Multicentre investigation of pathogenic bacteria and antibiotic resistance genes in Chinese patients with acute exacerbation of chronic obstructive pulmonary disease

Abstract

Objective

A prospective observational study to investigate the distribution and antimicrobial resistance of pathogenic bacteria in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in Beijing, China.

Methods

Patients with AECOPD were recruited from 11 general hospitals. Sputum specimens were cultured and bacteria identified. Antibiotic susceptibility was determined for each isolate, and presence of antibiotic resistance genes was evaluated using polymerase chain reaction.

Results

Pathogenic bacteria were isolated from 109/318 patients (34.28%); 124 isolates of 22 pathogenic bacterial species were identified, including Klebsiella pneumoniae (16.94%), Pseudomonas aeruginosa (16.94%), Acinetobacter baumannii (11.29%), Streptococcus pneumoniae (8.87%), and Staphylococcus aureus (7.26%). S. aureus was sensitive to tigecycline, teicoplanin, vancomycin and linezolid but resistant to penicillin and levofloxacin. K.pneumoniae, P. aeruginosa, A. baumannii and E. coli were susceptible to amikacin and cefoperazone.

Conclusions

K. pneumoniae and P. aeruginosa are the most common pathogenic bacteria in AECOPD cases in Beijing, China. Our antibiotic resistance findings may be helpful in selecting antibiotic therapy.

Keywords

Chronic obstructive pulmonary disease acute exacerbation bacteriology antibiotic resistance gene

Introduction

Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory illness worldwide¹ and is characterized by persistent airflow limitation that is usually progressive.² A large-scale epidemiological survey carried out between 2002 and 2004 found the prevalence of COPD in adults in China to be 8.2% (12.4% in men and 5.1% in women).³ Acute exacerbation of COPD (AECOPD) deteriorates pulmonary function and increases airway inflammation,⁴ and is a major cause of morbidity, mortality and reduced health-related quality-of-life in these patients.⁵ Guidelines produced by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) take into account the individual patient’s history of exacerbations when assessing symptomatology and risk of poor outcome.⁶ Bacterial infection contributes to AECOPD,^7–9 with 40–60% of patients harbouring bacterial pathogens during an acute exacerbation phase.¹⁰ Commonly isolated pathogens vary between geographical regions, with Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae common in Western countries,¹¹ Klebsiella pneumoniae and Pseudomonas aeruginosa frequently isolated in Taiwan,¹² and H. influenzae and P. aeruginosa in Hong Kong.¹³ To the best of our knowledge, there have been no large scale studies of bacterial infections in AECOPD in Beijing, China, however.

Early antibiotic treatment of AEOCPD can shorten recovery time, improve lung function and reduce the risk of treatment failure.⁶ Some studies have demonstrated statistically significant improvements with antibiotic therapy,^14,15 although it is generally prescribed on a empirical basis. Guidelines for antibiotic therapy of AECOPD have been published,¹⁶ but these cannot be applied on a worldwide basis due to geographical variations in antibiotic sensitivity and pathogen types. Antibiotic resistance is increasingly problematic, and judicious use of antibiotics is essential to prevent the emergence of multidrug-resistant bacteria. Information regarding antimicrobial resistance patterns and mechanisms will assist in determining the best antibiotic regimens for AECOPD treatment.

The aim of the present study was to conduct an epidemiological survey on the distribution, antimicrobial susceptibility and drug resistance genes of bacteria isolated from sputum samples of patients with AECOPD from hospitals in Beijing, China, to provide data that may contribute to rational treatment and management of AECOPD in this region.

Patients and methods

Study population

This prospective surveillance study recruited patients hospitalized with AECOPD between June 2013 and January 2014 in 11 general hospitals in Beijing, China (Chinese PLA 304 Hospital, Chinese PLA 306 Hospital, Chinese PLA 307 Hospital, Chinese PLA 309 Hospital, Chinese PLA Navy General Hospital, Chinese PLA Air Force General Hospital, Chinese PLA Second Artillery General Hospital, Chinese PLA General Hospital, Beijing Shijitan Hospital, Beijing Electric Power Hospital, Beijing Fengtai Hospital).

