Tulathromycin Solution for Parenteral Injection For Treatment Of Bovine
And Swine Respiratory Diseases

Microbiological Effects on Bacteria of Human Health Concern

A Qualitative Risk Estimation

  -----------------------------------------------------------------------
  As part of the New Animal Drug evaluation process for antimicrobial
  agents in livestock, the FDA/CVM may require a review by the Veterinary
  Medicine Advisory Committee. This document provides a public record of
  the Risk Estimation conducted by the Sponsor for injectable
  tulathromycin for therapeutic use in cattle and swine in accordance
  with FDA/CVM Guidance #152, entitled \"Evaluating the Safety of
  tulathromycin with regard to its Microbiological Effects on Bacteria of
  Human Health Concern\". This document is submitted as a part of the
  Human Food Safety Assessment. It summarizes proprietary data and
  information developed and generated by the Sponsor, as well as
  published data and information relevant to the Risk Estimation for the
  proposed therapeutic use of injectable tulathromycin in cattle and
  swine, consistent with Guidance #152.

  -----------------------------------------------------------------------

#  {#section .unnumbered}

September 15, 2004

# TABLE OF CONTENTS {#table-of-contents .unnumbered}

[ABbreviations 5](#abbreviations)

[GLOSSARY OF TERMS 6](#glossary-of-terms)

[EXECUTIVE SUMMARY 8](#executive-summary)

[Hazard Characterization 14](#hazard-characterization)

[Qualitative Risk ESTIMATION 17](#qualitative-risk-estimation)

[1. Release assessment 17](#release-assessment)

[1.1. Overview 17](#overview)

[1.2. Product description 18](#product-description)

[1.2.1. Product formulation 18](#product-formulation)

[1.2.2. Proposed conditions of use 18](#proposed-conditions-of-use)

[1.2.3. Drug substance description 18](#drug-substance-description)

[1.3. Mechanism and type of action 19](#mechanism-and-type-of-action)

[1.3.1. Mechanism of action 19](#mechanism-of-action)

[1.3.2. Type of action 20](#type-of-action)

[1.4. Spectrum of activity 21](#spectrum-of-activity)

[1.4.1. Spectrum of activity against foodborne organisms listed in
Guidance #152.
21](#spectrum-of-activity-against-foodborne-organisms-listed-in-guidance-152.)

[1.4.2. Effects of neutral and acidic pH on observed MIC
21](#effects-of-neutral-and-acidic-ph-on-observed-mic)

[1.4.3. Conclusions regarding susceptibility data
22](#conclusions-regarding-susceptibility-data)

[1.5. Pharmacokinetics and pharmacodynamics
22](#pharmacokinetics-and-pharmacodynamics)

[1.5.1. Absorption, distribution, metabolism, and elimination (ADME)
22](#absorption-distribution-metabolism-and-elimination-adme)

[1.5.2. Additional effects 24](#additional-effects)

[1.5.3. Pharmacodynamic properties 24](#pharmacodynamic-properties)

[1.6. Resistance mechanisms, genetics and location
25](#resistance-mechanisms-genetics-and-location)

[1.6.1. Known mechanisms of resistance in animal and human pathogens
25](#known-mechanisms-of-resistance-in-animal-and-human-pathogens)

[1.6.2. Location of resistance determinants
27](#location-of-resistance-determinants)

[1.6.3. In vitro activity of tulathromycin against human pathogens
containing characterized mechanisms of macrolide resistance
27](#in-vitro-activity-of-tulathromycin-against-human-pathogens-containing-characterized-mechanisms-of-macrolide-resistance)

[1.7. Occurrence and rate of transfer of resistance determinants
28](#occurrence-and-rate-of-transfer-of-resistance-determinants)

[1.7.1. Transferable resistance determinants
28](#transferable-resistance-determinants)

[1.7.2. Point mutations 29](#point-mutations)

[1.8. Resistance selection pressures
30](#resistance-selection-pressures)

[1.8.1. Information regarding cross-resistance to other antimicrobial
drugs approved in veterinary and human medicine
30](#information-regarding-cross-resistance-to-other-antimicrobial-drugs-approved-in-veterinary-and-human-medicine)

[1.8.2. Information regarding co-resistance to other antimicrobial drugs
approved in veterinary and human medicine
31](#information-regarding-co-resistance-to-other-antimicrobial-drugs-approved-in-veterinary-and-human-medicine)

[1.8.3. Conclusions regarding selection pressure
34](#conclusions-regarding-selection-pressure)

[1.9. Baseline prevalence of resistance
34](#baseline-prevalence-of-resistance)

[1.9.1. NARMS MIC data for Campylobacter
35](#narms-mic-data-for-campylobacter)

[1.9.2. Other MIC surveys of Campylobacter isolates
37](#other-mic-surveys-of-campylobacter-isolates)

[1.9.3. Conclusions regarding baseline MIC data for Campylobacter
38](#conclusions-regarding-baseline-mic-data-for-campylobacter)

[1.10. Other relevant data 39](#other-relevant-data)

[1.11. Overall Release Assessment Conclusion: \"Low\"
40](#overall-release-assessment-conclusion-low)

[2. Exposure Assessment 41](#exposure-assessment)

[2.1. Overview 41](#overview-1)

[2.2. Campylobacteriosis in man and its epidemiology
41](#campylobacteriosis-in-man-and-its-epidemiology)

[2.3. Exposure assessment for cattle
43](#exposure-assessment-for-cattle)

[2.3.1. Qualitative ranking of human consumption of beef in the US
43](#qualitative-ranking-of-human-consumption-of-beef-in-the-us)

[2.3.2. Qualitative ranking of Campylobacter contamination of beef
43](#qualitative-ranking-of-campylobacter-contamination-of-beef)

[2.3.3. Overall Beef Exposure Assessment Conclusion: \"Medium\"
44](#overall-beef-exposure-assessment-conclusion-medium)

[2.4. Exposure assessment for swine 44](#exposure-assessment-for-swine)

[2.4.1. Qualitative ranking of human consumption of pork in the US
44](#qualitative-ranking-of-human-consumption-of-pork-in-the-us)

[2.4.2. Qualitative ranking of Campylobacter contamination of pork
44](#qualitative-ranking-of-campylobacter-contamination-of-pork)

[2.4.3. Review of Campylobacter contamination in carcass and pork meat
44](#review-of-campylobacter-contamination-in-carcass-and-pork-meat)

[2.4.4. Overall Pork Exposure Assessment Conclusion: \"Medium\"
48](#overall-pork-exposure-assessment-conclusion-medium)

[3. Consequence assessment 48](#consequence-assessment)

[4. Overall Qualitative Risk estimation
48](#overall-qualitative-risk-estimation)

[4.1. Release Assessment Summary 48](#release-assessment-summary-1)

[4.2. Exposure assessment summary 48](#exposure-assessment-summary-1)

[4.3. Consequence assessment summary
48](#consequence-assessment-summary-1)

[4.4. Overall qualitative risk estimation is "High" for a "Critically
Important" drug
49](#overall-qualitative-risk-estimation-is-high-for-a-critically-important-drug)

[5. Risk Management considerations 49](#risk-management-considerations)

[5.1. Inherent properties of tulathromycin
49](#inherent-properties-of-tulathromycin)

[5.2. Extent of use 49](#extent-of-use)

[5.3. Approval and Risk Management Steps
49](#approval-and-risk-management-steps)

[6. Conclusion 50](#conclusion-1)

[References 51](#references)

TABLES

Table 1. Inhibitory activity of macrolides in a
transcription-translation assay using ribosomes isolated from *E.
coli*\* [20](#__RefHeading___Toc83018474)

Table 2. Tulathromycin MICs for selected bacterial isolates.
[21](#__RefHeading___Toc83018475)

Table 3. Effects of pH on tulathromycin activity
[22](#__RefHeading___Toc83018476)

Table 4. Genes, phenotypes, and bacterial hosts of resistance
determinants for the Macrolide-Lincosamide-Streptogramin group of
antimicrobial agents [26](#__RefHeading___Toc83018477)

Table 5. MICs of macrolides tested against *Campylobacter*
[36](#__RefHeading___Toc83018478)

Table 6. *Campylobacter* distribution among raw pork carcass surface
samples tested positive for *Campylobacter* in the US Nationwide Pork
Microbiological Baseline Data Collection Program.
[45](#__RefHeading___Toc83018479)

# ABbreviations {#abbreviations .unnumbered}

## ADME Absorption, distribution, metabolism, elimination {#adme-absorption-distribution-metabolism-elimination .unnumbered}

## ATCC American Type Culture Collection {#atcc-american-type-culture-collection .unnumbered}

## BRD Bovine respiratory disease {#brd-bovine-respiratory-disease .unnumbered}

## CDC United States Centers for Disease Control {#cdc-united-states-centers-for-disease-control .unnumbered}

## CFU Colony forming units {#cfu-colony-forming-units .unnumbered}

## ERS United States Department of Agriculture Economic Research Service {#ers-united-states-department-of-agriculture-economic-research-service .unnumbered}

## FDA/CVM United States Food & Drug Administration Center for Veterinary Medicine {#fdacvm-united-states-food-drug-administration-center-for-veterinary-medicine .unnumbered}

## FSIS United States Department of Agriculture Food Safety Inspection Service {#fsis-united-states-department-of-agriculture-food-safety-inspection-service .unnumbered}

## Guidance #152 United States Food & Drug Administration Center for Veterinary Medicine Guidance #152 entitled, \"Evaluating the Safety of Antimicrobial New Animal Drugs with Regard to Their Microbiological Effects on Bacteria of Human Health Concern\" \[Error: Reference source not found\] {#guidance-152-united-states-food-drug-administration-center-for-veterinary-medicine-guidance-152-entitled-evaluating-the-safety-of-antimicrobial-new-animal-drugs-with-regard-to-their-microbiological-effects-on-bacteria-of-human-health-concern-error-reference-source-not-found .unnumbered}

## MAC *Mycobacterium avium* complex {#mac-mycobacterium-avium-complex .unnumbered}

## MAI *Mycobacterium avium-intracellulare* {#mai-mycobacterium-avium-intracellulare .unnumbered}

## MPN Mean probable number {#mpn-mean-probable-number .unnumbered}

## MBC Minimal bactericidal concentration {#mbc-minimal-bactericidal-concentration .unnumbered}

## MIC Minimal inhibitory concentration {#mic-minimal-inhibitory-concentration .unnumbered}

## MLS~B~ Macrolide-lincosamide-streptogramin-component B {#mlsb-macrolide-lincosamide-streptogramin-component-b .unnumbered}

## NAHMS United States National Animal Health Monitoring System {#nahms-united-states-national-animal-health-monitoring-system .unnumbered}

## NARMS United States National Antimicrobial Resistance Monitoring System {#narms-united-states-national-antimicrobial-resistance-monitoring-system .unnumbered}

## NCCLS National Committee for Clinical Laboratory Standards {#nccls-national-committee-for-clinical-laboratory-standards .unnumbered}

## PAE Post-antibiotic effect {#pae-post-antibiotic-effect .unnumbered}

## RNA Ribonucleic acid {#rna-ribonucleic-acid .unnumbered}

## SRD Swine respiratory disease {#srd-swine-respiratory-disease .unnumbered}

## USDA United States Department of Agriculture  {#usda-united-states-department-of-agriculture .unnumbered}

# GLOSSARY OF TERMS {#glossary-of-terms .unnumbered}

The following terms have been defined by the US Food & Drug
Administration Center for Veterinary Medicine (FDA/CVM) in the Guidance
for Industry #152 (Guidance #152), entitled, \"Evaluating the Safety of
Antimicrobial New Animal Drugs with Regard to Their Microbiological
Effects on Bacteria of Human Health Concern\" \[[^1]\]. The definitions
below are copied verbatim from Guidance #152 and are the definitions
used in this document.

**Consequence assessment:** The consequence assessment describes the
relationship between specified exposures to a biological agent (the
hazardous agent) and the consequences of those exposures. For the
purposes of this risk assessment, FDA has decided that the potential
human health consequences of exposure to the defined hazardous agent may
be estimated qualitatively by considering the human medical importance
of the antimicrobial drug in question.

**Exposure assessment:** The exposure assessment describes the
likelihood of human exposure to the hazardous agent through food-borne
exposure pathways. The exposure assessment should estimate qualitatively
the probability of this exposure to bacteria of human health concern
through food-related pathways.

**Hazard:** Human illness, caused by an antimicrobial-resistant
bacteria, attributable to an animal-derived food commodity, and treated
with the human antimicrobial drug of interest.

**Hazardous agent**: Antimicrobial-resistant food-borne bacteria of
human health concern that are in or on a food-producing animal as a
consequence of the proposed use of the antimicrobial new animal drug.

**Hazard characterization:** The process by which one may identify the
hazard and the conditions that influence the occurrence of that hazard.
This is based upon drug-specific information, bacteria/resistance
determinant information, and the methodology for the determination of
"resistant" or "susceptible" bacteria.

**Release assessment:** The release assessment should describe those
factors related to the antimicrobial new animal drug and its use in
animals that contribute to the emergence of resistant bacteria or
resistance determinants (i.e., release of the hazardous agent) in the
animal. The release assessment should also estimate qualitatively the
probability that release of the hazardous agent would occur. For the
purposes of this assessment process, the boundaries of the release
assessment span from the point the antimicrobial new animal drug is
administered to the food-producing animal, to the point the animal is
presented for slaughter or the animal-derived food is collected.

**Risk:** The probability that human food-borne illness is caused by a
specified antimicrobial-resistant bacteria, is attributable to a
specified animal-derived food commodity, and is treated with the human
antimicrobial drug of interest.

**Risk estimation:** The overall estimate of the risk associated with
the proposed use of the drug in the target food-producing animals
following the integration of the release assessment, exposure assessment
and consequence assessment. The risk rankings represent the relative
potential for human health to be adversely impacted by the emergence of
antimicrobial resistance associated in a food-borne pathogen with the
use of the drug in food-producing animals

# EXECUTIVE SUMMARY {#executive-summary .unnumbered}

Tulathromycin is a macrolide antimicrobial agent proposed for
therapeutic use in treatment of swine respiratory disease (SRD) and
bovine respiratory disease (BRD). It is formulated for parenteral
injection as a single dose to provide a full course of therapy, and will
be available only by veterinary prescription.

Tulathromycin has a semi-synthetic, 15-membered macrolide ring
structure. It is a member of the triamilide subclass of macrolides
because it has three polar amine groups, which distinguish its structure
from other macrolides, including the azalides and ketolides.
Biochemical, microbiological, genetic and molecular studies have been
reviewed and accepted by the FDA/CVM that are relevant to
understanding: 1) the mechanism of action of tulathromycin, 2) the
activity of tulathromycin against macrolide-sensitive and resistant
organisms, 3) cross-resistance mechanisms and 4) potential of
tulathromycin to select for resistance development in foodborne
pathogens of bacteria associated with livestock.

Despite the unique structural features and pharmacokinetic properties of
tulathromycin, the bacterial mechanisms of resistance to this molecule
are consistent with those applicable to macrolides approved for various
uses in livestock for the past 30 years. Furthermore, the FDA/CVM has
reviewed and accepted data supporting that tulathromycin has a
cross-resistance profile comparable to that observed for the older
macrolides used in animal medicine, but *not* like the ketolide subclass
of macrolides more recently introduced for use in human medicine.
Tulathromycin has minimal inhibitory effect on protein synthesis by
erythromycin-resistant ribosomes. Furthermore, tulathromycin is not
active against macrolide-resistant bacteria (notably macrolide-resistant
*Streptococcus pneumoniae*) that are susceptible to telithromycin, a
member of the ketolide subclass of macrolides.

## Hazard Analysis Summary {#hazard-analysis-summary .unnumbered}

Tulathromycin has some *in vitro* activity against *E. coli, Salmonella*
and *Enterococcus* under standardized *in vitro* test conditions.
However, the activity against these organisms is pH dependent and
substantially diminished at the neutral to acidic pH that occurs in the
colonic contents and feces of animals. Thus, tulathromycin has low
potential to select for resistant organisms or resistance determinants
the colonic content and in feces.

Macrolides do not have label indications for treatment of infectious
disease due to *E. coli* or *Salmonella* in humans; most macrolides do
not have appreciable activity *in vitro* against these organisms;
neither quality control performance standards nor clinical
susceptibility breakpoints have been generated for macrolides for these
organisms; and macrolide resistance is not a component of national
surveillance or resistance of clinical isolates from humans.
Furthermore, macrolides are not a drug of choice for treatment of
enterococcal infections, due in part to the high macrolide resistance
rates in *Enterococcus* species documented since the 1970\'s \[[^2]\].
For these reasons, macrolide resistance in *E. coli, Salmonella*, and
*Enterococcus* is not addressed in this risk estimation.

Macrolides can be used for treatment of campylobacteriosis in patients
requiring antimicrobial therapy. Therefore the risk of
campylobacteriosis requiring macrolide therapy is considered in
accordance with Guidance #152 \[Error: Reference source not found\] as
follows:

THE HAZARD **(defined by Guidance #152 as \"Human illness, caused by an
antimicrobial-resistant bacteria, attributable to an animal-derived food
commodity, and treated with the human antimicrobial drug of
interest\")** being considered is campylobacteriosis, caused by a
macrolide-resistant *Campylobacter*, attributable to a food commodity
derived from swine or cattle, and treated with a macrolide.

THE HAZARDOUS AGENT (**defined by Guidance #152 as
\"Antimicrobial-resistant food-borne bacteria of human health concern
that are in or on a food-producing animal as a consequence of the
proposed use of the antimicrobial new animal drug.\"**) being considered
in this document is: macrolide-resistant food-borne *Campylobacter* that
are in or on cattle or swine as a consequence of the proposed use of
tulathromycin.

THE SPECIFIC RISK **(defined by Guidance #152 as \" The probability that
human food-borne illness is caused by a specified antimicrobial
resistant bacteria, is attributable to a specified animal-derived food
commodity, and is treated with the human antimicrobial drug of
interest\")** being considered is the probability that
campylobacteriosis is caused by a macrolide- resistant *Campylobacter*,
is attributable to a bovine- or swine-derived food commodity, and is
treated with a macrolide.

