assembled using the software SMRT portal v.3.2.0. Acquired antimicrobial resistance genes were identified using ResFinder 3.1. The genome of E. hirae strain CQP3-9 consists of one 2 695 881-bp chromosome, one 125 915-bp plasmid (pCQP3-9_1) and one 33 132-bp plasmid (pCQP3-9_2). The genome of CQP3-9 contains 2458 coding sequences and 89 RNA genes. The poxtA gene is the only linezolid resistance gene in CQP3-9, located on plasmid pCQP3-9_2 that co-harbours erm(B) (macrolide resistance), fexB (chloramphenicol and florfenicol resistance), and tet(M) and tet(L) (tetracycline resistance). Here we report for the first time the phenicol-oxazolidinone-tetracycline resistance gene poxtA in E. hirae, located on a plasmid that co-harbours erm(B), fexB, tet(L) and tet(M). The genome sequence of E. hirae CQP3-9 provides valuable information for the dissemination of poxtA among enterococci.

Development of a multiplex-PCR to simultaneously detect acquired linezolid resistance genes cfr, optrA and poxtA in enterococci of clinical origin

Linezolid-resistant enterococcus spp. are increasingly recognized by diagnostic laboratories. Resistance can be mediated by the expression of cfr, optrA or poxtA. We developed a multiplex-PCR to simultaneously detect all three genes. The PCR is suitable for microbiological diagnostics in order to restrict further spread of resistances in enterococci.

Novel linezolid resistance plasmids in Enterococcus from food animals in the USA.

To sequence the genomes and determine the genetic mechanisms for linezolid resistance identified in three strains of Enterococcus isolated from cattle and swine caecal contents as part of the US National Antimicrobial Resistance Monitoring System (NARMS) surveillance programme. Broth microdilution was used for in vitro antimicrobial susceptibility testing to assess linezolid resistance. Resistance mechanisms and plasmid types were identified from data generated by WGS on Illumina® and PacBio® platforms. Conjugation experiments were performed to determine whether identified mechanisms were transmissible. Linezolid resistance plasmids containing optrA were identified in two Enterococcus faecalis isolates and one Enterococcus faecium. The E. faecium isolate also carried the linezolid resistance gene cfr on the same plasmid as optrA. The linezolid resistance plasmids had various combinations of additional resistance genes conferring resistance to phenicols (fexA), aminoglycosides [spc and aph(3')-III] and macrolides [erm(A) and erm(B)]. One of the plasmids was confirmed to be transmissible by conjugation, resulting in linezolid resistance in the transconjugant. To the best of our knowledge, this is the first identification of linezolid resistance in the USA in bacteria isolated from food animals. The oxazolidinone class of antibiotics is not used in food animals in the USA, but the genes responsible for resistance were identified on plasmids with other resistance markers, indicating that there may be co-selection for these plasmids due to the use of different antimicrobials. The transmissibility of one of the plasmids demonstrated the potential for linezolid resistance to spread horizontally. Additional surveillance is necessary to determine whether similar plasmids are present in human strains of Enterococcus.

The human clone ST22 SCCmec IV methicillin-resistant Staphylococcus aureus isolated from swine herds and wild primates in Nepal: is man the common source?

Swine nasal samples [n=282] were collected from 12 randomly selected farms around Kathmandu, Nepal, from healthy animals. In addition, wild monkey (Macaca mulatta) saliva samples [n=59] were collected near temples areas in Kathmandu using a non-invasive sampling technique. All samples were processed for MRSA using standardized selective media and conventional biochemical tests. MRSA verification was done and isolates characterized by SCCmec, multilocus sequence typing, whole genome sequencing [WGS] and antibiotic susceptibilities. Six (2.1%) swine MRSA were isolated from five of the different swine herds tested, five were ST22 type IV and one ST88 type V. Four (6.8%) macaques MRSA were isolated, with three ST22 SCCmec type IV and one ST239 type III. WGS sequencing showed that the eight ciprofloxacin resistant ST22 isolates carried gyrA mutation [S84L]. Six isolates carried the erm(C) genes, five isolates carried aacC-aphD genes and four isolates carried blaZ genes. The swine linezolid resistant ST22 did not carry any known acquired linezolid resistance genes but had a mutation in ribosomal protein L22 [A29V] and an insertion in L4 [68KG69], both previously associated with linezolid resistance. Multiple virulence factors were also identified. This is the first time MRSA ST22 SCCmec IV has been isolated from livestock or primates.

Advances in novel linezolid resistance gene optrA

optrA, a transferrable linezolid resistance gene, was identified in a clinical strain of Enterococcus faecalis isolated in China in 2015. This gene is 1 968 bp in length, encoding 655 amino acids. And the sequence of these amino acids shares high homology with the amino acid sequence of ABC transporter, resulting in resistance to oxazolidinones (linezolid and tedizolid) and phenicols (florfenicol and chloramphenicol). Linezolid is an important oxazolidinone antibiotic used in the treatment of refractory infections such as infections caused by methicillin-resistant Staphylococcus aureus. Therefore, the appearance and prevalence of optrA gene have drawn worldwide attention. In this review, we focus on the discovery, epidemiology and molecular transmission mechanism of optrA gene. Key words: Linezolid; Resistance; optrA; Epidemiology; Molecular transmission mechanism

Prevalence and Abundance of Florfenicol and Linezolid Resistance Genes in Soils Adjacent to Swine Feedlots.

Florfenicol is extensively used in livestock to prevent or cure bacterial infections. However, it is not known whether the administration of florfenicol has resulted in the emergence and dissemination of florfenicol resistance genes (FRGs, including fexA, fexB, cfr, optrA, floR, and pexA) in microbial populations in surrounding farm environments. Here we collected soil samples for the detection of FRGs and the residue of florfenicol from six swine farms with the record of florfenicol usage. Quantitative polymerase chain reaction and metagenomic sequencing revealed a significantly higher relative abundance of FRGs in the soils adjacent to the three swine farms where florfenicol was heavily used compared with the other sites. Meanwhile, the detectable levels of florfenicol were also identified in soils from two of these three farms using ultra-performance liquid chromatography tandem mass spectrometry. It appears that amount of florfenicol used on swine farms and the spreading of soils with swine waste could promote the prevalence and abundance of FRGs, including the linezolid resistance genes cfr and optrA, in adjacent soils, and agricultural application of swine manure with florfenicol may have caused a residual level of florfenicol in the soils.