Week 7 Material Bacteriology II

what is a nosocomial infection

an infection that occurs during a hospital-based stay that was neither present nor in the prodromal (incubation) state when the patient entered the hospital

nosocomial UTI infections

33% of all nosocomial infections

E. coli is the most common organism

nosocomial lung infections

15% of nosocomial infections

Risk factors: advanced age, chronic lung disease, large volume aspiration, chest surgery, intracranial pressure monitoring devices, ICU hospitalization, intubation

Most common organisms causing nosocomial pneumonia: Gram-negative rods, S. aureus, M. catharralis

nosocomial surgical site infections

15% of all nosocomial infections

Most common agents: Gram positives, S. aureus and CNS, enterococci, Gram-negative rods, Candida

Risk factors: advanced age, obesity, infection at a remote site, malnutrition, diabetes, extended preoperative stay, extended surgery time

Nosocomial bloodstream infections

13% of all nosocomial infections

risk factors: age 1 year or younger or 60 years and older, malnutrition, immunosuppressive chemotherapy, loss of skin integrity, burns or bedsores, severe underlying illness, undwelling device, ICU stay, prolonged hospital say

Nosocomial infection transmission

Direct: contaminated food or intravenous solution

Indirect: from patient to patient via hands

Droplet: inhalation of droplets (>5 um in diameter) that cannot travel more than 3 feet (pertussis)

Airborne: inhalation of droplets (</= 5 um) that travel long distances (M. tuberculosis)

vector borne: insects and rats, rare mode in developed countries

antibiotic resistance in nosocomial infections

Nosocomial infections have changed due to the use and overuse of antibiotics

patients’ normal flora changes after hospitalization, become colonized with resistant organisms, it patients returned to nursing homes, resistant organisms can be transferred to other patients

risk factors for acquiring highly resistant organism include: prolonged hospitalization, prior antibiotic treatments

nosocomial infection prevention

Hand washing between patient contact is more effective

segregation of infected patients

wearing masks, gloves, and gowns

bagging of contaminated articles

cleaning room thoroughly after patient discharged

placing instructions on patients doors

types of bioterrorist evens

announce: overt

unannounced: covert

category A bioterror agents

Highest concern

variola major

bacillus anthracis

yersinia pestis

clostridium botulinum toxin

francisella tularensis

hemorrhagic fever viruses (filoviruses and arenaviruses)

category B bioterror agents

coxiella buretti

brucella spp.

burkholderia mallei

staphylococcal enterotoxin B

food and waterborne agents

ricin toxin

alphaviruses

category C bioterror agents

nipah virus

hantavirus

yellow fever virus

tickborne encephalitis virus

multidrug resistant mycobacterium tuberculosis

BSL-1

organism that do not cause disease in healthy humans and are of minimal potential hazard to lab personnel and the environment (ex. B. subtilis)

standard microbiology safety practices

no special safety equipment required

laboratory clothing recommended

sink for hand washing, open bench top resistant and impervious to water

BSL-2

organisms associated with human disease and pose moderate potential hazard (ex. shigella)

BSL-1 safety practices plus limited access to lab and extreme precautions with contaminated sharps

class I or II biological safety cabinet

appropriate personal protective equipment

BSL-1 facility requirements plus autoclave and eye wash

BSL-3

organism which pose serious or potentially lethal disease when inhaled (ex. M. tuberculosis)

BSL-2 safety practices plus controlled access to lab and all procedures conducted in biological safety cabinet

Class I or II biological safety cabinet

appropriate personal protective equipment

BSL-2 facility requirements plus negative airflow, air exhaust to outside, self-closing double doors

