Young Kyoung Park

Independent emergence of colistin-resistant Acinetobacter spp. isolates from Korea

High colistin resistance rates in Acinetobacter spp. were recently reported in Korean hospitals (J. Antimicrob. Chemother 2007;60:1163). In this study, we investigated if colistin-resistant Acinetobacter spp. isolates from Korean hospitals disseminated clonally or emerged independently. Multilocus sequence typing (MLST) analysis was performed for 58 colistin-resistant Acinetobacter spp. isolates: 8 isolates of the Acinetobacter baumannii subgroup A, 16 isolates of the A. baumannii subgroup B, and 34 isolates of the genomic species 13TU. A phylogenetic tree inferred from concatenated sequences of 7 MLST loci showed a clear distinction among the 3 Acinetobacter groups. In the MLST analysis, most colistin-resistant Acinetobacter spp. isolates showed different allele profiles at the 7 loci; that is, they belonged to different clones. Despite the clear distinction between the 3 Acinetobacter groups, interrelationships among the 3 groups were not consistent within the gene trees. In addition, some isolates showed clustering incongruent with their species or group identities in some gene trees. MLST analysis indicated that most colistin-resistant Acinetobacter spp. isolates from Korean hospitals arose independently. Considering the increasing use of colistin and the high recombination rate of Acinetobacter spp., independent but frequent emergence of colistin resistance in Acinetobacter spp. isolates is of great concern.

Correlation between overexpression and amino acid substitution of the PmrAB locus and colistin resistance in Acinetobacter baumannii

Relationships between the PmrAB two-component system and colistin resistance were investigated in Acinetobacter baumannii. The sequences of pmrA, pmrB and pmrC in 26 colistin-susceptible (ColS) and 7 colistin-resistant (ColR) A. baumannii isolates were determined. In addition, 30 ColR mutants (colistin minimum inhibitory concentration >64 mg/L) were selected in vitro from 10 ColS strains and the pmrA, pmrB and pmrC sequences of the in-vitro-selected ColR mutants were also determined. Expression of pmrA and pmrB was compared between the ColR mutants and their parent ColS strains using a quantitative real-time polymerase chain reaction method. Elevated expression of pmrA and pmrB genes was evident both in wild-type and in in-vitro-selected ColR strains. However, no amino acid differences in the pmrA, pmrB and pmrC genes were found between wild-type ColR and ColS isolates. Although six kinds of amino acid alterations in pmrB were identified in in-vitro-selected ColR mutants, no changes were found in some of the mutants. These findings indicate that increased expression of the PmrAB system is essential for colistin resistance in A. baumannii but that amino acid alterations might be only partially responsible for resistance.

Extreme Drug Resistance in Acinetobacter baumannii Infections in Intensive Care Units, South Korea

