christy king

Master of Public Health Veterinary Public Health Specialization

Environmental surveillance for extended spectrum β-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 µg/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-β-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 β-

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 was isolated from a hospital in North Carolina (Yigit et al., 2001). This particular K.

pneumoniae isolate expressed high levels of resistance to imipenem and meropenem (Yigit et al.,

2001). Since that finding, bacteria harboring KPC genes have been found all along the eastern

coast of the United States, typically isolated from hospital patients. These findings are important

because it suggests that there is a reservoir of resistance genes in the healthcare environment and

that these genes can be easily disseminated.

Because blaKPC has been identified and isolated from hospital patients in the United

States, we hypothesized that the same resistance gene is present in hospitalized patients in

Columbus, OH. If present in patients, then these bacterial resistance genes could easily enter the

sanitary sewer system at the hospitals, and be transported to the local sewage treatment facility.

Therefore, our main objective was to sample raw sewage from the Jackson Pike Wastewater

Treatment Plant, which directly collects sewage from hospitals in Columbus, OH, and screen the

sewage for carbapenemase-producing coliform bacteria.

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Review of the Literature: β-Lactamase Mediated Antimicrobial Resistance

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. When antibiotics lose their ability to

debilitate microbial growth, the patient that is affected may suffer. This can result in unwanted

health outcomes such as the loss of milk production in dairy cattle or even severe illness or death

of a family member. Because of this, it is important to know the epidemiology, characteristics,

and history of bacteria that contain genes that confer resistance to important antimicrobial drugs.

Antimicrobials play a critically important role in both human and veterinary medicine.

They are commonly applied therapeutically when bacterial infections are diagnosed, or applied

prophylactically when bacterial infections are expected but not yet present, as is common in

some populations of agricultural animals. The Food and Drug Administration (FDA) compiles an

annual report estimating the amount of antimicrobials sold or distributed for use in food-

producing animals. The most recent report based on information collected in 2011, reports that

about 13.8 million kilograms of antimicrobials were sold or distributed in the US for use in food-

producing animals (FDA, 2011). This indicates that veterinary antimicrobial use is extremely

common in livestock populations, and certainly warranted under appropriate medical

circumstances. This area of practice is of particular importance because there is overlap with

some antibiotic classes that are used in both human medicine and for animals intended to enter

the food supply. Because there is real concern of bacterial resistance to some of these classes of

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antibiotics that may be transmitted through food, strict, judicial use of pharmaceuticals must be

used in livestock animals as well as surveillance of antimicrobial use.

One class of antimicrobials that is frequently used in both human and veterinary medicine

are the cephalosporins. Cephalosporins are β-lactam antimicrobials which act “by inhibiting the

bacterial wall synthesis i.e. the inhibition of the transpeptidation reaction of the peptidoglycan

synthesis” (Shahid et al., 2009). Cephalosporins are often classified into four different

“generations” based on their antimicrobial properties. First-generation cephalosporins are

effective against most gram-positive cocci except for enterococci, and methicillin-resistant S.

aureus (MRSA) (Shahid et al., 2009). Second-generation cephalosporins target a wide range of

gram-negative bacteria while still boasting modest activity against gram-positive species (Marsh,

1984). Third-generation cephalosporins work well against Enterobacteriacea, including some β-

lactamase-producing strains (Shahid et al., 2009). Fourth-generation cephalosporins are active

against both gram-positive and gram-negative species, which are attributed to their “poor affinity

for and increased stability to the Bush group 1 β-lactams [cephalosporin-hydrolyzing β-

lactamases not inhibited by clavulanic acid], and to their more rapid penetration across the outer

membrane of bacterial cells” (Fung-Tomc, 1997). Together, third- and fourth-generation

cephalosporins are defined as extended-spectrum cephalosporins. There is also a fifth,

unrecognized group of cephalosporins that includes the drugs ceftaroline-fosamil and

ceftobiprole, which exhibit broader activity against gram-positive organisms such as MRSA

(Bassetti et al., 2013). Carbapenems are a related group of β-lactams that are more resistant to β-

lactamase inactivation than are other cephalosporins (Shahid et al., 2009).

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The use of cephalosporins is common in both human and veterinary medicine.

Cephalosporins can be used to treat “respiratory tract infections, skin infections, urinary tract

infections, joint infections and some bone infections caused by susceptible gram-positive

bacteria” in humans (Hornish and Kotarski, 2002). In veterinary medicine, first generation

cephalosporins, including cephalexin, cephalothin, and cefazolin, are commonly used to treat

mastitis in cows. First generation cephalosporins have narrow-spectrum activity and are effective

in treating infections caused by E. coli, Haemophilus influenzae, Klebsiella spp., or Proteus

mirabilis (Hornish and Kotarski, 2002). First generation cephalosporins are also useful in

companion animal medicine for treatment of canine S. intermedius Group skin infections and

urinary tract infections (Giguère, S., Prescott, John F., Dowling,Patricia M., 2013). Second

generation cephalosporins, such as cefuroxime, can also be used to treat mastitis (Hornish and

Kotarski, 2002). Cefoxitin, another second generation cephalosporin, is commonly used to treat

mixed aerobic-anaerobic infections which may be present in cases of aspiration pneumonia,

severe bite infections, gangrene, peritonitis, and pleuritis (Giguère, S., Prescott, John F.,

Dowling,Patricia M., 2013). Third and fourth generation cephalosporins are typically reserved

for use in human and veterinary medicine for bacterial infections resistant to earlier generations

of cephalosporins.