Diagnosis of COPD was based on GOLD guidelines.^6,13 AECOPD was considered if a patient with background COPD presented with an acute change of symptoms in at least one of dyspnoea, cough and sputum production.^6,13 Exclusion criteria were: (i) radiographic evidence of pneumonia; (ii) diagnosis of other pulmonary disease (e.g. bronchiectasis, diffuse panbronchiolitis, asthma, pulmonary tuberculosis, heart failure); (iii) exacerbation triggered by drugs, asbestos, sulphur dioxide or another toxic gas. Details of data collection procedures are shown in Table 1.

Table 1.

Data and sample collection schedule for an epidemiological study investigating pathogenic bacteria and antibiotic resistance in Chinese patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD).

Parameter	Baseline	Day 3	Day 14	End of therapy
Demographic data	Yes	–	–	–
General clinical data	Yes	Yes	–	Yes
X-radiography	Yes	–	–	–
Routine blood test	Yes	Yes	–	Yes
Inflammatory markers	Yes	Yes	–	Yes
Blood biochemical index	Yes	–	–	Yes
Arterial blood gas analysis	Yes	–	–	–
Pulmonary ventilation function + BDT	Yes	–	–	–
Patient’s condition	Yes	–	–	–
AECOPD treatment	Yes	Yes	–	Yes
Serodia-Myco II particle agglutination test	Yes	–	Yes	–
Collection and examination of lower respiratory tract secretions	Yes	–	–	Yes
Urinary pneumococcal antigen	Yes	–	–	Yes^a
Analysis of AECOPD treatment outcome	–	Yes	–	Yes

Patients with positive result at baseline.

BDT: bronchial dilation test.

The study was approved by the research ethics committee of Chinese PLA General Hospital and was undertaken according to principles outlined in the Declaration of Helsinki. All patients provided written informed consent.

Sample collection and culture

Fresh sputum samples were collected in sterile containers and processed as soon as possible (within 2 h) according to standard procedures by the designated microbiological laboratory at each research institution.¹⁷ Sputum samples containing <10 epithelial cells and/or >25 leukocytes per low-power field (×100) were accepted for subsequent analysis. At least two eligible consecutive samples were obtained from each participant. Sputum samples were inoculated onto blood agar plates, chocolate agar plates and Macconkey agar plates using standard techniques, and incubated at 37℃ in 5% carbon dioxide in air for 18–24 h.

Bacterial identification

Bacteria were identified via colony characteristics and biochemical reactions using standard microbiological methods or the VITEK® 2 system (BioMérieux, Marcy l’Etoile, France).¹⁸ Isolates of the same species from the same patient were considered a single isolate. Nonpathogenic micro-organisms including Neisseria, Streptococcus viridans, Corynebacterium and others were excluded from further analyses.

Streptococcus pneumoniae was identified via urinary pneumococcal antigen test (BinaxNOW® S. pneumoniae Antigen Card; Binax, Portland, ME, USA). Serodiagnosis of Mycoplasma pneumoniae was performed using Serodia®-Myco II particle agglutination test (Fujirebio, Tokyo, Japan), with titres ≥40 considered positive. Both tests were performed according to the manufacturers’ instructions.

Antibiotic susceptibility

Antibiotics used in drug susceptibility tests for Gram-negative bacteria included ampicillin, amoxicillin/clavulanate potassium, piperacillin/tazobactam, cefazolin, cefoxitin, ceftriaxone sodium, cefepime, aztreonam, ertapenem, imipenem, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, tigecycline, nitrofurantoin, cosulfamethoxazole (TMP/SMZ), cefoperazone and ceftazidime. Antibiotics used in drug susceptibility tests of the Gram-positive bacterium S. aureus included penicillin, levofloxacin, moxifloxacin, rifampicin, clindamycin, TMP/SMZ, tigecycline, teicoplanin, vancomycin and linezolid.

