Emergence and molecular characterization of multidrug-resistant Klebsiella pneumoniae isolates harboring bla_CTX-M-15 extended-spectrum β-lactamases causing ventilator-associated pneumonia in China

Introduction

Ventilator-associated pneumonia (VAP) is one of the most frequent hospital-acquired infections occurring in intubated and mechanically ventilated patients. The rate of VAP occurrence is reportedly 9%–27%, with mortality reaching 20%–50%.^1,2 Common causative pathogens of VAP include Gram-negative bacteria such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli and Gram-positive bacteria such as Staphylococcus aureus.^3–6 Extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae is a common organism of nosocomial infections that play an important role in VAP.^7,8

Some clinical isolates of pathogens produce ESBLs that are able to hydrolyze expanded-spectrum cephalosporins (eg, ceftriaxone, cefotaxime, and ceftazidime), aztreonam, and related oxyimino-β-lactams, but not carbapenems, and are inhibited by clavulanic acid and tazobactam.⁹ Genes encoding ESBLs, such as bla_SHV, bla_CTX-M, bla_TEM, bla_OXA, bla_PER, and bla_VEB, are usually located on large plasmids (>30 kb in size) that are highly mobile and often harbor resistance determinants for several unrelated classes of antimicrobials, such as aminoglycosides, trimethoprim/sulfonamides, tetracyclines, and chloramphenicol.¹⁰ Therefore, antibiotic therapy for treating such multidrug-resistant (MDR) pathogen infections is limited to a small number of expensive drugs.

CTX-M enzymes are among the most important ESBLs worldwide, with a clearly higher prevalence compared to other ESBLs. Indeed, emergence and outbreaks of the CTX-M enzyme have been described in bacteria from Africa, Europe, South America, and Asia.¹¹ At present, more than 90 CTX-M variants have been designated (http://www.lahey.org/Studies/other.asp), of which CTX-M-15 is the most prevalent variant around the world.¹² The global spread of bla_CTX-M-15 is largely due to E. coli of sequence type (ST) 131, IncFII plasmids, and the genetic platforms of the bla_CTX-M gene, such as ISCR1, ISEcp1, and IS26, which act as promoters for expression of various resistance genes and influence the mobilization of bla_CTX-M genes.^13,14

Many recent reports have indicated that VAP may be associated with multiresistant pathogens, such as P. aeruginosa and Gram-negative bacilli, characterized by the production of ESBLs.^15,16 However, limited data are available regarding the emergence and prevalence of bla_CTX-M-15-producing K. pneumoniae isolates from VAP cases in China. To better guide prevention efforts and clinical treatment of infections, this cross-sectional study was performed to investigate the antimicrobial resistance and molecular epidemiology of VAP caused by K. pneumoniae isolates producing ESBLs, especially bla_CTX-M-15, over a 3-year period.

Materials and methods

Clinical isolates and ESBL phenotype confirmation

A retrospective cross-sectional study was conducted at the intensive care unit (ICU) of the First Affiliated Hospital of Dalian Medical University, a 3,700-bed tertiary-care hospital with five ICU wards, from January 2013 to December 2015. K. pneumoniae strains were collected via endotracheal aspiration from mechanically ventilated patients with suspected pneumonia and stored at −80°C before use. VAP was defined as pneumonia occurring 48 hours or more after endotracheal intubation with at least two of the following criteria: fever greater than 38.3°C, leukocytosis or leucopenia, and purulent tracheal secretions (greater than 25 neutrophils observed per high-power field).¹⁷ In addition, one or more of the following criteria had to be met: new or persistent infiltrates on chest radiographs, the same microorganism isolated from pleural fluid and tracheal secretions, radiographic cavitation or histopathological demonstration of pneumonia, and positive cultures obtained from bronchoalveolar lavage (greater than 10⁴ colony forming units per mL).^18–20 A MicroScan WalkAway 96 Plus instrument (Siemens AG, Munich, Germany) was used for bacterial identification. Polymicrobial infections were excluded from analysis. All of the K. pneumoniae isolates were screened and confirmed using a double-disk synergy test for ESBL production.