Ceftiofur, a third-generation cephalosporin used only in veterinary medicine, is indicated

by label for use in swine, ruminants, and horses for respiratory disease (Hornish and Kotarski,

2002). It can also be used to treat metritis and foot rot in cattle (Hornish and Kotarski, 2002).

Extralabel ceftiofur use for “pasteurellosis in sheep and early mortality in chicks and turkey

poults” was common practice (Hornish and Kotarski, 2002). In 2008, the US FDA proposed

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legislation to limit the use of ceftiofur in food producing animals to exclude extralabel use.

However, this rule was withdrawn and a new rule was put in place in 2012 to prohibit the use of

cephalosporins for the use of disease prevention, inappropriate dosage levels and routes of

administration, and the use of products not approved in the major food species (FDA, 2012). The

American Veterinary Medical Association (AVMA) released a response to this ruling

specifically addressing the importance of antimicrobials for preventative use which stated,

“prevention of an infectious disease with antimicrobial therapy can potentially impact resistance

trends by decreasing the need and use of antimicrobials for herd-level treatment of disease at

higher doses, for longer duration, and with higher-potency classes” (AVMA, 2012).

Severely immunocompromised patients admitted to intensive care units in hospitals serve

as the most common infection source in human medicine (Giguère, S., Prescott, John F.,

Dowling,Patricia M., 2013). Because of this, extended-spectrum cephalosporins, especially 3rd

and 4th generation cephalosporins, are typically used to treat life-threatening diseases such as

serious infections caused by food-borne pathogens like Salmonella, Shigella and E. coli (Powers,

2015). The most common uses for cephalosporins in general in human medicine are for the

treatment of pneumonia, skin and soft tissue infections, sinusitis, urinary tract infections, and

otitis (Powers, 2015).

Meropenem, a carbapenem antibiotic, is commonly used to treat meningitis, urinary tract

infections, skin infections, septicemia, lower respiratory tract infections, and intra-abdominal

infections, because it is a highly effective broad-spectrum antibiotic (Bradley, 1997). Typically,

carbapenems are reserved as the last line of defense against severe bacterial infections because

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

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rods. The World Health Organization (WHO) described carbapenems as “critically important

antimicrobials” due to their “limited therapy for infections due to multi-drug resistance” and

because it “may result from transmission of Enterobacteriaceae including E. coli and Salmonella

spp. from non-human sources.” Therefore, resistance against these types of drugs as well as

cephalosporins that are more commonly used, is a serious public health threat. In 2013, the CDC

released a report concerning antibiotic resistance threats in the United States. In this report, the

CDC identified carbapenem-resistant Enerobacteriaceae (CRE) as one of three urgent threats

(CDC, 2013).

Antimicrobial resistance can occur by three different mechanisms; target/receptor

alteration, enzymatic modification, and/or restriction of drug accumulation (Poole, 2001). Target

modification varies widely depending on the specific organism and the corresponding antibiotic.

One such example of target modification is that of Streptococcus pneumoniae resistance to β-

lactam drugs. β-lactams target the penicillin binding proteins (PBPs) that help to catalyse murein

biosyntheis which is specific to bacteria (Hakenbeck et al., 1999). Because of this specificity to

murein biosyntheis, β-lactams can be effective against a wide range of bacteria due to the

similarity in the biosynthesis pathway. However, β-lactam resistance in S. pneumoniae is

propagated by the alteration of PBPs, acquired through point mutations (Hakenbeck et al., 1999).

Mutations in PBPs, specifically in the transpeptidase-penicillin binding domain, diminish affinity

to β-lactams. An increase in the number of mutations in one PBP directly contributes to the level

of resistance shown – the more mutations, the higher the resistance.

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The second type of bacterial resistance to antibiotics is restriction of drug accumulation.

There are several different ways that bacteria are able to limit the amount (or restrict entirely) of

antibiotic that enters through the cell wall. The most common method is through the use of an

efflux pump. Efflux pumps are active transporters that lie in the cytoplasmic membrane of most

cells. Efflux systems are capable of transporting and excreting antibiotics, biocides, dyes,

detergents, fatty acids, organic solvents and homoserine lactones in and out of the cell (Poole,

2001). E. coli use multidrug efflux systems that are part of the resistance-nodulation-division

(RND) family, which allow many drugs to enter the cell. This type of resistance mechanism is

intrinsic rather than mutationally-gained or through the acquisition of extrachromosomal DNA

(Poole, 2001). Some bacteria, including, E. coli, and P. aeruginosa, can have mutations within

local repressor genes which result in the over-expression of efflux pumps (Webber and Piddock,

2003). Over-expression of efflux pumps leads to resistance to various antimicrobials of clinical

importance such as fluoroquinolones.

