Xiaoting Hua

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.

Decreased susceptibility to tigecycline in Acinetobacter baumannii mediated by a mutation in trm encoding SAM-dependent methyltransferase

OBJECTIVES Acinetobacter baumannii is an important opportunistic pathogen and multidrug-resistant isolate. Although tigecycline is a potent antibiotic for treating infections with multidrug-resistant isolates, resistance is becoming a problem. This study aimed to explore the mechanism of tigecycline resistance in A. baumannii. METHODS A serial passage experiment was performed to collect isolates selected by tigecycline. The expression of efflux pumps was quantified in the final selected isolate, 19606-T8. The whole genome of 19606-T8 was sequenced and the putative mutations were confirmed using PCR and Sanger sequencing. A complementation experiment was performed to evaluate the contribution of the mutations to decreased susceptibility to tigecycline. The significance of a deletion mutation was further investigated in terms of growth rate and antibiotic susceptibilities. RESULTS We collected serial isolates by selective pressure of tigecycline, and designated them 19606-T1 to 19606-T8. The efflux pumps AdeABC, AdeFGH and AdeIJK were not overexpressed in 19606-T8, which had decreased susceptibility to tigecycline. Isolate 19606-T8 carried one deletion mutation in trm and three non-synonymous substitutions in msbA (A84V), lolA (P91L) and filC (N168K). The deletion mutation in trm (encoding S-adenosyl-L-methionine-dependent methyltransferase) resulted in decreased susceptibility to tigecycline as well as to minocycline and doxycycline. In complementation experiments, the MICs of tigecycline, minocycline and doxycycline in a tigecycline-resistant isolate were restored by complementation with wild-type trm. CONCLUSIONS Given that the deletion mutation in trm was associated with decreased susceptibility to tigecycline and that a wild-type trm plasmid could restore the susceptibility, trm is considered to play an important role in decreased susceptibility to tigecycline in A. baumannii.

Global transcriptional response of Acinetobacter baumannii to a subinhibitory concentration of tigecycline

Acinetobacter baumannii has emerged as a significant nosocomial pathogen worldwide. Multidrug resistance has limited the treatment options for A. baumannii. Tigecycline belongs to a new class of modified tetracycline antimicrobials known as glycylcyclines and has demonstrated good in vitro antimicrobial activity against multidrug-resistant Acinetobacter spp. However, tigecycline-resistant strains may be generated during treatment. To explore the response to tigecycline in A. baumannii, the transcriptional profile of A. baumannii was determined in the presence and absence of tigecycline. The results showed that a major facilitator superfamily (MFS) efflux pump and multiple transcriptional regulators possibly involved in the stress response and drug resistance were upregulated in response to tigecycline. Strong suppressors in the aerobic phenylacetate catabolic pathway and a number of ATP-binding cassette (ABC) transporters were also observed after exposure to tigecycline. Furthermore, A. baumannii showed a lower minimum inhibitory concentration of ceftazidime in the presence of tigecycline owing to downregulated OXA-23 and AmpC. These finding suggest that the response of A. baumannii to tigecycline is multifactorial and includes MFS family efflux pump transcriptional regulators and metabolic pathways, such as the phenylacetate catabolic pathway.

The Effect of Colistin Resistance-Associated Mutations on the Fitness of Acinetobacter baumannii

Acinetobacter baumannii had emerged as an important nosocomial and opportunistic pathogen worldwide. To assess the evolution of colistin resistance in A. baumannii and its effect on bacterial fitness, we exposed five independent colonies of A. baumannii ATCC 17978 to increasing concentrations of colistin in agar (4/5) and liquid media (1/5). Stable resistant isolates were analyzed using whole genome sequencing. All strains were colistin resistant after exposure to colistin. In addition to the previously reported lpxCAD and pmrAB mutations, we identified four novel putative colistin resistance genes: A1S_1983. hepA. A1S_3026, and rsfS. Lipopolysaccharide (LPS) loss mutants exhibited higher fitness costs than those of the pmrB mutant in nutrient-rich medium. The colistin-resistant mutants had a higher inhibition ratio in the serum growth experiment than that of the wild type strain in 100% serum. Minimum inhibitory concentration (MIC) results showed that the LPS-deficient but not the pmrB mutant had an altered antibiotic resistance profile. The compensatory mutations partially or completely rescued the LPS-deficient’s fitness, suggesting that compensatory mutations play an important role in the emergence and spread of colistin resistance in A. baumannii.

