A PERFECT STORM OF ANTIBIOTIC RESISTANCE
Section 14
Antibiotic Resistance in Agricultural Products. 9/09/2011
Back to Myth # 4
In 1999, M. Osterblad, et al., National Public Health Institute at Turku, Finland, reported on the
“Antimicrobial susceptibility of Enterobacteriaceae isolated from vegetables.” They said, “There is potential for the
normal faecal flora of humans to be augmented by resistant strains of bacteria, acquired from food. The frequency of
resistance in the aerobic Gram-negative faecal flora is often very high. The purpose of this study was to find out
whether food strains contribute to this resistance. One hundred and thirty-seven vegetable samples were studied, 48 of
Finnish origin, and 89 imported. From these samples, 535 different strains of bacteria belonging to the family
Enterobacteriaceae were isolated. Enterobacter spp. were most frequent, Escherichia coli was rare. Sensitivity testing
was undertaken only for isolates with different biotypes and antibiograms. No resistance was found to cefotaxime,
aztreonam, imipenem, gentamicin, nalidixic acid or ciprofloxacin. The frequency of trimethoprim resistance was 0.2%,
sulphamethoxazole resistance 1.3%, and tetracycline resistance 5.5%. These frequencies were much lower than those
found in faecal flora. Chloramphenicol and cefuroxime resistance was found in 12% and 14% of isolates, respectively.
The only statistically significant differences between the Finnish and imported strains were for these two; the Finnish
isolates were more resistant to cefuroxime, whereas the imported ones were more resistant to chloramphenicol.
Consequently, bacteria from vegetables are not responsible for the high prevalence of resistant Enterobacteriaceae in
faecal flora in Finland; they are in fact unusually susceptible to the antibiotics studied. Multiresistance profiles, typical of
strains associated with human activities, were not identified in these isolates.”
http://www.ncbi.nlm.nih.gov/pubmed/10350379
In many cases researchers do not discuss antibiotic resistance in the studies, even though as the case with E. coli 0157,
the use of antibiotics makes it even more deadly. Nor do they discuss the fact that the bacteria used in the research is a
weak modified laboratory strain with the dangerous toxic DNA removed. In the following 2004 study the researchers do
note they used an avirulent strain of E. coli O157:H7. Mahbub Islam, et al., University of Georgia, reported on the
“Persistence of Enterohemorrhagic Escherichia coli O157:H7 in Soil and on Leaf Lettuce and Parsley Grown in Fields
Treated with Contaminated Manure Composts or Irrigation Water.” They said, “Outbreaks of enterohemorrhagic
Escherichia coli O157:H7 infections associated with lettuce and other leaf crops have occurred with increasing
frequency in recent years. Contaminated manure and polluted irrigation water are probable vehicles for the pathogen in
many outbreaks. In this study, the occurrence and persistence of E. coli O157:H7 in soil fertilized with contaminated
poultry or bovine manure composts or treated with contaminated irrigation water and on lettuce and parsley grown on
these soils under natural environmental conditions was determined. Twenty-five plots, each 1.8 by 4.6 m, were used
for each crop, with five treatments (one without compost, three with each of the three composts, and one without
compost but treated with contaminated water) and five replication plots for each treatment. Three different types of
compost, PM-5 (poultry manure compost), 338 (dairy manure compost), and NVIRO-4 (alkaline-stabilized dairy manure
compost), and irrigation water were inoculated with an avirulent strain of E. coli O157:H7. Pathogen concentrations were
107 CFU/g of compost and 105 CFU/ml of water. Contaminated compost was applied to soil in the field as a strip at 4.5
metric tons per hectare on the day before lettuce and parsley seedlings were transplanted in late October 2002.
