Nanopore Sequencing for Detecting Bacteria in Bile and Preventing Surgical Site Infections in Patients Undergoing Surgery for Benign or Malignant Pancreatic Tumors
Phase: NA
Conditions: Benign Pancreatic Neoplasm, Malignant Pancreatic Neoplasm
Sponsor: Mayo Clinic

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“Answers in Hours” A Randomized Controlled Trial Using 
Microbiome Metagenomics for Bile Duct Cultures 
 
 
Principal Investigator 
 
 
 
 
 
 
Marina Walther-Antonio , Ph.D 
Surgical Research  
Mayo Clinic 
200 1st Street SW, Rochester, MN 55905 
507-284-2282  
Co-Principal Investigator(s)   
 
 
 
 
Funding Sponsor: 
Mark Truty, M.D. 
Division of Hepatobiliary and Pancreas Surgery 
Mayo Clinic 
200 1st Street SW, Rochester, MN 55905 
 
Mayo Clinic 
200 1st Street SW, Rochester, MN 55905 
 
Study Product: 
Nanopore Sequencing, GridION Sequencing Instrument 
Protocol Number: (IRBe) 
21-004234 
IDE Number: 
G220184 
 
 
Initial version: [2/28/2022] Version (1.0)  
Revision Date: [2/28/2023] Version (2.0) 
Revision Date: [9/21/2023] Version (3.0) 
 
 

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Table of Contents 
STUDY SUMMARY ................................................................................................................................5 
1 
INTRODUCTION ...........................................................................................................................6 
1.1 
BACKGROUND ...................................................................................................................................... 6 
1.2 
INVESTIGATIONAL DEVICE ................................................................................................................... 7 
1.3 
CLINICAL DATA TO DATE .................................................................................................................... 7 
1.4 
STUDY RATIONALE AND RISK ANALYSIS (RISKS TO BENEFITS RATIO) ................................................ 8 
1.4.1 
Study Rationale ....................................................................................................................8 
1.4.2 
Anticipated Risks .................................................................................................................9 
1.4.3 
Potential Benefits .............................................................................................................. 14 
1.5 
ANTICIPATED DURATION OF THE CLINICAL INVESTIGATION .............................................................. 15 
2 
STUDY OBJECTIVES ................................................................................................................. 10 
2.1 
HYPOTHESIS ....................................................................................................................................... 10 
2.2 
PRIMARY OBJECTIVE ......................................................................................................................... 10 
2.3 
SECONDARY OBJECTIVE .................................................................................................................... 10 
3 
STUDY DESIGN ................................................................ ERROR! BOOKMARK NOT DEFINED. 
3.1 
GENERAL DESIGN .............................................................................................................................. 16 
3.2 
PRIMARY STUDY ENDPOINTS ............................................................................................................. 12 
3.3 
SECONDARY STUDY ENDPOINTS ...................................................................................................... 222 
3.4 
PRIMARY SAFETY ENDPOINTS ............................................................................................................ 12 
4 
SUBJECT SELECTION, ENROLLMENT AND WITHDRAWAL .............................................. 22 
4.1 
INCLUSION CRITERIA ....................................................................................................................... 223 
4.2 
EXCLUSION CRITERIA ...................................................................................................................... 233 
4.3 
SUBJECT RECRUITMENT, ENROLLMENT AND SCREENING .................................................................. 13 
4.4 
EARLY WITHDRAWAL OF SUBJECTS............................................. ERROR! BOOKMARK NOT DEFINED. 
4.4.1 
When and How to Withdraw Subjects ................................................................................. 23 
4.4.2 
Data Collection and Follow-up for Withdrawn Subjects ....................................................... 13 
5 
STUDY DEVICE ......................................................................................................................... 13 
5.1 
DESCRIPTION ................................................................................................................................... 233 
5.2 
METHOD FOR ASSIGNING SUBJECTS TO TREATMENT GROUPS ......................................................... 244 
5.3 
MASKING/BLINDING OF STUDY ....................................................................................................... 244 
5.4 
STORAGE.................................................................................... ERROR! BOOKMARK NOT DEFINED.4 
5.5 
CLEANING/STERILIZATION PROCEDURES ................................... ERROR! BOOKMARK NOT DEFINED.4 
6 
STUDY PROCEDURES............................................................................................................... 15 
6.1 
ENROLLMENT VISIT ........................................................................................................................... 15 
6.2 
SAMPLE COLLECTION VISIT ............................................................................................................. 255 
6.3 
FOLLOW UP DATA COLLECTION. ..................................................................................................... 255 
7 
STATISTICAL PLAN ................................................................................................................ 266 
7.1 
SAMPLE SIZE DETERMINATION ........................................................................................................ 266 
7.2 
STATISTICAL METHODS ..................................................................................................................... 16 
8 
SAFETY AND ADVERSE EVENTS ............................................................................................ 17 
8.1 
DEFINITIONS ...................................................................................................................................... 17 
8.2 
RECORDING OF ADVERSE EVENTS ..................................................................................................... 19 
8.2.1 
Sponsor-Investigator Reporting, Notifying Mayo IRB ........................................................... 19 

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8.2.2 
Sponsor-Investigator Reporting: Notifying the FDA ............................................................. 20 
8.3 
MEDICAL MONITORING ..................................................................................................................... 21 
9 
DATA HANDLING AND RECORD KEEPING ........................................................................... 21 
9.1 
CONFIDENTIALITY.............................................................................................................................. 21 
9.2 
SOURCE DOCUMENTS....................................................................................................................... 341 
9.3 
CASE REPORT FORMS ........................................................................................................................ 22 
9.4 
RECORDS RETENTION ........................................................................................................................ 23 
10 
STUDY MONITORING, AUDITING, AND INSPECTING ......................................................... 23 
10.1 
STUDY MONITORING PLAN ................................................................................................................ 23 
10.2 
AUDITING AND INSPECTING ............................................................................................................. 383 
11 
ETHICAL CONSIDERATIONS .................................................................................................. 38 
12 
STUDY FINANCES ................................................................................................................... 394 
12.1 
FUNDING SOURCE ............................................................................................................................ 394 
13 
PUBLICATION PLAN .............................................................................................................. 394 
14 
REFERENCES ............................................................................................................................ 24 
15 
ATTACHMENTS .......................................................... 26ERROR! BOOKMARK NOT DEFINED. 
 
 
 
 
 
 

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LIST OF ABBREVIATIONS 
AE 
Adverse Event/Adverse Experience 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
DSMB 
Data and Safety Monitoring Board 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
ICU 
Intensive Care Unit 
IDE 
Investigational Device Exemption 
IRB 
Institutional Review Board 
NS 
Nanopore Sequencing 
PHI 
Protected Health Information 
PI 
Principal Investigator 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SOP 
Standard Operating Procedure 
SSI 
Surgical Site Infection 
TRAG 
Tissue Request Acquisition Group 
UADE 
Unanticipated Adverse Device Effect 
 
 
 

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Study Summary 
Title 
“Answers in Hours” A Randomized Controlled Trial Using 
Microbiome Metagenomics for Bile Duct Cultures 
Running Title 
“Answers in Hours” 
IRB Protocol 
Number 
21-004234 
Phase 
Pilot 
Methodology 
Randomized 1:1 Controlled Trial 
Overall Study 
Duration 
3 years 
Subject 
Participation 
Duration 
90 days  
Objectives 
Reduce the rate of bacterobilia driven surgical site infection (SSI) in 
patients undergoing pancreatic head resection by providing surgical 
team with Oxford Nanopore (ONT) microbial sequencing data in the 
post-operative setting. 
 
Reduce cost of care through reduction in SSI and improved antibiotic 
stewardship. 
Number of Subjects 
140 
Diagnosis and Main 
Inclusion Criteria 
Any adult, male or female, over age 18, undergoing 
pancreaticoduodenectomy or total pancreatectomy for benign or 
malignant indication.  
Study Device 
GridION Sequencing Instrument 
Reference therapy 
Standard culture techniques and prolonged peri-operative antibiotics  
Statistical 
Methodology 
For continuous variables, t-test will be performed and for binary 
variables chi-square or fisher’s exact testing will be performed as 
appropriate. Universal and multivariable linear regression adjustment 
will be performed if any baseline imbalances in demographic or 
clinical variables are detected 
 
 
 
 
 

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1 Introduction 
 
This document is a protocol for a human research study. This study will be carried out in 
accordance with the procedures described in this protocol, applicable United States 
government regulations and Mayo Clinic policies and procedures.  
1.1 
Background 
 
Postoperative infections, specifically surgical-site infections (SSI), are a significant source of 
morbidity in patients undergoing pancreatic head resection and have been reported in over 25% 
of patients 1,2. Infectious complications significantly increase the risk of delayed gastric 
emptying, pancreatic fistula, biliary fistula, abdominal collection, sepsis, acute respiratory 
failure, pulmonary complications, cardiovascular complications, and mortality 3–5. Patients 
who develop SSI are also less likely to receive adjuvant chemotherapy, more likely to 
experience delays in adjuvant therapy, and have reduced overall survival 6–9. Overall, 
complications arising from SSIs have been shown to have a direct effect on mortality, varying 
from 33% to 77% increased risk of mortality 10, and 75% of SSI-associated deaths are directly 
attributable to the SSI 11–13. 
 
The increased rates of morbidity and mortality associated with SSI have led to the current 
surgical perioperative antibiotic prophylaxis guidelines developed jointly by the American 
Society of Health-System Pharmacists, the Infectious Diseases Society of America, the 
Surgical Infection Society, and the Society for Healthcare Epidemiology of America which 
recommend the administration of cefazolin, cefoxitin, cefotetan, ceftriaxone, ampicillin-
sulbactam, clindamycin/ vancomycin + aminoglycoside or aztreonam or fluoroquinolone, or 
metronidazole + aminoglycoside or fluoroquinolone 60 – 120 minutes prior to surgical incision 
to reduce the risk of SSI during pancreatic/ biliary tract surgeries14. This has led to extensive 
‘blind’ use of broad-spectrum antibiotics which are associated with their own risks. Excessive 
usage of broad-spectrum antibiotics contributes towards the emergence of antimicrobial 
resistance (AMR), and their usage has been associated with acute adverse events15, and 
increased risk of developing sepsis 16, cancer 17–20 ,and heart disease21–24, as well as other 
antibiotic associated complicated such as Clostridium difficile infections 25–28. 
 
Many patients who undergo pancreatic head resection have obstructive jaundice prior to 
surgery due to compression or occlusion of the common bile duct. To alleviate these 
symptoms, patients often undergo preoperative biliary drainage, most commonly by insertion 
of a biliary stent. This creates a conduit between the biliary tree and the intestinal lumen, 
resulting in translocation of intestinal microbes into the normally sterile bile duct systems. 
Numerous studies have demonstrated and confirmed an association between preoperative 
biliary stenting and the occurrence of SSI 3,6–8,29, and as such, some institutions have opted to 
administer postoperative antibiotics30–32,32–35. In some cases, postoperative administration of 
broad-spectrum prophylactic antibiotics is performed without any assessment of the microbes 
present or sensitivities, largely due to the lack of available microbial testing infrastructure, 
large-volume, and nearby testing labs. In others, detection and characterization of biliary 
microbial contamination is currently achieved by performing standard bile cultures and 

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antimicrobial susceptibility testing (AST) on diverse polymicrobial community. Standard bile 
cultures, however, can take days to weeks to yield actionable results 36,37 and are typically 
received over the course of 2 – 4 days. Bile cultures are often polymicrobial, a mixture of 
anaerobic and aerobic species, carry multiple antimicrobial resistant (AMR) phenotypes, and 
contain fungal species  38–41. This contributes towards delays in timely results as aerobic 
cultures (up to 5 days to complete), anaerobic cultures (up to 14 days to complete), and fungal 
cultures (up to 24 days to complete) must be performed, and additional testing must be carried 
out to determine the different microbial species present in the sample before AST can be 
carried out. Results are often generated over the course of several days, and this temporal 
separation between the initial broad-spectrum treatment and the acquisition of all the 
diagnostic information often results in uncertainty and the administration of multiple empiric 
antimicrobials 42.  
 
At our institute the current standardized approach is administer a perioperative prophylaxis 
course of ceftriaxone plus metronidazole 60 minutes prior to surgery (Figure 1). Antibiotic 
treatment is continued for 5-days postoperatively for patients undergoing pancreatic head 
resection, and the selection and duration of this specific broad-spectrum prophylactic regiment 
is based on recommended surgical guidelines and our own historical SSI culture data 43. During 
surgery, bile swab and aspirate samples are collected and transferred to the Pathology lab to 
undergo standard bile cultures and AST. If the bile cultures are eventually negative for 
microbial growth, the patient will be taken off the broad-spectrum antibiotics following 
completion of the 5-day prophylactic regimen (Figure 1). If the bile cultures are positive, then 
antibiotic treatment will be extended for 10 days, and therapy is tailored based on organisms 
and resistances identified in the bile cultures (Figure 1). Introduction of this approach in 2014 
resulted in significant decrease in hospital stay, reduced the need for interventional radiology 
procedures, and decreased the risk of postoperative pancreatic fistula grade 43. Within the first 
year of introduction, our approach significantly decreased inpatient cost of care, decreased the 
total 30-day costs, and saved a total of $1.1 million , in addition to improving patient outcome 
43.  
 

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Figure 1: Schematic diagram of the current standardized perioperative prophylactic 
antibiotic treatment plan for patients undergoing total pancreatectomy and 
pancreaticoduodenectomy at our institution.  
 
Since the introduction of our standardized approach, we have improved antibiotic tailoring by 
developing methods of increasing detection of anaerobic species through our use of bile 
aspirate in addition to collection of bile swabs 38. However, similar to other institutions, it takes 
an average of 4 days for final bacterial culture results to be generated 44, and the majority of 
cultured microbial species have been found to exhibit multi-resistance phenotypes. This means 
that for the first 4-days post-surgery many of our patients at risk of developing SSI are 
receiving suboptimal antimicrobial therapy. This provides potential pathogens a window of 
opportunity to grow and propagate prior to introduction of tailored antimicrobial therapy. 
Rapid methods of detecting microbial species and antimicrobial resistance phenotypes are 
required to shorten this window of opportunity. Our data shows that in patients with positive 
bile cultures it took an average of 7 days to complete microbial identification and AST for 
bacterial and fungal species (range = 3 – 24 days), and it took 24 days to confirm the absence 
of biliary microbes (5 days for aerobic cultures, 14 days for anaerobic cultures, 24 days for 
fungal cultures). In particular, delays in detecting Candida species, was of concern due to the 
increased risk of SSI in patients with biliary candidiasis 45. In our own patient cohort, 17% of 
patients were positive for biliary candidiasis, and the microbe was the presumptive cause of 
22% of SSIs. In patients with biliary candidiasis, it took an average length of 4.6 days for 
fungal identification, and an additional 6 days for anti-fungal susceptibility testing to be 
complete. As anti-fungals are not part of the recommended perioperative prophylactic 
antimicrobial treatment course, it meant that that during the first 2 weeks post-surgery these 
patients were at increased risk of SSI due to untreated biliary candidiasis.  
 
A single diagnostic test that would enable simultaneous identification of bacterial species 
(aerobic, anaerobic, fastidious), fungal species, and antimicrobial resistance in an expeditated 

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fashion would be highly advantageous. It would allow for all the microbial information to be 
generated in a single report, enabling the administration of optimal tailored antibiotics in the 
early postoperative window without the need for continuous changes to the treatment regimen 
as new information becomes available. It would also enable early cessation of antimicrobial 
prophylaxis if no microbes are detected, potentially reducing the complications associated with 
use of broad-spectrum antibiotics. 
 
We hypothesized that use of metagenomic Oxford Nanopore (ONT) sequencing,  a real-time 
method of sequencing that has been demonstrated to detect microbial species in as little as  6 
hours 42,46,47, could be developed as a tool for rapid detection and characterization of biliary 
microbes. We have since developed methods of detecting intraoperative bile microbes, and 
have recently published the promising results of a prospective clinical trial that was performed 
to determine the feasibility and utility of ONT sequencing in detection of microbial species 
and antibiotic resistance genes using intra-operative bile from patients undergoing pancreatic 
head resection 44,48. Our preliminary data would suggest that not only is this technique feasible, 
but our ONT sequencing results highly correlate with traditional culture microbial 
identification and does so with a median time of 14 hours compared to 98 hours for traditional 
culture, a turnaround time improvement of 92%. Furthermore, the results of our study found 
that when standard cultures (SC) were negative (i.e. no further antibiotic therapy needed) the 
ONT perfectly correlated. This suggested that ONT could be used to identify patients with 
negative biliary microbes, enabling a more rapid cessation of unnecessary antimicrobial 
therapy in patients. This would improve antibiotic stewardship, a continual goal in surgical 
patient care. 
 
Given these promising preliminary results, we aim to reduce the rate of SSI while minimizing 
overutilization of antibiotic therapy in patients undergoing pancreatic head resection by 
providing supplemental ONT data in addition to standard clinical cultures. This will provide 
not only an important potential diagnostic, but will also elucidate microbial communities 
within contaminated bile, potentially leading to improved targeted approaches in the future. 
 
1.2 
Investigational Device 
 
The investigation device being utilized for this research study is the Oxford Nanopore 
Technologies (ONT) R9.4.1 flow cells and the ONT GridION MK1 Sequencing Instrument 
(Device number GXB03025).  ONT sequencing will be performed using MinKNOW (version 
21.11.7), MinKNOW Core (4.5.4), Bream (6.3.5) and Guppy (5.1.13) software. Microbial 
species identification will be achieved using Porechop (0.2.4), BBMap (35.85) and Centrifuge 
(1.0.4) software, and detection of antimicrobial resistance genes will be achieved using 
ResFinder 4.1 software49,50. The ResFinder 4.1 tool, developed and maintained by the Center 
for Genomic Epidemiology (CGE), was selected following research that demonstrated it to be 
as reliable as standard antimicrobial susceptibility testing (broth microdilution, disc diffusion) 
for detecting clinically relevant AMR phenotypes 51. Additionally, the tool utilizes a database 
of horizontally acquired chromosomal gene mutations that confer AMR, and thus use of 
ResFinder 4.1 will enable us to link predicted AMR phenotypes to specific microbial species. 

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To ensure that no potential resistance phenotypes are missed we will also use the 
PlasmidFinder (2.0.1)52 and CARD (CARD 2020)53 tools to check for any plasmid-acquired 
AMR phenotypes. 
 
If there are new releases of the software prior to the trial, the software will be updated to the 
latest versions and all samples in the trial will be sequenced and analyzed using the same 
software versions. 
 
 
1.3 
Intended use of Investigational Device  
 
The ONT GridION MK1 Sequencing Instrument (Device number GXB03025) will be used in 
conjunction with an optimized phenol chloroform DNA extraction to perform metagenomic 
sequencing on DNA extracted from intraoperative bile aspirate samples taken from patients 
undergoing total pancreatectomy and pancreaticoduodenectomy. The technology will utilize 
ONT sequencing and the data output will be basecalled read sequences in the fastq file format. 
Sequenced reads will be analyzed for microbial identification and AMR prediction.  
 
