Section 4

**Estimating quantity of fluoroquinolone resistant *Campylobacter*
contaminated chicken meat consumed**

+-------+-------------------------------------------+------------------+
| **Sym | **Description**                           | **Formula**      |
| bol** |                                           |                  |
+-------+-------------------------------------------+------------------+
| p~c~  | Total prevalence of *Campylobacter* among | Beta             |
|       | broiler carcasses                         | distribution     |
|       |                                           | based on data    |
+-------+-------------------------------------------+------------------+
| p~rc~ | Prevalence of fluoroquinolone resistant   | Beta             |
|       | *Campylobacter* among *Campylobacter*     | distribution     |
|       | contaminated broiler carcasses            | based on data    |
+-------+-------------------------------------------+------------------+
| p~p~  | Estimated prevalence of                   | = p~c~ \*p~rc~   |
|       | fluoroquinolone-resistant *Campylobacter* |                  |
|       | in broiler carcasses                      |                  |
+-------+-------------------------------------------+------------------+
| c     | Consumption of boneless domestically      | Data             |
|       | reared chickens in                        |                  |
|       |                                           |                  |
|       | U.S. per capita (lbs)                     |                  |
+-------+-------------------------------------------+------------------+
| V~c~  | Total consumption of boneless             | = c \* n~US~     |
|       | domestically reared chicken in U.S. (lbs) |                  |
+-------+-------------------------------------------+------------------+
| **V   | Total consumption of boneless,            | **=              |
| ~i~** | domestically reared chicken contaminated  | V~c~\***p**~p~** |
|       | with fluoroquinolone resistant            |                  |
|       | *Campylobacter* in U.S. (lbs)             |                  |
+-------+-------------------------------------------+------------------+

**Overview for Section 4**

This section estimates the burden of fluoroquinolone resistant
*Campylobacter* on chicken carcasses by multiplying the carcass
*Campylobacter* prevalence by the level of resistance in isolates from
chickens. An estimate of the proportion of domestically reared chicken
with fluoroquinolone resistant *Campylobacter* using food disappearance
data, less imports, was calculated to account for changes in chicken
consumption from year to year*.*

Parameters modeled include:

-   Total prevalence of *Campylobacter* among broiler carcasses

-   Prevalence of fluoroquinolone resistant *Campylobacter* among
    *Campylobacter* contaminated broiler carcasses

[Output:]{.underline} Estimated prevalence of fluoroquinolone-resistant
*Campylobacter* contaminated broiler carcasses

-   Consumption of boneless domestically reared chickens in U.S. per
    capita (lbs)

-   Total consumption of boneless domestically reared chicken in U.S.
    (lbs)

[Output:]{.underline} Total consumption of boneless, domestically reared
chicken contaminated with fluoroquinolone resistant *Campylobacter* in
U.S. (lbs)

**Parameter estimations**

[4.1 (p~c~) - Total prevalence of *Campylobacter* among broiler
carcasses]{.underline}

Approximately 200 broiler slaughter establishments were included in the
sample, representing 87% of all broiler slaughter establishments under
Federal inspection in 1994. The broilers slaughtered at these
establishments accounted for more than 99.9% of all broilers slaughtered
during the period. Sample size, to provide reasonable levels of
precision for a national prevalence, was estimated at 1200 samples. To
achieve this number of samples a random number of 1871 broiler carcass
samples were requested during the 52-week sampling period. Some samples
were not collected, some were collected but not analyzed and the total
number of samples providing laboratory results for the prevalence
estimate was 1297 samples (104). Sampling frame was based upon weekly
identification of randomly selected establishments using probabilities
for sample selection that were proportional to the slaughter volume of
the selected establishments, therefore those establishments slaughtering
a greater number of chickens were sampled more frequently than other
establishments. Sample delivery constraints resulted in the restriction
of sampling to first shifts, Monday through Thursday. Carcasses were
obtained from the drip line after the chill tank, the end point for
slaughter and evisceration and prior to further handling and processing.
Whole carcasses were randomly selected, and aseptically placed into a
sterile bag that was securely closed, double bagged, packed with a gel
pack and shipped to the laboratory via overnight delivery service. Only
samples received at temperatures between 0 to 10 degrees C (inclusive)
within one day of sample collection were analyzed. The analytical sample
was obtained from rinse fluid recovered after shaking the broiler
carcass in 400 ml of sterile Butterfield's Phosphate Diluent (104).
Isolation was achieved using Hunt's Enrichment Broth, incubating the
sample for 24 hours in a microaerophillic environment (5% O~2~ , 10%
CO~2~ and 85% N~2~), followed by streaking onto Modified *Campylobacter*
Charcoal Differential Agar for isolation of *Campylobacter* spp after
incubation at 42 degrees C for 24 hours (81). Tests to identify
*Campylobacter jejuni* and *coli* included wet mount examination,
glucose fermentation, catalase, nalidixic acid, and oxidase tests.
Nalidixic acid screening was performed to eliminate *Campylobacter* spp
other than *jejuni* and *coli* from the prevalence estimate. Since
fluoroquinolones were not licensed for use in poultry during the survey
period, it was assumed that the level of nalidixic acid resistant
isolates was low in 1994-5 because no selective pressure existed for
chickens to develop fluoroquinolone resistance. Therefore, the
prevalence estimate was unlikely to be affected by acquired resistance
and potential misclassification of *Campylobacter* species.

