When working on an anaerobic culture from an abdominal source (especially ruptured appy), how far should someone go in working up the anaerobes? There are likely to be many relatively insignificant anaerobes present and identification can take a long time. At one time we had a policy of looking for and identifying B. fragilis group and C. perfringens and all other flora went into mixed anaerobic flora. We have not had complaints from physicians but want to do right for the patient. RRR

Dealing Anaerobes from Abdomen This is a good question, and there would probably be a few different answers, depending on whom you ask. However, the extent of your work-up should be guided in part by local resistance patterns.

The most common anaerobic gram-negative rods isolated from the abdomen or intestine, including inflamed appendices, include

Bacteroides, Bilophila, Fusobacterium, Parabacteroides, Porphyromonas and Sutterella. Bacteroides fragilis is the main anaerobic organism

recovered from intra-abdominal infections and nearly all acute appendicitis cases. Many places are seeing increasing resistance in certain organisms, such as

Parabacteroides distasonis and Bacteroides thetaiotamicron of the B. fragilis group.

Other organisms which may warrant identification include Clostridium septicum (associated with gastrointestinal malignancy); C. ramosum, C.

innocuum, and C. clostridioforme (frequently resistant to antimicrobials); and C. perfringens. Other organisms to consider identifying fully include Finegoldia magna due to its pathogenicity and Peptostreptococcus anaerobius, but these organisms wouldnt be expected to be isolated very often from the intestinal

tract.

It is stated in the Anaerobic Gram-negative Rods chapter of the current 10th edition Manual of Clinical Microbiology that a definitive identification of anaerobic

isolates should be performed for all isolates from normally sterile body sites, isolates obtained from severely ill patients who are not responding to therapy, and

isolates for which prolonged treatment may be necessary.

Audrey N. Schuetz, MD, MPH, D(ABMM). Associate Director, Clinical Microbiology Laboratory. Assistant Professor, Pathology and Laboratory Medicine. Weill

Cornell Medical Center/NewYork-Presbyterian Hospital, New York, NY

2/10 Are bile stents an acceptable specimen for anaerobic culture? If not, why not? RRR

Culture of Bile Stent Bacteria colonize biliary stents, due to positioning of the stent near the duodenum and propensity for duodenal biliary reflux into the stent. Stents frequently are plugged up by biofilms which result from microbes which produce bacterial slime.

Cultures of biliary stents are not typically performed, since a positive culture likely reflects colonization of the stent. If performed, such cultures have

not typically been used to guide individual patient therapy but have been used for scientific studies of biofilms.

If performed, the technique of culturing the stent is meticulous. Various methods include scraping the intraluminal material immediately after removal (in

the endoscopy suite or in the operating room) and then placing into saline and agitating vigorously in a vortex before directly plating. Another

method involved freezing the stent at negative 20 degree Celsius, then longitudinally sectioning the stent under sterile conditions and scraping the ends of the

stent. Yet another method used was flushing the biliary stent during surgery and culturing the saline flush material. References (Molinari, Eur J Clin

Microbiol 1996;15:88 and Dowidar N, Scand J Gastroenterol, 1991;26:1137).

Most culture studies of biliary stents have grown aerobic organisms, such as those normally found in bile. A recent paper using molecular methods to

assess the biofilms of biliary stents has shown that a variety of organisms are present, including Fusobacterium spp., clostr idia, Veillonella spp., Streptococcus

angionosus, and bifidobacteria (Scheithauer, ISME Journal, 2009;3:797). However, these results were not used for guiding patient therapy.

4/10 XXX About the bactec machine, i would like to know if i can place a vial adaptor (such as those who Equshields produces and if it could get to inside otherwise .second, can i get empty vials for my study and fill them up with any kind of media i want? On the BACTEC website, there is some information on needle adapters, such as the Luer adapter, which is available for use with certain bottles. I dont know how similar it is to the adapter by Equashields Medical. As far as obtaining empty vials for your study, this would require discussion with BACTEC. It depends on the purpose of your study. If you are validating different anaerobic organisms and wish to use the BACTEC bottles for clinical purposes (according to FDA guidelines), you would need to use the broth provided in the bottles. If your study has another purpose, the company may work with you in another capacity. However, when you are validating the bottles for use, you are validating the bottles and the broth within the bottles. Another issue I see with adding your own broth after obtaining empty vials is guaranteeing the sterility of the process. It is very important that the broth be added into the bottles in a sterile manner.

5/11 Does anybody use a Flagyl disk on the primary plates of an anerobic culture? I understand this practice is widespread in Europe, but I have yet to see it used anywhere I worked in the US. A zone of inhibition is definitive for presence of anarobes, although due to increasing incidence of resistance among Bacteroides spp, absence of a zone cannot definitevley rule them out. RRR

MZ Disk on Anaerobic plate As you point out, resistance to metronidazole is increasing. Although metronidazole resistance remains rare for the B. fragilis group, it has been detected worldwide and has also been

observed spreading to other species. Additionally, there are other important anaerobes which may not test susceptible to metronidazole, including some Prevotella and many gram-positive anaerobic rods. Microaerophilic streptococci, Propionibacterium acnes and Actinomyces spp. are almost uniformly resistant. It might be a good idea for your laboratory to run an antibiogram of your anaerobes and examine resistance patterns.

Reference: Dubreuil and Odou. Anaerobic bacteria and antibiotics: What kind of unexpected resistance could I find in my laboratory

tomorrow? Anaerobe 2010;16:555-9.

6/11 How should one report sensitivity results of bacteria, e.g bacteroides, against Amoxicillin clavulanic acid and piperacillin tazobactam, if one antibiotic is sensitive and other is resistant? We usually report both resistant. Is it the right way to report? RRR

Sensi for Anaerobes Aug Vs Tzp For anaerobic suceptibility reporting, amoxicillin-clavulanate, piperacillin-tazobactam and any of the beta-lactam/beta-lactamase inhibitor combinations should be reported as tested, resistance to one does not indicate resistance to the other. In several longitudinal studies of antimicrobial susceptibility of Bacteroides spp. as well as other anaerobic gram negative organisms, piperacillin-tazobactam has better activity than amoxicillin-clavulanate. For the B. fragilis group carbapenems and piperacillin-tazobactam are still the most active agents.

