nsong Yu

Emergence of NDM-1-producing Acinetobacter baumannii in China

Sir, New Delhi metallo-b-lactamase (NDM) is a class B b-lactamase that confers resistance to virtually all b-lactams, including carbapenems. Initially reported in Sweden in Enterobacteriaceae isolates from a patient transferred from India, NDM-1 is currently spreading worldwide, including Belgium, especially in Enterobacteriaceae. Recently the presence of blaNDM-1 was also reported in Acinetobacter baumannii isolates from India, China and Germany, suggesting a broad host range distribution of this gene. – 4 Here, we report an NDM-1-producing A. baumannii detected in Belgium that was isolated from a patient following medical repatriation from Algeria. In early 2011, a young male patient was admitted to the emergency ward of an Algerian hospital for severe cranial and thoracic trauma following a road traffic accident. He presented with a Glasgow Coma Score (GCS) of 07/15 and was rapidly intubated, ventilated and transferred to the intensive care unit (ICU). While hospitalized in Algeria, he experienced three successive, distinct episodes of bloodstream infection, due to Acinetobacter spp., Candida albicans and Klebsiella pneumoniae, but no clinical information was available concerning the possible sources of these infections. In August, the patient was transferred, in a vegetative state (GCS of 02/15), to the ICU of a Belgian hospital. He was immediately placed in a single room with contact isolation precautions. Screening for asymptomatic intestinal carriage of carbapenemase producers was carried out upon admission, by rectal swabbing cultured by direct plating on ChromIDTM ESBL Agar (bioMérieux, Marcy l’Étoile, France). The recovered isolate was identified as A. baumannii (Ab 11314) by MicroFlex LT (Bruker Daltonik, Bremen, Germany). By disc diffusion, Ab 11314 was found to be multiresistant and synergy was observed in a double disc test using imipenem and EDTA (420 mg), suggesting involvement of a metallo-b-lactamase in resistance to b-lactams. Use of the CLSI microdilution method confirmed that the isolate was resistant to b-lactams including ceftazidime (MIC .64 mg/L), cefepime (MIC .64 mg/L), imipenem (MIC 4 mg/L; Etest MIC .32 mg/L), meropenem (MIC 4 mg/L; Etest MIC .32 mg/L), amikacin (MIC .32 mg/L) and ciprofloxacin (MIC .32 mg/L), and susceptible to tigecycline (MIC 0.125 mg/L), colistin (MIC 0.5 mg/L) and minocycline (MIC ,2 mg/L). Analysis of b-lactamase-coding genes by an MDR CT102 array (Check-Points, Wageningen, The Netherlands) and by PCR sequencing targeting metallo-b-lactamases and OXAcarbapenemases revealed the presence of blaNDM-1. Plasmid extraction was performed with a Qiagen plasmid kit, and this revealed a single 174 kb untypeable plasmid. This plasmid was efficiently electroporated and transferred by mating experiments to the A. baumannii recipient strain N9040364, and it conferred resistance to aminoglycosides (gentamicin, tobramycin and amikacin) only (data not shown). The blaNDM-1 gene was not associated with this plasmid suggesting that it was located in the chromosome. PCR mapping and sequencing revealed that blaNDM-1 was located between two direct repeats of the ISAba125 element in a transposon similar to the one in A. baumannii Ab 161/07 reported from Germany (accession no. HQ857107). Nevertheless, a PCR experiment performed with Ab 161/07 as a positive control revealed that in Ab 11314, in contrast, the mfs gene (a shikimate-transporter-coding gene belonging to the major facilitator superfamily) was not disrupted, indicating that the ISAba125 transposon was inserted elsewhere in the chromosome. By multilocus sequence typing (MLST) (http:// pubmlst.org/abaumannii/), A. baumannii Ab 11314 was shown to belong to sequence type (ST) 92 (allelic profile: 1-3-3-2-2-7-3), the founding genotype of clonal complex (CC) 92, which includes European clone 2 (EU2) and worldwide lineage 2 (WW2). CC92 is one of the most prevalent CCs worldwide, mostly represented by OXA-23-producing strains. Sequencing analysis of the intrinsic blaOXA-51-like and blaADC genes showed that this strain harboured blaOXA-64 and blaADC-26, neither of which was downstream of ISAba1 or ISAba125. These two intrinsic genes were identical to those recovered in the NDM-1-expressing A. baumannii Ab 161/07, but nevertheless presented a very different MLST profile (1-15-2-28-1-new allele-32), not related to any CC described up to now. This clinical isolate is the first characterized NDM-1-producing A. baumannii from Belgium. As yet, there have been only a few reports of NDM-producing A. baumannii, and the only case reported in Europe was from Germany in a patient transferred from Serbia in 2007. In the present case, the NDM-1-producing A. baumannii isolate originated from North Africa (Algeria) where NDM-1 has not yet been described in A. baumannii, although another single case of A. baumannii producing an NDM-1 variant (NDM-2) was recently reported from Egypt. This brief report further highlights the wide distribution of NDM-producing organisms around the world, their constant evolving spread Research letters

