Whole genome sequence revealed the fine transmission map of carbapenem-resistant Klebsiella pneumonia isolates within a nosocomial outbreak

Whole genome sequence revealed the fine transmission map of carbapenem-resistant Klebsiella... Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major cause of nosocomial infections worldwide. The transmission route of CRKP isolates within an outbreak is rarely described. This study aimed to reveal the molecular characteristics and transmission route of CRKP isolates within an outbreak of nosocomial infection. Methods: Collecting case information, active screening and targeted environmental monitoring were carried out. The antibiotic susceptibility, drug-resistant genes, molecular subtype and whole genome sequence of CRKP strains were analyzed. Results: Between October and December 2011, 26 CRKP isolates were collected from eight patients in a surgical intensive care unit and subsequent transfer wards of Beijing Tongren hospital, China. All 26 isolates harbored bla , bla , and bla genes, had the same or similar pulsed-field gel electrophoresis patterns, and KPC-2 SHV-1 CTX-M-15 belonged to the sequence type 11 (ST11) clone. By comprehensive consideration of genomic and epidemiological information, a putative transmission map was constructed, including identifying one case as an independent event distinct from the other seven cases, and revealing two transmissions starting from the same case. Conclusions: This study provided the first report confirming an outbreak caused by K. pneumoniae ST11 clone co-harboring the bla , bla , and bla genes, and suggested that comprehensive consideration of KPC-2 CTX-M-15 SHV-1 genomic and epidemiological data can yield a fine transmission map of an outbreak and facilitate the control of nosocomial transmission. Keywords: Carbapenemases, K. pneumoniae, KPC-2, Outbreak, Whole genome sequencing * Correspondence: zhj_0901@163.com; zhouhaijian@icdc.cn; chenchen1@ccmu.edu.cn; luxinxin2009@126.com Wenjun Sui, Haijian Zhou and Pengcheng Du contributed equally to this work. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China Beijing Key Laboratory of Emerging Infectious Diseases, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China Department of Clinical Laboratory, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 2 of 11 Background Active screening and targeted environmental monitoring Carbapenems remain the first-line therapeutic antimicro- Targeting KPC-2 K. pneumoniae, active screening and bials for severe infections caused by extended-spectrum environmental monitoring were carried out as measures β-lactamase (ESBL)-producing multidrug-resistant Enterobacte- of infection control between October 11th and October riaceae. However, the emergence of carbapenemase-mediated 27th. The method of active screening and targeted envir- resistance to all β-lactams, including carbapenems, is a major onmental monitoring was described previously [23]. In public health threat [1, 2]. Over the last decade, brief, for active screening, we collected samples from the carbapenem resistance, attributed to the production of nose, throat, groin, and axilla of each patient using ster- carbapenem-hydrolyzing β-lactamases, has been steadily ile cotton swabs (the sputum and stool samples were in- increasing among Enterobacteriaceae isolates, particularly cluded in our clinical samples), and a real-time PCR Klebsiella pneumoniae [3]. Carbapenem-resistant K. pneu- assay was used to screen all samples for bla [24]. The KPC moniae (CRKP) has emerged in many countries as a result first swab of each patient was taken within 48 h upon of intra-continental and inter-continental spread [4–6]. ICU admission. For targeted environmental monitoring, Clinically, the K. pneumoniae carbapenemase (KPC) six environmental sites on the bed sheet were sampled enzyme is one of the most prevalent carbapenemases. for each KPC-KP positive patient using contact plates The bla genes are predominantly plasmid encoded. (16 cm ; Qingdao Classical Biochemical Equipment, KPC In some cases, bla genes exist in particularly clones Qingdao, China). KPC (such as K. pneumoniae multilocus sequence type (ST) 258 and ST11, which have facilitated their rapid dissem- Bacterial identification and antimicrobial susceptibility ination. Since the first KPC-producing isolate was identi- Bacterial identification and antibiotic susceptibility test- fied from North Carolina, USA, in 1996 [7], the ing were initially performed using the VITEK-2 auto- occurrence of KPC-producing bacteria has been con- mated system (BioMérieux, France). Escherichia coli tinuously reported in other parts of the USA, Europe, ATCC 25922 was used as quality control strain for anti- South America, the Middle East, and Asia [5, 8–10]. biotic susceptibility testing. Susceptibility category was They have been associated with large nosocomial designated according to the Clinical and Laboratory outbreaks worldwide, including those in China. The Standards Institute (CLSI) breakpoints [22]. Susceptibil- CRKP outbreak isolates in China mostly carried bla ity to tigecycline was defined based on the criteria pro- KPC-2 [11–14]or bla [15–18]. KPC-producing members posed by the European Committee on Antimicrobial NDM-1 of the family Enterobacteriaceae have also been associated Susceptibility Testing-2011 (susceptible, minimum in- with high mortality rates, particularly among critically ill pa- hibitory concentration (MIC) ≤ 1 μg/ml). tients with a history of prolonged hospitalization [19–21]. Phenotypic screening for the presence of carbapene- Thesefactsstronglysuggest aneedfor theimplementation mase was performed using the modified Hodge test of adequate preventive measures to effectively control the (MHT). In addition, ESBL production was tested using spread of such pathogens. the Double-Disk Synergy Test (DDST), as recommended From October 8th to December 23th, 2011, a total of by the CLSI [22]. 12 CRKP strains were isolated from clinical samples of eight patients admitted to the surgical intensive care unit Determination of carbapenemase genes (SICU). Active screening and targeted environmental We screened carbapenem-resistance genes (bla , bla , KPC NMC monitoring were carried out between October 11th and bla , bla , bla , bla , bla , bla , bla , SME IMI GES NDM IMP VIM SPM October 27th, and eight CRKP were isolated from nose bla , bla , bla , bla , bla , GIM SIM OXA-48 OXA-51-like OXA-23-like and throat of inpatient in SICU, as well as environmen- bla , bla , in all strains as described previ- OXA-24-like OXA-58-like tal samples. Furthermore, six CRKP were retrieved from ously [5, 25, 26]. DNA sequencing was performed on both one case in subsequent transfer wards. In the present strands of the PCR amplification products. The results were study, we carried out a retrospective investigation of the compared and aligned with reference sequences using the molecular and genomic epidemiology of the outbreak. online BLAST database. Methods Pulsed-field gel electrophoresis (PFGE) Retrospective analysis of medical records We used the 1-day, standardized PFGE protocol for K. The medical records of patients from whom CRKP was pneumoniae [27]. Cell suspensions were placed in isolated were reviewed, including time of stay in SICU, polystyrene tubes (Falcon; 12 × 75 mm), and their optical bed site, data of the first CRKP isolation, and outcomes. density was adjusted to 3.8–4.0 using a Densimat photom- CRKP is defined as resistance to carbapenems according eter (BioMérieux, Marcy l’Etoile, France). K. pneumoniae to the Clinical and Laboratory Standards Institute (CLSI) slices were digested using 50 U per slice of XbaI(Takara, breakpoints [22]. Dalian, China) for 4 h at 37 °C, and electrophoresis was Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 3 of 11 performed using a CHEF-DRIII system (Bio-Rad Labora- teaching hospital in Beijing, China. Beijing Tongren tories, Hercules, CA, USA). Electrophoresis was run with Hospital receives an average of 3500–5000 outpatients a switch time of 6 s to 36 s for 18.5 h, and images were and emergency patients per day. SICU has 18 beds. captured using a Gel Doc 2000 system (Bio-Rad) and