Whole-plasmid analysis of NDM-1-producing Acinetobacter seifertii isolate and its fitness in several Acinetobacter species.

OBJECTIVES: The aim of this study was to characterize an NDM-1-producing Acinetobacter seifertii isolates from a patient in South Korea. METHODS: Antibiotic susceptibility testing and genotyping using multigene sequencing were performed and whole plasmid sequences were determined. RESULTS: The genotype of A. seifertii was ST1899 and was resistant to ceftazidime, trimethoprim-sulfamethoxazole, and piperacillin-tazobactam, in addition to carbapenem. blaNDM-1 was surrounded by the ISAba125 insertion sequence within the structure of Tn125 in the 47 kb-sized plasmid. The plasmid exhibited a structure similar to that of other plasmids of diverse Acinetobacter sp. found worldwide. Transconjugation and the growth curve indicated that the plasmid was adapted to A. seifertii rather than other closely related Acinetobacter sp. CONCLUSIONS: Acquisition of carbapenem resistance by horizontal transfer of the blaNDM-1-carrying plasmid from another Acinetobacter species was found with no growth defect.

Antibiotic Efficacy in Escherichia coli and Klebsiella pneumoniae Under Nutrient Limitation and Effectiveness of Colistin-Based Antibiotic Combinations to Eradicate Persister Cells.

Persister cells are responsible for recurrent or chronic infections resulting in antibiotic treatment failure. We aimed to investigate antibiotic efficacy in Escherichia coli and Klebsiella pneumoniae strains with limited metabolic activity. Bacterial cells cultured in nutrient-limited media showed characteristic persister phenotypes, including low intracellular ATP concentration, maintenance of antibiotic susceptibility, and an increase of (p)ppGpp levels. Amikacin showed no bactericidal activity under nutrient limitation conditions; however, metabolism-dependent ciprofloxacin exhibited metabolism-independent activity. The activity of colistin was metabolism-dependent, but it was retained under limited nutrient conditions. Nutrient limitation and antibiotic stress were related to the SOS response through recA expression in all four strains of E. coli and K. pneumoniae. However, the mRNA expression patterns of relA and spoT (associated with (p)ppGpp synthesis) and hpf and rpoS (downstream target genes of (p)ppGpp signaling) varied according to bacterial species, strain, and antibiotics, indicating diverse responses to nutrient stress in various persister cells. We also investigated the efficacy of antibiotic combinations to eradicate persister cells. As a result, colistin-based combinations were effective in the eradication of both E. coli and K. pneumoniae persister cells. In this study, persister cells were shown to be induced by metabolic stress, reducing antibiotic efficacy. We identified that combinations of colistin with amikacin or ciprofloxacin were effective to eliminate E. coli and K. pneumoniae persister cells.

Plasmids Carrying blaVIM-2 in Acinetobacter nosocomialis and A. seifertii Isolates from South Korea.

We identified nine Verona integron-encoded metallo-ß-lactamase-2 (VIM-2)-producing Acinetobacter nosocomialis (n = 8) and Acinetobacter seifertii (n = 1) isolates from South Korea and performed whole-plasmid sequencing for two A. nosocomialis isolates and one A. seifertii isolate. Genotyping, antibiotic resistance profiles, and whole plasmid sequences indicated clonal dissemination of the eight VIM-2-producing A. nosocomialis isolates. The plasmid-bearing blaVIM-2 in the A. seifertii isolate differed from those in the A. nosocomialis isolates.

Evolved resistance to colistin and its loss due to genetic reversion in Pseudomonas aeruginosa.

The increased reliance on colistin for treating multidrug-resistant Gram-negative bacterial infections has resulted in the emergence of colistin-resistant Pseudomonas aeruginosa. We attempted to identify genetic contributors to colistin resistance in vitro evolved isogenic colistin-resistant and -susceptible strains of two P. aeruginosa lineages (P5 and P155). Their evolutionary paths to acquisition and loss of colistin resistance were also tracked. Comparative genomic analysis revealed 13 and five colistin resistance determinants in the P5 and P155 lineages, respectively. Lipid A in colistin-resistant mutants was modified through the addition of 4-amino-L-arabinose; this modification was absent in colistin-susceptible revertant strains. Many amino acid substitutions that emerged during the acquisition of colistin resistance were reversed in colistin-susceptible revertants. We demonstrated that evolved colistin resistance in P. aeruginosa was mediated by a complicated regulatory network that likely emerges through diverse genetic alterations. Colistin-resistant P. aeruginosa became susceptible to the colistin upon its withdrawal because of genetic reversion. The mechanisms through which P. aeruginosa acquires and loses colistin resistance have implications on the treatment options that can be applied against P. aeruginosa infections, with respect to improving bactericidal efficacy and preventing further resistance to antibiotics.

Comparison of the Virulence-Associated Phenotypes of Five Species of Acinetobacter baumannii Complex.

In this study, we compared the virulence-associated factors of Acinetobacter baumannii complex species. Sixty-three isolates of five A. baumannii complex species, including 19 A. baumannii, 15 A. nosocomialis, 13 A. seifertii, 13 A. pittii, and 3 A. calcoaceticus isolates, were included in this study. For all isolates, biofilm formation, A549 cell adherence, resistance to normal human serum, and motility were evaluated. A. baumannii complex isolates showed diversity in biofilm formation, A549 cell adherence, and serum resistance, and no strong positive relationships among these virulence characteristics. However, A. seifertii showed relatively consistent virulence-associated phenotypes. In addition, A. baumannii clone ST110 exhibited consistently high virulence-associated phenotypes. Motility was observed in seven isolates, and all four A. baumannii ST110 isolates showed twitching motility. Although some inconsistencies in virulence-associated phenotypes were seen, high virulence characteristics were observed in A. seifertii, which has been mainly reported in Korea and shows high rates of colistin resistance.

pspK gene prevalence and characterization of non-typable Streptococcus pneumonia isolates from Asian countries.

