Research ArticleEmergence of New Delhi Metallo-Beta-Lactamase(NDM) and Klebsiella pneumoniae Carbapenemase (KPC)Production by Escherichia coli and Klebsiella pneumoniae inSouthern Vietnam and Appropriate Methods of DetectionA Cross-Sectional Study

Cuong Q Hoang 1 Hai D Nguyen1 Huy Q Vu2 Anh T Nguyen3 Binh T Pham 2Trung L Tran4 Hanh T H Nguyen2 Y M Dao5 Tuyet S M Nguyen6 Dung A Nguyen6Hang T T Tran2 and Lan T Phan1

1The Pasteur Institute Ho Chi Minh City 700000 Vietnam2Department of Medical Laboratory Science Faculty of Nursing and Medical Technology University of Medicine and PharmacyHo Chi Minh City 700000 Vietnam

3Molecular Biomedical Center for Diagnosis and Training University Medical Center Branch No 2Medical and Pharmacy University Hospital Ho Chi Minh City 700000 Vietnam

4College of Dentistry Yonsei University 50-1 Yonsei-ro Seodaemun-gu Seoul 03722 Republic of Korea5Department of Microbiology Dong Nai General Hospital Dong Nai Province 710000 Vietnam6Department of Microbiology Gia Dinh Peoplersquos Hospital Ho Chi Minh City 700000 Vietnam

Correspondence should be addressed to Cuong Q Hoang cuonghqpasteurgmailcom

Received 30 November 2018 Revised 7 March 2019 Accepted 31 March 2019 Published 23 April 2019

Academic Editor Anjali Joshi

Copyright copy 2019 CuongQHoang et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Carbapenemase-producing Enterobacteriaceae (CPE) are well known to cause many serious infections resulting in increasingmortality rate treatment cost and prolonged hospitalization Among the widely recognized types of carbapenemases New Delhi120573-lactamase (NDM) and Klebsiella pneumoniae carbapenemase (KPC) are the most important enzymes However in Vietnamthere are only scattered reports of CPE due to the lack of simple and affordable methods that are suitable to laboratory conditionsThis study aims to survey the characteristics of carbapenem-resistant E coli and K pneumoniae (CR-EK) at two hospitals inSouthern Vietnam and perform some simple methods to detect the two enzymesA total of 100 CR-EK strains were collected fromclinical isolates of Gia Dinh Peoplersquos Hospital and Dong Nai General Hospital Vietnam from November 2017 to May 2018 Thepatient-related information was also included in the analysis We conducted real-time polymerase chain reaction (PCR) ModifiedHodge Test (MHT) and combined disk test (CDT) on all isolates Carbapenemase-encoding genes were detected in 47 isolates(36 NDM 10 KPC and one isolate harboring both genes) The E coli strain carrying simultaneously these two genes was thefirst case reported here Most of isolates were collected from patients in ICU Infectious Disease Department and Departmentof Urologic Surgery Urine and sputum were two common specimens The true positive rate (sensitivity TPR) and specificity(SPC) of the imipenemndashEDTA (ethylen diamine tetra acetic acid) for NDM detection and the imipenemndashPBA (phenylboronicacid) for KPC detection on E coli were 938 971 and 667 957 respectively Meanwhile the imipenemndashEDTA forNDM detection and the imipenemndashPBA for KPC detection among K pneumonia achieved 905 100 and 100 929TPR and SPC respectively However MHT showed low sensitivity and specificity Our findings showed that CP-EK weredetected with high prevalence in the two hospitals We suggest that CDT can be used as a low-priced and accurate method ofdetection

HindawiBioMed Research InternationalVolume 2019 Article ID 9757625 9 pageshttpsdoiorg10115520199757625

2 BioMed Research International

1 Introduction

Antibiotic resistance is a tremendous health problem Thisincludes the resistance to carbapenems which was consid-ered as the last resort for Enterobacteriaceae infections [1]In 2017 the World Health Organization published a listof superbugs including carbapenem-resistant Enterobacte-riaceae Carbapenemase production and ESBLAmpC 120573-lactamase production coupled with porin loss or efflux pumpwere the common ways that Enterobacteriaceae becomeresistant to carbapenems [2] However the carbapenemaseproduction was more dangerous than the others [2] E coliand K pneumoniae are the most common pathogens inEnterobacteriaceae familyThese carbapenemasae-producingbacteria were found in many countries such as China[3] Parkistan [4] India [5] Turkey [6] Brazil [7] Mexico[8] Peru [9] and Greece [10] Through this mechanismNew Delhi Metallo-120573-lactamase-1 (NDM-1) was found inAsia with the highest frequency Klebsiella pneumoniae car-bapenemase (KPC) was the most popular enzyme causingcarbapenem resistance especially KPC-2 [1 11 12] Fur-thermore KPC-producing bacteria have spread all over theworld including Asia [11] Some gray literature also reportedabout KPC-producing Enterobacteriaceae in Vietnam [13ndash15] In other important views these two genes harboringbacteria were more dangerous and resistant to carbapenemwith a higher level than bacteria that harbored othertypes of carbapenemase-encoding genes (including OXA-48-like another popular carbapenemase present in Enter-obacteriaceae) [6 16ndash18] For instance these carbapenemase-producing bacteria result in severe infections with highmortality rate the infection caused by KPC-producing Kpnuemoniae was from 40 to 56 [16] and up to 88 forNDM-1 producing Enterobacteriaceae [6]Carbapenemmin-imum inhibitory concentrations (MICs) for these two typesof carbapeenemase producers in recent studies were higherthan carbapenemMICvalues againstOXA-48 type producers[17 18]

In another aspect these bacteria have spread rapidlythrough mobilisable genetic elements [19 20] for exampleplasmid IncX3 [21] IncAC2 [22] transposon Tn4401 [23]Tn125 [21] or class I Integron [24] These elements also playimportant roles in the existence of multiple genes and trans-porting various multidrug-resistant genes between bacterialspecies

In Vietnam the prevalence of these CPE is increasinglybeing reported in the hospital and aquatic environment [25ndash27] Data about carbapenemase-producing E coli and Kpneumoniaewere reported notably in Southern andNorthernVietnam Due to the lack of needed methods for screen-ing and detection there is an underestimation of CPE inother regions of Vietnam The requirement for low-pricedand efficient methods to screen or confirm carbapenemase-producing bacteria in laboratories which are likely to besuitable for low-resource settings in Vietnam is urgentToday the combinations of meropenem and varbobactamor imipenem and relebactam are taken into considerationas an option to treat infections caused by KPC-producingorganisms [28 29] Therefore it is vital to select an efficient

method to detect and characterize these carbapenemase-producing Enterobacteriaceae particularly in Vietnam

Among various methods PCR real-time PCR andDNA sequencing are the gold standards for carbapenemase-encoding genes detection but these methods have not beenwidely used in Vietnam due to the high cost Phenotypic testssuch as Modified Hodge Test and the combined disk test aresuitable because of the lower cost The combined disk test isbased on the synergy between metallo-120573-lactamases (MBLs)or KPC inhibitors (EDTA or PBA) and carbapenems Manystudies used these methods but none of them investigated Ecoli and K pneumoniae separately [30 31] The purpose ofthis study was to evaluate the characteristics of NDMKPC-producing E coliK pneumoniae at the hospitals in the SouthVietnam and to assess some simple methods for detectingthese bacteria in laboratory conditions

2 Materials and Methods

21 Study Design and Sample Collection The study wasdesigned as a cross-sectional study including all clinicalisolates from November 2017 to May 2018 The study wasconducted at the Molecular Biomedicine Laboratory in theDepartment of Medical Laboratory Science University ofMedicine and Pharmacy Ho Chi Minh City 100 clinicalisolate strains (50 E coli and 50 K pneumoniae) werecollected from the Microbiology Department of Gia DinhPeoplersquos Hospital andDong Nai General Hospital All of themwere nonsusceptible to one of the carbapenems (imipenemmeropenem or ertapenem) detected by the automatic sys-tems Patient data such as age sex specimen types andclinical wards were gathered to survey the characteristics ofpatients Sample list and relevant information are supplied inAnnex 1

22 Bacterial Isolates Fifty-nine isolates fromGia Dinh Peo-plersquos Hospital were identified and subjected to antimicrobialsusceptibility testing by using the Vitek 2 system (bioMerieuxVitek Inc Hazelwood MO USA) Furthermore IDAST of41 isolates from Dong Nai General Hospital was performedby using BD Phoenix (USA) From those hospitals asingle colony obtained from pure culture was inoculatedinto BHI (Heart Infusion Broth Himedia India) brothwith 20 glycerol (Xilong medical China) and stored at -20∘C to reculture for extracting DNA for real-time PCR andperforming phenotypic methods

23 Real-Time PCR

231 DNA Extraction Bacterial DNA was extracted byheat treatment One colony form pure isolates incubatedovernight on MacConkey agar (Merck KgaA DarmstadtGermany) was suspended in 200120583L of Tris-EDTApH 80Thesuspension was heated at 95∘C for 20 minutes followed bycentrifugation at 13000 revolutions perminute for 10minutesto collect 150120583L supernatant and stored at -20∘C for real-timePCR DNA quality was evaluated using NanoDrop 2000Spectrophotometer (ThemoFisher Scientific Wilmington

BioMed Research International 3

Table 1 Primers and probes used in this study

Oligonucleotide Nucleotide sequence 51015840ndash31015840 Origin Volume of reaction (nM) ReferencePrimers and probes for real-time PCRNDM-F Primer GAC CGC CCA GAT CCT CAA IDT 300NDM-R Primer CGC GAC CGG CAGGTT IDT 300NDM-probe HEX-TG GAT CAA GCA GGA GAT-ZENIBFQ IDT 200KPC-F Primer GGC CGC CGT GCA ATA C IDT 500KPC-R Primer GCC GCC CAA CTC CTT CA IDT 500 [32]KPC- probe 6FAM-TG ATA ACG CCG CCG CCAATT TGT-ZENIBFQ IDT 20016S rRNA-F Primer TGG AGC ATG TGG TTT AAT TCG A IDT 20016S rRNA-R Primer TGC GGG ACT TAA CCC AAC A IDT 20016S rRNA-probe Cy5-CA CGA GCT GAC GAC ARlowastC CAT GCA-IBRQ IDT 100Primers for DNA sequencingNDM-F-seq AGT CGC TTC CAA CGG TTT IDT 300

This studyNDM-R-seq CAT TGG CAT AAG TCG CAA TCC IDT 300KPC-F-seq GGT CAC CCA TCT CGG AAA IDT 500KPC-R-seq GGG ATG GCG GAG TTC AG IDT 500

Delaware USA) with the acceptable range for the ratio ofabsorbance at 260 nm280 nm from 16 to 18

232 Genotypic Detection of blaNDM blaKPC and 16SrRNA The genes were detected by real-time PCR usingTaqMan probes for all samples and were confirmed byDNA sequencing of selected positive samples The bacterialDNA was performed to screen for the presence of blaNDMand blaKPC The reaction used bacterial 16S rRNA gene asan internal control for DNA extraction and amplificationprocess The primers and probes used for blaNDM blaKPCand 16S rRNA were described by the Center of DiseaseControl and Prevention [32] To carry out real-time PCR 5120583LDNAwas added to a final volume of 20120583LMaster Mix whichincluded 1X PCR buffer 4 mM MgCl

2 02 mM dNTPs and

5 IU Taq DNA polymerase (Solgent Co Ltd South Korea)The concentration for each primers and probes is illustratedin Table 1 The negative control for PCR reaction is sterileDEPC-treated water DNA from GD018 which is a clinicalisolate that carries both blaNDM and blaKPC (confirmed byDNA sequencing) is used as a positive control

The real-time PCR conditions were chosen as followsEnzyme activation was achieved by primary heating step at95∘C for 15 minutes This was followed by 40 cycles of 95∘Cfor 15 seconds and 56∘C for 20 seconds Real-time PCR wasrun on CFX96 Touch Real-Time PCR Detection System(Bio-Rad Hercules California USA) Fluorescence signalwas detected after each cycle Positive sample was determinedwhen the automatically normalized fluorescence signal risesabove the threshold limit value calculated in each run A totalof 40 cycles takes 70 minutes

233 DNA Sequencing DNA extracts from selected pos-itive samples for blaNDM (GD013 GD002 and GD018)and blaKPC (GD011 GD16 and GD018) were sequencedusing the sequencing primers Initial PCR proceeded in theabove-mentioned conditions and PCR products were sent to

1-BASE (Singapore) for Sangerrsquos sequencing 16S rRNA genesfrom two samples GD013 and GD018 were also sequenced toconfirm the validity of the internal control primers