## Release Assessment Summary  {#release-assessment-summary .unnumbered}

Taking into account the factors outlined in Guidance #152 for the
release assessment, there is a \"Low\" probability that release of
macrolide-resistant *Campylobacter* will occur, for several reasons.
First, the microbiological activity of tulathromycin is substantially
diminished due to the neutral to acidic pH in colonic contents and in
feces and binding to fecal substrates \[[^3]\]. Second, macrolide
resistance in *Campylobacter* occurs by a mutational event in
*Campylobacter*, not by acquisition of a macrolide resistance gene(s)
and thus resistance transfer is not evident in this organism \[Section
1.7\]. Third, the observed frequency \[Error: Reference source not
found\] of mutation *in vitro* to tulathromycin- or other
macrolide-resistance for *Campylobacter* and other organisms tested (*E.
coli, Enterococcus* and *Salmonella*) was below the detection limits
expected for spontaneous mutation \[[^4],[^5]\]. Fourth, no unique
mechanisms of macrolide resistance have been detected, selected or
induced in the presence of tulathromycin in studies reviewed and
accepted by the FDA/CVM \[Error: Reference source not found\]. Fifth,
the proposed use of tulathromycin is consistent with appropriate
judicious use principles of veterinary medicine. Tulathromycin will be
administered by parenteral injection, under veterinary prescription
only, to individual animals requiring treatment due to bacterial
respiratory disease (cattle and swine) or at known high risk for
bacterial respiratory disease (cattle). It is not intended for whole
herd medication. The proposed single dose regimen provides a full course
of therapy, which diminishes the likelihood of recrudescence occurring
due to non-compliance by the user. A single dose regimen also reduces
overall stress to the production animal associated with repeated drug
administration. The product is intended for use consistent with
judicious use principles for a therapeutic antibacterial in cattle and
swine. Finally, bacterial respiratory disease is most frequently
encountered in animals, well before the animal is to be shipped from the
production site for slaughter, packaging, and entry into the retail food
chain.

Three macrolides (erythromycin, tylosin, and tilmicosin) are currently
approved for use in cattle and swine. Erythromycin is approved for use
in cattle and swine as an injectable formulation. Tylosin is approved
for use in swine and cattle as both an injectable formulation and as a
feed premix. Tilmicosin is approved for use in cattle as an injectable
formulation and as a feed premix in swine. Tulathromycin will be
available only as an injectable formulation. The selection pressure
exerted by tulathromycin is not expected to contribute substantially to
that of macrolide products currently available for use in livestock.

To date, macrolide resistance in *Campylobacter* appears to occur as a
result of a mutational event, and not from a gene transfer event
(Sections 1.7.1.1,1.7.2.3). The 1997-2001 data generated from the US
Centers for Disease Control (CDC) National Antimicrobial Resistance
Monitoring System (NARMS) program for enteric bacteria \[[^6]\] show
that the prevalence of macrolide resistance is only between 1-3% among
*Campylobacter* isolates from humans, with no apparent trends over time
and despite macrolide use in swine, cattle and poultry for over 30
years.

Therefore, the release assessment is qualitative ranked as a \"Low\"
probability that release of a macrolide-resistant *Campylobacter* would
occur.

## Exposure Assessment Summary {#exposure-assessment-summary .unnumbered}

Guidance #152 states, \"The exposure assessment describes the likelihood
of human exposure to the hazardous agent through food-borne exposure
pathways. The exposure assessment should estimate qualitatively the
probability of this exposure to bacteria of human health concern through
food-related pathways.\"

In this exposure assessment, the likelihood of human exposure to
macrolide-resistant *Campylobacter* through the food-borne route uses
the algorithm outlined in Table 5 of Guidance #152, copied in Figure 1
to integrate the ranking of food consumption and *Campylobacter*
contamination.

> Figure 1 CVM Guidance #152 Table 5 \[Error: Reference source not
> found\]: Possible process for ranking qualitatively the probability of
> human exposure to *Campylobacter* in a given food commodity:

  -------------------- ----------------- ---------------- ----------------
                       **Probability of                   
                       human exposure to                  
                       a given                            
                       bacteria**                         

                       **Amount of food                   
                       commodity (beef                    
                       or swine) being                    
                       consumed**                         

  **Amount of food     **High**          **Medium**       **Low**
  commodity                                               
  contamination**                                         

  **High**             **H**             **H**            **M**

  **Medium**           **H**             **M**            **L**

  **Low**              **M**             **L**            **L**
  -------------------- ----------------- ---------------- ----------------

**Cattle.** According to Guidance #152 \[Error: Reference source not
found\], the Exposure Assessment describing the likelihood of human
exposure to macrolide-resistant *Campylobacter* through consumption of
beef can be assigned by integrating the US national beef consumption
data (62.9 pounds/capita/year) listed in Table 2 of Guidance #152 and
ranked as \"High\", with *Campylobacter* contamination rate data for
cattle carcass and ground meat (0-4%) listed in Guidance #152 Table 4
and ranked as \"Low\".

Applying the \"High\" beef consumption ranking and the \"Low\"
*Campylobacter* contamination ranking to the algorithmic process
described in Table 5 in Guidance #152 (\[Error: Reference source not
found\]; copied in Figure 1 of this document), the Exposure Assessment
for human exposure to *Campylobacter* through beef consumption is
qualitatively ranked as \"Medium\".

**Swine.** According to Guidance #152, the exposure assessment
describing the likelihood of human exposure to macrolide-resistant
*Campylobacter* through consumption of pork meat can be assigned by
integrating US national pork meat consumption data (46.7
pounds/capita/year) listed in Table 2 of Guidance #152 and ranked as
\"High\" \[Error: Reference source not found\]) and using the
*Campylobacter* contamination rate data for pork carcasses (32%) listed
in Table 5 of Guidance #152 \[Error: Reference source not found\] and
ranked as \"High\". However, the Sponsor proposes that the contamination
rate of pork carcasses is *not* representative of *Campylobacter*
contamination rates of pork meat. Contamination rates of pork meat are
consistently much lower [\<]{.underline} 5% than that of pork carcasses
in the literature (see Section 2.4.3.5). Moreover, contamination rates
were 1% for pork chops in the 2002 and 2003 NARMS Retail Meat
Surveillance Program \[[^7]\]. Thus, the contamination rate for pork
meat should be qualitatively ranked as \"Low\".

Applying the \"High\" pork consumption ranking and the \"Low\"
*Campylobacter* contamination ranking of pork meat ([\<]{.underline}5%),
to the algorithmic process described in Table 5 in Guidance #152
(\[Error: Reference source not found\]; copied in Figure 1 of this
document), the Exposure Assessment for human exposure to *Campylobacter*
through pork consumption is qualitatively ranked as \"Medium\".

## Consequence Assessment Summary {#consequence-assessment-summary .unnumbered}

The Consequence Assessment for macrolide use in human medicine is
\"Critically Important \" because macrolides are used for treatment of
the foodborne pathogen, *Campylobacter,* associated with food-producing
animals and because they are important for use in treatment of
Legionnaire\'s disease, and prophylaxis and therapy for serious disease
due to *Mycobacterium avium* Complex (MAC)/*Mycobacterium
avium-intracellulare* (MAI), as presented in Appendix A of Guidance #152
\[Error: Reference source not found\].

## Overall Qualitative Risk Estimation is \"High\" for all \"Critically Important\" Drugs {#overall-qualitative-risk-estimation-is-high-for-all-critically-important-drugs .unnumbered}

Table 8 of Guidance #152 shows that all drugs ranked as \"Critically
Important\" also have an overall "High\" Risk Estimation, regardless of
exposure or release assessment.

## Approval and Management Considerations {#approval-and-management-considerations .unnumbered}

There are inherent characteristics of tulathromycin and its proposed use
that lower the concern for selection of macrolide-resistant
*Campylobacter.* First, tulathromycin activity is attenuated by the pH
and binding in the colonic contents and feces. Second, macrolide
resistance occurs in *Campylobacter* by chromosomal mutation and not by
gene acquisition, the latter of which is of greater concern for gut
flora. Third, macrolide resistance in *Campylobacter* isolates from
humans has remained [\<]{.underline} 3% with no obvious trends over time
in the NARMS surveys \[Error: Reference source not found\], after more
than thirty years of parenteral, oral, and topical use of macrolides use
in humans, companion animals and food animals. Fourth, the prevalence of
*Campylobacter* in pork and beef at retail is low (0-5%, Section 2.4).
Fifth, the microbiological, molecular, epidemiological, and historical
database strongly supports that pork and beef are not significant risk
factors for *Campylobacter* causing disease in humans (Section 2.2).

The extent of use limitations listed in Table 7 of Guidance #152
(reproduced in Figure 2) suggest that the proposed extent of use is
considered \"Low\", if the drug is intended for administration to
individual animals, *and* the duration of effective dose is
[\<]{.underline}21 days*.* This is the case of tulathromycin.
Tulathromycin is formulated only for parenteral injection and therefore
must be administered to individual animals.

> Figure 2. Guidance #152 Table 7 \[Error: Reference source not found\]:
> Possible process for ranking (High, Medium, Low) of extent of
> antimicrobial drug use in animals based on duration and method of
> administration.

  ---------------- ---------------- ------------------- -------------------
  **Duration of    **Intended                           
  use**            administration                       
                   to:**                                

                   **Individual     **Select groups or  **Flocks or herds
                   animals**        pens of animals**   of animals**

  **Short**\       L^1^             M^2^                H^3^
  (\<6 days)                                            

  **Medium**\      L                M                   H
  (6-21 days)                                           

  **Long\          M                H                   H
  **(\>21 days)                                         
  ---------------- ---------------- ------------------- -------------------

^1^Low, ^2^Medium, and ^3^High extent of use

Table 8 of Guidance #152 \[Error: Reference source not found\] lists
examples of potential risk management steps for \"Category I\" Drugs
having an overall \"High\" Risk Estimation. These steps may include any
one or a combination of the following considerations: 1) prescription
status, 2) low extent of use, 3) post approval monitoring (e.g., through
NARMS), 4) advisory committee review considerations and(or) 5)
extra-label use restrictions. The Sponsor proposes that: 1)
tulathromycin be approved under veterinary prescription; 2) the extent
of use is inherently low based on the proposed use and parenteral single
dosage; 3) this document is submitted as a component of the Veterinary
Medicine Advisory Committee review; 4) the existing NARMS program
provides monitoring for macrolide resistance in *Campylobacter* and 5)
no extra-label use restrictions should apply. The Sponsor proposes that
extra-label use restrictions are not required for approval, because
macrolides have been and are approved for therapeutic, safe use for a
variety of indications in poultry, swine, cattle and other animal
species.

# Conclusion {#conclusion .unnumbered}

With respect to microbial safety considerations, the proposed label uses
of tulathromycin include management considerations of prescription
status, inherent low extent of use due to parenteral single dose
administration, and Advisory Committee review. Macrolide resistance in
*Campylobacter* is currently monitored in the NARMS program. Therefore,
with these management considerations, approval of the proposed
indications for injectable tulathromycin in cattle and swine poses no
appreciable risk to public health with respect to microbial food safety.

# Hazard Characterization {#hazard-characterization .unnumbered}

Tulathromycin is a semi-synthetic, 15-membered ring macrolide containing
three polar amine groups. It is a member of the triamilide subclass of
macrolides, distinguishing it from other macrolides, including azalides
and ketolides.

The FDA/CVM has reviewed and accepted studies documenting that
tulathromycin has a mechanism of action and cross-resistance profile
that closely match those of erythromycin and tilmicosin as described in
Sections 1.4 and 1.6. Tulathromycin is a protein synthesis inhibitor
like other macrolides \[Error: Reference source not found; Section
1.3.1\]. It competes with erythromycin for binding sites on
macrolide-sensitive ribosomes and inhibits protein synthesis in a
transcription/translation assay of 30S ribosomal subunit preparations
\[Error: Reference source not found\]. Strains resistant to erythromycin
are also resistant to tulathromycin, with the following notable
exception. Unlike erythromycin, tulathromycin (like tilmicosin) is a
comparatively weak inducer of macrolide resistance due to *erm*B
\[Error: Reference source not found\].

There is extensive cross-resistance among tulathromycin, tilmicosin and
erythromycin as demonstrated by the minimal inhibitory concentrations
(MIC) of these drugs tested against gram-positive human pathogens with
known macrolide-resistance determinants \[Error: Reference source not
found\]. However, tulathromycin is not active against
macrolide-resistant *S. pneumoniae* that are susceptible to the
ketolide, telithromycin, used in human medicine. Therefore, the activity
of ketolides and their use in man should not be compromised by
tulathromycin use in animals.

In human medicine in the United States, macrolides are not used for
treatment of infections due to *Salmonella* or *E. coli.* NCCLS does not
list quality control performance standards or susceptibility breakpoint
criteria for erythromycin, azithromycin or clarithromycin for these
gram-negative enteric organisms \[[^8]\]. The NARMS program does not
monitor MICs or susceptibility of *Salmonella* isolates from humans
\[Error: Reference source not found\].

In humans, macrolides are used primarily for treatment of respiratory
tract infections (bronchitis, pneumonia, pharyngitis, sinusitis, ear
infections), and to a lesser extent for skin and urinary tract
infections caused by *Staphylococcus* and *Streptococcus* species. They
are also used to treat infections due to atypical organisms such as
*Legionella pneumophilia, Mycoplasma pneumoniae, Chlamydia pneumoniae,
Haemophilus influenzae* and *Moraxella catarrhalis, Mycobacterium avium*
complex and *Mycobacterium avium-intracellulare.* These organisms are
spread among humans via respiratory droplets and other secretions, and
acquisition of these disease-producing organisms by foodborne routes has
not been noted in the literature \[[^9],[^10],[^11],[^12],[^13],[^14]\].
Guidance #152 states that the risk estimation represents the relative
potential for human health to be adversely impacted by the emergence of
antimicrobial resistance associated in a food-borne pathogen with use of
a drug in food-producing animals. Thus these organisms are not the
subject of this document.

Macrolides are not generally used as therapeutic agents for treatment of
nosocomial *Enterococcus* infections, due to their limited activity
against these organisms. The NCCLS does not recommend routine (primary)
testing of macrolides against Group A, Group B and Group U enterococci,
and suggest selective, supplemental reporting of Group C enterococci
from humans \[Error: Reference source not found\]*.*

There are alternatives to conventional macrolides for treatment of
infections caused by respiratory pathogens and enterococci. Ketolides
retain activity against many types of erythromycin-resistant organisms
\[[^15],[^16][^17],[^18]\]. Other drug classes include streptogramins
(e.g., quinupristin/dalfopristin) and oxazolidones (e.g., linezolid)
both of which act by inhibiting protein synthesis. The latter drug
classes do not produce cross-resistance to conventional macrolides such
as erythromycin and azithromycin \[[^19],[^20]\] and bind to
macrolide-resistant ribosomes. Since the tulathromycin mechanism of
action and resistance are characteristic of the conventional macrolides,
bacterial cross-resistance to the latter drug classes is not expected or
likely.

There are concerns that use of macrolides in livestock can increase the
pool of transferable macrolide resistance determinants in *Enterococcus*
which may ultimately affect human therapeutic needs for treatment of
vancomycin-resistant and streptogramin-resistant *E. faecium*
\[[^21],[^22]\]. While tulathromycin is active against *Enterococcus*
species *in vitro*, its activity is highly attenuated at the neutral to
acidic pH normally encountered in colonic contents and in feces (See
Section 1.4.2). At these pH values, the activity of tulathromycin toward
these organisms is substantially attenuated, limiting the potential for
tulathromycin to exert selective pressure for resistance development or
transfer among these organisms. Furthermore, studies show that
tulathromycin binding occurs when tulathromycin is added to fecal
slurries (Section 1.5.1.3).

Considering this information, this risk estimation focuses on
campylobacteriosis caused by macrolide-resistant *Campylobacter.*
Tulathromycin is active *in vitro* against *Campylobacter.* Macrolides
and fluoroquinolones are the drugs typically used to treat
campylobacteriosis in patients in need of antimicrobial therapy.
Currently, macrolide resistance in *Campylobacter* isolated from humans
is low. In the NARMS reports \[Error: Reference source not found\], the
prevalence of macrolide resistance among *Campylobacter* isolates from
humans was between 1 and 3% from 1997-2001 \[Error: Reference source not
found\], with no discernable trends over time.

From the available information, and in context of Guidance #152 \[Error:
Reference source not found\], the qualitative risk assessment for
tulathromycin addresses the following:

THE HAZARD **(defined by Guidance #152 as \"Human illness, caused by an
antimicrobial-resistant bacteria, attributable to an animal-derived food
commodity, and treated with the human antimicrobial drug of
interest\")** being considered is campylobacteriosis, caused by a
macrolide-resistant *Campylobacter*, attributable to a food commodity
derived from swine or cattle, and (the patient is) treated with a
macrolide.

THE HAZARDOUS AGENT (**defined by Guidance #152 as
\"Antimicrobial-resistant food-borne bacteria of human health concern
that are in or on a food-producing animal as a consequence of the
proposed use of the antimicrobial new animal drug.\"**) being considered
in this document is: macrolide-resistant food-borne *Campylobacter* that
are in or on cattle or swine as a consequence of the proposed use of
tulathromycin.

THE SPECIFIC RISK **(defined by Guidance #152 as \" The probability that
human food-borne illness is caused by a specified antimicrobial
resistant bacteria, is attributable to a specified animal-derived food
commodity, and is treated with the human antimicrobial drug of
interest\")** being considered is the probability that
campylobacteriosis is caused by a macrolide-resistant *Campylobacter*,
is attributable to bovine- or swine-derived food commodity, and is
treated with a macrolide.

