Antimicrobial Susceptibilities of 930 Haemophilus

Microbiology Chemotherapy 2008;54:492–498 DOI: 10.1159/000160183

Received: April 28, 2008 Accepted after revision: July 27, 2008 Published online: October 3, 2008

Antimicrobial Susceptibilities of 930 Haemophilus influenzae Clinical Strains Isolated from the Island of Crete, Greece D.P. Kofteridis a G. Notas a S. Maraki b T. Anastasopoulos a G. Papazoglou a A.T. Zisiou a E. Mantadakis c G. Samonis a a

Infectious Diseases Unit, Division of Medicine, University of Crete and b Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, University Hospital of Heraklion, Heraklion, and c Department of Pediatrics, Democritus University of Thrace and University Hospital of Alexandroupolis, Alexandroupolis, Greece

Key Words Haemophilus influenzae ⴢ ␤-Lactamase ⴢ Antimicrobials ⴢ Antibiotic resistance ⴢ Antibiotic susceptibility

Abstract Background: Haemophilus influenzae is an important human pathogen. Materials and Methods: The purpose of the present retrospective study is to describe the antibiotic susceptibility to several common antibiotics of 930 consecutive clinical isolates of H. influenzae over the period of 1996–2005 in a tertiary general hospital on the island of Crete, Greece. Results: Overall, 9.5% of the isolates were ␤-lactamase producing. Resistance to ampicillin and amoxicillin-clavulanate was observed in 11 and 0.6% of the strains, respectively, remaining stable throughout the study period. Resistance to tetracycline increased from 1.6% in 1996 to 38% in 2005, while resistance to ciprofloxacin and ofloxacin was !1%. A significant decrease in resistance to trimethoprim-sulfamethoxazole was observed during the study period. No significant changes in resistance to other antimicrobials were seen. Conclusions: Amoxicillin-clavulanate and older quinolones remain potent agents against H. influenzae. Constant surveillance of antibiotic susceptibility of H. influenzae clinical isolates is important in order to guide appropriate empirical antibiotic therapy. Copyright © 2008 S. Karger AG, Basel

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Introduction

Haemophilus influenzae is an important human pathogen that causes a variety of infections including meningitis, epiglottitis, pneumonia, cellulitis, septic arthritis, otitis media, sinusitis, conjunctivitis, bacteremia, neonatal and maternal sepsis, and exacerbations of chronic obstructive pulmonary disease in adults [1–3]. This Gram-negative bacillus frequently colonizes the human upper respiratory tract, especially the nasopharynx, and is considered part of the normal respiratory flora [1]. Since most H. influenzae infections are the result of direct extension from the nasopharynx to the lower respiratory tract, distinction between colonization and infection is complicated and requires individualized assessment of clinical and microbiological data. In the presence of comorbidities, H. influenzae infections may be severe and associated with significant mortality, especially in patients with underlying lung diseases, hypogammaglobulinemia, hypocomplementemia, asplenia, T cell immunodeficiency and cancer [2, 4]. Resistance of H. influenzae to ␤-lactams is mediated by the production of ␤-lactamases (BL) or mutations in the penicillin-binding proteins (PBPs) [3, 5]. Furthermore, BL-negative ampicillin-resistant (BLNAR) isolates have been reported for more than 20 years, while BL-posDiamantis P. Kofteridis, MD Department of Internal Medicine, University Hospital of Heraklion P.O. Box 1352 GR–71 110 Heraklion, Crete (Greece) Tel. +30 2810 392 688, Fax +30 2810 392 359, E-Mail [email protected]

itive isolates resistant to amoxicillin-clavulanate (BLPACR) have also been identified [3, 6]. Most H. influenzae infections are treated empirically. An alarming increase in this organism’s antibiotic resistance, that varies from one geographical area to another and affects prognosis, has been observed [7]. Therefore, description of H. influenzae antibiotic resistance patterns in a given area and surveillance of changes in antibiotic susceptibility over time are essential for guiding appropriate empirical antibiotic therapy. The purpose of the current retrospective study is to describe the resistance of H. influenzae isolates derived from various clinical specimens obtained from patients cared for in the only tertiary general hospital on the island of Crete, Greece over the period of 1996–2005 to several antimicrobial agents, and the antibiotic susceptibility changes over time.

