Effects of Various Test Media on the Activities of 21 Antimicrobial Agents against Haemophilus influenzae

Michael R. Jacobs; Saralee Bajaksouzian; Anne Windau; Peter C. Appelbaum; Gengrong Lin; David Felmingham; Christine Dencer; Laura Koeth; Mendel E. Singer; Caryn E. Good

doi:10.1128/JCM.40.9.3269-3276.2002

DOI: 10.1128/JCM.40.9.3269-3276.2002

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FIG. 1.
Geometric mean MICs and upper and lower limits of 95% confidence limits (bars) for amoxicillin-clavulanic acid and cefprozil. Much less variation in mean MICs was found for amoxicillin-clavulanic acid ranges (0.7 to 1.4 μg/ml) for all methods and sites than for cefprozil (2.0 to 9.5 μg/ml). However, similar variations in the ranges of 95% confidence limits were noted for amoxicillin-clavulanate and cefprozil.

Tables

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TABLE 1.

Geometric mean MICs and standard deviations (doubling dilutions) for each method for agents with unimodal populations and >90% of endpoints on scale

Antimicrobial agent	MIC (μg/ml)^a
	Frozen MH HTM		In-house MH HTM						PML MH HTM		MH LHB NAD				IST LHB NAD		IST HTM
	Site 1		Site 1		Site 2		Site 3		Site 1		Site 1		Site 2		Site 3		Site 3
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Amox-clav acid^b	1.18	1.52	1.05	1.44	0.697	1.299	0.829	1.118	1.007	1.31	1.73	1.3	0.908	1.12	0.907	1.21	1	1.2
Azithromycin	1.602	0.84	0.938	0.872	0.525	0.728	0.674	0.769	0.683	0.821	0.859	0.98	0.693	0.693	0.717	0.745	0.747	0.713
Cefaclor	10.63	1.71	10.48	1.421	3.58	1.383	6.233	1.679	9.126	1.331	13.36	1.488	5.426	1.635	6.105	1.413	5.579	1.367
Cefdinir	ND^c	NA^a	0.536	1.176	0.291	1.06	0.48	0.736	0.514	1.118	0.595	1.234	0.409	1.104	0.566	0.672	0.525	0.537
Cefixime	0.042	1.002	0.051	1.008	0.0298	0.956	0.037	0.933	0.055	0.982	0.058	1.014	0.035	0.961	0.034	0.956	0.033	0.918
Cefprozil	7.835	1.65	7.568	1.489	2.809	1.36	3.63	1.676	6.148	1.398	9.514	1.54	4.691	1.728	3.074	1.562	3.458	1.444
Ceftriaxone	NA	NA	0.008	1.157	0.0058	0.945	0.0069	1.083	0.009	1.125	0.008	1.093	0.006	0.971	0.007	1.07	0.006	0.986
Cefuroxime	1.257	1.69	1.778	1.326	0.901	1.14	0.92	1.085	1.591	1.146	1.815	1.414	1.197	1.268	0.865	1.149	0.871	0.985
Chloramphenicol	0.547	1.38	0.768	1.213	0.4175	1.203	0.624	1.246	0.642	1.25	0.807	1.237	0.483	1.258	0.62	1.2	0.629	0.12
Ciprofloxacin	0.017	0.36	0.016	0.243	0.0158	0.2	0.016	0.243	0.016	0.293	0.016	0.243	0.016	0.2	0.017	0.356	0.019	0.468
Clarithromycin	8.112	0.899	6.869	0.894	5.278	0.791	7.21	0.796	5.897	0.833	6.021	0.911	5.696	0.81	7.111	0.805	7.013	0.8
Doxycycline	0.574	0.71	0.570	0.788	0.4234	0.878	0.582	0.786	0.574	0.779	0.547	0.825	0.384	0.874	0.758	0.829	1.094	0.787
Erythromycin	8.168	0.81	5.242	0.777	3.387	0.74	4.891	0.756	4.438	0.783	5.278	0.865	4.141	0.757	5.169	0.8	4.857	0.726
Gemifloxacin	0.009	0.45	0.008	0.243	0.008	0.243	0.008	0.243	0.008	0.14	0.008	0.1	0.008	0.1	0.008	0.261	0.008	0.367
Grepafloxacin	0.017	0.53	0.009	0.465	0.008	0.293	0.009	0.505	0.009	0.428	0.008	0.293	0.008	0.243	0.01	0.563	0.013	0.661
Levofloxacin	0.028	0.44	0.017	0.321	0.016	0.223	0.016	0.293	0.017	0.308	0.016	0.293	0.016	0.2	0.019	0.482	0.022	0.559
Ofloxacin	0.054	0.54	0.035	0.435	0.033	0.308	0.034	0.393	0.036	0.465	0.035	0.435	0.033	0.293	0.044	0.577	0.049	0.555
Trovafloxacin	0.018	0.63	0.0098	0.566	0.009	0.52	0.009	0.552	0.009	0.539	0.009	0.5	0.009	0.42	0.01	0.603	0.013	0.704

