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Colonisation of fluoroquinolone-resistant Haemophilus influenzae among nursing home residents in southern Taiwan
Journal of Hospital Infection 75 (2010) 304–308
Available online at www.sciencedirect.com
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Colonisation of fluoroquinolone-resistant Haemophilus influenzae among nursing home residents in southern Taiwan C.-M. Chang a, b, c, d, T.-L. Lauderdale e, H.-C. Lee a, c, d, N.-Y. Lee a, c, d, C.-J. Wu a, c, d, P.-L. Chen a, c, d, C.-C. Lee a, c, d, P.-C. Chen e, W.-C. Ko a, c, d, * a
Division of Infectious Diseases, Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan Division of Geriatrics and Gerontology, Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan Center of Infection Control, National Cheng Kung University Hospital, Tainan, Taiwan d Department of Medicine, National Cheng Kung University Medical College, Tainan, Taiwan e Division of Infectious Diseases, National Health Research Institutes, Zhunan, Taiwan b c
a r t i c l e i n f o
s u m m a r y
Article history: Received 24 November 2009 Accepted 24 December 2009 Available online 31 March 2010
Fluoroquinolones have been widely used to treat respiratory tract infections, but fluoroquinolone resistance in Haemophilus influenzae has remained rare. In 2007, prospective surveillance cultures of throat swabs and sputum were conducted every two months on 150 residents of four nursing homes in southern Taiwan. Forty-eight H. influenzae isolates were obtained from 30 (20%) residents. All isolates were non-b serotype and 27 (56.3%) possessed b-lactamases. Resistance to levofloxacin [minimum inhibitory concentration (MIC) >2 mg/mL] and moxifloxacin (MIC >1 mg/mL) was found in 20 (41.7%) and 21 (43.8%) isolates, respectively. High level levofloxacin and moxifloxacin resistance (MIC >32 mg/mL) was detected in 19 (39.6%) and 15 (31.3%) isolates, respectively. Among 150 residents, those with urinary catheterisation (P ¼ 0.018) and tracheostomy tubes (P ¼ 0.029) were independently associated with airway colonisation by moxifloxacin-resistant H. influenzae. Among 30 residents with carriage of H. influenzae, no factor was significantly associated with moxifloxacin resistance. Pulsedfield gel electrophoresis of the isolates revealed 14 distinct types. Two major clones accounted for 29 isolates, 27 of which were obtained from 13 residents in one nursing home. All but two of the fluoroquinolone-resistant isolates belonged to these two major clones. This study highlights the emergence of fluoroquinolone-resistant H. influenzae and its clonal spread among nursing home residents in southern Taiwan. Further studies on clinical implications and the extent of fluoroquinolone non-susceptibility and resistance are needed. Ó 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
Keywords: Fluoroquinolone Haemophilus influenzae Nursing home Resistance
Introduction Haemophilus influenzae is one of the major bacterial pathogens associated with community-acquired respiratory tract and otolaryngeal infections.1 Due to penicillin resistance in Streptococcus pneumoniae and to the lower pill burden and broad-spectrum antimicrobial activity, fluoroquinolones (FQs) have been widely used for respiratory tract infections in hospitals and long-term care
* Corresponding author. Address: Department of Internal Medicine, National Cheng Kung University Hospital, #138 Sheng Li Road, Tainan, 70403, Taiwan. Tel.: þ886 6 235 3535, ext. 3596; fax: þ886 6 275 2038. E-mail address: [email protected] (W.-C. Ko).
facilities.2 FQ-resistant H. influenzae isolates were first reported in 1993,3 but remained rare, as reflected by several surveillance studies. Before 2002, studies from the Far East, North America, Latin America, Europe and the USA revealed that they accounted for 0–0.3% of H. influenzae isolates.4–7 There have been sporadic case reports of FQ-resistant H. influenzae infections from diverse geographic locations.8–14 Among 457 H. influenzae isolates recovered between 2002 and 2004 in Japan, 12 (2.6%) FQ-resistant strains of multiple clones were identified from patients >58 years of age.8 A recent study of 31 H. influenzae isolates from intensive care units in 10 Taiwanese major teaching hospitals in 2005 revealed that 2 (6.5%) were FQ resistant.15 Furthermore, in 2004, a clonal outbreak of FQ-resistant H. influenzae in a long-term care facility in New York was reported.14
0195-6701/$ – see front matter Ó 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2009.12.020
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The present study was initiated to investigate antimicrobial susceptibilities, the genetic relatedness of H. influenzae isolates, and risk factors for airway carriage of FQ-resistant isolates among residents in nursing homes in southern Taiwan.
Applied Maths, Kortrijk, Belgium). A PFGE type was assigned to each strain in accordance with the criteria outlined by Tenover et al.20
Methods
Data were analysed with the Statistical Package for Social Sciences for Windows (SPSSWIN, Chicago, IL, USA, version 13.0). Continuous data, expressed as mean values SD), were compared by Student’s ttest or Mann–Whitney U-test. Categorical variables, expressed as numbers and percentages, were compared by c2-test or Fisher’s exact test. P < 0.05 was considered statistically significant. Variables with P < 0.1 in the univariate analysis of moxifloxacin-resistant H. influenzae colonisation were included in multivariate analysis.
