APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1987, p. 1241-12450099-2240/87/061241-05$02.00/0Copyright © 1987, American Society for Microbiology

Comparative Survival of Antibiotic-Resistant and -Sensitive FecalIndicator Bacteria in Estuarine Water

GARY W. PETTIBONE,'t SARA A. SULLIVAN,2 AND MICHAEL P. SHIARIS2*Environmental Science Program' and Department of Biology,2 University of Massachusetts, Boston Harbor Campus,

Boston, Massachusetts 02125

Received 28 April 1986/Accepted 10 March 1987

The survival of antibiotic-resistant and -sensitive strains of Escherichia coli, Enterococcus faecalis,Enterococcusfaecium, Streptococcus equinus, and two environmental isolates, AP17 and AQ62, was examinedin estuarine water. Each strain was rendered resistant to a combination of two antibiotics by serial passage inincreasing concentrations of antibiotics. Cultures were incubated in filter-sterilized estuarine water for up to 7days. Recovery was assessed by examining colony-forming ability on media with and without antibiotics. Noneof the antibiotic-resistant forms survived longer than its antibiotic-sensitive counterpart in estuarine water.Three of the resistant strains died off more rapidly than the antibiotic-sensitive wild type. Survival of the testbacteria in estuarine water was as follows: sensitive and resistant AQ62, resistant Escherichia coli < sensitiveEscherichia coli < resistant AP17 < resistant Enterococcus faecium < sensitive AP17, sensitive and resistantS. equinus < sensitive and resistant Enterococcus faecalis, sensitive Enterococcus faecium. The resultssupported the suggestion that fecal entercocci may serve as better indicators of fecal pollution than Escherichiacoli in marine ecosystems. Moreover, the results indicated that the use of antibiotic-resistant mutants to followthe fate of bacteria in the environment is inappropriate without adequate studies to ensure that resistant andwild-type strains react similarly to environmental stressors.

Fecal coliforms and fecal streptococci are accepted indi-cators of potential health hazards associated with fecalpollution. A standard methodology has been developed forthe routine detection and enumeration of these isolates (3).Indicator bacteria are not members of the indigenous flora ofsoil and aquatic ecosystems, hence they may be killed orinjured by environmental stresses (5, 15, 29, 32). Differentgroups of indicator bacteria are affected nonuniformly byenvironmental conditions (6, 30, 31, 42). Specifically, fecalstreptococci survive longer than fecal coliforms in seawaterand should be considered, perhaps, better indicators of fecalpollution for marine ecosystems (10, 14, 22, 38).

Ideally, it is desirable to follow the fate of these organismsdirectly in the environment. The recovery of experimentalbacteria from aquatic environments, however, is often hin-dered by the indigenous flora which may be present in muchhigher numbers. Antibiotic-resistant mutants of a test bac-terium can be selected from a mixed population of antibiotic-sensitive bacteria even if they are present in very lownumbers. This approach has been proposed for monitoringthe survival of genetically engineered bacteria in the envi-ronment (28) and the survival of bacteria in soils (12, 41),lake water (26, 37), river water (35), and sewage (26, 37).We compared the survival of several strains of fecal

streptococci and Escherichia coli, as well as antibiotic-resistant mutants derived from them, in estuarine water. Wediscuss problems associated with the methodology and con-clude that adequate measures must be taken to ensure thatantibiotic-resistant mutants are valid models for wild-typestrains in the environment.

* Corresponding author.t Present address: Department of Biology, State University Col-

lege at Buffalo, Buffalo, NY 14222.

