Sludge Watch ==> Sewage and antibiotic resistance profiles of sewage-related bacteria

[Maureen Reilly]

Sludgewatch Admin:

Let me draw your attention to the role of sewers in increasing the 
proportions of antibiotic resistant pathogens in sludge and sewerage ...  
Like the plague (yersinia pestis).  Note that this study is from 1984...and 
the issue of antibiotic resistant pathogens is worse every day.


"Some rare and potentially pathogenic species were isolated from chlorinated 
and
regrowth samples, including Yersinia enterocolitica, Yersinia pestis,
Pasteurella multocida, and Hafnia alvei. Our results indicate that
chlorination, while initially lowering the total number of bacteria in
sewage, may substantially increase the proportions of antibiotic-resistant,
potentially pathogenic organisms."





Appl Environ Microbiol. 1984 July; 48(1): 73–77.


Effect of chlorination on antibiotic resistance profiles of sewage-related
bacteria.
G E Murray, R S Tobin, B Junkins, and D J Kushner

This article has been cited by other articles in PMC.
Abstract
A total of 1,900 lactose-fermenting bacteria were isolated from raw sewage
influent and chlorinated sewage effluent from a sewage treatment plant, as
well as from chlorinated and neutralized dilute sewage, before and after a
24-h regrowth period in the laboratory. Of these isolates, 84% were
resistant to one or more antibiotics. Chlorination of influent resulted in
an increase in the proportion of bacteria resistant to ampicillin and
cephalothin, the increase being most marked after regrowth occurred
following chlorination. Of the other nine antibiotics tested, chlorination
resulted in an increased proportion of bacteria resistant to some, but a
decrease in the proportion resistant to the remainder.

Multiple resistance was found for up to nine antibiotics, especially in
regrowth populations. Identification of about 5% of the isolates showed that
the highest proportion of Escherichia coli fell in untreated sewage. Some
rare and potentially pathogenic species were isolated from chlorinated and
regrowth samples, including Yersinia enterocolitica, Yersinia pestis,
Pasteurella multocida, and Hafnia alvei. Our results indicate that
chlorination, while initially lowering the total number of bacteria in
sewage, may substantially increase the proportions of antibiotic-resistant,
potentially pathogenic organisms.



.............................


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http://aem.asm.org/cgi/reprint/48/1/73

