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Advances in Environmental Biology, 7(11) Oct 2013, Pages: 3549-3554
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Beta lactamase Producer Staphylococcus species in Iranian Foods
Shila Jalalpour
Molecular Medicine Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.
ARTICLE INFO
Article history:
Received 23August 2013
Received in revised form 24
September 2013
Accepted 29 September 2013
Available online 17 November 2013
Key words:
Staphylococcus aureus, Coagulase
Negative Staphylococci, β-lactamase,
Foods
ABSTRACT
Staphylococcal food poisoning occurs most often in foods that require hand
preparation, such as potato salad, ham salad, sandwich spreads, raw foods;
unpasteurized milk. S. aureus found on humans (skin, infected cuts, pimples, noses
and throats).When S. aureus is allowed to grow in foods, can produce a toxin that
causes illness. Although cooking destroys the bacteria, the toxin produced is heat
stable and may not be destroyed. Symptoms of Staphylococcus aureus food borne
illnesses are severe nausea, fever, abdominal cramps, vomiting and diarrhea. BetaLactamase confers penicillin resistance to various bacterial organisms by cleaving
the beta-lactam ring of penicillin's and cephalosporin antibiotics. The subject of this
study was survey of the prevalence of Beta lactamase enzyme in Staphylococcus
species isolated of foods in Isfahan city in Iran. This laboratory study was
performed during 2009-2010 years in Isfahan. In this study, 150 food samples
including, ketchup, mayonnaise, biscuits and chocolate were randomly sampled and
cultured on Blood Agar; and incubated for 24 hours. Identification of bacteria were
based on microbiological methods (staining, biochemical test). Identification of βlactamase, was performed with the Acidometric method with Penicillin G5000000
Unit and Phenol Red. A positive test was indicated by the mixture turning yellow in
color. From 150 samples collected, 142(95%) samples were contaminated with
Staphylococcus species. According to the acidometric result, the frequency of βlactamase in the Staphylococcus species was 112 (75%). Antibiotic resistance may
be intrinsic or result from either mutation of existing genetic material or from
acquisition of new genetic material. The results of this study represented vast spread
of beta lactamase in Staphylococcus species in food. Beta lactamase enzyme leads
to bacterial resistance against beta Lactame antibiotics.
© 2013 AENSI Publisher All rights reserved.
INTRODUCTION
Food borne illnesses are caused by eating food or drinking beverages contaminated with bacteria, parasites,
or viruses. Harmful chemicals can also cause food borne illnesses if they have contaminated food during
harvesting or processing. Food borne illnesses can cause symptoms that range from an upset stomach to more
serious symptoms, including diarrhea, fever, vomiting, abdominal cramps, and dehydration [15]. Most food
borne infections are undiagnosed and unreported, though the Centers for Disease Control and Prevention (CDC)
estimates that every year about 76 million people in the United States become ill from pathogens, or diseasecausing substances, in food. Of these people, about 5,000 die [3,17,26,27]. Harmful bacteria are the most
common cause of food borne illnesses. Some bacteria may be present on purchased foods. Such as raw foods are
the most common source of food borne illnesses because they are not sterile; examples include raw meat and
poultry that may have become contaminated during slaughter. Seafood may become contaminated during
harvest or through processing. One in 10,000 eggs may be contaminated with Salmonella inside the egg shell.
Products such as spinach, lettuce, tomatoes, sprouts, and melons can become contaminated with Salmonella,
Shigella, or Escherichia coli (E. coli) O157:H7. Contamination can occur during growing, harvesting,
processing, storing, shipping, or final preparation. Sources of products contamination are varied as these foods
are grown in soil and can become contaminated during growth or through processing and distribution.
Contamination may also occur during food preparation in a restaurant or a home kitchen [3,17,26,27]. When
food is cooked and left out for more than 2 hours at room temperature, bacteria can multiply quickly. Most
bacteria grow undetected because they don’t produce a bad odor or change the color or texture of the food.
Freezing food slows or stops bacteria’s growth but does not destroy the bacteria. The microbes can become
reactivated when the food is thawed. Refrigeration also can slow the growth of some bacteria. Thorough
cooking is needed to destroy the bacteria [4,18,22,23]. Staphylococcus aureus food poisoning is an illness that
Corresponding Author: Shila Jalalpour,Molecular Medicine Research Center, Rafsanjan University of Medical Sciences,
Rafsanjan, Iran.
E-mail:[email protected]
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Shila Jalalpour
Advances in Environmental Biology, 7(11) Oct 2013, Pages: 3549-3554
results from eating food contaminated with a toxin produced by this bacteria [17]. It’s often caused when a food
handler contaminates food products that are served or stored at room- or refrigerator temperature. Common
examples of such foods are desserts (especially custards and cream-filled or topped desserts), salads (especially
those containing mayonnaise, such as tuna salad, potato salad, and macaroni salad), poultry and other egg
products, and casseroles [1,2,22,29]. Staphylococcal food poisoning is caused by eating food contaminated with
Staphylococcus aureus. S. aureus is able to grow in a wide range of temperatures, pH (4.2 to 9.3) and sodium
chloride (salt) concentrations. These characteristics enable S. aureus to grow in a wide variety of foods and
conditions. Often this type of food poisoning occurs when cooked food is allowed to cool slowly and/or sit at
room temperature for some time. The warm food allows the S. aureus bacteria to grow. These bacteria produce
a toxin enterotoxin that remains in the food even when reheated. The symptoms are caused by the toxin not by
the bacteria themselves, hence staphylococcal food poisoning is sometimes called food intoxication'
[19,20,24,25,30]. Staphylococci are ubiquitous. These bacteria are present in air, dust, dirt, sewage, water, milk,
and food, or on food equipment, environmental surfaces, humans, and animals. Humans and animals are the
primary reservoirs. Staphylococci are present in the nasal passages and throats and on the hair and skin of 50
percent or more of healthy individuals. The colonization rate is even higher for those who work with or who
come in contact with sick individuals and hospital environments [1,2,21, 29,25]. The bacteria produce a toxin in
the food, which causes most of the symptoms. Risk factors include: Eating food that was prepared by a person
with a skin infection (these infections commonly contain Staphylococcus aureus bacteria), Eating food kept at
room temperature, Eating improperly prepared food, Eating the same food as someone who has symptoms,
Symptoms usually appear within 1- 6 hours after eating contaminated food. Usually, symptoms last only 2 days
or less, They may include: Nausea, Vomiting for up to 24 hours, Diarrhea, Loss of appetite, Severe abdominal
cramps, Abdominal distention, Mild fever [19,20,24,25,30].
The goal of treatment is to replace fluids and electrolytes (salt and minerals) lost by vomiting or diarrhea.
Antidiarrheal medications may be used, but are often not needed. Full recovery is expected. Recovery usually
occurs in 24 to 48 hours [24,25,30].
To avoid dehydration, you or your child should drink water and electrolyte solutions to replace fluids lost
by vomiting. A variety of pleasant-tasting electrolyte solutions are available over-the-counter. Solutions to try
for children: Pedialyte and Infalyte, Popsicles or Jello [19,20,24,25,30].
People with diarrhea who are unable to take fluids by mouth because of nausea or vomiting may need
intravenous fluids. This is true especially for small children, People taking diuretics (water pills) may need to
stop taking them during the acute episode. Ask your health care provider for instructions [19,20,24,25,30].
The antibiotic action is an environmental pressure; those bacteria which have a mutation allowing them to
survive will live on to reproduce. They will then pass this trait to their offspring, which will be a fully resistant
generation. Antibiotic resistance is the ability of a micro-organism to withstand the effects of an antibiotic. It is
a specific type of drug resistance [16]. Antibiotic resistance evolves naturally via natural selection through
random mutation, but it could also be engineered. SOS response of low-fidelity polymerases can also cause
mutation via a process known as programmed evolution. Once such a gene is generated, bacteria can then
transfer the genetic information in a horizontal fashion (between individuals) by plasmid exchange. If a
bacterium carries several resistance genes, it is called multiresistant or, informally, a superbug[5].