Alternatively, bacteria can utilize another method to restrict drug accumulation instead of

through the use of an efflux pump. Normally, the cell wall of gram-negative bacteria works as an

effective barrier to both hydrophilic and hydrophobic (Alekshun and Levy, 2007). However,

some species of bacteria have porin proteins, a result of genetic mutation. These porin proteins

act as a channel that can allow the entry/exit of antibiotics and other small molecules (Alekshun

and Levy, 2007). E. coli and P. aeruginosa are both examples of bacteria that have these porin

proteins (OmpF and OprD, respectively) (Alekshun and Levy, 2007). Both imipenem and

meropenem have the ability to enter and exit OprD (Alekshun and Levy, 2007).

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The third mechanism of bacterial resistance to antimicrobials is enzymatic modification

or elimination. β-lactamases are the primary mode of resistance to β-lactams. β-lactamases are

enzymes that inactivate antibiotics through the hydrolysis of the β-lactam ring (Thomson and

Smith, 2000). There are four classes of β-lactamases; Class A penicillinases, Class B metallo-β-

lactamases, Class C cephalosporinases and Class D oxacillinases (Thomson and Smith 2000).

This type of resistance is acquired through evolutionarily advantageous mutations or through the

collection of extrachromosomal DNA from the outside environment through transposons or

plasmids (Thomson and Smith 2000).

Bacterial acquisition of antimicrobial resistance genes can occur through two primary

routes: mutations or horizontal gene transfer. Spontaneous chromosomal mutations that

contribute to antibiotic resistance occur frequently in the microbial world. Spontaneous

mutations can be the result of mutagenesis with subsequent failure of DNA polymerase to correct

errors in the DNA strand. Accumulation of point mutations can lead to resistance, which is the

case for E. coli showing resistance to fluoroquinolones (Coates, 2012). E. coli accumulating

mutations in the Quinolone Resistance Determining Region (QRDR) are more likely to become

resistant to fluoroquinolones (Coates, 2012). Bacteria become stressed when exposed to

antimicrobials and this stress actually increases the genome mutation rate due to bacterial self-

damage (Kaufmann and Hung, 2010). This is significant because it exhibits the importance of

selective pressure in the evolution of antibiotic resistance. Mutations can also lead to over-

expression of resistance genes. Over-expression of the efflux pump is an example of how this

can contribute to antibiotic resistance. Mutations in the genome are important to consider

because these selected resistant elements can be passed vertically through bacterial progeny or

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passed laterally to susceptible bacteria through horizontal gene transfer. Vertical transmission of

resistant bacteria has been recorded in several instances in broiler operations. Enrofloxacin-

resistant E. coli clones were isolated from dead broilers and embryonated eggs during an

outbreak of colibacillosis in a Danish broiler production in 2006, suggesting that vertical

transmission of resistant E.coli clones in food animal populations is possible (Peterson et al.,

2006).

Horizontal gene transfer accounts for the majority of resistance genotypes that are found

(Nakamura et al., 2004). Genes can be transferred from one independent organism to another by

three different mechanisms: transformation, transduction, or conjugation. Transformation is the

uptake of DNA from the environment into the cell that causes genetic alteration. E. coli is able to

rapidly take up R-factor DNA from the environment as shown by Cohen et al. in 1972.

Transduction is the transfer of genetic material from a bacteriophage into a bacterium. This has

been demonstrated in one such manner by bacteriophages that encode the virulence determinant

for toxin production in V. cholerae (Ochman et al., 2000). Conjugation occurs when bacterial

cells transfer DNA through direct contact (Amabile-Cuevas and Chicurel, 1993). One cell acts as

a donor, while the other acts as the recipient. Resistance plasmids (R plasmids) can be

transferred via conjugation between pathogenic bacteria, which was first shown in E. coli and

Shigella by Ochiai et al. and Akiba et al. in 1959 and 1960.

Mobile plasmids that harbor resistance genes (or R plasmids) that encode enzymes which

break down antibiotics have existed for many years. In 1966, one of the first enzymes recorded

was a plasmid-mediated β-lactamase found in Greece that is known as TEM (Hawkey, 2008).

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This enzyme was isolated from a gram-negative organism and the resistance gene that encodes it

has since disseminated worldwide. The TEM β-lactamase gene has spread amongst a variety of

Enterobacteriaceae species as well as Pseudomonas aeruginosa, Haemophilus influenzae, and

Neisseria gonnorhoeae (Brunton et al., 1986). TEM-1 β-lactamase confers resistance to

penicillins and early cephalosporins by hydrolyzing the β-lactam ring (Salverda et al., 2010).

TEM-1 β-lactamase now has over 170 variants described, making it still one of the most

important and widely disseminated bacterial resistance enzymes.

SHV-1 is a similar β-lactamase with narrow-spectrum activity against penicillins and

cephalosporins that also appeared in the 1960’s (Blagui et al., 2008). This β-lactamase occurs in

high frequency in K. pneumoniae and is often a plasmid-encoded gene (Chaves et al., 2001).

Like the TEM, SHV-1 has also disseminated worldwide, being reported in the United States,

Switzerland, the United Kingdom, and Africa (Shaokat et al., 1987).