Vol. 48, No. 1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1984, p. 73-77
0099-2240/84/070073-05$02.00/0
Copyright © 1984, American Society for Microbiology
Effect of Chlorination on Antibiotic Resistance Profiles of Sewage-
Related Bacteria
G. E. MURRAY,' R. S. TOBIN, * B. JUNKINS,2 AND D. J. KUSHNER'
Biology Department, University of Ottawa, Ottawa, Ontario, Canada KIN 6N5,1 
and Health Protection Branch, Health
and Welfare Canada, Ottawa, Ontario, Canada KJA 0L22
Received 23 January 1984/Accepted 4 April 1984
A total of 1,900 lactose-fermenting bacteria were isolated from raw sewage 
influent and chlorinated sewage
effluent from a sewage treatment plant, as well as from chlorinated and 
neutralized dilute sewage, before and
after a 24-h regrowth period in the laboratory. Of these isolates, 84% were 
resistant to one or more antibiotics.
Chlorination of influent resulted in an increase in the proportion of 
bacteria resistant to ampicillin and
cephalothin, the increase being most marked after regrowth occurred 
following chlorination. Of the other nine
antibiotics tested, chlorination resulted in an increased proportion of 
bacteria resistant to some, but a decrease
in the proportion resistant to the remainder. Multiple resistance was found 
for up to nine antibiotics, especially
in regrowtb populations. Identification of about 5% of the isolates showed 
that the highest proportion of
Escherichia coli fell in untreated sewage. Some rare and potentially 
pathogenic species were isolated from
chlorinated and regrowth samples, including Yersinia enterocolitica, 
Yersinia pestis, Pasteurella multocida, and
Hafnia alvei. Our results indicate that chlorination, while j01jally 
lowering the total number of bacteria in
sewage, may substantially increase the proportions of antibiotic-resistant, 
potentially pathogenic organisms.
The occurrence of multiply antibiotic-resistant (MAR)
bacteria in both drinking water and wastewater has been
demonstrated in many studies (2, 3, 22, 23, 25) and is
considered an important potential health problem. Antibiotic
resistance in pathogens causes difficulty in effectively treating
human infections, but antibiotic resistance in organisms
which are not considered primary pathogens is also important
because of the ability of these organisms to transmit
resistance to other organisms by means of transmissible
resistance factors (R-factors) (8, 9, 21, 24).
In a survey of 193 healthy adults and children who were
not attending hospital and who had not recently received
antibiotics, 53% were found to carry antibiotic-resistant
coliforms in their feces, and in 61% of these coliforms
transmissible R-factors were demonstrated (15). Another
similar study indicated that 52% of patients entering hospital
for nonurgent surgical operations carried antibiotic-resistant
Escherichia coli. Approximately 60% of the resistant bacteria
possessed R-factors, with multiple resistance patterns
being more frequent than single ones (8). Other studies have
supported the view that intestinal bacteria carrying R-factors
are widespread in the human population (16, 19).
Wastewater treatments have been found to increase the
proportion of bacteria which carry R-factors (5, 6, 11, 14,
18). Furthermore, Shuval et al. (20) have shown that extensive
growth of both fecal and nonfecal coliforms occurred in
chlorinated samples, even though no coliforms were detected
immediately after chlorination. Under field conditions the
regrowth of coliforms in chlorinated effluent that had been
held for 3 days was inversely correlated with both the
residual chlorine in the reservoir and the initial number of
surviving coliforms. Laboratory experiments showed that
regrowth occurred after initial exposure to 11 ppm (11 ,ug/ml)
of chlorine, even in the absence of chlorine neutralization
(20).
Other workers (14) found that the proportion of antibioticresistant
coliforms increased from those in fecal material (0.1
to 1% of total coliforms being resistant) through urban
wastewater (10% resistant) to river water (50%) and finally
* Corresponding author.
to potable water, where 80% of any coliforms present were
antibiotic resistant. The increase in the proportions of antibiotic-
resistant bacteria has been attributed to R-factor transfer
(20). Multiple resistance in bacteria isolated from chlorine-
treated and untreated drinking water has been studied,
with the conclusion that treatment of raw water and its