↵ a For an agent with a geometric mean MIC of 4 μg/ml and an SD of one doubling dilution, the MIC range ±1 SD is 2 to 8 μg/ml and the MIC range ±2 SD is 1 to 16 μg/ml; if the SD is 0.5 doubling dilution, the MIC range ±1 SD is 2.8 to 5.7 μg/ml and the MIC range ±2 SD is 2 to 8 μg/ml.
↵ b Amox-clav acid, amoxicillin-clavulanic acid, shown as amoxicillin component.
↵ c ND, not determined.
d NA, not applicable due to >10% off-scale endpoints.

TABLE 2.

GEE results; estimated regression coefficients and statistical values of significance for comparison to standard

Antimicrobial agent	Estimated regression coefficients and significance^a
	Site-related differences^b				Method-related differences^c
	Site 2		Site 3		PML MH HTM		MH LHB NAD		IST LHB NAD		IST HTM
	Estimate	P value	Estimate	P value	Estimate	P value	Estimate	P value	Estimate	P value	Estimate	P value
Amox-clav acid^d	−0.4600	<0.0001	−0.2850	0.0934	0.4550	<0.0001	0.2600	<0.0001	0.1319	0.0046	0.2733	<0.0001
Azithromycin	−0.5550	<0.0001	−0.3175	0.0067	0.2575	0.0004	0.1550	<0.0001	0.0900	0.1346	0.1500	0.0188
Cefaclor	−1.4250	<0.0001	−0.6875	0.0005	1.2875	<0.0001	0.4750	<0.0001	−0.0300	0.6856	−0.1600	0.0690
Cefixime	−0.7786	<0.0001	−0.4607	0.0078	0.8893	<0.0001	0.2214	<0.0001	−0.1000	0.1556	−0.1571	0.0287
Cefprozil	−1.2250	<0.0001	−0.9575	<0.0001	1.0275	<0.0001	0.5350	<0.0001	−0.2400	0.0020	−0.0700	0.3047
Ceftriaxone	−0.5000	<0.0001	−0.2700	0.0901	0.5300	<0.0001	0.0100	0.7455	−0.0200	0.7387	−0.1100	0.0134
Cefuroxime	−0.7900	<0.0001	−0.8550	<0.0001	0.7520	<0.0001	0.2200	0.0001	−0.0900	0.0673	−0.0800	0.1122
Chloramphenicol	−0.8100	<0.0001	−0.2650	0.0974	0.5850	<0.0001	0.1400	0.0001	−0.0100	0.8083	0.0100	0.7629
Ciprofloxacin	−0.0200	0.0979	0.0000	1.0000	0.0500	0.0310	0.0000	1.0000	0.0800	0.0089	0.2300	<0.0001
Clarithromycin	−0.2300	<0.0001	0.1450	0.2044	0.0850	0.0821	−0.0400	0.2734	−0.0200	0.6947	0.0400	0.4917
Doxycycline	−0.4700	<0.0001	0.0100	0.9278	0.4600	<0.0001	−0.1000	0.0003	0.3800	<0.0001	0.9100	<0.0001
Erythromycin	−0.4900	<0.0001	−0.0300	0.7874	0.3200	<0.0001	0.1500	<0.0001	0.0800	0.1660	−0.0100	0.8694
Gemifloxacin	0.0000	1.0000	0.0000	1.0000	−0.0200	0.1531	−0.0300	0.0786	0.0100	0.3149	0.0400	0.0979
Grepafloxacin	−0.0750	0.0002	0.0925	0.1551	0.0775	0.0049	−0.0750	0.0002	0.0500	0.2218	0.4800	<0.0001
Levofloxacin	−0.0550	0.0056	−0.0175	0.6902	0.0475	0.0352	−0.0150	0.1758	0.2300	<0.0001	0.4500	<0.0001
Ofloxacin	−0.0750	0.0010	−0.0225	0.7053	0.1125	0.0004	−0.0050	0.7053	0.3500	<0.0001	0.5300	<0.0001
Trovafloxacin	−0.0450	0.0786	−0.0275	0.7171	−0.0125	0.6951	−0.1150	<0.0001	0.1200	0.0199	0.4200	<0.0001

