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Triple-sugar iron agar (TSI agar) is another example of a multi-test agar. It also tests for the production of hydrogen sulfide from amino acids. Phenol red is the pH indicator used in this test medium.
Procedure
- Obtain a slant of TSIA.
- Using an inoculating needle, stab your assigned organism into the butt of the TSIA slant. As you remove the inoculating needle, drag it in a zigzag pattern up the surface of the slant portion of the tube.
- Incubate the slant for 24-48 hours.
- After the incubation period, record any changes in the tube.
Interpretation
Review your results from Carbohydrate Fermentation and note the following:
- Slant color/butt color: Slant color indicates the fermentation of lactose and/or sucrose.
- Butt color indicates the fermentation of glucose.
- Production of gas: Agar shows bubbles or may split.
- Production of H2S: H2S formation is indicated by a blackening of the medium.
Left: A/A, H2S +, Gas +
Right: K/A, H2S +, Gas +
Biology 2420 serves an introduction into the microbial world. This course is a combination of both lecture and laboratory work. It includes the basic characteristics of bacteria, fungi, algae, and viruses. In addition, this course focuses on human diseases, the organisms that cause them, treatment and prevention. A laboratory section accompanies this course that will focus on identification of bacteria. Lab work will include microscopy, bacterial culture techniques as well as basic staining. This course is designed for nursing students but is applicable to all Allied Health Sciences. Introduction to Anatomy, Introduction to Physiology, or Anatomy and Physiology 1, with a minimum grade of C or better, (or equivalent with lab) is a prerequisite for this class. In addition, each student needs to demonstrate reading, writing, and mathematics proficiency as determined by the COMPASS or ASSET test, or by the statewide THEA test, or by providing an official transcript from another college.
Required Textbooks/Materials
Authors: Michael J. Leboffe & Burton E. Pierce
- Safety goggles or glasses with a safety rating of ANSI Z87.1.
- Old shirt or lab apron/coat
- Sharpie or glass marking pen
- Lab notebook
Course Rational
Course rational includes: (1) to adequately prepare students for the health science programs at ACC and professional work, (2) to develop your critical-thinking and problem-solving skills, and (3) to reinforce your abilities to follow directions and meet deadlines. Specific skills and competencies are expected of students who successfully complete this course including:
§ ability to identify various diseases and their possible causes
§ ability to observe phenomena and to record and analyze data
§ ability to infer from data
§ ability to demonstrate higher level thinking skills
§ ability to solve problems
§ ability to obtain information from graphs
§ ability to manipulate equipment
§ ability to work effectively in a group
§ ability to work safely in a lab setting
§ ability to follow directions
Instructional Methodology
Announcements and lecture notes will be posted on Blackboard. I will post updated lectures on Blackboard Monday of the week that they are scheduled. To be successful in this course, you will need to read the textbook in addition to the lecture notes, and complete assignments as posted on Blackboard or given in class. Some lectures will be assigned as homework to allow for discussion and critical thinking assignments in class. All assignments and exam scores will be posted on Blackboard but final grades will not be calculated using Blackboard.
I want you to succeed in this class. I will do what I can to help you. However I expect you to do your part too. I expect you to read the textbook assignments, to ask questions when you don&rsquot understand the material, to come visit me during my office hours to clarify class notes or other things you might need help with, and to spend a reasonable amount of time studying. Do not expect to be able to cram for the test. Microbiology is like a new language and it takes time to be able to learn it. Study with a study group on a regular basis.
Course Objectives
The common course objectives for Introduction to Microbiology are available at the departmental websitehttp://www.austincc.edu/biology/commoncourseobjectives.html.
Lectures In-Class and At-Home
I have provided copies of my lecture notes on Black Board to assist you in taking notes during lecture and studying for tests. Most lectures will be covered in class but there are a few &ldquoAt Home&rdquo lectures designated on the &ldquoLecture Schedule&rdquo handout. You will be required to read the assigned reading in the textbook and follow-up with the lecture notes that accompany the assigned reading. In-class assignment questions will often cover material found in the &ldquoAt Home&rdquo lectures so it is in your best interest to complete the lecture before completing an in-class assignment.
