Category: E. Coli

The recent E. coli outbreak in Romaine Lettuce has forced many restaurants, grocery stores, and households to pull the popular produce off the shelves. A recent discovery found that farmers harvesting the lettuce in the Central Coastal regions in northern and central California were responsible for the outbreak. Not only was the United States affected by this outbreak, but Canada was also. This outbreak likely happened due to the common harvesting patterns farmers in California had been used in order to help the lettuce grow. The contamination was discovered just before Thanksgiving as family members were purchasing entrees, sides, and desserts for the American holiday (Fox, 2018). So far, 43 people have been infected with E. coli in the United States, and one person has suffered from kidney failure (“Map of Reported Cases”, 2018).

The public has responded by removing romaine lettuce from menus, stores, and homes. With the supply of romaine lettuce was so low, the price of iceberg lettuce, and other lettuce increased up to 168% in the United States (Haigh, 2018). People still went out and continued to buy iceberg lettuce in order to prepare for holiday festivities. The CDC released an alert for those who have romaine lettuce from California to throw it away, or if they could not locate the origin to dispose of it anyways. Popular fast food chains such as Chick-Fil-A have posted their removal of the lettuce on their social media pages and within their restaurants (Gore, 2018). Local news stations have covered the outbreak which has lead to even more consumers getting rid of romaine lettuce completely and then focus on restocking the vegetable. Businesses and smaller organizations replaced their loss of romaine lettuce with iceberg lettuce, leaving the California farmers having to regain the trust of their once loyal consumers (Scipioni, 2018).

Analysis of Impact on the Organization

In addition to the outbreak concerning romaine lettuce, similar epidemics have occurred with other various food sources. The most recent to date occurred back on September 19, 2018 when Cargill Meat Solutions in Fort Morgan, Colorado issued a recall on its ground beef products (“E.coli”, 2018). The products packaged were shipped nationwide and labeled with and establishment number that would later be used to identify whether it was contaminated or not. According to the CDC, four states reported a total of 18 people who had been infected with E. coli with illness dates starting from July 5, 2018 to July 25 of the same year. Six of the 18 people were hospitalized, one reportedly developed hemolytic uremic syndrome, which is described as a type of kidney failure, and one person located in the state of Florida died (“E.coli”, 2018).

Similarly, in 2015, Mexican-style restaurant chain Chipotle Mexican Grill experienced two outbreaks of E. Coli within a span of a year, one larger epidemic and small outbreak that followed (“Multistate Outbreaks”, 2016). The larger began on December 1, 2015 when reports of E. coli strains became prominent in the Washington and Oregon areas. Interviews conducted led public health officials to believe that the source was a food restaurant all infected persons had recently dined at. As stated by the CDC, the larger epidemic infected a total of 55 people from 11 states, while the second, smaller outbreak infected only five (“Multistate Outbreaks”, 2016). No deaths were reported in either case.

As for key players who have been affected, which we assume are mainly businesses, have had an unexpected boost in sales. For example, USA Today claims that fast-food businesses are unlikely to be affected following the recall of romaine lettuce, stating that businesses will likely just replace the leafy green with an alternative (Meyer, 2018). A recent press statement by FDA Commissioner Scott Gottlieb, called for the public to dispose and destroy romaine lettuce in their homes, and the steps to take to prevent the consumption of the lettuce (Office of the Commissioner, 2018). ABC news puts the spotlight on a farm in St. Pete’s Warehouse Arts District that can guarantee “worry-free” romaine lettuce, stating that their produce is cultivated inside hydroponic shipping containers where the chance of E. coli growing is impossible (Hollenbeck, 2018).

The people who are impacted by this E.coli outbreak are those who consume romaine lettuce regularly. Thanks to a quick social media response, people have pulled the lettuce off of the shelves and disposing of it properly (Lupo, 2018). Zlati Meyer of USA Today even reported that those who ate just a few greens, have now found an excuse to avoid greens altogether (Meyer, 2018). Since the outbreak of romaine lettuce in mid November, the prices of iceberg lettuce have begun to rise. The prices have been reported to have increased in over 100% in the past month since the outbreak first occur (Haigh, 2018). Causing customers to pay way more than usual for their iceberg lettuce. In addition to those who became sick from consuming the contaminated lettuce. Those also impacted include businesses who produce romaine lettuce. As well as commercial businesses such as, restaurants and stores, who sell and use romaine lettuce regularly. Although it is reported that fast-food restaurants won’t be experiencing that much of an impact from the outbreak. Saying that restaurants will still gain business and that people will just avoid menu items involving lettuce (Meyer, 2018).

The recent E.coli outbreak of romaine lettuce began to generate news because of the rapid growth of people becoming ill in the United States from the outbreak. When the outbreak was first identified as a problem, 32 people were reported ill from the bacteria as of November 19, 2018 according to the CDC. Then as of November 26, 2018 the outbreak had increased to 43 reported illnesses (“E.coli”, 2018). Which was reported across 11 states in the country. The FDA also made a statement releasing that Canada has also been affected by the E.coli outbreak. Canadian health officials reported that only 22 people had been reported ill (“Office of the Commissioner”, 2018).

What will most likely happen in the future to prevent from more outbreaks, farmers will have to change the way that they harvest their crops. Causing stricter guidelines from the FDA to ensure healthy crops. They will also have to regain trust from those who consume their crops such as vendors, households, and businesses. Eventually, they will regain their trust back from consumers because people will only continue to buy the more expensive iceberg lettuce for so much longer. People will continue to go out and eat they may just choose to stay away from salads until the outbreak eventually dies down and they forget about it (Meyer, 2018).

PR Tie

As a business, losing the public’s trust is something that can occur within a matter of minutes, but to be able to gain their trust back is something that takes more time and work, and may not even work in the end when all is said and done. With the E.coli outbreak in romaine lettuce, for the people who became sick, and for those who bought the contaminated lettuce from the farms, it is difficult to gain back their trust. For restaurant owners, the relationship between the two is strained because the sales will drop as a result of contamination. From the farmers who who sold the contaminated produce to their customers, need to be open with their patrons and attempt to mend the customer relations that has taken a hit with this outbreak.

If you were to have a bag of M&M’s but you find out that 5 of the candies in the bag are contaminated, but the rest are clean, would you eat the candy or be safe and throw the bag away? Most would throw the bag away, as it is better to be safe than sorry when it comes to taking risks. This is how restaurants have reacted with the E.coli outbreak, as many of them who even have a slim chance of having contaminated lettuce have taken all the product out of their place of business and stopped selling the product till they can be sure that it is safe to sell it again (“Office of the Commissioner”, 2018). These places taking the lettuce away also has to do with the warnings from the FDA and the CDC, who have said that the romaine lettuce they have should be thrown away immediately to take extreme caution, as the outbreak has already affected nearly 50 people. This drastic overhaul of the lettuce is what crisis management looks like, as they are pulling out all the stops in order to ensure that no other people get the bacteria (“E.coli”, 2018).

Group Opinion

The families, and businesses have handled the E. coli outbreak well. With up to date news coverage, they have been aware of what to look for and to dispose of their romaine lettuce. The farmers who were responsible for the outbreak have also had to pull their produce from whom they were supplying it to.

The farmers could have prepared for an event like this with better harvesting patterns. The problems with the end of season harvest from lettuce that was grown in the summer must be fixed (Office of the Commissioner, 2018). They need to remove that aspect of their harvesting process out in order to prevent another potential outbreak of E. coli. Grocery stores could also let those know about the removal of romaine lettuce from their store due to the outbreak.

Some suggestions we have in order to make the key players prepare for the future is to always check the labels on their produce. Also, staying up to date with updates from the CDC and the FDA will be important to reduce consumption of future contaminations. For the grocery stores and other businesses, they have to make sure that the farmers they are purchasing produce from have sanitary and proper harvesting patterns in order to prevent another E.coli outbreak from happening once again, or possibly enforce more strict routine inspections of the facilities where these greens are produced.

Sources

1. Another romaine lettuce recall. This time, it may be from California. (n.d.). Retrieved November 30, 2018, from https://www.nbcnews.com/health/health-news/california-may-be-source-latest-romaine-lettuce-e-coli-outbreak-n939026
2. E.coli (Escherichia coli). (2018, November 26). Retrieved November 30, 2018, from https://www.cdc.gov/ecoli/2018/o157h7-11-18/index.html
3. E.coli (Escherichia coli). (2018, November 26). Retrieved November 30, 2018, from https://www.cdc.gov/ecoli/2018/o157h7-11-18/map.html
4. E.coli (Escherichia coli). (2018, September 20). Retrieved December 5, 2018, from https://www.cdc.gov/ecoli/2018/o26-09-18/index.html
5. Gore, L. (2018, November 21). Romaine lettuce E. coli outbreak: Chick-fil-A, other restaurants pulls salads over E. coli scare. Retrieved November 30, 2018, from https://www.al.com/news/2018/11/romaine-lettuce-e-coli-outbreak-chick-fil-a-other-restaurants-pulls-salads-over-e-coli-scare.html
6. Haigh, M. (2018, November 29). Iceberg lettuce prices soar as much as 168% after E. coli outbreak takes romaine off shelves. Retrieved November 30, 2018, from https://www.cnbc.com/2018/11/29/lettuce-prices-soar-amid-e-coli-outbreak-linked-to-romaine.html
7. Hollenbeck, S. (2018, November 29). Sales boost at St. Pete farm with ‘worry free’ romaine lettuce. Retrieved November 30, 2018, from https://www.abcactionnews.com/news/region-south-pinellas/st-petersburg/sales-boost-at-st-pete-farm-with-worry-free-romaine-lettuce
8. Lupo, L. (2018, November 26). Social Media Spread of Romaine Lettuce Ban Gets the Word Out. Retrieved December 3, 2018, from https://www.qualityassurancemag.com/article/social-media-spread-of-romaine-lettuce-ban-gets-the-word-out-/
9. Meyer, Z. (2018, November 29). Fast-food biz’s bottom line likely unaffected by E. coli romaine lettuce outbreak: Experts. Retrieved November 30, 2018, from https://www.usatoday.com/story/money/2018/11/29/fast-food-biz-likely-unaffected-e-coli-romaine-outbreak-experts/2130565002/
10. Meyer, Z. (2018, November 22). Romaine lettuce: Why it’s hard to keep it safe from E. coli and other bacteria. Retrieved December 4, 2018, from https://www.usatoday.com/story/money/2018/11/22/romaine-e-coli-outbreak-tracking-source-harder-than-you-think/2087905002/
11. Multistate Outbreaks of Shiga toxin-producing Escherichia coli O26 Infections Linked to Chipotle Mexican Grill Restaurants (Final Update). (2016, February 01). Retrieved December 5, 2018, from https://www.cdc.gov/ecoli/2015/o26-11-15/index.html
12. Office of the Commissioner. (2018, November 26). Press Announcements – Statement from FDA Commissioner Scott Gottlieb, M.D., on the current romaine lettuce E. coliO157:H7 outbreak investigation. Retrieved November 30, 2018, from https://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm626716.htm
13. Scipioni, J. (2018, November 30). The real winner from the romaine E.coli outbreak: Iceberg lettuce. Retrieved November 30, 2018, from https://www.foxbusiness.com/features/the-real-winner-from-the-romaine-e-coli-outbreak-iceberg-lettuce

