2014 Pathology Website

m1-1 . What is the MOST LIKELY diagnosis?

m1-2. The Gram stain from a skin pustule showed gram positive cocci in clusters. The organism grew on sheep blood agar (SBA) and was catalase positive. What is the most likely microorganism?

A. Staphylococcus aureus B. Staphylococcus epidermidis C. Streptococcus pyogenes D. Streptococcus pneumonia

Micro Case 2 (Path Slide 18) [ImageScope] [WebScope]

Clinical History: This 29-year-old male's illness began 10 weeks prior to death, with an episode of "flu". Two weeks later his urine became "smoky". He was found to have hematuria, albuminuria and elevated BUN (180 mg/dl). He died from a pulmonary embolus.

Image Gallery:

case study 2 microbiology

m2-1. What is the BEST diagnosis at the time of death?

m2-2. The throat culture obtained exhibited gram positive cocci in chains. It also showed beta-hemolysis on sheep blood agar (SBA) and was catalase negative. What was the most likely organism?

  • Streptococcus pyogenes
  • Streptococcus viridans
  • Staphylococcus aureus

m2-3. An elderly wheelchair bound man had a history of recurrent urinary tract infections. He presents with new onset of fever, chills, and confusion. Blood cultures were drawn. The organism grown was non-hemolytic, catalase negative, and PYR positive. Gram stain showed gram positive cocci. What is the most likely organism?

  • Staphylococcus epidermidis
  • Staphylococcus saprophyticus
  • Enterococcus species

m2-4. An elderly wheelchair bound man had a history of recurrent urinary tract infections. He presents with new onset of fever, chills, and confusion. Blood cultures were drawn. The organism grown was non-hemolytic, catalase negative, and PYR positive. Gram stain showed gram positive cocci. What is the MOST LIKELY organism?

Micro Case 3 (Path Slide 51) [ImageScope] [WebScope]

Clinical History: A 45-year-old male became ill approximately 2 to 3 weeks ago following an alcoholic spree. He had nausea, vomiting, dehydration, confusion and high fever. He died suddenly shortly after admission.

case study 2 microbiology

What is the MOST LIKELY diagnosis AND the likely causative agent?

m3-1. These images depict pneumonia in the stage of:

  • Red hepatization
  • Gray hepatization
  • Abscess formation

m3-2. Community acquired atypical pneumonia can be caused by which of the following organisms?

  • Legionella pneumophilia
  • Klebsiella pneumoniae
  • Mycoplasma pneumoniae
  • Pseudomonas aeruginosa

Micro Case 4 (Path Slide 195) [ImageScope] [WebScope]

Clinical History: A 67-year-old male had rheumatic heart disease for thirty years. Three months prior to death he began to have episodes of fever and chills accompanied by signs of worsening congestive heart failure. Splinter hemorrhages and purpuric skin rashes were noted three weeks before death.

case study 2 microbiology

m4-1. What is the MOST LIKELY diagnosis?

m4-2. Most cases that present with these findings are caused by:

  • Rickettsiae

m4-3. In this particular patient the MOST LIKELY causative organism was:

  • Group A Streptococci
  • Viridans Streptococci

m4-4. The organism MOST LIKELY to infect normal heart valves is:

  • Group B Streptococci

m4-5. The organism MOST OFTEN associated with infective endocarditis in IV drug abusers is:

m4-6. The heart valve MOST OFTEN affected by infective endocarditis in IV drug abusers is the:

  • Aortic valve
  • Mitral valve
  • Pulmonic valve
  • Tricuspid valve

m4-7. Which of these organisms are normal flora of the throat and are associated with dental caries, brain abscesses, and endocarditis?

  • Streptococcus pneumoniae
  • Peptostreptococcus species
ANSWER  

m4-8. A patient with suspected infectious endocarditis has a St. Jude’s prosthetic aortic valve and a fever of 38.6°C (101.5°F). Blood culture shows non-hemolytic, small, white colonies. The organism was Gram positive, catalase positive and coagulase negative. What is the MOST LIKELY organism?

  • Stapylococcus epidermidis

m4-9. A throat culture grows normal oropharyngeal flora. What alpha-hemolytic organism is most likely to be isolated on the blood agar?

A. Staphylococcus epidermidis B. Staphylococcus viridans C. Streptococcus pneumoniae D. Peptostreptococcus species

m4-10. A throat culture grows normal oropharyngeal flora. The coagulase test for the organism is positive. What is the genus and species of the organism?

A. Staphylococcus aureus B. Streptococcus agalactiae C. Streptococcus pyogenes D. Staphylococcus epidermidis

Micro Case 5

Clinical history: A 33-year-old female dairy farmer develops a severe headache and neck stiffness. On physical examination, her temperature is 38.2°C.  She has no papilledema.  A lumbar puncture is performed, and a Gram stain of the CSF obtained shows many short, gram-positive rods.

case study 2 microbiology

m5-1. Based on the clinical findings presented, what is the most likely causative agent in the case above?

m5-2. A sputum gram stain of an elderly person with cough and fever shows gram positive cocci in pairs. What is the most likely organism?

  • Haemophilus influenzae
  • Enterobacter species

m5-3. A 10-month-old child of a family from Mexico living in Durham was noted by his mother to have a grand mal seizure with shaking of arms and legs by the description given to the EMT. The EMTs found the child limp and unresponsive. In the emergency room the child’s fever was 39.5°C (103.1°F). Blood cultures and lumbar puncture were performed. CSF findings were as follows:

  • cell count of 4000
  • glucose 20mg/dl
  • protein 125mg/dl.

Gram stain showed PMNs and occasional Gram-negative coccobacillary organisms. The organism grew on chocolate agar but not on sheep blood agar or MacConkey’s agar. What is the most likely organism in this case?

  • Neisseria meningiditis
  • Moraxella catarrhalis
  • Streptococcus pneumonia

Micro Case 6 (Path Slide 123) [ImageScope] [WebScope]

Clinical History: A 25 year-old woman had pelvic pain, fever, and vaginal discharge for 3 weeks. On physical examination, she has lower abdominal adnexal tenderness and a painful, swollen left knee.  Laboratory studies show WBC count of 11,875/mm 3 with 68% segmented neutrophils, 8% bands, 18% lymphocytes, and 6% monocytes.

case study 2 microbiology

m6-1. What is the MOST LIKELY diagnosis AND the likely causative agent?

m6-2. What is a likely complication of this disease?

  • Ectopic pregnancy
  • Infertility
  • Tubo-ovarian abscess
  • ALL of the above

m6-3. What organisms are most likely to cause this disease?

m6-4. Which of the following statement about this disease is FALSE ?

  • It is usually associated with endometriosis
  • May be complicated by strictures and infertility
  • May also involve the adjacent ovary
  • May result in a hydrosalpinx
  • May be complicated by septicemia
  • ALL of the above statements are false regarding this disease.
  • NONE of the above statements are false regarding this disease.

m6-5. The following organisms are commonly responsible for pelvic inflammatory disease EXCEPT :

  • Aspergillus
  • Streptococcus
  • Staphylococcus

m6-6. A 25-year-old male presents with urethritis. The Gram stain shows intracellular gram positive cocci. What is the most likely organism?

  • Chlamydia trachomatis
  • Ureaplasma species
  • Neisseria gonorrhoeae
  • Haemophilus ducreyi

Micro Case 7 (Path Slide 9) [ImageScope] [WebScope]

Clinical History: A 51-year-old male had a " neurogenic bladder ", caused by a spinal cord tumor. He had multiple bladder infections which were treated with antibiotics. He had surgery to remove the tumor. Postoperatively, he developed fever and costovertebral angle tenderness which did not respond to antibiotics. He expired and an autopsy was performed.

case study 2 microbiology

m7-1. What is the BEST diagnosis?

  • Acute pyelonephritis
  • Acute glomerulonephritis
  • Chronic pyelonephritis
  • Chronic glomerulonephritis

m7-2 . ALL of the following are risk factors for this condition EXCEPT :

  • Reflux nephropathy
  • Congenital vesicoureteral reflux
  • Posterior urethral valves
  • Acetaminophen (Paracetamol) overdose
  • Urolithiasis

m7-3 . A urine culture from an 18-year-old woman with similar symptoms also grew a pure culture of more than 100,000 colonies/ml of an organism on sheep blood agar (SBA) and MacConkey’s agar. The gram stain also showed gram negative rods. What is the most likely organism?

  • Escherichia coli

Micro Case 8 ( Pathology Slide 39) [ImageScope] [WebScope]

Clinical History: 58-year-old African American female had been hemiplegic on the right side for 3 months prior to death. She developed malaise, fever and chills after visiting with her grandchildren. Her infection progressed. She developed dyspnea and expired.

Image Gallery

case study 2 microbiology

m8-1. Based on these clinical findings, what is the best diagnosis and likely causative agent ?

m8-2. What is the most likely cause of this disease in most adults?

M8-2. What organism would be most likely to cause a persistent infection in cystic fibrosis?

