Detection of Salmonella enterica Subpopulations

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2007, p. 7753–7756 0099-2240/07/$08.00⫹0 doi:10.1128/AEM.01228-07

Vol. 73, No. 23

Detection of Salmonella enterica Subpopulations by Phenotype Microarray Antibiotic Resistance Patterns䌤† Jean Guard-Bouldin,1* Cesar A. Morales,1 Jonathan G. Frye,2 Richard K. Gast,1 and Michael Musgrove1 U.S. Department of Agriculture, ESQRU,1 and U.S. Department of Agriculture, BEAR,2 950 College Station Road, Athens, Georgia 30605 Received 1 June 2007/Accepted 26 September 2007

Three strains of Salmonella enterica serotype Enteritidis were compared to Salmonella enterica serotype Heidelberg, Salmonella enterica serotype Newport, and Salmonella enterica serovar Typhimurium for growth in the presence of 240 antibiotics arranged within a commercial high-throughput phenotype microarray. The results show that antibiotic resistances were different for subpopulations of serotype Enteritidis separated only by genetic drift.

Salmonella enterica serotype Enteritidis is the only one of over 1,400 Salmonella enterica serotypes that efficiently contaminates the contents of eggs and causes disease in humans (4, 15, 20). It is a model serotype for evaluating how genetic drift impacts phenotype, because mutational mapping of genetically related pathotypes has been completed. Current estimates of the genetic distances between serotype Enteritidis subpopulations, between serotype Enteritidis phage types, and between serotype Enteritidis and other Salmonella serotypes with a common lipopolysaccharide core structure are estimated at one single-nucleotide polymorphism for every 10,000, 1,000, and 100 contiguous base pairs, respectively. A rapid screening method that detects the subpopulation biology of strains that appear to be clonal would facilitate epidemiological investigations that monitor the emergence of genetic drift that impacts human health. To identify antibiotics for this purpose, we used a commercially available high-throughput phenotype microarray (Phenotype MicroArray [PM]; Biolog, Inc.) to compare the antibiotic resistance patterns of three subpopulations of serotype Enteritidis to those of three other serotypes of S. enterica, namely serotype Heidelberg, serovar Typhimurium, and serotype Newport. Specifically, the strains evaluated were (i) egg-contaminating, biofilm-positive serotype Enteritidis phage type 4 strain ESQRU 22079 (serotype Enteritidis PT4) (17); (ii) biofilm-positive serotype Enteritidis phage type 13a strain ESQRU 21027, which does not contaminate eggs (BF serotype Enteritidis PT13a) (18); (iii) wild-type egg-contaminating serotype Enteritidis PT13a strain ESQRU 21046, which does not produce biofilm (wt serotype Enteritidis PT13a) (18); (iv) serotype Heidelberg strain ESQRU 23018, a human isolate from a rare egg-associated outbreak (13); (v) serotype Newport strain ESQRU 24012, which has multiple

antibiotic resistances; and (vi) serovar Typhimurium strain ESQRU 99187, which was isolated from the environment of chickens. Strains of these four serotypes were recovered from 50%, 53%, and 51% of laboratory-confirmed cases of salmonellosis in the United States for the years 2004, 2005, and 2006, respectively (26). PM analysis. PM testing was performed by Biolog’s PM Services group (Hayward, CA). The basic growth medium chemistry for PM analysis was published previously (3, 27). PM analysis was conducted in duplicate after incubation of the strains at 37°C for 48 h, and the data were analyzed by Student’s t test to confirm that the results were uniform between runs. The metabolism of D-serine was an internal control, because BF serotype Enteritidis PT13a has a 10-bp deletion in the serine deaminase gene (dsdA), and the algorithm for defining when wells were otherwise positive for growth above baseline based on the respiratory activity (RA) of the cells in the initial inoculum has been described previously (18). Raw data were obtained for 10 96-well master plates (Biolog PM plates 11 to 20), which included 240 antibiotics arranged as a dilution series across four wells (960 wells in total). Data were recorded as RAs and were filtered in Microsoft Excel to identify statistically significant differences by Student’s t test (see Table S1 in the supplemental material). Antibiotic susceptibilities were also tested by a conventional assay (Sensititre Microbiologic Systems, Westlake, OH) (12). Results. The conventional assay indicated that serotype Newport had multiple antibiotic resistances and that wt serotype Enteritidis PT13a was resistant to ampicillin and tetracycline (Table 1). Filtering of all PM data indicated that the results for five ␤-lactam antibiotics and sodium selenite showed significant (P ⬍ 0.001) differences between BF and wt serotype Enteritidis PT13a (Table 2). Wild-type serotype Enteritidis PT13a grew almost as well as serotype Newport in the ␤-lactam antibiotics amoxicillin, carbenicillin, and penicillin G in the PM (Table 2): specifically, serotype Newport had average RAs of 329.6, 296.0, and 345.6, and wt serotype Enteritidis PT13a had average RAs of 270.4, 274.3, and 289.8 for these compounds, respectively (see Table S1 in the supplemental