Contaminated irrigation water was applied only once on the plants as a treatment in five plots for each crop at the rate
of 2 liters per plot 3 weeks after the seedlings were transplanted. E. coli O157:H7 persisted for 154 to 217 days in soils
amended with contaminated composts and was detected on lettuce and parsley for up to 77 and 177 days, respectively,
after seedlings were planted. Very little difference was observed in E. coli O157:H7 persistence based on compost type
alone. E. coli O157:H7 persisted longer (by >60 days) in soil covered with parsley plants than in soil from lettuce plots,
which were bare after lettuce was harvested. In all cases, E. coli O157:H7 in soil, regardless of source or crop type,
persisted for >5 months after application of contaminated compost or irrigation water.”
http://ddr.nal.usda.gov/bitstream/10113/26385/1/IND43639771.pdf
In 2004, Sybille Boehme, et al., Robert Koch Institute at Wernigerode, reported on the “Occurrence of antibiotic-
resistant enterobacteria in agricultural foodstuffs.” They said, “Antibiotic-resistant bacteria or their corresponding
resistance determinants are known to spread from animals to humans via the food chain. We screened 20 vegetable
foods for antibiotic-resistant coliform bacteria and enterococci. Isolates were directly selected on antibiotic-containing
selective agar (color detection). Thirteen “common vegetables” (tomato, mushrooms, salad) possessed 104–107 cfu/g
vegetable of coliform bacteria including only few antibiotic-resistant variants (0–105 cfu/g). All seven sprout samples
showed a some orders of magnitude higher contamination with coliform bacteria (107–109 cfu/g) including a remarkable
amount of resistant isolates (up to 107 cfu/g). Multiple resistances (up to 9) in single isolates were more common in
sprout isolates. Resistant bacteria did not originate from sprout seeds. The most common genera among 92 isolates
were: 25 Enterobacter spp. (19 E. cloacae), 22 Citrobacter spp. (8 C. freundii), and 21 Klebsiella spp. (9 K.
pneumoniae). Most common resistance phenotypes were: tetracycline (43%), streptomycin (37%), kanamycin (26%),
chloramphenicol (29%), co-trimoxazol (9%), and gentamicin (4%). The four gentamicin-resistant isolates were
investigated in molecular details. Only three (chloramphenicol) resistant, typical plant-associated enterococci were
isolated from overnight enrichment cultures. In conclusion, a contribution of sprouts contaminated with multiresistant,
Gram-negative enterobacteria to a common gene pool among human commensal and pathogenic bacteria cannot be
excluded.” http://www.sproutnet.com/Research/occurrence_of_antibiotic.pdf
In 2006, Łucja Łaniewska-Trokenheim, et al, University of Warmia and Mazury in Olsztyn, reported on “ANTIBIOTIC
RESISTANCE OF BACTERIA OF THE FAMILY ENTEROBACTERIACEA ISOLATED FROM VEGETABLES – SHORT
REPORT.” They said, “The strains isolated were resistant to 8 out of 12 antibiotics used in the study. The highest
number of the strains demonstrated resistance to ampicilin, including: Hafnia sp., Citrobacter freundii, Enterobacter
aerogenes. – The experiment resulted in the isolation of 114 strains of Gram-negative bacteria of the family
Enterobacteriaceae. After the identification, the strains classified to the following genera: Citrobacter freundii – 21
strains, Enterobacter aerogenes – 41 strains, Erwinia carotovora – 5 strains, Escherichia coli – 5 strains, Hafnia sp. – 30
strains, Klebsiella sp. – 1 strain, Proteus vulgaris – 9 strains, Providencia sp. – 2 strains, and Serratia marcescens – 1
strain (Table 1). The following genera of bacteria were isolated from vegetables with the highest frequency:
Enterobacter aerogenes – 41 strains and Hafnia sp. – 30 strains as well as Citrobacter freundii – 21 strains. Due to
sporadic occurrence on vegetables, lower numbers of genera were determined for: Proteus vulgaris – 9 strains, Erwinia
sp. – 5 strains, Escherichia coli – 5 strains, Providencia sp. – 2 strains, as well as Klebsiella sp. and Serratia
marcescens – 1 strain each (Table 1). In the case of radish samples, 24 strains were isolated and classified to the
following genera: Hafnia sp. – 9 strains, Citrobacter freundii – 6 strains, Proteus vulgaris – 4 strains, Enterobacter
aerogenes – 3 strains, and Erwinia carotovora – 2 strains. From head lettuce samples, 22 strains were isolated and
classified to the following genera: Enterobacter aerogenes – 3 strains, Citrobacter freundii – 6 strains, Hafnia sp. – 2
strains, and Escherichia coli – 1 strain. In the case of iceberg lettuce, 16 strains were isolated that were predominated
by Enterobacter aerogenes – 12 strains. Next, 15 strains were isolated from chives, 13 strains form parsley root, 9
strains from wild celery, 8 strains from carrot, 6 strains from cabbage, and 2 strains from dill, including one strain of
Enterobacter aerogenes and another one of Hafnia sp.”
