Diversity of New Delhi metallo beta lactamase producing bacteria in China 1 2 3 4 Q1 5 6 7 8 9 10 11 12 Q2 13 14 15 16 17 18 Q3 19 20 International Journal of Infectious Diseases xxx (2016) xxx–xxx G[.]
G Model IJID 2836 1–4 International Journal of Infectious Diseases xxx (2016) xxx–xxx Contents lists available at ScienceDirect International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid Short Communication Diversity of New Delhi metallo-beta-lactamase-producing bacteria in China Q1 10 11 12 Q2 13 14 15 16 17 18 Q3 Xiaofeng Hua , Xuebing Xub , Xu Wangc, Wencheng Xued , Haijian Zhoue,f , Ling Zhangg,h , Qiuxia Maa,i , Rongtao Zhaoa , Guozheng Lia , Peng Lia , Chuanfu Zhanga , Yun Shia , Jian Wangj , Leili Jiaa , Rongzhang Haoa , Ligui Wanga , Dayang Zoua , Xuelin Liua , Shaofu Qiua,**, Hongbin Songa,** , Yansong Suna,* a Institute for Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China Shanghai Municipal Center for Disease Control & Prevention, Shanghai 200336, China c Center for Disease Control and Prevention of Shenyang Command, Shenyang 110034, China d Department of Transfusion, General Hospital of Shenyang Military Area Command, Shenyang 110016, China e State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China f Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, China g The Affiliated Chenggong Hospital of Xiamen University, Xiamen 361003, China h Central Laboratory, The 174th Hospital of the Chinese People’s Liberation Army, Xiamen 361003, China i Navy’s Center for disease control and prevention, PLA, Beijing 101113, China j Beijing Center for Disease Prevention and control, Beijing 100013, China b A R T I C L E I N F O A B S T R A C T Article history: Received December 2016 Received in revised form January 2017 Accepted January 2017 Corresponding Editor: Eskild Petersen, Aarhus, Denmark Objectives: The prevalence and dissemination of diverse NDM-producing bacteria in China was investigated Methods: We collected 1,162 isolates from cities during December 2013 May 2015 in China The NDM-positive strains as well as the NDM genotypes in these sample were detected via Vitek compact system (bioMérieux, France), 16S rRNA gene sequencing, PCR and an S1- pulsed-field gel electrophoresis assay and Southern blot hybridization The horizontal-transfer capability of the blaNDM gene was assessed by filter mating by using a standard E.coli J53 azide-resistant strain as the recipient Results: Three genotypes (NDM-1, NDM-3 and NDM-5) of NDM-producing bacteria were identified, among which the NDM-1-positive isolates were the most frequent one For the first time, we found NDM5-produing S.typhimurium and NDM-3-produing E.coli in China We also found that the NDM-positive (especially NDM-3 and NDM-5) strains were completely resistant to nearly all of the antimicrobial drugs utilized and blaNDM was mostly located on diverse plasmids with sizes ranging from 30 to 670 kb Conclusion: Various species of bacteria especially the enteric pathogens with diverse NDM genotypes had spread in China Hence, an ongoing surveillance of their dissemination is essential to prevent and control the spread of these organisms © 2017 The Authors Published by Elsevier Ltd on behalf of International Society for Infectious Diseases This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/) Keywords: NDM Diversity China 19 20 New Delhi metallo-beta-lactamase (NDM) producers are a major global health problem, which necessitates reliable detection * Corresponding author at: Department of Science and Technology, Academy of Military Medical Sciences, 27th Taiping Street, Haidian District, Beijing 100039, China ** Corresponding authors at: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, 20th Dongda Street, Fengtai District, Beijing 100071, China E-mail addresses: qiushf0613@hotmail.com (S Qiu), hongbinsong@263.net (H Song), sunys1964@hotmail.com (Y Sun) and surveillance methods 1,2 However, limited information is available regarding the prevalence and dissemination of NDM producers in China To better understand the current prevalence of the NDM-producing bacteria in China, we conducted a study Q4 during January 2013 to December 2015 A total of 1,162 isolates were obtained from patients admitted to the 20 