* Corresponding author. Mailing address: U.S. Department of Agriculture, Agricultural Research Service, Egg Safety and Quality Research Unit, 950 College Station Road, Athens, GA 30605. Phone: (706) 546-3446. Fax: (706) 546-3035. E-mail: Jean.Guard.Bouldin@ars .usda.gov. † Supplemental material for this article may be found at http://aem .asm.org/. 䌤 Published ahead of print on 26 October 2007. 7753

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TABLE 1. Antibiotic resistance patterns of serotypes of S. enterica by sensititer assay Antibiotic resistance pattern fora: Antibiotic compound

BF serotype Enteritidis PT13a (ESQRU 21027)b Resistance

Amikacin Amoxicillin-clavulanic acid Ampicillin Cefoxitin Ceftiofur Ceftriaxone Chloramphenicol Ciprofloxacin Gentamicin Kanamycin Nalidixic acid Streptomycin Sulfisoxazole Tetracycline Trimethoprimsulfamethoxazole a b

S S S S S S S S S S S S S S S

MIC 1 ⱕ1 ⱕ1 2 ⬍0.5 ⱕ0.25 4 ⱕ0.015 ⱕ0.25 ⱕ8 4 ⱕ32 32 ⱕ4 ⱕ0.12

wt serotype Enteritidis PT13a (ESQRU 21046)

Serotype Enteritidis PT4 (ESQRU 22079)

Resistance

MIC

Resistance

S S

1 8

S S

R S S S S S S S S S S R S

⬎32 1 0.5 ⱕ0.25 8 ⱕ0.015 ⱕ0.25 ⱕ8 4 ⱕ32 32 ⬎32 ⱕ0.12

S S S S S S S S S S S S S

MIC

Serotype Typhimurium (ESQRU 99187) Resistance

1 ⱕ1

S S

2 2 1 ⱕ0.25 4 ⱕ0.015 ⱕ0.25 ⱕ8 4 ⱕ32 32 ⱕ4 ⱕ0.12


Serotype Heidelberg (ESQRU 23018)

MIC 1 ⱕ1 ⱕ1 2 0.5 ⱕ0.25 4 ⱕ0.015 ⱕ0.25 ⱕ8 4 ⱕ32 32 ⱕ4 ⱕ0.12

Serotype Newport (ESQRU 24012)

Resistance

MIC

Resistance

MIC

S S

ⱕ0.5 ⱕ1

S R

1 32


ⱕ1 1 1 ⱕ0.25 8 ⱕ0.015 ⱕ0.25 ⱕ8 4 ⱕ32 ⱕ16 ⱕ4 ⱕ0.12

R R R I R S S S R R R R S

⬎32 ⬎32 ⬎8 16 ⬎32 0.25 ⱕ0.25 ⱕ8 ⬎32 ⬎64 ⬎256 ⬎32 ⱕ0.12

S, sensitivity; R, resistance; I, intermediate resistance. MICs are given in ␮g/ml. Strain numbers are given in parentheses.