Micro Case 9

Clinical history: A 52-year-old homeless, alcoholic man had a fever and a cough productive of thick sputum that worsened over several days. His temperature is 38.2°C.   Diffuse crackles are heard at the right lung base. Laboratory studies are as follows:

  • hemoglobin: 13.3 g/dL
  • hematocrit: 40%
  • platelet count: 291,8000/mm 3
  • WBC count: 13,240/mm 3 with 71 segmented neutrophils, 7% bands, 16% lymphocytes, and 6% monocytes.

case study 2 microbiology

m9-1. Based on these clinical findings, what is the likely causative agent?

m9-2. An 18-year-old Duke freshman presented to student health with severe headache, fever, and disorientation. A lumbar tap was performed with the following results:

  • cell count: 300 with 100% PMN
  • glucose: 10 mg/dl
  • protein: 100 mg/dl.

The gram stain of the spinal fluid revealed numerous PMNs with intracellular gram-negative diplococci. What is the most likely organism?

  • H aemophilus influenza
  • Neisseria meningitides

Micro Case 10

Clinical history: A 66-year-old man incurs extensive thermal burns to his skin and undergoes skin grafting procedures in the surgical intensive care unit.  Two weeks later, he has increasing respiratory distress.  Laboratory studies show hemoglobin of 13.1 g/dL, hematocrit 39.2%, platelet count 222,200/mm 3 , and WBC count 4520/mm 3 with 15% monocytes. A chest radiograph shows extensive bilateral infiltrates with patchy areas of consolidation. Image Gallery:

case study 2 microbiology

m10-1. Based on these clinical findings, what is the likely causative agent?

m10-2. A sputum specimen from a cystic fibrosis patient grew Gram negative rods on sheep blood agar and MacConkey’s agar. The organism was oxidase positive. What is the most likely organism?

Micro Case 11

Clinical history: A suspicious envelope arrived for sorting at rural post office. The envelope was opened and found to contain white powder. Approximately two days later, the postal worker who handled the letter developed cutaneous boils, which were and 1 to 5 cm in diameter with central necrosis and eschars. He and his wife also developed a mild nonproductive cough with fatigue, myalgia for 72 hours, followed by severe dyspnea, diaphoresis and cyanosis.  Temperature of 39.5°C, pulse 105/min, respiration 25/min, and blood pressure 85/45mm Hg.  Crackles were heard at the lung bases. A chest xray shows a widened mediastinum and small pleural effusions. WBC count of 13,130/mm 3 , hemoglobin 13.7g/dL, hematocrit 41.2%, MCV 91 um 3 , and platelet count 244,000/mm 3 . Both died despite antibiotic therapy. Several cattle, horses, and sheep on the postal worker's farm also died.

case study 2 microbiology

m11-2. In a somewhat related case, when Pharaoh did not heed Moses to the let the captive Hebrews go, a series of plagues fell upon the land of Egypt. In the fifth plague, large domesticated mammals including cattle, horses, and sheep died. This was followed by a plague in which the Egyptians developed cutaneous boils. Some developed a mild nonproductive cough associated with fatigue, myalgia, and low grade fever over 72 hours, followed by a rapid onset of severe dyspnea with diaphoresis and cyanosis. Despite antibiotic therapy with both ciprofloxacin and doxycycline (had they been available), many of those affected would die. Which of the following organisms is most likely to have produced these findings?

  • Bacillus anthracis
  • Herpes simplex virus
  • Mycobacterium leprae
  • Yersinia pestis

Micro Case 12 (UMich Slide 017) [ImageScope] [WebScope]

Clinical history : A 45-year-old woman is being treated in the hospital for pneumonia complicated by septicemia. She has required multiple antibiotics and was intubated and mechanically ventilated earlier in the course. On day 20 of hospitalization, she has abdominal distention. Bowel sounds are absent, and abdominal radiograph shows dilated loops of small bowel suggestive of ileus. She has a low volume of bloody stool.

case study 2 microbiology

m12-1. Based on these clinical findings, what is the likely causative agent?

m12-2. Which of the following are appropriate specimen samples for anaerobe culturing:

  • blood, spinal fluid, abscess aspirate
  • deep tissue biopsy, sputum, blood
  • cerebrospinal fluid, tissue and debridement from decubitus ulcer, bile

Micro Case 13

Clinical history: A 25-year-old man is involved in an accident in which he is ejected from the vehicle. He sustains a compound fracture of the left humerus and undergoes open reduction with internal fixation of the humeral fracture.  Several days later, he has marked swelling of the left arm and crepitus . 

case study 2 microbiology

m13-1. In a similar case, a middle aged woman with type 2 diabetes presents to the emergency room with a very painful right lower leg. She has a wound on that extremity and stated that she stumbled on a fallen tree limb in her yard. You observe that her lower leg is discolored and swollen with several areas of crepitus . The abscess fluid was sent to the microbiology laboratory for aerobic and anaerobic culture. The organism grew best on anaerobic cultures and was lecithinase positive. Gram stain shows gram positive rods and the organism show anaerobic growth on egg yolk agar. What is the most likely organism?

  • Fusobacterium nucleatum
  • Bacteroides fragilis
  • Clostridium perfringens
  • Peptostreptococcus spp.

Micro Case 14 (Path Slide 451) [ImageScope] [WebScope]

Clinical History: A 4-year-old female had a gradual onset of fever, productive cough, anorexia and diarrhea about eleven days prior to death. The breath sounds were harsh, and a few cracking rales were heard over the right base posteriorly.

case study 2 microbiology

m14-1. Based on these clinical findings, what is the likely causative agent?

m14-1. Which of the following is the BEST diagnosis?

  • Ghon complex
  • Miliary (disseminated) tuberculosis
  • Foreign body reaction to talcum powder
  • Atypical pneumonia
  • H1N1 influenza

Micro Case 15

Clinical history: A 35-year-old man with HIV complains that he has had a "bad" taste in his mouth and discoloration of his tongue for the past 6 weeks.

case study 2 microbiology

m15-1. What is the MOST LIKELY diagnosis?

m15-2. In a separate case, a blood culture from a neutropenic (<100 neutrophils/ul) 50-year-old woman on broad spectrum antibiotics grew a yeast. Tests for germ tubes were positive in the microbiology laboratory. What is the likely organism?

  • Candida albicans
  • Candida tropicalis
  • Candida pseudotropicalis
  • Candida parapsilosis

Micro Case 16

Clinical history: A 44-year-old diabetic woman developed facial pain over the past 24 hours.  She has become lethargic and obtunded. There is swelling with marked tenderness over the left and right maxilla, bilateral exophthalmos, diffuse abdominal pain, poor skin turgor, and dry mucous membranes. Her temperature is 37.7°C. She has tachycardia, but no murmurs, and tachypnea; the lung fields are clear.

case study 2 microbiology

m16-1. What is the MOST LIKELY diagnosis and the best treatment option?

m16-2. In a separate case, A 22-year-old with non-Hodgkins lymphoma was profoundly neutropenic after induction chemotherapy and developed fevers. Broad spectrum IV antibiotic therapy was administered, but fevers continued. Chest x-ray showed new bilaterial fluffy pulmonary infiltrates. A bronchoscopy was performed which showed hyaline, septate hyphae with acute-angle branching. What is the MOST LIKELY organism?

  • Blastomyces dermatitidis
  • Candida species
  • Aspergillus spp

Micro Case 17

Clinical history: A 50-year-old resident of Phoenix, Arizona, has a cough that has persisted for 1 month.  On physical examination, his temperature is 38.1°C. A chest radiograph shows 3.5-cm opacity with central cavitation in the right apical region.  An open lung biopsy is performed to exclude cancer.

case study 2 microbiology

m17-1. Which of the following organisms is MOST LIKELY to be responsible for these findings?

  • Aspergillus fumigates
  • Coccidioides immitis
  • Histoplasma capsulatum
  • Mycobacterium tuberculosis

Micro Case 18

Clinical history: For the past 3 weeks, a 52-year-old man has had a chronic cough with a low-grade fever. On physical examination, his temperature is 37.4°C.  A chest radiograph shows bilateral, scattered, 0.3- to 2-cm nodules in the upper lobes and hilar adenopathy.  A fine needle aspirate of one of the nodules shows inflammation with mononuclear cells, including macrophages that, with PAS or silver stains, show intracellular, 2- to 5-um, rounded, yeast-like organisms.

case study 2 microbiology

m18-1. Which of the following infectious diseases is MOST LIKELY to produce these findings?

  • Coccidioidomycosis
  • Candidiasis
  • Cryptococcosis
  • Histoplasmosis
  • Blastomycosis

Micro Case 19

Clinical history: For the past month, a 68-year-old patient has had painful oral abcesses, fever, and a cough productive of yellow sputum.  On physical examination, there is dullness to percussion at the left lung base. A chest radiograph shows areas of consolidation in the left lower lobe.  Despite antibiotic therapy, the course of the disease is complicated by abscess formation, and he dies.

case study 2 microbiology

m19-1. Based on these clinical findings, what is the BEST diagnosis AND the likely causative agent?

m19-2. In a separate case, a middle aged man presented to his physician with a persistent cough of two months following an extended overseas trip to visit relatives. He had also noted a 10 pound weight loss and night sweats. A sputum was sent to the microbiology laboratory for routine bacterial culture and AFB culture. Kinyoun stain of his sputum was positive. What is the likely causative agent?

