Comments on abstract, title, references

Title Antimicrobial Susceptibility to Azithromycin among Salmonella enterica Isolates from the United States Comments: A better rephrasing of the title would be: Susceptibility to the antimicrobial Azithromycin among Salmonella enterica Isolates from the United States

Abstract 1. Due to emerging resistance to traditional antimicrobial agents, such as ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol, azithromycin is increasingly used for the treatment of invasive Salmonella infections. Comments: Chloramphenicol is no longer considered as a traditional antimicrobial agent due to its severe side effects such as aplastic anemia.

1. In the present study, 696 isolates of non-Typhi Salmonella collected from humans, food animals, and retail meats in the United States were investigated for antimicrobial susceptibility to azithromycin. Seventy-two Salmonella enterica serotype Typhi isolates from humans were also tested. Comments: The 696 isolates of non-Typhi Salmonella collected from humans, food animals, and retail meats in the United States and the Seventy-two Salmonella enterica serotype Typhi isolates from humans that were tested for susceptibility to Azithromycin were collected by different teams from different places and at different times which may affect the uniformity of the testing results.

2. For each isolate, MICs of azithromycin and 15 other antimicrobial agents were determined by broth microdilution. Among the non-Typhi Salmonella isolates, azithromycin MICs among human isolates ranged from 1 to 32 g/ml, whereas the MICs among the animal and retail meat isolates ranged from 2 to 16 g/ml and 4 to 16 g/ml, respectively. Comments: It is paradoxical to see that the MICs of Azithromycin are of higher range among Salmonella isolates from human sources than the MICs for the same antimicrobial among Salmonella isolates from the non-human sources given the wide use of various antimicrobials as food additives at subtherapeutic doses which may lead to development of drug resistance.

3. The highest MIC observed in the present study was 32 g/ml, and it was detected in three human isolates belonging to serotypes Kentucky, Montevideo, and Para-typhi A. Based on our findings, we propose an epidemiological cutoff value (ECOFF) for wild-type Salmonella of <16 g/ml of azithromycin. Comments: It is paradoxical to see that the MICs of Azithromycin are of higher range than the MICs for the same antimicrobial among Salmonella isolates from the non-human sources given the wide use of various antimicrobials as food additives at subtherapeutic doses which may lead to development of drug resistance.

4. The susceptibility data provided could be used in combination with clinical outcome data to determine tentative clinical breakpoints for azithromycin and Salmonella enterica. Comments: Such conclusion should perhaps be based on a wider study and a larger number of Salmonella isolates.

Introduction: 1. Non-Typhi Salmonella is the second leading cause of foodborne illness in the United States (34). Each year, approximately 1.0 million people are infected with Salmonella, resulting in 19,000 hospitalizations and almost 400 deaths (34). Comments: The morbidity and fatality figures listed her are not compatible with data from the sited reference (34).

1. By performing antimicrobial susceptibility testing on 696 isolates of non-Typhi Salmonella isolated from humans, food animals, and retail meats in the United States, we provide data on the range of azithromycin MICs observed among Salmonella enterica isolates from the United States. Comments: A study with larger numbers of Typhoidal and non-typhoidal Salmonella isolates that more uniformly collected from human and non-human sources is probably warranted for the determination of the MICs of Azithromycin among Salmonella serotypes in the United States.

2. Due to widespread resistance to traditional first-line drugs, such as ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol, current recommendations suggest using a fluoroquinolone (e.g., ciprofloxacin) or an extended-spectrum cephalosporin (e.g., ceftriaxone) for treating invasive and severe Salmonella infections (16, 18). Comments: Chloramphenicol is no longer of the first line drugs to treat infection.

3. At present, no clinical azithromycin breakpoints have been defined for Enterobacteriaceae, including Salmonella, by either the CLSI or EUCAST (5, 6, 11). By performing antimicrobial susceptibility testing on 696 isolates of non-Typhi Salmonella isolated from humans, food animals, and retail meats in the United States, we provide data on the range of azithromycin MICs observed among Salmonella enterica isolates from the United States. We further present azithromycin MICs for 72 isolates of Salmonella serotype Typhi. This information could be combined with clinical outcome data to facilitate establishment of clinical azithromycin breakpoints for Salmonella

Comments: A larger study to determine the clinical azithromycin breakpoints. Such study should include larger number of Salmonella clinical isolates as well as salmonella isolates from several non-human sources based on a valid statistical representation of these isolates in the study.

MATERIALS AND METHODS

1. In 2008, 54 state and local public health laboratories participating in the National Antimicrobial Resistance Monitoring System (NARMS) forwarded every isolate of Salmonella serotype Typhi and every 20th non-Typhi Salmonella isolate from human clinical infections to the Centers for Disease Control and Prevention (CDC). Comments: Providing a rational for such sampling is helpful but is not included in this study.

