|Year : 2015 | Volume
| Issue : 5 | Page : 87-92
The dissemination mode of drug-resistant genes in Enterobacter cloacae
J Liu1, T Zeng2, G Su3, LY Lin4, Y Zhao5, WQ Yang2, WX Xie1, ZG Zhao5, GM Li5
1 Laboratory of Pathogenic Biology, Guangdong Medical College, Zhanjiang, China
2 School of Laboratory Science, Guangdong Medical College, Zhanjiang, China
3 Department of Emergency, Central People's Hospital of Zhanjiang, Zhanjiang, China
4 Cardio Thoracic Surgery of the Affiliated Hospital of Guangdong Medical College, Zhanjiang, China
5 Department of Microbiology and Immunology, Guangdong Medical College, Zhanjiang, China
|Date of Submission||28-Sep-2013|
|Date of Acceptance||07-May-2014|
|Date of Web Publication||6-Feb-2015|
Z G Zhao
Department of Microbiology and Immunology, Guangdong Medical College, Zhanjiang
Source of Support: None, Conflict of Interest: None
Background: Enterobacter cloacae (E. cloacae) infection has the highest mortality rate among Enterobacter infections. This study aimed to determine the prevalence and the transmission route of the class I integron, qnr genes, and CTX-M ESBLs genes in clinical isolates and to analyse the association between the prevalence of MDR genes and the antibiotic resistance of E. cloacae. Materials and Methods: The antibiotic susceptibility was tested the agar dilution method. The class I integron, qnr genes, and CTX-M ESBLs genes were detected by polymerase chain reaction (PCR). The prevalence data were analysed with the Chi-square test. Results: In the 100 clinical isolates, the class I integron-positive rate was 65%, with 12% on chromosome, 15% on plasmids and 38% on both. The positive rate of qnr genes was 37% with plasmid location. The positive rates for qnrA, qnrB and qnrS were 6%, 23% and 8%, respectively. The CTX-M ESBLs-positive rate was 34%. For CTX-M-1 ESBLs, 15% were on chromosome, 6% on plasmids and 4% on both; for CTX-M-9 ESBLs, 1% was on chromosome and 7% on plasmid; for CTX-M-25 ESBLs, 3% were on chromosome and 1% on plasmid. Conclusion: Antibiotic resistance genes may be horizontally and vertically disseminated among E. cloacae, which helps multidrug-resistant (MDR) strains of E. cloacae to be successful nosocomial agents.
Keywords: CTX-M ESBLs, Enterobacter cloacae, integron, qnr gene
|How to cite this article:|
Liu J, Zeng T, Su G, Lin L Y, Zhao Y, Yang W Q, Xie W X, Zhao Z G, Li G M. The dissemination mode of drug-resistant genes in Enterobacter cloacae. Indian J Med Microbiol 2015;33, Suppl S1:87-92
|How to cite this URL:|
Liu J, Zeng T, Su G, Lin L Y, Zhao Y, Yang W Q, Xie W X, Zhao Z G, Li G M. The dissemination mode of drug-resistant genes in Enterobacter cloacae. Indian J Med Microbiol [serial online] 2015 [cited 2020 Feb 22];33, Suppl S1:87-92. Available from: http://www.ijmm.org/text.asp?2015/33/5/87/150899
J Liu and T Zeng contributed equally to this research.
| ~ Introduction|| |
Enterobacter cloacae (E. cloacae), one of the most common human enteric gram-negative bacilli, is an important opportunistic pathogen known to cause nosocomial septicaemia, urinary tract and respiratory tract infections. In China, the infection rate of E. cloacae is the third highest behind Escherichia More Details coli and Klebsiella pneumoniae among enterobacteriaceae. There are increasing reports on the isolation of clinical multidrug-resistant (MDR) E. cloaca. 
Class I integron is a mutligene-capturing and disseminating system in bacteria, which has often been linked to multidrug resistance. Class I integron is a platform that assembles a large number of antibiotic resistance genes to integrate large multidrug-resistance cassettes into bacterial chromosome. Class I integron may appear in chromosome or plasmid. Plasmid-bearing class I integron could rapidly disseminate antibiotic-resistance genes throughout the species while antibiotic-resistance genes carried by chromosomal class I integron are generally restricted in particular phylogenetic lineages.  Once the antibiotic-resistance gene is anchored on chromosomal integron, it will be maintained for many years under antibiotic selection and will only be spread with the resistant bacteria.  Currently, the integron-associated antibiotic resistance is creating an increasing crisis in the management of bacterial infections and contributing to the emergence of MDR E. cloacae at clinical settings.
