Institutional outbreak involving multiple clades of IMP-producing Enterobacter cloacae complex sequence type 78 at a cancer center in Tokyo, Japan

This is a descriptive study of an institutional outbreak of CPE at a 700-bed cancer center in Tokyo, Japan. It provides care for patients with all type of malignancies. Annually, 17,500 patients are hospitalized with a mean of 12 days. All the CPE isolates between January 2014 and December 2017 were analyzed. In addition, clinical and epidemiological investigation was performed for all the patients who had CPE isolated.

Clinical information of the patients with CPE was extracted from the medical records retrospectively and included age, sex, type of malignancy and other comorbidities, date of the first isolation of CPE, type of sample from which CPE was isolated, hospitalized ward, department caring the patient, clinical significance of the CPE isolates (infection or colonization), use of antimicrobial agents within 30 days prior to the isolation of CPE, type and date of surgery within 90 days prior to the isolation, type of endoscopy within 90 days prior to the isolation, cancer chemotherapy within 90 days prior to the isolation, admission to the hospital during the study period and to other hospitals within a year prior to the isolation, and death within 30 and 90 days after the isolation of CPE. Additionally, the type of infection, antimicrobial treatment, necessity and achievement of source control, and prognosis were reviewed for the cases with infections caused by CPE. Infection and colonization were determined according to the CDC definition [12].

Routine bacterial identification and antimicrobial susceptibility testing were performed with MicroScan WalkAway (Beckman Coulter, Brea, CA, USA) at the hospital. The results of antimicrobial susceptibility testing were interpreted with CLSI M100-S17 guidelines in 2014, and with CLSI M100-S22 guidelines, in which lower breakpoints of cephalosporins and carbapenems for Enterobacterales were adopted, from 2015 through 2017 [13, 14]. Testing for carbapenemase production was performed on the isolates showing non-susceptibility against cefepime or any carbapenems. Non-susceptibility to cefepime was added to the screening criteria due to concerns about low sensitivity of the screening using non-susceptibility to carbapenems alone for the detection of IMP-producing Enterobacterales [15]. Carbapenemase production (focusing on MBLs) was confirmed with ceftazidime 30-μg disks (Eiken Chemical, Tokyo, Japan), imipenem 10-μg disks (Eiken Chemical, Tokyo, Japan), and sodium mercaptoacetate (SMA) 3-mg disks (Eiken Chemical, Tokyo, Japan). Isolates showing enlargement of inhibitory zone diameters around the ceftazidime disk or imipenem disk by > 5 mm when it was located adjacent to an SMA disk were determined to be carbapenemase-producing Enterobacterales [16, 17]. Additionally, modified carbapenem inactivation method (mCIM) according to CLSI M100-S27 guidelines was performed after April 2017 [18].