subsequent distribution selected for bacterial populations
resistant to several antibiotics (2, 3). The chlorination step
was thought to be involved in the selection of antibiotic
resistance.
The purpose of the present research was to determine the
extent which chlorination plays in the development of antibiotic
resistance. We studied the changes in antibiotic resistance
patterns after wastewater chlorination at a municipal
plant and also after laboratory chlorination and regrowth of
sewage-contaminated drinking water. The antibiotic resistance
patterns to 11 antibiotics were determined and analyzed.
A preliminary identification of a sample of the surviving
organisms was made.
MATERIALS AND METHODS
Description of bacterial populations studied. Four populations
of bacteria were used in this study. The following terms
are used to describe them. (i) The effluent population was
isolated from samples of chlorinated effluent obtained from
the Green Creek Sewage Treatment Plant, Ottawa, Ont.,
Canada. (ii) The influent population was isolated from raw
sewage influent above the treatment plant. (iii) The chlorinated
influent population was isolated from the influent raw
sewage which had been chlorinated in the laboratory. (iv)
The regrowth population was isolated from a sample of
chlorinated influent, following 24-h recovery (regrowth) after
laboratory chlorination.
Sample collection. Influent or effluent samples were collected
at 10:00 a.m. by plant personnel and held at 4°C during
transportation to the laboratory. The bacterial populations
were isolated from samples collected over the period February
through March 1981.
Wastewater treatment in the sewage treatment plant. The
influent remained in holding tanks for 1.5 h to allow gravity
sedimentation of particulate matter; coagulation was aided
73
74 MURRAY ET AL.
TABLE 1. Antibiotic disks used
Inhibition zone diam (including
Antibiotic Disk po- disk diamn) (mm) tency (,ug) Resis- Interme- Senstant
diate itive
Ampicillin 10 <12 12-13 >13
Cephalothin 30 <15 15-17 >17
Chloramphenicol 30 <13 13-17 >17
Gentamicin 10 - 13 >13
Kanamycin 30 <14 14-17 >17
Nalidixic acid 30 <14 14-18 >18
Nitrofurantoin 300 U <15 15-16 >16
Polymixin B 300 U <9 9-11 >11
Sulfonamides 300 <13 13-16 >16
Tetracycline 30 <15 15-18 >18
Trimethoprim 25 <11 11-15 >15
by the addition of 0.25 ppm (0.25 p.g/ml) of Percol 727 (Allied
Colloids, Rexdale, Ont., Canada). The influent was then
dosed with chlorine, such that after 20 min the residual
available chlorine was 0.5 mg/liter.
Isolation of effluent and influent populations. The samples
were shaken mechanically for 30 min at room temperature to
disrupt clumps of particulate material. Serial dilutions of
both influent and effluent samples were made with chlorinefree,
filter-sterilized tap water. Samples of these dilutions
were passed through membrane filters (0.45 p,m; Nuclepore
Corp., Pleasanton, Calif.) which were incubated on eosin
methylane blue agar (Difco Laboratories, Detroit, Mich.) at
37°C for 18 h, after which the colonies were counted and the
bacterial count in the original samples was calculated. Eosin
methylene blue agar was chosen as the initial medium on
which to isolate the sewage bacteria because of its selectivity
for lactose-fermenting strains (1). Such lactose-fermenting
(metallic sheen) colonies were then purified by streaking for
single colonies onto MacConkey agar (Difco).
Laboratory chlorination of influent. To part of the initial
10-fold dilution of influent, chlorine was added from a
standardized stock solution to give an initial dose of 1 mg of
total chlorine per liter in tap water (25). The sample was
allowed to stand at room temperature in the dark for 1 h, a
portion was withdrawn for chlorine measurement, and free
chlorine was neutralized in the remainder by the addition of
3 ml of 1% sodium thiosulfate (J. T. Baker Chemical Co.,
Phillipsburg, N.J.) per 100 ml of bacterial suspension. Target
bacteria survival varied from 10-3 to 10-4 in these chlorinated
samples. There was no detectable killing in an identical
nonchlorinated control sample.
Laboratory determination of chlorine levels. Total available
chlorine levels were measured by the N,N-diethyl-p-phenylenediamine
procedure, using the Hellige hand colorimeter,
in accordance with method 409F of the American Public
Health Association (1). Total available chlorine was 0.8 to
0;9 mg/liter in all experiments, the difference from that added
presumably being due to organic matter in the samples.
