Expansion of highly stable blaOXA-10 β-lactamase family within diverse host range among nosocomial isolates of Gram-negative bacilli within a tertiary referral hospital of Northeast India

The current study reports dissemination of highly stable blaOXA-10 family of beta lactamases among diverse group of nosocomial isolates of Gram-negative bacilli within a tertiary referral hospital of the northern part of India. In the current study, a total number of 590 Gram negative isolates were selected for a period of 1 year (i.e. 1st November 2011–31st October 2012). Members of Enterobacteriaceae and non fermenting Gram negative rods were obtained from Silchar Medical College and Hospital, Silchar, India. Screening and molecular characterization of β-lactamase genes was done. Integrase gene PCR was performed for detection and characterization of integrons and cassette PCR was performed for study of the variable regions of integron gene cassettes carrying blaOXA-10. Gene transferability, stability and replicon typing was also carried out. Isolates were typed by ERIC as well as REP PCR. Twenty-four isolates of Gram-negative bacilli that were harboring blaOXA-10 family (OXA-14, and OXA16) with fact that resistance was to the extended cephalosporins. The resistance determinant was located within class I integron in five diverse genetic contexts and horizontally transferable in Enterobacteriaceae, was carried through IncY type plasmid. MIC values were above break point for all the tested cephalosporins. Furthermore, co-carriage of blaCMY-2 was also observed. Multiple genetic environment of blaOXA-10 in this geographical region must be investigated to prevent dissemination of these gene cassettes within bacterial population within hospital settings.


Background
Expansion of β-lactamases in Gram-negative rods has been documented as most severe threat to the management of infectious diseases [1][2][3][4]. The ever-increasing use of antibiotics with the evolution of intrinsic and acquired resistance has led to the development of resistance mechanism in Gram-negative rods contributing to the expansion of several multi-drug resistance epidemics in hospital environment [1,3,5]. OXA-10 type was known to have narrow spectrum β-lactamase activity; although variant of this enzyme family has expandedspectrum activity [3,6,7]. It has been extensively associated with infection of Gram-negative bacteria in the last two decades restricting therapeutic options. These genes are often reported to be located within integron gene cassettes [3,4]. However, these rare types of beta lactamases are often unreported in the light of current incidence of New Delhi metallo beta lactamase and CTX-M types. The current study reports the dissemination of highly stable bla OXA-10 family among diverse group of nosocomial isolates of Gram-negative bacilli within a tertiary referral hospital of the northern part of India.

Screening and molecular characterization of β-lactamases
Isolates resistant to at least one of the expanded-spectrum cephalosporins (cefotaxime, ceftazidime, or ceftriaxone) were selected for the study. For amplification and characterization of β-lactamase genes, multiplex PCR was performed (T100, BioRad-USA) with set of five primers namely: bla CTX-M [8], bla TEM , bla OXA-10 , bla OXA-2 [9] and bla SHV [10] (Additional file 1: Table S1). Previously confirmed beta-lactamase genes were used as positive control which were obtained from Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. PCR was performed by using Go-Taq Green Master Mix (Promega, Madison, USA) and products were visualized in 0.5% Agarose gel. PCR product was purified using Gene JET PCR product purification kit (Thermo Scientific, Lithuania) and sequencing was done using Sanger's Method in Xcelris Lab Pvt Ltd in Ahmedabad, India.
PCR assay was also carried out for detection of AmpC genes in donor strains and transformants as described earlier [11]. Carbapenemase production in donor strains and transformants was tested by modified Hodge test, Imipenem-EDTA disc test [12] and boronic acid inhibition test [13] followed by PCR assay targeting bla OXA-48 [14] and bla OXA-23, -24/40 and -58 [15].

Study of genetic context and southern blot hybridization
Presence of integron was detected by integrase gene PCR [16]. To study the variable regions of integron gene cassettes carrying bla OXA-10 , two PCR assays were performed consequently: in one reaction 5′-CS and reverse primer of bla OXA-10 and in other reaction 3′-CS and forward primer of bla OXA-10 were used [9,16] (Additional file 1: Table S2). Purified PCR products were cloned on pGEM-T vector (Promega, Madison, USA) and further sequenced. To validate our study, Southern hybridization was performed on agarose gel by in-gel hybridization with the bla OXA-10 family specific probe labeled with Dig High Prime Labeling Mix (Roche, Germany) detection Kit. Plasmid DNA was separated on agarose gel and transferred to nylon membrane (Hybond N, Amersham, UK) and hybridized.

Transferability, PCR-based replicon typing and stability of bla OXA-10 family
Transformation was carried out using E. coli JM107 as recipient. Conjugation experiment was performed taking clinical isolates as donors and a streptomycin resistant E. coli-strain B (Genei, Bangalore) as recipient and transconjugants were selected on Luria-Bertani Agar plates containing cefotaxime (0.5 µg/ml) and streptomycin (800 µg/ml). Plasmid transfer was confirmed by colony PCR of transconjugants and transformants with the targeted primers [8,9]. Plasmid stability of all bla OXA-10 producers as well as their transformants was analyzed by serial passages method for consecutive 115 days without antibiotic pressure [17]. Colony PCR assay was carried out in the isolates after each passage. Incompatibility typing was carried out by PCR-based replicon typing targeting 18 different replicon types [18] among all the wild types and their transformants carrying bla OXA-10.

