Tigecycline Susceptibility and the Role of Efflux Pumps in Tigecycline Resistance in KPC-Producing Klebsiella pneumoniae

KPC-producing Klebsiella pneumoniae isolates have emerged as important pathogens of nosocomial infections, and tigecycline is one of the antibiotics recommended for severe infections caused by KPC-producing K. pneumoniae. To identify the susceptibility profile of KPC-producing K. pneumoniae to tigecycline and investigate the role of efflux pumps in tigecycline resistance, a total of 215 KPC-producing K. pneumoniae isolates were collected. The minimum inhibitory concentration (MIC) of tigecycline was determined by standard broth microdilution tests. Isolates showing resistance to tigecycline underwent susceptibility test with efflux pump inhibitors. Expression levels of efflux pump genes (acrB and oqxB) and their regulators (ramA, marA, soxS and rarA) were examined by real-time PCR, and the correlation between tigecycline MICs and gene expression levels were analysed. Our results show that the tigecycline resistance rate in these isolates was 11.2%. Exposure of the tigecycline-resistant isolates to the efflux pump inhibitor NMP resulted in an obvious decrease in MICs and restored susceptibility to tigecycline in 91.7% of the isolates. A statistically significant association between acrB expression and tigecycline MICs was observed, and overexpression of ramA was found in three tigecycline-resistant isolates, further analysis confirmed ramR mutations existed in these isolates. Transformation of one mutant with wild-type ramR restored susceptibility to tigecycline and repressed overexpression of ramA and acrB. These data indicate that efflux pump AcrAB, which can be up-regulated by ramR mutations and subsequent ramA activation, contributed to tigecycline resistance in K. pneumoniae clinical isolates.     

Suppression of stop codon UGA in acrB can contribute to antibiotic resistance in Klebsiella pneumoniae ATCC10031

We previously reported that Klebsiella pneumoniae MGH78578 exhibited higher resistance against various antimicrobials than K. pneumoniae ATCC10031. In this study, we showed that the plasmid, pKPN5, in K. pneumoniae MGH78578 played an important role in resistance against aminoglycosides, ampicillin, tetracycline, and chloramphenicol, while genome-derived β-lactamases and drug efflux pumps appeared to be more important in resistance to cloxacillin. acrAB, encoding a potent multidrug efflux pump, was cloned from K. pneumoniae MGH78578 and ATCC10031, to investigate reasons for the high drug resistance of K. pneumoniae MGH78578, and the results revealed that AcrAB from K. pneumoniae ATCC10031 conferred weaker drug resistance than AcrAB from K. pneumoniae MGH78578. DNA sequencing revealed that acrB from K. pneumoniae ATCC10031 carried the nonsense mutation, UGA, which was not found in acrB from K. pneumoniae MGH78578. However, acrB from K. pneumoniae ATCC10031 conferred slightly elevated resistant levels to several antimicrobials. The intact length of AcrB was detected in K. pneumoniae ATCC10031 by Western blot analysis, even though its quantity was small. Therefore, the stop codon UGA in acrB was thought to be overcome to some extent in this strain. We artificially introduced the nonsense mutation, UGA to the cat gene on pACYC184, and the plasmid also elevated the MIC of chloramphenicol in K. pneumoniae ATCC10031. These results suggest that a mechanism to overcome the nonsense mutation in acrB sustained resistance against a few β-lactams, dyes, and cholic acid in K. pneumoniae ATCC10031.     