In-Class Assignments and Homework
There will be in-class assignments given throughout the semester. They will be worth approximately 25-40 points. Most of the assignment will be completed in-class and turned in at the end of class. No make-ups are available for these assignments unless previously discussed with me.
Occasional outside of class homework assignments will be given. The number of assignments and how much they will be worth will be determined according to the needs of each class. Each homework assignment will be worth approximately 10-20 points each. Homework must be turned in by 12:00 am (midnight) of the date due. You can either e-mail homework assignments or turn them in on paper. Due dates will be given the day homework is assigned.
Consider the textbook reading assignments included in the lecture schedule as part of your assigned homework. It will help you to understand the material discussed in class and will contribute to your success in this class.
Case Studies (If there is time)
You may be given several short case studies to complete during the semester worth approximately 10-20 points each. Each case study will include the description of a disease that you may encounter in a clinical setting. Your job will be to determine the disease and write up a rationale. Case studies must be turned in by 12:00 am of the date due. You can either e-mail the case studies to me or turn them in on paper. Due dates will be given the day the case study is assigned.
There will be 4 lecture exams worth 100 points each. They will be made up of a combination of short answer essay, problem solving, diagramming, multiple-choice, true/false, and matching. Each exam will be a combination of written and computer based testing through black board and administered in the Testing Center. Exam dates are listed on the Lecture Schedule but are subject to change slightly. Any changes will be announced in class.
| Lab Schedule. Biol 2420. Schrass. Summer 2021 | |||||||
| week | date | Lab book | Topic | lab manual pages | Microbugz | Pearson's MicroLab Tutors/videos | Bb content |
| Week 1 | 2-Jun | Introduction to Lab and Microorganisms and Safety | 1-7 | Introduction Lab | Safety in the Microbiology Laboratory | ||
| Ex. 1-1 | Comparison of Hand-Cleansing Agents | p. 17 - 20 | Handscrubbing Handout | none | Practical 1: Hand Washing Lab | ||
| Ex. 2-1 | Ubiquity of Microorganisms | p. 57 - 62 | Ubiquity of Microorganisms (under Mirobial growth) | none | Practical 1: Ubiquity of Microorganisms | ||
| Week 2 | 7-Jun | Ex. 2-2 (Colonial and Cellular Handout) | Colony Morphology | p. 63 - 74 | Bacterial Data Sheet: Colonial and Cellular Morphology. Cultural Characteristics Lab | none | Practical 1: Colonial Morphology |
| Ex. 1-3 (Inoculation Handout on Microbugz) | Aseptic Transfers | p. 29 | Innoculation Handout | Aseptic Transfer of Bacteria | Practical 1: Aseptic Technique | ||
| Ex. 2-4 | Growth Patterns in Broth | p. 79 | Growth Patterns in broth Lab | Pratical 1: Growth Patterns in Broth | |||
| Ex. 1-4 | Streak Plate Method of Isolation Fig. 6.9 I the textbook | p. 41 | Streak Plate Lab | Streak Plate Technique | Practical 1: Streak Pate for Isolation | ||
| 9-Jun | Ex. 3-1 | Introduction to the Light Microscope | p. 143 - 160 | Microscope Use and Care Handout. Intro to Light Microscope Lab | Microscopy Penguin Prof Chater 4 in the textbook | Practical 1: Microscopy | |
| Ex. 3-3 | Examination of Eukaryotic Microbes | p. 161 | Examination of Eukaryotic Microbes Lab | ||||
