Characterization of Clinical Multidrug-Resistant Escherichia coli and Klebsiella pneumoniae Isolates,2007

Abstract

Various resistance mechanisms facilitate the emergence and spread of multidrug-resistance (MDR) phenotypes of Escherichia coli and Klebsiella pneumoniae. To elucidate the MDR mechanisms of E. coli and K. pneumoniae in China, we analyzed the antimicrobial susceptibilities of strains isolated from clinical samples in a large tertiary care hospital in Beijing, China, during 2007–2009 and characterized the isolates with a cefotaxime–ciprofloxacin–amikacin (CTX–CIP–AK) resistance pattern. In total, 98 and 52 clinical isolates of E. coli and K. pneumoniae, respectively, with a CTX–CIP–AK resistance pattern were subjected to antimicrobial susceptibility testing and screening of common β-lactamase genes, plasmid-mediated quinolone resistance (PMQR) genes, quinolone resistance-determining region (QRDR) substitutions, and 16S rRNA methylase genes by polymerase chain reaction amplification and DNA sequencing. Pulsed-field gel electrophoresis (PFGE) was used to determine the genetic relatedness of the isolates. Approximately 6.86% and 8.05% of the clinical E. coli and K. pneumoniae isolates, respectively, exhibited MDR phenotypes. The MDR K. pneumoniae isolates exhibited significantly higher ceftazidime resistance than the MDR E. coli isolates (90.4% vs. 76.5%, p=0.0339); a similar result was noted for piperacillin–tazobactam resistance (28.8% vs. 2%, p=0.0001). The common resistance determinants among the MDR E. coli and K. pneumoniae isolates were as follows: CTX-M (88.8% vs. 82.7%), PMQR genes (70.4% vs. 90.4%), gyrA mutations (100% vs. 90.4%), and 16S rRNA methylase genes (93.9% vs. 94.2%). Half (50%) of the MDR E. coli isolates belonged to phylogenetic group D, followed by group A (39.8%). For the E. coli isolates, 94 PFGE patterns and 23 clusters were identified, whereas 51 PFGE patterns and 11 clusters were identified for the K. pneumoniae isolates. Clinical E. coli and K. pneumoniae isolates seem to have a low prevalence of MDR phenotypes in China. The great genetic variation indicates a considerable transmission of common resistance determinants, including a high prevalence of QRDR substitutions in E. coli and K. pneumoniae.

Introduction

As the most prevalent enterobacterial pathogens, Escherichia coli and Klebsiella pneumoniae are responsible for various infections.¹⁰ Due to the extensive use of antimicrobial agents, strains of these enterobacteria isolated from clinical samples have been found to develop resistance to common antibiotics; some clinical isolates even presented a multidrug-resistant (MDR) phenotype (i.e., simultaneous resistance to various class of antibiotics, such as cephalosporins, fluoroquinolones, and aminoglycosides).^12,27,28,30 Consequently, other than carbapenems, the available therapeutic options for MDR E. coli and K. pneumoniae infections are limited.^11,12,27 Furthermore, once these organisms can produce carbapenemases, such as Klebsiella pneumoniae carbapenemase and New Delhi metallo-β-lactamase (NDM-1),²⁰ they will become pan-drug-resistant “superbugs” and pose a considerable threat to patient recovery and public health security. Therefore, surveillance of drug resistance and monitoring of the prevalence of resistance determinants are urgently required.

A growing body of literature evidences the mechanisms of drug resistance in clinical E. coli and K. pneumoniae isolates in China.^{16,17,28–30,32,33} Plasmid-mediated quinolone resistance (PMQR), β-lactamase, and 16S rRNA methylase genes have been identified as the genetic bases for bacterial drug resistance. However, little is known about the MDR mechanisms in clinical E. coli and K. pneumoniae strains in China. To address this issue, we analyzed the antimicrobial susceptibilities of E. coli and K. pneumoniae strains isolated from clinical samples in Beijing, China, during 2007–2009 and characterized the isolates with a cefotaxime–ciprofloxacin–amikacin (CTX–CIP–AK) resistance pattern. The prevalence of resistance determinants in these isolates was also investigated.