A five-year multicenter study of the susceptibility of the Bacteroides fragilis group isolates to cephalosporins, cephamins, penicillins, clindamycin, and metronidazole in the United States. Diagnostic Microbiology and Infectious Disease, Volume 18, Issue

4, April 1994, Pages 235-241

Kenneth E. Aldridge, Michael Gelfand, L. Barth Reller, Leona W. Ayers, Carl L. Pierson, Fritz Schoenknecht, Richard C. Tilton, Jeanette Wilkins, Amy Henderberg, Denise D. Schiro, Marlene Johnson, Aileen Janney, Charles V. Sanders

Increasing trends in antimicrobial resistance among clinically important anaerobes and Bacteroides fragilis isolates causing nosocomial infections: emerging resistance to carbapenems.

Antimicrob Agents Chemother. 2008 Sep;52(9):3161-8. Epub 2008 Jul 14.

Liu CY, Huang YT, Liao CH, Yen LC, Lin HY, Hsueh PR.

National survey on the susceptibility of Bacteroides fragilis group: report and analysis of trends in the United States from 1997 to 2004.

Antimicrob Agents Chemother. 2007 May;51(5):1649-55. Epub 2007 Feb 5.

Snydman DR, Jacobus NV, McDermott LA, Ruthazer R, Golan Y, Goldstein EJ, Finegold SM, Harrell LJ, Hecht DW, Jenkins SG, Pierson C, Venezia R, Yu V, Rihs J, Gorbach SL.

7/11 A.actinomycetemcomitans and H.actinomycetemcomitans. Same organism? Pathogenicity?

Yes, Actinobacillus actinomycetemcomitans and Haemophilus actinomycetemcomitans are the same organism. The organism is of uncertain taxonomic status as since 16S rRNA gene sequencing placed it more closely with the the genus Haemophilus. However, the proposed name change to Haemophilus was not approved by the International Committee on Systematic Bacteriology, the orgnization that officially approves such changes. Since the two are identical, the biochemical profile and pathogenicity remain the same. (P 353 Oxford)

12/8 What QC organisms are used to QC the antibiotics for the ID scheme for Nocardia? We currently use a N. farcinica and a N. asteroides but have no zone size limits. Should we be using KB Staph aureus and E. coli?

QC for Sensi of Actino/Nocardia The QC organisms for susceptibility testing of the aerobic actinomycetes, inclusive of Nocardia, include S. aureus ATCC 29213, P. aeruginosa ATCC 27853 and E. coli ATCC 35218 (for amoxicillin-clavulanic acid). The appropriate strains should be tested weekly or each day of use if testing performed more frequently then weekly. The acceptable ranges can be found in CLSI M100.

12/9 How do you distinguish Actinomyces from Propionibacterium?

D/D Prop. acnes & Actino The majority of clinical isolates of Propionibacterium are P. acnes and about 90% of these are catalase and indole-positive. The spot indole test may be accomplished with the spot indole reagent, para-dimethylaminocinnamaldehyde. All other species of Propis and all Actinos are indole-negative. In addition, most clinical isolates of Propis are catalase-positive while most Actinos are catalase-negative. P. avidum is catalase-positive and also produces a large zone of beta-hemolysis on blood agar. While these rapid tests will suffice in most instances, definitive identification of the genera requires

identification of end products of glucose metabolism: Propionibacteria produce major amounts of propionic acid while actinomyces produce major amounts of succinic with lactic acid present in some species. Fermentation of carbohydrates, presence of preformed enzymes and other biochemical tests are used for species identification. Tables of these reactions are published in the Manual of Clinical Microbiology 9th Ed. (ASM Press) and the Wadsworth Anaerobic Bacteriology Manual, 7th Ed. 1992 (Star Publishing Co.)

12/2/1If a gram positive branching bacillus does not grow on GC selective media can it be assumed that the isolate is not a Nocardia sp. (this question in reference to p.515 MCM 8th.Ed.)?

The use of selective media (such as Thayer-Martin, Legionella Selective media and others) is very effective for isolation of Nocardia species from specimens with mixed flora and may provide some evidence that the isolate may be a nocardia species. However, lack of growth on such media should not be used to rule out the nocardia. Original trials asked the question will the selective media help in ioslation, but did not didactically evaluate the growth capability of all strains. Conceivably, some strains may fail to grow and thus selective media should never be used alone.

12/2/Finish

Antimicrobial Testing

What is the definition of CRE Carbapenem-Resistant Enterobacteriaceae organism if your lab has not implemented the new breakpoints? We use (vitek ii) the following 3

rd generation cephalosporins: cefotaxime, ceftazidime, ceftriaxone; and the following

carbapenems: doripenem, ertapenem, imipenem, meropenem. RRR

CRE The CRE surveillance definition developed by the CDC as part of their CRE Toolkit is based on the 2012 CLSI breakpoints (M100 S22): Enterobacteriaceae meet the CRE definition is they test::

Intermediate or resistant to imipenem, or meropenem, or doripenem;

AND Resistant to all tested third generation cephalosporins (e.g., ceftriaxone, cefotaxime, and ceftazidime).

Some isolates of Morganella, Serratia, Providencia, Hafnia, Proteus, or Yersinia may have different resistance mechanisms that result in elevated imipenem MICs.

Therefore, isolates are considered CRE ONLY if they meet the following criteria:

Intermediate or resistant to at least two carbapenem antibiotics (imipenem, meropenem, or doripenem); AND

Resistant to all tested third generation cephalosporins.

If older CLSI breakpoints (pre-dating M100-S20) are being used to determine carbapenem susceptibility, consideration should be given to modifying the definition

to include ertapenem in order to increase sensitivity.

Can an Enterococcus gallinarum be Vancomycin susceptible? I got from a blood culture a PYR+ preliminary identified as Enterococcus spp. The Vitek 2 showed a Low Discrimination between E. casseliflavus and E. gallinarum. The final ID using pigment production and hippurate reaction was E. gallinarum but the Vitek 2 also showed susceptible to Vancomycin with a MIC =4.Would this Vancomycin susceptibility discard the E. gallinarum as final ID? If it wouldnt, could the Vancomycin interpretation be changed to Resistant? RRR

Isolates of Enterococcus gallinarum and E. casseliflavus/E. flavescens have an inherent, low-level resistance to vancomycin (J Clin

Microbiol. 1991 October; 29(10): 23352337,http://www.cdc.gov/HAI/settings/lab/VREClinical-Laboratory.html). They carry vanC genes that typically confer

vancomycin MICs of 2 to 16 g/ml. In your case, the vancomycin MIC is in range with what has been published.