Quinolone-resistance gene, qnrA, was firstly reported in America in 1998. Then qnrA, qnrB, qnrS, qnrC and qnrD have been identified in the enterobacteriaceae worldwide. In 2008, Jacoby et al., reported that the numbers of qnrA, qnrS and qnrB variants were 6, 4 and 20,  respectively. Some of the variants are carried on plasmids (such as qnrA1) while others on the chromosome (such as qnrA4)., The qnr genes encoding DNA gyrase, topoisomerase IV, outer membrane proteins and drug-efflux pumps in E. cloacae are involved in quinolone-resistance. It is also reported that qnr genes alone in E. cloacae strains could decrease quinolone susceptibility.  The qnr genes are widely distributed in E. cloacae strains and frequently linked to antibiotic-resistant outbreaks. 
In 1990, a new group of extended-spectrum β-lactamases (ESBLs) was described in Germany and was designated CTX-M (CefoTaXimase-Munich) type of ESBLs due to its preferential hydrolysis of cefotaxime.  The CTX-M family are classified into five groups: CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25. In China, CTX-M ESBLs are the most common factors causing resistance to β-lactamase antibiotics, which has led to an increasing concern about clinical management of related infections. 
Together with qnr genes and the plasmid-mediated integration mechanism, CTX-M ESBLs is responsible for the rapid horizontal and vertical dissemination of antibiotic-resistance genes among bacterial species. However, the relationship between the dissemination of CTX-M, integron and qnr on chromosome and plasmids and the antibiotic resistance of E. cloacae has rarely been analysed. The aim of this study was to determine the mode of drug-resistant gene transfer in E. cloacae, providing information for more prudent and efficient use of antimicrobial agents and a better control of MDR E. cloacae infections.
| ~ Materials and Methods|| |
Bacterial isolates collection
A total of 100 non-repetitive isolates were collected from June 2010 to July 2011 in the Central People's Hospital of Zhanjiang and the Affiliated Hospital of Guangdong Medical College. The integron-positive and integron-negative strains, CTX-M-positive and CTX-M-negative strains, qnrA-, qnrB-, qnrS-positive and qnr-negative strains were stored in our laboratory.
Chromosomal and plasmids DNA purification
At variable temperature, sodium dodecyl sulfate (SDS) plasmid elimination method was used to eliminate plasmid.  Agarose gel electrophoresis was used to confirmed result of plasmid elimination. Chromosomes and plasmid DNA were isolated using commercial kits (FastPure™ DNA kit Takara, Dalian, China and Plasmid Mini Kit, Qiagen GmbH, Hilden, Germany) following the manufacturers' protocols.
PCR amplification of integrons, qnr genes and CTX-M ESBLs and DNA sequencing
A series of primers were designed with Oligo7.27 Software and Premier5.0 Software for the detection of integrons, qnr genes and CTX-M ESBLs [Table 1]. The PCR products were analysed by a 1.5% agarose gel electrophoresis and the results were captured by INFINITY Gel documentation systems (Vilber Lourmat, France). According to the principle of random sampling,  two strains were chosen each with a gene-positive group for DNA sequencing in order to confirm PCR results. The PCR products were purified using a commercial kit (SUPREC™-01, Takara, Dalian, China). The purified DNA fragments were cloned into pCR2.1-TOPO (Life Technologies, Beijing, China). The sequencing results were compared to GenBank data with a Basic Local Alignment Search Tool (BLAST) search.
Antimicrobial susceptibility test and statistical analysis
The susceptibility of E. cloacae to ampicillin (AMP), Cefuroxime (CXM), cefotaxime (CTX), cefepime (FEP), cefoxitin (FOX), cefoperazone sulbactam (SCF), imipenem (IPM), aztreonam (ATM), norfloxacin (NOR), levofloxacin (LEV), ciprofloxacin (CIP), gentamicin (CN), Amikacin (AK) and trimethoprim-sulfamethoxazole (SXT) was tested by the agar dilution method,  and the results were interpreted according to the latest guidelines of the Clinical and Laboratory Standards Institute (CLSI).
