Escolar Documentos
Profissional Documentos
Cultura Documentos
8
0066-4804/08/$08.00⫹0 doi:10.1128/AAC.01674-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
The susceptibility to 14 antimicrobial agents and the mechanisms of aminopenicillin resistance were studied
in 197 clinical isolates of Haemophilus influenzae—109 isolated in 2007 (study group) and 88 isolated in 1997
(control group). Community antibiotic consumption trends were also examined. H. influenzae strains were
consecutively isolated from the same geographic area, mostly from respiratory specimens from children and
adults. Overall, amoxicillin resistance decreased by 8.4% (from 38.6 to 30.2%). -Lactamase production
decreased by 15.6% (from 33 to 17.4%, P ⴝ 0.01), but amoxicillin resistance without -lactamase production
increased by 7.1% (from 5.7 to 12.8%). All -lactamase-positive isolates were TEM-1, but five different
promoter regions were identified, with Pdel being the most prevalent in both years, and Prpt being associated
with the highest amoxicillin resistance. A new promoter consisting of a double repeat of 54 bp was detected.
Community consumption of most antibiotics decreased, as did the geometric means of their MICs, but
amoxicillin-clavulanic acid and azithromycin consumption increased by ca. 60%. For amoxicillin-clavulanic
acid, a 14.2% increase in the population with an MIC of 2 to 4 g/ml (P ⴝ 0.02) was observed; for azithromycin,
a 21.2% increase in the population with an MIC of 2 to 8 g/ml (P ⴝ 0.0005) was observed. In both periods,
the most common gBLNAR (i.e., H. influenzae isolates with mutations in the ftsI gene as previously defined)
patterns were IIc and IIb. Community consumption of trimethoprim-sulfamethoxazole decreased by 54%, while
resistance decreased from 50 to 34.9% (P ⴝ 0.04). Antibiotic resistance in H. influenzae decreased in Spain from
1997 to 2007, but surveillance should be maintained since new forms of resistances may be developing.
Haemophilus influenzae is an important human pathogen amino acid substitutions in penicillin-binding proteins (PBPs),
that causes community-acquired respiratory tract and invasive resulting in -lactamase-non-producing ampicillin resistance
infections. The increasing prevalence of antibiotic resistant (BLNAR) (7, 11, 27). In addition, the combination of -lacta-
phenotypes reported in this pathogen limits the choice of a mase production and alterations in the PBP3 has given rise to
suitable antimicrobial treatment. Antibiotic surveillance stud- a -lactamase-producing amoxicillin-clavulanic acid-resistant
ies are necessary to determine trends in national, regional, and (BLPACR) phenotype (13, 19, 26).
local susceptibility patterns and to effectively guide empirical Over the years, the number of antibiotics available for treat-
antimicrobial therapy. ing respiratory infections has changed considerably. For in-
The principal resistance mechanism to aminopenicillins de- stance, the use of trimethoprim-sulfamethoxazole, tetracy-
scribed in H. influenzae is -lactamase production, mainly of clines, and amoxicillin has declined in some countries, while
the TEM-1 type and sometimes of the ROB-1 type (16). The the use of new macrolides, quinolones, and amoxicillin-clavu-
TEM -lactamase gene can be associated with different pro- lanic acid has increased. However, the impact of these impor-
moter regions: P3, overlapping Pa/Pb, Pdel, which has a 135-bp tant changes on the antibiotic susceptibility of respiratory H.
deletion, and the newly described Prpt that includes a 54-bp influenzae has received little attention.
insertion in the promoter region (20, 24, 25). These promoters In Spain, high levels of antibiotic resistance have been re-
may have different affinities for RNA polymerase, altering ported, particularly in capsulated isolates of H. influenzae types
-lactamase expression and giving rise to different levels of b, e, and f (1, 3, 4). However, widespread vaccination in chil-
resistance to -lactam antibiotics (25). dren against H. influenzae type b was established in this country
The use of oral cephalosporins and amoxicillin-clavulanate in 1998 and, in recent years, the health authorities have imple-
has contributed to the spread of -lactam resistance due to
mented nationwide campaigns for the prudent use of antibiot-
ics, especially in children.