ASSUMPTION: If a carcass was positive for *Campylobacter*, the
predominant species isolated was *C. jejuni.*

The *Camplyobacter* prevalence estimate from the drip line was preferred
because at this point carcasses were ready for further processing and
had the least potential of human or other non-chicken sources of
contamination. Post-chiller sampling of carcasses takes into account the
cross-contamination from other chickens that occurs while in the chiller
that leads to carriage of many diverse strains of *Campylobacter* on a
single chicken product (92). The post-chiller location is a sampling
point that is repeatable, practical, and provides isolates for
susceptibility testing, closely linking these two parameters to provide
a better estimate of the level of resistance. This would be more
relevant for future surveys, when concurrent carcass prevalence and
susceptibility testing could be conducted, as is currently underway in
1999.

The prevalence of *Campylobacter* in chickens was estimated from a
1994-95 survey of **1,297** broiler carcass rinse samples at 88.2% of
carcasses, indicating that **1,144** carcasses tested positive (104).

The parameter was thus modeled as:

p~c~ = Beta(1144+1,1297-1144+1)

[4.2 (p~rc~) Prevalence of FQ resistant *Campylobacter* among
*Campylobacter* broiler carcasses]{.underline}

Isolates were collected in a pilot survey by USDA-FSIS, from October to
December 1998, from chicken carcass rinse samples (Section 4.1) and
cultured as described previously (81). If growth was evident, a single
colony was removed from the plate for susceptibility testing. A total of
128 *C. jejuni* isolates were collected from chicken carcasses for the
period. The isolates were speciated using the biochemical hippurate
assay and polymerase chain reaction (PCR) hippuricase primers to
identify hippurase negative *C. jejuni* (74). The proportion of
Fluoroquinolone resistant *C. jejuni* isolates was 9.4%
(**12**/**128**), and *C."*other" was 21.2% (14/66) (100). *C.* "other"
were hippurate negative isolates, that were not *jejuni* as identified
by PCR for the hippuricase gene and may include species such as *C.
lari* that are intrinsically resistant to quinolones.

In 1999, collection of isolates was continuous throughout the year and a
total of **481** *C. jejuni* isolates were obtained (101). Of these,
**45** isolates were resistant so the proportion of isolates that were
resistant was 9.4%.

The level of resistance to fluoroquinolone in *C. jejuni* from chickens
was used in the risk assessment because the greater proportion of human
disease, 92.7% in the *Campylobacter* Case Control Study, was due to
*C*. *jejuni*. *C. coli* were not clearly distinguished from the group
*C.* "other" which may have included *C. lari*, a species intrinsically
resistant to quinolones, therefore this precluded use of these isolates
in the risk assessment.

DISCUSSION: Limitations in determination of the level of fluoroquinolone
resistance in *Campylobacter* in 1998 included: the small number of
isolates collected, the lack of seasonal representation. In addition,
the presence of mixed colonies of organisms (*C. jejuni* and *C. coli*)
when selecting a single colony makes species identification more
complicated.