All CPE isolates detected first from each patient at the hospital were collected and analyzed at a research laboratory. Because susceptibility testing of carbapenems with drug concentration below 4 μg/mL was not performed at the hospital in 2014, antimicrobial susceptibility testing of all isolates was conducted again at the research laboratory with BD Phoenix NMIC/ID-208 panel (BD Diagnostics, Sparks, Maryland, USA) and results were interpreted according to CLSI M100-S27 guidelines [18]. Carriage of bla_IMP-1-group genes was screened by PCR as described previously [19]. Whole genome sequencing was performed with Illumina Miseq (Illumina, San Diego, California, USA). Genomic DNA libraries were prepared with Nextera XT DNA library preparation kit (Illumina) and were sequenced for 600 cycles (300-bp paired-end reads). Raw reads generated by Miseq were quality trimmed with Trimmomatic tool (version 0.38) and assembled using SPAdes (version 3.12.0). Since all CPE isolates were identified as ECC or related species by MicroScan WalkAway at the hospital, species and subspecies were determined by comparing the average nucleotide identity (ANI) of the genomes of study isolates with those of type strains of ECC using a threshold of > 96.5% at ANI Calculator of EZ BioCloud website (https://​www.​ezbiocloud.​net/​tools/​ani) [20]. Multilocus sequence typing (MLST), plasmid replicon typing, plasmid MLST, and screening of acquired resistance genes were conducted with MLST 1.8, PlasmidFinder 1.3, pMLST 1.4, and ResFinder 3.0, respectively, at Centers for Genomic Epidemiology website (https://​cge.​cbs.​dtu.​dk/​services/​). Structures of integrons were analyzed with Basic Local Alignment Search Tool (BLAST) (https://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi) using nucleotide sequences of contigs containing bla_IMP-1-group. Core-genome single nucleotide polymorphism (SNP)-based phylogenetic analysis of ST78 isolates of ECC was performed as described previously using genomic sequence of E. cloacae strain 109 belonging to ST78 (GenBank accession number NZ_CP020525) as the reference [6]. Conjugation experiments were carried out with filter mating methods using non-lactose-fermenting E. coli ML4909 strain (a rifampin-resistant mutant derived from E. coli K-12) as a recipient. Transconjugants were selected on Drigalski lactose agar supplied with moxalactam (16 μg/mL) and rifampin (100 μg/ml). The conjugation experiment was attempted three times for each donor isolate. Carriage of bla_IMP-1-group were confirmed with PCR and PCR-based replicon typing of the plasmids were performed for transconjugants [21].

All nucleotide sequences of draft genomes have been deposited in the NCBI database under BioProject accession number PRJDB9939.

In July 2014, CPE was detected from a hospitalized patient at the cancer center for the very first time. In total, 18 hospitalized patients were found to have CPE during the study period confirmed by clinical culture (n = 13) or surveillance culture (n = 5) (Table 1, Table S1). Seventeen and one isolates were identified as E. claocae and Cronobacter sakazakii, respectively, with MicroScan WalkAway. All isolates were positive for production of MBLs by the SMA disk testing. Date of first isolation of CPE from all but one (Patient-18) patients was clustered in a 16-months period (between July 2014 and Oct 2015). The mean age of 18 patients was 68.8 years. Sixteen patients (88.9%) were male. Seventeen patients (94.4%) had gastrointestinal malignancy. Six (33.3%), fourteen (77.8%), and sixteen (88.9%) patients had received chemotherapy within 90 days, surgery within 90 days, and antimicrobial therapy within 30 days of the first isolation of CPE, respectively. Although the patients were under the care of 7 different departments, Surgery-A (n = 7), Surgery-B (n = 4), and Medical Oncology-E (n = 3) departments were involved in the care of multiple patients. At the time of CPE isolation, the patients were located at one of five different wards or one intensive care unit (ICU). Six, five, and three patients were at ICU, Ward-V, and Ward-X, respectively. Fifteen patients (83.3%) received endoscopic procedure within 90 days of isolation of CPE. All six patients from whom CPE was isolated within 3 days after surgery (Patient-2, − 5, − 10, − 14, − 15, and − 16) received routine airway nebulization and frequent airway suctioning at ICU and bronchoscopy was also performed at operating rooms in two patients (Patient 5 and − 15). Thirteen (76%) of seventeen case patients between July 2014 and Oct 2015 had history of hospitalization at multiple wards before the isolation of CPE (Fig. 1).

A ward-wide active surveillance was performed twice at Ward-V (November 2014 and February 2015), three times at Ward-X (May, June, and July 2015), and once at Ward Y (November 2014). A total of 191 patients were screened and two patients were positive for carriage of CPE (Patient-7 and -12). Additionally, a patient with a history of admission in Ward-V was screened upon a later admission at Ward-X and was positive for the growth of CPE (Patient-9). Surveillance cultures performed at the discretion of primary physicians immediately after surgery without using selective media turned positive for carriage of CPE in two patients (Patient-2 and -6).