Antimicrobial susceptibility testing

The antimicrobial susceptibility of ESBL-producing K. pneumoniae (EPKP) isolates and recipient (J53Az^R) and transconjugant strains was determined using the standard broth microdilution method according to the recommendations of the Clinical and Laboratory Standards Institute.²¹ The following antimicrobial compounds were assessed: cefuroxime, cefotaxime, ceftazidime, cefepime, imipenem, aztreonam, amikacin, ciprofloxacin, levofloxacin, and tigecycline. E. coli ATCC 25922 was used as a reference strain. MDR K. pneumoniae strains were defined as acquired nonsusceptibility to at least one agent in three or more antimicrobial categories.

Molecular detection of resistance genes

To identify ESBL genes carried by the K. pneumoniae isolates, cell lysates were subjected to polymerase chain reaction (PCR) detection of bla_CTX-M, bla_SHV, bla_TEM, and bla_OXA genes. All PCR products amplified from β-lactamase genes were commercially sequenced, and subsequent searches in PubMed using the BLAST program (https://blast.ncbi.nlm.nih.gov/) were performed. Specific PCR assays were conducted as previously described to identify the possible association of bla_CTX-M-15 with ISEcp1 or with the IS26 insertion element.^13,22 Furthermore, plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrS, and aac(6¢)-Ib-cr) were confirmed by multiplex PCR using a previously described protocol.²³ Additional genes of antibiotic resistance, such as 16S rRNA methylase-encoding genes (armA, rmtB, and rmtC), were assessed to further characterize the identified strains.²⁴

Multidrug efflux pump gene expression

The expression of genes encoding the multidrug efflux pumps AcrB, OqxB, and KpgB and their global transcriptional regulators RarA and RamA in clinical EPKP isolates was assessed by quantitative reverse-transcription PCR using previously described oligonucleotide primers.²⁵ Total bacterial RNA was extracted using an E.Z.N.A.^TM bacterial RNA Kit (Omega Bio-Tek, Norcross, GA, USA) and was reverse transcribed to complementary cDNA using a PrimeScript RT Reagent Kit (Takara, Dalian, China) according to the manufacturer’s instructions. The cDNA was amplified using a SYBR^® Premix Ex TaqTM II Kit (Takara) and a Stratagene Mx3005P qPCR System (Stratagene Agilent, Santa Clara, CA, USA) with 40 cycles of 5 seconds at 95°C and 34 seconds at 60°C. Each strain was amplified in triplicate. The expression levels of each target gene were normalized to a housekeeping gene (rrsE). Data were analyzed using Agilent MxPro software based on the 2⁻DD^Ct method.

Plasmid analysis

S1-nuclease (Takara) digestion followed by pulsed-field gel electrophoresis (S1-PFGE) analysis was performed for all the EPKP isolates and transconjugants. For plasmid size estimation, comparison with the molecular weight marker Salmonella braenderup H9812 was performed. Plasmid replicons were determined using the PCR-based replicon typing scheme (PBRT) with 18 pairs in PCR for detecting F, FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, and FII replicons, as described by Carattoli et al.²⁶

Resistance transfer determination

To determine whether plasmids coding for antibiotic resistance enzymes can be transferred, conjugation experiments were performed with all isolates carrying bla_CTX-M-15 using a broth mating protocol. K. pneumoniae isolates were mated with the sodium azide-resistant E. coli strain J53Az^R. Transconjugants were selected on LB agar plates containing sodium azide (100 µg/mL) and cefotaxime (10 µg/mL). PCR amplification, antimicrobial susceptibility testing, and plasmid replicon typing were performed for all transconjugants to determine the presence of resistance determinants, antibiotic phenotypes, and incompatibility groups, respectively.

