Abstract
The emergence and spread of bacteria carrying the blaNDM-1 gene has become a worldwide concern. Here, we report eight cases of Klebsiella pneumoniae with blaNDM-1 in the neonatal ward of a teaching hospital in mainland China. Multilocus sequence typing showed that seven isolates were clonally related and confirmed them as sequence type 17 (ST17). One isolate belonged to ST433. These findings suggest continuous spread of blaNDM-1 in mainland China and emphasize the need for intensive surveillance and precautions.
TEXT
The emergence of carbapenem-resistant Gram-negative bacteria is a global threat. Antibiotics of the carbapenem family have been a mainstay for the treatment of antibiotic-resistant bacterial infections (1). Carbapenems can be hydrolyzed by bacterial enzymes, e.g., the New Delhi metallo-beta-lactamase NDM-1. NDM-1 makes bacteria resistant to a broad range of beta-lactam antibiotics, including those of the carbapenem family (2). NDM-1 was first detected in Escherichia coli and Klebsiella pneumoniae isolated from a Swedish patient of Indian origin in 2008 (3). Since then, infections associated with NDM-1-positive strains have been reported worldwide, including in India, the United Kingdom, the United States, Canada, Australia, France, Holland, China, Pakistan, Italy, Japan, and Spain (4). The majority of these reported cases were strains isolated from adult patients (5). Here, we report an outbreak of NDM-1-producing K. pneumoniae in the neonatal ward of a tertiary teaching hospital in mainland China.
In August 2012, a premature neonate was admitted to our hospital due to poor response after vaginal delivery. The patient developed neonatal sepsis and necrotizing enterocolitis. A carbapenem-resistant K. pneumoniae strain was isolated from the blood culture. There was no documented clinical history of the neonate's parents having a link to an area where NDM-1 is endemic. However, his mother suffered from acute appendicitis in the sixth month of pregnancy and was hospitalized in another hospital in Hunan. The neonate received a 5-day course of meropenem and a 3-day course of ciprofloxacin; however, no adequate clinical response was noted. His parents requested discharge from the hospital against medical advice, and the neonate subsequently died (case 1). In September 2012, another neonate had recurrent fever with cytomegalovirus infection and was also positive for a carbapenem-resistant K. pneumoniae isolate in blood culture. Ceftazidime was given for 10 days, after which the patient recovered (case 2). Successively, four cases were identified. In February 2013, there was also a preterm neonate who had decreased response after birth and displayed severe dyspnea. The patient was initially treated with mezlocillin-sulbactam. An isolate of K. pneumoniae was detected from the sputum. A combination of meropenem and cefoperazone-sulbactam was given for 10 days, and then the patient recovered and was discharged (case 7). By March 2013, eight cases had occurred in the neonatal ward in total. The carbapenem-resistant K. pneumoniae isolates were detected from blood and sputum samples individually. The clinical profiles of all patients are shown in Table 1.
TABLE 1.
Clinical characteristics of the neonates in the study
Case | Sexa | Pregnancy duration (wk) | Dates of hospital stay | Date isolate identified | Type(s) of infection | Antimicrobial therapyb | Clinical outcome |
---|---|---|---|---|---|---|---|
1 | M | 31 | 30 Aug–7 Sep 2012 | 1 Sep 2012 | Neonatal sepsis, necrotizing enterocolitis | MEM+CIP | Death |
2 | F | 39 | 9 Sep–11 Oct 2012 | 16 Sep 2012 | Neonatal sepsis, neonatal pneumonia | CAZ | Improvement |
3 | M | 30 | 23 Oct–28 Nov 2012 | 29 Oct 2012 | Neonatal respiratory distress syndrome, neonatal pneumonia | MEM | Improvement |
4 | M | 33 | 26 Oct–11 Nov 2012 | 7 Nov 2012 | Neonatal sepsis; neonatal pneumonia | MEM | Improvement |
5 | M | 28 | 29 Oct–6 Nov 2012 | 5 Nov 2012 | Neonatal respiratory distress syndrome, neonatal pneumonia | PIP-TZB+CAZ | Poor prognosis |
6 | M | 31 | 14 Nov 2012–12 Jan 2013 | 22 Dec 2012 | Neonatal sepsis, neonatal pneumonia | MEM | Improvement |
7 | F | 28 | 1 Feb–22 Mar 2013 | 18 Feb 2013 | Neonatal sepsis, neonatal pneumonia | MEM+CFP-TZB | Improvement |
8 | F | 33 | 1 Mar–14 Mar 2013 | 15 Mar 2013 | Neonatal pneumonia | CAZ | Improvement |
M, male; F, female.
MEM, meropenem; CIP, ciprofloxacin; CAZ, ceftazidime; PIP-TZB, piperacillin-sulbactam sodium; CFP-TZB, cefoperazone-sulbactam sodium.
All isolates were identified as K. pneumoniae by using the BD Phoenix automated microbiology system. Routine determination of antimicrobial susceptibilities was performed using the disk diffusion agar method according to the CLSI standards (6). It showed that all the isolates were resistant to beta-lactams, including carbapenems, but were susceptible to quinolones and aminoglycosides (Table 2). Production of carbapenemases was detected by the modified Hodge test (6), and carbapenemase production by all isolates was confirmed. Detection of blaNDM-1 was performed by PCR with designed primers NDM-1-F (5′-GGAAAACTTGATGGA-3′) and NDM-1-R (5′-TAAAACGCCTCTGTC-3′). The PCR products were sequenced and showed 100% identity with blaNDM-1 of K. pneumoniae that is deposited in GenBank with the accession number FN396876.1 (3). To clarify the mechanisms of carbapenem resistance, PCR screening and sequencing for other beta-lactamase genes (blaSHV, blaIMP, blaVIM, blaKPC-2, blaTEM-1, blaCTX-M-14, blaCTX-M-15, blaCMY-4, blaCMY-8, blaOXA-1, blaOXA-2, blaOXA-9, blaOXA-48, and blaOXA-181), fluoroquinolone resistance genes (qnrA, qnrB1, and qnrS), and aminoglycoside resistance genes [aac(3′), aac(6′), aph(3′), and armA] were performed (7, 8). The results revealed the presence of blaTEM-1 in addition to blaNDM-1 in all isolates. Seven isolates coharbored blaSHV-1, blaCMY-4, blaCTX-M-15, and qnrS. In isolate 7, only blaOXA-2 was detected in addition to blaNDM-1.
TABLE 2.
Antimicrobial susceptibilities of the eight NDM-producing K. pneumoniae isolates
Case | Sample type | ST | Resistance mechanisms | MIC(s) (mg/liter) ofa: |
||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
AMC | SAM | PIP | CAZ | CTX | CRO | FEP | IPM | MEM | LVX | CIP | AMK | GEN | ||||
1 | Blood | 17 | NDM-1, TEM-1, CTX-M-15, CMY-4, SHV-1, qnrS | >16/8 | >16/8 | >64 | >16 | >32 | >32 | >16 | ≥8 | >8 | ≤2 | ≤1 | ≤16 | ≤1 |
2 | Blood | 17 | NDM-1, TEM-1, CTX-M-15, CMY-4, SHV-1, qnrS | >16/8 | >16/8 | >64 | >16 | >32 | >32 | >16 | ≥8 | >8 | ≤2 | ≤1 | ≤16 | ≤1 |
3 | Sputum | 17 | NDM-1, TEM-1, CTX-M-15, CMY-4, SHV-1, qnrS | >16/8 | >16/8 | >64 | >16 | >32 | >32 | >16 | ≥8 | >8 | ≤2 | ≤1 | ≤16 | ≤1 |
4 | Sputum | 17 | NDM-1, TEM-1, CTX-M-15, CMY-4, SHV-1, qnrS | >16/8 | >16/8 | >64 | >16 | >32 | >32 | >16 | ≥8 | >8 | ≤2 | ≤1 | ≤16 | ≤1 |
5 | Blood | 17 | NDM-1, TEM-1, CTX-M-15, CMY-4, SHV-1, qnrS | >16/8 | >16/8 | >64 | >16 | >32 | >32 | >16 | ≥8 | >8 | ≤2 | ≤1 | ≤16 | ≤1 |
6 | Sputum | 17 | NDM-1, TEM-1, CTX-M-15, CMY-4, SHV-1, qnrS | >16/8 | >16/8 | >64 | >16 | >32 | >32 | >16 | ≥8 | >8 | ≤2 | ≤1 | ≤16 | ≤1 |
7 | Sputum | 433 | NDM-1, TEM-1, OXA-2 | >16/8 | >16/8 | ≤16 | >16 | >32 | >32 | >16 | 4 | 8 | ≤2 | ≤1 | ≤16 | ≤1 |
8 | Sputum | 17 | NDM-1, TEM-1, CTX-M-15, CMY-4, SHV-1, qnrS | >16/8 | >16/8 | >64 | >16 | >32 | >32 | >16 | ≥8 | >8 | ≤2 | ≤1 | ≤16 | ≤1 |
AMC, amoxicillin-clavulanic acid; SAM, ampicillin-sulbactam; PIP, piperacillin; CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxon; FEP, cefepime; IPM, imipenem; MEM, meropenem; LE, levofloxacin; CI, ciprofloxacin; AM, amikacin; GE, gentamicin.
Random amplified polymorphic DNA (RAPD) analysis (9) showed that seven of the K. pneumoniae strains belonged to the same clone. The clonal relationship was further analyzed using multiple locus sequence typing (MLST) according to protocols provided on the MLST websites (http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html). Seven of the K. pneumoniae isolates were defined as sequence type 17 (ST17), while one belonged to ST433. Neither ST belonged to the most common K. pneumoniae STs (ST14 and ST11) that are reported to harbor NDM-1 (10). Only one case of NDM-1-positive ST17 has been confirmed, in Guatemala (11).
The transferability of blaNDM-1 associated with plasmids was confirmed by broth mating conjugation assays, using E. coli K12J53 as the recipient strain. Transconjugants were selected on MacConkey agar plates containing sodium azide (100 μg/ml) and meropenem (0.25 μg/ml). Transconjugants revealed resistance to all beta-lactams and carbapenems. PCR and sequencing for transconjugants further confirmed the successful transfer of blaNDM-1 in all the isolates and cotransfer of blaCTX-M-15 in four isolates. This transmissibility with plasmids implies an alarming potential for rapid spread to diverse bacteria.
This report describes a neonatal outbreak of NDM-1-producing K. pneumoniae in mainland China. Since seven isolates analyzed in this outbreak were of the same clonal type, the first neonate was likely the source of this outbreak. His mother had a history of hospitalization during pregnancy and had high risk factors for fetal intrauterine infection. Although this organism was not detected from the mother, we could assume the NDM-1-producing K. pneumoniae might potentially have been colonized in small numbers in the mother's body and infected the fetus through the placental circulation or through the birth canal. Then, the isolate was transmitted between patients and caused nosocomial infection in the ward. In China, various blaNDM-1-carrying strains of the Enterobacteriaceae have been sporadically identified. The first blaNDM-1-positive K. pneumoniae isolate in China was identified in Hunan Province in 2012 (12). Now, this study reports more isolates in the same area. This indicates that Hunan province may be a reservoir of blaNDM-1 that may be unrelated to other areas of NDM endemicity, e.g., India. Among the cases in this study, six patients responded to treatment with ceftazidime and meropenem, which seems inconsistent with the resistance characteristics of the isolates. We considered that five isolates from sputum might be normal flora colonizing the respiratory tract rather than the real pathogen of infection.
After March 2013, more cases occurred, not only in the neonatal ward but spreading to other departments, including the intensive care unit, neurology ward, and pediatrics ward. Additionally, the blaNDM-1 gene was identified in Escherichia coli, Enterobacter cloacae, and Citrobacter isolates. It is uncertain whether the latter NDM-1-producing isolates were related to the neonatal ward or were imported from the outside. There were reports on NDM-producing bacteria originating from food animals (13), which indicated a reservoir in the community and connection to animals in China. The progenitor of the NDM-1 remains undefined. In its worrisome situation, the hospital implemented transmission-based precautions and enhanced environmental cleaning in order to prevent expanded spread. A new study has found that the human gut microbiota is a reservoir of antibiotic resistance genes, but little is known about their diversity and richness within the gut (14). Further studies are needed to confirm whether NDM-1 is spread from bacteria in the human gut due to excessive use of antibiotics. These findings highlight that epidemiological surveillance of multidrug-resistant microorganisms is indispensable for implementing appropriate interventions.
ACKNOWLEDGMENTS
We thank L. Yang (The Second XiangYa Hospital of Central South University, Changsha, Hunan, China) for reading the manuscript and making numerous valuable comments.
This work was supported by grants 81470133 and 81301988 from the China National Natural Scientific Foundation and grant 201310533-058 from the China National Innovation Experiment Program for University Students.
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