UNIVERSITY OF COLORADO

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UNIVERSITY OF COLORADO SCHOOL OF
MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY
TRAINING PROGRAM MICROBIOLOGY Alpha-hemolytic
Streptococci and Enterococci Topic 1 VRE
Screening
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CASE STUDIES AND TOPIC REVIEWS

• As a point of departure from the case study
  format presented before, the focus this week will
  be more informational than self-assessment.
• The purpose here is to introduce three topics as
  related to the non-beta hemolytic streptococci
  based on information presented either during past
  CACMLE Teleconferences or during the 2006 Micro
  in the Mountains conference.
• The topic reviews will include
• Screening for Vancomycin-Resistant Enterococci
• Emergence of penicillin-resistant pneumococci
• Clinical relevance of the viridans streptococci

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ENTEROCOCCUS VRE SCREEN
Presenter Claudia Hinnenbusch,
MT(ASCP) Clinical Microbiology UCLA Medical
Center, Los Angeles CACMLE Teleconference Oct.
24, 2001
A 38-year-old female was admitted to the UCLA
hospital for a liver transplant. Following
protocol, a rectal swab was collected to rule
out vancomycin-resistant Enterococcus sp.
Colonies isolated on Columbia colistin-naladixic
acid modified agar containing 10ug of vancomycin
were transferred to sheep blood agar. Dull gray,
opaque, alpha-hemolytic colonies were isolated
from the subculture after 24 (left) and 48
(right) hours incubation. A Vitek Gram positive
identification (GPITM) card gave a profile
number of 77367270530 Enterococcus
gallinarum/cassiloflavus. The susceptibility
result for vancomycin was 8 mcg/ml.
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ENTEROCOCCUS VRE SCREEN Recapitulation
The increase in the numbers of vancomycin-resistan
t enterococci has prompted the implementation of
surveillance programs in hospitals to monitor its
incidence and spread. Enterococcus faecium is
most common vancomycin resistant strain, having
acquired either the Van- A or the Van-B gene. E.
faecalis possessing these vancomycin-resistant
genes are less common. Isolates of E.
cassiloflavus and E. gallinarum, that have MIC
levels to vancomycin that are elevated (between 4
and 32 mcg/ml) are also encountered. Since this
low level resistance is intrinsic, such isolates
are not reported as VRE. Automated bacterial
identification systems are not always reliable in
identifying the enterococci to the species level.
Thus, when screening for VRE, one should accept
results from these systems only if they correlate
with the colony morphology and the known
antimicrobial profile of the organism in question.
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ENTEROCOCCUS VRE SCREEN Screening Protocol

• VRE Screening Protocol used at the UCLA Medical
  Center
• Patients being admitted for organ transplant have
  admission cultures, as well as cultures when they
  are transferred to another ward, or released.
  Cultures include a nasal swab to screen for MRSA,
  and a rectal swab to screen for VRE.
• Stool cultures from inpatients are only processed
  for VRE surveillance when the Infection Control
  team has given prior approval. They are rarely
  used to demonstrate clearance in patients
  previously found to have VRE.
• Since a correlation has been made showing an
  increased incidence of VRE in patients whose
  stool samples are positive for C. difficile
  toxin, stool samples positive for C. difficile
  are screened quarterly for the presence of VRE.
  In our institution, this has proven to be a
  cost-effective and efficient way to screen for
  VRE.
• NOTE IS THERE ANY SUCH PROTOCOL IN EFFECT AT
  UCHSC?