Tests were performed using the disk-diffusion method on Mueller–Hinton agar medium, with plates incubated for 16–20 h at 35℃ in room air. The re-identification of all bacterial isolates and minimal inhibitory concentration (MIC) determination were performed at the Department of Microbiology, Chinese PLA General Hospital, Beijing, China. MIC values were defined as sensitive (S), intermediate (I) or resistant (R), based on cut-off values defined by the Clinical and Laboratory Standards Institute.¹⁹ P. aeruginosa ATCC27853 and Escherichia coli ATCC25922 were used as control strains.

Antibiotic resistant gene amplification

The four most frequently isolated Gram-negative species were selected for polymerase chain reaction (PCR) detection of antibiotic resistance genes. Genomic DNA was extracted from each individual isolate (∼5 × 10⁶ cells per isolate) using a Blood and Cell Culture DNA Maxi kit (QIAGEN, Venlo, Netherlands) according to the manufacturer’s instructions, then stored at −20℃ until use. The PCR reaction mix comprised 10 µl 2 × GoldStar Best MasterMix (CWBIO company, Beijing, China), 2 µl each primer (Table 2), 2 µl DNA template, and 6 µl double-distilled water. The cycling programme involved preliminary denaturation at 94℃ for 10 min followed by 35 cycles of denaturation at 95℃ for 30 s, annealing (temperatures given in Table 2) for 30 s, and elongation at 72℃ for 60 s, followed by a final elongation step at 72℃ for 5 min. PCR was performed at the Pneumology Laboratory, Chinese PLA General Hospital Beijing, China, and products were sequenced by Beijing AuGCT company, Beijing, China.

Table 2.

Primer sequences for polymerase chain reaction of antibiotic resistance genes.

Gene	Primer sequence, 5′–3′		Annealing temperature, ℃	Product length, base pairs
Aminoglycoside resistance
aac(3)-II	Forward	ACTGTGATGGGATACGCGTC	52	237
aac(3)-II	Reverse	CTCCGTCAGCGTTTCAGCTA	52	237
aadA1	Forward	TGATTTGCTGGTTACGGTGAC	52	284
aadA1	Reverse	CGCTATGTTCTCTTGCTTTTG	52	284
aac6'-lb-cr	Forward	TATGAGTGGCTAAATCGA	52	394
aac6'-lb-cr	Reverse	CCCGCTTTCTCGTAGCA	52	394
aadB	Forward	GAGCGAAATCTGCCGCTCTGG	52	320
aadB	Reverse	CTGTTACAACGGACTGGCCGC	52	320
β-lactamase inhibitor resistance
blaTEM1	Forward	AGGAAGAGTATGAGTATTCAACA	52	535
blaTEM1	Reverse	CTCGTCGTTTGGTATGGC	52	535
blaSHV	Forward	GGTTATGCGTTATATTCGCC	52	867
blaSHV	Reverse	GGTTAGCGTTGCCAGTGC	52	867
blaDHA-1	Forward	AACTTTCACAGGTGTGCTGGGT	52	405
blaDHA-1	Reverse	CCGTACGCATACTGGCTTTGC	52	405
blaCTX-M-15	Forward	ATGGTTAAAAAATCACTGCGC	54	833
blaCTX-M-15	Reverse	TCGCGACGGCTTTCTGCCTT	54	833
Carbapenem resistance
oxa-2	Forward	CAGGCGCTGTTCGTGATGAGTT	52	233
oxa-2	Reverse	GCCTTCTATCCAGTAATCGCC	52	233
oxa-10	Forward	GTCTTTCGAGTACGGCATTA	52	702
oxa-10	Reverse	GATTTTCTTAGCGGCAACTTA	52	702
Outer membrane protein related
oprD	Forward	GCGCATCTCCAAGACCATG	56	193
oprD	Reverse	GCCACGCGATTTGACGGTG	56	193
Quinolone resistance
qnrA1	Forward	CAAGAGGATTTCTCACGCCAG	52	231
qnrA1	Reverse	GAACTCTATGCCAAAGCAGTTGG	52	231
qnrB	Forward	ATGGCTCTGGCACTCGTTGGCGA	56	681
qnrB	Reverse	CTAACCAATCACCGCGATGCCAAG	56	681
qnrS1	Forward	ATGGAAACCTACAATCATACATAT	56	657
qnrS1	Reverse	TTAGTCAGGATAAACAACAATACCC	56	657

Statistical analyses

Data were presented as mean ± SD or n (%).