Materials and Methods

Bacterial isolates

In 2007–2009, 2,346 and 994 individual E. coli and K. pneumoniae strains, respectively, were isolated from clinical samples, including sputum, blood, urine, abscesses, secretions, drainage fluids, bile, wounds, and catheters, in a 4,000-bed tertiary care hospital. All isolates were identified by colony morphology, biochemical testing, and VITEK 2 GN ID cards (bioMérieux, Inc.). Of these, 161 E. coli and 80 K. pneumoniae isolates exhibited a CTX–CIP–AK resistance pattern, as indicated by antimicrobial susceptibility testing and the Clinical and Laboratory Standards Institute (CLSI) standards.⁶ In early 2011, 98 E. coli and 52 K. pneumoniae isolates with a CTX–CIP–AK resistance pattern were subjected to phenotypic and genotypic characterization. E. coli ATCC 25922 and ATCC 35218 were used as the quality control strains for antimicrobial susceptibility testing. The sodium azide-resistant E. coli J53 strain was used as the recipient for conjugation testing.

No formal ethical approval was obtained to use the clinical samples, because they were collected during routine bacteriologic analyses in public hospitals, and the data were anonymously analyzed.

Antimicrobial susceptibility testing

Antimicrobial susceptibilities of the clinical isolates were determined by measuring the diameters of the zones of complete inhibition in the disk diffusion method. The minimum inhibitory concentrations (MICs) for the isolates and transconjugants were measured by using the agar dilution method. All susceptibility results were interpreted according to the 2010 CLSI performance standards.⁶

Screening of resistance determinants

β-lactamase genes, including bla_TEM, bla_SHV, bla_CTX-M (groups 1, 2, 8, 9, and 25/26), bla_PER, bla_VEB, bla_GES, and plasmid-mediated bla_AmpC, and PMQR genes, including qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxAB, and aac(6′)-Ib-cr were screened by multiplex polymerase chain reaction (PCR) methods as previously described.^3,7,15,19 PCR amplification was performed as previously described to screen 16S rRNA methylase genes, including armA, rmtA, rmtB, rmtC, and rmtD.⁸ Further, PCR amplification and DNA sequencing were performed to identify gyrA and parC mutations.⁹ Amplicons were sequenced in a DNA analyzer (3730xl; Applied Biosystems, Life Technologies).

Phylogenetic analysis

The phylogeny of the E. coli isolates was determined on the basis of the presence or absence of chuA, yjaA, and TspE4.C2 by using a triplex PCR method.⁵

Conjugation testing

Conjugation experiments were carried out in Luria–Bertani (LB) broth. Cultures of donor and recipient cells in the logarithmic phase (0.5 ml of each) were added to 4 ml of fresh LB broth and incubated overnight without shaking. Transconjugants were selected on a China blue agar plate containing AK (30 μg/ml) and sodium azide (170 μg/ml; Sigma-Aldrich Co.).

Pulsed-field gel electrophoresis

DNA fingerprints were obtained from pulsed-field gel electrophoresis (PFGE) profiles of genomic DNA digested with XbaI (New England Biolabs) according to the procedures developed by the US Centers for Disease Control and Prevention PulseNet program.²¹ The PFGE patterns were interpreted with BioNumerics software (Applied Maths NV) by using the Dice similarity coefficient. A tree indicating relative genetic similarity was constructed on the basis of the unweighted pair-group method of averages with a position tolerance of 1%. Clusters were defined as DNA patterns sharing ≥70% similarity (C1, C2, C3, etc.). Differences in genetic similarity by <5% were considered as representing subtypes within the main group.

Statistical analysis

Pearson's χ²-test (for frequencies of higher than five) or Fisher's exact test (for small contingency tables) was used in the Chinese High Intellectualized Statistical Software version 2010 to determine the significance of the prevalence values.

Results

Prevalence of MDR phenotypes

In 2007–2009, 6.86% (161/2,346) E. coli and 8.05% (80/994) K. pneumoniae isolates exhibited MDR phenotypes. Overall, 5.89%, 7.01%, and 7.65% of the clinical E. coli isolates and 8.46%, 8.12%, and 7.56% of the clinical K. pneumoniae isolates exhibited MDR phenotypes in 2007, 2008, and 2009, respectively.

