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For this semester, I was really sure that did well. But, maybe not for the Microbiology's lab. It is the scariest last paper. I admitted that I was not doing well. I did even better on my 1st semester lab.

By the way, thank you for all lecturers that taught us for this semester. Till we met again next next semester.

Love,
Balqis Mustazim

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I choose topic for my 3 Minute Talk was about Cholera which is an infectious watery diarrhea. If untreated it may lead to death. In Bahasa Malaysia, Cholera was known as taun. This disease was discovered by a German bacterialogist named Robert Koch. He studied the disease during epidemic in egypt.

For this 3MT, I think I did better than my Pecha Kucha last sem as I prepared well even that morning I got diarrhea. And, present my 3MT in unstable condition huhu. After all, all the presenter were very good.

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On the 12th week, we had game with Dr Irwan which was on Immunology topic. There were so many games that each group made. For examples, snake and ladder, bingo and also musical chair. My group made a game which was bingo. It was so fun and interesting game.

From my point of view, the musical chair game was the fantastic after all. It was really hard to play. But, it was the most interesting game. We all enjoyed the game very much.

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Mary Temple Grandin is an American professor of animal science at Colorado State University, consultant to the livestock industry on animal behavior, and autism spokesperson.

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Temple Grandin On Her Search Engine | Blank on Blank

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Temple Grandin.

Is a really great movie. She's an inspiration *love love love !

She also told the world that women can do what men can do. I also believe that great friends surround her make she trust herself more. It truly heart-breaking when people kept cursing her but, when she told about that to the person that understand her, she become more relax. Especially when her mom told her that she really love her, don't listen to what others thought about her.

Live your life, be yourself. You're pretty amazing.

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Our first class with Dr Irwan for this semester.

We had to do a 'research' on biotech company in groups. My group did on AB enzymes company.

This AB Enzymes is an industrial biotech company specialized in enzymes and their applications. The company is part of Associated British Foods and represented worldwide. Their offices are located in Brazil, China Germany, Finland, Singapore and USA.

Their products range from food grade enzymes for baking and beverages, grains and vegetable oil seeds processing, to enzymes for animal feed and technical applications: pulp & paper, textiles and laundry detergents.

Here, is the official website of the company:

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An innovative Company with long serving tradition

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Ibu asked me to fry the keropok lekor in the refrigerator. Bukak je peti tengok keropok lekor tu dah ada mold. I refused to fry that. And, my ibu said like this, " Selama belas tahun awak hidup ni, awak beli keropok kat kedai yang dia goreng tu, awak yakin ke yang keropok tu elok. Hidup leklok je awak ibu tengok sampai hari ni." I'm speechless lol. I did snap the pics of the mold but, the pics goneeeee! Maybe I'd delete unconditionally huhu.

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It is about my last paper syndrome. I was really homesick that time. Tetiba, dapat tahu ibu kemalangan. Lagi la rasa nak balik. Apa apapun syukur sebab ibu takde pape cuma kereta je rosak teruk. Alhamdulillah. Cuma trauma la sikit sebab baru je kemalangan.

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It quite funny as we so insecure when handling the fungi as we know it the endospore-forming and might cannot be killed. But, it fun when know that the penicilin that made was from the Penicillium chrysogenum is the hard texture fungi. I had post the picture of the fungi on instagram so, this is their response.

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First of all, we do the isolation.
From the isolation, we do gram staining.
The picture below is the result of my gram stain.
Next, we do 11 biochemical test which are:

starch hydrolysis,
lipid hydrolysis,
carbohydrate fermentation test,
indole test,
methyl red test,
Voges-Proskauer test,
citrate utilization test,
hydrogen sulfide test,
urease test,
litmus milk reaction,
nitrate reduction test,
catalase test and
oxidase test.

From the result of all the biochemical test, I'm sure that my unknown culture is Alcaligenes faecalis. Alcaligenes faecalis is a gram-negative, rod-shaped bacteria. The cultural characterics that can be seen when this bacteria were isolated in a slant are it is thin, white, spreading and the growth is viscous.

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It Yakult trip yeay.

We went to Yakult factory in Negeri Sembilan. It was so exciting and a day before, I did some research though on this company. Just a little luls! Yakult is a cultured drink which contains billion of probiotics. In a simple word, probiotics are the good bacteria for your body. Name of the probiotics in the yakult is Lactobacillus shirota strain.

And there, I know something that Yakult also have cosmetic products yo! And, the products are only avail at Japan . The price of the products also pretty expensive for a student like me to afford it uhuhu.

The next day, we went to INTI International University to hear a talk on A surgeon opinion about antibiotic resistant (SUPERBUG).

A little of the important advices that I got is, if you in flu, go see the doctor. Don't share your medicine with anyone. And, if you consume the antibiotic, don't forget to finish it all within the time the Dr asked.

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I suggest Bacillus anthracis. It is a gram-positive bacteria but had been stated in the article year 2001 as the King Agent of Bioterrorism. It is an endospore-forming bacteria. The endospores enter body through inhalation or breaks in skin. This Bacillus anthracis had been used as biological weapons in form of powdered and aerosol form in 5 states which are United Kingdom, Japan, United State, Russia and Iraq.

In 19th century, anthrax involved in several important medical development. The first vaccine containing live organism also discovered in this century which was Louis Pasteur's vet anthrax vaccine.

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A Review Emphasizing Surgical Treatment

Abstract

Objective

To familiarize surgeons with the specific complications of cutaneous, gastrointestinal, inhalation, and systemic infection with Bacillus Anthracis, which may require surgical treatment.

Summary Background Data

The recent cases of intentional exposure to Bacillus Anthracis in the United States make familiarity with the basic microbiology, clinical manifestations, diagnosis, treatment, and control of this disease essential if mortality and morbidity is to be minimized, particularly following mass exposure. Although the treatment of Bacillus Anthracis infection is primarily medical, there are specific surgical complications with which the surgeon should be familiar.

Methods

A review of the literature was undertaken, utilizing electronic databases on infection with Bacillus Anthracis, as well as consultation with experts in this field. Emphasis was placed on the diagnosis and treatment of complications of infection that might require surgical intervention.

Results

Cutaneous anthrax infection results in eschar formation and massive soft tissue edema. When involving the extremities, increased compartment pressure requiring fasciotomy may result. Primary infection of the gastrointestinal tract may result in oropharyngeal edema and respiratory compromise requiring a surgical airway. Direct involvement of the lower gastrointestinal tract can result in intestinal ulceration, necrosis, bleeding, and perforation, which would require surgical exploration and resection of affected segments. Systemic sepsis, most often associated with inhalation anthrax, can cause massive ascites, electrolyte derangements, and profound shock requiring aggressive fluid resuscitation and careful hemodynamic monitoring and respiratory support. Systemic anthrax infection can also lead to gastrointestinal involvement by hematogenous dissemination, resulting in complications and requiring surgical management similar to direct gastrointestinal infection.

Conclusions

Cutaneous, gastrointestinal, inhalation and systemic infection with Bacillus Anthracis can result in complications which would require familiarity with the pathogenesis and manifestations of this disease in order to recognize and treat promptly and successfully by surgical intervention.

The recent terrorist attacks in New York City and Washington D.C. and the subsequent cases of anthrax exposure and infection throughout the United States make the once-theoretical risk of a bioterrorist attack with aerosolized anthrax very real. It is estimated that 100 kg of anthrax spores released upwind of Washington D.C. would result in 130,000 to 3 million deaths; many of these patients would die undiagnosed and untreated. In a covert, large-scale release of anthrax, the initial deaths would result from a delay in recognizing the clinical manifestations of cutaneous, inhalation, or gastrointestinal disease and instituting appropriate treatment. To reduce the death rate, knowledge of the clinical manifestations, disease course, and medical as well as surgical treatment of anthrax infection is imperative.

Data regarding the diagnosis and management of anthrax comes from human case reports, mostly from foreign countries in which the disease is endemic, and experimental animal studies. The closest incident to a large-scale bioterrorist attack with aerosolized anthrax was the accidental release of the agent from a military microbiology facility at Sverdlovsk in 1979, from which limited information is available. There were at least 79 cases of anthrax infection, resulting in 68 deaths. With such limited data and individual clinical experience, not all recommendations are rigorously evidence-based but rather have been clinically derived, as well as based on established surgical and medical practices.

The Centers for Disease Control and Prevention (CDC) has begun a process of physician education to increase familiarity with the clinical manifestations and treatment, but there has been little discussion of the surgical aspects of anthrax infection. Although the primary treatment of anthrax infection is medical, surgical complications can occur, particularly if a large number of individuals are infected. Awareness of these complications and knowledge of their treatment are critical if the deaths and complications are to be limited. Aside from the surgical indications, it is likely that in the case of a large-scale anthrax exposure, all physicians, including surgeons, would play a pivotal role in diagnosing and treating these patients. As such, a review of intentional infection with aerosolized anthrax, emphasizing surgical management, seems timely.