To the Editor: Acinetobacter spp. have emerged as a cause of nosocomial infections, especially in intensive care units (ICUs). In South Korea, Acinetobacter spp. was ranked as the third most frequently found pathogen in ICUs (1). With the emergence of multidrug-resistant (MDR) or pandrug-resistant (PDR) isolates, few drugs are now available to treat MDR or PDR Acinetobacter infections; polymyxins are the only therapeutic option in many cases (2). Current polymyxin resistance rates among Acinetobacter isolates are low worldwide (3). We report the emergence of extreme drug resistance (XDR) in A. baumannii isolates from patients in ICUs of Samsung Medical Center in Seoul, South Korea. These isolates were resistant to all tested antimicrobial drugs, including polymyxin B and colistin, to which PDR isolates are normally susceptible. Sixty-three nonduplicate Acinetobacter spp. isolates were collected from the ICUs from April through November 2007. Species identification was performed based on partial RNA polymerase β-subunit gene sequences, amplified rDNA restriction analysis, and the gyrase B gene–based multiplex PCR method (3). Forty-four isolates were identified as A. baumannii: 9 as genomic species 3, six as genomic species 13TU, 2 as A. baumannii-like species, and 1 each as A. junnii and genomic species 10. In vitro susceptibility testing was performed and interpreted by using the broth microdilution method according to the Clinical and Laboratory Standards Institute guidelines (4). Colistin and polymyxin B resistances were defined as MIC >4 mg/L (4). MDR was defined as characterized by resistance to >3 classes of antimicrobial drugs, and PDR was defined as characterized by resistance to all antimicrobial drugs, regardless of colistin and polymyxin B susceptibility. XDR was defined as resistance to all antimicrobial drugs. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed for all PDR isolates according to previously described methods (5,6). Genes encoding oxacillinases, such as those classified as OXA-23-like, OXA-24/40-like, OXA-51-like, and OXA-58-like, were detected as previously described (7). PCR and sequence analyses were performed to detect and characterize the other antimicrobial resistance genes, according to methods reported (8). Of 63 Acinetobacter isolates, 31.7% and 34.9% were resistant to imipenem and meropenem, respectively. Of the 63 isolates, 27.0% and 30.2% were resistant to polymyxin B and colistin, respectively. For the other antimicrobial drugs, Acinetobacter spp. isolates showed antimicrobial resistance rates >50%. Nineteen isolates (30.2%), all belonging to A. baumannii, were PDR. Most of these PDR isolates (16/19, 84.2%) were collected from endotracheal aspirate, and others were from peritoneal fluid and sputum. When characterized by PFGE and MLST, all PDR isolates belonged to a single clone, ST22, and all contained the blaOXA-23 and blaOXA-66 genes. ISAba1 was detected upstream of blaOXA-23 and blaOXA-66 in all PDR isolates. In addition, most PDR isolates contained blaTEM-116, blaPER-1, and blaADC-29 genes. TEM-116 is a point mutant derivative of TEM-1, Val84→Ile. All β-lactamase genes were located on a plasmid. Also, ISAba1 was located at the upstream of all the blaADC, which was shown by PCR. However, none of the isolates had blaCTX-M, blaVEB, blaIMP, blaVIM, or blaGIM. Of the PDR isolates, 8 were resistant even to colistin and polymyxin B. These 8 isolates also showed resistance to tigecycline (MICs 4 mg/L). Thus, they were resistant to all antimicrobial drugs tested in this study and were considered to have XDR. The underlying diseases of the patients whose isolates were examined varied (Table). Although 2 isolates with XDR were colonizers, 6 caused infections. All but 1 patient was treated with mechanical ventilation before isolation of the pathogen. Number of hospital days before isolation of A. baumannii was 13–256 days, and the number of ICU days before isolation was 2–38 days. Four patients were immunocompromised, and 3 had bacteremia. Among the patients with infections characterized by XDR, the overall 30-day mortality rate was 66.7%, and the infection-related 30-day mortality rate was 50.0%. All 8 isolates with XDR showed common characteristics: ST22 containing OXA-23, OXA-66, TEM-116, PER-1, and ADC-29. Table Clinical characteristics of 8 patients infected with extremely drug-resistant Acinetobacter baumannii isolates, South Korea* We report the emergence of XDR in PDR A. baumannii isolates in South Korea. Characteristics of PDR A. baumannii isolates suggest that they spread from a single clone. A single A. baumannii strain with XDR might evolve from the prevailing PDR A. baumannii and could disseminate in the ICU, probably after contamination of the hospital environment and by nosocomial transmission. In South Korea, a high resistance rate to imipenem and meropenem in Acinetobacter spp. isolates may lead to extensive use of polymyxins (3). Thus, we can hypothesize that the most prevalent carbapenem-resistant, or MDR A. baumannii clone, became PDR and then evolved into clones with XDR by acquisition of polymyxin resistance caused by antimicrobial pressure. Our investigation showed a simultaneous emergence of resistance to all antimicrobial agents available, including colistin, polymyxin B, and tigecycline. XDR poses serious problems in the treatment of patients with A. baumannii infections, especially given the slow development of new antimicrobial agents.

KPC-Producing Extreme Drug-Resistant Klebsiella pneumoniae Isolate from a Patient with Diabetes Mellitus and Chronic Renal Failure on Hemodialysis in South Korea