  • Actinomyces spp
  • Nocardia spp.

Micro Case 20

Clinical history: A 50-year-old man post lung transplant was admitted to hospital with fever, chills and cough. Chest x-ray showed multiple small abscesses within a right middle lobe infiltrate. The patient had been treated with prednisone and azathioprine daily for rejection. Gram stain of a bronchoalveolar lavage of the right middle lobe shows gram positive rods. Modified acid fast stain shows partially acid fast bacilli.

case study 2 microbiology

m20-1. What is the most likely organism?

m20-2. Nocardia species are:

  • branching, gram negative bacilli
  • partially acid fast, beaded gram positive filaments
  • acid fast, branching gram negative filaments
  • partially acid fast, gram variable bacilli

Micro Case 21

Clinical history: An HIV positive male presented in clinic with confusion and disorientation. He had a fever 38.5°C and photophobia. His CD4 count was 80/ul. A lumbar puncture was performed. It showed 32 WBC/ul with 89% lymphocytes, and 6% monocytes, glucose of 22mg/dl, and protein of 89mg/dl. Gram stain showed yeast and India ink showed a thick capsule.

case study 2 microbiology

m21-1. Which of the following is the most likely pathogen?

  • Cryptococcus neoformans
  • Hemophilus infulenzae
  • West Nile virus

m21-2. In a separate case, A 35-year-old man who received kidney transplantation was being treated with cyclosporine, azathioprine, and high doses of corticosteroids.  While on this regimen, the patient began to experience headaches and became lethargic.  A clinical diagnosis of meningoencephalitis was made.  He died 7 days later.  Autopsy showed a gelatinous meningeal exudate, and on sectioning of the brain, multiple small cyst-like areas were seen.  Microscopic examination showed areas containing rounded structures with a prominent capsule that stained brightly with mucicarmine. 

case study 2 microbiology

What is the most likely organism?

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Educational Resource Review : Clinical microbiology case studies

Graphical abstract.

An external file that holds a picture, illustration, etc.
Object name is dlab006f1.jpg

LI, low-income countries; LMIC, low- and middle-income countries; HMI, high- and middle-income countries; HIC, high-income countries.

Resource web link:   http://clinmicro.asm.org/index.php/explore-the-profession/what-is-clinical-microbiology/418-clinical-microbiology-case-studies-clinical-microbiology-case-studies (Full classification scheme available at: http://bsac.org.uk/wp-content/uploads/2019/03/Educational-resource-review-classification-scheme.pdf )

WHO region and country (World Bank): Region of the Americas, USA (HIC)

Peer review commentary

These clinical microbiology case studies are available on the American Society for Microbiology website. Each of the case studies is humorously named and free to download and use. As of December 2020, there were 14 case studies on the website, with each case study available for individual download. Topics include malaria, Streptococcus pyogenes , Legionella , Salmonella and Nocardia , among others.

Each case study details a patient’s history and presentation, the action(s) of the doctor, lab test results, diagnosis and treatment, as well as the final patient outcome. The case studies are in PowerPoint (.ppt) format and contain, on average, 12 slides. Each case study was developed by a different author.

Overall, the case studies are straightforward and would be easy for teachers and other medical professionals to use as part of medical education. Contact information for each of the authors is provided at the end of the case study, should the educator want additional information before using the case study.

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Case Studies: Microbiology

All microbiology case studies.

A Bioinformatic Investigation of a Mysterious Meningoencephalitis

By Sari Matar, Dyan Anore, Basma Galal, Shawn Xiong

Sarah’s Stomach

By Kelli M. Kinlen, David M. Zuckerman

Molly’s Medical Mission Maladies

By William M. Kolling, Catherine D. Santanello

A New York State of Mind

By Samantha N. Jewell, Julian A. Brix

What’s in Your Food?

By Bwalya Lungu

A Fatal Bite

By Obidimma Ezezika, Mona Jarrah, Shawanah Rahman

Troubled Waters

By Sebastian A. Schormann, James E. Boyett, Samiksha A. Raut

New Tricks for Old Drugs

By Carlos C. Goller, Stefanie H. Chen, Melissa C. Srougi

Stuck on You

By Brenda F. Canine, Michael L. Dini, Breanna N. Harris

Making Better Poison Eaters

By Kelsie J. Anson, Briana N. Van Treeck, Jake J. Flood

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Clinical Case Study

You are a pathologist working in the lab at Daigger Memorial Hospital (located in central Oregon). The emergency room is sending you cultures collected from patients. Your job is to determine:

  • The pathogenic bacteria causing the disease
  • What disease is being presented
  • What is the best treatment given the patient’s unique symptoms and history

Review the patient assessment form provided. Consider the type of sample taken and determine the relevant primary and secondary symptoms. Generally, samples taken in a clinical setting contain multiple organisms. In this case study, the sample will contain one contaminant organism and one causative agent.

Note: Watch for allergies. These may affect the appropriate treatment you will prescribe for the patient.

For some diseases, an irregularity in pulse or respiration, for example, may be indicative of the seriousness of the illness or may even be a direct indicator of the disease presented. Therefore, it is important that you understand what is considered “Within Normal Limits” for all assessment areas on the patient assessment form. Familiarize yourself with the acceptable normal ranges for pulse, respiration, oxygen saturation, blood pressure, and temperature for the age group your patient falls within.

Clinical Case Study Flowchart »

Patient Assessment Form »

Normal Flora According to Body System

Adapted from: Strohl, W.A. et.al. Lippincott's Illustrated Reviews: Microbiology. Lippincott Williams & Wilkins. Baltimore, MD. 2001 and Forbes, B.A. et.al. Bailey and Scott's Diagnostic Microbiology. Eleventh Edition. Mosby. St. Louis, MO. 2002

Pathogenic Organisms According to Body System

Adapted from: Gilligan, P.H. et.al. Cases in Medical Microbiology and Infectious Diseases. 2nd ed. American Society for Microbiology, Washington, D.C. 1997. Murray, Patrick R. Editor. Manual of Clinical Microbiology. 8th ed. ASM Press. Washington, D.C. 2003. Strohl, W.A. et.al. Lippincott's Illustrated Reviews: Microbiology. Lippincott Williams & Wilkins. Baltimore, MD. 2001

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case study 2 microbiology

COVID-19 Response Case Studies

Aligned with our mission to advance the understanding and impact of microbiology, the Society reached out to our community of microbiologists to share their experiences in responding to SARS-CoV-2. We aim to showcase the perspective of scientists during the COVID-19 pandemic and the variety of roles adopted to mitigate the global crisis. 

Understanding how antiviral antibodies can activate natural killer cells to improve viral control

This is the first case study in our series that comes from a group of Project Investigators (PIs). Professor Ian Humphreys, Professor Eddie Wang, Professor Richard Stanton and Professor Alan Parker are from the Viral Immunology Research Group at Cardiff University, UK. Collectively, they describe how they responded to the COVID-19 pandemic, the challenges they faced and how basic microbiology and immunology are critical components of any pandemic response.

Water-Based Epidemiology – finding a needle in a very dirty haystack

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  • Open access
  • Published: 23 May 2024

Phenotypic and genotypic analysis of antimicrobial resistance and population structure of gastroenteritis-related Aeromonas isolates

  • Dana Sagas 1 , 2   na1 ,
  • Yizhak Hershko 1   na1 ,
  • Katia Levitskyi 3 ,
  • Merav Strauss 1 ,
  • Matan Slutzkin 3 ,
  • Bibiana Chazan 4 , 5 &
  • Amos Adler 2 , 3  

Annals of Clinical Microbiology and Antimicrobials volume  23 , Article number:  45 ( 2024 ) Cite this article

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Metrics details

The population structure and the correlation between antimicrobial resistance (AMR) phenotypes and genotypes in Aeromonas species isolated from patients with gastroenteritis are not well understood. The aims of the study were to: (1) investigate the antimicrobial susceptibility profiles of Aeromonas species isolated from patients with gastroenteritis; (2) explore the relationship between AMR genes and resistance phenotypes; and (3) describe the population structure of these isolates and provide evidence of transmission events among them.

This microbiological survey was performed at the Microbiology Laboratory of the Emek Medical Center in Afula, Israel. Cultivation of Aeromonas was attempted from stool samples that tested positive by PCR. Antimicrobial susceptibility testing (AST) was performed using the Sensititre GN3F microdilution panel. Whole genome sequencing (WGS) was done using the Illumina NextSeq500/550 system. Phylogenetic studies involved multi-locus sequence typing (MLST) and core genome (cg) MLST. Resistance mechanisms were identified using the Comprehensive Antibiotic Resistance Database and compared with the AST results.