MICROBIOLOGY

Bacillus anthracis is a gram-positive aerobic bacillus, with both a spore-forming and vegetative form. Anthrax spores can survive in their dormant state, typically in soil, for decades until they come into contact with a host through cutaneous breaks, inhalation, or ingestion. After the experimental release of anthrax spores by the British military on Gruinard Island during World War II, spores persisted and remained viable for 36 years until decontamination began in 1979. Anthrax spores are resistant to drying, heat, ultraviolet light, gamma radiation, and many disinfectants. Spores are induced to germinate into their vegetative form when they contact an environment rich in amino acids, nucleosides, and glucose, with a temperature at least 37°C and a CO ₂ concentration of at least 5%, as is found in the host animal or human. When nutrients are exhausted or the environment is unfavorable, sporulation occurs.

The pathogenicity of anthrax is due both to the exotoxins it secretes and the capsule it forms, each encoded by a separate plasmid (pX01 and pX02, respectively). PX01 codes for three toxin elements (protective antigen, lethal factor, and edema factor), which combine to form two binary toxins, edema toxin (edema factor + protective antigen) and lethal toxin (lethal factor + protective antigen). Edema toxin acts to upset cellular water homeostasis, which results in the massive edema characteristic of the disease; lethal toxin induces macrophage secretion of tumor necrosis factor-alpha and interleukin-1-beta, which mediate the systemic shock state. The vegetative anthrax capsule, encoded by pX02, inhibits immune cell phagocytosis and is itself weakly immunogenic, thus allowing the bacillus to circumvent the natural immune response. Both factors are necessary for full virulence. In addition, given the capacity for genetic manipulation of strains that may be used in bioterrorist attacks, antibiotic resistance may contribute to its virulence. As such, the Working Group on Civilian Biodefense recommended that when used as an agent of bioterrorism, anthrax strains should be presumed resistant to tetracycline and penicillin, and patients should be treated with ciprofloxacin until antibiotic sensitivity can be determined. The CDC, however, currently recommends either ciprofloxacin or doxycycline as first-line treatment.

CLINICAL MANIFESTATIONS

Cutaneous Anthrax

Cutaneous anthrax is the most common form of anthrax infection, accounting for 90% to 95% of the cases worldwide and all of the prior cases reported in the United States. Many of the exposures have been occupational and resulted from the handling of infected animals or laboratory materials. Spores are introduced through breaks in the skin; within 3 to 5 days, the skin bears the primary lesion marked by a painless, pruritic papule that 24 to 36 hours later forms an ulcer with accompanying vesicles. The area goes on to become necrotic, forming a black eschar resembling coal ( anthracos in Greek). Characteristic of anthrax, the involved soft tissue becomes markedly edematous and indurated, and regional lymphadenopathy may be noted (). The skin lesion may become secondarily infected, typically with staphylococci or streptococci, leading to purulence and cellulitis. Diagnosis in the setting of bioterrorism would rely on a high index of suspicion as well as the characteristic clinical presentation, with Gram stain and culture of fluid from the vesicle providing confirmation.

Medical treatment of localized cutaneous anthrax in the setting of bioterrorism, as recommended by the CDC, is oral ciprofloxacin or doxycycline. The Working Group on Civilian Biodefense recommends initial treatment with ciprofloxacin until sensitivity to doxycycline is established, at which time therapy can be adjusted. The recommended course is 60 days to prevent presumed inhalation of contacted spores, which appear to have prolonged latency in the respiratory tract. Steroids may be used as adjunctive therapy in patients with severe edema, especially in the head and neck region.

Although 80% to 90% of the cases of cutaneous anthrax are self-limited, associated death results primarily from systemic dissemination and elaboration of exotoxins. In cases of cutaneous anthrax with signs of systemic dissemination, extensive edema, or head and neck involvement, the CDC recommends treatment with intravenous ciprofloxacin along with one or two additional agents with in vitro activity against anthrax, including rifampin, vancomycin, penicillin, ampicillin, chloramphenicol, imipenem, clindamycin, or clarithromycin. Penicillin and ampicillin should not be used alone to treat anthrax because of concerns about beta-lactamase activity. When treated with antibiotics, the death rate of cutaneous anthrax is less than 1%, increasing to 10% to 20% without treatment.

Apart from antibiotics, treatment of localized cutaneous anthrax relies on routine local wound care with sterile moist dressings changed regularly, as well as elevation of the affected region to retard edema formation. Surgical debridement of the eschar is contraindicated until after the patient has been adequately treated with antibiotics because of the risk of systemic dissemination of the bacillus. Vesicular fluid becomes sterile 5 to 24 hours after treatment with antibiotics. After adequate medical treatment, excision of the eschar with skin grafting may be carried out, although smaller lesions typically heal without scarring. Because of the massive tissue edema associated with anthrax, large and circumferential lesions on the extremities could lead to compartment syndrome and vascular compromise. Any invasive measurement of compartment pressures or fasciotomy, if indicated, should avoid the primary lesion until adequate antibiotic treatment has been administered.

Gastrointestinal Anthrax

Gastrointestinal anthrax accounts for approximately 2.5% to 5% of naturally occurring cases worldwide and is associated with a death rate ranging from 25% to 60%. The gastrointestinal tract can be affected directly by the consumption of food contaminated with anthrax bacilli or by hematogenous spread from systemic disease, usually originating in the respiratory tract, as confirmed by autopsies performed on patients after accidental exposure at Sverdlovsk in 1979. In this series, 39 of the 42 patients had multiple submucosal hemorrhagic lesions in the gastrointestinal tract, particularly the small bowel, stomach, and colon. Mesenteric lymphadenitis was found in 9 of the 42. It is unknown how many other patients not included in the series had either primary or secondary gastrointestinal involvement, how many underwent surgery on the affected gastrointestinal tract, and what the overall death rate associated with gastrointestinal anthrax was in the Sverdlovsk incident.

In cases of direct exposure, spores can germinate in the oropharynx or the lower tract, with clinical manifestations occurring 2 to 5 days after ingestion. In the oropharyngeal form, the characteristic necrotic, ulcerated lesion with overlying eschar may be evident in the posterior pharynx, with accompanying cervical edema and lymphadenopathy. Pharyngeal edema may lead to dysphagia and airway compromise.

Similarly, in the lower gastrointestinal tract, spores can deposit at sites of mucosal disruption and germinate, usually in the terminal ileum and cecum, although cases affecting the stomach and duodenum have been reported. The spore thus appears to be unaffected by gastric acid. Once germination takes place in the wall of the gastrointestinal tract, macrophages engulf the vegetative form and are taken up by regional lymph nodes, leading to necrotizing, hemorrhagic mesenteric lymphadenitis as well as hematogenous and lymphatic dissemination. The primary point of entry becomes ulcerated, hemorrhagic, and necrotic. Gastrointestinal manifestations caused by hematogenous dissemination from another site of primary infection are marked by multifocal submucosal hemorrhages extending into the muscularis propria and scattered throughout the intestinal tract.

Patients typically present with fever and diffuse abdominal pain and tenderness. Rebound tenderness and signs of an acute abdomen are often present. Because of gastrointestinal ulceration and necrosis, hematemesis, coffee-ground emesis, and bloody diarrhea often develop, and intestinal perforation may occur. Patients go on to accumulate massive ascites within 2 to 4 days after the onset of abdominal pain. The ascitic fluid may be clear, blood-tinged, or turbid, and Gram stain demonstrates gram-positive bacilli. Systemic dissemination of the organism is accompanied by sepsis and shock.

Medical management of gastrointestinal anthrax consists of early institution of antibiotics, aggressive volume resuscitation with both crystalloid and colloid, and prompt correction of electrolyte imbalances. In the setting of bioterrorism, the CDC recommends intravenous ciprofloxacin or doxycycline and one or two additional antimicrobials, as listed above. Therapy can be further adjusted as antibiotic susceptibility data are available and the clinical situation permits. The recommended course is 60 days, but patients may be switched from intravenous to oral therapy once the clinical condition improves or if resources available do not allow for ongoing intravenous therapy. Oral-pharyngeal manifestations require airway management, with possible intubation or establishment of a surgical airway if edema precludes the former. Steroids may play a role in lessening the extent of pharyngeal edema.

Management of direct and hematogenous anthrax infections in the lower gastrointestinal tract involves both primary medical treatment and potential surgical resection of affected areas. Most previously reported patients with lower gastrointestinal tract anthrax have died of systemic shock before surgery could be performed; however, there are cases in which prompt recognition of peritonitis led to exploration and resection of nonviable portions of the bowel. 44,45,48,49,51 In addition, autopsy findings in patients with gastrointestinal anthrax have shown bowel perforation, gastric ulceration, and segments of necrotic intestine. Thus, gastrointestinal anthrax demands treatment of both the primary process and any resultant insults to the bowel.

