Genomic Analysis Of A KPC-2-Producing Klebsiella Pneumoniae ST11 Outbreak From A Teaching Hospital In Shandong Province, China.

PURPOSE: Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae bacteria causes nosocomial infections worldwide. However, KPC-producing K. pneumoniae outbreak has never been reported in Shandong Province, China. The purpose of our study was to elucidate the epidemiological and drug resistance mechanisms of KPC-producing K. pneumoniae strains collected from a large teaching hospital in Shandong during the outbreak. Moreover, we attempted to characterize the genetic environment and phylogenetic analysis of bla KPC-2 in outbreak isolates.
METHODS: We monitored a 64-day outbreak of infection in a general hospital in Shandong Province, and the bacteria causing the infection were all ST11-type K. pneumoniae. The genotype correlation of KPC-producing K. pneumoniae isolates was assessed by whole-genome sequencing (WGS) phylogenetic analysis. Subsequent studies included antibiotic susceptibility testing, multilocus sequence typing (MLST) and S1-pulsed-field gel electrophoresis (S1-PFGE), Southern blot hybridization.
RESULTS: From February 1, 2018 to April 5, 2018, 14 KPC-producing K. pneumoniae isolates from different wards were collected. All 14 isolates were resistant to carbapenems and carried the extended-spectrum β-lactamase (ESBL) gene as well as fosA, and sul genes. Whole-genome analysis showed that all 14 the outbreak isolates were all ST11 type. The bla KPC-2 carrying plasmids were all belong to IncFIIK2 type, and the size ranged from 94 kb to 368 kb.
CONCLUSION: As far as we know, this report first describes the genomics characterization of KPC-2-producing K. pneumoniae outbreak isolates from Shandong Province, China. In our study, these isolates appeared to be cloned, and ST11 K. pneumoniae was the major clone caused the outbreak. Therefore, routine surveillance of such strains in this region is urgently warranted.

Introduction

Bacterial resistance can reduce the effectiveness of antibiotics and increase the difficulty of treating infectious diseases, becoming a major problem affecting global public health.1 Carbapenems can be used to treat infections caused by various extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates. However, due to the irrational use of carbapenems, the emergence of carbapenem-resistant Enterobacteriaceae (CRE) has caused difficulties in clinical work.2 KPC enzymes hydrolyze carbapenems, not only that, but it is also the most important enzyme among class A carbapenemases.3 Infections caused by KPC-producing organisms are associated with high mortality rates up to 51%, which poses a huge challenge for clinical diagnosis and treatment.4,5

Klebsiellapneumoniae is a gram-negative pathogen and is the most common cause of hospital-acquired and community-acquired infections.6,7 It has been reported that the widespread of carbapenem-resistant K. pneumoniae (CRKP) is caused by horizontal transfer of mobile elements such as plasmids and insertion sequences.8 According to the Carbapenem-Resistant Enterobacteriaceae Network, pneumonia and bloodstream infections caused by carbapenem-resistant K. pneumoniae have a higher mortality rate. Carbapenem-resistant K. pneumoniae nonbacteremic infections can result in a 24.3% mortality rate.9

KPC-producing K. pneumoniae, which can lead to outbreaks of serious diseases globally,10,11 are rarely reported in Shandong Province, China. In this study, we identified 14 clinical K. pneumoniae strains carrying blaKPC-2, all of which belong to ST11. The aim of our study was to elucidate the epidemiological and drug resistance mechanisms of KPC-producing K. pneumoniae strains collected from a large hospital in Shandong during the outbreak. Moreover, we attempted to characterize the genetic environment and phylogenetic analysis of blaKPC-2 in outbreak isolates.

Materials And Methods

Sample Collection

From February 1, 2018 to April 5, 2018, we have collected strains producing carbapenemase from the laboratory of a large teaching hospital in Jinan, Shandong Province. Bacterial identification was conducted with both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) (Bruker Daltonik GmbH, Bremen, Germany). Identification of carbapenemase genes (blaKPC, blaNDM, blaOXA-48, blaVIM and blaIMP) using PCR and Sanger sequencing.5

Antibiotic Susceptibility Testing

Determine the minimum inhibitory concentrations (MICs) of antibiotics (Dalian Meilun Biotech Co., Ltd, Dalian, China) using agar dilution method: amikacin, aztreonam, cefotaxime, cefpirome, ciprofloxacin, ceftazidime, gentamicin, piperacillin-tazobactam, imipenem, meropenem, tobramycin, amoxicillin-clavulanic acid, chloramphenicol and fosfomycin.12 Tigecycline and polymyxin were determined by the broth microdilution method. Results were interpreted using the CLSI standards (). Controls were performed using Escherichia coli ATCC 25922 and K. pneumoniae ATCC 700603.

