Effects Antimicrobial Agents Fed to Chickens SomeGram- … · resistant Salmonella sp....

Vol. 50, No. 3APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1985, p. 638-6480099-2240/85/090638-11$02.00/0Copyright © 1985, American Society for Microbiology

Effects of Antimicrobial Agents Fed to Chickens on Some Gram-Negative Enteric Bacillit

STEPHEN W. MAMBERt* AND STANLEY E. KATZDepartment of Biochemistry and Microbiology, Cook College-Rutgers, The State University, New Brunswick,

New Jersey 08903

Received 4 February 1985/Accepted 28 May 1985

Total and antimicrobial agent-resistant aerobic and facultative anaerobic gram-negative enteric bacilli infecal samples of broiler chickens fed growth-promotional levels of antimicrobial agents were determinedquantitatively. Two 8-week studies were conducted utilizing groups of chickens fed antimicrobial-supplementedrations; the second study involved feed "pasteurization" as a means of minimizing colonization from the feed.Dilution/spread-plating/replica-plating techniques on selective media were used to obtain counts of totalorganisms and those resistant to tetracycline, chloramphenicol, streptomycin, ampicillin, or kanamycin. Thepredominant aerobic and facultative anaerobic gram-negative organism was Escherichia coli, which wasdetected in all samples at levels ranging from 105 to over 1010 CFU/g of feces. Less common were Proteusmirabilis, Klebsiella pneumoniae, and Pseudomonas sp., which varied in occurrence and levels from group togroup (range, <103 to 108 CFU/g). Resistance to all antimicrobials (except chloramphenicol in E. coli) wascommonly observed at incidences exceeding 103 CFU/g in the total populations. Colonization of the chickens'intestinal tracts by susceptible and resistant strains of E. coli, K. pneumoniae, and Pseudomonas sp. appearedto result from their presence in the environment of the newly hatched chickens. Ration pasteurization did affectP. mirabilis, which appeared to colonize from the feed. The results suggest that colonization by, andproliferation of, antimicrobial-resistant aerobic and facultative anaerobic gram-negative enteric bacilli inchicken intestinal tracts may be less dependent on selection through antimicrobial supplementation of theration than on their prevalence in environments from which they can colonize newborns.

The use of feeds containing low levels of antimicrobialagents for animal growth promotion and increased feedefficiency is economically beneficial to both the producerand the consumer (5, 7, 18, 21). However, such usage hasresulted in the development and maintenance of populationsof antimicrobial-resistant gram-negative enteric bacilli in theintestinal tracts of poultry and other animals. Many of theseorganisms owe their resistance to extrachromosomal geneticelements (R factors) which confer resistance to high concen-trations of one or more clinically useful antimicrobial agents(2, 4, 9, 14, 15, 23, 33, 35, 47). This has created concernsregarding potential animal and human health effects (2, 8, 9,14, 15, 19, 22, 31, 33, 36-38, 41-43, 46). Aside from themaintenance of a considerable reservoir of plasmid-bearingorganisms as part of the normal microbiota, concerns havebeen raised regarding the spread of pathogenic, resistantsalmonellae from animals to humans, as well as the coloni-zation of the human intestinal tract by relatively nonpatho-genic, antimicrobial agent-resistant, gram-negative entericbacilli such as Escherichia coli.

Fein et al. (17) and Levy et al. (26) studied the crossoverof antimicrobial-resistant E. coli from animals to humans.These studies support the belief that the use of antimicrobial-supplemented feeds are a factor contributing to the increasedprevalance of antimicrobial-resistant E. coli in human intes-tinal tracts. Enterobacteriaceae other than E. coli, such asSalmonella sp., and gram-negative enteric bacilli also mayhave a role in the problems of antimicrobial resistance.Outbreaks of human infections attributed to antimicrobial-

* Corresponding author.t Publication no. D-01112-1-85.t Present address: Warner-Lambert/Parke-Davis, Pharmaceutical

Research Division, Ann Arbor, MI 48105.

resistant Salmonella sp. of animal origin have been reported(2, 19). However, few studies of the levels and patterns ofantimicrobial resistance of organisms other than E. coli orSalmonella sp. in the feces of animals or humans have beenconducted.The purpose of this research was to conduct quantitative

studies of the commonly occurring aerobic and facultativeanaerobic gram-negative enteric bacilli in the intestinal tractsof broiler chickens resulting from the feeding of differentantimicrobial agents at growth promotion levels. The objec-tives were to determine (i) the levels of the most commonaerobic and facultative anaerobic gram-negative enteric ba-cilli in the feces of chickens, (ii) the antimicrobial resistancelevels and patterns of the most commonly occurring organ-isms, and (iii) if a given antimicrobial feed supplement hadany influence on the levels of both the total and antimicro-bial-resistant aerobic and facultative anaerobic gram-negative enteric bacilli encountered.

MATERIALS AND METHODS

Experimental design. Two separate 8-week studies wereconducted, using Vanguard broiler chickens (Meyer's Farm,Delaware, Pa.). All birds were banded and caged within 6 hof hatching and were not given feed or water until caged(cages used were starting/growing batteries from KuhlManufacturing Co., Flemington, N.J.).

(i) Study 1. Five groups of broilers (seven to eight birds pergroup) were fed a regular basal ration (Delaware ValleyFarmers' Cooperative Association, Inc., Flemington, N.J.)supplemented with 50 g of bacitracin, erythromycin, penicil-lin, streptomycin, or oxytetracycline per ton of feed (growthpromotion levels [16]). Two control groups were fed anunsupplemented basal ration. The five medicated and onecontrol group were housed in separate cages in one room,

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ENTERIC BACILLI OF CHICKENS FED ANTIMICROBIAL AGENTS 639

TABLE 1. Plating protocols for enumeration of total and antimicrobial-resistant organisms

Organisms Primary plating Incubation Typical colony Replica-plating Incubation Typical colonyenumerated agar e37C (h) morphology agart3tC (h) morphology

Lactose fermenters MacConkey 18-24 Brick red MacConkey 18-24 Brick redCitrate-positive MacConkey 18-24 Brick red Simmons citrate 18-24 Blue

lactose (nofermenters antimicrobials

added)Esculin-positive MacConkey 18-24 Brick red Bile-esculin (no 18-24 Black

lactose antimicrobialsfermenters added)

Urease-positive Nogrady 16-20 Blue, dark centered Nogrady 16-20 Blue, dark centeredenterobacteria

Esculin-positive Eller- 16-20 Convex, surrounded Eller-Edwards 16-20 Convex,enterobacteria Edwards by black-brown (modified) surrounded by

(modified) zone of esculin black-brown zonehydrolysis of esculin

hydrolysisAerobic XLD 18-24 Red, H2S negative XLD 18-24 Red, H2S negative

nonfermentersa Replica plates contained 25 jig of a given antimicrobial agent per ml.

whereas a second control group was housed in a separateroom.

(ii) Study 2. Eight groups of broilers (9 to 10 birds pergroup) were housed in one room and fed a basal ration thatwas "pasteurized" at 80°C for 48 h before being supple-mented. The mixer was cleaned between uses and washedwith 70% ethanol. Five groups were fed pasteurized rationscontaining the same levels of the aforementioned antimicro-bials, one control was fed an unsupplemented pasteurizedration, one group was fed the pasteurized ration supple-mented with 50 g of sulfamethazine per ton of feed, and onegroup was fed the pasteurized ration supplemented with 3 gof flavomycin per ton of feed (16). An additional three groupswere housed in a separate room: one control group was fedan unsupplemented regular basal ration, one group was fed aregular basal ration supplemented with 50 g of sulfa-methazine per ton of feed, and one group was fed a regularbasal ration supplemented with 3 g of flavomycin per ton offeed.