First Emergence of acrAB and oqxAB Mediated Tigecycline Resistance in Clinical Isolates of Klebsiella pneumoniae Pre-Dating the Use of Tigecycline in a Chinese Hospital

Tigecycline is one of the few therapeutic options for treating infections caused by some multi-drug resistant pathogens, such as Klebsiella pneumoniae. However, tigecycline-resistant K. pneumoniae has been discovered recently in China. From 2009 to 2013, nine tigecycline-resistant K. pneumoniae isolates were identified in our hospital. Six of nine strains were identified before using tigecycline. To investigate the efflux-mediated resistance mechanisms of K. pneumoniae, the expression of efflux pump genes (acrA, acrB, tolC, oqxA and oqxB) and pump regulators (acrR, marA, soxS, rarA, rob and ramA) were examined by real-time RT-PCR. Molecular typing of the tigecycline resistant strains was performed. ST11 was the predominant clone of K. pneumoniae strains, while ST1414 and ST1415 were novel STs. Efflux pump inhibitor (EPI)-carbonyl cyanide chlorophenylhydrazone (CCCP) was able to reverse the resistance patterns of 5 resistant K. pneumoniae strains. In comparison with strain A111, a tigecycline-susceptible strain (negative control), we found that the expression levels of efflux pump genes and pump regulators were higher in a majority of resistant strains. Higher expression levels of regulators rarA (2.41-fold, 9.55-fold, 28.44-fold and 18.31-fold, respectively) and pump gene oqxB (3.87-fold, 31.96-fold, 50.61-fold and 29.45-fold, respectively) were observed in four tigecycline resistant strains (A363, A361, A368, A373, respectively). Increased expression of acrB was associated with ramA and marA expression. To our knowledge, studies on tigecycline resistance mechanism in K. pneumoniae are limited especially in China. In our study, we found that both efflux pump AcrAB-TolC and OqxAB contributed to tigecycline resistance in K. pneumoniae isolates.     

Role of an acrR mutation in multidrug resistance of in vitro-selected fluoroquinolone-resistant mutants of Salmonella enterica serovar Typhimurium

Quinolone resistance in Salmonella spp. is usually attributed to both active efflux and mutations leading to modification of the target enzymes DNA gyrase and topoisomerase IV. Here, we investigated the presence of mutations in the efflux regulatory genes of fluoroquinolone- and multidrug-resistant mutants of Salmonella enterica serovar Typhimurium (S. Typhimurium) selected in vitro with enrofloxacin that both carried a mutation in the target gene gyrA and overproduced the AcrAB efflux pump. No mutations were detected in the global regulatory loci marRAB and soxRS for the four strains studied. A mutation in acrR, the local repressor of acrAB, was found for two ciprofloxacin-resistant selected-mutants, leading to duplication of amino acids Ile75 and Glu76. Complementation experiments with wild-type acrR showed that the mutation identified in acrR partially contributed to the increase in resistance levels to several unrelated antibiotics. The acrR mutation also contributed to acrAB overexpression as shown by RT-PCR. Thus, this study underlines the role of an acrR mutation, in addition to the mutation in gyrA, in the fluoroquinolone and multidrug resistance phenotype of S. Typhimurium mutants, through overexpression of acrAB.     

基于串联质谱标签法和平行反应监测技术的氟喹诺酮耐药沙门菌蛋白质组学分析

【背景】随着沙门菌对氟喹诺酮类药物的耐药性不断增强,对其耐药机理的研究显得尤为迫切和重要,蛋白质组学分析将为沙门菌的耐药机理研究提供新的靶点和方向。【目的】对鼠伤寒沙门菌诱导获得耐药性前后进行蛋白质组学分析,为深入研究沙门菌耐药机理奠定基础。【方法】用环丙沙星对鼠伤寒沙门菌ATCC13311进行耐药性诱导,利用串联质谱标签法(Tandem mass tag,TMT)对其耐药性进行差异蛋白的筛选和生物信息学分析,并选取15个差异蛋白进行平行反应监测(Parallel reaction monitoring,PRM)靶向蛋白验证。【结果】筛选出318个差异表达蛋白,其中上调159个,下调159个,涉及的KEGG通路主要包括细菌趋药性、ABC转运蛋白、双组分系统等;PRM定量到13个验证蛋白且变化趋势与TMT一致。【结论】通过TMT定量结合PRM靶向验证对鼠伤寒沙门菌耐药前后进行蛋白质组学分析,筛选出多个差异蛋白和代谢通路,包括外排泵相关蛋白、外膜蛋白、双组分相关蛋白及通路、细菌趋化性相关蛋白及通路等,为沙门菌氟喹诺酮类耐药机理的深入研究奠定了基础。     

Anti‐Campylobacter and resistance‐modifying activity of Alpinia katsumadai seed extracts

Aims

We tested extracts from Alpinia katsumadai seeds for anti‐Campylobacter activity and investigated the roles of the CmeABC and CmeDEF efflux pumps in Campylobacter resistance to these natural phenolics. Additionally, we investigated an A. katsumadai ethanolic extract (AlpE) and other plant extracts as putative efflux pump inhibitors on Campylobacter isolates and mutants in efflux pump genes.

