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Novel Tet(L) Efflux Pump Variants Conferring Resistance to Tigecycline and Eravacycline in Staphylococcus Spp.

ABSTRACT: Tigecycline is regarded as one of the few important last-resort antibiotics to treat complicated skin and intra-abdominal infections. Members of the genus Staphylococcus are zoonotic pathogens and pose a serious threat to public health. Tigecycline resistance in this species appears to be a rare phenomenon, and the mechanisms underlying tigecycline resistance have not been fully elucidated. Here, we report two novel variants of the tet(L) gene in Staphylococcus spp. from swine in China, designed as tet(L)_F58L and tet(L)_A117V. The tet(L)_F58L was located within a 18,720 bp chromosomal multidrug resistance gene cluster flanked by two copies of IS257 in Staphylococcus cohnii 11-B-312, while the tet(L)_A117V was located on a 6,292 bp plasmid in S. haemolyticus 11-B-93, which could be transferred to S. aureus by electrotransformation. Cloning of each of the two tet(L) variants into S. aureus RN4220 showed 16- or 8-fold increases in the minimal inhibition concentrations (MICs), which can fully confer the resistance to tigecycline (MICs from 0.125 to 2 mg/liter) and eravacycline (MICs from 0.125 to 1 or 2 mg/liter), but no increase in the MICs of omadacycline, compared with the MICs of the recipient strain S. aureus RN4220. In the in vivo murine sepsis and in the murine pneumonia models, an increase in CFU of S. aureus 29213_pT93 carrying the tet(L)_A117V was seen despite tigecycline treatment. This observation suggests that the tet(L)_A117V and its associated gene product compromise the efficacy of tigecycline treatment in vivo and may lead to clinical treatment failure. Our finding, that novel Tet(L) efflux pump variants which confer tigecycline and eravacycline resistance have been identified in Staphylococcus spp., requires urgent attention. IMPORTANCE Tigecycline and eravacycline are both important last-resort broad spectrum antimicrobial agents. The presence of novel Tet(L) efflux pump variants conferring the resistance to tigecycline and eravacycline in Staphylococcus spp. and its potential transmission to S. aureus will compromise the efficacy of tigecycline and eravacycline treatment for S. aureus associated infection in vivo and may lead to clinical treatment failure.

SUBMITTER: Wang N

PROVIDER: S-EPMC8653819 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Carbapenem-resistant Acinetobacter nosocomialis (CRAn) is a significant public health concern. Tigecycline non-susceptible CRAn (Tn-CRAn) isolates have emerged worldwide. Tigecycline resistance is mainly related to the overexpression of AdeABC efflux pump controlled by AdeRS two-component system (TCS). Two novel tetracycline derivatives, omadacycline and eravacycline, may present a treatment option for CRAn. This study investigated the in vitro antimicrobial activity of tigecycline, omadacycline and eravacycline against clinical CRAn isolates and the contribution of efflux pumps in their resistance. Eighty-nine clinical CRAn isolates, including 57 Tn-CRAn isolates were evaluated for minimum inhibitory concentrations (MICs) by the broth microdilution. The relationship between the antimicrobial resistance and efflux pump expression was assessed by their responses to the efflux pump inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). The contribution of the AdeABC efflux pump in their resistance was determined by the complementation of the AdeRS two-component system in wild-type, adeRS operon and adeB gene knockout strains. Among the 89 isolates, omadacycline and eravacycline MICs were correlated closely with those of tigecycline. They demonstrated improved potency, based on MIC90 values, by showing a 4 to 8-fold greater potency than tigecycline. The synergetic effects of tigecycline, omadacycline and eravacycline with NMP were observed in 57 (100%), 13 (22.8%), and 51 (89.5%) of Tn-CRAn isolates, respectively. Further analysis showed that the laboratory strain carrying the Type 1 adeRS operon increased the tigecycline, omadacycline and eravacycline MICs by 4-8-folds, respectively. Eravacycline demonstrated improved potency over tigecycline against populations of CRAn, including Tn-CRAn isolates. The over-expression of AdeABC efflux pumps was directly activated by the AdeRS two-component system and simultaneously reduced the susceptibilities of tigecycline, eravacycline, and omadacycline. Omadacycline and eravacycline MICs were correlated closely with those of eravacycline.

