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Beijing Children's Hospital, Capital Medical University, is a general pediatric teaching hospital with 970 beds. The hospital has more than 70, 000 admissions per year. All the patients were recruited as inpatients in Beijing Children's Hospital from January 2010 to December 2014.
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1, 730 strains of K. pneumoniae were isolated from nasopharyngeal secretions, tracheal aspirations, blood, pus secretions, mid-stream catheterized urine, pleural effusions, catheter and ascites. Routine methods and the Phoenix100 automated microbiology analyzer (BD Research Inc. USA) were employed to identify the bacterial strains.
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The CPM-non-susceptible isolates of K. pneumoniae were identified by disk diffusion method using imipenem or meropenem disks. Zone Diameter ≤ 22 mm was defined as non-susceptible to the drugs.
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Phenotypic test for the CPMase-producing strains was conducted by using the modified Hodge test and the double-disk synergy test (imipenem/ imipenem+EDTA and meropenem/meropenem+ EDTA).
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The Minimal Inhibition Concentration (MIC) values for 15 antibiotics were determined by a broth microdilution method (BD Research Inc. USA). The antibiotics were as follows: ampicillin, cefazolin, cefotaxime, ceftazidime, cefepime, aztreonam, amoxicillin-clavulanic, piperacillin-tazobactam, imipenem, meropenem, trimethoprim-sulfamethoxazole, gentamicin, amikacin, ciprofloxacin, and colistin. The interpretive break points of Clinical and Laboratory Standards Institute (CLSI) M100-S24 were used to interpret the MIC results for antimicrobial agents[14]. The Escherichia coli ATCC25922 and K. pneumoniae ATCC BAA-1705 strains were adopted as the standards for quality control (QC). WHONET 5.6 software (http://www.whonet.org/) was used to perform resistance analysis.
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To detect CPMase-producing genes in the CPM-non-susceptible strains, PCR amplification was performed on a PTC-100-type PCR instrument (MJ Research Inc. USA). The CPMase-producing genes included blaIMP, blaVIM, blaSPM, blaGIM, blaNDM-1, blaSIM, blaKPC, blaGES, blaIMI/NMC-A, blaSME, and blaOXA, and the primers were shown in Table 1 [12, 15-17]. The reaction system (total volume, 50 µL) consisted of: PCR Master Mix (2 × PCR buffer, 3 mmol/L Mg2+, 200 µmol/L dNTPs, and 0.1 U Taq DNA polymerase), 50 pmol each of the two primers, and DNA template obtained by heating a bacterial suspension. The reaction conditions were as follows: pre-denaturation at 95 ℃ for 3 min, followed by 35 amplification cycles of 95 ℃ for 1 min, 55 ℃ for 1 min and 72 ℃ for 1 min, with a final extension step of 72 ℃ for 5 min.
CPMase Sequence (5'to 3') Expected Size (bp) KPC15 P1: GCTACACCTAGCTCCACCTTC P2: GCATGGATTACCAACCACTGT 898 GES15 P1: ATGCGCTTCATTCACGCAC P2: CTATTTGTCCGTGCTCAGG 591 IMI/NMC-A15 P1: CAGAGCAAATGAACGATTTC P2: GGTACGCTAGCACGAATAC 879 SME15 P1: GTGTTTGTTTAGCTTTGTCGGC P2: GCAATACGTGATGCTTCCGC 642 IMP12 P1: GGAATAGAGTGGCTTAATTCTC P2: GTGATGCGTCYCCAAYTTCACT 361 VIM12 P1: CAGATTGCCGATGGTGTTTGG P2: AGGTGGGCCATTCAGCCAGA 523 GIM16 P1: TCGACACACCTTGGTCTGAA P2: AACTTCCAACTTTGCCATGC 477 SPM16 P1: AAAATCTGGGTACGCAAACG P2: ACATTATCCGCTGGAACAGG 271 SIM16 P1: TACAAGGGATTCGGCATCG P2: TAATGGCCTGTTCCCATGTG 570 NDM-112 P1: CAGCACACTTCCTATCTC P2: CCGCAACCATCCCCTCTT 292 OXA-51-like16 P1: TAATGCTTTGATCGGCCTTG P2: TGGATTGCACTTCATCTTGG 353 OXA-23-like16 P1: GATCGGATTGGAGAACC GA P2: ATTTCTGACCGCATTTCCAT 501 OXA-40-like16 P1: GGTTAGTTGGCCCCCTTA AA P2: AGTTGAGCGAAAAGGGGATT 246 OXA-58-like16 P1: AAGTATTGGGGCTTGTGCTG P2: CCCCTCTGCGCTCTACATAC 599 OXA-4817 P1: GCGTGGTTAAGGATGAACAC P2: CATCAAGTTCAACCCAACCG 438 OXA-18117 P1: ATGCGTGTATTAGCCTTATCG P2: AACTACAAGCGCATCGAGCA 888 Table 1. Primers used for PCR Amplification
The PCR products from the above reactions were purified and sequenced using the ABI PRISM TM377 with the dideoxy-mediated chain-termination method. Sequences obtained were compared with the preexisting sequences via NCBI BLAST to determine the type of CPMase. The nucleotide sequence containing the blaIMP-4, blaIMP-8, blaNDM-1, and blaKPC-2 open reading frame has been assigned the EMBO/GenBank accession number FJ384365.1, EU368856.1, and KP036457.1 and KR108243.1.
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SPSS 12.0 was used to perform regression analysis. Trends of resistance rates to each antimicrobial agent for microorganisms were performed using Chi-square statistics. A P < 0.05 was considered statistically significant.
Settings and Patients
Bacterial Strains
Screening of CPM Non-susceptible Strains
Phenotypic Analysis
Antimicrobial Susceptibility Testing
Identification of CPMase-producing Genes
Statistical Analysis
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During 2010-2014, 1, 730 non-duplicated strains of K. pneumoniae were isolated from different inpatients in our hospital. Among those strains, 179 (10.3%) were identified as non-susceptible (Zone Diameter ≤ 22 mm) to imipenem or meropenem via disk-diffusion method. The percentages of non-susceptible isolates were lower than 10% between 2010 and 2012, namely 7.8% (28/358) in 2010, 8.9% (37/415) in 2011, 7.4% (28/379) in 2012. However, it increased to 11.2% (40/356) in 2013 and further increased to 20.7% (46/222) in 2014.
The clinical features of CPM-non-susceptible K. pneumoniae isolates were shown in Table 2. The isolates were main obtained from Internal Medicine (39.7%, 71/179) and Haematology (29.6%, 53/179). Most of the patients were under 7 years old (76.0%, 136/179). The majority of the strains were isolated from tracheal aspirations (50.8%, 91/179) and blood (31.3%, 56/179). Notably, the isolates originated from blood increased from 14.2% to 41.3% during the study period.
Clinical Feature 2010(n = 28)n (%) 2011(n = 37)n (%) 2012(n = 28)n (%) 2013(n = 40)n (%) 2014(n = 46)n (%) Total(n= 179)n (%) Clinical units Internal medicine 11 (39.3) 24 (64.9) 8 (28.6) 14 (35.0) 14 (30.4) 71 (39.7) Haematology 6 (21.4) 9 (24.3) 8 (28.6) 15 (37.5) 15 (32.6) 53 (29.6) Intensive care unit 6 (21.4) 2 (5.4) 10 (35.6) 7 (17.5) 10 (21.8) 35 (19.6) Surgery 5 (17.9) 2 (5.4) 1 (3.6) 4 (10.0) 7 (15.2) 19 (10.6) Dermatology 0 0 1 (3.6) 0 0 1 (0.5) Gender Male 15 (53.6) 23 (62.6) 17 (60.7) 27 (67.5) 28 (60.9) 110 (61.5) Female 13 (46.4) 14 (37.4) 11 (39.3) 13 (32.5) 18 (39.1) 69 (38.5) Years < 1 m 2 (7.1) 5 (13.5) 6 (21.4) 3 (7.5) 2 (4.3) 18 (10.0) 1 m-1 y 12 (42.9) 16 (43.3) 8 (28.6) 17 (42.5) 18 (39.1) 71 (39.7) 1-6 y 9 (32.2) 8 (21.6) 5 (17.9) 8 (20.0) 17 (37.0) 47 (26.3) 7-12 y 2 (7.1) 4 (10.8) 9 (32.1) 8 (20.0) 8 (17.4) 31 (17.3) > 12 y 3 (10.7) 4 (10.8) 0 4 (10.0) 1 (2.2) 12 (6.7) Specimen site Tracheal aspirations 16 (57.2) 24 (64.9) 14 (50.0) 17 (42.5) 20 (43.5) 91 (50.8) Blood 4 (14.2) 7 (18.9) 10 (35.6) 16 (40.0) 19 (41.3) 56 (31.3) Midstream urine 4 (14.2) 4 (10.8) 1 (3.6) 2 (5.0) 3 (6.5) 14 (7.8) Pus secretions 1 (3.6) 1 (2.7) 1 (3.6) 4 (10.0) 1 (2.2) 8 (4.5) Nasopharyngeal secretions 1 (3.6) 1 (2.7) 0 1 (2.5) 3 (6.5) 6 (3.4) Catheter 2 (7.2) 0 1 (3.6) 0 0 3 (1.7) Ascites 0 0 1 (3.6) 0 0 1 (0.5) Table 2. Clinical Features of the Carbapenemase-producing K. pneumoniae Strains
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Phenotypic analysis based on modified Hodge test and double-disk synergy test showed that none of CPM-non-susceptible isolates of K. pneumoniae had negative results in both tests, indicating all of the 179 isolates were CPMase producers. Among them, 152 (84.9%) strains were positive in the modified Hodge test and 174 (97.2%) positive in the double-disk synergy test.
The results of the disk-diffusion experiments showed that 156 (87.1%) of K. pneumoniae isolates were meropenem resistant, 122 (68.1%) exhibited imipenem resistance, and 120 (67.0%) were resistant to both imipenem and meropenem.
The results of broth microdilution experiments conducted for all of the 179 isolates were shown in Table 3. The isolates demonstrated very high resistance rates against ampicillin (100%), cefazolin (100%), cefotaxime (100%), amoxicillin-clavulanic (100%), and ceftazidime (99.4%). There were only two drugs with susceptibility rates higher than 50%, namely amikacin (88.2%) and ciprofloxacin (65.9%). An increase of resistance was seen for imipenem and meropenem from 14.3% to 89.1% and from 67.9% to 100%, respectively. No isolate of K. pneumoniae was found resistant to colistin.
Antibiotics Total (n = 179) 2010 (n = 28) 2011 (n = 37) 2012 (n = 28) 2013 (n = 40) 2014 (n = 46) %R %I %R %I %R %I %R %I %R %I %R %I AM 100 0 100 0 100 0 100 0 100 0 100 0 CZ 100 0 100 0 100 0 100 0 100 0 100 0 CTX 100 0 100 0 100 0 100 0 100 0 100 0 CAZ 99.4 0.6 100 0 97.3 2.7 100 0 100 0 100 0 FEP 89.4 7.8 75.0 10.7 91.9 8.1 92.9 3.6 87.5 12.5 95.7 4.3 ATM 86.6 0 96.4 0 64.9 0 82.1 0 87.5 0 84.8 0 AMC 100 0 100 0 100 0 100 0 100 0 100 0 TZP 67.0 13.4 75.0 25.0 48.6 21.6 67.9 10.7 67.5 7.5 76.1 6.5 IMP 68.2 26.8 14.3 71.4 62.2 29.7 87.5 7.1 75.0 25.0 89.1 10.9 MEN 87.2 8.4 67.9 17.9 70.3 21.6 100 0 92.5 5.0 100 0 STX 76.0 0.6 75.0 0 75.7 0 85.7 3.6 82.5 0 65.2 0 GEN 68.7 1.1 50.0 0 70.3 2.7 75.0 3.6 65.0 0 67.4 0 ANK 10.1 1.7 7.1 3.6 18.9 5.4 10.7 0 25.0 0 10.9 0 CIP 20.7 13.4 28.6 0 25.0 5.6 25.0 28.6 15.0 25.0 15.2 8.7 CL 0 0 0 0 0 0 0 0 0 0 0 0 Note.AM, Ampicillin; CZ, Cefazolin; CTX, Cefotaxime; CAZ, Ceftazidime; FEP, Cefepime; ATM, Aztreonam; AMC, Amoxicillin-clavulanic; TZP, Piperacillin-Tazobactam; IMP, Imipenem; MEN, Meropenem; STX, Trimethoprim-sulfamethorazole; GEN, Gentamicin; ANK, Amikacin; CIP, Ciprofloxacin; CL, Colistin; R, resistant, I, intermediate. Table 3. Antimicrobial Resistance Profiles of K. pneumoniae Strains during the Study Period
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The PCR results showed that 166 (92.7%) strains harbored the CPMase-producing genes detected in this study. The blaIMP and blaNDM-1genes were present in 95 (57.2%) and 65 (39.2%) strains, respectively, and the other 6 (3.6%) strains had blaKPCgene.