The study included 67 patient-unique isolates. The species that were identified included A. caviae ( n  = 58), A. dhakensis ( n  = 3), A. media ( n  = 2), A. veronii ( n  = 2) and A. hydrophila ( n  = 2). Isolates were almost uniformly susceptible to amikacin, gentamicin, aztreonam, cefepime, ceftazidime, ciprofloxacin and meropenem. All isolates with the exception of 1–2 isolates were resistant to ampicillin, cefazolin and ampicillin-sulbactam which was compatible with the presence of the bla OXA genes. Variable resistance rates were observed to cefuroxime, cefoxitin, ceftriaxone, piperacillin-tazobactam that were not correlated with the presence of other β-lactamase genes. Resistance to tetracycline and trimethoprim-sulfamethoxazole correlated with the presence of tetA and sul1 , respectively. The population structure of A. caviae was highly diverse with the minority of the isolates (16/57) clustering into six defined sequence types. A cgMLST-based distance of four genes was found in one pair of isolates, suggesting common source transmission.

Conclusions

A. caviae is the dominant species related to gastroenteritis and is characterized by a diverse population structure, with almost no evidence for common-source transmission. Resistance rates to most antimicrobial agents were low and partially matched with the presence of resistance genes.

Introduction

Aeromonas species are capable of causing a variety of different infections, including gastro-intestinal and extraintestinal infections [ 1 ]. Defining the precise role of Aeromonas species in causing gastroenteritis (GE) proves challenging, particularly outside of isolated outbreaks. This challenge arises from the variability in clinical symptoms, the absence of a specific clinical profile, the presence of Aeromonas in asymptomatic individuals [ 2 ] and by the frequent isolation of other potential pathogens in the same stool sample [ 3 ]. Consequently, Aeromonas species are not consistently included in routine stool sample testing [ 4 ] and the available data on these infections remain relatively limited.

A previous study reported the rate of antimicrobial resistance (AMR) in Aeromonas isolated from stool samples but did not conduct an analysis of resistance mechanisms [ 4 ]. A recent study of Aeromonas isolated mainly from hepato-biliary infections did not find a correlation between AMR phenotype and resistance genes [ 5 ]. Furthermore, as Aeromonas gastroenteritis is a food-borne infection, understanding the population structure of these infections could aid in deciphering transmission dynamics within the community.

Our laboratory, serving both the hospital and the surrounding community, commenced testing for Aeromonas gastroenteritis in December 2021, providing us with an opportunity to address these questions. Thus, the study aimed to: (1) investigate the antimicrobial susceptibility profiles of Aeromonas species isolated from patients with gastroenteritis; (2) explore the relationship between AMR genes and resistance phenotypes; and (3) describe the population structure of these isolates and provide evidence of transmission events among them.

Setup and population

This microbiological survey study was performed at the Microbiology Laboratory of the Emek Medical Center (EMC) in Afula, Israel. The EMC laboratory serves as the regional laboratory for a population of about 0.5 million of Clalit Health Services members, living predominantly in rural settlements and small urban centers.

Whole-genome sequencing and bioinformatics analysis were performed at the Tel Aviv Sourasky Medical Center Microbiology laboratory.

Study design

The study was part of a prospective survey of bacterial gastroenteritis conducted between December 2021 until October 2022 [ 6 ]. All stool samples submitted to the EMC laboratory underwent PCR testing for Aeromonas according to the routine laboratory protocol. Samples that tested positive for Aeromonas by PCR were then cultured (details below), and one Aeromonas isolate per patient was included in the study.

The study was approved by the Ethical Review Board of the EMC.

Identification and cultivation of Aeromonas from stool samples

Stool samples were transported from the community clinics and were tested daily except on weekends, where part of each sample was suspended in ASL buffer (Qiagen, Hilden, Germany) and refrigerated at 4 °C until tested by PCR only (culturing was done from the original sample tube). Multiplex PCR for Aeromonas and other bacterial pathogens was performed as previously described [ 7 ]. Stool samples that were positive by PCR for Aeromonas were inoculated into alkaline peptone water 0.5 M NaCl with Cephalothin (10 mg/l) and incubated overnight [ 8 ] followed by sub-culturing onto SS agar plates (Hylabs, Rehovot, Israel). Presumptive Aeromonas colonies were identified by MALDI Biotyper Sirius system (Bruker Daltonics, Bremen, Germany) using the MBT IVD Library Revision software. Definite determination of species was based on whole genome sequencing as described below.

Antimicrobial susceptibility testing (AST)

AST were performed at the EMC laboratory using the Sensititre GN3F microdilution panel (Thermo Fisher Scientific) following the manufacturer’s instructions. AST included amikacin, ampicillin, ampicillin-sulbactam, aztreonam, cefazolin, cefepime, cefoxitin, ceftazidime, ceftriaxone, cefuroxime, ciprofloxacin, gentamicin, meropenem, piperacillin-tazobactam, tetracycline, tobramycin and trimethoprim-sulfamethoxazole (SXT). AST breakpoints were interpreted (when available) in accordance with the CLSI recommendations for Aeromonas species [ 9 ] or if absent (including ampicillin-sulbactam, aztreonam, cefazolin, cefepime, cefoxitin, cefuroxime and tobramycin), in accordance with the recommendations to Enterobacterales [ 10 ].

Whole genome sequencing and bioinformatic analyses

Whole genome sequencing (WGS) was done using the Illumina NextSeq500/550 system. Libraries were prepared using Illumina DNA Prep (Illumina,20,060,059). The IDT for Illumina DNA/RNA UD Indexes (Illumina, 20,027,213) were used to tagment the DNA libraries for sequencing. After sequencing of each library, FASTQ files were imported into CLC Genomics Workbench version 23.0.5(Qiagen, Denmark). Following sequencing, the reads underwent quality trimming and contigs were assembled using the CLC Genomics Workbench version 23 (Qiagen, Denmark), with the following sequences applied as a template: A. caviae NZ_AP022254, A. hydrophila CP000426, A. media CP118939, A. dhakensis CP000462 and A. veronii NZ_LKKE01000001-NZ_LKKD01000048. Identification of resistance mechanisms was conducted through a combined approach, which involved (i) annotating and identifying known acquired antibiotic-resistant genes using the Comprehensive Antibiotic Resistance Database (CARD) [ 11 ], with a cutoffs of 60% identity and 80% coverage; (ii) detection of nonsynonymous mutations of selected genes and examining their correlation with resistance traits using a multi-sequence alignment approach by AliView version 1.27. Identification of protein domain sites was done using the InterProScan software [ 12 ], in comparison with A. caviae WP_063864115. Species designation was based on the reported average nucleotide identity. This method involved comparing the genome sequence of the tested isolates with those of reference type strains accessible in the GenBank database [ 13 ].

Core genome (cg) multi locus sequence typing (MLST) scheme and phylogenetic analyses

We employed the chewBBACA [ 14 ] to develop two distinct whole-genome (wg) sequencing schemas, later refined into core genome (cg) schemas. The first schema encompassed 67 isolates representing five different Aeromonas species, and the second was exclusively tailored to the 58 isolates of A. caviae . Prodigal [ 15 ] (version 2.6.3) facilitated the identification of Coding DNA Sequences (CDS) for both schemas, while BLAST+ [ 16 ] (version 2.9.0) was used for conducting allelic comparisons. The initial phase involved constructing a wgMLST schema that included all CDS from the isolates, followed by the removal of paralogous alleles to establish the cgMLST schema. During the “CreateSchema” phase, each genome was annotated for pairwise comparisons, leading to an extensive all-against-all BLASTP search. The resulting BLAST score ratio (BSR) was calculated, with genes encoding identical or nearly identical proteins (BSR exceeding 0.6 by default) consolidated into a single database, representing alleles of the same locus.

For the A. caviae -specific schema, a cross-reference with the sequence types from PubMLST was also performed to enhance the contextual understanding of the isolates. The allelic profiles derived from the chewBBACA cgMLST schemas of both groups were then subjected to phylogenetic analysis using GrapeTree software (version 1.5.0). Neighbor Joining (NJ) and Minimum Spanning Trees were constructed for each schema, with the trees generated from the allelic profiles being visualized in iTOL [ 17 ]. This comprehensive approach allowed for a detailed exploration of the genetic relationships within and between the diverse Aeromonas spp., with a specific focus on the A. caviae isolates. Two isolates derived from two separate samples of the same patient were included in the analysis. As these two isolates were assumed to be directly related, they were included in order to provide an epidemiological control for the phylogenetic schema, i.e., the number of gene-differences that can be expected to reflect direct epidemiological linkage.

Data availability

This Whole Genome Shotgun project has been deposited in NCBI under BioProject accession number PRJNA1040111. In addition, all 58 A. caviae isolates were deposited in PubMLST under ID BIGSdb_20231207071129_2157505_94891.

Statistical analysis

MIC 50 and MIC 90 were calculated using RStudio software, version 4.1.2. Graphs were drawn using the ggplot2 package in R.

