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Distribution of integrons and phylogenetic groups among highly virulent serotypes of Klebsiella pneumoniae in a Chinese tertiary hospital
Journal Pre-proof Distribution of integrons and phylogenetic groups among highly virulent serotypes of Klebsiella pneumoniae in a Chinese tertiary hospital Wen-Jian Liao, Dan Li, Fangpeng Liu, Fang-Ling Du, Dan Long, Wei Zhang, Yang Liu
PII:
S2213-7165(19)30309-1
DOI:
https://doi.org/10.1016/j.jgar.2019.11.016
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JGAR 1103
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Journal of Global Antimicrobial Resistance
Received Date:
22 September 2019
Revised Date:
24 November 2019
Accepted Date:
27 November 2019
Please cite this article as: Liao W-Jian, Li D, Liu F, Du F-Ling, Long D, Zhang W, Liu Y, Distribution of integrons and phylogenetic groups among highly virulent serotypes of Klebsiella pneumoniae in a Chinese tertiary hospital, Journal of Global Antimicrobial Resistance (2019), doi: https://doi.org/10.1016/j.jgar.2019.11.016
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Distribution of integrons and phylogenetic groups among highly virulent serotypes of Klebsiella pneumoniae in a Chinese tertiary hospital
Wen-jian Liao1, Dan Li1, Fangpeng Liu1, Fang-ling Du2, Dan Long2, Wei Zhang1, Yang Liu2*
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1 Department of Respiratory Medicine, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, 330006, China.
2 Department of Clinical Microbiology, The First Affiliated Hospital of Nanchang University,
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Nanchang, Jiangxi, 330006, China
*Correspondence: Yang Liu, Department of Clinical Microbiology, The First Affiliated Hospital of
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Nanchang University, Nanchang, Jiangxi, 330006, China, Tel:086-13576091584
Fax:087-0791-
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Highlights
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88692748, Email:[email protected]
We detected a high prevalence of integrons(55.6%) among highly virulent serotypes (HVS)
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Klebsiella pneumoniae isolates in a Chinese tertiary hospital.
The variable regions of Class 1 integrons were polymorphic in our region.
The presence of integrons in HVS K.pneumoniae isolates resulted in increased antimicrobial resistance along with reduced virulence genes.
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Abstract Objectives: This study describes the distribution of integrons and phylogenetic groups among clinical highly virulent serotypes (HVS) Klebsiella pneumoniae isolates in a Chinese tertiary hospital. Methods: Class 1, 2, and 3 integrases were identified by polymerase chain reaction (PCR) among 90 clinical isolates of HVS Klebsiella pneumoniae. Antibiotic susceptibilities were examined by the disk diffusion method. MLST and PFGE were used to analyze the genotypes of these HVS Klebsiella
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pneumoniae isolates.
Results: Serotypes K1, K2, K20, K54, K57 accounted for 54.5%, 21.1%, 1.1%, 18.9%, and 4.4% of
the 90 isolates tested in this study. Among the 50 integron-positive isolates, 48(96%) and 2(4%) were
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classified as class 1(intI 1) an 2(intI 2) integrons, respectively. Gene cassettes encoding resistance to
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trimethoprim(dfr) and aminoglycosides(aac, aad) were found to be predominant in class 1 integrons. In
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additions, the most prevalent ST in HVS K.pneumoniae isolates was ST23 (49/90, 54.5%), followed by ST29 (11/90, 12.2%), ST86 (10/90, 11.1%), ST65(9/90, 10%), ST15(6/90, 6.7%), ST412(4/90, 4.4%),
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Conclusion:
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and ST34(1/90, 1.1%).
In summary, we found a high prevalence of integrons(55.6%) among highly virulent serotypes
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Klebsiella pneumoniae isolates in a tertiary hospital. Class 1 integrons were the most predominant and their variable regions were polymorphic in our area. The presence of integrons in HVS K.pneumoniae isolates resulted in increased antimicrobial resistance.
Keywords: Integrons; highly virulent serotypes; K.pneumoniae; drug resistance
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1. Introduction Klebsiella pneumoniae is one of the common pathogens of nosocomial infections including bloodstream infections, pneumonia, urinary tract infections, and liver abscesses[1]. With the extensive use of antibiotics, the increasing resistance of K. pneumoniae to antimicrobial agents is becoming a global concern. Many of the antibiotic-resistance genes are contained in discrete genetic elements
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known as integrons. Integrons are versatile gene acquisition systems that contribute to the prevalence
and horizontal transmission of antibiotics, disinfectants, and heavy metal resistance genes[2]. Based on
the nucleotide sequence of the integrase, three classes of integrons associated with drug resistance have
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been found. Class 1 integrons are the most common and wide-spread among bacterial species. More
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than 8000 gene cassette arrays have been identified in class 1 integrons[3].
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As described in previous studies, the capsule is an important virulence factor of K. pneumoniae. Capsule serotypes such as K1, K2, K20, K54, and K57 are found to be associated with serious
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infections, and such highly virulent serotypes (HVS) Klebsiella pneumoniae have been designated as hypervirulent Klebsiella pneumoniae[4]. Though multidrug-resistant HVS Klebsiella pneumoniae
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(MDR HVS K.pneumoniae) infections have only been reported in China[5,6], the prospect of MDR HVS Klebsiella pneumoniae undergoing wider dissemination is concerning. Thus, combined with the
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risk of horizontal gene transfer of integrons, MDR HVS Klebsiella pneumoniae is bound to become a major threat in nosocomial settings. In the present study, 90 HVS K.pneumoniae clinical isolates were collected from a tertiary hospital in Jiangxi province from 2016 to March 2018. Clinical data on each isolate was obtained; antimicrobial resistance patterns and integrons were identified and analyzed. Furthermore, The
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molecular typing of these isolates was performed by MLST and PFGE.