Step-Wise Increase in Tigecycline Resistance in Klebsiella pneumoniae Associated with Mutations in ramR, lon and rpsJ

Klebsiella pneumoniae is a gram-negative bacterium that causes numerous diseases, including pneumonia and urinary tract infections. An increase in multidrug resistance has complicated the treatment of these bacterial infections, and although tigecycline shows activity against a broad spectrum of bacteria, resistant strains have emerged. In this study, the whole genomes of two clinical and six laboratory-evolved strains were sequenced to identify putative mutations related to tigecycline resistance. Of seven tigecycline-resistant strains, seven (100%) had ramR mutations, five (71.4%) had lon mutations, one (14.2%) had a ramA mutation, and one (14.2%) had an rpsJ mutation. A higher fitness cost was observed in the laboratory-evolved strains but not in the clinical strains. A transcriptome analysis demonstrated high expression of the ramR operon and acrA in all tigecycline-resistant strains. Genes involved in nitrogen metabolism were induced in the laboratory-evolved strains compared with the wild-type and clinical strains, and this difference in nitrogen metabolism reflected the variation between the laboratory-evolved and the clinical strains. Complementation experiments showed that both the wild-type ramR and the lon genes could partially restore the tigecycline sensitivity of K. pneumoniae. We believe that this manuscript describes the first construct of a lon mutant in K. pneumoniae, which allowed confirmation of its association with tigecycline resistance. Our findings illustrate the importance of the ramR operon and the lon and rpsJ genes in K. pneumoniae resistance to tigecycline.

Genome Sequences of Two Multidrug-Resistant Acinetobacter baumannii Strains Isolated from a Patient before and after Treatment with Tigecycline

Acinetobacter baumannii is a Gram-negative bacterium which emerged as a significant nosocomial pathogen worldwide. To investigate the molecular basis of the tigecycline-resistant mechanism, we determined the genome sequences of two multidrug-resistant A. baumannii strains isolated from a patient before and after treatment with tigecycline.

Complete genome sequence of Acinetobacter baumannii XH386 (ST208), a multi-drug resistant bacteria isolated from pediatric hospital in China

Acinetobacter baumannii is an important bacterium that emerged as a significant nosocomial pathogen worldwide. The rise of A. baumannii was due to its multi-drug resistance (MDR), while it was difficult to treat multi-drug resistant A. baumannii with antibiotics, especially in pediatric patients for the therapeutic options with antibiotics were quite limited in pediatric patients. A. baumannii ST208 was identified as predominant sequence type of carbapenem resistant A. baumannii in the United States and China. As we knew, there was no complete genome sequence reproted for A. baumannii ST208, although several whole genome shotgun sequences had been reported. Here, we sequenced the 4087-kilobase (kb) chromosome and 112-kb plasmid of A. baumannii XH386 (ST208), which was isolated from a pediatric hospital in China. The genome of A. baumannii XH386 contained 3968 protein-coding genes and 94 RNA-only encoding genes. Genomic analysis and Minimum inhibitory concentration assay showed that A. baumannii XH386 was multi-drug resistant strain, which showed resistance to most of antibiotics, except for tigecycline. The data may be accessed via the GenBank accession number CP010779 and CP010780.

Abrp, a new gene, confers reduced susceptibility to tetracycline, glycylcine, chloramphenicol and fosfomycin classes in Acinetobacter baumannii

Acinetobacter baumannii, a non-fermenting gram-negative coccobacillus, is a major pathogen responsible for a variety of healthcare-associated infections, including pneumonia, urinary tract and bloodstream infections. Moreover, A. baumannii is associated with alarming increases in drug resistance rates to almost all available antibiotics leaving limited treatment options. Here, we characterize the biological functions of a novel gene, abrp, which encodes a peptidase C13 family. We demonstrate that the abrp is associated with decreased susceptibility to tetracycline, minocycline, doxycycline, tigecycline, chloramphenicol and fosfomycin. Deletion of abrp was able to increase cell membrane permeability and display slower cell growth rate. Results from the present study show that abrp plays an important role in conferring reduced susceptibility to different classes of antibiotics and cell growth in A. baumannii. The change of antibiotic sensitivities may result from modifications to the cell membrane permeability of A. baumannii.

Complete Genome Sequence of Klebsiella pneumoniae Sequence Type 17, a Multidrug-Resistant Strain Isolated during Tigecycline Treatment

ABSTRACT Klbesiella pneumoniae is one of the most important human pathogens and frequently causes many diseases. To facilitate the comparative genome analysis in tigecycline resistance mechanism, we report the complete chromosomal sequence of a multidrug-resistance K. pneumoniae strain before tigecycline treatment for reference genome.

Rapid emergence of high-level tigecycline resistance in Escherichia coli strains harbouring blaNDM-5 in vivo

Tigecycline (TIG) resistance is a growing concern because this antibiotic is regarded as one of the last resorts to treat infections caused by multidrug-resistant and extensively drug-resistant (XDR) bacteria. Information regarding TIG-resistant Escherichia coli isolates is scarce. In this study, we report the emergence of high-level TIG resistance in a longitudinal series of XDR E. coli isolates collected during TIG treatment. Whole-genome sequencing was performed for six E. coli strains harbouring bla(NDM-5) and genomic comparison revealed two amino acid substitutions. Mutation in rpsJ could be a significant factor conferring TIG resistance in these isolates. The fitness cost of TIG resistance in resistant strains was evaluated by determining the relative growth rate, indicating that TIG resistance reduced fitness by ca. 7%. This study is the first report to demonstrate high-level TIG resistance in E. coli in vivo. In addition, we report the first treatment-emergent minimum inhibitory concentration (MIC) development of TIG from 1mg/L to 64 mg/L in E. coli. Clinicians should be aware of the risk of an increase in the MIC of TIG under therapy.