Results

The study enrolled 326 patients, eight of whom were excluded (three refused to sign informed consent forms; five had congestive heart failure). The final study therefore included 318 patients (206 male/112 female; mean age 75.7 ± 9.6 years; age range 46–100 years). A total of 89 patients completed lung function tests; all other patients were unable to comply or their illness was too severe. Demographic and clinical data from the study population are shown in Table 3.

Table 3.

Clinical and demographic characteristics of 318 patients with acute exacerbation of chronic obstructive pulmonary disease included in an epidemiological study investigating pathogenic bacteria and antibiotic resistance in Beijing, China.

Characteristic	N (%) or mean ± SD
Male sex	206 (64.8)
Age, years	75.7 ± 9.6
Antibiotics in past 3 months	133 (41.8)
Comorbidities
Coronary heart disease	166 (52.2)
Diabetes mellitus	70 (22.0)
Hypertension	49 (15.4)
Cerebrovascular disease	32 (10.1)
Clinical symptoms
Fever	65 (20.4)
Cough	313 (98.4)
Dyspnoea	292 (91.8)
Pulmonary rales	287 (90.3)
Pulmonary function test findings (n = 89)
FEV₁/FVC%	41.4 ± 11.8
FEV₁, l	1.01 ± 0.43
FEV₁, % of predicted	50.9 ± 18.6
Infection indicators
Leukocytes, 10⁹/l	8.0 ± 3.4
Neutrophils, %	75.4 ± 12.8
C-reactive protein, mg/dl	40.9 ± 87.1
Procalcitonin, µg/dl	0.6 ± 2.7

FEV₁: forced expiratory volume in 1 s; FVC: forced vital capacity.

Pathogenic bacteria were isolated from the sputum of 109/318 patients (34.28%), with a total of 124 isolates of 22 pathogenic bacterial species identified (Table 4). Of these 124 isolates, 96 (77.42%) were Gram-negative, with the four most frequently isolated being K. pneumoniae (21/124; 16.94%), P. aeruginosa (21/124; 16.94%), A. baumannii (14/124; 11.29%), and E.coli (8/124; 6.45%). These four bacteria were selected for PCR analysis of antibiotic resistance genes.

Table 4.

Pathogenic bacteria isolated from patients with acute exacerbation of chronic obstructive pulmonary disease in Beijing, China.

Pathogenic bacterium	Total cases, n	Percentage of patients n = 318	Percentage of isolates n = 124
Klebsiella pneumoniae	21	6.60	16.94
Pseudomonas aeruginosa	21	6.60	16.94
Acinetobacter baumannii	14	4.40	11.29
Streptococcus pneumoniae	11	3.46	8.87
Staphylococcus aureus ^a	9	2.83	7.26
Escherichia coli	8	2.52	6.45
Haemolytic streptococcus	7	2.20	5.65
Mycoplasma pneumoniae ^b	6	1.89	4.84
Enterobacter cloacae	4	1.26	3.23
Stenotrophomonas maltophilia	3	0.94	2.42
Burkholderia cepacia	3	0.94	2.42
Acinetobacter	3	0.94	2.42
Pseudomonas putida	2	0.63	1.61
Enterobacter aerogenes	2	0.63	1.61
Alcaligenes xylosoxidans	2	0.63	1.61
Acinetobacter lwoffii	2	0.63	1.61
Klebsiella oxytoca	1	0.31	0.81
Serratia marcescens	1	0.31	0.81
Pseudomonas stutzeri	1	0.31	0.81
Proteus mirabilis	1	0.31	0.81
Acinetobacter junii	1	0.31	0.81
Shewanella putrefaciens	1	0.31	0.81

Data expressed as n or %.

Urinary pneumococcal antigen test (BinaxNOW®S. pneumoniae Antigen Card; Binax, Portland, ME, USA).

Serodia-Myco II particle agglutination test (Fujirebio, Tokyo, Japan).

All other bacteria isolated from sputum.

