THE WSTERN Journal of Medicine Refer to: Granoff DM, Basden M, Rockwell R: Ampicilin-resistant Hemophilus influenzae-Emergence in California's Central Valley. West J Med 128:101-105, Feb 1978

Ampicillin-Resistant Hemophilus influenzae Emergence in California's Central Valley DAN M. GRANOFF, MD; MILLIE BASDEN, RN, and RICHARD ROCKWELL, MA Fresno, California

Ampicillin-resistant Hemophilus influenzae had not been identified in Fresno, California, before June 1976. In the 12 months that followed, eight resistant type B strains and three resistant nontypable isolates were cultured from patients treated at two hospitals that provide nearly all of the acute pediatric inpatient care for the area. Two of the resistant strains were obtained from patients with invasive infections and represented 4.2 percent of Hemophilus influenzae isolated from blood, cerebrospinal fluid or joint aspirates during the 12 months. The remaining six resistant type B strains were obtained from 117 patients, and were the predominant organism in cultures of other sites, primarily respiratory secretions. In two of three patients infected with nontypable organisms, resistance appeared to emerge during therapy with ampicillin. Measurement of lactamase was a practical and accurate method for differentiating between ampicillin-sensitive and resistant strains. All ten of the lactamase-positive isolates tested had minimal inhibitory concentrations (MIC) for ampicillin of 15 ,g per ml, or less. In contrast 30 lactamase-negative strains had MIC's of 1.5 ,g per ml, or less, of ampicillin. Our results indicate that ampicillin resistance has become a significant problem in the Central Valley of California and probably the entire state.

CASES OF ampicillin-resistant Hemophilus influenzae were first reported in the United States in 1974 and were at first limited to a few localities.1-4 In subsequent years, clinical isolates resistant to ampicillin have been reported throughout the country and have necessitated changes in the recommendation of initial therapy of systemic heFrom the Department of Pediatrics, Valley Medical Center and University of California, San Francisco, Medical Education Program, Fresno. Submitted July 18, 1977. Reprint requests to: Dan M. Granoff, MD, Dept. of Pediatrics, Valley Medical Center of Fresno, 445 South Cedar Avenue,

Fresno, CA 93702.

mophilus infection.5 Despite this trend, cases of ampicillin-resistant hemophilus have been infrequently observed in California. In 1975 Overturf and his co-workers reported on their experience with antimicrobial susceptibility of Hemophilus influenzae at the Los Angeles County Hospital.6 These investigators could detect no significant change in susceptibility to ampicillin during the previous decade. However, in an addendum to their paper, they noted their first highly resistant isolate which was obtained from a child with THE WESTERN JOURNAL OF MEDICINE

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ABBREVIATIONS USED IN TEXT CIE= countercurrent immunoelectrophoresis MBC=minimal bactericidal concentration MIC = minimal inhibitory concentration

H. influenzae meningitis treated in Pasadena, California. Jacobsen and co-workers subsequently summarized data from 58 resistant isolates submitted to the Center for Disease Control through May 1975. Only two of these were from California. The purposes of the present report are to describe the emergence of ampicillin-resistant Hemophilus infiluenzae in Fresno County, California, and to confirm the' usefulness of testing for '/ lactamase as an adjunct to routine antimicrobial susceptibility testing. The data to be reported are representative of the pathogenic strains isolated in this community and indicate that ampicillin-resistance has become a significant problem in the Central Valley of California and probably the entire state.

Materials and Methods Fresno County has a population of approximately 450,000 and covers 6,000 square miles. The county is located in the Central Valley of California and surrounds the city of Fresno (population 165,000). A program of surveillance for ampicillin-resistant H. influenzae was begun at Valley Medical Center in September 1975. With the identification of the first resistant isolate in June 1976, this program was intensified by combining the clinical resources of Valley Medical Center and Valley Children's Hospital, the two hospitals in Fresno that provide nearly all acute pediatric inpatient care for the county and sparsely populated surrounding areas. Isolates of Hemophilus influenzae obtained from blood, cerebrospinal fluid or joint aspirates were systematically referred from the clinical laboratories of the two hospitals to the Infectious Disease Research Laboratory. When multiple isolates were received from a single patient, the results from one of the isolates, usually blood, are reported. In addition to receiving isolates from invasive infection, selected type B strains obtained from other sites, primarily respiratory secretions, were also sent to our laboratory. Some were selected randomly, and others were referred because there was a question of ampicillin-resistance on the basis of the disc agar diffusion test.