Antimicrobial susceptibility and minimum inhibitory concentration determination
Antimicrobial susceptibility of bla OXA-10 -harboring donor strains as well as transformants was determined by Kirby-Bauer disc diffusion method towards all nonβ-lactam antibiotics (Hi-Media, Mumbai, India) [19].
MICs of donor as well as transformants and transconjugants were also done against beta lactam groups on Muller Hinton agar (Hi-Media, Mumbai, India) plates by agar dilution method and results were interpreted as per CLSI recommendation [19].

Results and discussion
A total of 58.5% (n = 345) isolates were resistant to expanded-spectrum cephalosporins. Among them 24 showed amplification with bla OXA-10 primers and were further confirmed by sequencing as OXA-14 (n = 15), and OXA-16 (n = 9) derivatives (Table 1). Sequencing results confirmed the presence of resistance determinant within class I integron with five different types of genetic environment ( Fig. 1; Table 1). Upstream region of bla OXA-10 was occupied by dfrA12 (Type 1), dfrA17 (Type 5), dfrA1 (Type 3 and Type 2), dfrA7 (Type 4), arr2 (Type 2), aac A4 (Type 2), aad A5 (Type 4 and Type 5), while in downstream regions aad A2 (Type 1), aad A1 (Type 2 and Type 3), aac (6′)1b (Type 4), and qacE (Types 1-5) genes were present (Fig. 1). Plasmid encoding bla OXA-10 was successfully transferred in E. coli for Enterobacteriaceae, while in case of P. aeruginosa the attempt was not successful. Hybridization experiment revealed that bla OXA-10 carriage was plasmid mediated for Enterobacteriaceae. Replicon typing result established that bla OXA-10 was encoded within IncY type plasmid (Table 1) (Additional file 2: Figure S1). All isolates belonging Enterobacteriaceae family were found susceptible to tigecycline while Polymyxin B susceptibility was observed in P. aeruginosa. In case of Enterobacteriaceae, MICs of donor strain and transformants were observed above the breakpoint against cephalosporins, carbapenems, and monobactams (Table 2) and similar MIC pattern was too observed in P. aeruginosa (Table 3). The bla OXA-10 was highly stable and none of the isolates lost the gene till 115 serial passages. Modified Hodge test could detect carbapenemase activity in seven isolates and bla CMY-2 was also co-carried along with bla OXA-10 in four isolates (Table 1). DNA fingerprinting by ERIC (in E. coli-ERIC Types 1-9; Klebsiella spp.-ERIC Types 1-3; Proteus spp.-ERIC Type 1) ( Table 1; Additional file 2: Figure S2) and REP PCR (In P. aeruginosa-REP Types 1-11) ( Table 1; Additional file 2: Figure S3) was suggestive that diverse clonal types were present. So far, in India predominant types of beta-lactamases are CTX-M-15 and in recent years carbapenem therapy is compromised due to emergence of New Delhi Metallo beta-lactamase from this subcontinent. However, OXA type beta lactamases with extended spectrum activities are rarely reported [21,22]. Our data showed that the majority of the isolates were recovered from surgery ward (37.5%; n = 9) followed by medicine (33.33%; n = 8), pediatrics (16.66%; n = 4), and gynecology (12.5%; n = 3). Carriage of bla OXA-10 within integron with diverse genetic environment comprising different coexisting-resistant determinant shows its multiple sources of acquisition and complicacy while determining the therapeutic options. It was also found that bla OXA-10 was horizontally transferable in Enterobacteriaceae family which was supported by transformation and conjugation. However, unsuccessful transfer of bla OXA-10 in P. aeruginosa could be due to their plasmid not being replicated within E. coli recipient or a chromosomal location of the gene. High MIC against carbapenems could be due to presence of some gene types which was not amplified by our target primers. Capability of the organisms to retain the resistant gene even after withdrawal of antibiotic pressure underscores their vertical transfer and persistence in the cell, which possibly can be the reason of expansion of this resistance determinant within hospital environment as well as in community. High MICs of the donor strain as well as their transformants could be due to the coexistence of another β-lactamase enzyme as observed in the current study.

Conclusion
To the best of our knowledge this is the first report of gene cassette-mediated carriage of bla OXA-10 from India. Their acquisition and dissemination as well as adaptation against high antibiotic pressure in the hospital environment demands immediate measure to prevent transmission of these genetic vehicles, by the adoption of proper infection control measures and treatment policies.  Table S1. Oligonucleotides used as primers for amplification of different ESBL genes. Table S2. Primers used for characterization of integron.   DS  TF  DS  TF  DS  TF  DS  TF  DS  TF  DS  TF  DS  TF  DS  TF   2