Data regarding the presence of antibiotic resistance genes in K. pneumoniae, P. aeruginosa, A. baumannii and E. coli are shown in Table 5. The absence of the encoding gene of outer membrane protein D (oprD) and presence of aac6'-lb-cr was detected in all four bacteria. Full details of all other antibiotic resistance genes are shown in Table 5.

Table 5.

Presence of antibiotic resistance genes (detected via polymerase chain reaction) in the four most commonly isolated Gram-negative bacteria from the sputum of patients with acute exacerbation of chronic obstructive pulmonary disease in Beijing, China (n = 318).

Gene	Klebsiella pneumonia n = 21	Pseudomonas aeruginosa n = 21	Acinetobacter baumannii n = 14	Escherichia coli n = 8
β-lactamase inhibitor
blaTEM1	14.3	38.1	50.0	62.5
blaSHV	76.2	9.5	42.9	12.5
blaDHA-1	19.0	38.1	78.6	50.0
blaCTX-M-15	28.6	19.0	14.3	25.0
Outer membrane protein related
oprD	23.8	42.9	35.7	37.5
Carbapenem resistance
oxa-10	71.4	4.8	50.0	25.0
oxa-2	52.4	4.8	7.1	0.0
Aminoglycoside resistance
aadA1	14.3	28.6	21.4	50.0
aac(3)-II	71.4	33.3	78.6	87.5
aac6'-lb-cr	66.7	52.4	78.6	87.5
aadB	19.0	33.3	64.3	62.5
Quinolone resistance
qnrA1	9.5	33.3	0.0	25.0
qnrB	52.4	9.5	35.7	75.0
qnrS1	23.8	4.8	42.9	25.0

Data expressed as %.

Data regarding antibiotic susceptibility are shown in Table 6. K. pneumoniae had >50% susceptibility to all antibiotics tested, with the exception of ampicillin and nitrofurantoin. P. aeruginosa was sensitive to cefoperazone (61.9%) and amikacin (57.1%), with lower sensitivity to other antibiotics. A. baumannii was most sensitive to tigecycline (64.2%). Tigecycline and amikacin were the most effective antibiotics against E. coli (both 87.5%), followed by imipenem and cefepime (both 75.0%). All S. aureus isolates were sensitive to tigecycline, teicoplanin, vancomycin and linezolid, but resistant to penicillin and levofloxacin (data not shown).

Table 6.

Antibiotic susceptibility of the four most commonly isolated Gram-negative bacteria from the sputum of patients with acute exacerbation of chronic obstructive pulmonary disease in Beijing, China (n = 318).

Antibiotic	Klebsiella pneumonia n = 21			Pseudomonas aeruginosa n = 21			Acinetobacter baumannii n = 14			Escherichia coli n = 8
Antibiotic	R	I	S	R	I	S	R	I	S	R	I	S
ESBLs	14.3	0	85.7	/	/	/	/	/	/	37.5	0	62.5
Ampicillin	100	0	0	100	0	0	92.9	0	7.1	87.5	0	12.5
Amoxicillin/Clavulanate Potassium	42.9	0	57.1	100	0	0	100	0	0	37.5	0	62.5
Piperacillin/Tazobactam	23.8	9.5	66.7	28.6	28.6	42.9	50	0	50	12.5	25.0	62.5
Cefazolin	47.6	0	52.4	95.2	0	4.8	100	0	0	87.5	0	12.5
Cefoxitin	38.1	0	61.9	95.2	0	4.8	92.9	0	7.1	50	12.5	37.5
Ceftriaxone Sodium	42.9	0	57.1	95.2	0	4.8	57.1	14.3	28.6	87.5	0	12.5
Cefepime	14.3	0	85.7	47.6	9.5	42.9	57.1	0	42.9	25.0	0	75.0
Aztreonam	33.3	0	66.7	/	/	/	64.3	14.3	21.4	87.5	0	12.5
Ertapenem	14.3	0	85.7	/	/	/	/	/	/	37.5	0	62.5
Imipenem	14.3	0	85.7	71.4	4.8	23.8	50	0	50	25.0	0	75.0
Amikacin	9.5	0	90.5	38.1	4.8	57.1	35.7	21.4	42.9	12.5	0	87.5
Gentamicin	19.0	4.8	76.2	76.2	4.8	19.0	50	0	50	62.5	0	37.5
Tobramycin	9.5	9.5	81.0	66.7	0	33.3	42.9	7.1	50	50	12.5	37.5
Ciprofloxacin	19.0	0	81.0	57.1	9.5	33.3	50	7.1	42.9	75.0	0	25.0
Levofloxacin	14.3	4.8	81.0	61.9	9.5	28.6	35.7	14.3	50	62.5	12.5	25.0
Tigecycline	9.5	9.5	81.0	100	0	0	28.6	7.1	64.3	0	12.5	87.5
Nitrofurantoin	52.4	38.1	9.5	100	0	0	100	0	0	75.0	0	25.0
Cosulfamethoxazole (TMP/SMZ)	28.6	0	71.4	100	0	0	50	0	50	87.5	0	12.5
Cefoperazone	14.3	9.5	76.2	33.3	4.8	61.9	28.6	21.4	50	12.5	0	87.5
Ceftazidime	23.8	9.5	66.7	47.6	9.5	42.9	50	14.3	35.7	37.5	0	62.5