Antimicrobial susceptibility profiles of Aeromonas species

During the study period, Aeromonas species were isolated from 67 patient-unique stool samples and underwent WGS. The distribution of species was as follows: A. caviae ( n  = 58), A. dhakensis ( n  = 3), A. madia ( n  = 2), A. veronii ( n  = 2) and A. hydrophila ( n  = 2). Compared with WGS, MALDI-ToF correctly identified 52 of the 67 isolates (77.6%) with all A. dhakensis isolates misidentified as A. hydrophila . A second A. caviae isolate from an additional sample from the same patient was employed as a control for the phylogenetic analysis (see below). The results of the AST of the 58 A. caviae isolates are presented in Fig.  1 and table S1 . A. caviae isolates were uniformly susceptible to amikacin, gentamicin, aztreonam and cefepime. Also, with the exception of 1–2 isolates, all were susceptible to ceftazidime, ciprofloxacin and meropenem. In contrast, all isolates with the exception of 1–2 isolates were resistant to ampicillin, cefazolin and ampicillin-sulbactam. A more variable distribution of the MIC values was observed with the β-lactam antimicrobials cefuroxime, cefoxitin, ceftriaxone and piperacillin-tazobactam and with tetracycline and SXT.

figure 1

The MIC distributions of 12 antimicrobials for 58 A. caviae isolates. The red, orange and green dots indicate the resistant, intermediate and susceptible breakpoints, respectively. Black dots were used if no breakpoints were available. MIC 50 and MIC 90 values are indicated by black dotted and black dashed lines, respectively. Dot/dash lines indicate identical MIC 50 and MIC 90 values

Similar patterns of antimicrobials susceptibility profiles were observed with the non-caviae Aeromonas species (table S1 ).

Genotypic analysis of antimicrobial resistance in Aeromonas species

Tetracycline and SXT: The phenotypic-molecular correlations for tetracycline and SXT resistance are presented in Fig.  2 a. Resistance to tetracycline was related with the presence of the tet(A) gene, whereas SXT resistance was related to the presence of the sul1 gene. Two susceptible isolate also possessed the sul1 gene but had a relatively elevated MIC compared with the other susceptible isolates (2/38 vs. ≤0.5/9.5, respectively).

figure 2

Antimicrobial phenotypic-genotypic correlations of Aeromonas species. The correlation is presented in the context of a cgMLST-based neighbor-joining tree analysis. The phenotypic-genotypic correlations are presented from left to right for SXT, tetracycline and β-lactam antimicrobials, respectively

β-lactam antimicrobials: The phenotypic-molecular correlations for cefuroxime, cefoxitin, ceftriaxone, ceftazidime and piperacillin-tazobactam resistance are presented in Fig.  2 b. All A. caviae isolates harbored at least one bla OXA and bla MOX types genes, with the most common alleles being bla OXA−504 , bla MOX−12 and bla MOX−13 . Six isolates also harbored a second bla OXA gene allele. No correlation was observed between the allele type or the presence of more than one bla OXA gene allele and the presence of resistance to the β-lactam antimicrobials tested. Other bla OXA alleles alone or in addition to the bla OXA−504 gene were present in a 11-isolate phylogenetic cluster.

As mentioned above, almost all isolates were resistant to ampicillin, cefazolin and ampicillin-sulbactam which was compatible with the presence of the bla OXA genes. The single A. caviae isolate that was susceptible to all of these agents harbored both bla OXA−504 and bla MOX−13 . Comparative analysis of the bla OXA−504 gene protein sequence identified a S53Y substitution at the enzyme active site.

In species other than A. caviae , all isolates harbored one bla OXA gene of different alleles than A. caviae but only two isolates harbored bla MOX−9 . Other β-lactamase that were detected included bla cphA , bla ceps and bla AQU . As with A. caviae , the presence of the bla OXA gene correlated with the resistance to ampicillin, cefazolin and ampicillin-sulbactam but no correlation was found with the resistance phenotypes to the other β-lactam antimicrobials.

Phylogenetic analysis of Aeromonas caviae isolates

The cgMLST-based phylogenetic analysis of A. caviae isolates is presented in Fig.  3 and S1 . The population structure was highly diverse with the minority of the isolates (16/57) clustering into six defined sequence types (ST) (Fig.  3 and S2 ). Isolates 95,875 and 84,764 were obtained from the same patient and were included in the analysis as control. Both of them were identified as ST-2438 with only 7-gene difference in cgMLST analysis (figure S1 ). The numbers of cgMLST gene difference between isolates within the same ST were higher in most ST clusters with the exception of two ST-2429 isolates (from two patients), were the cgMLST gene difference was 4 genes.

figure 3

CgMLST-based neighbor-joining tree comprising 58 A. caviae isolates. Sequence types (ST) are marked in different colors according to the nomenclature of the 6-loci MLST scheme hosted on PubMLST

Our study of the molecular features of gastroenteritis-related Aeromonas species was initiated following the transition from culture to PCR-based diagnosis of bacterial gastroenteritis [ 6 ]. Prior to this transition, Aeromonas was not routinely tested in stool cultures in our laboratory (or anywhere in Israel) and thus the species distribution in our community was unknown. The predominant species in our study was A. caviae (58/67), with four other species accounting for the rest of the isolates. A. caviae was also reported as the predominant species (69%) in a previous study of Aeromonas gastroenteritis North-Western Israel [ 4 ], followed by A. veronii in 29%. Globally, four species account for most cases of gastroenteritis: A. hydrophila , A. caviae , A. veronii biovar.sobria , and A. trota ; A. caviae were the predominant species in most of the reviewed studies [ 2 ] We also identified three cases of A. dhakensis , a species that is rarely isolated from stool culture and is typically found in tropical areas [ 18 ].

AST results for the dominant species ( A. caviae ) showed relatively low variability, with the vast majority of isolates being either susceptible (e.g., ceftazidime, ciprofloxacin) or resistant to the tested antimicrobial drugs (e.g., ampicillin, cefazolin). More variability was found in several antimicrobial drugs, such as cefuroxime, ceftriaxone, tetracycline and SXT. Several previous studies [ 4 , 5 , 19 ] have demonstrated similar variability in susceptibility profiles, while others [ 19 ] have indicated nearly complete susceptibility. As we move into the current era, the diagnosis of bacterial gastroenteritis is increasingly shifting towards PCR rather than culture-based detection [ 6 ]. Consequently, Aeromonas gastroenteritis is also expected to be diagnosed more frequently through PCR in the future. Regretfully, AST reports are less likely to be available for the clinician [ 20 ] and the selection of antimicrobial treatment might be guided mostly based on pre-existing data. The cumulative data from our study and previous study suggests that among the oral agents, fluoroquinolones are probably the most appropriate choice [ 1 ].

Our study was aimed to explore the molecular mechanism beyond AMR in Aeromonas gastroenteritis-related isolates. Here, our goal was only partially achieved as we were able to provide an explanation for the uncommon resistance phenotypes to SXT and tetracycline ( sul1 and tetA/E , respectively) as previously described [ 21 , 22 ]. The picture regarding the β-lactam antimicrobials was more complex. All isolates harbored a bla OXA type genes, all A. caviae harbored a bla MOX type genes, while non- caviae Aeromonas species harbored additional types of β-lactamase. These genetic profiles matched with the resistance to ampicillin, cefazolin and ampicillin-sulbactam, as reported by some but not all studies [ 23 ]. However, we could not find an explanation for the few cases of ampicillin or cefazolin susceptibility with the exception of one case with a S53Y substitution in the bla OXA−504 gene protein sequence which could have possibly altered the activity of this enzyme. The S53Y substitution in the bla OXA−504 gene protein has never been reported before, thus necessitating validation of its biochemical effect in-vitro. We also could not identify a correlation between the genomic features and the resistance to 2nd - or 3rd -generation cephalosporin phenotypes. This lack of correlation was also reported by a previous study [ 5 ] and is likely the result of de-repression of the class C β-lactamase expression [ 24 ]. Thus, genomic studies are probably limited in their ability to decipher β-lactamase resistance in Aeromonas species which requires also a gene expression analysis.

Using the standard MLST schema as reference, we found that the majority of isolates did not cluster into specific ST. This is similar to the results of Aeromonas species isolates from various clinical sites [ 5 ]. In a study of A. veronii isolated from patients with gastroenteritis the isolates were defined as “closely related” [ 25 ]; However, given the differences in WGS methodology and the absence of MLST as a reference, evaluating this description in comparison with our results is challenging. Furthermore, through the use of cgMLST, we were able to illustrate that apart from one pair of isolates, the differences between isolates, even within the same ST, were considerable. This finding suggests that direct or common-source transmission is unlikely.

In addition to the previously mentioned limitations concerning the correlations between AMR phenotypes and genotypes, our study’s ability to comprehensively represent the bacterial population of Aeromonas gastroenteritis-related isolates may have been constrained. This limitation stemmed from culturing being performed only following a positive PCR result, and it was not always successful (merely 68 out of 283 PCR-positive cases, 24%). Additional possible cause for the low rate of culture positivity might have been less than optimal choice of the selective/enrichment media. A low rate of culture positivity versus PCR (0.34 versus 2.9, 8.5%) was also noted in a before-after study of the same PCR kit [ 7 ]. Consequently, we were unable to include all cases within the defined period, potentially hindering the identification of putative transmission chains. Despite these limitations, our study offers additional insights into the prevalence and mechanisms of AMR, as well as novel data regarding the population structure of Aeromonas gastroenteritis-related isolates.

