Infection Control & Hospital Epidemiology
cases, and MDR-PA was also isolated from a subset of cases. During the period from July 2014 to December 2014, case patients were retrospectively identified as persons with incident clinical cultures positive for (1) CR-KP resistant to ertapenem; (2) MDR- PA nonsusceptible to gentamicin, tobramycin, ciprofloxacin, levofloxacin, meropenem, aztreonam, and cefepime and suscep- tible to piperacillin and piperacillin/tazobactam; or (3) both organisms. Analyses of antibiotic susceptibility of all isolates were performed using minimal inhibitory concentration (MIC) and/or disc diffusion methods. These results were interpreted using Clinical and Laboratory Standards (CLSI) criteria. During the cluster’s peak period of October–November 2014,
25 unique case patients were identified, 19 (76%) of whom were exposed to the same bronchoscope (named B1; Olympus BF-160 Video Bronchoscope, Olympus, Tokyo, Japan). This finding prompted investigation of the potential association of B1 expo- sure with the cluster. All 9 MICU-circulating bronchoscopes were immediately taken out of service, were cultured according to previously published methods,2 and underwent borescopy to visualize the internal lumen and to identify structural defects and/ or deposits of biological material. Of 9 bronchoscopes, 8 were negative for bacterial growth and were returned to service. The B1 bronchoscope was positive for both CR-KP and MDR-PA. Bronchoscope reprocessing, including manual, enzymatic pre- cleaning (Steris, Mentor, OH), and automated high-level disin- fection (Custom Ultrasonics, Ivyland, PA) was assessed by UPMC infection prevention personnel to ensure compliance with current guidelines.10 No breaches in bronchoscopy technique or bronchoscope precleaning were detected. High-level disinfection (HLD) records were reviewed, and no failures were identified.
Bacterial genomics
PFGE was performed on 3 CR-KP B1 isolates and a subset of clinical CR-KP isolates according to published methods.6,11 Sub- sequent whole-genome sequencing (WGS) was performed on an expanded set of 28 isolates (14 CR-KP and 14 MDR-PA), including 4 isolates cultured from the B1 bronchoscope (3 CR-KP and 1 MDR-PA), to strengthen evidence for horizontal trans- mission. Genomic DNA samples were extracted using the Qiagen DNeasy Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. DNA samples were quantified by Qubit fluorimetry (Qubit fluorimeter, Thermo Fisher Scientific, Waltham, MA), and genomic libraries were prepared using a modified Illumina Nextera protocol (Illumina, San Diego, CA).12 Libraries were sized using an Agilent bioanalyzer (Agilent, Santa Clara, CA), normalized, pooled, and sequenced using an Illumina NextSeq500. FastQ reads were quality filtered and assembled using SPAdes
41
alternate allele proportion. A phylogenetic tree based on the aligned core SNPs was generated using RAxML version 8.2.9 software by running 100 bootstrap replicates under the general- ized time-reversible model (GTRCAT) and Lewis correction for ascertainment bias.15 Phylogenies were visualized using the python package ETE3. Isolates with <30 SNP differences were considered genetically related.
Clinical data
The electronic medical records of case patients were reviewed to assess clinical outcomes and epidemiology. Patients were classi- fied epidemiologically as “outbreak,”“possible outbreak,” or “pseudo-outbreak.” The “outbreak” classification was assigned to patients with isolates recovered from a clinical culture ≥1 day after B1 exposure (Table 1). The “pseudo-outbreak” classification included patients with isolates obtained from culture during exposure to B1 who had no subsequent recovery of outbreak- related isolates. The “possible outbreak” classification was assigned to patients whose death occurred within 10 days of B1 exposure such that subsequent recovery and epidemiologic clas- sification of isolates from clinical cultures could not be assessed. Genetically unrelated MDR-PA or CR-KP isolates were regarded as isolates unlikely to belong to a B1-related cluster.
Results Epidemiology
A total of 33 case patients with cultures positive for CR-KP and/ or MDR-PA from predominantly respiratory sources collected between July and December 2014 (Table 1) were identified and plotted over time (Fig. 1). Of these 33 case patients, 23 (69.7%) were exposed to bronchoscope B1. Of these 23 patients, 19 (82.6%) had cultures positive for MDR-PA and 11 (47.8%) had cultures positive for CR-KP. Upon removal of B1 from service, the number of patients with newly detected CR-KP or MDR-PA decreased.
MDR-PA epidemiology
version 3.10.1 software.13 Assemblies were annotated using Prokka version 0.1.1 software.14 Reads had a sequencing depth ranging from 47X to 256X, and assemblies had a median number of 75 contigs per sample, with an average assembly length of 7.1 Mbp for MDR-PA and 5.5 Mbp for CR-Kp. Sequence types were identified using SRST2.13 Reads were aligned to reference assembly, CR-KP112 and MDR-PA012 for K. pneumonia and P. aeruginosa genomes, respectively, using BWA-MEM version 0.7.12-r1039 software (
http://bio-bwa.sourceforge.net/). Single- nucleotide polymorphisms (SNPs) were identified using Snippy genome software with default parameters. Snippy uses Freebayes as the variant caller and filters out SNPs with <10 reads and 90%
Patient 1, who had clinical cultures positive for MDR-PA prior to B1 exposure, was identified as a potential transmission source (Fig. 1). Patient 1 was exposed to bronchoscope B1 3 days prior to the bronchoscope procedure of patient 4, who became culture positive for MDR-PA on the day of B1 exposure. Subsequently, 18 case patients with exposure to bronchoscope B1 developed MDR-PA positive cultures (Fig. 1).
CR-KP epidemiology
Patients 5 and 15 were considered putative index patients for contamination of B1 with CR-KP. Patient 5 was culture positive for CR-KP 100 days prior to B1 exposure, whereas patient 15 was culture positive for CR-KP 7 hours prior to B1 exposure. Both patients 5 and 15 had a bronchoscopy with B1 performed 11 days prior to patient 18—the first case patient who became culture positive for CR-KP on the same day of B1 exposure. Subse- quently, 9 patients with B1 exposure developed CR-KP–positive cultures (Fig. 1).
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