Project description:Transporters belonging to the chromosomally encoded resistance-nodulation-division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria. However, the cotransfer of large gene clusters encoding RND-type pumps from the chromosome to a plasmid appears infrequent, and no plasmid-mediated RND efflux pump gene cluster has yet been found to confer resistance to tigecycline. Here, we identified a novel RND efflux pump gene cluster, designated tmexCD1-toprJ1, on plasmids from five pandrug-resistant Klebsiella pneumoniae isolates of animal origin. TMexCD1-TOprJ1 increased (by 4- to 32-fold) the MICs of tetracyclines (including tigecycline and eravacycline), quinolones, cephalosporins, and aminoglycosides for K.pneumoniae, Escherichia coli, and Salmonella TMexCD1-TOprJ1 is closely related (64.5% to 77.8% amino acid identity) to the MexCD-OprJ efflux pump encoded on the chromosome of Pseudomonas aeruginosa In an IncFIA plasmid, pHNAH8I, the tmexCD1-toprJ1 gene cluster lies adjacent to two genes encoding site-specific integrases, which may have been responsible for its acquisition. Expression of TMexCD1-TOprJ1 in E. coli resulted in increased tigecycline efflux and in K. pneumoniae negated the efficacy of tigecycline in an in vivo infection model. Expression of TMexCD1-TOprJ1 reduced the growth of E. coli and Salmonella but not K. pneumoniaetmexCD1-toprJ1-positive Enterobacteriaceae isolates were rare in humans (0.08%) but more common in chicken fecal (14.3%) and retail meat (3.4%) samples. Plasmid-borne tmexCD1-toprJ1-like gene clusters were identified in sequences in GenBank from Enterobacteriaceae and Pseudomonas strains from multiple continents. The possibility of further global dissemination of the tmexCD1-toprJ1 gene cluster and its analogues in Enterobacteriaceae via plasmids may be an important consideration for public health planning.IMPORTANCE In an era of increasing concerns about antimicrobial resistance, tigecycline is likely to have a critically important role in the treatment of carbapenem-resistant Enterobacteriaceae, the most problematic pathogens in human clinical settings-especially carbapenem-resistant K.pneumoniae Here, we identified a new plasmid-borne RND-type tigecycline resistance determinant, TMexCD1-TOprJ1, which is widespread among K. pneumoniae isolates from food animals. tmexCD1-toprJ1 appears to have originated from the chromosome of a Pseudomonas species and may have been transferred onto plasmids by adjacent site-specific integrases. Although tmexCD1-toprJ1 still appears to be rare in human clinical isolates, considering the transferability of the tmexCD1-toprJ1 gene cluster and the broad substrate spectrum of TMexCD1-TOprJ1, further dissemination of this mobile tigecycline resistance determinant is possible. Therefore, from a "One Health" perspective, measures are urgently needed to monitor and control its further spread. The current low prevalence in human clinical isolates provides a precious time window to design and implement measures to tackle this.

