Incidence of carbapenem-resistant Escherichia coli ST2437 of clinical origin harbouring blaOXA-144 gene: a report from India.

AIMS: Carbapenem-resistant Escherichia coli has been categorized as a pathogen of critical priority by the World Health Organization as it is highly infectious with high mortality and morbidity rates and widespread transmission potential. Carbapenem resistance is primarily mediated by carbapenemase-encoding genes and, additionally, through intrinsic factors. In India, over the years, carbapenemase-encoding genes have been reported from diverse clinically significant pathogens. The present study identifies E. coli of clinical origin that harbours blaOXA-144. METHODS AND RESULTS: The study isolate was obtained from a tertiary referral hospital in northeast India. Carbapenemase production was investigated through culture on chromogenic agar and Rapidec Carba NP test as per manufacturer's instructions. Susceptibility of the isolate was performed by the Kirby-Bauer disc diffusion method and agar dilution method following CLSI guidelines. PCR targeting carbapenemase-encoding genes was performed, followed by transformation and conjugation experiments. Whole-genome sequencing of the isolate was done through the Illumina sequencing platform and the data were analysed using the Centre for Genomic Epidemiology database. BJD_EC180 is 6 919 180 bp in length and consists of six rRNA operons, 111 tRNA, and 6849 predicted protein-coding sequences. BJD_EC180 belonged to ST2437 and harboured the carbapenemase-encoding gene blaOXA-144 with ISAba1 upstream, along with multiple antibiotic resistance genes conferring clinical resistance towards beta-lactams, aminoglycosides, amphenicols, sulphonamides, tetracyclines, trimethoprim, and rifampin. CONCLUSIONS: Carbapenem-resistant E. coli harbouring blaOXA-144 associated with insertion sequence pose a serious health threat as their mobilization into carbapenem non-susceptible strains that will contribute to the resistance burden and therefore, needs urgent monitoring.

Evaluation of a novel chromogenic screening method for detection of carbapenem-resistant Escherichia coli.

Introduction. Early detection of carbapenem-resistant Escherichia coli (CREco), categorized as a critical priority pathogen by the World Health Organization (WHO), is crucial in optimizing therapeutic options and to thwart outbreaks in clinical settings.Gap statement. The need of the hour is a diagnostic method that can detect carbapenem resistance conferred by intrinsic or acquired carbapenem resistance mechanisms or both.Aim. The study investigates the performance of a novel screening chromogenic method for detection of CREco.Methodology. Carbapenem-susceptible (n=23) and non-susceptible (n=90) E. coli were used to investigate the efficiency of the blue chromogenic test. All of the isolates were received from a tertiary referral hospital in Silchar, India and subjected to the blue chromogenic test and observed for colour change. A colour change from colourless to blue is interpreted as a positive result. The test results were further compared with available methods for detection of carbapenem resistance conferred by carbapenemase production or other carbapenem resistance mechanisms.Results. The blue chromogenic test generated 100 % (CI: 95.98-100 %) sensitive and 100 % (CI: 85.75-100 %) specific results for the detection of CREco with no false-positive or false-negative results. Within 3 h after incubation, the test detects all CREco with carbapenemase activity. Additionally, the blue chromogenic test also positively detected E. coli harbouring carbapenemase variants and with efflux and porin activity, compared to other phenotypic-based approaches.Conclusion. The study highlights a novel method that is highly sensitive and specific, inexpensive, rapid and user-friendly for the detection of CREco. With the surge and expansion of CREco, this sensitive, specific, user-friendly and inexpensive method can be used in laboratories with limited facilities for early detection of CREco, thereby improving infection control along with averting future outbreaks.

Emergence of carbapenem-resistant enterobacterales co-harboring blaOXA-78 and blaOXA-58 from India.