| Ex. 3-11 | Wet Mount | p. 221 | Wet Mount and Hanging Drop Lab | ||||
| Week 3 | 14-Jun | Ex. 3-5 (Staining Chart) | Simple Stains | p. 185 | Stain Chart Handout . Simple Stains Lab . Bacterial Data Sheet: Colonial and Cellular Morphology | Smear Preparation | Practical 1: Simple Stain |
| Ex. 3-6 (Stain Protocol Handout) | Negative Stain | p. 191 | StainProtocols Handout. Negative Stain Lab | Practical 1: Negative Stain | |||
| Ex. 3-7 | Gram Stain | p. 195 | Gram Stain Lab | Gram Stain | Practical 1: Gram Stain | ||
| Ex. 3-10 | Endospore Stain | p. 215 | Endospore Lab | Practical 1: Endospore Stain | |||
| Ex. 3-8 | Acid-Fast Stains Catalyst U | p. 203 | Acid-Fast Stain Lab | Acid Fast Stain | Practical 1: Acid Fast Stain | ||
| Ex. 5-6 | Catalase Test | p. 321 | Catalase Test Lab | Practical 3: Staphylcoccus and Micrococcus Lab: Catalase Test | |||
| 16-Jun | no lab today so that students can take Exam I | ||||||
| Week 4 | 21-Jun | Ex. 3-13 (Morph Unknown Data Sheet) | Begin Morphological Unknown video on the dichotomous key | p. 229 | Morphological Unknown Lab and Data Sheet Handout | p. 119 textbook Dichotomous Key flow chart | Course Documentss: Projects: Unknown Project 1 - Morphological Unknown |
| 23-Jun | Ex. 2-7 | Fluid Thioglycollate Medium | p. 95 | Fluid Thioglycollate Broth Lab | B. Subtilus E.coli S. pyrgenes | Practical 1: Fluid Thioglycolate Medium | |
| Ex. 2-8 | Anaerobic Jar/Candle Jar | p. 99 | Anaerobic Jar. Candle Jar Lab | Practical 1: Anaerobic Jar | |||
| Ex. 5-28 (Motility Handout on Microbugz) | Motility Test | p. 437 | Motility Handout | Practical 1: Moility Media | |||
| Ex. 3-12 | Flagella Stain (Prepared Slides) | p. 225 | Flagella Stain Lab | Practical 1: Flagellar Stain | |||
| 28-Jun | Lab will not meet so that students can take Exam II | ||||||
| Week 5 | 30-Jun | Ex. 3-9 | Capsule Stain Catalyst U | p. 211 | Capsule Stain Lab | Practical 1: Capsule stain | |
| Importance of Biochemical Testing | Practical 2: Why are Biochemical Tests Important in Microbiology | ||||||
| Ex. 5-13 | Starch Hydrolysis (SA) | p. 361 | Practical 2: Starch Hydrolysis | ||||
| Ex. 5-17 | Gelatin Hydrolysis | p. 379 | Gelatinase Test Lab | Practical 2: Gelatin Hydrolysis | |||
| Ex. 5-14 | DNA Hydrolysis (DNase) | p. 367 | Dnase Test Lab | Practical 2: DNase Hydrolysis | |||
| Ex. 5-16 | Casein Hydrolysis (SMA) | p. 375 | Casease Test Lab | Practical 2: Casein Hydrolysis (Skim Milk Agar) | |||
| Ex. 5-15 | Lipid Hydrolysis (TRB) | p. 371 | Lipase Test Lab | Practical 2: Lipid Hydrolysis | |||
| Ex. 5-3 | Phenol Red Broth Catalyst U. | p. 303 | Phenol Red Lab | Practical 2: Carbohydrate Fermentaion Test and Phenol Red | |||
| 5-Jul | Holiday no classes are scheduled | ||||||
| Week 6 | 7-Jul | IMViC | Indole, Methyl Red, Vogues Proskauer | Practical 2: IMViC | |||
| Ex. 5-20 | SIM Medium | p. 393 | SIM Medium Lab | Sulfur Reduction Indole Production & Mobiity | Practical 2: SIM | ||
| Ex. 5-4 | Methyl Red and Voges-Proskauer Tests | p. 311 | Methyl Red and Voges-Prokauer Test Lab | Practical 2: MRVP | |||
| Ex. 5-9 | Citrate Test | p. 339 | Citrate Test Lab | Practical 2: Citrate Test | |||
| Ex. 5-8 | Nitrate Reduction Test | p. 333 | Nitrate Reduction Test Lab | Practical 2: Nitrate Redution Test | |||
| Ex. 5-18 | Urea Hydrolysis (Broth) | p. 383 | Urease Test Lab | Practical 2: Urea Hydrolysis | |||
| Enteric Labs | Practical 3: Enteric Lab Daily Handout | ||||||
| Ex. 5-7 | Oxidase Test | p. 327 | Oxidase Test Lab | Practical 3 Enteric Lab: Oxidase Test | |||