Resistance trends among clinical isolates in China reported from CHINET surveillance of bacterial resistance, 2005-2014

With the aim of gathering temporal trends on bacterial epidemiology and resistance from multiple laboratories in China, the CHINET surveillance system was organized in 2005. Antimicrobial susceptibility testing was carried out according to a unified protocol using the Kirby-Bauer method or automated systems. Results were analyzed according to Clinical and Laboratory Standards Institute (CLSI) 2014 definitions. Between 2005 and 2014, the number of bacterial isolates ranged between 22,774 and 84,572 annually. Rates of extended-spectrum β-lactamase production among Escherichia coli isolates were stable, between 51.7 and 55.8%. Resistance of E. coli and Klebsiella pneumoniae to amikacin, ciprofloxacin, piperacillin/tazobactam and cefoperazone/sulbactam decreased with time. Carbapenem resistance among K. pneumoniae isolates increased from 2.4 to 13.4%. Resistance of Pseudomonas aeruginosa strains against all of antimicrobial agents tested including imipenem and meropenem decreased with time. On the contrary, resistance of Acinetobacter baumannii strains to carbapenems increased from 31 to 66.7%. A marked decrease of methicillin resistance from 69% in 2005 to 44.6% in 2014 was observed for Staphylococcus aureus. Carbapenem resistance rates in K. pneumoniae and A. baumannii in China are high. Our results indicate the importance of bacterial surveillance studies.

Epidemiological characteristics and genetic structure of blaNDM-1 in non-baumannii Acinetobacter spp. in China

OBJECTIVES The goal of this study was to investigate the epidemiological characteristics and the surrounding genetic structure of bla(NDM-1) in non-baumannii Acinetobacter spp. in China. METHODS Non-baumannii Acinetobacter spp. were collected from 28 provinces in China and were screened for the presence of bla(NDM-1) using PCR. The following four methods were used to classify the Acinetobacter isolates: the Vitek 2 system, 16S-23S rRNA gene intergenic spacer sequencing, amplified rDNA restriction analysis and partial rpoB sequence analysis. An S1-PFGE assay and Southern blot hybridization were performed to determine the plasmid location of bla(NDM-1). The transferability of bla(NDM-1)-harbouring plasmids was confirmed by conjugation experiments and electrotransformation. The surrounding genetic structure of the bla(NDM-1) gene was analysed using a restriction endonuclease-based cloning approach and primer walking. RESULTS Among 726 non-baumannii Acinetobacter spp., nine isolates collected from six different provinces and assigned to seven different Acinetobacter spp. contained the bla(NDM-1) gene. None of these isolates was directly infectious to the patients or demonstrated an epidemiological importation from abroad. These bla(NDM-1) genes were located on plasmids that could be transferred to Escherichia coli J53 by conjugation and Acinetobacter baylyi ADP1 by electrotransformation. Seven of the nine strains shared a common genetic structure in which bla(NDM-1) was flanked by two copies of ISAba125. CONCLUSIONS The clinical challenge posed by bla(NDM-1) is currently minimal in China; however, more attention should be devoted to monitoring the dissemination of this gene due to its potential transferability via the ISAba125-associated transposon.

Prevalence of mcr-1 in Escherichia coli and Klebsiella pneumoniae recovered from bloodstream infections in China: a multicentre longitudinal study