Plasmid Characterization And Conjugation Assay

The plasmid was characterized by S1-PFGE, and the location of blaKPC was identified by Southern hybridization with digoxigenin-labelled blaKPC probe using the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics). Plasmid conjugation experiments by mating with E. coli J53 as a recipient strain. Next, the transconjugants were cultured on agar (OXOID, Hampshire, UK) medium supplemented with 200 mg/L sodium azide and 2 mg/L meropenem. Finally, MALDI-TOFMS was identified for transconjugants, and blaKPC was tested by PCR to ensure that the plasmid was successfully transferred to the recipient strain.

Whole-Genome Sequencing

Total DNA was obtained using an OMEGA Bacterial DNA Kit (Omega Bio-tek, Norcross, USA), followed by sequencing using Illumina HiSeq 4000-PE150 platform (Illumina, San Diego, CA, USA). We created genome sequence for 14 clinical K. pneumoniae isolates using SPAdes 3.11 by combining our Illumina sequencing reads. In addition, an online tool () was used to detect the acquired antimicrobial resistance genes in 14 isolates. The whole-genome sequences of the 14 isolates were deposited in GenBank under the following accession numbers: VJNW00000000-VJOJ00000000. The bacterial genome was annotated using the RAST server () and the transposon and IS elements were identified using the ISFinder database (). The genetic environment surrounding the carbe-genes were annotated using Easyfig 2.2.3. The presence of the virulence gene was identified by aligning the sequences of virulence factor from the database (). The gene sequence was uploaded to the PubMLST database () to determine the ST type of the isolate.

Phylogenetic Reconstruction And Analysis

Identification of core genomic single nucleotide polymorphisms SNPs on WGS data for 14 isolates was conducted by using the kSNP program.13 kSNP is a program based on the k-mer analysis. Kchooser is used to evaluate the optimal value of k-mer before kSNP is run. After the run of kSNP program, the output file was used for further analysis.14 The maximum likelihood tree of the core SNP matrix output of kSNP was generated by using iTOL ().

Results

Detection Of KPC-Producing K. Pneumoniae From Clinical Samples

The study population included all patients in the hospital, and the case patients were specimens producing carbapenem-resistant K. pneumoniae. Patients who were isolated from the first carbapenem-resistant K. pneumoniae were considered to be the source of transmission, and then carbapenem-resistant K. pneumoniae was continuously collected. The outbreak lasted for 64 days. A total of 14 carbapenem-resistant K. pneumoniae were collected from sputum (8/14), urine (5/14)and pus (1/14). After the bacteria were cultured and purified, single colonies were selected from the culture plates and identified with MALDI-TOF-MS. The isolates were recovered from the selected medium for PCR and sequencing, and the blaKPC gene was identified in all isolates, no other carbapenemase encoding genes were detected.

Clinical Characteristics Of 14 Carbapenem-Resistant Isolates

Clinical characteristics of 14 carbapenem-resistant isolates were summarized in . In this study, isolates were defined as nonduplicated strains only if they were isolated from different patients. Among them, there are 5 females and 9 males, aged between 45 and 88 years old. The patients came from 4 different wards, including 10 from intensive care unit (ICU), 2 from respiratory ward, 1 from neurosurgical ward and 1 from recovery unit. Ten of them had a history of infectious diseases.

Antibiotic Resistance Profiles Of 14 Carbapenem-Resistant Isolates

As shown in Figure 1, the antibiotic susceptibility test showed that all 14 isolates were resistant to piperacillin tazobactam, cefotaxime, ceftazidime, cefprozil, aztreonam, imipenem and meropenem, ciprofloxacin. Gentamicin (14%), tobramycin (14%) and amikacin (28%) have low sensitivity. Sensitivity to chloramphenicol (93%) and tigecycline (78%) was higher. Moreover, the experimental results showed that all 14 isolates were multi-drug resistant.

Figure 1

MICs were determined by agar dilution methods for all antibiotics except for colistin and tigecycline, for which broth microdilution was used. Results were interpreted using the CLSI guidelines. Pink indicates resistance, blue indicates mediation, and light yellow indicates sensitivity.