Regrowth population. The regrowth population was isolated
as above, from chlorinated influent which had been
allowed to stand at room temperature for 24 h after neutralization
of the chlorine by sodium thiosulfate. After this
recovery period the viable count increased 100-fold.
Determination of antibiotic resistance profiles. Antibiotic
resistance profiles were obtained for approximately 1,900
isolates. A single colony of each of the purified isolates from
the MacConkey agar plates was inoculated into 5 ml of
sterile broth and incubated at 37°C for 2 to 10 h to give an
optical density visually equal to a MacFarland no. 3 standard
(ca. 109 cells per ml). Mueller-Hinton agar (Difco) plates
were confluently seeded by swabbing with cotton (Q-tips;
Johnson & Johnson, Cheeseborough-Ponds, Markham, Ontario)
moistened with the culture. After the plates were dried
for 1 h, a semiautomatic dispenser was used to place up to 12
different antibiotic-impregnated disks on each plate. Diameters
of the inhibition zones surrounding the disks were
recorded after 18 h of incubation at 37°C. Bacterial isolates
were characterized as "resistant," "intermediate," or "sensitive"
according to the specifications for the disks provided
by the manufacturer (Pfizer Inc., New York, N.Y.) and
given in Table 1.
Identification of strains. After determination of antibiotic
resistance profiles, the strains were stored on the MacConkey
agar plates until all strains were thus characterized. One
isolate of every 10 from these MacConkey plates was
selected at random and provisionally identified by using the
Analytical Profile Index system (Analytab Products, Ayerst
Laboratories, Plainview, N.Y.).
Statistical methods. The date of sample collection was
treated as a blocking factor in the analysis. The difference
between the proportions of isolates which were resistant in
the different populations was examined for each antibiotic,
using the Mantel-Haenszel test for proportions in a blocked
experiment (17). Comparisons of proportion were made
between (i) influent and chlorinated influent, (ii) influent and
regrowth, (iii) influent and effluent, and (iv) regrowth and
TABLE 2. Comparison of antibiotic resistance in bacterial populations"
Influent (%) Effluent (Yc) Chlorinated influent (%) Regrowth (%)
Antibiotic' No. of No. of No. of
R I S isolates R I S isolates R I S isolates R I S isolates
AMP 68 6 25 692 73 6 20 190 77 8 14 723 83 5 12 258
CEPH 45 12 43 692 53 3 44 189 60 5 35 722 75 6 19 257
KAN 4 5 91 692 3 2 95 190 6 5 89 723 2 3 95 258
POL 1 8 90 692 0.5 0.5 99 190 2 8 91 723 0 1 99 258
GEN 1 99 692 1 99 188 2 98 723 1 99 258
TET 8 11 81 692 4 10 86 190 11 8 80 722 18 6 76 258
CHLOR 3 1 96 690 0 4 96 190 2 2 97 723 2 0.4 97 258
NIT 8 13 79 691 2 3 95 190 3 7 90 723 4 1 95 258
NAL 1 6 93 691 0.5 2 98 190 2 2 96 723 3 1 96 258
SUL 20 10 70 690 18 4 77 190 21 11 69 723 34 6 59 258
TRI 4 1 94 691 5 3 92 189 4 2 93 722 10 1 88 258
"R, Resistant; I. intermediate; S, sensitive.
b AMP, Ampicillin; CEPH, cephalothin; KAN, kanamycin; POL, polymixin B: GEN, 
gentamicin; TET. tetracycline: CHLOR, chloramphenicol; NIT,
nitrofurantoin; NAL, nalidixic acid; SUL, sulfonamides; TRI, trimethoprim.
APPL. ENVIRON. MICROBIOL.
EFFECT OF CHLORINATION ON ANTIBIOTIC RESISTANCE 75
s70
~60-
*50-
z
130-
20-
10
0 1 1b-
AMP CEPH KAN POL GEN
FIG. 1. Percentage of stra
chlorinated influent. Because of the exploratory nature of
the study, no attempt was made to control overall error rate
in the experiment. Significance was tested at P < 0.05.
RESULTS
About 84% of the 1,900 isolates were resistant to at least
one of the antibiotics tested: in particular, 79.5% in the
influent, 88.2% in the chlorinated influent, 87.5% in the
regrowth, and 81.2% in the effluent. Table 2 shows the
proportion resistant to each antibiotic for the different populations,
and Fig. 1 summarizes this information graphically.
The statistical significance of these results is shown in Table
3.
The proportion of bacteria resistant to ampicillin and
cephalothin increased significantly in the order influent,
effluent, chlorinated influent, and regrowth. For tetracycline,
nalidixic acid, sulfonamides, and trimethroprim the
regrowth population showed the highest incidence of resistance.
Relatively little resistance to other antibiotics appeared
in any of populations studied. At these low levels,
however, decreased resistance to kanamycin, chloramphenicol,
nitrofurantoin, gentamicin, polymixin, and nalidixic acid