# Qualitative Risk ESTIMATION {#qualitative-risk-estimation .unnumbered}

# Release assessment

## Overview

Guidance #152 states \[Error: Reference source not found\]: \"The
release assessment should describe those factors related to the
antimicrobial new animal drug and its use in animals that contribute to
the emergence of resistant bacteria or resistance determinants (i.e.,
release of the hazardous agent) in the animal. The release assessment
should also estimate qualitatively the probability that release of the
hazardous agent would occur. For the purposes of this assessment
process, the boundaries of the release assessment span from the point
the antimicrobial new animal drug is administered to the food-producing
animal, to the point the animal is presented for slaughter or the
animal-derived food is collected.\"

Taking into account the factors outlined in Guidance #152 for the
release assessment, there is a \"Low\" probability that release of
macrolide-resistant *Campylobacter* will occur, for several reasons.
First, the microbiological activity of tulathromycin is substantially
diminished due to the neutral to acidic pH in colonic contents and in
feces and binding to fecal substrates \[Error: Reference source not
found\]. Second, macrolide resistance in *Campylobacter* occurs by a
mutational event in *Campylobacter*, not by acquisition of a macrolide
resistance gene(s) and thus resistance transfer is not evident in this
organism \[Section 1.7\]. Third, the observed frequency \[Error:
Reference source not found\] of mutation *in vitro* to tulathromycin- or
other macrolide-resistance for *Campylobacter* and other organisms
tested (*E. coli, Enterococcus* and *Salmonella*) was below the
detection limits expected for spontaneous mutation \[Error: Reference
source not found,Error: Reference source not found\]. Fourth, no unique
mechanisms of macrolide resistance have been detected, selected or
induced in the presence of tulathromycin in studies reviewed and
accepted by the FDA/CVM \[Error: Reference source not found\]. Fifth,
the proposed use of tulathromycin is consistent with appropriate
judicious use principles of veterinary medicine. Tulathromycin will be
administered by parenteral injection, under veterinary prescription
only, to individual animals requiring treatment due to bacterial
respiratory disease (cattle and swine) or at known high risk for
bacterial respiratory disease (cattle). It is not intended for whole
herd medication. The proposed single dose regimen provides a full course
of therapy, which diminishes the likelihood of recrudescence occurring
due to non-compliance by the user. A single dose regimen also reduces
overall stress to the production animal associated with repeated drug
administration. The product is intended for use consistent with
judicious use principles for a therapeutic antibacterial in cattle and
swine. Finally, bacterial respiratory disease is most frequently
encountered in animals, well before the animal is to be shipped from the
production site for slaughter, packaging, and entry into the retail food
chain.

Three macrolides (erythromycin, tylosin, and tilmicosin) are currently
approved for use in cattle and swine. Erythromycin is approved for use
in cattle and swine as an injectable formulation. Tylosin is approved
for use in swine and cattle as both an injectable formulation and as a
feed premix. Tilmicosin is approved for use in cattle as an injectable
formulation and as a feed premix in swine. Tulathromycin will be
approved only as an injectable formulation. The selection pressure
exerted by tulathromycin is not expected to contribute substantially to
that of macrolide products currently available for use in livestock.

To date, macrolide resistance in *Campylobacter* appears to occur as a
result of a mutational event, and not from a gene transfer event
(Section 1.7.1.1,1.7.2.3). The 1997-2001 data generated from the US
Centers for Disease Control (CDC) National Antimicrobial Resistance
Monitoring System (NARMS) program for enteric bacteria \[Error:
Reference source not found\] show that the prevalence of macrolide
resistance is only between 1-3% among *Campylobacter* isolates from
humans, with no apparent trends over time and despite macrolide use in
swine, cattle and poultry for over 30 years.

Therefore, the release assessment is qualitative ranked as a \"Low\"
probability that release of a macrolide-resistant *Campylobacter* would
occur.

## Product description

### Product formulation

> **Active ingredients:** Tulathromycin in solution for parenteral
> injection

### Proposed conditions of use

> **Route of administration:** Parenteral injection in cattle and swine
>
> **Dosage regimen:** Administered as a single injection at the proposed
> dose that will provide a full course therapy (cattle or swine)
>
> **Proposed Indication**: Treatment of bacterial infections due to
> proposed label pathogens causing bovine respiratory disease (BRD) and
> control of respiratory disease in cattle at high risk of developing
> BRD, associated with target pathogens
>
> Treatment of bacterial infections due to proposed label pathogens
> causing swine respiratory disease (SRD)
>
> **Target Animal Species:** Swine and beef cattle (not for use in
> lactating dairy cattle or in pre-ruminant calves)
>
> **Proposed withdrawal time:** A pre-slaughter withdrawal time will be
> assigned by FDA/CVM.

### Drug substance description

> **Drug Class:** Macrolide
>
> **Macrolide subclass** Tulathromycin differs from other macrolides
> since it has three amino groups. Thus it has a chemical subclass
> designation of triamilide distinguishing its structure from azalides,
> ketolides, and other macrolides \[[^23],[^24]\]. Tulathromycin is used
> exclusively for veterinary medicine.
>
> **Molecular formula:** C~41~H~79~N~3~O~12~
>
> **Molecular weight** 806.23
>
> **Chemical tructure of tulathromycin**
>
> ![](media/image1.wmf)

## Mechanism and type of action 

### Mechanism of action

Macrolides inhibit protein synthesis by binding to bacterial ribosomes,
stimulating the dissociation of peptidyl-tRNA from the ribosome during
the translocation process \[Error: Reference source not
found,[^25],Error: Reference source not found\]. Translation inhibition
studies and ribosomal binding studies of tulathromycin, reviewed and
accepted by the FDA/CVM, show that the mechanism of action of the
triamilide to involve ribosomal binding, comparable to that observed for
other macrolides.

Tulathromycin was tested in transcription-translation assays for protein
synthesis by 30S ribosomal subunit preparations from
macrolide-susceptible and resistant *E. coli* (Table 1). The inhibitory
activity of tulathromycin was comparable to that determined for
erythromycin, tilmicosin and clarithromycin. None of these drugs
inhibited protein synthesis of 30S preparations from macrolide-resistant
ribosomes of *E. coli.*

+--------------------+-----------------------+-------------------------+
| []{#__RefHea       |                       |                         |
| ding___Toc83018474 |                       |                         |
| .anchor}Table 1.   |                       |                         |
| Inhibitory         |                       |                         |
| activity of        |                       |                         |
| macrolides in a    |                       |                         |
| transcr            |                       |                         |
| iption-translation |                       |                         |
| assay using        |                       |                         |
| ribosomes isolated |                       |                         |
| from *E. coli*\*   |                       |                         |
+--------------------+-----------------------+-------------------------+
| **Compound**       | **Mean IC~50~ (M)^†^  | **Mean IC~50~ (M)^†^    |
|                    | for                   | for macrolide-resistant |
|                    | macrolide-sensitive   | ribosome**              |
|                    | ribosome (std         |                         |
|                    | deviation)**          |                         |
+--------------------+-----------------------+-------------------------+
| Tulathromycin      | 0.44 (0.1)            | \>150                   |
+--------------------+-----------------------+-------------------------+
| Erythromycin       | 0.57 (0.17)           | \>150                   |
+--------------------+-----------------------+-------------------------+
| Tilmicosin         | 0.39 (.04)            | \>150                   |
+--------------------+-----------------------+-------------------------+
| Clarithromycin     | 0.64 (0.14)           | \>150                   |
+--------------------+-----------------------+-------------------------+
| Quinupr            | 0.99 (0.05)           | 0.53                    |
| istin/dalfopristin |                       |                         |
+--------------------+-----------------------+-------------------------+
| \*Determinations   |                       |                         |
| with               |                       |                         |
| eryt               |                       |                         |
| hromycin-sensitive |                       |                         |
| or --resistant 30S |                       |                         |
| subunits \[Error:  |                       |                         |
| Reference source   |                       |                         |
| not found,[^26]\]. |                       |                         |
|                    |                       |                         |
| †IC~50~ Drug       |                       |                         |
| concentration that |                       |                         |
| inhibits protein   |                       |                         |
| synthesis by 50%.  |                       |                         |
+--------------------+-----------------------+-------------------------+

These data show that the potency of tulathromycin toward
erythromycin-sensitive ribosomes is comparable to that of conventional
macrolides. The data also show that tulathromycin, tilmicosin, and
erythromycin are not active against erythromycin-resistant ribosomes.
Newer macrolide derivatives such as the ketolide, telithromycin
developed for human medicine, are generally active against resistant
ribosomes. Thus tulathromycin more closely resembles erythromycin and
tilmicosin rather than the ketolides or streptogramins in its activity
\[Error: Reference source not found,[^27]\].

Additional studies have further characterized tulathromycin binding to
macrolide-sensitive ribosomes. In a competitive ribosomal binding assay
measuring the displacement of ^14^C-erythromycin from the ribosome,
tulathromycin the observed equilibrium concentrations displacing 50% of
the bound radiolabel (i.e., EC~50~) for tulathromycin was 0.4 M,
compared to values of 1.5 and 0.78 M for non-labeled erythromycin and
tilmicosin, respectively) \[Error: Reference source not found\]. These
observations suggest that the tulathromycin binding site overlaps the
binding site of erythromycin.

Collectively, the results from these studies \[Error: Reference source
not found\], support that the tulathromycin mechanism of action involves
ribosomal binding, the same mechanism of action of macrolides that are
commonly used in veterinary and human medicine. The inability of
tulathromycin, erythromycin and tilmicosin to inhibit
transcription/translation in erythromycin-resistant ribosomes
distinguishes the mechanism from streptogramins and from ketolides.

### Type of action

Macrolide blocking of protein synthesis generally has a bacteriostatic
effect on the susceptible cell \[Error: Reference source not
found,Error: Reference source not found,Error: Reference source not
found\]. Macrolides are generally considered bacteriostatic agents
although bactericidal effects have been observed for some drug-bacterial
species combinations \[[^28]\]. Ultimately this characteristic is
dependent on the drug concentration, bacterial species and *in vitro*
testing conditions \[[^29],[^30]\].

MIC, MBC and time-kill kinetic studies \[Error: Reference source not
found\] show that bactericidal activity of tulathromycin against
*Campylobacter coli* and *jejuni* isolates from animals and
bacteriostatic activity against animal isolates of *Enterococcus, S.
aureus, E. coli* and *S. enterica* serotype Typhimurium \[Error:
Reference source not found\]. Bactericidal activity of tulathromycin has
also been observed for some of the targeted veterinary isolates \[Error:
Reference source not found\].

## Spectrum of activity 

Data reviewed and accepted by the FDA/CVM show that tulathromycin is a
broad-spectrum antibiotic, with *in vitro* activity against certain
gram-negative and gram-positive bacterial pathogens, including the
bacterial pathogens most commonly associated with bovine and swine
respiratory disease \[Error: Reference source not found\].

### Spectrum of activity against foodborne organisms listed in Guidance #152.

Table 2 lists the observed *in vitro* activity of tulathromycin against
*Enterococcus, Campylobacter, Salmonella* and *E. coli* tested at pH
7.2-7.4, consistent with NCCLS recommended standard test conditions
\[[^31]\].

+-------------------------+--------+-----------+-----------+--------+
| []{#__R                 |        |           |           |        |
| efHeading___Toc83018475 |        |           |           |        |
| .anchor}Table 2.        |        |           |           |        |
| Tulathromycin MICs for  |        |           |           |        |
| selected bacterial      |        |           |           |        |
| isolates.               |        |           |           |        |
+=========================+========+===========+===========+========+
| Microorganism           | No.    | MIC       |           |        |
|                         | s      | (g/ml)\*  |           |        |
|                         | trains |           |           |        |
+-------------------------+--------+-----------+-----------+--------+
|                         |        | Range     | MIC50     | MIC90  |
+-------------------------+--------+-----------+-----------+--------+
| *Campylobacter*         | 30     | 0.25 --   | 0.5       | 64     |
| species^†^              |        | 128       |           |        |
+-------------------------+--------+-----------+-----------+--------+
| *Enterococcus faecalis* | 9      | 4.0 -     | 8.0       | NA     |
|                         |        | \>128     |           |        |
+-------------------------+--------+-----------+-----------+--------+
| *Enterococcus faecium*  | 21     | 4.0 - \>  | 8.0       | \>128  |
|                         |        | 128       |           |        |
+-------------------------+--------+-----------+-----------+--------+
| *Enterococcus*          | 8      | 4.0 -     | 4.0       | NA     |
| species^‡^              |        | \>128     |           |        |
+-------------------------+--------+-----------+-----------+--------+
| *E. coli*               | 16     | 4.0 --    | 8.0       | 8.0    |
|                         |        | 8.0       |           |        |
+-------------------------+--------+-----------+-----------+--------+
| *Salmonella* spp.^§^    | 15     | 4.0 -     | 4.0       | 8.0    |
|                         |        | \>128     |           |        |
+-------------------------+--------+-----------+-----------+--------+
| \*Standard test         |        |           |           |        |
| conditions used         |        |           |           |        |
| consistent with NCCLS   |        |           |           |        |
| guidelines \[Error:     |        |           |           |        |
| Reference source not    |        |           |           |        |
| found\]. The pH of the  |        |           |           |        |
| bacteriological growth  |        |           |           |        |
| medium was adjusted to  |        |           |           |        |
| 7.2-7.4.                |        |           |           |        |
|                         |        |           |           |        |
| ^†^Isolates include: 2  |        |           |           |        |
| *C. fetus*, 13 *C.      |        |           |           |        |
| jejuni*, 15             |        |           |           |        |
| *Campylobacter*         |        |           |           |        |
| species.                |        |           |           |        |
|                         |        |           |           |        |
| ^‡^Isolates include: 1  |        |           |           |        |
| *E. avium*, 7 *E.       |        |           |           |        |
| gallinarium.*           |        |           |           |        |
|                         |        |           |           |        |
| ^§^Isolates include: 7  |        |           |           |        |
| *Salmonella             |        |           |           |        |
| choleraesuis*, 6        |        |           |           |        |
| *S.enterica s*erotype   |        |           |           |        |
| Dublin, 2 *S.enterica*  |        |           |           |        |
| serotype Enteritidis.   |        |           |           |        |
+-------------------------+--------+-----------+-----------+--------+

### Effects of neutral and acidic pH on observed MIC

The *in vitro* activity of tulathromycin is dependent on the initial pH
of the test medium. When the pH of the test medium was varied under
standard test conditions otherwise consistent with NCCLS guidelines, the
observed MICs were markedly higher at pH values at or below 7.0 compared
pH range (7.2-7.4) used in MIC tests consistent with NCCLS guidance
\[Error: Reference source not found\]. MIC results in Table 3 show that
even a small shift in the initial pH of the testing medium can have a
marked effect on the *in vitro* MIC values.

[]{#__RefHeading___Toc83018476 .anchor}Table 3. Effects of pH on
tulathromycin activity

+--------------------+-------+-------+-------+-------+-------+-------+
| *                  | *     |       |       |       |       |       |
| *Microorganism\*** | *Mean |       |       |       |       |       |
|                    | MIC   |       |       |       |       |       |
|                    | (     |       |       |       |       |       |
|                    | g/mL) |       |       |       |       |       |
|                    | at**  |       |       |       |       |       |
|                    | **p   |       |       |       |       |       |
|                    | H:†** |       |       |       |       |       |
+====================+=======+=======+=======+=======+=======+=======+
|                    | **    | **    | **    | **    | **    | **    |
|                    | 6.5** | 7.0** | 7.2** | 7.4** | 7.6** | 8.0** |
+--------------------+-------+-------+-------+-------+-------+-------+
| *E. coli ATCC      | \>128 | 18.4  | 4.59  | 2.0   | 2.0   | 2.0   |
| 25922*             |       |       |       |       |       |       |
+--------------------+-------+-------+-------+-------+-------+-------+
| *E. faecalis ATCC  | \>128 | 36.8  | 12.1  | 3.48  | 2.0   | 2.30  |
| 29212*             |       |       |       |       |       |       |
+--------------------+-------+-------+-------+-------+-------+-------+
| *S. aureus ATCC    | \>128 | 24.3  | 8.00  | 3.03  | 1.74  | 2.0   |
| 29213*             |       |       |       |       |       |       |
+--------------------+-------+-------+-------+-------+-------+-------+
| \* Quality control |       |       |       |       |       |       |
| isolates obtained  |       |       |       |       |       |       |
| from the American  |       |       |       |       |       |       |
| Type Culture       |       |       |       |       |       |       |
| Collection (ATCC). |       |       |       |       |       |       |
|                    |       |       |       |       |       |       |
| † Standard testing |       |       |       |       |       |       |
| conditions         |       |       |       |       |       |       |
| consistent with    |       |       |       |       |       |       |
| NCCLS methods were |       |       |       |       |       |       |
| used, except that  |       |       |       |       |       |       |
| pH of the culture  |       |       |       |       |       |       |
| medium was varied  |       |       |       |       |       |       |
| as indicated       |       |       |       |       |       |       |
| \[Error: Reference |       |       |       |       |       |       |
| source not         |       |       |       |       |       |       |
| found,Error:       |       |       |       |       |       |       |
| Reference source   |       |       |       |       |       |       |
| not found\].       |       |       |       |       |       |       |
+--------------------+-------+-------+-------+-------+-------+-------+

Similar pH effects were observed in two studies of *Fusobacterium* and
*Bifidobacterium* reviewed and accepted by FDA/CVM. The pH in colonic
contents and in feces is generally below 7.0
\[[^32],[^33],[^34],[^35]\], although not necessarily in high forage
diets in cattle \[Error: Reference source not found,[^36]\].

The pH phenomenon has been documented for other macrolides \[[^37]\]. It
is substantial in the case of tulathromycin due to the pK~a~ of the
three ionizable groups of the molecule \[Error: Reference source not
found\]. Azithromycin has two ionizable groups and erythromycin has one.
At neutral to acidic pH, the high positive charge of the molecule would
decrease lipid solubility, concomitantly reducing the ability of
tulathromycin to diffuse across cell membranes and reach the
intracellular target \[Error: Reference source not found,Error:
Reference source not found\].

### Conclusions regarding susceptibility data

Tulathromycin has a broad spectrum of activity *in vitro*, which is
substantially attenuated at a pH range normally found in the colonic
contents and feces of animals.

## Pharmacokinetics and pharmacodynamics

### Absorption, distribution, metabolism, and elimination (ADME)

Extensive pharmacokinetic and disposition data have been reviewed and
accepted by FDA/CVM for tulathromycin in cattle. Briefly, the
pharmacokinetic profile of tulathromycin administered as a single
injection at the proposed label dose is characterized by rapid and
extensive absorption followed by high distribution and slow elimination
\[[^38],[^39]\]. The maximum concentration in plasma (approximately 0.5
g/mL) is achieved approximately 30 minutes post-dosing. Peak plasma
concentrations are followed by a slow decline in systemic exposure with
an apparent elimination half-life of 90 hours in plasma. The
bioavailability of tulathromycin after parenteral administration in
cattle is approximately 90%. Plasma protein binding is low,
approximately 40%. The volume of distribution at steady-state determined
after intravenous administration is 11 L/kg.

Tulathromycin concentrations in lung homogenate are considerably higher
than those in plasma. There is strong evidence of substantial
accumulation of tulathromycin in neutrophils and alveolar macrophages,
although the *in vivo* concentration of tulathromycin at the site of
pulmonary infection has not been examined.