Project description:Several variants of the plasmid-carried tigecycline resistance gene cluster, tmexCD-toprJ, have been identified. This study characterized another novel variant, tmexC6D6-toprJ1b, located on the chromosome of environmental-origin Pseudomonas mendocina. TMexC6D6-TOprJ1 mediates resistance to multiple drugs, including tigecycline. The promoter activity of tmexC6D6-toprJ1b and negative transcriptional repression by the upstream regulator tnfxB6 are crucial for the expression of tmexC6D6-toprJ1b. tmexC6D6-toprJ1b was found in the plasmids or chromosomes of different Pseudomonas species from six countries. Two genetic backgrounds, class 1 integrons and int-carrying integrase units, were found adjacent to the tmexC6D6-toprJ1b gene cluster and might mediate the transfer of this novel efflux pump gene cluster in Pseudomonas. Further phylogenetic analysis revealed Pseudomonas as the major reservoir of tmexCD-toprJ variants, warranting closer monitoring in the future. IMPORTANCE Tigecycline is one of the treatment options for serious infections caused by multidrug-resistant bacteria, and tigecycline resistance has gained extensive attention. The emergence of a transferable tigecycline resistance efflux pump gene cluster, tmexCD-toprJ, severely challenged the efficiency of tigecycline. In this study, we identified another novel tmexCD-toprJ variant, tmexC6D6-toprJ1b, which could confer resistance to multiple classes of antibiotics, including tigecycline. Although tmexC6D6-toprJ1b was found only in Pseudomonas species, tmexC6D6-toprJ1b might spread to Enterobacteriaceae hosts via mobile genetic elements resembling those of other tmexCD-toprJ variants, compromising the therapeutic strategies. Meanwhile, novel transferable tmexCD-toprJ variants are constantly emerging and mostly exist in Pseudomonas spp., indicating Pseudomonas as the important hidden reservoir and origin of tmexCD-toprJ variants. Continuous monitoring and investigations of tmexCD-toprJ are urgent to control its spread.

Project description:Staphylococcus aureus is a versatile human commensal bacteria and pathogen that causes various community and hospital-acquired infections. The S. aureus efflux pump NorA which belongs to the major facilitator superfamily, confers resistance to a range of substrates. Many efflux pump inhibitors (EPIs) have been discovered, but none is clinically approved due to their undesirable toxicities. In this study, we have screened clinically approved drugs for possible NorA EPI-like activity. We identified six drugs that showed the best efflux pump inhibition in vitro, with a fractional inhibitory concentration index of ≤0.5, indicating synergism with hydrophilic fluoroquinolones. The mechanistic validation of efflux inhibitory potential was demonstrated in ethidium bromide-based accumulation and efflux inhibition assays. We further confirmed the functionality of EPIs by norfloxacin accumulation assay depicting more realistic proof of the conjecture. None of the EPIs disturbed membrane function or depleted the ATP synthesis levels in bacteria. Both raloxifene and pyrvinium displayed an increase in bactericidal activity of ciprofloxacin in time-kill kinetics, prolonged its post-antibiotic effect, and reduced the frequency of spontaneous resistant mutant development. The combination of EPIs with ciprofloxacin caused significant eradication of preformed biofilms. Moreover, in the murine thigh infection model, a single dose of pyrvinium combined with ciprofloxacin reduced the bacterial burden significantly compared to untreated control and ciprofloxacin alone, indicating the efficacy of the combination. Conclusively, this study represents approved drugs that can be repurposed and combined with antibiotics as NorA EPIs, having anti-biofilm properties to treat severe S. aureus infections at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus is a frequent pathogen bacterium and the predominant cause of worsened nosocomial infections. Efflux pumps contribute to drug efflux and are reportedly associated with biofilm formation, thereby promoting difficult-to-treat biofilm-associated S. aureus infections. One strategy to combat these bacteria is to reduce active efflux and increase pathogen sensitivity to existing antibiotics. Repurposing approved drugs may solve the classical toxicity issues with previous efflux pump inhibitors and help reach sufficient plasma concentrations. We describe the in silico-based screening of FDA-approved drugs that identified six different molecules able to inhibit NorA pump (Major Facilitator Superfamily). Our study highlights that these compounds bind to and block the activity of the NorA pump and increase the sensitivity of S. aureus and methicillin-resistant S. aureus to fluoroquinolones. These drugs combined with fluoroquinolones significantly reduced the preformed biofilms and displayed significant efficacy in the murine thigh infection model when compared to untreated control and ciprofloxacin alone.

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Novel Tet(L) Efflux Pump Variants Conferring Resistance to Tigecycline and Eravacycline in Staphylococcus Spp.

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