Selective media. For both studies, commercially availableselective media used were MacConkey agar and XLD agar(BBL Microbiology Systems, Cockeysville, Md.). ModifiedEller-Edwards medium had the followng composition:Oxgall, 10.0 g; esculin, 2.0 g; K2HPO4, 0.5 g; MgSO4, 0.2 g;NaCl, 0.2 g; MnSO4, 0.005 g; FeCl3, 0.005 g; ferric ammo-nium citrate, 0.5 g; crystal violet, 0.002 g; and agar, 15 g. Allingredients were added to 1.0 liter of deionized water andbrought to a boil to solubilize the ingredients. The mediumwas autoclaved at 121°C for 15 min. After cooling to 50°C, 10ml of a 20% solution of CaCO3 (autoclaved) was addedaseptically to the medium and mixed. The medium was thendispensed into petri plates. Nogrady agar (modified) had thefollowing composition: Sorenson phosphate buffer solution(Na2HPO4-KH2PO4 [0.067 M], pH 6.45), 450 ml; Tergitol 7agar (BBL), 330 g; nutrient broth (BBL), 8.0 g; NaCl, 5.0 g;lactose, 2.5 g; bromothymol blue, 0.1 g; neutral red, 0.03 g;and deionized water to bring the volume to 1.0 liter. Allingredients were mixed and boiled for 1 min. After cooling to50°C, 10 ml of a filter-sterilized urea solution, 250 mg/ml, and10 ml of a filter-sterilized solution of colistin, 1 mg/ml (SigmaChemical Co., St. Louis, Mo.), were added aseptically to themedium and mixed. The medium then was dispensed intopetri plates.

MacConkey agar was used to enumerate E. coli and otherlactose fermenters (3, 49). XLD agar was utilized to enumer-ate H2S-negative aerobic nonfermenters, encompassingPseudomonas sp., and the salmonellae (49). The selectiveagar of Nogrady (32), modified by the use of Tergitol 7 agaras a base and the addition of colistin (45), was used toenumerate urease-positive species of the genus Proteus,such as Proteus mirabilis. The nitrogen-deficient medium ofEller and Edwards (13) was modified by incorporation of theesculin reaction (12, 49) to facilitate detection and enumer-ation of Klebsiella pneumoniae and other members of thetribe Klebsielleae.

Differential and diagnostic media. Commercially prepareddifferential media were bile-esculin agar (Difco Laborato-ries, Detroit, Mich.), Simmons citrate agar (BBL), and triplesugar iron agar (BBL). Diagnostic systems for identifyinggram-negative enteric bacilli encountered were Pathotecoxidase test strips (General Diagnostics, Morris Plains,N.J.), the API-20 enteric system (Analytab Products, Inc.,Plainview, N.Y.), and the MORLOC system (Biotrol Corp.,Jamaica, N.Y.). The diagnostic systems were used as spec-ified by the respective manufacturers.

Sampling. Fresh fecal samples of 20 to 30 g were obtainedonce weekly from each group from the day after hatchingthrough 8 weeks. The samples were collected with pretaredsterile Mason jars and ethanol-flamed spatulas. In study 2, aday 0 sample was collected 6 h after hatching and before feedor water was administered. All fecal samples were stored onice and processed within 6 h of collection.Sample processing. For each sample, an appropriate

amount of Trypticase soy broth (BBL) was added to theMason jars to make a 10-1 dilution; the sample and brothwere immediately blended in an Osterizer blender whoseblender blade was ethanol flamed. Serial dilutions weremade in phosphate-buffered dilution water, pH 7.2 (1), and0.1-ml portions were then spread-plated onto MacConkey,Nogrady (modified), Eller-Edwards (modified), and XLDagar plates in duplicate.Enumeration of total and antimicrobial-resistant organisms.

All spread plates were incubated at 37°C for various periodsof time. Two plates containing well-defined colonies typicalof organisms surveyed were counted, and the results wereaveraged to obtain the count of the given organism per gram

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640 MAMBER AND KATZ

of feces. One plate (master plate) containing 20 to 300 typicalcolonies was replica plated (24). Replica plates contained25 ptg of tetracycline, chloramphenicol, streptomycin,ampicillin, or kanamycin per ml. Also, Simmons citrate andbile-esculin agar replica plates were used to detect citrate-positive and esculin-positive lactose fermenters, respec-tively. Replica plates were incubated at 37°C for variouslengths of time and all typical colonies were counted. Table1 summarizes the criteria used for enumerating total andantimicrobial-resistant organisms on the various media.The occurrence of antimicrobial-resistant organisms was

calculated as the number of typical colonies resistant to agiven antimicrobial agent divided by the number of typicalcolonies on the respective master plate. The number oforganisms resistant to the given antimicrobial agent per gramof feces was calculated.

Identification of colonies. Each week, one or two randomlyselected, well-isolated characteristic colonies were removedfrom each medium, streak/stab inoculated onto a triple sugariron agar slant, and incubated at 37°C for 18 to 24 h.Organisms producing an alkaline slant and no gas or H2Swere tested for oxidase activity and inoculated into anAPI-20 enteric. Organisms yielding an acid slant or butt orboth, gas, or H2S, or a combination of the three, were

inoculated into the MORLOC system.Further tests on selected isolates. Broth dilution antimicro-

bial susceptibility tests were performed (25). The ability totransfer antimicrobial resistance from selected donors tonalidixic acid- or rifampin-resistant E. coli strains or bothwas determined (29).

Quantitative examination of feed for total and fecalcoliforms, urease-positive enterobacteria, and Pseudomonasaeruginosa. Multiple-tube techniques (1) were used for de-termining the most probable number (MPN) of total andfecal coliforms and P. aeruginosa in samples of regular andpasteurized feed. The MPNs of urease-positive organismswere determined by a multiple-tube procedure, using brothprepared following the Nogrady agar formula.

RESULTS

Organisms in fecal samples. The gram-negative entericbacilli surveyed were E. coli, Proteus sp., Klebsiella sp.,Enterobacter sp., Salmonella sp., and Pseudomonas sp. Therationale for surveying these organisms in chicken fecalsamples was based on the findings of Levy et al. (26).

Limits of detection. Because of the nature of the samplematrix and the enumeration procedures, the organisms sur-veyed could not be detected below certain levels. The 10-'dilution of the fecal sample contained too much debris to bespread plated. Thus, spread-plating procedures did not allowthe detection of fewer than 10' CFU/g of feces. Replica-plating limits of detection were dependent on the number ofcolonies on the respective master plate.

TABLE 2. MPN determinations of total and fecal coliforms, P.aeruginosa, and urease-positive enterobacteria in feed samples

MPN/g of feedaOrganisms Regular Pasteurized

ration ration

Total coliforms 4.4 x 102 3.5 x 101

Fecal coliforms 9.4 x 101 1.5 x 101

P. aeruginosa 4.7 x 102 NDbUrease-positive enterobacteria >1.1 x 103 ND

a Average of six samples per determination.bND, Not determined.