Materials and Methods Bacterial Isolates and Identification Between January 1996 and December 2005, that is, over a 10year period, a total of 930 unique and consecutive H. influenzae clinical isolates were obtained from patients cared for in the University Hospital of Heraklion, Crete, Greece, from inpatients or patients examined in the outpatient clinics. Isolates were identified as H. influenzae on the basis of hemolytic reactions on horse blood agar, growth requirement for X and V factors, and by use of the API NH system (Biomérieux, Marcy l’Etoile, France) [3]. Serotyping was conducted by the slide agglutination method using type-specific antisera (Difco Laboratories, Detroit, Mich., USA). Stock cultures were prepared by using an absorbent bead system (Prolab Diagnostics, Austin, Tex., USA), and organisms were stored at –85 ° C. Subcultures were performed every year before testing. In vitro Susceptibility Studies Antimicrobial susceptibility testing was performed by the disk diffusion method, as described by the Clinical and Laboratory Standards Institute [8]. Haemophilus test media that fulfilled quality assurance criteria were used for antimicrobial susceptibility testing. The following antibiotics and concentrations were used: ampicillin, 2 and 10 ␮g; amoxicillin-clavulanate, 20 and 10 ␮g; chloramphenicol, 30 ␮g; tetracycline, 30 ␮g; ciprofloxacin, 5 ␮g; ofloxacin, 5 ␮g; trimethoprim-sulfamethoxazole (TMPSMX), 1.25/23.75 ␮g; rifampin, 5 ␮g. H. influenzae ATCC 49247 and ATCC 49766 served as quality control strains in each experiment. BL production was assayed by the nitrocefin test (Oxoid, Basingstoke, UK). BLNAR strains were defined as BL-negative strains that were resistant to ampicillin. Detection of BLNAR strains was accomplished by using 2 concentrations of ampicillin [9]. BLPACR strains were defined by production of BL and resistance to amoxicillin-clavulanate.

Antimicrobial Susceptibilities of 930 H. influenzae Strains

Table 1. Origin of H. influenzae isolates by clinical source, sex

(male vs. female) and age group (children vs. adults) Type of specimen

n

%

Sputum Nasopharyngeal specimens Bronchoalveolar lavage Pus Ophthalmic specimens Vaginal fluid Middle ear fluid Blood cultures Other1

362 331 83 49 30 29 17 9 20

38.9 35.6 8.9 5.3 3.2 3.1 1.8 1.0 2.2

Adults Men Women Total

430 190 620

69.4 30.6 100

Children Boys Girls Total

169 141 310

54.5 45.5 100

1 Urethral, intravenous catheter, tissue, ascitic fluid, prostatic fluid, intrauterine device.

Statistics Age group (children vs. adults), sex (male vs. female), serotype [serotypeable vs. nonserotypeable (NST)] and clinical source of the isolates were correlated with BL production. Serotype distribution of the clinical isolates was also correlated with age group. The number of serotypeable and NST strains isolated per study year was compared for significant differences. Susceptibility patterns to several antibiotics were correlated with serotype. Statistical analysis was performed using SPSS 14.0. ␹2 or Fisher’s exact test were used, as appropriate. p ! 0.05 was considered significant.

Results

A total of 930 strains of H. influenzae were isolated during the 10-year study period from an equal number of patients (1 clinical isolate per specimen per patient). Organisms were most commonly isolated from the lower respiratory tract and the nasopharynx. Specimens were obtained from 599 (64.4%) males and 331 (35.6%) females. Among specimens from males, 169 (28.2%) originated from boys aged 0–14 years and 430 (71.8%) from men 115 years of age. Among specimens from females, 141 (42.6%) originated from girls 0–14 years and 190 (57.4%) from women 115 years of age. Origin of all H. influenzae isolates by clinical source, sex and age group is shown in table 1. Chemotherapy 2008;54:492–498