24 Modified Hodge Test (MHT) The MHT was carriedout on all isolates following the Clinical amp LaboratoryStandards Institute (CLSI) guide on Mueller-Hinton agar(MHAndashBecton Diskinson US) using 10120583g meropenem(MEM) disks (Nam Khoa Biotech Vietnam) [33] Klebsiellapneumoniae ATCCBAAndash1705 and Klebsiella pneumoniaeATCCBAAndash1706 were used as a positive control and anegative control respectively

25 Combined Disk Test (PBAIPMndashEDTAIPM) Phenyl-boronic acid solution was prepared by dissolving 2g phenyl-boronic acid (PBA) (Sigma-Aldrich Steinheim Germany) in100mL solution containing 50mL distilled water and 50mLDimethyl Sulfoxide (Xilong medical China) EDTA 025Msolution prepared by mixing 1861g disodium ETDA2H

2O

in 200 mL of distilled water was adjusted to pH 80 byusing NaOH Both solutions were autoclaved at 121∘C for15 minutes Either 20120583L phenylboronic acid solution (400120583gPBA) or 10120583L EDTA solution (930120583g EDTA) was added on a10120583g imipenem disk (IPM) to get a PBAIPM or EDTAIPMdisk respectively These disks were used within 1 hour

A bacterial suspension with McFarland Equivalence Tur-bidity Standard 05 was streaked on MHA plate using aswab Three disks IPM EDTAIPM and PBAIPM wereplaced on the MHA plate with the distance from one diskto another being at least 24 mm Klebsiella pneumoniaeATCCBAAndash1705 and Klebsiella pneumoniae ATCCBAAndash1706 were used as a positive control and a negative controlrespectively An increase of 7 mm for EDTAIPM or 5mm forPBAIPM in the diameter of the zone of inhibition comparedto that of IPMwas considered as a positive result for NDMorKPC production respectively

4 BioMed Research International

Table 2 Distribution of diseases

Hospital

Disease

TotalPneumonia UTI Wound

InfectionAbnormalInfection Bacteremia Fever

SurgicalWoundInfection

Other

GD(N=59)

E 2 14 5 0 5 2 0 1 29K 13 9 2 1 2 2 1 0 30

DN(N=41)

E 3 10 3 0 0 1 2 2 21K 11 2 3 0 0 0 2 2 20

Total 29 35 13 1 7 5 5 5 100lowastE E coli K K pneumoniae GD Gia Dinh Peoplersquos Hospital DN Dong Nai General Hospital UTI Urinary Tract Infection

26 Statistical Analysis Data were entered using Excel 2010and all statistical analyses were carried out using SPSS version220 (IBM Corp 2013) We determined characteristics ofpatients carrying carbapenemase-producing bacteria with95 confidence interval (CI) odds ratio (OR)was calculatedChi-square and Fisher exact tests were used for categoricalvariables Independent sample t-test was used for comparingthe mean ages of the two groups

Real-time PCR was considered as a gold standardmethod Sensitivity and specificity negative predictive val-ues (NPV) and positive predictive values (PPV) were alsocalculated These values of MHT and CDT were comparedusing the nonparametric McNemarrsquos test P-values less than005 were considered as statistical significance

3 Results and Discussion

31 Results

311 Characteristics of Carbapenemase-Producing E coli andK pneumoniae at 2 Hospitals in the Southern Vietnam Ingeneral of 100 strains from clinical samples (50 isolatesof E coli and 50 isolates of K pneumoniae) 47 isolateswere positive by real-time PCR In those positive strains36 carried blaNDM 10 carried blaKPC and one carriedboth genes 583 (2136) strains carrying blaNDM and80 (810) strains carrying blaKPC were K pneumoniaeThe one carried both genes was E coli CRE were mainlyisolated from sputum (37) and urine (31) Samples werecollected from 13 different sites of ICU (14) Departmentof Urologic Surgery (14) Infectious Disease Department(17) Geriatrics Department (9) Respiratory Department(9) and some other departments (37) The majority ofthe study participants were male (55) The average age was669 (95CI 636ndash709) the youngest patient was 23 and theoldest onewas 94The proportion of people aged 60 years andolder was 69 the odd ratio of patients in the elder groupinfected by E coli or K pneumoniae having carbapenemase-encoding genes was 318 (95 CI 128ndash789) compared to theother group Furthermore in Dong Nai General Hospital thenumber of the elders infected by NDMKPC producers wassignificantly higher than that of the younger ones (p=0001)The prevalence of pneumonia was 290 the odd of CP-EK infected patients with pneumonia was 232 (95 CI

093ndash578) compared to CP-EK infected patients withoutpneumonia the odd of elders with pneumonia was 932 (95CI 205ndash4229) compared to the younger ones

Moreover data from our study indicated that the rate ofCPE is different between the two hospitals NDM-producingorganisms were the major pathogens in Gia Dinh Peo-plersquos Hospital (458) The prevalence of bacteria carryingblaNDM and blaKPC seems to be the same in Dong NaiGeneral Hospital (22 for blaNDMversus 195 for blaKPC)(Figure 1) Other characteristics of CRE infected patientswere described in Figures 1 and 2 Tables 2 and 3

312 Molecular Detection The conformity of three methodswas illustrated in Table 3

313 The Modified Hodge Test (MHT) All isolates wereevaluated by using theMHT and real-time PCRThismethodshowed low sensitivity and specificity compared to real-timePCR especially inK pneumoniae 38 (2353) of strains werepositive with the MHT whereas the real-time PCR resultswere negative 847 strains were real-time PCR positive butnegative for the MHT and 7 of those 8 strains carriedblaNDM The sensitivity and specificity for E coli and Kpneumoniaewere 722 688 and 891 381 respectivelyMoreover the PPV and NPV for E coli and K pneumoniaewere 565 815 and 667 727 respectively

314 Combined Disk Test In the same way all isolates weretested using combined disk test (using EDTA and PBA)

32 Discussion In our study the prevalence of NDM andKPC-producing bacteria in both hospitals is high Further-more the figures of NDM and KPC-producing EK amonginfected patients were different between the two hospitalswhich depend on areas and ages of patients (Figure 2)Probably the high rate of integron and gene cassette carryingE coli at Gia Dinh Peoplersquos Hospital (4565 strains carryingintergron gene and 32 of these 45 intergron harboring strainscarrying gene cassette) [34] led to coharboring of the twogenes in E coli strain In particular this is the first reportof the presence of E coli strain coharboring both genes inVietnam

The average age of patients and the proportion of eldergroup at Gia Dinh Peoplersquos Hospital were higher than these

BioMed Research International 5

Table 3 Particular characteristics of patients at 2 hospitals in this study

Characters Gia Dinh Peoplersquos Hospital(n=59)

Dong Nai General Hospital(n=41) P-value

Average age (95 CI) 715 (674 ndash 752) 604 (522 ndash 66) 0003Range 36 ndash 94 23 ndash 89Elder () 763 585 0079Positive rate () 508 415 0545Pneumonia rate () 254 341Odd of Outcome (PosNeg)between Pneumonia andthe other

079 714

Odd of Pneumonia(ElderYounger) 588 1891

Odd of Outcome(PositiveNegative) in Eldergroup the other

1045 15

Therapy departments ()

ICU (203)Geriatrics (153)

Department of Urologicsurgery (102) RespiratoryDepartment (85) and

other departments (457)

Infectious DiseaseDepartment (414)

Department of surgery(195) Respiratory

Department (98) ICU(49) and other

departments (244)K pneumoniae () 508 488

Gia Dinh people hospitalDong Nai general hospital

Negative blaNDM blaKPC blaNDM_blaKPC

100

Age

80

60

40

20

0

Figure 1 Different distribution of patientsrsquo age following genotype at 2 hospitals

figures for Dong Nai General Hospital In Dong Nai GeneralHospital people infected by non-NDMKPC-producing bac-teria were young most of whom were the group in the rangefrom40 to 60 (Figure 1) these young people could be infectedwith ESBLAmpC producing E coliK pneumoniae Anotherhypothesis might be that the isolates from these patientscould carry other carbapenemase-encoding genes such asblaOXA-48-like but these mechanisms cause a lower level ofresistance [2 17 18] These features could play an important

role in the difference of the odd of positive and negativeoutcome of the elder group between these 2 hospitals Theelders who suffered from pneumonia and infection with thetwo types of carbapenemase producers were more than theyounger ones especially in Dong Nai General Hospital Thereason for the difference between these two hospitals was stillunclear It could be explained that Gia Dinh Peoplersquos Hospitalwas established for a long time the severity of illnesswas quitedifferent compared with Dong Nai General Hospital

6 BioMed Research International

Table 4 The agreement of bacteria isolation real-time PCR and DNA sequencing results

Strains ID Isolation Real-time PCR DNA sequencingGD013 K pneumonia blaNDM 16s rRNA blaNDM K pneumoniaeGD002 E coli blaNDM blaNDMGD018 E coli blaNDM blaKPC 16S rRNA blaNDM blaKPC E coliGD011 K pneumoniae blaKPC blaKPCGD016 K pneumoniae blaKPC blaKPCDN011 K pneumoniae blaKPC blaKPC

Table 5 Combined disk test results in E coli and K pneumonia

Combined disk test Real-time PCR TPR ()95

SPC()

PPV()

NPV()

Accuracy()Species Pos Ne

NDMdetection

E coli(n=50)

Pos 15 1 938 971 938 971 960Neg 1 33

K pneumoniae(n=50)

Pos 19 0 905 100 100 936 96Neg 2 29

KPCdetection

E coli(n=50)

Pos 2 2 667 957 50 978 940Neg 1 45

K pneumoniae(n=50)

Pos 8 3 100 929 727 100 94Neg 0 39

lowastPos positive Neg negative

Gia dinh people hospital Dong Nai general hospitalgroup

negativeblaNDM

blaKPCblaNDM_blaKPC

100

Perc

enta

ge (

)

80

60

40

20

0

Figure 2 Different distribution of prevalence of CPE at 2 hospitals

Regarding the methods real-time PCR is an accurateand rapid method to detect the presence of carbapenemase-encoding genes However the cost associated with themethod is one of the major disadvantages that limits itsuse in common laboratories in Vietnam Among variousphenotypic methods MHT recommended in CLSI 2017 is asimple method [33] In this study SPC of MHT is low (2353K pneumoniae strainswere positive but negative for the real-time PCR) The possible reason for this is that other typesof carbapenemase may present in these bacteria However itcould be explained that AmpC ESBLs or strict requirements

of MHT process at each step may cause false positive results[35] Besides this method shows low sensitivity to NDM-producing Enterobacteriaceae (78 strains were found to havea false-negative result) [36 37] In our study the MHT resultsfor K pneumoniae are consistent with findings in recentstudies conducted byDariush Shokri et al [38] and RangnekaR aSeeM et al [39]Thismay be the reason why the CLSI 2018standard excluded MHT in the guideline [40]

In contrast CDT has been shown to be highly effec-tive The results of NDM detection by EDTA disk test forK pneumoniae (Table 4) were in accordance with findingsobtained by ParadimitrioundashOliveris et al [30] However itwas in contrast to findings reported by Dariush Shokri et al[38] This could be explained by the fact that Shokri carriedout his study including not only K pneumoniae but also Abaumanii and P aeruginosa The high rate of blaVIM in Paeruginosa may be the reason for false-negative results Astudy conducted by Ting-ting Qu et al in 2009 showed thatCDT also detected other types of carbapenemase includingVerona integron-borne metallo-120573-lactamase (VIM) whichwas the most widespread metallo-120573-lactamase in P aerug-inosa [41 42] Our results for KPC detection using PBAdisk test K pneumoniae (Table 4) were in accordance withfindings obtained by Athanasios Tsakris et al (using PBA-400120583g) [31] and Julio Zuniga et al (using 300120583g APBA) [43]The efficiency of PBA disk test in E coli was low particularlyin sensitivity and PPV because of the sample size of KPC-producing E coli only 3 strains with one of them being a falsenegative The results in Tables 5 and 6 illustrated the signifi-cantly higher specificity of combined disk test compared withMHT

BioMed Research International 7

Table 6 Comparison between the MHT and combined disk test

Bacteria Index NDM detection KPC detectionMHT EDTA McNemarlowast MHT Boronic McNemarlowast

E coli (n=50) TPR () 722 938 p=025 722 667 p=100SPC () 688 971 p=0006 688 957 plt0001

K pneumoniae (n=50) TPR () 897 905 p=100 897 100 NCSPC () 381 100 plt0001 381 929 plt0001

lowastmeans nonparametric McNemar test

4 Conclusions

The prevalence of CP-EK in Vietnam is high The lack ofsuitable methods to detect and differentiate these bacterialinfections has been the main obstacle for clinicians tochoosing an appropriate treatment During this study werecognized that CDT is reasonable and simple to detect thepresence of these important enzymes These features make ithighly applicable to all clinical laboratories in Vietnam

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Limitation In our study other genes such an OXA-48-likeESBL and AmpC 120573-lactamase were not included so thiscould affect our study results The only 10 KPC-producingorganisms in this studymay have effects on the results of PBAdisk test A lack of genotypic method to identify Ecoli and Kpneumoniae was also important

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors are grateful to Gia Dinh Peoplersquos Hospital(HCMC) and Dong Nai General Hospital for kindly provid-ing the clinical isolates and also to the staff of Pasteur Institutein Ho Chi Minh City for the comments and suggestions Wewould also like to thank Dr Vu Quang Huy the Head ofthe Department of Medical Laboratory Science University ofMedicine and Pharmacy Ho Chi Minh City for facilitatingthe study and thank Pham Thai Binh (Nam Khoa Biotech)Bui The Trung (Childrenrsquos Hospital no 2 HCMC) andNguyen Thi Phuong Thao (Dong Nai Medical College) forproviding the ATCC strains of bacteria and some reagents forthis study

Supplementary Materials

List of related information of patients and testing results weredetailed in Annex 1 Data analysis results testing images

reference and full-text of gray literature were described inAnnexes 2 3 4 5 respectively (Supplementary Materials)

References

[1] AU Khan LMaryam andR Zarrilli ldquoStructure Genetics andWorldwide Spread of New Delhi Metallo-120573-lactamase (NDM)a threat to public healthrdquo BMC Microbiology vol 17 no 1 2017

[2] X Jia W Dai W Ma et al ldquoCorrigendum carbapenem-resistant e cloacae in southwest china molecular analysis ofresistance and risk factors for infections caused by ndm-1-producersrdquo Frontiers in Microbiology vol 9 2018

[3] JWang X Yao J Luo L Lv Z Zeng and J-H Liu ldquoEmergenceof Escherichia coli coproducing NDM-1 and KPC-2 carbapen-emases from a retail vegetable Chinardquo Journal of AntimicrobialChemotherapy vol 73 no 1 pp 252ndash254 2018

[4] K M Day M Salman B Kazi et al ldquoPrevalence of NDM-1carbapenemase in patients with diarrhoea in Pakistan and eval-uation of two chromogenic culture mediardquo Journal of AppliedMicrobiology vol 114 no 6 pp 1810ndash1816 2013

[5] S Eshetie C Unakal A Gelaw B Ayelign M Endris and FMoges ldquoMultidrug resistant and carbapenemase producingEnterobacteriaceae among patients with urinary tract infectionat referral Hospital Northwest Ethiopiardquo Antimicrobial Resis-tance and Infection Control vol 4 no 1 article no 12 2015

[6] O Karabay M Altindis M Koroglu O Karatuna O AAydemir and A F Erdem ldquoThe carbapenem-resistant Enter-obacteriaceae threat is growing NDM-1 epidemic at a traininghospital in TurkeyrdquoAnnals of Clinical Microbiology and Antimi-crobials vol 15 no 1 2016

[7] I Rossi Goncalves M L Ferreira B F Araujo et al ldquoOutbreaksof colistin-resistant and colistin-susceptible KPC-producingKlebsiella pneumoniae in a Brazilian intensive care unitrdquoJournal of Hospital Infection vol 94 no 4 pp 322ndash329 2016

[8] P Torres-Gonzalez M Bobadilla-del Valle E Tovar-Calderonet al ldquoOutbreak Caused by Enterobacteriaceae HarboringNDM-1 Metallo-120573-Lactamase Carried in an IncFII Plasmid ina Tertiary Care Hospital in Mexico Cityrdquo Antimicrobial Agentsand Chemotherapy vol 59 no 11 pp 7080ndash7083 2015

[9] J Tamariz C Llanos C Seas et al ldquoDraft genome sequence ofthe first New Delhi metallo-120573-lactamase (NDM-1)-producingEscherichia coli strain isolated in Perurdquo Genome Announce-ments vol 6 no 13 2018

[10] H C Maltezou P Giakkoupi A Maragos et al ldquoOutbreak ofinfections due to KPC-2-producing Klebsiella pneumoniae in ahospital in Crete (Greece)rdquo Infection vol 58 no 3 pp 213ndash2192009

[11] L Dortet G Cuzon V Ponties and P Nordmann ldquoTrends incarbapenemase-producing Enterobacteriaceae France 2012 to2014rdquo Eurosurveillance vol 22 no 6 2017

BioMed Research International 3

Table 1 Primers and probes used in this study

Oligonucleotide Nucleotide sequence 51015840ndash31015840 Origin Volume of reaction (nM) ReferencePrimers and probes for real-time PCRNDM-F Primer GAC CGC CCA GAT CCT CAA IDT 300NDM-R Primer CGC GAC CGG CAGGTT IDT 300NDM-probe HEX-TG GAT CAA GCA GGA GAT-ZENIBFQ IDT 200KPC-F Primer GGC CGC CGT GCA ATA C IDT 500KPC-R Primer GCC GCC CAA CTC CTT CA IDT 500 [32]KPC- probe 6FAM-TG ATA ACG CCG CCG CCAATT TGT-ZENIBFQ IDT 20016S rRNA-F Primer TGG AGC ATG TGG TTT AAT TCG A IDT 20016S rRNA-R Primer TGC GGG ACT TAA CCC AAC A IDT 20016S rRNA-probe Cy5-CA CGA GCT GAC GAC ARlowastC CAT GCA-IBRQ IDT 100Primers for DNA sequencingNDM-F-seq AGT CGC TTC CAA CGG TTT IDT 300

This studyNDM-R-seq CAT TGG CAT AAG TCG CAA TCC IDT 300KPC-F-seq GGT CAC CCA TCT CGG AAA IDT 500KPC-R-seq GGG ATG GCG GAG TTC AG IDT 500

Delaware USA) with the acceptable range for the ratio ofabsorbance at 260 nm280 nm from 16 to 18

232 Genotypic Detection of blaNDM blaKPC and 16SrRNA The genes were detected by real-time PCR usingTaqMan probes for all samples and were confirmed byDNA sequencing of selected positive samples The bacterialDNA was performed to screen for the presence of blaNDMand blaKPC The reaction used bacterial 16S rRNA gene asan internal control for DNA extraction and amplificationprocess The primers and probes used for blaNDM blaKPCand 16S rRNA were described by the Center of DiseaseControl and Prevention [32] To carry out real-time PCR 5120583LDNAwas added to a final volume of 20120583LMaster Mix whichincluded 1X PCR buffer 4 mM MgCl

2 02 mM dNTPs and

5 IU Taq DNA polymerase (Solgent Co Ltd South Korea)The concentration for each primers and probes is illustratedin Table 1 The negative control for PCR reaction is sterileDEPC-treated water DNA from GD018 which is a clinicalisolate that carries both blaNDM and blaKPC (confirmed byDNA sequencing) is used as a positive control

The real-time PCR conditions were chosen as followsEnzyme activation was achieved by primary heating step at95∘C for 15 minutes This was followed by 40 cycles of 95∘Cfor 15 seconds and 56∘C for 20 seconds Real-time PCR wasrun on CFX96 Touch Real-Time PCR Detection System(Bio-Rad Hercules California USA) Fluorescence signalwas detected after each cycle Positive sample was determinedwhen the automatically normalized fluorescence signal risesabove the threshold limit value calculated in each run A totalof 40 cycles takes 70 minutes

233 DNA Sequencing DNA extracts from selected pos-itive samples for blaNDM (GD013 GD002 and GD018)and blaKPC (GD011 GD16 and GD018) were sequencedusing the sequencing primers Initial PCR proceeded in theabove-mentioned conditions and PCR products were sent to

1-BASE (Singapore) for Sangerrsquos sequencing 16S rRNA genesfrom two samples GD013 and GD018 were also sequenced toconfirm the validity of the internal control primers

24 Modified Hodge Test (MHT) The MHT was carriedout on all isolates following the Clinical amp LaboratoryStandards Institute (CLSI) guide on Mueller-Hinton agar(MHAndashBecton Diskinson US) using 10120583g meropenem(MEM) disks (Nam Khoa Biotech Vietnam) [33] Klebsiellapneumoniae ATCCBAAndash1705 and Klebsiella pneumoniaeATCCBAAndash1706 were used as a positive control and anegative control respectively

25 Combined Disk Test (PBAIPMndashEDTAIPM) Phenyl-boronic acid solution was prepared by dissolving 2g phenyl-boronic acid (PBA) (Sigma-Aldrich Steinheim Germany) in100mL solution containing 50mL distilled water and 50mLDimethyl Sulfoxide (Xilong medical China) EDTA 025Msolution prepared by mixing 1861g disodium ETDA2H

2O

in 200 mL of distilled water was adjusted to pH 80 byusing NaOH Both solutions were autoclaved at 121∘C for15 minutes Either 20120583L phenylboronic acid solution (400120583gPBA) or 10120583L EDTA solution (930120583g EDTA) was added on a10120583g imipenem disk (IPM) to get a PBAIPM or EDTAIPMdisk respectively These disks were used within 1 hour

A bacterial suspension with McFarland Equivalence Tur-bidity Standard 05 was streaked on MHA plate using aswab Three disks IPM EDTAIPM and PBAIPM wereplaced on the MHA plate with the distance from one diskto another being at least 24 mm Klebsiella pneumoniaeATCCBAAndash1705 and Klebsiella pneumoniae ATCCBAAndash1706 were used as a positive control and a negative controlrespectively An increase of 7 mm for EDTAIPM or 5mm forPBAIPM in the diameter of the zone of inhibition comparedto that of IPMwas considered as a positive result for NDMorKPC production respectively

4 BioMed Research International

Table 2 Distribution of diseases

Hospital

Disease

TotalPneumonia UTI Wound

InfectionAbnormalInfection Bacteremia Fever

SurgicalWoundInfection

Other

GD(N=59)

E 2 14 5 0 5 2 0 1 29K 13 9 2 1 2 2 1 0 30

DN(N=41)

E 3 10 3 0 0 1 2 2 21K 11 2 3 0 0 0 2 2 20

Total 29 35 13 1 7 5 5 5 100lowastE E coli K K pneumoniae GD Gia Dinh Peoplersquos Hospital DN Dong Nai General Hospital UTI Urinary Tract Infection

26 Statistical Analysis Data were entered using Excel 2010and all statistical analyses were carried out using SPSS version220 (IBM Corp 2013) We determined characteristics ofpatients carrying carbapenemase-producing bacteria with95 confidence interval (CI) odds ratio (OR)was calculatedChi-square and Fisher exact tests were used for categoricalvariables Independent sample t-test was used for comparingthe mean ages of the two groups

Real-time PCR was considered as a gold standardmethod Sensitivity and specificity negative predictive val-ues (NPV) and positive predictive values (PPV) were alsocalculated These values of MHT and CDT were comparedusing the nonparametric McNemarrsquos test P-values less than005 were considered as statistical significance

3 Results and Discussion

31 Results

311 Characteristics of Carbapenemase-Producing E coli andK pneumoniae at 2 Hospitals in the Southern Vietnam Ingeneral of 100 strains from clinical samples (50 isolatesof E coli and 50 isolates of K pneumoniae) 47 isolateswere positive by real-time PCR In those positive strains36 carried blaNDM 10 carried blaKPC and one carriedboth genes 583 (2136) strains carrying blaNDM and80 (810) strains carrying blaKPC were K pneumoniaeThe one carried both genes was E coli CRE were mainlyisolated from sputum (37) and urine (31) Samples werecollected from 13 different sites of ICU (14) Departmentof Urologic Surgery (14) Infectious Disease Department(17) Geriatrics Department (9) Respiratory Department(9) and some other departments (37) The majority ofthe study participants were male (55) The average age was669 (95CI 636ndash709) the youngest patient was 23 and theoldest onewas 94The proportion of people aged 60 years andolder was 69 the odd ratio of patients in the elder groupinfected by E coli or K pneumoniae having carbapenemase-encoding genes was 318 (95 CI 128ndash789) compared to theother group Furthermore in Dong Nai General Hospital thenumber of the elders infected by NDMKPC producers wassignificantly higher than that of the younger ones (p=0001)The prevalence of pneumonia was 290 the odd of CP-EK infected patients with pneumonia was 232 (95 CI

093ndash578) compared to CP-EK infected patients withoutpneumonia the odd of elders with pneumonia was 932 (95CI 205ndash4229) compared to the younger ones

Moreover data from our study indicated that the rate ofCPE is different between the two hospitals NDM-producingorganisms were the major pathogens in Gia Dinh Peo-plersquos Hospital (458) The prevalence of bacteria carryingblaNDM and blaKPC seems to be the same in Dong NaiGeneral Hospital (22 for blaNDMversus 195 for blaKPC)(Figure 1) Other characteristics of CRE infected patientswere described in Figures 1 and 2 Tables 2 and 3

312 Molecular Detection The conformity of three methodswas illustrated in Table 3

313 The Modified Hodge Test (MHT) All isolates wereevaluated by using theMHT and real-time PCRThismethodshowed low sensitivity and specificity compared to real-timePCR especially inK pneumoniae 38 (2353) of strains werepositive with the MHT whereas the real-time PCR resultswere negative 847 strains were real-time PCR positive butnegative for the MHT and 7 of those 8 strains carriedblaNDM The sensitivity and specificity for E coli and Kpneumoniaewere 722 688 and 891 381 respectivelyMoreover the PPV and NPV for E coli and K pneumoniaewere 565 815 and 667 727 respectively

314 Combined Disk Test In the same way all isolates weretested using combined disk test (using EDTA and PBA)

32 Discussion In our study the prevalence of NDM andKPC-producing bacteria in both hospitals is high Further-more the figures of NDM and KPC-producing EK amonginfected patients were different between the two hospitalswhich depend on areas and ages of patients (Figure 2)Probably the high rate of integron and gene cassette carryingE coli at Gia Dinh Peoplersquos Hospital (4565 strains carryingintergron gene and 32 of these 45 intergron harboring strainscarrying gene cassette) [34] led to coharboring of the twogenes in E coli strain In particular this is the first reportof the presence of E coli strain coharboring both genes inVietnam

The average age of patients and the proportion of eldergroup at Gia Dinh Peoplersquos Hospital were higher than these

BioMed Research International 5

Table 3 Particular characteristics of patients at 2 hospitals in this study

Characters Gia Dinh Peoplersquos Hospital(n=59)

Dong Nai General Hospital(n=41) P-value

Average age (95 CI) 715 (674 ndash 752) 604 (522 ndash 66) 0003Range 36 ndash 94 23 ndash 89Elder () 763 585 0079Positive rate () 508 415 0545Pneumonia rate () 254 341Odd of Outcome (PosNeg)between Pneumonia andthe other

079 714

Odd of Pneumonia(ElderYounger) 588 1891

Odd of Outcome(PositiveNegative) in Eldergroup the other

1045 15

Therapy departments ()

ICU (203)Geriatrics (153)

Department of Urologicsurgery (102) RespiratoryDepartment (85) and

other departments (457)

Infectious DiseaseDepartment (414)

Department of surgery(195) Respiratory

Department (98) ICU(49) and other

departments (244)K pneumoniae () 508 488

Gia Dinh people hospitalDong Nai general hospital

Negative blaNDM blaKPC blaNDM_blaKPC

100

Age

80

60

40

20

0

Figure 1 Different distribution of patientsrsquo age following genotype at 2 hospitals

figures for Dong Nai General Hospital In Dong Nai GeneralHospital people infected by non-NDMKPC-producing bac-teria were young most of whom were the group in the rangefrom40 to 60 (Figure 1) these young people could be infectedwith ESBLAmpC producing E coliK pneumoniae Anotherhypothesis might be that the isolates from these patientscould carry other carbapenemase-encoding genes such asblaOXA-48-like but these mechanisms cause a lower level ofresistance [2 17 18] These features could play an important

role in the difference of the odd of positive and negativeoutcome of the elder group between these 2 hospitals Theelders who suffered from pneumonia and infection with thetwo types of carbapenemase producers were more than theyounger ones especially in Dong Nai General Hospital Thereason for the difference between these two hospitals was stillunclear It could be explained that Gia Dinh Peoplersquos Hospitalwas established for a long time the severity of illnesswas quitedifferent compared with Dong Nai General Hospital

6 BioMed Research International

Table 4 The agreement of bacteria isolation real-time PCR and DNA sequencing results

Strains ID Isolation Real-time PCR DNA sequencingGD013 K pneumonia blaNDM 16s rRNA blaNDM K pneumoniaeGD002 E coli blaNDM blaNDMGD018 E coli blaNDM blaKPC 16S rRNA blaNDM blaKPC E coliGD011 K pneumoniae blaKPC blaKPCGD016 K pneumoniae blaKPC blaKPCDN011 K pneumoniae blaKPC blaKPC

Table 5 Combined disk test results in E coli and K pneumonia

Combined disk test Real-time PCR TPR ()95

SPC()

PPV()

NPV()

Accuracy()Species Pos Ne

NDMdetection

E coli(n=50)

Pos 15 1 938 971 938 971 960Neg 1 33

K pneumoniae(n=50)

Pos 19 0 905 100 100 936 96Neg 2 29

KPCdetection

E coli(n=50)

Pos 2 2 667 957 50 978 940Neg 1 45

K pneumoniae(n=50)

Pos 8 3 100 929 727 100 94Neg 0 39

lowastPos positive Neg negative

Gia dinh people hospital Dong Nai general hospitalgroup

negativeblaNDM

blaKPCblaNDM_blaKPC

100

Perc

enta

ge (

)

80

60

40

20

0

Figure 2 Different distribution of prevalence of CPE at 2 hospitals

Regarding the methods real-time PCR is an accurateand rapid method to detect the presence of carbapenemase-encoding genes However the cost associated with themethod is one of the major disadvantages that limits itsuse in common laboratories in Vietnam Among variousphenotypic methods MHT recommended in CLSI 2017 is asimple method [33] In this study SPC of MHT is low (2353K pneumoniae strainswere positive but negative for the real-time PCR) The possible reason for this is that other typesof carbapenemase may present in these bacteria However itcould be explained that AmpC ESBLs or strict requirements

of MHT process at each step may cause false positive results[35] Besides this method shows low sensitivity to NDM-producing Enterobacteriaceae (78 strains were found to havea false-negative result) [36 37] In our study the MHT resultsfor K pneumoniae are consistent with findings in recentstudies conducted byDariush Shokri et al [38] and RangnekaR aSeeM et al [39]Thismay be the reason why the CLSI 2018standard excluded MHT in the guideline [40]

In contrast CDT has been shown to be highly effec-tive The results of NDM detection by EDTA disk test forK pneumoniae (Table 4) were in accordance with findingsobtained by ParadimitrioundashOliveris et al [30] However itwas in contrast to findings reported by Dariush Shokri et al[38] This could be explained by the fact that Shokri carriedout his study including not only K pneumoniae but also Abaumanii and P aeruginosa The high rate of blaVIM in Paeruginosa may be the reason for false-negative results Astudy conducted by Ting-ting Qu et al in 2009 showed thatCDT also detected other types of carbapenemase includingVerona integron-borne metallo-120573-lactamase (VIM) whichwas the most widespread metallo-120573-lactamase in P aerug-inosa [41 42] Our results for KPC detection using PBAdisk test K pneumoniae (Table 4) were in accordance withfindings obtained by Athanasios Tsakris et al (using PBA-400120583g) [31] and Julio Zuniga et al (using 300120583g APBA) [43]The efficiency of PBA disk test in E coli was low particularlyin sensitivity and PPV because of the sample size of KPC-producing E coli only 3 strains with one of them being a falsenegative The results in Tables 5 and 6 illustrated the signifi-cantly higher specificity of combined disk test compared withMHT

BioMed Research International 7

Table 6 Comparison between the MHT and combined disk test

Bacteria Index NDM detection KPC detectionMHT EDTA McNemarlowast MHT Boronic McNemarlowast

E coli (n=50) TPR () 722 938 p=025 722 667 p=100SPC () 688 971 p=0006 688 957 plt0001

K pneumoniae (n=50) TPR () 897 905 p=100 897 100 NCSPC () 381 100 plt0001 381 929 plt0001

lowastmeans nonparametric McNemar test

4 Conclusions

The prevalence of CP-EK in Vietnam is high The lack ofsuitable methods to detect and differentiate these bacterialinfections has been the main obstacle for clinicians tochoosing an appropriate treatment During this study werecognized that CDT is reasonable and simple to detect thepresence of these important enzymes These features make ithighly applicable to all clinical laboratories in Vietnam

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Limitation In our study other genes such an OXA-48-likeESBL and AmpC 120573-lactamase were not included so thiscould affect our study results The only 10 KPC-producingorganisms in this studymay have effects on the results of PBAdisk test A lack of genotypic method to identify Ecoli and Kpneumoniae was also important

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors are grateful to Gia Dinh Peoplersquos Hospital(HCMC) and Dong Nai General Hospital for kindly provid-ing the clinical isolates and also to the staff of Pasteur Institutein Ho Chi Minh City for the comments and suggestions Wewould also like to thank Dr Vu Quang Huy the Head ofthe Department of Medical Laboratory Science University ofMedicine and Pharmacy Ho Chi Minh City for facilitatingthe study and thank Pham Thai Binh (Nam Khoa Biotech)Bui The Trung (Childrenrsquos Hospital no 2 HCMC) andNguyen Thi Phuong Thao (Dong Nai Medical College) forproviding the ATCC strains of bacteria and some reagents forthis study

Supplementary Materials

List of related information of patients and testing results weredetailed in Annex 1 Data analysis results testing images

reference and full-text of gray literature were described inAnnexes 2 3 4 5 respectively (Supplementary Materials)

References

[1] AU Khan LMaryam andR Zarrilli ldquoStructure Genetics andWorldwide Spread of New Delhi Metallo-120573-lactamase (NDM)a threat to public healthrdquo BMC Microbiology vol 17 no 1 2017

[2] X Jia W Dai W Ma et al ldquoCorrigendum carbapenem-resistant e cloacae in southwest china molecular analysis ofresistance and risk factors for infections caused by ndm-1-producersrdquo Frontiers in Microbiology vol 9 2018

[3] JWang X Yao J Luo L Lv Z Zeng and J-H Liu ldquoEmergenceof Escherichia coli coproducing NDM-1 and KPC-2 carbapen-emases from a retail vegetable Chinardquo Journal of AntimicrobialChemotherapy vol 73 no 1 pp 252ndash254 2018

[4] K M Day M Salman B Kazi et al ldquoPrevalence of NDM-1carbapenemase in patients with diarrhoea in Pakistan and eval-uation of two chromogenic culture mediardquo Journal of AppliedMicrobiology vol 114 no 6 pp 1810ndash1816 2013

[5] S Eshetie C Unakal A Gelaw B Ayelign M Endris and FMoges ldquoMultidrug resistant and carbapenemase producingEnterobacteriaceae among patients with urinary tract infectionat referral Hospital Northwest Ethiopiardquo Antimicrobial Resis-tance and Infection Control vol 4 no 1 article no 12 2015

[6] O Karabay M Altindis M Koroglu O Karatuna O AAydemir and A F Erdem ldquoThe carbapenem-resistant Enter-obacteriaceae threat is growing NDM-1 epidemic at a traininghospital in TurkeyrdquoAnnals of Clinical Microbiology and Antimi-crobials vol 15 no 1 2016

[7] I Rossi Goncalves M L Ferreira B F Araujo et al ldquoOutbreaksof colistin-resistant and colistin-susceptible KPC-producingKlebsiella pneumoniae in a Brazilian intensive care unitrdquoJournal of Hospital Infection vol 94 no 4 pp 322ndash329 2016

[8] P Torres-Gonzalez M Bobadilla-del Valle E Tovar-Calderonet al ldquoOutbreak Caused by Enterobacteriaceae HarboringNDM-1 Metallo-120573-Lactamase Carried in an IncFII Plasmid ina Tertiary Care Hospital in Mexico Cityrdquo Antimicrobial Agentsand Chemotherapy vol 59 no 11 pp 7080ndash7083 2015

[9] J Tamariz C Llanos C Seas et al ldquoDraft genome sequence ofthe first New Delhi metallo-120573-lactamase (NDM-1)-producingEscherichia coli strain isolated in Perurdquo Genome Announce-ments vol 6 no 13 2018

[10] H C Maltezou P Giakkoupi A Maragos et al ldquoOutbreak ofinfections due to KPC-2-producing Klebsiella pneumoniae in ahospital in Crete (Greece)rdquo Infection vol 58 no 3 pp 213ndash2192009

[11] L Dortet G Cuzon V Ponties and P Nordmann ldquoTrends incarbapenemase-producing Enterobacteriaceae France 2012 to2014rdquo Eurosurveillance vol 22 no 6 2017

4 BioMed Research International

Table 2 Distribution of diseases

Hospital

Disease

TotalPneumonia UTI Wound

InfectionAbnormalInfection Bacteremia Fever

SurgicalWoundInfection

Other

GD(N=59)

E 2 14 5 0 5 2 0 1 29K 13 9 2 1 2 2 1 0 30

DN(N=41)

E 3 10 3 0 0 1 2 2 21K 11 2 3 0 0 0 2 2 20

Total 29 35 13 1 7 5 5 5 100lowastE E coli K K pneumoniae GD Gia Dinh Peoplersquos Hospital DN Dong Nai General Hospital UTI Urinary Tract Infection

26 Statistical Analysis Data were entered using Excel 2010and all statistical analyses were carried out using SPSS version220 (IBM Corp 2013) We determined characteristics ofpatients carrying carbapenemase-producing bacteria with95 confidence interval (CI) odds ratio (OR)was calculatedChi-square and Fisher exact tests were used for categoricalvariables Independent sample t-test was used for comparingthe mean ages of the two groups

Real-time PCR was considered as a gold standardmethod Sensitivity and specificity negative predictive val-ues (NPV) and positive predictive values (PPV) were alsocalculated These values of MHT and CDT were comparedusing the nonparametric McNemarrsquos test P-values less than005 were considered as statistical significance

3 Results and Discussion

31 Results

311 Characteristics of Carbapenemase-Producing E coli andK pneumoniae at 2 Hospitals in the Southern Vietnam Ingeneral of 100 strains from clinical samples (50 isolatesof E coli and 50 isolates of K pneumoniae) 47 isolateswere positive by real-time PCR In those positive strains36 carried blaNDM 10 carried blaKPC and one carriedboth genes 583 (2136) strains carrying blaNDM and80 (810) strains carrying blaKPC were K pneumoniaeThe one carried both genes was E coli CRE were mainlyisolated from sputum (37) and urine (31) Samples werecollected from 13 different sites of ICU (14) Departmentof Urologic Surgery (14) Infectious Disease Department(17) Geriatrics Department (9) Respiratory Department(9) and some other departments (37) The majority ofthe study participants were male (55) The average age was669 (95CI 636ndash709) the youngest patient was 23 and theoldest onewas 94The proportion of people aged 60 years andolder was 69 the odd ratio of patients in the elder groupinfected by E coli or K pneumoniae having carbapenemase-encoding genes was 318 (95 CI 128ndash789) compared to theother group Furthermore in Dong Nai General Hospital thenumber of the elders infected by NDMKPC producers wassignificantly higher than that of the younger ones (p=0001)The prevalence of pneumonia was 290 the odd of CP-EK infected patients with pneumonia was 232 (95 CI

093ndash578) compared to CP-EK infected patients withoutpneumonia the odd of elders with pneumonia was 932 (95CI 205ndash4229) compared to the younger ones

Moreover data from our study indicated that the rate ofCPE is different between the two hospitals NDM-producingorganisms were the major pathogens in Gia Dinh Peo-plersquos Hospital (458) The prevalence of bacteria carryingblaNDM and blaKPC seems to be the same in Dong NaiGeneral Hospital (22 for blaNDMversus 195 for blaKPC)(Figure 1) Other characteristics of CRE infected patientswere described in Figures 1 and 2 Tables 2 and 3

312 Molecular Detection The conformity of three methodswas illustrated in Table 3

313 The Modified Hodge Test (MHT) All isolates wereevaluated by using theMHT and real-time PCRThismethodshowed low sensitivity and specificity compared to real-timePCR especially inK pneumoniae 38 (2353) of strains werepositive with the MHT whereas the real-time PCR resultswere negative 847 strains were real-time PCR positive butnegative for the MHT and 7 of those 8 strains carriedblaNDM The sensitivity and specificity for E coli and Kpneumoniaewere 722 688 and 891 381 respectivelyMoreover the PPV and NPV for E coli and K pneumoniaewere 565 815 and 667 727 respectively

314 Combined Disk Test In the same way all isolates weretested using combined disk test (using EDTA and PBA)

32 Discussion In our study the prevalence of NDM andKPC-producing bacteria in both hospitals is high Further-more the figures of NDM and KPC-producing EK amonginfected patients were different between the two hospitalswhich depend on areas and ages of patients (Figure 2)Probably the high rate of integron and gene cassette carryingE coli at Gia Dinh Peoplersquos Hospital (4565 strains carryingintergron gene and 32 of these 45 intergron harboring strainscarrying gene cassette) [34] led to coharboring of the twogenes in E coli strain In particular this is the first reportof the presence of E coli strain coharboring both genes inVietnam

The average age of patients and the proportion of eldergroup at Gia Dinh Peoplersquos Hospital were higher than these

BioMed Research International 5

Table 3 Particular characteristics of patients at 2 hospitals in this study

Characters Gia Dinh Peoplersquos Hospital(n=59)

Dong Nai General Hospital(n=41) P-value

Average age (95 CI) 715 (674 ndash 752) 604 (522 ndash 66) 0003Range 36 ndash 94 23 ndash 89Elder () 763 585 0079Positive rate () 508 415 0545Pneumonia rate () 254 341Odd of Outcome (PosNeg)between Pneumonia andthe other

079 714

Odd of Pneumonia(ElderYounger) 588 1891

Odd of Outcome(PositiveNegative) in Eldergroup the other

1045 15

Therapy departments ()

ICU (203)Geriatrics (153)

Department of Urologicsurgery (102) RespiratoryDepartment (85) and

other departments (457)

Infectious DiseaseDepartment (414)

Department of surgery(195) Respiratory

Department (98) ICU(49) and other

departments (244)K pneumoniae () 508 488

Gia Dinh people hospitalDong Nai general hospital

Negative blaNDM blaKPC blaNDM_blaKPC

100

Age

80

60

40

20

0

Figure 1 Different distribution of patientsrsquo age following genotype at 2 hospitals

figures for Dong Nai General Hospital In Dong Nai GeneralHospital people infected by non-NDMKPC-producing bac-teria were young most of whom were the group in the rangefrom40 to 60 (Figure 1) these young people could be infectedwith ESBLAmpC producing E coliK pneumoniae Anotherhypothesis might be that the isolates from these patientscould carry other carbapenemase-encoding genes such asblaOXA-48-like but these mechanisms cause a lower level ofresistance [2 17 18] These features could play an important

role in the difference of the odd of positive and negativeoutcome of the elder group between these 2 hospitals Theelders who suffered from pneumonia and infection with thetwo types of carbapenemase producers were more than theyounger ones especially in Dong Nai General Hospital Thereason for the difference between these two hospitals was stillunclear It could be explained that Gia Dinh Peoplersquos Hospitalwas established for a long time the severity of illnesswas quitedifferent compared with Dong Nai General Hospital

6 BioMed Research International

Table 4 The agreement of bacteria isolation real-time PCR and DNA sequencing results

Strains ID Isolation Real-time PCR DNA sequencingGD013 K pneumonia blaNDM 16s rRNA blaNDM K pneumoniaeGD002 E coli blaNDM blaNDMGD018 E coli blaNDM blaKPC 16S rRNA blaNDM blaKPC E coliGD011 K pneumoniae blaKPC blaKPCGD016 K pneumoniae blaKPC blaKPCDN011 K pneumoniae blaKPC blaKPC

Table 5 Combined disk test results in E coli and K pneumonia

Combined disk test Real-time PCR TPR ()95

SPC()

PPV()

NPV()

Accuracy()Species Pos Ne

NDMdetection

E coli(n=50)

Pos 15 1 938 971 938 971 960Neg 1 33

K pneumoniae(n=50)

Pos 19 0 905 100 100 936 96Neg 2 29

KPCdetection

E coli(n=50)

Pos 2 2 667 957 50 978 940Neg 1 45

K pneumoniae(n=50)

Pos 8 3 100 929 727 100 94Neg 0 39

lowastPos positive Neg negative

Gia dinh people hospital Dong Nai general hospitalgroup

negativeblaNDM

blaKPCblaNDM_blaKPC

100

Perc

enta

ge (

)

80

60

40

20

0

Figure 2 Different distribution of prevalence of CPE at 2 hospitals

Regarding the methods real-time PCR is an accurateand rapid method to detect the presence of carbapenemase-encoding genes However the cost associated with themethod is one of the major disadvantages that limits itsuse in common laboratories in Vietnam Among variousphenotypic methods MHT recommended in CLSI 2017 is asimple method [33] In this study SPC of MHT is low (2353K pneumoniae strainswere positive but negative for the real-time PCR) The possible reason for this is that other typesof carbapenemase may present in these bacteria However itcould be explained that AmpC ESBLs or strict requirements

of MHT process at each step may cause false positive results[35] Besides this method shows low sensitivity to NDM-producing Enterobacteriaceae (78 strains were found to havea false-negative result) [36 37] In our study the MHT resultsfor K pneumoniae are consistent with findings in recentstudies conducted byDariush Shokri et al [38] and RangnekaR aSeeM et al [39]Thismay be the reason why the CLSI 2018standard excluded MHT in the guideline [40]

In contrast CDT has been shown to be highly effec-tive The results of NDM detection by EDTA disk test forK pneumoniae (Table 4) were in accordance with findingsobtained by ParadimitrioundashOliveris et al [30] However itwas in contrast to findings reported by Dariush Shokri et al[38] This could be explained by the fact that Shokri carriedout his study including not only K pneumoniae but also Abaumanii and P aeruginosa The high rate of blaVIM in Paeruginosa may be the reason for false-negative results Astudy conducted by Ting-ting Qu et al in 2009 showed thatCDT also detected other types of carbapenemase includingVerona integron-borne metallo-120573-lactamase (VIM) whichwas the most widespread metallo-120573-lactamase in P aerug-inosa [41 42] Our results for KPC detection using PBAdisk test K pneumoniae (Table 4) were in accordance withfindings obtained by Athanasios Tsakris et al (using PBA-400120583g) [31] and Julio Zuniga et al (using 300120583g APBA) [43]The efficiency of PBA disk test in E coli was low particularlyin sensitivity and PPV because of the sample size of KPC-producing E coli only 3 strains with one of them being a falsenegative The results in Tables 5 and 6 illustrated the signifi-cantly higher specificity of combined disk test compared withMHT

BioMed Research International 7

Table 6 Comparison between the MHT and combined disk test

Bacteria Index NDM detection KPC detectionMHT EDTA McNemarlowast MHT Boronic McNemarlowast

E coli (n=50) TPR () 722 938 p=025 722 667 p=100SPC () 688 971 p=0006 688 957 plt0001

K pneumoniae (n=50) TPR () 897 905 p=100 897 100 NCSPC () 381 100 plt0001 381 929 plt0001

lowastmeans nonparametric McNemar test

4 Conclusions

The prevalence of CP-EK in Vietnam is high The lack ofsuitable methods to detect and differentiate these bacterialinfections has been the main obstacle for clinicians tochoosing an appropriate treatment During this study werecognized that CDT is reasonable and simple to detect thepresence of these important enzymes These features make ithighly applicable to all clinical laboratories in Vietnam

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Limitation In our study other genes such an OXA-48-likeESBL and AmpC 120573-lactamase were not included so thiscould affect our study results The only 10 KPC-producingorganisms in this studymay have effects on the results of PBAdisk test A lack of genotypic method to identify Ecoli and Kpneumoniae was also important

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors are grateful to Gia Dinh Peoplersquos Hospital(HCMC) and Dong Nai General Hospital for kindly provid-ing the clinical isolates and also to the staff of Pasteur Institutein Ho Chi Minh City for the comments and suggestions Wewould also like to thank Dr Vu Quang Huy the Head ofthe Department of Medical Laboratory Science University ofMedicine and Pharmacy Ho Chi Minh City for facilitatingthe study and thank Pham Thai Binh (Nam Khoa Biotech)Bui The Trung (Childrenrsquos Hospital no 2 HCMC) andNguyen Thi Phuong Thao (Dong Nai Medical College) forproviding the ATCC strains of bacteria and some reagents forthis study

Supplementary Materials

List of related information of patients and testing results weredetailed in Annex 1 Data analysis results testing images

reference and full-text of gray literature were described inAnnexes 2 3 4 5 respectively (Supplementary Materials)

References

[1] AU Khan LMaryam andR Zarrilli ldquoStructure Genetics andWorldwide Spread of New Delhi Metallo-120573-lactamase (NDM)a threat to public healthrdquo BMC Microbiology vol 17 no 1 2017

[2] X Jia W Dai W Ma et al ldquoCorrigendum carbapenem-resistant e cloacae in southwest china molecular analysis ofresistance and risk factors for infections caused by ndm-1-producersrdquo Frontiers in Microbiology vol 9 2018

[3] JWang X Yao J Luo L Lv Z Zeng and J-H Liu ldquoEmergenceof Escherichia coli coproducing NDM-1 and KPC-2 carbapen-emases from a retail vegetable Chinardquo Journal of AntimicrobialChemotherapy vol 73 no 1 pp 252ndash254 2018

[4] K M Day M Salman B Kazi et al ldquoPrevalence of NDM-1carbapenemase in patients with diarrhoea in Pakistan and eval-uation of two chromogenic culture mediardquo Journal of AppliedMicrobiology vol 114 no 6 pp 1810ndash1816 2013

[5] S Eshetie C Unakal A Gelaw B Ayelign M Endris and FMoges ldquoMultidrug resistant and carbapenemase producingEnterobacteriaceae among patients with urinary tract infectionat referral Hospital Northwest Ethiopiardquo Antimicrobial Resis-tance and Infection Control vol 4 no 1 article no 12 2015

[6] O Karabay M Altindis M Koroglu O Karatuna O AAydemir and A F Erdem ldquoThe carbapenem-resistant Enter-obacteriaceae threat is growing NDM-1 epidemic at a traininghospital in TurkeyrdquoAnnals of Clinical Microbiology and Antimi-crobials vol 15 no 1 2016

[7] I Rossi Goncalves M L Ferreira B F Araujo et al ldquoOutbreaksof colistin-resistant and colistin-susceptible KPC-producingKlebsiella pneumoniae in a Brazilian intensive care unitrdquoJournal of Hospital Infection vol 94 no 4 pp 322ndash329 2016

[8] P Torres-Gonzalez M Bobadilla-del Valle E Tovar-Calderonet al ldquoOutbreak Caused by Enterobacteriaceae HarboringNDM-1 Metallo-120573-Lactamase Carried in an IncFII Plasmid ina Tertiary Care Hospital in Mexico Cityrdquo Antimicrobial Agentsand Chemotherapy vol 59 no 11 pp 7080ndash7083 2015

[9] J Tamariz C Llanos C Seas et al ldquoDraft genome sequence ofthe first New Delhi metallo-120573-lactamase (NDM-1)-producingEscherichia coli strain isolated in Perurdquo Genome Announce-ments vol 6 no 13 2018

[10] H C Maltezou P Giakkoupi A Maragos et al ldquoOutbreak ofinfections due to KPC-2-producing Klebsiella pneumoniae in ahospital in Crete (Greece)rdquo Infection vol 58 no 3 pp 213ndash2192009

[11] L Dortet G Cuzon V Ponties and P Nordmann ldquoTrends incarbapenemase-producing Enterobacteriaceae France 2012 to2014rdquo Eurosurveillance vol 22 no 6 2017

BioMed Research International 5

Table 3 Particular characteristics of patients at 2 hospitals in this study

Characters Gia Dinh Peoplersquos Hospital(n=59)

Dong Nai General Hospital(n=41) P-value

Average age (95 CI) 715 (674 ndash 752) 604 (522 ndash 66) 0003Range 36 ndash 94 23 ndash 89Elder () 763 585 0079Positive rate () 508 415 0545Pneumonia rate () 254 341Odd of Outcome (PosNeg)between Pneumonia andthe other

079 714

Odd of Pneumonia(ElderYounger) 588 1891

Odd of Outcome(PositiveNegative) in Eldergroup the other

1045 15

Therapy departments ()

ICU (203)Geriatrics (153)

Department of Urologicsurgery (102) RespiratoryDepartment (85) and

other departments (457)

Infectious DiseaseDepartment (414)

Department of surgery(195) Respiratory

Department (98) ICU(49) and other

departments (244)K pneumoniae () 508 488

Gia Dinh people hospitalDong Nai general hospital

Negative blaNDM blaKPC blaNDM_blaKPC

100

Age

80

60

40

20

0

Figure 1 Different distribution of patientsrsquo age following genotype at 2 hospitals

figures for Dong Nai General Hospital In Dong Nai GeneralHospital people infected by non-NDMKPC-producing bac-teria were young most of whom were the group in the rangefrom40 to 60 (Figure 1) these young people could be infectedwith ESBLAmpC producing E coliK pneumoniae Anotherhypothesis might be that the isolates from these patientscould carry other carbapenemase-encoding genes such asblaOXA-48-like but these mechanisms cause a lower level ofresistance [2 17 18] These features could play an important

role in the difference of the odd of positive and negativeoutcome of the elder group between these 2 hospitals Theelders who suffered from pneumonia and infection with thetwo types of carbapenemase producers were more than theyounger ones especially in Dong Nai General Hospital Thereason for the difference between these two hospitals was stillunclear It could be explained that Gia Dinh Peoplersquos Hospitalwas established for a long time the severity of illnesswas quitedifferent compared with Dong Nai General Hospital

6 BioMed Research International

Table 4 The agreement of bacteria isolation real-time PCR and DNA sequencing results

Strains ID Isolation Real-time PCR DNA sequencingGD013 K pneumonia blaNDM 16s rRNA blaNDM K pneumoniaeGD002 E coli blaNDM blaNDMGD018 E coli blaNDM blaKPC 16S rRNA blaNDM blaKPC E coliGD011 K pneumoniae blaKPC blaKPCGD016 K pneumoniae blaKPC blaKPCDN011 K pneumoniae blaKPC blaKPC

Table 5 Combined disk test results in E coli and K pneumonia

Combined disk test Real-time PCR TPR ()95

SPC()

PPV()

NPV()

Accuracy()Species Pos Ne

NDMdetection

E coli(n=50)

Pos 15 1 938 971 938 971 960Neg 1 33

K pneumoniae(n=50)

Pos 19 0 905 100 100 936 96Neg 2 29

KPCdetection

E coli(n=50)

Pos 2 2 667 957 50 978 940Neg 1 45

K pneumoniae(n=50)

Pos 8 3 100 929 727 100 94Neg 0 39

lowastPos positive Neg negative

Gia dinh people hospital Dong Nai general hospitalgroup

negativeblaNDM

blaKPCblaNDM_blaKPC

100

Perc

enta

ge (

)

80

60

40

20

0

Figure 2 Different distribution of prevalence of CPE at 2 hospitals

Regarding the methods real-time PCR is an accurateand rapid method to detect the presence of carbapenemase-encoding genes However the cost associated with themethod is one of the major disadvantages that limits itsuse in common laboratories in Vietnam Among variousphenotypic methods MHT recommended in CLSI 2017 is asimple method [33] In this study SPC of MHT is low (2353K pneumoniae strainswere positive but negative for the real-time PCR) The possible reason for this is that other typesof carbapenemase may present in these bacteria However itcould be explained that AmpC ESBLs or strict requirements

of MHT process at each step may cause false positive results[35] Besides this method shows low sensitivity to NDM-producing Enterobacteriaceae (78 strains were found to havea false-negative result) [36 37] In our study the MHT resultsfor K pneumoniae are consistent with findings in recentstudies conducted byDariush Shokri et al [38] and RangnekaR aSeeM et al [39]Thismay be the reason why the CLSI 2018standard excluded MHT in the guideline [40]

In contrast CDT has been shown to be highly effec-tive The results of NDM detection by EDTA disk test forK pneumoniae (Table 4) were in accordance with findingsobtained by ParadimitrioundashOliveris et al [30] However itwas in contrast to findings reported by Dariush Shokri et al[38] This could be explained by the fact that Shokri carriedout his study including not only K pneumoniae but also Abaumanii and P aeruginosa The high rate of blaVIM in Paeruginosa may be the reason for false-negative results Astudy conducted by Ting-ting Qu et al in 2009 showed thatCDT also detected other types of carbapenemase includingVerona integron-borne metallo-120573-lactamase (VIM) whichwas the most widespread metallo-120573-lactamase in P aerug-inosa [41 42] Our results for KPC detection using PBAdisk test K pneumoniae (Table 4) were in accordance withfindings obtained by Athanasios Tsakris et al (using PBA-400120583g) [31] and Julio Zuniga et al (using 300120583g APBA) [43]The efficiency of PBA disk test in E coli was low particularlyin sensitivity and PPV because of the sample size of KPC-producing E coli only 3 strains with one of them being a falsenegative The results in Tables 5 and 6 illustrated the signifi-cantly higher specificity of combined disk test compared withMHT

BioMed Research International 7

Table 6 Comparison between the MHT and combined disk test

Bacteria Index NDM detection KPC detectionMHT EDTA McNemarlowast MHT Boronic McNemarlowast

E coli (n=50) TPR () 722 938 p=025 722 667 p=100SPC () 688 971 p=0006 688 957 plt0001

K pneumoniae (n=50) TPR () 897 905 p=100 897 100 NCSPC () 381 100 plt0001 381 929 plt0001

lowastmeans nonparametric McNemar test

4 Conclusions

The prevalence of CP-EK in Vietnam is high The lack ofsuitable methods to detect and differentiate these bacterialinfections has been the main obstacle for clinicians tochoosing an appropriate treatment During this study werecognized that CDT is reasonable and simple to detect thepresence of these important enzymes These features make ithighly applicable to all clinical laboratories in Vietnam

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Limitation In our study other genes such an OXA-48-likeESBL and AmpC 120573-lactamase were not included so thiscould affect our study results The only 10 KPC-producingorganisms in this studymay have effects on the results of PBAdisk test A lack of genotypic method to identify Ecoli and Kpneumoniae was also important

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors are grateful to Gia Dinh Peoplersquos Hospital(HCMC) and Dong Nai General Hospital for kindly provid-ing the clinical isolates and also to the staff of Pasteur Institutein Ho Chi Minh City for the comments and suggestions Wewould also like to thank Dr Vu Quang Huy the Head ofthe Department of Medical Laboratory Science University ofMedicine and Pharmacy Ho Chi Minh City for facilitatingthe study and thank Pham Thai Binh (Nam Khoa Biotech)Bui The Trung (Childrenrsquos Hospital no 2 HCMC) andNguyen Thi Phuong Thao (Dong Nai Medical College) forproviding the ATCC strains of bacteria and some reagents forthis study

Supplementary Materials

List of related information of patients and testing results weredetailed in Annex 1 Data analysis results testing images

reference and full-text of gray literature were described inAnnexes 2 3 4 5 respectively (Supplementary Materials)

References

[1] AU Khan LMaryam andR Zarrilli ldquoStructure Genetics andWorldwide Spread of New Delhi Metallo-120573-lactamase (NDM)a threat to public healthrdquo BMC Microbiology vol 17 no 1 2017

[2] X Jia W Dai W Ma et al ldquoCorrigendum carbapenem-resistant e cloacae in southwest china molecular analysis ofresistance and risk factors for infections caused by ndm-1-producersrdquo Frontiers in Microbiology vol 9 2018

[3] JWang X Yao J Luo L Lv Z Zeng and J-H Liu ldquoEmergenceof Escherichia coli coproducing NDM-1 and KPC-2 carbapen-emases from a retail vegetable Chinardquo Journal of AntimicrobialChemotherapy vol 73 no 1 pp 252ndash254 2018

[4] K M Day M Salman B Kazi et al ldquoPrevalence of NDM-1carbapenemase in patients with diarrhoea in Pakistan and eval-uation of two chromogenic culture mediardquo Journal of AppliedMicrobiology vol 114 no 6 pp 1810ndash1816 2013

[5] S Eshetie C Unakal A Gelaw B Ayelign M Endris and FMoges ldquoMultidrug resistant and carbapenemase producingEnterobacteriaceae among patients with urinary tract infectionat referral Hospital Northwest Ethiopiardquo Antimicrobial Resis-tance and Infection Control vol 4 no 1 article no 12 2015

[6] O Karabay M Altindis M Koroglu O Karatuna O AAydemir and A F Erdem ldquoThe carbapenem-resistant Enter-obacteriaceae threat is growing NDM-1 epidemic at a traininghospital in TurkeyrdquoAnnals of Clinical Microbiology and Antimi-crobials vol 15 no 1 2016

[7] I Rossi Goncalves M L Ferreira B F Araujo et al ldquoOutbreaksof colistin-resistant and colistin-susceptible KPC-producingKlebsiella pneumoniae in a Brazilian intensive care unitrdquoJournal of Hospital Infection vol 94 no 4 pp 322ndash329 2016

[8] P Torres-Gonzalez M Bobadilla-del Valle E Tovar-Calderonet al ldquoOutbreak Caused by Enterobacteriaceae HarboringNDM-1 Metallo-120573-Lactamase Carried in an IncFII Plasmid ina Tertiary Care Hospital in Mexico Cityrdquo Antimicrobial Agentsand Chemotherapy vol 59 no 11 pp 7080ndash7083 2015

[9] J Tamariz C Llanos C Seas et al ldquoDraft genome sequence ofthe first New Delhi metallo-120573-lactamase (NDM-1)-producingEscherichia coli strain isolated in Perurdquo Genome Announce-ments vol 6 no 13 2018

[10] H C Maltezou P Giakkoupi A Maragos et al ldquoOutbreak ofinfections due to KPC-2-producing Klebsiella pneumoniae in ahospital in Crete (Greece)rdquo Infection vol 58 no 3 pp 213ndash2192009

[11] L Dortet G Cuzon V Ponties and P Nordmann ldquoTrends incarbapenemase-producing Enterobacteriaceae France 2012 to2014rdquo Eurosurveillance vol 22 no 6 2017

6 BioMed Research International

Table 4 The agreement of bacteria isolation real-time PCR and DNA sequencing results

Strains ID Isolation Real-time PCR DNA sequencingGD013 K pneumonia blaNDM 16s rRNA blaNDM K pneumoniaeGD002 E coli blaNDM blaNDMGD018 E coli blaNDM blaKPC 16S rRNA blaNDM blaKPC E coliGD011 K pneumoniae blaKPC blaKPCGD016 K pneumoniae blaKPC blaKPCDN011 K pneumoniae blaKPC blaKPC

Table 5 Combined disk test results in E coli and K pneumonia

Combined disk test Real-time PCR TPR ()95

SPC()

PPV()

NPV()

Accuracy()Species Pos Ne

NDMdetection

E coli(n=50)

Pos 15 1 938 971 938 971 960Neg 1 33

K pneumoniae(n=50)

Pos 19 0 905 100 100 936 96Neg 2 29

KPCdetection

E coli(n=50)

Pos 2 2 667 957 50 978 940Neg 1 45

K pneumoniae(n=50)

Pos 8 3 100 929 727 100 94Neg 0 39

lowastPos positive Neg negative

Gia dinh people hospital Dong Nai general hospitalgroup

negativeblaNDM

blaKPCblaNDM_blaKPC

100

Perc

enta

ge (

)

80

60

40

20

0

Figure 2 Different distribution of prevalence of CPE at 2 hospitals

Regarding the methods real-time PCR is an accurateand rapid method to detect the presence of carbapenemase-encoding genes However the cost associated with themethod is one of the major disadvantages that limits itsuse in common laboratories in Vietnam Among variousphenotypic methods MHT recommended in CLSI 2017 is asimple method [33] In this study SPC of MHT is low (2353K pneumoniae strainswere positive but negative for the real-time PCR) The possible reason for this is that other typesof carbapenemase may present in these bacteria However itcould be explained that AmpC ESBLs or strict requirements

of MHT process at each step may cause false positive results[35] Besides this method shows low sensitivity to NDM-producing Enterobacteriaceae (78 strains were found to havea false-negative result) [36 37] In our study the MHT resultsfor K pneumoniae are consistent with findings in recentstudies conducted byDariush Shokri et al [38] and RangnekaR aSeeM et al [39]Thismay be the reason why the CLSI 2018standard excluded MHT in the guideline [40]

In contrast CDT has been shown to be highly effec-tive The results of NDM detection by EDTA disk test forK pneumoniae (Table 4) were in accordance with findingsobtained by ParadimitrioundashOliveris et al [30] However itwas in contrast to findings reported by Dariush Shokri et al[38] This could be explained by the fact that Shokri carriedout his study including not only K pneumoniae but also Abaumanii and P aeruginosa The high rate of blaVIM in Paeruginosa may be the reason for false-negative results Astudy conducted by Ting-ting Qu et al in 2009 showed thatCDT also detected other types of carbapenemase includingVerona integron-borne metallo-120573-lactamase (VIM) whichwas the most widespread metallo-120573-lactamase in P aerug-inosa [41 42] Our results for KPC detection using PBAdisk test K pneumoniae (Table 4) were in accordance withfindings obtained by Athanasios Tsakris et al (using PBA-400120583g) [31] and Julio Zuniga et al (using 300120583g APBA) [43]The efficiency of PBA disk test in E coli was low particularlyin sensitivity and PPV because of the sample size of KPC-producing E coli only 3 strains with one of them being a falsenegative The results in Tables 5 and 6 illustrated the signifi-cantly higher specificity of combined disk test compared withMHT

BioMed Research International 7

Table 6 Comparison between the MHT and combined disk test

Bacteria Index NDM detection KPC detectionMHT EDTA McNemarlowast MHT Boronic McNemarlowast

E coli (n=50) TPR () 722 938 p=025 722 667 p=100SPC () 688 971 p=0006 688 957 plt0001

K pneumoniae (n=50) TPR () 897 905 p=100 897 100 NCSPC () 381 100 plt0001 381 929 plt0001

lowastmeans nonparametric McNemar test

4 Conclusions

The prevalence of CP-EK in Vietnam is high The lack ofsuitable methods to detect and differentiate these bacterialinfections has been the main obstacle for clinicians tochoosing an appropriate treatment During this study werecognized that CDT is reasonable and simple to detect thepresence of these important enzymes These features make ithighly applicable to all clinical laboratories in Vietnam

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Limitation In our study other genes such an OXA-48-likeESBL and AmpC 120573-lactamase were not included so thiscould affect our study results The only 10 KPC-producingorganisms in this studymay have effects on the results of PBAdisk test A lack of genotypic method to identify Ecoli and Kpneumoniae was also important

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors are grateful to Gia Dinh Peoplersquos Hospital(HCMC) and Dong Nai General Hospital for kindly provid-ing the clinical isolates and also to the staff of Pasteur Institutein Ho Chi Minh City for the comments and suggestions Wewould also like to thank Dr Vu Quang Huy the Head ofthe Department of Medical Laboratory Science University ofMedicine and Pharmacy Ho Chi Minh City for facilitatingthe study and thank Pham Thai Binh (Nam Khoa Biotech)Bui The Trung (Childrenrsquos Hospital no 2 HCMC) andNguyen Thi Phuong Thao (Dong Nai Medical College) forproviding the ATCC strains of bacteria and some reagents forthis study

Supplementary Materials

List of related information of patients and testing results weredetailed in Annex 1 Data analysis results testing images

reference and full-text of gray literature were described inAnnexes 2 3 4 5 respectively (Supplementary Materials)

References

[1] AU Khan LMaryam andR Zarrilli ldquoStructure Genetics andWorldwide Spread of New Delhi Metallo-120573-lactamase (NDM)a threat to public healthrdquo BMC Microbiology vol 17 no 1 2017

[2] X Jia W Dai W Ma et al ldquoCorrigendum carbapenem-resistant e cloacae in southwest china molecular analysis ofresistance and risk factors for infections caused by ndm-1-producersrdquo Frontiers in Microbiology vol 9 2018

[3] JWang X Yao J Luo L Lv Z Zeng and J-H Liu ldquoEmergenceof Escherichia coli coproducing NDM-1 and KPC-2 carbapen-emases from a retail vegetable Chinardquo Journal of AntimicrobialChemotherapy vol 73 no 1 pp 252ndash254 2018

[4] K M Day M Salman B Kazi et al ldquoPrevalence of NDM-1carbapenemase in patients with diarrhoea in Pakistan and eval-uation of two chromogenic culture mediardquo Journal of AppliedMicrobiology vol 114 no 6 pp 1810ndash1816 2013

[5] S Eshetie C Unakal A Gelaw B Ayelign M Endris and FMoges ldquoMultidrug resistant and carbapenemase producingEnterobacteriaceae among patients with urinary tract infectionat referral Hospital Northwest Ethiopiardquo Antimicrobial Resis-tance and Infection Control vol 4 no 1 article no 12 2015

[6] O Karabay M Altindis M Koroglu O Karatuna O AAydemir and A F Erdem ldquoThe carbapenem-resistant Enter-obacteriaceae threat is growing NDM-1 epidemic at a traininghospital in TurkeyrdquoAnnals of Clinical Microbiology and Antimi-crobials vol 15 no 1 2016

[7] I Rossi Goncalves M L Ferreira B F Araujo et al ldquoOutbreaksof colistin-resistant and colistin-susceptible KPC-producingKlebsiella pneumoniae in a Brazilian intensive care unitrdquoJournal of Hospital Infection vol 94 no 4 pp 322ndash329 2016

[8] P Torres-Gonzalez M Bobadilla-del Valle E Tovar-Calderonet al ldquoOutbreak Caused by Enterobacteriaceae HarboringNDM-1 Metallo-120573-Lactamase Carried in an IncFII Plasmid ina Tertiary Care Hospital in Mexico Cityrdquo Antimicrobial Agentsand Chemotherapy vol 59 no 11 pp 7080ndash7083 2015

[9] J Tamariz C Llanos C Seas et al ldquoDraft genome sequence ofthe first New Delhi metallo-120573-lactamase (NDM-1)-producingEscherichia coli strain isolated in Perurdquo Genome Announce-ments vol 6 no 13 2018

[10] H C Maltezou P Giakkoupi A Maragos et al ldquoOutbreak ofinfections due to KPC-2-producing Klebsiella pneumoniae in ahospital in Crete (Greece)rdquo Infection vol 58 no 3 pp 213ndash2192009

[11] L Dortet G Cuzon V Ponties and P Nordmann ldquoTrends incarbapenemase-producing Enterobacteriaceae France 2012 to2014rdquo Eurosurveillance vol 22 no 6 2017

BioMed Research International 7

Table 6 Comparison between the MHT and combined disk test

Bacteria Index NDM detection KPC detectionMHT EDTA McNemarlowast MHT Boronic McNemarlowast

E coli (n=50) TPR () 722 938 p=025 722 667 p=100SPC () 688 971 p=0006 688 957 plt0001

K pneumoniae (n=50) TPR () 897 905 p=100 897 100 NCSPC () 381 100 plt0001 381 929 plt0001

lowastmeans nonparametric McNemar test

4 Conclusions

The prevalence of CP-EK in Vietnam is high The lack ofsuitable methods to detect and differentiate these bacterialinfections has been the main obstacle for clinicians tochoosing an appropriate treatment During this study werecognized that CDT is reasonable and simple to detect thepresence of these important enzymes These features make ithighly applicable to all clinical laboratories in Vietnam

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Limitation In our study other genes such an OXA-48-likeESBL and AmpC 120573-lactamase were not included so thiscould affect our study results The only 10 KPC-producingorganisms in this studymay have effects on the results of PBAdisk test A lack of genotypic method to identify Ecoli and Kpneumoniae was also important

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors are grateful to Gia Dinh Peoplersquos Hospital(HCMC) and Dong Nai General Hospital for kindly provid-ing the clinical isolates and also to the staff of Pasteur Institutein Ho Chi Minh City for the comments and suggestions Wewould also like to thank Dr Vu Quang Huy the Head ofthe Department of Medical Laboratory Science University ofMedicine and Pharmacy Ho Chi Minh City for facilitatingthe study and thank Pham Thai Binh (Nam Khoa Biotech)Bui The Trung (Childrenrsquos Hospital no 2 HCMC) andNguyen Thi Phuong Thao (Dong Nai Medical College) forproviding the ATCC strains of bacteria and some reagents forthis study

Supplementary Materials

List of related information of patients and testing results weredetailed in Annex 1 Data analysis results testing images

reference and full-text of gray literature were described inAnnexes 2 3 4 5 respectively (Supplementary Materials)

References

[1] AU Khan LMaryam andR Zarrilli ldquoStructure Genetics andWorldwide Spread of New Delhi Metallo-120573-lactamase (NDM)a threat to public healthrdquo BMC Microbiology vol 17 no 1 2017

[2] X Jia W Dai W Ma et al ldquoCorrigendum carbapenem-resistant e cloacae in southwest china molecular analysis ofresistance and risk factors for infections caused by ndm-1-producersrdquo Frontiers in Microbiology vol 9 2018

[3] JWang X Yao J Luo L Lv Z Zeng and J-H Liu ldquoEmergenceof Escherichia coli coproducing NDM-1 and KPC-2 carbapen-emases from a retail vegetable Chinardquo Journal of AntimicrobialChemotherapy vol 73 no 1 pp 252ndash254 2018

[4] K M Day M Salman B Kazi et al ldquoPrevalence of NDM-1carbapenemase in patients with diarrhoea in Pakistan and eval-uation of two chromogenic culture mediardquo Journal of AppliedMicrobiology vol 114 no 6 pp 1810ndash1816 2013

[5] S Eshetie C Unakal A Gelaw B Ayelign M Endris and FMoges ldquoMultidrug resistant and carbapenemase producingEnterobacteriaceae among patients with urinary tract infectionat referral Hospital Northwest Ethiopiardquo Antimicrobial Resis-tance and Infection Control vol 4 no 1 article no 12 2015

[6] O Karabay M Altindis M Koroglu O Karatuna O AAydemir and A F Erdem ldquoThe carbapenem-resistant Enter-obacteriaceae threat is growing NDM-1 epidemic at a traininghospital in TurkeyrdquoAnnals of Clinical Microbiology and Antimi-crobials vol 15 no 1 2016

[7] I Rossi Goncalves M L Ferreira B F Araujo et al ldquoOutbreaksof colistin-resistant and colistin-susceptible KPC-producingKlebsiella pneumoniae in a Brazilian intensive care unitrdquoJournal of Hospital Infection vol 94 no 4 pp 322ndash329 2016

[8] P Torres-Gonzalez M Bobadilla-del Valle E Tovar-Calderonet al ldquoOutbreak Caused by Enterobacteriaceae HarboringNDM-1 Metallo-120573-Lactamase Carried in an IncFII Plasmid ina Tertiary Care Hospital in Mexico Cityrdquo Antimicrobial Agentsand Chemotherapy vol 59 no 11 pp 7080ndash7083 2015

[9] J Tamariz C Llanos C Seas et al ldquoDraft genome sequence ofthe first New Delhi metallo-120573-lactamase (NDM-1)-producingEscherichia coli strain isolated in Perurdquo Genome Announce-ments vol 6 no 13 2018

[10] H C Maltezou P Giakkoupi A Maragos et al ldquoOutbreak ofinfections due to KPC-2-producing Klebsiella pneumoniae in ahospital in Crete (Greece)rdquo Infection vol 58 no 3 pp 213ndash2192009

[11] L Dortet G Cuzon V Ponties and P Nordmann ldquoTrends incarbapenemase-producing Enterobacteriaceae France 2012 to2014rdquo Eurosurveillance vol 22 no 6 2017

8 BioMed Research International

[12] C-R Lee J H Lee K S Park Y B Kim B C Jeong and S HLee ldquoGlobal dissemination of carbapenemase-producing Kleb-siella pneumoniae Epidemiology genetic context treatmentoptions and detection methodsrdquo Frontiers in Microbiology vol7 article no 895 2016

[13] T N Phuong Klebsiella pneumoniae carbapenemase producingKlebsiella pneumoniae molecular genetic characteristics Univer-sity of Science Vietnam National University Ho Chi Minh City2016

[14] P D Thai T D Linh N H Thu T H Hoang T H Son andT T V Phuong ldquoApply Southern-Blotting assay to detect KPC-plasmid of enterobacteriaceaestrains from clinical isolates fromhospitals in Hanoirdquo Vietnam Vietnam Journal of PreventiveMedicine vol 27 no 3 2017

[15] N H Thu T D Linh N T T Mai T V Phuong N H L Yenand T N Duong ldquoMolecular epidemiology of Klebsiella pneu-moniae carbapenemase - 2 producing Klebsiella pneumoniaeisolated from Saintpaul HospitalrdquoVietnam Journal of PreventiveMedicine vol 26 no 8 2016

[16] J A Ramos-Castaneda A Ruano-Ravina R Barbosa-Lorenzoet al ldquoMortality due to KPC carbapenemase-producing Kleb-siella pneumoniae infections Systematic review and meta-analysis Mortality due to KPC Klebsiella pneumoniae infec-tionsrdquo Infection vol 76 no 5 pp 438ndash448 2018

[17] V Studentova C C Papagiannitsis R Izdebski et al ldquoDetec-tion of OXA-48-type carbapenemase-producing Enterobacte-riaceae in diagnostic laboratories can be enhanced by additionof bicarbonates to cultivation media or reaction buffersrdquo FoliaMicrobiologica vol 60 no 2 pp 119ndash129 2015

[18] R Fattouh N Tijet A McGeer S M Poutanen R G Melanoand S N Patel ldquoWhat is the appropriate meropenem MIC forscreening of carbapenemase-producing Enterobacteriaceae inlow-prevalence settingsrdquo Antimicrobial Agents and Chemother-apy vol 60 no 3 pp 1556ndash1559 2016

[19] A Iyer E Barbour E Azhar et al ldquoTransposable elementsin Escherichia coli antimicrobial resistancerdquo Advances in Bio-science and Biotechnology vol 04 no 03 pp 415ndash423 2013

[20] P Nordmann T Naas and L Poirel ldquoGlobal spread of car-bapenemase producingEnterobacteriaceaerdquoEmerging InfectiousDiseases vol 17 no 10 pp 1791ndash1798 2011

[21] J Feng Y Qiu Z Yin et al ldquoCoexistence of a novel KPC-2-encoding MDR plasmid and an NDM-1-encoding pNDM-HN380-like plasmid in a clinical isolate of Citrobacter freundiirdquoJournal of Antimicrobial Chemotherapy vol 70 no 11 pp 2987ndash2991 2015

[22] T Bosch S PM Lutgens MH AHermans et al ldquoOutbreak ofNDM-1-producing klebsiella pneumoniae in a Dutch hospitalwith interspecies transfer of the resistance plasmid and unex-pected occurrence in unrelated health care centersrdquo Journal ofClinical Microbiology vol 55 no 8 pp 2380ndash2390 2017

[23] S Navon-Venezia K Kondratyeva and A Carattoli ldquoKlebsiellapneumoniae A major worldwide source and shuttle for antibi-otic resistancerdquo FEMS Microbiology Reviews vol 41 no 3 pp252ndash275 2017

[24] S Datta S Mitra P Chattopadhyay T Som S Mukherjee andS Basu ldquoSpread and exchange of bla NDM-1 in hospitalizedneonates role of mobilizable genetic elementsrdquo European Jour-nal of Clinical Microbiology amp Infectious Diseases vol 36 no 2pp 255ndash265 2017

[25] T Tada M Tsuchiya K Shimada et al ldquoDissemination ofCarbapenem-resistant Klebsiella pneumoniae clinical isolates

with various combinations of Carbapenemases (KPC-2 NDM-1 NDM-4 and OXA-48) and 16S rRNAMethylases (RmtB andRmtC) in Vietnamrdquo BMC Infectious Diseases vol 17 no 1 p467 2017

[26] R Isozumi K Yoshimatsu T Yamashiro et al ldquoBlaNDM-1-positive Klebsiella pneumoniae from environment VietnamrdquoEmerging Infectious Diseases vol 18 no 8 pp 1383ndash1385 2012

[27] T H Hoang H Wertheim N B Minh et al ldquoCarbapenem-resistant Escherichia coli and Klebsiella pneumoniae strainscontaining new delhi metallo-beta-lactamase isolated from twopatients in Vietnamrdquo Journal of Clinical Microbiology vol 51no 1 pp 373-374 2013

[28] G G Zhanel C K Lawrence H Adam et al ldquoCorrection toImipenemndashRelebactam and MeropenemndashVaborbactam TwoNovel Carbapenem-120573-Lactamase Inhibitor CombinationsrdquoDrugs vol 78 no 7 p 787 2018

[29] M P-WK D Bm J A Tm R Bc A Bs et al ldquoRelebactam isa Potent Inhibitor of the KPC-2 beta-Lactamase and Restoresthe Susceptibility of Imipenem Against KPC-Producing Enter-obacteriaceaerdquoAntimicrobial Agents and Chemotherapy 2018

[30] MChristofidouM Papadimitriou-Olivgeris E DAnastassiouet al ldquoPerformance of four different agar plate methods forrectal swabs synergy disk tests and metallo-120573-lactamase Etestfor clinical isolates in detecting carbapenemase-producingKlebsiella pneumoniaerdquo Journal of Medical Microbiology vol65 no 9 pp 954ndash961 2016

[31] A Tsakris I Kristo A Poulou et al ldquoEvaluation of boronicacid disk tests for differentiating KPC-possessing Klebsiellapneumoniae isolates in the clinical laboratoryrdquo Journal ofClinical Microbiology vol 47 no 2 pp 362ndash367 2009

[32] Control CfD Prevention ldquoMultiplex real-time PCR detection ofKlebsiella pneumoniae carbapenemase (KPC) and New Delhimetallo-120573-lactamase (NDM-1) genesrdquoAtlanta vol 500 no 6-72011

[33] Institute CLS ldquoPerformance standards for antimicrobial sus-ceptibility testingrdquo The Modified Hodge Test for SuspectedCarbapenemase Production in Enterobacteriaceae Clinical andLaboratory Standards Institute pp 108ndash113 2017

[34] V BichDADung BNA Pha andN SM Tuyet ldquoAntibioticresistance of escherichia coli in gia dinh peoplersquos hospitalrdquo HoChi Minh medical Journal vol 13 no 6 pp 253ndash257 2009

[35] S S Jeremiah V Balaji S Anandan and R D Sahni ldquoApossible alternative to the error prone modified Hodge test tocorrectly identify the carbapenemase producingGram-negativebacteriardquo Indian Journal of Medical Microbiology vol 32 no 4pp 414ndash418 2014

[36] H-K Kim J S Park H Sung and M-N Kim ldquoFurther mod-ification of the modified hodge test for detecting metallo-120573-lactamase-producing carbapenem-resistant Enterobacteri-aceaerdquo Annals of Laboratory Medicine vol 35 no 3 pp 298ndash305 2015

[37] B A Sultan E Khan F Hussain A Nasir and S Irfan ldquoEffec-tiveness of Modified Hodge Test to detect NDM-1 Carbapen-emases An experience from Pakistanrdquo Journal of the PakistanMedical Association vol 63 no 8 pp 955ndash960 2013

[38] D Shokri M R Khorasgani S M Fatemi and A Soleimani-Delfan ldquoResistotyping phenotyping and genotyping of newdelhimetallo-120573-lactamase (NDM) among gram-negative bacillifrom Iranian patientsrdquo Journal of Medical Microbiology vol 66no 4 pp 402ndash411 2017

[39] R Aseem ldquoApproach to carbapenemase detection in klebsiellapneumoniae in routine diagnostic laboratoriesrdquo Journal of

BioMed Research International 9

Clinical and Diagnostic Research vol 10 no 12 pp DC24ndashDC72016

[40] Institute CLS ldquoPerformance standards for antimicrobial sus-ceptibility testingrdquoModified Carbapenem Inactivation Methodsfor Suspected Carbapenemase Production in Enterobacteriaceaeand P aeruginosa pp 112ndash122 2018

[41] T Qu J Zhang J Wang et al ldquoEvaluation of phenotypic testsfor detection of metallo-120573-lactamase-producing pseudomonasaeruginosa strains in chinardquo Journal of Clinical Microbiologyvol 47 no 4 pp 1136ndash1142 2009

[42] D J Hong I K Bae I-H Jang S H Jeong H-K Kang and KLee ldquoEpidemiology and characteristics ofmetallo-120573-lactamase-producing Pseudomonas aeruginosardquo Journal of Infection andChemotherapy vol 47 no 2 pp 81ndash97 2015

[43] Z Julio CGerardo P Carlos andTMusharaf ldquoThe combined-disk boronic acid test as an accurate strategy for the detectionof KPC carbapenemase in Central Americardquo The Journal ofInfection in Developing Countries vol 10 no 3 pp 298ndash3032016

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 9

Clinical and Diagnostic Research vol 10 no 12 pp DC24ndashDC72016

[40] Institute CLS ldquoPerformance standards for antimicrobial sus-ceptibility testingrdquoModified Carbapenem Inactivation Methodsfor Suspected Carbapenemase Production in Enterobacteriaceaeand P aeruginosa pp 112ndash122 2018

[41] T Qu J Zhang J Wang et al ldquoEvaluation of phenotypic testsfor detection of metallo-120573-lactamase-producing pseudomonasaeruginosa strains in chinardquo Journal of Clinical Microbiologyvol 47 no 4 pp 1136ndash1142 2009

[42] D J Hong I K Bae I-H Jang S H Jeong H-K Kang and KLee ldquoEpidemiology and characteristics ofmetallo-120573-lactamase-producing Pseudomonas aeruginosardquo Journal of Infection andChemotherapy vol 47 no 2 pp 81ndash97 2015

[43] Z Julio CGerardo P Carlos andTMusharaf ldquoThe combined-disk boronic acid test as an accurate strategy for the detectionof KPC carbapenemase in Central Americardquo The Journal ofInfection in Developing Countries vol 10 no 3 pp 298ndash3032016

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom