Outline

• Next Generation Sequencing Overview Contemporary Cases Involving Bacteria • Clinical Cases of Emerging and Unusual Antimicrobial Resistance Patterns SCASM 2020 •Aeromonas hydrophila and Carbapenemases •Multi-drug Resistant Acinetobacter baumanii •Cefiderocol Utility and Resistance Dr. Evann Hilt, PhD, M(ASCP)CM Dr. Travis Price, PhD, M(ASCP)CM • Clinical Case involving an Outbreak Investigation • Challenging Culture-Negative Infectious Disease Cases

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Outline

• Next Generation Sequencing Overview • Clinical Cases of Emerging and Unusual Antimicrobial Resistance Patterns Next Generation Sequencing (NGS) •Aeromonas hydrophila and Carbapenemases for Clinical Microbiology •Multi-drug Resistant Acinetobacter baumanii •Cefiderocol Utility and Resistance • Clinical Case involving an Outbreak Investigation • Challenging Culture-Negative Infectious Disease Cases

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Next Generation Sequencing (NGS): What is it and how is it 1. 1. DNA Extraction different from past technology? 2. Fragmentation and Adapter 2. Ligation: randomly fragment the DNA 1st Generation = Sanger Sequencing and add adapters • Fluorescently-labeled nucleotides added to a DNA template 3. 3. Library Preparation: Use PCR to add indices to the DNA fragments • Performed on a single DNA fragment (“barcodes” to identify microbe) = • Sequence one gene from one microbe = 16S or rpoB gene sequencing Libraries 4. 4. Cluster Amplification: Libraries 2nd Generation = Next Generation Sequencing loaded onto sequencing chip and • Fluorescently-labeled nucleotides added to a DNA template amplified to form clusters • Performed on millions of DNA fragments at once 5. Sequencing: (Illumina MiSeq) Instrument takes pictures after • Sequence one gene or fragments from many microbes = “Broad-Range” PCR, Karius 5. fluorescently labelled nucleotides are • Sequence many genes from one microbe = Whole Genome Sequencing (WGS) added 6. Bioinformatic Analysis: Put the 6. fragmented sequences back together

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SCASM 407-014-20 Hilt/Price 1 1. 1. DNA Extraction Monday Next Generation Sequencing (NGS): How can we use it in the 2. Fragmentation and Adapter clinical microbiology lab? 2. Ligation: randomly fragment the DNA and add adapters Microbe Identification

Tuesday Culture Whole Genome 3. 3. Library Preparation: Use PCR to DNA Extraction Antimicrobial Resistance Prediction add indices to the DNA fragments (pure isolate) Sequencing (“barcodes” to identify microbe) = Epidemiology/Outbreak Investigation Libraries 4. Patient 4. Cluster Amplification: Libraries Wednesday Specimen loaded onto sequencing chip and amplified to form clusters Targeted Microbe Identification 5. Sequencing: (Illumina MiSeq) Amplicon 5. Instrument takes pictures after DNA Extraction Metagenomics fluorescently labelled nucleotides are added Shotgun Microbe Identification 6. Bioinformatic Analysis: Put the Friday 6. fragmented sequences back together

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Outline

• Next Generation Sequencing Overview • Clinical Cases of Emerging and Unusual Antimicrobial Resistance Patterns

•Aeromonas hydrophila and Carbapenemases Case 1 •Multi-drug Resistant Acinetobacter baumanii •Cefiderocol Utility and Resistance • Clinical Case involving an Outbreak Investigation • Challenging Culture-Negative Infectious Disease Cases

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Case 1-Clinical Presentation Case 1-Microbiology Data

Aeromonas hydrophila (9/23/19) • 63 year old female with a history of hypertension, • Aerobic/Anaerobic Culture- Antibiotic Class Antibiotic polycystic liver-kidney disease who was initially admitted MIC (MCG/mL) Interpretation • Gram-negative, catalase Piperacillin/Tazobactam >128 Resistant

to our medical center for liver transplant. Ceftriaxone >64 Resistant • The patient’s post-transplant course was complicated by Ceftazidime >32 Resistant • MALDI-TOF-Aeromonas spp. Ceftolozane/Tazobactam 8 No Inter Criteria bacteremia, cardiac arrest, and cerebral infarcts. Beta-Lactams Cefepime 4 Intermediate • Performed Broth Microdilution à Imipenem 1 Susceptible • In September 2019, the patient had a surgical drain with Meropenem 1 Susceptible milky drainage that was submitted for full microbiological Ertapenem >4 Resistant Ceftazidime/Avibactam <=2 No Inter Criteria workup • Based on these data, the clinician Gentamicin <=1 Susceptible treated the patient with Meropenem Aminoglycoside Tobramycin 4 No Inter Criteria Amikacin 16 Susceptible

Trimethoprime/Sulfonamide Trimethaprim/Sulfamethoxazole >4/80 Resistant

Colistin Colistin <=2 No Inter Criteria

Ciprofloxacin >4 Resistant Fluoroquinolone Levofloxacin 4 Intermediate

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SCASM 407-014-20 Hilt/Price 2 Aeromonas spp.-Organism and Epidemiology Case 1-Suscetibility Data

Aeromonas hydrophila (9/23/19) Aeromonas hydrophila (10/10/19) • Gram-negative facultative anaerobic, rod-shaped bacteria that morphologically • Two weeks later, the patient was B-1 B-2 Antibiotic Class Antibiotic resemble members of the family Enterobacteriaceae experiencing further drainage from a MIC (MCG/mL) Interpretation MIC (MCG/mL) Interpretation • Global distribution and isolated from a variety of aquatic environments including: surgical drain. Piperacillin/Tazobactam >128 Resistant >128 Resistant Ceftriaxone >64 Resistant >64 Resistant • Fresh water • This was submitted for culture again Ceftazidime >32 Resistant >32 Resistant Ceftolozane/Tazobactam 8 No Inter Criteria 16 No Inter Criteria • Brackish water and it grew another Beta-Lactams Cefepime 4 Intermediate >32 Resistant • Drinking water, including treated, well, and bottled Aeromonas hydrophila Imipenem 1 Susceptible >16 Resistant Meropenem 1 Susceptible >16 Resistant • Polluted waters Ertapenem >4 Resistant >4 Resistant

• Waste water effluent sludge Ceftazidime/Avibactam <=2 No Inter Criteria 4 No Inter Criteria • Contact with any fresh or brackish water body is the most common source of human Gentamicin <=1 Susceptible >16 Resistant Aminoglycoside Tobramycin 4 No Inter Criteria >16 No Inter Criteria

infection. Amikacin 16 Susceptible 32 Intermediate

Trimethaprim/Sulfametho Trimethoprime/Sulfonamide >4/80 Resistant >4/80 Resistant • Number of case studies report infection with Aeromonas hydrophila post https://commons.wikimedia.org/wiki/File:Aeromonas_hydrophila_colonies_01.jpg xazole

hemodialysis treatment (Ramsay AM et. al. JAMA. 1978; Lin SH et al. Am J of Kidney Diseases. 1996; Murata J et al. Intern Colistin Colistin <=2 No Inter Criteria >4 No Inter Criteria Med. 2006; Zhou Z et al. Infec Cont & Hospi Epi. 2013) Ciprofloxacin >4 Resistant >4 Resistant Fluoroquinolone Levofloxacin 4 Intermediate 2 Susceptible

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Next Generation Sequencing for Clinical Microbiology CphA-mediated Carbapenem Resistance in Aeromonas

Microbe Identification • CphA is a metallo-beta-lactams encoded by the cphA gene and is prevalent in Aeromonas spp. Culture Whole Genome DNA Extraction Antimicrobial Resistance Prediction (pure isolate) Sequencing • This is important since carbapenems are increasingly used as empirical therapy for serious infections • Conventional microbiology identification methods tend to fail at detecting this type of carbapenemase in Epidemiology/Outbreak Investigation Aeromonas Patient Detected cphA7 in both Specimen Aeromonas hydrophila isolates

Targeted Microbe Identification Amplicon DNA Extraction Metagenomics

Shotgun Microbe Identification

Sinclair et al. DMID. 2016

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Modified Carbapenem Inactivation Method (mCIM) and EDTA Carbapenem Inactivation Method (eCIM) Modified Hodge Test (MHT)

• Presence of carbapenemase •Positive test has been observed for • Klebsiella pneumoniae carbapenemase (KPC) • Metallo Beta lactamase (MBL) • SME-1 in Serratia marcescens

Sfeir MM. et al. JCM. 2019

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SCASM 407-014-20 Hilt/Price 3 Key Points

• Half of the A. hydrophila isolates tested falsely susceptible to imipenem and meropenem by the conventional BMD method despite clear carbapenemase production shown by strongly positive MHT and mCIM results. • These false susceptible results are very major errors (VMEs) because they mislead clinicians Case 2 to falsely believe that an ineffective antibiotic therapy is appropriate to administer. • Carbapenems are usually used empirically in treating Gram-negative bacteremia, as demonstrated in our cases. • The carbapenem treatment in our patient may have resulted in the selection of a sub-population of A. hydrophila that had an additional plasmid with antimicrobial resistance genes, or a hypothetical induction of the carbapenemase gene CphA, which led to extended resistance profile • RECOMMENDATION: Perform the mCIM and eCIM on Aeromonas isolates that report susceptible to carbapenems with conventional phenotypic broth microdilution methods

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Case 2-Clinical Presentations Case 2-Microbiology Data

• In December 2019, four patients were transferred from a local skilled • All four cultures grew the following organism: nursing facility within the same week (12/12-12/19) • Gram-negative coccobacillus •Patient 1- 73 year old female who presents with sepsis. • Catalase-positive, oxidase-negative, non-motile, and non-fermenting •Patient 2- 63 year old male who presents with low blood pressure concerning • Acinetobacter baumannii for sepsis. Patient Specimen Source Culture Result •Patient 3- 81 year old female who presents with developed worsening Patient 1 Blood Acinetobacter baumannii leukocytosis and hypothermia, with interval fever work up notable for Patient 2 Blood Acinetobacter baumannii consolidation on chest x-ray concerning for bacterial pneumonia. Patient 3 Blood Acinetobacter baumannii •Patient 4- 69 year old male who presents with anemia and septic shock. Patient 4 Tracheal Aspirate Acinetobacter baumannii

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Acinetobacter baumannii Case 2-Susceptibility Data