BACKGROUND: Carbapenem-Resistant Enterobacterales (CRE) has been categorized as pathogens of critical priority by World Health organization (WHO) as they pose significant threat to global public health. Carbapenemase production considered as the principal resistance mechanism against carbapenems and with the recent surge and expansion of carbapenemases and its variants among clinically significant bacteria in India, the present study reports expansion blaOXA-78 and blaOXA-58 of in CRE of clinical origin. METHODS: Bacterial isolates were collected from a tertiary referral hospital and identified through VITEK® 2 Compact automated System (Biomerieux, France). Rapidec® Carba NP (Biomerieux, France) was used to investigate carbapenemase production followed by antibiotic susceptibility testing through Kirby-Bauer Disc Diffusion method and agar dilution method. Class D carbapenemase genes were targeted through PCR assay followed by investigation of horizontal transmission of blaOXA-58 and blaOXA-78. Whole genome sequencing was carried out using Illumina platform to investigate the genetic context of blaOXA-58 and blaOXA-78 genes and further characterization of the CRE isolates. RESULTS: The carbapenem-resistant Escherichia coli (BJD_EC456) and Serratia marcescens (BJD_SM81) received during the study from the tertiary referral hospital were isolated from sputum and blood samples respectively. PCR assay followed by whole genome sequencing revealed that the isolates co-harbor blaOXA-58 and blaOXA-78, a variant of blaOXA-51. Horizontal transfer of blaOXA-58 and blaOXA-78 genes were unsuccessful as these genes were located on the chromosome of the study isolates. Transposon Tn6080 was linked to blaOXA-78 in the upstream region while the insertion sequences ISAba26 and ISCfr1 were identified in the upstream and downstream region of blaOXA-58 gene respectively. In addition, both the isolates were co-harboring multiple antibiotic resistance genes conferring clinical resistance towards beta-lactams, aminoglycosides, fluroquinolones, sulphonamides, tetracyclines. BJD_EC180 belonged to ST2437 while BJD_SM81 was of an unknown sequence type. The nucleotide sequences of blaOXA-78 (OQ533021) and blaOXA-58 (OQ533022) have been deposited in GenBank. CONCLUSIONS: The study provides a local epidemiological information regarding carbapenem resistance aided by transposon and insertion sequences associated blaOXA-78 and blaOXA-58 genes associated and warrants continuous monitoring to prevent their further dissemination into carbapenem non-susceptible strains thereby contributing to carbapenem resistance burden which is currently a global concern.

Transcriptional Response of vanB Operon in Staphylococcus aureus Against Vancomycin and Teicoplanin Stress.

Staphylococcus aureus is a global pathogen and is responsible for causing severe life-threatening infections. The current study was designed to investigate transcriptional expression of different core, regulatory, and accessory genes within vanB operon under differential exposure of vancomycin and teicoplanin. Four isolates selected for the study, were confirmed to harbour vanB gene in which three isolates showed MIC breakpoint above 16 µg/ml and one isolate above 8 µg/ml against vancomycin while teicoplanin showed higher MIC breakpoint as compared to vancomycin. Antibiotic susceptibility results showed that these isolates were susceptible towards imipenem and linezolid. Transcriptional expressional analysis of the core gene of vanB operon showed that expression of vanB is increased under vancomycin stress but is inversely proportional to increase in the concentration of the vancomycin while under teicoplanin stress the expression of vanB showed no significant pattern. Similar expressional pattern was found for vanH gene for both the glycopeptides. In case of vanX, expression was significantly increased at 1 µg/ml exposure of vancomycin, however, no pattern could be observed in case of teicoplanin stress. In case of regulatory gene, vanR, significant increase in expression was observed under vancomycin and teicoplanin stress of 1 µg/ml, however vanS, showed significant increase in the expression under 1 µg/ml of vancomycin. The accessory gene, vanY showed marginal increase in expression under both the antibiotic, while in case of vanW, the expressional pattern was found to be inversely proportional to the increasing antibiotic concentration.

Escherichia coli harbouring strAB with reduced susceptibility towards gentamicin and amikacin: a single centre study from India.

In this study we report the presence of streptomycin resistance gene strAB within clinical isolates of Escherichia coli where streptomycin is not used to treat Gram-negative infections. In total, 135 E. coli isolates were obtained for the study. PCR based detection of strAB was performed in the study isolates followed by assessment of horizontal transferability. Cloning of strAB was done in laboratory strain E. coli DH5α. Pre-cloning and post-cloning susceptibility of the strain was done for assessment of acquired resistance. Among tested isolates, 89 were found to harbour strAB and it was encoded within a IncI1 type plasmid. Cloning experiments revealed the strAB gene showed unusual non-susceptibility towards amikacin and gentamicin. The study highlighted that strAB, which has a role in streptomycin resistance, may also have a role in reduced susceptibility towards gentamicin and amikacin within a clinical setting.

Elevated expression of rsmI can act as a reporter of aminoglycoside resistance in Escherichia coli using kanamycin as signal molecule.