2. Material and methods 2.1 Bacterial isolates and antimicrobial susceptibilities A total of 620 nonduplicate K.pneumoniae clinical isolates were collected from the First Affiliated Hospital of Nanchang University in the southeastern region of China from 2016 to March 2018. K.pneumoniae isolates were identified by an automated Vitek II system (bioMerieux, Balmes-les-
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Grottes, France) and were further verified with 16S rRNA gene sequencing. Antibiotic susceptibilities were examined by the disk diffusion method on Mueller-Hinton agar according to the Clinical and
Laboratory Standards Institute (CLSI) guidelines[7]. Escherichia coli ATCC 25922 and K. pneumoniae
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ATCC 700603 were used as quality control.
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2.2 PCR detection of highly virulent serotypes, virulence genes, drug resistance genes, integrons, and
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variable regions
The DNA template was prepared using the boiling method in sterile distilled water for ten
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minutes. Six capsular serotypes( K1, K2, K5, K20, K54, K57) related to highly virulent strains, virulence genes(aerobactin, rmpA) were identified using polymerase chain reaction (PCR)
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amplification as described in previous papers[3,8]. Among the total 620 isolates of K.pneumoniae, 90 (14.5%) were identified as highly virulent serotypes. All highly virulent serotypes isolates were
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screened for the presence of integrase genes(intI1, intI2, and intI3) and variable regions by PCR[9]. PCR was also used to analyze ESBLs genes(CTX-M, SHV, TEM) and carbapenemase genes(KPC, IMP, VIM, NDM, OXA-48)[10]. PCR products were purified and sequenced, the sequences were analyzed with the BLAST program at the NCBI homepage (http://blast.ncbi,nlm.nih.gov/Blast .cgi). 2.3 PFGE and MLST
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All the HVS K.pneumoniae strains were subjected to PFGE after digestion with XbaI. The cluster cutoff line at 80% similarity was used to analyze the genetic relatedness. MLST was performed by the international K.pneumoniae MLST scheme (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Klebsiellapneumoniae.html), including seven house-keeping genes. The sequence types (STs) were determined using the MLST database. PFGE and
2.4 Statistical analysis
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MLST were performed as previously described[10].
SPSS software(version 25.0) was used for data analysis. Chi-square tests or Fisher’s exact tests
were used to test the association of a set of counts or frequencies between different K.pneumoniae data
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sets. All tests with a p-value <0.05 were taken as significant.
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3. Results
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3.1 Prevalence of integrons and clinical characteristics
Overall, 90 patients had proven acquisition of HVS Klebsiella pneumoniae isolates. Based on the
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presence of integrase genes, these HVS Klebsiella pneumoniae isolates were divided into two groups (integron-positive isolates and integron-negative isolates). PCR analysis of integron genes revealed that
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50 (55.6%) HVS Klebsiella pneumoniae isolates had integrase genes and 40 (44.4%) HVS Klebsiella pneumoniae isolates had no integrase genes. The integron-positive HVS Klebsiella pneumoniae isolates
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were mainly collected from departments of Neurology, Neurosurgery, Gastroenterology, Respiratory, NICU, and ICU, as shown in Table1. There was no significant difference in sex, age, department, use of invasive devices, surgery or major trauma, transfer, mortality in 30 days between the two groups. Patients infected with integron-positive isolates had higher ICU admission, diabetes prevalence than patients infected with integron-negative isolates (P<0.05). Interestingly pulmonary infections were
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more frequent in patients infected with integron-negative isolates than in patients infected with integron-positive isolates. 3.2 Detection of highly virulent serotypes, virulence genes, ESBLs genes, and carbapenemase genes Serotypes K1, K2, K20, K54, K57 accounted for 54.5%, 21.1%, 1.1%, 18.9%, and 4.4% of the 90 isolates tested; on the other hand, serotype K5 was not detected in this study. As shown in table2, there was no significant difference in integrons among highly virulent serotypes isolates. Interestingly the
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two virulence genes rmpA2 and aerobactin were more frequent in integron-negative isolates than in integron-positive isolates (P<0.05). PCR analysis of drug resistance genes revealed carbapenemase
genes(IMP, VIM, OXA-48) were not detected in the current study. There was no significant difference
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in ESBLs genes(SHV, TEM, CTX-M-14) and carbapenemase genes(KPC-2, NDM-1) between the two
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groups.
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3.3 Association between integrons and antimicrobial susceptibilities
The seventeen antibiotic susceptibility profiles are shown in Table 3. The association between
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integron carriage and antibiotic resistance was most significant for Cefuroxime, Aztreonam, Tobramycin, and Trimethoprim(P<0.01). In spite of finding no statistical significance, the other
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thirteen antibiotic resistance profiles seemed to be more frequent in integron-positive isolates than in integron-negative isolates.
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3.4 Analysis of integrase genes and gene cassettes Among the 50 integron-positive isolates, 48(96%) and 2(4%) were classified as class 1(intI 1) and
2(intI 2) integrons, respectively. No class 3 integrons were detected in this study. Variable regions could be amplified in 32（64%） integron-positive isolates. We identified 11 different gene arrays(AK)(figure 1) including those encoding resistance to trimethoprim(dfrA1, drfA5, dfrA12, drfA17,
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drfA27), aminoglycosides(aadAl, aadA2, aadA4, aadA5, aacA4, antI ), rifampin(arr3), quinolone(acc(6’)-Ib-cr), and hypothetical protein-coding genes(orfC, orf1, orf2, orf3). Nine gene arrays(A, C, D, E, F, G, H, I, J) were found in K1 serotype isolates. Four gene arrays(A, B, K, F) were found in K2 serotype isolates. Five gene arrays(A, C, D, F, H) were found in K54 serotype isolates. Only gene array F was detected in K20 serotype isolates. The polymorphism of gene cassettes in variable regions is detailed in figure 2.
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3.5 PFGE and MLST
The PFGE-based fingerprints of the HVS K.pneumoniae isolates displayed four different clusters (named A-D) using a similarity cutoff value of 80%. Cluster A comprised 49/90 isolates (54.4%),
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cluster B 19/90 isolates (21.1%), cluster C 17/90 (18.9%) and cluster D 4/90 isolates (4.4%). The
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MLST analysis distinguished a total of seven different STs. The most prevalent ST in HVS
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K.pneumoniae isolates was ST23 (49/90, 54.5%), followed by ST29 (11/90, 12.2%), ST86 (10/90, 11.1%), ST65(9/90, 10%), ST15(6/90, 6.7%), ST412(4/90, 4.4%), and ST34(1/90, 1.1%). We found a
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remarkable correlation between MLST and PFGE results, with 49 cluster A isolates belonging to ST23, 19 cluster B isolates belonging to ST65 and ST86, 17 cluster C isolates belonging to ST15 and ST29,
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and four cluster D isolates belonging to ST412, respectively. Interestingly, isolates belonging to clusters A, B, C, and D corresponded to highly virulent serotypes K1, K2, K54, and K57, respectively (Figure.