Extensive pharmacokinetic and disposition data have been reviewed and
accepted by FDA/CVM for tulathromycin in swine. Briefly, the
pharmacokinetic profile of tulathromycin administered as a single
injection at the proposed label dose is also characterized by rapid and
extensive absorption followed by high distribution and slow elimination
\[Error: Reference source not found\]. The maximum concentration in
plasma is approximately 0.6 g/mL, achieved approximately 30 minutes
post-dosing. Peak concentrations are followed by a slow decline in
systemic exposure with an apparent elimination half-life of
approximately 91 hours in plasma. The observed bioavailability of
tulathromycin after parenteral administration is approximately 88%.
Plasma protein binding is low, approximately 40%. The volume of
distribution at steady state determined after intravenous administration
is 13.2 L/kg.

Tulathromycin concentrations in lung homogenate are considerably higher
than those in plasma. There is strong evidence of substantial
accumulation of tulathromycin in neutrophils and alveolar macrophages,
although the *in vivo* concentration of tulathromycin at the site of
pulmonary infection has not been examined.

#### Metabolism 

The FDA/CVM has reviewed and accepted the metabolism files submitted by
the Sponsor. These data show that tulathromycin is not extensively
metabolized and is excreted in the urine and feces, mainly (\>90%) as
unchanged drug.

#### Elimination and tulathromycin residues in colonic contents post-treatment

Radiolabeled drug disposition studies in the target species have been
reviewed and accepted by FDA/CVM. In studies of radiolabeled
tulathromycin administered at the proposed dose to cattle and swine,
approximately 30% to 60% of the total dose was excreted via the
intestinal tract.

In cattle, fecal excretion and urinary excretion were comparable, each
comprising approximately half of the total drug-related residues in
excreta. The highest concentrations of drug residue (determined by
chemical assay) in colonic contents (approximately 8 μg/g) were observed
within the first day after dosing. Excreta residue levels gradually
dropped and by day 5 post-dose, the concentrations in colonic contents
had decreased to less than 1 μg/g \[Error: Reference source not found\].

For swine, approximately two-thirds of the total dose was recovered in
feces, with the highest concentrations (approximately 6.0 g/g) observed
on days 3 and 4-days after dosing. Total drug residues in excreta
gradually dropped and between day 6 and 12 post dose decreased to less
than 1 g/g \[Error: Reference source not found\].

#### Tulathromycin and fecal binding

Tulathromycin binds to fecal material. In equilibration fecal binding
studies \[Error: Reference source not found,[^40]\] wherein
tulathromycin was added to a fecal slurry prepared from four bovine
fecal samples, the observed adsorption coefficient (K~d~) of
tulathromycin was 23.3 (pH 6.7). A similar value was obtained for a
study of human fecal samples submitted to the FDA/CVM. The study showed
that significant percentages of tulathromycin bound to fecal solids that
readily sedimented at low speed centrifugation. Thus binding is another
abiotic factor, in addition to pH (Section 1.4.2), that will attenuate
the activity of tulathromycin entering the colon.

In addition to the equilibrium binding studies, the reduction in
antibacterial activity of tulathromycin in the presence of fecal
material was tested against various organisms. Tulathromycin activity
was reduced in *in vitro* studies wherein sterilized feces were added to
growth medium used for testing tulathromycin against *Enterococcus*, *E.
coli,* *Bifidobacterium* and *Fusobacterium*.

#### Tulathromycin and pH in the colon

Studies reviewed and accepted by the FDA/CVM demonstrate a substantial
attenuation of tulathromycin activity at pH [\<]{.underline}7.0, likely
due to the pK~a~ of the three ionizable amine groups of the molecule.
Whereas NCCLS guidelines specifies that the growth medium for MIC test
medium is to be set between 7.2-7.4 \[Error: Reference source not
found\], the pH of feces and colonic contents can range generally from
6.3 to 6.9 for swine and cattle (See Section 1.4.2). At these pH values,
tulathromycin activity against enteric organisms is markedly attenuated
(Section 1.4.2), lessening its ability to exert selective pressure for
organisms *in vivo*.

#### Conclusions regarding drug residue in the colonic contents and feces

The tulathromycin residues in the colonic contents and feces of animals
dosed parenterally is predominantly (\>90%) unchanged drug \[Error:
Reference source not found\]. Bacterial exposure to microbiologically
active drug is transient due to the single dose, parenteral
administration, and very low due to the abiotic factors such as pH
\[Section 1.4.2\] and binding \[Section 1.5.1.3\]) that affect the
ability of the drug to enter the bacterial cell upon entry of drug
residue in the colon.

### Additional effects 

Post-antibiotic effect (PAE) is defined as the persistent suppression of
bacterial growth *in vitro* after short exposure of bacteria to an
antimicrobial agent. The effect is dependent upon the type and
concentration of the antimicrobial agent, the bacterial species,
duration of exposure, and experimental conditions \[[^41]\].
Post-antibiotic effects have not been well-characterized, but a brief
PAE would be expected after exposure to tulathromycin.

### Pharmacodynamic properties

Although the relationship between tulathromycin and the characteristics
of its antimicrobial effects have not been characterized, macrolides, as
a class, tend to be primarily bacteriostatic, but may be bactericidal
against some pathogens \[[^42],[^43]\]. However, limited *in vitro* data
have indicated that tulathromycin is bactericidal against some
veterinary respiratory pathogens and against *Campylobacter jejuni* and
*C. coli*.

Macrolides also tend to exhibit concentration-independent killing; the
rate of bacterial eradication does not change once serum drug
concentrations reach two to three times the MIC of the targeted
pathogen. Under these conditions, the time that serum concentrations
remain above the MIC becomes a major determinant of antimicrobial
activity. Tulathromycin differs from many other macrolides in that it
has a long elimination half-life. This pharmacokinetic profile is
well-suited to the view that macrolides are time-dependent antimicrobial
agents and perform best when drug concentrations can be maintained above
inhibitory concentrations for the duration of therapy.

## Resistance mechanisms, genetics and location 

### Known mechanisms of resistance in animal and human pathogens 

There are three basic mechanisms of macrolide resistance. Target site
modification is common, and occurs either due to ribosomal RNA
methylation as a result of gene acquisition (e.g., ribosomal methylases
encoded by *erm*A, *erm*B, and *erm*C) or due to a sequence change in
the ribosome as a result of a mutational event. The *erm* genes alter a
site in the 23S ribosomal RNA needed for productive binding of
macrolides, lincosamides, and streptogramin B antibiotics (MLS~B~, see
Section 1.8.1.1). Likewise specific mutation leading to changes in the
ribosome can also impact binding \[Error: Reference source not
found,Error: Reference source not found,[^44],\]. An *erm* gene has not
been documented in macrolide-resistant isolates of *Campylobacter* (See
Section 1.7.2.3 for further discussion). The second basic mechanism is
drug inactivation, either due to phosphorylation of the 2'-hydroxy group
of the amino sugar (e.g., the phosphotransferases encoded by *mph*A and
*mph*B) or due to hydroxylation of the macrolide lactone ring (e.g., the
lactonases encoded by *ere*A and *ere*B). The third mechanism is drug
efflux (e.g., macrolide efflux pumps encoded by *msr*A and *mef*A).
Acquired genes involved in resistance to macrolides have been reviewed
by Roberts *et al.* \[[^45],[^46]\] and are summarized in Table 4.

[]{#__RefHeading___Toc83018477 .anchor}Table 4. Genes, phenotypes, and
bacterial hosts of resistance determinants for the
Macrolide-Lincosamide-Streptogramin group of antimicrobial agents

+----------+-----------+-----------+----------------------------------+
| Re       | R         | Gene\*    | Host Organism(s) Described\*     |
| sistance | esistance |           |                                  |
| mec      | Profile\* |           |                                  |
| hanism\* |           |           |                                  |
+----------+-----------+-----------+----------------------------------+
| R        | Macr      | *erm*(A)  | *Actinobacillus, Staphylococcus, |
| ibosomal | olide^R^, |           | Streptococcus*                   |
| RNA      | Linco     |           |                                  |
| me       | samide^R^ |           |                                  |
| thylase† | Streptogr |           |                                  |
|          | aminB^R^† |           |                                  |
|          |           |           |                                  |
|          | (         |           |                                  |
|          | Streptogr |           |                                  |
|          | aminA^S^) |           |                                  |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(B)  | *Actinobacillus, Clostridium,    |
|          |           |           | Enterococcus , Escherichia,      |
|          |           |           | Klebsiella, Neisseria,           |
|          |           |           | Pediococcus, Staphylococcus,     |
|          |           |           | Streptococcus, Wolinella*        |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(C)  | *Actinobacillus, Bacillus,       |
|          |           |           | Eubacterium, Lactobacillus,      |
|          |           |           | Neisseria, Staphylococcus,       |
|          |           |           | Streptococcus, Wolinella*        |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(D)  | *Bacillus*                       |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(E)  | *Streptomyces‡*                  |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(F)  | *Actinobacillus, Actinomyces,    |
|          |           |           | Bacteroides, Clostridium,        |
|          |           |           | Eubacterium, Fusobacterium,      |
|          |           |           | Gardnerella, Haemophilus,        |
|          |           |           | Neisseria, Porphyromonas,        |
|          |           |           | Prevotella, Peptostreptococcus,  |
|          |           |           | Selenomonas, Streptococcus,      |
|          |           |           | Staphylococcus, Treponema,       |
|          |           |           | Veillonella, Wolinella*          |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(G)  | *Bacillus, Bacteroides*          |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(H), | *Streptomyces‡*                  |
|          |           | (I), (N), |                                  |
|          |           | (O), (S), |                                  |
|          |           | (U), or   |                                  |
|          |           | (V)       |                                  |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(Q)  | *Actinobacillus, Clostridium,    |
|          |           |           | Streptococcus, Wolinella*        |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(R)  | *Aeromicrobium*                  |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(T)  | *Lactobacillus*                  |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(W)  | *Micromonospora*                 |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(X)  | *Corynebacterium*                |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(Y)  | *Staphylococcus*                 |
+----------+-----------+-----------+----------------------------------+
|          |           | *erm*(2)  | *Streptomyces‡*                  |
+----------+-----------+-----------+----------------------------------+
|          |           | *er       | *Streptomyces‡*                  |
|          |           | m*(30.31) |                                  |
+----------+-----------+-----------+----------------------------------+
| Tran     | Oleand    | *ole*(B), | *Streptomyces‡*                  |
| sporters | omycin^R^ | (C)       |                                  |
+----------+-----------+-----------+----------------------------------+
|          | Spir      | *srm*(B)  | *Streptomyces‡*                  |
|          | amycin^R^ |           |                                  |
+----------+-----------+-----------+----------------------------------+
|          | T         | *tlr*(C)  | *Streptomyces‡*                  |
|          | ylosin^R^ |           |                                  |
+----------+-----------+-----------+----------------------------------+
|          | Erythr    | *msr*(A)  | *Staphylococcus*                 |
|          | omycin^R^ |           |                                  |
+----------+-----------+-----------+----------------------------------+
|          | Stre      | *vga*(A), | *Staphylococcus*                 |
|          | ptogramin | (B)       |                                  |
|          | A^R^      |           |                                  |
+----------+-----------+-----------+----------------------------------+
|          | Erythr    | *mef*(A)  | *Acinetobacter, Corynebacterium, |
|          | omycin^R^ |           | Enterococcus , Neisseria         |
|          |           |           | Micrococcus, Staphylococcus,     |
|          |           |           | Streptococcus*                   |
+----------+-----------+-----------+----------------------------------+
| E        | Erythr    | *ere*(A)  | *Citrobacter, Enterobacter,      |
| sterases | omycin^R^ |           | Escherichia, Klebsiella*         |
+----------+-----------+-----------+----------------------------------+
|          | Erythro   | *ere*(B)  | *Escherichia, Klebsiella,        |
|          | mycin^R^‑ |           | Proteus*                         |
+----------+-----------+-----------+----------------------------------+
| Phosph   | Macr      | *mph*(A), | *Escherichia coli*               |
| orylases | olides^R^ | (B)       |                                  |
+----------+-----------+-----------+----------------------------------+
|          | Macr      | *mph*(C)  | *Staphylococcus*                 |
|          | olides^R^ |           |                                  |
+----------+-----------+-----------+----------------------------------+
| \*Table  |           |           |                                  |
| based on |           |           |                                  |
| Tables 2 |           |           |                                  |
| and 3 of |           |           |                                  |
| R        |           |           |                                  |
| eference |           |           |                                  |
| \[Error: |           |           |                                  |
| R        |           |           |                                  |
| eference |           |           |                                  |
| source   |           |           |                                  |
| not      |           |           |                                  |
| found\]  |           |           |                                  |
| and      |           |           |                                  |
| Table 2  |           |           |                                  |
| of       |           |           |                                  |
| R        |           |           |                                  |
| eference |           |           |                                  |
| \[Error: |           |           |                                  |
| R        |           |           |                                  |
| eference |           |           |                                  |
| source   |           |           |                                  |
| not      |           |           |                                  |
| found\]. |           |           |                                  |
| Even     |           |           |                                  |
| though   |           |           |                                  |
| they     |           |           |                                  |
| have     |           |           |                                  |
| ch       |           |           |                                  |
| emically |           |           |                                  |
| distinct |           |           |                                  |
| classifi |           |           |                                  |
| cations, |           |           |                                  |
| the      |           |           |                                  |
| MLS~B~   |           |           |                                  |
| agents   |           |           |                                  |
| are      |           |           |                                  |
| grouped  |           |           |                                  |
| together |           |           |                                  |
| because  |           |           |                                  |
| they     |           |           |                                  |
| have the |           |           |                                  |
| same     |           |           |                                  |
| m        |           |           |                                  |
| echanism |           |           |                                  |
| of       |           |           |                                  |
| action,  |           |           |                                  |
| binding  |           |           |                                  |
| compe    |           |           |                                  |
| titively |           |           |                                  |
| to       |           |           |                                  |
| ove      |           |           |                                  |
| rlapping |           |           |                                  |
| sites on |           |           |                                  |
| the 23S  |           |           |                                  |
| r        |           |           |                                  |
| ibosomal |           |           |                                  |
| RNA of   |           |           |                                  |
| the 50S  |           |           |                                  |
| subunit  |           |           |                                  |
| of the   |           |           |                                  |
| b        |           |           |                                  |
| acterial |           |           |                                  |
| r        |           |           |                                  |
| ibosome. |           |           |                                  |
|          |           |           |                                  |
| †R       |           |           |                                  |
| ibosomal |           |           |                                  |
| me       |           |           |                                  |
| thylases |           |           |                                  |
| confer   |           |           |                                  |
| re       |           |           |                                  |
| sistance |           |           |                                  |
| to       |           |           |                                  |
| Strep    |           |           |                                  |
| togramin |           |           |                                  |
| B        |           |           |                                  |
| co       |           |           |                                  |
| mponents |           |           |                                  |
| (e.g.,   |           |           |                                  |
| prist    |           |           |                                  |
| inamycin |           |           |                                  |
| IB,      |           |           |                                  |
| virgi    |           |           |                                  |
| niamycin |           |           |                                  |
| S,       |           |           |                                  |
| m        |           |           |                                  |
| ikamycin |           |           |                                  |
| B), but  |           |           |                                  |
| not to   |           |           |                                  |
| Strep    |           |           |                                  |
| togramin |           |           |                                  |
| A        |           |           |                                  |
| co       |           |           |                                  |
| mponents |           |           |                                  |
| (prist   |           |           |                                  |
| inamycin |           |           |                                  |
| IIA,     |           |           |                                  |
| virgi    |           |           |                                  |
| niamycin |           |           |                                  |
| M,       |           |           |                                  |
| m        |           |           |                                  |
| ikamycin |           |           |                                  |
| A and    |           |           |                                  |
| syn      |           |           |                                  |
| ergistin |           |           |                                  |
| A) or    |           |           |                                  |
| mixtures |           |           |                                  |
| of       |           |           |                                  |
| strep    |           |           |                                  |
| togramin |           |           |                                  |
| co       |           |           |                                  |
| mponents |           |           |                                  |
| A and B  |           |           |                                  |
| (e.g.,   |           |           |                                  |
| qu       |           |           |                                  |
| inuprist |           |           |                                  |
| in/dalfo |           |           |                                  |
| pristin, |           |           |                                  |
| Virgi    |           |           |                                  |
| niamycin |           |           |                                  |
| M/S).    |           |           |                                  |
|          |           |           |                                  |
| ‡These   |           |           |                                  |
| o        |           |           |                                  |
| rganisms |           |           |                                  |
| are used |           |           |                                  |
| in       |           |           |                                  |
| pr       |           |           |                                  |
| oduction |           |           |                                  |
| of       |           |           |                                  |
| antim    |           |           |                                  |
| icrobial |           |           |                                  |
| agents.  |           |           |                                  |
+----------+-----------+-----------+----------------------------------+

#### In vitro Induction of Macrolide Resistance 

Expression (production) of *erm* genes (MLS~B~ phenotype) in
gram-positive organisms is under either inducible or constitutive
control. Some macrolides serve to stimulate inducible resistance genes,
while other macrolides do not. For example, 14-membered ring macrolides
such as erythromycin stimulate expression of the inducible MLS~B~ genes,
while the 16-membered macrolides such as tilmicosin and tylosin do not.
Thus bacterial strains carrying inducible MLS~B~ are susceptible to
16-membered ring macrolides but are resistant to 14-membered ring
macrolides. Bacteria carrying inducible MLS~B~ genes, however, have
elevated MICs (i.e., resistance) to 16-membered ring macrolides when the
expression of these genes is already induced by 14-membered ring
macrolides at sub-MIC concentrations*.*

In studies reviewed and accepted by the FDA/CVM, tulathromycin only
weakly induced a measurable degree of resistance in a *S. aureus*
(pE194) strain containing a plasmid bearing an inducible *erm* gene.
Thus, this compound is not a potent inducer of resistance compared with
erythromycin. Tilmicosin was not active as an inducer under the test
conditions, either, consistent with earlier reports \[[^47]\]. The
clinical significance of these observations cannot be predicted from one
study. However, the three most commonly used macrolides in human health
(erythromycin, azithromycin, clarithromycin) can induce resistance in
gram-positive clinical isolates that possess inducible MLS~B~ (*erm*)
genes. Historically, clinical isolates are usually inducible, while over
the last decade, a significant percentage of macrolide-resistant
streptococcal and staphylococcal pathogens now demonstrate constitutive
*erm* expression conferring MLS~B~ resistance. It is possible that
macrolides that are able to induce these genes and elevate the MIC may
have favored the development of constitutive expression. If this is the
case, then the observation that tulathromycin is a weak inducer of this
gene lessen the concern for selection of constitutive resistance from
those isolates containing inducible *erm* genes.