• Opportunistic pathogen, specifically targets moist tissues such as

Patient 1 Patient 2 Patient 3 Patient 4 mucous membranes or areas of the skin that are exposed, either Antibiotic Class Antibiotics MIC (MCG/mL) Interpretive Criteria MIC (MCG/mL) Interpretive Criteria MIC (MCG/mL) Interpretive Criteria MIC (MCG/mL) Interpretive Criteria through accident or injury (Howard A. et al. Virulance 2012) Ampicillin/Sulbactam 8 Susceptible >32 Resistant >32 Resistant >32 Resistant Cefepime >32 Resistant >32 Resistant >32 Resistant >32 Resistant Ceftazidime >32 Resistant 16 Intermediate >32 Resistant 32 Resistant • Commonly associated with aquatic environments, it has been Beta-lactam Imipenem 16 Resistant >16 Resistant >16 Resistant >16 Resistant shown to colonize the skin and isolated in high numbers from the Meropenem 8 Resistant >16 Resistant >16 Resistant >16 Resistant Piperacillin/Tazobactam >128 Resistant >128 Resistant >128 Resistant >128 Resistant (Sebeny respiratory and oropharynx secretions of infected individuals Amikacin <=4 Susceptible <=4 Susceptible >64 Resistant >64 Resistant PJ et al. Clin Infect Dis 2008) Aminoglycoside Gentamicin >16 Resistant <=4 Susceptible >16 Resistant >16 Resistant • Considered a “red-alert” organism due to increase in resistance Tobramycin 16 Resistant <=1 Susceptible >16 Resistant >16 Resistant Trimethoprim/Sulfamethoxazole Trimethoprim/ Sulfamethoxazole >4/80 Resistant <=1/20 Susceptible <=1/20 Susceptible >4/80 Resistant Ciprofloxacin >4 Resistant 1 Susceptible >4 Resistant >4 Resistant to antibiotics Quinolone Levofloxacin >8 Resistant <=0.5 1 Susceptible >8 Resistant >8 Resistant Tetracycline Minocycline 1 Susceptible <=0.5 Susceptible 8 Intermediate >16 Resistant • Presence of inducible AmpC Colistin Colistin <=2 Susceptible <=2 Susceptible <=2 Susceptible <=2 Susceptible • Possess an intrinsic class D oxacillinase belonging to the OXA-51-like group • The most common enzymatic mode of carbapenem resistance is the https://www.southampton.ac.uk/medicine/news/2018/08/acinetobacter-baumannii-study.page All four A. baumanni isolates are multi-drug resistant production of oxacillinases encoded by genes of the blaOXA-23, blaOXA-40 and blaOXA-58-like lineage.

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SCASM 407-014-20 Hilt/Price 4 Next Generation Sequencing for Clinical Microbiology Case 2-Antibiotic Resistance Prediction

Microbe Identification Patient 1 Patient 2 Patient 3 Patient 4 Antibiotic Class Resistance Resistance Resistance Resistance Culture Whole Genome Prediction Prediction Prediction Prediction DNA Extraction Antimicrobial Resistance Prediction AMR Gene AMR Gene AMR Gene AMR Gene (pure isolate) Sequencing blaADC-25 R to cephalosporin blaADC-25 R to cephalosporin blaADC-25 R to cephalosporin blaADC-25 R to cephalosporin Beta-lactam blaOXA-223 R to carbapenem blaOXA-23 R to carbapenem blaOXA-23 R to carbapenem blaOXA-23 R to carbapenem blaOXA-237 R to carbapenem blaOXA-241 R to carbapenem blaOXA-66 R to carbapenem blaOXA-66 R to carbapenem Epidemiology/Outbreak Investigation aadA2 aph(3'')-Ib aadA1 ant(2'')-Ia aph(3')-VIa aph(3'')-Ib aph(3')-Ia aph(6)-Id aph(3')-Ia Patient Aminoglycoside Not Detected armA High-level R to all aph(3')-VIa Specimen aph(6)-Id armA High-level R to all Trimethoprim/ Sulfamethoxazole sul1 Not Detected sul1 sul1 Targeted Microbe Identification Amplicon Quinolone Not Detected Not Detected Not Detected aac(6')-Ib-cr DNA Extraction Metagenomics Tetracycline Not Detected Not Detected tet(B) R to minocycline tet(B) R to minocycline

Shotgun Microbe Identification Phenicol Not Detected Not Detected Not Detected catB8 mph(E) Macrolide Not Detected Not Detected Not Detected msr(E)

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Case 2-Comparison of Phenotypic and Genotypic Data Key Points