MATERIALS AND METHODS

Test bacteria. Pure cultures of Enterococcus faecalisATCC 19433, Enterococcus faecium ATCC 19434, Strepto-coccus equinus ATCC 9812, Escherichia coli ATCC 11775,and two environmental isolates, designated AP17 and AQ62,were used throughout the study. Strains AP17 and AQ62,isolated from Boston Harbor, Mass., waters on PSE (bileesculin azide) agar (GIBCO Diagnostics, Madison, Wis.)streaked from azide dextrose broth (GIBCO) enrichments,were identified as enterococci. They were catalase-negative,gram-positive cocci that grew in chains. They also grew at10°C, survived at 60°C for 10 mins, and grew at pH 9.6 in a

medium containing 6.5% NaCl. Based on further routinelaboratory tests, however, they could not be categorized as

one of the established species of Enterococcus (23).Selection for antibiotic resistance. Antibiotic-resistant bac-

teria were selected by the technique of Mallory et al. (28).Bacteria were transferred in liquid medium through a 10-foldseries of increasing concentrations of two antibiotics, begin-ning with a 1/1,000 dilution of the final antibiotic concentra-tion. Rifampin was dissolved in methanol, and all otherantibiotics were added to distilled water. Antibiotic solutionswere filter sterilized. Gram-positive cocci were renderedresistant to a combination of streptomycin (1,000 ,ug ml-')and rifampin (50 ,ug ml-'). Escherichia coli was renderedresistant to both kasugamycin (300 ,ug ml-') and novobiocin(100 ,ug ml-'). Care was taken to ensure the stability of theantibiotic resistance trait. Each of the antibiotic-resistantstrains was passed at least three times in an antibiotic-freemedium followed by inoculation into a medium containingthe appropriate combination of antibiotics. Growth of thebacteria in antibiotic-containing media demonstrated thestability of the trait. Antibiotic-sensitive bacteria were also

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FIG. 1. Effect of plate incubation period on recovery of antibi-otic-sensitive (A, A) and antibiotic-resistant (0, 0) Enterococcusfaecium during a 7-day exposure to filtered estuarine water. Plateincubation was for 24 h (open symbols) and 48 h (closed symbols).

passed at least three times in antibiotic-free media to ensuretheir continued sensitivity to the drugs.

Culture techniques. All strains were maintained at -85°Cin brain heart infusion (BHI) broth amended with 10 to 15%glycerol. Overnight cultures were mixed with the medium,and 1.0 ml was pipetted into cryotubes containing glassbeads. Excess medium was removed to leave the beadscoated, and the tubes were frozen. Bacteria were revived byaseptically placing one to two glass beads in BHI broth andincubating them at 35°C. Prior to experimentation, sensitiveand resistant strains were again streaked onto medium withand without antibiotics to reaffirm that the strains retainedresistance or sensitivity after storage.

Bacteria were grown in half-strength BHI broth supple-mented with 5.0 g of yeast extract liter-1 and 2.0 g ofdextrose liter-1 (BHIGYE) as described by Mallory et al.(28). When agar plates were used, BHIGYE was amendedwith 20 g of Bacto-Agar (Difco Laboratories, Detroit, Mich.)liter-1. Estuarine water (29.0%oo salinity) was collected fromBoston Harbor, filtered through a filter (pore size, 0.2 lim;Nuclepore Corp., Pleasanton, Calif.), and frozen in sterileglass containers for later use.

Bacterial cultures for experiments were grown inBHIGYE broth at 35°C for 18 h. A 1% (vol/vol) inoculumwas transferred to fresh broth and incubated for an addi-tional 18 h. These log-phase cultures were centrifuged at4,000 x g for 10 min. The pellet was washed twice withsterile estuarine water and suspended in 10 ml of sterileestuarine water to yield a stock suspension. Sterile estuarinewater was inoculated with stock suspension to an opticaldensity at 540 nm of 0.01 to provide a final suspension whichcontained between 106 and 107 CFU ml-1. Triplicate 50-mlvolumes of the final suspensions were transferred to 250-mlscrew-cap flasks, sampled for enumeration (time zero), andthen incubated at room temperature (21 to 24°C) on a rotaryshaker operated at 100 rpm.Enumeration of bacteria. Cultures were incubated in estu-

arine water for up to 7 days. At intervals, subsamples weretaken and serial dilutions were prepared in half-strengthBHIGYE broth. Appropriate dilutions were plated on tripli-cate on the surface of BHIGYE agar plates. Antibiotic-

resistant strains were also plated on triplicate BHIGYE agarplates containing antibiotics. Plates were incubated at 35°C.CFU were counted at 24 and 48 h. Incubation of platesbeyond 48 h did not increase the number of CFU. TheStudent's t test was used to determine statistical significancebetween the different treatments.