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2).
4. Discussion
In this study, five highly virulent serotypes, K1(49 isolates, 54.5%), K2(19 isolates, 21.1%), K20(1 isolate, 1.1%), K54(17 isolates, 18.9%), and K57(4 isolates, 4.4%) were detected among 90 isolates. Our results are consistent with the epidemiology of HVS K. pneumoniae from previous studies
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which indicate that K1 and K2 are the predominant capsular serotypes[11,12]. PCR analysis of virulence genes revealed that rmpA2 and aerobactin were more frequent in integron negative isolates than in integron positive isolates (P<0.05). This was in opposition to findings in a previous study[13].Because the virulence gene wcaG was located in the transferable regions of the chromosome and could spread through horizontal gene transfer by class 1 integrons. A reasonable explanation in this study was that virulence genes rmpA and aerobactin were on the intrinsic virulence plasmids of HVS
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K.pneumoniae isolates. The association between susceptibility to certain antibiotics in several HVS
K.pneumoniae STs and the presence of I-E* CRISPR-Cas systems has already been suggested[14]. In this sense, a plausible explanation for the association between the presence of virulence genes rmpA
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and aerobactin, and the lack of integrons could be the occurrence of I-E* CRISPR-Cas systems. Such
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thus maintaining antibiotic susceptibility.
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isolates could feature the I-E* CRISPR-Cas systems to clear foreign DNA fragments (e.g: integrons)
PCR analysis of 50 integron-positive isolates revealed that class 1 integrons were the most
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frequent as described in other studies concerning drug-resistant K. pneumoniae[15-17]. We also found a significant association between integron carriage and resistance to aminoglycosides and sulfonamides.
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Interestingly genes encoding resistance to aminoglycosides or sulfonamides were mostly located towards the 5’CS of integrons. Despite the evidently higher phenotypic resistance to cefuroxime and
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aztreonam in integron-positive HVS K. pneumoniae isolates this study, no gene cassette encoding resistance to cefuroxime and aztreonam was detected. This could be due to the fact that the resistance genes to cefuroxime and aztreonam were located outside the integrons. So drug resistance genes(ESBLs genes, and carbapenemase genes) in these isolates were further analyzed by PCR. Inspite of no significant difference in drug resistance genes(ESBL genes and carbapenemase genes) between
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the two groups, these drug resistance genes seemed to be more frequent in integron-positive isolates than in integron-negative isolates in our hospital, probably accounting for higher ICU admission of patients infected with integron-positive isolates. Certainly, specific mechanisms between the integronpositive isolates and beta-lactam antibiotics resistance need to be further explored. PCR analysis of variable regions revealed the existence of 11 different gene arrays (A-K); the most prevalent were I(F)(6/32, 18.8%) and I(H)(6/32, 18.8%), followed by I(A)(5/32, 15.6%) and
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I(C)(5/32, 15.6%), which may reflect the stability and transferability of these gene cassettes as reported in several previous studies[16,18,19]. Another interesting finding was that most I(F) class 1 integron positive HVS K. pneumoniae isolates carried more than three drug resistance gene cassettes
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simultaneously. Furthermore, the presence of ant, arr, and acc(6’)-Ib-cr in class 1 integrons combined
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with different cassettes, highlighting the plasticity of these genetic elements. All described above were
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in agreement with other studies from different geographical areas that showed a high polymorphism of integrons among K. pneumoniae isolates[20-22]. It was worth noting that 18(36%) of the integron-
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positive HVS K. pneumoniae isolates harbored empty integrons, as documented elsewhere[23]. These empty integrons may eventually capture new resistance genes (especially in hospital environments due
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to intense antibiotic selective pressure), or simply be the result of either defective integrases or defective integrase promoters.
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According to PFGE and MLST analysis, the 90 highly virulent serotypes K. pneumoniae
isolates were divided into 5 different clusters and 7different STs, respectively. Cluster A(ST23) was the most abundant, followed by Cluster B(ST65 and ST86), Cluster C(ST15 and ST29), Cluster D(ST412), and one ST34. As shown in Figure 2, our data revealed that integrons with different variable regions maybe could be transmissible by both horizontal transfer and clonal spread.
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5. Conclusion In summary, we found a high prevalence of integrons(55.6%) among HVS K.pneumoniae isolates in a tertiary hospital. Class 1 integrons were the most predominant and their variable regions were polymorphic in our area. The presence of integrons in HVS K.pneumoniae isolates resulted in increased antimicrobial resistance.
6. Ethics Statement
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The study has been evaluated by the Ethics Committee of the First Affiliated Hospital of
Nanchang University. Patients involved in the study were anonymized, no informed consent was required on account of being a retrospective study.
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7. Acknowledgments
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Financial support was provided by the National Natural Science Foundation of China (81860368).
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8. Author contributions
FLD and DL performed the laboratory measurements. YL and WJL made substantial contributions
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to conception and design. WZ and YL revised the manuscript critically for important intellectual content. FPL and DL participated in experimental design and data analysis. WJL drafted the
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manuscript. All authors read and approved the final manuscript.
9. Declarations
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Funding: Financial support was provided by the National Natural Science Foundation of China (81860368).
Competing Interests: The authors report no conflicts of interest in this work. Ethical Approval: The study has been evaluated by the Ethics Committee of the First Affiliated Hospital of Nanchang University.