Accordingly, the aims of the present study were (i) to deter-
* Corresponding author. Mailing address: Centro Nacional de Mi- mine the current antimicrobial susceptibility of clinical isolates
crobiologı́a, Instituto de Salud Carlos III, Carretera Pozuelo a Maja-
dahonda, 28220 Majadahonda, Madrid, Spain. Phone: (34) 91-822-
of H. influenzae in Spain compared to a control group of
3650. Fax: (34) 91-509-7966. E-mail: jcampos@isciii.es. isolates collected 10 years ago, before the vaccination against
䌤
Published ahead of print on 27 May 2008. H. influenzae type b was introduced; (ii) to compare trends of
2760
VOL. 52, 2008 DECREASING H. INFLUENZAE RESISTANCE 2761
antibiotic susceptibility in H. influenzae in relation to the evo- TABLE 1. Comparison between the H. influenzae study group
lution of antibiotic consumption by the community; and (iii) to isolated in 2007 and the control group isolated in 1997
study the prevalence and evolution of the most important No. of isolates (%) for: Fisher exact test
mechanisms of resistance to amoxicillin, paying special atten- Parameter
2007 (n ⫽ 109) 1997 (n ⫽ 88) OR (95% CI) P
tion to modifications in PBP3 and -lactamase genes and their
promoter regions. Age
Adults 35 (32.1) 32 (36.4) 0.83 (0.46–1.50) 0.55
Children 61 (56) 49 (55.7) 1.01 (0.57–1.78) 1.00
Unknown 13 (11.9) 7 (7.9) 1.57 (0.60–4.11) 0.48
MATERIALS AND METHODS
Bacterial isolates. The antibiotic susceptibility of 197 clinical isolates of H. Clinical source
influenzae was studied: 109 of them were isolated in 2007 and constituted the Respiratory 102 (93.6) 76 (86.4) 2.30 (0.86–6.12) 0.10
Conjunctivitis 43 (39.4) 37 (42) 0.90 (0.50–1.59) 0.77
study group, while 88, isolated in 1997, made up the control group. All were
Otitis 16 (14.7) 9 (10.2) 1.51 (0.63–3.60) 0.39
consecutively isolated from January to March from the same geographic area Sputum 5 (4.6) 3 (3.4) 1.36 (0.31–5.86) 0.73
covered by the university Hospital Gregorio Marañón, Madrid, Spain. This Other 37 (34) 24 (27.3) 1.37 (0.74–2.53) 0.35
hospital was chosen for the present study because it is one of the largest hospitals respiratorya
in Spain (1,700 beds), covering a large administrative health area (approximately Invasive 2 (1.8) 5 (5.7) 0.31 (0.06–1.60) 0.25
2); no differences were observed for the remaining nine anti- in 1997. In addition, a higher proportion of -lactamase posi-
biotics. For the azithromycin MICs, the 25th percentiles were tive isolates from 1997 had an amoxicillin MIC of ⱖ32 g/ml
1 g/ml (for 1997) and 2 g/ml (for 2007), while the 75th (P ⫽ 0.04) (Table 3). Nevertheless, while -lactamase produc-
percentiles were 2 g/ml (for 1997) and 4 g/ml (for 2007). For tion decreased by 15.5% from 1997 to 2007 (P ⫽ 0.01) (Table
amoxicillin MICs, the 75th percentiles were ⱖ64 g/ml (for 3), non--lactamase-mediated amoxicillin resistance increased
1997) and 4 g/ml (for 2007); for amoxicillin-clavulanic acid, by 7.1% (from 5.7% in 1997 to 12.8% in 2007). In fact, the
the 75th percentiles were 1 g/ml (for 1997) and 2 g/ml (for percentage of -lactamase-negative isolates with an amoxicillin
2007). Most importantly, the geometric mean of azithromycin MIC of ⱖ2 g/ml increased from 13.6% in 1997 to 28.9% in
increased 36.88% between 1997 and 2007, while it decreased 2007 (P ⫽ 0.04) (Table 3).
for amoxicillin (21.23%), cefaclor (15.30%), chloramphenicol (iii) Amoxicillin-clavulanic acid susceptibility. Resistance to
(14.29%), and trimethoprim-sulfamethoxazole (42.60%) (Ta- amoxicillin-clavulanic acid was not detected according to cur-
ble 2). A variation in the geometric mean of ⱖ14% was sta- rent breakpoints (6). However, the number of -lactamase-
tistically significant as determined by the unpaired t test. negative isolates with an amoxicillin-clavulanic acid MIC of ⱖ2
(ii) Amoxicillin susceptibility. A total of 30.2% of the 2007 g/ml was higher in 2007 (27.8%) than in 1997 (13.6%) (P ⫽
isolates had an amoxicillin MIC ⱖ 4 g/ml, compared to 38.6% 0.02) (Table 3).
