Abstract: Background and Objective: Drug resistance in bacteria is a challenge both in human and veterinary medicine. This study was conducted to characterize quinolone resistant determinants in Morganella morganii isolated from pet turtles. Materials and Methods: Antimicrobial susceptibility of twenty-two M. morganii isolates against nalidixic acid, ciprofloxacin, ofloxacin and levofloxacin was examined by disk diffusion assay and the Minimum Inhibitory Concentration (MIC). Substitutions of the Quinolone Resistance Determining Region (QRDR) and Plasmid Mediated Quinolone Resistance (PMQR) genes were detected using conventional PCR assays and sequencing. Results: Three isolates were resistant to the all tested quinolones and one isolate was resistant only to nalidixic acid. In QRDR substitution analysis, three isolates displayed the Ser463Ala, Ser464Tyr and novel Glu466Asp substitutions in gyrB and the Ser80Ile substitution in parC. Two isolates displayed only Ser463Ala substitution in gyrB. The unique PMQR gene detected was qnrD, which was found in 59% of the isolates. The aac-(6’)-Ib-cr gene variant was identified in 50% of the isolates. In addition, neighbor-joining phylogenetic tree derived using gyrB gene sequences exhibited two distinct clads comprising, first; present study isolates with a quinolone-resistant isolate of human clinical origin and second; isolates of environmental origin. Conclusions: All results suggest healthy pet turtles might serve as a potential reservoir for quinolone-resistant M. morganii due to the high prevalence of PMQR determinants, especially, qnrD and target gene alterations in QRDR together with a novel mutation in gyrB.
INTRODUCTION
Morganella morganii is a Gram-negative bacteria commonly found in the environment and in the intestinal tract of humans, mammals and reptiles as a part of the normal microflora1,2. It can involve in various infections including pneumonia, peritonitis, empyema, pericarditis, arthropathy, endophthalmitis, meningitis and wound infections in amphibians and reptiles3,4. It has been recorded that the studies related to M. morganii isolated from turtles are still scanty5. Meanwhile, the interest in wild animals as pets has increased over the past few years due to human curiosity and eccentricity. Several species of reptiles are bred as pets nowadays, especially exotic pet turtles have gained popularity in developed countries6,7.
However, pet turtles are well recognized as source of diverse pathogens and the direct and indirect contact associated contamination can transmit the pathogens to humans5. Over the years, many case studies have been conducted to assess the prevalence of M. morganii in human clinical cases, which usually caused nosocomial infections, particularly among the immunocompromised patients. Morganella morganii can lead to major clinical problems, such as wounds, urinary tract infections and septicaemia8,9. In a case study of Morganella infection, various groups of antibiotics including fluoroquinolones have been tested in vitro for the treatment2.
Quinolones are broad-spectrum antibiotics extensively used in human and veterinary medicine hence, has been resulted in rising levels of quinolone resistance. Morganella morganii from clinical and environmental sources have demonstrated increasing levels of quinolone resistance, which were frequently associated with the presence of resistance genes and related mechanisms9,10. In contrast, quinolone resistance mechanism is mediated by specific chromosomal mediated and Plasmid Mediated Quinolone Resistance (PMQR) genes. High level of quinolone resistance arises mainly due to chromosomally encoded mechanisms such as mutations in the Quinolone Resistance Determining Region (QRDR) of DNA gyrase and topoisomerase IV. The DNA gyrase is the primary target of quinolones, consisting of two subunits that are encoded by gyrA and gyrB genes. Topoisomerase IV is the secondary target of quinolones, which is also comprising of two subunits encoded by parC and parE genes11.
Plasmid Mediated Quinolone Resistance (PMQR) causatives include qnr-type pentapeptide proteins (qnrA, qnrB, qnrC, qnrD and qnrS) and aac(6')-Ib-cr conferring low-level of resistance to fluoroquinolones compared to chromosomally mediated resistance12,13. Prevalence of qnr genes could vary with the species of bacteria. Several past studies identified qnrD as more prevalent PMQR determinants in M. morganii isolates. The qnrD gene is a relatively uncommon PMQR gene, which has been detected in members of the Proteeae family from different origin14-16.
These resistance genes consisting plasmids and transposons are known as mobile genetic elements that can be transferred horizontally among distantly related lineages. Particularly, the water environment is more favorable for the transmission of resistant bacteria from animals to humans, thus, M. morganii as an opportunistic pathogen might be dangerous vector for the dissemination of antibiotic resistance genes through the aquatic environment9,11.
Therefore, the current study aimed to evaluate the quinolone resistance characteristics and related genetic background in pet turtle-borne M. morganii in order to advocate the awareness about the pathogen counting concerned to public health. This study also aimed to determine the quinolone susceptibility, occurrence of PMQR genes [qnrA, qnrB, qnrC, qnrD, qnrS and aac(6')-Ib-cr] and chromosomal QRDR mutations in gyrA, gyrB and parC in pet turtle-associated M. morganii.