con- The index case of this outbreak was identified on verted to TIFF files. The TIFF files were analyzed using October 8th, 2011. Following identification of second the BioNumerics version 5.1 software (Applied Maths, CRKP-infection case in the same room on October 10th, Kortrijk, Belgium). and a CRKP carrier on October 11th, an outbreak was declared. Between October 8 and December 23, a total Multilocus sequence typing (MLST) of 50 patients were admitted into the SICU and eight of MLST with seven genes (gapA, infB, mdh, pgi, phoE, them were found to have a CRKP infection or rpoB,and tonB) was performed on isolates according to colonization though routine clinical culture and active the protocol described on the K. pneumoniae MLST screening (Table 1). The timeline of patient admission website (http://bigsdb.pasteur.fr/). Alleles and STs were and CRKP isolation is showed in Fig. 1. assigned using the MLST database (http://bigsdb.pas- Among the eight patients, four were infected by CRKP teur.fr/klebsiella/klebsiella.html). Alleles and STs that and other four were CRKP carrier. Of the four infection had not been previously described were submitted to the cases, one died, one was discharged from the SICU, and curator of the database and were assigned new two were transferred to other wards and ultimately dis- designations. charged. It should be noted that cases 1 to 7 overlapped with at least three other cases when in the SICU, but Whole genome sequencing (WGS), detection of single case 8 did not overlap with any other case during SICU nucleotide polymorphisms (SNPs), and clustering analysis stay. The initial strains of each case were selected for WGS. CRKP detected twice from the the bed sheets of case 4 Bacterial strains were sequenced using Illumina sequen- during the outbreak. In addition to active screening and cing by constructing two paired-end (PE) libraries with targeted environmental monitoring, other stringent infection average insertion lengths of 500 bp and 2000 bp, re- prevention and control measures were implemented, includ- spectively. Sequences were generated using an Illumina ing contact precautions, strengthen of hand hygiene, GA IIx (Illumina Inc., San Diego, CA, USA). Raw data environmental cleaning (including changing the bed linen was processed in four steps, including removing reads more frequently for each CRKP-positive patient, and imme- with 5 bp of ambiguous bases, removing reads with diately stringent terminal sterilization with hydrogen perox- 20 bp of low quality (≤ Q20) bases, removing adapter ide after they were discharged from the ward or died), and contamination, and removing duplicated reads. Finally, enhanced antimicrobial stewardship were introduced. For 100× libraries were obtained with clean PE read data. the patients transferred to other wards, contact precautions Assembly was performed using SOAPdenovo v1.05 [28]. and follow-up screening were employed until they were dis- The whole-genome sequence of K. pneumoniae charged from our hospital. Since January 22, 2012 (the dis- HS11286 (GenBank accession: NC_016845.1) was used charge date of the last patients with CRKP colonization), as the reference sequence, and clean reads of sequenced over a period of 10 months, no further carbapenem-resistant isolates were mapped to the reference genome by bowtie Enterobacteriaceae (CRE) were isolated in the SICU. 2 software under the default parameters [29]. SNPs were then identified using Samtools [30] and combined to- Antibiotic susceptibility and characterization of resistance gether according to the reference. SNPs with low quality genes (read depth < 5) and those located within 5 bps on the All 26 CRKP isolates showed same results of antibiotic sus- same chromosome were removed to avoid the effect of ceptibility test. The MICs of meropenem, imipenem, and recombination, as described in our previous studies [31, ertapenem were ≥ 16 μg/ml, ≥ 16 μg/ml, and ≥ 8 μg/ml, re- 32]. The isolates were clustered and a heatmap was gen- spectively, for all isolates. All isolates were susceptible to erated using the heatmap package in R. The transmis- amikacin ((MIC ≤2 μg/ml), gentamicin (MIC ≤1 μg/ml), sion route was then reconstructed based on the tobramycin (MIC ≤1 μg/ml), and trimethoprim/sulfa- emergence of different SNPs in each isolate and the case methoxazol (MIC ≤1:19 μg/ml), and were resistant to information, including the onset time of infection and ampicillin (MIC ≥32 μg/ml), ampicillin/sulbactam (MIC the hospitalization time. ≥32 μg/ml), piperacillin (MIC ≥128 μg/ml), piperacillin/taz- obactam (MIC ≥128 μg/ml), cefazolin (MIC ≥64 μg/ml), Results cefotetan (MIC ≥64 μg/ml), ceftazidime (MIC ≥64 μg/ml), Outbreak descriptions ceftriaxone (MIC ≥64 μg/ml), cefepime (MIC ≥64 μg/ml), The outbreak occurred in the SICU of Beijing Tongren aztreonam (MIC ≥64 μg/ml), ciprofloxacin (MIC Hospital, a 1600-bed general tertiary care and university-affiliated ≥4 μg/ml), levofloxacin (MIC ≥8 μg/ml), nitrofurantoin Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 4 of 11 Table 1 Case descriptions involved in the outbreak of SICU, October to December 2011 Case No. Time of stay in SICU Date of the first isolation Type of specimen Infection /colonization Antimicrobial used before Antimicrobial used after Outcome 1 30/9/2011–17/10/2011 8/10/2011 sputum infection one course of VA one course of AK died 2 7/10/2011-14/10/2011 10/10/2011 abdominal drainage fluid infection one course of MEM one course of AK transfer to other ward 3 8/10/2011–17/10/2011 11/10/2011 nose colonization –– discharged 4 10/9/2011-16/12/2011 16/10/2011 throat infection one course of CAZ and two courses of Gn transfer to other ward 1 course of PIP/TAZ 5 14/10/2011–16/11/2011 18/10/2011 nose colonization one course of CIP one course of Ak discharged 6 16/10/2011–15/11/2011 21/10/2011 urine infection one course of CRO one course of GN discharged 7 24/10/2011–25/11/2011 27/10/2011 nose colonization one course of CRO one course of CRO discharged 8 20/12/2011–22/1/2012 23/12/2011 sputum colonization –– discharged VA Vancomycin, MEM meropenem, CAZ ceftazidime, PIP/TAZ piperacillin/tazobactam, CIP ciprofloxacin, CRO ceftriaxone, AK amikacin, GN gentamicin Case 2 was transferred to a general surgery ward and ultimately discharged from the hospital Case 4 was transferred to a geriatric ward and ultimately discharged from the hospital Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 5 of 11 Fig. 1 The timeline of patient admission and CRKP isolation. Shadows on the timeline represent the admitting’s duration of the case. Different wards are indicated in different colors. The red arrow indicates the isolation of the strains Fig. 2 Clustering of the 26 K. pneumoniae isolates based on PFGE patterns. The first positive cultures of each case are marked by an asterisk. The information of strain ID, PFGE type, patient ID, isolation date, ward, type of specimen, and MLST type is listed to the left of the patterns. ADF, abdominal drainage fluid; BAL, bronchoalveolar lavage Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 6 of 11 Fig. 3 Clustering of the 26 K. pneumoniae isolates based on MCG typing. The first positive cultures of each case are marked by an asterisk. The information of strain ID, patient ID, type of specimen, isolation date, and ward are listed to the left of the patterns. ADF, abdominal drainage fluid; BAL, bronchoalveolar lavage (MIC ≥512 μg/ml), and tigecycline (MIC ≥2 μg/ml). All interpretation criteria of PFGE patterns proposed by Ten- isolates were positive for carbapenemase and ESBL pro- over et al. [33], the first positive culture of cases 1, 2, 3, 5, duction by the MHT and DDST assays, respectively. We 6, 7 were the “same strain” and those of cases 4 and 8 further confirmed the presence and production of carba- were “closely-related strains”. penemases and ESBLs by PCR and sequencing. All 21 iso- Excepting for case 8, all six isolates showing different lates harbored the bla , bla ,and bla patterns to the dominant PFGE type were isolated from KPC-2 SHV-1 CTX-M-15 genes and tested negative for other antimicrobial resist- case 4, including three strains from urine, one from spu- ance genes (bla , bla , bla , bla , bla , tum, and two from the bed sheet. The two strains iso- NMC SME IMI GES NDM bla , bla , bla , bla , bla , bla , lated from the bed sheet (TRqt-47 and TRqt-48) of case IMP VIM SPM GIM SIM OXA-48 bla , bla , bla , bla , 4 showed one band that was different to the strain iso- OXA-51-like OXA-23-like OXA-24-like OXA-58-like and bla ). lated from the nose (TRqt-43) on the same day. It was TEM interesting that with increasing time, the PFGE patterns of the strains from case 4 also changed, especially Molecular subtyping analysis by PFGE and MLST isolates from December 2011. All the strains isolated in MLST indicated that all 26 isolates belonged to ST11. October showed dominant patterns or showed only one However, PFGE showed some diversity (Fig. 2). PFGE di- band different from the dominant pattern; two strains vided the 26 isolates into seven different PFGE types isolated in November (TR221, TR231-m) showed two (PT1–PT7). The dominant PFGE type (PT3) contained bands that were different from dominant pattern and an- 19 isolates. The other six PFGE types showed one to other strain isolated in December (TR258) showed four four bands that were different to the dominant PFGE bands that were different from dominant pattern. type (PT3). Except for cases 4 and 8, all the first positive cultures from each affected patients belonged to the dominant PFGE type (PT3). The isolates of case 4 (PT4) Comparison of outbreak isolates based on WGS-based SNPs and case 8 (PT2) showed one and three bands that were We performed WGS and MCG typing of the 26 CRKP different to those of PT3, respectively. Using the isolates, in hope of using the SNPs found in their Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 7 of 11 Fig. 4 Evolutionary relationships based on MCG typing of 26 CRKP isolates. Isolates from different patients are indicated in different colors. The first positive cultures of each case are marked by an asterisk genomes to determine putative transmission map of this different to the reference strain, respectively. All isolates outbreak. Genomic comparisons revealed a total of 32 from cases 1 and 3 had only one SNP difference to the MCG SNPs among the 26 isolates (Fig. 3). The evolu- reference strain. At the same time, two isolates from tionary relationships based on MCG typing of the 26 case 4, one isolate from case 6, and two isolates from CRKP isolates are outlined and presented in Fig. 4. case 7 had the same SNP profile and showed only one Five case 2 strains isolated in October showed the SNP difference to the reference strain. same SNP profile as the reference strain K. pneumoniae It is worth noting that four case 4 isolates, two from HS11286. The other case 2 strains isolated in early and the bed sheet (TRqt-47, TRqt-48) and two from urine late November had one and two SNPs that were (TR221, TR231-m), showed 7–10 SNP differences Fig. 5 Clustering of the eight first positive CRKP cultures of each case based on MCG typing. The matrix of the SNP differences is presented on the left of the cluster tree Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 8 of 11 the hospital. Thus Case 1 is unlikely to be the source. Case 3 checked in the SICU on October 8th, 2011, the same day of first CRKP isolation of case 1; however, case 3 checked in the SICU in the afternoon, and the CRKP-positive sample had already been collected from case 1 in the morning, which suggested that the CRKP of case 1 was not transmitted from case 3. Thus, case 2 was the most likely source of the outbreak isolates. Second, the isolates of cases 1 and 3 showed no SNP differences, suggesting direct transmission between them. However, it could not be confirmed that whether case 2 directly transmitted CRKP to case 1 or case 3. Third, the case 2’s isolate showed one SNP difference Fig. 6 Putative map of CRKP transmission during the outbreak. The to isolates of cases 4 and 7, but two SNP differences to transmission map was constructed with genetic and patient trace that of cases 5 and 6, suggesting that CRKP from case 2 data. Nodes represent patients, and arrows indicate a transmission was genomically closer to cases 4 and 7. However, case event directly from one patient to another 7’s hospital stay did not overlap with that of case 2 and the date of the first isolation of CRKP from case 7 was compared with the initial isolate (TR198) of case 4, and significantly later than the other cases; therefore, we de- another urine isolate of case 4 (TR258) isolated later had duced the case 7’s isolate was transmitted from case 2 only two SNP differences to TR198, suggesting that the through case 4. The isolates of cases 5 and 6 were trans- CRKP isolated from case 4 during the study period came mitted from case 4, as there was only one SNP between from a different source. isolates cases 5 and 6 and that from case 4, but showed The isolate of case 8 (TR262) showed 6–18 SNP differ- more SNPs differences to that of other cases. ences with other isolates and had five specific SNPs. To Consistent with the genomic and epidemiological in- determine whether case 8 is related to the other seven formation, the integrated map identified two transmis- cases, the initial isolates of each case were chosen and an- sions starting from case 2 (Fig. 6). The first transmission alyzed. Grouping the eight initial isolates on the basis of was directly from case 2 to case 1 or case 3, and a trans- the patterns of shared variants partitioned them into two mission was observed between cases 1 and 3. A second clusters (Fig. 5). Cluster 1 contained seven isolates of cases transmission from case 2 was predicted to go through 1 to 7, and cluster 2 consisted only of case 8. The pairwise case 4 before being transmitted to cases 5, 6, and 7. distances within cluster 1 were ≤ 3 SNP differences; in contrast, the distances between cluster 2 and cluster 1 iso- Discussion lates were 8 SNP differences. The within-cluster distance In this study, we described an outbreak caused by CRKP of cluster 1 was much smaller than the between-cluster in a SICU of a large university hospital in China. The distance, indicating considerable divergence between the CRKP isolates belonged to clone ST11, and coproduced two clusters. The epidemiological information of case 8 carbapenemase (KPC-2) and ESBLs (CTX-M-15 and indicated that they had not overlapped during their hos- SHV-1). ST11 is the dominant clone of KPC-producing pital stay with any of the other seven patients; therefore, K. pneumoniae in China and has also been reported we deduced that cases 1 to 7 probably shared the same sporadically in the rest of the world, including other transmission route and that case 8 seemed to be an inde- regions of Asia [34, 35], America [36], and Europe [37–39]. pendent event. KPC is the most common carbapenemase in K. pneumoniae and most of the KPC type in China is KPC-2 [40]. Similarly, Inference of most likely transmission route CTX-M-15 and SHV-1 are the main types of CTX-M-type Furthermore, the genomic and epidemiological informa- and SHV-type in China [41]. The coexistence of bla KPC-2 tion were integrated to construct the transmission route. with bla type genes in K. pneumoniae was previ- CTX-M-15 The most likely transmission route is shown in Fig. 6. ously reported in Bulgaria [42], Brazil [43], and China [44]. First, case 2 was suspected of being the source of the out- In a recent study, CRKP strains co-harboring the bla , KPC-2 break isolates because the CRKP from case 2 showed the bla ,and bla genes were found in several STs in- CTX-M-15 SHV same SNP patterns to reference strain K. pneumoniae cluding ST11; however, only sporadic strains were reported HS11286. This was also supported by the epidemiological in that study [44]. As far as we know, this is the first study to information. Case 1 admitted in the SICU on September report an outbreak caused by CRKP co-harboring the 30th, 2011, 9 days before the first CRKP was isolated, bla , bla ,and bla genes, which suggests KPC-2 CTX-M-15 SHV-1 which suggested that the CRKP of case 1 was acquired in that attention should be paid to the K. pneumoniae isolates Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 9 of 11 coproducing epidemic carbapenemases