↵ a Boldface, significant at >0.25-dilution difference. Regression coefficients are shown as doubling dilution differences above (positive values) or below (negative values) standard, and statistical values of significance (P values are compared to standard).
↵ b Compared to site 1.
c Compared to MH HTM.
↵ d Amox-clav acid, amoxicillin-clavulanic acid.

TABLE 3.

Paired t test P value results within sites, with results of MH HTM for each site as standard

Antimicrobial agent	Paired t test P value results within sites compared to MH HTM for each site^b
	Site 1		Site 2 MH LHB NAD	Site 3
	PML MH HTM	MH LHB NAD	Site 2 MH LHB NAD	IST LHB NAD	IST HTM
Amox-clav Acid^a	0.2410	0.0130	<0.0001	0.0060	<0.0001
Azithromycin	<<0.0001	0.0860	<<0.0001	0.1610	0.0200
Cefaclor	0.0059	0.0003	<<0.0001	0.7910	0.0960
Cefdinir	0.3060	0.0540	<<0.0001	<0.0001	0.0150
Cefixime	0.3200	0.0020	<0.0001	0.0390	0.0110
Cefprozil	<0.0001	0.0010	<<0.0001	0.0009	0.1980
Ceftriaxone	0.8199	0.5660	0.0960	0.7410	0.0160
Cefuroxime	0.0200	0.7270	<<0.0001	0.0700	0.1170
Chloramphenicol	<0.0001	0.1630	<0.0001	0.8100	0.7650
Ciprofloxacin	0.0109	0.0005	<0.0001	0.0004	<<0.0001
Clarithromycin	<0.0001	<0.0001	0.0336	0.6970	0.4950
Doxycycline	1.0000	0.0896	0.0005	<<0.0001	<<0.0001
Erythromycin	<0.0001	0.8290	<0.0001	0.1710	0.8700
Gemifloxacin	<0.0001	<<0.0001	<<0.0001	0.0118	<<0.0001
Grepafloxacin	0.8199	<<0.0001	<<0.0001	0.1310	<<0.0001
Levofloxacin	0.3680	0.0190	0.4410	<0.0001	<<0.0001
Ofloxacin	0.0517	0.7410	0.0019	<<0.0001	<<0.0001
Trovafloxacin	0.5930	<0.0001	<0.0001	0.0007	<<0.0001

↵ a Amox-clav acid, amoxicillin-clavulanic acid.
↵ b Critical values of significance were P < 0.0003, which was based on P < 0.05 with Bonferroni correction for multiple tests, and are shown in boldface. Additionally, P < 10⁻⁹ values were regarded as highly significant (shown as <<0.0001 values).

TABLE 4.