journal.pan.olsztyn.pl/fd.php?f=81
In 2007, T. J. Welch, et al., reported on the “Multiple Antimicrobial Resistance in Plague: An Emerging Public Health
Risk.” They said, “Antimicrobial resistance in Yersinia pestis is rare, yet constitutes a significant international public
health and biodefense threat. In 1995, the first multidrug resistant (MDR) isolate of Y. pestis (strain IP275) was
identified, and was shown to contain a self-transmissible plasmid (pIP1202) that conferred resistance to many of the
antimicrobials recommended for plague treatment and prophylaxis. Comparative analysis of the DNA sequence of Y.
pestis plasmid pIP1202 revealed a near identical IncA/C plasmid backbone that is shared by MDR plasmids isolated from
Salmonella enterica serotype Newport SL254 and the fish pathogen Yersinia ruckeri YR71. The high degree of
sequence identity and gene synteny between the plasmid backbones suggests recent acquisition of these plasmids from
a common ancestor. In addition, the Y. pestis pIP1202-like plasmid backbone was detected in numerous MDR
enterobacterial pathogens isolated from retail meat samples collected between 2002 and 2005 in the United States.
Plasmid-positive strains were isolated from beef, chicken, turkey and pork, and were found in samples from the following
states: California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New Mexico, New York and Oregon. Our
studies reveal that this common plasmid backbone is broadly disseminated among MDR zoonotic pathogens associated
with agriculture. This reservoir of mobile resistance determinants has the potential to disseminate to Y. pestis and other
human and zoonotic bacterial pathogens and therefore represents a significant public health concern. – As plasmid
pSN254 confers the Newport MDR-AmpC resistance phenotype, we sought to determine the occurrence and distribution
of the IncA/C plasmid backbone among 125 MDR Salmonella strains recovered from retail meats from 2002 to 2005
through the National Antimicrobial Resistance Monitoring System (NARMS see cite above), as well as a small collection
of E. coli strains recovered from food animals and Klebsiella isolates from ground turkey meat from Iowa. Using the repA
gene as marker, primary screening by PCR for the IncA/C replicon [13] revealed 70 repA-positive samples that were
subsequently probed with a panel of twelve PCR assays targeting backbone regions (shown in Fig. 1). These findings
indicated that IncA/C plasmid backbones were present in all repA-positive strains, in all meat types sampled, including
chicken, turkey, pork and ground beef and in all ten states that participate in the NARMS retail surveillance (Table 2).
Several IncA/C positive strains were found in California, New Mexico and Oregon, areas of the United States where Y.
pestis is endemic. IncA/C backbones were detected in representatives of S. enterica Newport, Heidelberg, Kentucky,
Dublin, Bredeney, and Typhimurium. In addition, screening of E. coli and Klebsiella MDR isolates revealed the presence
of this plasmid backbone in the nine tested Klebsiella isolates recovered from ground turkey collected in Iowa, and E.
coli isolated from a calf in North Dakota and a broiler chicken from Georgia (Table 2). A majority of S. Newport and S.
Heidelberg strains (9/14) and several Klebsiella isolates (3/9) were positive for all twelve markers, while other Salmonella
serotypes were positive for 50% or greater of the assayed loci (Table 2). ” http://www.plosone.org/article/info:doi%2F10.