There are two different microbial pathogens we are to discuss namely, Escherichia coli and Listeria monocytogenes that affect pregnant women and the unborn foetus upon eating (or coming into contacting) with infected meat. Listeria monocytogenes is a facultative anaerobic bacteria which causes the infection called Listeriosis. Escherichia coli is a bacteria that is commonly found in the lower intestine of warm blooded organisms, most E.coli strains are harmless, but some can cause serious food poisoning. Shiga toxin-producing E-coli (STEC) is a bacterium that can cause severe foodborne disease, its primary sources are raw or undercooked meat products, raw milk and faecal contamination of vegetables.

Diagnosis and Symptoms

Listeria Monocytogenes

A pathogenic microbial organism that can be found in raw meat, (uncooked vegetables and unpasteurized dairy products). Infection from the pathogen causes a diseases called Listeriosis. In pregnant woman the infection can start from the implantation of the foetus through birth. It can be devastating to the mother, sometimes leading to miscarriage/abortion and can be fatal to the unborn child.

Symptoms of the infected mother can be asymptomatic but the following are common: flu-like illness, fever, headache, backache, sore throat, muscle pains, vomiting, diarrhoea.

Diagnosis of listeria infection in pregnant women may be difficult due to the lack of GI symptoms normally associated with food-borne pathogens and non-specific symptoms of fever. Diagnosis can only be made by culturing the pathogenic micro-organism from blood, amniotic fluid, or spinal fluid. Vaginal or stool cultures are not helpful in diagnosis because some women are only carriers of the pathogen.

Escherichia Coli (E.coli)

E.coli is a bacteria that can be found naturally in GI and urinary tracts because it can be harmless and beneficial. Some strains of the bacteria can be pathogenic leading to serious infections in humans. One common infectious source of the pathogenic E.coli is an uncooked meat. This strain of E.coli may have severe effects such as miscarriage and low birth weight if its source infect a pregnant woman.

Symptoms during pregnancy: nausea, abdominal cramps, diarrhoea, vomiting, fatigue, dehydration, bloody urine, fever.

Stomach and kidney problems can be used as lead to the diagnosis of the pathogenic E.coli in pregnant woman. Continuous screening of the urine culture during pregnancy is the most common way in the diagnosis of the pathogenic strain of E.coli.

Treatment

Sometimes listeria affects people in a minor way, so medication is not required. For more dire cases antibiotics are the most common treatment, ampicillin can be used alone or in conjunction with another antibiotic, often gentamicin. If septicaemia or meningitis occur, the individual will be given intravenous antibiotics and require up to six weeks of care and treatment.

For an antibiotic to be effective against listeria, it must penetrate into the host cell and maintain high intracellular concentration. It must bind to the penicillin-binding protein 3 (PBP3) of listeria which causes cell death.

In pregnant women, the antibiotic must cross the placenta in adequate concentration. Penicillin, ampicillin and amoxillin block several PBPs and do not penetrate intracellularly. High doses are generally used to assure adequate penetration of the umbilical cord and placenta. 6g or more a day is recommended of ampicillin for treatment during pregnancy. This dosage provides adequate intracellular penetration and crosses the placenta in adequate amounts.

Nitrofurantoin and cephalosporins can be used to treat pregnant women who have been infected with E. coli. Nitrofurantoin is a good choice of antibiotic because of its high urinal concentration. Cephalosporins are well tolerated and adequately treat the important organism.

Control of Disease

Listeria is a name of a bacteria found in soil and water and some animals, including poultry and cattle. It can be present in raw milk and foods made from raw milk. Pregnant women have high risk of being affected by this bacteria but it can be prevented by the following:

• Do not drink raw (unpasteurized) milk, and do not eat foods that are made from unpasteurized milk.
• Wash hands, knives, countertops and cutting boards after handling and preparing uncooked food.
• Rinse raw produce thoroughly under running tap water before eating.
• Keep uncooked meats, poultry and sea food separate from vegetables, fruits, and cooked food and ready to eat food.
• Thoroughly cook raw food from animal sources such as meat, poultry or sea food to a safe internal temperature.
• Persons in higher risk groups should heat hot dogs, cold nuts and deli meat before eating them.

The prevention of E.coli infection requires control measures at all stages of food chain, from agricultural production on the farm to processing, manufacturing and preparation of foods in both commercial establishments and household kitchens.

Industry

The number of cases of disease might be reduced by various mitigation strategies for ground beef. Good hygienic slaughtering practices reduce contamination of carcasses by faeces, but do not guarantee the absence of STEC from products.

Education in hygienic handling of food for workers at farms, abattoirs and those involved in food production is essential to keep microbial contamination to a minimum. The only effective method of eliminating STEC from foods is to introduce bactericidal treatment such as heating and irradiation.

Household

Preventive measures for E. coli infection are similar to those recommended for other foodborne diseases, and also protect against foodborne diseases caused by STEC.

Conclusion

Most of severe diseases are caused by microorganisms, contamination can happen from slaughtering, handling, packaging, storage and transportation of food. Safety measures need to be taken in each and every step mentioned. Pregnant women and low immune system are the people that are at higher risk of these microorganisms. In the process of slaughtering, microbial contamination of carcass surfaces is unavoidable, while most of the micro floras transferred to the carcasses are non-pathogenic, there is possibility that pathogens such as E.coli and Listeria monocytogenes may be present and it presents one of the most critical safety challenges for the meat industry.

Escherichia coli is a gram-negative facultative bacteria belongs to family Enterobacteriaceae. E. coli is a cunning species encompassing the common vegetation in the intestinal tracts of the warm-blooded creatures (Afset et al., 2003). The main territory of E. coli has long been believed as the vertebrate gut since first termed as Bacterium coli community by a German pediatrician, Dr. Theodor Escherich, which he secluded from the waste material of a newborn patient (Escherich, 1885). E. coli is equally well-known as a pathogenic and non-pathogenic and a multipurpose pathogen of hominids, which is probably known to purpose more than 2 million expiries per annum by both gastric and extra-intestinal contagions (Nataro and Kaper, 1998). Well, human beings normally carry additional than a billion commensal E. coli prison cells in their intestine. In the surroundings outer the body, E. coli is usually originating in fecally polluted zones (Savageau, 1983). Conversely, approximately are non-pathogenic E. coli strains that are supposed to be mostly eco-friendly, as well as not of enteric origin (Ashbolt et al., 1997; Luo et al., 2011). E. coli is a genetically multipurpose species. Strains within single pathos group can initiate from genetically diverse backgrounds (Rasko et al., 2008; Ogura et al., 2009; Sahl et al., 2011; Cooper et al., 2014).

In accumulation, ever since terrestrial use is a monotonous process for the dumping of both animal (manure) and hominid remaining of waste origin (direct slurry), the existence of infectious E. coli has also been defined as unexpected longstanding existence in these substrates. E. coli plays a vital role as a pollution indicator however such as it is constantly existing in comparatively large extents each and every time waste material is present. E. coli is consequently well-thought-out a beneficial substitute of infectious E. coli (Brüssow, 2007). Conversely, as utmost infectious E. coli are lactase -ve, they are not noticed in ordinary water quality media recycled to compute E. coli. Human pathogenic strains typically take possession of other subconscious species asymptomatically. Based on their capability to be reason of the disease in souls, E. coli strains are segmented into following classifications: i) non-pathogenic E. coli, ii) intestinal pathogenic E. coli (IPEC) and iii) extra-intestinal pathogenic E. coli (ExPEC). ExPEC strains are consist of two pathovars: uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC) (Kaper et al., 2004; Smith et al., 2007). Conversely, afterward, ExPEC are less or not connected to aquatic infections. Aquatic diffusion includes the gastral pathogens which are also notorious as diarrheagenic E. coli. Based on their virulence elements, phenotype and pathology diarrheagenic, E. coli strains (Kaper et al., 2004). Commensal strains of E. coli are well known by the presence of virulence factors and also the presence of normal microflora. Pathogenic E. coli can be categorized into path0types by their virulence elements, composed with the category of disease. The six pathotypes able of generating gastric disease in souls are enter pathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), concisely devotee E. coli and enterohemorrhagic E. coli (EHEC). Some authors consider verotoxigenic E. coli (VTEC) to be the sixth pathotype, and EHEC to be a subset of VTEC (Beutin and Martin, 2012).

Nontoxic as well as nourishing diet is the significant to manage and sponsor for the well-being. Moreover, this by the modification in hominid diet routines, the way of life foodborne illnesses is screening a rising development, resultant in extraordinary illness and death through the sphere. It has been stated that the diarrheal sicknesses give rise to more than 50% of worldwide foodborne illness liability (Kirk et al., 2017). Amongst numerous foodborne pathogens, EHEC is a significant evolving pathogen accountable for digestive infections in offspring and aged individuals. The very low contagious extent of EHEC fluctuating from tens to hundreds association establishing components creates them vastly infectious and proliferations the danger of contagion. In human beings, EHEC reason of the contagions fluctuating from minor diarrhea to deadly obstacles, viz hemorrhagic colitis and hemolytic uremic syndrome (Hiraka et al., 2015; Garbaj et al., 2016). Milk is a vastly nutritive as well as healthy diet, it practices an important constituent of the hominid nourishment. It is well-known that ingesting of raw or contaminated milk assists as a prospective way of attaining EHEC contagion in human beings. Calves actuality basins for EHEC transfer these creatures in their intestine devoid of any obvious medical complaint.

