Outbreak of pulmonary infection caused by Klebsiella pneumoniae isolates harbouring bla IMP-4 and bla DHA-1 in a neonatal intensive care unit in China

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1 Journal of Medical Microbiology (2012), 61, DOI /jmm Outbreak of pulmonary infection caused by Klebsiella pneumoniae isolates harbouring bla IMP-4 and bla DH-1 in a neonatal intensive care unit in China Fangyou Yu, 1 Qunhua Ying, 2 Chun Chen, 3 Tingjian Li, 3 Baixing Ding, 4 Ying Liu, 3 Yuanyuan Lu, 3 Zhiqiang Qin, 5 Chris Parsons, 5 Cassandra Salgado, 5 Di Qu, 6 Jingye Pan 4 and Liangxing Wang 3 Correspondence Jingye Pan panjingye@hosp1.ac.cn Liangxing Wang wzyxywlx@163.com 1 Department of Laboratory Medicine, The First ffiliated Hospital of Wenzhou Medical College, Wenzhou , PR China 2 Department of Laboratory Medicine, Shaoxing Municipal Women and Children Hospital, Shaoxing , PR China 3 Department of Respiratory Medicine, The First ffiliated Hospital of Wenzhou Medical College, Wenzhou , PR China 4 Department of Intensive Care Unit, The First ffiliated Hospital of Wenzhou Medical College, Wenzhou , PR China 5 Division of Infectious Diseases, Department of Medicine, Medical University of South Carolina, Charleston, SC 29425, US 6 Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Institute of Medical Microbiology and Institutes of Biomedical Sciences, Shanghai Medical School of Fudan University, Shanghai , PR China Received 27 January 2012 ccepted 25 March 2012 Outbreaks caused by Klebsiella pneumoniae producing carbapenemases and other b- lactamases have been reported. Four neonates admitted to a neonatal intensive care unit (NICU) in a Chinese hospital developed respiratory infection while receiving intensive care. In all four cases, multidrug-resistant K. pneumoniae was isolated from multiple respiratory specimens, leading to additional characterization of these organisms and investigation of the local environment in the NICU. Multiple b-lactamase genes, including bla TEM-1, bla IMP-4, bla DH-1 and bla CTX-M-14, as well as the quinolone resistance gene qnrb4, were harboured by transferable plasmids from all four clinical isolates. Furthermore, PFGE confirmed that three of the four clinical isolates from the patients and three K. pneumoniae isolates collected from the hands of healthcare workers and an incubator in the NICU belonged to the same PFGE cluster, indicating that an outbreak due to multidrug-resistant K. pneumoniae carrying bla IMP-4 and bla DH-1 occurred in this NICU. s far as is known, this is the first report of the co-existence of bla IMP-4 and bla DH-1 in the same K. pneumoniae isolate. These data suggest that additional precautions are needed to prevent outbreaks of infection caused by multidrug-resistant K. pneumoniae resulting from environmental exposure in NICUs. INTRODUCTION Klebsiella pneumoniae frequently exhibits resistance to extended-spectrum cephalosporins due to the production of extended-spectrum b-lactamases (ESBLs) (Falagas & bbreviations: CLSI, Clinical and Laboratory Standards Institute; ESBL, extended-spectrum b-lactamase; KPC, Klebsiella pneumoniae carbapenemase; MBL, class B metallo-b-lactamase; NICU, neonatal intensive care unit. Karageorgopoulos, 2009). Carbapenems or fluoroquinolones are often used for the treatment of clinical infections caused by this organism. However, resistance of K. pneumoniae to carbapenems has been increasing (Espedido et al., 2005; Gupta et al., 2011), and this is often due to the production of carbapenemases, particularly K. pneumoniae carbapenemases (KPCs) and class B metallo-b-lactamases (MBLs) (Gupta et al., 2011; Queenan & Bush, 2007; Robledo et al., 2011; Zioga et al., 2010). KPCs, especially KPC-2, have G 2012 SGM Printed in Great Britain