Recently, it has been reported that some non-typable (NT) Streptococcus pneumoniae isolates from Korea and other countries contained a novel gene pspK in the capsular polysaccharide synthesis (cps) locus. In this study, we investigated the presence of pspK in 120 NT S. pneumoniae isolates from 12 Asian countries; isolate characteristics were also examined. The presence of pspK was assayed by PCR. Clonality of NT S. pneumoniae isolates containing pspK was investigated by MLST and PFGE. Antimicrobial susceptibility testing was performed and the structure of pspK was also determined. Nineteen NT isolates (15.8 %) were identified as containing pspK: two isolates from Korea, four from Vietnam, two from Hong Kong, eight from Thailand, and one each from Taiwan, the Philippines and Saudi Arabia. Seven isolates from Korea, Vietnam and Thailand were identified as ST1106, whereas just one or two belonged to ST310, ST393, ST10137, ST2754 or ST4136. All but one of the ST1106 NT isolates showed non-susceptibility to penicillin, and all isolates were resistant to cefuroxime, erythromycin, clindamycin and trimethoprim/sulfamethoxazole. The structure of pspK was similar amongst 20 isolates, which had a R1-R2-like region and a variable number of repeats in the repetitive region. However, one isolate (P05-11) from the Philippines lacked the R1-R2 region. NT S. pneumoniae isolates containing pspK were distributed across several Asian countries. Although MLST analysis suggested that most pspK-containing NT S. pneumoniae isolates may have emerged independently, ST1106 isolates with the selective advantage of antimicrobial resistance may have disseminated clonally throughout the countries.

Development of colistin resistance in pmrA-, phoP-, parR- and cprR-inactivated mutants of Pseudomonas aeruginosa.

OBJECTIVES: Colistin susceptibility in Pseudomonas aeruginosa is associated with a lipopolysaccharide (LPS) structure that is controlled by the modulation of several two-component regulatory systems. In this study, we attempted to elucidate the role of these two-component systems in the development of colistin resistance in P. aeruginosa. METHODS: pmrA-, phoP-, parR- or cprR-inactivated mutants were constructed from a colistin-susceptible P5 strain. Colistin-resistant mutants (P5R, P5ΔpmrA-R, P5ΔphoP-R, P5ΔparR-R and P5ΔcprR-R) were developed in vitro from a wild-type strain (P5) and pmrA-, phoP-, parR- or cprR-inactivated mutants by serial passage in colistin-containing media. Expression levels of the pmrA, phoP, parR, cprR and arnB genes were determined and amino acid alterations of two-component regulatory systems during development of colistin resistance were also investigated. RESULTS: While P5ΔpmrA-R, P5ΔparR-R and P5ΔcprR-R showed elevated expression of the phoP gene, the expression levels of the pmrA, parR and cprR genes were not different between gene-inactivated mutants and the adapted colistin-resistant mutants. P5ΔphoP-R showed no significant elevation in expression of any of the pmrA, parR or cprR genes. The arnB gene was overexpressed in all in vitro-selected colistin-resistant mutants compared with colistin-susceptible wild-type and gene-inactivated mutants. Three amino acid alterations in PhoQ and three in ParS were identified in induced colistin-resistant mutants. CONCLUSIONS: Our data suggest that individual two-component systems may not be essential for the acquisition of colistin resistance in P. aeruginosa. The PhoPQ two-component system may play a major role in the development of colistin resistance in our strains, but alternative or compensatory pathways may exist.

Genomic variations between colistin-susceptible and -resistant Pseudomonas aeruginosa clinical isolates and their effects on colistin resistance.

OBJECTIVES: The emergence of colistin-resistant Pseudomonas aeruginosa is becoming a serious concern worldwide. We investigated genetic variations involved in the acquisition and loss of colistin resistance in three clinical isogenic P. aeruginosa isolates (GKK-1, GKK-2 and GKK-3) recovered from a single patient and assessed their impacts on colistin resistance. METHODS: We applied whole genome sequencing technology to identify single nucleotide polymorphisms and insertions or deletions in two colistin-resistant isolates compared with a susceptible isolate. RESULTS: Thirty-seven non-synonymous mutations in 33 coding sequences were detected in the colistin-resistant isolates GKK-1 and GKK-3. Only one gene (PA1375) was significantly down-regulated in both colistin-resistant isolates; this gene encodes erythronate-4-phosphate dehydrogenase. Among the eight genes that were up-regulated in the colistin-resistant isolates, three encoded hypothetical proteins (PA1938, PA2928 and PA4541) and five were predicted to be involved in core biological functions, encoding a cell wall-associated hydrolase (PA1199), a response regulator EraR (PA1980), a sensor/response regulator hybrid (PA2583), a glycosyltransferase (PA5447) and an arabinose efflux permease (PA5548). All mutants with allelic replacement of these candidate genes, apart from one (PA1375), exhibited increases in colistin susceptibility, ranging from 2- to 16-fold. Colistin susceptibility decreased in complemented strains compared with the mutants; however, in three cases, resistance did not reach wild-type level. CONCLUSIONS: This study demonstrates genetic differences between P. aeruginosa isogenic isolates and identifies novel determinants that may be associated with the acquisition of colistin resistance. These findings will lay the foundation for a complete understanding of the molecular mechanisms of colistin resistance in P. aeruginosa.