Moreover, the pathogen be able to flourish well on numerous environmental places on farmhouse animals as well as their non-living surroundings, i.e. on fur of bodily, udder, cattle barn, aquatic, feed, manure, and soil (Msolo et al., 2016; Polifroni et al., 2012). Therefore, the danger from EHEC contagion is an endless trial cope by the animal supervisors. The main existence of EHEC amongst those calves that were foraged within grounds be able to credited to the polluted grassland wherever the dung was applied for cultivation persistence within the grounds. In the same way, (Hancock et al.,1997) have establish that the occurrence of EHEC was additional among those calves that were foraged on dung-applied grassland one-third when associated with animal that was retained on feedlot (82%). As extraordinary numeral of EHEC isolates were witnessed in the middle of the family circle who used only water for washing of udder and hands earlier extracting milk than those who used antibacterial solution (Neeta et al., 2014; Tegegne and Tesfaye, 2017). Correspondingly, who create that use of antibacterial agent to wash hand and udder considerably exaggerated the bacterial worth of raw milk.

EHEC strains also having locus of enterocyte effacement (LEE) however by means of twofold vital surplus components, Shiga toxin phage(s), as well as an EHEC mobile genetic elements, coding a hemolysin. Shiga toxin E. coli (STEC) which might or might not carry locus of enterocyte effacement (LEE) is predictable as an addition of EHEC with mutable medical consequences (Amézquita-López et al., 2017). In the pathogenesis of EHEC strains the existent of chromosomally determined locus of enterocyte effacement (LEE) shows a key role. The locus of enterocyte effacement (LEE) transmits genetic factor for the ascribing and destroying phenotype supporting microbial linkage as well as the devastation of hominoid gastric enterocytes (Robins‐Browne et al., 2004; Campellone, 2010; Clarke et al., 2003). Moreover, LEE coded genetic factor, an enormous integer of non-LEE effector genetic factor have been established on prophages and on integrative components in the chromosome of the EHEC (Ogura et al., 2008).

Shigatoxin E. coli strains have the incursion mobile genetic elements are the one pathogenic form as they share the similar way of cell incursion. The pathovars strains can be explained by one or a few virulence genetic factors (Müller et al., 2007; Vidal et al., 2005). But, the pathogenicity of EHEC is further difficult to explain by means of virulence genetic factors for example several genetic factors are associated (Köhler and Dorinda, 2011; Johnson and Russo, 2005). The achievement of virulence genetic factor is supposed to be responsible for an evolutionary corridor to pathogenicity. As a hereditarily varied assemblage, furthermost strains of Escherichia coli are mild commensals of animals and humans (Hartl and Dykhuizen, 1984; Selander et al., 1987). however, others are skilled of affecting either gastric or extra-intestinal sickness (Ørskov and Ørskov, 1992). Demonstration of medical symptomology and pathology seems to be diligently related with the tenure of definite virulence genetic factor arrangements in E. coli (Grauke et al., 2002; Law, 2000). Therefore, molecular approaches directing virulence elements are used to differentiate infectious deviations from commensal, non-pathogenic ubiquitous E. coli strains. Furthermore, the recognition of the plasmid-free in water bodies, as happens with shigatoxin phages, add an additional level of complication in identifying infectious, pathogenic E. coli (Tarr, P. I., Gordon, C. A., & Chandler, W. L. (2005). It is generally assumed, although with limited actual data, that fate and transport of fecal indicator E. coli is symptomatic of the intracellular, pathogenic E. coli’s ecological actions. commensal E. coli results can probably be generalized to be responsible for degrees of inactivation of the pathogenic associates. It is frequently non-pathogenic reserved in host guts. But, in the immunocompromised or gastric flora distressed hosts, or once the E. coli in the host guts turn out to be translocated, normal E. coli strains can cause related illnesses as a pathogenic one (Afset et al., 2003).

Various pathogenic E. coli are zoonotic pathogens, whereas others have beings as the only identified basin. Both assemblages are geologically universal and infections are described anywhere souls exist in and pathogenic E. coli are extent through the fecal-oral route, and communication is usually concluded: eating and drinking of polluted nutriments (wash away with fecally tainted water, or touched with deprived sanitation), consumption tainted water (or via recreational waters) or by person-to-person exchange (Pickering et al., 2012). Both might pollute water through nitrogenous waste from beings and for E. coli also from native creatures and flora and fauna. These pathogens pass in aquatic bodies through numerous habits, including manure run-offs, manure organizations which are not operational appropriately, subconscious manure runoff, and contaminated metropolitan rainstorm water overflow. Boreholes might be further susceptible to such pollution after overflowing, predominantly if the boreholes are narrow, have been tunneled or have been sunken by floodwater for long times. Existence of E. coli O157 (EHEC) and other serotypes carrying stx2 gene (virulence factor) in raw community manure and animal wastewater from several origins has been described (Shelton et al., 2006). In addition, since land application is a predictable process for the removal of both subconscious (manure) and hominid waste of fecal origin (direct deposition), the occurrence of pathogenic E. coli. The difficulty is that the assortment hereditary assembly of these strains, comprising most virulence genes encoded in plasmids that may be present or not, can make it hard to resolve commensal E. coli (Lim et al., 2010).

Molecular diagnosis helpful to diagnose or determine the biomarkers (virulence factors) which are responsible for the infectious diseases. These biomarkers are either toxins or proteins. In this research proposal, we have to explore these biomarkers by examining them in phylogenetic analysis and whole genome analysis for this purpose clustalw MEGAx and vista pipelines are considered. Sequences are retrieved by BLAST also determining the region of similarities and differences. Comparative analysis of pathogenicity factors between EHECs and commensals (non-pathogenic). Can enable us to identify DNA markers exclusively present in EHECs such DNA markers have utility in molecular diagnostic of EHEC strains versus commensal strains. Moreover, the phylogenetic analysis of these pathogenicity factors present in EHECs. Further reveals in EHECs and possible geographical distribution of these isolates. Finally, a full genome comparison between EHECs and commensals can further indicate the genetic difference between these two that can have prospects and DNA-based identification of EHECs.

New Zealand rivers – not so clean after all

Background

Tourism plays a big part in New Zealand’s export being a crucial part of the New Zealand economy. It is important for New Zealand to maintain a “100% pure” image as the Tourism New Zealand advert suggests. This is because countries are likely to purchase NZ exports if they think that it is coming from a ‘100% pure’ country. However, this “100% pure” image that Tourism New Zealand tries to portray might not be correct. It is shocking to find out that “more than 60 percent of monitored rivers in New Zealand are unsafe for swimming”(Stacy Kirk, 2013) and according to Charlie Mitchell, it is not safe to drink from half of the rivers in New Zealand due to E. coli contamination.

E. coli is a type of bacteria that is found in the intestines of humans and animals. E. coli affects many rivers in New Zealand, it is important that E. Coli/100mL of water is monitored in our rivers. When E. coli/100mL exceeds 550/100mL, it is deemed unsafe for swimming and drinking according to lawa (Land Air Water Aotearoa). E. coli can make its way into rivers from runoff water, sewage outflow causing caused by waste disposal in toilets and sinks and feces deposited by grazing cows or sheep. “More than 100,000 of treated waste is poured into the dirty Manawatu river each day” (Stuff, 2008), this is possible due to the lack of Government policies surrounding freshwater management. Back in 2010, Kiran Chug from Stuff said that ‘pollutants and toxic waste are still pouring into rivers’ even though the Government released a public statement two years prior ‘aimed at improving freshwater management’.

E. coli can be measured in two ways, using a microscope to count the units known as CFU (colony forming units) or estimating the units known as MPN (most probable number). The rivers in New Zealand were measured by NIWA and local councils. Some rivers could have been measured using the MPN method or the CFU method. This presents limitation as the rivers in New Zealand might not have been measured with the same method. Unpredictable weather could pose as another limitation. According to Environment Minister Amy Adams, heavy rain and wind “can churn up sediment…releasing pathogens back into the water.”

The response variable in this investigation is the number of E. coli/100mL, the explanatory variable is the year (2003 & 2013), the population is the New Zealand rivers in 2003 and 2013.

Question

I wonder what the difference is between the average number of E. coli/100mL in 2003 and the average number of E. coli/100mL in 2013, from New Zealand rivers in 2003 and 2013

Hypothesis

From the research that I have gathered, I hypothesize that there will be no difference in the average number of E. coli/100mL in New Zealand rivers from 2003 to 2013.

Purpose

The purpose of this investigation is to determine if there was a difference is between the average number of E. coli/100mL in 2003 and the average number of E. coli/100mL in 2013, from New Zealand rivers in 2003 and 2013, it would be important to conduct this investigation as it is important for New Zealand to maintain a ‘100% pure’ image. The results of this investigation could be used by the Government and the Ministry for the Environment as the results could indicate whether stricter freshwater policies need to be imposed.

Body:

• Graph of sample distributions
• Summary statistics
• Year Min Q1 Median Mean Q3 Max Std. Dev. n
• 2003 1 39 200 637.26 490 7500 1254.9 86
• 2013 1 28 110 421.33 375.6 17000 1271 303
• Features of sample distributions

Centre:

The two years above (2003 and 2013) shown on the box and whisker graph appears to be skewed to the right. In this case, since the mean value is significantly different from the median value, it appears that the mean has been affected by the shape. The median will give a more accurate representation of the average number of E. coli in 2003 and 2013. The 8 values past 3800 (3x Standard deviation) would affect the mean and therefore make the average less accurate. This is because the 8 values are extreme outliers

From the sample, the median number of E. coli/100mL in 2003 is 200/100mL whereas the median number of E. coli/00mL in 2013 was 110/100mL. The median number of E. coli in 2003 is 90/100mL more than the median number of E. coli in 2013. This means that in 2003, the rivers in New Zealand had more E. coli on average compared to the rivers in 2013 in the sample. I expected the median number of E. coli/100mL to be less than the median number of E. coli/100mL because of the cleaning initiatives. This makes sense because, in my research, I found out that the Manawatu council put aside $11 million in 2012 to “fix its water pollution issues” (Emma Horsely, 2012). There were also many initiatives like this between 2003 and 2013.