TABLE 3. Biochemical identification of isolates

Plating agar Morphology isolates Identity

MacConkey Brick red; typical 102 E. colilactose fermenter 1 K. pneumoniae'

1 Enterobactercloacaea

1 C. freundiiaMacConkey White; typical 4 Proteus

nonfermenter mirabilis2 Pseudomonas

sp.

Simmons citrate Blue; typical citrate 1 K. pneumoniae(replica plated positive 1 C. freundiifrom 4 ProteusMacConkey) mirabilisb

Bile-esculin Black (esculin 4 K. pneumoniae(replica plated positive) 1 Enterobacterfrom cloacaeMacConkey)

Nogrady Blue, dark-centered 59 Proteusmirabilis

Blue, mucoid 2 K. pneumoniae

Eller-Edwards Convex, surrounded 57 K. pneumoniae(modified) by black-brown 4 Enterobacter

zone of esculin cloacaehydrolysis 3 Enterobacter

agglomerans2 C. diversus

XLD Red, H2S negative 60 Pseudomonassp.,

P. aeruginosa,P. fluorescens

6 Salmonella sp.2 Acinetobacter

sp.a Shown to be citrate positive or esculin positive or both by comparison

with replica plates.bShown to be derived from white colonies on MacConkey agar.

Levels of antimicrobial-resistant organisms versus percentresistance. In similar studies (20, 28, 36, 37), the percentresistance of a given organism to various antimicrobials wasused. However, percent resistance may be misleading whentotal counts are high (>106 CFU/g). If such high total countsare common, substantial numbers of resistant organismsmay be present in the feces even if there is a decrease inpercent resistance by 90% or more. Thus, in this research,levels of total and antimicrobial-resistant organisms areexpressed as CFU per gram of feces rather than as percentresistance.MPN determinations and rationale for feed pasteurization.

The presence of salmonellae in animal feed has been docu-mented (30, 34). Other gram-negative enteric bacilli may bepresent in feeds and hence could colonize the chickens'intestinal tracts. Feed pasteurization thus was used in study2 to minimize the feed as a major vector for colonization byenvironmental gram-negative enteric bacilli.MPN results for total and fecal coliforms, P. aeruginosa,

and urease-positive enterobacteria indicated that the fourtypes of organisms were present in feed samples and that theurease-positive enterobacteria were present at the highestlevels (Table 2). Pasteurization decreased the levels of totaland fecal coliforms by a factor of 6 or more (Table 2).

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TABLE 4. Total and antimicrobial-resistant aerobic and facultative anaerobic gram-negative enteric bacilli in broiler chicken fecalsamples

Total Resistant to:

Organisms Study' - Tetracycline Chloramphenicol Streptomycin Ampicillin KanamycinIllnc (%)b CFU/g' |lnc(%)b CFU/g Inc (%)d CFU/g Inc(%) CFU/g Inc (%)d CFU/g Inc (%)d CFU/g

Lactose 1 63/63 3 x 105- 41/63 2 x 105- 1/63 1 x 108 63/63 3 x 105- 22/63 1 x 105- 20/63 2 x 105_fermenters (100) 8 x 1010 (65) 1 x 1010 (1.6) (100) 8 x 1010 (35) 1 x 109 (32) 8 x 108

2 99/99 1 x 107- 99/99 1 x 107- 1/99 7 x 106 99/99 9 x16- 59/99 2 x 105_ 99/99 2 x 106_(100) 1 x 1010 (100) 9 x 109 (1.0) (100) 9 x 109 (60) 5 x 109 (100) 5 x 109

Urease-positive 1 38/63 5 x 103- 28/38 3 x 103- 0/38 <103 24/38 1 x 104- 11/38 1 x 104- 10/38 3 x 1-enterobac- (60) 7 x 108 (76) 3 x 108 (0) (63) 3 x 107 (29) 3 x 107 (26) 8 x 106teria

2 31/99 1 x 103- 21/31 1 x 103- 7/31 1 x 103- 16/31 1 x 10W- 14/31 1 x 103- 18/31 3 x 10-(31) 8 x 107 (68) 3 x 107 (23) 4 x 106 (52) 3 x 107 (45) 2 x 107 (58) 7 x 107

Esculin-positive 1 54/63 1 x iO3- 5/54 3 x 104- 3/54 1 x 103- 7/54 2 x 103- 43/54 1 X 103- 1/54 6 x 103enterobac- (86) 7 x 108 (9.2) 2 x 107 (5.6) 1 x 104 (13) 4 x 106 (80) 3 x 108 (1.9)teria %

2 64/99 1 x 103- 26/64 5 x 103- 8/64 8 x 102- 17/64 1 x 103- 38/64 8 x 102- 35/64 3 x 103_(65) 9 x 107 (41) 1 x 107 (12) 1 x 105 (27) 3 x 107 (59) 2 x 107 (55) 8 x 107

Aerobic nonfer- 1 41/63 6 x 10W- 16/41 1 x 104- 8/41 1 x i04- 13/41 1 x 104- 29/41 3 x 103- 2/41 1 X 105_menters (65) 5 x 107 (39) 3 x 107 (20) 3 x 107 (32) 3 x 107 (71) 3 x 107 (4.9) 3 x 106

2 58/99 1 x 103- 42/58 1 x 103- 15/58 1 x 103- 34/58 1 x 103- 46/58 1 x 103- 11/58 1 x 103___________ (59) 6 x 107 (72) 3 x 107 (26) 3 x 107 (59) 4 x 106 (79) 3 x 107 (19) 4 x 105

a Seven groups of chickens were used in study 1, and 11 groups of chickens were used in study 2.b Inc, Incidence of total population: number of samples in which organisms were detected/total number of samples collected.' Range of CFU per gram of feces for all samples.d Inc, Incidence of resistant organisms in total population: number of samples in which resistant organisms were detected/number of samples in which total

organisms were present.

Biochemical identification of isolates. A total of 317 isolateswere identified by biochemical methods (Table 3). A total of97% of the 105 brick-red colonies isolated from MacConkeyagar were E. coli. Non-E. coli lactose fermenters, isolatedfrom MacConkey plates, Simmons citrate, or bile-esculinreplica plates, were K. pneumoniae, Enterobacter cloacae,and Citrobacterfreundii. All 59 dark-centered blue coloniesisolated from Nogrady agar plates were identified as Proteusmirabilis. Of 66 esculin-positive isolates on Eller-Edwards(modified) medium, 86% were K. pneumoniae; the otherisolates were Enterobacter cloacae, Enterobacter agglom-erans, or C. diversus. Sixty red, H2S-negative colonies onXLD agar plates were identified as Pseudomonas sp., P.aeruginosa, or P. fluorescens. Six XLD isolates were Sal-monella sp., and two were Acinetobacter sp.Enumeration of total and resistant organisms. The overall

incidences and levels of total and antimicrobial-resistantorganisms are summarized in Table 4.

(i) Lactose fermenters. The presence and levels of total andantimicrobial-resistant lactose fermenters in fecal samplesobtained from chickens in each 8-week study are shown inTable 5.

(a) Study 1. Lactose fermenters were present in all 63 fecalsamples taken from the seven groups; their numbers rangedfrom 3 x 105 to 8 x 1010 CFU/g of feces. Streptomycin-resistant lactose fermenters also were detected in allsamples, regardless of ration. Tetracycline resistance wasdetected in 65% of the 63 samples containing lactosefermenters, ampicillin resistance was detected in 35% of 63samples, and lactose fermenters resistant to kanamycin weredetected in 32% of the samples taken. Lactose fermentersresistant to chloramphenicol were detected in only onesample. Comparison of replica plates and subsequent bio-chemical identifications of isolates showed that lactosefermenters resistant to streptomycin, tetracycline, and ka-

namycin were almost exclusively E. coli. Ampicillin-resistant lactose fermenters were predominantly citrate-positive/esculin-positive organisms, notably K. pneumo-niae.