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Quinolone Resistance Determinants Among Serotype K1 highly virulent Klebsiella pneumoniae
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Eur J Clin Microbiol Infect Dis 2014, 33:365-369. [13] Derakhshan S, Najar PS, Bakhshi B: Association Between Presence of Virulence Genes and
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Antibiotic Resistance in Clinical Klebsiella Pneumoniae Isolates. Lab Med 2016, 47:306-311. [14] Li HY, Kao CY, Lin WH, Zheng PX, Yan JJ, Wang MC, et al. Characterization of CRISPR-Cas
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Klebsiella pneumoniae clinical and microbiota isolates belonging to different phylogenetic groups in Recife, State of Pernambuco. Rev Soc Bras Med Trop 2014, 47:165-169.
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[22] Karczmarczyk M, Abbott Y, Walsh C, Leonard N, Fanning S: Characterization of multidrugresistant Escherichia coli isolates from animals presenting at a university veterinary hospital. Appl Environ Microbiol 2011, 77:7104-7112. [23] Eftekhari N, Bakhshi B, Pourshafie MR, Zarbakhsh B, Rahbar M, Hajia M, et al: Genetic diversity
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Figure 1 Diagram of the 11 different gene arrays found in 32 variable regions of integron-positive
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isolates
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Figure 2 Pulsed-field gel electrophoresis (PFGE) patterns, K serotypes, STs, and integrons among
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90 clinical HVS K.pneumoniae isolates
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Table 1 Association between clinical characteristics and integron carriage in clinical isolates Clinical characteristics
Integron-positive isolates
Integron-negative isolates
(n=50)
(n=40)
Sex Male
35(70%)
24(60%)
Female Age (> 65 years)
15(30%) 35(70%)
16(40%) 23(57.5%)
Neurology Neurosurgery
8(16%) 6(12%)
Gastroenterology NICU Respiratory ICU
χ2
P-value
0.984
0.321
0.984 1.515
0.321 0.218
1(3%) 2(5%)
3.125 0.619
0.077 0.431
7(14%) 6(12%)
9(23%) 5(12%)
1.098 0
0.295 1
3(6%) 5(10%)
4(10%) 1(2%)
0.095 0.984
0.758 0.321
Hypertension Hemorrhage
11(22%) 6(12%)
6(15%) 4(10%)
0.711 0
0.399 1
Pulmonary infection Diabetes
8(16%) 7(14%)
14(35%) 0(0%)
4.344 4.277 9
0.037 0.039
Mechanical ventilation Endotracheal intubation
16(32%) 17(34%)
10(25%) 12(30%)
0.530 0.163
0.467 0.687
Tracheotomy Drainage tube
10(20%) 15(30%)
6(15%) 12(30%)
0.380 0
0.538 1
8(20%) 2(5%)
0.168 1.125
0.28 0.289
29(58%)
14(35%)
4.712
Surgery or Major trauma 29(58%) Transfer 12(24%)
24(60%) 8(20%)
0.037 0.206
0.03 0.848 0.65
Mortality in 30 days
8(20%)
0.206
0.65
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Comorbidity
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Use of invasive devices
Indwelling catheterization 15(30%) Central venous catheter 7(14%)
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ICU admission
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Department
12(24%)
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Statistically significant correlations (p < 0.05) are shown in bold font.
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Table 2 Association between virulence genes, ESBLs genes, carbapenemase genes, and integron carriage in clinical isolates of highly virulent serotypes K. pneumoniae Integron-positive isolates
Integron-negative isolates
(n=50)
(n=40)
2
20（50%）
P-value
K1
29(58%)
K2
8(12%)
K5
0
0
K20
1(2%)
0
0.8
K54
11(22%)
6(15%)
0.711
0.399
K57
1(2%)
3(7.5%)
1.583
0.319
11.222
0.001
3.95
0.047
0.512
0.474
0
1
0(0)
NA
0(0)
NA
NA NA
12(30%)
2.394
0.122
11(27.5%)
0.573
0.449
1.765
0.184
0
1
33(66%)
38(95%)
aerobactin
41(82%)
39(97.5%)
KPC-2
9(18%)
5(12.5%)
NDM-1
2(4%)
1(2.5%)
IMP
0(0)
NA
VIM
0(0)
OXA-48
0(0)
0(0)
NA
SHV
23(46%)
TEM
20(40%)
10(25%)
2.25
0.134
CTX-M-14
16(32%)
9(22.5)
1.0
0.317
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rmpA
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Statistically significant correlations (p < 0.05) are shown in bold font. NA: Not Applicable
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Table 3 Antimicrobial susceptibilities and integron carriage in clinical isolates
Antimicrobal susceptibility Total(n=90)
Integron-positive
Integron-negative
isolates (n=50)
isolates (n=40)
χ2
P-value
Cefazolin
32(35.6%)
15(30%)
17(42.5%)
1.515 0.218
Ceftazidime
62(68.9%)
34(68%)
28(70%)
0.041 0.839
Cefepime
66(73.3%)
33(66%)
33(82.5%)
3.094 0.079
Cefuroxime
61(65.6%)
27(54%)
34(85%)
9.778 0.002
Ampicillin
0(0)
0(0)
0(0)
0
Piperacillin tazobactam
76(84.4%)
38(76%)
34(85%)
1.125 0.289
Aztreonam
54(60%)
24(48%)
30(75%)
6.75
Gentamicin
72(80%)
36(72%)
34(75%)
2.173 0.140
Tobramycin
75(83.3%)
31(62%)
35(87.5%)
7.389 0.007
Amikacin
71(78.9%)
36(72%)
35(87.5%)
3.206 0.073
Levofloxacin
76(84.4%)
38(76%)
35(87.5%)
1.918 0.166
Ciprofloxacin
68(75.6%)
34(68%)
34(85%)
3.477 0.062
Trimethoprim
64(71.1%)
29(58%)
34(85%)
7.714 0.005
Ertapenem
72(80%)
37(74%)
35(87.5%)
2.531 0.112
Meropenem
74(82.2%)
40(80%)
34(85%)
0.38
Imipenem
75(83.3%)
41(82%)
34(85%)
0.144 0.704
Cefotaxime
60(66.7%)
32(64%)
28(70%)
0.36
1
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0.009
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Statistically significant correlations (p < 0.05) are shown in bold font.