The prevalence of extended-spectrum β-lactamases (ESBLs) has limited the available therapeutic options and necessitated the increased use of carbapenems against Klebsiella pneumoniae infections. In response to use of carbapenems, carbapenem resistance has developed and flourished. In 1996, a new β-lactamase termed “K. pneumoniae carbapenemase” (KPC) was reported in New York, NY (12). Subsequently, KPC-producing K. pneumoniae has been spread worldwide (9). However, it has not hitherto been reported in South Korea. We report a case of infection with a KPC-producing extreme drug-resistant (XDR) K. pneumoniae isolate in a tertiary care hospital in South Korea. A 65-year-old male patient who had diabetes mellitus and chronic renal failure on hemodialysis visited the emergency room because of persistent watery diarrhea. Upon admission, blood pressure was 80/55 mm Hg and body temperature was 37.8°C. Blood pressure became 100/65 mm Hg after a massive fluid administration. Sigmoidoscopy showed whitish dirty mucous membrane, compatible with pseudomembranous colitis. The patient was admitted to the intensive care unit under the diagnosis of sepsis due to pseudomembranous colitis. The patient received oral vancomycin because of refractoriness of metronidazole. On postadmission day 7, the patient exhibited vomiting and chest pain. An electrocardiogram and blood work revealed an acute myocardial infarction and ventricular tachycardia. An emergency angiography was performed uneventfully. Postoperatively, the patient displayed purulent sputum, tachypnea, and fever up to 39.5°C. A chest radiogram revealed infiltration on the right lower lobe. Meropenem was initiated to treat the hospital-acquired pneumonia. Six days later, a chest radiogram revealed expansion of the infiltration to the middle and upper lobes. The patient displayed a fever of 39.2°C and blood pressure of 70/40 mm Hg. The patient was treated as for septic shock with a mechanical ventilator, inotropics, and administration of broad-spectrum antibiotics including vancomycin and colistin. Gram stain of bronchial aspirates revealed one to three Gram-negative organisms per high-power field. The patients condition did not improve, and he was discharged with a poor prognosis. During hospitalization, antimicrobial susceptibility testing was performed by a broth microdilution method according to CLSI guidelines (3). The isolate, Kpn-DK2, was nonsusceptible to all antimicrobial agents tested (Table ​(Table1)1) and displayed intermediate resistance even to tigecyline (MIC of 4 mg/liter). The isolate could be defined as XDR (11). Kpn-DK2 was ESBL positive (3) but metallo-β-lactamase (MBL) negative (2). Kpn-DK2 was positive for blaTEM, blaSHV, and blaCTX-M but was negative for blaCMY, blaP99, blaVIM, blaIMP, and blaACT (5, 7) As a result of sequencing, Kpn-DK2 was proven to contain blaCTX-M-15 as an ESBL gene, and blaTEM-1 and blaSHV-11 were also evident. In addition, blaKPC-2 was also present, which is the first detection in South Korea. TABLE 1. Antimicrobial resistance of K. pneumoniae isolate Kpn-DK2 To investigate the genotype of Kpn-DK2, multilocus sequence typing (MLST) was performed as described previously (4), which revealed ST11. MLST has demonstrated that many KPC-producing K. pneumoniae isolates belong to ST258 (1, 6, 8, 10). Although ST11 KPC-producing K. pneumoniae isolates have not been previously reported, to our knowledge, ST258 and ST11 are single locus variants of each other that differ in the tonB allele, and both are widespread clonal groups. Notably, ST11 is the most common clone of ESBL-producing K. pneumoniae isolates in South Korea (unpublished data). Despite lack of evidence of the spread of KPC-producing isolates, it is worrisome that a KPC-producing K. pneumoniae isolate has been found in a common ESBL-producing clone in South Korea.

In vivo protection against ZIKV infection and pathogenesis through passive antibody transfer and active immunisation with a prMEnv DNA vaccine

Significant concerns have been raised owing to the rapid global spread of infection and disease caused by the mosquito-borne Zika virus (ZIKV). Recent studies suggest that ZIKV can also be transmitted sexually, further increasing the exposure risk for this virus. Associated with this spread is a dramatic increase in cases of microcephaly and additional congenital abnormalities in infants of ZIKV-infected mothers, as well as a rise in the occurrence of Guillain Barre’ syndrome in infected adults. Importantly, there are no licensed therapies or vaccines against ZIKV infection. In this study, we generate and evaluate the in vivo efficacy of a novel, synthetic, DNA vaccine targeting the pre-membrane+envelope proteins (prME) of ZIKV. Following initial in vitro development and evaluation studies of the plasmid construct, mice and non-human primates were immunised with this prME DNA-based immunogen through electroporation-mediated enhanced DNA delivery. Vaccinated animals were found to generate antigen-specific cellular and humoral immunity and neutralisation activity. In mice lacking receptors for interferon (IFN)-α/β (designated IFNAR−/−) immunisation with this DNA vaccine induced, following in vivo viral challenge, 100% protection against infection-associated weight loss or death in addition to preventing viral pathology in brain tissue. In addition, passive transfer of non-human primate anti-ZIKV immune serum protected IFNAR−/− mice against subsequent viral challenge. This study in NHP and in a pathogenic mouse model supports the importance of immune responses targeting prME in ZIKV infection and suggests that additional research on this vaccine approach may have relevance for ZIKV control and disease prevention in humans.

