Journal of Medical Microbiology (2012), 61, 984–989 Outbreak of pulmonary infection caused byKlebsiella pneumoniae isolates harbouring blaIMP-4and blaDHA-1 in a neonatal intensive care unit inChina Fangyou Yu,1 Qunhua Ying,2 Chun Chen,3 Tingjian Li,3 Baixing Ding,4Ying Liu,3 Yuanyuan Lu,3 Zhiqiang Qin,5 Chris Parsons,5Cassandra Salgado,5 Di Qu,6 Jingye Pan4 and Liangxing Wang3 1Department of Laboratory Medicine, The First Affiliated Hospital of Wenzhou Medical College, 2Department of Laboratory Medicine, Shaoxing Municipal Women and Children Hospital, 3Department of Respiratory Medicine, The First Affiliated Hospital of Wenzhou Medical College, 4Department of Intensive Care Unit, The First Affiliated Hospital of Wenzhou Medical College, 5Division of Infectious Diseases, Department of Medicine, Medical University of South Carolina, 6Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Institute of Medical Microbiology and Institutes of Biomedical Sciences, Shanghai Medical School of FudanUniversity, Shanghai 200032, PR China 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 fourcases, multidrug-resistant K. pneumoniae was isolated from multiple respiratory specimens,leading to additional characterization of these organisms and investigation of the localenvironment in the NICU. Multiple b-lactamase genes, including blaTEM-1, blaIMP-4, blaDHA-1 andblaCTX-M-14, as well as the quinolone resistance gene qnrB4, were harboured by transferableplasmids from all four clinical isolates. Furthermore, PFGE confirmed that three of the four clinicalisolates from the patients and three K. pneumoniae isolates collected from the hands of health-care workers and an incubator in the NICU belonged to the same PFGE cluster, indicating that anoutbreak due to multidrug-resistant K. pneumoniae carrying blaIMP-4 and blaDHA-1 occurred in thisNICU. As far as is known, this is the first report of the co-existence of blaIMP-4 and blaDHA-1 in thesame K. pneumoniae isolate. These data suggest that additional precautions are needed to prevent outbreaks of infection caused by multidrug-resistant K. pneumoniae resulting from ). Carbapenems or fluoroquino-lones are often used for the treatment of clinical infections Klebsiella pneumoniae frequently exhibits resistance to caused by this organism. However, resistance of K. pneumo- extended-spectrum cephalosporins due to the production of extended-spectrum b-lactamases (ESBLs) ), and this is often due to theproduction of carbapenemases, particularly K. pneumoniae Abbreviations: CLSI, Clinical and Laboratory Standards Institute; ESBL, carbapenemases (KPCs) and class B metallo-b-lactamases carbapenemase; MBL, class B metallo-b-lactamase; NICU, neonatal NICU outbreak of carbapenemase-producing K. pneumoniae been found in members of the Enterobacteriaceae worldwide Antimicrobial susceptibility testing. Antimicrobial susceptibilities were determined initially using GNS cards of the Vitek system to the family Enterobacteriaceae, KPCs have emerged in non- (bioMe´rieux). Multidrug resistance profiles were then furtherevaluated 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), dominant carbapenemase and has been recognized in many meropenem (10 mg), chloramphenicol (30 mg), tetracycline (30 mg), members of the Enterobacteriaceae, such as K. pneumoniae, trimethoprim/sulfamethoxazole (1.25/23.75 mg), amikacin (30 mg), Serratia marcescens, Escherichia coli and Enterobacter cloacae gentamicin (10 mg), ciprofloxacin (5 mg) and levofloxacin (5 mg).
An agar dilution method was used to determine MIC values including IMP and VIM, are commonly harboured by according to the criteria recommended by the Clinical and non-fermentative bacteria and have recently been iden- Laboratory Standards Institute E. coli ATCC 25922was used as a quality control strain for antimicrobial susceptibility tified in members of the Enterobacteriaceae worldwide (; ). MBLs often confer high-level resistance to all b- Detection of b-lactamases. A modified Hodge test was performed lactams except aztreonam and are not inhibited by to detect carbapenemases, as described previously A clavulanic acid, tazobactam or sulbactam. Although double-disc synergy test was designed to detect MBLs, as described by several IMP- and VIM-type MBLs have been described All the isolates studied were tested for ESBL in China, most were found in non-fermentative Gram- production by the CLSI-recommended confirmatory double-disccombination test negative bacilli (. Recent data indicatethat IMP-4 confers reduced susceptibility to carbapenems Detection of antimicrobial resistance determinants. Total DNA for K. pneumoniae isolated from patients in China ( was extracted by boiling. Potential antimicrobial resistance determi- nants, which included carbapenemase genes, ESBL genes, plasmid- clinical strains of K. pneumoniae carrying plasmid-borne borne ampC genes and plasmid-borne quinolone resistance determi- nants, were investigated by PCR and nucleotide sequencing, using IMP-4, blaSHV-12 and armA were found at a paediatric gene was also found to co-exist with blaKPC-2 in K.
ompK36 genes were determined by PCR and DNA sequencing with primers ompK35-F (59-ATGATGAAGCGCAATATTCTGGCAGTGG-39), ompK35-R (59-TCGGCTTTGTCGCCATTGCCGTCA-39), ompK36-F producing IMP-4 found in China were clonally unrelated (59-ATGAAAGTTAAAGTACTGTCCCTC-39) and ompK36-R (59- and occurred sporadically. An outbreak caused by multi- GTCGTCGGTAGAGATACCGGC-39). All amplicon sequences were drug-resistant K. pneumoniae harbouring bla compared with the sequences available in GenBank (http://www.
been found previously in China. In this report, we describe an outbreak of multidrug-resistant K. pneumo- Transfer of carbapenem resistance determinants. In order to niae infection in a neonatal intensive care unit (NICU) in determine whether carbapenem resistance was transferable in K.
China, as well as isolation of related organisms from pneumoniae isolates, a conjugation experiment was carried out in Luria–Bertani broth with E. coli J53 as the recipient, as describedpreviously Transconjugants were selected ontryptic soy agar plates containing sodium azide (100 mg ml21) for counterselection and imipenem (0.5 mg ml21) for plasmid-mediatedcarbapenem resistance selection.
Isolation and identification of bacterial strains. From October toDecember 2010, four neonates with asphyxia were admitted to the Determination of the flanking regions of the blaIMP-4 gene.
NICU of the 400-bed Shaoxing Municipal Women and Children Plasmid DNA of the transconjugants was extracted using a Plasmid Hospital in Shaoxing, eastern China. Of note, hospital admission for Midi kit (Qiagen) according to the manufacturer’s instructions.
all four patients (designated patients 1–4) overlapped. Before Purified blaIMP-bearing conjugative plasmids extracted from the admission to the NICU, pulmonary infections diagnosed by physical transconjugants were sequenced directly using a series of outward- examination and new findings consistent with pneumonia on chest directed primers specific for the locations next to the blaIMP-4 radiography were not found among the four patients. Bacterial isolates from sputum specimens growing over more than three-quarters of the plate by quantitative culture were considered to be PFGE. Genomic DNA was prepared from all tested K. pneumoniae responsible for the pulmonary infection. Bacterial isolates were isolates and cleaved with 40 U XbaI. Electrophoresis was performed identified by a Vitek-32 microbiology analyser (bioMe´rieux) accord- on 1 % agarose gels in 0.5 M Tris/borate/EDTA buffer on a CHEF- ing to the manufacturer’s instructions and additional biochemical Mapper XA PFGE system (Bio-Rad) for 24 h at 14 uC, with run tests. The initial K. pneumoniae isolates were screened for further conditions of 6 V cm21, a pulse angle of 120u and pulse times of investigation. After K. pneumoniae was isolated from patient 4, an 5–20 s. A l DNA ladder (Amersham Biosciences) was used as a outbreak control team was organized and infection control measures molecular mass marker and DNA bands were stained with were implemented. Environmental samples were obtained for culture ethidium bromide (0.5 mg ml21) prior to identification by by rubbing sterile polyester-fibre-tipped swabs moistened with sterile photography under UV light. Band profiles were interpreted by saline repeatedly over designated sites in the immediate vicinity of the the criteria of Patterns with a difference of no patients, including equipment used for their care and the fingers of more than three DNA bands were considered to belong to the same medical staff caring for the patients.
hospital on day 46. Although the isolates KpSX2, KpSX3and KpSX4 exhibited low-level resistance to imipenem, A suspected lung infection was diagnosed in patient 1 (aged patients 2, 3 and 4 were treated with intravenous imipenem 16 days), patient 2 (aged 17 days), patient 3 (aged 22 days) (20 mg kg21 every 12 h) for 10–14 days and left the and patient 4 (aged 14 days). K. pneumoniae isolates hospital on days 44, 47 and 40, respectively, after multi- designated KpSX1– KpSX4 were first recovered from drug-resistant K. pneumoniae was no longer isolated and sputum specimens of patients 1–4 following hospitalization the symptoms of lung infection had disappeared.
for 22, 22, 32 and 16 days, respectively. Subsequently, K.
pneumoniae isolates with identical antimicrobial resistance All tested isolates harboured int1, blaSHV, blaTEM, blaIMP, patterns were isolated from the sputum of each patient more blaDHA, blaCTX-M and qnrB4 genes detected by PCR. Addi- than three times. The gestational ages of the four patients tional sequencing of the amplified PCR products revealed ranged from 26 to 29 weeks and their weights were only the presence of blaSHV-11, blaTEM-1, blaIMP-4, blaDHA-1, 1050–1370 g both of these are risk factors for the blaCTX-M-14 and qnrB4 among these isolates. Therefore, co- acquisition of hospital-associated infections. Three K.
existence of multiple b-lactamase genes within each pneumoniae isolates designated KpE1, KpE2 and KpE3 were individual isolate could explain the resistance of these recovered from environmental samples, comprising two isolates to all b-lactams tested. To the best of our from the fingers of two different nurses and one from an knowledge, this is the first report of the co-existence of blaIMP-4 and blaDHA-1 in the same K. pneumoniae isolate.
Apart from multidrug-resistant K. pneumoniae, other Detection of multiple b-lactamases produced by members of multidrug-resistant pathogens, such as meticillin-resistant the Enterobacteriaceae in the clinical laboratory is challeng- Staphylococcus aureus, ESBL-producing Enterobacteriaceae, ing. The lack of ESBLs in the present study could be vancomycin-resistant enterococci and multidrug-resistant attributable to the masking effect of co-production of ESBLs, Acinetobacter baumannii, were not isolated from the AmpCs and carbapenemases. For example, co-production of clinical specimens including stools of the four investigated KPCs and MBLs masked the results of EDTA- or boronic patients. Each of the four clinical isolates and the three environmental isolates was susceptible to amikacin but carbapenemase-producing members of the Enterobacteria- resistant to cefazolin, ceftazidime, cefotaxime, cefepime, ceae also exhibit low-level resistance or even susceptibility to aztreonam, cefoxitin, gentamicin, tetracycline, chlor- amphenicol and trimethoprim/sulfamethoxazole decreased susceptibility of our isolates to ciprofloxacin could as determined by a disc diffusion test according to the be explained by the existence of qnrB4.
criteria of the Apart from KpSX1, whichexhibited no zones of inhibition, all tested isolates b-Lactam resistance could be transferred by conjugation exhibited similar zones of inhibition and MIC values for from all K. pneumoniae isolates to their recipients. All trans- IMP and MEM (According to the interpretive conjugants harboured int1, blaTEM-1, blaIMP-4, blaDHA-1, standards for IMP and MEM for Enterobacteriaceae blaCTX-M-14 and qnrB4 but not blaSHV-11. The E. coli transconjugants also exhibited relatively low MICs for IMP resistant to IMP and MEM. A modified Hodge test was and MEM that were insufficient to explain the carbapenem positive for all tested isolates, indicating that these isolates resistance exhibited by the parental isolates. Therefore, we produced carbapenemases. However, all tested isolates sought to identify additional mechanisms for carbepenem were negative for MBLs determined by a double-disc synergy test and negative for ESBLs determined by the IMP-4 MBL production combined with loss of outer- CLSI-recommended double-disc test ). Prior to membrane proteins confers high-level resistance to carba- the recovery of multidrug-resistant K. pneumoniae isolates, all four patients were treated with intravenous mezlocillin we sought to identify resistance determinants within the (75 mg kg21 every 12 h) plus cefmetazole (50 mg kg21 ompK35 and ompK36 genes for our K. pneumoniae isolates.
every 12 h) for preventing infections, and patient 1 was The nucleotide sequences of the ompK35 genes of all seven treated additionally with intravenous panipenem (20 mg isolates were identical to that of carbapenem-susceptible K.
kg21 every 12 h) after the suspected lung infection was pneumoniae ATCC 13883. A CAT mutation was observed found. Treatment with intravenous cephalosporins or car- at nt 160 in the ompK36 gene for isolate KpSX1, resulting bepenems may facilitate increased colonization by resistant in initiation of a stop codon at position 54 (CAGATAG) K. pneumoniae, which can subsequently cause infection.
for this strain. An early termination of translation caused After multidrug-resistant K. pneumoniae isolates were by the CAT mutation at nt 160 in the ompK36 gene might identified, patient 1 was treated with intravenous amikacin lead to the loss of OmpK36. These data indicated that high- (7.5 mg kg21 every 24 h) and levofloxacin (10 mg kg21 level resistance of KpSX1 to IMP and MEM may be due to every 24 h) for 10 days according to the results of the combination of IMP-4 and deficiency of the porin.
antimicrobial susceptibility testing. Thereafter, multidrug-resistant K. pneumoniae was not isolated and the symptoms We found that blaIMP-4 was located within a class I inte- of lung infection disappeared in patient 1, who left the gron whose order was int1-blaIMP-4-orfII-orfIII-qacED1-sul1.
Table 1. Phenotypic and genotypic characteristics of the K. pneumoniae clinical and environmental isolates DGA, Gestational age of patient.
dATM, Aztreonam; C, chloramphenicol; CAZ, ceftazidime; CEC, cefaclor; CTX, cefotaxime; CZ, cefazolin; FEP, cefepime; FOX, cefoxitin; GEN, gentamicin; IMP, imipenem; MEM, meropenem;SXT, trimethoprim/sulfamethoxazole; TE, tetracycline.
§+, Positive.
||AMC, Amoxicillin plus clavulanic acid; AMK, amikacin; CMZ, cefmetazole; LEV, levofloxacin; MEZ, mezlocillin; PAP, panipenem.
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