One of the most widely reported β-lactamases, AmpC β-lactamase, “mediates resistance

to cefazolin, cefoxitin, most penicillins, and β-lactamase inhibitor-β-lactam combinations”

(Jacoby, 2009). P. aeruginosa, Acinetobacter spp., and several members of the

Enterobacteriaceae encode AmpC β-lactamase by chromosomal genes. Some organisms that

cannot encode AmpC β-lactamase in this manner have the ability to acquire genes through

horizontal transfer to produce the same effect (Shahid et al, 2009). Such is the case for several

different species of bacteria like Klebsiella spp., E. coli, and Salmonella, that can confer

resistance to antimicrobials by acquiring mobile genes (Hanson, 2003). The plasmid-mediated

AmpC β-lactamase, blaCMY-2, is the most widely distributed in the Enterobacteriaceae family and

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is the most common extended-spectrum β-lactamase in the US (Jacoby, 2009; Sidjabat et al.,

2009). CMY-2 is important because it can threaten therapeutic options due to its ability

configure multi-drug resistance to the following antimicrobials; trimethoprim, chloramphenicols,

sulfonamide, and amioglycosides, due to it’s ability to carry multiple resistance genes (Jacoby,

2009).

This mobile resistance gene, blaCMY-2, has been identified in isolates from both humans

and animals. In 2000, the National Antimicrobial Resistance Monitoring System (NARMS)

identified ceftriaxone-resistant Salmonella carrying blaCMY-2, isolated from human patients

across the US (Dunne et al., 2000). National surveillance by NARMS of Salmonella carrying

blaCMY-2 was sparked in part by another study done in 1998 that isolated ceftriaxone-resistant

Salmonella Typhimurium from a 12-year-old boy in Nebraska. The ceftriaxone-resistant isolate

was found to be indistinguishable from one isolate from a herd of cattle owned by the boy’s

father (Fey et al., 2000). This finding was significant because it brought into question the role of

livestock animals in the transfer of antibiotic resistant pathogens to humans.

Since then, many studies have reported finding MDR bacteria carrying blaCMY-2 isolated

from livestock species in the US. In July 2000, the Salmonella Reference Center (SRC) at the

University of Pennsylvania School of Veterinary Medicine confirmed that a Salmonella Newport

isolate from a bovine was resistant to cephalosporins. Rankin et al. further investigated this

finding by phenotypically and genotypically characterizing 42 MDR Salmonella Newport

isolates submitted to the SRC that were collected from Pennsylvania, Maryland, New York and

New Jersey. These isolates were mostly of bovine origin, but also included equine, canine, and

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avian species and all isolates were found to be carriers of blaCMY-2 (Rankin et al., 2002). In 2005,

Alcaine et al. published a study that characterized 39 Salmonella isolates collected from cattle or

the environment in New York dairy herds. This study found 19 isolates that conveyed ceftiofur

resistance and also harbored blaCMY-2 (Alcaine et al., 2005). This mobile resistance genotype,

blaCMY-2, has also been identified in E. coli and Salmonella isolated form retail meat products

such as ground beef, beef steak, pork chops, and pork ribs purchased from grocery stores located

in Ohio and North Carolina (Mollenkopf et al., 2011).

Another β-lactamase gene that carries significant clinical interest belongs to the

extended-spectrum β-lactamase (ESBL) class and is identified as CTX-M. As a molecular class

A ESBL, CTX-M is active against extended-spectrum cephalosporins and monobactams, but not

cephamycins or carbapenems (Rossolini et al., 2008). This β-lactamase has been found in high

rates in K. pneumoniae and E. coli, isolated from hospital patients, among normal human

microflora, livestock populations, and even from companion animals (Rossolini et al., 2008).

“Typically, they derive from genes for TEM-1, TEM-2, or SHV-1 by mutations that alter the

amino acid configuration around the active site of these β-lactamases” (Paterson and Bonomo,

2005). CTX-M-type β-lactamases have been found in South America, Africa, the Far East,

Eastern Europe, Western Europe, and Northern America (Paterson and Bonomo, 2005). In fact,

clonal spread of CTX-M has been documented, specifically within Citrobacter freundii

(Gniadkowski et al., 1998). CTX-M-type β-lactamases are now considered to be the most

prevalent ESBL’s found in E. coli and K. pneumoniae isolated from humans (Rossolini et al.,

2008).

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Like blaCMY-2, blaCTX-M has been identified in MDR bacteria isolated from both humans

and animals. The first reported CTX-M resistance gene reported in the United States was

identified in a S. Typhimurium var. Copenhagen isolate from an infant recently adopted from

Russia (Zirnstein et al., 2000). In 2007, a paper published by Lewis II et al. looking at the

emergence of ESBLs at the University Health System in San Antonio, Texas stated, “CTX-M-

type ESBLs are now the most common ESBL type isolated from patients in our health care

system and may also be present but unrecognized in other U.S. locales.”

In livestock in the US, a similar phenomenon is occurring. CTX-M was first reported in

livestock in the US in 2010 from E. coli isolates harboring blaCTX-M-1 or blaCTX-M-79 (Wittum et

al., 2010). Another study screened 2,034 clinical isolates submitted for serotyping at the National

Veterinary Services Laboratory (NVSL) between October 2010 and June 2011 and identified 12

Salmonella isolates from turkeys, horses, and pigs from 6 US states that were harboring blaCTX-M-

1 (Wittum, Mollenkopf, and Erdman, 2012). Another published study reported finding blaCTX-M

in 1.6% of their samples from E. coli and K. pneumoniae originating from swine across 5 US

states (Mollenkopf et al., 2013). Also reported present in retail boneless chicken breasts

(Mollenkopf et al., 2014).