| 12-Jul | Ex. 5-21 | Triple Sugar Iron Agar (TSI)/Kligler Iron Agar | p. 401 | Triple Sugar Iron Agar Lab | |||
| Week 7 | Ex. 5-11 | Decarboxylation Test | p. 349 | Decarboxylase Test Lab | Practical 3 Enteric Lab: Decarboxylase Test | ||
| Ex. 5-12 | Phenylalanine Deaminase Test | p. 357 | Phenylalanine Deaminase Test Lab | Practical 3 Enteric Lab: Phenylalanine Deaminase Test | |||
| Ex. 4-6 | Eosin Methylene Blue Agar (EMB) | p. 267 | Eosin Methylene Blue Agar Lab | Practical 3 Enteric Lab" Eosin Methyleme Blue Agar | |||
| Ex. 4-7 | Hektoen Enteric Agar (HE) | p. 273 | Hektoen Enteric Agar Lab | Practical 3 Enteric Lab: Hektoen Enteric Agar | |||
| 14-Jul | Ex. 4-5 | MacConkey Agar (MAC) | p. 259 | MacConkey Agar Lab | Practical 3: Enteric Lab: MacConkey Agar | ||
| 19-Jul | lab will not meet so that students can take Exam III | ||||||
| Week 8 | Staphlococcus and Micrococcus Lab | Practical 3: Staphlococcus and Micrococcus Lab Daily Handout | |||||
| Ex. 5-25 | Blood Agar | p. 423 | Blood Agar Lab | Practical 3: Staphlococcus and Micrococcus Lab: Blood Agar | |||
| Ex. 4-1 | Phenylethyl Alcohol Agar (PEA) | p. 237 | Phenylethyl Alcohol Agar Lab | Practical 3: Staphlococcus and Micrococcus Lab: Phenylethyl Alcohol Agar | |||
| Ex. 4-4 | Mannitol Salt Agar (MSA) | p. 253 | Mannitol Salt Agar Lab | Practical 3: Staphlococcus and Micrococcus Lab: Mannitol Salt Agar | |||
| Streptococcus and Enterococcus | Practical 3: Directions for Streptococcus and Enterococcus lag | Practical 3: Streptococcus and Enterococcus: Daily Handout | |||||
| Ex. 4-3 | Bile Esculin Test | p. 249 | Practical 3: Streptococcus and Enterococcus: Bile Esculin handout and Bile Esculin Test | ||||
| Ex. 5-24 | Bacitracin Susceptibility Test (and Optochin A &P Disks) | p. 417 | Practical 3: Streptococcus and Enterococcus: Glood agar with A disc and P Disc | ||||
| 21-Jul | 6.5% Salt Tolerance Test | (Salt Tolerance Handout ) 6.5% Salt Tolerance Test | Practical 3: Streptococcus and Enterococcus: Salt Tolerance handout and Salt Tolerance test | ||||
| 26-Jul | Ex. 5-31 (Mixed Unknown Data Sheet) | Begin Mixed Unknown | p. 457 | Mixed Unknown Data Sheet | video at the end of PP | ||
| Week 9 | 28-Jul | HHMI Bacterial Identification lab | link to online virtual lab & worksheet is in the Bb Week by Week | ||||
| HHMI Immunology lab | |||||||
| Week 10 | no labs this week so that students can take exam IV and the final as well as complete their Mixed Unknown data sheets | ||||||
LECTURE COURSE OBJECTIVES
IN BIO 2420 students will learn to:
1. Discuss the germ theory of disease and its development.
2. Discuss aseptic procedures for the preparation of media and materials for the culturing and growth of microbes.
3. Enumerate and differentiate among the different groups of organisms included for study in microbiology.
4. Discuss and distinguish between eukaryotic and prokaryotic cell types.
5. Discuss energy acquisition and utilization by microbes and the function of enzymes in cellular activities.
6. Discuss microbial metabolism, including anabolic pathways and glycolytic, fermentative and respiratory catabolic pathways.
7. Discuss fundamental nucleic acid chemistry regarding the principle of complementarity, DNA replication, the genetic code, protein synthesis, metabolic regulation, and cellular reproduction.
8. Discuss microbial genetics including, but not restricted to sexual versus asexual means of reproduction, and transformation, transduction and conjugation in bacteria.