Availability of data and materials: The NGS datasets generated during the current study are available under BioProject accession number PRJNA1040111. In addition, all 58 A. caviae isolates were deposited in PubMLST under ID BIGSdb_20231207071129_2157505_94891.

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Dana Sagas and Yizhak Hershko contributed equally to the study.

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Clinical Microbiology, Emek Medical Center, Clalit Health Services, Afula, Israel

Dana Sagas, Yizhak Hershko & Merav Strauss

Department of Epidemiology and Preventative Medicine, School of Public Health, Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Dana Sagas & Amos Adler

Clinical Microbiology, Tel Aviv Sourasky Medical Center, 6 Weizmann Street, Tel Aviv, Israel

Katia Levitskyi, Matan Slutzkin & Amos Adler

Infectious Diseases Unit, Emek Medical Center, Clalit Health Services, Afula, Israel

Bibiana Chazan

Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa, Israel

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DS- data acquisition and interpretation; YH- data acquisition and interpretation, manuscript drafting; KL- data acquisition and interpretation; MS- data acquisition; MS- data acquisition and interpretation; BC-conception, study design and manuscript drafting; AA- conception and study design, data interpretation, manuscript drafting.

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Bitter taste TAS2R14 activation by intracellular tastants and cholesterol

  • Xiaolong Hu 1 , 2   na1 ,
  • Weizhen Ao 1 , 2   na1 ,
  • Mingxin Gao 3   na1 ,
  • Lijie Wu   ORCID: orcid.org/0000-0002-2253-0497 1   na1 ,
  • Yuan Pei   ORCID: orcid.org/0000-0003-4065-2540 1 ,
  • Shenhui Liu 1 , 2 ,
  • Yiran Wu   ORCID: orcid.org/0000-0001-6790-7473 1 ,
  • Fei Zhao 1 ,
  • Qianqian Sun 1 ,
  • Junlin Liu 1 ,
  • Longquan Jiang 1 , 2 ,
  • Xin Wang 1 , 2 ,
  • Yan Li 4 , 5 ,
  • Qiwen Tan 1 ,
  • Jie Cheng   ORCID: orcid.org/0000-0001-9972-2166 3 ,
  • Fan Yang   ORCID: orcid.org/0000-0002-3000-2336 3 ,
  • Chi Yang   ORCID: orcid.org/0000-0003-1630-5389 4 , 5 ,
  • Jinpeng Sun   ORCID: orcid.org/0000-0003-3572-1580 3 ,
  • Tian Hua   ORCID: orcid.org/0000-0002-6132-2068 1 , 2 &
  • Zhi-Jie Liu   ORCID: orcid.org/0000-0001-7279-2893 1 , 2  

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We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

  • Electron microscopy
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Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents 1,2 . TAS2R14 is also widely expressed in extra-gustatory tissues, suggesting its additional roles in diverse physiological processes and potential therapeutic applications 3 . Here, we present cryo-electron microcopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1 , coupling with different G protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A GPCRs. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis, and molecular dynamic simulations studies, illuminate the receptor’s broad-spectrum ligand recognition and activation via intricate multiple ligand-binding sites. Additionally, our study uncovers the specific coupling modes of TAS2R14 with gustducin and G i1 proteins. These findings should be instrumental in advancing our knowledge underlying bitter taste perception and its broader implications in sensory biology and drug discovery.

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These authors contributed equally: Xiaolong Hu, Weizhen Ao, Mingxin Gao, Lijie Wu

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iHuman Institute, ShanghaiTech University, Shanghai, China

Xiaolong Hu, Weizhen Ao, Lijie Wu, Yuan Pei, Shenhui Liu, Yiran Wu, Fei Zhao, Qianqian Sun, Junlin Liu, Longquan Jiang, Xin Wang, Qiwen Tan, Tian Hua & Zhi-Jie Liu

School of Life Science and Technology, ShanghaiTech University, Shanghai, China

Xiaolong Hu, Weizhen Ao, Shenhui Liu, Longquan Jiang, Xin Wang, Tian Hua & Zhi-Jie Liu

NHC Key Laboratory of Otorhinolaryngology, Qilu hospital and School of Basic Medical Sciences, Shandong University, Jinan, Shandong, China

Mingxin Gao, Jie Cheng, Fan Yang & Jinpeng Sun

Department of Oral Surgery, Shanghai Ninth People’s Hospital and College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai, China

Yan Li & Chi Yang

National Center for Stomatology and National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology and Research Unit of Oral and Maxillofacial Regenerative Medicine, Chinese Academy of Medical Sciences, Shanghai, China

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Supplementary video 1.

The 3D variability movies prepared from cryo-EM data of FFA-TAS2R14-Gi1 to illustrate the multiple conformation features of TM6.

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The MD simulated movie shows the unwinding process of TM6’s cytosolic end when AA was virtually depleted from the receptor.

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Hu, X., Ao, W., Gao, M. et al. Bitter taste TAS2R14 activation by intracellular tastants and cholesterol. Nature (2024). https://doi.org/10.1038/s41586-024-07569-9

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Carbapenem-resistant gram-negative bacterial infections and risk factors for acquisition in a Kenyan intensive care unit

  • Jane Wairimu Maina 2 ,
  • Jeniffer Munyiva Mutua 3 &
  • Abednego Moki Musyoki 1  

BMC Infectious Diseases volume  24 , Article number:  522 ( 2024 ) Cite this article

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Carbapenem-resistant Gram-negative bacteria (CR-GNB) are a critical public health threat globally; however, there are inadequate surveillance data, especially in intensive care units (ICU), to inform infection prevention and control in many resource-constrained settings. Here, we assessed the prevalence of CR-GNB infections and risk factors for acquisition in a Kenyan ICU.

A hospital-based cross-sectional study design was adopted, recruiting 162 patients clinically presenting with bacterial infection after 48 h of ICU admission, from January to October 2022 at the Nairobi West Hospital, Kenya. Demographics and clinical data were collected by case report form. The type of sample collected, including blood, tracheal aspirate, ascitic tap, urine, stool, and sputum depended on the patient’s clinical presentation and were transported to the hospital Microbiology laboratory in a cool box for processing within 2 h. The samples were analyzed by cultured and BD Phoenix system used for isolates’ identity and antimicrobial susceptibility.

CR-GNB infections prevalence was 25.9% (42/162), with Klebsiella pneumoniae (35.7%, 15/42) and Pseudomonas aeruginosa (26.2%, 11/42) predominating. All isolates were multidrug-resistant (MDR). P. aeruginosa and A. baumannii were 100% colistin-resistant, while K. pneumoniae (33.3%) was tigecycline-resistant. History of antibiotics (aOR = 3.40, p  = 0.005) and nasogastric tube (NGT) use (aOR = 5.84, p = < 0.001) were the risk factors for infection.

Our study highlights high MDR- and CR-GNB infections in ICU, with prior antibiotic exposure and NGT use as risk factors, and diminishing clinical value of colistin and tigecycline. In this study setting and beyond, strict implementation of antimicrobial stewardship programs and adherence to infection prevention and control through monitoring, evaluation and feedback are warranted to curb CR-GNB infections, especially among the risk groups.

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Carbapenem-resistant Gram-negative bacteria (CR-GNB) presents significant challenges in clinical practice [ 1 ]. These organisms are World Health Organization (WHO) critical priority pathogens, depending on the urgency with which new antibiotics are needed [ 2 ]. CR-GNB pathogens are often multidrug-resistant (MDR) with limited treatment options, increased morbidity, healthcare cost and mortality [ 2 ]. Carbapenems are considered antibiotics of ‘the last resort’ for infections caused by MDR-GNB. There are several mechanisms of carbapenem resistance, mainly limiting the uptake of a drug, modification of a drug target, inactivation of a drug, and active efflux of a drug [ 3 ].

Colistin and tigecycline are the first-line treatment options for infections caused by carbapenem-resistant (CR) bacteria, particularly in many resource-constrained countries with limited access to newer therapeutic options; however, uncertainties on their efficacy still exist due to emerging resistance, even when combined with other antimicrobials. The newer treatment options for CR bacterial infections, such as ceftazidime-avibactam, ceftolozane-tazobactam, meropenem-vaborbactam, imipenem-cilastatin-sulbactam, plazomicin, eravacycline, and cefiderocol, are faced with challenges of insufficient high-quality clinical data, delayed susceptibility testing methods approval, antibacterial spectra complexity, and acquisition costs [ 4 ].

Intensive care units (ICUs) are recognized as hotbeds for MDR pathogens acquisition and spread because the ICU-admitted patients are usually severely ill and have frequent invasive procedures, including intubation, mechanical ventilation, and vascular access. Frequently, ICU-admitted patients have reduced immunity following trauma, surgery, and sepsis and also due to impaired protective mechanisms that include cough and swallowing reflexes, gastric acid secretion, and normal flora [ 5 ]. The burden of ICU-acquired infections is higher in lower-middle-income countries (LMICs) compared to high-income countries [ 6 ]. Therefore, continuous and systematic-evidence-based surveillance, in line with the global action plan on AMR [ 7 ], is warranted in ICUs to inform infection prevention and control measures and optimized use of antimicrobials.