Antimicrobial susceptibilities

Among the 98 E. coli and 52 K. pneumoniae isolates with MDR phenotypes, most were susceptible to imipenem (98% vs. 96.2%) and meropenem (99% vs. 98.1%). These MDR K. pneumoniae isolates exhibited remarkably high ceftazidime (CAZ) resistance compared with the E. coli (90.4% vs. 76.5%, p=0.0339); the cefoxitin resistance was also high (73% vs. 62.2%, p=0.1704). Further, 52% and 92.3% of these E. coli and K. pneumoniae isolates, respectively, were unsusceptible (intermediate or resistance) to piperacillin–tazobactam.

Phylogenetic typing of MDR E. coli isolates

Most E. coli isolates with MDR phenotypes belonged to phylogenetic group D (50%), followed by group A (39.8%), group B1 (5.1%), and group B2 (5.1%).

Prevalence of resistance determinants

Table 1 lists the common resistance determinants. All isolates were negative for bla_PER, bla_VEB, bla_GES, rmtA, rmtC, rmtD, rmtE, and npmA. In addition, 56.1% (55/98) of the MDR E. coli isolates carried qepA and rmtB simultaneously, and 58.2% (32/55) of them were co-transferred by conjugation. Further, 44.2% (23/52) of the MDR K. pneumoniae isolates carried qnrB and armA simultaneously. All (26/26) of the DHA-producing MDR K. pneumoniae isolates were qnrB positive.

Table 1.

Prevalence (%) of Resistance Determinants Among Clinical Escherichia coli and Klebsiella pneumoniae Isolates with the Cefotaxime–Ciprofloxacin–Amikacin Resistance Pattern in China, 2007–2009

Resistance determinant	Escherichia coli n=98	Klebsiella pneumoniae n=52	p-Value
β-Lactamases genes
bla_CTX-M	88.8	82.7	0.2969
bla_TEM	93.9	75	0.0009
bla_SHV	1	92.3	0.0000
bla_DHA	1	50	0.0000
bla_CMY	12.2	3.8	0.1652
ESBL+^a	85.7	84.6	0.8563
QRDR substitutions
Single mutation in gyrA	1	69.2	0.0000
Double mutations in gyrA	99	21.2	0.0000
Single mutation in parC	91.8	82.7	0.0927
Double mutations in parC	8.2	1.9	0.2419
Mutations in both gyrA and parC	100	84.6	0.0003
PMQR genes
qnr	7.1	71.2	0.0000
aac(6′)-Ib-cr	16.3	51.9	0.0050
qepA	58.2	11.5	0.0000
oqxAB	16.3	61.5	0.0000
Total	70.4	90.4	0.0076
16S rRNA methylase genes
armA	10.2	59.6	0.0000
rmtB	83.7	38.5	0.0000
Total	93.9	94.2	0.7837

Phenotype detected by the double-disk synergy test.

ESBL, extended-spectrum β-lactamase; QRDR, quinolone resistance-determining region; PMQR, plasmid-mediated quinolone resistance.

Transferability of resistance determinants and antimicrobial susceptibility of transconjugants

In total, 74 (75.5%) and 31 (59.6%) transconjugants were obtained from the MDR E. coli and K. pneumoniae isolates, respectively. Among the MDR E. coli isolates, 32.2% (28/87) of bla_CTX-M, 42% (29/69) of the PMQR genes, and 56% (51/91) of the 16S rRNA methylase genes were located on transferable plasmids. Further, 60 of 74 (81.1%) transconjugants harbored at least two classes of these resistance determinants, and 14 of 74 (19.1%) transconjugants carried all three classes. For the MDR K. pneumoniae isolates, 34.9% (15/43) of bla_CTX-M, 14.6% (7/48) of bla_SHV, 30.8% (8/26) of bla_DHA, 34% (16/47) of the PMQR genes, and 38.8% (19/49) of the 16S rRNA methylase genes were located on transferable plasmids. Approximately 67.7% (21/31) of the transconjugants harbored at least two classes of these resistance genes, and 32.2% (10/31) of the transconjugants carried all three classes of resistance determinants.