It is always recommended to confirm the results. For species differentiation, motility and pigment tests can be done to distinguish among species phenotypically. E.

faecium and E. faecalis are non-motile, whereas E. gallinarum and E. casseliflavus/E. flavescens generally are motile. Most isolates of E.

casseliflavus/E. flavescens have a distinct yellow pigment. You already confirmed pigment production and the hippurate. Furthermore, if you have

doubts about the MIC you can always confirm by an E-test.

Joan-Miquel Balada-Llasat, PharmD. Ph.D. D(ABMM)

Associate Director, Clinical Microbiology

Assistant Professor, Clinical Pathology

The Ohio State University Wexner Medical Center

joan-miquel.balada-llasat@osumc.edu

We have a Vitek 2XL we use for identification and susceptibility testing. Is there any reference that addresses when to set a purity plate for isolates that are set up on the Vitek? I was trained to always set a purity check for every isolate, even QC organisms. Is this still considered a best lab practice, or are purity plates only set when the technologist feels there could me a mixed isolate?

Purity Plate significance on VitekYes, not only is it a best practice to set up a purity check for every isolate that is being tested for identification .and susceptibility testing, it is also a CAP requirement: MIC.21820 Pure Culture- Susceptibility Testing Phase II. Only single isolates or pure cultures are only used for final performance of antimicrobial susceptibility (i.e. no mixed susceptibilities). The evidence of compliance includes written procedure describing the use of pure isolates for susceptibility testing, including the use of purity plate verification. It is essential to have proof that the inoculum used for testing was pure, as if it were mixed, the test cannot be reported and must be repeated. Without continuing to perform purity checks on every isolate every time, you simply wouldnt be able to ensure that the test result is accurate. RRR

___________________________________________________________________________________________________________

4/290 If any one of the carbapenems is resistant should all the carbapenems be reported as resistant. For example, if ertapenem is 2 mcg/ml and meropenem is

(Ciprofloxacin S and Levofloxacin I) by the method used in the lab and by an alternative method (i.e. e-test) to confirm the results, just to be sure that is not

assay dependent.

7/290 We get frequent questions from our physicians and pharmDs regarding the relationship between cephalosporins, aztreonam, and piperacillin/tazobactam. Generally, the questions revolve around isolates that are resistant to the cephs and aztreonam but susceptible to pip/taz or vice versa. They believe that pip/taz should agree with these other drugs. What am I missing?

Piperacillin/tazobactam (P/T) and amoxicillin/clavulanate are stable against ESBLs, because the beta lactamases are inhibited by tazobactam and clavulanate (manifested in the double disc diffusion test where the third generation zone of inhibition is increased by clavulanate). Based on the MIC it should be OK to used them on ESBL-producing organisms, however why do we avoid the

use of P/T to treat infections caused by ESBL-producing organisms even when the MICs indicate susceptibility? (Can We Really Use -Lactam/-Lactam Inhibitor

Combinations for the Treatment of Infections Caused by Extended-Spectrum -Lactamase? Producing Bacteria? Clin. Inf. Dis. Vol54, issue 2). Some of the

reasons are:

1. Increase of P/T MICs when bacterial inocula reach 107 cfu/mL.

2. Presence of other mechanisms of -lactam resistance (SPICEM organisms) such as AmpC enzymes (eg, CMY-2, FOX-5, ACT-1), hyperproduction and specific

mutations of non-ESBL enzymes (eg, SHV or TEM), or additional ESBLs, may provide reduce the activity of P/T.

3. Pharmacokinetic/pharmacodynamic studies indicate that normal doses of P/T do not achieve therapeutic concentrations and this is associated with

unsatisfactory outcomes.

4. The use of P/T TzP for the treatment of infections caused by ESBL-producing organisms is limited and controversial (Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev 2005;18:657-86). More clinical relevant data is needed, especially to treat life

threatening infections.

COMMENT 1 I just reread my question and realized I left out a crucial part of it! The isolates we are questioned on are not ESBL producers. Often they are the SPACE bugs so AmpC is likely the issue. P/T is often Susceptible and we report it as such but it seems to cause concern.

Comment 2 Regarding your question; while organisms that produce inducible AmpC enzymes may be ineffectively treated by P/T, there is little clinical data to

verify this if isolates test susceptible in vitro. Kaye et al. found that P/Z use was not associated with the emergence of cephalosporin resistance in the treatment of

Enterobacter bacteremia (K.S. Kaye, S. Cosgrove, A. Harris, G.M. Eliopoulos, Y. Carmeli, Antimicrob Agents Chemother, 45 (2001), pp. 26282630). In another

study analyzing 377 episodes of Enterobacter bacteremia in adults, the only factor independently associated with a reduction in 30-day mortality was the early use

of P/Z (M. Marcos, A. Inurrieta, A. Soriano, J.A. Martinez, M. Almela, F. Marco et al. J Antimicrob Chemother, 62 (2008), pp. 397403). In summary, there is not

enough clinical evidence to avoid P/T use for susceptible SPICE organisms. The risk of clinical failure due to emergent resistance is in general small

8/209 * If you report 3rd and 4th generation Cephalosporins rutinary for Gram negative rods, is it necessary still to report Cefazolin? If yes, why is the reason of this

Reporting Cefazolin You should always include the report for Cefazolin (First Generation Cephalo). If the GNR strain is susceptible to 3rd and 4th generation Cephalosporins, you cannot predict that it will also be susceptible to Cefazolin., in fact it could be resistant. However, if it is resistant

to 3rd and 4th generation Cephalosporins, we can conclude that it will also be resistant to Cefazolin.

As well as suppressing penicillins, cephalosporins and aztreonam for ESBL positive organisms, should ampicillin/sulbactam or piperacillin/tazobactam be suppressed also?

If your laboratory has not yet adopted the current (2010) breakpoints for cephalosporin and aztreonam for the Enterobacteriaceae, then you should report all

ESBL-confirmed E. coli, K. pneumoniae and P. mirabilis as resistant to penicillins, cephalosporins and aztreonam, regardless of how they test to the individual

agents. This rule does not however apply to the beta-lactam/ beta-lactamase inhibitor combinations, which may be reported as they test. I realize this is confusing

to many people, and there are some who fee that beta-lactam / beta-lactamase inhibitor combinations should not be used to treat ESBL infections. So, I would

discuss this with your infectious diseases physicians, pharmacy and other key players before making a decision on how to report.

