Abstract
We investigated the molecular epidemiology and microbiological characteristics of 51 Escherichia coli isolates causing hospital-acquired pneumonia (HAP) in eight Asian areas. Sequence type 131 (ST131) was the most prevalent among E. coli isolates causing HAP, especially in South Korea, Thailand, and the Philippines. The current study showed that CTX-M-15-producing E. coli ST131 has emerged in and disseminated among patients with HAP in Asia. Our data suggest that this pandemic clone poses an important public health threat even in nosocomial infections.
Introduction
E
Hospital-acquired pneumonia (HAP) remains an important cause of both substantial morbidity and mortality among hospitalized patients. Increasing antimicrobial resistance in major pathogens causing HAP has aroused concern of the failure of antimicrobial treatment. 5 A previous surveillance study showed that the incidence of ESBL-producing E. coli (ESBL-EC) was 2.3% to 40% of cases of HAP (including ventilator-acquired pneumonia) in Asian countries. 6 However, the prevalence of E. coli ST131 among patients with HAP in Asian countries has not been well investigated. Therefore, we investigated the molecular epidemiology and microbiological characteristics of E. coli isolates causing HAP in Asian countries, focusing especially on ST131.
Materials and Methods
Escherichia coli isolates
As a part of a multinational study of Asian Network for Surveillance of Resistant Pathogens (ANSORP), 51 E. coli isolates were collected from patients with HAP in eight Asian areas during 2008 and 2009, including South Korea (n = 4), Hong Kong (n = 9), Taiwan (n = 4), Philippines (n = 4), Thailand (n = 26), Malaysia (n = 1), Singapore (n = 2), and India (n = 1). Among the 51 E. coli isolates, the most prevalent specimen source was sputum (n = 20, 39.2%), followed by endotracheal aspirate (n = 17, 33.3%), blood (n = 11, 21.6%), unknown (n = 2, 3.9%), and bronchoalveolar lavage fluid (n = 1, 2.0%). All participating hospitals were tertiary- or secondary-care teaching hospitals located in urban areas. Only the first isolate per patient was included in the study.
Antimicrobial susceptibility testing and ESBL characterization
In vitro antimicrobial susceptibility test (AST) of E. coli isolates was performed by the broth microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The 15 antimicrobial agents, including ampicillin, gentamicin, ceftazidime, cefotaxime, cefepime, aztreonam, ciprofloxacin, imipenem, meropenem, doripenem, cefoperazone/sulbactam, sulfamethoxazole/trimethoprim, piperacillin/tazobactam, colistin, and ceftobiprole, were tested. AST results were interpreted according to the CLSI breakpoints, as updated in 2015. 7 Multidrug-resistance (MDR) was defined as acquired nonsusceptibility to at least one agent in three or more antimicrobial categories. 8 ESBL activity was confirmed through a double-disk synergy test (ceftazidime, cefotaxime, and aztreonam minimal inhibitory concentration (MIC) of ≥2 mg/L) using BD BBL Sensi-Disc (BD Diagnostics, Sparks, MD). We also used polymerase chain reaction to amplify DNA, which we then sequenced using blaTEM, blaSHV, and blaCTX-M primers for the isolates with ESBL phenotype. 9
Molecular methods
Multilocus sequence typing (MLST) was performed for all E. coli isolates according to the Achtman scheme as previously described. 10 New sequence types were submitted to the MLST website and approved (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). ST131 isolates were also tested by allele-specific primers for allele 30 of fimH corresponding with the main fluoroquinolone resistance (FQ-R)-associated subset within ST131, the H30 subclone. 11
Statistical analyses
To compare the significance of differences in resistance between ST131 and non-ST131 isolates, the Fisher's exact t test was used to compare categorical variables. The SPSS statistics package (version 11.5 software; SPSS, Inc., Chicago, IL) was used for analyses.
Results and Discussion
ST131 (n = 19, 37.2%) was the most prevalent type, followed by ST23 (n = 3, 5.8%), ST69 (n = 3, 5.8%), ST95 (n = 3, 5.8%), ST38 (n = 2, 3.9%), and ST73 (n = 2, 3.9%), which together accounted for 62.7% of isolates (Table 1). Except for ST131, most STs were represented by one to three E. coli isolates. A high frequency of ST131 was observed in Thailand (13/26, 50.0%), South Korea (2/4, 50.0%), the Philippines (2/4, 50.0%), and Hong Kong (2/9, 22.2%). Among the ESBL-producing isolates, 14 (14/26, 53.8%) isolates belonged to ST131. H30 subclone, which is recently emerged FQ-R-associated lineage within ST131, accounted for 92.8% (13/14) to 94.7% (18/19) of ST131 isolates within FQ-R and ESBL groups, respectively.
Others include ST117, 161, 216, 773, 1607, 1837, 4477, 5019, 5020, 5021, and 5022.
KR, South Korea; HK, Hong Kong; TW, Taiwan; PH, Philippines; TH, Thailand; MY, Malaysia; SG, Singapore; IN, India; CC, clonal complex; ESBLs, extended-spectrum beta-lactamases; ST, sequence type.