Paired t test P value results of MH HTM results between sites

Antimicrobial agent^a	Paired t test P value results of MH HTM results between sites^b
Antimicrobial agent^a	Site 1 vs. site 2	Site 1 vs. site 3	Site 2 vs. site 3
Amox-clav acid^a	<<0.0001	0.0551	0.1703
Azithromycin	<<0.0001	0.0005	0.0010
Cefaclor	<<0.0001	0.0003	0.0002
Cefdinir	<<0.0001	0.2551	<0.0001
Cefixime	<<0.0001	0.0007	0.0249
Cefprozil	<<0.0001	<0.0001	0.0753
Ceftriaxone	<<0.0001	0.0814	0.0837
Cefuroxime	<<0.0001	<0.0001	0.8497
Chloramphenicol	<<0.0001	0.0729	0.0004
Ciprofloxacin	0.1583	1.0000	0.5298
Clarithromycin	<<0.0001	0.5465	<0.0001
Doxycycline	<<0.0001	0.7883	0.0002
Erythromycin	<<0.0001	0.3757	<0.0001
Gemifloxacin	1.0000	1.0000	1.0000
Grepafloxacin	0.0004	0.3096	0.0011
Levofloxacin	0.0136	0.6570	0.2873
Ofloxacin	0.0075	0.7407	0.3384
Trovafloxacin	0.0705	0.6019	0.6982

↵ a Amox-clav acid, amoxicillin-clavulanic acid.
↵ b Critical values of significance were P < 0.0003, which was based on P < 0.05 with Bonferroni correction for multiple tests, and are shown in boldface. Additionally, P < 10⁻⁹ values were regarded as highly significant (shown as <<0.0001 values).

TABLE 5.

Paired t test P value results for LHB-supplemented media

Antimicrobial agent	Paired t test P value results for LHB-supplemented media^b
Antimicrobial agent	Site 1 vs site 2	Site 1 vs site 3	Site 2 vs site 3
Amox-clav acid^a	<0.0001	0.0332	0.9460
Azithromycin	0.0008	0.0367	0.6595
Cefaclor	<<0.0001	<0.0001	0.3890
Cefdinir	<<0.0001	0.6150	0.0003
Cefixime	<<0.0001	<0.0001	0.7870
Cefprozil	<<0.0001	<<0.0001	0.0041
Ceftriaxone	<<0.0001	0.0825	0.2351
Cefuroxime	<<0.0001	<0.0001	0.0078
Chloramphenicol	<<0.0001	0.0186	0.0301
Ciprofloxacin	0.1583	0.0590	0.0177
Clarithromycin	0.1583	0.0495	0.0059
Doxycycline	<<0.0001	0.0001	<<0.0001
Erythromycin	<0.0001	0.7962	0.0039
Gemifloxacin	1.0000	0.1583	0.1583
Grepafloxacin	0.0832	0.0003	<0.0001
Levofloxacin	0.0246	0.0001	<0.0001
Ofloxacin	0.0042	<0.0001	<0.0001
Trovafloxacin	0.4823	0.0070	0.0016

↵ a Amox-clav acid, amoxicillin-clavulanic acid.
↵ b MN LHB NAD in sites 1 and 2; IST LHB NAD in site 3. Critical values of significance were P < 0.0003, which was based on P < 0.05 with Bonferroni correction for multiple tests, and are shown in boldface. Additionally, P < 10⁻⁹ values were regarded as highly significant (shown as <<0.0001 values).

TABLE 6.

Percentages of MICs within one or two dilutions from reference method based on test site and medium