The transconjugants carrying armA or rmtB exhibited similar AK susceptibility to the armA- and rmtB-negative transconjugants. The CTX susceptibility of the transconjugants was significantly affected by the production of CTX-M. CTX MIC₅₀ and MIC₉₀ of the CTX-M-producing transconjugants were 32 and 256 mg/l, respectively, whereas those of the CTX-M-negative strains were 2 and 64 mg/l, respectively. However, the CAZ susceptibility of the transconjugants was rarely affected by CTX-M production: CAZ MIC₅₀ of the CTX-M-producing and CTX-M-negative transconjugants was 4 and 2 mg/l, respectively, and MIC₉₀ of these transconjugants was 64 and 32 mg/l, respectively. The CIP MIC of the transconjugants was slightly affected by the PMQR genes.

Genetic relatedness

Among the 98 MDR E. coli isolates, 94 PFGE patterns and 23 clusters were identified; more than one isolate was found in four PFGE patterns. Among the 52 MDR K. pneumoniae isolates, 51 PFGE patterns and 11 clusters were identified; more than one isolate was found in one PFGE pattern (Fig. 1).

FIG. 1.

Dendrogram of patterns generated by pulsed-field gel electrophoresis of multidrug-resistance Klebsiella pneumoniae isolates. G1: group 1; G9: group 9.

Discussion

In this study, the clinical E. coli isolates (6.86%) exhibited a lower prevalence of MDR phenotypes than did the clinical K. pneumoniae isolates (8.05%). However, from 2007 to 2009, the E. coli isolates presented increased MDR prevalence (5.89%–7.68%), whereas the K. pneumoniae isolates showed decreased MDR prevalence (8.46%–7.79%). Considering that both types of MDR isolates were sourced from the same antibiotic use scenarios, the reason for the differential prevalence needs further analysis.

The great genetic variation of the MDR isolates indicates the considerable transmission of common resistance determinants. Despite a similar prevalence of bla_CTX-M between the two species (88.8% vs. 82.7%), the MDR E. coli isolates exhibited lower CAZ resistance than did the MDR K. pneumoniae isolates. This difference could be explained by the high prevalence of the SHV-type enzyme among MDR K. pneumoniae isolates, which exhibit strong hydrolytic activity against CAZ compared with CTX, rather than by the effect of CTX-M, because this enzyme slightly affected the CAZ MIC of the transconjugants.²² Another explanation is that more than half of the MDR K. pneumoniae isolates produced DHA and CMY, which can efficiently hydrolyze cephamycins,^2,13 whereas only a few MDR E. coli isolates produced these enzymes (Table 1). Further, the high prevalence of the plasmid-mediated AmpC-type enzymes contributed to the reduced susceptibility to the β-lactam/β-lactamase inhibitor combinations, such as piperacillin–tazobactam, because of the functional traits of AmpC enzymes that are not inhibited by β-lactamase inhibitors but hydrolyze piperacillin significantly.^1,2,13 Almost half of the MDR E. coli isolates were susceptible to piperacillin–tazobactam, because of the low prevalence of AmpC-type β-lactamases, whereas only 7.7% of the MDR K. pneumoniae isolates were susceptible to this agent.

In this study, the absolute fluoroquinolone resistance was the result of the high prevalence of QRDR substitutions, because most MDR isolates possessed at least 1 QRDR mutation in both gyrA and parC. However, the two species exhibited significant differences in the prevalence of these substitutions (Table 1). The low frequency of QRDR substitutions in the MDR K. pneumoniae isolates could partly be explained by the high prevalence of qnr (71.2%), because Qnr reduces the amount of enzyme–DNA complexes, which are the target of fluoroquinolones, and protects QRDR domains from mutation.^4,25,26,31 PMQR genes were highly prevalent among the MDR E. coli (70.4%) and K. pneumoniae isolates (90.4%) and played an important role in their fluoroquinolone-resistant phenotype. For example, K. pneumoniae strains E9108, E9193, E9194, E9196, and E9218 did not have either gyrA or parC mutations. The mechanisms for their fluoroquinolone-resistant phenotype might include efflux pumps or membrane impermeability, or the generation of synergistic resistance effects with different PMQR genes.¹⁸ In comparison with previous reports,^15,32 our study showed a higher prevalence of PMQR genes, indicating a considerable impact of PMQR mechanisms in QRDR substitutions in MDR E. coli and K. pneumoniae strains. In addition, due to the coexistence of PMQR genes with extended-spectrum β-lactamase (ESBL) or carbapenemase genes and their cross-species/genera transferability,¹⁸ a high prevalence of PMQR mechanisms could facilitate the spread of MDR isolates. In this study, qepA exhibited a strong association with rmtB among the MDR E. coli isolates; a close relationship was observed between qnrB and armA and bla_DHA among the MDR K. pneumoniae isolates.