Study design and population From February through November 2007, a surveillance study was prospectively conducted in Tainan City at four nursing homes that do not provide acute care services. One of the nursing homes is hospital-affiliated. The number of beds ranged from 34 to 70 (median: 50). Written informed consents were obtained from participating residents or their proxies before their enrolment. The study was approved by the Institutional Review Board of the National Cheng Kung University Hospital, Taiwan. Demographic and clinical data were obtained from the nursing records. Age, gender, underlying medical diseases, antimicrobial use within three months, Barthel index of activities of daily living, and the presence of feeding tube, indwelling bladder catheter, or tracheostomy, were recorded on a case record form.16 The Charlson Comorbidity Index was used to assess the degrees of comorbidity.17 Isolation, identification, and antimicrobial susceptibility testing Microbiological specimens were collected by a study nurse every two months. Sputum specimens were obtained by expectoration or laryngeal suction of the residents. Nasal and throat swab cultures were collected with a BD CultureSwabÔ (BD Diagnostic Systems, Franklin Lakes, NJ, USA). The specimens were streaked onto trypticase soy agar with 5% sheep blood, chocolate II agar and Columbia CNA agar with 5% sheep blood within a day of collection. The plates were incubated at 37 C in an atmosphere containing 5% CO2 for 48 h. Colonies on chocolate agar plates showing typical morphology (i.e. smooth, greyish, and wet colonies) were selected. H. influenzae were identified based on Gram stain, positive catalase and oxidase reactions, and the requirement of X and V factors for growth. The isolates were preserved in trypticase soy broth with 15% glycerol for further studies. The disc diffusion method was performed following Clinical and Laboratory Standards Institute (CLSI) guidelines.18 Haemophilus Test Medium Agar (Becton, Dickinson & Co., MD, USA) plates were inoculated with each bacterial suspension (adjusted to a McFarland 0.5 standard) and were incubated at 35 C for 16–18 h. Standard discs of 11 antimicrobial agents were tested.19 Minimum inhibitory concentrations (MICs) of levofloxacin and moxifloxacin were determined by Etest (AB Biodisk, Solna, Sweden). Currently, the rare occurrence of H. influenzae isolates with reduced susceptibility to fluoroquinolones precludes defining a category other than ‘‘susceptible’’ by CLSI.18 For simplicity, the above isolates were described as ‘‘resistant’’, without the category of ‘‘non-susceptible’’. H. influenzae ATCC 49247 was used as a control strain for susceptibility testing. Capsular type b was identified by the slide agglutination method. The b-lactamase activity was tested with a nitrocephin disc.
Statistical analysis
Results Characteristics of isolates Sputum, nasal or throat swab cultures were obtained from 150 (74%) of 201 participating residents at four nursing homes (NH-A, B, C, and D). During a 10 month period, 33 (22%) residents were positive for H. influenzae in their airways at least once. In total, of 62 H. influenzae isolates were isolated from sputum (41 isolates), throat swabs (19), and nasal swabs (2). Ten isolates could not be subcultured, so 52 isolates from 30 residents were studied. Eight isolates were simultaneously obtained from sputum and throat cultures of four individuals and had identical PFGE patterns, so only one of each pair was analysed, leaving 48 isolates from 30 residents for further study. None of the isolates belonged to serotype b. Antimicrobial susceptibility The results of antimicrobial susceptibility testing with the disc diffusion method are shown in Table I. The 27 (56.3%) ampicillinresistant isolates all exhibited b-lactamase activity. Only one isolate without b-lactamase activity was intermediately resistant to ampicillin (Etest MIC 2 mg/mL). All 48 isolates were susceptible to ampicillin–sulbactam, amoxicillin–clavulanate, ceftriaxone, cefpodoxime, and ertapenem. Whereas 94% of isolates were susceptible to cefuroxime, only 44% and 19% were susceptible to clarithromycin and trimethoprim–sulfamethoxazole, respectively. The MIC50, MIC90 and MIC ranges of the 48 isolates were 0.5, >32 and 0.012 to >32 mg/mL for levofloxacin, and 0.75, >32 and 0.016 to >32 mg/mL for moxifloxacin. Overall, 21 isolates were resistant to levofloxacin (MIC >2 mg/mL) and 21 to moxifloxacin (MIC > 1 mg/mL). One isolate (P5025) was susceptible to levofloxacin (MIC ¼ 1 mg/mL) but resistant to moxifloxacin (MIC ¼ 4 mg/ mL). For the remaining in-vitro FQ-susceptible isolates based on the CLSI interpretive criteria, an additional 13 (27.1%) and 14 (29.5%) strains had higher levofloxacin and moxifloxacin MICs (0.125 mg/ mL), respectively. Of note, high level levofloxacin and moxifloxacin non-susceptibility (MIC >32 mg/mL) was detected in 19 (39.6%) and 15 (31.3%) isolates, respectively. Compared with moxifloxacinsusceptible isolates, moxifloxacin-resistant isolates were more likely to be resistant to clarithromycin (57.1% vs 11.1%, P ¼ 0.003) and trimethoprim-sulfamethoxazole (95.2% vs 70.4%, P ¼ 0.035).
Pulsed-field gel electrophoresis Risk factors for colonisation of H. influenzae Molecular typing of the genomic DNA was performed by pulsedfield gel electrophoresis (PFGE). Preparation of DNA plugs and subsequent digestion by SmaI was done following the standardised protocols of PulseNet from the CDC website. Cluster analysis of the PFGE banding patterns was performed by the unweighted pair group method with arithmetic averages with the Jaccard coefficient, and the dendrogram was prepared using BioNumeric software (Version 4.5,
Compared to residents without colonisation of H. influenzae on univariate analysis, carriers of H. influenzae were significantly associated with chronic lung diseases (P < 0.001), malignancy (P ¼ 0.001), tube feeding (P < 0.001), urinary catheterisation (P ¼ 0.031), tracheostomy (P < 0.001), poor functional status (P ¼ 0.001), and living at Nursing Home (NH) A (P < 0.001) (detailed data not shown). Compared
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Table I Antimicrobial susceptibility of 48 Haemophilus influenzae isolates, as determined by disc diffusion method Total isolates (N ¼ 48)
LVX AMP SAM AMC ETP CXM CRO CPD CLR SXT
MXF resistant (N ¼ 21)
MXF susceptible (N ¼ 27)
S (%)
I (%)
R (%)
S (%)
I (%)
R (%)
S (%)
I (%)
R (%)
27 (56.3) 20 (41.6) 48 (100) 48 (100) 48 (100) 45 (93.8) 48 (100) 48 (100) 21 (43.8) 9 (18.8)
– 1 (2.1) 0 0 0 2 (4.2) 0 0 12 (25) 0
21 (43.8)a 27 (56.3) 0 0 0 1 (2.1) 0 0 15 (31.3) 39 (81.3)
1 (4.8) 12 (57.1) 21 (100) 21 (100) 21 (100) 18 (85.7) 21 (100) 21 (100) 5 (23.8) 1 (4.8)
– 1 (4.8) 0 0 0 2 (9.5) 0 0 4 (19.0) 0
20 (95.2)a 8 (38.1) 0 0 0 1 (4.8) 0 0 12 (57.1) 20 (95.2)
27 (100) 8 (29.6) 27 (100) 27 (100) 27 (100) 27 (100) 27 (100) 27 (100) 16 (59.3) 8 (29.6)
–
0 19 (70.4) 0 0 0 0 0 0 3 (11.1) 19 (70.4)
0 0 0 0 0 0 0 8 (29.6) 0
MXF, moxifloxacin; S, susceptible; I, intermediate; R, resistant; LVX, levofloxacin; AMP, ampicillin; SAM, ampicillin–sulbactam; AMC, amoxicillin–clavulanate; ETP, ertapenem; CXM, cefuroxime; CRO, ceftriaxone; CPD, cefpodoxime; CLR, clarithromycin; SXT, trimethoprim–sulfamethoxazole. a Non-susceptibility to levofloxacin.