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Table 2. Distribution of H. influenzae serotypes by study year

Year

NST

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

33 68 93 99 45 86 29 40 66 72

20 22 17 20 5 15 3 8 13 13

9 11 18 18 5 16 4 5 9 7

1 2 4 4 1 1 0 0 0 1

0 0 2 2 1 0 1 0 0 2

1 3 2 3 2 3 0 1 3 1

0 2 4 3 2 3 0 0 2 4

64 108 140 149 61 124 37 54 93 100

631

136

102

14

8

19

20

930

All years

Serotype a

Serotype b

Among the 930 isolates, 88 (9.5%) produced BL. Among strains isolated from adults, 60 (9.7%) produced BL, while 28 (9%) isolates from children produced BL (p = 0.81). BL production did not correlate with sex. Among the 599 isolates from males, 60 (10%) produced BL, while among the 331 isolates from females, 28 (8.5%) produced BL (p = 0.48). BL production did not correlate with serotype, either. Among the 631 NST isolates, 55 (8.7%) produced BL, while among the 299 serotypeable isolates, 33 (11%) produced BL (p = 0.28). NST H. influenzae accounted for almost two thirds of the isolates (631, 67.8%). Among typeable isolates, there were 136 (14.6%) serotype a, 102 (11%) serotype b, 14 (1.5%) serotype c, 8 (0.9%) serotype d, 19 (2%) serotype e and 20 (2.2%) serotype f. The number of serotypeable versus NST isolates was similar by study year (p = 0.6; table 2). Among the 620 (66.7%) isolates derived from adults, 427 (68.8%) were NST, 86 (13.87%) were serotype a, 65 (10.5%) serotype b, 10 (1.6%) serotype c, 7 (1.13%) serotype d, 11 (1.8%) serotype e, and 14 (2.3%) serotype f. Among the remaining 310 (33.3%) isolates originating from children and neonates, 204 (65.8%) were NST, 50 (16.1%) were serotype a, 37 (11.9%) serotype b, 4 (1.3%) serotype c, 1 (0.3%) serotype d, 8 (2.6%) serotype e and 6 (1.9%) serotype f. Serotype distribution did not differ by age group (adults vs. children, p = 0.69). Ampicillin-resistant isolates represented 11%, while amoxicillin-clavulanate-resistant ones only 0.6%. Overall, 15 (1.6%) BLNAR and 4 (0.43%) BLPACR isolates were identified. BLNAR strains were isolated from sputum (8 samples) and from the nasopharynx (7 samples). Among the 15 BLNAR isolates, 13 were NST and only 2 were serotypeable. BLNAR isolates were not more likely to be 494

Chemotherapy 2008;54:492–498

Serotype c

Serotype d

Serotype e

Serotype f

Total

NST than other strains (p = 0.16). BLPACR isolates developed from sputum (2 samples), bronchoalveolar lavage and vaginal fluid (1 sample each). Two of the 4 BLPCAR isolates were NST, 1 was serotype b and the other was serotype f. BLPACR isolates were also not more likely to be NST than the remaining isolates (p = 0.59). Minor changes in the percentage of BL-producing isolates were observed during the study period. Significantly more ophthalmic specimens were BL positive (36.7%, p ! 0.001), compared to isolates from other sources. Less than 1% of the isolates displayed resistance to quinolones, only 0.4% were resistant to chloramphenicol and 2.5% to rifampin, with no changes over time. TMP-SMX resistance was common and seen in 337 isolates (36.2%). It was observed in 217 of 631 NST (34.3%) and 120 of 299 (40.1%) serotypeable isolates (p = 0.09). A significant increase in sensitivity of Haemophilus strains to TMP-SMX during the study period was observed (p ! 0.05; fig. 1). Although initially only 9.5% of the isolates were resistant to tetracycline, a significant increase in resistance to this agent was observed, especially during the last 2 years of the study, reaching 38% in 2005 (p ! 0.01; fig. 2). Sputum isolates were significantly less resistant to tetracycline (3.9%, p ! 0.001), while those isolated from vaginal fluid were more resistant to this agent (27.6%, p ! 0.001). Thirty-seven isolates were recovered from the genital tract, that is, vagina, urethra, prostatic fluid and intrauterine devices. These isolates were found to produce BL more frequently than isolates from other sources (18.9 vs. 9.5%, p ! 0.05) and were significantly more resistant to chloramphenicol (8.1 vs. 0.3%, p ! 0.001), tetracycline (37.8 vs. 8.6%, p ! 0.01) and rifampin (16.2 vs. 4.6%, p ! 0.01). Antibiotic sensitivities to the antimicrobials tested are shown in table 3. Kofteridis et al.