The middle 50% of E. coli/100mL in 2013 falls within the middle 50% of E. coli/100mL. This means that the difference between the medians might not be statistically significant as the boxes 2013 box overlaps with the 2003 box. This means that there may not be a difference between 2003 and 2013 back in the population.

Spread:

The number of E. coli/100mL varied a lot in 2003 and 2013. In 2003, the number of E. coli varied between 1 and 7500. Whereas the number of E. coli in 2013 varied between 1 and 17000. However, 2013 varied more than in 2003. There could have been many factors at play here such as floods in certain areas, river locations (urban, pastoral, and native areas), and rivers with sewage outflows.

Rivers that pass-through Wellington are considered as urban rivers and some rivers that pass-through Christchurch is considered as pastoral. According to Stats NZ “E.coli levels are 22 times higher in urban areas and 9.5 times higher in pastoral rivers compared with rivers in native forest areas”. This means that the number of E.coli could vary due to the river location.

Also, heavy rainfall in regions could also cause sewage overflow and an increase in runoff water which could increase E.coli/100mL.

In the sample, the middle 50% of the number of E. coli/100mL in 2003 was between 39 and 490, with an Inter-Quartile range of 451. On the other hand, the middle 50% of the number of E. coli/100mL in 2013 was between 28 and 375.6, an interquartile range of 347.6. This is surprising because I thought that the overlap between the 2 years would not be that big. This is because, over the years, the ministry of environment has tried to improve the river quality in New Zealand.

Shape:

The 2003 group is skewed to the right, the 2013 group also has the same skew. This means that both groups are asymmetrically distributed. There were a few rivers that exceeded 4000/100mL causing the upper 25% to have more variation than the lower 75% for both groups. However, the upper 25% of the 2013 group had more variation than the upper 25% of the 2003 group. This is because the Whareroa stream in 2013 had 17000 E. coli /100mL. it is also important to note that different methods could have been used to determine the number of E. coli/100mL such as the CFU and the MPN method. The rivers that were measured using the MPN method would have been estimates. This means that back in the population we might not have the same results. I noticed that both groups are unimodal and have modes that are close to 0, suggesting that majority of New Zealand rivers in both 2003 and 2013 were safe for swimming (during the time that the data was collected) as it did not exceed the 550/100mL mark. This is surprising as some articles wrote about 60% of New Zealand rivers not safe for swimming.

Unusual features:

There are 4 extremely large numbers of E. coli/100mL in each group exceeding 3800 E. coli/100mL (3 times the standard deviation). The highest recorded number of E. coli/100mL at 17,000 E. coli/100mL was from the Whareroa stream recorded in January 2013. This is an interesting figure as it was 31 times over the 550/100mL limit. Many factors could have played into the measurement of E. coli in this river. Upon research, the Whareroa stream is close to a farm and the data was collected on one of the hottest months in Wellington history (Jo Moir, 2013). There were also 7 other rivers that had an unusually large number of E. coli/100mL.

The Whareroa stream is located in Wellington and according to Tracks NZ, there is a farm close to the stream. The data was collected on one of the hottest months in Wellington history. This might have caused the livestock to drink from the stream

Graph of bootstrap distribution of the difference

State the Confidence Interval

In this investigation, the purpose was to find out if there was a difference between the average number of E. coli/100mL in 2003 and the average number of E. coli/100mL in 2013, from New Zealand rivers in 2003 and 2013. It would be more appropriate to use the median over the mean to measure the average number of E. coli/100mL as there were a few unusual values.

1,000 bootstrap resamples were taken from the sample and from these resamples, 95% of these confidence intervals will contain the actual difference between the median number of E. coli/100mL in 2003 and 2013 back in the population. The bootstrap interval helps us factor in sampling error

If I was to get another random sample of rivers in New Zealand, I would get different means, medians and etc. The difference of the medians that I got (90/100mL) is not enough to draw a conclusion that back in the population there is a difference between the medians in 2003 and 2013. This is why I need to consider sampling variation to determine whether the difference in medians in my sample was significant.

The confidence interval for the median difference between the 2 years (2003 and 2013) is from -10/100mL and 149.8/100mL. This means that the number of E. coli/100mL for both 2003 and 2013 is likely to be between -10/mL and 149.8/100mL.

Interpretation of the confidence interval of the difference

The width of the confidence interval indicates that the values are pretty much the same, meaning that there is a little variation in the sample. This is because both groups have big samples sizes

From the confidence interval shown above, I cannot make a call that there will be a difference in E. coli/100mL in 2003 and E. coli/100mL in 2013 as there is not enough evidence provided. This is because the confidence interval includes 0. This means that back in the population, the median E. coli/100mL in 2013 could be bigger, smaller, or the same as the median E. coli/100mL in 2003. I cannot make a call because it is probable that the difference between the median of the number of E. coli/100mL in 2003 and 2013 is 0.

Conclusion

The purpose of this investigation was to determine if there was a difference is between the average number of E. coli/100mL in 2003 and the average number of E. coli/100mL in 2013. In my research, I found out that New Zealand wasn’t as clean as the “100% pure” campaign claimed to be. I also found out that a lot of harmful things are pumped into our rivers every day and during 2003-2013 not much was done by the government to impose strict freshwater management. I predicted that there wouldn’t be a difference between the median number of E. coli/100mL in 2003 and 2013 due to the lack of Government intervention.

The results from the bootstrap graph show that there is not enough evidence to conclude there was a difference between the medians of the average number of E. coli/100mL in both groups. This is because the confidence interval contains 0. If I was to get another random sample, I would have different summary statistics, but I might be able to determine if there was a difference is between the average number of E. coli/100mL in 2003 and the average number of E. coli/100mL in 2013.

To expand this research, it would be interesting to investigate the average number of E. coli/100mL in other countries and if there has been an improvement from 2003 to 2013. The New Zealand Government has not been placing enough importance on its rivers and failure to recognize that many rivers are polluted will cause more of a problem in the future. Perhaps, other countries might also have an issue with lack of Government intervention concerning river cleanliness.

Bibliography

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E. coli Commensal

Escherichia coli (E. coli) is a Gram-negative, facultative anaerobic, rod-shaped bacterium of the genus Escherichia (Tenaillon et al. 2010).. It is commonly found in the lower intestine of warm-blooded organisms (Secher T 2016). E. coli is an extremely diverse bacterial species which forms part of the gut microbiome, a term which describes the ecological community of commensal, symbiotic and pathogenic microorganisms found in the intestine (Bull M J et al., 2014). Throughout life, the population of E. coli usually settles around 107–108 colony-forming units per gram of faeces (Secher T 2016). The E. coli population of the intestines usually includes a set of durable core strains, as well as temporary transients that vary with health, nutrition, infection and antibiotic exposure (Blount 2015). E. coli grows in the thin mucous layer that lines the gut where it competes with many other microorganisms for nutrients. This results in it being a non-fastidious organism, with a wide-ranging diet (Blount 2015). E. coli is the primary aerobic microorganism in the gastrointestinal tract (Tenaillon et al. 2010). As E. coli is a facultative anaerobe it is able to optimise growth based on the availability of oxygen. It is able to utilise both anaerobic and microaerobic respiration in order to survive in the low oxygen environment in the intestine (Tenaillon O 2010). Although E. coli competes with other microorganisms for resources such as surface area and nutrition, it is suggested to also share a mutualistic relationship with some. It aids anaerobic commensals in the gut by consuming any oxygen that may enter the gut, helping the maintain an anaerobic environment. In exchange, it is believed that E. coli may benefit from the breakdown of mucosal polysaccharides and dietary fibres by obligate anaerobes in the intestines (Tenaillon O 2010). E. coli is one of the first commensal microorganisms that the human infant gains exposure to. This is thought to occur via exposure to maternal faecal microbiota during childbirth. Interestingly, this exposure appears to be reducing in industrialised countries due to more stringent hospital hygiene standards and an increase in the number of caesarean sections being performed (Nowrouzian et al. 2003). Although the relationship between E. coli and the host is often considered to be commensal in which one party notably benefits and the other is neither helped nor harmed, there is some evidence that E. coli provides some succour to the host in the form of the production of vitamins K and B12, as well as the competitive exclusion of pathogens from the gut (Katouli 2010) (Hudault, Guignot and Servin 2001). In return, the human intestinal tract provides E. coli with a steady supply of carbon and energy sources, a comfortable environment with a moderate pH and temperature as well as protection against certain stresses and transport and dissemination facilities.

E. coli Pathogenic

However, E. coli is not entirely harmless, and some strains comprise several major foodborne pathogens. E. coli is an organism that frequently crosses the line between commensalism and pathogenicity (Leimbach, Hacker and Dobrindt 2013). This is due in part to its highly flexible genome. E. coli carries between 4.5 and 5.5 million base pairs of DNA but fewer than half of all genes encoded are conserved among all members of this species (Figler and Dudley 2016). Sequencing information estimates that the core genome of E. coli comprises less than 20% of the more than 16,000 genes in the E. coli pan-genome (Blount 2015). As E. coli is able to acquire genetic variation through horizontal gene transfer, it is enabled to adapt to particular niches, improve its metabolic capacity and assimilate virulence factors (Blount 2015). This gives rise to the numerous pathotypes of E. coli which pose a substantial threat to both human health and the economy. Per year it is estimated that E. coli infections cause 2 million deaths in humans (Russo and Johnson 2003). E. coli is conventionally serotyped based on three types of antigens; somatic (O), capsular (K) and flagellar (H) (Figler and Dudley 2016). E. coli O157 and O104 are among the most widespread foodborne pathogens.