We aimed to design and analyse expressional response of endogenous and exogenous 16S rRNA methyl transferase genes under sub inhibitory concentration stress of different clinically relevant aminoglycoside antibiotics in Escherichia coli to identify an endogenous marker. One hundred twenty nine aminoglycoside resistant E. coli of clinical origin were collected for detection of 16S rRNA methyl transferase genes by PCR assay and each gene type was cloned within E. coli JM107. Parent isolates were subjected to plasmid elimination by SDS treatment. Expression analysis of both acquired and endogenous 16S rRNA methyl transferase genes were performed by quantitative real-time PCR in clones and parent isolates under aminoglycoside stress (4 mg/L). Majority of the isolates were harbouring rmtC (35/129), followed by rmtB (32/129), rmtA (21/129), rmtE (13/129), armA (11/129) rmtF (9/129) and rmtH (8/129). Plasmid was successfully eliminated for all the isolates with 6% of SDS. Expression analysis indicates that kanamycin, tobramycin and netilmicin stress could increase the expression of 16S rRNA methyltransferese genes. In the presence of kanamycin stress the expression of rsmI was consistently elevated for all the wild type isolates and clones tested. Except for isolates harbouring rmtB and rmtC expression of rsmE and rsmF was increased in the presence of all aminoglycosides. For all the cured mutants it was apparently observed that expression of endogenous methyl transferases were marginally increased. Elevated expression of constitutive rsmI can be used as a potential biomarker for detection of acquired 16S rRNA methyl transferase mediated aminoglycoside resistance by using sub inhibitory concentration of kanamycin as signal molecule.

Colistin exposure enhances expression of eptB in colistin-resistant Escherichia coli co-harboring mcr-1.

Colistin resistance has increased due to the increasing and inappropriate use of this antibiotic. The mechanism involves modification of lipid A with phosphoethanolamine (PEtN) and/or 4-amino-4deoxy-L-arabinose (L-Ara4N). EptA and eptB catalyze the transfer of phosphoethanolamine to lipid A. In this study, gene network was constructed to find the associated genes related to colistin resistance, and further in vitro validation by transcriptional analysis was performed. In silico studies showed that eptB gene is a highly interconnected node in colistin resistance gene network. To ascertain these findings twelve colistin-resistant clinical isolates of Escherichia coli were selected in which five were harboring the plasmid-mediated mcr-1. Screening for colistin resistance was performed by broth microdilution (BMD) method and Rapid polymyxin NP test. PCR confirmed the presence of the eptA and eptB genes in all isolates and five isolates were harboring mcr-1. Transcriptional expression in five isolates harboring mcr-1, showed an enhanced expression of eptB when exposed under sub-inhibitory colistin stress. The present study for the first time highlighted genetic interplay between mcr-1 and eptA and eptB under colistin exposure.

Subinhibitory concentration stress of colistin enhanced PhoPQ expression in Escherichia coli harboring mcr-1.

The increased and inappropriate use of colistin led to the emergence of its resistance among Gram-negative bacterial isolates and the most common mechanism of colistin resistance in Gram-negative bacteria is the modification of the lipopolysaccharide mediated by two-component regulatory systems, PhoPQ and PmrAB. The aim of the present study was to investigate the transcriptional expression of the PhoPQ system against colistin stress in clinical isolates of Escherichia coli with colistin-resistant phenotype. Six colistin-resistant E. coli isolates were obtained from Silchar Medical College and Hospital, Silchar that were of clinical origin and received for routine culture and sensitivity testing. Screening for colistin resistance was done by broth microdilution method and further screened for the presence of the different types of plasmid-mediated colistin resistance mcr genes namely, mcr-1 to mcr-10 by polymerase chain reaction (PCR). The screened positive isolates were subjected to PCR assay targeting phoP and phoQ genes and their expression was measured by quantitative real-time PCR. The results of this study revealed that two E. coli isolates (TS2 and TS4) were found to carry the mcr-1 gene. PhoP and PhoQ gene amplification was observed in all the isolates. Transcriptional analysis showed that the isolates harboring the mcr-1 gene showed an enhanced level of expression in the PhoP, PhoQ genes in the presence of a subinhibitory concentration of colistin whereas no significant expression was observed for the isolates which were devoid of the mcr gene. This study demonstrates the involvement of mcr-1 in the PhoPQ system in clinical isolates of colistin-resistant E. coli which will help in designing a molecular marker for detecting colistin-resistant E. coli and contribute to the assessment of resistance burden and infection control strategy.

Report of a carbapenemase gene blaIMP-4 in multi-drug resistant Escherichia coli from sewage water: A threat on clinical-environmental interphase.