RESULTS

Effect of plate incubation time and medium on colonyformation. Both antibiotic-resistant and -sensitive strainswere examined for their ability to form colonies on agarmedia during extended incubation in estuarine water. Duringthe first 2 days, plates incubated for 48 h did not havesignificantly higher counts than those incubated for 24 h(<5% increase in CFU). With increasing exposure to estua-rine water, however, differences in the number of CFUbetween 24 and 48 h of incubation on spread plates becameapparent (Fig. 1), indicating that an increasing period ofrecovery on the agar medium was necessary with longerexposure to seawater.

Differences in colony morphology were also evident be-tween antibiotic-resistant and -sensitive strains, dependingon whether the plating medium contained antibiotics. Esch-erichia coli demonstrated the most noticeable disparitiesamong the test bacteria (Fig. 2). The gram-positive cocci alsoproduced smaller colonies when they were plated on antibi-otic agar and with increased exposure to the estuarine water.The incorporation of antibiotics into the plating medium

had a strain-dependent effect on the recovery of antibiotic-resistant bacteria exposed to estuarine water (Table 1). Onlytwo of the six resistant strains, Enterococcus faecium andAP17, showed lower recovery on the antibiotic medium thanthe antibiotic-free medium after a 2-day exposure to estua-rine water. The difference between the two media becamemore pronounced with increasing exposure time to estuarinewater, as illustrated for resistant Enterococcus faeciumisolates (Fig. 3).

Survival of bacterial strains in estuarine water. The antibi-otic-sensitive and -resistant strains of the six indicator spe-cies were suspended in estuarine water for 6 days andenumerated at days 0, 2, 4, and 6. For all organisms exceptEnterococcus faecium and Enterococcus faecalis, CFU de-

FIG. 2. Colony development of antibiotic-resistant and -sensi-tive strains of Escherichia coli after 48 h of incubation of plates at35°C. Sensitive (A) and resistant (B) Escherichia coli isolates onantibiotic-free medium and resistant Escherichia coli isolates onmedium containing kasugamycin and novobiocin (C) are shown.

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TABLE 1. Effect of antibiotics in the plating medium on therecovery of antibiotic-resistant indicator organisms after

a 2-day exposure to filtered estuarine watera

% Recovery onc:Strainb

Antibiotic-free medium Antibiotic medium

Enterococcus faecium 4.4 1.4dEnterococcus faecalis 27 32Streptococcus equinus 11 12AP17 0.48 0.34eEscherichia coli <0.01 <0.01AQ62 <0.01 <0.01

a Plates were incubated at 35°C for 48 h.b All strains were resistant.c Percent recovery was calculated as follows: [(CFUday o - CFUday 2)/

CFUday ol X 100.d Percent recovery was significantly different (a < 0.05) on the two media.ePercent recovery was significantly different (a < 0.01) on the two media.

survival experiment (Fig. 1). Survival of the test bacteriawas as follows: sensitive and resistant AQ62, resistantEscherichia coli < sensitive Escherichia coli < resistantAP17 < resistant Enterococcus faecium < sensitive AP17,sensitive and resistant S. equinus < sensitive and resistantEnterococcus faecalis < sensitive Enterococcus faecium.

In no case did the antibiotic-resistant form of a speciessurvive longer than its antibiotic-resistant form in estuarinewater. Of the six species examined, the antibiotic-resistantand -sensitive strains of two, Enterococcus faecalis and S.equinus, behaved similarly. The antibiotic-resistant forms ofEnterococcus faecium, AP17, and Escherichia coli, on theother hand, did not survive as well as their antibiotic-sensitive counterparts. Antibiotic-sensitive and -resistantforms of AQ62 could not be compared due to their rapid dieoff in estuarine water.