Six patients had infections due to CPE (Table 2). Although one patient died of an intraabdominal infection following intestinal perforation on the 11th day of the onset of the infection, infections in other patients were cured without relapse. Two other patients died within 90 days due to reasons unrelated to the carriage of CPE (Table S1).

The patients with a history of isolation of CPE were cared for in a private room under contact precautions according to the infection prevention protocol of the hospital. After July 2014 when the isolation of CPE from patients hospitalized in different wards was documented, occurrence of an institutional outbreak of CPE was notified to the all hospital staffs and strict compliance to the infection prevention protocol was enforced. In addition, direct observation of hand hygiene compliance was initiated by infection preventionists and the data were fed back to each hospital department. Compliance to the infection prevention protocol was thoroughly checked especially at the wards where the patients with CPE was hospitalized. Sampling from hospital environment was not performed. Compliance to the cleaning and disinfection protocol of endoscopes was confirmed and bacterial cultures of the relevant endoscopes, including bronchoscopes for the operating rooms and ICU and duodenoscope, were negative for the growth of CPE.

All CPE isolates were non-susceptible to cefepime and all but one isolates were non-susceptible to piperacillin-tazobactam with BD Phoenix NMIC/ID-208 panel. Three isolates were susceptible to aztreonam. Although most of the isolates were non-susceptible to carbapenems, MIC of > 4 μg/mL for imipenem and meropenem was observed only in two isolates and one isolate, respectively. Most of the isolates was susceptible to non-β-lactam antibiotics tested (Table 3).

Fourteen of eighteen CPE isolates were identified as E. hormaechei by ANI and other isolates were E. hormaechei subsp. steigerwaltii (n = 2), E. asburiae (n = 1), and E. xiangfangensis (n = 1) (Table 3). All E. hormaechei isolates were ST78 and isolated between July 2014 and August 2015. While one isolate (TUM17942) carried bla_IMP-11, all other isolates carried bla_IMP-1. As expected, all isolates carried chromosomal ampC genes, but acquired genes for extended-spectrum β-lactamases (ESBL), AmpC, and carbapenemases other than bla_IMP were not identified in any isolates. Plasmid replicon for IncHI2 was documented in 16 isolates and six of these (TUM14648, TUM14652, TUM17945, TUM17946, TUM17947, TUM17949) were pMLST-ST1 (smr0119:1-smr0018:1). While remaining 10 isolates also had allele 1 of smr00119, smr00118 had 0.3% difference from allele 1 in four isolates (TUM14647, TUM17939, TUM17943, TUM17944), were not fully sequenced in two isolates (TUM14654. TUM17940), and were non-typable in four isolates (TUM14658, TUM14792, TUM14797, TUM17948).

All isolates carrying bla_IMP-1 had In316 (intI1-bla_IMP-1-aac(6′)-IIc-sul1)-like structure. In 7 isolates (TUM17941, TUM17943, TUM17945, TUM17946, TUM17947, TUM17948, and TUM17949), nucleotide sequences of In316 were completely preserved and other isolates had single nucleotide difference (n = 5) or had fragmentation of the structure into multiple contigs (n = 5).

ST78 isolates were divided into three clades by core-genome (4,257,370 bp, 82.8% of the genomic sequence of the reference strain) based SNP analysis (Fig. 2). All patients from whom isolates of clade A were identified had a history of admission at ICU, Ward-V, or Ward-Y. On the other hand, all patients from whom isolates of clade C were identified had a history of multiple admission at Ward-X (Fig. 1).

In 13 isolates, bla_IMP was successfully transferred into recipient E. coli cell by conjugation experiment (Table 3). All transconjugant were positive for PCR using primers for bla_IMP-1-group. TUM17942 yielded transconjugants positive for IncL/M by PCR-based replicon typing. Transconjugants of the remaining isolates were positive for IncHI2 by PCR-based replicon typing, which suggested the location of bla_IMP-1 on IncHI2 plasmids.