Acquired carbapenemases pose a significant role in the dissemination of antimicrobial-resistant Enterobacteriaceae and in this study we have identified the occurrence of blaIMP-4 in E. coli isolate from a sewage outfall located nearby a secondary health Centre. It was found to co-existed with blaCTX-M-15 located within a self-conjugable plasmid of IncF type. The current study underscores environment as a potential reservoir of carbapenem resistance and the need of the hour is to track and check dissemination of resistance in environment, human and agricultural settings.

Transcriptional expression of secondary resistance genes ccdB and repA2 is enhanced in presence of cephalosporin and carbapenem in Escherichia coli.

BACKGROUND: The issue of carbapenem resistance in E.coli is very concerning and it is speculated that cumulative effect of both primary resistance genes and secondary resistance genes that act as helper to the primary resistance genes are the reason behind their aggravation. Therefore, here we attempted to find the role of two secondary resistance genes (SRG) ccdB and repA2 in carbapenem resistance in E. coli (CRE). In this context influential genes belonging to secondary resistome that act as helper to the primary resistance genes like blaNDM and blaCTX-M in aggravating ß-lactam resistance were selected from an earlier reported in silico study. Transcriptional expression of the selected genes in clinical isolates of E.coli that were discretely harboring blaNDM-1, blaNDM-4, blaNDM-5, blaNDM-7 and blaCTX-M-15 with and without carbapenem and cephalosporin stress (2 µg/ml) was determined by real time PCR. Cured mutants sets that were lacking (i) primary resistance genes, (ii) secondary resistance genes and (iii) both primary and secondary resistance genes were prepared by SDS treatment. These sets were then subjected to antibiotic susceptibility testing by Kirby Bauer disc diffusion method. RESULTS: Out of the 21 genes reported in the in silico study, 2 genes viz. repA2 and ccdB were selected for transcriptional expression analysis. repA2, coding replication regulatory protein, was downregulated in response to carbapenems and cephalosporins. ccdB, coding for plasmid maintenance protein, was also downregulated in response to carbapenems except imipenem and cephalosporins. Following plasmid elimination assay increase in diameter of zone of inhibition under stress of both antibiotics was observed as compared to uncured control hinting at the reversion of antibiotic susceptibility by the-then resistant bacteria. CONCLUSION: SRGs repA2 and ccdB help sustenance of blaNDM and blaCTX-M under carbapenem and cephalosporin stress.

Extensively Drug-Resistant Hypervirulent Klebsiella pneumoniae From a Series of Neonatal Sepsis in a Tertiary Care Hospital, India.

The recent emergence of multidrug-resistant (MDR) Klebsiella pneumoniae with hypervirulent traits causing severe infections and considerable mortality is a global cause for concern. The challenges posed by these hypermucoviscous strains of K. pneumoniae with regard to their optimal treatment, management, and control policies are yet to be answered. We studied a series of extensively drug-resistant (XDR) and hypervirulent K. pneumoniae ST5235 isolates with resistance to carbapenems and polymyxins causing neonatal sepsis in a tertiary care hospital in India. A total of 9 K. pneumoniae isolates from 9 cases of neonatal sepsis were studied with respect to their clinical relevance, antimicrobial susceptibility profile, presence of extended spectrum ß lactamase (ESBL) production, and responsible genes, carbapenemases (classes A, B, and D), and aminoglycoside-resistant genes. Hypervirulence genes encoding hypermucoid nature, iron uptake, and siderophores were detected by multiplex PCR. The plasmid profile was studied by replicon typing. Isolates were typed by multilocus sequence typing (MLST) and enterobacterial repetitive intergenic consensus (ERIC) PCR to study the sequence types (STs) and clonal relation, respectively. The neonates in the studied cases had history of pre-maturity or low birth weight with maternal complications. All the cases were empirically treated with piperacillin-tazobactam and amikacin followed by imipenem/meropenem and vancomycin and polymyxin B as a last resort. However, all the neonates finally succumbed to the condition (100%). The studied isolates were XDR including resistance to polymyxins harboring multiple ESBL genes and carbapenemase genes (bla NDM and bla OXA-48). Hypervirulence genes were present in various combinations with rmpA/A2 genes present in all the isolates. IncFI plasmids were detected in these isolates. All belonged to ST5235. In ERIC PCR, 6 different clusters were seen. The study highlighted the emergence and burden of XDR hypervirulent isolates of K. pneumoniae causing neonatal sepsis in a tertiary care hospital.

Expressional Pattern of psm-mec System in Methicillin-Resistant Staphylococcus aureus Under Oxacillin Stress.

The psm-mec element and other regulatory factors such as sarA, agrA, and RNAIII are responsible for maintaining the genetic framework for enhanced virulence of MRSA. psm-mec is found predominantly in the staphylococcal cassette chromosome (SCCmec). sarA, agrA, and RNAIII control gene expression to facilitate adaptation in certain environment. Genome-wide approaches have shown that expression of virulence factors is frequently regulated at transcriptional, translational level, and mRNA degradation level. In this study, transcriptional responses of psm-mec gene in accordance with other regulatory factors sarA, agrA, and RNAIII were observed under normal conditions as well as when exposed to 2 µg/ml and 6 µg/ml of oxacillin stress. One-way t-test was carried out for analysing RQ values obtained through real-time PCR. This study showed downregulation of psm-mec gene and upregulation of other regulatory genes at lower concentration of oxacillin. However, this was reverse when exposed against higher concentration of oxacillin. It was observed from the study that the expression of virulence factors were dependent on each other under different concentration of oxacillin. Thus, this study highlights that psm-mec, sarA, agrA, and RNAIII gene are under direct control of antibiotic pressure in a concentration-dependent manner.

Correction to: Expansion of acquired 16S rRNA methytransferases along with CTX-M-15, NDM and OXA-48 within three sequence types of Escherichia coli from northeast India.

An amendment to this paper has been published and can be accessed via the original article.

Expansion of acquired 16S rRNA methytransferases along with CTX-M-15, NDM and OXA-48 within three sequence types of Escherichia coli from northeast India.

BACKGROUND: This study aimed to identify ten different 16S rRNA methyltransferase genes (rmtA, rmtB, rmtC, rmtD, armA, rmtF, npmA, rmtH, rmtE and rmtG) and their coexisting ESBL and carbapenemase with the emergence of three E.coli clones within a single study centre. METHODS: A total of 329 non-duplicate E.coli isolates were studied to detect the presence of 16S rRNA methyltransferases along with ß-lactamases (TEM, SHV, OXA, VEB, GES, PER,CTX-M types, NDM, OXA-48,VIM, IMP and KPC) using PCR assay. Horizontal transferability were validated by transformation and conjugation analysis. Plasmid incompatibility typing and MLST analysis was also performed. RESULTS: A total of 117 isolates were found to be resistant to at least one of the aminoglycoside antibiotics. It was observed that 77 (65.8%) were positive for 16S rRNA methyltransferases. Among them thirty nine isolates were found to harbour only blaCTX-M-15, whereas combination of genes were observed in three isolates (blaVEB+ blaCTX-M-15 in 2 isolates and blaPER + blaCTX-M-15 in 1 isolate). blaNDM and blaOXA-48 like genes were found in 23 and 9 isolates, respectively. All the resistance genes were conjugatively transferable, and incompatibility typing showed multiple 16S rRNA methyltransferase genes were originated from a single Inc. I1 group. MLST analysis detected 3 clones of E.coliST4410, ST1341 and ST3906. CONCLUSION: The present study identified emergence of three clones of E.coli, resistant to aminoglycoside -cephalosporin- carbapenem. This warrants immediate measures to trace their transmission dynamics in order to slow down their spread in clinical setting.

Occurrence of diverse aminoglycoside modifying enzymes with co-existing extended-spectrum-ß-lactamases within Enterobacteriaceae isolated in India.

OBJECTIVE: The present study describes aminoglycoside modifying enzymes (AMEs) among clinical isolates with coexisting extended spectrum beta-lactamases. METHODOLOGY: A total of 227 non duplicate enterobacterial isolates were collected and identified from patients who were admitted to different wards or attended OPD of a tertiary referral hospital of North-East India. Isolates were initially screened for antimicrobial susceptibility testing followed by PCR based screening of aminoglycosides modifying enzymes and co-existing ESBLs and carbapenemases. Horizontal transferability, incompatibility typing and stability of plasmids were also analyzed. RESULTS: Diverse types of AMEs were observed namely; ant(3″)-I, ant(4')-Ia, aac(3)-IIc, ant(3')-I, aac(6')-Ib, ant(2″)-Ia and aac(6'). Majority of the AME positive isolates harboured blaTEM followed by blaCTX-M-15 and a combination of blaTEM and blaCTX-M-15 were also observed. Nine isolates were found to harbour carbapenemases genes. AME genes were found to be located within a self conjugative plasmid of Inc FIA, IncY, IncN, IncFIB and IncA/C incompatibility types. It was observed that most AME genes were stable over 50 days of serial passages whereas aph(3')-Via and aph(3')-IIb were completely lost within 50 days. CONCLUSION: This study underscores the co-existence of AMEs and ESBLs within enterobacteriaceae which emphasize a reassessment of combination therapy in the health settings.