and ESBLs, especially was the source of CRKP in this outbreak; (iii) there the outbreak strains described here belonging to the epi- were two transmissions starting from case 2. demic ST11 clone. Given the easy of transfer and acquisition of carbape- Conclusions nemase and ESBLs genes, measures must be imple- Though this study is a retrospective study and thus the mented to control the outbreak and avoid nosocomial results of the WGS could not be used to control the transmission [2, 45]. Agodi et al. reported that cleaning nosocomial transmission. However, our data clearly and disinfection of the ICU, segregation of affected pa- showed that WGS and MCG typing could reveal the de- tients, barrier nursing, and strict compliance with hand tails of transmission within a CRKP nosocomial out- hygiene procedures led to containment of an outbreak of break. In the future, real-time genomic sequencing and KPC-producing K. pneumoniae [46]. In our study, we analysis of an outbreak should be carried out and the applied active screening and targeted environmental findings could be used to control outbreaks. monitoring to combat the secondary transmission of Abbreviations imported KPC clones in the SICU. Except for the two CLSI: Clinical and Laboratory Standards Institute; CRE: carbapenem-resistant index cases, we found that six patients acquired this Enterobacteriaceae; CRKP: Carbapenem-resistant K. pneumoniae; pathogen during their hospital stay, two of them with DDST: Double-Disk Synergy Test; ESBL: extended-spectrum β-lactamase; KPC: K. pneumoniae carbapenemase; MHT: modified Hodge test; urinary tract infections. Some reports stated that the MIC: minimum inhibitory concentration; MLST: Multilocus sequence typing; rectum was the most sensitive sampling site for univer- PFGE: Pulsed-field gel electrophoresis; SICU: surgical intensive care unit; sal screening of CRE [47]. However, during the period of SNP: detection of single nucleotide polymorphism; ST: sequence type; WGS: Whole genome sequencing our study, the nose, throat, and sputum were also sensi- tive sites to detect CRE, which might suggest, indirectly, Funding the important role of the respiratory tract in dissemin- This work was supported by grants from the National Natural Science Foundation of China (NSFC) [grant number 81371861], the Priority Project on ation during the outbreak. Therefore, it is important to Infectious Disease Control and Prevention [grant number 2012ZX10004215] apply active screening with nose, throat, and sputum from the Ministry of Science and Technology of the People’s Republic of sampling to detect hospital-acquired cases early during China and the Capacity-building project for pathogenic bacteria monitoring from Health and Family Planning Commission of the People’s Republic of an outbreak. China [grant number 131031102000150003]. There is no role of the funding Additionally, the genetic relatedness between the body in the design of the study and collection, analysis, and interpretation of strains judged by the interpretation criteria proposed by data and in writing the manuscript. Tenover et al. [33], we drew two additional inferences Availability of data and materials concerning the strains from case 4. First, with increasing This WGS project has been deposited at GenBank under the Bioproject ID time, the PFGE patterns of strains from case 4 changed, PRJNA313004, accession numbers LUVP00000000-LUGR00000000. suggesting that genome mutations occurred in the bac- Authors’ contributions teria in vivo for case 4, which was also proved by whole XL and HZ designed the study. WS, LW, MW, YH and JH carried out the data genome sequencing and comparison. Second, the two collection, active screening and target environmental monitoring. WS, HZ, strains from the bed linen of case 4 were isolated from TQ and HR carried out the experiments. WS, HZ, PD, TQ and CC performed the data analysis. WS, HZ and XL wrote the manuscript. All authors read and the same patient at the same time. However the PFGE approved the final version of the manuscript. patterns of the two strains were different. This result may be explained if the two isolates contaminated the Ethics approval and consent to participate bed linen at different times. This study was approved by the scientific and ethics committees of Beijing Tongren Hospital. All clinical specimens from patients were collected for Using PFGE, the strain isolated from case 8 was a diagnostic testing in hospitals at the request of the attending doctors. The “closely-related strain” to the other strains. However, active screening and targeted environmental monitoring was carried out in case 8 showed weak epidemiological relevance to the the case of an emergency investigation and in accordance with the recommendations of “Emergency treatment plan for hospital infection other cases, because this case did not overlap with outbreak in Tongren Hospital” and “2007 Guideline for Isolation any other case during their SICU stay. So we could Precautions: Preventing Transmission of Infectious Agents in Healthcare not judge the relationship between case 8 and the Settings (https://www.cdc.gov/infectioncontrol/pdf/guidelines/isolation- guidelines.pdf)”. All experiments were performed in accordance with other cases. We further used WGS-SNP analysis to relevant guidelines and regulations. The consent of the patients for the study the population structure of CRKP isolates from active screening of specimens, including K. pneumoniae detection, was eight cases to reveal the relationships among them. obtained verbally by medical staff in the hospital. The medical records were considered as legal documents. Furthermore, to protect patient WGS-SNP divided the isolate from case 8 far from privacy, the hospital set up a patient privacy and medical record the strains of other cases. By combination of the epi- management system according to the criminal procedure law, civil demiological information and molecular results, we procedure law, tort liability law, and medical malpractice law in China. Excepting for the patient’s doctor, no one can enter the management deduced that (i) cases 1 to 7 were on the same trans- system. Although no written informed consent was provided by mission route and that case 8 was an independent patients, the above measures are sufficient to protect the patients’ event; (ii) case 2, but not case 1 (the index patient), privacy. Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 10 of 11 Competing interests 14. Zhan L, Wang S, Guo Y, Jin Y, Duan J, Hao Z, et al. Outbreak by Hypermucoviscous The authors declare that they have no competing interests. Klebsiella pneumoniae ST11 isolates with Carbapenem resistance in a tertiary Hospital in China. Front Cell Infect Microbiol. 2017;7:182. https://doi.org/10.3389/fcimb.2017.00182. Publisher’sNote 15. Yu J, Wang Y, Chen Z, Zhu X, Tian L, Li L, et al. Outbreak of nosocomial Springer Nature remains neutral with regard to jurisdictional claims in NDM-1-producing Klebsiella pneumoniae ST1419 in a neonatal unit. J Glob published maps and institutional affiliations. Antimicrob Resist. 2016;8:135–9. https://doi.org/10.1016/j.jgar.2016.10.014. 16. Jin Y, Shao C, Li J, Fan H, Bai Y, Wang Y. Outbreak of multidrug Author details resistant NDM-1-producing Klebsiella pneumoniae from a neonatal Department of Clinical Laboratory, Beijing Tongren Hospital, Capital Medical unit in Shandong Province, China. PLoS One. 2015;10:e0119571. University, Beijing 100730, China. State Key Laboratory for Infectious Disease https://doi.org/10.1371/journal.pone.0119571. Prevention and Control, National Institute for Communicable Disease Control 17. Zheng R, Zhang Q, Guo Y, Feng Y, Liu L, Zhang A, et al. Outbreak of and Prevention, Chinese Center for Disease Control and Prevention, Beijing plasmid-mediated NDM-1-producing Klebsiella pneumoniae ST105 among 102206, China. Collaborative Innovation Centre for Diagnosis and Treatment neonatal patients in Yunnan, China. Ann Clin Microbiol Antimicrob. 2016;15: of Infectious Diseases, Hangzhou 310003, China. 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Whole genome sequence revealed the fine transmission map of carbapenem-resistant Klebsiella pneumonia isolates within a nosocomial outbreak