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UNIVERSITY OF COLORADO SCHOOL OF
MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY
TRAINING PROGRAM MICROBIOLOGY Alpha-hemolytic
Streptococci and Enterococci Topic 2
Penicillin Resistant Pneumococci
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Abbreviated Identification of Streptococcus
pneumoniae
SUMMARY Mucoid alpha-hemolytic
colonies on Blood agar Lancet-shaped,
gram-positive cocci in pairs, with halo Bile
Soluble Susceptible to Optochin (P
Disk) Streptococcus pneumoniae
Perform antimicrobial susceptibility test
WHY IS THIS NECESSARY?
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Antibiotic Resistant Pneumococcus
A respiratory isolate was found to have no zone
of inhibition around the 1ug oxacillin disk (6mm)
in a screening disk diffusion test. It was
reported out as penicillin resistant. As a
follow up, a routine disk antibiotic
susceptibility test panel was performed. As
illustrated in the photograph, the isolate was
found to be susceptible to other antibiotics
(cefuroxime, tetracycline, erythromycin,
chloramphenicol, and ceftriaxone). The 15mm in
diameter zone around the disk at the 1 oclock
position indicates intermediate resistance to SXT.
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Antibiotic Resistant Pneumococcus
Emergence of world-wide resistance to pneumococci
South Africa, Baraguanath Hospital, Johannesburg
(Am J Dis Child 146920-923, 1992) revealed
resistance in 40 of community acquired and 80
of hospital acquired isolates of S. pneumoniae
recovered from 83 children with meningitis and/or
bacteremia. South Africa Witwaters-rand
University Of a study population of 4766
consecutive isolates of S. pneumoniae recovered
from blood and CSF during the period 1979-1986,
the average penicillin resistance climbed from
3.8 in 1979 to 14.2 in 1986. 92.2 of
serogroups were 6 or 19. Spain Bellvitge
Principes Espanya Hospital, Barcelona 23 rate
of resistance among 66 episodes of pneumococcal
meningitis (Am J Med 84839-846,1988). Spain
Hospital General Gregorio Mara-nor, Madrid 42.5
of strains isolated from 139 patients were
non-susceptible (Clin Infect Dis 14427-425,
1992).
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Emergence of world-wide resistance to pneumococci
Africa Nairobi, Kenya. Study populationof
150 S. pneumoniae isolates from HIV-positive
patients, 19 were resistant with MICs ranging
from 0.12 0.25ug/ml. Rare serotype 14 in 18
of cases. Hungary, Heim Pal Childrens Hospital,
Budapest. Epidemiologic study revealed 58 of
all S. pneumoniae isolates to be resistant to
penicillin (70 of isolates recovered from
children.) (J Infect Dis 163542-548,
1991). Houston, Baylor college of Medicine. Of
95 isolates of S. pneumoniae, 34 were susceptible
at lt0.1ug/ml, 42 were intermediate at 0.1 1.0
ug/ml and, 19 were resistant at gt2ug/ml (12.1
of all isolates). (Antimicrob Agents Chemother
361703-1707. 1992). Atlanta, Georgia, CDC.
Pneumococcal Surveillance Working Group (Facklam,
et al. J Infect Dis 1631273-1278, 1991) Study
population5,459 isolates of S. pneumoniae
submitted from 35 hospitals during the period
1979 to 1987. Overall resistance was 5 at MICs
gt0.1ug/ml.
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Emergence of world-wide resistance to pneumococci
These accounts were considered quite worrisome
at the time. The high prevalence of
penicillin-resistant S. pneumoniae in certain
locales undoubtedly represents antibiotic
pressure, where antibiotic therapy has been
administered virtually without restriction. Recent
studies reveal that resistance has increased in
many parts of the world to 15 - 35 depending on
the geographic region. (Whitney, et al. NEJM
3431917-1924, 2000) Thus it is currently
recommended that S. pneumoniae isolates from
blood, CSF, and other closed body sites, and from
treatment failures, should be tested routinely
for susceptibility to penicillin. Guidelines Susc
eptible MIC lt0.06 ug/ml (oxacillin zones
gt20mm) Relatively resistant MIC 0.12 1.0
ug/ml (oxacillin zones lt19mm) Resistant MIC
gt1.0 ug/ml (oxacillin testing cannot distinguish
between relatively resistant and resistant).
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UNIVERSITY OF COLORADO SCHOOL OF
MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY
TRAINING PROGRAM MICROBIOLOGY Alpha-hemolytic
Streptococci and Enterococci Topic 3 Viridans
Streptococci
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Case Study Human Meningitis

• The case is that of a 59 y.o. male farmer with
  sudden onset of fever, and confusion
• Peripheral Blood count
• 12,800 wbcs/mm3 (73 neutrophils 12 bands)
• Cerebrospinal Fluid
• 3520 wbcs/mm3 (100 neutrophils)
• Glucose lt1 mg/deciliter
• Protein 368 mg/deciliter

Illustration of gray white, alpha-hemolytic
colonies recovered on sheep blood agar from
spinal fluid sediment after 48 hours
incubation. The isolate was identified
as Streptococcus suis
Case presented by William M. Janda, Ph.D.
D(ABMM) Assoc Prof Dept. Pathology Director
Clinical Microbiolog Laboratory University of
Illinois Medical Center Chicago, Illinois Micro
in the Mountains 2006
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Case Study Human Meningitis
Based on the clinical history and the laboratory
findings, the patient was empirically started on
ceftriaxone and vancomycin. When the isolate was
identified as Streptococcus suis, the therpapy
was switches to IV ampicillin. The patient
complained of lower back pain. MRI studies
revealed diskitis and osteomyelitis of L3 and
L4. The patient was discharged after 13 days to
complete a 6-week course of IV ampicillin and
oral clindamycin. This was the first reported
case in the United States. (NEJM 35413-25, 2006)
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Streptococcus suis Characteristics

• a-hemolytic streptococcus
• No growth at 10oC or 45oC
• LAP-Positive
• Esculin hydrolysis Positive
• Growth, 6.5 NaCl-Negative
• Arginine Dihydrolase-Positive
• Hippurate hydrolysis, urease, acetoin production
  Negative
• Acid from glucose, maltose, sucrose, and lactose
• No acid from mannitol, sorbitol, or ribose
• Included in the API Strep data base

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Streptococcus suis Epidemiology

• Although found worldwide, there has never been a
  case reported in the U.S. (until 2006!!!)
• Two cases reported in Canada
• Majority of human disease reported in Asia
  (Thailand, China, and Hong Kong)
• Third most common agent of bacterial meningitis
  in Hong Kong
• Human cases reported from the Netherlnds,
  Denmark, Great Britain, France, Belgium, Germany,
  and Sweden
• Human infections most common in those who work
  directly with swine or in the manufacture of pork
  products
• Abattoir and slaughterhouse workers, pig farmers,
  meat inspectors, veterinarians

