A national collaborative study of the in vitro activity of oral cephalosporins and loracarbef (LY 163892)

Pathology (1997), 29, pp. 79-83

A NATIONAL COLLABORATIVE STUDY OF THE IN VITRO ACTIVITY OF ORAL CEPHALOSPORINS AND LORACARBEF (LY 163892) R. A. V.

BENN*,

C. J.

FERNANDESt, G.

R.

NIMMO:j: AND THE AUSTRALIAN GROUP FOR THE

STUDY OF ANTIMICROBIAL RESISTANCE (AGAR)1j[

Department of Microbiology, Royal Prince Alfred Hospital, NSW*, Microbiology Department Royal North Shore Hospital, NSWt, and the Department of Microbiology, Princess Alexandra Hospital, Qld*

Summary

MATERIALS AND METHODS

A national collaborative study involving the laboratories of 17 Australian hospitals examined the in vitro activity of loracarbef, cefaclor, cephalexin, amoxycillin and amoxycillin/clavulanate against 2661 recently isolated common bacterial pathogens. Loracarbef was the most active agent against Escherichia coli (MICgo = 1 mg/I) and had activity comparable to other agents against Klebsiella pneumoniae and Proteus mirabilis. Like the oral cephalosporins, it had no activity against species of Enterobacter and Serratia. ~-Iactamase-producing Staphylococcus aureus and Haemophilus influenzae were moderately sensitive to loracarbef (MIC 9o = 8 mg/I for both species). Streptococcus pneumoniae was moderately sensitive to loracarbef (MIC go = 2 mg/I) but strains which were insensitive to penicillin were often highly resistant.

Bacterial strains

Key words: Loracarbef, cefaclor, cephalexin. Accepted 24 September 1996

INTRODUCTION Loracarbef is a carbacephem analogue of cefaclor, a ~-lactam antibiotic from which it differs only in the substitution of carbon for sulphur at position one of the dihydrothiazine ring. l The result is an orally absorbed compound, the first of a new class of ~-lactam antibiotics, which has greater chemical stability,2 a longer plasma half life3 and greater resistance to degradation by plasmid-mediated ~-lactamases4 than its predecessor. A large national collaborative study of the activity of loracarbef was undertaken in Australia in 1991-1992 using an agar dilution technique. The study, conducted using common pathogens collected by hospital laboratories, also looked at the activity of comparable orally absorbed ~-lactams.

'l[The following members of AGAR were involved: P. Collignon and 1. Bell, Woden Valley Hospital, ACT; R. Munro and D. Daley, Liverpool Hospital, NSW; A. Vickery and B. Yan, Royal Prince Alfred Hospital, NSW; G. Gilbert and R. Mallon, Westmead Hospital, NSW; R. Haski, P. Catenach and L. Hunt, Eli Lilly Australia, NSW; J. Tumidge aud C. Coutant, Monash Medical Centre, Vic; J. Spicer and C. Frauklin, The Alfred Hospital, NSW; J. Andrew and K. Stockmann, St Vincent's Hospital, Vic; G. Hogg and M. Easton, Royal Children's Hospital, Vic; K. Ott, Royal Hobart Hospital, Tas; 1. Lim and B. Winter, Institute of Medical and Veterinary Sciences, SA; H. Pruul, Flinders Medical Centre, SA; K. Karthigasu and L. Mulgrave, Queen Elizabeth 1I Medical Centre, WA; D. McGechie, M. Toohey andG. Francis, Frellumtle Hospital, WA; K. Christiansen, Royal Perth Hospital, WA; V. Asche, Royal Darwin Hospital, NT; J. Bull, Princess Alexandra Hospital, Qld; and J. Faoagali, Royal Brisbane Hospital, Qld.