Methods and Results

AlpE showed antimicrobial activity against sensitive and multidrug‐resistant Campylobacter isolates. CmeB inactivation resulted in the greatest reduction in resistance, while cmeF and cmeR mutations produced only moderate effects on minimal inhibitory concentrations (MICs). The chemical efflux pump inhibitors additionally reduced MICs in isolates and mutants, confirming that active efflux is an important mechanism in resistance to AlpE, with additional contributions of other efflux systems. A notable decrease in resistance to tested antimicrobials in the presence of subinhibitory concentrations of AlpE confirms its modifying activity in Campylobacter spp.

Conclusions

AlpE is important anti‐Campylobacter source of antimicrobial compounds with resistance‐modifying activity. At least two of the efflux systems are involved in the resistance to A. katsumadai antimicrobial seed extracts.

Significance and Impact of the Study

This is the first report of antimicrobial and resistance‐modifying activity of AlpE from A. katsumadai seeds, demonstrating its potential in the control of Campylobacter in the food chain.     

An efflux inhibitor of the MacAB pump in Salmonella enterica serovar Typhimurium

Multidrug efflux pumps play an important role in bacterial multidrug resistance by actively excreting antibiotics. The ATP-binding cassette–type drug efflux pump MacAB was originally reported as a macrolide-specific pump. MacAB is also known to be required for the virulence of Salmonella enterica serovar Typhimurium following oral infection in mice. Here, we performed a screening of inhibitors of Salmonella MacAB and found a compound that increased the susceptibility of a MacAB-expressing strain to macrolides. It was previously reported that MacAB is required to resist peroxide-mediated killing in vitro and that a supernatant of wild-type Salmonella rescues the growth defect of a macAB mutant in H₂O₂. In this study, we also found that the MacAB inhibitor reduced the ability of the supernatant to rescue Salmonella cells in H₂O₂. This compound could lead to a better understanding of the function of MacAB.     

Exposure of Salmonella enterica serovar Typhimurium to high level biocide challenge can select multidrug resistant mutants in a single step

Background

Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux.

Methodology/Principal Findings

Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide.

Conclusions/Significance

These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations.     

Efflux systems in Serratia marcescens

A widespread bacterium Serratia marcescens (family Enterobacteriaceae) is an opportunistic pathogen and exhibits multiple drug resistance. Active removal of antibiotics and other antimicrobials from the cells by efflux systems is one of the mechanisms responsible for microbial resistance to these compounds. Among enterobacteria, efflux systems of Escherichia coli and Salmonella enterica ser. Typhimurium have been studied most extensively. Few efflux systems that belong to different families have been reported for S. marcescens. In this review, we analyzed available literature about S. marcescens efflux systems and carried out the comparative analysis of the genes encoding the RND type systems in different Serratia species and in other enterobacteria. Bioinformatical analysis of the S. marcescens genome allowed us to identify the previously unknown efflux systems based on their homology with the relevant E. coli genes. Identification of additional efflux systems in S. marcescens genome will promote our understanding of the physiology of these bacteria, will detect new molecular mechanisms of resistance, and will reveal their resistance potential.     