Data presented as %.

R, resistant; I, intermediate; S, sensitive; ESBLs, extended spectrum β-lactamases.

Antibiotic susceptibility determined according to guidelines of the Clinical and Laboratory Standards Institute.¹⁹

Discussion

Pathogenic bacteria were found in 34.3% of patients with AECOPD in the present study, based on sputum culture. Others have reported infection rates of 37.4% in China,²⁰ 32.3% in Hong Kong,²¹ 54.7% in Italy,²² and 51.7% in the UK (London).⁷ The primary pathogenic bacteria detected in the present study were K. pneumoniae (16.94%), P. aeruginosa (16.94%), A. baumannii (11.29%) and S. pneumoniae (8.87%), indicating that Gram-negative bacteria may be the predominant cause of AECOPD in Beijing. These findings are consistent with those from Indonesia, Malaysia and Taiwan,^11,23 but substantially different from Hong Kong, South Korea and the Philippines,²³ and Western countries,¹⁰ where the microbiology of community-acquired pneumonia (H. influenzae, S. pneumoniae and M. catarrhalis) was responsible for AECOPD.

Regional variation in bacterial flora has several possible explanations. First, differences in living habits, dietary habits and antibiotic use may affect the distribution of pathogenic bacteria. Although it is unclear whether antibiotic selection pressure is a factor in the prevalence of pathogenic bacteria, care should be taken in the choice of antibiotic therapy for people with AECOPD. Secondly, the severity of AECOPD varied between studies. Severe AECOPD is associated with a lowered immune response as well as hospitalization, both of which benefit bacterial colonization. COPD severity is an important determinant of micro-organism type,²⁴ and enteric Gram-negative bacilli and P. aeruginosa may be more frequently isolated from patients with more severe expiratory airflow limitation.²⁵ The distribution of pathogenic bacteria in patients with a single AECOPD episode has been shown to be different from that in patients with two or more episodes, such that H. influenzae was associated with a single episode and Enterobacteriaceae species were only isolated from patients with several exacerbations.²⁶ Finally, sputum culture and strain identification methods varied between studies, and included quantitative, semiquantitative and qualitative culture, biochemical studies, PCR and automatic microbial identification systems. It has been shown that the use of PCR to detect Chlamydia pneumoniae in sputum samples could overestimate its frequency.²⁶

The four primary Gram-negative bacteria species detected in this study are opportunistic pathogens, which always result in a long term disease course in AECOPD.²² These bacteria were shown to have vastly different antibiotic resistance profiles. K. pneumoniae was >50% susceptible to all antibiotics used in this study (except ampicillin and nitrofurantoin), a finding that is similar to others.^20,23 In contrast, P. aeruginosa was only >50% susceptible to cefoperazone and amikacin. Notably, the susceptibility of P. aeruginosa to imipenem was 23.8%, which is significantly lower than reported values of 74.8% in 2003 and 70.5% in 2008, in China.²⁷ A. baumannii is a major pathogen in nosocomial infections, and its antibiotic resistance is an increasing problem. Imipenem has been shown to be the most powerful antimicrobial agent against A. baumannii,²⁷ but half of A. baumannii strains were resistant to imipenem in the present study, and tigecycline was most effective. We found that the most effective antibiotics against E. coli were amikacin, tigecycline and cefoperazone, followed by cefepime and imipenem, but others have reported 100% sensitivity to imipenem, followed by amikacin and ceftazidime.²⁰ It is clear that pathogenic bacteria have different antibiotic sensitivity profiles in different countries or regions, possibly as a result of different drug-resistance mechanisms.