Subsequent sequencing of the 166 PCR-positive strains further identified the genotypes of the respective CPMase genes. Among the 95 strains harboring the blaIMP gene, 92 (96.8%) and 3 (3.2%) strains were IMP-4 and IMP-8 producers, respectively. The sequencing results also confirmed that the 65 strains with PCR positive results of blaNDM-1 gene were NDM-1 producers. In addition, all sequenced KPC producers (6 strains) had blaKPC-2 gene.
During our study period from 2010 to 2014, the most predominant CPMase-producing isolates were IMP-4 producers (51.4%, 92/179). NDM-1 producer also occupied a very large percentage (36.3%, 65/179). Only 3.4% (6/179) and 1.7% (3/179) were IMP-8 and KPC-2 producers, respectively. The annual distributions revealed an increase of NDM-1 producers and decrease of IMP-4 producers from 2010 to 2014 (Table 4).
Genotype 2010 (n = 28)n(%) 2011 (n = 37)n(%) 2012 (n = 28)n(%) 2013 (n = 40)n(%) 2014 (n = 46)n (%) Total (n = 179)n(%) IMP-4 type 21 (75.0) 29 (78.4) 14 (50.0) 15 (37.5) 13 (28.3) 92 (51.4) IMP-8 type 3 (10.8) 0 0 0 0 3 (1.7) NDM-1 type 2 (7.1) 4 (10.8) 11 (39.3) 19 (47.5) 29 (63.0) 65 (36.3) KPC-2 -type 0 1 (2.7) 0 2 (5.0) 3 (6.5) 6 (3.4) Undetermined 2 (7.1) 3 (8.1) 3 (10.7) 4 (10.0) 1 (2.2) 13 (7.2) Table 4. Distribution of Different Genotypes from 2010 to 2014
The results of broth microdilution experiments indicated that different CPMase producers had different profiles of antimicrobial resistance (Table 5). Compare to IMP-4 producers, the isolates from KPC-2 producers had higher resistant rates frequencies in terms of resistance to imipenem (P = 0.017), amikacin (P = 0.010) and ciprofloxacin (P= 0.013). There were also significant differences in the frequencies of drug resistance between the isolates from NDM-1 producers and IMP-4 producers for piperacillin-tazobactam (P = 0.000), imipenem (P = 0.000) and meropenem (P = 0.000). The isolates from KPC-2 producers and NDM-1 producers had significantly higher rates of drug resistance to antibiotics than those from the IMP-4 producers (P < 0.05).
Antimicrobial Agent IMP-4 type (92) IMP-8 type (3) NDM-1 type (65) KPC type (6) Undetermined (13) %R %I %R %I %R %I %R %I %R %I AM 100 0 100 0 100 0 100 0 100 0 CZ 100 0 100 0 100 0 100 0 100 0 CTX 100 0 100 0 100 0 100 0 100 0 CAZ 98.9 1.1 100 0 100 0 100 0 100 0 FEP 82.6 13.0 66.7 33.3 100 0 100 0 84.6 7.7 ATM 84.8 0 100 0 89.2 0 100 0 69.2 0 AMC 100 0 100 0 100 0 100 0 100 0 TZP 46.7 18.5 100 0 89.2 7.7 83.3 16.7 76.9 7.7 IMP 50.0 42.4 0 66.7 96.9 3.1 100 0 53.9 38.4 MEN 78.3 16.3 66.7 0 100 0 100 0 84.6 0 STX 69.6 0 33.3 0 84.6 1.5 66.7 0 84.6 0 GEN 64.1 1.1 66.7 0 72.3 1.5 83.3 0 76.9 0 ANK 12.0 2.2 0 0 4.6 1.5 50 0 7.7 0 CIP 21.7 9.8 33.3 0 10.8 21.5 66.7 0 30.8 7.7 CL 0 0 0 0 0 0 0 0 0 0 Note.AM, Ampicillin; CZ, Cefazolin; CTX, Cefotaxime; CAZ, Ceftazidime; FEP, Cefepime; ATM, Aztreonam; AMC, Amoxicillin-clavulanic; TZP, Piperacillin-Tazobactam; IMP, Imipenem; MEN, Meropenem; STX, Trimethoprim-sulfamethorazole; GEN, Gentamicin; ANK, Amikacin; CIP, Ciprofloxacin; CL, Colistin. R, resistant, I, intermediate. Table 5. Antimicrobial Resistance Profiles of Different Genotypes of K. pneumoniae Strains