Mutations of Helicobacter pylori associated with fluoroquinolone resistance in Korea

Background and Aim: Fluoroquinolone resistance of Helicobacter pylori is known to be dependent on mutations in the QRDR of gyrA. This study was performed to investigate the distribution of gyrA point mutations and to evaluate the impact of the mutations on second‐line H. pylori eradication therapy. Methods: After H. pylori isolation from gastric mucosal specimens, fluoroquinolone resistance was examined using the agar dilution method. DNA sequencing of the QRDR of gyrA was performed in 89 fluoroquinolone‐resistant and 27 fluoroquinolone‐susceptible isolates. Transformation experiments were performed to confirm mutations in the resistant strains. The eradication rates of moxifloxacin‐containing triple therapy were evaluated depending on the resistance of fluoroquinolone. Results: The gyrA mutations were detected in 75.3% (55 of 73 strains) of the primary resistant strains and 100% (16 strains) of the secondary resistant strains. The most common mutations were Asp‐91 (36.0%) and Asn‐87 (33.7%). The MIC values in the transformed strains differed depending on the gyrA mutations, N87, and D91. Six patients with fluoroquinolone‐resistant strains received moxifloxacin‐containing triple therapy as the second‐line therapy, and two of three patients with Asn‐87 mutations (66.7%) failed in the eradication. By contrast, three patients with Asp‐91 mutations had successful eradication treatment. Conclusions: Fluoroquinolone resistance of H. pylori was caused by gyrA Asn‐87 and Asp‐91 point mutations. The Asn‐87 mutation seems to be an important determinant of failure of fluoroquinolone‐containing triple eradication therapy based on eradication results.     

Development of quinolone resistance and prevalence of different virulence genes among Shigella flexneri and Shigella dysenteriae in environmental water samples

The purpose of this study was to find out the mechanism of quinolone resistance in Shigella sp. isolated from environmental water samples from various parts of Kolkata, India. Out of 196 Shigella sp. isolated from 2014 to 2017, we selected 32 Shigella isolates for antimicrobial susceptibility tests. The minimum inhibitory concentrations (MIC) for quinolones ranged from 30 to 50 μg ml⁻¹ for ofloxacin, 5–20 μg ml⁻¹ for ciprofloxacin and 20–30 μg ml⁻¹ for norfloxacin. A few amino acid changes were found in quinolone resistance determining region (QRDR) of gyrA. Mutations in gyrA lead to a higher increment of MIC of quinolones. Among the plasmid-mediated (PMQR) quinolone resistance genes investigated, qnrB and aac(6')-lb-cr genes were found in all isolates. qnrA and qnrS were found in 25% and 62% of the isolates, respectively. ipaH gene was found in all of the isolates followed by the presence of other virulence genes ial, sen and stx1. Almost all the isolates having high MICs showed efflux pump activity in drug accumulation assay. All the mechanisms may or may not be present in a single strain. Several types of efflux pumps, presence of PMQR genes and mutations in drug target site of QRDR region may play the crucial role for resistance in our isolates.     

Determination of the Efflux Pump-Mediated Resistance Prevalence in Pseudomonas aeruginosa, Using an Efflux Pump Inhibitor

In Gram negative bacteria, fluoroquinolone resistance is acquired by target mutations in topoisomerase genes or by reducing the permeation of drugs due to the increase in expression of endogenous multidrug efflux pumps that expel structurally unrelated antimicrobial agents. An ongoing challenge is searching for new inhibitory substances in order to block efflux pumps and restore the antibiotic drugs susceptibility. In this research, we sought to investigate the interplay between ciprofloxacin and an efflux pump inhibitor (EPI), phenyl alanine arginyl β-naphtylamide (PAβN), to determine the prevalence of efflux pump overexpression in clinical isolates of Pseudomonas aeruginosa. Ciprofloxacin was tested at different concentrations (256–0.25 μg/ml) with a fixed concentration of PAβN (50 μg/ml). The isolates susceptibility profiles were analyzed by disc diffusion and agar dilution methods using 10 antibiotic discs and 4 powders. It was found that in the presence of PAβN, resistance to ciprofloxacin was inhibited obviously and MIC values were decreased. The comparison between subgroups of P. aeruginosa isolates with different resistance profiles indicates that efflux pump overexpression (EPO) is present in 35% of ciprofloxacin resistant isolates with no cross resistance and in variable frequencies among isolates showing cross resistance to other tested antibiotics: gentamicin (31%), ceftazidime (29%), and imipenem (18%). Altogether, these results imply that PAβN maybe effective to restore the fluoroquinolone drugs susceptibility in clinical treatment procedures. Results also show that increased use of a fluoroquinolone drug such as ciprofloxacin can affect the susceptibility of P. aeruginosa to other different antipseudomonal agents.     