• Three different multi-drug resistant strains are found in the four patients Patient 1 Patient 2 Patient 3 Patient 4 Antibiotic Class Antibiotics MIC (MCG/mL) Interpretive Criteria MIC (MCG/mL) Interpretive Criteria MIC (MCG/mL) Interpretive Criteria MIC (MCG/mL) Interpretive Criteria Ampicillin/Sulbactam 8 Susceptible >32 Resistant >32 Resistant >32 Resistant transferred from local skilled nursery facility Cefepime >32 Resistant >32 Resistant >32 Resistant >32 Resistant Ceftazidime >32 Resistant 16 Intermediate >32 Resistant 32 Resistant Beta-lactam Imipenem 16 Resistant >16 Resistant >16 Resistant >16 Resistant Meropenem 8 Resistant >16 Resistant >16 Resistant >16 Resistant • The three strains had different antimicrobial resistance gene profiles Piperacillin/Tazobactam >128 Resistant >128 Resistant >128 Resistant >128 Resistant Amikacin <=4 Susceptible <=4 Susceptible >64 Resistant >64 Resistant corresponding to different sensitivity patterns. Aminoglycoside Gentamicin >16 Resistant <=4 Susceptible >16 Resistant >16 Resistant Tobramycin 16 Resistant <=1 Susceptible >16 Resistant >16 Resistant Trimethoprim/Sulfamethoxazole Trimethoprim/ Sulfamethoxazole >4/80 Resistant <=1/20 Susceptible <=1/20 Susceptible >4/80 Resistant Ciprofloxacin >4 Resistant 1 Susceptible >4 Resistant >4 Resistant Quinolone • Patient 3 and 4 shared the same strain, which is most resistant and Levofloxacin >8 Resistant <=0.5 1 Susceptible >8 Resistant >8 Resistant Tetracycline Minocycline 1 Susceptible <=0.5 Susceptible 8 Intermediate >16 Resistant Colistin Colistin <=2 Susceptible <=2 Susceptible <=2 Susceptible <=2 Susceptible derived from Asia; however, there are substantial genetic difference to Agrees with Genotypic Disagrees with Genotypic rule out a direct relatedness between the two isolates.

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Clinical Presentation

• 55y female w/ hx of cryptogenic cirrhosis due to NASH, complicated by ascites, hepatic encephalopathy, and hepatorenal syndrome presented for liver transplant Case 3 evaluation • Course was complicated by multiple seizures requiring endotracheal intubation and subsequent septic shock with respiratory and blood cultures growing carbapenem-resistant E. coli harboring New Delhi Metallo-beta-lactamase (NDM) • She was put on Ceftazidime/Avibactam and Aztreonam therapy • Patient expired 3 days after positive blood cultures

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SCASM 407-014-20 Hilt/Price 5 MIC (ug/ml) (interpretation) Amikacin ≤4 (S) Carbapenem-Resistant Enterobacterales (CRE) Susceptibility Testing Aztreonam >32 (R) Cefazolin >32 (R) • Carbapenemases produced by CRE: • Modified Hodge: + Cefepime >32 (R) Ceftazidime >32 (R) • Class A (e.g., Klebsiella pneumoniae carbapenemases [KPC]) • mCIM: + (no zone) Ceftazidime-Avibactam >32 (R) • Class B (e.g., New Delhi metallo-β-lactamases [NDM]) • eCIM: + (zone) Ceftriaxone >64 (R) • Class D (e.g., OXA-type carbapenemases) • Carba-R: blaNDM Ciprofloxacin >4 (R) • Current β-lactam/β-lactamase inhibitor combination drugs are not active against Colistin ≤2 (I) Ertapenem >4 (R) Class B and have limited activity against Class D carbapenemases Gentamicin ≤1 (S) • Other potential agents (e.g., aminoglycosides, polymyxins, tigecycline) are Imipenem 16 (R) associated with toxicities and increasing resistance Levofloxacin >8 (R) Meropenem >16 (R) • Managing patients with serious infections caused by CRE presents a significant Piperacillin-Tazobactam >128 (R) challenge! Tigecycline 0.5 (S) Tobramycin 16 (R)

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MIC (ug/ml) (interpretation) Amikacin ≤4 (S) Susceptibility Testing Aztreonam >32 (R) Cefiderocol Cefazolin >32 (R) • Modified Hodge: + Cefepime >32 (R) • Novel Siderophore Cephalosporin • mCIM: + (no zone) Ceftazidime >32 (R) Ceftazidime-Avibactam >32 (R) • Siderophore: iron-chelating molecules • eCIM: + (zone) Ceftriaxone >64 (R) produced by bacteria to facilitate • Carba-R: blaNDM Ciprofloxacin >4 (R) Colistin ≤2 (I) transport of iron into the cell Cefiderocol Disk Diffusion Ertapenem >4 (R) Gentamicin ≤1 (S) Imipenem 16 (R) Levofloxacin >8 (R) Meropenem >16 (R) Piperacillin-Tazobactam >128 (R) Tigecycline 0.5 (S) Tobramycin 16 (R)

Trimethoprim-Sulfa. >4/80 (R) 33 34 33 34

Cefiderocol Cefiderocol

• Sideromycin: siderophore molecule covalently linked to a chemical moiety with antibacterial activity • 1980s • GR69153 (Cefetecol) • M-14659 (Anti-pseudomonal) • 1990s • S-9096 (Anti-GNR activity) • 2015 • S-649266 (Cefiderocol)

Sato and Yamawaki. Cefiderocol: Discovery, Chemistry, and In Vivo Profiles of a Novel Cephalosporin. CID. 2019. Zhanel et al. Cefiderocol: A Siderophore Cephalosporin with Activity Against Carbapenem-Resistant and Multidrug-Resistant Gram-Negative Bacilli. Drugs. 2019.