1.4 
Clinical Data to Date 
 
This study will use data that has been collected under the following research study: 
“Comparative Analysis of Bile Microorganisms in Patients Undergoing Major Hepatobiliary 
Operations”, IRB# 19-011419 and published in two peer-reviewed journals 44,48. In brief, these 
studies involved analysis of intraoperative bile aspirate samples collected from 42 patients 
undergoing pancreatic head resection (total pancreatectomy, n = 9; pancreaticoduodenectomy, 
n = 33) at Mayo Clinic, Rochester (see Table 1 for patient characteristics). Metagenomic ONT 
sequencing was performed on bile aspirates to identify microbial species (bacterial, fungal) 
and antibiotic resistance genes. Sequencing results were then compared to bile cultures 
performed as part of Mayo Clinic’s standard of care clinical practice  to determine how ONT 
sequencing compared to current ‘Gold Standard’ culture practices 44,48.  
 
To determine the optimum ONT protocol for detection of microbial species and antibiotic 
resistance genes present in the bile aspirates, different methods of DNA extraction, use of host 
DNA depletion, and use of different AMR gene databases were evaluated 48. In total, 
intraoperative biliary microbes were detected in 54.7% (23/42) of patients undergoing total 
pancreatectomy or pancreaticoduodenectomy using SC techniques. This included 35.7% 
(15/42) of patients with positive bacterial cultures, 2.4% (1/42) of patients with positive fungal 
cultures, and 16.7% of patients with positive fungal and bacterial cultures. The male sex, use 
of biliary instrumentation, and performance of vascular reconstruction during surgery was 
significantly correlated with the detection of biliary microbes. 
 
Table 1: Clinical characteristics of patients included in our preliminary study 
 

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Clinical feature 
Total 
Biliary microbes + 
Biliary microbes - 
P value 
Age, mean (IQR) 
61 (22 - 71) 
66.3 (46.3 – 81.3) 
58.0 (18 – 82.6) 
0.0858 
BMI, mean (IQR) 
26.4 (19 – 40.4) 
26.3 (19 – 40.4) 
26.4 (19.7 – 36.2) 
0.9295 
Sex, % male (n) 
54.8 (23) 
38.1 (16) 
16.7 (7) 
0.0375 
Surgery indication, % (n) 
 
 
 
0.892 
   Pancreatic ductal 
   adenocarcinoma 
45 (29) 
50 (21) 
19.0 (8) 
 
   Intraductal Papillary Mucinous 
   Neoplasm             
7.1 (3) 
0 (0) 
7.1 (3) 
 
   Neuroendocrine tumors 
11.9 (5) 
2.4 (1) 
9.5 (4) 
 
   Other 
11.9 (5) 
2.4 (1) 
9.5 (4) 
 
ASM Score, mean (IQR) 
2.6 (1 – 4) 
2.7 (2 – 4) 
2.5 (1 – 3) 
0.1874 
Type of surgery 
 
 
 
0.9716 
   Total pancreatectomy, % (n)       
21 (9) 
11.9 (5) 
9.5 (4) 
 
   Pancreaticoduodenectomy, % (n) 
79 (33) 
42.9 (18) 
35.7 (15) 
 
Biliary instrumentation, % (n) 
52 (22) 
52.4 (22) 
0 (0) 
<0.0001 
Vascular reconstruction, % (n) 
38 (16) 
31.0 (13) 
7.1 (3) 
0.0079 
BMI, body mass index; IQR, interquartile range 
 
Comparison of our optimized ONT sequencing protocol SC results revealed that our ONT 
protocol had identified 100% of biliary microbial contamination cases, detected 75% of 
cultured bacterial species and 76% of cultured fungal species, and predicted 81% of antibiotic 
resistance phenotypes observed using SC results 44,48. With regards to detection of common 
biliary pathogens 3,54–57, our ONT protocol improved detection of biliary candidiasis, detected 
100% of cases of biliary Escherichia coli and biliary Klebsiella, and detected 83% of biliary 
Enterobacter, 76.5% of biliary Enterococcus, 93.8% of biliary Streptococcus. However, ONT 
did not detect one case of Pseudomonas infection and one case of Staphylococcus aureus, 
indicating as proposed in this study that SC techniques should be run alongside ONT protocols 
to ensure that no microbial species are missed. As for detection of pathogens of high clinical 
concern (i.e. ESBL, CRE species), ONT similar detection rates compared to SC, and 
potentially improved detection of E. coli, K. oxytoca, K. pneumoniae, and Citrobacter freundii. 
Overall, ONT improved detection of microbial species (particularly with regards to anaerobic 
species and fungal species) and predicted a significantly higher number of antibiotic resistance 
phenotypes than were identified using SC. Significantly, the results turnaround for ONT 
sequencing was just 14 hours compared to an average turnaround time of 98 hours for SC.  On 
average, it took 4.8 days (range = 1 – 15 days) to complete microbial identification and an 
additional 2 days (range = 0 – 9 days) to complete AST for the culture positive samples. Delays 
greater than 5 days in completing microbial identification occurred in samples with multiple 
aerobic species of the same genus (Enterococcus spp., Klebsiella spp., Streptococcus spp.), 
anaerobic species (Schaalia (Actinomyces) turicensis, Finegoldia magna, Veillonella sp., 
Actinomyces johnsonii, Parvimonas micra), and/ or fungal species Saccharomyces cerevisiae, 
Candida spp.), and on average, optimal antimicrobial therapy was delayed for 7 days. Use of 
a test that would enable microbial identification and prediction of AMR phenotypes within 
24hrs post-surgery, would, therefore, have significantly shortened the delay in patients 
receiving optimized tailored antimicrobial therapy post-surgery. 
 
Table 2: Detection of common biliary pathogens and pathogens of high clinical concern 
using standard culture (SC) and our Oxford Nanopore sequencing (ONT) protocol 

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Detection of common biliary microbes  
Microbial species 
Patients with positive 
SC result, n (%) 
Patients with positive ONT 
sequencing result, n (%) 
Patients with positive SC & 
ONT sequencing result, n (%) 
Enterobacter spp. 
6 (14.3) 
6 (14.3) 
5 (11.9) 
Enterococcus spp. 
17 (40.5) 
13 (31.0) 
13 (31.0) 
Escherichia coli 
3 (7.1) 
5 (9.5) 
3 (7.1) 
Klebsiella spp. 
11 (26.2) 
14 () 
11 (26.2) 
Pseudomonas spp. 
1 (2.4) 
0 (0) 
0 (0) 
Staphylococcus 
aureus 
1 (2.4) 
0 (0) 
0 (0) 
Streptococcus 
spp. 
16 (38.1) 
19() 
15 (35.7) 
Candida spp. 
5 (11.9) 
7 (16.7) 
5 (11.9) 
Detection of pathogens of high clinical concern 
Enterobacter 
aerogenes 
0 (0) 
0 (0) 
0 (0) 
Enterobacter 
cloacae 
5 (11.9) 
6 (14.3) 
4 (9.5) 
Escherichia coli 
3 (7.1) 
5 (11.9) 
3 (7.1) 
Haemophilus 
influenzae 
0 (0) 
0 (0) 
0 (0) 
Klebsiella oxytoca 
7 (16.7) 
10 (23.8) 
7 (16.7) 
Klebsiella 
pneumoniae 
4 (9.5) 
7 (16.7) 
4 (9.5) 
Kluyvera species 
0 (0) 
0 (0) 
0 (0) 
Neisseria 
gonorrhoeae 
0 (0) 
0 (0) 
0 (0) 
Proteus mirabilis 
0 (0) 
0 (0) 
0 (0) 
Pseudomonas 
aeruginosa 
1 (2.4) 
0 (0) 
0 (0) 
Salmonella 
enterica 
0 (0) 
0 (0) 
0 (0) 
Citrobacter 
freundii 
4 (9.5) 
6 (14.3) 
3 (7.1) 
Serratia 
marcescens 
3 (7.1) 
1 (2.4) 
1 (2.4) 
 
 
SSI occurred in 21.4% (9/42) patients within 30 days post-surgery, and an additional two 
patients developed SSI 34 days post-surgery and 42 days post-surgery, respectively. In total, 
44% (4/9) of these patients had positive intraoperative SC and ONT results, and 22% (2/9) of 
patients had positive post-operative abdominal fluid cultures. In the cases where post-operative 
organisms were detected, Candida albicans was the most commonly detected (2/4 patients) 
followed by Enterococcus faecalis (1/4 patients), highlighting the importance of improved 

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detection of Candida species in these patients. Comparison of SC and ONT predictive power 
found no difference with regards to identifying patients at risk of SSI. 
 
 
1.5 
Study Rationale and Risk Analysis (Risks to Benefits Ratio) 
1.5.1 Study Rationale 
 
SSIs are one of the most common and costly of hospital-acquired infections within the US, 
accounting for 20% of all hospital-acquired infections13.  A single SSI increases hospital stay 
by an average of 9.7 days and accounts for a 2- to 11-fold increase in the risk of mortality 13. 
Patients with pancreatic cancer are clinically vulnerable and have a particularly high risk of 
SSI following pancreatic head resection, with global incidence rates ranging from 25 – 45% of 
patients undergoing the procedure 1,2. The occurrence of a SSI increases the rate of mortality 
and the need for additional invasive procedures, and it can result in delay or failure to complete 
adjuvant chemotherapy 9,58. 
 
Rapid etiologic diagnosis of biliary microbial contamination can facilitate timely and rational 
post-operative antimicrobial therapy, reducing the risk of a SSI developing. SCs, however, can 
take many days to return actionable results36,37 and are typically received over the course of 4-
7 days. Bile cultures are often polymicrobial, a mixture of anaerobic and aerobic species, carry 
multiple antibiotic resistant phenotypes, and contain fungal species  38–41. This contributes 
towards delays in timely results, and the temporal separation between the initial broad-
spectrum treatment and the acquisition of all the diagnostic information often results in 
uncertainty and the administration of multiple empiric antimicrobials42. 
 
Next-generation sequencing platforms, such as Illumina and ION Torrent, have been widely 
used in the identification and characterization of microbial species, including previous 
characterization of the bile microbiome in healthy and diseased states 59,60. However, in 
practice, these technologies require at least 16 hours for the sequencing run alone and analysis 
of the sequencing data can only be performed once the sequence run has concluded47,61. 
 
ONT sequencing has been demonstrated to successfully characterize microbial populations 
present in a range of clinical samples and situations in as little as 6 hours 42,46,47,62–77. Moreover, 
we have previously demonstrated that ONT sequencing provides a rapid, comprehensive, and 
accurate profile of microbial pathogens and antibiotic resistance phenotypes present in 
intraoperative bile aspirates matching SC techniques 44,48. Collectively, this suggests that the 
incidence rate of SSIs occurring in patients undergoing pancreatic head resection may 
potentially be reduced by the use of ONT sequencing to rapidly characterize intraoperative 
biliary microbial contamination and guide early targeted antimicrobial therapy post-surgery, 
more rapidly than SCs Additionally, use of ONT sequencing would improve antibiotic 
stewardship by reducing the use of broad-spectrum antibiotics, and patients would benefit 
either by receiving a more targeted antimicrobial approach or by being taken off unnecessary 
broad-spectrum antibiotics earlier which currently is not possible, subsequently reducing the 

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risk of clinically relevant antibiotic-associated adverse events. Clinical risk to the patient is 
minimal. 
1.5.2 Anticipated Risks 
 
ONT sequencing will be utilized alongside current SCs. We will use SC to ensure that the care 
patients receive will be no worse than current practice. In other words, culture will ensure that 
ONT only improves patient care and outcome. In the event that ONT results and culture 
assessment contradict, the care plan will prioritize the results of standard culture in the event 
of treatment failure (Table 3). The risks associated with participation in this study are no greater 
than for those patients not on the study also undergoing identical operations. The only 
therapeutic interventions this trial includes is alteration or cessation of postoperative 
antimicrobial therapy based on either ONT or SCs. In order to minimize any theoretical risks 
in the ONT arm, such as missed microbial species and/or antibiotic resistance phenotype or 
ineffective antibiotic regimen, SC data will be utilized in both arms and antibiotic therapy will 
be adjusted if SCs suggest broader coverage (Table 3). There will be no alteration in the 
standard clinical postoperative care of patients in this study.    
 
Table 3: Incorporation of ONT results in clinical management decisions 
 
ONT Result 
Clinical management decision 
A negative ONT result 
Discontinue antibiotics after 24 hours of peri-
operative coverage 
A positive ONT result for a single pathogen typical of 
biliary or gastrointestinal flora 
Adjust and/or narrow antibiotic therapy to cover 
single pathogen. If single pathogen deemed clinically 
insignificant 
(ie. 
lactobacillus), 
discontinue 
antibiotics after 24 hours of peri-operative coverage. 
A positive ONT result for multiple pathogens 
Adjust and/or narrow antibiotic therapy to cover all 
pathogens, excluding those deemed clinically 
insignificant. If all pathogens are considered clinically 
insignificant, discontinue antibiotics after 24 hours of 
peri-operative coverage. 
A positive ONT result positive for rare or unexpected 
flora 
Adjust and/or narrow antibiotic therapy to cover rare 
or unexpected flora, excluding those deemed 
clinically insignificant. 
ONT results different from SC 
Adjust and/or narrow antibiotic therapy to cover 
pathogens found in ONT results, excluding those 
deemed clinically insignificant. SC results to only be 
considered in the event of treatment failure with 
antibiotic therapy targeted towards ONT results. 
 
1.5.3 Potential Benefits 
 
Potential benefits associated for patients who participate in this study include:  
 
• Earlier diagnosis of biliary microbial contamination 
• Earlier targeted antimicrobial therapy post-surgery 

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o Reduces window of opportunity for pathogenic species to propagate and cause 
infection 
o Reduced risk of SSIs 
o Potential candidate for earlier chemotherapy 
• Earlier cessation of unnecessary antimicrobial therapy following negative result of 
biliary aspirates 
o Cost saving from stoppage of IV antibiotic administration 
o Potential candidate for earlier chemotherapy 
o Reduced gut microbiome disruption and decreased risk of adverse events 
following antibiotics 
• Wider impact – reduction of broad-spectrum antibiotic usage, subsequently 
improving antibiotic stewardship and avoid antibiotic associated complications 
 
The technology will improve antibiotic stewardship by rapidly identifying which patients 
require antimicrobial therapy, reducing time spent on broad-range antimicrobial therapy, and 
decreasing the risk of emerging resistance by ensuring that patients who do require 
antimicrobial therapy are on an optimal treatment regimen. Patients will directly benefit 
through early diagnosis of biliary microbial contamination. This will enable patients who 
require antimicrobial therapy to receive early targeted antimicrobial therapy post-surgery, 
potentially reducing the risk of infection by decreasing the window of opportunity for 
pathogenic species to propagate.  Patients who do not require antimicrobial therapy will benefit 
by being taken off therapy earlier, reducing patient exposure to broad-spectrum antibiotics and 
the risk of adverse events and long-term health impacts associated with unnecessary 
antimicrobial therapy. 
 
The data from this study will provide an important scientific characterization of the biliary 
flora in patients undergoing pancreatic head resection. Given the high risk of infection, better 
characterization of these microbes using untargeted approaches will provide a more complete 
picture of potential pathogen risks on a population level. The scientific knowledge gained in 
this study may influence future studies and therapeutic possibilities, especially with regard to 
underassessed species such as fungi and obligate anaerobes. 
 
1.6 
Anticipated Duration of the Clinical Investigation 
 
The anticipated duration of this study is 2 years. Direct participation for participants lasts until 
surgical specimens have been obtained. Additional follow-up data will be collected from the 
medical record for up to 90 days following the procedure.  
 
2 Study Objectives 
 
2.1 Hypothesis:  
 

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The use of ONT sequencing, a novel metagenomic technique, will result in faster 
identification of organisms from intra-operatively collected bile samples and allow for early 
treatment of biliary microbial contamination. Supplementing SC with ONT sequencing data 
will allow patients to be treated earlier in the post-operative setting with tailored antibiotics, 
which will reduce the rate of biliary microbial contamination related SSIs in patients 
undergoing pancreatic head resection as compared to SC alone.  
 
2.2 
Primary Objective 
 
To improve antibiotic stewardship (reducing duration of peri-operative prophylactic antibiotic 
regimen, reducing administration of broad-spectrum antibiotic) by providing surgical team 
with rapid ONT sequencing data in the early post-operative setting. 
 
Success Criteria: Significantly reduced antibiotic regimen duration and use of broad-spectrum 
antibiotics, corrected for key co-variates (p<0.1). 
 
2.2 Secondary Objective 
 
To reduce the cost of care through reduction in SSI and improved antibiotic stewardship. 
 
Success Criteria: Significantly reduced rates of SSI and reduced total hospital costs, 
corrected for key co-variates (p<0.1). 
2.1 
General Design 
 
This will be a prospective 1:1 randomized controlled trial with the following study arms: 
 
Arm 1: Patient Group 1 
 
Standard Care Only. Patients will undergo intra-operative bile aspiration (as is 
routinely performed) with SC of the aspirate. All patients will receive standard 
prophylactic antibiotics according to practice standards and will be continued for up 
to 5 days as is routine. If bile cultures are positive, antimicrobials will be continued 
for a complete 10-day course of antibiotics (Figure 2). 
 
Arm 2: Patient Group 2 
 
Standard Care + ONT. Patients will undergo intra-operative bile aspiration (as is 
routinely performed) with standard cultures AND ONT sequencing of the aspirate.   All 
patients will receive standard prophylactic antibiotics according to practice standards. 
ONT sequencing results will be made available to providers through electronic medical 
record (EMR) and team electronic notifications. Patients with negative ONT results 
will be taken off the standard prophylactic antibiotics and patients with positive ONT 
results will have antimicrobial therapy adjusted according to ONT sequencing results. 
However, if SCs are positive or suggest alternative/broader coverage then these results 

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will guide therapy and not ONT results. If bile cultures are positive, antimicrobials will 
be continued for a complete 10-day course of antibiotics (Figure 2). 
 
 
Figure 2: A schematic diagram of our proposed study to investigate the use of Oxford 
nanopore sequencing to detect and characterize biliary microbial contamination 
 
The study will include patients undergoing elective pancreaticoduodenectomy or total 
pancreatectomy for any clinical indication. Patients will be excluded from the study if they are 
< 18 years of age, enrolled in any similar clinical trials involving use of peri-operative 
antibiotics, are undergoing distal pancreatectomy, or undergoing emergency total 
pancreatectomy or emergency pancreaticoduodenectomy (Table 4). Following recruitment to 
the study, patients will be randomly assigned intraoperatively/post-operative to either the 
control group (Arm 1) or the interventional group (Arm 2). We will use stratified 
randomization with age (below 65 years vs 65 years and above), sex, operation type 
(pancreaticoduodenectomy vs total pancreatectomy), biliary stenting (yes vs no), and patient 
ASA score as stratification factors. 
 
Table 4: Patient inclusion and exclusion criteria for the clinical trial 
 
Inclusion Criteria 
• > 18 years of age 
• Scheduled for elective total pancreatectomy or 
pancreaticoduodenectomy 
Exclusion Criteria 
• < 18 years of age 

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• Emergency total pancreatectomy or 
pancreaticoduodenectomy 
• Patients scheduled for distal pancreatectomy 
• Women who are pregnant 
• Patients without the cognitive capacity to consent  
• Patients enrolled in similar clinical trials involving use of 
perioperative antibiotics 
 
Bile will be collected via bile duct aspiration. Bile aspiration will be performed with a 16-
gauge needle and 3 cc syringe prior to bile duct division (Figure 3), and aspirated material will 
be aseptically transferred into sterile aerobic and anaerobic transport vials. Samples will be 
concurrently transported to the Pathology lab and the microbiome lab to undergo SC  
techniques and ONT sequencing, respectively. Standardization of bile collection and surgical 
procedure will be assured as all surgeries will be performed by the same lead surgeon, Dr. 
Mark Truty. Surgical variables that have been associated with increased SSI risk will be 
recorded. This will include patient sex, age, BMI, ASA score, the volume of blood loss, 
whether the patient had a biliary stent inserted prior to surgery, closure technique, and whether 
vascular reconstruction was performed during surgery. 
 
 
 
Figure 3: Collection of intraoperative bile aspirate during pancreatic head resection (total 
pancreatectomy, pancreaticoduodenectomy)  
 
SCs obtained from intraoperative bile aspiration for both study arms will be performed in 
compliance with clinical standards. This is routinely performed by the Pathology lab and no 
changes to these protocols are necessary (see Supplementary Materials S1 – S6)  for culture 
methods and AST protocols). Results will be reported in the EMR which will be accessible by 
the clinical team, as is the current clinical practice. ONT sequencing for those assigned to 
Study Arm 2 will be performed on a 1cc liquid bile sample collected fresh from the operating 
room after specimen release from the Pathology lab. Approval from our institutional TRAG 

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(Tissue Request Acquisition Group), will be requested for managing of intraoperative samples 
and will notify the study team of sample receipt. After collection, microbial DNA will be 
extracted and sequenced in preparation for reading per protocols 44,48. In brief, Porechop and 
BBMap will be used to remove adaptor sequences and low complexity reads. The filtered reads 
will then be aligned using Centrifuge in order to determine species identification. Bile aspirates 
will be declared microbial positive when 500 or more microbial reads are detected. 
Additionally, 50 reads per million (RPM) ratio (RPM-r), defined as RPM-r = RPMsample / 
RPMnegative control , will be used as a minimum threshold to reduces biases caused by different 
sequencing depth and mitigate concerns regarding potential microbial contamination, as 
described in previous studies 47,73,78,79. Relative abundance of the microbial species detected 
will then be determined and high abundant species (species with a relative abundance greater 
than 1%) will then be reported to the care team. The sequenced reads will also be used to 
identify acquired chromosomal AMR genes and putative AMR phenotypes using ResFinder 
4.151. A minimum coverage of 95% and a minimum sequence identity of 95% will be used, 
and predicted phenotype and source organism of the identified AMR gene will be reported to 
the care team. To ensure that no potential resistance phenotypes are missed we will also use 
the PlasmidFinder and CARD tools with the default settings to check for any plasmid-acquired 
AMR phenotypes. This will be a critical step as CRE and ESBL genes are located on plasmids. 
However, due to these genes being located on plasmids, use of PlasmidFinder and CARD will 
not be used to declare presence of CRE- and/or ESBL-organisms but instead inform the 
Surgical Clinical Pharmacy and the Pathology laboratory about their potential presence in the 
patient sample 
 
After sequencing data is gathered, samples will be destroyed. The data results from the ONT 
sequencing testing will be entered into EMR as a research clinic note for documentation and 
results will additionally be emailed to the medical providers on the care team. Decisions on 
antibiotic regimen will be made with the assistance of Surgical Clinical Pharmacy with changes 
in regimens as deemed appropriate based on standard cultures and ONT results, as described 
in Table 5. A negative ONT result will result in discontinuation of perioperative antibiotics 
after 24 hours while a positive ONT result will be used to guide early targeted antibiotic therapy 
(Table 5). With regards to early discontinuation of antibiotics, we are confident that this will 
not adversely impact patient care as our preliminary data demonstrated that ONT resulted in 
no false negative calls. However, as this is a new technology, SC will be performed alongside 
ONT, and if microbial growth is detected in the cultures, the clinician will have the option of 
reverting patient care to current standard of practice. This option will also be available in the 
event of treatment failure in patients with positive ONT results (Table 5).  
 
Given that currently clinically available tests will be available to the care team with the 
additional ONT sequencing as supplemental data there will be minimal risk to subjects 
participating in this study. 
 
Table 5 Incorporation of ONT results in clinical management decisions 
 
ONT Result 
Clinical management decision 
A negative ONT result 
Discontinue antibiotics after 24 hours of peri-operative 
coverage 

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A positive ONT result for a single pathogen 
typical of biliary or gastrointestinal flora 
Adjust and/or narrow antibiotic therapy to cover single 
pathogen. 
If 
single 
pathogen 
deemed 
clinically 
insignificant (ie. lactobacillus), discontinue antibiotics 
after 24 hours of peri-operative coverage. 
A positive ONT result for multiple pathogens 
Adjust and/or narrow antibiotic therapy to cover all 
pathogens, 
excluding 
those 
deemed 
clinically 
insignificant. If all pathogens are considered clinically 
insignificant, discontinue antibiotics after 24 hours of 
peri-operative coverage. 
A positive ONT result positive for rare or 
unexpected flora 
Adjust and/or narrow antibiotic therapy to cover rare or 
unexpected flora, excluding those deemed clinically 
insignificant. 
ONT results different from SC 
Adjust and/or narrow antibiotic therapy to cover 
pathogens found in ONT results, excluding those deemed 
clinically insignificant. SC results to only be considered 
in the event of treatment failure with antibiotic therapy 
targeted towards ONT results. 
 
 
Databases Used 
For the analysis of our endpoints, we are relying on sequence homology for two main tasks, 
mainly, (1) the identification of species and (2) identification of AMR phenotypes. For species 
identification, we use the refseq genome database (downloaded on 12/18/2020) with all data 
from the following higher order taxa groups included: human, protozoa, viral, bacterial, and 
fungal. For the identification of AMR phenotypes, we use the ResFinder database (downloaded 
on 06/04/2021, version 4.1) which contains the acquired chromosomal resistance genes, 
ensuring we know which species is carrying a given resistance gene. For plasmid AMR gene 
identification, we will use the PlasmidFinder-specific database (downloaded on 
7.14.2021/version 2.0.1) and the CARD data (downloaded on 07/07/21, version CARD 2020). 
These databases undergo regular updates since they were first developed. To ensure the 
analysis remains consistent throughout the study, databases will not be updated for the duration 
of the study. In the unlikely event that we require a software/ database update in order to run 
these tools (compatibility issues with future institutionally required OS update, inability to 
restore databases from archives after computational disaster), we will validate any newer 
versions of the database and/or algorithms using the data and culture gold standards from our 
published study and recently collected data and will only proceed if we have comparable 
performance in accuracy for species and AMR identification. 
 
Patient Follow-up and Safety Monitoring 
 
Direct study participation will last until surgical samples have been collected. Follow-up data 
will be collected from the medical record for 90 days following the surgical procedure in order 
to satisfy the study objectives. Event monitoring will be daily while inpatient and weekly 
following patient discharge (Figure 3). Adverse events will be assessed daily, including 
occurrence of SSI, type of SSI, and severity of SSI (Table 6). Serious Adverse events will be 
reported to the IRB following Mayo Clinic policies and FDA guidances.  In the event of a SSI, 
we will treat and monitor appropriately using standard care procedures which include 
additional culture testing, drainage, and imaging. 

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Table 6: Classes of surgical site infections that will be assessed and recorded 
 
Surgical Site Infection Class 
Surgical Site Infection definition 
Superficial incisional 
Only skin and subcutaneous tissue involvement 
Deep incisional 
Facia or muscular layers   
Organ space 
Involvement of any part of the body that was opened or 
manipulated during the surgical procedure 
 
 
The overall outcome of the study will be statistically assessed every 2 weeks and any 
statistically significant differences between the study arms will be reported to the data safety 
board (p-value < 0.05 for the primary outcome or SSI rate). Criteria for stopping the study will 
include significantly worse patient outcomes (p<0.05) in Arm 2 of the clinical trial (Table 7), 
significant deviations from the protocol as determined by IRB, or any other safety issues 
identified by the data safety monitoring board. 
 
 
Screening
Randomization
Study Arm 1
(Standard 
Cultures)
Study Arm 2
(Standard 
Cultures + ONT 
Sequencing)
Event Monitoring
(Daily while 
inpatient, weekly 
once discharged 
for up to 90 days)
 
 
Figure 3: A schematic figure of the clinical trial protocol 
 

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Table 7: Stopping Rules-Clinical criteria for stopping the clinical trial 
 
Patient Outcome 
Criteria for ending the clinical trial 
Mortality 
Rates of mortality significantly higher in ARM 2 of the 
clinical trial (P < 0.05) 
Morbidity 
Rates of morbidity significantly high in Arm 2 of the clinical 
trial (P < 0.05) 
SSI 
Rates of SSI significantly higher in ARM 2 of the clinical trial 
Antibiotic adverse events 
Number of antibiotic adverse events significantly higher in 
ARM 2 of the clinical trial (P < 0.05) 
Pancreatic fistula 
Rates of pancreatic fistula significantly higher in ARM 2 of 
the clinical trial (P < 0.05) 
Biliary fistula 
Rates of biliary fistula significantly higher in ARM 2 of the 
clinical trial (P < 0.05) 
Intra-abdominal abscess 
Rates of intra-abdominal abscess significantly higher in ARM 
2 of the clinical trial (P < 0.05) 
Pulmonary complications 
Rates of pulmonary complications significantly higher in 
ARM 2 of the clinical trial (P < 0.05) 
Cardiovascular complications 
Rates of cardiovascular complications significantly higher in 
ARM 2 of the clinical trial (P < 0.05) 
 
Data Safety Monitoring Board 
The Department of Surgery Data Safety Monitoring Board will be responsible for analysis of 
the results on a bi-yearly basis (every 6 months) in order to evaluate outcomes as the is 
enrolling.  
 
2.2 
Primary Study Endpoints 
 
• Reduced administration of antibiotics with a non-significant (p>0.05) effect on SSI 
rate 
2.3 
Secondary Study Endpoints 
 
• Cost effectiveness (reduced total hospital costs) 
• Timeliness of sample analysis 
• Reduction in SSI 
 
 
3 Subject Selection, Enrollment and Withdrawal 
 
Any adult, male or female, over age 18, undergoing pancreaticoduodenectomy or total 
pancreatectomy for benign or malignant indication.  
 
Target accrual: 140 patients.  

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3.1 
Inclusion Criteria 
• >18 yr M or F 
• Undergoing pancreaticoduodenectomy or total pancreatectomy for any benign or 
malignant indication with informed consent 
3.2 
Exclusion Criteria 
• Women who are pregnant 
• Patients who are institutionalized or incarcerated 
• Patients without the cognitive capacity to consent  
• Patients undergoing emergency pancreaticoduodenectomy or total pancreatectomy 
• Patients enrolled in similar clinical trials involving use of perioperative antibiotics 
 
3.3 
Subject Recruitment, Enrollment and Screening 
 
Patients scheduled for pancreaticoduodenectomy or total pancreatectomy for benign or 
malignant indication at Mayo Clinic Rochester will be approached by the study team prior to 
their surgery if they meet the enrollment criteria. Recruitment methods may include electronic 
invitations to the study through the patient portal or email. After receipt of the initial study 
invitation, a study coordinator will contact the patient by phone to review the study and answer 
any questions. If the patient is interested in participating, the study coordinator will consent 
the patient via paper or electronic consenting methods.  
3.3.1 When and How to Withdraw Subjects 
 
Participants will be withdrawn from the study if they no longer meet enrollment requirements 
or wish to withdrawal consent. If a participant chooses to withdraw their consent, no 
additional testing or data collection will be performed.  
 
3.3.2 Data Collection and Follow-up for Withdrawn Subjects 
 
Additional data collection during the follow-up period will not occur for subjects who have 
been withdrawn from the research study.  
 
4 Study Device 
4.1 
Description 
 
Please refer to the manufacturing information/instructions for use provided by Oxford 
Nanopore Technologies for full device description. Briefly, the GridION MK1 is a ONT 
sequencing device that will be used to examine microbial DNA in bile cultures from subjects 
undergoing pancreatic head resection. The same GridION instrument (device ID GXB03025) 

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will be used to test each sample. Serial number/lot number information for the sterile, single 
time use flow cells/ reagents will be recorded on a device accountability log. MinKnow, 
Guppy, Porechop, BBMap, Centrifuge, and ResFinder 4.1 software will be used for data 
analysis. The GridION device will be stored in a secure laboratory and only be accessible to 
trained study staff. In the event the GridION needs replacement, we will contact the 
manufacturer for a replacement and validate performance using residual DNA from our 
published studies. A GridION instrument will be considered validated if microbial species are 
detected in the expected proportions. We will then record the new GridION serial number and 
date put into service for this study.   
 
ONT sequencing and analysis will only be performed by our Postdoctoral Fellow trained in 
the technology, and there are no medical or surgical procedures involved in the use of the 
device 
4.2 
Method for Assigning Subjects to Treatment Groups 
 
Two potential confounding variables exist in this patient population: operation type 
(pancreaticoduodenectomy vs total pancreatectomy) and pre-operative biliary stenting. 
Therefore, to eliminate potential bias between arms, patients will be stratified according to 
operation type (pancreaticoduodenectomy/total pancreatectomy) and biliary stenting (Y/N). 
Stratified patients will be assigned to arms by the study team using Medidata Rave with pre-
determined assignments. 
 
Given the prospective nature of this study, it will include subjects previously diagnosed with a 
specific disease and add newly diagnosed subjects in the future as patients are accrued.  
4.3 
Masking/Blinding of Study 
 
This is an open, randomized trial. Subjects and study team members will not be blinded.  
4.4 
Storage 
The GridION sequencing instrument will be located in Dr.Walther-Antonio ’s lab. The 
device will only be accessible by study staff. 
 
4.5 
Cleaning/Sterilization Procedures (optional) 
 
DNA extraction and sequencing library preparation will be performed in a UV biosafety 
cabinet category 2 hood to ensure sterile conditions throughout the library preparation steps. 
Prior to sequencing Flush Buffer will be loaded into the ONT R9.4.1 flow cells in order to 
remove the storage solution prior to use. To ensure that no cross-contamination occurs, a new, 
sterile flow cell will be used for each patient. 
 

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5 
Study Procedures 
5.1 
Enrollment Visit 
 
At the time of enrollment, the informed consent will be reviewed with the participant and 
informed consent will be obtained.  
 
Consented patients will be de-identified in compliance with the Health Insurance Portability 
and Accountability Act of 1996 (HIPAA) and patient identifiers will be stored electronically 
with access limited to members of the research team on a need-to-know basis. Patient 
demographics and baseline collection information will be gathered from the participant’s 
medical record and documented in the Medidata Rave database. Demographics and baseline 
collection data collection will include age, sex, indication for surgery, ASA, major-
comorbidities, pre-operative biliary stenting/manipulation, and pre-surgical cholangitis, etc.  
 
 
5.2 
Sample Collection Visit 
 
Following enrollment procedures, 2 cc of residual bile collected during surgery will be 
aliquoted into cryovial and placed on ice (not frozen). It will be picked up by the study team 
at the time of collection for ONT sequencing. Tissue Request Acquisition Group (TRAG) 
approval will be requested for managing of samples. The sample will be aliquoted from the 
clinical collection and so collection will not add additional time to the surgery procedure. 
Participants will be randomized in Medidata Rave to a study arm based on operation type and 
pre-operative biliary stenting using a dynamic stratification. Participants will be randomized 
in a 1:1 ratio. 
 
5.3 
Follow up Data Collection 
 
Outcomes will be abstracted from the medical record for 90-days post-operatively and will 
include outcomes of primary endpoints that include: duration of perioperative antibiotic 
regimen, total number of antibiotics administered, and administration of broad-spectrum 
antibiotics vs narrow-spectrum antibiotics. Secondary endpoints will include SSI rate, SSI 
severity (superficial, deep, organ/space, bloodstream, or urine), and total hospital costs. 
Standard post-pancreatectomy surgical outcomes will also be collected including: total hospital 
length of stay (days), ICU length of stay (days), need for and type or interventional procedures, 
pancreas-specific complications (pancreatic leak/grade, delayed gastric emptying, and post 
pancreatectomy hemorrhage) hospital readmission, and death. In addition, all culture results 
and ONT sequencing results will be captured and compared. Additional clinical data will be 
gathered if deemed appropriate for outcome analysis. Patients will be assigned a study 
identification number at the beginning of the study to aide in securing identifiable patient 
information. All patient information will be stored in a secure Medidata Rave database that 

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will be accessed by designated study members only. Statistical analysis will be performed to 
compare outcomes between study arms as described in analysis section of this application. 
 
6 Statistical Plan 
6.1 
Sample Size Determination 
 
Data for power analysis based on pilot study data (IRB# 19-011419). Power analysis is based 
on improvement in antibiotic stewardship with the following assumptions: 
- 
Treatment groups are random and independent 
- 
Microbial detection positivity rate using standard cultures = 54%  
- 
Microbial detection positivity rate using nanopore sequencing = 77%  
Analytical method: 
- 
Sample size per group = 66 (total N = 132), Target size=70 per arm, total N=140 
- 
Power = 80%, alpha (type I error) = 0.05, beta (type II error) = 0.2 
6.2 
Statistical Methods 
 
Descriptive Statistics 
 
The primary analysis of this study will be a modified intention to treat analysis (ITT), 
performed to determine how use of ONT changed patient treatment, i.e. did use of ONT 
improve antibiotic stewardship. Proportions of patients with detection of microbes of interest 
will be estimated along with exact 95% binomial confidence intervals. Comparisons of the 
proportions between the two study arms will be compared using Fisher’s exact test or Chi-
square test as appropriate. We will calculate the time from randomization to first microbial 
detection within a patient. We will report the mean difference in time to first microbial 
detection within 96 hours post randomization in both treatment arms, together with a 95% 
confidence interval. Comparison of time to first microbial detection within 96 hours post 
randomization, will be performed using Wilcoxon rank sum test or two sample t-test as 
appropriate. If there are any baseline imbalances in demographic or clinical variables between 
the two groups, we will perform the analysis using univariable and multivariable linear 
regression adjusting for these variables with imbalances as possible confounders. Duration of 
antibiotics, total number of different antibiotics, and broad-spectrum antibiotics vs narrow-
spectrum antibiotics administered in the two arms will be compared to determine if use of ONT 
improved antibiotic stewardship by significantly reducing the duration of perioperative 
antibiotics and use of broad-spectrum antibiotics 
 
Secondary analysis will evaluate SSI’s will be evaluated and classified using ACS NSQIP 
definitions and data collected will include mortality rate, SSI incidence rate, ICU length of 
stay, total length of hospital stay, need for interventional procedures, and hospital readmission. 
Statistical analysis will be performed by the study team to determine if use of ONT 
significantly improved patient outcome compared to patients who received standard of care 

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only. The secondary endpoint of the study is a binary endpoint i.e. whether patients develop 
SSI (yes, no). Association between the secondary outcome and predictor variables will be 
assessed using univariable and multivariable logistic regression.  
 
In both primary and secondary analysis, the following variables will be assessed for inclusion 
in the regression models 
 
• Age at time of surgery DOB 
• BMI at time of surgery RAVE calculate 
• Sex 
• Diabetes mellitus co-morbidity at time of surgery (clarifications needed for data 
collection) 
• Cancer diagnosis 
• ASA score at time of surgery 
• Administration of chemotherapy prior to surgery (from what timepoint or date of last 
chemo?) 
• Presence of biliary stent at time of surgery  
• Blood loss during surgery 
• Length of surgery (skin to skin, open to close?) 
• Vascular reconstruction (arterial, venous) during surgery 
• Closure technique 
• Pancreatic leak risk score (when done?  In record?) 
• Surgical drains (what is needed) 
• Post-op TPN 
 
Variables with p-values less than 0.15 will be considered as potential candidates for a 
multivariable model. We will report odds ratios and 95% confidence interval. The area under 
the receiver operating characteristic (ROC) curve will be estimated as a measure of the ability 
of the model to discriminate between patients who had SSI within 90 days versus those who 
did not. An area under the ROC estimate of 0·7–0·8 will be regarded as acceptable, 0·8–0·9 
will be regarded as excellent, and more than 0·9 will be regarded as outstanding. 
 
We will also assess cost effectiveness of using ONT compared to SC. To achieve this, we will 
compare the cost to perform SC testing and ONT testing against costs associated with post-
operative patient care. This will include the total cost to administer perioperative antibiotics, 
costs associated with length of hospital stay, and the costs associated with the need of 
additional procedures and tests associated with SSI (i.e. post-operative SC and antibiotic 
susceptibility testing, additional antibiotics, wound debridement, abdominal abscess drainage, 
CT scan, US scan). 
 
For continuous variables, t-test will be performed and for binary variables chi-square or 
fisher’s exact testing will be performed as appropriate. Additional analysis will be performed 
as necessary to determine differences in study arms. Should additional analysis beyond the 
scope of our abilities is necessary then a statistician will be employed. 

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Inpatient related costs associated with each intervention arm will be analyzed and compared 
with the assistance of the Robert D. and Patricia E. Kern Center for Science of Health Care in 
order to understand the economic impact of each intervention arm. 
 
Handling of Missing Data 
 
Missing data could arise from lost samples or from a catastrophic failure of computer storage 
and backup systems. If any of these were to occur before an intervention could be determined 
using ONT sequencing, we will note these events and revert the patient to Standard of Care 
group.  
 
Primary Hypothesis:  
 
The use of ONT sequencing, a novel metagenomic technique, will result in more rapid 
identification of organisms in intra-operatively collected bile samples and allow for earlier 
treatment of biliary microbial contamination. Supplementing SC with ONT sequencing data 
will allow patients to be treated earlier in the post-operative setting with tailored antibiotics, 
which will reduce the rate of biliary microbial contamination related SSIs in patients 
undergoing pancreatic head resection as compared to standard cultures alone and will improve 
antimicrobial stewardship by avoiding unnecessary use of antibiotics.  
 
7 Safety and Adverse Events 
7.1 
Definitions 
 
Unanticipated Adverse Device Effect (UADE) 
A UADE is any serious adverse effect on health or safety or any life-threatening problem or 
death caused by, or associated with, a device if that effect, problem or death was not previously 
identified in nature, severity, or degree of incidence in the investigational plan or IDE 
application (including a supplementary plan or application), or any other unanticipated serious 
problem associated with a device that relates to the rights, safety, or welfare of subjects. 
 
Adverse Effect (Event) 
Any untoward medical occurrence in a subject involved in clinical study of an investigational 
device; regardless of the causal relationship of the problem with the device or, if applicable, 
other study related treatment(s). 
 
Associated with the investigational device: There is a reasonable possibility that the adverse 
effect may have been caused by the investigational device. 
 
Life-threatening adverse effect: Any adverse effect that places the subject, in the view of 
either the investigator or the sponsor, at immediate risk of death from the effect as it occurred.  

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It does not include a reaction that, had it occurred in a more severe form, might have caused 
death. 
 
Serious adverse effect: An adverse effect is considered “serious” if, in the view of either the 
investigator or the sponsor, it results in any of the following outcomes:  
• death  
• a life-threatening AE 
• inpatient hospitalization or prolongation of existing hospitalization 
• a persistent or significant disability/incapacity 
• a congenital anomaly/birth defect. 
 
Unanticipated adverse effect: Any adverse effect, the nature, specificity, severity, or 
frequency of which is not consistent with the risk information in the clinical study protocol or 
elsewhere in the current IDE application. 
 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a preexisting 
condition.  At the end of the study, any new clinically significant findings/abnormalities that 
meet the definition of an adverse event must also be recorded and documented as an adverse 
event. 
 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization should be 
documented and reported as an unanticipated adverse device effect unless specifically 
instructed otherwise in this protocol.  Any condition responsible for surgery should be 
documented as an adverse event if the condition meets the criteria for an adverse event. 
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as 
an adverse event in the following circumstances:  
• 
Hospitalization or prolonged hospitalization for diagnostic or elective surgical 
procedures for a preexisting condition.  Surgery should not be reported as an outcome 
of an adverse event if the purpose of the surgery was elective or diagnostic and the 
outcome was uneventful 
• 
Hospitalization or prolonged hospitalization required to allow efficacy measurement 
for the study 
• 
Hospitalization or prolonged hospitalization for therapy of the target disease of the 
study, unless it is a worsening or increase in frequency of hospital admissions as judged 
by the clinical investigator. 
 
Post-study Adverse Event 
All unresolved adverse events should be followed by the investigator until the events are 
resolved, the subject is lost to follow-up, or the adverse event is otherwise explained.  At the 
last scheduled visit, the local investigator should instruct each subject to report, to the local 
investigator, any subsequent event(s) that the subject, or the subject’s personal physician, 
believes might reasonably be related to participation in this study.  The local investigator 

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should notify the study regulatory sponsor of any death or adverse event occurring at any time 
after a subject has discontinued or terminated study participation that may reasonably be 
related to this study.  The sponsor should also be notified if the local investigator should 
become aware of the development of problems, cancer or of a congenital anomaly in a 
subsequently conceived offspring of a subject that has participated in this study. 
 
Preexisting Condition 
A preexisting condition is one that is present at the start of the study.  A preexisting condition 
should be recorded as an adverse event if the frequency, intensity, or the character of the 
condition worsens during the study period. 
 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRTSO) 
Any unanticipated problem or adverse event that meets all of the following three criteria: 
• 
Serious: Serious problems or events that results in significant harm, (which may be 
physical, psychological, financial, social, economic, or legal) or increased risk for the 
subject or others (including individuals who are not research subjects). These include: 
(1) death; (2) life threatening adverse experience; (3) hospitalization - inpatient, new, 
or prolonged; (4) disability/incapacity - persistent or significant; (5) birth 
defect/anomaly; (6) breach of confidentiality and (7) other problems, events, or new 
information (i.e. publications, DSMB reports, interim findings, product labeling 
change) that in the opinion of the local investigator may adversely affect the rights, 
safety, or welfare of the subjects or others, or substantially compromise the research 
data, AND 
• 
Unanticipated: (i.e. unexpected) problems or events are those that are not already 
described as potential risks in the protocol, consent document, not listed in the 
Investigator’s Brochure, or not part of an underlying disease. A problem or event is 
"unanticipated" when it was unforeseeable at the time of its occurrence. A problem or 
event is "unanticipated" when it occurs at an increased frequency or at an increased 
severity than expected, AND 
• 
Related: A problem or event is "related" if it is possibly related to the research 
procedures. 
 
7.2 
Recording of Adverse Events 
 
All adverse events occurring during the study period must be recorded.  All observed or 
volunteered adverse effects (serious or non-serious) and abnormal test findings, regardless of 
the treatment group if applicable or suspected causal relationship to the investigational device 
or if applicable other study treatment or diagnostic product(s) will be recorded in the subjects’ 
case history.  For all adverse effects sufficient information will be pursued and or obtained as 
to permit; an adequate determination of the outcome, an assessment of the casual relationship 
between the adverse effect and the investigational device or, if applicable other study treatment 
or diagnostic product.  The clinical course of each event should be followed until resolution, 
stabilization, or until it has been ultimately determined that the study treatment or participation 

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is not the probable cause.  Serious adverse events that are still ongoing at the end of the study 
period must be followed up, to determine the final outcome.  Any serious adverse event that 
occurs after the study period and is considered to be at least possibly related to the study 
treatment or study participation should be recorded and reported immediately. 
 
Causality and severity assessment 
The sponsor-investigator will promptly review documented adverse effects and abnormal test 
findings to determine 1) if the abnormal test finding should be classified as an adverse effect; 
2) if there is a reasonable possibility that the adverse effect was caused by the investigational 
device or other study treatments; and 3) if the adverse effect meets the criteria for a serious 
adverse effect. 
 
If the sponsor-investigator’s final determination of causality is “unknown and of questionable 
relationship to the investigational device or other study treatments,” the adverse effect will be 
classified as associated with the use of the investigational device or other study treatments for 
reporting purposes.  If the sponsor-investigator’s final determination of causality is “unknown 
but not related to the investigational device or other study treatments,” this determination and 
the rationale for the determination will be documented in the respective subject’s case history. 
7.1 
Sponsor-Investigator Reporting of Unanticipated Adverse Device Effects and 
Unanticipated Problems 
 
 
When an adverse event has been identified, the study team will take appropriate action 
necessary to protect the study participant and then complete the Study Adverse Event 
Worksheet and log.  The sponsor-investigator will evaluate the event and determine the 
necessary follow-up and reporting required. 
 
 
The sponsor-investigator will promptly review documented Unanticipated Adverse Device 
Effects and as necessary shall report the results of such evaluation to FDA within 10 working 
days and Mayo IRB within 5 working days of initial notice of the effect.  Thereafter the 
sponsor-investigator will submit such additional reports concerning the effect as requested. 
 
 
 
7.1.1  
7.1.2 Sponsor-Investigator Reporting, Notifying Mayo IRB 
 
The sponsor-investigator will report to the Mayo IRB any UPIRTSOs and Non-UPIRTSOs 
according to the Mayo IRB Policy and Procedures. 
 

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7.1.3 Sponsor-Investigator Reporting: Notifying the FDA 
 
The sponsor-investigator will report to the FDA all unanticipated adverse device effects 
according to the required reporting timelines, formats and regulations. 
 
The sponsor-investigator will submit a completed FDA Form 3500A to the FDA’s Center for 
Devices and Radiological Health for any observed or reported adverse effect that is determined 
to be an unanticipated adverse device effect.  A copy of this completed form will be provided 
to the DSMB and all participating sub-investigators. 
 
The completed FDA Form 3500A will be submitted to the FDA as soon as possible and, in no 
event, later than 10 working days after the sponsor-investigator first receives notice of the 
adverse effect. 
 
If the results of the sponsor-investigator’s follow-up evaluation shows that an adverse effect 
that was initially determined to not constitute an unanticipated adverse device effect does, in 
fact, meet the requirements for reporting; the sponsor-investigator will submit a completed 
FDA Form 3500A as soon as possible, but in no event later than 10 working days, after the 
determination was made. 
 
For each submitted FDA Form 3500A, the sponsor-investigator will identify all previously 
submitted reports that that addressed a similar adverse effect experience and will provide an 
analysis of the significance of newly reported adverse effect in light of any previous, similar 
report(s). 
 
Subsequent to the initial submission of a completed FDA Form 3500A, the sponsor-
investigator will submit additional information concerning the reported adverse effect as 
requested by the FDA. 
 
Reporting Process 
Unanticipated Adverse Device Effect reports will be submitted on FDA Form 3500A. 
The contact information for submitting reports is: 
 
Food and Drug Administration 
Center for Devices and Radiological Health 
Document Mail Center - WO66-G609 
10903 New Hampshire Avenue 
Silver Spring, Maryland 20993-0002 
 
Deviations from the investigational plan. 
 
The sponsor-investigator shall notify Mayo IRB (see 21 CFR 56.108(a) (3) and (4)) of any 
deviation from the investigational plan to protect the life or physical well-being of a subject in 
an emergency. Such notice shall be given as soon as possible, but in no event later than 5 

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working days after the emergency occurred. Except in such an emergency, prior approval by 
the sponsor-investigator is required for changes in or deviations from a plan, and if these 
changes or deviations may affect the scientific soundness of the plan or the rights, safety, or 
welfare of human subjects, FDA and IRB notification in accordance with 21 CFR 812.35(a) 
also is required. 
7.2 
Medical Monitoring and Data Safety Monitoring Board 
The Mayo Clinic Department of Surgery Data Safety Monitoring Board will serve as the 
monitoring committee for this study. The function of a DSMB is to review data and endpoints 
on a timeline set forth by the Data Safety Monitoring Plan (DSMP) in the IRB approved 
protocol with members independent of the investigators and direct study team.   
 
 
 
It is the responsibility of the sponsor-investigator to oversee the safety of the study.  This safety 
monitoring will include careful assessment and appropriate reporting of adverse events as 
noted above, as well as the construction and implementation of a site data and safety-
monitoring plan (see Section 10 Auditing, Monitoring and Inspecting).  Medical monitoring 
will include a regular assessment of the number and type of serious adverse events. 
 
Patient’s will be monitored 24/7 by a team composed of the consultant surgeon, general surgery 
residents, advanced nurse practitioners, and physician assistants throughout their hospital stay. 
Patients will also be monitored and cared for by the primary care team composed of nurses, 
nurse assistants, pharmacists, and other consulting services in order to best take care of the 
patient.  Diagnostics tests to evaluate for SSI will include physical examination, laboratory 
testing, and imaging such as computed tomography (CT) and ultrasound (US). Patients with 
complicated hospital courses will return for a clinical visit after approximately 2 weeks with 
cross sectional CT imaging and laboratory testing with further follow up visits as clinically 
indicated. In the instance of an uneventful hospital course, patient’s will return at 4 weeks from 
their date of discharge for a clinical visit with CT imaging and laboratory testing. After this 
first routine follow-up appointment with surgery, patients will continue to follow with medical 
oncology and with the surgical clinic on an as needed basis. 
 
8 Data Handling and Record Keeping 
8.1 
Confidentiality 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  
Those regulations require a signed subject authorization informing the subject of the following: 
• What protected health information (PHI) will be collected from subjects in this study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research subject to revoke their authorization for use of their PHI. 
 

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In the event that a subject revokes authorization to collect or use PHI, the investigator, by 
regulation, retains the ability to use all information collected prior to the revocation of subject 
authorization.  For subjects that have revoked authorization to collect or use PHI, attempts 
should be made to obtain permission to collect at least vital status (long term survival status 
that the subject is alive) at the end of their scheduled study period. 
8.2 
Source Documents 
 
ONT sequenced reads will be stored as fastq and fast5 files. Detection of microbial species 
will be stored as original ckraken files,in excel spreadsheets, and in Medidata Rave. 
Information of antimicrobial resistance genes detected in the ONT sequencing data will be 
stored as fsa, txt, excel files, and Medidata Rave. Original records of patient outcome will be 
stored in clinical charts (hospital records), and extrapolated patient clinical data will be stored 
in excel spreadsheets and Medidata Rave. 
 
Source data comprise all information, original records of clinical findings, observations, or 
other activities in a clinical trial necessary for the reconstruction and evaluation of the trial.  
Source data are contained in source documents.  Examples of these original documents, and 
data records include: hospital records, clinical and office charts, laboratory notes, memoranda, 
subjects’ diaries or evaluation checklists, pharmacy dispensing records, recorded data from 
automated instruments, copies or transcriptions certified after verification as being accurate 
and complete, microfiches, photographic negatives, microfilm or magnetic media, x-rays, 
subject files, and records kept at the pharmacy, at the laboratories, and at medico-technical 
departments involved in the clinical trial. When applicable, information recorded on the CRF 
shall match the Source Data recorded on the Source Documents. 
8.3 
Case Report Forms 
 
A Case Report Form (CRF) will be completed for each subject enrolled into the clinical study.  
The investigator-sponsor will review, approve and sign/date each completed CRF; the 
investigator-sponsor’s signature serving as attestation of the investigator-sponsor’s 
responsibility for ensuring that all clinical and laboratory data entered on the CRF are 
complete, accurate and authentic. 
 
The study case report form (CRF) is the primary data collection instrument for the study.  All 
data requested on the CRF must be recorded.    If a space on the CRF is left blank because the 
procedure was not done or the question was not asked, “N/D” will be entered in RAVE.  If the 
item is not applicable to the individual case, write “N/A”.  All entries should be printed legibly 
in ink on the Source Document Worksheet.   
 
Data Handling and Record Keeping  
Confidentiality  
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA). 

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Those regulations require a signed subject authorization informing the subject of the 
following:  
• What protected health information (PHI) will be collected from subjects in this study  
• Who will have access to that information and why  
• Who will use or disclose that information  
• The rights of a research subject to revoke their authorization for use of their PHI.  
(This information is contained within the Mayo IRB Informed Consent Template Section 
14) In the event that a subject revokes authorization to collect or use PHI, the 
investigator, by regulation, retains the ability to use all information collected prior to the 
revocation of subject authorization. For subjects that have revoked authorization to 
collect or use PHI, attempts will be made to obtain permission to collect at least vital 
status (long term survival status that the subject is alive) at the end of their scheduled 
study period.  
  
Source Documents  
Source data is all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial.  
Source data are contained in source documents. Examples of these original documents, and 
data records include: hospital records, clinical and office charts, laboratory notes, 
memoranda, subjects’ diaries or evaluation checklists, pharmacy dispensing records, 
recorded data from automated instruments, copies or transcriptions certified after 
verification as being accurate and complete, microfiches, photographic negatives, microfilm 
or magnetic media, x-rays, subject files, and records kept at the pharmacy, at the 
laboratories, and at medico-technical departments involved in the clinical trial.  
  
Case Report Forms  
  
Data will be captured at each participating site by qualified study staff who will perform 
primary data collection from source-document reviews to electronic case report forms 
(eCRF) via Medidata Rave, the information technology endorsed by Mayo Clinic’s Clinical 
Trial Management System (CTMS) as described in Appendix/Attachment (please fill in  
Appendix/Attachment location info within protocol). Data reported in the eCRF derived 
from source documents should be consistent with the source documents or the discrepancies 
should be explained. Data will be entered for this study utilizing one or a combination of the 
following methods:  
1. 
Data may be captured electronically, without use of paper.  
2. 
Data may be transcribed from the Electronic Medical Record (EMR-an 
electronic source that must be available for review) into an EDC system, without use 
of paper.  
3. 
Data may be captured on paper (considered source documentation) and 
transcribed into the EDC system, BUT paper documentation must be retained and 
available for review.  

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• 
Data Management  
Study sites will transcribe subject source data into eCRFs using Medidata Rave. The 
Medidata  
Rave system is compliant with 21 CFR (Code of Federal Regulations) Part 11 FDA (Food 
and Drug Administration) requirements. Edit checks, electronic queries, and audit trails are 
built into the system to ensure accurate and complete data collection and security. Data will 
be transmitted via the internet from investigational sites to a central hosting site, utilizing 
state-of-the-art encryption mechanisms to ensure security and confidentiality.  
  
• 
Data Processing  
All data is entered into electronic case report forms (eCRF’s) through the Medidata Rave 
system. Case report forms are automatically rolled out based on a predetermined, visit based 
schedule to improve study staff workflow and data quality. Data is exported nightly to a 
secure FTP for analysis and reporting.  
  
• 
Data Security and Confidentiality  
The Medidata Rave database access model is role based and fully auditable at the study, 
form, and field levels. Data is de-identified whenever possible and the ability to update data 
is limited to necessary staff. Access is managed by the Mayo Clinic Research Service 
Center, under a controlled and monitored access request system. Metadata’s platform 
specifically supports Electronic Record and Electronic Signature (ER/ES) requirements, 
including US 21 CFR part 11.  
  
• 
Data Quality Assurance/Data Clarification Process  
Each eCRF contains edit checks and custom functions to ensure the highest possible data 
quality. Only necessary eCRF’s are available for data entry to reduce the possibility of 
erroneous entry.  
  
• 
Data Clarification Process  
The edit checks and custom functions on the eCRF’s trigger queries requesting the attention 
of appropriate study staff. The fields are marked in pink to allow study staff to quickly 
identify the data fields that require attention or actions. Additionally, secure email 
notifications are sent for adverse event tracking and monitoring.  
Clinical Trial Management Systems (CTMS)  
  
CTMS is the Mayo Clinic Research Committee-endorsed institutional resource for clinical 
data management. CTMS is a robust institutional effort initiated in 2010 to address emerging 
changes within the data and statistical coordinating centers affiliated with NCI-funded 
cooperative groups. In 2010, NCI selected Medidata Rave® (http://www.mdsol.com/) as the 
required data collection tool for all cooperative studies. To capitalize on Mayo Clinic and the 
NCI’s investment in Medidata Rave®, Mayo Clinic formalized a three-tier data management 
infrastructure with the Medidata Rave® product as the premier system.  
  

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Medidata Rave® is a product for multi-center clinical trials conducted under 21 CFR Part 11 
requirements. This web-based system provides ease of use coupled with an integrated 
randomization module (Medidata Balance™), custom reporting, robust data validation 
routines, and straightforward integration with SAS.  
  
• 
Electronic Data Capture: Medidata Rave® allows for data collection in 
multisite studies. During the course of the data entry into Medidata Rave®, the 
system provides real-time within-case report form (CRF) and inter-CRF data 
consistency verification. Medidata Rave® is flexible in nature so that all data 
can be entered even if “required” fields and or other consistency checks 
requirements are not satisfied. The system uses an internal “flagging” or 
“query” system to distinguish the valid from the invalid data thereby ensuring 
compliance with the FDA guidance document “Computerized Systems Used in 
Clinical Trials.” All data discrepancy issues are tracked and audited by the 
system to ensure the highest quality data is available for analysis and study 
reporting.  
  
Contained within the CTMS initiative at Mayo Clinic is a diverse set of 
administrative and technical personnel to support the development and 
implementation of clinical trials in Medidata Rave®. While the time necessary to 
program Rave’s electronic case report forms (eCRFs) has been directly budgeted, 
the CTMS initiative supports protocol independent activities such as 
software/server maintenance, data standards, institutional system integrations, 
SAS data, and training of study personnel through institutional resources.  
  
The dedicated VPN connection between Mayo Clinic and Medidata provides the 
conduit for data connectivity. Clinical trial data hosted in Medidata is accessible 
when needed for SAS using the SAS On Demand Connection, in combination 
with Mayo Clinic’s SAS Pipeline program, which creates a common and direct 
combination of the metadata (labels, formats, etc.) and data (raw values) into SAS 
datasets on a scheduled (nightly) basis. This process removes the need to 
separately label and format the entire clinical trial database separately in SAS.  
  
• 
Medidata Balance™: Randomization encounters challenges in complex 
multisite clinical studies in which random assignment to study drug must be 
completed prior  
 
Data Processing 
 
Data processing will be performed by the study investigator, study coordinators and finally by 
a qualified statistician.  Given that this is a single center study, there will not be any transfer 
of data.  All data pertinent to the study will be stored in a central location accessible to only 
study personnel. 
 
 

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8.4 
Records Retention 
 
The sponsor-investigator will maintain records and essential documents related to the conduct 
of the study.  These will include subject case histories and regulatory documents. 
 
The sponsor-investigator will retain the specified records and reports during the study and for 
the longer of the following; 
1. As outlined in the Mayo Clinic Research Policy Manual –“Retention of and Access to 
Research Data Policy” http://mayocontent.mayo.edu/research-policy/MSS_669717,  
 
9 Study Monitoring, Auditing, and Inspecting 
9.1 
Study Monitoring Plan 
 
The investigator will allocate adequate time for such monitoring activities.  The Investigator 
will also ensure that the monitor or other compliance or quality assurance reviewer is given 
access to all the study-related documents and study related facilities (e.g. pharmacy, diagnostic 
laboratory, etc.), and has adequate space to conduct the monitoring visit. 
 
9.2 
Auditing and Inspecting 
The sponsor-investigator will permit study-related monitoring, audits, and inspections by the 
IRB, the monitor, and government regulatory agencies, of all study related documents (e.g., 
source documents, regulatory documents, data collection instruments, study data etc.).  The 
sponsor-investigator will ensure the capability for inspections of applicable study-related 
facilities (e.g., pharmacy, diagnostic laboratory, etc.). 
 
Participation as a sponsor-investigator in this study implies acceptance of potential inspection 
by government regulatory authorities and applicable compliance offices. 
 
 
 
10 Ethical Considerations 
This study is to be conducted according to United States government regulations and 
Institutional research policies and procedures. 
 
This protocol and any amendments will be submitted to a properly constituted local 
Institutional Review Board (IRB), in agreement with local legal prescriptions, for formal 
approval of the study.  The decision of the IRB concerning the conduct of the study will be 
made in writing to the sponsor-investigator before commencement of this study. 
 
All subjects for this study will be provided a consent form describing this study and providing 
sufficient information for subjects to make an informed decision about their participation in 

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this study.  This consent form will be submitted with the protocol for review and approval by 
the IRB for the study.  The formal consent of a subject, using the Approved IRB consent form, 
must be obtained before that subject undergoes any study procedure.  The consent form must 
be signed and dated by the subject or the subject’s legally authorized representative, and the 
individual obtaining the informed consent. 
 
11 Study Finances 
11.1 Funding Source 
 
The research study is supported by institutional funds provided in the form of a Transform the 
Practice award from the Center for Individualized Medicine, Mayo Clinic. 
 
12 Publication Plan 
 
Link to protocol registration site for ClinicalTrials.gov:  https://register.clinicaltrials.gov/ . 
13 References 
 
 
1. 
Suragul, W. et al. Predictors of surgical site infection after pancreaticoduodenectomy. 
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14 Supplementary Materials 
 
S1. Standard cultures performed on the bile samples 
 
 Swab samples were inoculated into thioglycolate broth (thio), chocolate blood agar (CBA), 
tryptic soy agar with 5% sheep blood (BAP), eosin-methylene blue agar and 
Columbia CNA agar with 5% sheep blood, colistin and nalidixic acid (CNA). Bile fluid 
aspirate samples were aseptically removed from the transport vial and one aliquot of fluid 
placed onto each of chocolate blood agar, BAP, eosin-methylene blue agar, and CNA culture 
media for aerobic culture. Aerobic culture plates were incubated at 35°C in 5% CO2 and 
examined daily for growth for up to 5 days. Anaerobic culture media were inoculated in parallel 
and 
included 
CDC 
anaerobe 
5% 
sheep 
blood, phenylethyl 
alcohol blood agar, 
laked Brucella blood agar with kanamycin and vancomycin and a BAP used as an 
aerotolerance screen. 
 
S2: Protocol for the identification of aerobic cultures 
 
PROCEDURE: 
 
1. Culture reading schedule:  
a. Based on the time the culture was set up, read cultures in two batches 
beginning at 8:00 a.m. and 12:30 p.m. to allow for a minimum incubation time 
of 16 hours. At 8:00 a.m. move plates cultured by the midnight tech (found on 
the bottom shelf of the incubator) to the appropriate a.m. shelf to complete 
their incubation. Culture set up Initial examination 8:00 a.m. - 4:00 p.m. 4:00 
p.m. – Midnight, Midnight- 8:00 a.m. 8:00 a.m., the next day 12:30 p.m., the 
next day 8:00 a.m., the next day  
b. The schedule for reading urine cultures is found in Urine Culture [011327].  
c. The schedule for reading stool cultures is found in Stool Culture [011342].  
2. Initial examination of GEN and GENS cultures: Open the DGEN1 work list. Sort the 
work list by last name to separate bench I and bench II orders.  
a. Separate all positive cultures from the negative cultures.  
b. Review the requested test order(s), patient demographics, medical record 
number, ordered tests and special instructions from any paperwork received, 
and match with the plate identification on day 1. File all negative culture 
paperwork on day one at the bench. 
c. Mark all negative orders as the plates and thioglycollates are placed in a rack 
for further incubation.  
• If the order only has GC-Lect plates, RLABL the culture and 
reincubate the plate with the bench subcultures.  
• Body fluids incubated in Bactec bottles will not have paper 
work or media at the bench. Open the order from the worklist 
to check that the media setup is bottles or check the FX 
instruments to be sure there are bottles incubating before you 
mark an order as negative. d. Add the “No growth to date” test 

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comment to all of the selected negative orders. The orders will 
be removed from the work list when the work list is refreshed.  
• Choose “Define TC”. Select appropriate test (e.g., GEN). 
Select the NGTD test comment from the keypad. Choose 
preliminary status, and add results.  
• If the culture is positive at a later date the “No growth to date” 
comment should be Hidden by placing a (?) before the 
comment.  
3. Initial examination of SPUT/SPUTS, CGAS, CGBS, GC and GEN/GENS cultures 
from a cornea donor source (held 7 days).  
a. Add the RLABL media to the order and use the sticker to label a culture 
divider. Aerobic Culture Examination and Documentation Guidelines [PROC 
011336.021]  
b. All orders that have had the RLABL media added will be removed from the 
DGEN1 work list when you refresh.  
c. Negative cultures are reincubated in the bench subculture reincubate racks and 
looked at daily.  
4. Negative culture re-examination of GEN/GENS cultures (Days 2-5):  
a. Perform work in the BSC wearing safety glasses and gloves.  
b. Examine reincubated plates daily and discard negative plates at 5 days.  
c. Examine sub plates from body fluids subcultured from Bactec bottles daily.  
• Reincubate RCBA plates subcultured from body fluid bottles 
that flag as a false positive (NOS) for a total of 48 hours before 
tossing as negative.  
• Result the RLABL media to move the culture back to the 
worklist. The order will be finalized on day 5 from the DGEN2 
work list.  
d. Examine thioglycollate broths daily and discard negative broths at 5 days.  
• Return negative brain heart infusion broths containing foreign 
body specimens (e.g., bullets, glass shards, metal fragments 
(not surgical hardware) to Initial Processing. Specimens will be 
saved in the Initial Processing area for one year for medico-
legal purposes.  
• Return heart valves to be saved to Initial Processing area for 
final disposition (some valves may be forwarded to the 
Pathology Lab) after culture workups are complete. Refer to 
Initial Processing procedures for details.  
e. For positive plates and thioglycollates: Verify by Gram stain that the organism 
is not a select agent (e.g. Brucella sp., Francisella sp., etc.) before removing 
plates from the BSC for workup. See Biosafety Policy for the Bacteriology 
Lab [036885].  
• Initiate a Pathogen Alert Worksheet [051193] if a select agent 
or highly infectious organism is suspected.  
f. RLABL and workup any positive cultures. If a highly infectious or select 
agent is suspected keep the plates in the BSC.  

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g. Bench I personnel will open the DGEN2 work list after bench I and II have 
finished the reincubate process.  
1) Mark all orders that are 2-5 days old. NOTE: For day 5 cultures, if 
the received date is later than the collected date, wait a full 5 days 
after the received date before you mark the order.  
2) Autoresult the orders:  
• The “No growth to date” test comment will be updated in the 
final report for 2-4 day old cultures with a preliminary status. 
Orders will stay on the worklist.  
• In the electronic workcard a test comment of “?This order was 
updated and reported after X days” will document that the 
culture media was reviewed daily. X= day reviewed.  
3) The “No growth after 5 days of incubation” test comment will be 
added to the 5 day old cultures with a final status. These orders will 
be removed from the worklist when the worklist is refreshed.  
5. Workup of GEN/GENS cultures on Day 1:  
a. Enter the order using Result Entry.  
b. Add the RLABL media to the order and use the sticker to label a culture 
divider. All orders that have RLABL media added will be removed from the 
DGEN1 work list when you refresh.  
c. Recheck the DGEN1 work list. The work list should be empty. Troubleshoot 
any remaining orders.  
d. Keep all positive paperwork with the pending culture and file at the bench 
when the culture is finalized.  
e. Review the requested test order(s), patient demographics, medical record 
number, ordered tests and special instructions from any paperwork received, 
and match with the plate identification each day the culture is worked on.  
• Notate sterile collection circumstances in the Common Media 
Comments. “OR collected specimen” should be noted by IP in 
the Order Comments; however Ultrasound (US) and computed 
tomography (CT) guided collections will need to be manually 
identified and notated 
f. Check the Order Comments and Culture Comments: This is where 
information communicated from Initial Processing (i.e. “OR collected”) will 
be found. Copy the comments into the culture work card as a Common Media 
Comment.  
g. Compare the computer-generated order number to the media labels used to 
identify the solid/liquid media inoculated for the culture each time the culture 
is reviewed or there is work performed on the culture.  
h. Review direct Gram stain result, if applicable. If the direct Gram stain does 
not correlate with the growth on the culture, the direct Gram stain should be 
reviewed.  
•  Refer to Gram Stain [011334] for instructions on resolving 
Gram stain delta checks.  

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i. The core benches will check the DGSD and DGSDN resulting worklists daily 
to resolve any orders that qualify.  
j.  Document work in the electronic workcard. The date is entered by the 
system. Continue to record colonial morphology, quantitation, order 
additional tests and record results. Most of these choices will be provided by 
using the keyboards set up in the system. Additional information on LIS entry 
can be found in SCC SoftMic Functions – Bacteriology Laboratory [046139].  
k. Reincubate all plates on positive cultures to provide a total of 2 days of 
incubation.  
l. Hold final report on orders that have been referred to a specialty area e.g. 
RMYC, RSPL, RANA until the referral media has been resulted.  
m. Identify all aerobic organisms according to Organism Identification [011332].  
n. Perform susceptibility testing once every 7 days per organism per source 
unless specifically requested by phone to perform more often.  
o. Refer to the appropriate source specific procedure for further workup 
instructions.  
p. Charge for testing performed by adding the bill only charges under the RBILL 
media.  
6. Workup of Multiple Positive Cultures  
a. Determine that a patient has multiple cultures from the same anatomical 
source. A “left” and a “right” site cannot be considered the same.  
b. When multiple cultures from a patient yield isolates with identical colonial 
morphology, it is only necessary to identify the isolate from one culture. The 
identity of morphologically similar isolates from subsequent cultures may, 
therefore, be presumed. This applies only to cultures from the same, 
consecutive, or every other day if there are fresh plates for comparison of 
morphologies. Exceptions: Isolates of gram positive-cocci (except S. aureus) 
or gram positive-bacilli may utilize the same by morphology comparison only 
after 2 orders are verified to be the same identification by Bruker testing.  
c. Document in the workcard that the isolate is “same by morphology as” and 
record the order number of the culture/isolate that was identified. “Same by 
morph” is approved as shorthand for the above phrase.  
d. Do not bill for identification of isolates that are identified by morphology 
comparison.  
7. Thioglycollate workup: NOTE: If growth in thioglycollate is questionable, do not 
stain until day 5 to minimize the chance of contaminating the broth.  
a. Thioglycollate workup when primary media is positive: Gram stain any 
positive thioglycollate broth. Subculture to appropriate aerobic media if stain 
suggests growth of an organism not already recovered on plated media. 
Consider additional subculture media (i.e., CBA, BCYE) or special incubation 
conditions. “***CT” = CT guided “***US” = ultrasound Aerobic Culture  
b. Thioglycollate workup when primary media is negative for organism seen in 
thio Gram stain: If only thioglycollate broth is positive anaerobic organisms 
could be present, Gram stain the broth and subculture both aerobically and 
anaerobically (RBAPS and RCBAS, RSBS). Take the RSBS to initial 

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processing and place in the CO2 holding jar (lid up). Set the timer for two 
hours and confirm that the CO2 is flowing.  
1) Review aerobic subculture after 1 day of incubation, work up aerobic 
growth if present. Reincubate negative plates for 1 additional day.  
2) Anaerobe lab will return the RSBS to the bench after 2 days of 
incubation. If anaerobic growth is present proceed with identification 
according to Organism Identification [011332].  
8. Workup of plates referred to the bench from the Anaerobe lab:  
a. Anaerobe lab will order a ^RXANA media under the general culture and place 
the plate in the “XANAE LAB” general bench rack.  
b. General bench staff will compare the morphology to their culture plates.  
• If the isolate is the same as the aerobic culture record a “same 
as” statement under the RXANA plate.  
• If the isolate is different from the aerobic culture order a 
RBWU# under the RXANA plate and proceed with workup 
based on the categories in 1 and 2 above. Anaerobe lab will 
provide the isolate quantity. If the quantity is 1-4+ consult with 
management on what to report.  
c. The RXANA media must be resulted before the General culture can be 
finalized.  
d. Aerobic isolates reported after the general culture is finalized will be flagged 
as an additional report by the LIS.  
9. Completion of specimens referred to anaerobe Lab from the bench.  
a. ^RANA is ordered on all specimens submitted to the anaerobe lab for workup. 
Submit the ^RANA sticker to anaerobe lab when submitting an isolate from 
the general bench.  
b. If no ANAE(S) is ordered, the anaerobe staff will enter a Common Media 
Comment under the aerobic culture “Wait for anaerobes” to indicate that 
processing is in progress. The aerobic culture should be held on the bench 
referral wall until anaerobe processing is complete.  
• When complete, the anaerobe finalizer will review the work, 
result the RANA media, add a common media comment of 
“Anaerobe work-ups complete”, write “ANAE complete” on 
the culture divider and return it to the bench. The culture can 
then be finalized.  
c. If an ANAE(S) culture is ordered, then the referral will be processed under the 
ANAE(S) and the general bench can finalize their culture.  
10. Media labeling guidelines:  
 

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a. Label subculture plates with the media label and note the WORKUP number and date.  
b. Label biochemical tubes with the media label and note the WORKUP number and date. For biochemical sets (multiple 
tubes inoculated at the same time, placed in rows in a rack), label the first tube in the set and place subsequent tubes in 
a row; labeling is not required. 
 