### Location of resistance determinants

The genes encoding ribosomal methylases, macrolide transporters, and
macrolide hydrolases and esterases have been documented in the
chromosome and on non-chromosomal genetic elements \[Error: Reference
source not found,Error: Reference source not found\].

### In vitro activity of tulathromycin against human pathogens containing characterized mechanisms of macrolide resistance 

Tulathromycin, erythromycin and tilmicosin were tested against a battery
of clinical bacterial isolates containing known macrolide resistance
genes \[Error: Reference source not found\]. Strains were predominantly
gram-positive organisms of human origin. Genes encoding the common
*ermB* determinant, as well as strains with efflux-mediated resistance
to erythromycin were studied. In general, tulathromycin was less active
(i.e., higher MIC\'s) than erythromycin against the
macrolide-susceptible gram-positive organisms.

The constitutively expressed *ermB* and *ermC* resistance determinant,
commonly found in clinical isolates of *Staphylococcus* and
*Streptococcus*, conferred cross-resistance to erythromycin, tilmicosin
and tulathromycin. However, erythromycin was inactive against the
*Staphylococcus aureus* isolate containing an inducible *erm* gene,
while tulathromycin and tilmicosin retained activity. The observed
inactivity of tulathromycin against this strain is consistent with a
weak potential to induce such genes, as observed in *Staphylococcus
aureus strain* RN4220(pE194) containing a plasmid-mediated, inducible
MLS~B~ resistance determinant (data reviewed and accepted by FDA/CVM;
See Section 1.6.1.1).

Strains containing the efflux resistance determinant, *mef*A, were
resistant to all three drugs, although the change in MIC was not as
substantial as observed for the *erm*-containing isolates.

From these studies reviewed and accepted by FDA/CVM, tulathromycin
resembles erythromycin in terms of the degree to which its activity is
affected by known macrolide resistance determinants, with the exception
of its comparatively weak potential to induce erm genes that are subject
to induction by erythromycin. Thus, these *in vitro* studies support the
premise that tulathromycin, in terms of its cross-resistance profile,
has no difference in potential to select for macrolide-resistance than
macrolides such as erythromycin and tilmicosin currently in use in
veterinary medicine.

## Occurrence and rate of transfer of resistance determinants 

### Transferable resistance determinants 

The *erm* gene can be transferred among bacteria by conjugation,
transposition, and by transduction \[Error: Reference source not
found\]. Researchers have observed *erm* genes encoded on mobile
elements among streptococcal isolates from animals in the early 1980\'s
\[Error: Reference source not found,Error: Reference source not found\].
There are, in theory, an unlimited number of plasmid, transposon or
chromosomal vectors that could serve as mechanisms for transfer. This is
also the case for other macrolide resistance genes including
transporters, esterases and hydrolases.

Preliminary studies reviewed and accepted by the FDA/CVM have been
conducted to measure the frequency of transfer of plasmid-mediated
macrolide resistance when equal concentrations of tulathromycin,
erythromycin or tilmicosin are used as the selective agent \[Error:
Reference source not found\]. The transfer system utilized the
self-transmissible plasmid (pAM1) from *E. faecalis* JH2-2 transferred
to macrolide-susceptible *E. faecalis* OG1X. The plasmid pAM1 \[[^48]\]
encodes a constitutive MLS~B~ gene. Plasmid transfer was detected at a
high frequency of 1-2 x 10^-2^ when any of the macrolides were used for
counter-selection at 50 g/ml. The original MICs of the recipient strain
to all three compounds used were 2-4 g/ml. Therefore, an equivalent
selective pressure was placed on the recipient with all three
macrolides.

These limited data concerning the cell-to-cell transfer of tulathromycin
resistance support that the transfer frequency of a known
plasmid-encoded macrolide resistance gene is comparable whether
tulathromycin or currently marketed macrolides are used in
counter-selection. Since only one system generated transfer frequency
data, it is not possible to make broad conclusions regarding the
relative risk of resistance transfer between foodborne bacteria in the
intestinal tracts of livestock or humans. It is not clear how many
mating pairs and plasmid types (or what experimental conditions for
testing) would be needed to predict selection pressure *in vivo.*
However, given the similar mechanism of action and spectrum of
tulathromycin compared with other macrolides, it is expected that the
selective pressure for resistance transfer will be similar for
macrolides already used in poultry, swine, and cattle. Additionally, at
the neutral to acidic pH of the colonic contents and feces, the
microbiological activity is attenuated.

####  Campylobacter

In molecular examination of macrolide-resistant isolates, the resistance
acquisition is demonstrated to be due to point mutations in the
chromosome of *Campylobacter* (Section 1.7.2.3). Thus studies of
transfer rates of this gene are not applicable to this organism.
Transferable elements encoding macrolide resistance have not been
documented.

### Point mutations

#### Characterization of field isolates for studies of point mutation frequencies

A collection of 57 isolates of *E. coli, Salmonella, Enterococcus*, and
*Campylobacter* were used in pure culture studies to estimate the rates
of mutation to macrolide resistance. The isolates were screened for
presence eight common macrolide resistance determinants (*erm*A,
*erm*B[^49], *erm*C, *ere*A, *mph*A, *mph*B, *msr*A and *mef*A) by
molecular and phenotypic methods. The analyses supported that there was
an absence of inducible macrolide resistance genes (known or
uncharacterized) in the strains tested \[Error: Reference source not
found\]. Thus this collection of isolates was deemed suitable for
evaluating mutation frequencies *in vitro* (Section 1.7.2.2).

#### Mutation frequencies 

The FDA/CVM has reviewed and accepted a study submitted by the Sponsor
wherein the frequency of mutation of *E. coli, Salmonella, Enterococcus*
and *Campylobacter* were determined *in vitro* by recognized
microbiological methods. The collections of isolates described above
were plated in pure cultures on medium containing inhibitory
concentrations of tulathromycin to select for growth of spontaneous
mutants resistant to the triamilide. Resistant mutants were not
detected. Since at least 10^8^ to 10^9^ colony-forming units (CFU) of
each susceptible strain were plated, the frequency of spontaneous
mutants resistant to tulathromycin was less than 10^-8^ or less than
10^-9^.

These results suggest that the frequency of mutation to tulathromycin
occurs *in vitro* are very low, and are consistent with low frequency of
occurrence of spontaneous mutations in ribosomal genes observed with
other macrolides \[Error: Reference source not found,Error: Reference
source not found\].

#### Campylobacter

The frequency of resistant mutants in *C. jejuni/C. coli* strains
exposed to concentrations of tulathromycin equal to 4- to 8-times their
MIC was the mutation rate expected for spontaneous mutation (Section
1.7.2.2).

To date, all macrolide resistance has been documented to occur by
mutation in the chromosome encoding ribosomal RNA of *Campylobacter*
\[[^50],[^51],[^52]\]. There are a few reports of macrolide-resistance
due to mutation in efflux pumps in *Campylobacter* (Section 1.8.1.3)*.*
Jensen and Aarestrup \[Error: Reference source not found\] described a
collection of 54 cattle and swine isolates of *C. coli* that were
resistant to erythromycin (MIC \> 8 g/ml). All of the resistant strains
contained a mutation in position 2230 of the 23S ribosomal DNA, a change
consistent with spontaneous mutation. These results are consistent with
the low frequency of mutation observed to tulathromycin in the studies
described in Section 1.7.2.2. Spontaneous mutation in chromosomally
encoded ribosomal genes is the most likely mechanism by which clinically
relevant macrolide resistance occurs in *Campylobacter.* This is
consistent with the observed rates of low macrolide resistance in
essential genes *Campylobacter*.

## Resistance selection pressures

### Information regarding cross-resistance to other antimicrobial drugs approved in veterinary and human medicine 

All phenotypic, biochemical, and molecular studies support that
tulathromycin, erythromycin and tilmicosin have similar profiles with
respect to known resistance determinants of *Enterococcus,*
*Streptococcus* and *Campylobacter,* with the exception of *erm* genes
that are inducible. Unlike the human use macrolides (erythromycin,
clarithromycin and azithromycin), both tilmicosin and tulathromycin (as
well as the lincosamides and the streptogramins) are poor inducers of
expression of inducible *erm* genes. Cells harboring constitutively
expressed *erm* genes are cross-resistant to the entire MLS~B~ group of
molecules (\[Error: Reference source not found,Error: Reference source
not found\] see Section 1.8.1.1).

Macrolides have been used extensively in human and animal medicine for
years (see Section 1.8.2.2). Rates of macrolide resistance in
*Enterococcus* have been high since the 1970\'s in the United States
\[Error: Reference source not found\]. Therefore, the impacts of
tulathromycin use in view of the widespead and prolonged macrolide use
can be very difficult, if not impossible, to project because selective
pressure has already occurred and continues due to other macrolide use
practices.

#### Macrolide-Lincosamide-Streptogramin B (MLS~B~) group classification and cross resistance

Even though they have chemically distinct classifications[^53], the
MLS~B~ agents are grouped together because they have the same mechanism
of action, binding competitively to overlapping sites on the 23S
ribosomal RNA of the 50S subunit of the bacterial ribosome \[Error:
Reference source not found,Error: Reference source not
found,[^54],[^55]\]. Cross-resistance to MLS~B~ (but not streptogramin
component A) can occur due to mutation or due to acquisition of an *erm*
gene resulting in changes in the 23S ribosomal RNA. Note that other
types of acquired resistance mechanisms (e.g., change in cell
permeability, drug inactivation) do not confer complete cross-resistance
among the macrolides, lincosamides and streptogramins \[Error: Reference
source not found,Error: Reference source not found\]. Constitutively
expressed *erm* genes are the only genes that confer complete
cross-resistance to the MLS~B~ group (but not streptogramin mixtures).
*Erm* genes, notably *erm*B, have been demonstrated in enterococci, but,
to date, *erm* genes have not been demonstrated in *Campylobacter.*

#### MLS~B~ Resistance in Gram-positive cocci from humans

Macrolide resistance due to an *erm* gene has been documented since the
1950\'s among human isolates, soon after the introduction of
erythromycin for use \[Error: Reference source not found,Error:
Reference source not found\]. At first the resistance was characterized
as being inducible by erythromycin (and other 14-membered ring
macrolides), but constitutive resistance conferring cross-resistance
among the MLS~B~ components was also detected.

Rollins *et al*. \[[^56]\] documented macrolide resistance and
multi-resistant streptococcal isolates in swine, chickens and humans
from collections obtained from human patients, and healthy animals in
1979 and 1980. Subsequent studies by LeBlanc *et al*. in 1986 \[[^57]\]
showed that selected isolates of Group D streptococci contained DNA that
was consistent with sequence homology for resistance to erythromycin,
kanamycin and streptomycin. These studies at minimum demonstrate that
*erm* genes have been broadly disseminated since as early as the 1980\'s
if not before this time point in the US.

Given that the dissemination was already high 20 years ago, it is
difficult to dissect the relative contribution of any antimicrobial use
pattern, including proposed tulathromycin use, to influence the pattern
of such resistance determinants in commensal or pathogenic organisms of
man or animals.

#### Campylobacter

*Campylobacter* isolates that are sensitive to erythromycin are also
sensitive to tulathromycin under standard test conditions. Tulathromycin
MICs for macrolide-sensitive *Campylobacter* species in a sample of 30
strains ranged from 0.25 to 1 g/ml \[Error: Reference source not
found\]. *Campylobacter* resistant to erythromycin had high MICs to
tulathromycin (32 to 128 g/g). This cross-resistance with other
macrolides is consistent with the mechanism of action of tulathromycin
of binding to the ribosome (See Section 1.3).

In addition, an efflux pump found recently in *Campylobacter* isolates
confers elevated MICs not only to macrolides, but also to other
antimicrobial agents, including tetracyclines, fluoroquinolones, to
other antimicrobial classes, as well as other types of compounds, such
as dyes, etc. \[[^58],[^59], [^60]\] Therefore it is feasible that a
number of different antimicrobial agents and other types of compounds
used in animal medicine may exert selective pressure for *Campylobacter*
having higher macrolide MICs.

### Information regarding co-resistance to other antimicrobial drugs approved in veterinary and human medicine 

#### Consideration of the extent of use of the proposed product

##### Extent of use of tulathromycin 

Tulathromycin has a pharmacokinetic profile that produces a rapid onset
of action, high concentrations of active drug in target tissues and a
long duration of action \[Error: Reference source not found\].

-   **Duration of administration - Tulathromycin** is administered as a
    single injection for the proposed disease indications on the label.

-   **Individual vs. small groups vs. flocks/ herds -** As an
    injectable, tulathromycin is an individual animal treatment. It is
    administered to cattle with clinical signs related to BRD and to
    animals identified to be at high risk of developing BRD. In swine,
    it is administered to animals with signs of SRD.

-   **Dosage -** The FDA/CVM has reviewed and accepted data regarding
    the efficacy of tulathromycin administered at the proposed dosage in
    a single dose.

-   **Treatment period** **-** Tulathromycin is to be administered as a
    single injection treatment for the disease indications on the label.

-   **Use pattern -** Tulathromycin provides an alternative for or
    replaces existing treatments. Its use is not expected to expand the
    use of antimicrobial agents for animals suffering from BRD or SRD.
    This product will not change the incidence of BRD, nor will it
    change the criteria feedlot managers and veterinary consultants use
    to determine if arriving cattle are at risk of developing BRD. The
    proposed uses of tulathromycin are consistent with judicious use
    principles outlined by the FDA/CVM in collaboration with the
    American Veterinary Medical Association \[[^61]\],[^62]\].

Tulathromycin provides a complete course of treatment with a single
injection in both cattle and swine. This is particularly important since
handling associated with multiple injections increases stress on the
animal and increases costs associated with disease and disease therapy.
The pharmacokinetic/pharmacodynamic profile of tulathromycin enables
this single injection treatment regime, assuring compliance for a full
course of therapy. Approval also increases the range of products from
which to choose. Approval of tulathromycin represents a major advance in
the treatment of livestock respiratory disease.

#### Extent of use of macrolides in cattle and swine

The macrolides tylosin and erythromycin are available for use in swine
in oral and parenteral formulations, and tilmicosin is also available as
an oral formulation for swine. Historically, tylosin is the most
commonly used macrolide agent in swine for a wide variety of indications
in including pneumonia, erysipelas, weight gain/feed efficiency,
controlling swine dysentery and *Mycoplasma* arthritis*.* Tylosin has
been approved for use since 1961. Tylosin also is approved for a number
of poultry indications. Tilmicosin has been one of the most widely used
injectable antimicrobials in cattle for the treatment of BRD and for the
treatment of cattle at high risk of BRD following its approval in 1992.

Frequently organisms carrying an *erm* gene are multi-resistant to other
drug classes in addition to the MLS~B~ group \[Error: Reference source
not found,Error: Reference source not found,[^63],[^64],[^65]\] and
therefore the use of a number of antimicrobial agents may exert
selective pressure for an *erm* resistance determinant. However, the
existing, widespread use of macrolides in cattle and swine is detailed
below.

##### Extent of use of macrolides in cattle

The United States Department of Agriculture/National Animal Health
Monitoring System (USDA NAHMS) conducted a survey of beef feedlot
production sites in 1999 in 12 states representing 84% of the US feedlot
inventory \[Error: Reference source not found\]. Approximately 42% of
all feedlot cattle received tylosin orally in the feed or water as a
health or production tool, with cattle remaining on the product for an
average of 138-145 days, depending on arrival body weight. In the same
survey, injectable macrolides were the primary treatment in over
two-thirds of the cattle that received antimicrobial treatment upon
arrival, and injectable macrolides were the primary initial treatment
for nearly one-third of cattle requiring treatment for BRD.

The same USDA NAHMS survey of 1999 \[[^66]\] indicated that BRD is the
overwhelmingly predominant disease condition in cattle arriving at the
feedlot, with nearly 15% of the cattle showing signs of BRD after
arrival, currently estimated at 25-27 million cattle. Vogel \[[^67]\]
estimated mortality attributable to BRD at between 65 and 79% of all
types of death in the feedlot. Loneragan *et al.* \[[^68]\] estimated
that respiratory disease counted for 57.1% of all feedlot deaths,
accounting for approximately 7 deaths per 100 cattle (99.8%) in
feedlots. In addition, the 1999 USDA NAHMS survey \[[^69]\] indicated
that 19% of all cattle received to a feedlot receive an injectable
antibiotic, and that approximately 10.4% of all arriving cattle are
treated on arrival with some antibiotic \[Error: Reference source not
found\]. Nearly all feedlots surveyed used some form of antimicrobial as
treatment for BRD, with feedlots having more than one antimicrobial as
potential initial treatment. Over 31% of surveyed feedlots used
tilmicosin as the primary initial treatment for BRD; florfenicol,
tetracyclines, and ceftiofur were used as the primary initial treatment
in 21.9%, 21.6%, and 6% of feedlots, respectively \[Error: Reference
source not found\].

##### Extent of use of macrolides in swine

The USDA NAHMS conducted a survey of swine production sites in 17 states
representing 94 percent of the US pig inventory and 92 percent of US
pork producers with 100 or more swine during the survey period Dec 31,
2000-May 31, 2001. Among sites housing nursery pigs, 82.7% of the sites
fed antibiotics for growth promotion or disease prevention
\[[^70],[^71]\]. Among these sites, 37.8% reported feeding a macrolide
\[Error: Reference source not found,Error: Reference source not found\].
Macrolide (tylosin) use among grower finisher sites was reportedly
higher in the survey: 56.3% of the sites reported used in feed, 30.7% of
the sites by injection, and 4% of the sites in water.

Respiratory disease was ranked as the number one producer-identified
cause of mortality in both nursery pigs and grower/finisher swine,
responsible for 28 percent of nursery deaths and 40 percent of
grower/finisher mortality \[[^72]\]. In addition, two separate bacterial
respiratory diseases (*Mycoplasma* pneumonia and *Actinobacillus
pleuropneumoniae*) were ranked in the top ten disease problems in
grower/finisher swine. Injectable antibiotics represent a fraction of
the overall therapeutic antibiotic use in swine, according to the Doane
Animal Health Marketing Survey data (provided by Doane to the sponsor).
As such, injectable penicillin commands nearly two-thirds of the market
share (in terms of doses), with injectable oxytetracycline, tylosin,
lincomycin, and ceftiofur also being used. In NAHMS studies \[[^73]\],
one third of surveyed swine production sites used antimicrobials in
grower/finisher swine, primarily to treat/control respiratory disease.
Although entire pens are treated, the duration of therapy was
approximately 4-6 days. Approximately 27% of production sites used
antibiotics in feed to treat respiratory disease, compared with nearly
38% that use antibiotics in feed that use antibiotics in feed for
disease prevention. The duration of treatment in fed for treatment f
respiratory disease ranged from 15-39 days.