sentinel hospitals in eight cities located in China, including Wuhan, Shanghai, Beijing, Xiamen, Haikou, Shenyang, Jinan, Changsha and Suzhou The informed consent of all participants was obtained prior to enrollment Identification of the isolates to the species level was carried out by using the Vitek compact http://dx.doi.org/10.1016/j.ijid.2017.01.011 1201-9712/© 2017 The Authors Published by Elsevier Ltd on behalf of International Society for Infectious Diseases This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Please cite this article in press as: X Hu, et al., Diversity of New Delhi metallo-beta-lactamase-producing bacteria in China, Int J Infect Dis (2017), http://dx.doi.org/10.1016/j.ijid.2017.01.011 21 22 23 24 25 26 27 28 29 30 31 G Model IJID 2836 1–4 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 X Hu et al / International Journal of Infectious Diseases xxx (2016) xxx–xxx system (bioMérieux, France) and 16S rRNA gene sequencing PCR was used to screen the b-lactamase genes and PCR product sequencing results revealed the genotype of blaNDM Antimicrobial susceptibility testing was performed by Vitek compact according to the Clinical and Laboratory Standards Institute An S1-pulsedfield gel electrophoresis (PFGE) assay and Southern blot hybridization were performed to determine the location of blaNDM genes by using specific blaNDM digoxigen-labeled probes (Roche) All the assays were performed as previously described 3,4 The horizontaltransfer capability of the blaNDM gene was assessed by filter mating using a standard Escherichia coli J53 azide-resistant strain as the recipient We detected blaNDM in 45 (3.9%) of the 1,162 isolates and the NDM-producing bacteria were identified as E coli (14, 31.1%), Klebsiella pneumoniae (12, 26.7%), Acinetobacter calcoacelicus (9, 20.0%), Salmonella enteritidis (4, 8.9%), Enterobacter cloacae (3, 6.7%), Salmonella typhimurium (1, 2.2%), Citrobacter freundii (1, 2.2%) and Klebsiella oxytoca (1, 2.2%) (Figure 1) Moreover, the blaNDM-positive isolates were from Hainan (8), Beijing (8), Wuhan (7), Xiamen (7), Shanghai (5), Jinan (4), Shenyang (3), Changsha (2) and Suzhou (1) Klebsiella pneumoniae, E coli and A calcoacelicus were found in most areas; however, S entertidis and S typhimurium were only found in Shanghai (Figure 1) Among the 45 blaNDM-positive isolates, 36 (80.0%) were NDM-1 positive, (17.8%) were NDM-5 positive and (2.2%) was NDM-3 positive (Figure 1) As shown in Figure 1, NDM-1 was found among K pneumoniae, A calcoacelicus, E coli, E cloacae and S entertidis isolated from the eight cities in China Moreover, NDM5 was found among E coli and S typhimurium, while NDM-3 was only found in E coli isolated from Beijing Among the Figure Southern blot-based dendrogram, plasmid-S1 profiles and antimicrobial resistance profile of the 45 NDM-producing isolates from the indicated eight cities in China The dendrogram was constructed based on the unweighted pair-group method using average linkages and pair-wise Dice coefficients Results from the Southern blot were clustered The NDM gene present within the indicated bacterial strains with the horizontal line was located on the plasmids, the NDM (AhSZ001) gene of the bacterial strains without the horizontal line was located on the chromosome, and the line showed the size and position of the plasmids PFGE of S1-digested plasmid The horizontal lines identify the plasmid map of the plasmids; Marker: H9812 Antimicrobial susceptibility was performed by disk fusion Black indicates resistance; gray indicates intermediate; white indicates susceptible Antibiotic abbreviations are: ampicillin (AMP), amoxicillin/clavulanic acid (AMC), piperacillin (PIP), cefazolin (FAZ), ceftazidime (CAZ), ceftriaxone (CTR), cefepime (FEP), aztreonam (AZT), imipenem (IMI), meropenem (MEC), amikacin (AMI), gentamicin (GEN), ciprofloxacin (CIP), levofloxacin (LVX), Tetracycline (TET), nitrofurantoin (NIT) and trimethoprim/sulfamethoxazole (SXT) The strain number, isolate date, species, NDM genotype of the strains and the detailed information of the specimen including the year, region, clinical ward and source are shown Please cite this article in press as: X Hu, et al., Diversity of New Delhi metallo-beta-lactamase-producing