Several genes are known to encode the ability to produce carbapenemase. The most well-

known and widely dissemninated genes include blaKPC, blaNDM-1, blaIMP, blaVIM, and blaOXA.

NDM-1, IMP, and VIM are Class B carbapenemases, whereas KPC is Class A and OXA is Class

D. Class B carbapenemases are distinguished as metallo-β-lactamases. Class A carbapenemases

can be chromosome encoded or plasmid encoded (Nordmann, Patrice, 2011). They are partially

inhibited by clavulanic acid, but still hydrolyze carbapenems (Nordmann, Patrice, 2011). Class B

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metallo-β-lactamases are usually integron-encoded and their activity is inhibited by EDTA

(Nordmann, Patrice, 2011). This class hydolyzes all β-lactams except aztreonam (Nordmann,

Patrice, 2011). Class D enzymes are plasmid encoded, weakly hydrolyze carbapenems, and their

activity is not inhibited by EDTA or clavulanic acid (Nordmann, Patrice, 2011).

One of the very first Class A carbapenemases found was a KPC in 1996 which was

isolated from a hospital in North Carolina (Yigit et al., 2001). This particular K. pneumoniae

isolate expressed high levels of resistance to imipenem and meropenem (Yigit et al., 2001).

Since that finding, bacteria harboring KPC’s have been found all along the eastern coast of the

United States. Through the Meropenem Yearly Susceptibility Test Information Collection

(MYSTIC) Program, a study was performed by Deshpande et al in 2006 looking at the past 6

years of data for surveillance of blaKPC. Researchers detected blaKPC in K. pneumoniae and C.

freundii from medical centers in New York and Delaware and in E. coli from hospitals in New

York and Ohio (Deshpande et al, 2006). KPC-1 has been identified in K. pneumoniae isolated

from a hospital in North Carolina (Yigit et al, 2001). KPC-2 and 3 were identified in K.

pneumoniae from hospitals in Massachusetts (Hossain et al., 2004). KPC findings have also been

reported in Greece, China, Israel, Puerto Rico, and Columbia (Nordmann et al., 2009; Navon-

Venezia et al., 2009).

NDM-1 (New Delhi Metallo-betalactamase-1), a class B metallo-β-lactamase, was first

discovered in a Swedish patient of Indian origin who was traveling to New Delhi, India (Yong et

al., 2009). A carbapenem-resistant K. pneumoniae strain carrying blaNDM-1 on plasmids of

varying sizes caused a urinary tract infection to develop (Kumarasamy et al., 2010). This

21

carbapenem resistance gene has since been found in Enterobacteriaceae in India, Pakistan, and

the United Kingdom, and has been widely disseminated in drinking water and the environment

(Kumarasamy et al., 2010).

Current literature on ESBLs cultured from sewage treatment plants in the US is limited.

In 2003, a study was performed looking at antibiotic-resistant bacteria isolated from wastewater

and river bank water from Mainz, Germany. The study identified vancomycin-resistant

enterococci and beta-lactam-hydrolysing Enterobacteriaceae harboring vanA and ampC

respectively (Schwartz et al., 2003). Another study published in 2007 identified E. coli resistant

to ampicillin, cephalothin, nalidixic acid, and tetracycline along the east coast of Australia, but

did not further characterize these isolates genotypically (Watkinson et al., 2007). However,

another study done in Australia in 2009 sampled 5 different sewage treatment plants and tested

for the presence of ESBL E. coli and found 61.1% of their isolates to be positive. From those

isolates, both blaCTX-M and blaTEM were identified (Reinthaler et al., 2010). In 2010, samples

were collected from wastewater in Algiers region, Algeria and screened for ESBL K.

pneumoniae. The study identified 84 isolates which all harbored blaCTX-M-1 and several virulence

factors such as entB, uge, and wabG, among others (Atmani et al., 2015). Because of the lack of

literature on Enterobacteriaceae harboring blaKPC isolated from wastewater treatment plants in

the US, this project will contribute significantly to the knowledge base of resistance genes

present in aquatic environments.

22

Materials and Methods

Source of the isolates. Samples were collected from the Jackson Pike Waste Water

Treatment Plant located in Columbus, OH from June and August of 2011 and 2012 and from

May to July of 2014. Incoming, untreated wastewater (influent) was the target for sample

collection. At the time of sample collection, two 50 mL falcon tubes were filled with primary

influent, transported at ambient temperature to our research laboratory, and separated into 6, 1

mL aliquots per 50 mL falcon tube creating a total of 12 samples per plant visit. The Jackson

Pike Waste Water Treatment Plant was visited 30 times over the span of 4 summers, giving a

total of 369 samples. Samples were initiated for processing the same day for culture and isolation

of extended-spectrum cephalosporin resistant coliform bacteria.

Bacterial culture. For the recovery of coliform bacteria resistant to extended-spectrum

cephalosporins, 1 mL of influent was added to 9 mL of MacConkey broth containing either

cefotaxime 4 ug/ml or meropenem 1 ug/ml. For each visit, half of the samples (6, 1 mL aliquots)

were processed in MacConkey broth containing cefotaxime 4 ug/ml, and the other half of the

samples (6, 1 mL aliquots) were processed in MacConkey broth containing meropenem 1 ug/ml.