Antimicrobial agent	% MICs within 1 dilution/2 dilutions from reference method
	In-house MH HTM			PML MH HTM (Site 1)	MH LHB NAD		IST LHB NAD (Site 3)	IST HTM (Site 3)
	Site 1	Site 2	Site 3	PML MH HTM (Site 1)	Site 1	Site 2	IST LHB NAD (Site 3)	IST HTM (Site 3)
Amox-clav acid^a	97/100	82/98	82/93	98/100	91/100	89/99	81/92	81/94
Amoxicillin	95/100	74/91	80/90	97/98	86/100	86/97	80/90	72/86
Ampicillin	100/100	83/100	79/100	100/100	100/100	93/100	84/89	87/95
Azithromycin	91/100	40/92	69/95	71/97	97/98	70/94	68/93	73/95
Cefaclor	91/98	49/84	67/97	86/97	80/95	71/87	67/86	67/85
Cefprozil	97/99	47/81	90/98	90/98	81/96	67/89	43/73	52/78
Ceftriaxone	99/100	96/100	100/100	100/100	100/100	99/100	90/100	91/100
Cefuroxime	81/97	86/97	72/90	72/90	87/99	91/99	70/89	72/90
Chloramphenicol	87/100	90/100	95/100	95/100	82/100	92/100	93/96	94/96
Ciprofloxacin	99/100	100/100	99/100	99/100	99/100	96/99	99/100	100/100
Clarithromycin	99/100	96/100	99/100	99/100	98/100	96/100	94/98	93/98
Doxycycline	99/100	99/100	97/100	97/100	97/99	96/100	85/96	67/96
Erythromycin	98/100	67/99	88/100	88/100	95/99	87/99	88/96	83/96
Gemifloxacin	64/97	41/98	41/92	41/92	12/80	5/67	75/97	87/98
Grepafloxacin	79/99	54/99	81/100	81/100	41/98	23/98	95/100	99/100
Levofloxacin	99/100	78/100	99/100	99/100	98/100	74/100	95/100	98/99
Ofloxacin	100/100	100/100	100/100	100/100	99/100	98/100	99/100	99/100
Trim./Sulfa.^b	71/92	89/96	52/83	52/83	74/92	54/84	65/78	69/91
Trovafloxacin	71/94	66/91	85/99	68/94	61/91	52/90	90/100	96/100

↵ a Amox-clav acid, amoxicillin-clavulanic acid.
↵ b Trim./Sulfa., trimethoprim-sulfamethoxazole.

TABLE 7.

MIC₅₀ and MIC₉₀ for each method

Antimicrobial agent	MIC₅₀/MIC₉₀ (μg/ml)
	Frozen MH HTM (Site 1)	In-house MH HTM			PML MH HTM (Site 1)	MH LHB NAD		IST LHB NAD (Site 3)	IST HTM (Site 3)
	Frozen MH HTM (Site 1)	Site 1	Site 2	Site 3	PML MH HTM (Site 1)	Site 1	Site 2	IST LHB NAD (Site 3)	IST HTM (Site 3)
Amox-clav acid^a	1/8	1/4	0.5/2	1/2	1/4	1/4	1/2	1/4	1/4
Amoxicillin	8/>16	4/>16	2/>16	2/>16	4/>16	4/>16	2/>16	2/>16	4/16
Ampicillin	2/>16	2/>16	1/>16	1/>16	2/>16	2/>16	1/>16	1/>16	1/>16
Azithromycin	2/4	1/2	0.5/1	0.5/1	1/1	1/2	0.5/2	1/1	1/1
Cefaclor	16/64	8/32	4/8	4/32	8/32	16/64	4/32	8/16	4/16
Cefdinir	N/A	0.5/2	0.25/1	0.5/1	0.5/1	0.5/2	0.25/1	0.5/1	0.5/1
Cefixime	0.03/0.12	0.06/0.12	0.03/0.06	0.03/0.06	0.06/0.12	0.06/0.12	0.03/0.06	0.03/0.06	0.03/0.06
Cefprozil	8/32	8/32	2/8	4/16	4/32	8/32	4/32	4/32	4/16
Ceftriaxone	0.008/0.03	0.008/0.03	0.004/0.015	0.004/0.015	0.008/0.015	0.008/0.015	≤0.004/0.015	≤0.004/0.03	≤0.004/0.015
Cefuroxime	1/8	2/4	1/2	1/2	1/4	1/8	1/4	1/2	1/2
Chloramphenicol	0.5/1	0.5/1	0.25/0.5	0.5/1	0.5/1	0.5/1	0.5/0.5	0.5/1	0.5/1
Ciprofloxacin	0.015/0.03	0.015/0.015	0.008/0.015	0.015/0.015	0.015/0.015	0.008/0.015	0.008/0.015	0.015/0.03	0.015/0.03
Clarithromycin	8/16	8/16	4/8	8/16	8/8	8/16	8/16	8/8	8/8
Doxycycline	0.5/1	0.5/1	0.5/1	0.5/2	0.5/1	0.5/1	0.25/1	0.5/2	1/4
Erythromycin	8/16	4/8	4/4	4/8	4/8	4/8	4/8	4/8	4/8
Gemifloxacin	0.008/0.015	0.004/0.008	0.004/0.004	0.004/0.008	0.004/0.008	0.002/0.004	0.002/0.004	0.004/0.008	0.008/0.008
Grepafloxacin	0.015/0.015	0.008/0.015	0.008/0.008	0.008/0.015	0.008/0.015	0.004/0.008	0.004/0.008	0.008/0.015	0.015/0.03
Levofloxacin	0.03/0.03	0.015/0.015	0.015/0.015	0.015/0.015	0.015/0.015	0.015/0.015	0.015/0.015	0.015/0.03	0.015/0.03
Ofloxacin	0.06/0.06	0.03/0.06	0.03/0.03	0.03/0.06	0.03/0.06	0.03/0.06	0.03/0.03	0.03/0.06	0.06/0.06
Trim./Sulfa.^b	0.12/>4	0.12/>4	0.06/>4	0.12/>4	0.25/>4	0.03/>4	0.03/4	0.03/4	0.12/4
Trovafloxacin	0.015/0.03	0.008/0.015	0.008/0.015	0.008/0.015	0.008/0.015	0.008/0.015	0.008/0.015	0.008/0.015	0.015/0.015