PFGE and multilocus sequence typing (MLST)

The genetic relatedness of the identified K. pneumoniae isolates was examined by PFGE and MLST. DNA was extracted and digested with 45 U XbaI (Takara) for 2 hours at 37°C. PFGE was performed for the EPKP isolates using a CHEF-DRIII apparatus (Bio-Rad Laboratories, Hercules, CA, USA) as previously described.²⁷ MLST analysis was conducted by sequencing fragments of seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB), and STs were assigned using the K. pneumoniae MLST website (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae).

Ethical clearance

The collection of K. pneumoniae in this study was part of routine hospital laboratory procedure. This retrospective study was performed using samples for secondary use, free of the need for informed consent and ethics committee approval.

Results

Antimicrobial susceptibility testing

From January 2013 to December 2015, 89 mechanically ventilated patients were identified with K. pneumoniae-induced VAP in our tertiary-care hospital. The patients, 60 (67.4%) of whom were male and 29 (32.6%) were female, had a mean ± SD age of 56.0±14.0 years. Among the patients, 30 clinical K. pneumoniae isolates were screened and confirmed for ESBL production (30/89, 33.71%). High-level resistance to cephalosporins (cefuroxime [28/30, 93.33%; MIC range: 8 to >512 µg/mL], ceftazidime [29/30, 96.67%; MIC range: 8–512 µg/mL], cefotaxime [28/30, 93.33%; MIC range: 2–512 µg/mL], and cefepime [24/30, 80.00%; MIC range: 4–128 µg/mL]) as well as noteworthy resistance to fluoroquinolones (ciprofloxacin [26/30, 86.67%; MIC range: 1–256 µg/mL], levofloxacin [23/30, 76.67%; MIC range: 2–256 µg/mL]), and aminoglycosides (amikacin [16/30, 53.33%; MIC range: 4 to >512 µg/mL]) were found. All 30 EPKP isolates exhibited a MDR phenotype and were examined in subsequent experiments. Their susceptibility profiles for ten antimicrobial agents are shown in Table 1.

Table 1 Resistance features of the ESBL-producing K. pneumoniae isolates, transconjugants, and recipients Notes: aIsolates 6C, 8C, 19C, 20C, 22C, 24C, 26C, and 30C are transconjugants. Abbreviations: ESBL, extended-spectrum β-lactamase; K. pneumoniae, Klebsiella pneumoniae; MIC, minimum inhibitory concentration; CFX, cefuroxime; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; IPM, imipenem; ATM, aztreonam; AMK, amikacin; LEV, levofloxacinb; CIP, ciprofloxacin; TGC, tigecycline; “–”, negative.

Identification of antibiotic resistance genes and the bla_CTX-M-15 genetic context

Of the 30 EPKP isolates, bla_SHV, bla_CTX-M, bla_OXA, and bla_TEM were identified in 21, 21, 1, and 20 isolates, respectively. Among the ESBL genes detected in this study, the bla_CTX-M-15 allele was the most prevalent (19/30, 63.33%), followed by bla_SHV-28 (2/30, 6.67%) and bla_TEM-53 (2/30, 6.67%). DNA sequence analysis revealed the insertion of ISEcp1 48 bp upstream of the start codon of the 15 CTX-M-15 genes (except EPKP4, EPKP12, EPKP16, and EPKP27). However, PCR amplification with primers specific for IS26 was negative for all K. pneumoniae isolates carrying bla_CTX-M-15. Regarding β-lactamases other than ESBLs, bla_SHV-11 (16/30, 53.33%) and bla_TEM-1 (16/30, 53.33%) alleles predominated; other minor β-lactamases, including bla_CTX-M-14, bla_CTX-M-22, bla_OXA-10, bla_TEM-1b, bla_SHV-33, and bla_SHV-27, were also detected. For PMQR genes, 17 isolates were found to carry qnrB, 21 isolates qnrS, and two other variants such as qnrA or aac-(6¢)-Ib-cr. Resistance genes for other antibiotics included armA in 17 isolates and rmtB in two (Table 1).