(b) Study 2. Lactose fermenters were present in all 99 fecalsamples taken from the 11 groups, with numbers rangingfrom 107 to 1010 CFU/g. Lactose fermenters resistant tostreptomycin, tetracycline, and kanamycin also were de-tected in all 99 samples (100% recovery). Lactose fermentersresistant to ampicillin were less common, detected in 60% ofthe samples. As in study 1, resistance to chloramphenicolwas detected only in one sample. Comparison of replicaplates and subsequent biochemical identifications of isolatesshowed that lactose fermenters resistant to streptomycin,tetracycline, kanamycin, and ampicillin in study 2 fecalsamples were almost exclusively citrate-negative/esculin-negative organisms (E. coli).

E. coli organisms were detected at a level of 8.8 x 108CFU/g in a day 0 fecal sample collected 6 h after hatchingand before any feed or water was given. E. coli organismsresistant to streptomycin, tetracycline, kanamycin, andampicillin were present at levels of 8.8 x 108 (100%), 8.8 x108 (100%), 1.3 x 108 (15%), and 8.3 x 107 (10%) CFU/g inthe day 0 sample, respectively.

(ii) Urease-positive enterobacteria. The presence and levelsof total and antimicrobial-resistant urease-positive entero-bacteria (Proteus mirabilis) in fecal samples obtained fromchickens in each 8-week study are shown in Table 6.

(a) Study 1. Urease-positive enterobacteria were presentin 38 of 63 fecal samples; their numbers ranged from 5 x 103to 7 x 108 CFU/g. Urease-positive enterobacteria weredetected in one of nine (11%) and none of nine (0%) fecalsamples from the groups fed bacitracin- and tetracycline-supplemented rations, respectively. Urease-positive entero-bacteria resistant to tetracycline and streptomycin were

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TABLE 5. Incidence and range of total and antimicrobial-resistant lactose fermenters

Total lactose Lactose fermenters resistant to:fermenters

Study Groupa Tetracycline Chloramphenicol Streptomycin Ampicillin KanamycinInCb CFU/g'7 dC dCincd CFU/g incd CFU/g Incd CFU/g Incd CFU/g Incd CFU/g

1 1 9/9 4 x 107- 4/9 3 x 106- 0/9 e 9/9 1 x 107- 2/9 3 x 10- 1/9 9 x 105

9/9 5 x 106-8 x 1010

9/9 2 x 106_9 x 109

9/9 1 X 107-1 x 1010

9/9 2 x 10W-1 x 1010

9/9 3 x 105-3 x 109

9/9 5 x 10o-8 x 109

1 x 101O

4/9 2 x 1O6_1 x 109

6/9 2 x 105_6 x 107

8/9 3 x 106-1 x 109

8/9 3 x 106-3 x 108

8/9 3 x 107-2 x 109

3/9 4 x 105-1 x 108

0/9

0/9

2 x 1010

9/9 4 x 106_8 x 1010

9/9 3 x 105-7 x 109

1/9 1 x 108 9/9 6 x 107-1 x 1010

0/9

0/9

0/9

9/9 1 X 107-7 x 109

9/9 3 x 105-2 x 109

9/9 4 x 106-8 x 109

1 x 109

5/9 4 x 105_1 x 109

2/9 1 x 105_3 x 105

7/9 9 x 106_1 x 109

2/9 1 x 107-1 x 108

2/9 2 x 105-8 x 105

3/9 2 x 105_2 x 106

5/9 2 x 106_8 x 108

4/9 4 x. 105-3 x 108

1/9 1 X 105 5/9 3 x 107-S x 108

3/9 8 x 105-9 X 107

0/9

2 8 9/9 2 x 108-B-Cont.3 4 x 109

9 9/9 1 X 108_B-FM 8 x 109

9/9 4 x 107-3 x 109

9/9 3 x 10'-1 x 108

9/9 5 x 107-6 x 109

9/9 1 x 108-7 x 109

9/9 3 x 107-7 x 109

9/9 2 x 10'-4 x 109

9/9 3 x 10'-6 x 109

9/9 1 x 107-1 x 1010

9/9 8 x 10V-5 x 109

9/9 2 x 108_3 x 109

9/9 1 x W_8 x 109

9/9 3 x 107-2 x 109

9/9 2 x 107-4 x 109

9/9 1 x 107-5 x 109

9/9 9 x 107-5 x 109

9/9 2 x 107-2 x 109

9/9 2 x 107-2 x 109

9/9 2 x 107-4 x 109

9/9 1 X 107-9 x 109

9/9 3 x 107-4 x 109

0/9

0/9

0/9

0/9

0/9

0/9

0/9

0/9

0/9

0/9

9/9 6 x 108_3 x 109

- 9/9 1 x 108-8 x 109

9/9 3 x 107-2 x 109

9/9 2 x 107-7 x 109

9/9 1 x 107-6 x 109

- 9/9 1 x 108-6 x 109

9/9 2 x 107-2 x 109

9/9 1 X 107-2x 109

9/9 1 x 107-6x 109

9/9 9 x 106-9 x 109

1/9 7 x 106 9/9 3 x 107-4 x 109

3/9 9 x 106_3 x 107

4/9 1 x 106_3 x 107

6/9 3 x 106-6 x 108

7/9 3 x 106_4 x 108

4/9 3 x 105-1 X 108

9/9 3 x 10-5 x 109

3/9 1 x 107-3 x 108

6/9 4 x 106-1 X 108

7/9 1 x 106-2 x 109

3/9 2 x 105-1 x 108

7/9 9 x 105-1 x 109

9/9 5 x 106-1 x 109

9/9 9 x 106_5 x 109

9/9 1 x 107-8 x 108

9/9 2 x 10C-3 x 109

9/9 2 x 106_7 x 108

9/9 7 x 107-5 x 109

9/9 3 x 107-1 x 109

9/9 2 x 107-2 x 109

9/9 2 x 107-3 x 109

9/9 8 x 106_1 x 109

9/9 2 x 107-2 x 109

a B-Cont., Control group(s) fed unsupplemented basal ration; B-BC, group fed basal ration with 50 g of bacitracin per ton of feed; B-EM, group fed basal rationwith 50 g of erythromycin per ton of feed; B-PC, group fed basal ration with 50 g of penicillin per ton of feed; B-SM, group fed basal ration with 50 g ofstreptomycin per ton offeed; B-TC, group fed basal ration with 50 g of oxytetracycline per ton offeed; B-FM, group fed basal ration with 3 g offlavomycin per tonoffeed; B-SU, group fed basal ration with 50 g of sulfamethazine per ton offeed; PB-Cont., group fed unsupplemented pasteurized basal ration; PB-BC, group fedpasteurized basal ration with 50 g of bacitracin per ton offeed; PB-EM, group fed pasteurized basal ration with 50 g of erythromycin per ton offeed; PB-PC, group

fed pasteurized basal ration with 50 g of penicillin per ton of feed; PB-SM, group fed pasteurized basal ration with 50 g of streptomycin per ton of feed; PB-TC,group fed pasteurized basal ration with 50 g of oxytetracycline per ton of feed; PB-FM, group fed pasteurized basal ration with 3 g of flavomycin per ton of feed;PB-SU, group fed pasteurized basal ration with 50 g of sulfamethazine per ton of feed.

b Inc, Incidence of total population: number of samples in which organisms were detected/total number of samples collected.C Range of CFU per gram of feces for all samples.d Inc, Incidence of resistant organisms in total population: number of samples in which resistant organisms were detected/number of samples in which total

organisms were present.'-, Below detectable levels.