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0.538
0.549
PII:
S2213-7165(19)30309-1
DOI:
https://doi.org/10.1016/j.jgar.2019.11.016
Reference:
JGAR 1103
To appear in:
Journal of Global Antimicrobial Resistance
Received Date:
22 September 2019
Revised Date:
24 November 2019
Accepted Date:
27 November 2019
Please cite this article as: Liao W-Jian, Li D, Liu F, Du F-Ling, Long D, Zhang W, Liu Y, Distribution of integrons and phylogenetic groups among highly virulent serotypes of Klebsiella pneumoniae in a Chinese tertiary hospital, Journal of Global Antimicrobial Resistance (2019), doi: https://doi.org/10.1016/j.jgar.2019.11.016
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Distribution of integrons and phylogenetic groups among highly virulent serotypes of Klebsiella pneumoniae in a Chinese tertiary hospital
Wen-jian Liao1, Dan Li1, Fangpeng Liu1, Fang-ling Du2, Dan Long2, Wei Zhang1, Yang Liu2*
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1 Department of Respiratory Medicine, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, 330006, China.
2 Department of Clinical Microbiology, The First Affiliated Hospital of Nanchang University,
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Nanchang, Jiangxi, 330006, China
*Correspondence: Yang Liu, Department of Clinical Microbiology, The First Affiliated Hospital of
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Nanchang University, Nanchang, Jiangxi, 330006, China, Tel:086-13576091584
Fax:087-0791-
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Highlights
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88692748, Email:[email protected]
We detected a high prevalence of integrons(55.6%) among highly virulent serotypes (HVS)
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Klebsiella pneumoniae isolates in a Chinese tertiary hospital.
The variable regions of Class 1 integrons were polymorphic in our region.
The presence of integrons in HVS K.pneumoniae isolates resulted in increased antimicrobial resistance along with reduced virulence genes.
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Abstract Objectives: This study describes the distribution of integrons and phylogenetic groups among clinical highly virulent serotypes (HVS) Klebsiella pneumoniae isolates in a Chinese tertiary hospital. Methods: Class 1, 2, and 3 integrases were identified by polymerase chain reaction (PCR) among 90 clinical isolates of HVS Klebsiella pneumoniae. Antibiotic susceptibilities were examined by the disk diffusion method. MLST and PFGE were used to analyze the genotypes of these HVS Klebsiella
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pneumoniae isolates.
Results: Serotypes K1, K2, K20, K54, K57 accounted for 54.5%, 21.1%, 1.1%, 18.9%, and 4.4% of
the 90 isolates tested in this study. Among the 50 integron-positive isolates, 48(96%) and 2(4%) were
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classified as class 1(intI 1) an 2(intI 2) integrons, respectively. Gene cassettes encoding resistance to
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trimethoprim(dfr) and aminoglycosides(aac, aad) were found to be predominant in class 1 integrons. In
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additions, the most prevalent ST in HVS K.pneumoniae isolates was ST23 (49/90, 54.5%), followed by ST29 (11/90, 12.2%), ST86 (10/90, 11.1%), ST65(9/90, 10%), ST15(6/90, 6.7%), ST412(4/90, 4.4%),
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Conclusion:
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and ST34(1/90, 1.1%).
In summary, we found a high prevalence of integrons(55.6%) among highly virulent serotypes
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Klebsiella pneumoniae isolates in a tertiary hospital. Class 1 integrons were the most predominant and their variable regions were polymorphic in our area. The presence of integrons in HVS K.pneumoniae isolates resulted in increased antimicrobial resistance.
Keywords: Integrons; highly virulent serotypes; K.pneumoniae; drug resistance
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1. Introduction Klebsiella pneumoniae is one of the common pathogens of nosocomial infections including bloodstream infections, pneumonia, urinary tract infections, and liver abscesses[1]. With the extensive use of antibiotics, the increasing resistance of K. pneumoniae to antimicrobial agents is becoming a global concern. Many of the antibiotic-resistance genes are contained in discrete genetic elements
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known as integrons. Integrons are versatile gene acquisition systems that contribute to the prevalence
and horizontal transmission of antibiotics, disinfectants, and heavy metal resistance genes[2]. Based on
the nucleotide sequence of the integrase, three classes of integrons associated with drug resistance have
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been found. Class 1 integrons are the most common and wide-spread among bacterial species. More
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than 8000 gene cassette arrays have been identified in class 1 integrons[3].
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As described in previous studies, the capsule is an important virulence factor of K. pneumoniae. Capsule serotypes such as K1, K2, K20, K54, and K57 are found to be associated with serious
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infections, and such highly virulent serotypes (HVS) Klebsiella pneumoniae have been designated as hypervirulent Klebsiella pneumoniae[4]. Though multidrug-resistant HVS Klebsiella pneumoniae
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(MDR HVS K.pneumoniae) infections have only been reported in China[5,6], the prospect of MDR HVS Klebsiella pneumoniae undergoing wider dissemination is concerning. Thus, combined with the
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risk of horizontal gene transfer of integrons, MDR HVS Klebsiella pneumoniae is bound to become a major threat in nosocomial settings. In the present study, 90 HVS K.pneumoniae clinical isolates were collected from a tertiary hospital in Jiangxi province from 2016 to March 2018. Clinical data on each isolate was obtained; antimicrobial resistance patterns and integrons were identified and analyzed. Furthermore, The
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molecular typing of these isolates was performed by MLST and PFGE.
2. Material and methods 2.1 Bacterial isolates and antimicrobial susceptibilities A total of 620 nonduplicate K.pneumoniae clinical isolates were collected from the First Affiliated Hospital of Nanchang University in the southeastern region of China from 2016 to March 2018. K.pneumoniae isolates were identified by an automated Vitek II system (bioMerieux, Balmes-les-
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Grottes, France) and were further verified with 16S rRNA gene sequencing. Antibiotic susceptibilities were examined by the disk diffusion method on Mueller-Hinton agar according to the Clinical and
Laboratory Standards Institute (CLSI) guidelines[7]. Escherichia coli ATCC 25922 and K. pneumoniae
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ATCC 700603 were used as quality control.