to 138 residents without moxifloxacin-resistant isolates, those with moxifloxacin-resistant H. influenzae carriage were more likely to have chronic lung diseases (P ¼ 0.001), malignant neoplasm (P ¼ 0.008), tube feeding (P ¼ 0.002), urinary catheterisation (P ¼ 0.001), and tracheostomy tubes (P < 0.001), and were also more often living at NHA (P ¼ 0.002). On multivariate analysis, residents with urinary catheterisation [odds ratio (OR): 9.20, 95% confidence interval (CI): 1.47– 57.53, P ¼ 0.018] and tracheostomy tubes (OR: 7.82, 95% CI: 1.24–49.28, P ¼ 0.029) were significantly associated with airway colonisation of moxifloxacin-resistant H. influenzae (detailed data not shown). Twelve (40%) of 30 residents with airway carriage of H. influenzae were colonised with moxifloxacin-resistant isolates at least once during the study. Colonisation was more likely in residents with tracheostomy tubes on univariate analysis (P ¼ 0.061) (Table II). On multivariate analysis, no variable was significantly associated with colonisation of moxifloxacin-resistant H. influenzae (data not shown). Patterns of pulsed-field gel electrophoresis Analysis of the PFGE patterns of these 48 isolates revealed 14 distinct types (clones) (A–N) (Figure 1). Two major PFGE types, Table II Comparisons of clinical characteristics between nursing home residents with airway carriage of moxifloxacin-susceptible and moxifloxacin-resistant Haemophilus influenzae Variablesa
Moxifloxacin Susceptible (N ¼ 12)
Age (years) Charlson comorbidity index Barthel index Male Underlying diseases Hypertension History of stroke Chronic lung diseases Diabetes mellitus Malignant neoplasm Dementia History of myocardial infarction Chronic kidney disease Tube feeding Urinary catheterisation Tracheostomy tube Number of drugs Hospitalisation during previous 3 months Antimicrobial use during previous 3 months Living at hospital-affiliated nursing home Living at nursing home A a b
P-values
Resistant (N ¼ 18)
75.8 9.7 73.7 14.1 0.621 3.1 1.6 3.3 0.8 0.666 7.8 10.5 19.2 28.4 0.130 7 (38.9) 4 (33.3) >0.999b 15 (83.3) 14 (77.8) 8 (44.4) 7 (38.9) 4 (22.2) 2 (11.1) 1 (5.6) 1 (5.6) 18 (94.4) 3 (16.7) 5 (27.8) 6.6 2.8 1 (5.6) 1 (5.6) 1 (5.6) 10 (55.6)
9 (75.0) 9 (75.0) 8 (66.7) 2 (16.7) 4 (33.3) 1 (8.3) 1 (8.3) 0 14 (91.7) 6 (50.0) 8 (66.7) 6.5 1.6 4 (33.3) 1 (8.3) 1 (8.3) 10 (83.3)
Data are presented as mean SD or number of patients (%). Fisher’s exact test.