bacteria in China, Int J Infect Dis (2017), http://dx.doi.org/10.1016/j.ijid.2017.01.011 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 G Model IJID 2836 1–4 X Hu et al / International Journal of Infectious Diseases xxx (2016) xxx–xxx Table Antimicrobial resistance profiles of the NDM-producing bacteria Antimicrobial drug % Resistance of blaNDM-positive isolates Total (n = 45) Ampicillin Piperacillin Cefazolin Ceftazidime Ceftriaxone Cefepime Aztreonam Imipenem Amoxicillin/clavulanic acid Meropenem Gentamicin Sulfamethoxazole/trimethoprim Nitrofurantoin Ciprofloxacin Levofloxacin Tetracycline Amikacin NDM-1 (n = 36) NDM-5 (n = 8) NDM-3 (n = 1) R I S R I S R I S R I S 100.0 100.0 100.0 100.0 100.0 100.0 91.1 88.9 82.2 77.8 75.6 68.9 51.1 53.3 48.9 44.4 33.3 0 0 0 6.7 6.7 2.2 13.3 0 31.1 8.9 6.7 2.2 0 0 0 2.2 4.4 15.6 8.9 24.4 31.1 17.8 37.8 44.4 55.56 64.4 100.0 100.0 100.0 100.0 100.0 100.0 88.9 88.9 77.8 72.2 72.2 63.9 58.3 44.4 38.9 36.1 33.3 0 0 0 8.3 5.6 2.8 16.7 0 27.8 11.2 8.3 0 0 0 0 2.8 5.6 19.4 11.1 27.8 36.1 18.9 44.4 52.8 63.9 66.7 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 87.5 100.0 12.5 87.5 87.5 87.5 37.5 0 0 0 0 0 0 50.0 0 12.5 0 0 0 0 0 12.5 37.5 12.5 12.5 12.5 50.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 0 0 0 0 100.0 0 0 0 0 0 0 0 0 0 0 100.0 0 100.0 100.0 *R, resistant; I, intermediate; S, susceptible 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 NDM-5 positive E coli, three were from Shenyang, two from Beijing, and two from Jinan; one NDM-5 positive S typhimurium was from Shanghai As shown in Table 1, of the NDM-1 positive strains, 72.2% were resistant to meropenem and gentamicin, 63.9% to sulfamethoxazole/trimethoprim, 58.3% to nitrofurantoin, and 44.4% to ciprofloxacin The NDM-3-positive strain was resistant to meropenem, gentamicin, nitrofurantoin, ciprofloxacin and levofloxacin Of the NDM-5 positive strains, 100% were resistant to meropenem and sulfamethoxazole/trimethoprim, 87.5% to gentamicin and ciprofloxacin, and 12.5% to nitrofurantoin blaNDM was mostly located on diverse plasmids with sizes ranging from 30 to 670 kb Herein, we found that among the 45 blaNDM-positive strains, 44 strains’ blaNDM genes were located on the plasmids, and strain’s blaNDM gene was located on the chromosome (Figure 1) Moreover, we identified nine strains that simultaneously contained two plasmids with blaNDM-1, and the strains were K pneumoniae (five), E cloacae (one), A calcoacelicus (one), E coli (one) and K oxytoca (one) (Figure 1) Results of the conjugal transfer assay indicated that the plasmids harboring the blaNDM-1 gene were successfully transferred into E coli J 53 Furthermore, we observed that several NDM-positive isolates contained blaNDM on the chromosome Three genotypes of blaNDM were identified in our study, including blaNDM-1, blaNDM-3 and blaNDM-5, among which NDM-1-positive bacteria was the most prevalent type NDM was mostly found among E coli and K pneumoniae , and our findings align with previous studies Notably, we also found the emergence of NDM-3-produing E coli and NDM-5-produing S typhimurium, which was reported for the first time in China Previous reports have indicated that S enterica has the potential to act as a reservoir for carbapenemase genes within the community and to lead to the transmission of carbapenem resistance via the food chain The NDM-1 and NDM-5 positive S enterica have been found in many countries including the United Kingdom, Pakistan and China 6–8 Herein, we identified NDM-5 positive S typhimurium and NDM1 positive S enteritidis, indicating the potential prevalence of NDMpositive typhoidal Salmonella Of particular concern is that the NDM-positive bacteria that causes infection currently has limited antimicrobial choices and requires a risk and benefit analysis of available agents We also found that the NDM-positive (especially NDM-3 and NDM-5) strains were completely resistant to nearly all of the antimicrobial drugs utilized in this study Additionally, blaNDM-1 was previously reported to be located either on the chromosome or on plasmids 10 Our study indicated that NDM-1, NDM-3 and NDM-5 genes were mostly located on plasmids of different sizes We also found that plasmids with similar size