B-Cont.1 2 x 1010

2B-BC

3B-EM

4B-PC

5B-SM

6B-TC

7B-Cont.2

10B-SU

11PB-BC

12PB-EM

13PB-PC

14PB-SM

15PB-TC

16PB-Cont.1

17PB-FM

18PB-SU



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TABLE 6. Incidence and range of total and antimicrobial-resistant, urease-positive enterobacteria

Total Urease-positive enterobacteria resistant to:Study Groupa

urease-positiveStudy Groupa enterobacteria Tetracycline Chloramphenicol Streptomycin Ampicillin Kanamycin

Incb CFU/gc Incd CFU/g Incd CFU/g Incd CFU/g Incd CFU/g Incd CFU/g

1 1 8/9 2 x 10- 7/8 5 x 104- 0/8 _e 5/8 1 x 104- 1/8 1 x 104 3/8 1 x 105-B-Cont.1 4 x 107 3 x 107 2 x 107 8 x 106

1/9 3 x 105 1/1 2 x 104 0/1 1/1 2 x 104 1/1 1 X 105 1/1 3 x 104

9/9 7 x 104-1 x 108

5/9 1 X 105_2 x 106

8/9 1 x 105-7 x 108

6/9 2 x io4-3 x 107

2/5 3 x io4-2 x 106

7/8 8 x 105-3 x 108

0/9

0/5

0/8

- 7/9 2 x 104-3 x 107

2/5 1 x 104-1 x 106

5/8 8 x 105-2 x 107

3/9 3 x 104-3 x 107

2/5 9 x 105-2 x 106

2/8 1 x 105_1 X 106

2/9 1 x 105-1 X 106

1/5 6 x 105

2/8 1 x 106'-1 X 106

0/9

7/9 5 x 103-1 X 107

5/7 3 x 103-1 X 107

0/7 4/7 6 x 104-1 X 107

2/7 1 x 104-5 x 105

1/7 6 x 104

3/9 1 x 103-2 x 107

8/9 6 x 104-8 x 107

6/9 1 x 103_3 x 107

1/3 2 x 107

8/8 5 x 1i4-3 x 107

5/6 4 x 105_2 x 107

0/3 1/3 2 x 107 1/3 2 x 107 1/3 2 x 107

3/8 1 x 105-4 x 106

3/6 3 x io4-3 x 106

7/8 4 x 105_3 x 107

3/6 7 x 104-1 X 107

5/8 4 x 105_1 X 107

2/6 1 x 106_1 X 106

7/8 5 x 104-2 x 107

5/6 6 x 105-1 X 107

0/9

0/9

4/9 1 x 103-1 X 104

0/9 -

0/9

16 5/9 1 x 103-PB-Cont.1 3 x 105

17 3/9 6 x 10W-PB-FM 8 x 106

4/5 1 x 103-3 x 105

3/3 1 x 104-8 x 106

1/5 1 X 103

0/3

2/5 8 x 1i4-3 x 105

3/3 1 x 104-S x 106

3/5 1 x 103-2x 105

3/3 5 x 103-7 x 106

2/5 3 x io4-2 x 105

3/3 5 x io4-7 x 107

2/9 1 x 103-1 X 103

0/2 0/2 0/2 0/2

See footnotes to Table 5.

detected in 74 and 63% of 38 fecal samples, respectively.Urease-positive enterobacteria resistant to ampicillin andkanamycin were less common, present in 29 and 26% of thesamples, respectively. Chloramphenicol resistance was notobserved in any sample.

(b) Study 2. Urease-positive enterobacteria were detectedin 31 of 99 fecal samples taken from study 2 groups; theirnumbers ranged from 1 x 103 to 8 x 107 CFU/g. Urease-positive enterobacteria were detected in only 19% of 72samples taken from the eight groups fed the pasteurized

basal rations and were undetected in all samples from groupsfed the pasteurized ration supplemented with bacitracin,erythromycin, streptomycin, or oxytetracycline. Urease-positive enterobacteria resistant to tetracycline, streptomy-cin, kanamycin, ampicillin, and chloramphenicol were de-tected in 68, 52, 58, 45, and 23% of 31 fecal samples,respectively. Urease-positive enterobacteria were absentfrom the day 0 fecal sample.

(iii) Esculin-positive enterobacteria. Table 7 shows thepresence and levels of total and antimicrobial-resistant

2B-BC

3B-EM

4B-PC

5B-SM

6B-TC

7B-Cont.2

2 8B-Cont.3

9B-FM

10B-SU

11PB-BC

12PB-EM

0/413PB-PC

14PB-SM

15PB-TC

0/4 0/4 0/4 0/4

18PB-SU

0/2

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TABLE 7. Incidence and range of total and antimicrobial-resistant, esculin-positive enterobacteriaTotal Esculin-positive enterobacteria resistant to:

esculin-positiveStudy Groupa enterobacteria Tetracycline Chloramphenicol Streptomycin Ampicillin Kanamycin

Incb CFU/gc Incd CFU/g Incd CFU/g Incd CFU/g Incd CFU/g Incd CFU/g1 1 9/9 1x103- 0/9 e 0/9 0/9 - 6/9 5 x 103- 0/9

B-Cont.1 6 x 108 2 x 108

8/9 1 x 10'-9 x 106

8/9 2 x 103-3 x 106

9/9 2 x 104-7 x 108

5/9 1 x 10'-1 X 106

6/9 6 x 103-2x 105

9/9 2 x 1i4-6 x 106

0/8

0/8

1/8 1 x 103 1/8 1 x 104 6/8 6 x 103-2 x 106

0/8

2/9 5 x 106_2 x 107

0/5

0/6

0/9

0/5

- 0/6

3/9 3 x 104-3 x 105

0/8

2/9 2 x 103-4x 106

1/5 5 x 105

0/6

2/9 6 x 103-1 X 104

7/8 1 x 103-4 x 105

8/9 4 x 105-3 x 108

4/5 9 x 102-1 X 106

3/6 2 x 103-4 x 104

3/9 6 x 103-1 X 105

9/9 6 x 103-1 X 105

0/8

1/8 6 x 103

0/9

0/5

0/6

0/9

2 8B-Cont.3

6/9 1 x 103-2 x 106

1/6 1 x 104 1/6 2 x 104 0/6 4/6 6 x 104-2 x 106

2/6 7 x 104-2 x 105

6/9 5 x 103-2 x 107

5/6 5 x 103-1 X 106

2/6 3 x 103_3 x 104

4/6 1 x 105-3 x 105

5/6 3 x 103-2 x 107

4/6 5 x 103-1 x 107

6/9 3 x 104-3 x 107

7/9 1 x 10'-1 X 106

3/6 1 x 104-2 x 106

1/7 1 x 106

2/6

0/7

1 x i-1 x l0o

0/6

0/7

5/6 2 x 104-9 x 106

2/7 4 x 103-1 X 106

4/6 2 x 104-1 X 107

3/7 5 x 10'-1 X 106

12PB-EM

13PB-PC

14PB-SM

15PB-TC

16PB-Cont. 1

17PB-FM

18PB-SU

5/9 2 x 103-9 X 107

9/9 2 x 103-3 x 107

4/9 1 x 103-3 x 107

3/9 1 x 10'-S x 104

5/9 1 X 103-1 X 104

6/9 1 x 103-4 x 106

7/9 1 x 103-3 x 105

3/5 6 x 103-S x 106

5/9 1 x 104-1 X 107

2/4 5 x 105_S x 106

1/3 5 x 104

1/5 5 x 104

2/6 8 x 105-3 x 106

2/7 9 x 103-2 x 104

1/5

1/9

0/4

0/3

0/5

1/6

0/7

See footnotes to Table 5.