A Chi-square test was used for the comparison of dichotomous variables as integrons, qnr genes and CTX-M ESBLs on E. cloacae resistance.
| ~ Results|| |
Characterisation of integrons
Among the 100 E. cloacae isolates, 65 possessed class I integron (65.00%), of which 12 had class I integron on the chromosomal DNA (12.00%) which was approximately 18.46% of all the positive isolates; 15 isolates had class I integron on the plasmid DNA (15.00%) which was approximately 23.08% of all the positive isolates; 38 isolates had class I integron located on both the chromosome and the plasmid (38%) which was approximately 58.46% of all the positive isolates. The size of PCR products from 12 positive isolates was verified on agrose gel [Figure 1]. Overall, class I integron detected in 50.00% isolates with chromosomal location and in 53.00% isolates with plasmid location.
|Figure 1: The electrophotogram of PCR product of class I integrons. 1: Positive control; 2: Negative control; and 3-12: Clinical samples PCR = Polymerase chain reaction|
Click here to view
Analysis of qnr gene
A total of 37 (37.00%) out of the 100 isolates were positive for the qnr genes. The numbers of positive isolates for qnrA, qnrB and qnrS were 6.00% (6/100), 23.00% (23/100) and 8.00% (8/100), respectively. All qnr genes were located on plasmid. None of qnrC or qnrD was detected. Class I integrons were carried by 78.38% (29/37) of qnr-positive isolates. A total of 50.00% (3/6) qnrA-positive isolates contained class I integrons, with one on chromosome, one on plasmid and one on both. Twenty out of 23 (86.96%) qnrB-positive isolates were class I integron-positive, four on chromosome, six on plasmid and ten on both. There were 75.00% (6/8) class I integron-positive qnrS-containing isolates, one on the chromosome, two on plasmid and three on both.
CTX-M ESBLs gene analysis
There were 34 CTX-M-positive isolates detected, of which the position and the status of other resistance genes are summarised in [Table 2].
|Table 2: Detection of CTX‑M genes and positive isolates carrying other resistance genes|
Click here to view
Results of antimicrobial susceptibility testing
The results of antimicrobial susceptibility test are shown in [Table 3]. A Chi-square test was used for analysing the difference between groups. The resistance rates to CXM, CTX, ATM, CIP, CN, AK and SXT of the integron-positive group were significantly higher than those of the integron-negative group (P < 0.05). The resistance rates to CTX, NOR, LEV, CIP, AK and SXT of the qnr genes-positive group were significantly higher than those of the qnr genes-negative group (P < 0.05). The resistance rates to CXM, CTX, SCF, ATM, NOR, CIP and SXT of the CTX-M genes-positive group were significantly higher than those of the CTX-M genes-negative group (P < 0.05).
| ~ Discussion|| |
In this study, the rate of E. cloacae class I integron-positive isolates was 65.00%, which is consistent with previous study.  The rates of plasmid-carrying and chromosomal class I integron were similar (53.00% vs. 50.00%) while 38.00% of class I integron-positive isolates had dual localisation, which suggests that resistant genes could be transferred horizontally and vertically. Under optimal environmental conditions, the integron-mediated resistance genes could quickly spread among E. cloacae and result in outbreaks of MDR bacterial infection. An outbreak of E. cloacae carrying integron-transmitted chromosomae-integrated VEB-3 beta-lactamase has been reported in China.  With strong selective pressure and increased integration of integrons,  it is hypothesised that resistant genes could quickly spread among class I integron-carrying bacteria.