Clinical laboratories are crucial in generating antibiograms for evidence-based antimicrobial selection by clinicians and AMR surveillance. However, these facilities in many resource-limited countries are inadequate and lack the capacity for microbiology, with the majority of patients likely to receive inappropriate antibiotic prescriptions. A recent study in Kenya shows that only 0.1% of 1505 patients in 14 public hospitals were treated based on antibiograms, and 46.4% inappropriately received antibiotics [ 8 ]. This clinical practice has critical implications for the emergence and spread of MDR organisms. MDR pathogens, including CR strains, are a growing health problem in Kenya [ 9 , 10 ]; however, data on CR-GNB, especially in our ICUs, is limited. Here, we determined the prevalence CR-GNB infections and risk factors for acquisition among patients admitted to the ICU.

Study area, design, and population

We conducted this cross-sectional study between January and October 2022 among consecutive patients admitted to the intensive care unit (ICU) at the Nairobi West Hospital (NWH), Kenya. The NWH is a 400-bed capacity (including 18 ICU beds) private tertiary hospital that receives patient referrals from all over the country. The study included patients clinically presenting with a bacterial infection (persistent fever, swollen lymph nodes, chills and sweats, confusion, cloudy and smelly urine, increased heart rate, difficulty breathing, persistent cough, vomiting, diarrhoea, wound that is red, hot, swollen, or has pus) and excluded those admitted for less than 48 h. The treating physician’s clinical judgement guided the type of sample collected from each patient. The authors sought informed written consent from each participant through a close relative or a legally appointed family representative and followed the Declaration of Helsinki, observing the well-being of patients and prompt sharing of results with the treating clinicians. The Kenyatta University Ethical Review Committee granted the study ethical clearance (Protocol no. PKU/2395/11,531).

Data and clinical samples collection

The participants’ demographic and clinical presentation data were collected using a case report form. The clinical sample types depended on the patient’s clinical presentation and were collected following standard bacteriological procedures [ 11 ], as previously described by Maina and others [ 12 ]. A qualified nurse collected the tracheal aspirate and ascitic tap samples into sterile containers. Swab samples were collected using sterile swabs (Delta lab, Spain), whereas urine samples were collected aseptically from a catheter collection port using a needle into 20 mL sterile screw-capped universal containers (Delta lab, Spain). Stool and sputum samples were collected into a sterile polypots (Delta lab, Spain). For blood samples, we obtained 8–10 mL of participants’ blood using a needle and syringe into BD BACTEC™ Blood Culture Media (BD Diagnostics, Sparks, MD, USA). All samples were uniquely labeled, blood cultures held at room temperature and other samples in a cool box and transported to the NWH Microbiology laboratory for processing within 2 h [ 12 ].

Isolation, identification and antimicrobial susceptibility testing

Bacterial isolation followed the standard operating procedures in bacteriology [ 13 ]. We inoculated stool samples on MacConkey agar and Xylose Lysine Deoxycholate agar (XLD agar), while pus swabs, sputum, catheter tips, and tracheal aspirates on MacConkey agar, sheep blood agar, and chocolate blood agar (all culture media from Hi Media Laboratories LLC, India); and incubated overnight at 37 °C under both ambient air and 5% CO2 conditions. Urine samples were inoculated onto cysteine–lactose electrolyte-deficient agar (CLED) (HI Media Laboratories LLC, India) and incubated overnight at 37 °C. Blood samples were cultured in the BD BACTEC™ Automated Blood Culture System (BD Diagnostics, Sparks, MD, USA) at 36 °C for up to 5 days, and positive-flagged samples were sub-cultured on MacConkey agar, sheep blood agar, and chocolate agar, (Hi Media Laboratories LLC, India), then incubated overnight at 37 °C under both ambient air and 5% CO2 conditions [ 12 ].

The isolates’ identification and antimicrobial susceptibility testing were performed using the BD Phoenix system (BD Diagnostics, Sparks, MD, USA) and following the manufacturer’s instructions. Antibiotics selection and interpretation of the isolates susceptibility followed the CLSI guidelines [ 13 ]. The tested antibiotics were amoxicillin/clavulanic acid (4/2–16/2 µg/ml), ampicillin (4–16 µg/ml), piperacillin/tazobactam (4/4–64/4 µg/ml), trimethoprim/sulfamethoxazole (1/19–4/76 µg/ml), nitrofurantoin (16–64 µg/ml), gentamicin (2–8 µg/ml), amikacin (8–32 µg/ml), ceftriaxone (1–32 µg/ml), cefazolin (4–16 µg/ml), cefotaxime (4–16 µg/ml), ceftolozane/tazobactam (1/4–8/4 µg/ml), ceftazidime (2–16 µg/ml), cefepime (1–16 µg/ml), tigecycline (1–4 µg/ml), ciprofloxacin (0.5–2 µg/ml), levofloxacin (1–4 µg/ml), meropenem (0.5–4 µg/ml), ertapenem (0.25–2 µg/ml), imipenem (0.25–4 µg/ml) and colistin (1–4 µg/ml). Pseudomonas aeruginosa (ATCC 27,853) and Escherichia coli (25,922) were used as the standard control organisms [ 12 ].

We defined carbapenem resistance as resistance to either ertapenem (≥ 2 µg/ml), imipenem (≥ 4 µg/ml), or meropenem (≥ 4 µg/ml), whereas resistance to either ceftriaxone (≥ 4 µg/ml) or ceftazidime (≥ 16 µg/ml) as third-generation cephalosporin resistance [ 12 , 13 ]. Isolates resistant to three or more antibiotic classes were considered multidrug-resistant (MDR) [ 9 ]. Multiple antibiotic resistance indices (MARI) were calculated as a/b, where a = number of antibiotics isolate was resistant to, b = the total number of antibiotics tested [ 12 , 14 ].

Statistical analysis

This study analyzed the data using the Statistical Package for the Social Sciences (SPSS) version 17.0 for Windows (IBM SPSS Statistics, IBM Corporation, Armonk, NY, USA). We analyzed data for normality and presented in figures and tables, categorical data in frequencies and percentages, and continuous data in means, medians, and interquartile ranges. Authors used binomial logistic regression analysis to determine the association between CR-GNB infections and patients’ socio-demographic and clinical characteristics. Any association with p -value ≤ 0.2 were further analyzed by multinomial logistic regression, with the statistical significance level set at p  < 0.05 (95% Confidence Interval (95% CI)) and statistically significant associations bolded in Table 4  [ 12 ].

Demographic and clinical characteristics of patients with GNB infections

The majority of the patients with Gram-negative bacteria (GNB) infections were: aged between 40 and 60 years old (46.7%), males (58.9%), not referred from other healthcare facilities (62.2%), and had: a history of antibiotic use (76.7%), invasive procedure (74.4%), and prior hospitalization history (62.2%) but not in ICU (98.9%), Table  2 .

Distribution of CR-GNB in clinical samples

In this study, 90 out of 162 (55.6%) patients had non-replicate Gram-negative bacterial infections. The prevalence of carbapenem-resistant Gram-negative bacteria (CR-GNB) was 46.7% (42/90), with Klebsiella pneumoniae (35.7%, 15/42) and Pseudomonas aeruginosa (26.2%, 11/42) as the most prevalent isolates overall, Fig.  1 . In urine samples, K. pneumoniae predominated (6/15, 40%), while Acinetobacter baumannii (42.9%, 3/7) and Pseudomonas aeruginosa (41.7%, 5/12) were the most common pathogen in blood and pus swab samples, respectively. All Acinetobacter baumannii (5/5, 100%), 85% of Pseudomonas aeruginosa (11/13) and 54% of K. pneumoniae (15/28) were carbapenem-nonsusceptible, Fig.  1 .

figure 1

Distribution of CR-GNB. % percentage , No . number , CR carbapenem-resistant , CS carbapenem-sensitive

Antimicrobial susceptibility profiles of CR-GNB

We observed third-generation cephalosporins resistance in all isolates of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa , and Acinetobacter baumannii , Table  3 . Aminoglycosides resistance ranged from 25 to 100%, being highest among A. baumannii isolates. Acinetobacter baumannii and P. aeruginosa displayed 100% colistin resistance, Table  4 . Additionally, A. baumannii showed 100% resistance to quinolones (LVX, CIP) tested but, together with E. coli , remained susceptible to tigecycline. Resistance to tigecycline was observed in K. pneumoniae (33.3%), P. aeruginosa (87.5%), Table  3 . This study did not present the antimicrobial susceptibility profiles of Stenotrophomonas maltophilia, Klebsiella oxytoca , and Klebsiella aerogenes because they were only one isolate each.

MDR phenotypes

All the CR-GNB isolates were multidrug-resistant (MDR) and had multiple antibiotic resistance indexes ranging from 0.55 to 1.0, Table 4 .

Factors associated with carriage of CR-GNB

Patients with a history of antibiotic use were three (3) times more likely to have CR-GNB isolate compared to those without the history (aOR = 3.40, 95% CI: 1.97–11.89, p  = 0.005), while those using NG tube were about six (6) times more likely to be diagnosed with GNB infection (aOR = 5.84, 95% CI: 2.16–15.79, p = < 0.001), Table  4 .