## Unquantified Issues in the Assessment of the Prevalence of Resistance in Campylobacter isolates 

Other problems were raised with the isolation and susceptibility testing
of *Campylobacter*. Lack of agreement of MIC susceptibility test results
occurs in up to 10% (2/20) of isolates subjected to repeat testing in
one study (personal communication P. Fedorka-Cray). One explanation of
the inconsistency is that the single colony may be composed of multiple
isolates and that all isolates in the mixed colony may not have the same
potential to survive storage, freezing, re-culture and testing. The
effect of selecting a colony with multiple isolates decreases the
reliability of susceptibility testing. The species of each reported
isolate was confirmed by PCR of the *hipp-O* and *ceu* genes to identify
*C. jejuni* and *C. coli* respectively.

In addition to the problem mentioned in the paragraph above, many varied
*Campylobacter* colonies are present on a culture plate. The selection
of a single colony from a plate of diverse colonies provides a "plate
average," and the level reported will consistently underestimate the
true carcass prevalence because a plate may carry resistant and
susceptible isolates. A survey conducted in 1998-9 using a selective
media for fluoroquinolone resistance indicated that of retail chicken
products from which *Campylobacter* had been isolated, 24.5% (15/61)
carried fluoroquinolone resistant isolates (82). Therefore, the actual
prevalence of chicken carcasses carrying resistance may be much higher
than the estimate obtained from testing a single isolate. Use of a
quinolone-containing screening media would provide a better estimate of
the true carcass prevalence and may give an indication of load of
fluoroquinolone resistant *Campylobacter* on chicken. This may be a more
accurate method to use to assess the impact of resistant pathogens.

The three issues described above; the lack of reliability of in
identification of *Campylobacter* species using biochemical assay and
the lack of accuracy in the determination of the level of resistance
using a single isolate leading to an underestimation of the level of
resistance in chicken carcasses are issues that are currently not
quantifiable. These issues need to be better characterized and methods
developed to allow more meaningful assessments of their impact on both
human and foodborne isolates. This risk assessment determined the
measurable risk, limiting the model to those parameters for which data
were relevant, valid and available.

The parameter was thus modelled as:

1998: p~rc~ = Beta(12+1,128-12+1)

1999: p~rc~ = Beta(45+1,481-45+1)

[4.3]{.underline} (p~p~) - [Estimated prevalence of
fluoroquinolone-resistant *Campylobacter* in broiler
carcasses]{.underline}

This parameter is calculated as:

p~p~ = p~c~ \*p~rc~

[4.4 (c) - Consumption of boneless domestically reared chickens in U.S.
per capita (lbs)]{.underline}

An annual value representing measurable human exposure to chicken in the
United States less product sent for rendering, product diverted for pet
food, exports, water added during processing and imports was the pounds
of boneless broiler food disappearance, which in 1998 was 50.8 lbs per
capita (102, 113).

1998 c = 50.8 lbs

1999 c = 54.3 lbs

[4.5 (V~c~) -- Total consumption of boneless domestically reared
chickens in U.S. per capita (lbs)]{.underline}

This parameter is calculated as:

V~c~ = c \* n~US~

[4.6 (**V~i~**) - Total consumption of boneless, domestically reared
chicken contaminated with fluoroquinolone resistant *Campylobacter* in
U.S. (lbs)]{.underline}

This parameter is calculated as:

V~i~ = V~c~ \*p~rc~

It represents the amount of boneless product contaminated with
fluoroquinolone resistant *Campylobacter* consumed in the U.S. in the
year. Figure 4.1 shows the uncertainty distribution for **V~i~**.

  ----------- ----------------- -------------- -------------- --------------
  **Year**    **Model output**  **5^th^        **Mean**       **95^th^
                                percentile**                  percentile**

  **1998**    V~I~              7.34E+08       1.21E+09       1.77E+09

  **1999**    V~I~              9.68E+08       1.24E+09       1.54E+09
  ----------- ----------------- -------------- -------------- --------------

![](media/image1.wmf){width="6.009027777777778in"
height="4.208333333333333in"}

**Figure 4.1**. Uncertainty distribution for **V~i~**.

# Section 4 Summary

The mean estimated value for pounds of boneless chicken carrying
fluoroquinolone resistant *Campylobacter* consumed in 1998 is
1,210,000,000 pounds. The 5^th^ percentile estimate is 734,000,000 and
the 95^th^ percentile estimate is 1,770,000,000 pounds. In 1999, the
mean estimated value was 1,240,000,000 pounds with a 5^th^ percentile of
968,000,000 and a 95^th^ percentile of 1,540,000,000. Relative
contributions of the various components of the model to the model
uncertainty will be presented in Section 5, Sensitivity Analysis.