Note that if you are able to verify the current breakpoints for the cephalosporins and aztreonam, you can avoid this situation, as you may report all

beta-lactams as they test, without worrying about ESBL presence.

290 / 2/1 Are all resistance mechanism inducible? If not, can you recommend a good reference for determining which are and which arent?

Inducible Resistance Some mechanisms of resistance are inducible, while others are constitutive. An example of inducible resistance is inducible clindamycin resistance conferred by the erm gene; this is what you are looking for when you do the D-test. The Manual of Clinical Microbiology is a good resource for reviewing mechanisms of resistance.

290 /2/2 Can MICs for parenteral cefuroxime be used to interpret oral cefuroxime as S,I,or R?

MIC of Cefuroxime MICs are based on PK-PD properties. Depending on the route of administration the MIC used for susceptible/intermediate and resistance may change. Based on the CLSI M100-S22 for Enterobacteriacea and Staphylococcus spp., cefuroxime breakpoints interpretations are as follows:

S I R (g/ml)

Parental 8 16 32 Based on a dosage regimen of 1.5 g every 8 H

Oral 4 8-16 32

Joan-Miquel Balada-Llasat, PharmD. Ph.D. D(ABMM)

Associate Director, Clinical Microbiology

Assistant Professor, Clinical Pathology

The Ohio State University Wexner Medical Center

Phone: (614) 257-2785

joan-miquel.balada-llasat@osumc.edu

Comment 1 So, I could call an E.coli with an MIC of 8 susceptible to parenteral cefuroxime and intermediate to oral cefuroxime regardless of whether we tested

cefuroxime axetil or cefuroxime sodium?

Comment 2/answer Cefuroxime sodium is the drug tested in vitro, I would use these MIC interpretations for oral and parenteral dosage. The acetoxyethyl ester

prodrug of cefuroxime (effective orally), has a modification to allow absorption, its activity depends on in vivo hydrolysis and release of cefuroxime to the

bloodstream.

A linezolid resistant and mecA positive coagulase negative staphylococcus was isolated from a cellulitis patient who underwent linezolid therapy. The isolate is resistant to chloramphenicol, rifampicin, cotrimaxazole, gentamycin etc. Moreover this is showing an unusual susceptibility pattern:Erythromycin- sensitive, Clindamycin- resistant, Streptogramin (Quinupristin-Dalfopristin) sensitive. Could this be due to drug inactivation by lnu A / lnu B? Can we rule out the possibility of rRNA methylase / efflux pumps for this rare pattern? Can there be any other mechanisms for this pattern?

Linezolid Resistance in CONS A difficult question As you point out, there are a number of mechanisms that could account for an Erythromycin-S, Clindamycin-R Staphylococcus including both inactivation (lnu genes) and efflux (vga, lsa genes). However, I am most concerned with the first

result that you listed: Linezolid resistance.

The incidence of Linezolid resistance (LR) in Coagulase-negative Staphylococci is 28-times that of S. aureus (LRSA) {Gu B et al. J Antimicrob

Chemother, 2012}. Resistance to Linezolid can result from mutations occurring in the 23S rRNA binding sites, as well as in the peptide translocation center of the

ribosome. Some of these mutations can also confer resistance to Clindamycin. However, most concerning is resistance due to acquisition of a plasmid-borne

ribosomal methyltransferase known ascfr (chloramphenicol florfenicol resistance), the product of which adds a methyl group at the C-8 position of the 23S rRNA

nucleotide A2503. This gene was originally detected on a multi-resistance plasmid from a bovine strain of S. sciuri, but has also subsequently mobilized to

human Staphylococci (including S. aureus). The presence of cfr in Staphylococcus species is associated with a low fitness cost and outbreaks due to clonal

spread of cfr-containing strains have been described (Bonilla H et al, CID, 2012; Morales G et al. CID 2012).

In terms of the susceptibility results that you described, cfr also confers resistance to Clindamycin, but not to Erythromycin (although some plasmids

harboring cfr also habor erm and other resistance genes). Given the multidrug resistant pattern you described for this isolate, I would be most concerned about

a cfr mechanism. However, confirming the presence of this gene would require PCR that most likely would have to be performed in a specialty laboratory.

290/2/4 I have found in E. faecium and E. faecalis in the Vitek 2 with Vancomycin resistent pattern and Ampicillin susceptible confirmed by KB. I know the resistance mechanisms are different between these drugs, but is it a normal or common pattern in those organisms? must be necessary confirm those results from the Vitek 2 with a KB?

Amp/Vanc Resistance in Enteroc As you mentioned the mechanisms are different, however there is a high correlation with vancomycin and ampicillin resistance in Enterococcus faecium strains. This is based on the fact that these strains are nosocomialy transmitted. E.

faecium strains are often resistant to ampicillin or vancomycin or both. In our institution most of the E. faecium strains are ampicillin resistant, if

they test susceptible to ampicillin we confirm the result by checking the purity plate and then we repeat the susceptibility test.

Is there any reason not to report Nitrofurantoin on ESBL organisms?

Nitrofurantoin has a broad spectrum of activity against a variety of Gram positive and Gram negative bacteria, such as Staphylococci and E. coli. However, Pseudomonas spp. and Proteus spp. are resistant to this antimicrobial. The mechanism of

action of nitrofurantoin has not been fully described. Strains of bacteria that are resistant to nitrofurantoin have been shown to possess

reduced nitroreductase activity. Nitrofurantoin is a synthetic antibacterial agent that is indicated for use for treatment of uncomplicated

cystitis. Therefore, regardless of an organisms ESBL status, this antimicrobial should only be reported on organisms isolated from

urine specimens. Nitrofurantoin is not a beta-lactam antibiotic and ESBL enzymes have no activity against

nitrofurantoin. The ESBL status of an organism is irrelevant to reporting of nitrofurantoin.

291/2/6 We use the Vitek 2 for AST and often get results for E. coli where cefazolin is S and Cefixime is I/R. Is this an acceptable result? At the moment we check the cefazolin by KB disk diffusion and it often is S. In this case we are not reporting the cefixime as the first generation is S. How are others handling this?