BACKGROUND Polymyxin antibiotics are used as last-resort therapies to treat infections caused by multidrug-resistant Gram-negative bacteria. The plasmid-mediated colistin resistance determinant MCR-1 has been identified in Enterobacteriaceae in China. We did this study to investigate the prevalence of the mcr-1 gene in clinical isolates from patients with bloodstream infections in China. METHODS Clinical isolates of Escherichia coli and Klebsiella pneumoniae were collected from patients with bloodstream infections at 28 hospitals in China, then screened for colistin resistance by broth microdilution and for the presence of the mcr-1 gene by PCR amplification. We subjected mcr-1-positive isolates to genotyping, susceptibility testing, and clinical data analysis. We established the genetic location of mcr-1 with Southern blot hybridisation, and we analysed plasmids containing mcr-1 with filter mating, electroporation, and DNA sequencing. FINDINGS 2066 isolates, consisting of 1495 E coli isolates and 571 K pneumoniae isolates were collected. Of the 1495 E coli isolates, 20 (1%) were mcr-1-positive, whereas we detected only one (<1%) mcr-1-positive isolate among the 571 K pneumoniae isolates. All mcr-1-positive E coli and K pneumoniae isolates were resistant to colistin, with minimum inhibitory concentrations values in the range of 4-32 mg/L, except for one E coli isolate that had a minimum inhibitory concentration less than or equal to 0·06 mg/L. All 21 mcr-1-positive isolates were susceptible to tigecycline and 20 isolates (95%) were susceptible to the carbapenem and β-lactamase inhibitor combination piperacillin and tazobactam. One mcr-1-positive E coli isolate also produced NDM-5, which confers resistance to beta-lactam antibiotics. The 21 mcr-1-positive isolates were clonally diverse and carried mcr-1 on two types of plasmids, a 33 kb IncX4 plasmid and a 61 kb Inc12 plasmid. The 30 day mortality of the patients with bloodstream infections caused by mcr-1-positive isolates was zero. INTERPRETATION mcr-1-positive isolates from bloodstream infections were rare, sporadic, and remained susceptible to many antimicrobial agents. E coli, rather than K pneumoniae, was the main host of the mcr-1 gene. Further studies are needed to clarify the clinical impact of this novel resistance gene. FUNDING National Natural Science Foundation of China.

Wide distribution of CC92 carbapenem-resistant and OXA-23-producing Acinetobacter baumannii in multiple provinces of China

Carbapenem-resistant Acinetobacter baumannii has spread rapidly across China and is currently reported to be a worldwide nosocomial menace. In light of its increasing clinical significance, this study aimed to investigate the molecular epidemiology of carbapenem-resistant and carbapenem-susceptible isolates obtained from multiple provinces of China. Antimicrobial susceptibility was determined by disk diffusion assay according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Multilocus sequence typing (MLST) was used to investigate the molecular epidemiology of the isolates. In addition, a minimum spanning tree algorithm was applied to cluster sequence types (STs) into clonal complexes (CCs) in order to analyse their evolutionary relatedness. Generally, the average rate of resistance to most antibiotics in carbapenem-resistant isolates was extremely high (>85%), except for minocycline (20.45%). Analysis of MLST data confirmed that the genetic background of carbapenem-resistant isolates was relatively simple, with ST92 being the most prevalent clone, occurring in every province, followed by ST138, ST75 and ST381. In contrast, carbapenem-susceptible isolates had a more diverse genetic background. Furthermore, the most widely distributed CC of carbapenem-resistant isolates was bla(OXA-23)-like-producing and predominantly CC92, which incorporate ST136 and its several single-locus variants. Interestingly, isolates belonging to CC92 possessed higher antibiotic resistance rates compared with other STs. Overall, these observations suggest a wide distribution of carbapenem-resistant and bla(OXA-23)-like-producing clone CC92, especially ST92, ST75 and ST138, as the principal reason for the rapidly increasing carbapenem resistance rate in China.

High Incidence and Endemic Spread of NDM-1-Positive Enterobacteriaceae in Henan Province, China

ABSTRACT The emergence and spread of New Delhi metallo-β-lactamase 1 (NDM-1)-producing carbapenem-resistant Enterobacteriaceae (CRE) present an urgent threat to human health. In China, the blaNDM-1 gene has been reported mostly in Acinetobacter spp. but is rarely found in Enterobacteriaceae. Here, we report a high incidence and endemic spread of NDM-1-producing CRE in Henan Province in China. Sixteen (33.3%) of the 48 CRE isolates obtained from patients during June 2011 to July 2012 were positive for blaNDM-1, and the gene was found to be carried on plasmids of various sizes (∼55 to ∼360 kb). These plasmids were readily transferrable to recipient Escherichia coli by conjugation, conferred resistance to multiple antibiotics, and belonged to multiple replicon types. The blaNDM-1-positive CRE isolates were genetically diverse, and six new multilocus sequence typing (MLST) sequence types were linked to the carriage of NDM-1. Five of the isolates were classified as extensively drug-resistant (XDR) isolates, four of which also carried the fosA3 gene conferring resistance to fosfomycin, an alternative drug for treating infections by CRE. In each blaNDM-1-positive CRE isolate, the blaNDM-1 gene was downstream of an intact ISAba125 element and upstream of the bleMBL gene. Furthermore, gene environment analysis suggested the possible transmission of blaNDM-1-containing sequences from Acinetobacter spp. to Klebsiella pneumoniae and Klebsiella oxytoca. These findings reveal the emergence and active transmission of NDM-1-positive CRE in China and underscore the need for heightened measures to control their further spread.