The beta lactam antibiotics enters the bacterium via the porin and binds to the penicillin-binding proteins
(PBP's),and inhibiting cell wall synthesis. There are several ways that the bacterium can side-step this attack,
this mechanisms are as follows:
1. Diminished transport into the bacterium; The bacterium on the left has lost the porin that normally
transports the antibiotic into the cell. In a normal environment, such a loss would be a disaster for the bacterium
as it would lose its competitive edge over other bacteria. 2. An altered binding site; This bacterium has a slightly
altered PBP [5,6].The PBP can still carry out its function, but is no longer inhibited by the penicillin, which
cannot bind to it. The bacterium carries on unscathed. 3. Enzymes that break down the antibiotic; This is an
important method of resistance for many bacteria. Beta lactams are for example broken down by beta
lactamases. There is a whole host of beta lactamases, and new ones seem to be discovered every day. Although
more attention has been focussed on beta lactamases than on other enzymes that destroy antibiotics, there are
many such enzymes that break down aminoglycosides, chloramphenicol, and so on [5,6]. Beta-lactamases are
enzymes (EC 3.5.2.6) produced by some bacteria and are responsible for their resistance to beta-lactam
antibiotics like penicillins, cephalosporins (are relatively resistant to beta-lactamase), cephamycins, and
carbapenems (ertapenem). These antibiotics have a common element in their molecular structure: a four-atom
ring known as a beta-lactam. The lactamase enzyme breaks that ring open, deactivating the molecule's
antibacterial properties [5-7]. Beta-lactam antibiotics are typically used to treat a broad spectrum of grampositive and gram-negative bacteria. Beta-lactamases produced by gram-positive organisms are usually secreted
[5-7].
Penicillinase is a specific type of β-lactamase, showing specificity for penicillins, again by hydrolysing the
beta-lactam ring. Molecular weights of the various penicillinases tend to cluster near 50kD (12-14).
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Penicillinase was the first β-lactamase to be identified: it was first isolated by Abraham and Chain in 1940 from
gram-negative E. coli even before penicillin entered clinical use but penicillinase production quickly spread to
bacteria that previously did not produce it or only produced it rarely. Penicillinase-resistant beta-lactams such as
methicillin were developed, but there is now widespread resistance to even these [5-7].
The subject of this study was survey prevalence Beta lactamase enzyme in Staphylococcus species isolated
of foods In Isfahan city in Iran.
MATERIALS AND METHODS
Sampling:
This laboratory study was performed during 2009-2010 years in Islamic Azad University Shahreza branch
in Isfahan. In this study, a total of 150 food samples including, ketchup, mayonnaise, biscuits and chocolate
were randomly selected.
Bacterial strains:
The Bacteria were identified on the basis of colony morphology, Gram stain reaction, spore formation, and
biochemical tests with the BioMerieux database system.
The specimens grew on sheep blood incubated at 37°C under aerobic conditions (5,28).
A Gram stain was first performed to guide the way, which showed a typical gram-positive bacteria, cocci,
in clusters. The isolates were cultured on mannitol salt agar, which was a selective medium with 7–9% NaCl
that allows S. aureus to grow, producing yellow-colored colonies as a result of mannitol fermentation and
subsequent drop in the medium's pH. Furthermore, for differentiation on the species level, catalase (positive for
all Staphylococcus species), coagulase (fibrin clot formation, positive for S. aureus), DNAse (zone of clearance
on nutrient agar), lipase (a yellow color and rancid odor smell), and phosphatase (a pink color) tests were all
done. For staphylococcal food poisoning, phage typing were performed to determine the staphylococci
recovered from the food [5-7, 28].
Detection Beta Lactamase:
Acidometric test is a Diagnostic test for the rapid detection of the β-lactamase in bacteria.
This test is based on hydrolysis of the β-lactam ring, which results in the production of penicilloic acid.
This process caused acidification of the bacterial suspension, and changed the colour of the acidobasic indicator
phenol red. The result of the reaction is very fast. The red color of this indicator represent negative test and the
yellow color of this indicator represent positive test (Figure 1) [8-11].
Fig. 1: Beta Lactamase Test
Results:
In present study that was performed with laboratory method during of 9 month in 2009-2010 years in
Isfahan A total 150 food samples including, ketchup, mayonnaise, biscuits and chocolate, randomly selected and
were study. Specimen transfer to microbiology laboratory and in sterile condition, cultured on sheep blood
incubated at 37 °C under aerobic conditions. According results from 150 food samples, all of ketchup,
mayonnaise, biscuits, chocolate samples (100%) was contaminated with Staphylococcus species.