CZ Sensitive but CFM Resistant The fact that you are observing higher MICs for Cefixime (third generation cephalosporin) than for Cefazolin (first generation cephalosporin) is unusual. I would question the Vitek result. I suggest to test Cefixime by a different susceptibility method and also check the susceptibilities to other third generation cephalosporins such as

Ceftriaxone or Ceftazidime.

291/2/7 Would you report snesi results of Coamoxiclav (augmentin) and ticra/tazobactam (tazocin) for an ESBL producer? In certain practices only tazocin is reported but not augmentin . What is the basis for that? startt

Tzp - for ESBL producers Based on the M100-S22 CLSI document, for all confirmed ESBL-producing strains, for the labs that do not use current cephalosporin and aztreonam interpretative criteria, the test interpretation should be reported as resistant for all penicillins, cephalosporins, and

aztreonam. If current cephalosporin and aztreonam interpretative criteria are used, they should be reported as tested. Furthermore, it recommends reporting

ESBL-producing isolates as susceptible to -lactam/-lactamase inhibitor combination antimicrobials, like piperacillin-tazobactam, when they test as such in the

laboratory. However, reports of clinical failures have led to recommend a carbapenem antimicrobial for all ESBL infections (J. Clin. Microbiol. 39:2206-2212; Drugs

63:353-365).

Gavin et al (Antimicrob Agents Chemother. 2006 June; 50(6): 22442247) assessed infections caused by extended-spectrum--lactamase-producing Escherichia

coli or Klebsiella spp. treated with piperacillin-tazobactam to determine if the susceptibility breakpoint predicts outcome. Treatment was successful in 10 of 11

nonurinary infections from susceptible strains and in 2 of 6 infections with MICs of >16/4 g/ml. All six urinary infections responded to treatment regardless of

susceptibility.

In conclusion, while urinary tract infections caused by piperacillin-tazobactam-susceptible ESBL producers may be successfully

treated, it has been some failure for other more serious infections, arguing that the breakpoints should be reviewed.

291/2/8 If I am using the new 2012 CLSI breakpoints for Cephalosporins and I dont have to confirm the ESBL tested strains with KB, do I have to report the MIC or area disk tested without to change the interpretation for Cephalosporins in ESBL isolates? also, using those new breakpoints, any Enterobacteriaceae tested as Resistent or Intermediate to 3rd generation of Cephalosporins (Enterobacter, Serratia, Morganella, etc) can be reported as ESBL?

Cephalosporins and ESBL The new CLSI guidelines focus on the minimum inhibitory concentration for various bug-drug combinations rather than detection of mechanisms of resistance. The new breakpoints are more sensitive for detection of cephalosporin resistance in Enterobacteriaceae. However, although laboratories are not required to confirm the

presence of ESBLs, they may still do so if they wish. The new guidelines state: If the current cephalosporin, aztreonam, and

carbapenem breakpoints are used, ESBL and/or Modified Hodge testing are not required. However, these tests may be used to confirm the presence of a resistance mechanism of epidemiologic significance.

Im not sure I understand the second part of your question, but I or R to cepahlosporins in the Genera you list (Enterobacter, Serratia,

etc.) has no specific correlation to ESBL. These organisms have chromosomal, inducible AmpC. Cephalosporin resistance in these

isolates may be due to a variety of mechanisms, including AmpC, ESBL, or even carbapenemase, depending on the rest of the

resistance profile. 3rd generation cephalosporin resistance in these organisms does not rule in or rule out the presence of an ESBL.

291/2/9 How is it that Enterobacter, Serratia, Citrobacter and other SPICE group organisms, being resistant to 3rd generation of Cephalosporins, should be called ESBL? I have found few strains of Enterobacter with a suggestive pattern of AmpC producer, but since the CLSI does not have protocols or procedures to confirm ESBL or AmpC phenotypes on these genera, how should they be reported? In the same way, should an E. coli strain shown an AmpC pattern and a negative confirmatory KB double-disk diffusion test (less than 5mm and all of them resistant) be called ESBL

ESBL & 3rd Gen Cephalo In order to detect the presence of either or both of the AmpC and ESBL enzymes, the CLSI published revised interpretive guidelines in 2010. In your email, you raise the example of SPACE-MP organisms having de-repressed AmpCs and an E. coli with a pattern of resistance suggestive of the presence of an AmpC beta-

lactamase (and not surprisingly, a negative ESBL test). It is these very issues that speak to the rationale behind the revision of the breakpoints which are focused not on the detection of specific resistance mechanisms themselves, but rather, on the minimum inhibitory concentration (MIC) of the organism to a given agent. Indeed, Kohner et al (J Clin Microbiol. 2009. 47(8): 24192425) demonstrated that the majority of strains tested harboring an ESBL, pAmpC or both and ESBL &

pAmpC had MICs to 3rd generation cephalosporins that were in the non-susceptible range using the revised break-points (but in the

susceptible range with the older breakpoints).

So the answer to your question depends on whether or not you are using the revised breakpoints. Using the revised breakpoints, you

would just report the antibiotic results as is with no further need for ESBL testing. However, if you are using the breakpoints from 2009

or before, ESBL testing should continue to be performed with the appropriate editing of the results for cephalosporins, aztreonam or

penicillins to resistant when an ESBL is detected.

But in the E. coli case you describe, you would not be able to detect the presence of an ESBL using the CLSI procedure even

if this isolate were an ESBL producer. And therein lies one of the advantages of the revised breakpoints

292/2/10 Is is possible for an enteric gram negative rod to have an ESBL AND a carbapenemase? How can I easily define ESBL and carbapenemase for non-microbiology staff?

Difference between ESBL & -lactamases It is possible for Enterobacteriaceae organisms to have an ESBL and a carbapenemase, it is well known that these organisms harbor other mechanisms of -lactam resistance such as porin

mutations and encode other -lactamases including ESBLs and AmpCs.

It is important to take into consideration that KPC -lactamases are inhibited by clavulanic acid, if using the disc

diffusion assay for ESBL detection the carbapenemase producers organisms may be positive.

In a simplistic way I would define ESBL organisms as those organisms that are resistant to all betalactams antibiotics but

carbapemems;

Carbapenamese producing organisms to be resistant to all betalactams (including penicillins, cephalosporins,

monobactam and carbapemens).

292/3/2 Should a P. aeruginosa only non-susceptible to Carbapenems and susceptible to all others antimicrobial classes (including Ceftazidime and Cefepime) be called MDRO? I have gotten few strains being only Intermediate or Resistante to Imipenem or Meropenem in Vitek II and confirmed by KB using the CLSI 2012 and still they are called MDRO.