0.660b >0.999b 0.284b 0.249b 0.678b >0.999b >0.999b >0.999b >0.999b 0.102b 0.061b 0.952 0.128b >0.999b >0.999b 0.235b
clones A (19 isolates) and B (10 isolates), accounted for 60.4% of all isolates, and 27 of these 29 isolates were from 13 residents of nursing home A (NH-A). Four subtypes of major clone A showed variable moxifloxacin MICs (0.38 to >32 mg/mL). Isolates of subtypes A and A3 had decreased susceptibility to moxifloxacin (MIC 0.125 mg/mL), while 8 of the 12 isolates in subtypes A1 and A2 had high moxifloxacin MICs (>8 mg/mL). All ten isolates of clone B were highly resistant to levofloxacin and moxifloxacin (>32 mg/ mL), and all were from NH-A. Among the remaining 19 isolates (clones C-N), 10 were isolated from other nursing homes, and only two were not susceptible to moxifloxacin. Isolates with similar PFGE patterns from different nursing homes (clone D from NH-A and NH-C, clone F from NH-A and NH-B) were also found. Discussion Our study revealed that nearly half the H. influenzae isolates recovered from nursing home residents in Taiwan were resistant to FQs, and 12 (8%) of the 150 residents had been colonised by the FQresistant strains at one point. Such a substantial prevalence of FQresistant and high-level-resistant H. influenzae (MIC >32 mg/mL) in the airway carriage and polyclonal spread have not been reported before. Furthermore, of 210 clinical isolates of H. influenzae found at our hospital in 2007, 31 (14.8%) and 34 (16.2%) were resistant to levofloxacin and moxifloxacin, respectively (by disc diffusion method, unpublished data). These findings highlight the emerging role of FQ-resistant H. influenzae in healthcare facilities. Multivariate analysis showed that carriers of moxifloxacinresistant H. influenzae were associated with urinary catheterisation and tracheostomy tubes. Among carriers of H. influenzae, no significant factors were associated with the presence of moxifloxacin resistance except tracheostomy tubes (albeit borderline statistical significance). Thus, the significant factors of urinary catheterisation and tracheostomy tubes among all residents might only reflect the association with H. influenzae airway carriage rather than moxifloxacin resistance. The reason that our study failed to identify significant variables associated with moxifloxacin nonsusceptibility may be due in part to the limited number of cases with H. influenzae airway carriage. Among moxifloxacin-resistant H. influenzae isolates, two major clones (A and B) were found within NH-A, indicating their clonal spread in this long-term care facility. Although the multivariate analysis failed to support an association of NH-A and FQ-resistant H. influenzae colonisation, these two clones can be regarded as endemic because of geographic correlation. In fact, 34 (70.8%) of 48 isolates and 20 of 30 residents with H. influenzae colonisation came from NH-A where the residents were more likely to have chronic lung diseases and tracheostomy tubes, factors associated with increased
100
80
60
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Strain P1582 S1062 S1552 S1193 S5145 S1452 S1192 S1812-1 S1312 S1521 S1444 S1311 S1195 S1454 P1603 N1771 N1341 P3115 P5025 S1153 P3172 P0221 P0223 P1423 P5283 S1303 S1274 S1814 S1275 P1545 S3045 S1442 S1523 S1534 S1445 S1255 S1535 S1525 S1061 S1812 S1313 S1064 S1455 S1743 S5322 P1543 S0404 S5095
307
Nursing home A A A A B A A A A A A A A A A A A C B A C D D A B A A A A A-C C A A A A A A A A A A A A A B A D B
MIC of MXF 0.016 0.016 0.016 0.016 0.032 >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 0.023 0.023 0.064 4 0.094 0.25 0.75 0.75 0.125 0.016 0.5 0.5 0.5 0.5 0.75 0.75 0.19 0.5 16 12 >32 >32 16 0.38 >32 >32 3 24 0.38 0.016 0.023 >32 0.023
PFGE Pattern C C1 C2 C3 M B B B B B B B B B1 B2 D D D1 H I G E E J K A A A A A A A1 A1 A1 A1 A1 A1 A1 A2 A2 A2 A2 A2 A3 F F L N
Figure 1. Dendrogram of 48 Haemophilus influenzae isolates from four nursing homes. Strain identification: the first letter denotes specimen source (S, sputum; P, pharyngeal swab; N, nasal swab); the middle three digits are the nursing home resident number, and the last digit is the surveillance culture order (from first to fifth surveillance cultures). MIC, minimum inhibitory concentration; MXF, moxifloxacin. Pulsed-field gel electrophoresis (PFGE) pattern: isolates sharing >80% similarity were assigned an alphabet pattern name followed by a number for subtype.
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colonisation rates of H. influenzae irrespective of FQ resistance. Inadequate implementation of infection control measures and poor hygiene in tracheostomy care at NH-A might contribute to more frequent colonisation since NH-A residents with tracheostomy tubes were associated with colonisation of H. influenzae more than those in the other three nursing homes [18 (23.1%) vs 3 (2.4%), P < 0.001]. Although more than half of the H. influenzae isolates in the present study were susceptible to FQs according to the CLSI criteria, 25% and 29.5% of susceptible strains had higher MICs (0.125 mg/mL) of levofloxacin and moxifloxacin, respectively. Identification of these isolates raises clinical concerns regarding the therapeutic efficacy of FQ treatment and warrants further investigation. Previous studies have reported that H. influenzae isolates with reduced susceptibility to FQs carried a single point mutation in GyrA, which might be the initial step to the development of clinically significant resistance in Gram-negative bacteria.13,21,22 It has been demonstrated that infections due to Salmonella spp. with reduced FQ susceptibility led to a higher clinical failure rate if treated with FQs.23 Therefore, there is increasing likelihood of evolution toward FQ resistance in H. influenzae isolates. The increased use of levofloxacin and moxifloxacin has led to concern about the emergence of resistance. Indeed, the prevalence of levofloxacin-resistant pneumococcal isolates in Hong Kong increased from 5.5% in 1998 to 13.3% in 2000, and in Taiwan the prevalence of ciprofloxacin-resistant Salmonella enterica serotype Choleraesuis isolates increased from 0 in 1999 to 60% in 2001.24,25 As the selective antimicrobial pressure of FQ continues, it would not be surprising to observe the isolation frequency of FQ-resistant H. influenzae increase. A recent study on an outbreak of FQ-resistant H. influenzae in a long-term care facility in New York reported that previous levofloxacin use was associated with infection with a resistant strain.14 High level resistance might be induced by repeated treatment with FQs in patients with chronic lung diseases.26–28 Although no significant association with previous FQ use was found in our study, the emergence of H. influenzae isolates with high level resistance to levofloxacin and moxifloxacin is still a reminder that FQs should be used cautiously. In summary, our study reported the emergence of polyclonal levofloxacin/moxifloxacin-resistant H. influenzae isolates among nursing home residents. Further studies on the resistance mechanism and the prevalence of FQ-resistant H. influenzae in other healthcare facility populations are warranted. Acknowledgements We would like to thank M.M.-H. Lin for collecting specimens and E.P.-C. Wu for laboratory assistance. Conflict of interest statement None declared. Funding sources This study was supported by research grants from the Center for Disease Control, Taiwan (DOH96-DC-1010), National Cheng Kung University Hospital (NCKUH 9702038), and National Health Research Institutes (98A1-CLPP01-014). References 1. Murphy TF. Haemophilus infections. In: Mandell GL, Bennett JE, Dolin R, editors. Mandell, Douglas, and Benett’s principles and practice of infectious diseases. 6th ed. New York: Churchill Livingstone; 2005. p. 2661–2669. 2. Goldstein IJC, Garabedian-Ruffalo SM. Widespread use of fluoroquinolones versus emerging resistance in pneumococci. Clin Infect Dis 2002;35: 1505–1511.
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Colonisation of fluoroquinolone-resistant Haemophilus influenzae among nursing home residents in southern Taiwan C.-M. Chang a, b, c, d, T.-L. Lauderdale e, H.-C. Lee a, c, d, N.-Y. Lee a, c, d, C.-J. Wu a, c, d, P.-L. Chen a, c, d, C.-C. Lee a, c, d, P.-C. Chen e, W.-C. Ko a, c, d, * a
Division of Infectious Diseases, Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan Division of Geriatrics and Gerontology, Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan Center of Infection Control, National Cheng Kung University Hospital, Tainan, Taiwan d Department of Medicine, National Cheng Kung University Medical College, Tainan, Taiwan e Division of Infectious Diseases, National Health Research Institutes, Zhunan, Taiwan b c
a r t i c l e i n f o
s u m m a r y
Article history: Received 24 November 2009 Accepted 24 December 2009 Available online 31 March 2010
Fluoroquinolones have been widely used to treat respiratory tract infections, but fluoroquinolone resistance in Haemophilus influenzae has remained rare. In 2007, prospective surveillance cultures of throat swabs and sputum were conducted every two months on 150 residents of four nursing homes in southern Taiwan. Forty-eight H. influenzae isolates were obtained from 30 (20%) residents. All isolates were non-b serotype and 27 (56.3%) possessed b-lactamases. Resistance to levofloxacin [minimum inhibitory concentration (MIC) >2 mg/mL] and moxifloxacin (MIC >1 mg/mL) was found in 20 (41.7%) and 21 (43.8%) isolates, respectively. High level levofloxacin and moxifloxacin resistance (MIC >32 mg/mL) was detected in 19 (39.6%) and 15 (31.3%) isolates, respectively. Among 150 residents, those with urinary catheterisation (P ¼ 0.018) and tracheostomy tubes (P ¼ 0.029) were independently associated with airway colonisation by moxifloxacin-resistant H. influenzae. Among 30 residents with carriage of H. influenzae, no factor was significantly associated with moxifloxacin resistance. Pulsedfield gel electrophoresis of the isolates revealed 14 distinct types. Two major clones accounted for 29 isolates, 27 of which were obtained from 13 residents in one nursing home. All but two of the fluoroquinolone-resistant isolates belonged to these two major clones. This study highlights the emergence of fluoroquinolone-resistant H. influenzae and its clonal spread among nursing home residents in southern Taiwan. Further studies on clinical implications and the extent of fluoroquinolone non-susceptibility and resistance are needed. Ó 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
Keywords: Fluoroquinolone Haemophilus influenzae Nursing home Resistance
Introduction Haemophilus influenzae is one of the major bacterial pathogens associated with community-acquired respiratory tract and otolaryngeal infections.1 Due to penicillin resistance in Streptococcus pneumoniae and to the lower pill burden and broad-spectrum antimicrobial activity, fluoroquinolones (FQs) have been widely used for respiratory tract infections in hospitals and long-term care
* Corresponding author. Address: Department of Internal Medicine, National Cheng Kung University Hospital, #138 Sheng Li Road, Tainan, 70403, Taiwan. Tel.: þ886 6 235 3535, ext. 3596; fax: þ886 6 275 2038. E-mail address: [email protected] (W.-C. Ko).
facilities.2 FQ-resistant H. influenzae isolates were first reported in 1993,3 but remained rare, as reflected by several surveillance studies. Before 2002, studies from the Far East, North America, Latin America, Europe and the USA revealed that they accounted for 0–0.3% of H. influenzae isolates.4–7 There have been sporadic case reports of FQ-resistant H. influenzae infections from diverse geographic locations.8–14 Among 457 H. influenzae isolates recovered between 2002 and 2004 in Japan, 12 (2.6%) FQ-resistant strains of multiple clones were identified from patients >58 years of age.8 A recent study of 31 H. influenzae isolates from intensive care units in 10 Taiwanese major teaching hospitals in 2005 revealed that 2 (6.5%) were FQ resistant.15 Furthermore, in 2004, a clonal outbreak of FQ-resistant H. influenzae in a long-term care facility in New York was reported.14
0195-6701/$ – see front matter Ó 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2009.12.020
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The present study was initiated to investigate antimicrobial susceptibilities, the genetic relatedness of H. influenzae isolates, and risk factors for airway carriage of FQ-resistant isolates among residents in nursing homes in southern Taiwan.
Applied Maths, Kortrijk, Belgium). A PFGE type was assigned to each strain in accordance with the criteria outlined by Tenover et al.20
Methods
Data were analysed with the Statistical Package for Social Sciences for Windows (SPSSWIN, Chicago, IL, USA, version 13.0). Continuous data, expressed as mean values SD), were compared by Student’s ttest or Mann–Whitney U-test. Categorical variables, expressed as numbers and percentages, were compared by c2-test or Fisher’s exact test. P < 0.05 was considered statistically significant. Variables with P < 0.1 in the univariate analysis of moxifloxacin-resistant H. influenzae colonisation were included in multivariate analysis.