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FEBRUARY 1978 * 128 * 2

Strains cultured from mucous membrane sites were studied only if the organism was the predominant isolate in the specimen. After isolation on chocolate agar, H. influenzae was identified by methods previously described,8 except that antigenic typing was done by slide agglutination instead of the Quellung reaction. Antimicrobial susceptibility of Hemophilus influenzae was done by the clinical laboratory using Kirby-Bauer disc agar diffusion.9 However, chocolatized sheep blood agar was used instead of supplemented clear media. Upon receipt of hemophilus isolates in the Infectious Disease Research Laboratory, /3 lactamase production was measured using the method of Escamilla.10 Specific antigenic typing of the positive isolates was confirmed by slide agglutination, or countercurrent immunoelectrophoresis (CIE) " in the presence of antisera types a-f (Hyland Laboratories, Costa Mesa, California). Antimicrobial susceptibility was studied by broth dilution techniques for ten of the 11 isolates identified as positive for ,B lactamase, and for 30 ,B lactamase-negative 'isolates, including those initially recorded by the clinical laboratory as possibly resistant to ampicillin. We used brain-heart infusion broth supplemented by 5 percent Fildes extract (Difco Laboratories) and an innoculum of bacteria in log phase growth carefully diluted to a final concentration of 104 colony forming units per ml. The minimal inhibitory concentration (MiC) was recorded after 24 hours incubation at 35 'C.12 The minimal bactericidal concentration (MBC) was obtained by subculturing 24-hour broth cultures onto chocolate agar and determining the concentration of ampicillin that resulted in 100 percent reduction of bacterial growth. A known ampicillin-sensitive strain, and an ampicillin-resistant Hemophilus influenzae type B strain (MIc, 62 ,ug per ml) obtained from the Center for Disease Control were tested in each assay.

Results During the 12 months ending June 15, 1977, eight Hemophilus influenzae type B strains were isolated at the two hospitals and confirmed by our laboratory as ,B lactamase positive or resistant to ampicillin (Table 1). They represented 4.8 percent of all type B strains identified at the two institutions. There were 48 patients with invasive hemophilus infections who had organisms identified in blood, cerebrospinal fluid or joint aspirates. Of

HEMOPHILUS INFLUENZAE TABLE 1.-Hemophilus influenzae Type B Isolated at Two Hospitals in Fresno County, California (June 15, 1976-June 14, 1977)

Specimen

1i Lactainiase Total Percenzt Niumtber of Nunmber NminI/ber of Tested Positive Total Isolates

Blood, cerebrospinal fluid or joint aspirate 48 Other .............. 117

42 62

2 6

(4.2) (5.1)

TOTAL ........... 165

104

8

(4.8)

these, 42 were tested for /8 lactamase and 2 were positive. The six isolates not tested were from cultures obtained during the first six weeks of the study and were inadvertently not sent to our laboratory. One of these isolates was recorded as resistant to ampicillin. In all, 117 patients had Hemophilus influenzae type B as the predominant organism in cultures of other sites, primarily respiratory secretions. A total of 62 isolates were assayed for /3 lactamase activity and 6 were positive. The remaining 55 isolates were not tested for /8 lactamase but were reported as sensitive to ampicillin by the clinical laboratory. Three ampicillin-resistant, nontypable Hemophilus influenzae isolates were also identified during this period. However, nontypable Hemophilus influenzae were not systematically examined and for this reason we cannot estimate the incidence of ampicillin-resistance among these strains. It is of interest that two of the nontypable strains werefrom patients initially infected with ampicillinsensitive organisms. One patient had chronic bronchitis and the original isolate was grown in nearly pure culture from sputum. The second