Pseudo as MDR There are many different definitions for multi-drug resistant organism (MDRO). I assume that the MDRO label was applied to this organism by the Vitek2 software? Regardless, the literal interpretation for MDRO is for an organism to

be resistant to more than one antimicrobial agent, but generally this term is applied to bacteria that are resistant to three or more

antimicrobial classes. However, in some cases, bacteria are labeled MDR based on susceptibility to one key antimicrobial agent often

associated with co-resistance to other agents, such as here, with carbapenem resistance in Pseudomonas aeruginosa. Recently, there

was a proposal for international definitions of MDR among various bacteria. I refer you to this article.Nasrullah U have this article..: Magiorakos et

al. 2012 Clin Microb Infect 18:268. Per this groups definition, MDR in P. aeruginosa is non-susceptible to >=1 agent in >=3

antimicrobial categories. The categories included are: aminoglycosides (gentamicin, tobramycin, amikacin), the antipseudomonal carbapenems (imipenem, meropenem, doripenem), antipseudomonal cephalosporins (ceftazidime, cefepime), antipseudomonal fluoroquinolones (ciprofloxacin, levofloxacin), antipseudomonal penicillins + beta-lactamase inhibitors (ticarcillin-clavulanate, piperacillin-tazobactam), monobactams (aztreonam), fosfomycin, and the polymyxins. Based on this definition, your isolate would not qualify as MDR.

One thing to consider is which carbapenems you are testing in this case is the isolate in question resistant to imipenem AND

meropenem? Or are you only testing imipenem? In P. aeruginosa, loss of the OprD outer membrane porin, in combination with this

organisms chromasomal AmpC, leads to imipenem resistance, whereas meropenem (and cefepime and ceftazidime) are less

dependent on OprD for cell entry. So, if the MIC to imipenem is elevated (you say non-susceptible, I assume this is R?) but

meropenem is susceptible, this might be a loss/downregulation of OprD.

292/3/3 If we are testing a Pseudomonas aeruginosa against Pip/Tazo Etest. We generally use QC E. coli 35218. Should we also be doing QC on Pseudo 27853 and ATCC E. coli 25922 or is 35218 sufficient?

QC Strain - for Pseudo Sensi The E. coli ATCC 35218 is the QC strain recommended and sufficient when testing -lactam/ -lactamase inhibitors against P. aeruginosa, it is not required as routine user QC testing.

292/3/4 I got in the Vitek II a possible K. pneumoniae ESBL alert, when I confirmed it by diffusion disk KB using Ceftazidime and Ceftriaxone with Clavulanic Acid, the areas were less than 5mm and all the disks tested resistant. I setup a new KB using Cefepime, Amp/Sulb, Aztreonam and TZP and only the Cefepime disk was susceptible. I read that one of the difference between AmpC and inhibitor-resistant beta-lactamase was the resistance to Tazobactam. Could it be an AmpC producer strain because its resistance to TZP? Is it possible to use the IMP-CAZ or FOX-TZP disk method to induce the production of AmpC when it is suspect even without CLSI guidelines about it?

AmpC beta-lactamase The resistance pattern you describe certainly is suspicious for an AmpC beta-lactamase (resistant to 3rd generation cephalosporins, Aztreonam & Pipericillin-Tazobactam, but susceptible to Cefepime). Because Klebsiella pneumoniae does not harbor a chromosomal ampC gene (Bergstrom S et al. J Bacteriol. 1993. 155:1297-1305), a plasmid-ampC (pAmpC) is more likely. Plasmid-borne ampCgenes are constitutively expressed, and

thus no induction of expression would be observed with the Fox-Tzp or Imi-Caz disks. This is in contrast to organisms

like Enterobacter species, Citrobacter species, etc. where a blunting of the Tzp and Caz zones would be expected if an inducible ampC were present.

It is possible that the isolate could also harbor an ESBL despite the Cefepime result obtained: In a study by Kohner et al (J Clin Micro.

2009. 47(8): 24192425), approximately 50% of E. coli andKlebsiella isolates harboring both an ESBL and pAmpC had Cefepime MICs

in the susceptible range (MIC8). Although a number of inhibitors (e.g. boronic acid, cloxacillin, etc.) have been published to enable

detection of ESBLs in the presence of an expressed AmpC beta-lactamase, no CLSI-approved methods have been published.

Furthermore, the CLSI currently recommends that ESBL testing only be performed for infection control purposes where necessary

when the current Enterobacteriaceae breakpoints are used. Regardless of whether an ESBL is present in this particular isolate or

whether the presence of a pAmpC were confirmed, this isolate would certainly be considered multi-drug resistant and contact

precautions for the patient may be warranted depending on your institutions infection control policies.

292/3/5 (It is a difficult question) We have been followed and documenting few Serratia marcescens strains with a discrepant pattern on Carbapenems and Cehalosporins. I have asked this before about the possible explanation about having Imipenem I or R and Meropenem and the rest of 3rd and 4th generation of Cephalosporins S. The gold standart to determine the presence of SME is a KPC PCR but we dont count with possibility to make a KPC PCR to each S. marcescens and we are looking for how deal with these strains about if to call it CRE and to change all the Cephalosporins to resistant in these cases. We are looking for to test some of these strains to determine the presence of SME and documenting their pattern through Vitek II and KB, but How could this case be directed? is it possible to use the phenotypical pattern of the SME positive strains to create a protocol for upcoming report of these strains? if those strains are SME positive, should they be called CRE and change the susceptibility pattern of all the Carbapenems and Cephalosporins?

SME Vs CRE (It is a difficult question). This is in fact a very complicated issue (as I am sure you are well aware!) I would like to re-iterate what Dr. Buchan mentioned before, in that the phenotype you are observing (eg, I or R to carbapenems

but S to 3rd and 4th generation cephalosporins) may be caused by multiple mechanisms, including carbapenemase such as SME,

chromosomally encoded AmpC enzymes, porin mutations, or a combination of these. Unfortunatly, there is no good phenotypic test to

confirm SME vs. KPC vs. AmpC/porin loss or other mechanisms. Some suggest looking at the relative in vitro MICs to imipenem

(usually high) versus ertapenem (usually low) as an indication SME might be at play (Carrer et al. 2008 International J Antimicrobial

Agenets 31:181-2). However, the only real way to determine this would be to perform a SME-specific PCR. Performing a PCR for

blaKPC will not help you resolve the question as to whether these isolates harbor SME, but rather tell you only if the isolates in question

harbor a KPC gene: SME is a different carbapenemase altogether.