Antimicrobial Resistance Genes

We present the data for the antimicrobial resistance genes in Table 1. The results of the analysis indicated that all isolates carried blaKPC-2. Figure 2 shows the genetic environment surrounding the blaKPC-2. There are four distinct genetic environments in the isolates carrying blaKPC-2 in this study. Thirteen isolates (92%) carried blaCTX-M-65, 12 isolates (85%) carried blaSHV-11and 9 isolates carried blaTEM-1B (64%). All isolates carried fosA, and sul- genes, encoding fosfomycin- and sulphonamide-, resistance. Moreover, a high prevalence of tetracycline resistance gene (57% isolates) was also observed. In addition, 14 isolates also carry resistance genes such as oqxA, oqxB, mphA, tetA and tetM (Table 1).

Table 1

Antibiotic Resistance Genes, STs, Plasmid Type And Plasmid Size Of KPC-Producing K. Pneumoniae Isolates

Isolate	Source	Antibiotic Resistance Genes	MLST	Plasmid Type	Plasmid Size
2C1	Sputum	blaCTX-M-65, fosA6, sul2, aph(3’’)-Ib, aph(6)-Id, blaSHV, blaCTX-M-3, qnrS1, sul1, aph(3’)-Ia, qnrB2, aac(3)-Iid, mph(A), aadA16, dfrA27, ARR-3, aac(6’)Ib-cr	ST11	IncFⅡK2	94 kb
3C1	Sputum	dfrA1, ant(3’’)-Ia, erm(42), ant(3’’)-Ia, sul1, sul2, blaSHV-11, fosA6	ST11	IncFⅡK2	94 kb
3C2	Sputum	rmtB, blaTEM-1B, blaSHV-11, blaCTX-M-65, fosA6, ant(3’’)-Ia, sul1	ST11	IncFⅡK2	140 kb
3C3	Sputum	blaCTX-M-65, oqxA, oqxB, blaTEM-1B, rmtB, blaSHV-11, fosA3, fosA6, sul1, ant(3’’)-Ia, blaCTX-M-15	ST11	IncFⅡK2	140 kb
3C9	Urine	blaSHV-11, fosA6, ant(3’’)-Ia, sul1, rmtB, blaTEM-1B, blaCTX-M-65	ST11	IncFⅡK2	138.9 kb
3C11	Sputum	mph(A), aph(6)-Id, aph(3’’)-Ib, sul2, tet(A), fosA6, catA2, blaCTX-M-65, dfrA12, aadA2, sul1, blaSHV-11, blaTEM-1B, rmtB, aph(3’)-Ia	ST11	IncFⅡK2	336.5 kb
3C17	Urine	erm(42), ant(3’’)-Ia, sul1, blaCMY-2, fosA3, blaCTX-M-65, blaTEM-1B, rmtB, dfrA1, sul2, fosA6	ST11	IncFⅡK2	160 kb
3C18	Sputum	blaSHV-12, ant(3’’)-Ia, sul1, fosA6, rmtB, blaTEM-1B, blaCTX-M-65	ST11	IncFⅡK2	135 kb
3C21	Urine	sul1, aadA2, dfrA12, blaSHV-11, blaCTX-M-65, aac(3)-Iid, fosA6	ST11	IncFⅡK2	140 kb
3C23	Sputum	fosA3, sul1, aac(6’)Ib-cr, ARR-3, dfrA27, aadA16, aph(3’’)-Ib, aph(6)-Id, blaCTX-M-65, rmtB, sul2, aadA2, blaTEM-141, blaSHV-11, fosA6, oqxB, oqxA	ST11	IncFⅡK2	368 kb
3C25	Sputum	aadA2, blaTEM-141, sul1, blaCTX-M-15, aac(6’)Ib-cr, ARR-3, dfrA27, aadA16, aph(6)-Id, aph(3’’)-Ib, rmtB, sul2, blaCTX-M-65, oqxA, oqxB, blaSHV-11, fosA3	ST11	IncFⅡK2	140 kb
3C29	Urine	blaTEM-1B, rmtB, blaCTX-M-65, ant(3’’)-Ia, sul1, blaSHV-11, fosA6	ST11	IncFⅡK2	150 kb
4C2	Urine	fosA3, fosA6, dfrA14, sul2, aph(3’’)-Ib, aph(6)-Id, qnrS1, sul1, aadA2, blaCTX-M-65, rmtB, blaTEM-1B, blaSHV-11	ST11	IncFⅡK2	94 kb
4C5	Pus	blaSHV-11, rmtB, blaTEM-1B, sul1, ant(3’’)-Ia, blaCTX-M-65, fosA6, fosA3	ST11	IncFⅡK2	150 kb