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Advances in Environmental Biology, 7(11) Oct 2013, Pages: 3549-3554
According results of this study from 150 sample,142 (95%) sample was contaminated with Staphylococcus
species. According to Acidometric result frequency β-lactamase in the Staphylococcus species was 112 (75%).
77% strain of Staphylococcus aureus and 73% coagulase negative Staphylococcus produce β-lactamase.(Diag 1)
Respectively 85%, 80%, 70, 70% and 70% of Staphylococcus species isolated from ketchup, mayonnaise,
biscuits, chocolate samples, can produce β-lactamase.(Diag 2)
Diagram. 1:Beta lactamase in Coagulase negative and positive Staphylococcus isolated from foods
Diagram. 2: Beta lactamase in Staphylococcus spp. isolated from foods
Discussion:
More than 90 percent of the cases of food poisoning each year are caused by Staphylococcus aureus,
Salmonella, Clostridium perfringens, Campylobacter, Listeria monocytogenes, Vibrio parahaemolyticus,
Bacillus cereus, and Entero-pathogenic Escherichia coli. These bacteria are commonly found on many raw
foods. Normally a large number of food-poisoning bacteria must be present to cause illness. Food borne illness
can be prevented by controlling the initial number of bacteria present, preventing the small number from
growing ,destroying the bacteria by proper cooking and avoiding re-contamination. The onset of symptoms in
staphylococcal food poisoning is usually rapid and in many cases severe, depending on individual susceptibility
to the toxin, the amount of contaminated food eaten, the amount of toxin in the food ingested, and the general
health of the victim [4,23]. It is important to prevent the contamination of food with Staphylococcus before the
toxin can be produced. S. aureus is often present on skin, under fingernails, in the nose and throat, in cuts,
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abrasions, boils, and abscesses. S. aureus can also be found on contaminated surfaces and food preparation
utensils. The following can help prevent staphylococcal food poisoning:
Washing hands and under fingernails vigorously with soap and water before handling and preparing food;
Not preparing food if a person has a nose or eye infection; Not preparing or serving food for others if a person
has wounds or skin infections on the hands or wrists; Keeping kitchens and food-serving areas clean and
sanitized; Keeping hot foods hot (over 140°F) and cold foods cold (40°F or under); Especially if the food is
stored for more than 2 hours; Storing cooked food in a wide, shallow container and refrigerating as soon as
possible [4,23]
Antibiotic resistance can also be introduced artificially into a micro-organism through transformation
protocols. This can be a useful way of implanting artificial genes into the micro-organism [13]. Antibiotic
resistance is a consequence of evolution via natural selection or programmed evolution. The antibiotic action is
an environmental pressure; those bacteria which have a mutation allowing them to survive will live on to
reproduce. They will then pass this trait to their offspring, which will be a fully resistant generation [13,5].
Several studies have demonstrated that patterns of antibiotic usage greatly affect the number of resistant
organisms which develop. Overuse of broad-spectrum antibiotics, such as second- and third-generation
cephalosporins, greatly hastens the development of methicillin resistance, even in organisms that have never
been exposed to the selective pressure of methicillin per se (thus the resistance was already present). Other
factors contributing towards resistance include incorrect diagnosis, unnecessary prescriptions, improper use of
antibiotics by patients, and the use of antibiotics as livestock food additives for growth promotion (5).
Staphylococcal resistance to penicillin is mediated by penicillinase (a form of β-lactamase) production: an
enzyme that cleaves the β-lactam ring of the penicillin molecule, rendering the antibiotic ineffective.
Penicillinase-resistant β-lactam antibiotics such as methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, and
flucloxacillin are able to resist degradation by staphylococcal penicillinase (5). Spread of S. aureus (including
MRSA) is through human-to-human contact, although recently some veterinarians have discovered that the
infection can be spread through pets, with environmental contamination thought to play a relatively unimportant
part. Emphasis on basic hand washing techniques are, therefore, effective in preventing the transmission of S.
aureus (5).
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