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Abstract

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major cause of nosocomial infections worldwide. The transmission route of CRKP isolates within an outbreak is rarely described. This study aimed to reveal the molecular characteristics and transmission route of CRKP isolates within an outbreak of nosocomial infection. Methods: Collecting case information, active screening and targeted environmental monitoring were carried out. The antibiotic susceptibility, drug-resistant genes, molecular subtype and whole genome sequence of CRKP strains were analyzed. Results: Between October and December 2011, 26 CRKP isolates were collected from eight patients in a surgical intensive care unit and subsequent transfer wards of Beijing Tongren hospital, China. All 26 isolates harbored bla , bla , and bla genes, had the same or similar pulsed-field gel electrophoresis patterns, and KPC-2 SHV-1 CTX-M-15 belonged to the sequence type 11 (ST11) clone. By comprehensive consideration of genomic and epidemiological information, a putative transmission map was constructed, including identifying one case as an independent event distinct from the other seven cases, and revealing two transmissions starting from the same case. Conclusions: This study provided the first report confirming an outbreak caused by K. pneumoniae ST11 clone co-harboring the bla , bla , and bla genes, and suggested that comprehensive consideration of KPC-2 CTX-M-15 SHV-1 genomic and epidemiological data can yield a fine transmission map of an outbreak and facilitate the control of nosocomial transmission. Keywords: Carbapenemases, K. pneumoniae, KPC-2, Outbreak, Whole genome sequencing * Correspondence: zhj_0901@163.com; zhouhaijian@icdc.cn; chenchen1@ccmu.edu.cn; luxinxin2009@126.com Wenjun Sui, Haijian Zhou and Pengcheng Du contributed equally to this work. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China Beijing Key Laboratory of Emerging Infectious Diseases, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China Department of Clinical Laboratory, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 2 of 11 Background Active screening and targeted environmental monitoring Carbapenems remain the first-line therapeutic antimicro- Targeting KPC-2 K. pneumoniae, active screening and bials for severe infections caused by extended-spectrum environmental monitoring were carried out as measures β-lactamase (ESBL)-producing multidrug-resistant Enterobacte- of infection control between October 11th and October riaceae. However, the emergence of carbapenemase-mediated 27th. The method of active screening and targeted envir- resistance to all β-lactams, including carbapenems, is a major onmental monitoring was described previously [23]. In public health threat [1, 2]. Over the last decade, brief, for active screening, we collected samples from the carbapenem resistance, attributed to the production of nose, throat, groin, and axilla of each patient using ster- carbapenem-hydrolyzing β-lactamases, has been steadily ile cotton swabs (the sputum and stool samples were in- increasing among Enterobacteriaceae isolates, particularly cluded in our clinical samples), and a real-time PCR Klebsiella pneumoniae [3]. Carbapenem-resistant K. pneu- assay was used to screen all samples for bla [24]. The KPC moniae (CRKP) has emerged in many countries as a result first swab of each patient was taken within 48 h upon of intra-continental and inter-continental spread [4–6]. ICU admission. For targeted environmental monitoring, Clinically, the K. pneumoniae carbapenemase (KPC) six environmental sites on the bed sheet were sampled enzyme is one of the most prevalent carbapenemases. for each KPC-KP positive patient using contact plates The bla genes are predominantly plasmid encoded. (16 cm ; Qingdao Classical Biochemical Equipment, KPC In some cases, bla genes exist in particularly clones Qingdao, China). KPC (such as K. pneumoniae multilocus sequence type (ST) 258 and ST11, which have facilitated their rapid dissem- Bacterial identification and antimicrobial susceptibility ination. Since the first KPC-producing isolate was identi- Bacterial identification and antibiotic susceptibility test- fied from North Carolina, USA, in 1996 [7], the ing were initially performed using the VITEK-2 auto- occurrence of KPC-producing bacteria has been con- mated system (BioMérieux, France). Escherichia coli tinuously reported in other parts of the USA, Europe, ATCC 25922 was used as quality control strain for anti- South America, the Middle East, and Asia [5, 8–10]. biotic susceptibility testing. Susceptibility category was They have been associated with large nosocomial designated according to the Clinical and Laboratory outbreaks worldwide, including those in China. The Standards Institute (CLSI) breakpoints [22]. Susceptibil- CRKP outbreak isolates in China mostly carried bla ity to tigecycline was defined based on the criteria pro- KPC-2 [11–14]or bla [15–18]. KPC-producing members posed by the European Committee on Antimicrobial NDM-1 of the family Enterobacteriaceae have also been associated Susceptibility Testing-2011 (susceptible, minimum in- with high mortality rates, particularly among critically ill pa- hibitory concentration (MIC) ≤ 1 μg/ml). tients with a history of prolonged hospitalization [19–21]. Phenotypic screening for the presence of carbapene- Thesefactsstronglysuggest aneedfor theimplementation mase was performed using the modified Hodge test of adequate preventive measures to effectively control the (MHT). In addition, ESBL production was tested using spread of such pathogens. the Double-Disk Synergy Test (DDST), as recommended From October 8th to December 23th, 2011, a total of by the CLSI [22]. 12 CRKP strains were isolated from clinical samples of eight patients admitted to the surgical intensive care unit Determination of carbapenemase genes (SICU). Active screening and targeted environmental We screened carbapenem-resistance genes (bla , bla , KPC NMC monitoring were carried out between October 11th and bla , bla , bla , bla , bla , bla , bla , SME IMI GES NDM IMP VIM SPM October 27th, and eight CRKP were isolated from nose bla , bla , bla , bla , bla , GIM SIM OXA-48 OXA-51-like OXA-23-like and throat of inpatient in SICU, as well as environmen- bla , bla , in all strains as described previ- OXA-24-like OXA-58-like tal samples. Furthermore, six CRKP were retrieved from ously [5, 25, 26]. DNA sequencing was performed on both one case in subsequent transfer wards. In the present strands of the PCR amplification products. The results were study, we carried out a retrospective investigation of the compared and aligned with reference sequences using the molecular and genomic epidemiology of the outbreak. online BLAST database. Methods Pulsed-field gel electrophoresis (PFGE) Retrospective analysis of medical records We used the 1-day, standardized PFGE protocol for K. The medical records of patients from whom CRKP was pneumoniae [27]. Cell suspensions were placed in isolated were reviewed, including time of stay in SICU, polystyrene tubes (Falcon; 12 × 75 mm), and their optical bed site, data of the first CRKP isolation, and outcomes. density was adjusted to 3.8–4.0 using a Densimat photom- CRKP is defined as resistance to carbapenems according eter (BioMérieux, Marcy l’Etoile, France). K. pneumoniae to the Clinical and Laboratory Standards Institute (CLSI) slices were digested using 50 U per slice of XbaI(Takara, breakpoints [22]. Dalian, China) for 4 h at 37 °C, and electrophoresis was Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 3 of 11 performed using a CHEF-DRIII system (Bio-Rad Labora- teaching hospital in Beijing, China. Beijing Tongren tories, Hercules, CA, USA). Electrophoresis was run with Hospital receives an average of 3500–5000 outpatients a switch time of 6 s to 36 s for 18.5 h, and images were and emergency patients per day. SICU has 18 beds. captured using a Gel Doc 2000 system (Bio-Rad) and con- The index case of this outbreak was identified on verted to TIFF files. The TIFF files were analyzed using