There are five major foodborne E. coli pathotypes. These are Enteroaggregative E. coli, (EAEC), Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), Enterotoxigenic E. coli (ETEC) and Enterohemorrhagic E. coli (EHEC), (Yang et al. 2017). Pathogenic E. coli infection usually causes severe diarrhoea. Other symptoms include abdominal pain, nausea, emesis and fever (Yang et al. 2017). Strains such as EHEC and EAEC can cause haemolytic uremic syndrome a condition characterised by thrombocytopenia, microangiopathic haemolytic anaemia and acute renal failure (Naylor et al. 2003). E. coli can also cause extraintestinal diseases such as urinary tract infections, neonatal meningitis and sepsis (Poolman and Wacker 2016).

Pathogenic E. coli have a number of virulence mechanisms available to them. The most significant of these is known as the locus of enterocyte effacement (LEE) and is comprised of a cluster of virulence genes on the chromosomal pathogenicity island (PAI) (Yang et al. 2017). This locus encodes a type III secretion system. The type III secretion system transports virulence factors to the host epithelial cells. The pathogenic mechanism of E. coli is characterised by attachment and effacement of the host epithelium (Gaytan et al. 2016).

In order to affect this pathogenicity in the human, however, E. coli has a long journey of survival that involves the utilisation of a vast number of attributes, all of which are controlled by its highly active genome. The first barrier to infection in the human is survival outside of the human.

E. coli Environmental

One of the factors which make certain E. coli strains such important and formidable pathogens is the ability to survive in extraneous environments. As a commensal intestinal lodger, E. coli by design must regularly enter the outside environment in faecal waste discharged by humans and animals. The extensive dispersal of E. coli in the environment is exacerbated through human activity in the form of fertiliser made from animal wastes, wastewater from abattoirs and factory farms and effluent from water treatment plants and sewage schemes (Ishii and Sadowsky 2008). Although it was previously believed that E. coli did not survive well outside of the host, recent studies have refuted this and have shown that it can persist for long periods of independent of its host and may indeed become part of the indigenous flora of its new environment, a process called naturalisation (Jang et al. 2017). However, the external environment is much less stable than the mammalian host and there is huge variation in factors such as nutrition, temperature, oxygen, moisture, pH, and/or the surrounding microbial community (Blount 2015). E. coli has been able to acquire certain features which enable it to survive in such unforgiving environments. One such adaptation is the development of persister variants, which are metabolically inactive dormant versions of the E. coli strain (Shah et al. 2006). Although most E. coli strains are non-pathogenic, there do exist strains with the potential to cause significant harm to the human host. Many of these are acquired from environmental sources, either through the ingestion of unsanitary water, colonised vegetables or contaminated meat. E. coli is known to colonize leafy green vegetables such as spinach and lettuce, causing food poisoning outbreaks such as the outbreak of E. coli O157:H7 in 2006 linked with spinach and the outbreak of STEC O104 in 2011 associated with fenugreek seeds. The most recent outbreak which occurred in late 2018 was of STEC O157:H7 and was linked to romaine lettuce. Comment by Siobhan Ladden: https://www.cdc.gov/ecoli/2018/o157h7-11-18/index.html

Pathogenic E. coli colonising the human from an outside source must first overcome barriers mounted both explicitly and ambiguously by the human host. Before it reaches the intestine, where it thrives and proliferates it must first survive the passage through the upper and mid gastrointestinal tract.

During its passage to the intestinal tract, E. coli must first pass through extremely acidic conditions in the stomach. In order to do so intact, it must survive an extremely low pH of between 1.5 and 3.0.(Kanjee and Houry 2013). Imperative to E. coli’s survival in the stomach is its outer membrane. This is the first barrier the acid must breach in order to cause injury to the organism. The membrane of E. coli is highly adaptive and can respond to acid stress in a number of ways. E. coli can change the composition its membranes in order to prevent the entry of damaging acid. E. coli achieves this by increasing the concentration of cyclopropane fatty acids and reducing the concentration of unsaturated lipids. Additionally, the entry of acid may be decreased by obstruction of the outer membrane porins (OMPs) through the binding of polyphosphate to the OMPs. In the periplasm, the chaperone proteins HdeA and HdeB protect substrate proteins from aggregation and upon return to a neutral pH aid in substrate refolding (Ding et al. 2015). In the cytoplasm, the Hsp31 chaperone binds to and stabilizes unfolded intermediates until the stress is alleviated and then allows the proteins to refold either naturally or through ATP-dependent chaperone systems (Ding et al. 2015). Additionally, an amino acid decarboxylase system helps maintain pH homeostasis in the cytoplasm. This consists of a cytoplasmic decarboxylase, which converts its substrate into a related amine and an antiporter, which exchanges the imported amino acid for the cytoplasmic amine produced (Kern et al. 2007). Protection of DNA under acid stress is handled by the DNA-binding Dps (DNA-binding protein from starved cells) protein which binds to and protects DNA (Ding et al. 2015).

The survival of intestinal bacteria such as E. coli is reliant on its ability to survive not only the acidic environment of the stomach but also the high concentration of up to 30mM of bile salts in the human intestinal tract. It has been speculated that the pathogenicity of such enteric bacteria is dependent on its ability to survive and grow in the presence of bile salts. Bile is a fluid manufactured in the liver and stored in the gallbladder. In humans, primary bile acids consist of cholate and chenodeoxycholate. Later, the acids are conjugated to either glycine or taurine via amide bonds for secretion (Thanassi, Cheng and Nikaido 1997). Bile acids function in the emulsion of lipids to aid digestion. As well as in digestion, bile salts aid in the body’s defence against microbial pathogens. This is illustrated by the fact that the small intestine, which maintains high concentrations of bile acids, typically contains very scarce bacterial populations (Merritt and Donaldson 2009). Unconjugated bile salts can traverse both the outer and inner membranes of Gram-negative bacteria and accrue in the cell cytoplasm. Here, bile salts may destroy bacteria through a number of mechanisms, namely the disruption of cell membrane integrity, oxidative stress, DNA damage, upregulation of RNA secondary structure formation, and denaturation of cellular proteins. Central to the resistance of E. coli to bile are two important multidrug efflux systems AcrAB-TolC and EmrAB-TolC (Wang et al. 2017). Efflux pumps such as these are mechanisms which appear to remove toxic bile salts from the cytoplasm after they have penetrated the cell membrane. Other contributors to bile resistance include the protein YdhE of the multidrug and toxic compound extrusion (MATE) family, and the YdgEF small multidrug resistance (SMR) protein (Paul et al. 2014). A 2014 study proposed that a small multidrug resistance transporter called MdtM promotes persistence of E. coli in bile salts through the catalysis of secondary active transport of bile salts out of the cell cytoplasm. A functional cooperation between this proposed system and the AcrAB-TolC system discussed above results in an enhanced resistance of E. coli to bile (Paul et al. 2014)

E. coli Transcriptional Regulators

The initial step in gene expression occurs when particular segment of DNA is copied into RNA in order to be translated into a protein sequence and is known as transcription. Transcriptional regulators help to control the expression of certain genes involved in this process. Regulators can work by either activating or repressing single or operonic genes. Transcription regulators allow the RNA polymerase access to promotors which allows for response to environmental changes. This can happen by positive or negative regulation (Flores-Bautista et al. 2018). It is estimated that less than a tenth of all genes in E. coli function as transcription regulators by directly binding to DNA, though other proteins may indirectly participate in regulation of transcription (Perez-Rueda and Collado-Vides 2000).

E. coli has a large number of regulatory genes, due to its ability to adapt to a wide-ranging group of environments. This is important as it needs to be able to survive both inside the mammalian gut and in the extraneous environment. This helps to shape it as a significant pathogen, as it can survive the journey from one mammal to another, via soil and in turn food. It must be able to survive under a diverse range of environments with a huge range in temperature, pH and osmolality.

The most common type of transcriptional regulator in the prokaryotes are the LsyR type transcriptional regulators (LTTRs)(Maddocks and Oyston 2008). They were first identified by Henikoff et al in the 1980s and are believed to include over 40,000 members (Knapp and Hu 2010). As the LTTR family comprises such a large membership, it is no surprise that the genes it regulates encompass a vast number of functions. Some of these include genes involved in virulence, metabolism, transport, detoxification and motility. LTTRs are also highly involved in the biosynthesis of amino acid pathways (Perez-Rueda and Collado-Vides 2000). In E. coli some well-characterised LTTRs include ArgP which regulates arginine transport (Nandineni and Gowrishankar 2004), CynR which regulates cyanate detoxification, (Sung and Fuchs 1992), CysB which regulates Cysteine biosynthesis (van der Ploeg et al. 1997), LrhA which functions in motility and chemotaxis (Lehnen et al. 2002), LysR which regulates lysine biosynthesis (Stragier and Patte 1983), OxyR, involved in the oxidative stress response to H2O2 (Farr and Kogoma 1991) and QseA involved in quorum sensing (Sperandio, Torres and Kaper 2002).

LTTRs are highly structurally conserved and most comprise of about 330 amino acids. They contain a co-factor binding domain at the C terminus and a helix-turn-helix (HTH) motif at the N terminus. These domains afford a means of binding to DNA. The LTTRs make up a unique group of HTH-containing transcriptional regulators, as the HTH is situated 20-90 amino acids from the N terminus irrespective of whether it is activating or repressing transcription. By contrast, the HTH is situated at the C terminus in transcriptional activators and at the N terminus in transcriptional repressors (Maddocks and Oyston 2008). For this reason, LTTRs are known as dual regulators (Maddocks and Oyston 2008).

E. coli Y genes

E. coli is one of the most well-characterised microorganisms, due to its central role as a model organism in microbiology and molecular biology (Blount 2015). However, the function of many of its genes remains unknown. Less than 67% of E. coli’s protein-encoding genes appear with a recognised function in the HAMAP (High-quality Automated and Manual Annotation of Proteins) database (Pedruzzi et al. 2015). A 2018 study reported that 1563 of 4653 unique E. coli genes lack direct experimental evidence of function (Ghatak et al. 2019). A total of 131 of these have absolutely no evidence of function. An additional 304 of these genes (6.6%) are pseudogenes or phantom genes ((Flores-Bautista et al. 2018, Ghatak et al. 2019). Traditionally, genes which lack an annotated function have been referred to as “y-genes”. The lack of experimentally characterised function of so many genes is in part due to the recent explosion in protein sequencing technology and bioinformatics. The increase in biological data has led to a huge backlog of sequenced genes for which the function has not yet been elucidated experimentally. Many genes have hypothetical functions, derived by comparing sequences and structures between proteins with experimentally characterised functions and proteins with only theoretical functions (Flores-Bautista et al. 2018).