2 NICU outbreak of carbapenemase-producing K. pneumoniae been found in members of the Enterobacteriaceae worldwide (Gupta et al., 2011; Nordmann et al., 2009). In addition to the family Enterobacteriaceae, KPCs have emerged in nonfermentative bacteria (Cuzon et al., 2011; Robledo et al., 2011; Villegas et al., 2007). In China, KPC-2 is a predominant carbapenemase and has been recognized in many members of the Enterobacteriaceae, such as K. pneumoniae, Serratia marcescens, Escherichia coli and Enterobacter cloacae (Cai et al., 2008; Wei et al., 2007; Wu et al., 2010). MBLs, including IMP and VIM, are commonly harboured by non-fermentative bacteria and have recently been identified in members of the Enterobacteriaceae worldwide (Maltezou, 2009; Queenan & Bush, 2007; Robledo et al., 2011). MBLs often confer high-level resistance to all b- lactams except aztreonam and are not inhibited by clavulanic acid, tazobactam or sulbactam. lthough several IMP- and VIM-type MBLs have been described in China, most were found in non-fermentative Gramnegative bacilli (Walsh et al., 2005). Recent data indicate that IMP-4 confers reduced susceptibility to carbapenems for K. pneumoniae isolated from patients in China (Chen et al., 2009; Mendes et al., 2008; Wei et al., 2011).Two clinical strains of K. pneumoniae carrying plasmid-borne bla IMP-4, bla SHV-12 and arm were found at a paediatric centre in Shanghai, China (Liu et al., 2009). The bla IMP-4 gene was also found to co-exist with bla KPC-2 in K. pneumoniae isolates (Chen et al., 2009; Mendes et al., 2008; Wei et al., 2011). The K. pneumoniae isolates producing IMP-4 found in China were clonally unrelated and occurred sporadically. n outbreak caused by multidrug-resistant K. pneumoniae harbouring bla IMP-4 has not been found previously in China. In this report, we describe an outbreak of multidrug-resistant K. pneumoniae infection in a neonatal intensive care unit (NICU) in China, as well as isolation of related organisms from environmental sources. METHODS Isolation and identification of bacterial strains. From October to December 2010, four neonates with asphyxia were admitted to the NICU of the 400-bed Shaoxing Municipal Women and Children Hospital in Shaoxing, eastern China. Of note, hospital admission for all four patients (designated patients 1 4) overlapped. Before admission to the NICU, pulmonary infections diagnosed by physical examination and new findings consistent with pneumonia on chest radiography were not found among the four patients. Bacterial isolates from sputum specimens growing over more than threequarters of the plate by quantitative culture were considered to be responsible for the pulmonary infection. Bacterial isolates were identified by a Vitek-32 microbiology analyser (biomérieux) according to the manufacturer s instructions and additional biochemical tests. The initial K. pneumoniae isolates were screened for further investigation. fter K. pneumoniae was isolated from patient 4, an outbreak control team was organized and infection control measures were implemented. Environmental samples were obtained for culture by rubbing sterile polyester-fibre-tipped swabs moistened with sterile saline repeatedly over designated sites in the immediate vicinity of the patients, including equipment used for their care and the fingers of medical staff caring for the patients. ntimicrobial susceptibility testing. ntimicrobial susceptibilities were determined initially using GNS cards of the Vitek system (biomérieux). Multidrug resistance profiles were then further evaluated by a disc diffusion test using commercial discs containing cefazolin (30 mg), cefotaxime (30 mg), ceftazidime (30 mg), cefepime (30 mg), aztreonam (30 mg), cefoxitin (30 mg), imipenem (10 mg), meropenem (10 mg), chloramphenicol (30 mg), tetracycline (30 mg), trimethoprim/sulfamethoxazole (1.25/23.75 mg), amikacin (30 mg), gentamicin (10 mg), ciprofloxacin (5 mg) and levofloxacin (5 mg). n agar dilution method was used to determine MIC values according to the criteria recommended by the Clinical and Laboratory Standards Institute (CLSI, 2011). E. coli TCC was used as a quality control strain for antimicrobial susceptibility testing. Detection of