1371%2Fjournal.pone.0000309
In 2010, M.P. Falomir, D. Gozalbo and H. Rico, Universitat de València, reported on “Coliform bacteria in fresh
vegetables: from cultivated lands to consumers.” They said, “Coliforms were isolated in 50% out of the 60 samples
analyzed, although only one isolate was identified as Escherichia coli (Table 1). The identified species (n: 45) included
enterobacteria: Klebsiella pneumoniae (n: 5), Klebsiella oxytoca (n: 10), Serratia marcescens (n: 1), Serratia rubidaea
(n: 1), Enterobacter cloacae (n: 20), Kluyvera ascorbata (n: 2), and Pantoea agglomerans (n: 3), as well as other
bacterial species: Acinetobacter baumannii (n: 1) and Stenotrophomonas maltophilia – Fresh vegetables normally carry
natural non-pathogenic epiphytic microorganisms, but during growth, harvest, transportation and further handling the
produce can be contaminated with pathogens from animal and human sources. As most of these produce are eaten]\
without further processing, their microbial content may represent a risk factor for the consumer’s health and therefore a
food safety problem. The consumption of fresh vegetables has been increasing in recent years, and since the early
1990s the reported outbreaks associated with consumption of fresh vegetables have grown steadily. Most of the
reported outbreaks of gastrointestinal disease linked to the fresh produce have been associated with
bacterial contamination, particularly with members of the Enterobacteriaceae family. In addition, the presence of
antibiotic resistances both in epiphytic and pathogenic microorganisms in fresh vegetables may contribute to horizontal
spreading of resistances among bacterial populations. In this study we have determined the presence of coliform
bacteria as well as their antibiotic susceptibilities in fresh vegetables, as an indicator of their microbiological quality and
their potential as a risk factor for consumer’s health. Samples of several fresh vegetables (n: 116) (i) collected directly
from cultivated lands, (ii) from supermarkets and greengrocer’s shops in Valencia city (Spain) (including samples of
ready-to eat four range vegetables), (iii) as well as ready-to-eat salads (n: 16) served in dinning halls of a nursery and a
primary school (including fresh vegetables used as ingredients to prepare the salads) were analyzed. Coliforms and
other enterobacterial species were isolated in a significant proportion of individual vegetable samples (average >50%),
whereas this proportion increased in ready-to-eat salads (100%); the identified isolates included mainly species
belonging to Klebsiella, Enterobacter, and other genera (Serratia, Citrobacter, Kluyvera, Pantoea, Flavimonas, Hafnia,
and others), as well as four identified as Escherichia coli. Susceptibility of isolates to eleven common chemotherapeutic
agents was tested. Most isolates were resistant to ampicillin, and to amoxicillin/clavulanic acid; although resistances to
other chemotherapeutic agents were rare, some isolates showed multiresistance to 3-5 agents. Therefore, microbial
contamination of fresh vegetables with opportunistic pathogens can be considered as a food safety concern, as
consumption of these produce may represent a potential risk for the consumer’s health, particularly in debilitated or
immunocompromised individuals. Since bacteria serving as a reservoir for resistance determinants may have great
influence on resistance gene transfer in natural habitats, such as the human colon, the presence of antibiotic-resistant
bacteria in fresh vegetables may constitute an additional food safety concern.” http://www.formatex.