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2.2 PCR detection of highly virulent serotypes, virulence genes, drug resistance genes, integrons, and
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variable regions
The DNA template was prepared using the boiling method in sterile distilled water for ten
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minutes. Six capsular serotypes( K1, K2, K5, K20, K54, K57) related to highly virulent strains, virulence genes(aerobactin, rmpA) were identified using polymerase chain reaction (PCR)
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amplification as described in previous papers[3,8]. Among the total 620 isolates of K.pneumoniae, 90 (14.5%) were identified as highly virulent serotypes. All highly virulent serotypes isolates were
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screened for the presence of integrase genes(intI1, intI2, and intI3) and variable regions by PCR[9]. PCR was also used to analyze ESBLs genes(CTX-M, SHV, TEM) and carbapenemase genes(KPC, IMP, VIM, NDM, OXA-48)[10]. PCR products were purified and sequenced, the sequences were analyzed with the BLAST program at the NCBI homepage (http://blast.ncbi,nlm.nih.gov/Blast .cgi). 2.3 PFGE and MLST
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All the HVS K.pneumoniae strains were subjected to PFGE after digestion with XbaI. The cluster cutoff line at 80% similarity was used to analyze the genetic relatedness. MLST was performed by the international K.pneumoniae MLST scheme (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Klebsiellapneumoniae.html), including seven house-keeping genes. The sequence types (STs) were determined using the MLST database. PFGE and
2.4 Statistical analysis
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MLST were performed as previously described[10].
SPSS software(version 25.0) was used for data analysis. Chi-square tests or Fisher’s exact tests
were used to test the association of a set of counts or frequencies between different K.pneumoniae data
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sets. All tests with a p-value <0.05 were taken as significant.
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3. Results
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3.1 Prevalence of integrons and clinical characteristics
Overall, 90 patients had proven acquisition of HVS Klebsiella pneumoniae isolates. Based on the
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presence of integrase genes, these HVS Klebsiella pneumoniae isolates were divided into two groups (integron-positive isolates and integron-negative isolates). PCR analysis of integron genes revealed that
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50 (55.6%) HVS Klebsiella pneumoniae isolates had integrase genes and 40 (44.4%) HVS Klebsiella pneumoniae isolates had no integrase genes. The integron-positive HVS Klebsiella pneumoniae isolates
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were mainly collected from departments of Neurology, Neurosurgery, Gastroenterology, Respiratory, NICU, and ICU, as shown in Table1. There was no significant difference in sex, age, department, use of invasive devices, surgery or major trauma, transfer, mortality in 30 days between the two groups. Patients infected with integron-positive isolates had higher ICU admission, diabetes prevalence than patients infected with integron-negative isolates (P<0.05). Interestingly pulmonary infections were
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more frequent in patients infected with integron-negative isolates than in patients infected with integron-positive isolates. 3.2 Detection of highly virulent serotypes, virulence genes, ESBLs genes, and carbapenemase genes Serotypes K1, K2, K20, K54, K57 accounted for 54.5%, 21.1%, 1.1%, 18.9%, and 4.4% of the 90 isolates tested; on the other hand, serotype K5 was not detected in this study. As shown in table2, there was no significant difference in integrons among highly virulent serotypes isolates. Interestingly the
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two virulence genes rmpA2 and aerobactin were more frequent in integron-negative isolates than in integron-positive isolates (P<0.05). PCR analysis of drug resistance genes revealed carbapenemase
genes(IMP, VIM, OXA-48) were not detected in the current study. There was no significant difference
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in ESBLs genes(SHV, TEM, CTX-M-14) and carbapenemase genes(KPC-2, NDM-1) between the two
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groups.
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3.3 Association between integrons and antimicrobial susceptibilities
The seventeen antibiotic susceptibility profiles are shown in Table 3. The association between
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integron carriage and antibiotic resistance was most significant for Cefuroxime, Aztreonam, Tobramycin, and Trimethoprim(P<0.01). In spite of finding no statistical significance, the other
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thirteen antibiotic resistance profiles seemed to be more frequent in integron-positive isolates than in integron-negative isolates.
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3.4 Analysis of integrase genes and gene cassettes Among the 50 integron-positive isolates, 48(96%) and 2(4%) were classified as class 1(intI 1) and
2(intI 2) integrons, respectively. No class 3 integrons were detected in this study. Variable regions could be amplified in 32（64%） integron-positive isolates. We identified 11 different gene arrays(AK)(figure 1) including those encoding resistance to trimethoprim(dfrA1, drfA5, dfrA12, drfA17,
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drfA27), aminoglycosides(aadAl, aadA2, aadA4, aadA5, aacA4, antI ), rifampin(arr3), quinolone(acc(6’)-Ib-cr), and hypothetical protein-coding genes(orfC, orf1, orf2, orf3). Nine gene arrays(A, C, D, E, F, G, H, I, J) were found in K1 serotype isolates. Four gene arrays(A, B, K, F) were found in K2 serotype isolates. Five gene arrays(A, C, D, F, H) were found in K54 serotype isolates. Only gene array F was detected in K20 serotype isolates. The polymorphism of gene cassettes in variable regions is detailed in figure 2.
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3.5 PFGE and MLST
The PFGE-based fingerprints of the HVS K.pneumoniae isolates displayed four different clusters (named A-D) using a similarity cutoff value of 80%. Cluster A comprised 49/90 isolates (54.4%),
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cluster B 19/90 isolates (21.1%), cluster C 17/90 (18.9%) and cluster D 4/90 isolates (4.4%). The
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MLST analysis distinguished a total of seven different STs. The most prevalent ST in HVS
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K.pneumoniae isolates was ST23 (49/90, 54.5%), followed by ST29 (11/90, 12.2%), ST86 (10/90, 11.1%), ST65(9/90, 10%), ST15(6/90, 6.7%), ST412(4/90, 4.4%), and ST34(1/90, 1.1%). We found a
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remarkable correlation between MLST and PFGE results, with 49 cluster A isolates belonging to ST23, 19 cluster B isolates belonging to ST65 and ST86, 17 cluster C isolates belonging to ST15 and ST29,
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and four cluster D isolates belonging to ST412, respectively. Interestingly, isolates belonging to clusters A, B, C, and D corresponded to highly virulent serotypes K1, K2, K54, and K57, respectively (Figure.