patient was an infant with otitis media who had heavy Hemophilus influenzae grown from middle ear drainage. In both patients, the organisms were initially sensitive to ampicillin when tested by disc agar diffusion, and were negative for /3 lactamase. Treatment with ampicillin resulted in poor clinical responses, and repeat cultures 30 days later in the patient with bronchitis, and 10 days later in the patient with otitis media, showed ampicillin-resistant Hemophilus influenzae positive for ,/ lactamase. Further data on the three nontypable and eight type B ampicillin-resistant hemophilus are shown in Table 2. Ampicillin-resistant strains were detected in Fresno throughout the year but more than half of the isolates were observed during the most recent three months. Resistant Hemophilus organisms were isolated from patients of all ages (range: 1 month to 63 years). There were two adults in the group and one of the two had bacteremia caused by a confirmed, ampicillinresistant organism. It is possible that there were other adults in Fresno County with resistant hemophilus infection during the 12 months because our surveillance was primarily directed at childhood infections. Although we initially defined ampicillin-resistant Hemophilus influenzae as organisms with an MIC greater than 3 jtg per ml, all ten of the /3 lactamase-positive isolates studied by tube dilution techniques had MIC's of 15 ,ug per ml or greater. The MBC'S were one tube dilution higher in five of the isolates and two tubes higher in the remaining five of the group. Thirty Hemophilus influenzae type B isolates negative for jB lactamase production were selected

TABLE 2.-fl Lactamase-Producing Isolates of Hemophilus influenzae Identified at Two Hospitals in Fresno County, California (June 15, 1976-June 14, 1977) Patient

Age of

Date

Patient

1.... 6/15/76 2/2 years 2. ... 7/19/76 13 months 3. ... 7/27/76 6 months 4. 9/29/76 6 years 11/17/76 11 months 5. 6. ... 4/ 4/77 63 years 7. ... 4/13/77 5 months 4/19/77 1 month 8. 9.... 5/10/77 13 years 10. ... 5/20/77 10 months 11. ... 6/13/77 56 years ...

...

...

Source

Tracheal aspirate Throat Cerebrospinal fluid Sputum Ear Blood

Eye Eye Sputum* Ear* Sputum

MBC = minimal bactericidal concentration MIC = minimal inhibitory concentration

*Initial isolates

were

Type

MIC ,ug/ml

B B B B B B NT B NT NT B

31 62 ND 62 31 31 31 15 15 31 31

MBC

gg/ml

125 125 ND 250 125 62 62 62 31 125 62

ND = not done NT = nontypable

ampicillin-sensitive and negative for f8 lactamase.

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for further study of ampicillin susceptibility by tube dilution techniques. Twelve had MIC's of less than 0.35 jug per ml, 16 had MIC's of 0.7 ug per ml and two isolates had MIC's of 1.5 Mag of ampicillin per ml. No 8 lactamase-negative isolate was detected that was resistant to ampicillin. Standardized Kirby-Bauer disc diffusion sensitivity testing as done by the clinical laboratory was a reasonable, but not invariably accurate, method for determining ampicillin-sensitive and resistant hemophilus isolates. False resistance was reported in nine isolates, or 5.8 percent of susceptible strains. False susceptibility was reported in one of the ampicillin-resistant isolates (zone size, 21 mm) and occurred in the adult with bacteremia who had four positive blood cultures. The initial report in this patient led to continuation of ampicillin therapy for four days, during which the patient's condition deteriorated. All strains sensitive or resistant to ampicillin encountered during the 12 months were susceptible to chloramphenicol.