In June of 2012, MacConkey broth containing cefotaxime 4 ug/ml was determined to be an

effective initial screening process for coliform bacteria resistant to extended-spectrum

cephalosporins and all samples processed after this time went through this initial screening

phase. After overnight incubation at 37°C, this broth was streaked to MacConkey agar

containing meropenem 1 ug/ml. In 2014, a new protocol for screening coliform bacteria resistant

to extended-spectrum cephalosporins was put into place. The new method starts with the same

initial screening phase of raw sewage into MacConkey broth containing cefotaxime 4 ug/ml.

23

However, after overnight incubation at 37°C, this broth was streaked to Drigalski agar which

contains ertapenem, cloxacillin, and zinc sulfate which screens for carbapenemase-producing

members of the family Enterobacteriaceae (Nordmann et al., 2012). The new method was tested

alongside the old method to evaluate similarity in ability to screen for coliform bacteria resistant

to extended-spectrum cephalosporins and there was no difference after testing 20 samples.

Lactose positive and indole positive isolates were confirmed as E. coli by PCR (Bayardelle and

Zafarullah, 2002).

Isolate Characterization. Identification of bacterial pathogens was done by using the

Sensititre® ARIS 2X automated system at the Ohio Department of Agriculture as per their

protocols. Some of the isolates also underwent further genetic analysis by PCR, looking to

identify the presence of OXA or KPC genes (Gröbner et al., 2009)(Moland et al., 2003).

Conjugation experiments were performed in order to establish the transmissibility of plasmids

harboring blaCTX-M using donor E. colis: 2993 C, UPB 38 and UPB 41 and E. coli 29522 as a

negative control (Gebreyes, Thakur, 2005). Additionally, isolates that tested positive for the

OXA gene on PCR were bidirectionally sequenced to assess DNA-binding surface amino acid

substitutions (Genewiz, South Plainfield, NJ).

Antimicrobial susceptibility testing. Antimicrobial susceptibility of isolates to meropenem

and imipenem was performed on all samples up until 2014 by Kirby Bauer susceptibility testing,

following CLSI guidelines. Minimum inhibitory concentrations (MICs) to a panel of 26

antimicrobial drugs important to human and veterinary medicine were generated for some

isolates using a semi-automated broth microdilution system (V2AGNF and ESB1F MIC plates,

24

Sensititre Sensitouch, TREK Diagnostic Systems, Cleveland, OH) following Clinical and

Laboratory Standards Institute guidelines.

Carbapenemase Detection. A Modified Hodge Test was used to test for the production of

carbapenemase as set by guidelines provided by the CDC (Jesudason et al., 2005). Metallo-β-

lactamase detection was performed using the EDTA double disk diffusion method as outlined by

Arakawa et al. in 2000. In 2014, a newer test was used to identify carbapenemase production

called the Carba NP test (Nordmann et al. in 2012).

25

Results

A total of 369 untreated wastewater influent samples were collected over a three-year

time period from the Jackson Pike facility, starting in June of 2011. Samples were collected from

June and August of 2011 and 2012 and from May to July of 2014. Table 1 summarizes the rate

of recovery of isolates with reduced susceptibility to meropenem from these samples, and the

proportion with confirmed carbapenemase production. From this table, of the total 369 samples,

194 (52.6%) produced isolates on MacConkey agar plates containing meropenem, indicating

reduced susceptibility to carbapenems. 78 (21.1%) isolates tested positive for carbapenemase

production via the Modified Hodge Test. In addition, 49 (13.3%) isolates tested positive for

carbapenemase production via the Carba NP test. Confirmed carbapenemase producers were

defined as isolates testing positive on both the Modified Hodge Test and the Carba NP test,

isolates collected in 2014 that were positive on just Carba NP, or isolates that were collected

prior to 2014 that had tested positive on only the Modified Hodge Test and are no longer viable

and unavailable for confirmation using CarbaNP. Therefore, the number of confirmed

carbapenemase producers was calculated to be 88 (23.8%). The highest prevalence of confirmed

carbapenemase producers was found in August of 2012 with a rate of 21 out of 24 (87.5%)

samples producing isolates confirmed positive for carbapenemase production.

In 2011, 225 samples were collected of which 93 (41.3%) contained isolates that

expressed reduced susceptibility to meropenem. Of those, 22 (23.7%) isolates were confirmed to

be carbapenemase producers, representing 9.8% of the 225 original samples. In 2012, 108

samples were collected and 65 (60.2%) contained isolates that expressed reduced susceptibility

to meropenem. Of those, 54 (83.1%) isolates were confirmed to be carbapenemase producers,

26

representing 50% of the original samples. In 2014, 36 samples were collected and all contained

isolates that expressed reduced susceptibility to meropenem. Of those, 12 (33.3%) isolates were

confirmed to be carbapenemase producers. Table 2 depicts the confirmed carbapenemase

producing coliform bacteria sorted by species. Raoultella Ornithinolytica had the highest

proportion of isolates that produced carbapenemase with a prevalence of 29 out of 88 (33.0%)

total isolates. E. coli had the second highest prevalence of carbapenemase producers with 22 of

88 (25%) total isolates confirmed.