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2).
4. Discussion
In this study, five highly virulent serotypes, K1(49 isolates, 54.5%), K2(19 isolates, 21.1%), K20(1 isolate, 1.1%), K54(17 isolates, 18.9%), and K57(4 isolates, 4.4%) were detected among 90 isolates. Our results are consistent with the epidemiology of HVS K. pneumoniae from previous studies
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which indicate that K1 and K2 are the predominant capsular serotypes[11,12]. PCR analysis of virulence genes revealed that rmpA2 and aerobactin were more frequent in integron negative isolates than in integron positive isolates (P<0.05). This was in opposition to findings in a previous study[13].Because the virulence gene wcaG was located in the transferable regions of the chromosome and could spread through horizontal gene transfer by class 1 integrons. A reasonable explanation in this study was that virulence genes rmpA and aerobactin were on the intrinsic virulence plasmids of HVS
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K.pneumoniae isolates. The association between susceptibility to certain antibiotics in several HVS
K.pneumoniae STs and the presence of I-E* CRISPR-Cas systems has already been suggested[14]. In this sense, a plausible explanation for the association between the presence of virulence genes rmpA
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and aerobactin, and the lack of integrons could be the occurrence of I-E* CRISPR-Cas systems. Such
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thus maintaining antibiotic susceptibility.
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isolates could feature the I-E* CRISPR-Cas systems to clear foreign DNA fragments (e.g: integrons)
PCR analysis of 50 integron-positive isolates revealed that class 1 integrons were the most
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frequent as described in other studies concerning drug-resistant K. pneumoniae[15-17]. We also found a significant association between integron carriage and resistance to aminoglycosides and sulfonamides.
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Interestingly genes encoding resistance to aminoglycosides or sulfonamides were mostly located towards the 5’CS of integrons. Despite the evidently higher phenotypic resistance to cefuroxime and
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aztreonam in integron-positive HVS K. pneumoniae isolates this study, no gene cassette encoding resistance to cefuroxime and aztreonam was detected. This could be due to the fact that the resistance genes to cefuroxime and aztreonam were located outside the integrons. So drug resistance genes(ESBLs genes, and carbapenemase genes) in these isolates were further analyzed by PCR. Inspite of no significant difference in drug resistance genes(ESBL genes and carbapenemase genes) between
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the two groups, these drug resistance genes seemed to be more frequent in integron-positive isolates than in integron-negative isolates in our hospital, probably accounting for higher ICU admission of patients infected with integron-positive isolates. Certainly, specific mechanisms between the integronpositive isolates and beta-lactam antibiotics resistance need to be further explored. PCR analysis of variable regions revealed the existence of 11 different gene arrays (A-K); the most prevalent were I(F)(6/32, 18.8%) and I(H)(6/32, 18.8%), followed by I(A)(5/32, 15.6%) and
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I(C)(5/32, 15.6%), which may reflect the stability and transferability of these gene cassettes as reported in several previous studies[16,18,19]. Another interesting finding was that most I(F) class 1 integron positive HVS K. pneumoniae isolates carried more than three drug resistance gene cassettes
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simultaneously. Furthermore, the presence of ant, arr, and acc(6’)-Ib-cr in class 1 integrons combined
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with different cassettes, highlighting the plasticity of these genetic elements. All described above were
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in agreement with other studies from different geographical areas that showed a high polymorphism of integrons among K. pneumoniae isolates[20-22]. It was worth noting that 18(36%) of the integron-
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positive HVS K. pneumoniae isolates harbored empty integrons, as documented elsewhere[23]. These empty integrons may eventually capture new resistance genes (especially in hospital environments due
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to intense antibiotic selective pressure), or simply be the result of either defective integrases or defective integrase promoters.
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According to PFGE and MLST analysis, the 90 highly virulent serotypes K. pneumoniae
isolates were divided into 5 different clusters and 7different STs, respectively. Cluster A(ST23) was the most abundant, followed by Cluster B(ST65 and ST86), Cluster C(ST15 and ST29), Cluster D(ST412), and one ST34. As shown in Figure 2, our data revealed that integrons with different variable regions maybe could be transmissible by both horizontal transfer and clonal spread.
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5. Conclusion In summary, we found a high prevalence of integrons(55.6%) among HVS K.pneumoniae isolates in a tertiary hospital. Class 1 integrons were the most predominant and their variable regions were polymorphic in our area. The presence of integrons in HVS K.pneumoniae isolates resulted in increased antimicrobial resistance.
6. Ethics Statement
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The study has been evaluated by the Ethics Committee of the First Affiliated Hospital of
Nanchang University. Patients involved in the study were anonymized, no informed consent was required on account of being a retrospective study.
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7. Acknowledgments
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Financial support was provided by the National Natural Science Foundation of China (81860368).
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8. Author contributions
FLD and DL performed the laboratory measurements. YL and WJL made substantial contributions
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to conception and design. WZ and YL revised the manuscript critically for important intellectual content. FPL and DL participated in experimental design and data analysis. WJL drafted the
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manuscript. All authors read and approved the final manuscript.
9. Declarations
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Funding: Financial support was provided by the National Natural Science Foundation of China (81860368).
Competing Interests: The authors report no conflicts of interest in this work. Ethical Approval: The study has been evaluated by the Ethics Committee of the First Affiliated Hospital of Nanchang University.
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Antibiotic Resistance in Clinical Klebsiella Pneumoniae Isolates. Lab Med 2016, 47:306-311. [14] Li HY, Kao CY, Lin WH, Zheng PX, Yan JJ, Wang MC, et al. Characterization of CRISPR-Cas
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[18] Zeighami H, Haghi F, Hajiahmadi F: Molecular characterization of integrons in clinical isolates of betalactamase-producing Escherichia coli and Klebsiella pneumoniae in Iran. J Chemother 2015, 27:145-151.
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[19] Li B, Hu Y, Wang Q, Yi Y, Woo PC, Jing H, et al: Structural diversity of class 1 integrons and their
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Klebsiella pneumoniae clinical and microbiota isolates belonging to different phylogenetic groups in Recife, State of Pernambuco. Rev Soc Bras Med Trop 2014, 47:165-169.