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Streptococcus suis The Pigs Group B
Streptococcus

• Pathogen of swine
• Transmitted from aymptomatic sows to their
  newborn
• Rapidly fatal disease in piglets (sepsis,
  meningitis, pneumonia)
• Sporadic disease in humans
• Meningitis is the most serious manifestation
• Human fatality rate of 5-10
• Serotype 2 (of 35 serotypes) responsible for vast
  majority of human disease

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Streptococcus suis Clinical Disease

• Human infections enter through breaks in skin,
  the nasopharynx, or the gastrointestinal tract
• Influenza-like prodrome with rapid development
  of bacteremia and meningitis
• High rate of cochlear-vestibular involvement
  resulting in ataxia, dizziness
• Cranial nerve involvement leads to hearing loss
• Complications associated with bacteremic
  dissemination
• Arthritis, spondylodiscitis, endophthalmitis,
  peritonitis, pneumonia, and endocarditis

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Sichuan, China Outbreak July-August, 2005

• 215 human cases reported among farmers exposed
  during the slaughter of pigs
• 28 developed toxic shock syndrome
• Sepsis (24), meningitis (48) or both
• 62 mortality
• Ribotyping revealed that the same strain was
  reponsible for all cases

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Streptococcus suis
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Viridans Streptococcal Groups

• Group II Sanguis Group
• S. sanguinis (3 biotypes), S. parasanguinis, S.
  gordonii, S. sinensis
• Group III Mitis Group
• S. mitis, S. oralis, S. cristatus, S. peroris, S.
  infantis, S. australis, S. oligofermentans
• Group IV Mutans Group
• S. mutans, S. sobrinus, S. cricetus, S. downei,
  S. ratti, S. macacae, S. ferus
• Group V Salivarius Group
• S. salivarius, S. vestibularis, S. infantarius,
  S. thermophilus, S. hyointestinalis
• Group VI Anginosus Group (also included in
  beta-hemolyic group)
• S. constellatus subspecies, S. anginosus, S.
  intermedius
• Group VII Bovis Group
• S. bovis sensu stricto, S. gallolyticus, S.
  infantarius, S. suis.

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Viridans Group Streptococci

• Found in the upper respiratory tract and the
  urogenital tract
• Endocarditis
• 30-40 of cases due to viridans streptococci
• Usually isolated from multiple blood cultures
• Occur in patients with pre-existing valvular
  disease
• Also associated with infection of prosthetic
  valves
• Complications may include multi-valve infection,
  mitral valve aneurysms, paravalvular abscesses,
  and glomerulonephritis
• S. mitis, S. sanguis, S. oralis, S. gordonii, S.
  mutans, S. salivarius, S. vestibularis, and S.
  sinensis

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Viridans Streptococcal Bacteremia

• Prolonged bacteremia with viridans streptococci
  in neutropenic pediatric and adult patients now
  recognized as a distinct clinical entity
• Associated with aggressive cytotoxic chemotherapy
  given for treatment of leukemias, lymphomas,
  solid tumors, and bone marrow transplantation
• Risk factors
• Administration of high doses of cytotoxic agents
  (esp. cytarabine)
• Presence of mucosal ulcerations secondary to
  chemotherapy/radiation (oral mucositis)
• Absence of previous antimicrobial therapy
• Severe neutropenia
• May also be complicated by development of ARDS,
  hypotension, shock, and endocarditis

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Viridans Streptococcal Susceptibility Testing
In past years, the viridans streptococci were
generally susceptible to penicillin, ampicillin,
and most other antimicrobial agents. More
recently, resistance has developed against
penicillins, cephalosporins, aminoglycosides, and
other classes of antibiotics. In a study of 211
viridans streptococci recovered from blood
cultures, 38 were resistant to penicillin (MICs
gt0.25ug/ml) and 41 were resistant to
erythromycin (Potgeiter, et al. 1992. Eur J Clin
Microbiol Infect Dis 11543-546). These strains
remained susceptible to cephalosporins, imipenem,
and vancomycin. In a second follow-up study
(Antimicrob Agents Chemother 372740-2742, 1993)
4 strains of S. mitis were resistant to
penicillin (MICs16-32ug/ml), and two
demonstrated high-level gentamicin resistance
(MIC gt2000 ug/ml). These gentamicin resistant
strains contained the same structural gene that
codes for gentamicin resistance in E. faecalis
and E. faecium, integrated into the chromosome
and not the plasmid.
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Abbreviated Identification of Viridans
Streptococci
Small, dry, gray, alpha-hemolytic colonies on
sheep blood agar
Gram positive cocci in long chains
Optochin resistant Bile insoluble
PYR Negative
VP Positive Acid from mannitol/sorbitol
Arginine dihydrolase Positive
VP Pos mannitol/sorbitol Negative
Chemically inert
S. mutans
S. sanguius
S. mitis
S. salivarius