 
PROCEDURE: Use the following table(s) to identify possible pathogenic bacteria from patient specimens. The primary testing scheme 
should be utilized whenever feasible. The secondary testing should be utilized when the primary testing scheme is inconclusive or 
unavailable. Refer to Bruker MALDI-TOF MS Reporting-Scheme-Bacteriology [048215] for MALDI-TOF acceptance ranges and 
resulting guidelines. 
 
Organisms suspected of being a select agent Identification of Francisella spp., Brucella spp., Burkholderia mallei/pseudomallei, Bacillus 
anthracis, Yersinia pestis, and N. meningitidis can be performed by MALDI-TOF when plate preparation steps are performed in the 
BSC. Refer isolate to Special Procedures Laboratory if any of these organisms are suspected. • Isolates received with a request to rule 
out B. cereus Biovar anthracis must be submitted to MDH in order for the biovar to be determined. • Alert a member of the management 
team when a select agent or N. meningitidis is suspected. 
 
S2: Table 1 
 
Gram-negative bacilli  
Suspected 
Organism 
Identification  
Morphology  
Primary Testing  
Secondary Testing H  
Comments  
Acinetobacter spp.  
 
MALDI-TOF  
Blue centers on EMB:  
Strip oxidase (-)  
TSI (K/K)  
Report Acinetobacter spp.  
 
Brucella spp.  
Francisella spp.  
Filmy growth at 24 hours  
See comments  
Perform all in BSC:  
stain tiny-cb  
Tape all plates  
MALDI-TOF  
Refer 
entire 
culture/paperwork to SPL  
 
Perform all plate 
manipulation in BSC, see 
Fastidious Fermenting Gram-
negative Bacilli / Gram-
negative Cocci Identification 
[034753] or LRN flowcharts 
(see link above). Notify QC 

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and Supervisor of all isolates 
reported.  
Gram-negative 
bacillus, 
resembling Capnocytophaga 
spp.  
Spreading “oil slick” yellow 
colonies on CBA or BA  
MALDI-TOF  
Stain: long thin gnb with 
pointed ends,  
Drop oxidase (-)  
Catalase (-)  
Prelim 
Gram-negative 
bacillus, 
resembling 
Capnocytophaga spp.  
 
Citrobacter spp.  
Darkened agar on BA  
MALDI-TOF  
TSI (A/A, H2S)  
Tube indole (-)  
Report C. freundii 
complex  
TSI (K/A)  
Tube indole (+)  
Report C. koseri  
Refer to SPL if secondary 
testing is inconclusive.  
Foul odor is characteristic  
Eikenella corrodens  
Pitting agar  
MALDI-TOF  
Stain: small gnb  
Drop oxidase (+)  
Catalase (-)  
Prelim 
Gram-negative 
bacillus  
Bleach-like 
odor 
is 
characteristic  
 
Gram-negative bacilli  
Suspected 
Organism 
Identification  
Morphology  
Primary Testing  
Secondary Testing H  
Comments  
Escherichia coli  
 
Green Metallic Sheen on 
EMB  
Non-mucoid  
Quick indole (+)  
OR  
MALDI-TOF if the BA is 
discolored indicating a 
different identification  
e.g., 
Citrobacter 
spp., 
Morganella morganii  
MALDI-TOF  
MALDI-TOF does not 
differentiate between E. coli 
and Shigella spp.  
Indole odor is characteristic  
Document 
the 
listed 
morphology in the LIS if using 
biochemical testing  
Beta-hemolytic on BA  
(Mucoid or Non-mucoid)  
Quick indole (+)  
Strip oxidase (-)  
OR  
MALDI-TOF if the BA is 
discolored indicating a 
different identification  
e.g., 
Citrobacter 
spp., 
Morganella morganii  
MALDI-TOF  

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Lactose positive on EMB and 
non-hemolytic on BA  
MALDI-TOF  
Quick indole (+)  
Strip oxidase (-)  
PYR (-)  
Lactose negative on EMB and 
non-hemolytic on BA  
MALDI-TOF  
Urine sources:  
Quick indole (+)  
Strip oxidase (-)  
MUG (+)  
Haemophilus spp.  
Tan on CBA  
No growth on BA  
MALDI-TOF  
Stain: small gnb,  
Drop oxidase (+)  
Prelim 
Gram-negative 
bacillus  
AST needs both genus and 
species to send susceptibilities  
Proteus spp.  
Swarming (+/-)  
MALDI-TOF  
Ornithine (+) Tube indole 
(-)  
Report Proteus mirabilis  
Ornithine (-) Tube indole 
(-)  
Report Proteus penneri  
Ornithine (-) Tube indole 
(+)  
Report 
Proteus 
hauseri/vulgaris  
Preliminary report:  
Swarming: “Gram-negative 
bacillus resembling Proteus” 
(RPRO)  
Non-swarming: 
“Gram-
negative bacillus”  
Pseudomonas aeruginosa  
“Fringy” and beta-hemolytic  
Plus a third characteristic: 
Silver 
sheen, 
obvious 
grape/corn 
chip 
odor 
or 
pigment production  
Strip oxidase (+)  
MALDI-TOF  
Pseudomonas otitidis resembles  
P. aeruginosa in morphology. 
Use caution when using odor as 
a definitive characteristic; P. 
otitidis is more stinky.  
Document 
the 
listed 
morphology in the LIS if using 
primary testing  
 
Gram-negative bacilli  
Suspected Organism Identification  
Morphology  
Primary Testing  
Secondary Testing H  
Comments  
 
“Watery” and beta-
hemolytic  
Plus a third 
characteristic: Silver 
sheen, obvious grape/corn 
chip odor, or pigment 
production  
Strip oxidase (+)  
MALDI-TOF  
If watery isolate has no 
odor or non-hemolytic and 
MALDI-TOF is 
inconclusive, refer to SPL 
for 16S rRNA gene 
sequencing to rule out 
possible Inquilinus 
limosus.  

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Document 
the 
listed 
morphology in the LIS if 
using primary testing  
Stenotrophomonas maltophilia  
Gun-metal gray colonies 
on BA  
MALDI-TOF  
Strip oxidase (-),  
TSI (K/K),  
Lysine with oil overlay (+)  
Report Stenotrophomonas 
maltophilia  
AST needs both genus and 
species 
to 
send 
susceptibilities.  
Gram-negative bacilli, not listed 
above  
 
MALDI-TOF  
 
 
 
H When no secondary testing is listed and MALDI-TOF does not provide a satisfactory identification, refer to SPL if full identification is required per the 
source specific SOP.  
 
 
S2: Table 2 
 
Staphylococci A,C  
Suspected Organism Identification  
Morphology  
Primary Testing  
Secondary Testing H  
Comments  
Micrococcus spp.  
Yellow pigmented  
MALDI-TOF  
Sterile sources:  
Stain: gpc in 
tetrads/clusters,  
Prelim 
Gram-positive 
cocci  
 
Rothia mucilaginosa  
Sticky  
MALDI-TOF  
Sterile sources:  
Stain: gpc in 
tetrads/clusters,  
Prelim 
Gram-positive 
cocci  
 
Staphylococcus aureus  
 
Typical:  
Large White/Creamy,  
Usually beta-hemolytic  
Stain: gpc in 
tetrads/clusters  
Staphaurex (+)  
OR  
MALDI-TOF  
 
 
Atypical:  
Clear grayish  
(cell wall deficient)  
MALDI-TOF  
Stain: gpc in 
tetrads/clusters,  
Staphaurex (+)  
Report 
Staphylococcus 
aureus  
 
Staphylococcus, coagulase negative  
White,  
Usually non-hemolytic  
MALDI-TOF G  
Stain: gpc in 
tetrads/clusters,  
 

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Staphaurex (-)  
Report 
Staphylococcus, 
coagulase negative  
Staphylococcus lugdunensis  
White/Creamy,  
Non-hemolytic or slightly 
beta-hemolytic  
MALDI-TOF  
Stain: gpc in 
tetrads/clusters,  
Staphaurex (- or +)  
PYR (+)  
Tube ORN w/oil (+)  
Report 
Staphylococcus 
lugdunensis  
 
Staphylococcus saprophyticus  
White,  
Non-hemolytic  
Grows on EMB  
MALDI-TOF  
Stain: gpc in 
tetrads/clusters,  
Staphaurex (- or +)  
Prelim 
Gram-positive 
cocci, 
resembling 
Staphylococcus  
 
Aerococcus spp.  
Alpha-hemolytic,  
Whitish  
MALDI-TOF  
Sterile source:  
Stain: gpc resembling 
Staph  
Prelim 
Gram-positive 
cocci  
Aerococcus spp. may be 
PYR (+) and resemble 
Enterococcus spp. but 
AST will usually be Pen 
(S).  
 
Urine:  
Stain from plate gpc 
resembling staph  
Stain from THIO broth 
resembles Staph  
Prelim 
Gram-positive 
cocci  
Gram-positive cocci, not listed above  
 
MALDI-TOF  
 
 
 
A Multiple positive cultures of GPC (except S. aureus) on all sources: if morphology is identical on multiple orders, use MALDI-TOF to identify the first two 
orders. If the same genus and species is confirmed on both orders you may use the “same by morph” rule to report additional orders. If different identifications 
are found do not “same by morph” any orders.  
C Perform MALDI-TOF or refer all vancomycin resistant Gram-positive cocci (or coccobacillus) as a possible Leuconostoc (notification by AST reporter).  
G Species level identification of isolates by MALDI-TOF is optional when susceptibilities are not being performed.  
H When no secondary testing is listed and MALDI-TOF does not provide a satisfactory identification, refer to SPL if full identification is required per the source 
specific SOP.  
 
 
 
 

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S2: Table 3 
 
 
Streptococci A, B, C  
Suspected Organism Identification  
Morphology  
Primary Testing  
Secondary Testing H  
Comments  
Abiotrophia/ Granulicatella spp.  
Alpha-hemolytic  
Growth on CBA  
No growth on BA  
MALDI-TOF  
Sterile sources:  
Stain: gpc in pairs/chains or 
pleomorphic,  
Catalase (-)  
Prelim 
Gram-positive 
cocci 
 
Enterococcus spp.  
Alpha-hemolytic  
Non-hemolytic  
Occasionally 
Beta-
hemolytic  
MALDI-TOF  
Stain: gpc in pairs/chains,  
Catalase (-)  
PYR (+)  
[If Beta: StrepPro (D+)]  
Report Enterococcus spp.  
If PYR (-) consider 
testing by MALDI-
TOF if you suspect S. 
bovis group  
Streptococcus pneumoniae  
Alpha-hemolytic  
Sunken center  
Dimpled  
Dime/checker shaped  
Stain: gpc lancet shaped in pairs, or short chains  
Catalase (-)  
Deoxycholate (+)  
Perform Optochin/Taxo P disk (Susceptible, zone >14 mm) if 
deoxycholate is questionable  
OR  
MALDI-TOF 
  
Streptococcus bovis group (SBOVI)  
Alpha-hemolytic  
Non-hemolytic  
MALDI-TOF  
 
S. 
bovis 
group 
resembles 
Enterococcus 
spp. 
morphologically but is 
PYR (-) 
Streptococcus viridans group  
Alpha hemolytic  
Non-hemolytic  
MALDI-TOF  
Stain: GPC in pairs/chains  
and  
Catalase (-), PYR(-)  
Perform MUG test when 
isolated in multiple cultures 
from bloods and any CSF 
culture. If MUG(+), refer to 
Perform deoxycholate 
if 
morphology 
questionable 
for 
S. 
pneumoniae 
from 
bloods, 
CSF, 
respiratory and isolates 

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SPL for 16S rRNA gene 
sequencing to rule out S. 
suis.  
Report 
Streptococcus 
viridans group  
from the neck and 
above.  
Streptococcus suis  
Alpha hemolytic,  
Non-hemolytic,  
May be mucoid  
MALDI-TOF  
 
 
Streptococcus, beta hemolytic, non-
groupable (SBHNG)  
Beta hemolytic  
MALDI-TOF  
Stain: gpc in pairs and 
chains,  
Catalase (-)  
StrepPro: (A), (B), (C), (D), 
(F), (G) all (=)  
Report SBHNG 
  
Streptococcus 
pyogenes 
(Streptococcus, group A) E  
Beta-hemolytic,  
Alpha-prime (mixture of 
beta 
and 
non-beta 
colonies)  
MALDI-TOF  
Stain: gpc in pairs and 
chains,  
Catalase (-),  
StrepPro (A+) and one other 
group (-)  
PYR (+)  
Report 
Streptococcus 
pyogenes  
Report 
as 
Streptococcus 
pyogenes regardless of 
testing method.  
Streptococcus agalactiae 
(Streptococcus, group B)  
 
Beta-hemolytic  
MALDI-TOF  
Stain: gpc in pairs and 
chains,  
Catalase (-),  
StrepPro (B+) and one other 
group (-)  
Report 
Streptococcus 
agalactiae  
Report as 
Streptococcus 
agalactiae regardless 
of testing method.  
 
Non-hemolytic  
MALDI-TOF  
Stain: gpc in pairs and 
chains,  
Catalase (-)  
StrepPro (B+) and one other 
group (-)  
Prelim 
Gram-positive 
cocci, 
resembling 
Streptococcus  
Streptococcus dysgalactiae  
(Streptococcus, group C)  
Beta-hemolytic  
MALDI-TOF  
Stain: gpc in pairs and 
chains,  
Catalase (-)  
 

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StrepPro (C+) and one other 
Group (-)  
Report 
Streptococcus, 
group C  
Streptococcus anginosus group  
(Streptococcus, group F)  
Beta-hemolytic  
May have buttery odor D  
MALDI-TOF  
Stain: gpc in pairs and 
chains,  
Catalase (-)  
StrepPro (F+) and one other 
group (-)  
Report 
Streptococcus, 
group F  
 
Streptococcus dysgalactiae  
(Streptococcus, group G)  
Beta-hemolytic  
MALDI-TOF  
Stain: gpc in pairs and 
chains,  
Catalase (-)  
StrepPro (G+) and one other 
group (-)  
Report 
Streptococcus, 
group G  
 
Gram-positive cocci, not listed above  
 
MALDI-TOF  
 
 
 
A Multiple positive cultures of GPC (except S. aureus) on all sources: if morphology is identical on multiple orders, use the MALDI-TOF to identify the first 
two orders. If the same genus and species is confirmed on both orders you may use the “same by morph” rule to report additional orders. If different 
identifications are found do not “same by morph” any orders.  
B Perform MALDI-TOF or refer to SPL all vancomycin resistant Gram-positive cocci (or coccobacillus) as a possible Leuconostoc (notification by AST 
reporter).  
C Perform MALDI-TOF or refer all vancomycin resistant Gram-positive cocci (or coccobacillus) as a possible Leuconostoc (notification by AST reporter).  
D Buttery odor is valid only from a blood agar plate, not a chocolate plate.  
E Streptococcus anginosus is usual microbiota in respiratory and genital/rectal sources. It can be beta-hemolytic (usually small colonies with “butterscotch” 
odor) and Strep Pro A(+). S. anginosus will be PYR (-) but S. pyogenes will be PYR (+).  
H When no secondary testing is listed and MALDI-TOF does not provide a satisfactory identification, refer to SPL if full identification is required per the source 
specific SOP.  

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S2: Table 4 
 
 
Gram-positive bacillus F  
 
Suspected Organism Identification  
Morphology  
Primary Testing  
Secondary Testing H  
Comments  
Bacillus spp.  
Beta-hemolytic  
(morph rules out  
B. anthracis)  
MALDI-TOF  
Stain : Large gpb/gvb,  
with spores +/-  
Catalase (+)  
See comments  
Refer to SPL for 
Species Identification 
if:  
• _CSF culture is 
positive and direct 
Gram stain is positive 
with Bacillus spp.  
• _Other sterile 
sources have multiple 
cultures positive with 
Bacillus spp.  
 
Large spore forming aerobic Gram-
positive bacillus, not Bacillus cereus 
or Bacillus anthracis (SAG NCA)  
Non-hemolytic  
Perform in BSC  
MALDI-TOF  
Perfom in BSC until B. 
anthracis is ruled out with 
MOT (+)  
Catalase (+), MOT (+),  
EYA (-/NG), Esculin slant 
for spores (+)  
Reincubate esculin slant and 
stain for spores, from both 
the esculin slant and the 
oldest BA plate, for up to 5 
days. 
MALDI-TOF: 
B. 
anthracis ruled out and 
MALDI does not provide a 
genus level Report Soft 
code SAG NCA  
Large Gram-positive bacillus 
resembling  
B. anthracis  
Non-hemolytic and  
Sticky, ground glass 
morphology  
Tape plates and Refer to 
SPL to rule out possible B. 
anthracis  
 
Perform in BSC  
MOT (-), EYA (+ 
Lecithinase)  
Arcanobacterium haemolyticum  
Beta-hemolytic  
MALDI-TOF  
Stain: small Gram-positive 
bacillus,  
Catalase (-),  
Prelim 
Small 
Gram-
positive bacillus  
 
Corynebacterium spp. /  
Coryneform, this category might 
include:  
Trueperella spp.,  
Brevibacterium casei,  
Rothia spp.,  
 
 
Non-hemolytic, usually 
small white, can be dry 
or yellow  
 
Sterile source:  
MALDI-TOF  
Stain: small Gram-positive 
bacillus,  
Catalase (+),  
Motility (-)  
Prelim 
Small 
Gram-
positive 
bacillus, 
Motility performed to 
rule out Listeria spp.  

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Dermabacter hominis,  
Corynebacterium otitidis,  
Curtobacterium spp.,  
Microbacterium spp.,  
Other small Gram-positive bacillus  
resembling 
Corynebacterium or small 
Gram-positive bacillus as 
appropriate  
Lower respiratory sources 
when predominant 
organism:  
MALDI-TOF to rule out 
possible 
C. 
pseudodiphtheriticum  
Stain: coryneform,  
Catalase (+),  
Urea (+),  
Refer to SPL to rule out 
possible 
C. 
pseudodiphtheriticum  
 
 
 
Urine sources when 
predominant or >105:  
MALDI-TOF to rule out 
possible Corynebacterium 
group F1, C. urealyticum, or 
C. regelii  
Stain: coryneform,  
Catalase (+),  
Urea (=) Report Urogenital 
microbiota  
Urea (+)  
Report Small Gram-
positive bacillus, 
resembling 
Corynebacterium, urease 
producer  
 
 
 
Other, non-sterile sources:  
Stain: gpb or coryneform,  
Catalase (+)  
OR  
MALDI-TOF  
 
Refer to source 
specific SOPs for 
reporting guidance.  
 