material) (18). Both wt serotype Enteritidis PT13a and serotype Newport grew in sodium selenite (RA ⬎ 50), whereas BF serotype Enteritidis PT13a did not (Table S1 in the supplemental material). Overall, sensitivity to the ␤-lactam antibiotics differentiated between monomorphic strains of serotype Enteritidis as well as did the control compound D-serine, which links an easily observable phenotype to a known genomic lesion—a 10-bp deletion (bp 3880104 to 3880113 within the reference genome of serotype Enteritidis PT4). Two other findings for the compounds in Table 2 were that the monomorphic BF serotype Enteritidis PT13a strains were significantly different from all other strains and that wt serotype Enteritidis PT13a had resistances that were more similar to those of serotype Newport than could be detected by the conventional assay (Table 2). The sensitivity of wt serotype Enteritidis PT13a to amoxicillin-clavulanic acid in the conventional assay is more appropriately reported as intermediate, because values were eight times higher than those for any of the other Salmonella strains that were sensitive (Table 1). Thirty-six compounds distinguished monomorphic PT13a strains from dimorphic serotype Enteritidis PT4 and from all

other serotypes (Table 3). Serovar Typhimurium was somewhat unique, because its sensitivity was intermediate to that of monomorphic and dimorphic strains of serotype Enteritidis (Table 3). Serovar Typhimurium grew more like monomorphic serotype Enteritidis PT13a strains in 14 (38.9%) of the 36 compounds, whereas serotype Heidelberg and serotype Newport resembled monomorphic strains in their growth in only 8.3% and 11.1% of the compounds, respectively. These results suggest that serotype Heidelberg and serotype Newport grew more like dimorphic serotype Enteritidis PT4 than like the monomorphic strains. The conventional assay for antibiotic resistances suggested that wt serotype Enteritidis PT13a was resistant to tetracycline. However, there was no evidence of this resistance in the PM (Table 4). In contrast, serotype Newport was resistant to tetracycline antibiotics (P ⬍ 0.01) in both the conventional and PM assays. The resistance of wt serotype Enteritidis PT13a to tetracycline thus appears to be overstated by the conventional assay. Differences in antibiotic concentrations and in the media used to assess resistance could account for the variation between assays.

TABLE 2. Differentiation of S. enterica subpopulations and serotypes by antibiotic compounds that differentiate between monomorphic subpopulations of serotype Enteritidis P of similarity to wt serotype Enteritidis PT13aa Compound name

Concn range of antibiotic (␮g/ml)

PM plate no.

Amoxicillin Carbenicillin D-Serine Oxacillin Penicillin G Phenethicillin Sodium selenite

0.1–10 1.3–130 347–34,700 13–1,300 1–100 18–1,800 30–3,000

11C 14A 17A 12B 12B 19 16A

BF serotype Enteritidis PT13a

Serotype Enteritidis PT4

Serotype Typhimurium

Serotype Heidelberg

Serotype Newportb

0.000 0.000 0.000 0.000 0.000 0.000 0.000

0.006 0.052 0.004 0.379 0.014 0.061 0.060

0.001 0.003 0.731 0.292 0.006 0.019 0.682

0.026 0.108 0.213 0.546 0.083 0.182 0.005

0.000 0.500 0.240 0.355 0.000 0.247 0.069

A probability (P) of ⬍0.001 indicates a significant difference. Serotype Newport has multiple antibiotic resistances; thus, it has an origin of antibiotic resistance that is fundamentally different to that of serotype Enteritidis that correlates with single-nucleotide polymorphisms. a b

ANTIBIOTIC RESISTANCES OF SALMONELLA SUBPOPULATIONS

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TABLE 3. Differentiation of S. enterica subpopulations and serotypes by antibiotic compounds that screen for dimorphism P of similarity to wt serotype Enteritidis PT13aa Concn range of antibiotic (␮g/ml)

Compound name

2,4-Diamino-6,7diisopropylpteridine 3,5-Diamino-1,2,4-triazole (guanazole) 5-Fluoroorotic acid 5-Fluoro-5⬘-deoxyuridine ␤-Chloro-L-alanine Azathioprine Cefamandole Cefsulodin Ceftriaxone Cesium chloride Cetoperazone Chelerythrine Chlorhexidine Chromium chloride D-Cycloserine Diamide Erythromycin Fusidic acid Glycine L-Glutamic-␥-hydroxamate Moxalactam Myricetin Nitrofurantoin Oleandomycin Oxolinic acid Phosphomycin Piperacillin Rifamycin SV Sodium bromate Sorbic acid Spectinomycin Sulfanilamide Tinidazole Triclosan Trifluoperazine Trifluorothymidine

PM plate no.