Because there are scant data to direct specific principles, knowledge of the effects of anthrax on the gastrointestinal tract and good surgical judgment must serve as the guides to management. Instances of ongoing sepsis or progressive abdominal pain and tenderness in patients treated promptly with antibiotics and restoration of intravascular volume would certainly be suspicious for gastrointestinal injury and should be managed as any other case of ischemic or perforated bowel. Also, progressive gastrointestinal bleeding refractory to medical treatment should be managed by localization of the bleeding source and surgical resection, because blood loss will exacerbate the severe hypovolemia characteristic of anthrax and will lead to an increased death rate.

Inhalation Anthrax

Inhalation anthrax accounts for 2.5% to 5% of the cases occurring sporadically; however, it is believed that inhalation anthrax will be the first manifestation to present after the release of aerosolized spores. All 42 patients reported in the autopsy series after accidental aerosolized release at Sverdlovsk showed evidence of inhalation anthrax, and 64 of the 79 patients with presumed inhalation anthrax reported from the incident died, suggesting that it may be a primary cause of death in a bioterrorist attack. In other reports, inhalation anthrax was found to be nearly uniformly fatal. The pulmonary system is the mode of entry of the 1- to 5-μm anthrax spores, but it is not the site of primary pathology. Rather than causing a pneumonia, the bacilli are taken up by alveolar macrophages and transported to mediastinal and peribronchial lymph nodes, where germination continues, resulting in hemorrhagic mediastinitis. From there, the vegetative form of the bacillus spreads systemically. A focus of hemorrhagic pneumonitis often develops, possibly related to the portal of systemic entry from the pulmonary system.

The incubation period from the time of exposure to the development of first symptoms, usually fever, nonproductive cough, myalgia, and malaise, is highly variable. Based on the Sverdlovsk cases, the modal incubation period was 10 days, with symptoms developing 2 days to 6 weeks after presumed exposure, suggesting prolonged viability of the spore form in the lungs. It is estimated that the dose of inhaled anthrax spores sufficient to result in 50% deaths would be 2,500 to 55,000. The pathognomonic sign on a chest radiograph in a previously healthy patient with symptoms of inhalation anthrax is a widened mediastinum (). Pleural effusions may also be seen. With 1 to 3 days of the onset of initial symptoms, patients go on to develop symptoms of local progression and systemic dissemination with dyspnea, stridor, diaphoresis, fever, chills, and frank shock. Death occurred a mean of 3 days after the onset of symptoms in the Sverdlovsk series.

Diagnosis of inhalation anthrax in the setting of bioterrorism is based on a high index of suspicion as well as nonspecific symptoms that mimic a viral upper respiratory infection and widened mediastinum on chest radiograph. Although Gram stain and culture of sputum is unlikely to yield the diagnosis, initial confirmation of clinical suspicion can be obtained by Gram stain and culture of pleural fluid showing gram-positive bacilli. Gram stain and culture of the peripheral blood and/or cerebrospinal fluid may also yield a diagnosis after systemic dissemination has occurred. The CDC recommends intravenous ciprofloxacin or doxycycline and one or two additional antimicrobials, as listed above, for the treatment of inhalation anthrax in the setting of bioterrorism. Therapy can be further adjusted as antibiotic susceptibility data are available and the clinical situation permits. The recommended course is 60 days, but patients may be switched from intravenous to oral therapy once the clinical condition improves or if resources available do not allow for ongoing intravenous therapy. Postexposure prophylaxis is based on the risk of exposure. Ciprofloxacin or doxycycline administered orally is recommended by the CDC until sensitivity can be determined. Prophylaxis should be continued for 60 days.

Anthrax Sepsis and Shock

Systemic anthrax infection is marked by hypotension, anoxia, and initial respiratory alkalosis followed by respiratory and metabolic acidosis, all consistent with the shock state. Also, electrolyte derangements include hypocalcemia, hypoglycemia, and hyperkalemia. It is likely that shock is mediated by toxin-induced macrophage secretion of tumor necrosis factor-alpha and interleukin-1-beta. Aggressive resuscitation with crystalloid and colloid, correction of electrolyte and acid-base abnormalities, and administration of intravenous antibiotics are the foundations for treating systemic anthrax sepsis. Invasive hemodynamic monitoring may be used to guide volume replacement and ensure its adequacy. Pressor support may also play a role in the management of anthrax sepsis as an adjunct to adequate resuscitation. Many of these patients develop oliguric or anuric renal failure and severe respiratory distress and should be supported appropriately.

In addition, patients should be monitored and treated for secondary gastrointestinal or meningeal involvement. Inhalation anthrax is the typical primary source in patients developing systemic dissemination that affects the gastrointestinal tract. Anthrax meningitis more commonly occurs after hematogenous or lymphatic dissemination from skin infections, although it can also occur in the setting of primary gastrointestinal or inhalation anthrax. In the autopsy series from Sverdlovsk, approximately half of the patients had meningeal involvement, presumably after inhalation anthrax. Anthrax meningitis may present with fever, fatigue, myalgia, nausea, vomiting, seizures, agitation, and delirium. Rapid neurologic decline and death may follow 1 to 6 days after the onset of illness. The cerebrospinal fluid is often bloody and usually stains for gram-positive bacilli, which can provide support for the clinical diagnosis. Besides antibiotics, surviving patients have also been treated with steroids.

DIAGNOSIS

Microbiologic studies are essential not only for confirmatory diagnosis of anthrax but also for antibiotic susceptibility testing in the setting of bioterrorism. Gram stain of fluid from vesicular lesions, ascitic fluid, pleural effusions, cerebrospinal fluid, and blood obtained after systemic dissemination will usually show gram-positive bacilli. Culture with appropriate bacteriologic testing can further confirm the clinical diagnosis. The microbiology laboratory should be notified that anthrax is clinically suspected, because identification of Bacillus species on Gram stain or in cultures is usually regarded as a contaminant.

Serologic and immunologic testing using enzyme-linked immunosorbent assay or indirect microhemagglutination for bacterial exotoxins or titers of antibody to protective antigen and capsular components may be used, but antibodies are not present until late in the course of the infection. A skin test using an extract from an attenuated strain of B. anthracis is available and is diagnostic in 82% of patients 1 to 3 days and in 99% of patients 4 weeks after the onset of symptoms. Polymerase chain reaction techniques used to amplify markers specific to B. anthracis exist but are not widely available outside research and government laboratories.

VACCINATION

There are currently two vaccines available for immunization against anthrax: an inactivated cell-free extract produced in the United States and a human live attenuated vaccine produced and used in the former Soviet Union. Because of the residual virulence of a live attenuated strain of B. anthracis, this form has been deemed unsuitable for use in the United States. Anthrax Vaccine Adsorbed (AVA) is a noninfectious filtrate from the culture of an attenuated strain of B. anthracis that uses the protective antigen exotoxin to confer immunologic protection. The vaccine was licensed by the U.S. Food & Drug Administration in 1970 and has been used to vaccinate individuals with high-risk occupational exposure to anthrax as well as U.S. military forces. Six doses are required at 0, 2, and 4 weeks and 6, 12, and 18 months, with annual boosters thereafter. Few serious adverse events have been associated with the vaccine, with mild local reactions occurring in approximately 30% of those receiving the vaccine and systemic reactions consisting of malaise, lassitude, fever, and chills in less than 0.2%. The efficacy of AVA has been established largely from animal studies because the high death rate associated with anthrax would preclude any human challenge studies, although there is anecdotal data from vaccinated and nonvaccinated individuals in high-risk occupations. The cumulative data suggest that immunization with AVA affords most subjects immunity against a lethal challenge with aerosolized anthrax. There is currently no recommendation for mass immunization against anthrax. However, in the event of a bioterrorist event, vaccination is recommended concurrently with antibiotics for both prophylaxis and treatment.

INFECTION CONTROL

Human-to-human contagion has not been reported, suggesting that the primary infectious form of anthrax is the spore. From case reports of patients with anthrax undergoing surgery, there is no evidence that special precautions were taken. In addition, there are no distinct guidelines for protection of the surgical team while operating on patients with anthrax. Standard universal precautions appear at this time adequate for protection. Instruments and materials contaminated during an operation on a patient with anthrax should be autoclaved or incinerated, as is the usual practice. There is no specific recommendation of antibiotic prophylaxis or vaccination for healthcare workers exposed to patients with anthrax.

CONCLUSIONS

Although bioterrorism-associated anthrax infections reported in the United States have to date been limited, preparation for further isolated infections as well as possible large-scale exposure seems essential. As such, surgeons should be familiar with both the medical and more specific surgical aspects of diagnosing and treating anthrax in its localized and disseminated forms. Because there is no way to detect an intentional, covert release of anthrax, only heightened clinical suspicion based on understanding the pathogenesis of B. anthracis will be effective in combating the disease.

Footnotes

Correspondence: Lisa Colletti, MD, 2922H Taubman, Box 0331

Ann Arbor, MI 48109-0331.

E-mail: colletti@umich.edu

Accepted for publication February 28, 2002.

References

1. Office of Technology Assessment, US Congress. Proliferation of Weapons of Mass Destruction. Washington, DC: US Government Printing Office; 1993;53-55. Publication OTA-ISC-559.

