Christy King

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  • 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

  • 2

    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.

  • 3

    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.

  • 4

    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

  • 5

    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

  • 6

    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-

  • 7

    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