Carbapenem-resistant Gram-negative bacteria (CR-GNB) are an eminent global health challenge because of the limited treatment options and high mortality rates [ 1 , 15 ]. The burden of CR-GNB is disproportionately higher in low and middle income countries (LMICs) compared to high-income countries [ 3 ]. Here in this study, the prevalence of CR-GNB was 46.7%, higher than previously reported in East Africa [ 16 ], the United States [ 17 ], and Central Asia and Europe [ 18 ]. These study findings suggest a higher burden of CR-GNB, in our study setting, possibly due to limited alternative therapeutic options, widespread and irrational use of carbapenem antibiotics, and the failure of existing treatments.

In the current study, carbapenem-resistant Klebsiella pneumoniae (CRKP, 35.7%, 15/42) and carbapenem-resistant Pseudomonas aeruginosa (CRPA, 26.2%, 11/42) were the most the prevalent isolates overall. Similarly, CRPA (4/42, 9.5%) and CRKP predominated in urine, while carbapenem-resistant Acinetobacter baumannii (CRAB, 42.9%, 3/7) was the most common pathogen in blood samples. In general, all Acinetobacter baumannii (5/5, 100%), 85% of Pseudomonas aeruginosa (11/13) and 54% of K. pneumoniae (15/28) were carbapenem-nonsusceptible. Our study findings corroborate with others among ICU-admitted patients in Egypt [ 19 ], South Korea [ 20 ]), Fuzhou, and China. K. pneumoniae, P. aeruginosa and A. baumannii are opportunistic pathogens known for their high frequency and diversity of antimicrobial resistance genes and are well adapted to hospital environments, where they frequently cause severe infections in hospitalized and immunodeficient persons.

The incidence of infections caused by third-generation cephalosporin-resistant (3GCR) and CR-GNB in ICU patients is rising [ 21 ]. We observed third-generation cephalosporins resistance (3GC-R) in all carbapenem-resistant Escherichia coli (CREc), CRKP, CRPA, and CRAB isolates, corroborating studies among ICU-admitted patients in Egypt [ 22 , pp. 2011–2017]. 3GC-R was higher than reported in six German university hospitals [ 21 ]. The current study findings may suggest antibiotics overprescription, selecting resistant strains. Further, CRAB and CRPA, in our study, displayed 100% colistin resistance. Syed and others documented similar high colistin-resistance levels in ICUs at tertiary care hospitals in Karachi, Pakistan [ 23 ]. Colistin is considered ‘the drug of the last resort’ for many CR-GNB infections, with its frequent use in agriculture and pisciculture considered the cause of the rising resistance. In Kenya, 13% of farmers in Kiambu County used colistin in poultry feeds [ 24 ]. Colistin resistance is a growing problem, especially in developing countries, necessitating stringent infection control and comprehensive antimicrobial stewardship policies.

In the current study, K. pneumoniae (33.3%) and P. aeruginosa (87.5%) were resistant to tigecycline, but CRAB and CREc remained susceptible. P. aeruginosa is known to be intrinsically resistant to tigecycline, and in E. coli and K. pneumoniae , the resistance occurs due to AcrAB efflux pump overexpression [ 25 ]. The pharmacokinetic/pharmacodynamic properties of tigecycline and colistin suggest that these antimicrobial agents are among the most effective options in vitro in combating CR-GNB among critically ill patients with difficult-to-treat infections. The emergence of resistance to these antibiotics is, therefore, a significant issue that needs to be addressed, considering that the newly approved antimicrobial agents, such as ceftazidime/avibactam and meropenem/vaborbactam, are costly and are not integrated yet into routine antimicrobial susceptibility testing, are expensive, which restricts the available options for effectively treating CR-GNB infections [ 4 ], especially in resource-constrained settings.

All the CR-GNB isolates were multidrug-resistant (MDR) and had multiple antibiotic resistance (MAR) indexes ranging from 0.55 to 1.0. The MAR index is the ratio of antibiotics number an isolate is resistant to the total number of antibiotics used in susceptibility testing. The index is a good tool for health risk assessment. It helps to determine whether the isolates are from a region of high or low antibiotic use, with a MAR index greater than 0.2 indicating a ‘high-risk’ source of contamination [ 9 ]. This high MDR rates result in a substantial healthcare burden and are associated with increased mortality rates.

Patients with a history of antibiotic use were three times more likely to have CR-GNB isolate, consistent with other studies [ 26 , 27 ] in China. Antibiotic exposure results in gut microbiota dysbiosis, involving a reduction in the diversity of gut microbiota, alterations in the abundance and gene expression, protein activity, and gut metabolome, compromised colonization resistance to invading harmful bacteria, and the emergence of antibiotic-resistant microbes [ 28 ]. This emphasizing the importance of implementing and upholding stringent antimicrobial stewardship practices to reduce overuse and reliance on specific antibiotic classes, especially carbapenems. We found patients using NG tubes to be six (6) times more likely to be diagnosed with GNB infection. Intubation disrupts the body’s natural protective barrier, allowing pathogens to invade, adhere and form biofilms in the inner surface of the tubes. The bacteria delivered to the gut by contaminated feeding tubes may lead to dysbiosis and poses significant health risks [ 29 ].

This study is subject to some limitations. First, the study utilized clinical laboratory-based CR-GNB testing using surveillance data, which may not accurately detect individuals who are carriers of these bacteria. Laboratory-based surveillance data may only capture cases where patients have symptomatic infections or during routine screening. This study may have missed colonized or asymptomatic carriers of CR-GNB, even though they can still contribute to the transmission and spread of CR within healthcare settings. Secondly, this was a single-centre study. As such, the findings may not accurately reflect the rate of CR in the region. However, the snapshot of cases reported is informative and can be very useful in estimating and comprehending the burden of carbapenem resistance in the hospital setting. Implementing comprehensive infection control measures, including active surveillance and screening of high-risk individuals, to effectively identify and manage carriers of CR-GNB can prevent transmission and outbreaks.

Our study highlights high MDR- and CR-GNB infections in ICU, with prior antibiotic exposure and NGT use as risk factors, and diminishing clinical value of colistin and tigecycline. In this study setting and beyond, strict implementation of antimicrobial stewardship programs and adherence to infection prevention and control through monitoring, evaluation and feedback are warranted to curb CR-GNB infections, especially among risk groups.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We acknowledge the Nairobi West Hospital Microbiology Laboratory staff, Jeff Kwasi, Daniel Warui and Christopher Wambua, for their technical support during sample collection and processing.

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case study 2 microbiology

Expanding the horizons of levan: from microbial biosynthesis to applications and advanced detection methods

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Levan, a β-(2,6)-linked fructose polymer, exhibits diverse properties that impart versatility, rendering it a highly sought-after biopolymer with various industrial applications. Levan can be produced by various microorganisms using sucrose, food industry byproducts and agricultural wastes. Microbial levan represents the most potent cost-effective process for commercial-scale levan production. This study reviews the optimization of levan production by understanding its biosynthesis, physicochemical properties and the fermentation process. In addition, genetic and protein engineering for its increased production and emerging methods for its detection are introduced and discussed. All of these comprehensive studies could serve as powerful tools to optimize levan production and broaden its applications across various industries.

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This research was supported by the King Mongkut’s Institute of Technology Ladkrabang (KMITL) under a Doctoral Scholarship [KDS2022/014].

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Wang, S., Wu, B. & Todhanakasem, T. Expanding the horizons of levan: from microbial biosynthesis to applications and advanced detection methods. World J Microbiol Biotechnol 40 , 214 (2024). https://doi.org/10.1007/s11274-024-04023-w

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13: Putting it all Together—Case Studies in Microbiology

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  • Joan Petersen & Susan McLaughlin
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In this lab, a series of stations will be set up around the room, each of which will present a microbiology case study. Some background information will be given to you, along with organisms on slides, growth media, etc. This is your opportunity to put together everything that you have learned about microbiology this semester. You will be expected to integrate the various pieces of information that are provided to answer some questions about the situation and come up with a diagnosis. The case studies will not be graded, and you will have an opportunity to work together to solve these problems. All of these activities will be good practice for your lab practical—try to enjoy the process!

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    This paper highlights the fundamental role of integrating different geomatics and geophysical imaging technologies in understanding and preserving cultural heritage, with a focus on the Pavilion of Charles V in Seville (Spain). Using a terrestrial laser scanner, global navigation satellite system, and ground-penetrating radar, we constructed a building information modelling (BIM) system to ...

  29. 13: Putting it all Together—Case Studies in Microbiology

    In this lab, a series of stations will be set up around the room, each of which will present a microbiology case study. Some background information will be given to you, along with organisms on slides, growth media, etc. This is your opportunity to put together everything that you have learned about microbiology this semester. You will be ...

  30. Microbiology Lab Exam 2 (Case Study Exercise 10)

    Quiz yourself with questions and answers for Microbiology Lab Exam 2 (Case Study Exercise 10), so you can be ready for test day. Explore quizzes and practice tests created by teachers and students or create one from your course material.