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Abstract

Multidrug-resistant-ESBL-producing E. coli are on high emergence in UTIs and other infections. Such E. coli are big challenge and responsible for serious threats to healthcare professionals, treatment failures, increased morbidity and mortality, and render beta-lactam antibiotics ineffective. A total of 9391 uropathogenic E. coli (non-repetitive) were isolated and identified on the basis of standard biochemical reactions. The antibiotic susceptibility testing was accomplished by the Kirby-Bauer disc diffusion method by following Clinical Laboratory Standard Institute (CLSI) guidelines. MICs of various antibiotics against ESBL-producing uropathogenic E. coli were evaluated by agar dilution method using guidelines of Clinical Laboratory Standard Institute (CLSI). ESBL production of E. coli was detected by Double Disc Synergy Test (DDST). Among 93901 of uropathogenic E. coli, 5916 (63%) were ESBLs producers. They were 100% resistant to beta-lactams, Ampicillin (AMP), Cefotaxime (CTX), 85% to Ciprofloxacin (CIP), 96% to Nalidixic acid (NA), 81% to Aztreonam (ATM), 81% to Trimethoprim/Sulfamethoxazole (SXT), and 58% to Amoxicillin-Clavulanate (AMC), 22% to Piperacillin-Tazobactam (TZP), 22% to Cefoperazone-Sulbactam (SCF), 21% to Nitrofurantoin (F), 16% to Amikacin (AK), 4% to Fosfomycin (FOS) and 2% to Imipenem (IPM). None of the ESBLs-producing E. coli was found resistant to Polymyxin B (PB). Results of MICs were in agreement with the antibiotic disc diffusion results. The objective of the study was to determine the prevalence of ESBL-producing E. coli, co-existence of antibiotics resistance, and to evaluate antibiotics’ effectiveness.

Keywords: ESBLs, MDR, E. coli, UTIs

Introduction

Multidrug-resistant (MDR) E. coli commonly produce beta-lactamase enzymes to withstand various kinds and generations of beta-lactam antibiotics (1). Extended-spectrum β-lactamase (ESBLs) arbitrated resistance is a rising healthcare issue (2). These enzymes confer resistance to Aztreonam, Oxyimino beta-lactams, and 1st to 4th generations of cephalosporins but can be hindered by the clavulanate, sulbactam, and tazobactam (3). ESBLs are frequently present in both members of Enterobacteriaceae and non- Enterobacteriaceae and are emerged by mutations in plasmid-mediated TEM1, TEM2, and SHV genes (4, 5). However, PER, CTX-M, VEB, and GES are recently evolved ESBLs (6). The ordinary antibiotic sensitivity screening in various clinical labs is unsuccessful to identify the ESBL producers E. coli as they might display false susceptible zone to Ceftazidime, Cefotaxime, and Ceftriaxone (7). The wide distribution of spread of ESBLs producing E. coli strains in hospitals and community reduces the use β-lactam antibiotics, leading to severe treatment failures, obliged to imply extended-spectrum and high-priced antibiotics (3). Co-existence of non-beta-lactams resistance genes (Trimethoprim-Sulfamethoxazole, tetracycline, aminoglycosides, and fluoroquinolones) has been reported in ESBLs producing E. coli which further causes therapeutic failure (6, 7). This study was planned to determine the prevalence of ESBL-producing E. coli, co-existed antibiotics resistance in a hospital, and to evaluate antibiotics’ effectiveness against MDR-ESBLs-producing uropathogenic E. coli.

Methodology

During five years, a total of 9391 uropathogenic E. coli (non-repetitive) were isolated and identified on the basis of standard biochemical tests (8). The antibiotic sensitivity test was accomplished by the Kirby-Bauer disc diffusion method by following Clinical Laboratory Standard Institute (CLSI) guidelines (9). The following antibiotic discs were implied; Ampicillin 10µg (AMP), Cefotaxime 30µg (CTX), Amoxicillin+clavulanic acid 20/10µg (AMC), Pipracillin+tazobactam 100/10µg (TZP), Cefoperazone+sulbactam 105µg (SCF), Aztreonam 30µg (AZT), Imipenem 10µg (IPM), Ciprofloxacin 5µg (CIP), Amikacin 30µg (AK), Trimethoprim/sulfamethoxazole 25/23.75µg (SXT) and Polymyxin B 300µg (PB).

Determination of Minimum inhibitory concentrations (MICs)

MICs of Ampicillin (AMP), Amikacin (AK), Ceftazidime (CAZ), Cefotaxime (CTX), Ciprofloxacin (CIP), and Imipenem (IPM) were determined against urinary isolates of E. coli by incorporating twofold increasing concentration of antibiotics in molten Muller Hinton agar by using the guidelines of Clinical Laboratory Standard Institute (CLSI). For the standardization of experiment, quality control strains E. coli ATCC25922 were used (10).

Screening for ESBLs

Third-generation cephalosporin-resistant E. coli were subjected to ESBL screening by double disc synergy test (DDST). Standardized 0.5 McFarland suspension of E. coli was prepared in normal saline. Isolated colonies of previously screened GNB were picked by straight wire and dispensed in normal saline tubes then swabbed on MHA plates to prepare the lawn of culture. Disc of amoxicillin-clavulanate (20/10µg) in the centre, cefotaxime (30µg) disc on one side, and aztreonam (30µg) on the other side were dispensed about 20-25mm apart to each other. Plates were incubated at 37°C for 18-24 hours under aerobic conditions. The expansion of cefotaxime and aztreonam zone adjacent to the amoxicillin-clavulanate disc was considered as ESBLs positive (11). E. coli ATCC 25922 was used as negative controls.

Figure 1: Double disc synergy test showing extension of Cefotaxime and Aztreonam zones toward amoxicillin clavulanate

Results

Among 93901 of uropathogenic E. coli, 5916 (63%) were found ESBLs producers. E. coli resistant to third-generation cephalosporins as determined by disc diffusion method, agar dilution method, and further confirmed by double disc synergy test (DDST) as indicated in Fig. 1 (a and b). Figures show patterns of inhibition zone extension of Cefotaxime (CTX) and Aztreonam (ATM) toward amoxicillin plus clavulanate hence, considered ESBL positive. ESBL-producing E. coli was found highly prevalent in urine specimens (63%) Fig. 2.

Fig. 2 Frequency of ESBLs-producing uropathogenic E. coli versus Non-ESBLs producing E. coli

ESBL-producing E. coli isolates were 100% resistant to beta-lactams, Ampicillin (AMP), Cefotaxime (CTX), 85% to Ciprofloxacin (CIP), 96% to Nalidixic acid (NA) (for only urinary isolates), 81% to Aztreonam (ATM), 81% to Trimethoprim/Sulfamethoxazole (SXT), and 58% to Amoxicillin-Clavulanate (AMC). Decreased resistance rates were noticed as 22% to Piperacillin-Tazobactam (TZP), 22% to Cefoperazone-Sulbactam (SCF), 21% to Nitrofurantoin (F), 16% to Amikacin (AK), 4% to Fosfomycin (FOS) and 2% to Imipenem (IPM). None of the ESBLs-producing E. coli was found resistant to Polymyxin B (PB) as shown in Fig. 3.

Fig. 3 Antibiotic resistance profiles of ESBL-producing uropathogenic E. coli

Representatives of ESBL-producing E. coli isolates (n=50), 10 from each of the 5 years were selected for determination of minimal inhibitory concentrations (MICs) against Ampicillin (AMP), Amikacin (AK), Ceftazidime (CAZ), Cefotaxime (CTX), Ciprofloxacin (CIP), Fosfomycin (FOS) and Imipenem (IPM). Results of MICs were interpreted according to guidelines of CLSI. Fig. 3 depicts lower MICs of Imipenem (IPM), Amikacin (AK), and Fosfomycin (FOS) against isolates of ESBL-producing E. coli, and higher MICs for Ampicillin (AMP), Cefotaxime (CTX), Ciprofloxacin (CIP) and Ceftazidime (CAZ). Accordingly, none of the isolates was found susceptible to Ampicillin (AMP), Cefotaxime (CTX), Ceftazidime (CAZ), and Ciprofloxacin (CIP).