6. Manchee RJ, Stewart WD. The decontamination of Gruinard Island. Chem Br. July 1988;690-691.

8. Makino S, Sasakawa C, Uchida I, et al. Cloning and CO ₂-dependent expression of the genetic region for encapsulation from Bacillus anthracis. Mol Microbiol 1988; 2: 371-376.

12. Leppla SH. The anthrax toxin complex. In: Alouf J, Freer JH, eds. Sourcebook of bacterial protein toxins. London: Academic Press, 1991: 277-302.

19. Makino SI, Uchida I, Terakado N, et al. Molecular characterization and protein analysis of the [ cap region, which is essential for encapsulation in Bacillus anthracis. J Bacteriol 1989; 171: 722-730. PMC free article]

29. Edwards MS. Anthrax. In: Feigin RD, Cherry JD, eds. Textbook of pediatric infectious diseases, 3rd ed, Vol. 1. Philadelphia: WB Saunders, 1992: 1053-1056.

30. Lew D. Bacillus anthracis (anthrax). In: Mandell GL, Bennett JE, Dolin R, eds. Principles and practices of infectious disease. New York: Churchill Livingstone, 2000: 2215-2220.

32. Brachman PS, Kaufmann A. Anthrax. In: Evans AS, Brachman PS, eds. Bacterial infections of humans. New York: Plenum, 1998.

56. Defense Intelligence Agency. Soviet Biological Warefare Threat. Washington, DC: US Dept of Defense, 1986. Publication DST-161OF-057-86.

66. Penn CC, Klotz SA. Anthrax. In: Gorbach SL, Bartlett JG, Blacklow NR, eds. Infectious diseases, 2nd ed. Philadelphia: WB Saunders, 1998: 1575-1578.

67. Parry JM, Turnbull PCB, Gibson JR. A color atlas of Bacillus species. London: Wolfe Medical, 1983: 272.

68. Turnbull PCB, Kramer JM. Bacillus. In Balows A, ed. Manual of clinical microbiology, 5th ed. Washington, DC: American Society for Microbiology, 1991: 296-303.

79. Klevytskya A, Schupp J, Price LB, et al. A multiplexed fluorescent PCR genotyping system based on ten highly informative single-locus markers for B. anthracis strain identification. Presented at the Third International Conference on Anthrax. Plymouth, England, 1998.

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88. 50 Federal Register 51058. 1985.

93. Fellows PF, Linscott MK, Ivins BE. Anthrax vaccine efficacy against Bacillus anthracis strains of diverse geographical origin. Presented at 3rd International Conference on Anthrax, September 1998. Plymouth, England.

96. Ivins BE, Fellows PF, Pitt MLM, et al. Efficacy of a standard human anthrax vaccine against Bacillus anthracis aerosol spore challenge in rhesus monkeys. Salisbury Med Bull 1996; 87: 125-126.

97. Pitt MLM, Ivins BE, Estep JE, et al. Comparison of the efficacy of purified protective antigen and MDPH to protect non-human primates from inhalation anthrax. Salisbury Med Bull 1996; 87: 130.

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Bacillus Anthracis as an Agent of Bioterrorism: A Review Emphasizing Surgical Treatment

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A Review Emphasizing Surgical Treatment To familiarize surgeons with the specific complications of cutaneous, gastrointestinal, inhalation, and systemic infection with Bacillus Anthracis, which may require surgical treatment. The recent cases of intentional exposure to Bacillus Anthracis in the United States make familiarity with the basic microbiology, clinical manifestations, diagnosis, treatment, and control of this disease essential if mortality and morbidity is to be minimized, particularly following mass exposure.

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This day, we discuss about the dichotomous key that we did. Here, I wanna share about bacteria that in the breast milk other than Lactobacillus sp.

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ABSTRACT

Objective:

The objective of this work was to study the lactobacilli and bifidobacteria population in human milk of healthy women, and to investigate the influence that several factors (including antibioteraphy during pregnancy and lactation, country and date of birth, delivery mode, or infant age) may exert on such population.

Methods:

A total of 160 women living in Germany or Austria provided the breast milk samples. Initially, 66 samples were randomly selected and cultured on MRS-Cys agar plates. Then, the presence of DNA from the genera Lactobacillus and Bifidobacterium, and from most of the Lactobacillus and Bifidobacterium species that were isolated, was assessed by qualitative polymerase chain reaction (PCR) using genus- and species-specific primers.

Results:

Lactobacilli and bifidobacteria could be isolated from the milk of 27 (40.91%) and 7 (10.61%), respectively, of the 66 cultured samples. On the contrary, Lactobacillus and Bifidobacterium sequences were detected by PCR in 108 (67.50%) and 41 (25.62%), respectively, of the 160 samples analyzed. The Lactobacillus species most frequently isolated and detected was L salivarius (35.00%), followed by L fermentum (25.00%) and L gasseri (21.88%), whereas B breve (13.75%) was the bifidobacterial species most commonly recovered and whose DNA was most regularly found. The number of lactobacilli- or bifidobacteria-positive samples was significantly lower in women who had received antibiotherapy during pregnancy or lactation.

Conclusions:

Our results suggest that either the presence of lactobacilli and/or bifidobacteria or their DNA may constitute good markers of a healthy human milk microbiota that has not been altered by the use of antibiotics.

antibiotics, Keywords: Bifidobacterium, human milk, Lactobacillus

Bacterial gut colonization in early life is a process that exerts a short-, medium-, and long-term influence on the health status of a host, and that involves bacteria arising from different sources ( 1); among them, culture-dependent studies have revealed that human milk is a source of live staphylococci, streptococci, lactic acid bacteria, bifidobacteria, propionibacteria, corynebacteria, and closely related Gram-positive bacteria to the infant gut ( 2). Several studies have shown that there is a mother-to-infant transfer of bacterial strains belonging, at least, to the genera Lactobacillus, Staphylococcus, Enterococcus, and Bifidobacterium through breast-feeding ( 3- 7). In fact, human milk constitutes 1 of the main sources of bacteria to the breast-fed infant gut since a baby consuming approximately 800 mL/day of milk would ingest between 1 × 10 ⁵ and 1 × 10 ⁷ bacteria daily ( 8). It has been suggested that exposure of the breast-fed infant to such a wealth of bacterial phylotypes through breast-feeding may exert beneficial effects against several diseases ( 9). Breast-feeding has been shown to improve infant health outcomes lowering the risk of respiratory and gastrointestinal tract infections, necrotizing enterocolitis, otitis media, and allergic disease and to prevent later health problems such as inflammatory bowel disease, obesity, and type 2 diabetes mellitus ( 10).

The application of culture-independent molecular techniques, and particularly those based on 16S rRNA genes, allowed a complementary biodiversity assessment of the human milk microbiome. The use of such techniques confirmed the dominance of staphylococci and streptococci, the relatively frequent presence of lactic acid bacteria and bifidobacteria, and the existence of DNA belonging to other bacterial groups, such as some Gram-negative bacteria ( 5, 11- 13). Recently, the first microbiome study focused on human milk was published and the results indicated that milk bacterial communities were generally complex ( 9). Among the hundreds of operational taxonomic units detected in the milk of every woman, only 9 ( Streptococcus, Staphylococcus, Serratia, Pseudomonas, Corynebacterium, Ralstonia, Propionibacterium, Sphingomonas, and Bradyrhizobiaceae) were present in every sample from every woman. On the contrary, milk bacterial community was generally stable over time within an individual ( 9).

In contrast to staphylococci, streptococci, corynebacteria, or propionibacteria, which seem to be widespread in human milk, the presence of lactobacilli and bifidobacteria seems to be more variable among women ( 9, 11, 14, 15). Such variability may be the consequence of isolation difficulties, owing to fastidious growth and incubation requirements, or may be the result of the technical bias associated to molecular studies. It could also be because of host peculiarities; it has been suggested that the human milk microbiome is influenced by several factors that significantly skew its composition ( 16). In this context, the objective of this study was to assess whether demographic or clinical factors, such as country and date of birth, infant age, delivery mode, or antibiotherapy during pregnancy and lactation, may exert an influence on the bifidobacterial and lactobacillic population present in the breast milk of healthy women.

METHODS

Subjects and Sampling

A total of 160 healthy women participated in the study and provided a sample of breast milk. Women were recruited to cover a moderately wide area of central Europe from randomly chosen regions in Germany and Austria to represent southern and eastern (Germany) and western and eastern (Austria) parts of both countries, which included both rural and urban settings. Recruitment was carried out by midwives, who were contacted initially via the HiPP Scientific sales force (Pfaffenhofen, Germany). All of the volunteers gave written informed consent to the protocol, which was approved by the ethical committee of Hospital Clínico (Madrid, Spain). The milk samples were collected in a sterile tube by manual expression using sterile gloves. Previously, nipples and mammary areola were cleaned with soap and sterile water and soaked in chlorhexidine. The first drops (∼500 μL) were discarded. All of the samples were kept frozen until delivery to the laboratory. All of the women filled a questionnaire designed to collect information on demographic characteristics and some other factors, such as mode of delivery, anesthesia during labor, or antibiotherapy during pregnancy and lactation (Table ).