Figure 2

The genetic environment of the blaKPC-2 gene in K. pneumoniae was isolated from clinical sources. The arrows represent the direction of transcription. The red open reading frame (ORF) indicates the blaKPC-2 gene, the pink ORF indicates the mobile element, the yellow ORF indicates other resistance genes or enzymes and the gray ORF indicates other genes or genes of unknown function. (A) The genetic environment of blaKPC-2 is similar in isolates 2C1, 3C1, 3C2, 3C9, 3C17, 3C18, 3C29 and 4C5. (B) The isolates 3C3, 3C23 and 3C25 all carry blaKPC-2 and share the identical genetic environment surrounding the same gene. (C) The isolate 3C21 and the isolate 4C2 have the same genetic environment. (D) The genetic environment of the isolate 3C11 carrying the blaKPC-2 gene.

Molecular Characteristics Of 14 Clinical Isolates

MLST analysis found that 14 clinically derived carbapenemase-producing K. pneumoniae were all ST11. S1-PFGE and Southern blot analysis demonstrated that the plasmid size carrying blaKPC-2 ranged from 94 kb to 368 kb (Table 1, ). By comparison of the plasmid sequences, 14 isolates belonged to the IncFIIK2 type plasmid. The results of the conjugation experiments showed that only the plasmid of the isolate 3C25 was successfully transferred to the E. coli J53. The virulence-related genes detected in 14 isolates included ybtX (100%, 14/14), mrk (93%, 13/14), fim (93%, 13/14), entB (93%, 13/). 14), entC (93%, 13/14), entF (93%, 13/14), irp1 (93%, 13/14), ybtE (0.7%, 1/14), ybtP (0.7%, 1/14) and entD (0.7%, 1/14) (). Phylogenetic analysis based on k-mer algorithm shows that 3C9 and 3C17, 3C18 and 3C29, 3C3 and 3C23 have certain phylogenetic relationships (Figure 3). Moreover, their blaKPC-2 gene has the same genetic environment (Figure 2).

Figure 3

SNP analysis of KPC-2-producing K. pneumoniae isolates, performed using kSNP. The maximum likelihood analysis of the core SNP matrix output for kSNP is performed in iTOL.

Discussion

We reported the outbreak of ST11 KPC-producing K. pneumoniae for 2 months in a general hospital in Shandong Province, China. All 14 isolates belonged to ST11 and carried the KPC-2-encoded IncFIIK2 plasmid. ST11 KPC-producing K. pneumoniae isolates are very common in China.15 In Shandong Province, there are few reports about KPC-producing K. pneumoniae causing outbreaks in the teaching hospital. Previously, we report the discovery of a KPC-2-producing Raoultella ornithinolytica isolate from well water in rural Shandong, but the gene characteristics were not described in detail.16 In this study, we describe the genetic characteristics of blaKPC-2, similar to other parts of China, and can be mediated through different molecular mechanisms.17,18 Recent reports have shown a clear correlation between the K. pneumoniae ST11 and IncFII-like plasmid. This result indicates that the IncFII-like plasmid may promote the spread of the blaKPC gene in K. pneumoniae ST11 in China.19 In this work, 14 CRKP strains were isolated from a large teaching hospital in Shandong Province in two months. MIC results (Figure 1) demonstrated that 14 isolates exhibited multidrug resistance.20 All the 14 isolates were resistant to meropenem and imipenem, and the whole-genome sequencing results showed that all isolates carried blaKPC-2, indicating that the drug-resistant phenotype was consistent with the genotype. Moreover, we have also found other drug resistance genes, such as blaCTX-M-65 and blaTEM-1 blaSHV-11 encoding β-lactam resistance; mphA encoding macrolide resistance; aac(6’)Ib-cr and aph(3’)- Ia encodes aminoglycoside resistance; sul1, sul2 and sul3 encode sulfonamide resistance; oqxA and oqxB encode fluoroquinolone resistance; fosA encodes fosfomycin resistance; tetA and tetM encode tetracycline resistance; and dfrA1 encodes trimethoprim resistance.21 These findings illustrate the multi-drug resistant phenotype of these K. pneumoniae isolates.