Table 3 summarizes the results of KPC gene identification using PCR. A total of 83

isolates that showed reduced susceptibility to meropenem were lost to further work-up due to

lack of viability, and so were not available for additional testing. Out of our 88 confirmed

carbapenemase producers, only 49 isolates were available for further genotypic characterization.

Out of these 49 isolates, 43 (89.6%) were positive for the KPC gene. The majority of these KPC-

positive isolates (14 of 43 (32.6%)) were identified as Klebsiella group 47 (now known as

Raoultella Ornithinolytica).

Table 4 shows the results of Kirby Bauer disk diffusion susceptibility testing sorted by

date of recovery and species of bacteria. Of the 155 isolates tested, 51 (32.9%) were resistant to

meropenem, 22 (14.2%) showed intermediate resistance to meropenem, and 82 (52.9%) were

susceptible to meropenem. The highest number of isolates shown to be resistant to meropenem

via Kirby Bauer Susceptibility testing was found in July of 2012, which corresponds to the

highest number of confirmed carbapenemase producers recovered in a month. E. coli was found

to be the most frequently resistant to meropenem out of all of the species tested with 19 of 51

(37.3%) isolates being classified as resistant.

27

Discussion

From 2011 to 2014, 88 coliform bacteria recovered from 369 wastewater samples

(23.8%) tested positive for carbapenemase production. The frequency at which these isolates

were recovered suggests that there is a common source, such as a hospital, that is responsible for

the dissemination of these resistance genes, specifically in this case, blaKPC. Out of the 49

confirmed carbapenemase producers that were tested for the KPC gene, 43 (89.6%) isolates were

positive. This is not surprising as the first report of KPC was in a K, pneumoniae isolate from

patients in a North Carolina hospital in 1996 (Yigit et al, 2001). Since then, multiple reports of

KPC indicate that this carbapenemase gene has become endemic in many healthcare settings in

the United States.

Many reports of KPC-producing organisms have been pathogens isolated from clinical

submissions in medical centers. One study, performed by The Meropenem Yearly Susceptibility

Test Information Collection (MYSTIC) Program, recovered “51 strains of Enterobacteriaceae

over a five-year period (from 1999-2005) with increased imipenem and meropenem MIC values

(≥2 µg/mL)” (Deshpande et al, 2006). Each positive KPC-producing strain was collected at a

medical facility located across the US that included the following states; New York, Arkansas,

Delaware, and Ohio. Another study recovered KPC-producing K. pneumoniae from a medical

center in Texas from 3 different patients between 2009 and 2010 (Hirsch et al, 2011). KPC-

producing Enterobacteriaceae isolates have been recovered in at least 33 states and have been

reported across the world (Kitchel et al, 2009). Reports have surfaced for KPC-producing

Enterobacteriaceae from Brazil, China, Colombia, France, Greece, India, Israel, Norway,

Scotland, and Sweden (Kitchel et al, 2009). All KPC-producing isolates were recovered in

28

medical facilities, suggesting that hospitals, intensive care units (ICUs), and other patient-

orientated facilities may actually serve as a common source for blaKPC. Because blaKPC has been

previously identified and reported in the US, environmental dissemination of blaKPC likely occurs

since we were also able to recover and isolate KPC-producing organisms from the Jackson Pike

Wastewater Treatment Plant in Columbus, Ohio.

We found that the KPC gene was present in a variety of gram-negative organisms, which

is consistent with reports from the current literature. In Puerto Rico, the KPC gene was detected

in E. coli, K. pneumoniae, P. aeruginosa, and Acinetobacter baumannii found in hospitals

(Robledo et al, 2011). The KPC gene as also been detected in C. freundii isolated from patients

in Shanghai, China (Li et al, 2011). K. pneumoniae, Enterobacter cloacae, and Enterobacter

aerogenes isolated from patients in hospitals located in Pakistan and the United States have been

shown to carry the KPC gene (Pesesky et al, 2015). The KPC gene has also been identified in

Raoutella ornithinolytica isolated from patients in Ontario, Canada (Tijet et al, 2014). Klebsiella

oxytoca and Serratia marcescens isolated from medical centers in Arkansas and New York,

respectively, have also been shown to carry the KPC gene (Deshpande et al, 2006). This suggests

that the KPC gene is on a plasmid that has a broad host range which is important when we

consider how it plays a role epidemiologically.

In addition to the KPC gene being found in gram-negative organisms, we were also to

recover a gram-positive organism, Streptococcus uberis, carrying this gene. S. uberis carrying

the KPC gene has not been previously reported in the literature. This finding suggests that there

is plasmid exchange between gram-positive and gram-negative species. However, this finding is

29

not too important clinically because we don’t often treat gram-positive infections with extended-

spectrum cephalosporins.