There are several antibiotic resistance mechanisms in Gram-negative bacterium, including the presence of extended spectrum β-lactamases and antibiotic modifying enzymes, and the loss of porins.²⁸ Based on these mechanisms, we evaluated the presence 14 antibiotic resistant genes in four Gram-negative bacteria strains, in order to examine the relationship between drug resistance genes and antibiotic susceptibility. β-lactamase inhibitor resistant genes blaTEM1, blaSHV, blaDHA-1 and blaCTX-M-15 were detected in most A. baumannii and E. coli isolates, which were resistant to first- and second-generation cephalosporins and sensitive to some third- and fourth-generation cephalosporins. It is possible that blaTEM1, blaSHV, blaDHA-1 and blaCTX-M-15 might be responsible for this resistance to first- and second-generation cephalosporins. Conversely, although some P. aeruginosa isolates were positive for blaTEM1, blaSHV, blaDHA-1 and blaCTX-M-15, they were also resistant to first- and second-generation cephalosporins, suggesting the possible existence of alternative resistance mechanisms in P. aeruginosa. The outer membrane protein oprD regulates the entry of carbapenems,²⁹ and the absence of oprD was the most prevalent mechanism of resistance in P. aeruginosa in the present study. Although OprD was absent from the largely imipenem-resistant P. aeruginosa isolates in the current study, it was also not present in imipenem-sensitive K. pneumoniae, A. baumannii and E. coli isolates. Similar results were found with oxa-10 and oxa-2, which were detected in most imipenem-sensitive K. pneumoniae isolates, but not in imipenem-sensitive A. baumannii and E. coli isolates. This finding suggests that oxa-10 and oxa-2 are not responsible for imipenem resistance in K. pneumoniae.

Plasmid-mediated qnr genes can facilitate bacterial spread and are mainly present in K. pneumoniae and E. coli.³⁰ In the present study, 52.4% of K. pneumoniae isolates and 75.0% of E. coli isolates were positive for qnrB. Interestingly, the aminoglycosides resistance genes, aac6'-lb-cr and aac(3)-II, were detected in all four Gram-negative bacteria in the present study, but all isolates were highly susceptibility to amikacin. It is possible that the amikacin resistance mechanism in Beijing is not related to aac6'-lb-cr and aac(3)-II.

The present study had two main limitations. First, the majority of patients (229/318) did not complete lung-function tests during the study period. It was therefore not possible to evaluate the relationship between the severity of airway obstruction and the distribution of pathogenic bacteria. Secondly, sensitive techniques such as PCR were not used for the identification of bacteria in sputum samples, some of which (e.g. M. pneumoniae, C. pneumoniae and Legionella) require special culture media and methods, and are therefore difficult to identify. To our knowledge, this is the first multicentre study of bacteriology in patients with AECOPD performed in Beijing. The strengths of our study were its prospective nature, with a relatively large sample size, and the inclusion of tests for antibiotic susceptibility and drug-resistance genes.

In conclusion, we found that K. pneumoniae and P. aeruginosa are the most common pathogenic bacteria in AECOPD in Beijing. Amikacin and cefoperazone provide sufficient antibacterial activity against the four main Gram-negative bacilli isolated from these patients (K. pneumoniae, P. aeruginosa, A. baumannii and E. coli), but the multidrug resistance of P. aeruginosa and A. baumannii warrants attention. These findings may be helpful in selecting antibiotic therapy for AECOPD in Beijing.

Footnotes

Acknowledgement

We thank our colleagues in PLA General Hospital who provided technical help.

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This work was supported by a Special Fund for Health-scientific Research in the Public Benefit of Ministry of Health grant (201002008).

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