Study design and population From February through November 2007, a surveillance study was prospectively conducted in Tainan City at four nursing homes that do not provide acute care services. One of the nursing homes is hospital-affiliated. The number of beds ranged from 34 to 70 (median: 50). Written informed consents were obtained from participating residents or their proxies before their enrolment. The study was approved by the Institutional Review Board of the National Cheng Kung University Hospital, Taiwan. Demographic and clinical data were obtained from the nursing records. Age, gender, underlying medical diseases, antimicrobial use within three months, Barthel index of activities of daily living, and the presence of feeding tube, indwelling bladder catheter, or tracheostomy, were recorded on a case record form.16 The Charlson Comorbidity Index was used to assess the degrees of comorbidity.17 Isolation, identification, and antimicrobial susceptibility testing Microbiological specimens were collected by a study nurse every two months. Sputum specimens were obtained by expectoration or laryngeal suction of the residents. Nasal and throat swab cultures were collected with a BD CultureSwabÔ (BD Diagnostic Systems, Franklin Lakes, NJ, USA). The specimens were streaked onto trypticase soy agar with 5% sheep blood, chocolate II agar and Columbia CNA agar with 5% sheep blood within a day of collection. The plates were incubated at 37 C in an atmosphere containing 5% CO2 for 48 h. Colonies on chocolate agar plates showing typical morphology (i.e. smooth, greyish, and wet colonies) were selected. H. influenzae were identified based on Gram stain, positive catalase and oxidase reactions, and the requirement of X and V factors for growth. The isolates were preserved in trypticase soy broth with 15% glycerol for further studies. The disc diffusion method was performed following Clinical and Laboratory Standards Institute (CLSI) guidelines.18 Haemophilus Test Medium Agar (Becton, Dickinson & Co., MD, USA) plates were inoculated with each bacterial suspension (adjusted to a McFarland 0.5 standard) and were incubated at 35 C for 16–18 h. Standard discs of 11 antimicrobial agents were tested.19 Minimum inhibitory concentrations (MICs) of levofloxacin and moxifloxacin were determined by Etest (AB Biodisk, Solna, Sweden). Currently, the rare occurrence of H. influenzae isolates with reduced susceptibility to fluoroquinolones precludes defining a category other than ‘‘susceptible’’ by CLSI.18 For simplicity, the above isolates were described as ‘‘resistant’’, without the category of ‘‘non-susceptible’’. H. influenzae ATCC 49247 was used as a control strain for susceptibility testing. Capsular type b was identified by the slide agglutination method. The b-lactamase activity was tested with a nitrocephin disc.
Statistical analysis
Results Characteristics of isolates Sputum, nasal or throat swab cultures were obtained from 150 (74%) of 201 participating residents at four nursing homes (NH-A, B, C, and D). During a 10 month period, 33 (22%) residents were positive for H. influenzae in their airways at least once. In total, of 62 H. influenzae isolates were isolated from sputum (41 isolates), throat swabs (19), and nasal swabs (2). Ten isolates could not be subcultured, so 52 isolates from 30 residents were studied. Eight isolates were simultaneously obtained from sputum and throat cultures of four individuals and had identical PFGE patterns, so only one of each pair was analysed, leaving 48 isolates from 30 residents for further study. None of the isolates belonged to serotype b. Antimicrobial susceptibility The results of antimicrobial susceptibility testing with the disc diffusion method are shown in Table I. The 27 (56.3%) ampicillinresistant isolates all exhibited b-lactamase activity. Only one isolate without b-lactamase activity was intermediately resistant to ampicillin (Etest MIC 2 mg/mL). All 48 isolates were susceptible to ampicillin–sulbactam, amoxicillin–clavulanate, ceftriaxone, cefpodoxime, and ertapenem. Whereas 94% of isolates were susceptible to cefuroxime, only 44% and 19% were susceptible to clarithromycin and trimethoprim–sulfamethoxazole, respectively. The MIC50, MIC90 and MIC ranges of the 48 isolates were 0.5, >32 and 0.012 to >32 mg/mL for levofloxacin, and 0.75, >32 and 0.016 to >32 mg/mL for moxifloxacin. Overall, 21 isolates were resistant to levofloxacin (MIC >2 mg/mL) and 21 to moxifloxacin (MIC > 1 mg/mL). One isolate (P5025) was susceptible to levofloxacin (MIC ¼ 1 mg/mL) but resistant to moxifloxacin (MIC ¼ 4 mg/ mL). For the remaining in-vitro FQ-susceptible isolates based on the CLSI interpretive criteria, an additional 13 (27.1%) and 14 (29.5%) strains had higher levofloxacin and moxifloxacin MICs (0.125 mg/ mL), respectively. Of note, high level levofloxacin and moxifloxacin non-susceptibility (MIC >32 mg/mL) was detected in 19 (39.6%) and 15 (31.3%) isolates, respectively. Compared with moxifloxacinsusceptible isolates, moxifloxacin-resistant isolates were more likely to be resistant to clarithromycin (57.1% vs 11.1%, P ¼ 0.003) and trimethoprim-sulfamethoxazole (95.2% vs 70.4%, P ¼ 0.035).
Pulsed-field gel electrophoresis Risk factors for colonisation of H. influenzae Molecular typing of the genomic DNA was performed by pulsedfield gel electrophoresis (PFGE). Preparation of DNA plugs and subsequent digestion by SmaI was done following the standardised protocols of PulseNet from the CDC website. Cluster analysis of the PFGE banding patterns was performed by the unweighted pair group method with arithmetic averages with the Jaccard coefficient, and the dendrogram was prepared using BioNumeric software (Version 4.5,
Compared to residents without colonisation of H. influenzae on univariate analysis, carriers of H. influenzae were significantly associated with chronic lung diseases (P < 0.001), malignancy (P ¼ 0.001), tube feeding (P < 0.001), urinary catheterisation (P ¼ 0.031), tracheostomy (P < 0.001), poor functional status (P ¼ 0.001), and living at Nursing Home (NH) A (P < 0.001) (detailed data not shown). Compared
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Table I Antimicrobial susceptibility of 48 Haemophilus influenzae isolates, as determined by disc diffusion method Total isolates (N ¼ 48)
LVX AMP SAM AMC ETP CXM CRO CPD CLR SXT
MXF resistant (N ¼ 21)
MXF susceptible (N ¼ 27)
S (%)
I (%)
R (%)
S (%)
I (%)
R (%)
S (%)
I (%)
R (%)
27 (56.3) 20 (41.6) 48 (100) 48 (100) 48 (100) 45 (93.8) 48 (100) 48 (100) 21 (43.8) 9 (18.8)
– 1 (2.1) 0 0 0 2 (4.2) 0 0 12 (25) 0
21 (43.8)a 27 (56.3) 0 0 0 1 (2.1) 0 0 15 (31.3) 39 (81.3)
1 (4.8) 12 (57.1) 21 (100) 21 (100) 21 (100) 18 (85.7) 21 (100) 21 (100) 5 (23.8) 1 (4.8)
– 1 (4.8) 0 0 0 2 (9.5) 0 0 4 (19.0) 0
20 (95.2)a 8 (38.1) 0 0 0 1 (4.8) 0 0 12 (57.1) 20 (95.2)
27 (100) 8 (29.6) 27 (100) 27 (100) 27 (100) 27 (100) 27 (100) 27 (100) 16 (59.3) 8 (29.6)
–
0 19 (70.4) 0 0 0 0 0 0 3 (11.1) 19 (70.4)
0 0 0 0 0 0 0 8 (29.6) 0
MXF, moxifloxacin; S, susceptible; I, intermediate; R, resistant; LVX, levofloxacin; AMP, ampicillin; SAM, ampicillin–sulbactam; AMC, amoxicillin–clavulanate; ETP, ertapenem; CXM, cefuroxime; CRO, ceftriaxone; CPD, cefpodoxime; CLR, clarithromycin; SXT, trimethoprim–sulfamethoxazole. a Non-susceptibility to levofloxacin.
to 138 residents without moxifloxacin-resistant isolates, those with moxifloxacin-resistant H. influenzae carriage were more likely to have chronic lung diseases (P ¼ 0.001), malignant neoplasm (P ¼ 0.008), tube feeding (P ¼ 0.002), urinary catheterisation (P ¼ 0.001), and tracheostomy tubes (P < 0.001), and were also more often living at NHA (P ¼ 0.002). On multivariate analysis, residents with urinary catheterisation [odds ratio (OR): 9.20, 95% confidence interval (CI): 1.47– 57.53, P ¼ 0.018] and tracheostomy tubes (OR: 7.82, 95% CI: 1.24–49.28, P ¼ 0.029) were significantly associated with airway colonisation of moxifloxacin-resistant H. influenzae (detailed data not shown). Twelve (40%) of 30 residents with airway carriage of H. influenzae were colonised with moxifloxacin-resistant isolates at least once during the study. Colonisation was more likely in residents with tracheostomy tubes on univariate analysis (P ¼ 0.061) (Table II). On multivariate analysis, no variable was significantly associated with colonisation of moxifloxacin-resistant H. influenzae (data not shown). Patterns of pulsed-field gel electrophoresis Analysis of the PFGE patterns of these 48 isolates revealed 14 distinct types (clones) (A–N) (Figure 1). Two major PFGE types, Table II Comparisons of clinical characteristics between nursing home residents with airway carriage of moxifloxacin-susceptible and moxifloxacin-resistant Haemophilus influenzae Variablesa
Moxifloxacin Susceptible (N ¼ 12)
Age (years) Charlson comorbidity index Barthel index Male Underlying diseases Hypertension History of stroke Chronic lung diseases Diabetes mellitus Malignant neoplasm Dementia History of myocardial infarction Chronic kidney disease Tube feeding Urinary catheterisation Tracheostomy tube Number of drugs Hospitalisation during previous 3 months Antimicrobial use during previous 3 months Living at hospital-affiliated nursing home Living at nursing home A a b
P-values
Resistant (N ¼ 18)
75.8 9.7 73.7 14.1 0.621 3.1 1.6 3.3 0.8 0.666 7.8 10.5 19.2 28.4 0.130 7 (38.9) 4 (33.3) >0.999b 15 (83.3) 14 (77.8) 8 (44.4) 7 (38.9) 4 (22.2) 2 (11.1) 1 (5.6) 1 (5.6) 18 (94.4) 3 (16.7) 5 (27.8) 6.6 2.8 1 (5.6) 1 (5.6) 1 (5.6) 10 (55.6)
9 (75.0) 9 (75.0) 8 (66.7) 2 (16.7) 4 (33.3) 1 (8.3) 1 (8.3) 0 14 (91.7) 6 (50.0) 8 (66.7) 6.5 1.6 4 (33.3) 1 (8.3) 1 (8.3) 10 (83.3)
Data are presented as mean SD or number of patients (%). Fisher’s exact test.
0.660b >0.999b 0.284b 0.249b 0.678b >0.999b >0.999b >0.999b >0.999b 0.102b 0.061b 0.952 0.128b >0.999b >0.999b 0.235b
clones A (19 isolates) and B (10 isolates), accounted for 60.4% of all isolates, and 27 of these 29 isolates were from 13 residents of nursing home A (NH-A). Four subtypes of major clone A showed variable moxifloxacin MICs (0.38 to >32 mg/mL). Isolates of subtypes A and A3 had decreased susceptibility to moxifloxacin (MIC 0.125 mg/mL), while 8 of the 12 isolates in subtypes A1 and A2 had high moxifloxacin MICs (>8 mg/mL). All ten isolates of clone B were highly resistant to levofloxacin and moxifloxacin (>32 mg/ mL), and all were from NH-A. Among the remaining 19 isolates (clones C-N), 10 were isolated from other nursing homes, and only two were not susceptible to moxifloxacin. Isolates with similar PFGE patterns from different nursing homes (clone D from NH-A and NH-C, clone F from NH-A and NH-B) were also found. Discussion Our study revealed that nearly half the H. influenzae isolates recovered from nursing home residents in Taiwan were resistant to FQs, and 12 (8%) of the 150 residents had been colonised by the FQresistant strains at one point. Such a substantial prevalence of FQresistant and high-level-resistant H. influenzae (MIC >32 mg/mL) in the airway carriage and polyclonal spread have not been reported before. Furthermore, of 210 clinical isolates of H. influenzae found at our hospital in 2007, 31 (14.8%) and 34 (16.2%) were resistant to levofloxacin and moxifloxacin, respectively (by disc diffusion method, unpublished data). These findings highlight the emerging role of FQ-resistant H. influenzae in healthcare facilities. Multivariate analysis showed that carriers of moxifloxacinresistant H. influenzae were associated with urinary catheterisation and tracheostomy tubes. Among carriers of H. influenzae, no significant factors were associated with the presence of moxifloxacin resistance except tracheostomy tubes (albeit borderline statistical significance). Thus, the significant factors of urinary catheterisation and tracheostomy tubes among all residents might only reflect the association with H. influenzae airway carriage rather than moxifloxacin resistance. The reason that our study failed to identify significant variables associated with moxifloxacin nonsusceptibility may be due in part to the limited number of cases with H. influenzae airway carriage. Among moxifloxacin-resistant H. influenzae isolates, two major clones (A and B) were found within NH-A, indicating their clonal spread in this long-term care facility. Although the multivariate analysis failed to support an association of NH-A and FQ-resistant H. influenzae colonisation, these two clones can be regarded as endemic because of geographic correlation. In fact, 34 (70.8%) of 48 isolates and 20 of 30 residents with H. influenzae colonisation came from NH-A where the residents were more likely to have chronic lung diseases and tracheostomy tubes, factors associated with increased
