UNIVERSITY OF COLORADO SCHOOL OF MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM

1. UNIVERSITY OF COLORADO SCHOOL OF MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM MICROBIOLOGY Alpha-hemolytic Streptococciand Enterococci Topic 1: VRE Screening

2. 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

3. 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 = Enterococcusgallinarum/cassiloflavus. The susceptibility result for vancomycin was 8 mcg/ml.

4. ENTEROCOCCUS:VRE SCREEN Recapitulation The increase in the numbers of vancomycin-resistant 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.

5. 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?

6. UNIVERSITY OF COLORADO SCHOOL OF MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM MICROBIOLOGY Alpha-hemolytic Streptococciand Enterococci Topic 2: Penicillin Resistant Pneumococci

7. 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?

8. 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 o’clock position indicates intermediate resistance to SXT.

9. Antibiotic Resistant Pneumococcus Emergence of world-wide resistance to pneumococci South Africa, Baraguanath Hospital, Johannesburg (Am J Dis Child 146:920-923, 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 84:839-846,1988). Spain: Hospital General Gregorio Mara-nor, Madrid: 42.5% of strains isolated from 139 patients were “non-susceptible” (Clin Infect Dis 14:427-425, 1992).

10. Emergence of world-wide resistance to pneumococci Africa: Nairobi, Kenya. Study population—of 150 S. pneumoniae isolates from HIV-positive patients, 19% were resistant with MIC’s ranging from 0.12 – 0.25ug/ml. Rare serotype 14 in 18% of cases. Hungary, Heim Pal Children’s 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 163:542-548, 1991). Houston, Baylor college of Medicine. Of 95 isolates of S. pneumoniae, 34 were susceptible at <0.1ug/ml, 42 were intermediate at 0.1 – 1.0 ug/ml; and, 19 were resistant at >2ug/ml (12.1% of all isolates). (Antimicrob Agents Chemother 36:1703-1707. 1992). Atlanta, Georgia, CDC. Pneumococcal Surveillance Working Group (Facklam, et al. J Infect Dis 163:1273-1278, 1991): Study population—5,459 isolates of S. pneumoniae submitted from 35 hospitals during the period 1979 to 1987. Overall resistance was 5% at MIC’s >0.1ug/ml.

11. 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 343:1917-1924, 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: Susceptible: MIC <0.06 ug/ml (oxacillin zones >20mm) Relatively resistant: MIC 0.12 – 1.0 ug/ml (oxacillin zones <19mm) Resistant: MIC >1.0 ug/ml (oxacillin testing cannot distinguish between relatively resistant and resistant).

12. UNIVERSITY OF COLORADO SCHOOL OF MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM MICROBIOLOGY Alpha-hemolytic Streptococciand Enterococci Topic 3: Viridans Streptococci

13. 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 wbc’s/mm3 (73% neutrophils; 12% bands) Cerebrospinal Fluid: • 3520 wbc’s/mm3 (100% neutrophils) • Glucose: <1 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

14. 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 354:13-25, 2006)

15. Streptococcus suis: Characteristics • α-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

16. 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

17. 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 seriousmanifestation • Human fatality rate of 5-10% • Serotype 2 (of 35 serotypes) responsible for vast majority of human disease

18. 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

19. 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

20. Streptococcus suis

21. 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. bovissensu stricto, S. gallolyticus, S. infantarius, S. suis.

22. 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

23. 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

24. 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 (MIC’s >0.25ug/ml) and 41% were resistant to erythromycin (Potgeiter, et al. 1992. Eur J Clin Microbiol Infect Dis 11:543-546). These strains remained susceptible to cephalosporins, imipenem, and vancomycin. In a second follow-up study (Antimicrob Agents Chemother 37:2740-2742, 1993) 4 strains of S. mitis were resistant to penicillin (MIC’s16-32ug/ml), and two demonstrated high-level gentamicin resistance (MIC >2000 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.

25. 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. salivarius S. mitis