esculin-positive enterobacteria encountered ifl the fecal sam-

ples from chickens in each 8-week study.(a) Study 1. Esculin-positive enterobacteria were detected

in 54 of 63 fecal samples, with numbers ranging from 1 x 103to 7 x 108 CFU/g. Esculin-positive enterobacteria were

detected in 56 and 67% of the 18 samples taken from thegroups fed streptomycin- and tetracycline-supplemented ra-

tions, respectively, and in >89% of the 45 samples takenfrom the other five groups. Esculin-positive enterobacteria

8 x 102 3/5 2 x 103-3 x 107

1X 103 2/9 7 x 104-2 x 10'

1/4 5 x 105

1/3 5 x 104

1/5 6 x 104

4 x 103 3/6 1 x 104-1 X 105

2/7 1 x 103-8x 103

3/5 8 x 102-2 x 107

7/9 2 x 104-2 x 107

4/5 2 x 103-8 x 107

7/9 3 x 103-1 X 107

1/4 5 x 105 2/4 5 x 105-

3 x 107

1/3 5 x 104 1/3 5 x 104

2/5 7 x 1i4-1 x 105

3/6 5 x 103-3 x 106

5/7 1 x 103-9 X 104

2/5 9 x 103_7 x 104

2/6 8 x 105_4 x 106

4/7 1 x 10"-2 x 105

resistant to ampicillin were detected in 80% of 54 fecalsamples. Occurring less commonly were esculin-positiveenterobacteria resistant to streptomycin, tetracycline, kana-mycin, and chloramphenicol, which were detected in 13, 9,2, and 6% of the 54 samples, respectively.

(b) Study 2. Esculin-positive enterobacteria were detectedin 64 of 99 fecal samples, with numbers ranging from 1 x 103to 9 x 107 CFU/g. Esculin-positive enterobacteria were

detected in all 9 samples from the group fed the penicillin-

2B-BC

3B-EM

4B-PC

5B-SM

6B-TC

7B-Cont.2

9B-FM

10B-SU

11PB-BC

APPL. ENVIRON. N41CROBIOL.


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TABLE 8. Incidence and range of total and antimicrobial-resistant, aerobic nonfermenters

Total Aerobic nonfermentors resistant to:Groupa aerobic

Study nonfermenters Tetracycline Chloramphenicol Streptomycin Ampicillin Kanamycin

Incb CFU/gc Incd CFU/g Incd CFU/g Incd CFU/g Incd CFU/g Incd CFU/g

1 1 5/9 5 x 104- 1/5 6 x 105 0/5 e 2/5 8 x 105- 3/5 1 x 105- 0/5B-Cont.1 5 x 107 1 X 106 1 X 106

6/9 1 x 104-1 X 107

3/6 8 x 104-1 X 107

2/6 7 x 105-6 x 106

1/6 5 x 106 2/6 1 x 104-8 x 106

5/9 2 x 105-8 x 106

7/9 6 x 104-2 x 107

5/9 6 x 103_3 x 107

7/9 1 x io4-8 x 105

6/9 1 x io4-7 x 106

1/5 2 x 105

3/7 2 x 106_1 X 107

2/5 2 x 106-3 x 107

3/ 1 x 104-3 x 105

3/6 2 x 10W-8 x 105

2/5 1 x 105-2 x 106

1/7 1 x 107

1/5 3 x 107

2/7 1 x 104

0/6

2/5 8 x 105-6 x 106

3/5 5 x 105-3 x 106

1/7 9 x 105 5/7 3 x 103-9 X 106

3/5 4 x 106-3 x 107

5/5 3 x 103-3 x 107

1/7 3 x 104 7/7 1 x 104-3 x 105

3/6 1 x 104-8 x 105

4/6 1 x 1i4-4 x 106

0/5

1/7 1 x 105

0/5

0/7

0/6

4/9 1 x 103-2 x 107

3/4 2 x 103-1 X 107

1/4 5 x 103 1/4 1 x 105 2/4 5 x 103-3 x 106

6/9 1 x 103-8 x 106

5/9 1 x 10W-4 x 105

6/9 3 x 104-2 x 106

5/9 1 X 103-6 x 107

5/9 4 x 103-8 x 106

4/9 4 x 103-4 x 106

6/9 1 x 103-3 x 106

6/9 1 x 103-2 x 106

5/9 1 X 103-6 x 106

6/9 1 x 103-3 x 106

2/6 4 x 106-7 x 106

3/5 1 x 103-3 x 105

5/6 3 x 103-1 X 106

4/5 4 x 103_3 x 107

4/5 1 x 103-6 x 106

3/4 1 x 103-4 x 106

5/6 1 x 103-2 x 106

4/6 4 x 104-2 x 106

4/5 1 x 103-4 x 106

5/6 8 x 103-3 x 106

1/6 7 x 106

1/5 3 x 105

2/6 3 x 103-3 x 104

1/5 3 x 107

1/5 3 x 104

1/4 3 x 106

1/6 8 x 105

1/6 1 x 104

1/5 1 X 103

4/6 1 x 103-2 x 106

2/6 7 x 103-2 x 106

2/5 2 x 103-4 x 105

3/6 2 x 103-6 x 105

3/5 4 x 105-3 x 106

3/5 8 x 103-4 x 106

3/4 5 x 105-3 x 106

3/6 1 x 104-7 x 105

6/6 1 x 103-2 x 106

4/5 2 x 1i4-3 x 105

4/6 9 x 103-2 x 105

3/6 7 x 103-7 x 106

4/5 1 x 103-3 x 105

6/6 1 x 103-1 X 106

4/5 4 x 103-3 x 107

4/5 4 x 103-5 x 106

4/4 1 x 103-4 x 106

5/6 1 x 103-3 x 106

6/6 1 x 103-2 x 106

3/5 9 x 103S x 106

5/6 8 x 103-2 x 106

1/6 1 x 103

2/5 1 x 103-3 x 105

2/6 3 x 103-1 X 104

1/5 4 x 105

1/5 8 x 103

0/4

0/6

1/6 1 x 104

1/5 1 X 104

1/6 2 x 105

a-e See footnotes to table 5.

supplemented pasteurized ration, but were present in only 44and 33% of 18 samples from the groups fed the streptomycin-and tetracycline-supplemented pasteurized rations, respec-tively. Resistance to ampicillin and kanamycin was detectedin 59 and 55% of 64 fecal samples, respectively. Esculin-positive enterobacteria resistant to tetracycline and strepto-mycin were detected in 41 and 27% of 64 samples,respectively. Esculin-positive enterobacteria resistant tochloramphenicol were less common, being detected in only

12% of the samples. Esculin-positive enterobacteria werenot detected in the day 0 fecal sample.