BSL-4

organisms with life threatening potential and transmission by aerosol or of unknown risk of transmission (ex. Ebola)

maximum containment, special clothing shower upon exit, separate building, special engineering design

route of infection- food

potentially significant route of delivery

secondary to either purposeful or accidental exposure to aerosol

route of infection- water

capacity to affect large numbers of people

dilution factor

water treatment may be effective in removal of agents

route of infection- respiratory

inhalation of spores, droplets and aerosols

aerosols are most effective delivery method

advantages of biologic as weapons

infectious via aerosol, organisms fairly stable in environment, susceptible civilian populations, high morbidity and mortality, person to person transmission (smallpox, plague, VHF), difficult to diagnose and/or treat, easy to obtain, inexpensive to produce, potential for dissemination over large geographic area, creates panic, can overwhelm medical services, perpetrators escape easily

level A laboratory

BSL-2 lab with a certified class II biological safety cabinet, BSL-1 microbiology practices, directed by competent scientists, personnel specifically trained in handling pathogenic agents.

role is to rule out critical biological agents and refer to higher level laboratory

if announced: notify FBI, and the PHL, based on consultation, test and refer

if unannounced: rule out, if unable to rule out call the nearest level B lab

laboratory risk for bioterrorism agents

B. anthracis: BSL-2, low risks

Y. pestis: BSL-2, medium risk

brucella spp: BSL-2/3, high risk

F. tularensis: BSL-2/3, high risk

botulinum toxin: BSL-2, medium risk

smallpox: BSL-4, high risk

viral hemorrhagic fever: BSL-4, high risk

plague epidemiology

US averages 13 cases per year

30% of cases are in Native Americans in the southwest.

15% case fatality

most cases occur in summer

bubonic, septicemic, and pneumonic

Yersinia pestis specimen selection

bubonic: bubo, lymph node aspirate

septicemic: blood, obtain three sets 10-30 min apart

pneumonic: sputum, bronchial washings

yersinia pestis specimen inoculation

inoculate routine plating media and make thin smear for DFA

use Wayson only if DFA is unavailable, Wayson stain is not diagnostic must confirm by DFA and mouse inoculation

yersinia pestis characteristics

small, gram negative bipolar coccobacilli

Wayson stain is pink-blue cells with a closed safety pin looks

BHI broth will have little chunks in it

Botulism

Diagnosis of botulism is made clinically

Health care providers suspecting botulism should contact their State Health Department

Infective dose: 0.001 µg/kg

Incubation period: 18 - 36 hours

Dry mouth, double vision, droopy eyelids, dilated pupils n Progressive descending bilateral muscle weakness & paralysis. Respiratory failure and death

Mortality 5-10%, up to 25%

Level A Procedures for Botulism Event

Properly collected specimens are to be referred to designated testing laboratories

Prior to the shipment of any botulism associated specimen, the designated laboratory must be notified and approved by the State Health Department

Clinical specimens to be collected: Serum, Gastric contents or vomitus, Feces or return from sterile water enema, Wound tissue

Botulism toxins are extremely poisonous

Minute quantities acquired by ingestion, inhalation, or by absorption can cause death

All materials suspected of containing toxin must be handled with CAUTION

anthrax Epidemiology

Primarily a disease of herbivorous animals such as sheep, cattle, goats, and horses

Humans acquire the infection accidentally in agricultural or industrial setting, During processing of hides or animal hair, gains access through cuts or inhalation

Anthrax Clinical Manifestations

Cutaneous anthrax begins 2 to 5 days after inoculation of spores (95%), lesion starts as an erythematous papule that progresses into an ulcerative black eschar or “malignant pustule”

Pulmonary anthrax (rare) is acquired by inhalation of spores, malaise, mild fever, nonproductive cough follows

Gastrointestinal (very rare)