9. Discuss methods utilized in biotechnology as applied to microbes, including the role of microbes and biotechnology in industrial microbiology, the pharmaceutical and food industries, and systematic and diagnostic microbiology.
10. Discuss symbiotic relationships, including commensal, mutualistic, and parasitic relationships among hosts and microbes.
11. Discuss disease processes, the cellular structures, metabolic and genetic activities, and biological and chemical agents employed by microbes in colonizing, infecting, invading, and causing diseases in hosts.
12. Discuss the processes, both nonspecific and specific, employed by hosts in resisting the onslaught of infectious diseases.
13. Discuss the principles and methods of diagnosing diseases, identifying disease-causing agents, and tracking and enumerating diseases around the world.
14. Discuss the signs, symptoms, etiology, course, prevention, control, diagnosis, and treatment for the most common infectious diseases of all organ systems of the human.
Main text
Methods
Source of animals
Sick pigs were owned by individual farmers, who contacted The Animal Biomedical and Molecular Biology Laboratory of Udayana University reporting sickness and mortality in their piggery. They agreed for their sick and dead animals to be included in the study. The farms were located across Bali Province (Table 1).
During January to July 2018, 30 suspected S. suis cases were recorded. The inclusion criteria were acute illness with at least one clinical sign of neurological disorder, reddish skin discoloration and arthritis. The animals were not treated with antibiotics.
Freshly dead animals were necropsied. Organs with clear pathological lesions were collected in Stuart Transport Medium (CM0111 Oxoid) and buffered formalin. Isolation and biochemical characterization were conducted according standard protocol [16]. The tissues from one animal were pooled and extracted. The suspension was plated on a 5% defibrinated sheep blood agar plate. The plate was incubated at 37 °C for 18–24 h. Some suspected colonies were Gram stained and grown in triple sugar iron agar and sulfide indole motility media. Other tests were catalase, oxidase, citrate, methyl red, Voges–Proskauer (VP), glucose and lactose tests. Three suspected colonies were injected to separate tryptic soy broth (Sigma Aldrich MFCD00132536) and incubated at 37 °C for 18–24 h. DNA was isolated using 10% chelex-100 (Biorad, CA) [17, 18]. The glutamate dehydrogenase (GDH) and recombination/repair protein (recN) gene fragments were amplified using polymerase chain reaction (PCR) using published specific primer sets for S. suis [19, 20]. The GDH and recN fragments were sequenced by Apical Scientific Sequencing (Malaysia) using an automatic chain termination method. Serotyping was conducted using PCR to detect the cps1I gene for serotype 1 and 14 and the cps2I gene for 2 and 1/2 as recommended [21].
Tissue was processed and stained with hematoxylin and eosin (H&E) staining based on a published protocol [22].
Results
Out of 30 cases, 8 were indicative for S. suis and showed small and non-hemolytic colonies that were Gram positive with coccus-chain forming appearance. Only positive cases are described further in this manuscript. The epidemiological and clinical data of the presumably positive cases are presented in Table 1. Listed from the most frequent, clinical signs were reddish skin discoloration, anorexia, nasal exudate, diarrhea, limping, eye exudate, lethargy, swollen joints, shivering, weakness, cough, lack of appetite, depression, dyspnea, tremor, fever and snot. Animal were aged 3–12 months. The cases were from Denpasar, Gianyar, Tabanan and Karangasem regencies.
The results of Gram staining and biochemical tests of suspected colonies from the eight suspected S. suis infections are presented in Table 2. All isolates were Gram (+), coccus form, short chained and positive in acid slant, catalase and lactose, but negative in VP test.
After electrophoresis of PCR products (not shown), positive samples showed a single band of around 700 bp for GDH and 350 bp for recN. The GDH sequences of five isolates of S. suis from our study were identical. The blast result showed a percentage identity (PID) of 99.09% to human isolates from Bali Indonesia as published previously [10]. The sequences of recN of two isolates show a PID of 100% to human isolates, while other two each have 98.5%. Upon PCR serotyping, two isolates were confirmed to be serotype 2 or 1/2. Two cps2I sequences of animal isolates in our study are completely identical to those of human isolates [10]. The GenBank Acc. No. of GDH, recN and cps2I for serotype 2 or 1/2 are MN334770–MN334774, MN520294–MN520297, and MN520292–MN520293, respectively.