was observed after chlorination or regrowth or both (Fig. 1;
Table 3).
Occurrence of multiple resistance. Table 4 shows the
proportion of each population resistant to one to nine
antibiotics. The mean value of multiple antibiotic resistance
was significantly higher in the regrowth population at 2.34
antibiotics per isolate than it was in the chlorinated effluent
(1.89), influent (1.64), and effluent (1.61).
Resistance to certain antibiotics appeared to be linked.
For example, over 90% of the isolates resistant to four or
more antibiotics were resistant to ampicillin, cephalothin,
sulfonamides, and tetracycline or to ampicillin, cephalothin,
sulfonamides, and trimethoprim. In the group multiply resistant
to four or more antibiotics, resistance to nitrofurantoin
and nalidixic acid was associated with one another in 88% of
the isolates where resistance to one of them was detected.
Strains identified in the populations. Table 5 shows the
proportions of bacterial species identified in each of the
populations. This information applies to the 70% of strains
which survived storage after determination of their antibiotic
resistance profiles. Thirty-five percent of these failed to key
out on the Analytical Profile Index system. Consequently,
the species distribution we found may differ somewhat from
7Z777 WLUENT
EFFLUENT
CHLORINATED INFLUENT
_REGROWTH
TET CHLOR NIT NAL SUL
ins resistant to each antibiotic.
the environmental distribution. However, as expected, most
species were coliforms or related species in the family
Enterobacteriaceae. Most interesting is the decrease in the
proportion of E. coli strains identified in the chlorinated
influent, regrowth, and effluent populations compared with
the nonchlorinated influent.
DISCUSSION
The proportions of bacteria resistant to at least one
antibiotic ranged from 79.5% in the influent to 87.5% in the
TABLE 3. Patterns of incidence of resistance
Antibiotic
Ampicillin
Cephalothin
Kanamycin
Polymixin B
Gentamicin
Significant
differences
(P < 0.05)"
CI > I
R > I
R > CI
Cl > I
R > I
R > CI
CI > R
I>R
CI > R
I > R
I> E
Tetracycline CI > I
R > I
R > Cl
Chloramphenicol I > E
I > R
Nitrofurantoin I > Cl
Nalidixic acid R > Cl
Trimethoprim R > CI
Sulfonamides R > Cl
R > I
a I, Influent; CI, chlorinated influent; R, regrowth; E, effluent.
VOL. 48, 1984
76 MURRAY ET AL.
TABLE 4. Percentage of strains resistant to zero, one, or more
antibiotics
No. of antibiotics % of strains resistant in given population
to which bacteria
exhibited Influent Chlorinated Regrowth Effluent
resistance influent
0 20.5 11.8 12.5 18.8
1 30.9 26.5 10.9 30.6
2 26.3 35.3 34.6 29.0
3 13.8 17.1 24.5 15.6
4 5.4 6.5 13.2 4.3
5 1.6 1.5 1.6 1.6
6 1.2 1.1 1.2 0.0
7 0.1 0.0 0.8 0.0
8 0.1 0.0 0.8 0.0
9 0.1 0.1 0.0 0.0
Population (n) 689 720 254 186
Mean resistance 1.65 1.90 2.33 1.61
regrowth samples. These values are comparable to those
obtained by Armstrong et al., who found proportions of up
to 87% in river water and 84.5% in clear well water resistant
to one or more antibiotic (2), and with the findings of
LeClerc and Mizon (14), who found that up to 80% of
coliforms in potable water were antibiotic resistant.
While our study was in progress, Armstrong et al. (2)
published comparisons between standard plate count isolates
from source river water and drinking water produced
by flash mixing with chlorine. They found a significant
increase in the proportions of MAR bacteria. They also
studied antibiotic resistance profiles of isolates that survived
laboratory chlorination of river water, resulting in an 800-
fold decrease in the standard plate count population, and
found no increase in the proportion of MAR strains in these
isolates. In our study we found a significant increase in the
percentage of strains multiply resistant to two or three
antibiotics when influent was chlorinated in the laboratory
and a marginal increase when influent was compared with
effluent which had been treated at the sewage treatment
plant (Table 3).
Differences between our results and those of Armstrong et
al. (2) may be more apparent than real: in our study only
lactos,e-fermenting bacteria isolated from eosin methylene
blue plates were studied, so our populations are more
representative of coliforms, and in our laboratory chlorination
procedures we observed a 10-fold-greater degree of
bacterial death by chlorination than that observed by Armstrong
et al. (2, 3). It is possible that chlorinated coliform
populations are more likely to develop MAR strains than