I can tell you that SME is relatively rare (see for example, Castanheira, M. et al. 2008. Antimicrob. Agents Chemother. 52:570-573) and

so I would be surprised if you have multiple isolates that harbor the gene coming through your lab. As an example, at UCLA we

encountered a number of S. marcescens with the unusual carbapenem-R, cephalosporin-S phenotype between 2010-2011. When

investigated, none of 18 strains harbored SME genes as determined by a SME-PCR specific PCR. The relative infrequency of SME

may relate to a significant fitness cost associated with acquiring the SME-1 signal sequence (Marciano et al. 2007 Genetics 176:4:

2381-92). Our experience from the past indicates that when multiple isoaltes with SME are encountered, this may be related to

nosocomial spread. Five S. marcescens isolates that produced SME-2 were isolated at UCLA in 1992, and pulsed-field gel

electrophoresis analysis revealed that all of the isolates were the same strain ( Queenan, A.M. et al. 2000. Antimicrob. Agents

Chemother. 44:3035-3039).

So the question remains on how to deal with these isolates. I would suggest this is a discussion to be had with key stakeholders at your

institution, such as Infection Control, Infectious Diseases specialists and Infectious Diseases Pharmacists. While these isolates test

susceptible to the cephalosporins in vitro, there is no evidence that these drugs will work in vivo. No studies have addressed this issue

(probably because such isolate are so uncommon), although case reports suggest cefepime may be an option (Fairfax et al. 2011

Diagnostic Microbol and Infecti Dis 71:3: 325-6). Regardless, from my perspective the most conservative thing to do would be to edit

the cephalosporins to resistant in these cases. You might consider sending some of the isolates to a reference lab to additional testing

(such as SME PCR), as you suggested. Finally from an infection control perspective, I would suggest using the current CLSI

breakpoints to define carbapenem resistance, and call the isolates with carbapenem resistance CRE.

292/3/6 Why are there no CLSI breakpoints for viridans strep and trimeth/sulfa?

Why cot NOT used for Strep viridians The clinical laboratory in consultation with infectious disease, pharmacy as well as the pharmacy and therapeutics and infection control committees should select the most appropriate antimicrobial

to test and to report and are ultimely responsible for the selection of antibiotics to be used in different infections. CLSI use voluntary

consensus standards and include antibiotic interpretations that have proven efficacy by acceptable in vitro test performance against the

pathogen, clinical efficacy, prevalence of resistance, FDA clinical indications for use, and current consensus recommendations for first-

choice and alternative drugs.

There are only trimethoprim-sulfamethoxazole (cotrimoxazole, TMP-SMX) breakpoints and FDA approval use agaisnt Streptococcus

pneumoniae , however increasing resistance is well documented with resistance rates of 18 to 26% (J Clin Microbiol. 1999 January;

37(1): 215217). Research susceptibility studies using the S. pneumoniae breakpoints and E-test have shown very high rates of

Viridans Streptococcus resistance to TMP-SMX (68%; J Clin Microbiol. 1999 June; 37(6): 18761880). Based on the fact that B-lactams are a great first

choice for treatment, the pharmacokinetics, and the high level of resistance, the use of TMP-SMX is not recommended to treat Viridans

Streptococcus infections.

_________________________________________________________________________________________________________________________________

Recently I have performed manual sensitivity for Pseudomonas aeroginosa isolated form Blood and when it comes to reading I have noticed that piperacillin tazobactam has D shape from Imipenem side (distance between Imipenem and TZP is 30 mm) . This shape looks like gram positive ICR strains. Is it antibiotics antagonism? Does it have any clinical significance?

Inducible expression of AmpC beta-lactamase The result that you observed with this isolate was most likely due to inducible expression of the P. aeruginosa AmpC beta-lactamase. Many isolates of certain enteric (Serratia marcescens, Providencia spp, Citrobacter freundii, Enterobacter spp, Morganella morganii), and non-enteric organisms (P. aeruginosa, Aeromonas) can up-regulate expression of their chromosomally-encoded ampC genes in response to sub-inhbitory concentrations of certain beta-lactam antibiotics. Although Imipenem is a strong inducer of AmpC expression, it is not a good substrate for the enzyme and is therefore relatively resistant to hydrolysis. This is why you saw a blunting of the TZP zone, but did not see a blunting of the Imipenem zone itself.

Interestingly, the combination of TZP with Imipenem was shown to be the most sensitive antibiotic combination for the detection of

inducible AmpC expression in a previously published study (Dunne & Hardin. J Clin Micro. 2005. 43(12):5945-5949). The AmpC beta-

lactamase can become stably over-expressed when mutations are acquired in the regulators of ampC expression (so-called de-

repressed AmpC strains). Resistance by this mechanism can occur in some patients receiving certain broad-spectrum cephalosporins

(e.g. ceftriaxone) for serious infections with the aforementioned organisms. However, there is no CLSI-approved method for the

detection of inducible AmpC expression and no consensus about how to report antibiotics when an isolate is found to have inducible

AmpC expression. It is also important to keep in mind that the AmpC beta-lactamase has also been mobilized onto a plasmid,

conferring high-level expression of this enzyme to organisms that acquire the plasmid.

There are some excellent reviews available on AmpC beta-lactamases (e.g. Jacoby GA. Clin Micro Rev. 2009. 22:161-182, Thomson KS. J Clin Micro. 2010. 48(4):1019-25).

For the purpose of determining MDRO status, are all beta-lactam drugs considered one class are they broken up into multiple classes? For example, are cefepime, zosyn, and aztreonam considered to be one class or three? What about the varying cephalosporin generations? Would cefazolin, cetriaxone, and cefepime be one class or three?

Classes in MDROs This is a great question and unfortunately there is a lack of consensus, not only on the definition of MDRO but also in how to divide the Beta lactams. Based on CLSI, Beta lactams can be divided in 5 different classes:

Penicillins, Cephems, Monobactams, Carbapenems, and Beta lactam-beta lactamase combinations. For the purpose of MDRO definition, in our laboratory we divide the Beta lactams in these 5 classes. However, different labs use different

criteria, although in most cases Carbapenems are always considered a different class. I would suggest getting a clear understanding

from your Infection Control Group in how to interpret the definition of MDRO and which classes of antibiotics should be included.