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SCASM 407-014-20 Hilt/Price 6 Cefiderocol Fetroja (Cefiderocol)

“Trojan Horse” Strategy

Zhanel et al. Cefiderocol: A Siderophore Cephalosporin with Activity Against Carbapenem-Resistant and Multidrug-Resistant Gram-Negative Bacilli. Drugs. 2019. www.fetroja.com

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Fetroja (Cefiderocol) Cefiderocol: Susceptibility Testing

• FDA approved within the past year for patients 18 years or older with: • Complicated urinary tract infections (cUTIs), including pyelonephritis caused by the following susceptible Gram-negative microorganisms: E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa, and E. cloacae complex. • Hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia, caused by the following susceptible Gram-negative microorganisms: A. baumannii complex, E. coli, E. cloacae complex, K. pneumoniae, P. aeruginosa, and S. marcescens. • IV only; Concentrates in the urine • Insignificantly affected by efflux pumps and porin channel mutations • Intrinsic structural stability against all three classes of Carbapenemases

Sato and Yamawaki. Cefiderocol: Discovery, Chemistry, and In Vivo Profiles of a Novel Cephalosporin. CID. 2019. https://www.fda.gov/drugs/development-resources/cefiderocol-injection Ito-Horiyama et al. Stability of Novel Siderophore Cephalosporin S-649266 Against Clinically Relevant Carbapenemases. Antimicrob Agents Chemother. 2016. Poirel et al. Stability of Cefiderocol Against Clinically Significant Broad-Spectrum Oxacillinases. Int J Antimicrob Agents. 2018. 39 40 39 40

Cefiderocol: Susceptibility Testing Cefiderocol: Susceptibility Profiles

M100, 30th Edition, 2020 Jacobs et al. ARGONAUT-I: Activity of Cefiderocol (S-649266), a Siderophore Cephalosporin, against Gram-Negative Bacteria, Including Carbapenem-Resistant Nonfermenters and Enterobacteriaceae with Defined Extended-Spectrum Beta-Lactamases and Carbapenemases. Antimicrob Agents Chemo. 2019.

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SCASM 407-014-20 Hilt/Price 7 MIC (ug/ml) (interpretation) Amikacin ≤4 (S) Cefiderocol: Susceptibility Profiles Susceptibility Testing Aztreonam >32 (R) Cefazolin >32 (R) • Modified Hodge: + Cefepime >32 (R) • mCIM: + (no zone)What is the mechanismCeftazidime of >32 (R) Ceftazidime-Avibactam >32 (R) • eCIM: + (zone) Ceftriaxone >64 (R) • Carba-R: blaNDM resistance?Ciprofloxacin >4 (R) Colistin ≤2 (I) Cefiderocol Disk Diffusion Ertapenem >4 (R) Gentamicin ≤1 (S) Imipenem 16 (R) Levofloxacin >8 (R) Meropenem >16 (R) Piperacillin-Tazobactam >128 (R) Tigecycline 0.5 (S) Kazmierczak et al. In vitro activity of cefiderocol, a siderophore cephalosporin, against a recent collection of clinically relevant carbapenem-non-susceptible Gram-negative Tobramycin 16 (R) bacilli, including serine carbapenemase- and metallo- β-lactamase-producing isolates (SIDERO-WT-2014 Study). Int J of Antimicrobial Agents. 2018.

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Cefiderocol: Mechanisms of resistance? Key Points

Porin channel • Cefiderocol is a novel siderophore cephalosporin with potent antimicrobial activity Modified outer mutations membrane and intrinsic structural stability to carbapenemases (including metallo-β-lactamases) transporters • FDA approved cUTIs, hospital-acquired and ventilator-associated bacterial pneumonia β-lactamase(s) for certain susceptible Gram-negative microorganisms • There are FDA and CLSI interpretive criteria for certain Gram-negative microorganisms

• Testing by broth microdilution requires specialized iron-depleted CAMHB Modified • Resistance to Cefiderocol is believed to be rare periplasmic Modified binding proteins penicillin-binding • The mechanism of resistance is currently unclear proteins

Zhanel et al. Cefiderocol: A Siderophore Cephalosporin with Activity Against Carbapenem-Resistant and Multidrug-Resistant Gram-Negative Bacilli. Drugs. 2019.

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Outline

• Next Generation Sequencing Overview • Clinical Cases of Emerging and Unusual Antimicrobial Resistance Patterns

•Aeromonas hydrophila and Carbapenemases Case 4 •Multi-drug Resistant Acinetobacter baumanii •Cefiderocol Utility and Resistance • Clinical Case involving an Outbreak Investigation • Challenging Culture-Negative Infectious Disease Cases

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SCASM 407-014-20 Hilt/Price 8 Clinical Presentation Clinical Presentation: Micro Results

• 71y male w/ hx of pulmonary fibrosis, respiratory failure, coronary artery disease, • Tissue specimen: • Other micro results: and tricuspid valve regurgitation s/p double lung transplant and tricuspid valve •Bacterial culture: Coagulase-neg Staph •MTB Quant Gold: Neg repair (03/18), presents (10/19) w/ 30d history of swelling and redness near the •Anaerobic culture: No growth •Galactomannan EIA: Neg left chest scar from surgery •Fungal culture: No growth •Resp PCR Panel: Neg • Social hx: lives in Reno, Nevada, no recent travel, works in factory, pet dog •Nocardia culture: No growth • Tissue collected for micro during debridement of the surgical site •AFB culture and stain: • Ziehl-Neelsen stain positive • Culture: Mycobacterium avium complex (MAC)