other bacteria or that increase in the number of MAR strains
appears only after the survival rate falls to 10-3 or less. The
difference in the incidence of MAR strains arising in our
laboratory studies compared with those found after wastewater
treatment at the sewage treatment plant probably
reflects differences between chlorination and recovery
which occur in the two environments. Despite such differences,
both treatments increase the incidence of antibiotic
resistance in survivors, but the effect on MAR incidence is
more pronounced in laboratory-chlorinated samples.
Though increased resistance, notably to ampicillin, cephalosporin,
tetracycline, and sulfanilimide, was the most striking
finding, resistance to some antibiotics decreased on
chlorination (see Results). There is, thus far, no clear
relation between mode of antibiotic action and pattern of
TABLE 5. Identified strains"
Frequency in populations
Species Influent Chlorinated Regrowth Effluent
influent
Klebsiella ozaenae 0 2 0 0
Klebsiella oxytoca 7 8 2 2
Klebsiella pneumoniae 8 7 5 2
Aeromonas hydrophila 6 11 3 2
Escherichia coli 12 1 0 1
Enterobacter cloacae 1 0 1 0
Enterobacter aglomerans 0 2 1 1
Yersinia pestis 0 1 0 0
Yersinia enterocolitica 0 0 0 1
Pasteurella multocida 0 1 1 0
Serratia liquifaciens 0 1 0 0
Hafnia alvei 0 0 0 2
Citrobacter freundii 0 1 1 0
Shigella boydii 1 1 0 0
Unidentifiable 20 19 2 3
"Strains identified were selected at random (every 10th numbered isolate)
from isolates that survived storage after determination of antibiotic 
resistance
profiles.
resistance development. Among the antibiotics to which
resistance increased after chlorination, ampicillin and cephalothin
act on cell wall synthesis and sulfanilamide and
trimethoprim act on folic acid synthesis and metabolism,
respectively. Tetracycline, to which resistance increases,
and chloramphenicol, gentamicin, and kanamycin, to which
it decreases, act on different aspects of ribosomal function
(10). Nitrofurantoin, to which resistance decreases after
chlorination, has been reported to affect translation initiation
and to cause DNA damage (13).
It is not clear whether chlorination selects or induces
changes in antibiotic resistance in bacterial populations.
Several workers have suggested that the fecal coliforms,
which are generally more antibiotic resistant than other
coliforms, may have a survival advantage in natural and
treated wastewaters (4, 5, 7, 11).
Armstrong et al. (2) suggested, without specifying the
mechanisms, that stress-tolerant strains selected by chlorination
would be more antibiotic resistant. It is uncertain
whether specifically chlorine-resistant coliforms exist (12),
and certainly no physiological linkage between resistance to
chlorine and that to antibiotics suggests itself. However, the
possibility that resistant or MAR strains survive chlorination
better than sensitive strains could be tested directly. Another
possibility, that chlorination helps in the transfer of
antibiotic resistance plasmids to the surviving population of
bacteria, is also open to experimental investigation.
Although most coliforms are usually considered as harmless
indicators of water quality, strains of MAR bacteria that
colonize the intestinal tract of humans or animals could
transfer their resistance to intestinal commensals and in turn
to drug-sensitive pathogens (9, 21, 24). The maximum removal
of MAR bacteria from sewage before discharge to the
environment and prevention of their contamination of drinking
water are obviously highly desirable. However, whereas
chlorination initially lowers the total number of bacteria, it
may substantially increase the proportions of those resistant
to one or more antibiotics and thus facilitate the transfer of
resistance to other, possibly pathogenic, strains.
ACKNOWLEDGMENTS
We express our thanks to members of the Biology Department of
Ottawa University for their support, especially Becky Wallace,
APPL. ENVIRON. MICROBIOL.
EFFECT OF CHLORINATION ON ANTIBIOTIC RESISTANCE 77
Peter Lomax. and Gillian Johnson for their help in culturing and
identifying bacterial strains. We thank Donald Warburton for editorial
assistance.
This work was carried out under contract to the Department of
Supplies and Services. Canada (contract 1SU80-00323). Partial
support was provided from a grant awarded to D.J.K. by the Natural
Sciences and Engineering Research Council of Canada.
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