b-lactamases. modified Hodge test was performed to detect carbapenemases, as described previously (CLSI, 2011). double-disc synergy test was designed to detect MBLs, as described by Peleg et al. (2005). ll the isolates studied were tested for ESBL production by the CLSI-recommended confirmatory double-disc combination test (CLSI, 2011). Detection of antimicrobial resistance determinants. Total DN was extracted by boiling. Potential antimicrobial resistance determinants, which included carbapenemase genes, ESBL genes, plasmidborne ampc genes and plasmid-borne quinolone resistance determinants, were investigated by PCR and nucleotide sequencing, using previously published primers (Bradford et al., 2004; Robicsek et al., 2006; Yu et al., 2007). The DN sequences of the ompk35 and ompk36 genes were determined by PCR and DN sequencing with primers ompk35-f (59-TGTGGCGCTTTCTGGCGTGG-39), ompk35-r (59-TCGGCTTTGTCGCCTTGCCGTC-39), ompk36-f (59-TGGTTGTCTGTCCCTC-39) and ompk36-r (59- GTCGTCGGTGGTCCGGC-39). ll amplicon sequences were compared with the sequences available in GenBank ( ncbi.nlm.nih.gov/genbank/). Transfer of carbapenem resistance determinants. In order to determine whether carbapenem resistance was transferable in K. pneumoniae isolates, a conjugation experiment was carried out in Luria Bertani broth with E. coli J53 as the recipient, as described previously (Wang et al., 2003). Transconjugants were selected on tryptic soy agar plates containing sodium azide (100 mg ml 21 ) for counterselection and imipenem (0.5 mg ml 21 ) for plasmid-mediated carbapenem resistance selection. Determination of the flanking regions of the bla IMP-4 gene. Plasmid DN of the transconjugants was extracted using a Plasmid Midi kit (Qiagen) according to the manufacturer s instructions. Purified bla IMP -bearing conjugative plasmids extracted from the transconjugants were sequenced directly using a series of outwarddirected primers specific for the locations next to the bla IMP-4 gene. PFGE. Genomic DN was prepared from all tested K. pneumoniae isolates and cleaved with 40 U XbaI. Electrophoresis was performed on 1 % agarose gels in 0.5 M Tris/borate/EDT buffer on a CHEF- Mapper X PFGE system (Bio-Rad) for 24 h at 14 uc, with run conditions of 6 V cm 21, a pulse angle of 120u and pulse times of 5 20 s. l DN ladder (mersham Biosciences) was used as a molecular mass marker and DN bands were stained with ethidium bromide (0.5 mg ml 21 ) prior to identification by photography under UV light. Band profiles were interpreted by the criteria of Tenover et al. (1995). Patterns with a difference of no more than three DN bands were considered to belong to the same PFGE type

3 F. Yu and others RESULTS ND DISCUSSION suspected lung infection was diagnosed in patient 1 (aged 16 days), patient 2 (aged 17 days), patient 3 (aged 22 days) and patient 4 (aged 14 days). K. pneumoniae isolates designated KpSX1 KpSX4 were first recovered from sputum specimens of patients 1 4 following hospitalization for 22, 22, 32 and 16 days, respectively. Subsequently, K. pneumoniae isolates with identical antimicrobial resistance patterns were isolated from the sputum of each patient more than three times. The gestational ages of the four patients ranged from 26 to 29 weeks and their weights were only g (Table 1); both of these are risk factors for the acquisition of hospital-associated infections. Three K. pneumoniae isolates designated KpE1, KpE2 and KpE3 were recovered from environmental samples, comprising two from the fingers of two different nurses and one from an incubator in the NICU. part from multidrug-resistant K. pneumoniae, other multidrug-resistant pathogens, such as meticillin-resistant Staphylococcus aureus, ESBL-producing Enterobacteriaceae, vancomycin-resistant enterococci and multidrug-resistant cinetobacter baumannii, were not isolated from the clinical specimens including stools of the four investigated patients. Each of the four clinical isolates and the three environmental isolates was susceptible to amikacin but resistant to cefazolin, ceftazidime, cefotaxime, cefepime, aztreonam, cefoxitin, gentamicin, tetracycline, chloramphenicol and trimethoprim/sulfamethoxazole (Table 1) as determined by a disc diffusion test according to the criteria of the CLSI (2011). part from KpSX1, which exhibited no zones of inhibition, all tested isolates exhibited similar zones of inhibition and MIC values for IMP and MEM (Table 1). ccording to the interpretive standards for IMP and MEM for Enterobacteriaceae recommended by the CLSI (2011), all isolates were resistant to IMP and MEM. modified Hodge test was positive for all tested isolates, indicating that these isolates produced carbapenemases. However, all tested isolates were negative for MBLs determined by a double-disc synergy test and negative for ESBLs determined by the CLSI-recommended double-disc test (CLSI, 2011). Prior to the recovery of multidrug-resistant K. pneumoniae isolates, all four patients were treated with intravenous mezlocillin (75 mg kg 21 every 12 h) plus cefmetazole (50 mg kg 21 every 12 h) for preventing infections, and patient 1 was treated additionally with intravenous panipenem (20 mg kg 21 every 12 h) after the suspected lung infection was found. Treatment with intravenous cephalosporins or carbepenems may facilitate increased colonization by resistant K. pneumoniae, which can subsequently cause infection. fter multidrug-resistant K. pneumoniae isolates were identified, patient 1 was treated with intravenous amikacin (7.5 mg kg 21 every 24 h) and levofloxacin (10 mg kg 21 every 24 h) for 10 days according to the results of antimicrobial susceptibility testing. Thereafter, multidrugresistant K. pneumoniae was not isolated and the symptoms of lung infection disappeared in patient 1, who left the hospital on day 46. lthough the isolates KpSX2, KpSX3 and KpSX4 exhibited low-level resistance to imipenem, patients 2, 3 and 4 were treated with intravenous imipenem (20 mg kg 21 every 12 h) for days and left the hospital on days 44, 47 and 40, respectively, after multidrug-resistant K. pneumoniae was no longer isolated and the symptoms of lung infection had disappeared. ll tested isolates harboured int1, bla SHV, bla TEM, bla IMP, bla DH, bla CTX-M and qnrb4 genes detected by PCR. dditional sequencing of the amplified PCR products revealed the presence of bla SHV-11, bla TEM-1, bla IMP-4, bla DH-1, bla CTX-M-14 and qnrb4 among these isolates. Therefore, coexistence of multiple b-lactamase genes within each individual isolate could explain the resistance of these isolates to all b-lactams tested. To the best of our knowledge, this is the first report of the co-existence of bla IMP-4 and bla DH-1 in the same K. pneumoniae isolate. Detection of multiple b-lactamases produced by members of the Enterobacteriaceae in the clinical laboratory is challenging. The lack of ESBLs in the present study could be attributable to the masking effect of co-production of ESBLs, mpcs and carbapenemases. For example, co-production of KPCs and MBLs masked the results of EDT- or boronic acid-based testing in one study (Zioga et al., 2010). Some carbapenemase-producing members of the Enterobacteriaceae also exhibit low-level resistance or even susceptibility to carbapenems (Espedido et al., 2005; Liu et al., 2009). The decreased susceptibility of our isolates to ciprofloxacin could be explained by the existence of qnrb4. b-lactam resistance could be transferred by conjugation from all K. pneumoniae isolates to their recipients. ll transconjugants harboured int1, bla TEM-1, bla IMP-4, bla DH-1, bla CTX-M-14 and qnrb4 but not bla SHV-11. The E. coli transconjugants also exhibited relatively low MICs for IMP and MEM that were insufficient to explain the carbapenem resistance exhibited by the parental isolates. Therefore, we sought to identify additional mechanisms for carbepenem resistance in these isolates. IMP-4 MBL production combined with loss of outermembrane proteins confers high-level resistance to carbapenems in Klebsiella oxytoca (Chen et al., 2009). Therefore, we sought to identify resistance determinants within the ompk35 and ompk36 genes for our K. pneumoniae isolates. The nucleotide sequences of the ompk35 genes of all seven isolates were identical to that of carbapenem-susceptible K. pneumoniae TCC CT mutation was observed at nt 160 in the ompk36 gene for isolate KpSX1, resulting in initiation of a stop codon at position 54 (CGTG) for this strain. n early termination of translation caused by the CT mutation at nt 160 in the ompk36 gene might lead to the loss of OmpK36. These data indicated that highlevel resistance of KpSX1 to IMP and MEM may be due to the combination of IMP-4 and deficiency of the porin. We found that bla IMP-4 was located within a class I integron whose order was int1-bla IMP-4 -orfii-orfiii-qaced1-sul Journal of Medical Microbiology 61

4 987 Table 1. Phenotypic and genotypic characteristics of the K. pneumoniae clinical and environmental isolates Strain Patient/ sample BW (g)* G (weeks)d Disc diffusion zone (mm) MIC (mg l 1 ) ntimicrobial resistanced PFGE Resistance gene int1 ntimicrobials for treatment IMP MEM IMP MEM bla qnr KpSX1 Patient CTX, CZ, C, SXT, TE, TM, CZ, KpSX2 Patient CTX, CZ, C, SXT, TE, TM, CEC, KpSX3 Patient CTX, CZ, C, SXT, TE, TM, CZ, KpSX4 Patient CTX, CZ, C, SXT, TE, TM, CZ, KpE1 Fingers CTX, CZ, C, SXT, TE, TM, CZ, KpE2 Fingers CTX, CZ, C, SXT, TE, TM, CZ, KpE3 Incubator CTX, CZ, C, SXT, TE, TM, CZ, B B4 + MEZ, CMZ, CZ, MC, PP, MK, LEV B4 + MEZ, CMZ, CZ, MC, PP, IMP B4 + MEZ, CMZ, CZ, IMP B4 + MEZ, CMZ, IMP *BW, Birth weight of patient. DG, Gestational age of patient. dtm, ztreonam; C, chloramphenicol; CZ, ceftazidime; CEC, cefaclor; CTX, cefotaxime; CZ, cefazolin; FEP, cefepime; FOX, cefoxitin; GEN, gentamicin; IMP, imipenem; MEM, meropenem; SXT, trimethoprim/sulfamethoxazole; TE, tetracycline. +, Positive. MC, moxicillin plus clavulanic acid; MK, amikacin; CMZ, cefmetazole; LEV, levofloxacin; MEZ, mezlocillin; PP, panipenem. B4 + B4 + B4 + NICU outbreak of carbapenemase-producing K. pneumoniae

5 F. Yu and others The flanking structure of bla IMP-4 was therefore identical to the nucleotide sequence of a class I integron harbouring bla IMP-4 (GenBank accession no. FJ384365) described previously in Shanghai near to Shaoxing, China (Liu et al., 2009). The genetic relatedness of all K. pneumoniae isolates was also evaluated using PFGE. These results showed that two different band patterns, designated types and B, were identified for these isolates. PFGE type accounted for three clinical isolates (KpSX1, KpSX2 and KpSX3) and all three environmental isolates, whilst KpSX4 exhibited PFGE type B (Fig. 1). These data indicated that, with the exception of KpSX4, all of the isolates recovered in this study were closely related. Our data complement those reported in recent outbreaks caused by K. pneumoniae producing carbapenemases and other b-lactamases as described elsewhere (Kassis-Chikhani et al., 2010; Souli et al., 2010; Steinmann et al., 2011). Ultimately, all four patients were isolated in single-bed rooms, where strict contact precautions were implemented. ll NICU personnel were provided with additional training regarding standard precautions for prevention of infections in this setting, including appropriate hand hygiene and meticulous environmental cleaning. In addition, incubators, telephones, personal computers and door handles were cleaned with approved environmental disinfectants. Since the implementation of these procedures, multidrugresistant K. pneumoniae isolates have not been isolated from patients or environmental sources in this NICU. We recommend early implementation of outbreak investigations with identification of two or more clinical cases of multidrug-resistant K. pneumoniae in the NICU setting in order to prevent additional cases and morbidity associated with these clinically challenging infections. 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