Antimicrobial resistance rates to ceftazidime, cefotaxime, cefepime, aztreonam, and ciprofloxacin were significantly higher in ST131 than in non-ST131 isolates (p < 0.05) (Table 2). All of the isolates were susceptible to carbapenems. The overall rate of MDR in E. coli isolates was 76.4%. The most common pattern of MDR was resistance to ampicillin, ceftazidime, cefotaxime, and ciprofloxacin (58.9%). MDR was more prevalent in ST131 isolates (n = 17, 89.4%) than non-ST131 isolates (n = 22, 68.7%). In particular, a high prevalence of MDR in ST131 was observed in South Korea (100%), Hong Kong (100%), Thailand (92.3%), and the Philippines (50%).
CPZ/SBT, cefoperazone/sulbactam; SMX/TMP, sulfamethoxazole/trimethoprim; PIP/TAZ, piperacillin/tazobactam.
NA, not applicable; breakpoint has not been determined yet by CLSI.
CLSI, Clinical and Laboratory Standards Institute; MIC, minimal inhibitory concentration; ST131, sequence type 131.
The major ESBL types of E. coli isolates causing HAP were CTX-M-15 (n = 13, 50.0%), followed by CTX-M-14 (n = 5, 19.2%), CTX-M-27 (n = 2, 7.7%), CTX-M-57 (n = 5, 7.7%), CTX-M-3 (n = 1, 3.8%), and CTX-M-65 (n = 1, 3.8%). TEM-1 and SHV β-lactamases (SHV-1 and SHV-11) were found in 13 (50.0%) and 2 isolates (7.7%), respectively. CTX-M-15 was the most dominant ESBL gene type (n = 10, 52.6%), followed by CTX-M-27 (n = 2, 10.5%), CTX-M-14 (n = 1, 5.2%), and CTX-M-3 (n = 1, 5.2%), among the ST131 isolates. In particular, CTX-M-15 was more frequent in ST131 isolates (n = 10, 52.6%) than in non-ST131 isolates (n = 3, 9.3%; p < 0.001).
We report a high prevalence of ST131 among E. coli isolates causing HAP in Asian countries, especially in South Korea (n = 2, 50%), Thailand (n = 13, 50%), and the Philippines (n = 2, 50%). Although only a few E. coli isolates could be included and the distribution of isolates was not balanced among the different countries, ST131 was the most prevalent in HAP in Asian countries. Previous studies have already revealed that intra-abdominal infection and urinary tract infections are common in patients with ESBL-EC in Asia.12–14 To our knowledge, this is the first reported study to analyze the prevalence of E. coli ST131 among patients with HAP in Asian countries.
In this study, CTX-M-15 was the predominant ESBL in E. coli isolates from South Korea, Hong Kong, Thailand, and India. CTX-M-15-producing E. coli ST131 isolates showed high resistance rates to most antimicrobial agents except the carbapenems and colistin. Unlike the TEM- and SHV-derived ESBLs, CTX-M enzymes are not limited to nosocomial infections, and their potential for spread in communities has been of great concern.15,16
The current study showed that CTX-M-15-producing E. coli ST131 has emerged and disseminated in patients with HAP in the Asian region. Our data suggest that this pandemic clone poses an important public health threat even in nosocomial infections such as HAP. Focused attention on ST131 with continuous surveillance of the emergence and dissemination of this strain is warranted to prevent its further spread.
Footnotes
Acknowledgments
The authors thank all investigators of the Asian Network for Surveillance of Resistant Pathogens (ANSORP) Study Group who participated in this study for their dedication and contribution. The members who participated in this study in the ANSORP are as follows: Hyuck Lee, Dong-A University Hospital, Busan, Korea; Sook-In Jung, Chonnam National University Hospital, Gwangju, Korea; Camilla Rodrigues, Hinduja Hospital, Mumbai, India; Li Yang Hsu, National University Hospital, Singapore; Tan Kah Kee, Jenny Tong May Geok, and Suhailah Md Hanapiah, Hospital Tuanku Jaafar, Seremban, Malaysia; Thomas M. K. So, SureCare Medical Centre, Kowloon, Hong Kong; Wai-Keung Kwan, Yan Chai Hospital, Hong Kong; Maria Paz Mateo, Veterans Memorial Medical Center, Quezon City, Philippines; Mediadora Saniel, The Medical City, Pasig, Philippines; Jien-Wei Liu, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Taipei, Taiwan; Po-Ren Hsueh, National Taiwan University, Taipei, Taiwan; Visanu Thamlikitkul, Siriraj Hospital, Bangkok, Thailand; Anan Chongthaleong, Chulalongkorn University Hospital, Bangkok, Thailand; Piroon Mootsikapun, Khon Kaen University Hospital, Khon Kaen, Thailand; Prasit Tharavichitkul, Chiang Mai University Hospital, Chiang Mai, Thailand; Sarayut Lucien Geater, Songkla University Hospital, Songkhla, Thailand; Jirachai Waiwarawut, Chonburi Hospital, Chonburi, Thailand; Suthat Rungruanghiranya, Srinakharinwirot University, Bangkok, Thailand; and Pinyo Horsin, Bumrungrad International Hospital, Bangkok, Thailand.
This study was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. HI12C0756). Bacterial isolates were obtained from the Asian Bacterial Bank (ABB) of the Asia Pacific Foundation for Infectious Diseases (APFID).
Disclosure Statement
No competing financial interests exist.