↵ a Amox-clav acid, amoxicillin-clavulanic acid.
↵ b Trim./Sulfa., trimethoprim-sulfamethoxazole.

TABLE 8.

Ranges and mean percentages of susceptible isolates for all methods based on NCCLS and PK-PD breakpoints

Antimicrobial agent	NCCLS			PK-PD
Antimicrobial agent	Susceptible breakpoint (μg/ml)	% Susceptible range	% Susceptible mean	Susceptible breakpoint (μg/ml)	% Susceptible range	% Susceptible mean
Amoxicillin-clavulanic acid^a	4	89.0-99.0	96.9	2	75.0-96.0	85.7
Amoxicillin	NA^b	NA	NA	2	41-57	49.3
Ampicillin	1	44.0-57.0	50.3	NA	NA	NA
Azithromycin	4	All at 100	100	0.12	0.0-6.0	2.2
Cefaclor	8	47.0-90.0	67.7	0.5	0.0-3.0	0.78
Cefdinir	1	79.0-100.0	93.5	0.5	61.0-87.9	74.2
Cefixime	1	All at 100	100	1	All at 100	100
Cefprozil	8	52.0-91.0	73.2	1	1.0-27.0	13.2
Ceftriaxone	2	All at 100	100	NA	NA	NA
Cefuroxime	4	83.0-99.0	93.8	1	47.0-75.8	62.7
Chloramphenicol	2	92.9-93.0	93.0	NA	NA	NA
Ciprofloxacin	1	All at 100	100	1	All at 100	100
Clarithromycin	8	77.0-96.0	90.3	0.25	All at 0	0
Doxycycline	NA	NA	NA	0.25	78.0-89.0	80.9
Erythromycin	NA	NA	NA	0.25	All at 0	0
Gemifloxacin	NA	NA	NA	1	All at 100	100
Grepafloxacin	0.5	All at 100	100	1	All at 100	100
Levofloxacin	2	All at 100	100	1	All at 100	100
Ofloxacin	2	All at 100	100	1	All at 100	100
Trim./Sulfa.^c	0.5	71.0-82.0	76.0	NA	NA	NA
Trovafloxacin	1	All at 100	100	1	All at 100	100