DISCUSSION

clined to less than 1% of the initial bacterial suspension aftera 4-day exposure. Concurrent with decreasing recovery was

an increase in the variance of CFU among the replicateflasks, as described by the calculated coefficient of variance.For example, the coefficient of variance for sensitiveEnterococcus faecium CFU increased from 25% at day 0 to163% at day 7 (actual recoveries are illustrated in Fig. 1).These data were typical of all bacteria tested.Although experiments were carried out for 6 days, recov-

eries after 2 days of exposure to estuarine water were chosento represent comparative survival among the bacterial (Table2). Die-off rates could not be calculated to compare survivalamong the bacteria tested because the number of CFUtypically did not decrease linearly and because some bacte-ria displayed a time lag before their viable counts decreased.All species of Streptococcus and Enterococcus, except forAQ62, survived longer than Escherichia coli based on recov-

ery on agar plates. Sensitive Enterococcus faecium isolateswere least affected by the estuarine water, as demonstratedby a 2-day lag during which the number of CFU increased.Similar results were obtained with this strain in estuarinewater, other than with the batch used in the comparative

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La)

10

;2 3.-.4-

TIME (DAYS)FIG. 3. Effect of antibiotics in the plating medium on the recov-

ery of resistant Enterococcus faecium isolates exposed to filteredestuarine water. Symbols: 0, no antibiotics in medium; 0, strepto-mycin and rifampin in medium.

With the exception of one environmental isolate, strepto-cocci and enterococci were recovered for longer periodsthan Escherichia coli from filtered estuarine water. Theseresults are in agreement with reports that fecal streptococcipersist longer in seawater than do fecal coliforms (7, 14, 34).However, in most of the studies the survival of the individualspecies that comprise the fecal streptococcus group, whichis heterogeneous in composition, has not been addressed.Although we used only single strains to represent eachspecies, differences in survival among the fecal streptococciprobably reflect this genetic heterogeneity.Of the bacteria tested, Enterococcus faecium demon-

strated the most resistance to estuarine water under theconditions of exposure. Slantez and Bartley (38) reportedthat Enterococcus faecium survived longer than other fecalstreptococci in seawater, but they also observed an increasein the CFU of Escherichia coli after 2 days as opposed to therapid die off of Escherichia coli reported by others (9, 16)and in this study (Table 2.) Perhaps the lower salinity of theseawater allowed the growth of Escherichia coli. Seawater isa chemically complex and variable milieu, and results withbacteria recovered from seawater are highly dependent onthe location and time of sampling. Therefore, while thecomparative values (percent recovery) of survival are useful,these values should not be taken as absolute values forestuarine water. We observed that survival values changedconsiderably from one batch of estuarine water to another.

TABLE 2. Recovery of antibiotic-sensitive and -resistantindicator bacteria on antibiotic-free medium after a 2-day

exposure to filtered estuarine watere

% Recovery forb:

Strain Antibiotic-sensitive Antibiotic-resistantstrain strain

Enterococcus faecium 118 4.4CEnterococcus faecalis 31 27Streptococcus equinus 16 11AP17 11 0.48cEscherichia coli 0.09 <0.01dAQ62 <0.01 <0.01

a Plates were incubated at 35°C for 48 h.b See Table 1, footnote c, for the equation used to calculate percent

recovery.c Significant difference (ca < 0.01) between percent recovery of antibiotic-

sensitive and -resistant strains.d Significant difference (a < 0.05) between percent recovery of antibiotic-

sensitive and -resistant strains.

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1244 PETTIBONE ET AL.