30

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. Classification N MacConkey w/

Meropenem Modified Hodge Test Positive

Carba NP Positive

Confirmed Carbapenemase Producers

Total Samples 369 194, (52.6%) 78, (21.1%) 49, (13.3%) 88, (23.8%)

2011 Samples 225 93, (41.3%) 23, (10.2%) 3, (1.3%) 22, (9.8%)

Jun-11 36 20, (55.6%) 1, (2.8%) 0, (0.0%) 1, (2.8%)

Jul-11 74 37, (50.0%) 13, (17.6%) 0, (0.0%) 13, (17.6%)

Aug-11 115 36, (31.3%) 9, (7.8%) 3, (2.6%) 7, (6.1%)

2012 Samples 108 65, (60.2%) 55, (50.9%) 34, (31.5%) 54, (50.0%)

Jun-12 36 5, (13.9%) 4, (11.1%) 4, (11.1%) 4, (11.1%)

Jul-12 48 37, (77.1%) 30, (62.5%) 21, (43.8%) 29, (60.4%)

Aug-12 24 23, (95.8%) 21, (87.5%) 9, (37.5%) 21, (87.5%)

2014 Samples 36 36, (100.0%) - 12, (33.3%) 12, (33.3%)

May-14 12 12, (100.0%) - 3, (25.0%) 3, (25.0%)

Jun-14 12 12, (100.0%) - 6, (50.0%) 6, (50.0%)

Jul-14 12 12, (100.0%) - 3, (25.0%) 3, (25.0%)

*Confirmed carbapenemase producers represent isolates that tested positive on both the Modified Hodge est and the Carba NP test, isolates collected in 2014 that were positive on just Carba NP, or isolates that were collected prior to 2014 that had tested posiive on only the Modified Hodge Test and are no longer viable. Dashes indicate that isolates were not tested using the Modified Hodge Test.

31

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. Species N Modified Hodge

Test Positive Carba NP Positive

Confirmed Carbapenemase Producers

KPC Posiive

Total Samples 369, (100%) 78, (21.1%) 49, (13.3%) 88, (23.8%) 43, (11.7%)

Citrobacter freundii

17, (4.6%) 7, (9.0%) 6, (12.2%) 7, (8.0%) 5, (11.6%)

Escherichia coli

57, (15.5%) 23, (29.5%) 4, (8.2%) 22, (25.0%) 4, (9.3%)

Raoultella Ornithinolytica

36, (9.8%) 30, (38.5%) 17, (34.7%) 29, (33.0%) 14, (32.6%)

Kluyvera ascorbata

1, (0.3%) 1, (1.3%) 0, (0.0%) 1, (1.1%) 0, (0.0%)

Raoultella terrigena

2, (0.5%) 1, (1.3%) 1, (2.0%) 1, (1.1%) 1, (2.3%)

Serratia plymuthica

7, (1.9%) 5, (6.4%) 4, (8.2%) 5, (5.7%) 4, (9.3%)

*Confirmed carbapenemase producers represent isolates that tested positive on both the Modified Hodge Test and the Carba NP test, isolates collected in 2014 that were positive on just Carba NP, or isolates that were collected prior to 2014 that had tested positive on only the Modified Hodge Test and are no longer viable. This table only includes gram-negative bacteria. There were 23 isolates that were either gram-positive or lacked identification that were not included in this table.

32

Table 3. KPC PCR test results sorted by species. Species Total Samples

Tested for KPC via PCR

Total PCR KPC Positive Isolates

Total Samples 49, (100%) 43, (89.6%)

Citrobacter freundii 6, (12.2%) 5, (11.6%)

Escherichia coli 4, (8.2%) 4, (9.3%)


17, (34.7%) 14, (32.6%)

Raoultella terrigena 1, (2.0%) 1, (2.3%)

Serratia plymuthica 4, (8.2%) 4, (9.3%)

Streptococcus uberis 5, (10.2%) 3, (7.0%)

*12 isolates were not species identified and are not included in this table.

33

Table 4. Kirby Bauer susceptibility results using Meropenem discs sorted by date and species for coliform bacteria recovered from untreated wastewater influent. Classification N Resistant Intermediate Susceptible

Total Samples 155 51, (32.9%) 22, (14.2%) 82, (52.9%)

Jun-11 20 0, (0.0%) 0, (0.0%) 20, (24.4%)

Jul-11 37 11, (21.6%) 3, (13.6%) 23, (28.0%)

Aug-11 36 13, (25.5%) 3, (13.6%) 20, (24.4%)

Jun-12 5 5, (9.8%) 0, (0.0%) 0, (0.0%)

Jul-12 34 16, (31.4%) 11, (50.0%) 7, (8.5%)

Aug-12 23 6, (11.8%) 5, (22.7%) 12, (14.6%)

Aeromonas hydrophilia

1 0, (0.0%) 0, (0.0%) 1, (1.2%)

Citrobacter freundii

17 5, (9.8%) 1, (4.5%) 11, (13.4%)

Enterobacter gergoviae

1 1, (2.0%) 0, (0.0%) 0, (0.0%)

Escherichia coli

57 19, (37.3%) 5, (22.7%) 33, (40.2%)


33 14, (27.5%) 8, (36.4%) 11, (13.4%)

Kluyvera ascorbata

1 1, (2.0%) 0, (0.0%) 0, (0.0%)

Pseudomonas fluorescens

2 2, (4.0%) 0, (0.0%) 0, (0.0%)

Raoultella terrigena

2 1, (2.0%) 0, (0.0%) 1, (1.2%)

Serratia plymuthica

7 3, (6.0%) 2, (9.1%) 2, (2.4%)

34

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