It is very difficult to clarify the transmission events between patients based only on epidemiological data.22 Combining genetic information with clinical epidemiological information can explain the spread of outbreak strains. Based on phylogenetic analysis and SNP differences, we can divide the 14 isolates into three branches, each representing a single transmission event (Figure 3). Combined with patient inpatient department and SNP differences, the transmission of pathogens from one patient to another was demonstrated. For example, 3C3 and 3C23, 3C18 and 3C29 have a certain affinity with each other, but from different departments, not only that, they also have the same genetic environment. The 14 isolates in this study had shorter sampling intervals and the same plasmid carrying blaKPC-2, belonging to the same clone, and having a certain relationship with each other, indicating that the outbreak occurred in a short period of time.23 When an outbreak occurs, the patient and the environment should be disinfected immediately, and the patient should be given appropriate treatment, and if necessary, the patient should be isolated, which can effectively contain the outbreak.

By analyzing virulence genes, all isolates carry the mrk operon, encoding the genes for yersiniabactin (irp1, irp2, fyuA and ybtAEPQSTUX), however, the rmpA or rmpA2 genes were absent from all isolates (Figure S2), both of which encode a high mucus phenotype and serve as high mark of virulence. The mrk operon encodes type 3 fimbriae, a virulence factor prevalent in Streptococcus pneumoniae.24 Type 3 fimbriae can not only mediate biofilm formation but also enhance bacterial adhesion to medical devices. Moreover, type 3 fimbriae may be an important factor in the formation of biofilm-associated infections, which can enter the host and persist in the clinical environment.25,26 Yersiniabactin is an iron carrier that helps bacteria gain the ability to chelate iron from infected host cells.27 In this study, all isolates carried the yersiniabactin genes pose challenges for clinical treatment.

Previous investigations have documented the diversity of blaKPC-harboring plasmids, which include IncFII, IncN, IncL/M, IncR and ColE1 groups, ranging in size from 10 to 300 kb.28–31 IncF replicons can be divided into FIA, FIB, FIC and FII groups, wherein the IncFII plasmid family exists in various Enterobacter species and plays an important role in the spread of antibacterial resistance genes such as blaKPC.32,33 In addition, the FII replicons can be divided into different subtypes, including FIIY, FIIK and FIIS, generating a number of compatible variants for overcoming the incompatibility barrier with obtaining plasmids.34 In our study, all 14 isolates belonged to the IncFIIK2 type plasmid. IncFIIK2 was a common blaKPC-harboring plasmids, reported in the United States, Israel, the United Kingdom, Italy and Colombia.32 Unlike previous studies, plasmid sizes in this study ranged from 94 kb to 368 kb, which was relatively large. The results of the conjugation assay showed that the plasmid binding in this study was difficult and the binding efficiency was less than 10%. The results correlated not only with plasmid size, but also with the presence of mobile genetic elements.35Although the 14 isolates were all in the IncFIIK2 type plasmid, the size of the plasmid carrying the blaKPC-2 was different, indicating that the blaKPC-2 in the hospital may be of various origins and spread in hospitals for many years. It is necessary to conduct a long-term retrospective genomic study of KPC-producing K. pneumoniae throughout the hospital to elucidate the evolution of KPC-producing K. pneumoniae in the hospital.

In addition, we investigated the genetic environment surrounding blaKPC-2 (Figure 2) and the results suggest that mobile genetic elements may promote the transmission of the blaKPC-2 gene. Although the genetic structure of these bacteria is different, the genetic background of blaKPC-2 is relatively similar in all plasmids. The blaKPC-2 genes were located in the same genetic context, the insertion sequence ISKpn27 is located upstream, and ISKpn6 is located downstream, except for 3C11, which is the same as previously reported.36

Conclusion

We first reported an outbreak of ST11-type KPC-2-producing K. pneumoniae in a large hospital in Shandong Province in a short period of time, although it is prevalent in China. We presented the genomic characteristics of blaKPC-2 positive K. pneumoniae isolates through whole-genome sequencing. All 14 isolates carrying the blaKPC-2 gene and have four different types of genetic environments. All isolates carried the virulence genes pose challenges for clinical treatment. It is now necessary to carry out routine genomic monitoring of such plasmids to effectively curb the spread and spread of resistant bacteria in this area.