The role of RamA on the development of ciprofloxacin resistance in Salmonella enterica serovar Typhimurium

Active efflux pump is a primary fluoroquinolone resistant mechanism of clinical isolates of Salmonella enterica serovar Typhimurium. RamA is an essential element in producing multidrug resistant (MDR) S. enterica serovar Typhimurium. The aim of the present study was to elucidate the roles of RamA on the development of ciprofloxacin, the first choice for the treatment of salmonellosis, resistance in S. enterica serovar Typhimurium. Spontaneous mutants were selected via several passages of S. enterica serovar Typhimurium CVCC541 susceptible strain (ST) on M-H agar with increasing concentrations of ciprofloxacin (CIP). Accumulation of ciprofloxacin was tested by the modified fluorometric method. The expression levels of MDR efflux pumps were determined by real time RT-PCR. In ST and its spontaneous mutants, the ramA gene was inactivated by insertion of the kan gene and compensated on a recombinant plasmid pGEXΦ(gst-ramA). The mutant prevention concentration (MPC) and mutant frequencies of ciprofloxacin against ST and a spontaneous mutant in the presence, absence and overexpression of RamA were tested. Four spontaneous mutants (SI1-SI4) were obtained. The SI1 (CIP MICs, 0.1 mg/L) without any target site mutation in its quinolone resistant determining regions (QRDRs) and SI3 (CIP MICs, 16 mg/L) harboring the Ser83→Phe mutation in its QRDR of GyrA strains exhibited reduced susceptibility and resistance to multidrugs, respectively. In SI1, RamA was the main factor that controlled the susceptibility to ciprofloxacin by activating MdtK as well as increasing the expression level of acrAB. In SI3, RamA played predominant role in ciprofloxacin resistance via increasing the expression level of acrAB. Likewise, the deficiency of RamA decreased the MPCs and mutant frequencies of ST and SI2 to ciprofloxacin. In conclusion, the expression of RamA promoted the development of ciprofloxacin resistant mutants of S. enterica serovar Typhimurium. The inhibition of RamA could decrease the appearance of the ciprofloxacin resistant mutants.     

Role of mutations in parC and gyrA in forming resistance of Mycoplasma hominis to fluoroquinolones

The set of the laboratory strain M. hominis H-34 mutants resistant to fluoroquinolones (ciprofloxacin-Cfl, lomefloxacin-Lfl, ofloxacin-Ofl) was obtained by selection in broth medium. The mutation was found in the quinolone resistance-determining region (QRDR) of A subunit of topoisomerase IV gene (parC) and new mutations were found in QRDR of genes encoding the A subunit of DNA gyrase (gyrA) in M. hominis mutants resistant to various concentrations of the Cfl, Lfl and Ofl. After multistep selection of the obtained mutants at constant concentrations of Cfl additional mutation Ser83 to Trp was revealed. No mutations in parE and gyrB were found. Mutations in parC for laboratory strain M. hominis H34 appeared at lower antibiotic concentrations than in gyrA. All mutations in gyr A were associated with mutations in parC. This confirms the previous data that topoisomerase IV is the primary target of Cfl and Ofl and suggests that it is the primary target of Lfl. Some M. hominis mutants selected at Ofl without any substitution in QRDRs were shown to be insensitive to Cfl and of Lfl. Studies of cross-resistance of the selected M. hominis mutants showed that their resistance to various fluoroquinolone concentrations could not depend on any mutations in QRDR of topoisomerase IV and DNA gyrase genes and suggests involvement of other unknown molecular mechanisms specific for Mycoplasmas.     

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of < 0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full‐length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane.