Emergence of Carbapenemase Encoding Genes in Proteus Species in Tertiary Care Hospital of Bangladesh

Keywords: Proteus, blaOXA-484, blaOXA-66, carbapenemases

Along with di erent carbapenemase encoding genes, in recent year class D OXA enzymes are documented in Proteus spp which are not common in Enterobacteriaceae. e dissemination of plasmids, transposons and integrons among bacteria and species playing roles for this dissemination. So, this study was designed to observe the emergence and distribution of di erent classes of carbapenemase encoding genes among imipenem resistant Proteus spp. isolated from tertiary care hospital in Bangladesh. Total 15 imipenem resistant Proteus isolates were included in this study, which were collected from wound swab, pus, urine and blood samples. Identi cation was done by culture and biochemical test and antibiotic susceptibility test was done by disc di usion method. MIC of imipenem (g/ml) was done among imipenem resistant P. mirabilis by agar dilution method. blaKPC, blaNDM-1, blaVIM, blaIMP, blaOXA-48 like, blaOXA-23 like, blaOXA-51 like, blaOXA-58 like carbapenemase encoding genes were detected among imipenem resistant Proteus spp. by PCR and sequencing of blaOXA-48 like, blaOXA-51 like gene done by capillary method to compare the sequences with the same gene, available in gene bank. Among 15 imipenem resistant isolates blaNDM-1 (26.67%), blaKPC (20%), blaVIM (20%), blaOXA-484 (20%) were predominant carbapenemase encoding genes followed by blaOXA-66(6.67%). 
is study nds that blaOXA-484 gene and blaOXA-66 class D carbapenemase encoding genes are emerging in Proteus spp. and may play a contributing factor in developing carbapenem resistance. 

INTRODUCTION

e genus Proteus, are commonly implicated pathogens in hospitals and as a cause of community acquired infections. 1 is pathogen has various mode of transmission and causes infections in di erent anatomical sites. 2 It ranks as 3rd cause of health care associated infections. 3 Carbapenem antibiotics have been used as a last resort to treat infections caused by multidrug resistant gram negative bacteria till date. 4 But in recent years rampant global spread of carbapenem resistant Enterobacteriaceae members becoming a threat, including Proteus. 5 Acquisition of carbapenemase enzymes and loss of porins are the main mechanism of resistance to this group of antibiotic. 6 e most common carbapenemases include veronica integrin metallo-beta-lactamases types (VIM), imipenemase (IMP) types, Klebsiella pneumoniae carbapenemase (KPC), oxacillinase-48 (OXA-48), and New Delhi metallobeta-lactamase-1 (NDM-1), encoded by carbapenem resistance determining genes blaVIM, blaIMP,blaKPC, blaOXA-48 and blaNDM, respectively. 7  study was designed to evaluate the carbapenem resistance pattern along with distribution of genes encoding carbapenem resistance among Proteus spp. isolated from tertiary care hospital in Bangladesh. 

MATERIALS AND METHODS

is cross sectional analytic type of study was conducted in the Department of Microbiology, Dhaka Medical College, Dhaka, from January 2016 to December 2016. Ethical clearance was taken from the ethical review committee of the institution. A total 310 wound swabs, pus, urine and blood samples were isolated from of clinically suspected infected patients in Dhaka Medical College Hospital. 

IDENTIFICATION OF PROTEUS SPECIES

Allthe wound swab, pus, urine and blood samples, collected from the patients were inoculated on blood agar and Mac Conkey's agar media and incubated at 37ºC aerobically for 24 hours. Primary blood culture was done in Trypticase Soy Broth followed by subculture on blood and MacConkey's agar media. 
en proteus spp. were identi ed by characteristic swarming growth and shy smell on blood agar media, non-lactose fermenting colony on MacConkey's agar media. It was glucose fermenter, oxidase negative, urease positive on biochemical reactions. 

ANTIMICROBIAL SUSCEPTIBILITY TEST

Antimicrobial susceptibility testof isolated Proteus spp. was performed using Kirby-Bauer disc di usion method. Commercially available antibiotic disc (OxoidLtd, Basingstoke, United kindom) 8 such as ceftazidime (30 μg), cefuroxime (30μg), ceftriaxone (30μg), cefoxitin (30μg), cefepime (30μg), imipenem (10μg), amoxiclav (amoxicillin and clavulanic acid) (20/10 μg), cipro oxacin (5 μg), amikacin (30 μg), gentamicin (10 μg), piperacillin-ZZZZ (25μg), ampicillin (10 μg), doxycycline hydrochloride (30 μg) were used. Zones of inhibition were interpreted according to CLSI guidelines (CLSI, 2015). 8 

Screening for carbapenemases by the disc di usion technique

Screening for carbapenem-resistance was determined using the Kirby Bauer disc di usion method with a 10 mg imipenem disc. ree to ve well isolated colonies of test organisms were emulsi ed into 3 mL of sterile normal saline. e turbidity of the suspension was compared with the 0.5 McFarland turbidity standard and the suspension was incubated on Mueller Hinton agar plates at 37°C for 24 hours. An inhibition zone of 19mm diameter around the imipenem disc was considered resistant, 20 to 22 mm indicated intermediate and 23 mm was considered sensitive. 8 

Phenotypic detection of carbapenemase producers

All the isolates showing reduced susceptibility to imipenem (zone diameter ˂19 mm) were tested for carbapenemase production using the modi ed Hodge test. Brie y, a lawn culture (0.5 McFarland) of E. coli 25922 was streaked on a Mueller Hinton agar plate.A 10µg imipenem disc was placed in the center of the agar plate. e test isolates were streaked in a straight line from the edge of the disc to the edge of the plate and were incubated overnight. A positive test was indicated by a cloverleaf-like indentation at the intersection of the test organism and the standard strain, within the zone of inhibition of the carbapenem antibiotic. 8  detection of MBL production was performed by the double-disc synergy test. 9 Minimum inhibitory concentration (MIC) of imipenem was done among imipenem resistant Proteusby agar dilution method in Muller-Hinton media and CLSI guidelines was followed for the interpretation. 

Minimum inhibitory concentration (MIC) of imipenem among imipenem resistant Proteus:


Molecular characterization of carbapenem resistance genes

By polymerase chain reaction (PCR), Class A serine carbapenemase (blaKPC), Ambler class B metallo beta lactamases (blaNDM-1, blaVIM, blaIMP), Ambler class D (blaOXA-48 like, blaOXA-51 like, blaOXA-58 like, blaOXA-23 like) genes detection wascarried out among imipenem resistant Proteusisolates. 

Preparation of the bacterial pellets:

Aloopful of bacterial colony was taken into a falcon tube, containing trypticase soy broth and overnight incubated at 37ºc temperature. 
en the tubes were centrifuged at 4,000g for 10 minutes and supernatant was discarded. A small amount of sterile trypticase soy broth was added into the falcon tubes with pellets and mixed evenly. In 2-3 microcentrifuge tubes, equal amount of bacterial suspension was taken and centrifuged at 4,000 rpm for 10 minutes. 
e supernatant was discarded and the microcentrifuge tube containing bacterial pellets were kept at -20ºc until DNA extraction. 
Bacterial DNA was extracted by the boiling method. 10 . ree hundred µl of distilled water was added into microcentrifuge tube, containing bacterial pellets and vortexed until mixed. e tubes were boiled for 10 minutes in a heat block and placed immediately into ice kept for 5 minutes. Centrifugation was done at 14,500g for 6 minutes, 10µl supernatant was used for PCR. 

Ampli cation of DNA:

e cycling parameters followed in this study was as follows: initial denaturation at 95°C for 10 minutes, then 30 cycles of denaturation at 95°C for one minute, annealing at 58°c for blaKPC, 52°C (for bla IMP and blaVIM), 58°C (for blaNDM-1) and 52°C for (blaOXA-48 like, blaOXA-51 like, blaOXA-58 like, blaOXA-23 like) for 45 seconds, extension at 72°C for iminute, and nal extension at 72°C for 10 minutes. 

Visualization of ampli ed products:

e ampli ed DNA were loaded into a 1.5% agarose gel, electrophoreses done at 100 volts for 35 minutes, stained with 1% ethidium bromide and visualized under UV light. 

Procedure of DNA sequencing:

For sequencing of bacterial DNA, puri cation of ampli ed PCR products were done by using DNA puri cation kit (FAVORGEN, Biotech Corp). Puri ed PCR products were sent to Malayasia (1s‫ݐ‬ BATCH Laboratories) and sequencing was done by capillary method on ABI PRISM 3500. en the sequenced DNA was compared with data in Gene Bank. 

Statistical analysis:

Data were analyzed by using Microsoft Excel (2007) software. 

RESULTS

Table I shows the distribution of P. mirabilis and P. vulgaris isolated from di erent samples. Total of 42 (13.55%) Proteus spp. were isolated from 310 wound swab, blood and urine samples. Among 42 proteus spp. 32 (76.19%) were P. mirabilis and 10 (23.81%) were P. vulgaris. blaOXA-51 like 0 (0.00) 0 (0.00) 0 (0.00) 1 (6.67) 1 (6.67) 
blaOXA-58 like 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 
blaOXA-23 like 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) e DNA sequence of the ampli ed PCR product of blaOXA-51 like gene (Figure 5) was 99% identical with class D beta-lactamase OXA-66, found in Acinetobacter baumanniistrain AM8, which is available in the gene bank (accession number KY923052). blaOXA-51 like gene had mutation at 28, 404 position (Figure 6). 

DISCUSSION

Very limited studies have been documented on proteus mediated infections, demographics of related patients and over all antibiotic resistance pattern of Proteus spp. ough the availability of a wide range antimicrobials of di erent categories, Proteus spp. mediated increased resistance to antimicrobials are documented. In recent years, researchers are giving attention to Proteus spp. because of high occurrence in nosocomial infections and expanding pro le of antibiotic resistance. 
In this study, highest proportion of imipenem resistant Proteus isolates were multidrug resistant and showed increased resistance to cipro oxacin, ceftriaxone, cefepime, gentamicin, amoxiclav and sensitive to piperacillintazobactam (40%) in narrow range. Out of 15 imipenem resistant Proteus spp., 20% isolates were positive for blaKPC gene. Single blaKPC-2 positive P. mirabilis was reported in the studies of Pilato et al. (2016) and Cabral et al. (2014), respectively.11,12 Possibility of this gene transmission may be due to horizontal transmission by transposons, the mobile genetic elements which can transfer from one bacterium to another. 13 ll now only a few numbers of NDM-1 producing P. mirabilis has been detected in di erent studies). e proportion of MBL producers from di erent studies including the present one suggests that the prevalence of MBL producers varies on geographical distribution and time. 16 e fth of P. mirabilis were positive for blaOXA-48 like gene and validated by sequencing as blaOXA-484 gene. Inconsistent to present ndings 23.3% OXA-48 like positive Proteus spp., were reported by Fursovaet al., (2015) which was close to the present nding. 17 Since most clinical microbiology laboratories do not test for the presence of OXA-48 like enzymes and the associated phenotype (i.e. low-level carbapenem resistance) may be di cult to recognize, the incidence of OXA-48-like gene positive carbapenem resistant Enterobacteriaceae is likely underestimated. 
In this study, base sequence of the PCR product of OXA-48 like gene which was 99% identical to the Klebseilla pneumoniae H141920513 (blaOXA-484) gene which is available in the gene bank (accession number NG_ 049766.1). e OXA-484 gene had mutation at 108 position (Figure 4). In the present study, single blaOXA-51 like positive P. mirabilis was detected by PCR and the result was validated by sequencing. Sequencing result con rmed bla0XA-66 variant (Table 6). It is to be noted that in a recent study by Osterbladet al. (2016) reported, Acinatobactor type class D carbapenemaseblaOXA-23 gene in P. mirabilis. 18 base sequence of PCR product of OXA-51 like gene was 99% identical to the Acinatobactorbaumannii strain AM8 blaOXA-66 (blaOXA-66) gene which is available in the gene bank (Accession number KY923052.1). blaOXA-66 gene had mutation at 28 and 404 position (Figure 6). 

CONCLUSIONS

In this study blaKPC, blaVIM, blaNDM-1, blaOXA-484 and blaOXA-66 were predominant carbapenemase encoding genes among imipenem resistant proteus. Both blaOXA-484 and blaOXA-66 were the new variant of class D carbapenemase encoding genes among imipenem resistant Proteus spp. is study re ects that blaNDM-1 positive proteus are increasing and blaOXA-484 and blaOXA-66 are emerging in Bangladesh. Further study should be carried out to detect the resistant genes and proper implementation of antimicrobial policies infection control programs will surely limit the rapid dissemination of this type of infection. 

Figure 1 :

 1 Figure 1: Photograph of gel electrophoresis of ampli ed DNAof blaOXA-48 like geneand blaOXA-51 like gene. 


Figure 1

 1 Figure 1 is the photograph of gel electrophoresis of amplied DNAof blaOXA-48 like geneand blaOXA-51 like gene. Here lane 4 shows the ampli ed DNA of 597bp for blaOXA-48 like gene and lane 6 shows the ampli ed DNA of 435bp for blaOXA-51 like gene. Lane 1 is for negative control without DNA. Lane 2 and lane3 are for negative sample. Lane 7and lane8 are blank. 


Figure 2 :

 2 Figure 2: Photograph of gel electrophoresis of ampli ed DNA of blaKPC, blaVIM and blaNDM-1 gene. 


Figure 2 Figure 3 .Figure 4 :

 234 Figure 2 shows photograph of gel electrophoresis of amplied DNA of blaKPC, blaVIM and blaNDM-1 gene. Here lane 3 shows the ampli ed DNA of 264 bp for blaNDM-1 gene. Lane 6 shows ampli ed DNA of 390 bp for gene and lane 7 shows ampli ed DNA of 498 bp for blaKPC gene. Lane1 is for negative control without DNA. Lane 2and lane 4 is for negative sample. Lane 8 is blank. 


Figure 5 :

 5 Figure 5: DNA sequence of ampli ed PCR product of blaOXA-51 like gene using speci c primer. 


Figure 4 shows

 4 Figure 4 shows DNA sequencing of blaOXA-48 like and blaOXA-51 like gene was done. e DNA sequence of the amplied PCR product of OXA-48 like gene (Figure3) which was found 99% identical to Klebseilla pneumoniae H141920513blaOXA gene for OXA-48 family class D beta-lactamase OXA-484, which is available in the gene bank (accession number NG_049766.1). blaOXA-48 like gene had mutation at 108 position 


Table I : Distribution of P. mirabilis and P. vulgaris isolated from di erent samples.

 I 
	Organism	Wound swab	Pus	Urine	Blood	Total
		n (%)	n (%)	n (%)	n (%)	n (%)
	P. mirabilis	18 (69.23)	6 (85.71)	7 (87.50)	1 (100.00)	32 (76.19)
	P. vulgaris	8 (30.77)	1 (14.29)	1 (12.50)	0 (0.00)	10 (23.81)
	Total	26 (100.00)	7 (100.00)	8 (100.00)	1 (100.00)	42 (100.00)

Table II : Distribution of imipenem resistant Proteus spp. in di erent samples. (N = 15)

 II Proteus spp. were isolated from di erent samples and 8 imipenem resistant Proteus spp. were included from department of microbiology of DMC, which were isolated from di erent samples. 


	Organism	Wound swab	Pus	Urine	Blood	Total
		n (%)	n (%)	n (%)	n (%)	n (%)
	P. mirabilis	8 (53.33)	3 (20.00)	2 (13.33)	1 (6.67)	14 (93.33)
	P. vulgaris	0 (0.00)	1 (6.67)	0 (0.00)	0 (0.00)	1 (6.67)
	Total	8 (53.33)	4 (26.67)	2 (13.33)	1 (6.67)	15 (100.00)

Out of 42 proteus spp.7 (16.67%)were imipenem resistant.As the sample of imipenum resistant isolates is insu cient, additional 8 imipenem resistant proteus were collected from department of Microbiology of the Dhaka Medical College to detect the carbapenemase encoding genes among them.So total 15 imipenem resistant proteus spp.were included for further study (TableII).MIC of imipenem among these 15 imipenem resistant Proteus spp.ranged from 4 to 64g/ml and highest proportion (26.67%) had MIC 16g/ml.N = Total number of bacteria.n=Totalnumber of imipenem resistant bacteria."*"= 7 imipenem resistantTableIIIshows distribution of class metallo-β-lactamase encoding genes in Proteus spp. detected by PCR method. Among 15 imipenem resistant Proteus spp. 3(20%) were positive for blaKPC, 3 (20%) for blaVIM, 4 (26.67%) for blaNDM-1, 3 (20%) for blaOXA-48 like and one (6.67%) for blaOXA-51 like gene. No isolates were found positive for blaIMP, blaOXA-23 like and blaOXA-58 like gene. 
Table III : Distribution of class metallo-β-lactamase encoding genes in Proteus spp. detected by PCR method (N= 15).

 III 
	Genes	Wound swab	Pus	Urine	Blood	Total
		n (%)	n (%)	n (%)	n (%)	n (%)
	blaKPC	2 (13.333)	0 (0.00)	1 (6.67)	0 90.00)	3 (20.00)
	blaVIM	2 (13.33)	1 (6.67)	0 (0.00)	0 (0.00)	3 (20.00)
	blaIMP	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)
	blaNDM-1	2 (13.33)	1 (6.67)	1 (6.67)	0 (0.00)	4 (26.67)
	bla0XA-48 like	1 (6.67)	1 (6.67)	1 (6.67)	0 (0.00)	3 (20.00)



  ). blaOXA-48 like gene had mutation at 108 position Comparison of DNA sequence of the ampli ed PCR product of blaOXA-51 like gene and Acinetobacter baumannii strain AM8 blaOXA-66 (blaOxa-66) gene. 


	Score 984 bits (1090) Query 13TCCGATGTGGGCATATCCATATTCATCGCAAAAAACCACACATTATCATCAAGTTCAACC 72 Expect Identities Gaps Strand 0.0 556/561 (99%) 3/561 (0%) Plus/Minus |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 734 TCCGATGTGGGCATATCCATATTCATCGCAAAAAACCACACATTATCATCAAGTTCAACC 675 Query 73 CAACCAACCCACCAGCCAATCTTAGGTTCGATTCTAGTCGAGTATCCCGTTTTAGCCCGA 132 |||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||| Sbjct 674 CAACCAACCCACCAGCCAATCTTAGGTTCGATTCCAGACGAGTATCCCGTTTTAGCCCGA 615 Query 133 ATAATATAGTCGCCATTGGCTTCGGTCAGCATGGCTTGTTTCACGATGCGCTGACTACGC 192 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 614 ATAATATAGTCGCCATTGGCTTCGGTCAGCATGGCTTGTTTCACGATGCGCTGACTACGC 555 Query 193 TCAGAAACGTGCAGCTTGTTGTGATACAGCTTGCGTAAAAAAGCGATTTGCTGGGTAGCC 252 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 554 TCAGAAACGTGCAGCTTGTTGTGATACAGCTTGCGTAAAAAAGCGATTTGCTGGGTAGCC 495 Query 253 GAAATGCGAATACCACCATCGAGCCAAAAACTGTCTACATTGCCCGAGATATCCTCATTG 312 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 494 GAAATGCGAATACCACCATCGAGCCAAAAACTGTCTACATTGCCCGAGATATCCTCATTG 435 Query 313 CCATAATCGAAGGCGTGCAGCATTTTACTCATACGTGCCTCACCAATTTGGCGGGCAAAT 372 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 434 CCATAATCGAAGGCGTGCAGCATTTTACTCATACGTGCCTCACCAATTTGGCGGGCAAAT 375 Query 373 TCTTGATAAACAGGCACAACTGAGTACTTCATCGCGGTAATTAAGTCATGGTCACGATTC 432 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 374 TCTTGATAAACAGGCACAACTGAGTACTTCATCGCGGTAATTAAGTCATGGTCACGATTC 315 Query 433 CAAGCGGCGATATCACGCGTCTGTCCATCCCACTTAAAGACTTGGTGTTCATCCTTAACC 492 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 314 CAAGCGGCGATATCACGCGTCTGTCCATCCCACTTAAAGACTTGGTGTTCATCCTTAACC 255 Query 493 ACGCCCAAATCGAGGGCGATCAAGCTATTGGGAATTTTAAAGGTAGATGCGGGTAAAAAT 552 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 254 ACGCCCAAATCGAGGGCGATCAAGCTATTGGGAATTTTAAAGGTAGATGCGGGTAAAAAT 195 Expect IdenƟƟes Gaps 655 bits(726) 0.0 391/396(99%) 3/396(0%) Query 18CTTGAGCACCGTAAGGCAACCACCACAGAAGTATTGTAAGATGGGATGGTaaaaaaaGGT 77 |||||||||| |||||||||||||||||||||||| |||| ||||||||||||||||||| Sbjct CTTGAGCACCATAAGGCAACCACCACAGAAGTATT-TAAG-TGGGATGGTAAAAAAAGGT 365 Query 78 TATTCCCAGAATGGGAAAAGGACATGACCCTAGGCGATGCCATGAAAGCTTCCGCTATTC 137 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 366 TATTCCCAGAATGGGAAAAGGACATGACCCTAGGCGATGCCATGAAAGCTTCCGCTATTC 425 Query 138 CAGTTTATCAAGATTTAGCTCGTCGTATTGGACTTGAGCTCATGTCTAAGGAAGTGAAGC 197 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 426 CAGTTTATCAAGATTTAGCTCGTCGTATTGGACTTGAGCTCATGTCTAAGGAAGTGAAGC 485 Query 198 GTGTTGGTTATGGCAATGCAGATATCGGTACCCAAGTCGATAATTTTTGGCTGGTGGGTC 257 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 486 GTGTTGGTTATGGCAATGCAGATATCGGTACCCAAGTCGATAATTTTTGGCTGGTGGGTC 545 Query 258 CTTTAAAAATTACTCCTCAGCAAGAGGCACAGTTTGCTTACAAGCTAGCTAATAAAACGC 317 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 546 CTTTAAAAATTACTCCTCAGCAAGAGGCACAGTTTGCTTACAAGCTAGCTAATAAAACGC 605 Query 318 TTCCATTTAGCCAAAAAGTCCAAGATGAAGTGCAATCCATGCTATTCATAGAAGAAAAGA 377 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 606 TTCCATTTAGCCAAAAAGTCCAAGATGAAGTGCAATCCATGCTATTCATAGAAGAAAAGA 665 Query 378 ATGGAAACAAAATATACGCAAAAAGGGGGTTGGGGA 413 |||||||||||||||||||||||| | ||||||||| Figure 6: Score Sbjct 666 ATGGAAACAAAATATACGCAAAAA-GTGGTTGGGGA 700
	Query 553 GCTT-GTTCGGCCCCGTTAAA 572
	|||| ||||| |||||| ||
	Sbjct 194 GCTTGGTTCG--CCCGTTTAA 176
	CGTNCTTGAGCACCGTAAGGCAACCACCACAGAAGTATTGTAAGATGGGATGGTAAAAAAA
	GGTTATTCCCAGAATGGGAAAAGGACATGACCCTAGGCGATGCCATGAAAGCTTCCGCTATT
	CCAGTTTATCAAGATTTAGCTCGTCGTATTGGACTTGAGCTCATGTCTAAGGAAGTGAAGCG
	TGTTGGTTATGGCAATGCAGATATCGGTACCCAAGTCGATAATTTTTGGCTGGTGGGTCCTTT
	AAAAATTA CTCCTCAGCAAGAGGCACAGTTTGCTTACAAGCTAGCTAATAAAACGCTTCCAT
	TTAGCCAAAAAGTCCAAGATGAAGTGCAATCCATGCTATTCATAGAAGAAAAGAATGGAAA
	CAAAATATACGCAAAAAGGGGGTTGGGGAAA

14,15 In

  the present study, 26.67% blaNDM-1 producing P. mirabilis were detected by PCR. Qin et al. (2015) and Girlichet al. (2015) reported single XDR P. mirabilis harboring blaNDM-1 gene, respectively. 





  14,15Asian continent serves as the major reservoir of NDM-1 producers, with around 58.15% abundance of NDM-1 variant distributed mostly in China and India.In this study, 20% P. mirabilis were positive for blaVIM gene. On the other hand, Vourliet al. (2006) reported, 100% isolated proteus were blaVIM positive. 



Acknowledgements:

Department of Microbiology, Dhaka Medical college, Dhaka provided nancial and laboratory support to perform this study. 


Competing interests:

ere is no con ict of interest.
Dr S M Shamsuzzaman, Dr Md Nahiduzzamane Shazzad, Shamsuzzama Sm, Shazzad Mn, Shahin Ma
Emergence of Carbapenemase Encoding Genes in Proteus Species in Tertiary Care Hospital of Bangladesh