October 8th, 2011. Following identification of second the BioNumerics version 5.1 software (Applied Maths, CRKP-infection case in the same room on October 10th, Kortrijk, Belgium). and a CRKP carrier on October 11th, an outbreak was declared. Between October 8 and December 23, a total Multilocus sequence typing (MLST) of 50 patients were admitted into the SICU and eight of MLST with seven genes (gapA, infB, mdh, pgi, phoE, them were found to have a CRKP infection or rpoB,and tonB) was performed on isolates according to colonization though routine clinical culture and active the protocol described on the K. pneumoniae MLST screening (Table 1). The timeline of patient admission website (http://bigsdb.pasteur.fr/). Alleles and STs were and CRKP isolation is showed in Fig. 1. assigned using the MLST database (http://bigsdb.pas- Among the eight patients, four were infected by CRKP teur.fr/klebsiella/klebsiella.html). Alleles and STs that and other four were CRKP carrier. Of the four infection had not been previously described were submitted to the cases, one died, one was discharged from the SICU, and curator of the database and were assigned new two were transferred to other wards and ultimately dis- designations. charged. It should be noted that cases 1 to 7 overlapped with at least three other cases when in the SICU, but Whole genome sequencing (WGS), detection of single case 8 did not overlap with any other case during SICU nucleotide polymorphisms (SNPs), and clustering analysis stay. The initial strains of each case were selected for WGS. CRKP detected twice from the the bed sheets of case 4 Bacterial strains were sequenced using Illumina sequen- during the outbreak. In addition to active screening and cing by constructing two paired-end (PE) libraries with targeted environmental monitoring, other stringent infection average insertion lengths of 500 bp and 2000 bp, re- prevention and control measures were implemented, includ- spectively. Sequences were generated using an Illumina ing contact precautions, strengthen of hand hygiene, GA IIx (Illumina Inc., San Diego, CA, USA). Raw data environmental cleaning (including changing the bed linen was processed in four steps, including removing reads more frequently for each CRKP-positive patient, and imme- with 5 bp of ambiguous bases, removing reads with diately stringent terminal sterilization with hydrogen perox- 20 bp of low quality (≤ Q20) bases, removing adapter ide after they were discharged from the ward or died), and contamination, and removing duplicated reads. Finally, enhanced antimicrobial stewardship were introduced. For 100× libraries were obtained with clean PE read data. the patients transferred to other wards, contact precautions Assembly was performed using SOAPdenovo v1.05 [28]. and follow-up screening were employed until they were dis- The whole-genome sequence of K. pneumoniae charged from our hospital. Since January 22, 2012 (the dis- HS11286 (GenBank accession: NC_016845.1) was used charge date of the last patients with CRKP colonization), as the reference sequence, and clean reads of sequenced over a period of 10 months, no further carbapenem-resistant isolates were mapped to the reference genome by bowtie Enterobacteriaceae (CRE) were isolated in the SICU. 2 software under the default parameters [29]. SNPs were then identified using Samtools [30] and combined to- Antibiotic susceptibility and characterization of resistance gether according to the reference. SNPs with low quality genes (read depth < 5) and those located within 5 bps on the All 26 CRKP isolates showed same results of antibiotic sus- same chromosome were removed to avoid the effect of ceptibility test. The MICs of meropenem, imipenem, and recombination, as described in our previous studies [31, ertapenem were ≥ 16 μg/ml, ≥ 16 μg/ml, and ≥ 8 μg/ml, re- 32]. The isolates were clustered and a heatmap was gen- spectively, for all isolates. All isolates were susceptible to erated using the heatmap package in R. The transmis- amikacin ((MIC ≤2 μg/ml), gentamicin (MIC ≤1 μg/ml), sion route was then reconstructed based on the tobramycin (MIC ≤1 μg/ml), and trimethoprim/sulfa- emergence of different SNPs in each isolate and the case methoxazol (MIC ≤1:19 μg/ml), and were resistant to information, including the onset time of infection and ampicillin (MIC ≥32 μg/ml), ampicillin/sulbactam (MIC the hospitalization time. ≥32 μg/ml), piperacillin (MIC ≥128 μg/ml), piperacillin/taz- obactam (MIC ≥128 μg/ml), cefazolin (MIC ≥64 μg/ml), Results cefotetan (MIC ≥64 μg/ml), ceftazidime (MIC ≥64 μg/ml), Outbreak descriptions ceftriaxone (MIC ≥64 μg/ml), cefepime (MIC ≥64 μg/ml), The outbreak occurred in the SICU of Beijing Tongren aztreonam (MIC ≥64 μg/ml), ciprofloxacin (MIC Hospital, a 1600-bed general tertiary care and university-affiliated ≥4 μg/ml), levofloxacin (MIC ≥8 μg/ml), nitrofurantoin Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 4 of 11 Table 1 Case descriptions involved in the outbreak of SICU, October to December 2011 Case No. Time of stay in SICU Date of the first isolation Type of specimen Infection /colonization Antimicrobial used before Antimicrobial used after Outcome 1 30/9/2011–17/10/2011 8/10/2011 sputum infection one course of VA one course of AK died 2 7/10/2011-14/10/2011 10/10/2011 abdominal drainage fluid infection one course of MEM one course of AK transfer to other ward 3 8/10/2011–17/10/2011 11/10/2011 nose colonization –– discharged 4 10/9/2011-16/12/2011 16/10/2011 throat infection one course of CAZ and two courses of Gn transfer to other ward 1 course of PIP/TAZ 5 14/10/2011–16/11/2011 18/10/2011 nose colonization one course of CIP one course of Ak discharged 6 16/10/2011–15/11/2011 21/10/2011 urine infection one course of CRO one course of GN discharged 7 24/10/2011–25/11/2011 27/10/2011 nose colonization one course of CRO one course of CRO discharged 8 20/12/2011–22/1/2012 23/12/2011 sputum colonization –– discharged VA Vancomycin, MEM meropenem, CAZ ceftazidime, PIP/TAZ piperacillin/tazobactam, CIP ciprofloxacin, CRO ceftriaxone, AK amikacin, GN gentamicin Case 2 was transferred to a general surgery ward and ultimately discharged from the hospital Case 4 was transferred to a geriatric ward and ultimately discharged from the hospital Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 5 of 11 Fig. 1 The timeline of patient admission and CRKP isolation. Shadows on the timeline represent the admitting’s duration of the case. Different wards are indicated in different colors. The red arrow indicates the isolation of the strains Fig. 2 Clustering of the 26 K. pneumoniae isolates based on PFGE patterns. The first positive cultures of each case are marked by an asterisk. The information of strain ID, PFGE type, patient ID, isolation date, ward, type of specimen, and MLST type is listed to the left of the patterns. ADF, abdominal drainage fluid; BAL, bronchoalveolar lavage Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 6 of 11 Fig. 3 Clustering of the 26 K. pneumoniae isolates based on MCG typing. The first positive cultures of each case are marked by an asterisk. The information of strain ID, patient ID, type of specimen, isolation date, and ward are listed to the left of the patterns. ADF, abdominal drainage fluid; BAL, bronchoalveolar lavage (MIC ≥512 μg/ml), and tigecycline (MIC ≥2 μg/ml). All interpretation criteria of PFGE patterns proposed by Ten- isolates were positive for carbapenemase and ESBL pro- over et al. [33], the first positive culture of cases 1, 2, 3, 5, duction by the MHT and DDST assays, respectively. We 6, 7 were the “same strain” and those of cases 4 and 8 further confirmed the presence and production of carba- were “closely-related strains”. penemases and ESBLs by PCR and sequencing. All 21 iso- Excepting for case 8, all six isolates showing different lates harbored the bla , bla ,and bla patterns to the dominant PFGE type were isolated from KPC-2 SHV-1 CTX-M-15 genes and tested negative for other antimicrobial resist- case 4, including three strains from urine, one from spu- ance genes (bla , bla , bla , bla , bla , tum, and two from the bed sheet. The two strains iso- NMC SME IMI GES NDM bla , bla , bla , bla , bla , bla , lated from the bed sheet (TRqt-47 and TRqt-48) of case IMP VIM SPM GIM SIM OXA-48 bla , bla , bla , bla , 4 showed one band that was different to the strain iso- OXA-51-like OXA-23-like OXA-24-like OXA-58-like and bla ). lated from the nose (TRqt-43) on the same day. It was TEM interesting that with increasing time, the PFGE patterns of the strains from case 4 also changed, especially Molecular subtyping analysis by PFGE and MLST isolates from December 2011. All the strains isolated in MLST indicated that all 26 isolates belonged to ST11. October showed dominant patterns or showed only one However, PFGE showed some diversity (Fig. 2). PFGE di- band different from the dominant pattern; two strains vided the 26 isolates into seven different PFGE types isolated in November (TR221, TR231-m) showed two (PT1–PT7). The dominant PFGE type (PT3) contained bands that were different from dominant pattern and an- 19 isolates. The other six PFGE types showed one to other strain isolated in December (TR258) showed four four bands that were different to the dominant PFGE bands that were different from dominant pattern. type (PT3). Except for cases 4 and 8, all the first positive cultures from each affected patients belonged to the dominant PFGE type (PT3). The isolates of case 4 (PT4) Comparison of outbreak isolates based on WGS-based SNPs and case 8 (PT2) showed one and three bands that were We performed WGS and MCG typing of the 26 CRKP different to those of PT3, respectively. Using the isolates, in hope of using the SNPs found in their Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 7 of 11 Fig. 4 Evolutionary relationships based on MCG typing of 26 CRKP isolates. Isolates from different patients are indicated in different colors. The first positive cultures of each case are marked by an asterisk genomes to determine putative transmission map of this different to the reference strain, respectively. All isolates outbreak. Genomic comparisons revealed a total of 32 from cases 1 and 3 had only one SNP difference to the MCG SNPs among the 26 isolates (Fig. 3). The evolu- reference strain. At the same time, two isolates from tionary relationships based on MCG typing of the 26 case 4, one isolate from case 6, and two isolates from CRKP isolates are outlined and presented in Fig. 4. case 7 had the same SNP profile and showed only one Five case 2 strains isolated in October showed the SNP difference to the reference strain. same SNP profile as the reference strain K. pneumoniae It is worth noting that four case 4 isolates, two from HS11286. The other case 2 strains isolated in early and the bed sheet (TRqt-47, TRqt-48) and two from urine late November had one and two SNPs that were (TR221, TR231-m), showed 7–10 SNP differences Fig. 5 Clustering of the eight first positive CRKP cultures of each case based on MCG typing. The matrix of the SNP differences is presented on the left of the cluster tree Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 8 of 11 the hospital. Thus Case 1 is unlikely to be the source. Case 3 checked in the SICU on October 8th, 2011, the same day of first CRKP isolation of case 1; however, case 3 checked in the SICU in the afternoon, and the CRKP-positive sample had already been collected from case 1 in the morning, which suggested that the CRKP of case 1 was not transmitted from case 3. Thus, case 2 was the most likely source of the outbreak isolates. Second, the isolates of cases 1 and 3 showed no SNP differences, suggesting direct transmission between them. However, it could not be confirmed that whether case 2 directly transmitted CRKP to case 1 or case 3. Third, the case 2’s isolate showed one SNP difference Fig. 6 Putative map of CRKP transmission during the outbreak. The to isolates of cases 4 and 7, but two SNP differences to transmission map was constructed with genetic and patient trace that of cases 5 and 6, suggesting that CRKP from case 2 data. Nodes represent patients, and arrows indicate a transmission was genomically closer to cases 4 and 7. However, case event directly from one patient to another 7’s hospital stay did not overlap with that of case 2 and the date of the first isolation of CRKP from case 7 was compared with the initial isolate (TR198) of case 4, and significantly later than the other cases; therefore, we de- another urine isolate of case 4 (TR258) isolated later had duced the case 7’s isolate was transmitted from case 2 only two SNP differences to TR198, suggesting that the through case 4. The isolates of cases 5 and 6 were trans- CRKP isolated from case 4 during the study period came mitted from case 4, as there was only one SNP between from a different source. isolates cases 5 and 6 and that from case 4, but showed The isolate of case 8 (TR262) showed 6–18 SNP differ- more SNPs differences to that of other cases. ences with other isolates and had five specific SNPs. To Consistent with the genomic and epidemiological in- determine whether case 8 is related to the other seven formation, the integrated map identified two transmis- cases, the initial isolates of each case were chosen and an- sions starting from case 2 (Fig. 6). The first transmission alyzed. Grouping the eight initial isolates on the basis of was directly from case 2 to case 1 or case 3, and a trans- the patterns of shared variants partitioned them into two mission was observed between cases 1 and 3. A second clusters (Fig. 5). Cluster 1 contained seven isolates of cases transmission from case 2 was predicted to go through 1 to 7, and cluster 2 consisted only of case 8. The pairwise case 4 before being transmitted to cases 5, 6, and 7. distances within cluster 1 were ≤ 3 SNP differences; in contrast, the distances between cluster 2 and cluster 1 iso- Discussion lates were 8 SNP differences. The within-cluster distance In this study, we described an outbreak caused by CRKP of cluster 1 was much smaller than the between-cluster in a SICU of a large university hospital in China. The distance, indicating considerable divergence between the CRKP isolates belonged to clone ST11, and coproduced two clusters. The epidemiological information of case 8 carbapenemase (KPC-2) and ESBLs (CTX-M-15 and indicated that they had not overlapped during their hos- SHV-1). ST11 is the dominant clone of KPC-producing pital stay with any of the other seven patients; therefore, K. pneumoniae in China and has also been reported we deduced that cases 1 to 7 probably shared the same sporadically in the rest of the world, including other transmission route and that case 8 seemed to be an inde- regions of Asia [34, 35], America [36], and Europe [37–39]. pendent event. KPC is the most common carbapenemase in K. pneumoniae and most of the KPC type in China is KPC-2 [40]. Similarly, Inference of most likely transmission route CTX-M-15 and SHV-1 are the main types of CTX-M-type Furthermore, the genomic and epidemiological informa- and SHV-type in China [41]. The coexistence of bla KPC-2 tion were integrated to construct the transmission route. with bla type genes in K. pneumoniae was previ- CTX-M-15 The most likely transmission route is shown in Fig. 6. ously reported in Bulgaria [42], Brazil [43], and China [44]. First, case 2 was suspected of being the source of the out- In a recent study, CRKP strains co-harboring the bla , KPC-2 break isolates because the CRKP from case 2 showed the bla ,and bla genes were found in several STs in- CTX-M-15 SHV same SNP patterns to reference strain K. pneumoniae cluding ST11; however, only sporadic strains were reported HS11286. This was also supported by the epidemiological in that study [44]. As far as we know, this is the first study to information. Case 1 admitted in the SICU on September report an outbreak caused by CRKP co-harboring the 30th, 2011, 9 days before the first CRKP was isolated, bla , bla ,and bla genes, which suggests KPC-2 CTX-M-15 SHV-1 which suggested that the CRKP of case 1 was acquired in that attention should be paid to the K. pneumoniae isolates Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 9 of 11 coproducing epidemic carbapenemases and ESBLs, especially was the source of CRKP in this outbreak; (iii) there the outbreak strains