(iv) Aerobic nonfermenters on XLD agar. Table 8 showsthe presence and levels of total and antimicrobial-resistantaerobic nonfermenters in fecal samples obtained from chick-ens in each 8-week study.

(a) Study 1. Aerobic nonfermenters were detected in 41 of63 fecal samples, with numbers ranging from 6 x 103 to 5 x

107 CFU/g. Aerobic nonfermenters resistant to ampicillin

2B-BC

3B-EM

4B-PC

5B-SM

6B-TC

7B-Cont.2

1/6 3 x 106

2 8B-Cont.3

1/4 1 x 103

9B-FM

10B-SU

11BP-BC

12PB-EM

13PB-PC

14PB-SM

15PB-TC

16PB-Cont.1

17PB-FM

18PB-SU

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646 MAMBER AND KATZ

TABLE 9. Broth dilution MICs for selected isolates from chickenfecal samples

MIC (,ug/ml)Isolate Tetracy- Strepto- Ampi- Kana-

cline mycin cillin mycin

E. coli1 62.5 250 <1.95 2502 62.5 62.5 <1.95 125

Proteus mirabilis1 62.5 125 <1.95 31.22 31.2 31.2 <1.95 31.2

K. pneumoniae <1.95 <1.95 125 <1.95P. fluorescens <1.95 <1.95 62.5 <1.95

were detected in 71% of 41 samples. Resistance to tetracy-cline and streptomycin was detected in 39 and 32% of the 41samples, respectively. Resistance to chloramphenicol andkanamycin was less common, occurring in 19 and 5% of 41samples, respectively.

(b) Study 2. Aerobic nonfermenters were detected in 58 of99 fecal samples, at numbers ranging from 1 x 103 to 6 x 107CFU/g. Aerobic nonfermenters resistant to ampicillin, tetra-cycline, and streptomycin were detected in 79, 72, and 59%of the 58 samples, respectively. Resistance to chloramphen-icol and kanamycin was detected in 26 and 19% of 58samples, respectively. Aerobic nonfermenters were not de-tected in the day 0 fecal sample collected from the newlyhatched chickens.

Detection of salmonellae on XLD agar. H2S-negativesalmonellae were isolated only six times; five isolates werein fecal samples from study 1 chickens, whereas one isolatewas found in fecal samples from study 2 chickens. Exami-nation of replica plates showed two of the six isolates to beresistant to ampicillin, but susceptible to the other fourantimicrobials to which resistance was tested. No H2S-positive red colonies, indicative of Salmonella sp. (3, 49),were observed on XLD plates.

Antimicrobial susceptibility tests and conjugation experi-ments. Two E. coli isolates obtained from the study 2 day 0fecal sample, plus two randomly selected Proteus mirabilis,one K. pneumoniae, and one P. fluorescens isolate, weresubmitted to broth dilution susceptibility tests. Results areshown in Table 9. The E. coli and Proteus mirabilis strainswere resistant to tetracycline, streptomycin, and kanamycin,whereas the K. pneumoniae and P. fluorescens wereampicillin resistant.

Conjugative transfer experiments showed that the E. coliisolates could readily transfer resistance to tetracycline andstreptomycin, but not kanamycin, to recipient E. coli strains.No transferable resistance was observed when E. coli recip-ients were used with the four non-E. coli isolates.

Further susceptibility/resistance transfer experimentswere beyond the scope of this study. However, three mul-tiple resistant E. coli isolates were preserved by lyophiliza-tion.

DISCUSSIONQualitatively, the aerobic and facultative anaerobic gram-

negative enteric bacilli of chickens found in this study werecomparable to the findings of others (10, 26). Quantitatively,E. coli was the most dominant of the aerobic and facultativeanaerobic gram-negative enteric bacilli. This compared fa-vorably with other reports (39, 40, 44). Quantitative levels ofboth E. coli and Proteus mirabilis were similar to thosereported by Levy et al. (26), who found the predominantaerobic gram-negative organisms in the feces of young

chickens fed a regular basal ration to be E. coli (80 to 90%)and Proteus mirabilis (10 to 20%).Feed supplementation with antimicrobial agents appeared

to have little influence on the occurrence and levels of bothantimicrobial-susceptible and antimicrobial-resistant gram-negative enteric bacilli. Strains of antimicrobial-resistantgram-negative enteric bacilli were present regardless ofration (regular, pasteurized, antimicrobial supplemented, orunsupplemented). With respect to population dynamics,increases or decreases in levels of antimicrobial-resistantorganisms were dependent on the levels of total organismspresent. In many cases, the data indicated that decreases inthe relative incidence of resistant organisms (percent resist-ance) from one week to the next did not reflect a decrease inthe number of resistant organisms. Rather, such relativedecreases in percent resistance reflected relative increases inthe number of total organisms.The data indicate that environmental sources are impor-

tant factors in the colonization of the chicken's intestinaltract. Colonizing organisms could come from the immediateenvironment of the newly hatched chicks, the shell, feed,water, or other environmental sources. Whereas egg sur-faces are often contaminated by microorganisms, microbialcontamination does not occur within eggs laid by healthychickens (6). This was verified experimentally by dissecting18- to 19-day-old fertile eggs and assaying for the presence ofbacteria. No aerobic or facultative anaerobic bacteria werefound in samples plated on Trypticase soy agar (BBL) orMacConkey agar plates or inoculated into tubes containinglauryl sulfate tryptose broth (BBL).The immediate environment of the newly hatched chick-

ens used in these studies included the egg surfaces, incuba-tor surfaces, the animal handlers, the ambient air, andstraw-lined boxes used for transport of the newborns fromthe breeder's farm to the poultry farm. During the 6 h thatpassed between hatching and caging, the newborns were notgiven any feed or water. Yet, exposure to E. coli must haveoccurred, as evidenced by the presence of 8.8 x 108 E. coliin the day 0 fecal sample collected from the surfaces of theshipping boxes at the beginning of study 2. Because of thefacts cited above, it is believed that the most probable sourceof these E. coli was the environmental surfaces exterior tothe egg.For calculation of exponential bacterial growth, N, =

N02', where No is the initial population density, N, is thedensity after t generation times, and one generation timeequals 20 min for E. coli. If the number of E. coli in the day0 fecal sample are evaluated, N, = N(6h) = 1.1 x 108 and 2'= 218 = 262,144; No = (1.1 x 108) / (2.6 x 105) = 4.2 x 102.Thus, at least 420 E. coli as the initial colonizing dose wouldbe required to obtain 8.8 x 108 CFU/g of feces. The data forthe day 0 fecal sample also indicated that several strains ofsingle or multiple antimicrobial-resistant E. coli were theinitial colonizers; 100% of the E. coli for the day 0 samplewere tetracycline and streptomycin resistant (8.8 x 108),whereas 15% (1.3 x 108) were kanamycin resistant and 10%(8.3 x 107) were ampicillin resistant. Additional rapid colo-nization of the chicks (within 1 day of hatching) fromenvironmental sources occurred by the time the day 1samples were obtained. This is evidenced by the presence ofsome antimicrobial-susceptible strains of E. coli in the day 1fecal samples from the study 2 chickens. Thus, initial colo-nization of the chicks in both studies by antimicrobial-susceptible and -resistant strains of E. coli appears to haveoccurred from the immediate environment of hatching.

Strains of antimicrobial-susceptible and -resistant K.