High AK susceptibility has been reported for clinical E. coli and K. pneumoniae isolates.^12,28 As a semisynthetic aminoglycoside, AK is rarely inactivated by common aminoglycoside-modifying enzymes,¹⁴ making it a good indicator of the presence of 16S rRNA methylase genes in clinical gram-negative isolates.⁸ In this study, armA and rmtB played a major role in AK resistance, because most MDR isolates contained either one or both of these genes. However, 6.1% and 5.8% of the MDR E. coli and K. pneumoniae isolates, respectively, tested negative for the known 16S rRNA methylase genes, indicating that other mechanisms also confer AK resistance in E. coli and K. pneumoniae, such as unknown 16S rRNA methylase variants or several isozymic forms of aminoglycoside phosphotransferase.^23,24 This finding was confirmed by conjugation testing, because the transconjugants not carrying armA or rmtB also exhibited high-level AK resistance.

In conclusion, the clinical E. coli and K. pneumoniae isolates exhibited diverse phenotypic characteristics because of the differential prevalence of common resistance determinants. Despite the relatively low prevalence in this study, a larger-scale epidemiological investigation on the prevalent characteristics of clinical MDR E. coli and K. pneumoniae isolates and further research on the mechanisms of resistance transmission are warranted to prevent the spread of MDR strains.

This study has several limitations. First, the retrospective investigation was carried out with limited information of the strains isolated from clinical samples. Inadequate sample collection and handling and, especially, etiological diagnoses based on the subjective analyses of patients affect the ability to perform randomized clinical trials, which may be a major source of selection bias when investigating the prevalence of MDR phenotypes in clinical isolates. Second, only 98 E. coli and 52 K. pneumoniae isolates were further analyzed in this study. The loss of strains because of inadequate preservation and contamination could lead to bias. Third, a detailed analysis is required to understand the mechanisms related to MDR phenotypes, such as the Inc-groups of resistance plasmids, sequence type of MDR isolates, and, especially, variant types of resistance determinants. Fourth, the selection of transconjugants on AK only (and not on other antibiotics) could result in a significant underestimation of the contribution of conjugative plasmids to the resistance phenotype.

Footnotes

Acknowledgment

This research was supported by grant 2008ZX10004-001-C from the Ministry of Science and Technology, China.

Authors' Contributions

J.Y. conducted the molecular genetic studies and drafted the manuscript. Y.Z., L.Y., and Y.L. participated in the strain isolation and antimicrobial susceptibility testing. W.W. participated in the molecular genetic studies. W.Z. participated in the sequencing of resistance determinants. Z.C. participated in the PFGE analysis. L.H. helped design of the study. All authors read and approved the final manuscript.

Disclosure Statement

The authors declare that they have no competing interests.

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Characterization of Clinical Multidrug-Resistant Escherichia coli and Klebsiella pneumoniae Isolates,2007–2009,China

Abstract

Introduction

Materials and Methods

Bacterial isolates

Antimicrobial susceptibility testing

Screening of resistance determinants

Phylogenetic analysis

Conjugation testing

Pulsed-field gel electrophoresis

Statistical analysis

Results

Prevalence of MDR phenotypes

Antimicrobial susceptibilities

Phylogenetic typing of MDR E. coli isolates

Prevalence of resistance determinants

Transferability of resistance determinants and antimicrobial susceptibility of transconjugants

Genetic relatedness

Discussion

Footnotes

Acknowledgment

Authors' Contributions

Disclosure Statement

References