TABLE 3. Antimicrobial susceptibility comparison between the H. influenzae study group isolated in 2007 and the control group isolated in 1997
No. of isolates (%) for: Fisher exact test
Parameter % Change
2007 (109) 1997 (88) OR (95% CI) Pd
Amoxicillin resistance (MIC ⱖ 4 g/ml) 33 (30.2) 34 (38.6) –8.4 0.69 (0.38–1.25) 0.23
-Lactamase-positive isolates 19 (17.4) 29 (32.9) –15.5 0.43 (0.22–0.83) 0.01
Amoxicillin resistance in -lactamase- 14 (12.8) 5 (5.7) ⫹7.1 1.99 (0.68–5.85) 0.31
negative isolates
Amoxicillin, MIC ⱖ 2 g/mla 26 (28.9) 8 (13.6) ⫹15.3 2.60 (1.08–6.20) 0.04
Amoxicillin, MIC ⱖ 32 g/mlb 15 (78.9) 29 (100) –21.1 2.45 (1.02–5.90) 0.04
Amoxicillin-clavulanic acid, MIC ⫽ 2 to 25 (27.8) 8 (13.6) ⫹14.2 0.05 (0.003–1.16) 0.02
4 g/mlc
gBLNAS 60 (55) 43 (48.9) ⫹6.1 1.30 (0.73–2.25) 0.40
gBLPAR 16 (14.7) 17 (19.3) -4-.6 0.72 (0.34–1.52) 0.44
gBLNAR 30 (27.5) 16 (18.2) ⫹9.3 1.71 (0.86–3.40) 0.13
gBLPACR 3 (2.8) 12 (13.6) –15.4 0.13 (0.036–0.45) 0.0004
Azithromycin, MIC ⫽ 2 to 8 g/ml 95 (87.2) 58 (66) ⫹21.2 3.50 (1.72–7.20) 0.0005
Trimethoprim-sulfamethoxazole, 38 (34.9) 44 (50) –15.1 0.53 (0.30–0.95) 0.04
MIC ⱖ 4 g/ml
Trimethoprim-sulfamethoxazole and 16 (14.7) 23 (26.1) –11.4 0.49 (0.24–1.00) 0.04
amoxicillin simultaneous resistance
a
This values includes intermediate and resistant isolates (9). Total -lactamase-negative isolates: 90 in 2007 and 59 in 1997.
b
Total -lactamase-positive isolates: 19 in 2007 and 29 in 1997.
c
Total -lactamase-negative isolates: 90 in 2007 and 59 in 1997.
d
Significant values are indicated in boldface.
VOL. 52, 2008 DECREASING H. INFLUENZAE RESISTANCE 2763
equal to the Prpt promoter, in such a way that blaTEM-2Prpt and azithromycin experienced an important increase of ca.
seems to have three promoters. The new promoter and the 60% (Table 5).
promoter corresponding to Prpt are likely stronger than the Simultaneous variation on antibiotic consumption and anti-
coexisting P3 promoter as the ⫺35 regions are a closer match biotic susceptibility is presented in Fig. 3. For a number of
to the consensus sequence (Fig. 2). antibiotics (amoxicillin, cefaclor, cefuroxime, cefixime, cla-
The highest geometric mean for amoxicillin was observed rithromycin, and cotrimoxazole), both antibiotic consumption
for isolates harboring the Prpt promoter (56.42 g/ml), fol- and antimicrobial resistance decreased over the study period.
lowed by Pdel promoter (44.44 g/ml) and Pa/Pb promoter In contrast, amoxicillin-clavulanic acid and azithromycin expe-
(41.50 g/ml) (data not shown); for amoxicillin-clavulanic acid, rienced parallel increases in antibiotic consumption and anti-
the geometric means were 1.13, 1.29, and 0.96 g/ml for the biotic geometric means (Fig. 3).
Prpt, Pdel, and P3 promoters, respectively (data not shown).
Antibiotic consumption. A decrease in antibiotic consump- DISCUSSION
tion at the community level was observed for amoxicillin,
cefaclor, cefuroxime, cefixime, cotrimoxazole, and clarithromycin Several observations of clinical and epidemiological interest
(Table 5); conversely, amoxicillin plus -lactamase inhibitor can be made from the data presented here. First, the propor-
FIG. 2. Promoter region (bp 1 to 208) of blaTEM (23). Underlining represents region in P3 which is replicated once in Prpt and twice in 2Prpt.
*, Point of insertion in P3 to create 2Prpt (text in boldface represents inserted sequences). Boxed regions represent ⫺10 and ⫺35 sequence of
promoter P3, circled regions represent ⫺10 and ⫺35 sequence of promoter Prpt, and dotted circles represent new ⫺10 and ⫺35 regions in 2Prpt.