A total of 2661 recent isolates included in the study were collected in late 1991 and early 1992 from 17 teaching hospitals in all Australian states and territories. Only single isolates of one species were included from individual patients and all isolates were judged to have been clinically significant. Cultures were identified using standard bacteriological methods. Antimicrobial agents All strains were tested for susceptibility to loracarbef (LOR), cefaclor (CCR), cephalexin (CLX), amoxycillin (AMX), amoxycillin/clavulanate 2: 1 (AMC) and penicillin (PEN). All antibiotics were tested over the range of 0.25-64 mg/l except for pencillin which was tested at 0.008--4 mg/1. Laboratory standards of loracarbef, cefaclor and cephalexin with known potencies were supplied by EIi Lilly Australia Pty Ltd. Susceptibility tests For all species except Haemophilus infiuenzae, minImUm inhibitory concentrations (MIC) of antibiotics were determined by an agar dilution procedure using Iso-Seusitest agar (Oxoid, Basingstoke, UK) supplemented with 5% 'freeze/thaw' lysed horse blood. Strains of H. injiuenzae were tested on Hemophilus Test Medium. s Preparation of inocula, the inoculation and incubation of plates and the determination of endpoints were performed according to the NCCLS method. 6 Staphylococcus aureus (ATCC 25923 and ATCC 29213), Staphylococcus epidermidis (ATCC 14990), Escherichia coli (ATCC 25922) and Enterobacter cloacae (ATCC 13047) were included in each replica plate. (3-lactamase production by H. injiuenzae and S. aureus was tested by the nitrocefin method.

RESULTS The activity of five antibiotics agains the Enterobacteriaceae is shown in Table 1. As determined by the MIC 9o, loracarbef was four times more active than cefaclor, and eight times more active than either cephalexin or amoxycillinJclavulanate, against 318 isolates of E. coli. Although not displayed in the table, the activity of amoxycillin was bimodal; 175 E. coli had MICs in the range 0.25-32 mg/l while the rest were not inhibited by the highest concentration (64 mg/l) used in the study. When clavulanic acid was combined with amoxycil1in the MIC 90 was 8 mg/l. While the activity of loracarbef against Klebsiella pneumoniae was comparable to that against E. coli, some strains of K. pneumoniae displayed high level resistance which is further analysed in Table 4. Among the other Enterobacteriacea tested, Proteus mirabilis had sensitivities similar to E. coli, while 20 isolates of Salmonella species were somewhat more resistant. Serratia marcescens and Enterobacter cloacae were mostly resistant. The data for Citrobacter species reveal the relative susceptibility of C. diversus and relative resistance of C. freundii.

0031-3025/97/010079-5 © 1997 Royal College of Pathologists of Australasia

80

Pathology (1997), 29, February

BENN etal.

TABLE

1

Activity of loracarbef and other antibiotics against Enterobacteriaceae

Organism (number tested)

Range

MIC9Q MICso (mgll)

Escherichia coli (318)

LOR CCR CLX AMX AMC

0.25->64 0.25->64 2->64 0.5->64 0.5-64

0.5 1 4 8 4

Klebsiella pneumoniae (254)

LOR CCR CLX AMX AMC

0.25->64 0.25->64 1- >64 2->64 0.5-32

0.5 1 4 >64 2

Proteus mirabilis (217)

LOR CCR CLX AMX AMC

0.25->64 0.5->64 2->64 0.25->64 0.25-32

1 I 8 0.5 0.5

Serratia marcescens (28)

LOR CCR CLX AMX AMC

16->64 64->64 >64->64 16->64 16->64

>64 >64 >64 64 64

> > > > >

64 64 64 64 64

Enterobacter cloacae (56)

LOR CCR CLX AMX AMC

0.25->64 1->64 4->64 2->64 1- >64

32 64 64 > 64 32

> > > > >

64 64 64 64 64

Citrobacter diversus (24)

LOR CCR CLX AMX AMC

0.25->64 0.25-64 1->64 32->64 1-64

Citrobacter freundii (27)

LOR CCR CLX AMX AMC

0.5->64 1->64 1->64 2- >64 2->64

Salmonella species (20)