Diverse aminoglycoside phosphotransferase types conferring aminoglycoside resistance in Enterobacteriaceae: A single-centre study from Northeast India.

The present study investigates the molecular basis of aph-mediated aminoglycoside resistance and their transmission dynamics in a tertiary care hospital of Northeast India. Two hundred forty one isolates (230 Escherichia coli and 11 Klebsiella pneumoniae) were collected and screened for aminoglycoside resistance genes. Various aph types were amplified using polymerase chain reaction (PCR) assay. Plasmid incompatibilty, horizontal transferability and ERIC-PCR based typing were carried out for all the positive isolates. Among them, 67 isolates showed the presence of aph gene. Aph (3")-IIIa and aph (3')-Via were predominant and horizontally transferable. All the plasmids were of incompatibility I1 group. Twenty-eight different haplotypes of E. coli were found harbouring aph gene types. This study was able to identify diverse aph types in a single centre and their corresponding phenotypic trait.

Association of glycerol kinase gene with class 3 integrons: A novel cassette array within Escherichia coli.

BACKGROUND: Integrons are genetic elements which are known for their role in capturing and spreading of antibiotic resistance determinants among Gram-negative bacilli. So far, there is no study regarding Class 3 integron and their genetic organisation in India. OBJECTIVE: This study investigates the occurrence of Class 3 integron and their gene cassette array among Escherichia coli. MATERIALS AND METHODS: In this study, a total of 200 E. coli isolates were collected from indoor and outdoor patients from Silchar Medical College and Hospital during September 2015 to February 2016. Detection of the integrase genes and gene cassettes within the Class 3 integron was performed by polymerase chain reaction which was further analysed by sequencing. RESULTS: Twenty-seven isolates were found to harbour Class 3 integron. Sequencing of the gene cassettes and whole Class 3 integron revealed the presence of nine different types of cassettes array, out of which the arrangement with glycerol kinase gene cassette was found to be the most prevalent. Arrangement with blaCTX-Mgene cassette was also detected in few isolates. CONCLUSION: This study provides epidemiological profiling of Class 3 integrons in this geographical area. The data generated in this study are helpful in infection control programme, anti-infective research and search for epidemiological markers.

Occurrence of Acquired 16S rRNA Methyltransferase-Mediated Aminoglycoside Resistance in Clinical Isolates of Enterobacteriaceae within a Tertiary Referral Hospital of Northeast India.

The methylation of a ribosomal target leads to a high level of resistance to all clinically relevant aminoglycoside antibiotics, so early detection of these resistance determinants will help to reduce the incidence of treatment failures as well as lessen the dissemination rate. Here, we characterized different 16S rRNA methyltransferases responsible for aminoglycoside resistance and their epidemiological background in clinical isolates of Enterobacteriaceae in a tertiary referral hospital in India. All aminoglycoside-resistant isolates were screened for different 16S rRNA methyltransferases by PCR assay, and incompatibility typing of the conjugable plasmid harboring resistance genes was performed by PCR-based replicon typing. An assay for the stability and elimination of these resistance plasmids was performed. The coexistence of extended-spectrum ß-lactamases and metallo-ß-lactamases was also detected, and the heterogeneity of these isolates was determined by enterobacterial repetitive intergenic consensus PCR. The PCR assay revealed the presence of armA, rmtA, rmtB, rmtC, and rmtD in single and multiple combinations, and these were carried by a diverse group of Inc plasmids. Plasmids harboring these resistance determinants were highly stable and maintained until the 55th serial passage, but SDS treatment could easily eliminate the plasmids harboring the resistance determinants. The coexistence of blaTEM, blaPER, blaGES, and blaSHV, as well as blaVIM and blaNDM, within these isolates was also detected. Strains with different clonal patterns of aminoglycoside resistance were found to spread in this hospital setting. We observed that the 16S rRNA methyltransferase genes were encoded within different Inc plasmid types, suggesting diverse origins and sources of acquisition. Therefore, the present study is of epidemiological importance and can have a role in infection control policy in hospital settings.