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[21] Chang CY, Fang YT, Tsai SM, Chang LL, Yu WL: Characterization of class 1 integrons and gene cassettes in clinical isolates of Klebsiella pneumoniae from Taiwan. Diagn Microbiol Infect Dis
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[22] Karczmarczyk M, Abbott Y, Walsh C, Leonard N, Fanning S: Characterization of multidrugresistant Escherichia coli isolates from animals presenting at a university veterinary hospital. Appl Environ Microbiol 2011, 77:7104-7112. [23] Eftekhari N, Bakhshi B, Pourshafie MR, Zarbakhsh B, Rahbar M, Hajia M, et al: Genetic diversity
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of Shigella spp. and their integron content. Foodborne Pathog Dis 2013, 10:237-242.
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Figure 1 Diagram of the 11 different gene arrays found in 32 variable regions of integron-positive
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isolates
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Figure 2 Pulsed-field gel electrophoresis (PFGE) patterns, K serotypes, STs, and integrons among
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90 clinical HVS K.pneumoniae isolates
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Table 1 Association between clinical characteristics and integron carriage in clinical isolates Clinical characteristics
Integron-positive isolates
Integron-negative isolates
(n=50)
(n=40)
Sex Male
35(70%)
24(60%)
Female Age (> 65 years)
15(30%) 35(70%)
16(40%) 23(57.5%)
Neurology Neurosurgery
8(16%) 6(12%)
Gastroenterology NICU Respiratory ICU
χ2
P-value
0.984
0.321
0.984 1.515
0.321 0.218
1(3%) 2(5%)
3.125 0.619
0.077 0.431
7(14%) 6(12%)
9(23%) 5(12%)
1.098 0
0.295 1
3(6%) 5(10%)
4(10%) 1(2%)
0.095 0.984
0.758 0.321
Hypertension Hemorrhage
11(22%) 6(12%)
6(15%) 4(10%)
0.711 0
0.399 1
Pulmonary infection Diabetes
8(16%) 7(14%)
14(35%) 0(0%)
4.344 4.277 9
0.037 0.039
Mechanical ventilation Endotracheal intubation
16(32%) 17(34%)
10(25%) 12(30%)
0.530 0.163
0.467 0.687
Tracheotomy Drainage tube
10(20%) 15(30%)
6(15%) 12(30%)
0.380 0
0.538 1
8(20%) 2(5%)
0.168 1.125
0.28 0.289
29(58%)
14(35%)
4.712
Surgery or Major trauma 29(58%) Transfer 12(24%)
24(60%) 8(20%)
0.037 0.206
0.03 0.848 0.65
Mortality in 30 days
8(20%)
0.206
0.65
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Comorbidity
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Use of invasive devices
Indwelling catheterization 15(30%) Central venous catheter 7(14%)
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ICU admission
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Department
12(24%)
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Statistically significant correlations (p < 0.05) are shown in bold font.
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Table 2 Association between virulence genes, ESBLs genes, carbapenemase genes, and integron carriage in clinical isolates of highly virulent serotypes K. pneumoniae Integron-positive isolates
Integron-negative isolates
(n=50)
(n=40)
2
20（50%）
P-value
K1
29(58%)
K2
8(12%)
K5
0
0
K20
1(2%)
0
0.8
K54
11(22%)
6(15%)
0.711
0.399
K57
1(2%)
3(7.5%)
1.583
0.319
11.222
0.001
3.95
0.047
0.512
0.474
0
1
0(0)
NA
0(0)
NA
NA NA
12(30%)
2.394
0.122
11(27.5%)
0.573
0.449
1.765
0.184
0
1
33(66%)
38(95%)
aerobactin
41(82%)
39(97.5%)
KPC-2
9(18%)
5(12.5%)
NDM-1
2(4%)
1(2.5%)
IMP
0(0)
NA
VIM
0(0)
OXA-48
0(0)
0(0)
NA
SHV
23(46%)
TEM
20(40%)
10(25%)
2.25
0.134
CTX-M-14
16(32%)
9(22.5)
1.0
0.317
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rmpA
-p
1
Jo
ur
na
Statistically significant correlations (p < 0.05) are shown in bold font. NA: Not Applicable
18
Table 3 Antimicrobial susceptibilities and integron carriage in clinical isolates
Antimicrobal susceptibility Total(n=90)
Integron-positive
Integron-negative
isolates (n=50)
isolates (n=40)
χ2
P-value
Cefazolin
32(35.6%)
15(30%)
17(42.5%)
1.515 0.218
Ceftazidime
62(68.9%)
34(68%)
28(70%)
0.041 0.839
Cefepime
66(73.3%)
33(66%)
33(82.5%)
3.094 0.079
Cefuroxime
61(65.6%)
27(54%)
34(85%)
9.778 0.002
Ampicillin
0(0)
0(0)
0(0)
0
Piperacillin tazobactam
76(84.4%)
38(76%)
34(85%)
1.125 0.289
Aztreonam
54(60%)
24(48%)
30(75%)
6.75
Gentamicin
72(80%)
36(72%)
34(75%)
2.173 0.140
Tobramycin
75(83.3%)
31(62%)
35(87.5%)
7.389 0.007
Amikacin
71(78.9%)
36(72%)
35(87.5%)
3.206 0.073
Levofloxacin
76(84.4%)
38(76%)
35(87.5%)
1.918 0.166
Ciprofloxacin
68(75.6%)
34(68%)
34(85%)
3.477 0.062
Trimethoprim
64(71.1%)
29(58%)
34(85%)
7.714 0.005
Ertapenem
72(80%)
37(74%)
35(87.5%)
2.531 0.112
Meropenem
74(82.2%)
40(80%)
34(85%)
0.38
Imipenem
75(83.3%)
41(82%)
34(85%)
0.144 0.704
Cefotaxime
60(66.7%)
32(64%)
28(70%)
0.36
1
re
-p
ro of
0.009
Jo
ur
na
lP
Statistically significant correlations (p < 0.05) are shown in bold font.
19
0.538
0.549