A panel of histological pictures of confirmed cases is depicted in Fig. 1. The histopathologies of confirmed cases were similar with varying severities. The prominent pictures were congestion in the brain, meningitis, bronchopneumonia, endocarditis, myocarditis, pericarditis, erosion and enteritis along the gastrointestinal tract with obvious depletion of Payer patches, hemorrhagic hepatitis and glomerulonephritis, as well as lymphoid depletion, hemorrhage and accumulation of inflammatory cells in the spleen. Dominant inflammatory cell infiltration in those tissues was neutrophil and macrophage.
Histopathological pictures of various tissues of confirmed S. suis infection in pig cases in Bali, Indonesia, 2018. Panel a1–2 are brain and meninges showing meningitis a3–4 are lung tissues showing bronchopneumonia b1–2 are myocardium showing endocarditis b3–4 are myocardium showing pericarditis c1–2 are liver showing congestion and hepatitis c3–4 are intestine showing hemorrhagic enteritis with depletion of Payer patches d1–2 are kidney showing hemorrhagic glomerulonephritis d3–4 are spleen showing perifollicular infiltration of inflammatory cells and hemorrhage H&E stained. Magnifications in column 1 and 3 are ×100 Column 2 and 4 are ×400
Discussion
We confirmed that S. suis does present and cause illness in pigs in Bali. Human cases confirmed at Bali Referral Hospital [10] must have originated from pigs. Streptococcus suis meningitis is a global zoonotic community-acquired bacterial meningitis [1, 2, 4]. The bacterium is of extra importance in Asia as human outbreaks are related to traditional pork consumption practices [5, 6]. The practice of consuming a delicacy of raw pork with raw blood is also common in Bali.
In this study, we carefully selected suspected cases, especially those of acute cases with no history of antibiotic medication. The cases occurred in pigs under 1 year old. Although S. suis can be isolated from sick and healthy pigs at various ages [23], clinical manifestation seems more frequent in young animals [24]. A study in Canada [25] showed that S. suis was more frequently isolated from pigs aged between 5 and 10 weeks.
We recorded clinical signs in our cases involving many organs of central nervous, respiratory, urogenital and circulatory systems and gastrointestinal tract. Recorded clinical signs of S. suis infection in the literature can indeed be multi-organ. The signs can be pyrexia, lack of appetite, depression, nasal discharge, dyspnea, tremors, seizures, incoordination, unusual stances (such as sitting like a dog), inability to stand, paddling, opisthotonos, convulsions, nystagmus, skin disease, swollen limbs and death [26]. In some cases, the disease goes per-acute and ends with sudden death without obvious signs [26]. Although septicemia and meningitis are the most striking manifestations of the disease, endocarditis, pneumonia and arthritis have been reported [27]. Another review article also reported that the disease syndromes caused by S. suis in swine include arthritis, meningitis, pneumonia, septicemia, endocarditis, polyserositis, abortion and abscesses [28]. In a recent experimental infection, affected pigs presented clinical signs of anorexia, depression, fever, glazed eyes, reddened mucous membranes, severe nervous symptoms (incoordination, lateral prostration, paddling, opisthotonos, convulsions and lameness in the posterior limbs) [29].
Our suspected cases were from all regencies in Bali Province. However, the confirmed cases were from four regencies, namely Tabanan, Denpasar, Gianyar and Karangasem. Considering that Bali is a small island of 5.600 km 2 with a high population density (http://www.baliprov.go.id/v1/geographi) and understanding the free movement of animals in the province, we assume that S. suis is distributed throughout the province. The morbidity, mortality and case fatality rates in our study were 18.7%, 8.4% and 44.9%, respectively. Morbidity, mortality and case fatality rates of S. suis in pigs vary [30]. Therefore, we assumed that our observation on the case epidemiology is plausible.