info/microbiology2/1175-1181.pdf
In 2011, S.A. Hassan, et al., Taif University, reported on “Bacterial Load of Fresh Vegetables and Their Resistance to
the Currently Used Antibiotics in Saudi Arabia.” They said, “This study was carried out to describe the bacterial load and
the occurrence of some disease-causing enteric bacteria on raw vegetables sold in Saudi markets. The study further
aimed to analyze antibiotic resistance rates, production of extended-spectrum beta lactamase, and plasmid carriage
among bacterial population of raw vegetables. Results revealed that none of them contained Bacillus cereus,
Salmonella, and Escherichia coli O157:H7. However, Staphylococcus aureus and Shigella were detected in 11.8% and
4.4% of the samples, respectively. The bacterial loads ranged from 3 to 8 log(10) CFUg(-1) for aerobic bacteria and 1 to
4 log(10) CFUg(-1) for coliforms as well as Enterobacteriaceae. The isolates exhibited resistance in decreasing order for
ampicillin (76.5%), cephalothin (69.5%), trimethoprime-sulfamethoxazole (36.7%), aminoglycosides (21.9%), tetracycline
(17.2%), fluoroquinolones (17.2%), amoxycillin-clavulanic acid (13.3%), and chloramphenicol (7.8%). Maximum
resistance to extended-spectrum beta-lactam antibiotics occurred in 14.8% of isolates and the production of extended-
spectrum beta-lactamase was achieved by 2.3% of isolates. Multiple resistances to four or more antimicrobial agents
along with plasmid with varied sizes were documented. These investigations indicate the occurrence of antibiotic
resistance and plasmid carriage among bacterial isolates populating raw vegetables.” http://www.ncbi.nlm.nih.
gov/pubmed/21612423
In 2011, Amanda J. Deering, et al., Purdue University at West Lafayette, reported on the “Identification of the Cellular
Location of Internalized Escherichia coli O157:H7 in Mung Bean, Vigna radiata, by Immunocytochemical Techniques.”
They said, “Escherichia coli O157:H7 has been associated with numerous outbreaks involving fresh produce. Previous
studies have shown that bacteria can be internalized within plant tissue and that this can be a source of protection from
antimicrobial chemicals and environmental conditions. However, the types of tissue and cellular locations the bacteria
occupy in the plant following internalization have not been addressed. In this study, immunocytochemical techniques
were used to localize internalized E. coli O157:H7 expressing green fluorescent protein in germinated mung bean (Vigna
radiata) hypocotyl tissue following contamination of intact seeds. An average of 13 bacteria per mm3 were localized
within the sampled tissue. The bacteria were found to be associated with every major tissue and corresponding cell type
(cortex, phloem, xylem, epidermis and pith). The cortical cells located on the outside of the vascular bundles contained
the majority of the internalized bacteria (61%). In addition, the bacteria were localized primarily to the spaces between
the cells (apoplast) and not within the cells. Growth experiments were also performed and demonstrated that mung bean
plants could support the replication of bacteria to high levels (107 CFU per plant) following seed contamination and that
these levels could be sustained over a 12-day period. Therefore, E. coli O157:H7 can be internalized in many different
plant tissue types after a brief seed contamination event, and the bacteria are able to grow and persist within the plant.”
http://www.ncbi.nlm.nih.gov/pubmed?term=10.4315%2F0362-028X.JFP-11-015[aid]&cmd=DetailsSearch
Also in 2011, Amanda J. Deering, et al., Purdue University at West Lafayette, reported on the “Examination of the
Internalization of Salmonella serovar Typhimurium in Peanuts, Arachis hypogaea, Using Immunocytochemical
Techniques.” They said, “A variety of products of plant origin, such as tomatoes, melons, peppers and peanuts, have
been implicated in Salmonella spp. associated outbreaks in recent years. Although these bacteria have been found to
internalize within some plants associated with foodborne-related outbreaks, the internalization in peanut plants has not
been examined to date. To investigate internalization and where the bacteria localize within the plant, intact peanut
seeds were contaminated with Salmonella serovar Typhimurium expressing green fluorescent protein (GFP) for 30 min.
and immunocytochemical techniques were used to localize the bacterium within the stem tissue of 16-day-old peanut
plants. An average of 13.6 bacteria/mm3 were localized within the sampled tissue. The bacteria were found to be
associated with every major tissue (cortical, vascular, epidermal and pith) and corresponding cell type. The cortical cells
located to the outside of the vascular bundles contained the majority of the Salmonella cells (72.4%). Additional growth
experiments demonstrated peanut seedlings could support the reproduction of Salmonella to high levels (109
CFU/plant) after 2 days following seed contamination. Together, these results show that Salmonella Typhimurium can
internalize within many different plant tissue types after a brief seed contamination event and that the bacteria are able
to grow and persist within the plant.”
http://www.sciencedirect.com/science/article/pii/S0963996911000846
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