Discussion Beginning in June 1976 ampicillin-resistant Hemophilus influenzae emerged as important pathogens in Fresno County, California. Resistant Hemophilus organisms accounted for two and possibly three cases of invasive disease. In addition, several days after the admission to hospital of the patient in our initial index case, Hemophilus influenzae meningitis developed in a close playmate of this patient during a trip to Kern County, California. The isolates from the playmate were repeatedly tested at the local hospital using established disc agar diffusion methods,9 and were found to be resistant to ampicillin (zone size, 13 mm). This patient's isolates were not included in our results because the child was admitted to hospital outside of Fresno County. However, she is of particular interest because of the clustering of the two cases. Disc agar diffusion sensitivity testing has been previously reported to be an accurate method for differentiating between ampicillin sensitive and resistant Hemophilus influenzae.9" 2 Chocolate agar, which was employed by the clinical laboratories during our study, was found to be less reliable for sensitivity testing of hemophilus than supplemented clear media such as Mueller Hinton. When grown on chocolate agar, resistant strains showed a gradation of growth around the ampicillin disc and the endpoint of the zone 104

FEBRUARY 1978 * 128 * 2

was more difficult to determine. It was also more difficult to see small amounts of growth in the inhibiting zone, the resistant colonies being more apparent on clear media. We experienced similar difficulties with our sensitivity cultures on chocolate agar and no longer employ this medium for sensitivity testing. The observation that nearly all ampicillinresistant Hemophilus influenzae produce /8 lactamase allows us to use this property as a method for detecting resistant organisms. A number of practical, rapid and accurate enzymatic assays have been described for detection of this enzyme,10" 3-'5 and the use of one of these circumvents many of the technical difficulties encountered in traditional antimicrobial susceptibility testing of Hemophilus influenzae.'2 Our experience supports the previous recommendation that clinical laboratories routinely test for this enzyme as an adjunct to standard Kirby-Bauer disc sensitivity testing.9" 15 Emergence of ampicillin-resistant, Hemophilus influenzae in patients receiving ampicillin therapy is rare and, to our knowledge, has not been previously reported in patients with otitis media or purulent bronchitis. However, the cases of two patients with Hemophilus influenzae type B meningitis have been described in which the original infections were caused by ampicillin-sensitive organisms, and in which relapses were associated with ampicillin-resistant, /B lactamase positive strains.16"17 It is possible that the original infections in these patients as well as in our patients were caused by a mixed population of resistant and sensitive Hemophilus influenzae, and that initial sensitivity testing and enzymatic assays failed to detect the small proportion of resistant organisms. A third patient with Hemophilus influenzae meningitis has been reported in whom the original cerebrospinal fluid culture showed a mixed population of ampicillin-resistant and sensitive organisms.2 Previous reports of ampicillin-resistant Hemophilus influenzae infection have described localized outbreaks caused by resistant organisms,'l-3 or have presented data from isolates studied at the Center for Disease Control,7 or a large university hospital'8 where the frequency of resistant organisms may have been biased by patient referral patterns. Despite these differences, the incidence of ampicillin-resistance found in the present study corresponds closely to reports from other centers, and to data recently gathered at

HEMOPHILUS INFLUENZAE

the Center for Disease Control (Clyde Thornsberry, personal communication). However, it should be noted that we did not test for ,B lactamase in all of the hemophilus strains isolated by the clinical laboratories. For this reason the 4.8 percent incidence of ampicillin resistance reported for Fresno (Table 1) is a minimal figure, since some resistant organisms may have gone unrecognized. At present our data support the recommendation of the American Academy of Pediatrics that chloramphenicol be employed in the initial therapy of suspected systemic Hemophilus influenzae type B infections. Results of antimicrobial sensitivity testing and assays of / lactamase production should be utilized by clinicians in deciding whether or not to continue therapy with ampicillin or chloramphenicol. REFERENCES 1. Tomeh MO, Starr SE, McGowan JE Jr., et al: Ampicillinresistant Haemophilus influenzae type B infection. JAMA 229:295297, Jul 15, 1974 2. Khan W, Ross S, Rodriquez W, et al: Haemophilus influenzae type B resistant to ampicinin. JAMA 229:298-301, Jul 15, 1974 3. Center for Disease Control: Ampicillin-resistant Hemophilus influenzae-Texas. Morbidity Mortality Weekly Rep 23:99, Mar 16, 1974 4. Schiffer MS, Schneerson R, MacLowry J, et al: Clinical,

bacteriological and immunological characterisation of ampicillinresistant Haemophflus influenzae type b. Lancet 2:257-259, Aug 3, 1974