LOR CCR CLX AMX AMC

0.25->64 1->64 2->64 0.5->64 0.5->64

0.5 1 4 64 2 4 16 32 > 64 32 0.5 1 4 I 1

Table 2 displays the activity of the test antibiotics against staphylococci, streptococci, H. injluenzae, Moraxella catarrhalis and certain other pathogens. Loracarbef was a little more active than cefaclor or cephalexin against S. aureus, regardless of ~-lactamase production, but none of the cephalosporins was as active as amoxycillinlclavulanate. Table 5 analyses these data in greater detail. About 10% of penicillinase-producing S. aureus displayed high level resistance to all ~-lactams, ie; they were multi- or methicillinresistant s. aureus (MRSA). The activity of loracarbef against streptococci was similar to that of the cephalosporins which were tested. Of the 217 isolates of s. pneumoniae included in the study, 21 were inhibited by penicillin only at concentrations of 0.125 mg/l or greater. Table 3 compares the activities of the studied antibiotics against these penicillin-insensitive isolates and reveals the relatively poor activity of loracarbef and the cephalosporins. Examination of 21 strains of Listeria monocytogenes (Table 2) revealed a sensitivity pattern similar to that of S. pneumoniae, ie; greater susceptibility to penicillins that to cephalosporins. Table 6 shows the activity of the test agents against H. injluenzae. Loracarbef was somewhat more active than cefaclor against ~-lactamase producing strains.

1 4 8 > 64 8 2 4-8 8 > 64 8 2

4 16 32 2

16 32 8 > 64 16 > > > > >

64 64 64 64 64 16 64 64 64 32

DISCUSSION The activity of loracarbef against this large and diverse collection of Australian isolates of clinically encountered bacteria is not unlike that recorded in studies performed in other countries. 4 ,7,8,11-16 ~-lactamase-producing strains of E. coli, S. aureus and H. injluenzae are sensitive to loracarbef, K. pneumoniae is mostly sensitive, while Gram negatives such as S. marcescens, E. cloacae and C. freundii which produce certain chromosomal ~-lactamases, often in response to hospital administration of the newer cephalosporins, are resistant. In this study loracarbef was somewhat more active than cephalexin, cefaclor and amoxycillin/clavulanate against E. coLi, about half of which were resistant to high concentrations of amoxycillin and which were presumably ~-lactamase­ producing. Loracarbef is stable in the presence of TEM-l ~-lactamases7 but it is hydrolysed by TEM-2 ~-lactamases,8 both of which may be produced by E. coli. Greater frequency of TEM-l ~-lactamases (or the absence of TEM-2 ~-lacta­ mases) among Australian strains of E. coli might account for its good activity in this study. Loracarbef was the most active agent against 254 isolates of K. pneumoniae. Nevertheless, 26 (10.2%) were not inhibited by 2 mg/l of loracarbef and these isolates were even

IN VITRO ACTIVITY OF CEPHALOSPORlNS AND LORACARBEF TABLE

2

Activity of loracarbef and other antibiotics against Gram positive and respiratory pathogens

Organism (number tested)

MIC90 MICso (mg/I)

Range 0.5-2 0.5-32 1-4 <0.25-1 <0.25-0.5 0.008-0.5

1 2 2 <0.25 <0.25 0.03

2 4 4 <0.25 <0.25 0.06

Staphylococcus aureus ( - ) (217)

LOR CCR CLX AMX AMC PEN

Staphylococcus aureus ( + ) (331)

LOR CCR CLX AMX AMC PEN

0.5--64 0.25-64 2-64 0.25-64 0.25-32 0.125->8

2 2 4 2 0.5 2

8 8 8 16 2 >8

Haemophilus injluenzae ( - ) (241)

LOR CCR CLX AMX AMC PEN

0.25-64 0.25-64 0.25-64 0.25-8 0.25-4 0.008-8

2 2 16 0.5 0.5 0.5

4 8 32 1 I 1

Haemophilus injluenzae ( + ) (114)

LOR CCR CLX AMX AMC PEN

0.25-64 0.25-64 1-64 1-64 0.25-8 0.06->8

Streptococcus pneumoniae (217)

LOR CCR CLX AMX AMC PEN

0.25->64 0.25->64 0.25->64 0.25-4 0.25-4 0.015-4

0.5-1 0.5 2 <0.25 <0.25 0.008

Streptococcus pyogenes (239)

LOR CCR CLX AMX AMC PEN

0.25-2 0.25-4 0.25-4 0.25-0.25 0.25-0.25 0.008-0.125

0.25 0.25 0.5 0.25 0.25 0.008

0.25 0.5 1 0.25 0.25 0.015

Streptococcus agalactiae (250)

LOR CCR CLX AMX AMC PEN

0.25-2 0.25-4 0.5-8 0.25-0.25 0.25-0.25 0.008-0.125

1 1 2 0.25 0.25 0.Q3

2 2 4 0.25 0.25 0.03

Moraxella catarrhalis (87)

LOR CCR CLX AMX AMC PEN

0.25-64 0.25-64 0.25-64 0.25-16 0.25-4 0.015-> 8

0.5 0.5 1-2 1 0.25 4

4 2-4 2-4 4-8 0.25 8

Listeria monocytogenes (21)

LOR CCR CLX AMX AMC PEN

8-16 8-16 32-32 0.25-0.25 0.25-0.25 0.125-0.5

8 8 32 0.25 0.25 0.125

16 16 32 0.25 0.25 0.25

1 4 8 16 1 >8

8 16 >64 >64 4 >8 2 2 4 <0.25 <0.25 0.06

- = beta-lactamase negative; + = beta-lactamase positive. Antibiotics other thau penicillin were not tested at concentrations lower than 0.25 mgll.

TABLE

PEN LOR CCR CLX AMX AMC

3

Activity of autibiotics against 21 strains of penicillin-insensitive Streptococcus pneumoniae (penicillin MIC of 0.125 mg/l or greater) 0.125

0.25

0.5

5

8 1*

1 1 3

2 5 5

l3* 12*

1 2

3 2

2

4

3 2 3 5 2 3

2 2 5 2 2

8

16

32

1 2

2 1 2

1 2

64

*Antibiotics other than penicillin were not tested at concentrations lower than 0.25

>64 7 5 7

mg/I.

81

82

Pathology (1997), 29, February

BENN et al.

TABLE

4

Activity of cephalosporins and amoxycillinlclavulanate against 254 isolates of Klebsiella pneumoniae Minimal inhibitory concentration (mgll)

LOR CCR CLX AMX AMC

TABLE

0.25

0.5

48 5

143 102 1 16

5

34 76 2 104

2

4

8

16

32

64

>64

3 35 28 1 68

7 8 138

7 6 59 6 17

4 7 18 28 14

2 7 3 68 5

2 1 1

4 7 5 150

30

Activity of antibiotics against Staphylococcus aureus Minimal inhibitory concentration (mgll) n

~0.25

0.5

2

4

8

16

32

64

168

41 171

48

8

3

2

25

88

103 154

23 74

23

6

1 3

25

4

143 122

70 160

20

8 70

102

70

22

16

21

13

20 139

69

17

4

19

3

0

8 39

68

67

*

*

*

*

LOR

-ye +ve

217 331

8 2

CCR

-ye +ve

217 331

2 1

CLX

-ye +ve

217 331

AMX

-ye +ve

217 331

199 3

10 14

AMC

-ye +ve

217 330

197 78

PEN

-ye +ve

217 331

209 11

72

44

50

28

*96 isolates were not inhibited by penicillin at 4 mgl1, the highest concentration tested.

TABLE

6

Activity of test agents against Haemophilus injluenzae Minimal inhibitory concentration (mgll) Blac

n

0.25

0.5

2

4

8

16

32

64

LOR

-ye +ve

241 114

2 2

23 15

77 39

96 27

31 18

7 8

2 2

2 1

1 2

CCR

-ye +ve

241 114

2 1

5 3

23 12

97 39

49 22

52 20

9 12

1 3

3 2

CLX

-ye +ve

241 114

2 1

4

24 14

61 41

91 27

36 17

22 14

AMX

-ye +ve

241 114

52

136

39 4

8 3

5 22

17

16

10

42

AMC

-ye +ve

241 114

59 9

93 21

72 44

11 21

6 16

3

PEN

-ye +ve

241 114

107 2

96 1

26 3

9 1

2 5

*

*

*

*

1

*

*Penicillin was not tested above 4 mg/l.

more resistant to cefaclor and cephalexin. Some may have harboured extended spectrum ~-lactamases, a form of resistance now widely reported in this country.9 Kitzis et al. 10 noted that 15 oral ~-lactams including loracarbef were affected by the presence of these enzymes in K. pneumoniae, although some high level resistance could not be explained by the presence of ~-lactamases alone. Cao et al.,4 in a study based on 30 isolates from New York, also noted some strains with loracarbef MICs equal to, or greater than, 16 mg/I. Like the cephalosporins, loracarbef was considerably less active than the penicillins against isolates of S. aureus which did not produce p..lactamase, a relatively large number of which were included in this study. On the other hand, the stability of loracarbef in the presence of staphylococcal p-Iactamase is well demonstrated by the fact that MICs are about the same in ~-lactamase-producing strains. Cao et at.4

found that loracarbef was more stable than cefaclor but less stable than cephalexin in the presence of the p-Iactamases of S. aureus. Loracarbef and cefaclor are at least fourfold less active than amoxycillin against non p-lactamase-producing H. inJluenzae. Nevertheless, good stability of loracarbef in the presence of the ~-lactamase produced by H. injluenzae is suggested by the similarity of the MICs of loracarbef and amoxycillin/clavulanate for those strains and by the small difference in MIC 90 between strains of H. inJluenzae which produce p-lactamase (MIC9o = 8 mgll) and those which do not (MIC9o = 4 mg/l). The activity of loracarbef against H. inJluenzae should be considered in relation to achievable blood levels. Nelson et at. is found mean peak loracarbef blood levels of 12.6 mg/l and 18.7 mgll after infant doses of 7.5 mg/kg and 15 mg/kg, respectively. Clearly it would be

IN VITRO ACTIVITY OF CEPHALOSPORINS AND LORACARBEF

necessary to give larger oral doses of loracarbef to ensure therapeutic levels for the majority of our ~-lactamase­ producing isolates. The activity of loracarbef against S. pneumoniae (MIC 90 = 2 mg/l) was less than that of penicillin (MIC 90 = 0.06 mg/l) and amoxycillin (MIC 90 < 0.25 mg/l). When insensitivity to penicillin was defined as an MIC of 0.125 mg/l or greater, this study found 21 out of217 (9.6%) strains of S. pneumoniae to be insensitive, with just two of the 17 participating hospitals being responsible for detecting ten of these isolates. A recent Australian survey found an overall rate of 2% penicillin insensitivity with a range of 0-9%.9 Against such strains this study found that the oral cephalosporins and loracarbef had poor activity, a finding which has been noted by others. 17 •19 The clinical role of loracarbef in Australian medical practice has yet to be determined, but its potential could be predicted by a consideration of the in vitro data generated by this study together with published pharmacokinetic data. Studies in adult volunteers have found mean peak serum levels of loracarbef after 400 mg oral doses to be in the order of 13-18 mg/l. 20•21 More than 90% of the dose is eliminated unchanged via the kidneys, yielding urine levels of greater than 200 mg/l in the first four hours and of at least 40 mg/l 8-12 hours after administration. 21 Judging by its activity against a large collection of common Enterobacteriaceae, loracarbef would be an appropriate first line agent for community acquired urinary tract infection. It would also be a suitable alternative to amoxycillinlclavulanate for pediatric respiratory tract infections in areas where penicillin-insensitive pneumococci are not a problem. ACKNOWLEDGEMENT The Australian Group for the study of Antimicrobial Resistance (AGAR) acknowledges Eli Lilly Australia for its support of this investigation and its continuing support for the study of antibiotic resistance in this country. Address for correspondence: RA. V.B., Department of Microbiology, Royal

Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia.

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