TABLE 1

Count and Identification of Bacteria in the Samples

Adequate dilutions of 66 randomly selected milk samples were spread onto agar plates of Man, Rogosa, and Sharpe (Oxoid, Basingstoke, UK) supplemented with l-cysteine (0.5 g/L) (MRS-Cys) for isolation of lactobacilli and bifidobacteria. The plates were incubated for 48 hours at 37°C anaerobically (85% nitrogen, 10% hydrogen, 5% carbon dioxide) in an anaerobic workstation (MINI-MACS; DW Scientific, Shipley, UK).

After incubation and counting, 10 isolates from each culture medium were randomly selected and identified at the species level by classical morphological and biochemical tests. In addition, all the Gram-positive isolates with morphology compatible with that of lactobacilli or bifidobacteria were selected and identified at the genus level by classical morphological and biochemical tests and by demonstration of fructose-6-phosphate phosphoketolase activity in cellular extracts. Identification at the species level was performed by MALDI-TOF (Vitek MS; BioMerieux, Marcy l'Etoile, France) or by polymerase chain reaction (PCR) sequencing of a 470-bp fragment of the 16S rRNA gene using primers pbl16 (5′-AGAGTTTGATCCTGGCTCAG-3′) and mlb16 (5′-GGCTGCTGGCACGTAGTTAG-3′) ( 17). The PCR conditions were as follows: 96°C for 30 seconds, 48°C for 30 seconds, and 72°C for 45 seconds (40 cycles) and a final extension at 72°C for 4 minutes. The amplicons were purified using the Nucleospin Extract II kit (Macherey-Nagel, Düren, Germany) and sequenced at the Genomics Unit of the Universidad Complutense de Madrid, Spain. The resulting sequences were used to search sequences deposited in the EMBL database using the BLAST algorithm, and the identity of the isolates was determined on the basis of the highest scores (≥98%).

Somatic Cell Count (SCC) in the Samples

SCC was performed with a DeLaval cell counter DCC (DeLaval International AB, Tumba, Sweden), using single-use cell counter cassettes and instructions provided by the manufacturer. The cassette that contains small amounts of a DNA-specific stain (propidium iodide) is used to collect the sample. A piston carries the milk sample toward a counting window that is exposed to an LED light source. The fluorescence signal given by the cell nuclei is recorded as a digital image that is subjected to automated image analysis.

Bacterial DNA Isolation From the Milk Samples

Initially, a fraction of the breast milk samples (1 mL) was centrifuged at 7150 g for 20 minutes. Then, total DNA was isolated from the pellets using the QIAamp DNA Stool Mini Kit (QIAgen, Hilden, Germany) following a protocol described previously ( 11). DNA was eluted in 20 μL of buffer AE (provided in the kit), and the purified DNA extracts were stored at −20°C.

Qualitative PCR Assays

Genus-specific detection of DNA from the genera Lactobacillus or Bifidobacterium was accomplished using the primers and PCR conditions reported by Collado et al ( 18). At the species level, a 2-step multiplex PCR assay was used to detect DNA from L fermentum, L gasseri, L plantarum, L reuteri, L rhamnosus, and L salivarius, using species-specific primers and PCR conditions previously reported ( 19). In parallel, the presence of DNA from L casei/ L paracasei was assessed using the primers and PCR conditions described by Chagnaud et al ( 20).

PCR detection of DNA from B longum, B infantis, B dentium, and B gallicum was carried using the primers and PCR conditions described by Matsuki et al ( 21), whereas the presence of DNA from B adolescentis, B angulatum, B bifidum, B breve, B catenulatum, and B pseudocatenulatum was assessed using those reported by Matsuki et al ( 22). Finally, PCR detection of DNA from B lactis was performed using the species-specific PCR assay developed by Ventura et al ( 23).

Each PCR assay included DNA extracted from a reference strain of each targeted species (positive control). Amplicons were analyzed by electrophoresis (90 V, 1 hour) on 1% agarose gels. Subsequently, the gels were stained and bands were visualized in a Gel-Doc system (Bio-Rad, Hercules, CA).

Statistical Analysis

Quantitative data were expressed as the mean and 95% confidence interval (CI) of the mean or, when they were not normally distributed, as the median and interquartile range. A correlation analysis was performed to test the relation between bacterial counts in MRS-Cys and SCCs. Proportions were compared using χ ² statistics, including the Fisher exact test and the Freeman-Halton test for contingency tables greater than 2 × 2. Differences were considered significant at P  < 0.050. SAS version 9.2 (SAS Institute Inc, Cary, NC) was used to carry out the analyses cited above.

RESULTS

Characteristics of the Lactating Women Participating in the Study

The 160 women enrolled in this study had a mean age of 31.82 years (95% CI 31.10-32.54 years) and their breast-fed baby had a gestational age of 39.72 weeks (ranging from 29 to 44 weeks). The median number of children was 1 (n = 104, 65.00% of participants), and only 11 women (6.88%) had 3 or more children. Moreover, 21.87% of the infants were born by cesarean section, more than one-third of the women (35.22%) received anesthesia during delivery, and 40.62% of the women had received antibiotherapy during pregnancy and/or lactation (Table ). Most of the participating women were exclusively breast-feeding their babies whereas 32.08% did it partially. Regarding the time of sampling, most of the breast milk samples (73.75%) were collected from women during the second to fourth week of lactation, whereas 10.00% were obtained during the first week and the remaining 15.63% after the first month of lactation. Most of the women who gave samples during the first month of lactation were exclusively breast-feeding their infants (75.37%), whereas this percentage descended notably in samples obtained after the first month of lactation (28.00%). Other demographic and clinical characteristics, such as the month of delivery, nationality and origin of the mother (urban or rural), and place where the samples were obtained, are summarized in Table .

Bacterial and SCCs

Bacterial growth was observed in 58 of the 66 milk samples inoculated on MRS-Cys agar plates. The mean (95% CI) value of bacterial counts obtained in such medium was 1.63 (1.49-1.77) log ₁₀ colony-forming units (CFU)/mL and ranged between 1.0 and 2.7 log ₁₀ CFU/mL (Fig. ). In those breast milk samples where bacterial growth was observed, the mean (95% CI) value of SCCs was 36.67 (35.96-41.37) cells per microliter and ranged between 23 and 68 cells per microliter, whereas in the rest of the samples (n = 8) where bacterial growth was undetectable, lower SSCs values (mean value of 27.16 cells/μL) were observed. A weak but statistically significant correlation was noted between bacterial counts in MRS-Cys plates and SSCs ( r = 0.395, P  = 0.002), as shown in Figure . Overall, the bacterial count and SCC values found in these breast milk samples indicated that none of the women were experiencing mastitis when the samples were collected.

Bacterial Identification

In relation to Gram-positive cocci, Staphylococcus spp were isolated from 51 samples (77.27%); Staphylococcus epidermidis was detected in all 51 samples, whereas other coagulase-negative staphylococci were isolated from <25% of the samples (data not shown). Staphylococcus aureus could not be detected in any sample. Streptococci were also isolated from 40 samples (60.61%) and most of them belonged to the species Streptococcus mitis, Streptococcus salivarius, or Streptococcus parasanguinis (data not shown).

Lactobacilli and bifidobacteria could be isolated from 27 (40.91%) and 7 (10.61%) samples of breast milk, respectively (Fig. ). The mean (95% CI) values of bacterial counts for Lactobacillus and Bifidobacterium were 1.11 (0.99-1.23) and 0.96 (0.76-1.16) log ₁₀ CFU/mL, respectively. Identification of the isolates at the species level revealed that they belonged to the following species: B breve, B longum, L casei, L fermentum, L gasseri, L gastricus, L plantarum, L reuteri, L rhamnosus, L salivarius, and L vaginalis. The species most frequently found was L salivarius that was isolated from 9 milk samples (13.64% of the 66 cultured samples), followed by L fermentum (7 samples, 10.61%), L gasseri (6 samples, 9.09%), and B breve (5 samples, 7.58%) (Table ). Usually, only 1 species of either lactobacillus or bifidobacteria was present in an individual breast milk sample, although 2 different species were isolated from 7 samples, and B longum, L gastricus, and L reuteri were detected simultaneously in 1 sample (Table ).

TABLE 2

PCR Assessment of Lactobacilli and Bifidobacteria Diversity in the Milk Samples

The presence of lactobacilli and bifidobacterial DNA alone or in combination was analyzed by PCR using species-specific primers in 160 breast milk samples, and it was confirmed in 113 (70.60%) samples (Fig. A). More specifically, Lactobacillus sequences (alone or in combination with Bifidobacterium sequences) and Bifidobacterium sequences (alone or in combination with Lactobacillus sequences) were detected in 108 and 41 samples (67.50% and 25.62% of total samples), respectively, whereas both genera were present simultaneously in 36 samples (22.50% of total samples) (Fig. A).