Ultimely, the reporting of MDRO should be used for Epidemiology and infection control purposes.

Hi Nasrullah You have this article.Read it. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance 2012

294/4/1 I have found 4 strains of Serratia marcescens and 5 of Enterobacter aerogenes with a commond susceptibility pattern; Cefazolin R/I or S, Ceftriaxone S, Ceftazidime S and Cefepime S; but the Carbapenems has a discrepancy between Meropenem S and Imipenem R in all of them. After verify these results using a KB diffusion disk and the CLSI 2012, the susceptibility pattern has been the

same. I have read about the SME in those organisms as the possible cause of Imipenem R; when this result is gotten do those organism have to be called CRE? calling them CRE all the Carbapenems and Cephalosporins tested as Susceptible have to be changed to Resistant? How those organisms could be interpreted and reported?

SME Difficult Question . The study of beta-lactamase enzymes, the organisms carrying them, and interpretation of susceptibility results continues to expand and evolve with the discovery of new beta-lactamase enzymes and increased understanding

of PK/PD properties associated with the different beta-lactams.

The strains you report (4 Serratia marcescens and 5 Enterobacter aerogenes) share a common susceptibility pattern; Cefazolin R/I or

S, Ceftriaxone S, Ceftazidime S, Cefepime S, Imipenem R, Meropenem S; but are likely the result of different resistance mechanisms.

S. marcescens

As you allude to in your question, this pattern in S. marcescens is likely the result of an SME enzyme. These are Ambler class A serine beta-lactamases. SME-1 was the first SME reported and demonstrates a broad spectrum of activity against beta-lactams including penicillins, cephalosporins, carbapenems and aztreonam. Subsequently, two additional SME enzymes (SME-2, SME-3) have been described that differ from SME-1 by single amino acid substitutions. These substitutions result in up to a 100-fold lower

affinity (and thus reduced hydrolysis) for a number of substrates including the extended spectrum cephalosporins, and meropenem

while retaining high level activity against first and second generation cephalospoirins and imipenem. This susceptibility pattern is

consistent with the pattern you report and may indicate the presence of SME-2 or SME-3 in your strains. Such enzymed are typically

not inducible and are chromosomally encoded, thus do not present a risk for spread of these resistance determinants to other bacteria.

Several case reports of S. marcescens strains containing SME-2 or SME-3 suggest that these strains remain susceptible to third and

fourth generation cephalosporins including ceftazidime and cefepime despite demonstrated resistance to imipenem. However, other

mechanisms of resistance may produce a similar susceptibility profile in the family Enterobacteriacae including chromosomally encoded

(inducible) AmpC enzymes, porin mutations, or a combination of mechanisms. Therefore without molecular or nucleic acid sequence

confirmation of SME type it is most appropriate to treat as a CRE and rely on alternative (non-beta-lactam) antibiotics if

available/appropriate.

E. aerogenes

The susceptibility pattern you describe for four E. areogenes isolates is also typical of some of the Ambler class A beta-lactamases.

In Enterobacter species the enzymes NMC-A and IMI-1 would be the most likely candidates. These enzymes are chromosomally

located and are expressed at a low basal level in the absence of an inducing agent. Inducing agents for these enzymes include

imipenem and cefoxitin. Therefore, these strains can demonstrate in vitro resistance to imipenem while appearing phenotypically

susceptible to ertapenem, meropenem, and other extended spectrum cephalosporins. Characteristics that further support the presence

of NMC or IMI include the growth of isolated colonies inside the zone of inhibition upon disk diffusion or Etest for carbapenems. These

colonies are not observed when tested in the presence of clavulanic acid. Such strains should also demonstrate inducible resistance

as a flattening of the meropenem or ertapenem zone of inhibition proximal to an imipenem or cefoxitin disk in a standard double disk

diffusion assay (D-zone test).

As with Serratia spp., Enterobacter spp. can also inducible AmpC enzymes or other beta-lactamases that make correlation of a specific

resistance mechanism to a specific susceptibility pattern difficult. Because of the inducible nature of these carbapenemease enzymes

(NMC, IMI) these strains should be regarded as CRE and alternative (non-beta-lactam) antibiotic therapies should be pursued.

294/4/2 For Multidrug resistant Acinetobacter, we have noticed that Amikacin result is suppresed from VITEK II . Do we still have to confirm Amikacin if both Gentamicin and Tobramycin are resistant?

Amika, Genta, Tobra for Acineto Amikacin results for Acinetobacter cannot be extrapolated from tobramycin or gentamicin results. That is to say, just because the tobramycin or gentamicin is resistant it does not necessarily mean the amikacin will also be resistant, and if an amikacin results is needed, you must test amikacin. A couple of

points about this:

If you refer to the CLSI M100 S22 document, Table 1A, agents are listed in a single box when interpretive results and clinical efficacy

are similar. Amikacin is in a box seperate from gentamicin and tobramycin, and has a different breakpoint (eg,

However, it is less common to find resistance to Carbapenems. Class A carbapenemases (SME) have been identified in Serratia

marcescens strains (Antimicrob Agents Chemother. 1990 May; 34(5):755-8). SME carbapenemases have a broad hydrolysis spectrum that includes penicillins, 1st and 2ndgeneration cephalosporins, aztreonam, and carbapenems. These carbapenemases are not KPC (KPC confer resistance to most Beta lactams including 3rd and 4th generation Cephalosporins), and they do not confer resistance to 3rd and 4th generation Cephalosporins. Based on the information provided, it does not look that the S. marcescens strains described are KPC, however it would suggest to

perform the KPC PCR (feel free to send the strains to my attention and I can test them for you if you do not offer the test). Previous

publications have shown that SME hydrolyze preferably Imipenem, but they still confer lower resistance to Meropenem and Doripenem

(Antimicrob Agents Chemother. 2006 October; 50(10): 34853487), this is not consistent with your observations. Ideally, the presence

of SME should be confirmed by molecular methods.

-

Joan-Miquel Balada-Llasat, PharmD. Ph.D. D(ABMM) Associate Director, Clinical Microbiology Assistant Professor, Clinical Pathology

The Ohio State University Wexner Medical Center, University Hospital East, 1492 East Broad Street, Columbus, OH 43205

Phone: (614) 257-2785, joan-miquel.balada-llasat@osumc.edu