Anthrax Laboratory Diagnosis

Large gram-positive bacilli in short chains

Nonhemolytic, white to gray on sheep’s blood agar

"Medusa head” appearance

Lack of motility

Penicillin inhibition zone

Capsule formation

“STICKY” consistency on SBA

catalase-positive

Aerobic spore formation

Inhalational Anthrax

Infective dose = 8,000 - 15,000 spores

Incubation period = 1-6 days

Duration of illness = 3-5 days

Fever, malaise, and fatigue

Short period of improvement = up to 2 days

Abrupt respiratory distress…death <24hrs

No person-to-person transmission

Anthrax Specimen Selection

Inhalation: Sputum and Blood

Cutaneous: Vesicles and Eschar

Gastrointestinal: Stool and Blood

Francisella tularensis

Plague-like disease in rodents (California), Deer-fly fever (Utah), Glandular tick fever (Idaho and Montana) Market men’s disease (Washington, DC), Rabbit fever (Central States), O’Hara’s disease (Japan)

Poorly staining, tiny Gram-negative coccobacilli

Fastidious, requires cysteine for robust growth: Cysteine Heart Agar (CHA) is ideal, BYCE can be used

tularemia

Contagious --- no

Infective dose --- 10-50 organisms

Incubation period --- 1-21 days (average=3-5 days)

Duration of illness --- ~2 weeks

Mortality --- treated : low, untreated: moderate

Persistence of organism ---months in moist soil n Vaccine efficacy --- good, ~80%

Brucellosis

Zoonotic disease caused by any of 4 Brucella spp.: abortus, melitensis, suis, and canis

Systemic infection characterized by an undulant fever pattern

Relatively rare in the U.S. with approximately 100 cases/year

The most commonly reported laboratory-associated bacterial infection

Infective dose = 10 -100 organisms

Incubation period = 5 days - > 6 months

Duration of illness = weeks to months

Fever, profuse sweating, malaise, headache and muscle/back pain

No person-to-person transmission

Mortality: < 5% n Stable organisms

Brucellosis transmission

Ingestion: The most common mode of transmission

Direct skin contact/puncture: Occupational hazard for farmers, butchers and veterinarians

Aerosols: Highly infectious

Brucella spp. Specimen Selection

Serum

Blood or bone marrow

Tissue (spleen, liver)

Brucella spp. Key Level A Lab Tests

Colonial morphology on SBA, Fastidious ¨Visible growth may take 48 - 72 hrs, Small (0.5-1.0mm), convex, glistening, Non-hemolytic and non-pigmented

Oxidase

Urea hydrolysis: B. suis & B. canis ~15 min, B. abortus & B. melitensis ~24hr

What is a zoonosis?

A disease that occurs predominantly in vertebrate animals and is transmitted to humans through direct contact with an infected animal or its excretions, or by an insect vector that feeds on animals.

Name two zoonoses associated with livestock.

Campylobacter (chicken) and Salmonella (chicken/eggs).

What is Bacillus anthracis commonly known as?

Anthrax, also referred to as Woolsorters Disease.

What disease is caused by Brucella?

Brucellosis.

What is Francisella tularensis associated with?

Tularemia.

What zoonotic disease is associated with rodents?

Leptospirosis, caused by Leptospira interrogans.

What are two vector-borne zoonoses?

Rickettsiae and Borrelia burgdorferi (Lyme Disease).

What is Yersinia pestis known for causing?

Plague.

What are the two bacterial zoonoses associated with Streptobacillus and Spirillum?

Streptobacillus moniliformis and Spirillum minor.

What are the general features of Streptobacillus moniliformis?

Long, thin, pleomorphic, gram-negative, aligns in chains, and forms 'L' forms.

How is Streptobacillus transmitted to humans?

Through a bite from a rat or ingestion of contaminated milk.

What is the incubation period for Streptobacillus moniliformis?

Less than 10 days.

What are common symptoms of zoonotic diseases caused by Streptobacillus?

Abrupt onset, ulceration at the bite site, lymphadenopathy, high fever, headache, rash, and recurrent fevers.

What is the treatment for infections caused by Streptobacillus?

Penicillin.

What is the typical laboratory diagnosis method for Spirillum?

Darkfield microscopy of blood or infected tissue.