MATERIALS AND METHODS
Bacterial isolates: Twenty-two isolates of M. morganella were obtained from fecal samples of 6 commercially available pet turtle species namely, Chinese stripe-necked turtles (Ocadia sinensis), yellow-bellied sliders (Trachemys scripta scripta), river cooters (Pseudemys concinna concinna), Northern Chinese softshell turtles (Pelodiscus maackii), African sideneck turtles (Pelusios castaneus) and common musk turtles (Sternotherus odoratus). Turtles were purchased randomly through pet shops in Korea and had an average weight of 15±2 g, carapace diameter of 40±5 mm and were under 4 weeks of age. All turtles did not show any clinical signs of disease and confirmed not to have a treatment history hence, considered as healthy.
Bacterial identification using 16S rRNA gene amplification and sequencing: Genomic DNA was extracted from presumptively identified M. morganii isolates by Chelex 100 extraction method and PCR for 16S rRNA was performed using universal primers; 12F and 1492R. Amplicons were sequenced and tested for the similarity using BLAST algorithm of NCBI database so as to confirm the species status.
Determination of quinolone susceptibility and minimum inhibitory concentrations: Antibiotic susceptibility to nalidixic acid, ciprofloxacin, ofloxacin and levofloxacin was determined by disc diffusion on Mueller Hinton agar (MBcell Ltd., Seoul, Korea) using OXOID™ antibiotic disks (Oxoid Co. Ltd., Seoul, Korea). Testing was confirmed by duplicating and the resistance profiles (resistant, intermediate, or susceptible) were assigned using criteria described by Clinical and Laboratory Standards Institute (CLSI)17. The Minimum Inhibitory Concentrations (MIC) were determined using broth microdilution method containing nalidixic acid (1-512 μg mL–1), ciprofloxacin, ofloxacin and levofloxacin (0.06-32 μg mL–1). Resistance breakpoints (resistant, intermediate, or susceptible) were assigned using criteria described by CLSI17.
Detection of mutations in quinolone resistance-determining region (QRDR): The QRDRs of all isolates were examined by amplifying and sequencing gyrA (441 bp), gyrB (300 bp) and parC (204 bp) genes using primers (Table 1) described by Lascols et al.18. The PCR amplifications were conducted in 50 μL volumes consisting of 20 μL of Quick Taq® HS DyeMix (TOYOBO, Japan), 1 μL of 20 pmol μL–1 each primer and 2 μL of the template under standard conditions. The PCR products were analyzed by electrophoresis on 2% (w/v) agarose gels. Amplimers were purified using Expin™ PCR SV kit (GeneAll®, Korea) and sequenced by Cosmogenetech Co. Ltd, Daejeon, Korea. The gyrA, gyrB and parC nucleotide sequences obtained from the present study were aligned and compared with published reference sequences using Mutation Surveyor V5.0.1 (SoftGenetics LLC, USA) in order to identify amino acid substitutions.
Detection of quinolone resistance genes: Total DNA (2 μL) was subjected to PCR for amplifications of qnrA, qnrB, qnrS, qnrC and qnrD genes using primers (Table 1) described by Adachi et al.19 and Dasgupta et al.20. The DNA fragments were detected by electrophoresis in a 2% (w/v) agarose gels. The presence of the aac(6')-Ib-cr gene was investigated by PCR using primers (Table 1) described by Park et al.21 and the presence of cr variant was investigated by sequencing and blasting in NCBI database.
Phylogenetic comparison of gyrB sequences with published sequences: The sequences derived for gyrB gene region were analyzed and a neighbor-joining phylogenetic tree was obtained with 1000 bootstrap replications.
| Table 1: | Primers used for amplification of quinolone resistance genes |
| Table 2: | Details of the gyrB gene sequences of M. morganii downloaded from NCBI database for the phylogenetic analysis |
For the analysis, four previously published gyrB sequences of M. morganii clinical and environmental isolates were obtained from the GenBank database (Table 2) and MEGA6 sequence analyzing software was used for aligning and construction of the phylogenetic tree.
RESULTS
16S rRNA gene-based identification: A subsequent BLAST search after 16S rRNA sequencing indicated a 99-100% match to M. morganii sequences available in GenBank which confirmed 22 isolates as M. morganii.
Quinolone susceptibility and MICs of isolates: Eighteen out of 22 (82%) isolates of M. morganii were susceptible to quinolones; nalidixic acid, ciprofloxacin, ofloxacin and levofloxacin both in disk diffusion test and MIC. Only 3 isolates were resistant to all quinolones tested in this study while one isolate was resistant to only nalidixic acid. Nalidixic acid MICs of resistant strains ranged from 32>256 μg mL–1, while ofloxacin, ciprofloxacin and levofloxacin MICs of resistant strains were ranged 16-32 μg mL–1 (Table 3).