2. Similarly, non-Typhi Salmonella isolates from retail meats (chicken breasts, ground turkey, ground beef, and porkchops) were submitted by the states participating in the Foodborne Diseases Active Surveillance Network (FoodNet) for analysis at the U.S. Food and Drug Administration Center for Veterinary Medicine (FDA-CVM). Non-Typhi Salmonella isolates from food animals were obtained from carcass rinsates (chicken), carcass swabs (turkey, cattle, and swine), and ground products (chicken, turkey, and beef). Animal samples were collected by the U.S. Department of Agriculture’s (USDA) Food Safety Inspection Service (FSIS) from federally inspected slaughter and processing plants throughout the United States and forwarded to the USDA in Athens, GA, for further analysis. Comments: Samples were collected by different groups, from several sources, and at different times. This may lead to variations in the antimicrobial testing results.

3. In addition, all isolates were tested for susceptibility to 15 antimicrobial agents included on the NARMS Gram-negative panel (amikacin, ampicillin, amoxicillin-clavulanic acid, ceftiofur, ceftriaxone, cefoxitin, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, sulfamethoxazole, streptomycin, trimethoprim-sulfamethoxazole, and tetracycline) and interpreted according to CLSI standards, where available (5).

Comments: It would have been of value to have the classes of antimicrobial resistance profiles for these antimicrobials compared to the Azithromycin MICs study in this paper.

1. Additional susceptibility testing with azithromycin Etest (bioMe´rieux, Inc., NC) strips was performed according to the manufacturer’s instructions on Mueller-Hinton II agar plates incubated at 37°C for 16 to 20 h. An MIC histogram was constructed, and the MIC50 value, representing the MIC at which the growth of 50% of the population is inhibited, was calculated for each of the four sample groups (Salmonella serotype Typhi from humans and non-Typhi Salmonella from humans, retail meats, and animals). The histograms were further inspected to identify the wild-type MIC distribution,

Comments: Results of the ETest and the Sensitizer testing for MICs are not clearly presented in the result section.

Results:

1. In 2008, 2,379 isolates of non-Typhi Salmonella were submitted to the CDC, 1,326 to the USDA, and 495 to the FDACVM as part of the NARMS program for enteric bacteria. At each agency, a subset of 232 isolates was randomly chosen to be tested for antimicrobial susceptibility to azithromycin. In addition, 72 isolates of Salmonella serotype Typhi were included. Among the Salmonella serotype Typhi isolates, 49 (68.1%) were isolated from blood cultures and 16 (22.2%) from stool. The sources of the remaining 7 isolates were not provided by the submitting laboratory.

Comments: Rational for the selection of the 232 subsets of Salmonella isolates could have been useful.

1. The sources of the remaining 7 isolates were not provided by the submitting laboratory. Among the 232 non-Typhi Salmonella isolates randomly selected from human isolate submissions, Salmonella enterica serotypes Enteritidis (19.0%) and Typhimurium (13.4%) were most common. Of these, 84.1% were isolated from stool cultures and 9.1% from blood cultures. The remaining 16 isolates were isolated from urine, other sources, or non-specified sources. Among the isolates randomly selected for analysis from food animal submissions, Salmonella enterica serotypes Kentucky (16.4%), Heidelberg (9.5%), and Montevideo (7.3%) were most common, and among the isolates randomly selected from retail meats, Salmonella enterica serotypes Heidelberg (19.6%), Hadar (16.8%), and Typhimurium variant O:5(10.8%) predominated.

Comments: There are clear differences between the Salmonella serotypes from human and non-human sources. DISCUSSION 1. Clinical breakpoints are necessary to detect emerging and changing patterns of resistance and to guide clinicians in the selection of effective antimicrobial therapy. The first step toward defining clinical breakpoints is to collect relevant data, including (i) pharmacodynamic data of the drug, (ii) pharmacological properties of the drug,(iii) clinical outcome data, and (iv) microbiological data, i.e., MIC data for the specific pathogen in question (7, 35). In this paper, we provide MIC data for 696 isolates of non-Typhi Salmonella and 72 isolates of Salmonella serotype Typhi that could contribute to establishing susceptibility breakpoints for Salmonella and azithromycin.

Comments: Well stated as a contribution to the establishment of the susceptibility breakpoints for Salmonella and azithromycin. Elsewhere in the manuscript, it was stated that the susceptibility breakpoints for Salmonella and azithromycin was established. Needs correction.

1. Additional studies would be required to confirm the accuracy of the proposed ECOFF and determine whether isolates displaying an MIC value of 32 ug/ml belong to the wild-type distribution. Whether NWT isolates should be classified as clinically resistant remains to be determined following the accumulation of clinical endpoint data.

Comments: Such conclusion supports our initial comments that further studies are required to establish a valid susceptibility breakpoints for Salmonella and azithromycin.