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Mycobacterium avium complex (MAC): Clinical Presentation Mycobacterium avium complex (MAC): Taxonomy

• MAC is the most clinically encountered group of slow-growing NTM in US • M. arosiense • M. intracellulare • Typical presentation: Pulmonary disease • M. avium • M. marseillense •Risk Factors: COPD, Bronchiectasis, CF, smoking, immunosuppressed • M. avium subsp. avium • M. paraintracellulare •Symptoms: non-specific; chronic cough, fever, hemoptysis • M. avium subsp. hominissuis • M. timonense • Disseminated infection • M. avium subsp. paratuberculosis • M. vulneris •Risk Factors: HIV/AIDS, immunosuppression/immune defects • M. avium subsp. silvaticum •Symptoms: fever, anorexia, hepatomegaly, diarrhea, abdominal pain • M. bouchedurhonense • M. youngonense • Osteomyelitis • M. chimaera • Cervical Lymphadenitis (children) • M. colombiense

Kasperbaur Resp Crit Care Med 2008 Havlik JID 1992, Benson CID, 1993

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Mycobacterium avium complex (MAC): Reservoir Mycobacterium avium complex (MAC): Lab Identification

• Environmental organisms: water, soil, plants, animals (birds, farm animals) • Slow-growing (>7 days) NTM •River water • Runyon Classification: Nonchromogen (non-pigmented) •Drinking water/plumbing • Smooth colonies •Hospital water systems •Heater-cooler units (HCUs) and aerosols

Iivanainen Appl Environ Microbiol 1993 von Reyn Lancet 1994 Falkinham Appl Environ Microbiol 2001 Lande EID 2019 53 54 53 54

SCASM 407-014-20 Hilt/Price 9 Mycobacterium avium complex (MAC): Lab Identification Mycobacterium avium complex (MAC): Lab Identification

• AccuProbe (Hologic) • Biochemical/Phenotypic Tests: •Fluorescently labeled ssDNA probe complementary to the 16S rRNA of a •Colony morphology: M. colombiense (rough) specified target organism •Nitrate reduction: M. arosiense (positive) •Hybridization is read and quantitated using a luminometer (signal above an •Growth temperature: M. chimera (growth at 25C); M. arosiense (growth at 42C) indicated threshold indicates a positive ID) • MALDI-TOF MS •VITEK MS: M. avium (no subsp.), M. intracellulare, M. chimaera • DNA Sequencing •Multilocus sequence typing (MLST) •Targeted: rpoB gene 16S rRNA gene is almost identical among MAC species à AccuProbe cannot differentiate the species

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Clinical Presentation: Micro Results 4 Cases of M. chimaera infection between 09/2018-11/2020

• Tissue specimen: • 71y male w/ hx of pulmonary fibrosis, respiratory failure, coronary artery disease, and •Bacterial culture: Coagulase-neg Staph tricuspid valve regurgitation s/p bilateral lung transplant and tricuspid valve repair •Anaerobic culture: No growth (03/18), presents w/ 30d history of swelling and redness near the left chest scar from surgery: Mycobacterium chimaera •Fungal culture: No growth •Nocardia culture: No growth • 70y male w/ hx of pulmonary fibrosis, s/p bilateral lung transplant and tricuspid •AFB culture and stain: annuloplasty (03/18), presents w/ sternal wound infection: Mycobacterium chimaera • Ziehl-Neelsen stain positive • 19y male w/ hx of idiopathic dilated cardiomyopathy s/p heart transplant (09/18) • Culture: Mycobacterium avium complex (MAC): Mycobacterium chimaera presents for post-surgical evaluation; surgical cultures positive: Mycobacterium chimaera • 24y male w/ hx of congenital aortic stenosis s/p aortic valve replacement (09/19), presents with 1 month of hemoptysis, cough, fevers, night sweats, and weight loss: Mycobacterium chimaera

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4 Cases of M. chimaera infection between 09/2018-11/2020 M. chimaera infections and Heater-Cooler Units (HCUs)

• 71y male w/ hx of pulmonary fibrosis, respiratory failure, coronary artery disease, and • First reported in 2015: 6 cardiac implant patients tricuspid valve regurgitation s/p bilateral lung transplant and tricuspid valve repair (2008-2012) that developed M. chimaera endocarditis, (03/18), presents w/ 30d history of swelling and redness near the left chest scar from graft infections, and/or bacteremia; HCUs grew the surgery: Mycobacterium chimaera outbreak strain of M. chimaera • 70y male w/ Allhx of4 pulmonarypatients fibrosis,had cardiac s/p bilateral surgery lung transplant at RRMC and tricuspid • Several cases reported worldwide since 2015 annuloplastyUCLA (03/18), between presents w/ 03/2018 sternal wound-09/2019. infection: Mycobacterium chimaera • The CDC and FDA released information on potential • 19y male w/ hx of idiopathic dilated cardiomyopathy s/p heart transplant (09/18) contamination of 3T HCUs used in cardiac bypass presents for post-surgical evaluation; surgical cultures positive: Mycobacterium procedures • Patients exposed to the bacteria can develop general chimaera and nonspecific symptoms that can often take months • 24y male w/ hx of congenital aortic stenosis s/p aortic valve replacement (09/19), to develop (night sweats, muscle aches, weight loss, presents with 1 month of hemoptysis, cough, fevers, night sweats, and weight loss: fatigue, fever)