Two distinct clones of carbapenem-resistant Acinetobacter baumannii isolates from Korean hospitals

We investigated the characteristics of 48 carbapenem-resistant Acinetobacter baumannii isolates collected from 5 tertiary care hospitals in Korea by multilocus sequencing typing, pulsed-field gel electrophoresis, and polymerase chain reaction amplification of the antimicrobial resistance determinants. We identified 2 distinct main clones of carbapenem-resistant A. baumannii isolates, which showed different antimicrobial resistance profiles and are also differentiated by the kinds of oxacillinase (OXA) carbapenemases and Acinetobacter-derived cephalosporinase (ADC) beta-lactamases. One main clone, ST22:A, had 27 carbapenem-resistant isolates (56.3%), showed high polymyxin B and colistin resistances (33.3% and 37.0%, respectively), and contained both bla(OXA-51-like) and bla(OXA-23-like) genes and the bla(ADC-29) or bla(ADC-30) gene. In contrast, the other main clone, ST28:B, included 15 isolates (31.3%), showed complete susceptibilities to polymyxin B and colistin, and contained only the bla(OXA-51-like) gene and bla(ADC-31) or bla(ADC-32) genes. The distribution of these main carbapenem-resistant A. baumannii clones did not relate to locality, indicating that they are widespread in Korean hospitals. In addition, we found new types of PER beta-lactamases, PER-6.

Changes in antimicrobial susceptibility and major clones of Acinetobacter calcoaceticus–baumannii complex isolates from a single hospital in Korea over 7 years

Acinetobacter species have emerged as opportunistic nosocomial pathogens in intensive care units. Epidemic spread and outbreaks of multidrug-resistant or carbapenem-resistant Acinetobacter baumannii infections have been described worldwide. Species distribution, antimicrobial resistance and genotypes were investigated for Acinetobacter species isolates collected from a single institution in Korea over 7 years. Two hundred and eighty-seven Acinetobacter species isolates were collected from patients with bloodstream infections in one Korean hospital from 2003 to 2010. Most of them belonged to the Acinetobacter calcoaceticus-baumannii complex (94.4 %). The most frequently isolated species was A. baumannii (44.2 %), followed by Acinetobacter nosocomialis (formerly Acinetobacter genomic species 13TU) (34.1 %). The proportion of A. baumannii increased significantly from 2008 to 2010 (40.4 to 50.0 %). From 2008, imipenem and meropenem resistance rates increased significantly compared with 2003-2007 (12.9 % and 20.5 %, respectively, to 41.4 % and 41.5 %, respectively). An increased carbapenem resistance rate between the two periods was identified more clearly amongst A. baumannii isolates. Polymyxin-resistant A. baumannii isolates emerged in 2008-2010, despite the availability of few isolates. The increase of carbapenem resistance in A. baumannii might be due to the substitution of main clones. Although ST92 and ST69 were the most prevalent clones amongst A. baumannii in 2003-2007 (47.8 % and 15.9 %, respectively), ST75 and ST138 had increased in 2008-2010 (39.7 % and 25.9 %, respectively). Although ST92 showed moderate resistance to carbapenems, most ST75 and ST138 isolates were resistant to carbapenems. All ST75 and ST138 isolates, but only one ST92 isolate, contained the bla(OXA-23-like) gene. Increased carbapenem resistance in Acinetobacter species and A. baumannii isolates might be due to the expansion of specific carbapenem-resistant clones.

A Single Clone of Acinetobacter baumannii, ST22, Is Responsible for High Antimicrobial Resistance Rates of Acinetobacter Spp. Isolates That Cause Bacteremia and Urinary Tract Infections in Korea

We investigated the characteristics of a total of 96 Acinetobacter spp. isolates that were shown to cause bacteremia and urinary tract infections (UTIs) from 10 university hospitals located in various regions of Korea from November 2006 to August 2007. The antimicrobial susceptibilities of these isolates were determined using a broth microdilution method, and the species were identified using molecular identification. In addition, we performed multilocus sequence typing for Acinetobacter baumannii subgroup A isolates. A. baumannii subgroup A was the most prevalent in patients with both bacteremia (32 isolates, 53.3%) and UTIs (20 isolates, 55.6%), followed by Acinetobacter genomic species 13TU (15.0% and 27.8% in bacteremia and UTIs, respectively). A. baumannii subgroup B and Acinetobacter junii were found exclusively in isolates causing bacteremia (seven and five isolates, respectively). Among 96 Acinetobacter spp. isolates, 19.8% were resistant to imipenem and 25.0% were resistant to meropenem. Most carbapenem-resistant A. baumannii isolates contained PER or oxacillinase-23-like enzymes (65.2% and 78.3%, respectively). In addition, 13.5% were resistant to polymyxin B and 17.7% were resistant to colistin. A. baumannii subgroup A isolates (52 isolates, 54.2%) showed higher resistance rates to most antimicrobial agents than other species, but not to colistin. Among A. baumannii subgroup A isolates, ST22 was the most prevalent genotype (33 isolates, 63.5%) and showed higher resistance rates to all antimicrobial agents than the other genotypes. In addition, four out of five polymyxin-resistant A. baumannii group A isolates belonged to ST22. Thus, dissemination of the main clone of A. baumannii, ST22, may contribute to the high resistance rates of Acinetobacter isolates to antimicrobials, including carbapenems, in Korea.

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.

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.