The Lactobacillus species most frequently found was L salivarius (56 samples, 35.00% of total samples), followed by L fermentum (40 samples, 25.00%) and L gasseri (35 samples, 21.88%) (Fig. B). Other lactobacilli species detected using this approach were L reuteri (11.88%), L plantarum (10.63%), L rhamnosus (8.13%), and L casei (4.38%). Regarding bifidobacteria, B breve DNA was the most frequently found and it was present in 21 (13.75%) of the analyzed samples. B longum and B lactis were detected only in 7 samples (4.38%), each. Another 7 breast milk samples contained other species of bifidobacteria (Fig. B).

Globally, there was great interindividual variability regarding the lactobacilli and bifidobacterial PCR profile. In fact, up to 52 different species combinations were found among the 113 breast milk samples in which lactobacilli and/or bifidobacteria could be detected (Fig. ). A total of 31 women displayed a profile that was not shared with any other recruited woman. The profiles of most of the samples comprised only 1 or 2 different species (38 and 43 samples, respectively) (Fig. ). The profile comprising only L salivarius DNA was shared by 13 of the analyzed samples (representing 8% of total samples), whereas the combination of L fermentum with either L salivarius or L gasseri was present in 9 and 8 human milk samples, respectively (∼5% of total samples). In contrast, 8 breast milk samples contained DNA from 4 different Lactobacillus and Bifidobacterium species. A detailed analysis of all the combinations of lactobacilli and bifidobacterial species that were detected by PCR is presented in Figure .

Taken as a whole, there was a good correspondence between isolation by culture technique and PCR detection of lactobacilli or bifidobacteria, both at the genus and the species level in the 66 breast milk samples that were seeded on MRS-Cys plates (Table ). Lactobacilli DNA was detected in 50 samples, and viable lactobacilli were isolated from approximately half of them (26 samples). The proportion of samples in which bifidobacteria were isolated by culture and in which their DNA was detected by PCR was lower (6 samples of 18 positives for bifidobacteria by PCR; Table ). B lactis was detected only using molecular techniques (in 2 milk samples).

TABLE 3

Influence of Demographic and Clinical Parameters in the Detection of Lactobacilli and Bifidobacteria in Breast Milk

Associations between the presence or absence of lactobacilli or bifidobacterial DNA and relevant demographic or clinical characteristics of the women participating in the study were investigated in this study. Most of the analyzed samples (73.75%) were collected from women during a period that extended from the second to the fourth week after delivery, except 16 samples (10.00%) obtained during the first week and 25 samples (15.63%) provided by women who had been breast-feeding for more than 1 month. No differences were found regarding the distribution of bacteria belonging to either the genus Lactobacillus or Bifidobacterium or to their respective species between samples taken during the first week, from the second to the fourth week or after the first month of breast-feeding (Tables and ).

TABLE 4

TABLE 5

The number of milk samples positive for lactobacilli or bifidobacteria were significantly lower in those women who had received antibiotherapy during pregnancy or lactation (χ ² test; P = 0.000 and P = 0.037 for lactobacilli and bifidobacteria, respectively) (Tables and ). The administration time of the antibiotic (pregnancy, delivery, or lactation) did not modify the frequency of samples with Lactobacillus, Bifidobacterium or their respective species, although it would be advisable to analyze a higher number of subjects to draw a conclusive evidence of this aspect. Lactobacilli were also less frequently detected in the breast milk of women who had been subjected to cesarean section and/or had received anesthesia during delivery although, in such cases, the differences were not statistically significant (χ ² test, P = 0.059 and P  = 0.097, respectively) (Table ).

To further investigate the influence of these factors on the diversity of the Lactobacillus and Bifidobacterium species detected in these human milk samples, an analysis was carried out considering the presence or absence of DNA from the different species (Tables and ). L fermentum and L salivarius DNA was detected only in 13.85% and 16.92%, respectively, of the samples obtained from women who had received antibiotherapy, whereas the percentage ascended to 32.63% and 47.37%, respectively, in the case of women who had not been treated with antibiotics (χ ² test, P = 0.007 and <0.001, respectively). Similarly, L plantarum was present in 14 milk samples (14.74%) obtained from women who did not take antibiotics but it was detected only in 3 women (4.62%) of the group that received antibiotherapy, although the difference was not statistically significant (Fisher test, P = 0.065). Detection of L fermentum and L salivarius was also higher in women who had their babies by vaginal delivery than in those that had them by cesarean section, although the differences did not reach statistical significance ( P = 0.088 and P = 0.098, respectively). Receiving anesthesia during delivery had a similar impact in the presence of DNA from L fermentum and L salivarus in breast milk; the presence of these lactobacilli species was found more frequently in women who did not receive anesthesia during delivery, although the difference was statistically significant only for L salivarius ( P = 0.068 and P = 0.007, respectively). Similarly, a higher frequency of B breve, B lactis, and B longum was observed in milk samples from women with a vaginal delivery but, again, the differences were not statistically significant.

Finally, the association between the presence or absence of viable lactobacilli and bifidobacterium and relevant clinical parameters in the 66 milk samples that had been cultured was investigated. There was a tendency indicating that the proportion of milk samples containing viable lactobacilli, particularly L fermentum and L salivarius, was higher in the group of women who did not receive antibiotics during pregnancy and/or breast-feeding compared with the group that received antibiotherapy, but the association did not reach significant values (Table S1). The associations between the presence of Lactobacillus and Bifidobacterium DNA and some clinical parameters that have been previously described were, however, confirmed in this smaller group of milk samples (Table S1).

DISCUSSION

In the last years, culture-dependent methods have shown that breast milk is a source of commensal, mutualistic, and probiotic bacteria with the ability to influence the initial colonization of the infant gut ( 2, 24). In this study, a low number of viable bacteria (<10 ³ CFU/mL) was found in most of the samples, in agreement with previous analyses ( 25). This fact reflects the hygienic collection and proper storage of the samples and, together with the low SCC level of milk samples, indicates that participating women did not experience mastitis. Higher counts are usually related to non-hygienic sampling, improper storage of the samples, and/or use of contaminated milk pumps for sampling collection or mastitis ( 26, 27)

Although the main target of this study were lactobacilli and bifidobacteria, coagulase-negative staphylococci and viridans streptococci could be isolated from 51 (77.27%) and 40 (60.61%), respectively, of the 66 cultured samples. Staphylococci and streptococci constitute the dominant culturable bacteria in human milk, and related DNA sequences of both genera are also the prevailing ones in this biological fluid, albeit with substantial interindividual differences ( 4, 8, 9, 11, 14, 16, 18, 28). In spite of this, staphylococci and streptococci have received a marginal attention regarding their role in the human mammary gland and in the early colonization of the infant gut, although they could be useful to reduce the acquisition of undesired pathogens, particularly in infants exposed to hospital environments ( 7, 28- 30).

With respect to lactobacilli and bifidobacteria, they could be isolated from 27 (40.91%) and 7 (10.61%) of the 66 cultured samples, respectively. Such bacterial groups constitute a subdominant culturable population in human milk ( 3, 5- 8, 31), and are particularly attractive because of its use as probiotics. It is noteworthy that lactobacilli and bifidobacteria are difficult to isolate because their low concentration in human milk is close to the detection limit for culture methods ( 5). All of the lactobacilli and bifidobacterial species isolated in this study have already been described in human milk ( 3, 5- 8). Particularly, strains belonging to the species L fermentum, L salivarius, L gasseri, B breve, and B longum can be transferred to the infant gut through breast milk ( 3, 6, 7).

In this work, qualitative PCR analysis revealed the presence of lactobacilli and bifidobacterial DNA in 108 (67.50%) and 41 (25.62%), respectively, of the 160 samples analyzed. Globally, there was a good correspondence between isolation of viable bacteria and PCR detection of lactobacilli or bifidobacterial DNA, at both the genus and the species level. The higher number of samples from which lactobacilli or bifidobacterial DNA was amplified compared with those from which viable bacteria were isolated by culture methods was an expected result, owing to the low concentrations of lactobacilli and bifidobacteria in human milk that hinder their detection (as stated before) and to the lack of discrimination between DNA from live or dead bacteria of PCR assays. Human milk contains a wide range of free bacterial DNA signatures, which may contribute to program the neonatal immune system ( 25).

Previous culture-independent assessments of the bacterial diversity of human milk have shown the presence of lactobacilli and bifidobacterial DNA ( 5, 11- 14, 16, 18, 25). In contrast, some pyrosequencing-based studies have found a low relative abundance of DNA from lactobacilli and/or bifidobacteria ( 9, 14, 32). Differences in genetic, cultural, environmental, or dietary factors ( 9, 14), or technical bias associated with culture-independent methods (differential lysis among different bacterial strains, species, genera, and phylotypes when obtaining bacterial DNA from the biological sample and/or differential amplification rate because of the selected primers for pyrosequencing) could explain these controversial results. In fact, in a previous study, the application of universal primers or Lactobacillus-specific primers to a set of human milk samples showed different results ( 11, 12). Therefore, standardization of procedures to be able to compare results provided by different studies focused on the same type of biological material would be advisable.

In this study, the influence of several factors on the detection of lactobacilli and bifidobacterial DNA by PCR was also assessed. The factor that exerted the strongest influence on the presence of lactobacilli or bifidobacteria was the administration of antibiotherapy to mothers during pregnancy or lactation. More specifically, detection of lactobacilli or bifidobacterial DNA in the milk samples using genus- and some species-specific ( L fermentum and L salivarius) primers was significantly lower in those women who had received antibiotherapy during such periods. Lactobacilli were also less frequently detected in the breast milk of women who had been subjected to cesarean section, probably owing to the antibiotherapy associated to such surgery. It has been long known that antibiotics are responsible for dysbiosis processes in the human microbiota, leading to antibiotic-associated diarrhea and gastroenteritis, urogenital, and oral infections ( 33). It is becoming evident that antibiotherapy during pregnancy, intrapartum, and lactation alters the maternal microbiota, a fact that may have negative consequences for infant health ( 34). An increased risk of asthma exacerbation and hospitalization, requiring inhaled corticosteroids, in children if mothers used antibiotics during pregnancy, supports a role for bacterial ecology in pre- or perinatal life for the development of asthma ( 35).

The decrease in the lactobacilli and bifidobacterial populations of breast milk may have negative consequences for breast-fed infants because they are important members of the human gut microbiota in early life. Infants with delayed colonization or decreased numbers of these bacteria may be more susceptible to a variety of gastrointestinal or allergic conditions ( 36). Recently, a comprehensive analysis of the fecal microbiota in infants with colic, as compared with control infants, revealed that bifidobacteria and lactobacilli were significantly reduced in infants with colic. Moreover, the colic phenotype correlated positively with specific groups of Proteobacteria but negatively with bacteria belonging to the Firmicutes phyla, which includes some lactobacilli and canonical groups known to produce butyrate and lactate ( 37). Interestingly, several trials have shown that infants with infantile colic benefit from the administration of a Lactobacillus strain, claimed to be of human milk origin ( 38). Therefore, it is not strange that most reviews and meta-analysis have repeatedly confirmed the beneficial effects of some probiotic lactobacilli or bifidobacteria strains for the prevention or treatment of antibiotic-associated conditions ( 39, 40).

Among the bacteria isolated from human milk, species such as L gasseri, L salivarius, L reuteri, L fermentum, or B breve are considered among those with probiotic potential and enjoy the Qualified Presumption of Safety status conceded by the European Food Safety Authority (EFSA). In contrast to other bacteria, these seem to be uniquely adapted to reside in the human digestive tract and to interact with us in symbiosis from the time we are born ( 24). Some studies have shown that human milk lactobacilli may play several roles in the infant gut, such as the increase in the production of functional metabolites such as butyrate, which is the main energy source for colonocytes and a relevant compound in the modulation of intestinal function ( 2). As a result, they improve the intestinal habit, with an increase in fecal moisture, and in stool frequency and volume ( 41). They can also contribute to the reduction of the incidence and severity of infections in the breast-fed infant by different mechanisms ( 42, 43). Recently, the administration of L fermentum CECT5716, a strain isolated from human milk, in a prebiotic containing follow-on formula to infants during an intervention period of 6 months led to 46% ( P = 0.032), 27% ( P = 0.026), and 30% ( P = 0.003) reduction in the incidence rates of gastrointestinal infections, upper respiratory tract infections, and total number of infections, respectively ( 44). Consistently, the same probiotic L fermentum CECT5716 added to a prebiotic containing infant formula also led to a significant reduction of incidence rates of gastrointestinal infections (−71%, P = 0.018) in infants ages 1 to 6 months ( 41). Breast milk bacteria may also participate in the correct maturation of the infant immune system because some strains are able to modulate both natural and acquired immune responses in animal models and humans ( 45- 47).

Finally, the antibiotic-associated loss of lactobacilli and bifidobacteria in milk may also have negative consequences for breast health because of the overgrowth of mastitis-causing agents ( 48). In fact, multiresistance to antibiotics is a common feature among clinical staphylococci involved in such conditions ( 49). This explains why this condition used to be elusive to antibiotic therapy, and why it constitutes one of the main reasons to cease breast-feeding ( 50). In this context, the development of new strategies based on selected probiotic lactobacilli isolated from human milk is an efficient alternative for mastitis treatment ( 27). This suggests that human milk lactobacilli may play important roles in mammary homeostasis.

In conclusion, the results of this study confirm that lactobacilli and bifidobacteria are common members of the human milk microbiota of women who did not receive antibiotics during pregnancy or lactation. Therefore, the presence of such bacteria may be a marker of a healthy non-antibiotic-altered human milk microbiota, and this should be taken into account when defining a criterion standard of breast milk. As a consequence, administration of selected human milk lactobacilli or bifidobacteria to pregnant or lactating women receiving antibiotics, or to their infants, may constitute an attractive approach to restore the natural bacterial ecosystem existing in human milk.

Supplementary Material
Supplemental Digital Content:
Treatment of Jaundice in Colonial America

In 17th century colonial America was a letter written to Governor John Winthrop (1588-1649) of Massachusetts by his physician friend, Edward Stafford, recommending saffron for treating jaundice. Using John Gerard's Herball (1597) as a source, Stafford in 1643 suggested the following recipe:

For the yellow Jaundise or Jaunders - Boyle a quart

sweet milke, dissolve therein as much bay-salt, or

fine Salpeter, as shall make it brackish in taste and

putting Saffron in a fine linen clout, rubb it into

the Milke, until the Milke be very yellow; and give it the [child] to drink.

The recipe was based on the popular Doctrine of Signatures ( similia similubus curantur), which subscribed to the notion that the use of a drug must resemble the disease. Thus, walnut shells were used for head injuries, bear's grease for baldness, turmeric or saffron for jaundice, powdered mummy for aging, thistle for a stitch, and rust or red wine for anemia. The Doctrine was a favorite of Paracelsus and would become the basis of Hahnemann's homeopathy.

- Contributed by Angel R. Colón, MD

Footnotes

This work was supported by a research contract funded by HiPP. Know-how derived from the FUN-C-FOOD (Consolider-Ingenio 2010) and AGL2010-15420 projects from the Ministerio de Economía y Competitividad (Spain) was applied in this study.

The authors report no conflicts of interest.

REFERENCES

29. Iwase T, Uehara Y, Shinji H, et al. Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization. 2010; 465:346-349

47. Pérez-Cano FJ, Dong H, Yaqoob P. In vitro immunomodulatory activity of Lactobacillus fermentum CECT5716 and Inmunobiology 2010; Lactobacillus salivarius CECT5713: two probiotic strains isolated from human breast milk. 12:996-1004

50. World Health Organization Mastitis: Causes and Management. Geneva:WHO; 2000

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Lactobacilli and Bifidobacteria in Human Breast Milk: Influence of Antibiotherapy and Other Host and Clinical Factors

# data[].attributes.attachment_link.description
The objective of this work was to study the lactobacilli and bifidobacteria population in human milk of healthy women, and to investigate the influence that several factors (including antibioteraphy during pregnancy and lactation, country and date of birth, delivery mode, or infant age) may exert on such population.

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We were divided into 8 groups. My group got to do on Gram-Positive bacteria. We made a concept map on that topic using coggle app.

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There are too many expectations to this new semester. First of all, I want to change myself to be a better person. Achieve the dreams especially to make my parents proud with my own result as I did during primary and secondary school as well. My pointer for first sem is a big regret to myself as other coursemate got better pointer. However, I will try my best for this new semester.
I really hope I could manage time better than last sem. It was a regret when I can't manage my time well as I involved with PALAPES. I did like to join PALAPES since primary school when I was involved in an army camp. We were taught to make singgang batu using batu dalam sungai tu. The taste was incredibly same with singgang ikan . Seriously, it was damn sad when I had to quit PALAPES because of my leg's injuries.

Fighting for the new journey

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My adopted microbes for my PK project is about Bioluminescent. There're two people that presented as same as me but not to same at all. Syahida presented about the Vibrio Ficheri which is the bacterial bioluminiscence that live inside the Bobtail Hawaiian squid meanwhile, Tharashini presented about "Glow in The Dark."

Actually my PK is more on researches and about the dinoflagellate. There is a microbiologist that I admired named Dr. Siouxsie Wiles which also bioluminescent ethuasiast. She deals with this microbes. Specifically, she makes them glow. Why? To save lives.

She said in a talk in TEDxchristchurch, "Out of every four people who are admitted into a hospital overnight in New Zealand are there because of infectious microbes. We are running out of medicines to treat these infections."

At Wiles's lab - the Bioluminescent Superbugs Lab at University Auckland - she and others work to make that task easier, using the light-producing bacteria created via bioluminescent organisms. They use the glowing bacteria like mini torches by putting them inside of animals and they use sensitive cameras to see where they are, how many there are and how changes over time without having to euthanize the animals.

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I interview about the multi-degrading microbes which called Burkholderia cepacia strain DRY27 that can degrade both acrylamide and diesel. The level range of temperature from 10 - 40°C.