Erysipelothrix 
rhusiopathiae/tonsillarum  
Alpha-hemolytic  
MALDI-TOF  
Stain: small, slender gpb,  
Prelim Small Gram-
positive bacillus  
 
Gardnerella vaginalis  
Small 
white 
non-
hemolytic on CNA  
MALDI-TOF  
Stain: small Gram +/- 
bacillus,  
Prelim Gram-negative 
bacillus  
 
Gardnerella vaginalis  
Small 
white 
non-
hemolytic on CNA  
MALDI-TOF  
Stain: small Gram +/- 
bacillus,  
Prelim Gram-negative 
bacillus  
 
F Multiple positive cultures on all sources: if morphology is identical on multiple orders, use the MALDI-TOF to identify the first two orders. If the same 
genus and species is confirmed on both orders you may use the “same by morph” rule to report additional orders. If different identifications are found do not 
“same by morph” any orders.  
H When no secondary testing is listed and MALDI-TOF does not provide a satisfactory identification, refer to SPL if full identification is required per the source 
specific SOP. 

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S2: Table 5 
 
Miscellaneous  
Suspected 
Organism 
Identification  
Morphology  
Primary Testing  
Secondary Testing H  
Comments  
Moraxella catarrhalis  
Sticky “hockey puck”,  
Grey/white opaque coloring  
Growth on BA  
Perform in BSC:  
Stain: gnc (if performed)  
MALDI-TOF  
 
 
Neisseria gonorrhoeae  
Clear, tan colony  
No growth on BA  
Perform in BSC:  
Stain: gnc (if performed)  
MALDI-TOF  
See Comment:  
 
Refer to Neisseria 
gonorrhoeae Culture 
[011279]  
If patient is < 15 years old 
Refer to SPL for further 
testing  
 
Neisseria meningitidis  
Large, clear, tan colony  
Grows on BA  
Perform in BSC:  
Stain: gnc (if performed)  
MALDI-TOF  
 
Perform all work, 
including susceptibilities, 
in a BSC with suspect N. 
meningitidis. Notify QC 
and Supervisor of all 
isolates reported.  
Yeast  
Domed, Waxy Feelers  
Gram stain, unless “feelers” 
present  
Refer to Mycology if source 
is sterile, blood, catheter tips. 
(RMYC) per  
Referral 
of 
Fungal/Yeast 
Isolates to the Mycology Lab 
[011338]  
Yeast  
 
Filamentous Fungi  
Fuzzy  
 
Refer to Mycology from all 
sources except respiratory 
and stool. (RMYC) per  
Referral 
of 
Fungal/Yeast 
Isolates to the Mycology Lab 
[011338]  
Filamentous Fungi  
Mycobacterium, Nocardia, or 
other aerobic actinomycete 
(e.g., Gordonia, Rhodococcus, 
Streptomyces, Tsukamurella)  
Sticky  
Chalky/crusty  
Stain:  
beaded and/or branching 
Gram-positive bacillus  
or beaded filamentous Gram-
positive bacillus  
Refer 
to 
Mycology/TB 
(RTBR)  
Rapid growing 
Mycobacterium, Nocardia, 
or other aerobic 
actinomycete  
Musty odor is characteristic  

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H When no secondary testing is listed and MALDI-TOF does not provide a satisfactory identification, refer to SPL if full identification is required per the source 
specific SOP.  
 
S2: Table 6 
 
Anaerobic Organisms  
Suspected 
Organism 
Identification  
Morphology  
Primary Testing  
Secondary Testing  
Comments  
Aerotolerant non-spore 
forming anaerobes  
e.g., 
Actinomyces 
spp., 
Cutibacterium spp.  
Alpha-hemolytic,  
Non-hemolytic  
Small white  
Stain: small gpb may be 
Coryneform or branchy  
Catalase (+/-),  
aerobic growth is (-)/(wk+)  
 
Refer to anaerobe lab 
(RANA) if:  
• _ANAE(S)/ ACT is 
pending  
• Non-swab specimen from 
blood, bone, joint or central 
nervous system (CNS)  
 
Report 
as 
“non-spore 
forming 
Gram-positive 
bacillus 
not 
further 
identified”  
(NGPB-NFI)  
 
• Sterile source, ANAE/ 
ACT not ordered  
• Nonsterile source, isolated 
<48  
 
or when pure/predominant 
and reporting is preferred  
 
Report with usual 
microbiota if:  
• Isolate > 48 hours from 
nonsterile source 
 
 
 

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Obligate anaerobes  
 
Any isolate(s) from blood, 
bone, joint or central nervous 
system (CNS) culture in pure 
or mixed culture  
or  
Pure cultures from sterile 
sources 
acceptable 
for 
anaerobic culture  
Appropriate aerobic and anaerobic subcultures:  
Aerobic growth (-), Anaerobic growth (+)  
Refer to Anaerobe lab (RANA) for species level identification 
and reporting  
See specific source SOP for 
further guidelines.  
Anaerobes are reported 
under the general culture if 
a separate anaerobe culture 
is not ordered.  
C. tertium and Letptotrichia 
sp. should be treated as 
obligate 
anaerobes 
regardless of growth.  
Thio with pure culture of 
anaerobe 
from 
a 
source 
unacceptable for anaerobic 
culture (includes swabs)  
Appropriate aerobic subcultures:  
Aerobic growth on CBA (-) and BA (-),  
Report anaerobe as: Possible anaerobic organism isolated in 
broth only, not further identified (PANFI)  
Thio with mixed culture 
(more than one anaerobe, or 
mixture 
of 
aerobes 
and 
anaerobes) from source other 
than bone, joint, or CNS  
Appropriate aerobic subcultures:  
Aerobic growth (-),  
Report anaerobe(s) as: Organism in mixed culture from broth 
only, possible anaerobe present-not further identified (OPMC)  
 
 
Back-up Identification of Enteric Gram Negative Bacilli  
 
Order a work-up, quantitate, and describe the organism as usual. Order the long set media ^RTSI, ^RLYS, ^RCIT, ^RUREA, ^RORN, 
^RMOT, ^RIND, ^RMALN. If Klebsiella sp.is suspected add ^RMR and ^RVP. If Morganella sp or Providencia sp is suspected add 
^RLIA. Inoculate media, labeling the first tube in the rack with the date and a small, RLABL sticker. Incubate the media set in room air 
at 35oC. Read tubes after 16 to 24 hours of incubation.  
 
Interpret tube reactions using Table 7 of this SOP. Any organisms falling outside the descriptions in Table 1 must be referred to the 
Special Procedures Laboratory for further identification. Add the RSPL test to the workcard. 
 
 
 
S2: Table 7 
Organism 
TSI 
Lysine 
 
Citrate 
 
Urea 
 
Ornithine 
 
MotilityB 
 
Indole 
 
Malonate 
 
Comments 
 
slant 
butt 
gas 
H2S 
Shigella sp.  
K  
A  
=  
=  
=  
=  
=  
+/=  
=  
=  
=  
Must 
be 
referred 
for 
serological confirmation  

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Edwardsiella 
tarda  
K  
A  
+  
+  
+  
=  
=  
+  
+  
+  
=  
  
Escherichia 
coli  
A/K  
A  
+/=  
=  
+/=  
=  
=  
+  
+  
+  
=  
  
Salmonella 
sp.  
K  
A  
+/=  
+  
+  
+/=  
=  
+/=  
+  
=  
=  
Must 
be 
referred 
for 
serological confirmation  
Citrobacter 
freundii  
K/A  
A  
+  
+  
=  
+  
+/=  
+/=  
+  
=  
+/=  
  
Citrobacter  
A  
A  
+  
=  
=  
+  
+/=  
+  
+  
+  
+  
  
  
(diversus) 
koseri  
Klebsiella sp.  
A/K  
A  
+  
=  
+/=  
+  
+  
=  
=  
+/=  
+  
Do strip oxidase on LYS= to 
rule out Aeromonas sp.  
Add MR/VP  
Enterobacter 
cloacae  
A/K  
A  
+  
=  
=  
+  
+/=  
+  
+  
=  
+  
Do strip oxidase to rule out 
Aeromonas sp.  
Refer if organism has GMS  
Klebsiella 
(Enterobacter
) aerogenes  
A  
A  
+  
=  
+  
+  
=  
+  
+  
=  
+/=  
Do DNase if malonate = to 
rule out Yersinia sp.  
Yersinia sp.  
K/A  
A  
+/=  
=  
+/=  
+  
+/=  
+  
+  
=  
=  
Must 
be 
referred 
for 
confirmation  
Morganella 
morganii  
K  
A  
+  
=  
+/=  
=  
+  
+  
+  
+  
=  
LIA R/A  
Add LIA  
Providentia 
sp.  
K/A  
A  
+  
=  
=  
+  
+/=  
=  
+  
+  
=  
LIA R/A  
Add LIA  
Must 
be 
referred 
for 
confirmation  
A Charts in Gram-Negative Bacilli Identification [008515] may be consulted as necessary to attempt to distinguish between the species within a genus.  
B Non-motile variants occur in all motile species.  

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S3: Aerobic Antimicrobial Susceptibility testing 
 
PROCEDURE: 
 
A. Initial Set Up of ZMMLS, SUSC2, CARNP Samples  
1. Remove samples to be processed from all packaging material.  
a. Discard all packaging material in a red biohazard bag.  
b. Match up client paperwork, if available, to the respective sample.  
2. In Result Entry, scan the bar code for the sample.  
a. Verify patient information on the sample labels and available 
paperwork match.  
b. Review the following areas for any information pertaining to 
organism identification and specific testing requested.  
1)  Client provided paperwork if available.  
• Free text information relevant to testing in the 
Common media comment field. Include “Per 
paperwork” to indicate the origin of the 
information.  
2) Labels on sample. o Free text information relevant to 
testing in the   Comment media comment field. Include 
“Per slant” to indicate the origin of the information.  
3) Site field. 
4) All comment fields including Order comments, Culture 
comments and Micro Order Entry comments.  
• Copy and paste any relevant information from 
Order comment, Culture comments or Micro Order 
Entry comments into the Common Media comment 
field. 
c. Add media RLABL to the order. 
• This can be done either using the Add Media button or from the 
RBILL keypad in the workcard.  
d. In workcard view under the original media RMMLS, add the media 
code that corresponds to the type of media received from the client. 
 
e. Using the ASTWU keypad under the media received, add the 
appropriate subculture media and also add a bacterial workup. The 
subculture media ordered will depend upon the identification 
provided by the client. 
 
S3: Table 1 
 
Identification provided by client 
Subculture media to 
be used 
 
Additional comments 
 
Gram-negative bacilli (Enterobacterales, 
Nonfermenters, Pasteurella spp.) 
BA, EMB 
 
Add CBA as needed 
 

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Haemophilus 
spp. 
and 
HACEK 
organisms 
(Haemophilus 
spp., 
Aggregatibacter 
actinomycetemcomitans, 
Cardiobacterium 
hominis, 
Eikenella 
corrodens and Kingella kingae) 
 
 
 
 
CBA, BA, EMB 
 
 
N. meningitidis/Gram-negative cocci 
CBA, BA 
 
Tape all plates 
 
N. gonorrhoeae 
CBA 
 
Gram-positive bacteria 
BA 
Add CBA as needed 
Campylobacter spp. 
CBA 
Incubated in Campy jar using  
campy atmosphere. 
Helicobacter pylori 
BA 
Incubated in Campy jar with moistened 
towel using campy atmosphere. H. 
pylori media (HP media) can also be 
added. 
 
f. Under the work up media, add appropriate susceptibility testing. 
(RSENS, RDAPT, RTIG, RCANP, etc).  
• Indicate any specific antimicrobials requested or information 
that should be written on the BAMS tube at the time that 
susceptibilities are picked.  
g. Enter appropriate information into the isolate tab. Do not verify at 
this time. NOTE: This step does not have to be performed at the time 
of ordering work in the workcard. However, it does need to be 
completed before susceptibility results can be sent from BAMS to 
the MIC tab. 
• Entering from the isolate tab:  
a) Organism number.  
b) Appropriate panel (MIC, KB and/or BP tab).  
c) Organism identification,  
d) Isolate comment–all MCL isolates will have 
comment, “Organism identified by client.” added.  
e) Media ID–enter appropriate bacterial workup.  
• Entering from the workcard, under bacterial workup  
a) Using the DBWU1 keypad, select >DAST  
b) Using the DAST keypad, add ^;RSENS;&OIBC under 
the work up media  
c) Add organism identification code after the “^”, but 
before the first “;” (e.g ^PSEAER;RSENS;&OIBC)  
d) Select “Ok”  
e) Select “Yes” for the Micro Results Entry- “Do you 
want to add the isolate?”  
h. Save order and print labels.  
 
3. Prepare culture divider. 
a. Sticker the culture divider using the large RLABL sticker. 
b. Write organism identification on the divider. 

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c. Write any specific antimicrobials requested on the divider 
4. Label client sample with the small sticker of the media received. (i,e., 
RSLNT)  
5. Label subculture plates and susceptibility tube with remaining stickers.  
6. All client samples are subcultured in the Biological Safety Cabinet (BSC) 
using appropriate personal protective equipment. (Lab coat, gloves pulled 
over the cuff of the lab coat and safety eyewear.) Refer to the Biosafety Policy 
for the Bacteriology Lab [036885] for additional guidance. (e.g., International 
clients).  
7. Incubate subculture plates at appropriate atmosphere and temperature.  
8. Any sample submitted without an organism identification, should not be 
subcultured until the isolate identification is provided by the client.  
a. Submit a case ticket to MCL.  
b. Set the sample and labeled divider aside while waiting for a case 
resolution.  
9. Client samples are filed in racks on the shelves at the end of the AST bench.  
B. Initial Set Up of Samples with a Combination of ZMMLS and Molecular Lab Testing 
Related to Antimicrobial Resistance (KPNRP, OXVRP, GNRG) Aerobic 
Antimicrobial Susceptibility Testing Initial Setup and Workflow Information [PROC 
061663.004]  
1. Isolates with Molecular Lab testing will only be delivered to AST when there 
is also a ZMMLS test ordered. Samples that only have Molecular testing 
ordered will be delivered to Molecular Lab directly.  
2. Remove samples to be processed from all packaging material. a. Discard all 
packaging material in a red biohazard bag. b. Match up client paperwork, if 
available, to the respective sample.  
3. In Order Entry, scan the bar code for the sample.  
a. Verify patient information on the sample labels and available 
paperwork match.  
b. Review the order comment field for organism identification 
information.  
c. Print 3 copies of the standard label. Stickers are used for the 
following.  
1) Culture divider.  
2) BA (also write organism identification on plate).  
3) EMB.  
d. Save order. 
e. Any sample submitted without an organism identification, should not 
be subcultured until the isolate identification is provided by the 
client.  
1) Submit a case ticket to MCL.  
2) Set the sample and labeled divider in the remedy box 
while waiting for a case resolution.  
C. ZMMLS/SUSC2/CARNP: Day 2  
1. Gather all subculture plates and culture dividers set up the previous day.  

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2. Examine all subculture plates in the BSC. Plates will not be removed from 
BSC until potential highly infectious organisms can be ruled out. Refer to the 
Biosafety Policy for the Bacteriology Lab [036885] for criteria that must be 
met before plates can be removed from BSC.  
3. For cultures that have been deemed safe to remove from the BSC, open the 
workcard view of Result entry.  
a. Answer the subculture media with “Growth” or “No Growth”. If 
growth is weak it may be necessary to tape and reincubate plates. 
This should also be indicated in the workcard.  
b. Under the Bacterial workup add the following.  
1) Colony morphology  
i. Use descriptors consistent with Organism 
Identification [011332].  
2) Additional sub culture plates.  
3) Additional AST testing requested/appropriate due to 
organism identification.  
c. Date stamp RSENS media with the date susceptibility testing will be 
performed.  
d. Date stamp any additional testing and media ordered in the 
workcard.  
e. Save culture.  
i. Isolate will remain unverified and no status is applied to the 
order until the susceptibility results are ready to be reported.  
f. Culture dividers are filed on the Reporting side for resulting the next 
day.  
g. Sub plates will be saved at the end of the bench for 1 week.  
 
D. ZMMLS in Combination with Molecular Testing Related to Antimicrobial Resistance 
(KPNRP, OXVRP, GNRG)-Day 2   
1. Follow the steps outlined above in ZMMLS/SUSC2/CARNP:Day 
2. BA plates are delivered to the Molecular bench for testing. 
 
 
S4: Anaerobic cultures 
 
 
 PROCEDURE: 
 
Processing and Incubation  
 
1. Initial Processing staff will incubate inoculated THIO and TSA plate in CO0155772 at 
35°C.  
2. All other anaerobe media will be placed into a CO2 holding jar, lid up, for 2 hours or 
until the jar is  full. 

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3. Jars will be incubated in the Anaerobe Laboratory incubator at 35°C until the 
morning of day 3. 
 
24 hour Pre-examination of cultures for aerobic growth 
1. The afternoon of the day after inoculation, remove the THIOs and TSA plates from 
the incubator and separate the positive THIOs and TSA plates from those with no 
growth. 
2. If a THIO appears positive and the aerobic plate is negative, order RLABL and an 
aerotolerance subculture in SoftLabMic. Inoculate subculture mediaand incubate the 
TSA in ambient atmosphere, the CBA in CO2atmosphere and the CDC plate in 
anaerobic atmosphere. 
3. If a primary TSA plate has growth, and there are no organisms being processed under 
the GEN(S)culture, subculture the primary TSA morphology(ies) to TSA plate(s)and 
incubate in CO2atmosphere. 
 
 
48 hour Primary Examination 
1. Unpack thetwo-dayold GasPak
®jars. Check anaerobic indicators on remaining jars, 
and documenting TotalQC. 
   O Used jars will need to be bleached before being returned to circulation.  
2. Separate out ANAE(S)/ACT, GEN(S), GENB, IDENT, B, and CFRC(S) CBA plates. 
O Return IDENT and CFRC(S) plates to SPL. 
      O Return GENS thio and isolate subs to their respective benches. 
O Subculture plates from Bactec bottles (xBTAN) will be evaluated by ANAE lab 
regardless   of source; GENS body fluids, GENB tissues, and B/BBLD blood 
products. 
3. Separate positive anaerobe cultures from negative. 
O Note that if any one of the primary media(THIO, SBS, RB,PEA, LVK, or TSA) has 
growth the culture is considered positive.  
O Segregate duodenal cultures (source: SMBA) from other cultures. These will be 
processed separately. 
4. ANAE(S)/ACT cultures(other than SMBA’s)that are “No growth” are marked and 
auto-resulted from the DANAR resulting worklist by scanning a representative 
anaerobe plate from each culture and selecting the ‘Auto-result’ function.  
 
5. Refresh the DANAR worklist to eliminate all no growth cultures. 
6. Place negative plates into metal canisters and incubate on the heated side of the 
anaerobic chamber until plates are 7 days old. These will be checked Monday, 
Wednesday, and Friday’s for growth. 
7. Incubate negative THIOs in the non-CO235ºC incubator for 14 days and check daily 
for growth.  
8. SIBO cultures: 
O Process according to Quantitative Culture for Small Intestine Bacterial Overgrowth 
(SIBO)[012095]. 
9. Scan the remaining positive specimens in order to add an RLABL to the order.  