Differential expression of efflux pump genes

The levels of differential gene expression of the assayed multidrug efflux pumps and their global regulators among the 30 EPKP isolates are presented in Figure 1. Real-time PCR analysis showed uniformly high expression levels of acrB (1.80- to 4.00-fold) and ramA (2.12- to 17.68-fold) of the RamA/AcrB pathway in seven isolates (EPKP3, EPKP4, EPKP13, EPKP18, EPKP20, EPKP23, and EPKP24). Twelve isolates (EPKP3, EPKP4, EPKP6, EPKP9, EPKP10, EPKP17, EPKP18, EPKP20, EPKP22, EPKP25, EPKP28, and EPKP29) exhibited simultaneously high expression levels of oqxB (1.08- to 16.77-fold) and rarA (1.18- to 84.50-fold) genes. In addition, 13 isolates (EPKP1, EPKP3, EPKP6, EPKP11, EPKP13, EPKP14, EPKP17, EPKP18, EPKP20, EPKP21, EPKP22, EPKP26, and EPKP27) showed upregulation of the kpgB gene (2.07- to 18.53-fold).

Figure 1 Differential gene expression of multidrug efflux pumps and global regulators among the 30 EPKP isolates. Notes: Fold change in the expression of genes encoding (A) the multidrug efflux pump AcrB; (B) the multidrug efflux pump OqxB; (C) the multidrug efflux pump KpgB; (D) the global regulator RamA; and (E) the global regulator RarA. Fold changes in gene expression were determined after normalizing to that of the 16S rRNA gene (rrsE) in each strain and then comparing the expression of each gene with corresponding genes in the tigecycline-susceptible Klebsiella pneumoniae strain ATCC 13883. Abbreviations: EPKP, ESBL-producing K. pneumoniae; ESBL, extended-spectrum β-lactamase.

Plasmid analysis

We examined the plasmid profiles of 30 EPKP isolates by S1-PFGE analysis. As shown in Figure 2, 25 different plasmid profiles were observed among the 30 EPKP isolates (ranging from ~70 to 320 kb). Fourteen strains (EPKP2, EPKP4, EPKP6, EPKP7, EPKP10, EPKP12, EPKP13, EPKP16, EPKP17, EPKP21, EPKP24, EPKP27, EPKP28, and EPKP30) harbored single plasmids of different sizes, and eleven strains (EPKP1, EPKP3, EPKP5, EPKP8, EPKP9, EPKP11, EPKP19, EPKP20, EPKP22, EPKP26, and EPKP29) harbored two to four plasmids. Conversely, five isolates (EPKP14, EPKP15, EPKP18, EPKP23, and EPKP25) contained no detectable plasmid elements.

Figure 2 S1 endonuclease pulsed-field gel electrophoresis analysis of plasmids from the 30 VAP-inducing EPKP isolates in China. Notes: (A) “M” indicates the molecular weight marker; Lanes 1–11 show the plasmid profiles of isolates EPKP1–EPKP11, respectively; (B) Lanes 12–22 show the plasmid profiles of isolates EPKP12–EPKP22, respectively; and (C) Lanes 23–30 show the plasmid profiles of isolates EPKP23–EPKP30, respectively. Abbreviations: VAP, ventilator-associated pneumonia; EPKP, ESBL-producing Klebsiella pneumoniae; ESBL, extended-spectrum β-lactamase.

Resistance transfer and PBRT

The resistance profiles of the eight transconjugants were similar to those of the bla_CTX-15-producing K. pneumoniae donor strains, demonstrating the transfer of antimicrobial resistance, including the ESBL phenotype. In addition, resistance to several non-β-lactam-based antimicrobial compounds, such as fluoroquinolones, was also cotransferred along with β-lactam resistance; in contrast, resistance to tigecycline was not transferred. For the transconjugants, the most commonly detected resistance genes included bla_CTX-M-15 (n=6), bla_TEM-1 (n=3), qnrS (n=4), and qnrB (n=4), while the ISEcp1 element was detected in six isolates (Table 1).