emerged in different strains and that the plasmid was able to be transferred into E coli isolates, suggesting that different types of blaNDM genes continue to be transferred horizontally by different plasmids Interestingly, nine of the bacterial strains had two plasmids that simultaneously contained blaNDM-1, which may contribute to the high drug resistance of these strains In summary, we found that numerous types of harmful bacteria, especially the enteric pathogens with diverse types of NDM, have spread in China An ongoing surveillance is warranted in efforts to minimize future infections 105 Finding Statement 119 The present study was funded by the National Key Research and Q5 Development Plan of China (2016YFC1200700 and AWS15J006), the National Natural Science Foundation of China (Grant numbers 81473024 and 81673237) 120 Potential conflicts of interest 124 All authors: no conflicts Ethical approval for the study was obtained from the Hospital Research Ethics Committee at each hospital Written consent was obtained from all patients for biological sample collection for routine investigation according to the hospitals’ protocols 125 References 131 Cornaglia G, Giamarellou H, Rossolini GM Metallo-beta-lactamases: a last frontier for beta-lactams? The Lancet Infectious diseases 2011;11:381–93 Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study The Lancet Infectious diseases 2010;10:597–602 Fu Y, Du X, Ji J, Chen Y, Jiang Y, Yu Y Epidemiological characteristics and genetic structure of blaNDM-1 in non-baumannii Acinetobacter spp in China The Journal of antimicrobial chemotherapy 2012;67:2114–22 Borgia S, Lastovetska O, Richardson D, Eshaghi A, Xiong J, Chung C, et al Outbreak of carbapenem-resistant enterobacteriaceae containing blaNDM1Ontario, Canada Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 2012;55:e109–117 Please cite this article in press as: X Hu, et al., Diversity of New Delhi metallo-beta-lactamase-producing bacteria in China, Int J Infect Dis (2017), http://dx.doi.org/10.1016/j.ijid.2017.01.011 106 107 108 109 110 111 112 113 114 115 116 117 118 121 122 123 126 127 128 129 130 132 133 134 135 136 137 138 139 140 141 142 143 G Model IJID 2836 1–4 144 145 146 147 148 149 150 151 152 153 X Hu et al / International Journal of Infectious Diseases xxx (2016) xxx–xxx Walsh TR, Weeks J, Livermore DM, Toleman MA Dissemination of NDM1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study The Lancet Infectious diseases 2011;11:355–62 Day MR, Meunier D, Doumith M, de Pinna E, Woodford N, Hopkins KL Carbapenemase-producing Salmonella enterica isolates in the UK The Journal of antimicrobial chemotherapy 2015;70:2165–7 Irfan S, Khan E, Jabeen K, Bhawan P, Hopkins KL, Day M, et al Clinical isolates of Salmonella enterica serovar Agona producing NDM-1 metallo-beta-lactamase: first report from Pakistan Journal of clinical microbiology 2015;53:346–8 Huang J, Wang M, Ding H, Ye M, Hu F, Guo Q, et al New Delhi metallo-betalactamase-1 in carbapenem-resistant Salmonella strain, China Emerging infectious diseases 2013;19:2049–51 Rogers BA, Sidjabat HE, Silvey A, Anderson TL, Perera S, Li J, et al Treatment options for New Delhi metallo-beta-lactamase-harboring enterobacteriaceae Microbial drug resistance 2013;19:100–3 10 Chen Y, Zhou ZH, Jiang Y, Yu YS Emergence of NDM-1-producing Acinetobacter baumannii in China J Antimicrob Chemoth 2011;66:1255–9 Please cite this article in press as: X Hu, et al., Diversity of New Delhi metallo-beta-lactamase-producing bacteria in China, Int J Infect Dis (2017), http://dx.doi.org/10.1016/j.ijid.2017.01.011 154 155 156 157 158 159 160 161 ... baumannii in China J Antimicrob Chemoth 2011;66:1255–9 Please cite this article in press as: X Hu, et al., Diversity of New Delhi metallo-beta-lactamase-producing bacteria in China, Int J Infect Dis... 2012;55:e109–117 Please cite this article in press as: X Hu, et al., Diversity of New Delhi metallo-beta-lactamase-producing bacteria in China, Int J Infect Dis (2017), http://dx.doi.org/10.1016/j.ijid.2017.01.011... genotype of the strains and the detailed information of the specimen including the year, region, clinical ward and source are shown Please cite this article in press as: X Hu, et al., Diversity of New