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a. Select Define MC from the right-hand menu.  
b. Click OK in the select tests window to define the media comment under test  
ANAE(S)/ACT.  
c. Type in RBILL in the select media window followed by OK.  
d. In the DBILL window select RLABL from the keypad and then OK.  
e. Select Add Results from the right-hand menu.  
f. In the upper tool bar select Refresh. This will print a card label foreach positive 
specimen. 
10. Investigate any specimens remaining on the DANAR work list and address 
accordingly. 
O Common causes of ‘missing’ cultures include. 
       1) Ordered but not acceptable (e.g. swab) with test not yet credited. 
                    2) Set up close to midnight and closed in the wrong day’s jar. 
                    3) Placed into bottles with test not yet credited. 
                    4) Ordered but accidentally not set up. 
 •When this occurs an evaluation of specimen quality and viability needs to be 
assessed prior to  determining whether the culture can be set-up. 
 
 
S5: Anaerobic Antimicrobial Susceptibility testing 
 
PROCEDURE: 
 
Preparation of Agar Dilution Plates 
  
1. Remove eight 200mL, eight 150mL bottles, and six 100mL bottles and bottles of 
Brucella agar from the walk-in refrigerator on Friday afternoon. Loosen bottle caps and 
set on counter in AST lab for Sunday evening lab personnel to melt the following 
morning. For non-routine antibiotic plates, technologists may melt agar at any time using 
the microwave oven in Antimicrobial Susceptibility Lab.  
2. Cool freshly melted agar in a 50°C water bath (at least 30 minutes).  
3. Remove seventeen 10mL aliquots of laked sheep blood from the -20°C freezer on 
Monday morning and allow to thaw at room temperature.  
4. Prepare dilutions according to the dilution schemes found in Attachment 1 and 
Attachment 2.  
 
Table 2: Antibiotics and Test Concentrations  
Drug  
Abbr  
Concentrations (mcg/mL)  
Amoxicillin-Clavulanate  
ANAMC  
4/2, 8/4  
Ampicillin-Sulbactam  
ANSAM  
0.5/0.25, 1/0.5, 2/1, 4/2, 8/4, 16/8, 32/16  
Cefotaxime  
ANCTX  
16, 32  
Ceftriaxone  
ANCRO  
16, 32  
Ciprofloxacin  
ANCIP  
1,2  
Clindamycin  
ANCC  
2, 4  
Ertapenem  
ANETP  
4, 8  

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Imipenem  
ANIPM  
0.03, 0.06, 0.12, 0.25, 0.5, 4, 8, 16  
Meropenem  
ANMEM  
4, 8  
Metronidazole  
ANMET  
2, 4, 8, 16  
Minocycline  
ANMI  
4, 8  
Moxifloxacin  
ANMX  
2, 4  
Penicillin  
ANP  
0.5,1  
Piperacillin-Tazobactam  
ANTZP  
16/4, 64/4  
Rifampin  
ANRIF  
0.03, 0.06, 0.12, 0.25  
Vancomycin  
ANVA  
0.5,1,2,4  
 
 
1. Add 5 mL of laked sheep blood per 100 mL of Brucella agar.  
2. Prepare each dilution plate by adding the indicated volume of antimicrobial. After 
thorough mixing, pour agar contents into 6 petri dishes which have been labeled with the 
antimicrobial abbreviation code, the concentration and date on the lowest concentration 
plate of any given drug. If bubbles form on the surface of the media, immediately flame 
the surface with a Bunsen burner. Place the lid ajar and allow media to solidify.  
3. Rinse out the 100 mL media bottles, ensure there are no traces of blood in the bottle or 
cap, and return to Media Lab black bins for processing.  
4. Store agar dilution Brucella blood agar plates in stacks of dilution sets at 2-8°C (anaerobe 
refrigerator) for up to 7 days. 
• Exceptions; 
a) Amoxicillin-Clavulanate plates must beused on the day 
of preparation 
b) Imipene plates must be used within 3 days of 
preparation 
 
 
 

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Table 3: Routine Antimicrobial Testing Performed Based on Organism 
Group  
The antimicrobials listed (except vancomycin) are included on the routine 
panel. Those marked with ‘X’ are routinely reported.  
Specimen source and site limitations are noted in column headings.  
Clindamycin 
(not urine or CSF) 
Ertapenem 
(not CSF) 
Metronidazole 
Moxifloxacin 
Penicillin 
Piperacillin-
Tazobactam 
MinocyclineA 
(not CSF) 
VancomycinA 
 
•  β-lactamase positive anaerobic gram-negative bacilli 
•  Bacteroides sppB  
•  Parabacteroides sppB  
•  Phocaeicola sppB  
 
X 
X 
X 
 
 
X 
 
 
 
•  β-lactamase negative anaerobic gram-negative bacilli 
•  Anaerobic cocci  
•  Non-spore forming gram positive bacilli (other than those listed below)  
 
X 
 
X 
 
XF 
 
 
 
 
•  Large spore-forming GPB (such as Clostridium, Lachnoanaerobaculum, 
Paraclostridium, Enterocloster, etc)  
 
X 
X 
X 
 
X 
X 
 
 
 
•  Cutibacterium, Propionimicrobium, Arachnia, and Propionibacterium 
speciesC  
 
 
 
 
X 
XF 
 
X 
 
 
•  Actinomyces, Schaalia, Gleimia, Pauljensenia, Buchananella, Bowdeniella, 
Winkia speciesC,D  
 
X 
 
 
 
XF 
 
 
 
 
•  Staphylococcus saccharolyticusA,E  
 
X 
 
X 
 
X 
 
 
 

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•  Clostridioides difficile recovered from feces/intestinal isolates  
 
 
 
X 
 
 
 
 
X 
 
A – There will be no interpretive categories reported 
B - β-lactamase testing not performed on Bacteroides, Parabacteroides, or Phocaeicola spp 
C – Metronidazole has little or no activity against these organisms 
D – Add isolate comment of ‘This organism routinely demonstrates resistance to metronidazole’ 
E – Add isolate comment of ‘Susceptibility testing performed anaerobically’ 
F – Ertapenem and piperacillin-tazobactam will be released on isolates that test as penicillin  resistant 
 
Patient Testing 
 
1. Assess subculture plates at 24-48 hours of incubation for sufficient growth and purity. Confirm that appropriate QC organisms and 
a sufficient number of Schaedler’s broth tubes are available. Broth must spend a minimum of 4 hours reducing in chamber prior to 
use.  
2. Order ^RANS in the workcard for each patient isolate that will be set up. Ensure that the organism is entered into the isolate tab 
with associated RANAD panel.  
3. Setup/Modify Worksheet  In BAMS, create a board utilizing the appropriate panel(s) and name it ANA (if more than one board 
will be set up, number the boards sequentially; e.g. ANA1, ANA2).  
a. See Reporting Anaerobic Antimicrobial Susceptibilities with the BAMS System [058400] for information on utilizing 
the BAMS system.  
b. See Attachment 4 for panel descriptions.  
c. In the associated worksheet, assign the isolates to positions corresponding to the order on the Seed Plate Worksheet. 
Print a copy to retain.  
4. Inoculation of plates  
NOTE: Inoculated antimicrobial plates must be closed into anaerobic jars within 30 minutes from the time the Schaedler’s broth is 
removed from the anaerobic chamber.  
 

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a. Remove antimicrobial plates as needed for the day’s susceptibilities from the 
refrigerator and allow to equilibrate to room temperature. See Table 3 for 
routine susceptibilities performed per organism group. NOTE: If testing a 
swarming Clostridium sp, an additional SB plate per anaerobe jar will be 
required.  
b. Working in the unheated side of the chamber, suspend organisms in 
Schaedler’s broth using the Grant bio Densitometer to adjust the broth to a 
turbidity of 0.5-0.6 McFarland.  
c. Attach the replicator pin holder to the support arm.  
d. Pipet India ink into well #1 of the seed plate to serve as an orientation marker. 
Fill well approximately halfway.  
e. Pipet an aliquot of each control and patient sample into its assigned well using 
a plastic transfer pipet. 1) Fill wells approximately half full. Overfilling may 
cause mixing due to splashing.  
i. 2) Do not fill wells with spreading Clostridium sp at this time.  
f. Inoculate plates in the following order: 1) SB control plate(s) (omit spreading 
Clostridium, if applicable).  
i. Special drugs, if requested but not to be reported on the swarming 
Clostridium  
ii. Fill well(s) with spreading Clostridium sp isolate(s).  
iii. Stamp second SB control plate.  
iv. Remaining susceptibility plates.  
g. Inspect each plate to verify that each position has been inoculated. If no 
inoculation has occurred, a 10 mcL calibrated loop may be used to inoculate that 
spot or the organism must be retested. Do not re-stamp plates.  
h. Allow inoculated media to stand (without inverting) to permit the moisture to 
absorb into the agar.  
i. Perform a purity check by streaking 1 mcL from each well to half of a CDC 
blood agar plate.  
j. Place plates in a GasPak jar with indicator and Anaero-Pak without tipping or 
inverting the plates. Incubate at 33-37°C for 42-48 hours.  
k. Remove the India ink from the seed block with a transfer pipet followed by a 
swab. Place block and pins into the ethanol soaking pans on the cart in AST. 
Safety eyewear is required while handling alcohol. Soak the seed plate and 
replicator pin holder in 70% ethyl alcohol for a minimum of 30 minutes. Evening 
shift will scrub seed plate and replicator pin holder with a brush and detergent, 
rinsing  
 
 
 
 
 
 
 
 
 

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REPORTING/INTERPRETING RESULTS: 
  
Reading the Plates  
 
See Reporting Anaerobic Antimicrobial Susceptibilities with the BAMS System [058400].  
1. Read plates  
a. Retrieve the worksheet from the “to read” shelf and:  
i. Update the identifications on genus-level and Gram stain only listings 
as possible.  
ii. Update any anatomic source listed as SIT or OTHER- AST results 
may not be released without a confirmed source.  
b. Examine purity plates to verify pure isolates were tested. Mixes are invalid 
and must be retested.  
c. Examine the control plate(s) for growth. 
i.  If multiple jars were utilized, compare the control plates to one 
another. If each of the control plates appears to be growing at the same 
strength the whole board may be compared to a single plate. If the 
plates appear to have different levels of growth, then the antibiotics 
plates must be compared to the control plate associated with that jar.  
ii. Failure of an organism to grow on the control plate is termed a Growth 
Control Failure (GCF). No antibiotics will be reported for that isolate. 
Re-testing may ensue depending on organism identification. See 
procedural notes for exceptions.  
d. Read the endpoint as that concentration where a marked change occurs 
in the appearance of growth as compared to control plate inoculum. The 
marked change in growth might be a change from confluent growth to a haze, 
<10 tiny colonies, or one to three normal-sized colonies. See Attachment 3 for 
pictorial reference.  
2. Confirm that all QC organisms are within range.  
3. Assess isolate antibiotic patterns to ensure that confirmatory testing is not required. 
See Table 4. 
a. Organisms which are resistant to antibiotics where the ‘Expected Result’ is 
susceptible should be evaluated prior to reporting.  
b. If it is determined that an isolate demonstrates an unusual resistance pattern, 
additional testing should be conducted to ensure that the result is clinically 
accurate. Testing may include, but is not limited to, confirmation of ID and/or 
confirmation of AST result by repeat testing.  
c. Consultation of the Mayo Antibiogram App can assist in determining if an 
organism is demonstrating an unusual level of resistance. 
http://mayoweb.mayo.edu/antibiogram/antibiogram.html  
4. Assess β-lactamase results in conjunction with the penicillin result.  
5. In rare cases an organism may be β-lactamase negative but penicillin resistant. In 
these cases, repeat the β-lactamase testing to confirm a negative result and report 
ertapenem and piperacillin-tazobactam in addition to the routine antimicrobials.  

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6. Bacteroides, Parabacteroides, and Phocaeicola spp. are expected to be β-lactamase 
positive, however rare species may not possess β-lactamase enzymes. Organisms in 
these genera that present as penicillin susceptible should be investigated. Perform a β-
lactamase test, and if the result is negative, report the β-lactamase result and penicillin 
result instead of ertapenem and piperacillin-tazobactam.  
7. Send results. 
 
Reporting Results  
1. Refer to Table 3 for specifics on which antimicrobials to report and Table 4 for their 
interpretations. The MIC and interpretation will be displayed in the final report. These 
tables also list expected results. Further antibiogram information can be obtained 
from:  
o Mayo Clinic Antimicrobial Therapy Quick Guide  
o http://mayoweb.mayo.edu/antibiogram/antibiogram.html  
2. β-lactamase negative Porphyromonas spp. are routinely reported as “Additional 
susceptibility testing not performed per laboratory criteria -  this organism is 
predictable susceptible to penicillin” in conjuction with the β-lactamase result. 
Susceptibility testing may be attempted if phoned request is received.  
3. Organisms that have been repeated for confirmatory purposes, but which fail to grow 
for additional testing should be reported as “Growth not adequate for additional 
susceptibilities.” (&BASN). If AST pattern is suspect, consultation with management, 
the Microbiology Fellows, or the Lab Director should be considered. 
4. Organisms that do not grow on the BAMS control plates should be repeated once 
more before adding an isolate comment of “Unable to perform susceptibility testing. 
Organism did not grow on test medium.” (&BUPS) 
o Do not repeat susceptibility testing on Porphyromonas spp. or slow growing 
pigmented anaerobic gram negative bacilli that fail to grow on the control media on 
the first attempt. These organisms generally do not grow rapidly enough to achieve 
readable results. Add “Unable to perform susceptibility testing. Organism did not 
grow on control media” (&BUPS) isolate comment. 
 
 
 

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Table 4: Reporting Guidelines for Anaerobic Bacteriaa  
Antibiotic 
(Soft code) 
Additional Testing / 
Notes  
Report on:  
Interpretation (μg/mL)  
Expected Results  
S  
I  
R  
Clindamycin  
(ANCC)  
• Do not report on CSF 
sources 
• Do not report on 
URINE sources 
• Morphologies other than small Gram-positive 
bacilli resembling Cutibacterium 
≤2 
4 
>4 
 
Metronidazole 
(ANMET)  
• Verify purity and/or 
aerotolerance on 
organisms that are 
metronidazole resistant 
• Confirm resistance on 
anaerobic organisms 
other than small non-
sporeforming Gram-
positive bacilli before 
releasing result 
• Morphologies other than small Gram-positive 
bacilli resembling Cutibacterium sp, 
Actinomyces sp, and related genera 
≤8 
16 
>16 
• Most obligate anaerobic 
organisms should be 
metronidazole susceptible 
• Staph. saccharolyticus 
and aerotolerant GPB such 
as Actinomyces sp, should 
be resistant 
Penicillin  
(ANP)  
 
•Morphologies other than β-lactamase positive 
gram negative bacilli 
• Do not release on organisms that are β-
lactamase positivec 
 
≤0.5 
1 
>1 
• Susceptible 
• Clostridium and 
Eggerthella sp may be 
resistant 
• Anaerobic GNC are 
commonly intermediate or 
resistant 
Ertapenem  
(ANETP)  
• Do not report on CSF 
sources 
• Repeat testing to 
confirm resistance before 
reporting 
• Freeze organism if 
resistant 
• β-lactamase positive anaerobic gram negative 
bacillic 
• Penicillin resistant organisms 
≤4 
8 
>8 
Susceptible 
Piperacillin-
Tazobactam  
 
• β-lactamase positive anaerobic gram negative 
bacillic 
≤16/4 
64/4 
>64/4 
Susceptible 

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(ANTZP)  
• Penicillin resistant organisms 
Moxifloxacin  
(ANMX)  
• Freeze organism if 
resistant 
• Cutibacterium sp 
• Propionimicrobium sp 
• Propionibacterium sp 
• Arachnia sp 
≤2 
4 
>4 
Susceptible 
Minocycline  
(ANMI)  
• Do not report on CSF 
sources 
• Freeze organism if 
resistant 
• Cutibacterium sp 
• Propionimicrobium sp 
• Propionibacterium sp 
• Arachnia sp 
No interpretive categories 
exist, report MIC onlyb 
≤4 
 
Interpretation Codes 
S: Susceptible 
I: Intermediate 
R: Resistant 
N: Non susceptible 
D: Susceptible dose dependent 
No interpretation: Add the test comment }MYST “There are no established interpretive guidelines for agents reported without 
interpretations.” 
 
Table 4: Reporting Guidelines for Anaerobic Bacteriaa 
Antibiotic  
(Soft code)  
Additional Testing / Notes  
Report  
Interpretation (μg/mL)  
Expected 
Results  
S  
I  
R  
Amoxicillin-
Clavulanate 
(ANAMC)  
 
By special request only  
≤4/2  
8/4  
>8/4  
Ampicillin-
Sulbactam  
(AMSAM)  
By special request only  
≤4/2  
16/8  
≥32/16  
Cefotaxime  
(ANCTX)  
By special request only  
≤16  
32  
>32  
Ceftriaxone  
By special request only  
≤16  
32  
>32  

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(ANCRO)  
• Organisms which are Penicillin susceptible 
and Ceftriaxone resistant should be verified 
before reporting  
Ciprofloxacin  
(ANCIP)  
• Do not report on CSF sources  
By special request only  
No interpretive 
categories 
exist, 
report 
MIC onlyb  
Imipenem  
(ANIPM)  
• Do not report on CSF sources  
By special request only  
≤4  
8  
>8  
Meropenem  
(ANMEM)  
• Repeat testing to confirm resistance before 
reporting  
• Freeze organism if resistant  
By special request only  
≤4  
8  
>8  
Rifampin  
(ANRIF)  
• C. acnes only  
By special request only  
No interpretive categories exist, report MIC 
onlyb f  
≤0.03  
Vancomycin  
(ANVA)  
• Organisms other than C. acnes & 
Clostridioides difficile  
By special request only  
No interpretive categories exist, report MIC onlyb  
ECVd  
ECVe  
• Cutibacterium acnes & Clostridioides 
difficile  
By special request only  
<2  
≥4  
a Staphylococcus saccharolyticus should have MIC’s reported without interpretive criteria.  
b Add comment “There are no interpretive guidelines for agents reported without interpretations.” Into report.  
c The criterion “β-lactamase positive” refers to organisms that have tested positive for β-lactamase, and to organisms that are presumed to be β-lactamase positive 
(eg. Bacteroides and Parabacteroides spp).  
d “This MIC is consistent with the Epidemiological Cutoff Value (ECV) observed in isolates WITHOUT acquired resistance; however, correlation with treatment 
outcome is unknown. Infectious Disease consult is suggested.” Otherwise there is no interpretation.  
e “This MIC is consistent with the Epidemiological Cutoff Value (ECV) observed in isolates WITH acquired resistance; however, correlation with treatment 
outcome is unknown. Infectious Disease consult is suggested.” Otherwise there is no interpretation.  
f “The range of MIC values of C. acnes isolates presumed to lack resistance mechanisms is <= 0.03 mcg/mL.”