Plasmid replicon typing showed that in the K. pneumoniae isolates carrying bla_CTX-M-15, the plasmids had different replicons, including IncFIC (n=11), IncFIB (n=8), IncFIA (n=2), IncF (n=1), IncFII (n=1), IncK (n=7), and IncL/M (n=1) (Figure 3). However, PCR replicon typing of the transconjugants identified only three replicons, IncFIB, IncFIC, and IncFII, which were present in both donors and transconjugants and were associated with the transfer of the ESBL phenotype (Table 2).

Figure 3 Genetic relatedness, plasmid size, and replicon type of the 30 VAP-inducing EPKP isolates in China. Note: Dendrogram of patterns generated by PFGE. Abbreviations: MLST, multilocus sequence typing; ST, sequence type; –, not detected; VAP, ventilator-associated pneumonia; EPKP, ESBL-producing K. pneumoniae; ESBL, extended-spectrum β-lactamase; PFGE, pulsed-field gel electrophoresis.

Table 2 Antibiotic resistance genes and plasmid replicon types in transferable blaCTX-15-producing Klebsiella pneumoniae donors and their transconjugants Notes: aIsolates EPKP6, 8, 19, 20, 22, 24, 26, and 30 are original isolates; EPKP6C, 8C, 19C, 20C, 22C, 24C, 26C, and 30C are transconjugants. Abbreviations: ESBL, extended-spectrum β-lactamase; PMQR, plasmid-mediated quinolone resistance.

PFGE and MLST of isolates

The 30 EPKP isolates were assigned to 20 distinct PFGE clusters sharing ≥80% band similarity as well as eleven ST types (ST11, ST15, ST37, ST65, ST268, ST304, ST716, ST828, and ST1049), including two new STs (ST2321 and ST2322) (EPKP3 [02-01-02-01-03-01-25] and EPKP19 [03-20-01-01-01-01-04]). The most prevalent ST was ST304 (n=15, 50%), followed by ST716 (n=3, 10%) and ST37 (n=3, 10%). No clear relationship between replicon and sequence type was observed among the isolates identified in this study (Figure 3).

Discussion

This is one of only a few studies performed to date investigating the antimicrobial resistance and molecular epidemiology of K. pneumoniae carrying bla_CTX-M-15-caused VAP in China. The prevalence of ESBL reported herein is a matter of concern because MDR pathogens causing infectious diseases are common in this area, limiting therapeutic options for treating severe infections often associated with a poor outcome. Indeed, the incidence of EPKP is increasing among patients receiving mechanical ventilation in the ICU of our tertiary-care hospital. The prevalence rate of ESBL among K. pneumonia isolates causing VAP was 33.71%. Despite the high prevalence of ESBLs reported in this study, it is lower compared with the prevalence of those causing device-associated infections among children in a pediatric ICU of other medical centers.²⁸

MIC determinations showed all EPKP isolates to be highly resistant to cephalosporins, with noteworthy resistance to fluoroquinolones and aminoglycosides also observed. As shown in Table 1, although the MDR phenotype reported in our studied isolates is frequently associated with ESBL producers, the concurrent combination of different mechanisms, such as PMQR gene expression, 16S rRNA methylase production, and differential expression of multidrug efflux pump genes may lead to this phenotype. The co-presence of ESBL genes and other resistance determinants on the same plasmid is reported regularly.²⁹ In agreement with our findings, PMQR has been associated with bla_CTX-M genes, with genes conferring resistance to aminoglycosides and tetracycline and other bla genes being found on the same plasmids as carrying bla_CTX-M.³⁰

Over the past decade, predominant CTX-M-type ESBLs have been described globally, including China, South Korea, and many other countries.^11,31,32 In fact, together with bla_CTX-M-14, bla_CTX-M-15 is currently the most common variant detected worldwide in clinically important Gram-negative bacteria.^33,34 In this study, we identified the CTX-M-15 enzyme as the most prevalent ESBL in VAP patients. The insertion sequence ISEcp1 has previously been shown to play an important role in the mobilization and expression of genes encoding bla_CTX-M,^13,35 therefore, linkage of bla_CTX-M-15 with ISEcp1 was assessed and shown to be present in all but four of the K. pneumoniae isolates carrying the bla_CTX-M-15 gene. The presence of internal sequences such as IS26, which is related to the transmission of β-lactamase genes, such as DHA-1, CFE-1, ACC-1, and SHV-2a, is typically found for the IncFII plasmid.^33,36 Regardless, PCR amplification of the IS26 gene was negative for all 19 CTX-M-15 genes.