Acrylamide used in paper industries,  agricultural and also oil in mineral processing in form of polymer acrylamide which is called polyacrylamide. It also used in paper industries, it will produce the monomer and waste. In agriculture, the acrylamide used in production of decomposes. It cannot control as they will absorb into the soil. So, we need this microbes to remove acrylamide as it have potential as neurotoxicant so, need to remove them from the environment.

Dr Mohd Tarmizi bin Yusof explained to us, the advantages of Burkholderia cepacia strain DRY27 is suitable for application in the tropics as the level range of temperature for it to react is from 10 - 40°C.  Burkholderia cepacia strain DRY27 is locally isolated so it is easier to find this microbes to be used as some has experimented that, paper used to absorb the oil in the river.

Currently, there are no multi-degrading microbes have been reported as commersialised.

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On the topic Fungi, my interest is about this toadstool mushroom,

Amanita muscaria as i think it has a beautiful name such as human name. This mushrooom is one of the most dangerous mushroom. Dr Wan also make a joke about if you all have a child later, you named her as Amanita lol.

If your friend have eaten this poisonous mushroom in the forest, what will you do?

Give him/her charcoals as when we sent him/her to ER, the doctor will give him/her charcoals in a form of tablets.

How can you know if him/her has eaten this poisonous mushroom? So here, I share a website about this.

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Mushroom Poisoning Syndromes - North American Mycological Association

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There are many different types of mycotoxins. Of 14 distinctive types of mushroom poisoning found worldwide, so far about 10 distinctive patterns of reactions to mycotoxins have been observed in North America.

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We had our class in computer lab for this topic, Protist. We discuss about this topic in the group and divide the parts to the note for each group members. I did the part Pyrrophyta.

Pyrrophyta or known as Dinoflagellate. The single-celled dinoflagellates are bioluminescent. When conditions are right, dinoflagellates bloom in dense layers at the surface of the water, causing the ocean to take on reddish-brown color in daylight and a sparkly sheen as they move in the wave at night. When they're eaten, the toxic dinoflagellates accumulate in high concentrations in larger fish and filter feeding shellfish. And when marine mammals or people eat these organisms, it can cause sickness or even death.

Here, I share a website about the news of Algal Bloom.

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This time our streaming is quite special as Choy was also there hehe. Choy is a very kind person and he guide us well to make sure we're not feel burden with this project. Thank you Choy! This is our last video conference. A bit sorry as my lappy gone wrong at the end of the video.

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It quite difficult to have the next streaming video as the members were busy with their university activity as they also on their week test. But, Alhamdulillah we managed to do the project well. All of the member cooperate to make sure the things finished and we enjoyed it as well.

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I'm one of the member from team 20. Our adopted microbe is Lactobacillus Bulgaricus. This bacteria is a gram positive bacteria. The reason I suggested this bacteria to my group members as one day when I ate a yogurt, I've an instinct to take a look at the ingredients part of the marigold yogurt since before I didn't notice if there is a part that write about what the microbes used to ferment the yogurt at the yogurt's container. The other members agreed to adopt this microbe as they said, "Why not we adopt this Lactobacillus Bulgaricus as we all love yogurt".

Lactobacillus Bulgaricus probiotics and functional foods are made in Bulgaria. This video is about the technology of preparation, the team, the equipment, the list of products: foods, probiotics, synbiotics, cosmetics.

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Lactobacillus Bulgaricus probiotics and functional foods are made in Bulgaria. This video let you know the technology of preparation, the team, the equipment, the list of products: foods, probiotics, synbiotics, cosmetics

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For thhis entry, I would like to share the infographic about Microscope.

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Sept 23, 2016 @DKU

Our first microbiology conference with other university. Definitely, I'm sooooo excited. The purpose of the conference is about out MEGA project which was the Virtual Microbes. The project in collabs, UPM-UM-UKM-UMT . We definitely met our other friends from the Microbes World!

And, this picture is definitely us with that excited (kind of jakun actually lol) faces. Great time ahead looking with this cute aliens.

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Virtual lab is a very interesting website to learn as I became more understand about this gram staining.

Gram staining used to view cellular morphology and for diagnostic purpose which is about what type of cell wall a bacterium has.

Gram positive = thicker peptidoglycan layer and no outer membrane
Gram negative =  thinner peptidoglycan and an outer membrane.

Reagents used for the staining are crystal violet (purple), iodine, ethanol, safranin (pink).

Function of the reagents :
- crystal violet used as primary stain ; +ve stain and it stains the cell wall purple
- iodine used as mordant and it combines with crystal violet to form an insoluble complex that gets trapped in the thicker peptidoglycan layers. If forget, the ethanol will decolorized the gram positive cells`
- ethanol used as decolorizer. If forget, everything will have crystal violet on it either gram positive or negative.
- safranin used as counterstain. It is a simple +ve stain that provides dye for decolorized cells for gram -ve. It stain all cells, but only -ve ones actually appear pink. If forget, we difficult to visualize and identify gram -ve organism and their thin cells may have bound enogh through crystal violet and will misidentified with gram +ve.

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In last lecture, I had find some information about the history of vaccination which is about Edward Jenner that inoculated his own son to discovered the vaccine for swinepox. Here, I'll share about it. In 1789, Jenner decided (just after he had been elected as a Fellow of the Royal Society) to try immunizing (or what he thought was immunizing) his ten month old son, Edward, Jr, and two of his neighbor’s servants, by inoculating them with swinepox.

He had learned well from his famous teacher, Dr John Hunter, that one will learn more by “trying the experiment” rather than by just speculating about it. So Jenner performed the experiment by making a small scratch on the servants’ and the baby’s arms with a lancet and then infecting the scratch “with matter from a pustule of the baby’s nurse, who had caught the swinepox infection.” Eight days later baby Edward took sick and developed sores, but he did eventually recover.

Then, two years later, Jenner again challenged his son with smallpox again, this time, with unhappy results. This time there was a reaction, and a severe one. But he quickly recovered, and a year later Jenner inoculated him with smallpox once again.

Unfortunately, however, in the years following these experiments, young Edward “became a sickly child and exhibited signs of mild mental retardation,” likely due to neurological damage.

Jenner’s son died at age 21 from tuberculosis, which has been linked to the smallpox vaccine. Despite his death, Jenner continued testing his vaccine on many others producing varying results such as vaccine-induced smallpox and vaccine-induced tuberculosis.

Jenner, in 1798, formulated a new vaccine, which combined “horse-grease” and cow-pox matter as he had proclaimed his first formulation in 1796 as having “no protective virtue.” His new vaccine was met with public disdain and disgust and his experiments failed. He was ridiculed. Jenner then returned to promote his original formulation.

By 1807, he convinced the Royal College of Physicians and the British Parliament that his once defunct and admittedly unprotective vaccine was safe and effective, and as well could produce large revenues. Not trusting the scientific solutions being offered, some countries banned variolation. Yet, quite surprisingly, Jenner’s vaccine became compulsory and mandatory in many other countries. The smallpox vaccine was widely used until 1979 when the World Health Organization (WHO) declared smallpox eradicated from the face of the earth.

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I would like to share some knowledge that my group members and I had present. I'm one of the member from group 7. We presented on the topic The Fluorescence Microscopy. Here, I would like to share a video that explain more details about the fluorescence microscopy. It usually use in medical microbiology and microbial ecology studies. Hope you'll enjoy the video and understand more about the this microscopy

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Talking about the basics of Fluorescence microscopy, photobleaching, quantum yield, stokes shift

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On Thursday, 14092016, I had learnt about the different between procaryotes which are only Bacteria and Archaea and eucaryotes which are the other microbes. What eucaryotes have but procaryotes don't have are, nucleus, mitochondria, golgi apparatus and endoplasmic reticulum. Besides, on that day, my group and I had to discuss about archaea. Archaea is a single-celled microbes. Its cell wall lack peptidoglycan(polymer consists of sugars and amino acids that form outside the plasma membrane) but they have pseudopeptidoglycon. Meanwhile, archaea can survive in extreme conditions(high temperature, high pressure, low pH) as they have more stable stable membrane chemistry. Its amazing right?! . I really enjoy the class with Dr Wan very much as the group formed make we cooperate with each other to search more about the microbes that we had learn and share the information to the class. We also can know either the information that we searched true or not as the website maybe mistype or stated the wrong facts about the microbes.

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Assalamualaikum and hye! For my first reflective journal, I would like to talk about my first impression about Microbiology. I was exposed to this course when I came to Faculty of Biotech's booth at edufair last March which is organized at UPM Serdang. I think this course is very interesting when it come to occupation sectors. Moreover, I really amazed with the specific name of the microbes and also their harmful and benefits to us. As I attended my first Microbiology 1 with Dr Wan, I never doubt my first expectation about this course. Dr Wan really make me more curious and increased my spirit to explore more. I hope, I will get along with this course till the end. I will try my best to pass with flying colours even I'm the only one from this course that stay in KOSASS.