What is the habitat of Streptobacillus?

Oropharynx of rats and other rodents.

What are the two cycles of plague transmission?

Urban cycle and sylvatic cycle.

What is the primary cause of massive epidemics of plague?

Infected fleas biting domestic rats.

What are the symptoms of bubonic plague?

Buboes, bacteremia, and seeding of the lungs, liver, and spleen.

What is the laboratory diagnosis for Yersinia pestis?

Direct microscopic examination of aspirates from buboes or sputum, showing gram-negative bacilli.

What type of colonies does Yersinia pestis grow on sheep's blood agar?

Nonhemolytic, smooth, and slightly opaque colonies.

What is the incubation period for Spirillum?

Greater than 10 days.

What are the common sources of infection for zoonotic diseases?

Direct contact with infected animals, bites, or ingestion of contaminated food or water.

What are the risk factors associated with zoonotic infections?

Occupational exposure, contact with animals, and environmental factors.

Antibiotics

Substances that inhibit the growth of or destroy microorganisms.

Resistance

The ability of microorganisms to withstand the effects of antibiotics.

Mechanisms

Processes by which antibiotics exert their effects or by which resistance is developed.

Marcia A.

Author or contributor related to the topic of antibiotics and resistance.

Antimicrobial chemotherapy

The use of chemicals to inhibit or kill microorganisms in or on the host

Selective toxicity

The agent used must inhibit or kill the microorganism without harming the host

Antimicrobial chemotherapeutic chemicals

Chemicals synthesized in the lab which can be used therapeutically on microorganisms

Bacteriostatic

Inhibit bacterial growth

Bacteriocidal

Kill the targeted organisms

Broad spectrum

Effective against BOTH gram positive and gram negative bacteria

Narrow spectrum

Effective against one type of organism

Opportunistic infections

Destroy normal flora

Yeast infections

Infections caused by yeast

Ulcerative colitis

A chronic inflammatory bowel disease

Drug toxicity

Adverse effects caused by drugs

Allergic reactions

Immune responses to substances that are usually harmless

Select for resistant microorganisms

Encouragement of the growth of microorganisms that are resistant to treatment

Cell wall biosynthesis

The process of forming the cell wall of bacteria

Folate synthesis

The process of producing folate, essential for DNA biosynthesis

DNA replication

The process of copying DNA

RNA transcription

The process of synthesizing RNA from a DNA template

mRNA translation

The process of translating mRNA into a protein

Beta-lactam antibiotics

Bind to penicillin binding proteins (PBP) on cell wall and interrupt cell wall synthesis

Natural Penicillin

Penicillin G, effective against gram-positive bacteria

Semi-synthetic penicillins

Methicillin, nafcillin, which are not inactivated by penicillinase

Beta lactamase inhibitors

Clavulanic acid and sulbactam, used to protect penicillins from degradation

Cephalosporins

Antibiotics that are resistant to penicillinase and have a broad spectrum

Carbapenems

Imipenem, which resist beta-lactamase activity and inhibit peptidoglycan synthesis

Glycopeptides

Do not bind to penicillin binding proteins but bind to cell wall precursors to prevent cell wall development

Inhibition of Folate Synthesis

Involves drugs like sulfamethoxazole and trimethoprim that are broad spectrum when combined.

tuberculosis infections

Infections caused by the bacterium Mycobacterium tuberculosis.

mRNA translation interference

The disruption of the process by which messenger RNA is translated into proteins.

Inhibit protein synthesis

Prevent the formation of proteins necessary for bacterial growth and function.

Agents that block translation

Substances that interfere with the process of translating mRNA into proteins.

30s subunit

A component of the bacterial ribosome that can be targeted by antibiotics to inhibit translation.

50s subunit

Another component of the bacterial ribosome involved in peptide bond formation and tRNA release.

Aminoglycosides

A class of antibiotics that bind irreversibly to the 30s subunit, causing misreading of mRNA.