described here belonging to the epi- were two transmissions starting from case 2. demic ST11 clone. Given the easy of transfer and acquisition of carbape- Conclusions nemase and ESBLs genes, measures must be imple- Though this study is a retrospective study and thus the mented to control the outbreak and avoid nosocomial results of the WGS could not be used to control the transmission [2, 45]. Agodi et al. reported that cleaning nosocomial transmission. However, our data clearly and disinfection of the ICU, segregation of affected pa- showed that WGS and MCG typing could reveal the de- tients, barrier nursing, and strict compliance with hand tails of transmission within a CRKP nosocomial out- hygiene procedures led to containment of an outbreak of break. In the future, real-time genomic sequencing and KPC-producing K. pneumoniae [46]. In our study, we analysis of an outbreak should be carried out and the applied active screening and targeted environmental findings could be used to control outbreaks. monitoring to combat the secondary transmission of Abbreviations imported KPC clones in the SICU. Except for the two CLSI: Clinical and Laboratory Standards Institute; CRE: carbapenem-resistant index cases, we found that six patients acquired this Enterobacteriaceae; CRKP: Carbapenem-resistant K. pneumoniae; pathogen during their hospital stay, two of them with DDST: Double-Disk Synergy Test; ESBL: extended-spectrum β-lactamase; KPC: K. pneumoniae carbapenemase; MHT: modified Hodge test; urinary tract infections. Some reports stated that the MIC: minimum inhibitory concentration; MLST: Multilocus sequence typing; rectum was the most sensitive sampling site for univer- PFGE: Pulsed-field gel electrophoresis; SICU: surgical intensive care unit; sal screening of CRE [47]. However, during the period of SNP: detection of single nucleotide polymorphism; ST: sequence type; WGS: Whole genome sequencing our study, the nose, throat, and sputum were also sensi- tive sites to detect CRE, which might suggest, indirectly, Funding the important role of the respiratory tract in dissemin- This work was supported by grants from the National Natural Science Foundation of China (NSFC) [grant number 81371861], the Priority Project on ation during the outbreak. Therefore, it is important to Infectious Disease Control and Prevention [grant number 2012ZX10004215] apply active screening with nose, throat, and sputum from the Ministry of Science and Technology of the People’s Republic of sampling to detect hospital-acquired cases early during China and the Capacity-building project for pathogenic bacteria monitoring from Health and Family Planning Commission of the People’s Republic of an outbreak. China [grant number 131031102000150003]. There is no role of the funding Additionally, the genetic relatedness between the body in the design of the study and collection, analysis, and interpretation of strains judged by the interpretation criteria proposed by data and in writing the manuscript. Tenover et al. [33], we drew two additional inferences Availability of data and materials concerning the strains from case 4. First, with increasing This WGS project has been deposited at GenBank under the Bioproject ID time, the PFGE patterns of strains from case 4 changed, PRJNA313004, accession numbers LUVP00000000-LUGR00000000. suggesting that genome mutations occurred in the bac- Authors’ contributions teria in vivo for case 4, which was also proved by whole XL and HZ designed the study. WS, LW, MW, YH and JH carried out the data genome sequencing and comparison. Second, the two collection, active screening and target environmental monitoring. WS, HZ, strains from the bed linen of case 4 were isolated from TQ and HR carried out the experiments. WS, HZ, PD, TQ and CC performed the data analysis. WS, HZ and XL wrote the manuscript. All authors read and the same patient at the same time. However the PFGE approved the final version of the manuscript. patterns of the two strains were different. This result may be explained if the two isolates contaminated the Ethics approval and consent to participate bed linen at different times. This study was approved by the scientific and ethics committees of Beijing Tongren Hospital. All clinical specimens from patients were collected for Using PFGE, the strain isolated from case 8 was a diagnostic testing in hospitals at the request of the attending doctors. The “closely-related strain” to the other strains. However, active screening and targeted environmental monitoring was carried out in case 8 showed weak epidemiological relevance to the the case of an emergency investigation and in accordance with the recommendations of “Emergency treatment plan for hospital infection other cases, because this case did not overlap with outbreak in Tongren Hospital” and “2007 Guideline for Isolation any other case during their SICU stay. So we could Precautions: Preventing Transmission of Infectious Agents in Healthcare not judge the relationship between case 8 and the Settings (https://www.cdc.gov/infectioncontrol/pdf/guidelines/isolation- guidelines.pdf)”. All experiments were performed in accordance with other cases. We further used WGS-SNP analysis to relevant guidelines and regulations. The consent of the patients for the study the population structure of CRKP isolates from active screening of specimens, including K. pneumoniae detection, was eight cases to reveal the relationships among them. obtained verbally by medical staff in the hospital. The medical records were considered as legal documents. Furthermore, to protect patient WGS-SNP divided the isolate from case 8 far from privacy, the hospital set up a patient privacy and medical record the strains of other cases. By combination of the epi- management system according to the criminal procedure law, civil demiological information and molecular results, we procedure law, tort liability law, and medical malpractice law in China. Excepting for the patient’s doctor, no one can enter the management deduced that (i) cases 1 to 7 were on the same trans- system. Although no written informed consent was provided by mission route and that case 8 was an independent patients, the above measures are sufficient to protect the patients’ event; (ii) case 2, but not case 1 (the index patient), privacy. Sui et al. Antimicrobial Resistance and Infection Control (2018) 7:70 Page 10 of 11 Competing interests 14. Zhan L, Wang S, Guo Y, Jin Y, Duan J, Hao Z, et al. Outbreak by Hypermucoviscous The authors declare that they have no competing interests. Klebsiella pneumoniae ST11 isolates with Carbapenem resistance in a tertiary Hospital in China. Front Cell Infect Microbiol. 2017;7:182. https://doi.org/10.3389/fcimb.2017.00182. Publisher’sNote 15. Yu J, Wang Y, Chen Z, Zhu X, Tian L, Li L, et al. Outbreak of nosocomial Springer Nature remains neutral with regard to jurisdictional claims in NDM-1-producing Klebsiella pneumoniae ST1419 in a neonatal unit. J Glob published maps and institutional affiliations. Antimicrob Resist. 2016;8:135–9. https://doi.org/10.1016/j.jgar.2016.10.014. 16. Jin Y, Shao C, Li J, Fan H, Bai Y, Wang Y. Outbreak of multidrug Author details resistant NDM-1-producing Klebsiella pneumoniae from a neonatal Department of Clinical Laboratory, Beijing Tongren Hospital, Capital Medical unit in Shandong Province, China. PLoS One. 2015;10:e0119571. University, Beijing 100730, China. State Key Laboratory for Infectious Disease https://doi.org/10.1371/journal.pone.0119571. Prevention and Control, National Institute for Communicable Disease Control 17. Zheng R, Zhang Q, Guo Y, Feng Y, Liu L, Zhang A, et al. Outbreak of and Prevention, Chinese Center for Disease Control and Prevention, Beijing plasmid-mediated NDM-1-producing Klebsiella pneumoniae ST105 among 102206, China. Collaborative Innovation Centre for Diagnosis and Treatment neonatal patients in Yunnan, China. Ann Clin Microbiol Antimicrob. 2016;15: of Infectious Diseases, Hangzhou 310003, China. 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Journal

Antimicrobial Resistance & Infection ControlSpringer Journals

Published: Jun 1, 2018

References

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