Master of Public Health Veterinary Public Health Specialization

Environmental surveillance for extended spectrum -lactamase genes in Enterobacteriaceae in an urban

municipal wastewater treatment plant influent

Christy King, B.S.

Thomas Wittum, MS, PhD, Joshua Daniels, DVM, PhD Diplomate ACVM, Jiyoung Lee, MS, PhD

Submitted in Partial Completion of Requirements for the Master of Public Health Degree at

The Ohio State University

April, 2016

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Abstract

In response to ever increasing use of antibiotics, bacteria are evolving resistance to

critical frontline antimicrobial drugs that treat potentially deadly invasive gram-negative and

gram-positive infections. The most serious threat is bacteria that are resistant to carbapenem

drugs because carbapenem drugs are typically used as our last line of defense against

antimicrobial resistant organisms. Bacteria may gain this resistance by acquiring mobile genes

that confer the ability to produce enzymes that inactivate the antibiotic. Two

genes, blaKPC and blaNDM-1, are known to encode bacterial ability to produce carbapenemase.

While blaKPC is known to be commonly present in the US healthcare system, blaNDM-1 is

primarily disseminated in India and Southeast Asia. Because of the frequency of international

travel we hypothesized that blaNDM-1 could be present in Ohio wastewater treatment plants. The

purpose of this study was to determine if carbapenem-resistant, coliform bacteria were present in

Columbus wastewater, and to fully characterize those isolates and their resistance mechanisms.

We collected 369 samples of untreated sewage water at the Jackson Pike Wastewater Plant,

Columbus, OH between June and August of 2011 and 2012 and from May to July of 2014.

Samples were collected during the summer months as a result of availability and convenience.

Using selective media, we identified 194 (52.6%) samples with suspect colonies that grew in the

presence of 1 g/ml of meropenem. Of these, 51 (32.9%) were classified as meropenem resistant

using Kirby-Bauer disk diffusion assay and 19 of those isolates were also confirmed to be E.

coli using biochemical tests and PCR. These isolates were resistant to most of the 26 drugs on

our MIC panels using microbroth dilution. Carbapenemase production was verified for 78

isolates using the Modified Hodge test. Overall, 88 isolates were confirmed carbapenemase

producers with verification through either the Modified Hodge Test or by the Carba NP Test.

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However, none of the isolates were positive on the EDTA Double Disk Diffusion test, indicating

absence of metallo--lactamase production. 49 isolates were tested for the presence of blaKPC via

PCR, with 43 (89.6%) isolates returning with positive identification. The most common species

of bacteria found to carry this gene was Klebsiella Group 47 (now known as Raoultella

Ornithinolytica). Our detection of these isolates suggests the presence of a reservoir of important

mobile carbapenem resistance genes for pathogens. This kind of resistance poses a large threat if

it was to be introduced into a population of humans that are more susceptible to infection and

cannot fight a multi-drug resistant bacterial infection. Patients in a hospital setting have been

identified as one such population as resistant gram-negative bacteria such as E. coli and

Klebsiella pneumoniae can behave opportunistically in hospital environments. This risk is a

major concern in the field of public health and is an urgent threat in they eyes of the CDC.

Therefore, surveillance for antimicrobial resistance is an important part of education, awareness,

and prevention in the public health sector.

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Table of Contents

Abstract ............................................................................................................................................2

List of Tables ...................................................................................................................................5

Introduction......................................................................................................................................6

Review of the Literature ..................................................................................................................8

Materials and Methods...................................................................................................................22

Source of the Isolates .................................................................................................................22

Bacterial Culture .......................................................................................................................22

Isolate Characterization ............................................................................................................23

Antimicrobial Susceptibility Testing ..........................................................................................23

Carbapenemase Detection .........................................................................................................24

Results............................................................................................................................................25

Discussion ......................................................................................................................................27

Tables.............................................................................................................................................30

References......................................................................................................................................34

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List of Tables

Table 1. Summary of carbapenemase producing coliform bacteria recovered from untreated wastewater influent showing the total number of isolates that grew on MacConkey agar with reduced susceptibility to meropenem.............................................................................................30

Table 2. Carbapenemase producing bacteria recovered from untreated wastewater influent listed by species and separated by Modified Hodge Test, Carba NP test, confirmed carbapenemase producers, and KPC .......................................................................................................................31

Table 3. KPC PCR test results sorted by species...........................................................................32

Table 4. Kirby Bauer susceptibility results using Meropenem discs sorted by date and species for coliform bacteria recovered from untreated wastewater influent. .................................................33

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Introduction

Antimicrobial resistance has become an issue of great public health importance. The

availability of appropriate and effective antibiotics in human and veterinary medicine can mean

life or death for patients with invasive bacterial infections (Roberts et al., 2009). When

antibiotics lose their ability to debilitate microbial growth, the patient that is affected may suffer.

This can result in adverse health outcomes such as the loss of milk production in dairy cattle or

even severe illness or death of a family member. Typically, carbapenem antimicrobials are

reserved as the last line of defense against severe bacterial infections in human beings because

they are active against almost all aerobic or anaerobic gram-positive or gram-negative cocci or

rods (Craig, 1997). Therefore, resistance against these types of drugs as well as the extended-

spectrum cephalosporin antimicrobials that are more commonly used, is a serious public health

threat.

Several bacterial genes are known to encode the ability to produce carbapenemase, a -

lactam-hydrolyzing enzyme that can hydrolyze penicillins, cephalosporins, monobactams, and

carbapenems (Queenan and Bush, 2007). Carbapenemases are divided up into several different

classes, consisting of Class A, B, and C (Poirel et al., 2007). Class A carbapenemases of note

include IMI-1, IMI-2, SME-1, SME-2, SME-3, KPC-1, KPC-2, KPC-3, SHV-1, and TEM-1

(Poirel et al., 2007). All Class A carbapenemases are inhibited by clavulanic acid making them

readily identifiable using double-disk synergy testing with imipenem (Poirel et al., 2007). Class

B carbapenemases consist of a mixture of beta-lactamases and metallo-beta-lactamases (Poirel et

al., 2007). Class B carbapenemases of note include IMP-1, VIM-1, and VIM-2. Class B

carbapenemases typically work against a broad spectrum of antimicrobials including expanded-

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spectrum cephalosporins and carbapenems (Poirel et al., 2007). Metallo-beta-lactamases are

susceptible to EDTA inhibition and are not inhibited by clavulanic acid (Poirel et al., 2007).

Class D carbapenemases consist of carbapenem-hydrolyzing beta-lactamases or oxcillinases

(Poirel et al., 2007). Some Class D carbapenemases of note include OXA-23 and OXA-27. OXA

carbapenemases have been reported worldwide and demonstrate carbapenemase activity mainly

in Acinetobacter baumanii (Poirel et al., 2007).

The most well-known and widely disseminated genes include blaKPC, blaNDM-1, blaIMP,

blaVIM, and blaOXA. One of the very first Class A carbapenemases found was a KPC in 1996

which wa