The gyrA, gyrB and parC QRDR substitution analysis: Alterations in the gyrB QRDR were observed in 23% (5/22) of the isolates. Three quinolone-resistant isolates displayed the Ser463Ala, Ser464Tyr and novel Glu466Asp substitutions while, 2 isolates exhibited only Ser463Ala substitution. The Ser80Ile substitution in QRDR of parC occurred only in 14% (3/22) of the isolates, where 86% (19/22) of the isolates were harboring wild-type parC. Three isolates which were resistant to all tested quinolones demonstrated gyrB and parC QRDR substitutions simultaneously (Table 3).
| Table 3: | Quinolone susceptibility pattern and genetic characteristics of turtle-borne M. morganii isolates |
NA30: Nalidixic acid (30 μg), CIP5: Ciprofloxacin (5 μg), OFX5: Ofloxacin (5 μg), LVF5: Levofloxacin (5 μg), S: Susceptible, I: Intermediate, R: Resistant, wt: Wild-type, MIC: Minimum inhibitorty concentration, QRDR: Quinolone resistance determining region, PMQR:Plasmid mediated quinolone resistance |
|
Detection of qnrA, qnrB, qnrS, qnrC, qnrD and aac(6')-Ib-cr genes: The qnrD gene appeared to be the most prevalent where 59% (13/22) of the total isolates produced amplimers for the qnrD but, no positive amplification for qnrA, qnrB, qnrS, qnrC was detected in any of the isolates. Although the aac(6')-Ib was amplified in all the isolates, only 50% (11/22) of them were confirmed as cr variant by sequencing (Table 3).
Phylogenetic comparison of gyrB sequences: Phylogenetic tree derived by analyzing and comparing the gyrB gene sequences obtained by present study and the published NCBI sequences is illustrated in Fig. 1. Neighbor-joining phylogenetic tree indicate two distinct clads comprising first; present study isolates together with a quinolone resistant M. morganii human clinical isolate and the second; environmental isolates of M. morganii.
| Fig. 1: | Neighbor-joining phylogenetic tree derived by analyzing M. morganii gyrB sequences obtained from the current study and downloaded from NCBI |
| Sequences referred to AB972375.1, DQ360899.1, KF732712.1 and HM122057.1 were obtained from NCBI public database and the rest of the sequences were acquired from the current study, 1, 2: Major clads | |
DISCUSSION
The trade of pet turtles has generalized with increasing number of aquariums, pet shops, online shops and pet cafes. As the pet trade thrives, careless management of pet shops, as well as high rearing density, can be the cause of emerged potential zoonotic bacteria6. Pet turtles have been reported as a reservoir for several opportunistic pathogenic bacteria, such as Salmonella spp., Citrobacter spp. and Aeromonas spp.1,19. But, there have been very few reports on M. morganii isolated from pet turtles. Epidemiological studies have revealed that M. morganii is frequently isolated from human clinical cases of nosocomial bacterial infections with emerged resistance mechanisms against quinolones9.
In the present study, 22 isolates were identified as M. morganii. The majority of these pet turtle-associated M. morganii isolates demonstrated a lower frequency of resistance to quinolones. In MIC break points of this study, three isolates demonstrated resistance to nalidixic acid, ciprofloxacin, ofloxacin and levofloxacin while only one isolate showed resistance to only nalidixic acid. In contrast, 18 (82%) isolates were susceptible for all tested quinolones. Similar outcomes have been reported earlier, indicating that quinolone-susceptible isolates (75%) predominated in M. morganii isolated from patients22. Albornoz et al.23 also reported M. morganii clinical isolates showing 42% resistance to quinolones.
A specific primer that can be used to amplify the gyrA of M. morganii has not been reported so far. The studies of Mazzariol et al.24 and Yaiche et al.10 could not amplify gyrA in M. morganii and it was suggested that this is due to the unknown genome characteristics of this bacterium. However, Ser463Ala, Ser464Tyr and novel Glu 466Asp substitutions in gyrB were detected in all 3 quinolone-resistant isolates while Ser463Ala substitution was detected in 2 quinolone-susceptible isolates. None of the other susceptible isolates had mutations. To date, none of animal associated M. morganii was described with gyrB mutations. This study described for the first time, a new gyrB mutation in M. morganii. In Gram-negative bacilli, gyrB mutations are rare and they have been described at amino acid positions 426, 431, 447, 463, 464 and 46618. The Ser463Ala substitution has been reported in a clinical isolate of M. morganii in Tunisia10.
The substitution of Ser80Ile in parC was observed in only 3 quinolone-resistant isolates. All the quinolone-susceptible isolates did not show any substitution in parC which aligns with the results of previous study23. Double target substitutions of QRDR (gyrB-parC) were detected in 12% (3/22) of the isolates. Substitutions of Ser463Ala, Ser464Tyr and novel Glu466Asp in the gyrB QRDR coupled with Ser80Ile in the parC QRDR conferred high levels of quinolone resistance in M. morganii isolates. More or less similar outcome was reported, in which clinical strain of M. morganii harbored two gyrB substitutions (Ser463Ala, Ser464Tyr) and one parC substitution (Ser80Ile)10. However, several studies reported only single substitution in gyrB coupled with a parC substitution22,23.
The qnr proteins obstruct the action of quinolones on bacterial DNA gyrase and topoisomerase IV. Generally, the aac(6')-Ib gene causes the resistance to aminoglycosides, but aac(6')-Ib-cr encodes a variant of the aminoglycoside acetyltransferase which can confer to reduced susceptibility of quinolones11. The PMQR determinants lead to low-level resistance compared to QRDR mutations although, their dissemination between bacteria and the simultaneous presence of two or more resistance determinants in the same microorganism has an additive effect of increasing quinolone MIC values25,26.
This study reports the high prevalence of qnrD gene in pet turtle-associated M. morganii strains. Especially, 59% (13/22) of M. morganii of current study harbored qnrD gene while, the qnrD gene has been reported frequently in M. morganii clinical isolates recovered from patients16,24. With regards to the horizontal transfer of PMQR determinants between human and turtle flora, that similarity points out a potential public health risk. The aac(6')-Ib amplimer could be obtained from all the M. morganii isolates and 50% (11/22) isolates were confirmed as cr variants. Interestingly, all the resistant isolates harbored both aac(6')-Ib-cr and qnrD genes. Similar results have been detected earlier where aac(6')-Ib-cr and qnr genes together conferring the quinolone resistance of clinical M. morganii isolates22,27.
Phylogenetic analysis of gyrB sequences clearly interprets the genetic similarity of M. morganii isolated from pet turtles with the quinolone-resistant human clinical isolate and how they diverge from environmental isolates. Meanwhile, quinolone-resistant isolates were clustered more closely with the quinolone-resistant human clinical isolate. Only four reference sequences could be used to construct the phylogenetic tree due to less availability of nucleotide sequences of M. morganii gyrB gene in GenBank.
As a whole, it is evident that pet turtle-associated M. morganii are harboring PMQR genes and mutations in the QRDR and their phenotypical expression is also pronounced. In emphasis, only three strains were resistant to all tested quinolones while harboring gyrB and parC mutations in QRDR and qnrD and aac(6')-Ib-cr PMQR genes concurrently. It suggests that the gene expression mechanisms might be involved positively in modulating the final MIC28.
In pet shops, rearing aquatic animals including ornamental fish and turtles with high density in closed systems can pave the way for the emergence of antibiotic resistance due to horizontal transfer of genetic elements. This can be a possible reason for the high prevalence of PMQR genes in both quinolone-resistant and susceptible M. morganii isolates. Previous studies have demonstrated a high prevalence of PMQR genes in aquatic environments19,29. Although the exposure of antimicrobials causes the emergence of antimicrobial resistance, the natural evolutionary response has correlated with complex and interlinking drivers which have not revealed completely. On the other hand, acquisition of antimicrobial resistance mechanisms does not necessarily compromise microbial fitness. Worldwide clonal spread and long-term persistence of resistant bacteria are also seen in the absence of direct antibiotic selection pressure30,31.
CONCLUSION
This is the first study describing quinolone resistance determinants in M. morganii strains isolated from pet turtles with a high prevalence of qnrD gene. A novel mutation (Glu 466Asp) in the QRDR of the gyrB gene was detected. Profoundly, pet turtle may represent a potential source of quinolone-resistant M. morganii due to the high prevalence PMQR genes [qnrD and aac(6')-Ib-cr] and mutations in the QRDR of gyrB and parC. These findings suggest that studies associated with quinolone resistance, particularly, the genetics-based resistance of M. morganii worth considering significant. To gain more perceptivity into the molecular characterization of quinolone-resistant M. morganii isolates, other possible mechanisms of resistance should also be investigated deeply.
SIGNIFICANT STATEMENT
The present study evaluated the genetic context related to quinolone resistance of M. morganii isolated from several species of pet turtles reared under laboratory conditions. The outcomes pointed out the pet turtles posing a potential public health risk due to high prevalence of plasmid mediated qnrD and target gene alterations in chromosomal QRDR.
ACKNOWLEDGMENTS
This study was supported by Basic Science Research Program through the National Research Foundation of Korea and funded by the Ministry of Education (Grant No. NRF-2015R1D1A1A01060638).