Our microbiological data confirmed S. suis. Gram staining, chain formation and biochemical characterization shows that identified isolates were Gram (+), coccus with grape-like or short chain. All were acid slant, catalase and lactose positive, while VP test was negative. S. suis is an encapsulated gram-positive bacterial coccus that occurs singly, frequently in pairs, or occasionally in short chains [3]. Most strains are alpha-hemolytic on bovine and sheep blood agar plates after 24 h of incubation at 37 °C [3]. Four tests are used for a presumptive identification of S. suis, that is, no growth in 6.5% NaCl agar, a negative VP test and production of acid in trehalose and salicin broths [25, 31]. The VP test is critical in differentiating S. suis from other Streptococcus species [15].
Final confirmation was made using PCR of GDH and recN. Both gene fragments are proposed as a system for reclassifying S. suis or as a specific PCR system for S. suis [19, 20]. We have established the system for S. suis detection from human and animal samples at Udayana University, Bali. The GDH blast result showed a PID of 99.09%, while four recN sequences showed PIDs of 98.5% and 100% to human isolates from Bal [10]. Upon PCR serotyping, two isolates were positive with primer sets for serotype 2 or 1/2 but not those of serotype 1 and 14. Only two primer sets were available in our lab. The readable sequences were identical to the S. suis serotype 2 and 1/2 Cps2I gene Ref. [21].
Although we carefully selected the cases with inclusion criteria of suspected cases that were acute and had at least one clinical signs of neurological disorder, reddish skin discoloration and arthritis, as well as no history of antibiotic treatment, only 8 of 30 suspected cases were confirmed as S. suis infection. The animals were not treated with antibiotics. The negative cases might have been caused by other infectious agents such as Haemophilus parasuis, pseudorabies or Escherichia coli [32].
Prominent histologic pictures were congestion in the brain and meningitis, bronchopneumonia, myocarditis, erosion and enteritis along the gastrointestinal tract, hemorrhagic liver and kidney. Dominant inflammatory cell infiltration in those tissues was neutrophilic. Gross and microscopic findings of S. suis include one or more of fibrinous polyserositis, fibrinous or hemorrhagic bronchopneumonia, purulent meningitis, myocardial necrosis, focal myocarditis and valvular endocarditis [30]. Moreover, meningoencephalitis, a striking lesion, has been found in China [29]. Meningitis histology in one case (Fig. 1, panel A1–2) resembles meningitis as described by that group.
This study was the result of passive surveillance in which the owners contacted our laboratory reporting sickness and mortality in their piggeries. The study did not investigate the link of animal cases to human infections. Active surveillance should be conducted to determine the overall risk factors and elucidate the link between animal isolates and human cases. That the GDH sequences of animal isolates are not identical to those of human isolates and the variation of recN and the confirmed serotype of 2 or 1/2 in two out of eight isolates expands our knowledge that the circulating bacteria might vary in the province. This should be mapped as it will benefit understanding of human transmission.
In conclusion, Streptococcus suis has been confirmed in sick pigs in Bali, Indonesia. Two isolates were confirmed to be serotype 2 or 1/2. Further research is needed to elucidate the risk factors for human infection and to map the distribution of S. suis in Indonesia. The fragment of GDH or recN might be used to map infections in Indonesia. Vaccines can be developed using inactivated strain [33] to reduce economic losses and the risk of human infection, including among domestic and international travelers.
2.3 Surgical Wound Classification
The surgical lesion has been classified into 4 classs:Table 2.3 Surgical Wound ClassificationClass ICleanAn clean secret agent lesion in which no redness is encountered and the respiratory, alimental, venereal, or cleanurinary piece of land is non entered. In add-on, clean lesions are chiefly closed and, if necessary, drained with closed drainage.
Operative incisional lesions that follow nonpenetrating ( blunt ) injury should be included in this class if they meet the standards.Class IIClean-ContaminatedAn operative lesion in which the respiratory, alimental, venereal, or urinary piece of lands are entered under controlled conditions and without unusual taint. Specifically, operations affecting the bilious piece of land, appendix, vagina, and oropharynx are included in this class, provided no grounds of infection or major interruption in technique is encountered.Class IIIContaminatedOpen, fresh, inadvertent lesions. In add-on, operations with major interruptions in unfertile technique ( e.