90

40 35

Resistant isolates (%)

Sensitive isolates (%)

80 70 60 50 40

30 25 20 15 10 5 0

30

–5 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

Fig. 1. Sensitivity to TMP-SMX during the study period.

Fig. 2. Resistance to tetracycline during the study period.

Table 3. In vitro activity of antibiotics against H. influenzae clinical isolates (1996–2005)

Sensitivity, %

Ampicillin Amoxicillin-clavulanate Chloramphenicol Tetracycline Ciprofloxacin Ofloxacin TMP-SMX Rifampin

CLSI interpretative breakpoints (zone diameters), mm

S

I

R

S

I

R

88.9 99.4 99.6 90.6 99.8 99.7 62.3 97.4

0.0 0.0 0.0 6.2 0.0 0.0 1.4 1.6

11.1 0.6 0.4 3.2 0.2 0.3 36.3 1

≥22 ≥20 ≥29 ≥29 ≥21 ≥16 ≥16 ≥20

19–21 – 26–28 26–28 – – 11–15 17–19

≤18 ≤19 ≤25 ≤25 – – ≤10 ≤16

CLSI = Clinical and Laboratory Standards Institute; S = sensitive; I = intermediate; R = resistant.

Discussion

Resistance to antimicrobials remains a growing problem worldwide with great impact on the therapeutic practices and cost of medical treatment. Furthermore, resistance may substantially vary by region. Hence, knowledge of the local resistance patterns in a given geographical area is of utmost importance for selection of appropriate empirical antibiotic therapy. In the present study, we evaluated the antibiotic susceptibility patterns of a large number of H. influenzae clinical strains isolated from patients cared for in the only Antimicrobial Susceptibilities of 930 H. influenzae Strains

tertiary general hospital of the island of Crete over a 10year period. Antimicrobial resistance of H. influenzae remains an issue mainly for non-type b strains, since after the introduction of a conjugate vaccine against H. influenzae type b (Hib) in children, serious infections due to Hib have been virtually eliminated. This vaccine has also dramatically changed the epidemiology of the bacteremic disease, since prior to its widespread implementation, Hib accounted for 195% of bacteremias in children, while nowadays bacteremia is more common in those 665 years old [10, 11]. At the present time, life-threatening inChemotherapy 2008;54:492–498