In our study, PBRT and conjugation experiments showed that among the clinical K. pneumoniae isolates from mechanically ventilated patients, IncFIB, IncFIC, and IncFII replicons were present in the transconjugants and the bla_CTX-M-15 gene was co-transferred to the recipient strain with bla_TEM and qnr genes and the ISEcp1 element. These results indicate that IncF-related plasmids carrying bla_CTX-M-15 are a major vehicle mediating the local prevalence of resistance determinants in K. pneumoniae isolates. Nonetheless, it was previously reported that the bla_CTX-M-15 gene can also be found in IncN, IncR, IncFIIk, or IncL/M types.^11,37–39

Regarding our S1-PFGE analysis, the existence of five EPKP isolates (EPKP14, EPKP15, EPKP18, EPKP23, and EPKP25) containing no plasmid suggests that these “plasmid-encoded” resistance genes have been integrated into the chromosome. In addition, smaller plasmids may not have been detected by S1-PFGE analysis.⁴⁰ Further studies are needed to investigate other resistance genes possibly carried on smaller plasmids.

A previous retrospective study of 49 mechanically ventilated patients in tertiary hospitals in China showed that ST23 was dominant among hypervirulent K. pneumoniae strains,⁴¹ yet ST304 was the most prevalent in our collection of “classic” K. pneumoniae isolates (n=15, 50.00%). To the best of our knowledge, this is the first report of ST304 in K. pneumoniae, and no clear relationship between replicon and sequence type was observed among the current isolates. This result suggests that this is not a result of the dissemination of particular clones but rather is due to the spread of multiple specific clones and/or mobile genetic elements.

The emergence of MDR pathogens as causative agents of VAP has resulted in a greater administration of inappropriate initial antimicrobial therapies, defined as an antimicrobial regimen that lacks in vitro activity against the isolated organism(s) responsible for the infection.⁴² Our data highlights the importance of continuous surveillance of both resistant isolates and genetic elements of resistance to monitor the emergence and trends of ESBL-producing isolates to promote adequate therapeutic strategies for managing MDR bacterial infections.

The present study has several limitations. First, because this study was a retrospective analysis and only limited VAP patient information was available, the study focused on the molecular characterization of the prevalence of genes among clinical EPKP. Another limitation of the study included a lack of analysis of other resistance-related determinants, such as the outer-membrane permeability of EPKP isolates. Further studies are needed to address these limitations.

Conclusion

Although ESBL-producing members of Enterobacteriaceae have been reported in China, very limited data are available regarding the susceptibility and molecular characterization of K. pneumoniae isolates from mechanically ventilated patients. This study highlights the emergence of ESBLs, particularly the CTX-M-15 type, in K. pneumoniae-induced VAP in Chinese hospitals. We showed that the bla_CTX-M-15 gene was cotransferred with the bla_TEM and qnr genes and the ISEcp1 element, conferring a high level of resistance to most antibiotics tested. All transconjugants were associated with IncFIB, IncFIC, and IncFII plasmids.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant number 81401697), the Doctoral Start-up Foundation of Liaoning Province (grant number 201501159), the Postdoctoral Research Foundation of China (grant number 2015M571314), and the Program for High-Level Entrepreneurial and Innovative Talents of Dalian, China (2017RQ120). This work was also supported by the Liaoning Provincial Program for Top Discipline of Basic Medical Sciences.

Disclosure

The authors report no conflicts of interest in this work.

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