Fig. 3: MICs of various antibiotics against ESBL-producing E. coli

Discussions

Extended-spectrum beta-lactamases (ESBLs) appear frequently in Enterobacteriaceae e.g. Escherichia coli. These enzymes are clinically significant because they inactivate penicillins, monobactams, and cephalosporins; important antibiotics, given as first-line antibiotics to treat many seriously patients of UTIs. Delayed detection and improper treatment of deadly infections generated by ESBL producers with cephalosporin has been related with increased mortality. Many ESBL-producing E. coli are multi-drug resistant to non-beta-lactams like quinolones, Trimethoprim-Sulfamethoxazole, and aminoglycosides reducing the treatment choices (12). The sudden rise of ESBL-producing bacteria is an increasing problem and has been characterized as a pandemic (13). In this scenario, proper and correct detection of ESBL is mandatory in clinical microbiology laboratory (14). According to Kumar et al. 2014, 66% of E. coli in blood and 55% of E. coli in urine were recognized as ESBL-producers (15). In another study accomplished in Islamabad, Pakistan, reported high prevalence rate 53% of ESBL-producing E. coli in urine. While higher frequency of UTI-associated ESBL-producing E. coli (63%) was found in present study. Antibiotic-resistant profiles of ESBL-producing E. coli of urine revealed the instant failure of beta-lactams (Ampicillin and Cefotaxime), quinolones (Nalidixic acid and Ciprofloxacin), Aztreonam and Trimethoprim-Sulfamethoxazole. Hence, these antibiotics are not good choice for the treatment of Uropathogenic ESBLs-producing E. coli. Such antibiotic resistance pattern is due to the co-existence of ESBLs and other antibiotic resistance genes such as quinolones and Trimethoprim-Sulfamethoxazole on plasmid or on E. coli chromosome. The high prevalence of increased emergence and evolution of such MDR-ESBLs producing E. coli is due to the selective pressure of low levels of cephalosporins and other antibiotics, misuse of antibiotics, and natural selection of antibiotic-resistant mutants (16). However, low resistance rates were noticed as 22% to Piperacillin-Tazobactam (TZP), 22% to Cefoperazone-Sulbactam (SCF), 21% to Nitrofurantoin (F), 16% to Amikacin (AK), 4% to Fosfomycin (FOS) and 2% to Imipenem (IPM). None of the ESBLs-producing E. coli was found resistant to Polymyxin B (PB). Piperacillin-Tazobactam and Cefoperazone-Sulbactam are satisfactory but few ESBLs type can hinder their activity. The most effective antibiotic treatment choices for uropathogenic ESBLs-producing E. coli are Polymyxin B, Imipenem, Fosfomycin, and Amikacin in sequence (17, 18, 19). The findings of this study are in agreement with that of Taneja and Sharma, 2008 and Ahmed et al. 2015 (14, 18). The antibiotic resistance patterns of representatives of ESBL-producing E. coli were verified or counter-checked by MICs that displayed the higher MICs values against Ampicillin, Cefotaxime, Ciprofloxacin, and Ceftazidime and lower MICs values against Imipenem, Amikacin and Fosfomycin Fig. 3. The results point out the therapeutic importance of Imipenem, Fosfomycin, and Amikacin against uropathogenic ESBL-producing E. coli (20).

Conclusions

The increase in emergence of MDR-ESBL type uropathogenic E. coli was recognized which could be the consequence of overuse of cephalosporins. The best treatment options for UTIs associated MDR-ESBL producing E. coli are Polymyxin B, Imipenem, Fosfomycin, and Amikacin.

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21. AMP 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 5050 50 50 50 50 50 50 50 45 35 35 AK 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 50 45 40 40 25 20 15 5 5 5 5 4 CAZ 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 50 50 50 50 50 50 40 40 35 30 30 25 CIP 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 50 50 50 50 50 45 40 40 35 35 30 25 CTX 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 50 50 50 50 50 50 45 45 45 35 35 30 FOS 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 48 40 35 30 30 25 25 20 5 2 1 1 IPM 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 50 35 35 15 5 5 5 5 5 4 2 2 Conc. of antibiotics

Abstract:

Escherichia coli is a gram-negative bacterium that is found in the human digestive system. Staphylococcus epidermidis is a gram-positive coccus-shaped bacterium commonly found on the human skin. In this study, we looked at the metabolism and physiology of both organisms on differential and selective media. Bacteria were grown and observed on Tryptic Soy Agar (TSA), Phenylethyl Agar (PEA), Mannitol Salt Agar (MSA), and MacConkey’s Agar (MAC). E. coli was able to grow on TSA, MSA, and MAC, and was partially inhibited on the PEA plate. On the MAC plate, the surrounding media was a hot pink color due to lactose fermentation. E. coli grew yellow on the MSA plate, which suggests that it can survive in high salt conditions and ferment mannitol, however, we believe a false positive result is due to inoculation technique error. The growth of E. coli was inhibited on the PEA plate because phenylethyl alcohol inhibits Gram-negative organisms by breaking down the outer membrane of the cell wall. S. epidermidis was able to grow on the TSA, PEA, and MSA plates. No growth was observed on the MAC plate because the bile salts break down the cytoplasmic membrane in Gram-positive organisms. S. epidermidis was able to grow on PEA because Gram-positive organisms do not contain an outer membrane in the cell wall structures, so phenylethyl alcohol cannot interfere with DNA synthesis. S. epidermidis grew pink on the MSA plate because it is a halotolerant organism and cannot ferment mannitol unlike other staphylococci such as S. aureus.

Introduction:

Escherichia coli is a gram-negative rod-shaped bacterium that is between 1.1-6 μmeters long. It is a facultative anaerobe, meaning that it can have a respiratory and fermentative metabolism. It is commonly found in the lower part of the intestines in both humans and other mammals. They are considered an opportunistic pathogen. Certain strains of E. coli are known to cause digestive issues in infants, humans, and animals alike (Bergey 1984). E. coli can ferment glucose and many other carbohydrates, and produce pyruvate as a by-product. This pyruvate can be broken down further into acetic, lactic, and formic acids. E. coli can ferment lactose, and some strains can ferment D-mannitol (Bergey 1984).

The mtlA gene in E. coli encodes for enzyme IIMtl. This enzyme converts D-mannitol to D-mannitol-1-phosphate (Figure 1). D-mannitol-1-phosphate is produced endogenously by E. coli, even if the cells are able to grow on another carbon source. The mtlD gene encodes a dehydrogenase, which converts D-mannitol-1-phosphate to fructose-6-phosphate. mtlR is the repressor gene for the D-mannitol operon. Internalized D-mannitol is the effector. Mutations of MtlA prevent growth of E. coli on D-mannitol. Expression of the MTL operon is dependent on the cAMP-CRP complex (Neidhardt et al. 1987).IIMtl

Dehydrogenase

• mtlD
• mtlA
• Fructose-6-phosphate
• D-mannitol-1-phosphate
• D-mannitol

Figure 1. Simple depiction of D-mannitol fermentation and mtl operon shown by E. coli. Structures were created using ChemDraw software.

The lac operon in E. coli is well-studied. The lac-operon in E. coli consists of 3 genes: lacZ, lacY, and lacA gene (Figure 2). The lacZ gene encodes for ß-galactosidase, which cleaves lactose into both galactose and glucose. The lacY gene is responsible for encoding the enzyme permease, which transports lactose into E. coli cells. Transport of lactose by the integral cytoplasmic membrane LacY protein depends on the membrane potential of the cell. The LacA gene is the third gene in the lac operon. The LacA gene encodes for transacetylase, which is the enzyme that is responsible for adding an acetyl CoA group to ß-galactosidase. Studies have found that the lacA gene is not necessary for the normal function of the lac operon system (Neidhardt et al. 1987).

If no lactose is present in the cell, the lac repressor is bound to the lac operator (lacO), which prevents RNA polymerase from binding to the lac promoter (lacP). Transcription cannot occur, so no mRNA is created (Figure 2a) (Neidhardt et. al 1987). If lactose is present and there is sufficient cAMP in the cell, the repressor is inactivated, and the lac operon is expressed. In order for lactose to enter the cell, it must be transported by Lac-permease. The presence of lactose causes an allosteric change of the repressor, allowing the repressor to detach from lacO and allow RNA polymerase to bind to lacP. Transcription is initiated 38 bp before the lacZ gene (Neidhardt et. al 1987). Transcription occurs as usual, and eventually, translation of the final mRNA product, which will produce the enzymes ß-galactosidase, permease, and transacetylase from the lacZ, lacY, and lacA genes, respectively (Figure 2b).

Figure 2. Overview of the lac operon in E. coli cells. Photo used from 2011 Pearson Campbell biology textbook.

Staphylococcus epidermidis is a gram-positive bacterium that is roughly 0.5-1.5 μmeter in size. It is a facultative anaerobe and generally non-motile (Bergey 1984). Staphylococci grow in grape-like clusters (Foster 1996). Staphylococci can be distinguished from other coccus-shaped bacteria such as streptococci and micrococci by performing a catalase test. In this test, you take a culture of bacteria either on a plate or a slide and immerse them with hydrogen peroxide. If the culture bubbles, then you have a catalase-positive bacterium. Both streptococcus and micrococcus are catalase-negative.

• S. epidermidis is the most common species of staphylococcus found on the human skin (Fey and Olson 2010). It is frequently found on the head, in the nose, and around the armpits. Previous epidemiological studies have shown that humans can carry anywhere from 10 to 24 different strains of S. epidermidis at a time (Kloos and Musselwhite 1975). S. epidermidis can be transferred through common contact.
• S. epidermidis is a coagulase-negative staphylococci (CoNS). It lacks the enzyme coagulase, which coagulase-positive staphylococci (such as Staphylococcus aureus) have (Otto 2009). It is now known that some strains of S. aureus are actually coagulase-negative too. There are over 30 species of CoNS, but S. epidermidis is the most common. Coagulase is not an enzyme, but rather an extracellular protein. It binds to prothrombin in the host to form a complex known as staphylothrombin. Thrombin has protease activity, which allows it to convert fibrinogen to fibrin. Thrombin can bind and form clots in blood plasma after it is incubated with an S. aureus broth culture. This is essentially how you test for coagulase in the lab (Foster 1996).
• S. epidermidis is known to produce biofilms, especially on foreign objects in the body such as implants and catheters. (4). These biofilms consist of teichoic acid, extracellular DNA, polysaccharide intracellular adhesion (PIA), and proteinaceous factors (Bhp, Aap, and Embp) (Fey and Olson 2010). S. epidermidis does not bind to fibrinogen on foreign objects in the body, but rather to fibronectin (Foster 1996). S. epidermidis can produce “slime” in the body on the objects it attaches to. This slime is primarily composed of teichoic acid, which is commonly found in the cell walls of gram-positive bacteria (Foster 1996).
• Unlike S. aureus, S. epidermidis is benign to its host. It does not produce virulence factors, but rather is a skin commensal (Foster 1996, Otto 2009). It is more difficult to diagnose a CoNS infection, due to the lack of virulence factors (Foster 1996). Among CoNS, S. epidermidis is the most common bacteria for skin infections (Otto 2009). S. epidermidis is also known to be resistant to many types of antibiotics, including methicillin (Foster 1996, Otto 2011). Methicillin-resistant-Staphylococcus-epidermidis (MRSE) contain the metA gene, which encodes for a penicillin-binding protein (PBP2a) to decrease its affinity to methicillin (Otto 2011).
• S. epidermidis has eight different sodium ion exchangers, and six transport systems for osmoprotectants, which allows the bacterium to handle extreme salt concentration and osmotic pressure (Rogers and Fey 2009). S. epidermidis can produce poly-γ-glutamic acid (PGA) and PNAG/PIA, which are exopolymers that help the bacterium to survive under its host’s immune defense. PGA is a pseudopolymer that originates from genes on the cap locus. PGA allows for S. epidermidis to grow under high salt concentrations. PGA is found in other CoNS but is absent from S. aureus. Many halotolerant bacteria contain PGA (Otto 2009). S. epidermidis does not ferment mannitol, but some other species of CoNS can ferment the sugar (Sah et al. 2018). S. epidermidis can also produce acid from lactose via metabolism of the D-tagatose-6-phosphate pathway (Bergey 1984).