Mycobacterium chimaera https://www.cdc.gov/hai/outbreaks/heater-cooler.html Sax CID 2015 Gotting JHI 2016 59 Svensson EID 2017 60 59 60

SCASM 407-014-20 Hilt/Price 10 M. chimaera infections and Heater-Cooler Units (HCUs) Next Generation Sequencing for clinical microbiology

Microbe Identification

Whole • First reported in 2015: 6 cardiac implant patients Culture (2008-2012) that developed M. chimaera endocarditis, DNA Extraction Genome Antimicrobial Resistance Prediction (pure isolate) Sequencing graft infections, and/or bacteremia; HCUs grew the outbreak strain of M. chimaera Epidemiology/Outbreak Investigation • Several cases reportedIf the worldwide heater since-cooler 2015 devices are the source of Patient Specimen • The CDC and FDA releasedthe M. information chimaera on potential, then all 4 patient isolates contamination of 3T HCUs used in cardiac bypass Targeted procedures should be genetically identical. Microbe Identification Amplicon • Patients exposed to the bacteria can develop general DNA Extraction Metagenomics and nonspecific symptoms that can often take months to develop (night sweats, muscle aches, weight loss, Shotgun Microbe Identification fatigue, fever)

https://www.cdc.gov/hai/outbreaks/heater-cooler.html Sax CID 2015 Gotting JHI 2016 Svensson EID 2017 61 62 61 62

Next Generation Sequencing for clinical microbiology Next Generation Sequencing for clinical microbiology

Base pair differences between M. chimaera genomes • The genome size of M. chimaera is M. chimaera genomes about 6 million base pairs M. chimaera genomes

• Using NGS, we can obtain the Patient 1 Patient 2 Patient 3 Patient 4 sequence of the entire genome Patient 1 - 2 2 2 • If the isolates are all from the same source, there should be < 15 base Patient 2 2 - 2 2 pair differences among them. Patient 3 2 2 - 2 Patient 4 2 2 2 -

approx. 6x106 base pairs long approx. 6x106 base pairs long

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Key Points Outline

• M. avium complex (MAC) is a slow-growing NTM usually associated with • Next Generation Sequencing Overview pulmonary infections, but can also cause disseminated infection, osteomyelitis, and lymphadenopathy • Clinical Cases of Emerging and Unusual Antimicrobial Resistance Patterns • The main reservoir for MAC is water sources •Aeromonas hydrophila and Carbapenemases • There are several species within MAC; speciation is difficult and requires MALDI- •Multi-drug Resistant Acinetobacter baumanii TOF MS or DNA sequencing •Cefiderocol Utility and Resistance • There is an ongoing outbreak of M. chimaera from HCUs that can be acquired during cardiac surgeries • Clinical Case involving an Outbreak Investigation • NGS can be used to aid in outbreak investigation by comparing the genomic • Challenging Culture-Negative Infectious Disease Cases relatedness of microorganisms

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SCASM 407-014-20 Hilt/Price 11 Clinical Presentation

• 43y female with vasculitis, hypertension, ESRD s/p kidney transplant (2015), left upper extremity DVT, and hypothyroidism who presents with diffuse joint swelling and pain with fevers Case 5 • Social hx: originally from Mexico, no recent travel, no pets or animal exposure, no tobacco/alcohol/drug use, sexually active but no past hx of STIs • Joint fluid and wrist biopsy were collected for micro workup

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Clinical Presentation: Micro Results Next Generation Sequencing for clinical microbiology

Microbe Identification • Joint fluid, wrist specimen: • Other micro results: Culture Whole Genome •Bacterial culture and stain: •Brucella PCR: Not Detected DNA Extraction Antimicrobial Resistance Prediction (pure isolate) Sequencing • Gram stain: No bacteria seen •Bartonella henselae IgM-neg, IgG-neg • Culture: No growth •Q-fever Antibodies-neg Epidemiology/Outbreak Investigation •Anaerobic culture: No growth •MTB Quant Gold: Neg Patient •Fungal culture: No growth Specimen •AFB culture: No growth

•MTB PCR: Not Detected Targeted Microbe Identification •GC/CT PCR: Not Detected Amplicon DNA Extraction Metagenomics

Shotgun Microbe Identification

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Next Generation Sequencing for clinical microbiology Next Generation Sequencing for clinical microbiology

• 16S rDNA: ribosomal RNA gene • 16S rDNA: ribosomal RNA gene • Identification of bacteria (not AFB) • Identification of bacteria (not AFB) • rpoB: RNA Polymerase subunit beta gene • rpoB: RNA Polymerase subunit beta gene

• Identification of bacteria (including AFB) • Identification of bacteria (including AFB) • ITS: Internal Transcribed Spacer – region • ITS: Internal Transcribed Spacer – region between 18S and 26S rDNA genes between 18S and 26S rDNA genes

• Identification of fungi • Identification of fungi

• Joint Fluid, wrist: Mycoplasma hominis

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SCASM 407-014-20 Hilt/Price 12 Mycoplasma hominis: clinical presentation Mycoplasma hominis: lab diagnosis