Dissemination of a clone carrying a fosA3-harbouring plasmid mediates high fosfomycin resistance rate of KPC-producing Klebsiella pneumoniae in China

Fosfomycin has been proposed as an adjunct to other active agents for treating KPC-producing Klebsiella pneumoniae infections. This study aimed to investigate the prevalence of fosfomycin resistance and plasmid-mediated resistance determinants among KPC-producing K. pneumoniae isolates from clinical samples in China. In total, 278 KPC-producing and 80 extended-spectrum β-lactamase (ESBL)-producing (non-KPC-producing) clinical K. pneumoniae isolates were collected in 12 hospitals from 2010 to 2013. Fosfomycin susceptibility testing was carried out using the agar dilution method. Phylogenetic clonal patterns were revealed by pulsed-field gel electrophoresis (PFGE). Isolates were screened for plasmid-mediated fosfomycin resistance genes (fosA, fosA3 and fosC2) by PCR amplification. A plasmid was completely sequenced by next-generation sequencing. The fosfomycin resistance rate in KPC-producers (60.8%; 169/278) was significantly higher than in ESBL-producers (12.5%; 10/80). In addition, 94 KPC-producing isolates were positive for fosA3 and most of them were clonally related. A 23939-bp plasmid (pFOS18) co-harbouring fosA3 and bla(KPC-2) was completely sequenced, revealing that the fosA3 gene was flanked by two copies of IS26; however, bla(KPC-2) was located on a Tn3-Tn4401 integration structure. Although the fosA3 and blaKPC-2 genes are located on different transposon systems, they are able to spread together worldwide through plasmid transfer. Dissemination of the clone carrying the fosA3-harbouring plasmid mediates the high fosfomycin resistance rate of KPC-producing K. pneumoniae in China. Fosfomycin as an alternative option for treating infections caused by KPC-producing K. pneumoniae should not be recommended in hospitals in which fosfomycin-resistant clonal dissemination is emerging.

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.

Characterization of a Novel Plasmid Type and Various Genetic Contexts of blaOXA-58 in Acinetobacter spp. from Multiple Cities in China

Background/Objective Several studies have described the epidemiological distribution of bla OXA-58-harboring Acinetobacter baumannii in China. However, there is limited data concerning the replicon types of bla OXA-58-carrying plasmids and the genetic context surrounding bla OXA-58 in Acinetobacter spp. in China. Methodology/Principal Findings Twelve non-duplicated bla OXA-58-harboring Acinetobacter spp. isolates were collected from six hospitals in five different cities between 2005 and 2010. The molecular epidemiology of the isolates was carried out using PFGE and multilocus sequence typing. Carbapenemase-encoding genes and plasmid replicase genes were identified by PCR. The genetic location of bla OXA-58 was analyzed using S1-nuclease method. Plasmid conjugation and electrotransformation were performed to evaluate the transferability of bla OXA-58-harboring plasmids. The genetic structure surrounding bla OXA-58 was determined by cloning experiments. The twelve isolates included two Acinetobacter pittii isolates (belong to one pulsotype), three Acinetobacter nosocomialis isolates (belong to two pulsotypes) and seven Acinetobacter baumannii isolates (belong to two pulsotypes/sequence types). A. baumannii ST91 was found to be a potential multidrug resistant risk clone carrying both bla OXA-58 and bla OXA-23. bla OXA-58 located on plasmids varied from ca. 52 kb to ca. 143 kb. All plasmids can be electrotransformed to A. baumannii recipient, but were untypeable by the current replicon typing scheme. A novel plasmid replicase named repAci10 was identified in bla OXA-58-harboring plasmids of two A. pittii isolates, three A. nosocomialis isolates and two A. baumannii isolates. Four kinds of genetic contexts of bla OXA-58 were identified. The transformants of plasmids with structure of IS6 family insertion sequence (ISOur1, IS1008 or IS15)-ΔISAba3-like element-bla OXA-58 displayed carbapenem nonsusceptible, while others with structure of intact ISAba3-like element-bla OXA-58 were carbapenem susceptible. Conclusion The study revealed the unique features of bla OXA-58-carrying plasmids in Acinetobacter spp. in China, which were different from that of Acinetobacter spp. found in European countries. The diversity of the genetic contexts of bla OXA-58 contributed to various antibiotics resistance profiles.