Materials and methods:

S. epidermidis and E. coli were aseptically transferred from slants onto four different plates according to the protocol. All organisms were grown on a TSA plate and then grown on Mannitol Salt Agar, Phenylethyl Alcohol, and MacConkey’s Agar plates. Media was inoculated using aseptic technique according to protocol. The bacteria were spread onto the plates in a zigzag fashion. The plates were sectored with a permanent marker and labeled with a number that was assigned to each organism tested (Table 1). The plates were incubated at 37°C. Observations were made and recorded into a lab notebook at 24 and 48 hours.

1. TSA

• M. luteus
• S. epidermidis
• S. aureus
• B. subtilis
• E. coli
• E. aerogenes
• P. aeruginosa

Unknown #14

Unknown #15

1. PEA

• M. luteus
• S. epidermidis
• E. coli
• E. aerogenes

Unknown #14

Unknown #15

1. MSA

• M. luteus
• S. epidermidis
• S. aureus
• B. subtilis
• E. coli

Unknown #14

Unknown #15

1. MAC

• S. epidermidis
• E. coli
• E. aerogenes
• P. aeruginosa

Unknown #14

Unknown #15

Table 1. Organisms used for this experiment were labeled onto each plate according to this chart. Unknown assigned organisms were also tested on each plate.

Tryptic Soy Agar (TSA):

Tryptic Soy Agar (TSA) is a complex media that is produced by enzymatic digestion of casein and soybean meal. It contains the basic nutrients needed for most organisms to grow. All organisms that were used in this experiment are able to grow on the TSA media, so it was used as a control plate.

Phenylethyl Alcohol (PEA):

Phenylethyl Alcohol (PEA) is a selective media only. It contains many of the same components that TSA media contains such as casein, soybean meal, sodium chloride, and agar. The only other component the media contains is phenylethyl alcohol. The media partially inhibits the growth of Gram-negative organisms. Phenylethyl alcohol interferes with DNA synthesis and melts the outer membrane of Gram-negative organisms, which selects for Gram-positive organisms to grow. This plate was compared to the TSA plate as a reference.

Mannitol Salt Agar (MSA):

Mannitol Salt Agar (MSA) is a selective and differential media. The media contains 7.5% NaCl, mannitol, phenol red, and peptones. MSA selects for any organism that is able to grow in high salt concentrations (halotolerant organisms). The mannitol salt agar plate is primarily used to distinguish different Staphylococcus species. It differentiates organisms that are able to ferment mannitol. Organisms that are able to ferment mannitol will appear yellow on the plate. This color change is due to the organism producing an acid from fermentation, which changes the phenol red indicator to yellow. Organisms that are able to grow on the media, but do not ferment mannitol appear a pink/red color on the media due to peptone breakdown. Yellow on the plate indicates a pH that is < 6.9, whereas pink indicates a pH > 8.4.

MacConkey’s Agar (MAC):

MacConkey’s Agar (MAC) is a selective and differential media that contains crystal violet, lactose, peptones, bile salts, and neutral red. The media selects for Gram-negative organisms. Crystal violet and bile salts in the media inhibit the growth of any Gram-positive organisms. This media differentiates organisms that are able to ferment lactose. The neutral red dye is a pH indicator that turns red/pink below a pH of 6.8 and remains colorless with a pH above 6.8. Organisms that are able to ferment lactose, will appear a pink/red color on the media. This red color change is due to acid accumulating from lactose fermentation, which changes the neutral red dye indicator to hot pink/red. Non-lactose fermenting organisms that are able to grow on the media will appear colorless.

Observation/Results:

After incubation, observations were made and recorded at both 24 and 48 hours for all plates and organisms tested. A scoring indicator was created to compare growth from the PEA and TSA plates (Table 2).

Figure 4. TSA Plate after 24 hours of incubation at 37°C. Organism #2 is E. coli.

Figure 3. TSA Plate after 24 hours of incubation at 37°C. Organism #5 is S. epidermidis.

Tryptic Soy Agar (TSA) Plate:

After 24 hours, both S. epidermidis and E. coli grew white on the TSA plate. The E. coli grew smooth, round with an entire edge. After 48 hours, E. coli grew more. S. epidermidis did not grow anymore after 48 hours.

Phenylethyl Alcohol (PEA) Plate:

After incubating for 24 hours, S. epidermidis grew white. The bacterium grew a similar amount compared to the TSA plate. E. coli only partially grew white on the plate. There was not as much growth as compared to the TSA plate. After 48 hours, S. epidermidis grew more and E. coli looked the same as the 24-hour observations.

Mannitol Salt Agar (MSA) Plate:

After 24 hours, S. epidermidis grew a faint pinkish color on the MSA plate. E. coli grew yellow. The medium surrounding both S. epidermidis and E. coli was pink in color. After 48 hours, there was significantly more growth for E. coli and S. epidermidis. S. epidermidis was more clearly hot pink, whereas after 24 hours the bacteria were only faintly pink in color. The medium surrounding the E. coli colonies was yellow after 48 hours, whereas it was still a pink color with yellow colonies after 24 hours.

MacConkey’s Agar (MAC) Plate:

After 24 hours, E. coli grew hot pink, and the medium surrounding the colonies was also hot pink in color. S. epidermidis did not grow on the plate. After 48 hours, E. coli grew more and S. epidermidis still did not grow. The E. coli had a pink halo surrounding the growth after 48 hours.

Discussion/conclusions:

Both organisms were able to grow on the TSA plate. This is because tryptic soy agar is a complex media that supplies the basic necessary nutrients that allow most organisms to grow.

• S. epidermidis was able to grow on the PEA plate because phenylethyl alcohol is selective for Gram-positive organisms. S. epidermidis is a Gram-positive bacterium. Phenylethyl alcohol interferes with DNA synthesis and breaks down the outer membrane of Gram-negative organisms. Since Gram-positive bacteria do not have an outer membrane layer to their cell wall, just a thick layer of peptidoglycan, PEA does not inhibit their growth. As seen in FIGURE ??? and TABLE S. epidermidis grew just as much on the TSA plate as the PEA plate.

On the Mannitol Salt plate, S. epidermidis was able to grow because it is a halotolerant bacterium. S. epidermidis can produce PGA, which allows it to grow under high salt conditions (Otto 2009). This is why we see growth of S. epidermidis on the MSA plate (FIGURE ). S. epidermidis does not ferment mannitol, unlike other staphylococci such as S. aureus, which is why it grew pink on the MSA plate. If it were able to ferment the mannitol, the organism would produce an acid which would change the phenol red indicator dye to a yellow color. As a reference, S. aureus is able to ferment mannitol, and this is why it appeared yellow on the MSA plate (FIGURE XX Organism #3).

• S. epidermidis was not able to grow on the MacConkey’s Agar plate (FIGURE!!). This is because the crystal violet and bile salts in the MAC media inhibit the growth of Gram-positive organisms. The media selects from Gram-negative organisms to grow, and since S. epidermidis is a Gram-positive organism, it is not able to grow on the MacConkey’s Agar. The cytoplasmic membrane is directly beneath the peptidoglycan layer in the cell wall of a Gram-positive bacterium such as S. epidermidis. This cytoplasmic membrane is bile-sensitive, and when introduced to bile, the cell is not able to grow and survive (Neidhardt et al. 1987). The bile in the medium penetrated to the cytoplasmic membrane of S. epidermis, preventing growth from occurring on the MacConkey’s Agar plate.

E. coli was only able to partially grow on the PEA plate in comparison to the TSA plate (FIGURE AND TABLE!). This is because E. coli is a Gram-negative bacterium. Phenylethyl alcohol breaks down the permeability of cell membrane barriers in Gram-negative bacteria (Silver and Wendt 1966). When exposed to phenylethyl alcohol, large amounts of potassium inside the cell are leaked outward. Inhibition of DNA synthesis also occurs when Gram-negative bacteria are exposed to phenylethyl alcohol. This phenomenon, however, is due to the cell membrane barrier breaking down (Silver and Wendt 1966).

E. coli was able to grow on the Mannitol Salt Agar plate (Figure XX). The media surrounding the organism appeared yellow (FIGURE). This suggests that E. coli is able to survive in a high salt condition and ferment mannitol. Although E. coli is able to ferment mannitol into mannitol-1-phosphate by the enzyme IIMtl, the literature suggests that E. coli is unable to tolerate such high salt concentrations. We suspect that a false positive result occurred on our Mannitol Salt Agar plate. This could have been due to contamination from improper aseptic technique, or due to the concentration of salt in the MSA plate being too low. E. coli cells should dehydrate and should not survive the high concentration of salt in the MSA plate.

E. coli was able to grow on the MacConkey’s Agar plate. The media surrounding the organism appeared hot pink (FIGURE!!!!) Since E. coli is a Gram-negative organism, it is able to grow on MacConkey’s Agar. The crystal violet and bile salts that prevent Gram-positive organisms such as S. epidermidis from growing on the media, do not inhibit the growth of Gram-negative organisms such as E. coli. This is due to the outer membrane of the cell wall in Gram-negative organisms, which is fairly resistant to bile. It provides a protection for the inner cytoplasmic membrane, which is highly sensitive to bile (Neidhardt et al. 1987). The pink color indicates lactose fermentation. E. coli is a known excellent lactose fermenter. This color change is due to acid accumulating from the fermentation, changing the neutral red pH indicator to a red/pink color. This indicates that the pH is below 6.8.

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