Our study showed that the qnr-positive rate was 37%, which is consistent with results observed in Shanghai (38.60%) and higher than those observed in Korea.  The leading resistant gene was qnrB in our study, whereas qnrA was reported as the predominant gene elsewhere,  suggesting that the infective strains vary at different time and location. Moreover, 78.38% qnr-positive isolates were also positive for class I integrons, indicating that integron plays an important role in the dissemination of qnr genes. Class I integron and other mobile elements such as ISCR1 promote the transmission of qnr genes among bacteria. ,,
We identified CTX-M-1, CTX-M-9 and CTX-M-25 ESBLs isolates with CTX-M-1 ESBLs being the dominant subtype. The location of CTX-M was different among the three groups. CTX-M-1 ESBLs were mostly identified on the chromosome with few located on plasmids or both locations. Those results were contradictory to a previous report that the main location for CTX-M-1 ESBLs was on plasmid.  Meanwhile, the CTX-M ESBLs-positive isolates could harbour more than one type of CTX-M ESBLs. It has been shown that some CTX-M-1 ESBLs-positive isolates harbour CTX-M-25 ESBLs as well. In those isolates CTX-M-1 ESBLs could be detected on both the chromosome and plasmids, whereas CTX-M-25 ESBLs genes were found on the chromosome only, which might be resulted from a high mutation rate and a mobile and fluid genetic background of CTX-M-1 ESBLs genes.  CTX-M-9 ESBLs genes were solely located on the chromosome or plasmid and were not found to coexist with any other type of CTX-M ESBLs. Our data suggested that CTX-M ESBLs could horizontally and vertically disseminate among bacterial species. With the emergence of CTX-M ESBLs on both the chromosome and plasmids, a close attention should be paid to the drug resistance caused by CTX-M ESBLs.
Some of the CTX-M ESBLs-positive isolates were also found to be positive for class I integron. In recent years, it has been reported that CTX-M-1, CTX-M-9 and CTX-M-25 could spread with class I integrons in China, Korea and Israel. ,, The presence of CTX-M ESBLs-positive isolates with class I integrons embedded in both the chromosome and plasmid suggests an integron-based dissemination.
We detected 8 qnr-positive isolates from the CTX-M ESBLs-positive isolates. The qnrA, qnrB and qnrS genes were found in the CTX-M-1 ESBLs-positive isolates. The qnrB gene was detected in the CTX-M-9-positive isolates. We also detected qnrA genes in CTX-M-25 ESBLs-positive isolates harbouring CTX-M-1 ESBLs. The coexistence of CTX-M and qnr genes in the same isolates has been documented before.  The overuse or misuse of antimicrobial agents in clinical and veterinary settings facilitates the emergency and spreading of drug resistance, which requires adequate regulatory attention and vigilant monitoring.
Statistical analyses suggested that antibiotic resistance was closely related to the presence of class I integrons, qnr genes and CTX-M ESBLs. However, some antibiotic resistance did not show significant differences upon the presence of resistant genes, which implies that there might be other drug-resistance mechanisms or that antibiotic-resistance genes could stay silenced until needed. 
Interestingly, β-lactamase antibiotics, such as CTX, showed significantly different susceptibilities between qnr-positive and qnr-negative groups. The resistance to quinolone type antibiotics, such as NOR and CIP, was strongly associated with CTX-M genes. The reason for the difference may be that qnr genes and CTX-M ESBLs are embedded in the same E. cloacae isolate. The susceptibility to sulfonamide antibiotics SXT was affected by the presence of integron, qnr genes and CTX-M genes, whereas qnr-positive isolates show less susceptibility for the aminoglycoside antibiotics AK. Considering that class I integrons were positive in 78.38% of qnr-positive isolates and 35.29% of CTX-M ESBLs-positive isolates and they contained aminoglycoside and trimethoprim antibiotics-resistance genes, class I integrons had a strong association with antibiotics resistance. 
In conclusion, MDR could disseminate by both horizontal and vertical transmission of E. cloacae, which makes drug-resistant E. cloacae strains successful nosocomial agents. Attention should be paid to class I integrons, qnr genes and CTX-M ESBLs-associated antibiotic resistance. The detection of those resistance elements would provide direction for clinical antibiotic usage.
| ~ Acknowledgements|| |
We acknowledge the financial support from the National natural science fund for youths (81201831), Special Fund for Science and Technology, Department of Treasure of Zhanjiang City (2013A01007), Guangdong medical college on the project (M2012005 Zhanjiang Science and Technology Bureau (2012C3106022) and Youth Fund of Guangdong Medical College (Q2010015).
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[Table 1], [Table 2], [Table 3]