g. , unfastened cardiac massage ) or gross spillage from the GI piece of land, and scratchs in which ague, non-purulent redness is encountered are includedin this class.Class IVDirty-InfectedOld traumatic lesions with maintained devitalized tissue and those that involve bing clinical infection or perforated entrails. This definition suggests that the beings doing postoperative infection were present in the operative field before the operation.( Alicia J. Mangram, April 1999 )
A figure of patient-related and procedure-related factors have been shown in univariate or multivariate analyses to act upon the hazard of SSIsTable 2.4 Hazard factors that impacting the SSIPatient related factorsProcedure related factorsAge, e.g. advanced age and utmost ageNutritional position, e.g.
malnutrition and recent weight lossUncontrolled blood sugar degree, e.g. Diabetes MellitusSmokingFleshinessAltered immune position, e.g. HIV/AIDS, chronic steroid usage, old chemo/radio-therapyLength of pre-operative infirmary stayCo-existent infection at other portion of the organic structureAlcoholismColonization with microorganisms ( peculiar Staphylococcus aureus )CancerDuration of surgical chaparralSkin asepsisPreoperative shavePreoperative tegument readyingDuration of operationAntimicrobial prophylaxisOperating room airingInadequate sterilisation of surgical instrumentsForeign stuff in the surgical siteSurgical drainsSurgical techniquehapless hemostasisfailure to kill dead infinitetissue injury( Alicia J. Mangram, April 1999 )Harmonizing to the survey at 1996, it shows that mild perioperative hypothermia, which is common during major surgery, may advance surgical-wound infection by triping thermoregulatory vasoconstriction, which decreases hypodermic O tenseness ( ANDREA KURZ, May 1996 ) . Reduced degrees of O in tissue impair oxidative violent death by neutrophils and diminish the strength of the healing lesion by cut downing the deposition of collagen ( ANDREA KURZ, May 1996 ) . Hypothermia besides straight impairs immune map ( ANDREA KURZ, May 1996 ) .
However, there are still missing of clinical informations to back up that the patient ‘s organic structure temperature will increase the hazard of SSI.Hyperglycemia impairs the unsusceptibility system of the patient. High blood sugar lead to non-enzymatic glycation of proteins that can demobilize the Immunoglobulin G ( IgG ) by diminishing complement arrested development and increase collagenase activity ( Hennessey et al. , 1991 ) . Hyperglycemia besides impaired the leucocyte maps which lead to detain of chemotaxis, impair phagocytosis and hinder bacteriocidal activity ( Mowat and Baum, 1971 Chang, 1979 ) .
Characterization of the unusual non-thiosulfate-reducing Edwardsiella tarda isolated from eel (Anguilla japonica) farms
Unusual Edwardsiella tarda, that do not produce hydrogen sulfide (H2S) from thiosulfate, was isolated from diseased eels (Anguilla japonica) in varying farming sites. In this study, we examined the biochemical characteristics of H2S production from the bacterium E. tarda. Diseased eels, along with pond water, were sampled from eight eel farms. All isolates were used for biochemical characterization and 16S rRNA and fimbrial gene sequencing. The phs gene of some isolates were also sequenced. H2S production was analyzed from either thiosulfate (S2O3 2 − )- or sulfite (SO3 − )-containing media. DNA analysis identified all isolates as E. tarda. Of the 17 E. tarda isolates, 11 were unable to reduce thiosulfate to H2S, while all isolates produced H2S from sulfite. In addition, non-thiosulfate-reducing E. tarda were isolated from four of the eight sampled eel farms. In media containing sulfate (SO4 2 − ) and in media without thiosulfate or sulfite, no H2S production was observed. In addition, glucose and galactose appeared to repress thiosulfate reduction in all strains, whereas these different carbon sources did not affect sulfite reduction. This indicates that thiosulfate and sulfite reduction pathways do not overlap in E. tarda. For this reason, careful attention should be made when identifying or differentiating non-thiosulfate-reducing E. tarda using H2S production characteristics.
Highlights
► Unusual Edwardsiella tarda was isolated from diseased Japanese eels. ► Of the 17 E. tarda isolates, 11 were unable to reduce thiosulfate to H2S. ► All isolates produced H2S from sulfite. ► Non-thiosulfate-reducing E. tarda were isolated from 4 of the eight sampled eel farms. ► Careful attention should be made when identifying this unusual bacteria.