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pneumoniae and Pseudomonas sp., although not detected inthe day 0 fecal sample taken from the study 2 chickens, alsoappeared to have colonized from the environment immediateto the newborns. The evidence for this is the presence ofesculin-positive enterobacteria and aerobic nonfermenters in17 of 18 and 10 of 18 day 1 fecal samples, respectively, andtheir common presence at levels exceeding 103 CFU/g insamples from all groups, regardless of ration. Strains ofantimicrobial-susceptible and -resistant Proteus mirabilis,by comparison, appeared to colonize primarily from thefeed, rather than from the environment. The evidence forthis includes their absence in the day 0 fecal sample collectedin study 2, their absence in most of the 18 day 1 samplescollected from the groups of chickens, and, most important,their presence in few or no samples at levels exceeding 103CFU/g of feces from groups fed the pasteurized rations instudy 2. Also, Proteus mirabilis were present at higher levelsthan either coliforms or Pseudomonas sp. in feed samplesassayed by multiple-tube fermentation tests (Table 2).The colonization by, and proliferation of, strains of anti-

microbial-susceptible and -resistant aerobic and facultativeanaerobic gram-negative enteric bacilli in chickens' intesti-nal tracts is influenced by host physiological factors, envi-ronmental factors, and microbial interactions within theintestinal tract (11). Such factors may be as important asantimicrobial feed supplementation. On& exception to theminimal effects of feed supplementation was the apparentrole of antimicrobials in preventing or minimizing coloniza-tion by, and proliferation of, strains of Proteus mirabilis. Inthese studies, Proteus mirabilis was not detected in anysamples from the group fed the regular basal ration supple-mented with oxytetracycline and in only one sample fromthe group fed the bacitracin-supplemented regular ration. Aspreviously noted, Levy et al. (26) found both E. coli andProteus mirabilis to be the predominant Enterobacteriaceaein young chickens. However, once the intestinal populationshad been established, feeding tetracycline resulted in thepredomination of E. coli, despite the original presence of 10to 20% tetracycline-resistant strains of Proteus mirabilis.Levy et al. (26) suggested that "E. coli have a selectiveadvantage in the intestinal tract over P. mirabilis." Both thebacitracin- and tetracycline-supplemented rations used inthese studies minimized the presence of Proteus mirabilis inthe intestinal tracts. The Proteus mirabilis organisms en-countered were frequently tetracycline resistant. However,it appears that exposure to tetracycline-supplemented (orbacitracin-supplemented) rations more logically would causethe effect, rather that the "selective advantage" of E. coliover Proteus mirabilis in the chickens' intestinal tracts assuggested by Levy et al. (26). In contrast to the effects oftetracycline on Proteus mirabilis, penicillin supplementationof the ration appeared to enhance the proliferation ofesculin-positive enterobacteria (K. pneumoniae) in the intes-tinal tracts of the animals. This indicates that antimicrobialsupplementation of the ration also may have a role inpromoting colonization by, and proliferation of, a particulargenus or species of bacteria.The presence of antimicrobial-resistant E. coli in the day 0

sample taken from study 2, as well as in all chicken fecalsamples, is not a unique occurrence. Kiser et al. (23) foundtetracycline-resistant E. coli in 1-day-old chickens whichwere never given tetracycline in the feed, and the resistantstrains continued to be recovered up to 9 weeks of agewhether or not tetracycline-supplemented feed was used.Linton et al. (28) found high levels of antimicrobial-resistantcoli-aerogenes bacteria in calves not given antimicrobials

and were "convinced that similarly high incidences of resist-ance occur frequently." Linton et al. (28) considered thesefindings "as a reflection of the situation existing in the fieldat the present time."Can colonization of the chickens' intestinal tracts by

aerobic and facultative anaerobic gram-negative enteric ba-cilli, such as E. coli, be minimized? Holmberg et al. (19)emphasized "the need for more prudent use of antimicrobi-als in both human beings and animals." Both the SwannCommittee Report (46) and the FDA Task Force (15) sug-gested the use of antimicrobial agents which are not used forhuman or veterinary chemotherapeutic purposes. Two anti-biotics, bacitracin and flavomycin (14, 48), were used toexamine this concept in study 2. Strains of single andmultiple resistant E. coli were found at high levels in all fecalsamples collected in study 2, regardless of ration. Thus, therestriction of various antimicrobials, or the use of agentssuch as flavomycin, reported to be effective against R-factor-bearing E. coli in other studies (48), are not necessarily theanswer to the problem, if only because of the ubiquitouspresence of antimicrobial-resistant gram-negative entericbacilli in agricultural environments.

Conclusions. The results indicate that antimicrobial-resistant strains of aerobic and facultative anaerobic gram-negative enteric bacilli which colonize the intestinal tracts ofchickens are not necessarily selected for by antimicrobialsupplementation of animal feed. Instead, colonization by,and proliferation of, antimicrobial-resistant strains may bedependent on their common presence in environments fromwhich they can colonize the intestinal tracts of newlyhatched chickens. The data suggest that those organisms,whether antimicrobial susceptible or resistant, which werethe first colonists became members of the "normal" intesti-nal microbiota. They then could proliferate in the intestinaltract for extended periods of time, whether or not theanimals were given antimicrobial-supplemented feeds.Because the proliferation of antimicrobial-resistant organ-

isms appeared to be related more to their mere presence inanimal environments than as the result of antimicrobialselective pressures, the current nature of the problem maybe an epidemiological one. Linton (27) stated that, as a resultof the use of antimicrobials in the animal environment andtheir subsequent selection for antimicrobial-resistant strainsthat can compete with susceptible strains during coloniza-tion of animal intestinal tracts, "it is the norm to find thelarger proportion of Escherichia coli in pigs, poultry andcalves resistant to one or more therapeutically useful antibi-otics whether or not the animals are receiving antibiotics."The data from these studies support and extend the aboveobservation to gram-negative bacilli other than E. coli,notably Proteus mirabilis, K. pneumoniae, and Pseudomo-nas sp. At this point in time, the restriction of antimicrobialuse in animal feeds may have little effect on the levels ofsuch strains in the animals' intestinal tracts. Research shouldbe conducted on methods of minimizing initial colonizationby strains of antimicrobial-resistant E. coli and other gram-negative enteric bacilli as well as new animal produc-tion/husbandry procedures.

ACKNOWLEDGMENTThis research was supported by state funds.We wish to thank Fabien Smith for typing the manuscript.

LITERATURE CITED1. American Public Health Association. 1971. Standard methods for

the examination of water and wastewater, 13th ed. AmericanPublic Health Association, Washington, D.C.

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2. Anderson, E. S. 1968. The ecology of transferable drug resistance in the Enterobacteria. Annu. Rev. Microbiol. 22:131-180.

3. BBL Microbiology Systems. 1968. BBL manual of products andlaboratory procedures, 5th ed. BBL Microbiology Systems,Cockeysville, Md.

4. Benveniste, R., and J. Davies. 1973. Mechanisms of antibioticresistance in bacteria. Annu. Rev. Biochem. 42:471-506.

5. Bird, H. R. 1969. Biological basis for the use of antibiotics inpoultry feeds, p. 31-41. In The use of drugs in animal feeds.National Academy of Sciences Publ. 1679. National Academyof Sciences, Washington, D.C.

6. Board, R. G. 1969. The microbiology of the hen's egg. Adv.Appl. Microbiol. 11:245-281.

7. Burg, R. W. 1982. Fermentation products in animal health.ASM News 48:460-463.

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