VOL. 52, 2008 DECREASING H. INFLUENZAE RESISTANCE 2765
TABLE 5. Community antimicrobial consumption in Spain over long periods of time altering the antibiotic consumption
Consumption by the community has important consequences on the antibi-
(DDD/1,000 otic susceptibility of H. influenzae. A decrease in antibiotic
inhabitants/ Variation
WHO ATC code Antibiotic
(%)
resistance is only occasionally observed in the scientific litera-
day) in:
ture, but it is well documented here for amoxicillin (a 36.9%
1997 2006 decrease in consumption, a 8.4% decrease in resistance, and a
J01C -Lactam antibacterials, 15.5% decrease in -lactamase production) and trimethoprim-
penicillins sulfamethoxazole (a 54.1% decrease in consumption and a
J01CA04 Amoxicillin 6.5 4.1 –36.9 15.1% decrease in resistance). In addition, simultaneous resis-
J01CR02 Amoxicillin plus -lactamase 4.4 7.2 ⫹63.6
inhibitor tance to amoxicillin and trimethoprim-sulfamethoxazole de-
creased by 11.4%.
J01D Cephalosporins Furthermore, in addition to the decreasing prevalence of
J01DC04 Cefaclor 0.4 0.1 –75.0 -lactamase, the H. influenzae population with an amoxicillin
J01DC02 Cefuroxime 1.2 1.0 –16.7
MIC of ⱖ32 g/ml decreased. These results are in accordance
FIG. 3. Comparison of percentages of variation between community antibiotic consumption and antibiotic activity in H. influenzae (Spain, 1997
to 2007). Drug abbreviations: AMX, amoxicillin; AMC, amoxicillin-clavulanic acid; CEC, cefaclor; CXM, cefuroxime; CFM, cefixime; CLR,
clarithromycin; AZM, azithromycin; and SXT, trimethoprim-sulfamethoxazole.
2766 GARCÍA-COBOS ET AL. ANTIMICROB. AGENTS CHEMOTHER.
dance with data previously reported by us (11), the vast ma- Moxon. 1994. PCR for capsular typing of Haemophilus influenzae. J. Clin.
Microbiol. 32:2382–2386.
jority of the gBLNAR isolates in the present study had amino 9. Fleischmann, R. D., M. D. Adams, O. White, R. A. Clayton, E. F. Kirkness, A. R.
acid substitutions at the Lys-Thr-Gly motif, the most common Kerlavage, C. J. Bult, J. F. Tomb, B. A. Dougherty, J. M. Merrick, K. McKenney,
being Asn-526 to Lys. However, in the present study no amino G. Sutton, W. FitzHugh, C. Fields, J. D. Gocayne, J. Scott, R. Shirley, L. Liu, A.
Glodek, J. M. Kelley, J. F. Weidman, C. A. Phillips, T. Spriggs, E. Hedblom,
acid substitutions were found at positions 385 and 389, previ- M. D. Cotton, T. R. Utterback, M. C. Hanna, D. T. Nguyen, D. M. Saudek, R. C.
ously found to be related to a decreased susceptibility to cefo- Brandon, L. D. Fine, J. L. Fritchman, J. L. Fuhrmann, N. S. M. Geoghagen,
taxime and cefixime (11, 22). C. L. Gnehm, L. A. McDonald, K. V. Small, C. M. Fraser, H. O. Smith, and J. C.
Venter. 1995. Whole-genome random sequencing and assembly of Haemophilus
As in other reports (20, 24), in our -lactamase-positive influenzae Rd. Science 269:496–512.
isolates the most prevalent promoter was Pdel. The geometric 10. Fluit, A. C., A. Florijn, J. Verhoef, and D. Milatovic. 2005. Susceptibility of
mean for amoxicillin was higher for the Pdel promoter than for European -lactamase-positive and -negative Haemophilus influenzae iso-
lates from the periods 1997/1998 and 2002/2003. J. Antimicrob. Chemother.
the Pa/Pb promoter, as reported by Tristram et al. (24). In our 56:133–138.
study, the Prpt promoter presented the highest geometric 11. Garcı́a-Cobos, S., J. Campos, E. Lázaro, F. Román, E. Cercenado, C. Garcı́a-
Rey, M. Pérez-Vázquez, J. Oteo, and F. de Abajo. 2007. Ampicillin-resistant
means for amoxicillin and cefaclor, suggesting that it may be non--lactamase-producing Haemophilus influenzae in Spain: recent emer-
associated with increased levels of resistance to these two an- gence of clonal isolates with increased resistance to cefotaxime and cefixime.