When incubated in filtered estuarine water taken in Decem-ber (29.%oo salinity), sensitive Enterococcusfaecalis isolatesdid not die off after a 2-day incubation (data not shown);however, when water sampled in May of the following yearwas used (30.7%o salinity), sensitive Enterococcus faecalisisolates decreased by approximately 70% in 2 days (Table 2).Although the salinity did not vary greatly, other chemicalconstituents such as chlorine (8), toxic organic compounds(4), metals (2, 13, 25), and organometals (24, 36, 43) canaffect the survival of indicators in aquatic ecosystems. Theeffects of various environmental factors on Escherichia colihave been described (5, 8, 9, 15, 16, 20), but few analogousstudies have been conducted for the fecal streptococci.The persistence of S. equinus in filtered estuarine water is

in sharp contrast to the rapid die off of S. equinus andStreptococcus bovis in freshwater (18, 19, 30) and filteredseawater (38). 5. bovis and S. equinus are probably justdifferent strains of the same species (27). Therefore, onlyone was used here.The two isolates from Boston Harbor, AP17 and AQ62,

were not as resistant as Enterococcus faecium andEnterococcus faecalis to exposure in estuarine water. Thismay reflect a difference between environmental isolates andAmerican Type Culture Collection (Rockville, Md.) strains.However, survival of sensitive AP17 isolates was compara-ble to survival of sensitive S. equinus isolates, and the use ofAmerican Type Culture Collection strains is valid to dem-onstrate the effects of estuarine water on fecal streptococci.The means by which AP17 and AQ62 got into Boston Harborare not known, and based on routine biochemical tests theyare not members of well-described enterococcal species.More work is needed to describe the types of streptococcithat are found in seawater.Comparison of the survival of antibiotic-sensitive and the

counterpart antibiotic-resistant strains of fecal pollution in-dicator bacteria indicated that there are potential difficultiesin the use of antibiotic-resistant mutants for the assessmentof survival in the environment. With the advent of geneticengineering and the increasing likelihood of the deliberaterelease of genetically engineered bacteria into the environ-ment (40), there is an increasing need to determine thedispersal and survival of specific organisms in the environ-ment. The antibiotic-resistant mutants employed by us andothers (37) to assess survival under environmental condi-tions are probably a different class of antibiotic-resistantstrains than the R-factor strains which have been examinedfor survival (1, 17, 21, 39). Rather, antibiotic resistancelikely arose from DNA point mutations which resulted in analteration of antibiotic target sites in the cell or in a de-creased permeability of the mutants to compounds similar tothe antibiotics (33). The inherent assumption in the use ofsuch mutants in survival experiments is that they respond inthe same manner to environmental stress as the wild types.Park (35) concluded that this approach would be useful sinceantibiotic-resistant mutants inoculated into a laboratory me-dium grow at rates equal to those of antibiotic-sensitive wildtypes. Environmental stress to enteric bacteria, however, isprobably minimal in a rich growth medium. In contrast, weconclude that significant differences in survival may occurbetween the parent and mutant strains under environmentalconditions. Indeed, Novick (33) has described such antibi-otic-resistant mutants as "evolutionary cripples."The use of antibiotic-resistant mutants would have merit

for the assessment of survival if the wild type and theantibiotic-resistant mutant were to behave similarly to envi-ronmental stress, as in the case of the S. equinus and

Enterococcus faecalis strains used in this study. Cautionshould be taken because differences in colony morphologyon antibiotic medium and the increasing recovery time ofcolony formation are signs that the antibiotic-resistant mu-tants are more stressed that the wild types. The antibiotic-resistant strains may still be viable but nonculturable onmedia, as described by Colwell et al. (11). We conclude thatthe use of antibiotic-resistant mutants to follow the fate ofbacteria in the environment is inappropriate without ade-quate preliminary studies to ensure that resistant and wild-type strains react similarly to environmental stressors.

ACKNOWLEDGMENTS

We thank Barbara MacGregor for assistance in the isolation andcharacterization of the gram-positive cocci from Boston Harbor. Wealso thank J. J. Cooney for critical review of the manuscript andDoug Jambard-Sweet, Robin Brissette, and Bobby Wong for tech-nical assistance during this study.

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