5. Committee on Infectious Diseases: Ampicillin-resistant strains of Hemophilus influenzae type b. Pediatrics 55:145-146, Jan 1975 6. Overturf GD, Wilkins J, Leedom JM et al: Susceptibility of Hemophilus influenzae, type b, to ampicillin at Los Angeles County/ University of Southern California Medical Center-A reappraisal after ten years. J Pediatr 87:297-300, Aug, 1975 7. Jacobson JA, McCormick JB, Hayes P et al: Epidemiologic characteristics of infections caused by ampicillin-resistant Hemophilus imfluenzae. Pediatrics 58:388-391, Sep 1976 8. Granoff DM, Roskes S: Urinary tract infection due to Hemophilus influenzae, type b. J Pediatr 84:414-416, Mar 1974 9. Center for Disease Control: Current trends ampicillin-re-

sistant Haemophilus influenzae. Morbidity Mortality Weekly Rep 24:205-206, Jun 14, 1975 10. Escamilla J: Susceptibility of Haenzophilus influenzae to ampicillin as determined by use of a modified, one-minute betalactamase test. Antimicrob Agents Chemother 9:196-198, Jan 1976 11. Myhre EB: Typing of Hemophilus influenzae by counterimmunoelectrophoresis. Acta Pathol Microbiol Scand (B) 82:164-

166, Apr 1974 12. Thornsberry C, Kirven LA: Antimicrobial susceptibility of Haemophilus influenzae. Antimicrob Agents Chemother 6:620-624, Nov 1974 13. Thornsberry C, Kirven LA: Ampicillin resistance in Haemophilus influenzae as determined by a rapid test for beta-lactamase production. Antimicrob Agents Chemother 6:653-654, Nov 1974 14. Catlin BW: lodometric detection of Haemophilus influenzae Beta-lactamase: Rapid presumptive test for ampicillin resistance. Antimicrob Agents Chemother 7:265-270, Mar 1975 15. Scheifele DW, Syriopoulou VP, Harding AL, et al: Evaluation of a rapid beta-lactamase test for detecting ampicillin-resistant straint of Hemophilus influenzae type b. Pediatrics 58:382-386, Sep 1976 16. Delage G, DeClerck Y, Lescop J, et al: Heinophilus influenzae type b infections: Recurrent disease due to ampicillinresistant strains. J Pediatr 90:319-320, Feb 1977 17. Albritton WL, Hammond G, Hoban S, et al: Ampicillinresistant H. influenzae subdural empyema following successful treatment of apparently ampicillin-sensitive H. influienzae meningitis. J Pediatr 90:320-321, Feb 1977 18. McGowan JE, Terry PM, Nahmias AJ: Susceptibility of Haemophilus influenzae isolates from blood and cerebrospinal fluid to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. Antimicrob Agents Chemother 9:137-139, Jan 1976

Blood Loss in the Colon DR. CLOUSE: .... most of the blood losses from the colon, about 90 percent of them, are from diverticula and about 75 percent of the blood losses from diverticula are from the right side of the colon rather than the left. So the concept of doing a left colectomy blindly is, I think, probably not justified before angiography. A patient has to be losing probably 0.5 to 1.0 ml of blood a second for bleeding from the gastrointestinal tract to be shown easily by angiography." -MELVIN E. CLOUSE, MD, Boston, citing a paper by William Joseph Casarella of Columbia-Presbyterian Medical Center, New York City. Extracted from Audio-Digest Surgery, Vol. 23, No. 16, in the Audio-Digest Foundation's subscription series of tape-recorded programs. For subscription information: 1577 East Chevy Chase

Drive, Glendale, CA 91206.

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Ampicillin-resistant Hemophilus influenzae.

THE WSTERN Journal of Medicine Refer to: Granoff DM, Basden M, Rockwell R: Ampicilin-resistant Hemophilus influenzae-Emergence in California's Central...
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