### Conclusions regarding selection pressure

The impact of tulathromycin use on *Campylobacter* is expected to be
minor due to the abiotic factors in the colonic content and in feces
(i.e., pH, binding), which attenuate the microbiological activity by pH.
Given the similarities in mechanism and resistance profiles with
macrolides used in animal medicine for therapeutic and growth promotion
uses, and the fact that tulathromycin is to be administered once as a
single dose regimen, parenterally by prescription, only to individuals
under veterinary prescription, there is no expectation that
tulathromycin use will add significant additional selection pressure for
resistance emergence in campylobacter, or other organisms.

Macrolides have been and are available for use in animals in injectable,
oral and topical formulations. They have been approved for more than 30
years for therapeutic, metaphylactic and growth promotant indications in
poultry, swine and cattle. However the observed rate of macrolide
resistance in *Campylobacter jejuni* isolated from humans is 1-3% since
1989, with no trends over time (Table 5). Mobile genetic elements
encoding macrolide resistance have not been reported in *Campylobacter.*
Therefore, given this historical perspective and experimental data, it
is reasonable to propose that macrolide use in general and tulathromycin
use specifically for therapeutic use in cattle and swine production at
times of respiratory infections are not expected to have a significant
impact on release of macrolide-resistant organisms. The experimental
evidence that tulathromycin does not select for unique macrolide
resistance determinants in *Campylobacter* shows that tulathromycin has
no higher likelihood for selection of resistant organisms than
macrolides already in use.

## Baseline prevalence of resistance

*Campylobacter*, including macrolide-resistant strains, are found in
cattle and swine on the farm and at slaughter. However as discussed
fully in Section 2, contamination of beef and pork products with
*Campylobacter*, either macrolide-sensitive or macrolide-resistant, is
infrequent.

The NARMS surveillance reports for *Campylobacter* isolated from animals
are collections of poultry isolates \[[^74]\]. However, US national
surveys of macrolide resistance among *Campylobacter* isolates in cattle
and swine are lacking. This lack of emphasis on *Campylobacter*
contamination from beef and pork may be due in part to epidemiological
reports suggesting that risk factors for campylobacteriosis due to beef
or pork is low \[104, 112, 113, 114\], compared to risk factors of
consumption of poultry or, to a lesser extent, raw milk.[^75]

In examining the resistance surveillance programs for resistance in
*Campylobacter*, it is important to recognize that 1) various programs
have used different sampling strategies and isolation procedures, which
may or may not include use of antimicrobial agents for isolation of
*Campylobacter* from animal or meat sources; 2) NCCLS performance
standards for susceptibility testing of *Campylobacter* have only
recently issued in 2003; and testing can vary among various surveys due
to differences in test systems; 3) NCCLS has not established macrolide
breakpoints for *Campylobacter*, and the macrolide breakpoints and test
media for other organisms (e.g., anaerobes vs. aerobic cocci) are
substantially different \[Error: Reference source not found\].

The NARMS program uses and E-test to monitor drug MICs of
*Campylobacter* isolates from humans \[Error: Reference source not
found\]. Although performance standards are not published by the NCCLS,
the E-test method used is consistent across years in the NARMS program,
permitting comparisons of MICs from one year to the next.

### NARMS MIC data for Campylobacter

The United States NARMS program includes surveillance of *Campylobacter*
for humans and for animals. Table 5 summarizes MIC data for
*Campylobacter* isolated from humans in the United States under this
program \[Error: Reference source not found,[^76],92\]. *Campylobacter*
isolates show 1-2% prevalence of macrolide resistance (erythromycin
MIC[\>]{.underline} 8 g/mL by E-test methods) among *C. jejuni* isolates
(n= 209-365 isolates/year). These rates were comparable to an earlier
1992 survey of roughly 332 isolates tested by E-test \[Error: Reference
source not found\]. The numbers of isolates from *C. coli* from humans
are few, making the analysis of trends over time difficult; however, the
prevalence of macrolide-resistance in *C. coli* is numerically higher
for than for *C. jejuni*, which has been similarly documented worldwide
\[Error: Reference source not found\].

<table>
<colgroup>
<col style="width: 5%" />
<col style="width: 6%" />
<col style="width: 8%" />
<col style="width: 9%" />
<col style="width: 6%" />
<col style="width: 6%" />
<col style="width: 6%" />
<col style="width: 8%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 4%" />
<col style="width: 9%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 7%" />
</colgroup>
<thead>
<tr class="header">
<th colspan="15"><span id="__RefHeading___Toc83018478"
class="anchor"></span>Table 5. MICs of macrolides tested against
<em>Campylobacter</em></th>
</tr>
<tr class="odd">
<th rowspan="2"><strong>Ref</strong></th>
<th rowspan="2"><strong>Source</strong></th>
<th rowspan="2"><strong>Test method</strong></th>
<th rowspan="2"><strong>Location</strong></th>
<th rowspan="2"><strong>Species</strong></th>
<th rowspan="2"><strong>Survey date</strong></th>
<th rowspan="2"><strong>N</strong></th>
<th colspan="4"><strong>Erythromycin MIC (g/mL)*</strong></th>
<th colspan="4"><strong>Azithromycin MIC (g/mL)*</strong></th>
</tr>
<tr class="header">
<th><strong>Range</strong></th>
<th><strong>MIC<sub>50</sub></strong></th>
<th><strong>MIC<sub>90</sub></strong></th>
<th><strong>%R*</strong></th>
<th><strong>Range</strong></th>
<th><strong>MIC<sub>50</sub></strong></th>
<th><strong>MIC<sub>90</sub></strong></th>
<th><strong>%R**</strong></th>
</tr>
<tr class="odd">
<th><strong>92</strong></th>
<th>Human</th>
<th>microbroth dilution</th>
<th>19 sentinel county labs</th>
<th><em>C. jejuni</em></th>
<th>1989-1990</th>
<th>286</th>
<th>NR</th>
<th>NR</th>
<th>NR</th>
<th>1%</th>
<th>NR</th>
<th>NR</th>
<th>NR</th>
<th>1%</th>
</tr>
</thead>
<tbody>
<tr class="odd">
<td>Error: Reference source not found,92</td>
<td>Human</td>
<td>E-test</td>
<td>CDC sentinel sites</td>
<td><em>C. jejuni, coli</em></td>
<td>1992</td>
<td>332 (preliminary)</td>
<td>NR</td>
<td>NR</td>
<td>NR</td>
<td>2%‡</td>
<td>NR</td>
<td>NR</td>
<td>NR</td>
<td>NR</td>
</tr>
<tr class="even">
<td rowspan="10">Error: Reference source not found,92</td>
<td rowspan="10"><p>Humans</p>
<p>Human</p></td>
<td rowspan="10">E-test</td>
<td rowspan="10">CDC sentinel sites</td>
<td rowspan="5"><em>C. jejuni</em></td>
<td>2001</td>
<td>365</td>
<td><u>&lt;</u>0.38-<u>&gt;</u>48</td>
<td>0.75</td>
<td>1.5</td>
<td>2%</td>
<td><u>&lt;</u>0.064-<u>&gt;</u>8</td>
<td>0.125</td>
<td>0.25</td>
<td>2%</td>
</tr>
<tr class="odd">
<td>2000</td>
<td>306</td>
<td><u>&lt;</u>0.38-<u>&gt;</u>48</td>
<td>0.75</td>
<td>1.5</td>
<td>1%</td>
<td><u>&lt;</u>0.064-<u>&gt;</u>8</td>
<td>0.125</td>
<td>0.25</td>
<td>2%</td>
</tr>
<tr class="even">
<td>1999</td>
<td>294</td>
<td><u>&lt;</u>0.38-<u>&gt;</u>48</td>
<td>0.75</td>
<td>1.5</td>
<td>2%</td>
<td><u>&lt;</u>0.064-<u>&gt;</u>8</td>
<td>0.125</td>
<td>0.375</td>
<td>3%</td>
</tr>
<tr class="odd">
<td>1998</td>
<td>297</td>
<td><u>&lt;</u>0.38-<u>&gt;</u>48</td>
<td>0.75</td>
<td>2.25</td>
<td>2%</td>
<td><u>&lt;</u>0.064-<u>&gt;</u>8</td>
<td>0.188</td>
<td>0.375</td>
<td>1%</td>
</tr>
<tr class="even">
<td>1997</td>
<td>209</td>
<td><u>&lt;</u>0.38-<u>&gt;</u>48</td>
<td>1.0</td>
<td>3</td>
<td>1%</td>
<td><u>&lt;</u>0.064-<u>&gt;</u>8</td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td rowspan="5"><em>C. coli</em></td>
<td>2001</td>
<td>17</td>
<td><u>&lt;</u>0.38-<u>&gt;</u>48</td>
<td>0.75</td>
<td>4.5</td>
<td>6%</td>
<td><u>&lt;</u>0.064-<u>&gt;</u>8</td>
<td>0.375</td>
<td>0.75</td>
<td>6%</td>
</tr>
<tr class="even">
<td>2000</td>
<td>12</td>
<td><u>&lt;</u>0.38-<u>&gt;</u>48</td>
<td>1.0</td>
<td>4.5</td>
<td>8%</td>
<td><u>&lt;</u>0.064-<u>&gt;</u>8</td>
<td>0.375</td>
<td>1</td>
<td>8%</td>
</tr>
<tr class="odd">
<td>1999</td>
<td>20</td>
<td><u>&lt;</u>0.38-<u>&gt;</u>48</td>
<td>1.0</td>
<td>3</td>
<td>10%</td>
<td><u>&lt;</u>0.064-<u>&gt;</u>8</td>
<td>0.188</td>
<td>0.75</td>
<td>10%</td>
</tr>
<tr class="even">
<td>1998</td>
<td>8</td>
<td><u>&lt;</u>0.38-<u>&gt;</u>48</td>
<td>NA</td>
<td>NA</td>
<td>12%</td>
<td><u>&lt;</u>0.064-<u>&gt;</u>8</td>
<td>NA</td>
<td>NA</td>
<td>12%</td>
</tr>
<tr class="odd">
<td>1997</td>
<td>6</td>
<td><u>&lt;</u>0.38-<u>&gt;</u>48</td>
<td>NA</td>
<td>NA</td>
<td>0%</td>
<td><u>&lt;</u>0.064-<u>&gt;</u>8</td>
<td>NA</td>
<td>NA</td>
<td>0%</td>
</tr>
<tr class="even">
<td colspan="15"><p>*Erythromycin resistance defined as MIC
<u>&gt;</u>8;</p>
<p>** Azitrhomycin Resistance defined as MIC <u>&gt;</u> 2 g/mL</p></td>
</tr>
</tbody>
</table>

Currently, the NARMS program for antimicrobial susceptibility testing of
*Campylobacter* isolates from animals only monitors poultry isolates
\[Error: Reference source not found\]. The erythromycin resistance (MIC
[\>]{.underline}8g/mL) rates for poultry isolates of *C. jejuni*
(n=128-590 isolates/year) ranged from 0.2-5.1% for years 1998-2003 with
no obvious trend over time \[[^77]\]. The erythromycin resistance (MIC
[\>]{.underline}8g/mL) rates for poultry isolates of *C. coli* (n=63-288
isolates/year) ranged from 11-23% for years 1998-2003 with no obvious
trend \[Error: Reference source not found\].

The NARMS surveillance program does not currently monitor susceptibility
profiles of *Campylobacter* isolated from cattle or swine \[Error:
Reference source not found,Error: Reference source not found\]. A
literature search did not reveal published US national surveys of
antimicrobial susceptibility profiles of *Campylobacter* species
isolated from swine. However, Englen *et al.* \[[^78]\] reported a
survey of antimicrobial sensitivity of *Campylobacter* isolated from
healthy feedlot cattle in the US. Isolates were obtained from feedlot
cattle as part of the 1999 USDA NAHMS monitoring survey of health
feedlot cattle \[Error: Reference source not found,Error: Reference
source not found\]. Fecal samples were collected from cattle by state
veterinarians at feedlots in states representing 90% of the cattle fed
in the US. The prevalence of macrolide resistance (MIC [\>]{.underline}
8 by E-test method), was 2.2% of the *C. jejuni* (n=92) and 7.7% (n=26)
of *C. coli* isolates obtained in the study. The report did not provide
information regarding the total number of feedlots or cattle sampled to
obtain the 118 isolates in the study. Among the four total isolates of
the 118 isolate collection that were resistant to erythromycin, two *C.
jejuni* and one *C. coli* isolates were multi-resistant to at least
three other drug classes.

Ge *et al.* reported the MIC values of *Campylobacter* isolated from
retail meats collected in Washington, DC \[[^79]\], showing 17%
erythromycin resistance among isolates from poultry. Of other meat
samples analyzed for *Campylobacter* in the same study, only three of
181 pork samples, and 1 of 182 retail beef samples were positive
\[[^80]\] for this organism. The authors did not report on the MICs of
the four pork and beef isolates in the collection. White *et al*.
presented a preliminary report on the macrolide resistance among
*Campylobacter* isolates from the FDA/CVM Retail Meat Sentinel Site
Survey \[Error: Reference source not found\]. Results of surveys and
reports from the EU are consistent with data on *Campylobacter*
susceptibility patterns in the US, as surveyed in Section 1.9.2.

### Other MIC surveys of Campylobacter isolates

#### Isolates from humans

There are few published surveys of macrolide resistance in
*Campylobacter* in the United States besides those reported via the
NARMS program \[Error: Reference source not found\]. Nachamkin *et al*.
\[[^81]\] reported 2% overall erythromycin resistance among 142 isolates
from patients from Pennsylvania collected 1982-92, and fluctuated from
0-5% among 297 patients 1995-2001, with 3.5% recorded in 2001 (E-test
method, \[[^82]\]).

In Canada, Gaudreau and Gilbert reported 0% erythromycin resistance (MIC
[\>]{.underline} 8 g/mL tested by agar dilution) for 291 isolates
collected from patients in Montreal, Quebec 1985-6, 1992-3 and 1995-6
\[[^83]\]. In a later report by the same authors, yearly resistance
rates varied from 1 to 12% in 1998-2001 (51-72 isolates/year), with no
statistically significant trend detected \[[^84]\]. These reports are
consistent with the rate reported for Canada earlier by Karamali, where
erythromycin resistance (MIC [\>]{.underline} 8 g/mL by agar dilution)
was 0.5% among *C. jejuni* tested isolated from Canada before 1981
\[[^85]\].

Wagner *et al*. \[[^86]\] recorded macrolide resistance among *C.
jejuni* isolates from Germany from 1998-2001, using a standard method.
The prevalence rate of erythromycin resistance (MIC [\>]{.underline} 8
g/mL) was 4.2% (n=144). Luber *et al*. \[[^87]\] recorded micro-broth
dilution results of 1.5% (n=68) and 0% (n=65) erythromycin resistance
(MIC [\>]{.underline} 8 g/mL) in 1991 and 2001, respectively, from *C.
jejuni* isolates from patients in Berlin. *C. coli* isolates were
higher, with 7.1% (n=14) and 29.4% (n=17) resistance to erythromycin in
1991 and 2001, respectively. Krausse & Ullmann \[[^88]\] reported yearly
results of *C. jejuni* and *C. coli* isolates; erythromycin MIC values
[\>]{.underline} 8 g/mL fluctuated between 0-2% in survey periods
1980-82 (n=30), 1997-8 (n=93), 1999-2000 (n=85) and 2001 (n=99) in
Germany.

#### Isolates from animals

Van Looveren *et al*. \[[^89]\] reported that 67% of the 61 isolates of
*C. coli* from swine in Belgian slaughterhouses in 1998 were
erythromycin resistant (MIC [\>]{.underline}8 g/mL, tested by agar
dilution). Macrolide resistance rates of *C. jejuni* isolates from
poultry (n= 285) were 6.3% and 8.6% in broilers and layers,
respectively. The macrolide MIC values of the four swine isolates of *C.
jejuni* were not reported.

#### Macrolide resistance in C. jejuni vs. C. coli

The higher prevalence rates of macrolide resistance of *C. coli*
compared to *C. jejuni* have been recognized since *Campylobacter*
susceptibility reports first began in the late 70\'s and early 80\'s,
soon after methods were developed for routine culture of this fastidious
genus in the laboratories worldwide \[Error: Reference source not
found,[^90],[^91],Error: Reference source not
found,[^92],[^93],[^94],[^95][^96]\]. Engberg *et al*. \[Error:
Reference source not found\] provided a listing of surveys reported
since 1989 of azithromycin and erythromycin resistance in
*Campylobacter* isolates from humans. Although the reported resistance
rates ranged from 0-11% for *C. jejuni*, and from 0-68% for *C. coli*,
no conclusions can be drawn regarding trends or baseline prevalence,
since the reports used different sampling methods, sensitivity test
methods, and criteria for scoring resistance. The authors noted trends
for fluoroquinolones, but did not draw conclusions for trends in
macrolides. The reasons for the differences in prevalence of macrolide
resistance among *Campylobacter* species is not clear. Macrolide
resistance is higher in *C. coli* isolates from poultry than *C. jejuni*
in poultry (NARMS report 2003\[[^97]\]). Studies are needed to better
understand this observation.

### Conclusions regarding baseline MIC data for Campylobacter

The prevalence rates of macrolide resistance are between 1-3% among all
human isolates of *Campylobacter* in the NARMS program 1997-2001, with
no apparent trends over time. The macrolide resistance rates of *C.
jejuni* vary similarly (1-3%; 209-365 isolates/year). The rates for *C.
coli* from humans are more difficult to estimate (0-12%, n= 6-20
isolates/year) than *C. jejuni* rates because the frequency of *C. coli*
isolation is so low among humans in the United States (as observed in
many other countries). *C. jejuni* isolates outnumber *C. coli* isolates
by more than 10-fold in the NARMS program for surveillance of human
*Campylobacter* isolates. Nonetheless, overall macrolide resistance
rates are low despite decades of macrolide and lincosamide use humans,
swine and other food-producing animal species in the United States.

Prevalence data are insufficient in the United States to determine
national rates of resistance among *Campylobacter* isolates from swine
and cattle. No United States national surveys have been published for
isolates from swine. The 1999 survey of feedlot cattle \[Error:
Reference source not found\] yielded an overall rate of 3% erythromycin
resistance among *Campylobacter* isolates.

Macrolide resistance has been observed to often be \>20% among most
surveys of *C. coli* whether isolated from swine or other animal species
in studies conducted in other parts of the world, and in all reports of
*C. coli* conducted since methods were developed to culture these
organisms in the late 1970\'s. However, *C. coli* represent a minority
of cases causing campylobacteriosis in surveys of human clinical
isolates in the United States and elsewhere in the world.

## Other relevant data

In an early exploratory study[^98] the potential for tulathromycin to
eliminate the carrier state of *Salmonella typhimurium* in growing swine
was evaluated. Swine were challenged with a strain of *S. typhimurium*
with a tulathromycin MIC of 1.56 g/ml and were later dosed once
intramuscularly with tulathromycin at either 10 or 15 mg/kg body weight
(10 pigs/group); a control group received a single IM dose of saline.
Subsequent to treatment, fecal samples were collected from pigs for
*Salmonella* analyses through 28 days post-treatment. Based on the
quantities of *Salmonella* shed and the proportion of pigs shedding any
salmonellae, it was concluded that *Salmonella* shedding in the groups
of pigs treated with either dosage of tulathromycin were similar to the
control pigs. Separate excretion studies with tulathromycin in pigs
showed residues in feces of 10 to 70 g/g in the first three days
following an IM dose of 2.5 mg/kg; thus exposure of salmonellae to
residues following dosing regimen of 10 and 15 mg/kg in the exploratory
study would be expected to be substantially larger. Despite a challenge
strain MIC of 1.56 g/ml and the relatively high expected drug exposure,
salmonellae populations were apparently not impacted by the
tulathromycin treatments. This observation is consistent with an
expected lack of bioactivity of drug residues in the colon, even in the
absence of passage through the stomach.

This study does not address the resistance selection *per se*. However,
this study supports the expectation that the tulathromycin MICs
determined for pure cultures of enteric bacteria under standard test
conditions does not predict the potency of this drug *in vivo* in the
colon contents or in feces. Other factors not taken into account in the
MIC test, such as neutral to acidic pH and binding to fecal particles
are expected to attenuate the activity of tulathromycin \[Sections
1.4,1.5.1.3\]. The observation that S*almonella* shedding in this
experimental swine model was not appreciably affected *in vivo* when
tulathromycin was administered supports that tulathromycin
microbiological activity in the colonic contents and feces is highly
attenuated, lessening the risk for resistance selection in the gut.

## Overall Release Assessment Conclusion: \"Low\"

The following table summarizes the components of the release assessment
requested in Guidance #152.

+----------------+-----------------------------------------------------+
| Relevant       | Extent to which relevant factors favor resistance   |
| parameters     | emergence                                           |
+================+=====================================================+
|                | Comments/conclusions regarding factors              |
+----------------+-----------------------------------------------------+
| Mechanism of   | Inhibits protein synthesis by binding to the        |
| activity       | bacterial ribosome.                                 |
|                |                                                     |
|                | Bacteriostic or bactericidal. Bactericidal against  |
|                | *Campylobacter*                                     |
+----------------+-----------------------------------------------------+
| Spectrum of    | Broad spectrum .                                    |
| activity       |                                                     |
+----------------+-----------------------------------------------------+
| Ph             | -Rapid,extensive absorption,                        |
| armacokinetics |                                                     |
|                | -High volume of distribution,slow elimination.      |
|                |                                                     |
|                | -Plasma protein binding is low (40%)                |
|                |                                                     |
|                | -Maximum plasma concentration 30 min after dose     |
|                |                                                     |
|                | -Bioavailability is high (88-90%)                   |
+----------------+-----------------------------------------------------+
| Ph             | -Macrolides may exhibit post-antibiotic effects.    |
| armacodynamics | Thus the potential exists for continued activity    |
|                | for a short period after the drug is removed.       |
+----------------+-----------------------------------------------------+
| Resistance     | -3 mechanisms: 1) target (ribosomal RNA binding     |
| mechanisms     | site) modification, 2) drug efflux, 3) drug         |
|                | inactivation.                                       |
|                |                                                     |
|                | -Chromosomal mutation of ribosomal DNA is most      |
|                | relevant to macrolide resistance in                 |
|                | *Campylobacter.*                                    |
+----------------+-----------------------------------------------------+
| Resistance     | -Resistance transfer important for enterococci,     |
| transfer       | streptococci, many organisms                        |
|                |                                                     |
|                | -No reports to date of macrolide-resistance         |
|                | transfer in *Campylobacter* (all                    |
|                | macrolide-resistance characterized to date due to   |
|                | mutation)                                           |
+----------------+-----------------------------------------------------+
| Selection      | -Total residue (microbiologically active and        |
| pressure       | inactive) in colonic content and feces is           |
|                | attenuated due to abiotic factors: 1) neutral to    |
|                | acidic pH of colonic contents and feces 2) binding  |
|                | to fecal substrates.                                |
|                |                                                     |
|                | \- Macrolides have been approved and in use in      |
|                | cattle, swine, poultry for over 30 years.           |
|                |                                                     |
|                | -Tulathromycin is administered to individual        |
|                | animals                                             |
|                |                                                     |
|                | -Tulathromycin is to be administered by veterinary  |
|                | perscription only                                   |
|                |                                                     |
|                | -The single-dose regimen enhances the potential for |
|                | user compliance related to full course treatment,   |
|                | and reduces animal stress associated with repeated  |
|                | dosing at the animal production site.               |
|                |                                                     |
|                | -Alternative therapies (including other macrolides) |
|                | are available. Tulathromycin use is expected to     |
|                | displace existing macrolide use, and not increase   |
|                | macrolide or antibiotic use.                        |
+----------------+-----------------------------------------------------+
| Baseline       | *-C. jejuni* resistance to macrolides is            |
| prevalence of  | [\<]{.underline}3% among human isolates tested *in  |
| resistance     | vitro*, with no trends over time during NARMS       |
|                | surveillance*.*                                     |
|                |                                                     |
|                | *-C. coli* isolates from swine have higher          |
|                | percentages of macrolide resistance, which has been |
|                | documented worldwide since the time *Campylobacter* |
|                | was first cultivated and tested.                    |
|                |                                                     |
|                | *-C. coli* represents \<10 % of isolates from       |
|                | humans (NARMS data)                                 |
+----------------+-----------------------------------------------------+
| Other factors  | -Respiratory diseases requiring treatment generally |
|                | occurs during early stage of production, long       |
|                | before animals are ready for entry into the food    |
|                | chain.                                              |
+----------------+-----------------------------------------------------+

*Campylobacter jejuni* and *C. coli* is found in cattle and swine as
part of the intestinal flora and will be exposed to tulathromycin where
therapy is needed. Selection of macrolide resistance by spontaneous
mutation would occur at a very low frequency. These mutational events
are deemed of low significance, as evidenced by the long use of
macrolides in animals, and low prevalence rates of macrolide resistance
in *Campylobacter* in humans.

Macrolide resistance has not been demonstrated to be transferable in
*Campylobacter.* After more than three decades of macrolide use in
animals and in humans, the rates of macrolide resistance in
*Campylobacter* isolated from humans remains low in the US. Thus
macrolide use in livestock, companion animals and in man has not had a
significant impact on the level of pre-existing macrolide resistance in
*Campylobacter.*

The overall conclusion is that there is a "Low" probability of release
of macrolide-resistant *Campylobacter* as a result of tulathromycin use.

# Exposure Assessment

## Overview

Table 5 of FDA/CVM Guidance #152 \[Error: Reference source not found\]
(Figure 1), provides a possible process for ranking qualitatively the
probability of human exposure to a given bacteria in food commodities
based on national surveys of food commodity consumption in the United
States and food commodity contamination rate data. The algorithm used to
rank overall exposure, based on these rates is copied in Figure 1.

Using the default values provided in Guidance #152 for beef (consumption
rate of \"High\" and *Campylobacter* contamination rate of \"Low\"), the
overall exposure assessment for beef is \"Medium\", based on this
algorithm.

Using the default values for pork (consumption rate of \"High\" and
C*ampylobacter* contamination carcass contamination rate of \"High\",
the overall exposure assessment for pork is \"High\". However,
*Campylobacter* epidemiological evidence, *Campylobacter* contamination
rate data in carcass and at retail, and processing in pork shows that
the contamination rate of retail pork meat is consistently low
(contamination rates in retail pork \<5% \[Section 2.4.3.3\]). By
applying the \"Low\" contamination rate categorization found in Guidance
#152 and using its algorithm for ranking the probability of human
exposure, the overall exposure assessment for pork is categorized as
\"Medium\".

These qualitative exposure assessments of \"Medium\" for human exposure
to *Campylobacter* via beef or pork are conservatively high estimates.
Epidemiological studies indicate the primary risk factors for
campylobacteriosis in humans, for both outbreaks and sporadic cases are
consumption of raw milk/milk products and untreated surface water, as
well as handling and consumption of raw or undercooked poultry. The
observed frequency of isolation rates from beef and pork meats at retail
are consistently low.

## Campylobacteriosis in man and its epidemiology

The principle reservoir for *Campylobacter* is the alimentary tract of
wild and domesticated animals and birds. In the NARMS program from
1997-2001 for monitoring *Campylobacter* isolated from humans, from 8 to
20 *C. coli* isolates were profiled per year, compared to 209-365 *C.
jejuni* isolates are per year \[Error: Reference source not found\].
This ratio of species in humans is consistent with surveys of
*Campylobacter* in humans worldwide. *C. jejuni* is most frequently
isolated in cases of campylobacteriosis in humans and estimated to be
the predominant species responsible *Campylobacter* gastroenteritis
\[[^99]\].

*C. jejuni* is predominantly associated with poultry and cattle, whereas
*C. coli* is predominantly associated with swine \[Error: Reference
source not found\]. Ingestion or preparation of poultry or poultry
products, non-pasteurized milk or milk products, contaminated water or
zoonotic exposure are frequently listed among the highest risk factors
generally for campylobacteriosis due to *C. jejuni* \[[^100]\]. Risks
for sporadic cases, accounting for the majority of cases, also include
handling/consumption of raw or undercooked poultry as a primary factor.
Poultry products have generally been implicated as the primary vehicle
for transmission of foodborne *Campylobacter* to humans \[Error:
Reference source not found,[^101],[^102]\].

Although *C. coli* is prevalent on surface samples of swine carcasses
\[Error: Reference source not found,[^103],[^104]\], it is apparent that
the contamination rates of *Campylobacter* are substantially lower
during pork meat processing and at retail (see Section 2).
Epidemiological studies support this observation. There are very few
documented cases of human infection by *Campylobacter* from pork
products and an abundance of documented cases of infection from poultry
products and, and to a lesser extent, raw milk \[Error: Reference source
not found,[^105]\]. *C. jejuni* is the species most frequently isolated
from poultry products \[Error: Reference source not found,Error:
Reference source not found, Error: Reference source not found, Error:
Reference source not found\]. In a summary of outbreaks with a known
etiology involving pork between 1990-1997 in the USA, only 2% of
foodborne illness due to pork had an etiology associated with
*Campylobacter* \[[^106]\]*.* Frequently pork consumption is not
mentioned as a risk factor for *Campylobacter* disease in humans
\[Error: Reference source not found\]. In a recent study examining the
risk of transmission of *Campylobacter* from swine to man, the author
concluded that transmission of *C. jejuni* was non-evident, and pork
consumption was a very low risk for *C. coli* \[Error: Reference source
not found\].

Gillespie *et al.* \[[^107]\] used a case-case analysis to compare *C.
coli* infections with cases of *C. jejuni* infection to generate
hypotheses for infection based on sentinel surveillance information in
England and Wales. They postulated risk factors for *C. coli* to be
consumption of bottled water, ingestion of pâté (frequently pork liver
in the UK) or meat pies (meat source unknown), based on this analysis.
Neither pâté nor meat pies are commonly prepared from pork in the United
States diet.

Recent molecular analysis of *C. coli* isolates from swine and poultry
suggest that host specificity of strains of *C. coli*, which may in the
future help to better understand the epidemiology of *C. coli*
\[[^108]\]. A Swedish case control study identified an odds ratio for
"bone-in" pork, but the authors cautioned that further study is needed
to confirm this statistic \[[^109]\]. In a report by the CDC in 1992 on
the epidemiology of *C. jejuni*, pork products were not listed as a risk
factor, with poultry, eggs, raw milk, poultry, and drinking water as
primary factors \[Error: Reference source not found\]. In a study of
*Campylobacter* contamination of thigh/breast of poultry, and ovine,
bovine and porcine liver, in an area surrounding a reference laboratory
in the UK, the *C. coli* strain characteristics of isolates from porcine
liver were not comparable to the human isolates, leading the authors to
postulate that pork is a relatively minor contributor to human
campylobacteriosis \[[^110]\]. While the most predominant serotype
identified among human isolates was found in all food sources, the
authors noted that this serotype was the least frequently isolated from
pig liver as compared to the other types of meats surveyed. Although the
Nielson *et al.* suggests that cattle are a major source of human
infections due to serotype overlap, other epidemiological studies do not
identify beef consumption as a major risk factor for campylobacteriosis
in humans \[[^111],[^112],[^113]\].

Munroe *et al.* \[ [^114]\] isolated *C. jejuni* and *C. coli* from
feces of chicken, cattle and swine. Whereas 96% of the chicken isolates
were identified as *C. jejuni* serotypes frequently isolated from human
cases of enteritis, the *Campylobacter* isolated from swine feces were
97% *C. coli* and did not belong to serotypes common to human isolates
\[[^115]\]. Similarly, Nielson *et al.* \[[^116]\] observed that 94% of
human *Campylobacter* diarrhea isolates were *C. jejuni,* and *C. coli*
was isolated from patients at only a 6% rate. The investigators
concluded that swine were not an important source of human
*Campylobacter* infections because of the differences in serotypes
between human disease and swine fecal isolates \[Error: Reference source
not found\]. The authors reported overlap of serotypes of isolates from
humans and those from live poultry and from live cattle among Danish
isolates. The authors suggested that poultry and cattle are sources of
campylobacter in humans, noting the relatively high prevalence of
*Campylobacter* at retail, and suggesting that other foods should be
considered. The authors reported overlap of serotypes of isolates from
humans and those from live poultry and from live cattle.

Based on these epidemiological studies and reviews, the exposure of
humans to *Campylobacter* via beef and pork is relatively low.

## Exposure assessment for cattle

### Qualitative ranking of human consumption of beef in the US

In Guidance #152 \[Error: Reference source not found\], beef consumption
in the United States is qualitatively ranked as \"High\" (64.5
lb/capita/year), based on the most recent data available from the USDA
Economic Research Service (ERS) \[[^117]\].

### Qualitative ranking of Campylobacter contamination of beef

Guidance #152 \[Error: Reference source not found\] lists qualitative
rankings for beef contamination by *Campylobacter,* based on the USDA
Food Safety Inspection Service (FSIS) Nationwide Beef Microbiological
Baseline Data collection program \[[^118],[^119],[^120]\]. These
rankings are \"Low\" for ground beef (0% contamination \[Error:
Reference source not found\]), cows/bulls (1% contamination \[Error:
Reference source not found\]) and steers/heifer (4% contamination
\[Error: Reference source not found\]) samples. These contamination
results are consistent with recent data generated by the FDA/CVM in the
new NARMS sentinel site studies of retail meat \[Error: Reference source
not found\]. Among the 642 and 809 ground beef samples tested for
*Campylobacter* for years 2002 (6 sentinel sites) and 2003 (8 sentinel
sites), only 1% of the samples from retail ground beef each year yielded
positive results for *Campylobacter.* In Guidance #152, Table 5 provides
one means to qualitatively rank human exposure to *Campylobacter*, based
on these national data.

### Overall Beef Exposure Assessment Conclusion: \"Medium\" 

The application of the USDA ERS beef consumption data (ranked \"High\"
per capital consumption), the beef contamination rate data (ranked
\"Low\"), based on FSIS data \[Error: Reference source not found,Error:
Reference source not found,Error: Reference source not found\], and the
NARMS Sentinel Site Retail Meat contamination rate data \[Error:
Reference source not found\] to this ranking process yields an overall
exposure assessment of \"Medium\" for human exposure to *Campylobacter*
through consumption of beef, based on the process described in Table 5
of Guidance #152 (Figure 1).

## Exposure assessment for swine

### Qualitative ranking of human consumption of pork in the US

In Guidance #152 \[Error: Reference source not found\], pork consumption
in the United States is qualitatively ranked as \"High\" (48.2
lb/capita/year), based on the most recent data available from the ERS
\[Error: Reference source not found\].

### Qualitative ranking of Campylobacter contamination of pork

#### Default contamination values based on pork carcass data

Guidance #152, qualitatively ranks the contamination rate of pork by
*Campylobacter* as \"High\", based on the USDA FSIS Baseline Data
Collection Program for Market Hogs in 1995-6 \[Error: Reference source
not found,Error: Reference source not found\]. The FSIS sampling in this
baseline study included only carcasses sampled after 12 hours chilling,
and before completion of the pork processing steps for retail
distribution. These processing are very detrimental to the survival of
*Campylobacter* as detailed below (Section 2.4.3.3, 2.4.3.4, 2.4.3.5).
Thus the baseline study used in Guidance #152 to estimate pork
contamination rates by *Campylobacter* substantially overestimates the
exposure of humans to *Campylobacter* by pork at retail. This is
corroborated by retail meat studies as discussed below, as well as the
epidemiological studies cited in Sections 2.2 and 2.4.3.5.

### Review of Campylobacter contamination in carcass and pork meat

#### Campylobacter in swine 

*C. coli,* but not *C. jejuni,* is frequently found in the
gastrointestinal tracts of swine. Surveys show 60-100% detection of
*Campylobacter* species in feces of swine herds in various parts of the
world \[Error: Reference source not found,[^121],[^122]\]. In a national
swine study coordinated by the United States NAHMS program, the
predominant *Campylobacter* species isolated from 1057 fecal samples was
*C. coli*, detected in 69% of the fecal samples (n=1057) while *C.
jejuni* was detected in only 0.3% of samples \[Error: Reference source
not found, [^123]\]. Burch estimated from a review various surveys of
swine, mainly in Europe, that 96% of *Campylobacter* isolated from pig
feces was *C. coli,* and only 4% were *C. jejuni* \[Error: Reference
source not found\]. Nielson *et al.* observed that among 316 fecal
samples collected from 19 swine slaughterhouses in Denmark, 46% were
positive for *Campylobacter,* 95% of which were speciated as *C. coli*,
and only 4% as *C. jejuni* \[Error: Reference source not found\].