100
80
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Strain P1582 S1062 S1552 S1193 S5145 S1452 S1192 S1812-1 S1312 S1521 S1444 S1311 S1195 S1454 P1603 N1771 N1341 P3115 P5025 S1153 P3172 P0221 P0223 P1423 P5283 S1303 S1274 S1814 S1275 P1545 S3045 S1442 S1523 S1534 S1445 S1255 S1535 S1525 S1061 S1812 S1313 S1064 S1455 S1743 S5322 P1543 S0404 S5095
307
Nursing home A A A A B A A A A A A A A A A A A C B A C D D A B A A A A A-C C A A A A A A A A A A A A A B A D B
MIC of MXF 0.016 0.016 0.016 0.016 0.032 >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 0.023 0.023 0.064 4 0.094 0.25 0.75 0.75 0.125 0.016 0.5 0.5 0.5 0.5 0.75 0.75 0.19 0.5 16 12 >32 >32 16 0.38 >32 >32 3 24 0.38 0.016 0.023 >32 0.023
PFGE Pattern C C1 C2 C3 M B B B B B B B B B1 B2 D D D1 H I G E E J K A A A A A A A1 A1 A1 A1 A1 A1 A1 A2 A2 A2 A2 A2 A3 F F L N
Figure 1. Dendrogram of 48 Haemophilus influenzae isolates from four nursing homes. Strain identification: the first letter denotes specimen source (S, sputum; P, pharyngeal swab; N, nasal swab); the middle three digits are the nursing home resident number, and the last digit is the surveillance culture order (from first to fifth surveillance cultures). MIC, minimum inhibitory concentration; MXF, moxifloxacin. Pulsed-field gel electrophoresis (PFGE) pattern: isolates sharing >80% similarity were assigned an alphabet pattern name followed by a number for subtype.
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colonisation rates of H. influenzae irrespective of FQ resistance. Inadequate implementation of infection control measures and poor hygiene in tracheostomy care at NH-A might contribute to more frequent colonisation since NH-A residents with tracheostomy tubes were associated with colonisation of H. influenzae more than those in the other three nursing homes [18 (23.1%) vs 3 (2.4%), P < 0.001]. Although more than half of the H. influenzae isolates in the present study were susceptible to FQs according to the CLSI criteria, 25% and 29.5% of susceptible strains had higher MICs (0.125 mg/mL) of levofloxacin and moxifloxacin, respectively. Identification of these isolates raises clinical concerns regarding the therapeutic efficacy of FQ treatment and warrants further investigation. Previous studies have reported that H. influenzae isolates with reduced susceptibility to FQs carried a single point mutation in GyrA, which might be the initial step to the development of clinically significant resistance in Gram-negative bacteria.13,21,22 It has been demonstrated that infections due to Salmonella spp. with reduced FQ susceptibility led to a higher clinical failure rate if treated with FQs.23 Therefore, there is increasing likelihood of evolution toward FQ resistance in H. influenzae isolates. The increased use of levofloxacin and moxifloxacin has led to concern about the emergence of resistance. Indeed, the prevalence of levofloxacin-resistant pneumococcal isolates in Hong Kong increased from 5.5% in 1998 to 13.3% in 2000, and in Taiwan the prevalence of ciprofloxacin-resistant Salmonella enterica serotype Choleraesuis isolates increased from 0 in 1999 to 60% in 2001.24,25 As the selective antimicrobial pressure of FQ continues, it would not be surprising to observe the isolation frequency of FQ-resistant H. influenzae increase. A recent study on an outbreak of FQ-resistant H. influenzae in a long-term care facility in New York reported that previous levofloxacin use was associated with infection with a resistant strain.14 High level resistance might be induced by repeated treatment with FQs in patients with chronic lung diseases.26–28 Although no significant association with previous FQ use was found in our study, the emergence of H. influenzae isolates with high level resistance to levofloxacin and moxifloxacin is still a reminder that FQs should be used cautiously. In summary, our study reported the emergence of polyclonal levofloxacin/moxifloxacin-resistant H. influenzae isolates among nursing home residents. Further studies on the resistance mechanism and the prevalence of FQ-resistant H. influenzae in other healthcare facility populations are warranted. Acknowledgements We would like to thank M.M.-H. Lin for collecting specimens and E.P.-C. Wu for laboratory assistance. Conflict of interest statement None declared. Funding sources This study was supported by research grants from the Center for Disease Control, Taiwan (DOH96-DC-1010), National Cheng Kung University Hospital (NCKUH 9702038), and National Health Research Institutes (98A1-CLPP01-014). References 1. Murphy TF. Haemophilus infections. In: Mandell GL, Bennett JE, Dolin R, editors. Mandell, Douglas, and Benett’s principles and practice of infectious diseases. 6th ed. New York: Churchill Livingstone; 2005. p. 2661–2669. 2. Goldstein IJC, Garabedian-Ruffalo SM. Widespread use of fluoroquinolones versus emerging resistance in pneumococci. Clin Infect Dis 2002;35: 1505–1511.
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