Serotype Heidelberg

Serotype Newportb

0.7–70

12B

0.353

0.001

0.000

0.001

0.003

83–8,300

18C

0.076

0.000

0.000

0.000

0.000

12B 18C 17A 18C 17A 17A 11C 13B 17A 14A 19 16A 15B 16A 11C 15B 13B 16A 13B 18C 14A 15B 13B 16A 14A 16A 18C 16A 12B 16A 18C 18C 13B 18C

0.418 0.007 0.268 0.043 0.171 0.120 0.355 0.838 0.960 0.598 0.013 0.009 0.613 0.530 0.047 0.041 0.001 0.082 0.024 0.261 0.100 0.053 0.812 0.256 0.030 0.650 0.027 0.000 0.087 0.596 0.020 0.236 0.054 0.040

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.000 0.000 0.000 0.000 0.009 0.002 0.000 0.000 0.000 0.000 0.000 0.000 0.002 0.000 0.000 0.000 0.000 0.005 0.000 0.001 0.005 0.000 0.000 0.001 0.000

0.001 0.000 0.020 0.000 0.000 0.000 0.001 0.004 0.000 0.000 0.000 0.000 0.011 0.011 0.030 0.002 0.000 0.000 0.000 0.011 0.013 0.466 0.000 0.000 0.000 0.456 0.056 0.010 0.000 0.020 0.405 0.000 0.095 0.000

0.000 0.002 0.000 0.000 0.000 0.000 0.000 0.003 0.000 0.000 0.000 0.000 0.000 0.010 0.000 0.000 0.000 0.000 0.000 0.003 0.000 0.001 0.000 0.000 0.000 0.000 0.003 0.000 0.000 0.003 0.300 0.000 0.000 0.000

0.003 0.000 0.000 0.000 0.000 0.000 0.000 0.002 0.000 0.000 0.000 0.000 0.000 0.004 0.016 0.000 0.000 0.000 0.000 0.001 0.002 0.036 0.000 0.000 0.000 0.000 0.001 0.000 0.000 0.000 0.132 0.000 0.005 0.000

1–100 0.7–70 26–2,600 6–600 0.03–3 0.9–90 0.002–0.2 14–1,400 0.007–0.7 0.6–60 0.006–0.6 6–600 0.3–30 2.6–260 0.7–70 11–1,100 36–3,600 0.5–50 0.01–1 2–200 0.08–8 13–1,300 0.01–1 0.17–17 0.07–7 3–300 210–21,000 15–1,500 0.3–30 3.6–360 11–1,100 0.023–2.3 6–600 0.07–7

A P of ⬍0.01 indicates a significant difference. Serotype Newport has multiple antibiotic resistances; thus, it has an origin of antibiotic resistance that is fundamentally different to that of serotype Enteritidis that correlates with single-nucleotide polymorphisms. a b

Conclusions. Several recent antibiogram studies of field isolates suggest that serotype Enteritidis is developing some antibiotic resistances (2, 5–11, 14, 16, 19, 21–25). However, genomic resequencing has not detected any differences in the mar regulon of strains of serotype Enteritidis. If the chromosome of serotype Enteritidis bacteria is currently evolving an-

tibiotic resistances from small-scale evolutionary events, then an assay for subpopulation biology using antibiotics could help identify genetic drift that impacts public health (1). The results from these analyses improved the interpretation of resistances to the ␤-lactam and tetracycline classes of antibiotics by combining information from both the PM and conventional assays.

TABLE 4. Differentiation of S. enterica subpopulations and serotypes by the tetracycline class of antibiotic compounds P of similarity to wt serotype Enteritidis PT13aa Compound name

Concn range of antibiotic (␮g/ml)

PM plate no.

Chlortetracycline Oxytetracycline Rolitetracycline Tetracycline

0.25–25 0.25–25 0.2–20 0.08–8

11C 20B 13B 12B




Serotype Heidelberg

Serotype Newportb

0.063 0.149 0.216 0.556

0.739 0.514 0.831 0.297

0.528 0.406 0.694 0.780

0.723 0.276 0.475 0.081

0.000 0.000 0.000 0.000

A P of ⬍0.01 indicates a significant difference. Serotype Newport has multiple antibiotic resistances; thus, it has an origin of antibiotic resistance that is fundamentally different to that of serotype Enteritidis that correlates with single-nucleotide polymorphisms. a b

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