#### Pork carcass contamination

Given the relatively high prevalence rates of *C. coli* observed in
studies of swine feces, it is not surprising that carcass data would
also suggest a high prevalence of *Campylobacter.* In a study conducted
in Iowa in1999, the average carcass contamination level was 9% (Table 10
of Reference \[Error: Reference source not found\]). In the 1995-1996
National Pork Microbiological Baseline Data Collection Program for
Market Hogs, *Campylobacter* was detected in 32% of 2112 samples of
carcasses \[[^124]\], with detection levels of [\<]{.underline} 0.03
cells/cm^2^ of surface area sampled (1 cm thick samples) (Table 6).

+-----------------------+-----------+---------------------------------+
| []{#__Ref             |           |                                 |
| Heading___Toc83018479 |           |                                 |
| .anchor}Table 6.      |           |                                 |
| *Campylobacter*       |           |                                 |
| distribution among    |           |                                 |
| raw pork carcass      |           |                                 |
| surface samples       |           |                                 |
| tested positive for   |           |                                 |
| *Campylobacter* in    |           |                                 |
| the US Nationwide     |           |                                 |
| Pork Microbiological  |           |                                 |
| Baseline Data         |           |                                 |
| Collection Program.   |           |                                 |
+=======================+===========+=================================+
| Quantitative cell     | Projected | No Samples\* testing positive   |
| count range\*         | MPN/200   | at this level of pathogen       |
|                       | cm^2^     | burden\*                        |
| (MPN/cm^2^)\*         |           |                                 |
+-----------------------+-----------+---------------------------------+
|                       |           |                                 |
+-----------------------+-----------+---------------------------------+
| [\<]{.underline}0.03  | [\<]{.un  | 508                             |
|                       | derline}6 |                                 |
+-----------------------+-----------+---------------------------------+
| 0.03 - 0.30           | 6-60      | 113                             |
+-----------------------+-----------+---------------------------------+
| 0.31-3.0              | 60-600    | 13                              |
+-----------------------+-----------+---------------------------------+
| 3.1-30.0              | 600-6000  | 4                               |
+-----------------------+-----------+---------------------------------+
| \>30.0§               | \>6000    | 1                               |
+-----------------------+-----------+---------------------------------+
| Total positive        |           | 639                             |
| samples               |           |                                 |
+-----------------------+-----------+---------------------------------+
| Total negative        |           | 1473                            |
| samples               |           |                                 |
+-----------------------+-----------+---------------------------------+
| Total samples tested  |           | 2112                            |
+-----------------------+-----------+---------------------------------+

The National Research Council and Institute of Medicine reported that
10% of carcasses were scored positive for *Campylobacter* (Table 5-3 of
Reference \[[^125]\]). In a Norwegian study, 36% of swine carcasses were
positive in 2002 (Table 5 of Reference \[Error: Reference source not
found\]). The Norwegian isolates were predominantly *C. coli;* and *C.
jejuni* was not isolated from swine carcass (Table 4 of Reference
\[Error: Reference source not found\]). In a Canadian study of swine at
slaughter, *C. coli*, *C. jejuni* and *C. laridis* isolated from swine
feces accounted for 97%, 2% and 1% of the isolates, respectively
\[[^126]\], and the isolation rates of the same three *Campylobacter*
species were lower from diaphragm specimens of the carcasses before cold
storage, 20%, 2%, and 1%, respectively. These studies all corroborate
the relatively high pork carcass contamination rates, but the detection
limits for studies will vary, and in the case of the US survey, the
limit of detection was very low.

#### Campylobacter in carcasses does not represent exposure to consumers

The carcass contamination rates recorded in the National Pork
Microbiological Baseline Data Collection Program in market hogs \[Error:
Reference source not found\] represent a high estimate of actual
consumer exposure rates to *Campylobacter.* The carcasses in this study
were sampled after 12 h cooler chilling, which marked a natural break in
a pork processing operation, and provided a window of time for
convenient sampling by the USDA FSIS. Typical hog slaughter plants use a
scalding procedure to remove all hair from the carcass, leaving the skin
intact on the carcass during this stage of the chilling process. The
skin still remains on the carcass during this sampling time.
*Campylobacter* species were recovered from 32% of the 2112 carcasses
sampled using a selective enrichment procedure from a 60 cm^2^ composite
sample of the carcass surface (1 cm deep cuts for sampling) from samples
obtained from the jowl, belly and ham, followed by a selective,
bacterial enrichment procedure for *Campylobacter.* If a sample tested
positive it was then reanalyzed using a standard mean probable number
bacteriological enumeration procedure to determine the number of
organisms per cm^2^ of carcass surface area (Table 6). These samples
were taken before removal of skin. Typically after chilling (the
sampling time for the *Campylobacter* survey), the carcass is broken
into primal cuts such as the belly, shoulder, ham and loin. The primal
cuts are further processed by removal of the skin and excess fat. The
skin and fat do not accompany either the primal or commercial fresh pork
products into the market place. Fresh pork skin is typically not
consumed as an edible tissue. Most skin and associated subcutaneous fat
trimmed from the carcass are rendered at high temperature to produce
lard and other cooked products. During and after final processing of
pork, primal cuts are exposed to cold and aerobic environments that are
hostile to the survival of *Campylobacter* \[Error: Reference source not
found,Error: Reference source not found,[^127]\]*.* Exposure to air and
removal of the skin should greatly decrease exposure to *Campylobacter*
for reasons described above. This is supported by both the low
prevalence of Campylobacter on beef carcass (hide removed before
sampling, Section 2.3.1) and low prevalence of *Campylobacter*
contamination (\<5%) of fresh pork products sampled at retail (See
Section 2.4.3.5).

#### Low survival rates of Campylobacter survival from carcass to retail

Survival rates of *Campylobacter* are adversely impacted by exposure to
air, low temperatures (freezing) and desiccation. This may explain the
low contamination rates in retail pork (Section 2.4.3.5). Several review
articles describe the inability of these organisms to survive the
environmental conditions encountered on swine carcasses \[Error:
Reference source not found,Error: Reference source not found,[^128]\],
demonstrating the sensitivity of *Campylobacter* to oxygen and pH
extremes, their sensitivity to drying by forced air chilling and
freezing. These conclusions are supported by field observations \[Error:
Reference source not found,Error: Reference source not found,Error:
Reference source not found\]. Therefore, because of *Campylobacter*
environmental sensitivity, it is expected that observed carcass
contamination rates in the sampling scheme used by the USDA Food Safety
Inspection Service would be lower than the contamination rates observed
at retail.

These conclusions are supported by field observations. In one study, the
contamination rates of *Campylobacter* on carcasses dropped from 9%
after slaughter, to 0% after chilling \[[^129]\]. In another study
\[Error: Reference source not found\], 56.7% of carcasses were positive
for *C. coli,* but after blast-freezing only 1.7% of the same carcasses
were positive. While *Campylobacter* was detected in 100% and 80% of
rectal and colonic samples, respectively \[[^130]\] in a recent study by
the USDA of 30 swine carcasses (sub sample of 360) through processing,
only 33% of the carcasses were positive for *Campylobacter* immediately
after exsanguination, 0% after polishing, 7% before chilling and 0%
after overnight chilling \[Error: Reference source not found\]. *C.
jejuni* represented only 1% of the *Campylobacter* isolates.

In a study where *C. coli* was inoculated onto pork meat at 3000
CFU/cm^2^ and subjected to blast chilling, counts were reduced to
undetectable levels even when bacterial growth enrichment methods were
used in attempts to recover *Campylobacter* \[Error: Reference source
not found\].

Thus, the likelihood of *Campylobacter* survival post carcass sampling
is low, and would help explain why retail meat sampling indicate low
prevalence rates for *Campylobacter*.

#### Retail pork meat contamination 

The carcass contamination levels summarized above for *Campylobacter*
are much higher than what is observed in raw pork products at retail. In
a survey of 59 retail meat stores in the Washington DC area in 2001,
pork products had a *Campylobacter* prevalence rate of 1.7% \[Error:
Reference source not found\] among 181 samples surveyed. In a
Netherlands survey in 2001, 0% of 524 pork samples and 0.4% of
255-minced pork/beef samples tested positive \[[^131]\]. In a study
conducted at Colorado State University wherein meat samples were
collected from 24 retail stores across the United States, 1.3% of 384
samples were positive \[[^132],[^133]\]. The highest contamination rates
occurred in ground pork products at 3.1% \[Error: Reference source not
found,Error: Reference source not found\]. The authors of the latter
study concluded that *Campylobacter* was the least frequent pathogen
found on retail pork samples \[Error: Reference source not found\].
Alketruse *et al*. \[Error: Reference source not found\] noted lowest
contamination rates in pork samples compared to beef and poultry. In the
most recent preliminary data reported for the NARMS Sentinel Site Retail
Meat surveys \[Error: Reference source not found\], the prevalence of
*Campylobacter* isolated from pork chops was 1% in survey years 2002
(n=613 pork chops sampled, 6 sentinel sites) and 2003 (n=829 pork chops
sampled, 8 sentinel sites).

Collectively, these studies repeatedly demonstrate that although there
can be high prevalence rates of *Campylobacter* in swine feces and soon
after slaughter on carcass with skin on, the organism does not persist
through the pork meat processing and survive to retail.

#### Qualitative ranking of Campylobacter contamination of pork as \"Low\"

Guidance #152 \[Error: Reference source not found\] lists a
*Campylobacter* contamination rate of 32% in market hogs (Table B3 of
Appendix B of Reference \[Error: Reference source not found\]). While
these numbers might be expected, given the high prevalence of *C. coli*
in live swine, the 32% value is a very high overestimate of the
contamination rates in raw pork meat at retail, based on the data above.
As summarized in Section 2.4.3.2, the actual abundance of
*Campylobacter* cells in these positive samples was very low. Most of
these positive samples had quantitative cell levels [\<]{.underline}
0.03 *Campylobacter* cells per cm^2^ as estimated by the most probable
number method (Table 6). Furthermore, data summarized in Sections
2.4.3.3, 2.4.3.4, and 2.4.3.5 support the low contamination rates at
retail.

Qualitatively, the risk of ingestion of *Campylobacter* is "Low",
without further processing or cooking. The survival characteristics of
*Campylobacter* in pasteurized milk, and cooked meat suggest that
*Campylobacter* is rapidly inactivated by minimal pasteurization
\[[^134]\] (60C for 80 sec) or cooking meat to an internal temperature
of 70C \[[^135]\]. In addition, curing and cooking of processed pork
products will also help eliminate contamination by most organisms
including *Campylobacter.*

This qualitative ranking of "Low" exposure is further corroborated by
the fact that *Campylobacter* isolated from humans is generally *C.
jejuni*, and not the predominant *C. coli* organisms typically isolated
from swine, and that epidemiologically, pork meat is not listed as a
major risk factor for *Campylobacter.*

#### Qualitative ranking of Campylobacter contamination of pork is \"Low\"

Guidance #152 \[Error: Reference source not found\] contains non-binding
recommendations regarding risk assessment. According to Guidance #152,
the qualitative determination that *Campylobacter* of pork is \"High\".
The Sponsor proposes that this default contamination rate is an
overestimate of the true overall exposure assessment to the consumer,
based on the following application of the data summarized in Exposure
Assessment Section 2.4.2). Rather, the qualitative prevalence ranking of
*Campylobacter* in pork derived food commodities is \"Low\" because
studies measure contamination rates much less than 5% in pork retail
meats \[Error: Reference source not found,Error: Reference source not
found,Error: Reference source not found,Error: Reference source not
found\] as discussed above.

### Overall Pork Exposure Assessment Conclusion: \"Medium\" 

The application of the ERS pork consumption data (ranked \"High\" per
capita consumption) and the retail pork contamination rate (based on the
literature summarized in Section 2.4.3, including the NARMS Sentinel
Site Retail Meat contamination rate data for pork \[Error: Reference
source not found\]) to this ranking process yields an overall exposure
assessment of \"Medium\" for human exposure to *Campylobacter* through
consumption of pork, based on the process described in Table 5 of
Guidance #152 (Figure 1).

#  Consequence assessment

The Consequence Assessment of macrolides in human medicine is
\"Critically Important \" because macrolides are used for treatment of
the foodborne pathogen, *Campylobacter,* associated with food-producing
animals and because they are important for use in treatment of
Legionnaire\'s disease, and prophylaxis and therapy for serious disease
due to *Mycobacterium avium* Complex (MAC) and *Mycobacterium
avium-intracellulare* (MAI), as presented in Appendix A of Guidance #152
\[Error: Reference source not found\].

# Overall Qualitative Risk estimation 

## Release Assessment Summary 

The Release Assessment is ranked as \"Low\" probability that
macrolide-resistant *Campylobacter* will emerge or be selected as
consequence of the proposed use of tulathromycin, as outlined in Section
1.

## Exposure assessment summary

For cattle and for swine, the Exposure Assessment is a \"Medium\"
probability that humans will be exposed to *Campylobacter* as a result
of exposure to food products derived from cattle or swine, as outlined
in Section 2.

## Consequence assessment summary

The Consequence Assessment of macrolides in human medicine is
\"Critically Important \" because macrolides are used for treatment of
the foodborne pathogen, *Campylobacter,* associated with food-producing
animals and because they are important for use in treatment of
Legionnaire\'s disease, and prophylaxis and therapy for serious disease
due to *Mycobacterium avium* Complex (MAC) and *Mycobacterium
avium-intracellulare* (MAI), as presented in Appendix A of Guidance #152
\[Error: Reference source not found\].

## Overall qualitative risk estimation is "High" for a "Critically Important" drug

The three qualitative assessments above (\"Low\" Release Assessment;
\"Medium\" Exposure Assessment, \"Critically Important\" Consequence
Assessment) can be integrated consistent with Table 6 of Guidance #152
\[Error: Reference source not found\].

All \"Critically Important Drugs\" result is an Overall Risk Estimation
of \"High\" regardless of what the ranking result is for the Release and
Exposure Assessments.

# Risk Management considerations

## Inherent properties of tulathromycin 

There are inherent characteristics of tulathromycin and its proposed use
that lower the potential for concern for selection of
macrolide-resistant *Campylobacter.* First, tulathromycin antibacterial
activity in the colonic content and feces is limited due to the pH in
these microbial environments, lessening selective pressure during the
transient period in which it occurs in the gut post dose. Second,
macrolide resistance occurs in *Campylobacter* by chromosomal mutation
and not by gene-acquisition. Third, macrolide resistance in
*Campylobacter* isolates from humans has remained [\<]{.underline} 3%
with no obvious trends over time in the NARMS surveys despite widespread
macrolide use in humans, companion animals and food animals. Fourth, the
prevalence of any *Campylobacter* in United States pork and beef at
retail is low (0-5%) and the microbiological, molecular,
epidemiological, and historical data base supports that pork and beef is
not a major risk factor for *Campylobacter* causing disease in humans.

## Extent of use

The extent of use limitations listed in Table 7 of Guidance #152 (Figure
2) suggest that the extent of use is considered \"Low\" if the drug is
intended for administered to individuals, and the duration of effective
dose is \<21 days. Therefore, tulathromycin (a branded proprietary
product) use in the treatment of BRD and SRD by individual animal
injection is consistent with a \"Low\" extent of use. Extralabel use may
be considered under the guidance of a veterinarian, within the context
of a valid veterinarian/client/patient relationship. Alternative drug
therapies (tilmicosin, ceftiofur, florfenicol) are available for
comparable indications.

## Approval and Risk Management Steps

Examples of potential risk management steps for \"Category I\" drugs
having an overall \"High\" Risk Estimation are listed in Table 8 of FDA
Guidance #152 \[Error: Reference source not found\], copied below.

> Figure 3 Table 8 of Guidance #152 \[Error: Reference source not
> found\]Examples of potential risk management steps associated with the
> approval of antimicrobial new animal drugs in food-producing animals
> based on the level of risk (high, medium, or low).

  ---------------- ------------------ ------------------- ----------------
  **Approval       **Category 1       **Category 2        **Category 3
  conditions**     (High)**           (Medium)**          (Low)**

  Marketing        Rx                 Rx/VFD              Rx/VFD/OTC
  Status^1^                                               

  Extra-label use  ELU Restrictions   Restricted in some  ELU permitted
  (ELU)                               cases^3^            

  Extent of use^2^ Low                Low, medium         Low, medium,
                                                          high

  Post-approval    Yes                Yes                 In certain cases
  monitoring\                                             
  (e.g., NARMS)                                           

  Advisory         Yes                In certain cases^3^ No
  committee review                                        
  considered                                              
  ---------------- ------------------ ------------------- ----------------

> ^1^Prescription (Rx), Veterinary Feed Directive (VFD),
> Over-The-Counter (OTC)
>
> ^2^See Table 7 for characterization of extent of use

^3^These risk management steps may be appropriate for certain Category 2
drugs that were ranked critically important for consequence assessment
**and** ranked "high" for release **or** exposure assessment

The Sponsor proposes that tulathromycin should be approved as a
veterinary prescription product. The extent of use will be inherently
low, based on the proposed use and parenteral single dosage. This
document is submitted as a component of the Advisory Committee review.
Extralabel use restrictions are not required for this approval, because
macrolides have been approved and used extensively for a variety of
indications in poultry, swine, cattle and other animal species for
decades. The proposed uses of tulathromycin are consistent with
judicious use principles outlined by the FDA/CVM in collaboration with
the American Veterinary Medical Association \[Error: Reference source
not found,Error: Reference source not found\]. Furthermore, the database
reviewed and accepted by the FDA/CVM supports that tulathromycin has a
similar mechanism of action as conventional macrolides approved for
animal use, and has resistance and cross-resistance profiles common to
approved macrolides. Tulathromycin has no more potential to select for
unique resistance mechanisms that would compromise macrolide use in
humans than any of the macrolides already used in animal medicine and
that the selection pressure of existing macrolides use in animals is
substantially more than any incremental pressure that use of this
product might provide.

# Conclusion

With respect to microbial safety considerations, the proposed label uses
of tulathromycin include management considerations of prescription
status, inherent low extent of use due to parenteral single dose
administration, and Advisory Committee review. Macrolide resistance in
*Campylobacter* is currently monitored in the NARMS program. Therefore,
with these management considerations, approval of the proposed
indications for injectable tulathromycin in cattle and swine poses no
appreciable risk to public health with respect to microbial food safety.

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