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vasive infections due to Hib have almost disappeared from Greece, a country that has offered a nationwide and highly effective infant immunization program against Hib since 1994 [12]. In the current study, approximately 11% of the isolates were Hib, with no difference between adults and children. Resistance to aminopenicillins of H. influenzae strains is mainly due to enzymatic hydrolysis by BL and to a lesser degree to changes in the PBPs [5]. BL-mediated ampicillin resistance against H. influenzae was first discovered in 1974 [13], and soon became a major concern, with the non-Hib strains increasing their ampicillin resistance by 1–3% per year to a level of 36.4% in the USA by 1996 [14]. A similar increase in BL production in some European countries, such as the UK and France between 1992 and 2001 has also been reported [14]. However, recent data from Europe and the USA have shown a decrease in BL-producing strains of H. influenzae [15, 16]. In the present study, 9.5% of the isolates produced BL and this remained constant over the entire study period from 1996 to 2005, comparable to other concurrent European studies [14]. The increased frequency of BL-positive strains from ophthalmic specimens is also similar to previous studies from the USA [17]. In the present study, 11% of the strains were resistant to ampicillin, a percentage much higher than that reported in the SENTRY project (0–3.2%; 1997–1999) [18], but similar to that reported for Europe (10%; 2004–2005) [7]. Only 0.6% of the tested isolates were resistant to amoxicillin-clavulanate, a widely prescribed antibiotic in Greece. Similar findings have been reported showing resistance rates to this antibiotic in the range of 0–2.4% [15, 18–22]. BLNAR strains have become an increasing problem in Japan, where today they account for around 30% of all H. influenzae strains [13, 23]. Although their global prevalence is low, their incidence is on the rise [14, 19]. In a recent multicenter study from the USA, only 3.3% of the isolates were BLNAR [15]. In the same study, 0.4% of the isolates were BLPACR, possibly due to altered PBP-3, that is, the mechanism of resistance for BLNAR strains. BLPACR strains were first reported in 1997 [6]. In the present report, BLNAR isolates represented only 1.6%, while BLPACR were only 0.43% of the tested isolates. Moreover, BLNAR and BLPACR strains were not more likely to be NST than other strains. Quinolones have been shown to be extremely effective against H. influenzae [1, 14, 18–22, 24, 25]. Our data confirm this, showing very low rates of resistance to ciprofloxacin and ofloxacin, although no newer quinolones were tested. 496


Increased resistance of H. influenzae to sulphonamides has been observed in several studies, reaching 50% in some countries, including a gradual increase in TMP-SMX resistance [15, 17–22]. An interesting finding of the present study is the significant decrease in resistance of H. influenzae strains to TMP-SMX over the last years. Additionally, low resistance rates to this antibiotic have been reported in our area for other Gram-negative uropathogens [26]. The decreased use of TMP-SMX due to highly publicized reports in the lay press for anaphylactic reactions and deaths may be a possible explanation for this ‘rebound’ in sensitivity. Unfortunately, precise data indicating the consumption of TMP-SMX in Crete during the study period are unavailable [26]. However, a recent Finnish study has shown no significant connection between TMP-SMX use and resistance among H. influenzae clinical isolates [27]. H. infuenzae has been reported to be extremely susceptible to chloramphenicol, with most countries reporting resistance rates between 0.4 and 2.8%, apparently due to the low current use of this antimicrobial agent [15, 18, 19, 22]. Likewise, only 0.4% of the strains in our study were resistant to chloramphenicol. Rifampin was widely used as adjuvant treatment for Hib meningitis in the mid-1980s [28] and for bacterial eradication in cases of throat colonization by Hib strains [29]. However, its current use is rather minimal. The 9.5% resistance to tetracycline in our study is similar to the 10.6% found in Athens in 1988 [30], but higher than in other international studies reporting rates between 0.4 and 3.3% [6, 15, 18, 19, 22]. Interestingly, an increase in resistance to tetracycline was observed over the study years, which is not easily explained, considering that this agent is infrequently used nowadays. H. influenzae in the genital tract has been linked to vaginitis, bartholinitis, intrauterine devices-related endometritis, septic abortions, urethral syndrome and neonatal early-onset H. influenzae sepsis [31–34]. Several of these reports have shown that the H. influenzae strains isolated in these settings are resistant to one or more of the antibiotics commonly used against sexually transmitted diseases (including tetracycline) and resistant to ampicillin. Strains isolated from the genital tract in the present study produced BL more frequently than those from other areas and were significantly more resistant to chloramphenicol, tetracycline and rifampin. Hence, quinolones seem to be the most appropriate agents for the empirical treatment of genitourinary infections due to H. influenzae.

Kofteridis et al.

In conclusion, in the island of Crete, Greece, H. influenzae remains highly susceptible to amoxicillin-clavulanate and older quinolones. Resistance to ampicillin during the period 1996–2005 remained stable and resistance to TMP-SMX decreased, while that to tetracyclines increased. Our study emphasizes the need for continuous

surveillance of the evolution of antibacterial resistance in our community over time in order to guide clinicians in empirically choosing the most appropriate antibiotic therapy for the variety of infections caused by H. influenzae.

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