• Mollicute: group of small rod-shaped bacteria with no cell wall Mycoplasma Mycoplasma Ureaplasma hominis pneumoniae spp. • Typically have sterols in the cell membrane to provide structure • No cell wall à cannot visualize by Gram stain; intrinsically resistant to β- Location GU, Resp Resp GU, Resp lactams Urea - - + • Mycoplasma spp., Ureaplasma spp., plant pathogens • M. hominis Arginine + - - • Commonly associated with urogenital infections (cystitis, pyelonephritis, A8 agar, A8 Agar Culture/Media SP4 10B broth urethritis, PID) SP4+Arginine M. hominis: “Fried egg” appearance Ureaplasma: brown colony • Neonatal colonization after vaginal delivery: typically transient; sepsis, meningitis Colony Morphology “Fried egg” Spherical Pinpoint • Less common infections: pneumonia, endocarditis, septic arthritis and prosthetic joint infections Incubation Time 2-4 days 3-4 weeks 2-4 days • Typically occurs after invasive procedures and in immunocompromised patients

S.A. Hogenhout, in Encyclopedia of Microbiology (Third Edition), 2009 Waites KB, Katz B, Schelonka RL. Mycoplasmas and ureaplasmas as neonatal pathogens. Clin Microbiol Rev. 2005 Taylor-Robinson, D, Infections due to species of Mycoplasma and Ureaplasma: an update. Clin Infect Dis. 1996 73 74 73 74

Key Points

• Mollicutes are a group of bacteria with no cell wall and require specialized conditions to grow making detection difficult • M. hominis is found in the GU and respiratory tracts and can cause infections in these sites typically in immunocompromised patients Case 6

• Can be transmitted by sexual contact or during childbirth

• Dissemination beyond these sites usually occurs following invasive procedures or surgery • NGS can be used to target conserved genes from microorganisms directly from patient specimens to aid in culture-negative infections

• Will also detect commensal organisms – requires careful interpretation

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Case 6-Clinical Presentation Case 6-Microbiology Data

• 15 year old female with past medical history of double outlet right ventricle, who had a full biventricular surgical repair in 2008 • April 2020-Presented to the ER with 3 days of fever (T-max 102.9), well controlled with Tylenol and ibuprofen. Patient denies any other symptoms at this time. • On cardiology exam non-focal with stable murmur; however, labs reveal elevated inflammatory markers and echo with evidence of possible vegetation suggestive of endocarditis

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SCASM 407-014-20 Hilt/Price 13 Next Generation Sequencing for Clinical Microbiology Karius Digital Culture™ Test -NGS Test for Blood

Microbe Identification Blood-based next generation sequencing • Detect > 1,250 bacteria, DNA viruses, and eukaryotic pathogens, including yeasts, molds, and protozoa Culture Whole Genome DNA Extraction Antimicrobial Resistance Prediction (pure isolate) Sequencing • Culture-negative infection • Infection with fastidious or unculturable organisms • Identify pathogens in the setting of immunosuppression Epidemiology/Outbreak Investigation • Deep infection that requires an invasive biopsy for diagnosis Specimen Collection Patient • BD Vacutainer® Plasma Preparation Tube (PPT) – draw volume 5 mL of whole blood, fill tube completely • Separate plasma within 6 hours – Spin @ 1,100 RCF for 10 minutes at room temperature Specimen • Package the tube with separated plasma as is • Do not open the tube or transfer the plasma into a separate tube • Packaging and transport materials are supplied by Karius – send-out bench should have the kits Targeted Ordering Microbe Identification Amplicon • Karius does not accept specimens on the weekend, so if drawn late in the week, add comment to have send-out bench separate plasma and freeze DNA Extraction Metagenomics • PPT is accepted up to 92 hours at room temperature…in theory could send Friday and accept on Monday @ RT Time to Result: 1 business day- time from sample receipt Shotgun Microbe Identification

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Advantages of NGS Future of Clinical Microbiology

Microbe Identification

Culture Whole Genome • Faster turn around time for slower growing organisms DNA Extraction Antimicrobial Resistance Prediction (pure isolate) Sequencing •Results are reported before phenotypic results (saves weeks) Epidemiology/Outbreak Investigation • Safer Protocol, reduces need for BSL-3 if required Patient • Species or subspecies identification Specimen • Antibiotic resistance information Targeted Microbe Identification •A single procedure to examine multiple drug resistant genes Amplicon DNA Extraction Metagenomics •High confidence and consistency in results Shotgun Microbe Identification

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Acknowledgements

Clinical Microbiology Laboratory Bacteriology, AFB, Mycology Sections Infection Prevention Staff QUESTIONS? Omai Garner, PhD Ruel Marisol, CLS Sr. Specialist Shaun Yang, PhD Allison Tsan, CLS Sr. Specialist Suki Chandrasekaran, PhD Sierra Smith, CLS Sr. Specialist Nathan Clemons, PhD Nancy Kwan, CLS Benjamin von Bredow, PhD Victoria Anikst, CLS Edwin Kamau, PhD Ayrton Dayo, CLS Henry Hu, CLS Cinthia Flores, CLS Allison Nomura, CLS Cathrine Chong, CLS Kat Ngyuen, CLS Research & Development Section Previous UCLA CPEP Fellows Marisol Trejo Deisy Contreras, PhD Catherine Le Susan Realegeno, PhD Won Jae Seo Paige Larkin, PhD

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