research article spread of tem, vim, shv, and ctx-m...

Research ArticleSpread of TEM VIM SHV and CTX-M 120573-Lactamasesin Imipenem-Resistant Gram-Negative Bacilli Isolatedfrom Egyptian Hospitals

El sayed Hamdy Mohammed1 Ahmed Elsadek Fakhr2 Hanan Mohammed El sayed2

Said abd Elmohsen Al Johery2 and Wesam Abdel Ghani Hassanein1

1Department of Botany Faculty of Science Zagazig University Zagazig 44511 Egypt2Department of Medical Microbiology amp Immunology Faculty of Medicine Zagazig University Zagazig 44511 Egypt

Correspondence should be addressed to El sayed Hamdy Mohammed anashamdy25yahoocom

Received 26 August 2015 Revised 9 December 2015 Accepted 14 December 2015

Academic Editor Giuseppe Comi

Copyright copy 2016 El sayed Hamdy Mohammed et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Carbapenem-resistant Gram-negative bacilli resulting from 120573-lactamases have been reported to be an important cause ofnosocomial infections and are a critical therapeutic problem worldwideThis study aimed to describe the prevalence of imipenem-resistant Gram-negative bacilli isolates and detection of 119887119897119886VIM 119887119897119886TEM 119887119897119886SHV 119887119897119886CTX-M-1 and 119887119897119886CTX-M-9 genes in these clinicalisolates in Egyptian hospitals The isolates were collected from various clinical samples identified by conventional methods andconfirmed by API 20E Antibiotic susceptibility testing was determined by Kirby-Bauer technique and interpreted according toCLSI Production of 119887119897119886VIM 119887119897119886TEM 119887119897119886SHV and 119887119897119886CTX-M genes was done by polymerase chain reaction (PCR) Direct sequencingfrom PCR products was subsequently carried out to identify and confirm these 120573-lactamases genes Out of 65 isolates (461)Escherichia coli (262) Klebsiella pneumoniae and (107) Pseudomonas aeruginosa were identified as the commonest Gram-negative bacilli 33(508) were imipenem-resistant isolates 22 isolates (667) carried 119887119897119886VIM 24(727) had 119887119897119886TEM and 5(15)showed 119887119897119886SHV while 12(36) 6(182) and 0(000) harbored 119887119897119886CTX-M-1 119887119897119886CTX-M-9 and 119887119897119886CTX-M-825 respectively There is ahigh occurrence of 120573-lactamase genes in clinical isolates and sequence analysis of amplified genes showed differences betweenmultiple SNPs (single nucleotide polymorphism) sites in the same gene among local isolates in relation to published sequences

1 Introduction

Gram-negative bacilli are a heterogeneous group of Gram-negative bacteria that are common commensals infectiousagents and also sometimes referred to as ldquonightmare bacteriardquo[1] Hospital acquired infections due to Gram-negative bacilliare a leading cause of morbidity andmortality worldwide [2]

Carbapenem a member of the 120573-lactam family has abroad spectrum of activity and is stable tomost 120573-lactamasesThese properties make carbapenem an important therapeu-tic option for treating serious infections involving resis-tant strains of Enterobacteriaceae anaerobes Pseudomonasaeruginosa and Acinetobacter spp [3] although carbapen-ems including imipenem and meropenem are often usedas ldquoantibiotics of last resortrdquo when patients with infections

become severely ill or are suspected of harboring resistantbacteria [4] However carbapenem-resistant Gram-negativebacilli isolates were increasingly reported worldwide [5]

This resistance may be attributed to presence of metallo-120573-lactamase in bacteria such as IMP (Imipenemase) VIM(Verona-Integron metallo-120573-lactamase) [6] and extendedspectrum 120573-lactamases (ESBLs) such as SHV TEM andCTX-M [7]

For establishment of appropriate antimicrobial therapyand control of the spread of drug resistant Gram-negativebacilli the PCR-based detection methods of resistant genesshow the bioinformatics analysis of their molecular diversityand evolution becoming increasingly important [8]

This work aimed to study distribution of imipenem-resistant Gram-negative isolates and shed focused light on

Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2016 Article ID 8382605 15 pageshttpdxdoiorg10115520168382605

2 International Journal of Microbiology

Table 1 Primer name primer sequences and expected amplicon size of amplified DNA products

PCR name 120573-lactamase targeted Primer name Sequence (51015840-31015840) Amplicon size (bp)

VIM VIM variants including VIM-1 andVIM-2

VIM-for GATGGTGTTTGGTCGCATA 390VIM-rev CGAATGCGCAGCACCAG

TEM TEM variants including TEM-1 andTEM-2

TSO-T-for CATTTCCGTGTCGCCCTTATTC 800TSO-T-rev CGTTCATCCATAGTTGCCTGAC

SHV SHV variants including SHV-1 TSO-S-for AGCCGCTTGAGCAAATTAAAC 713TSO-S-rev ATCCCGCAGATAAATCACCAC

CTX-Mgroup 1

Variants of CTX-M group 1 includingCTX-M-1 CTX-M-3 and CTX-M-15

CTXMGp 1-for TTAGGAARTGTGCCGCTGYAb688

CTXMGp 12 rev CGATATCGTTGGTGGTRCCATb

CTX-Mgroup 9

Variants of CTX-M-9 includingCTX-M-9 and CTX-M-14

CTX-9-F TCAAGCCTGCCGATCTGGT 561CTX-9-R TGATTCTCGCCGCTGAAG

CTX-Mgroup 825

CTX-M-8 CTX-M-25 CTX-M-26 andCTX-M-39 to CTX-M-41

CTX-825-F AACRCRCAGACGCTCTACb326

CTX-825-R TCGAGCCGGAASGTGTYATb

bY = T or C R = A or G S = G or C

some genes encoding beta-lactamase enzymes responsible forsuch resistance in Zagazig University Hospitals in Egypt

2 Material and Methods

21 Bacterial Isolates Clinical isolates of Gram-negativebacilli including Escherichia coli (119899 = 30) Klebsiella pneu-moniae (119899 = 17) Pseudomonas aeruginosa (119899 = 7) Proteusmirabilis (119899 = 2) Citrobacter freundii (119899 = 1) Acinetobacterbaumanii (119899 = 3) and Enterobacter cloacae (119899 = 4) werecollected fromblood urine pus and sputum specimens fromhospitalized patients in ZagazigUniversityHospitals in Egyptfrom January 2013 toMarch 2014These clinical samples wereprocessed by plating on blood agar and MacConkey agar[9] A growth temperature of 44∘C was used sometimes toconfirm the identity of these isolates and the identified strainswere stored in glycerol (20 VV) at 70∘C and subculturedseveral times to be viable All isolates were identified bystandard biochemical tests [10] and confirmed by API 20E(BioMerieux Marcy lrsquoEtoile France)

22 Antibiotic Susceptibility Testing The susceptibility testingof studied isolates was performed by disc diffusion method(modified Kirby-Bauer method) using Muller-Hinton agar(Becton Dickinson MA USA) and interpreted accordingto the Clinical Laboratory Standard Institute (CLSI) guide-lines [11] The antibiotic disks used imipenem (IPM 10 120583g)amikacin (AK 30 120583g) ciprofloxacin (CIP 5 120583g) piperacillin(PRL 100 120583g) cefoperazonesulbactam (CES 10 + 5 120583g)cefoxitin (FOX 30 120583g) and cefotaxime (CTX 30120583g) whichwere placed 15mm away from the central disc and the plateswere incubated for about 18ndash24 hrs at 37∘C

23 Molecular Detection of 120573-Lactamase Genes by PCR Fordetection of 120573-lactamase genes responsible for imipenem-resistance rapid genomic DNA was prepared from aboutfive colonies heated in 100mL distilled water (95∘C for10min) followed by a centrifugation step of cell suspension at

12000 rpm for 5min then supernatant was taken as a sourceof template DNA PCR amplification was carried out by usingDNA thermal cycler (Biometra Singapore) using a specificprimer for 119887119897119886VIM 119887119897119886TEM 119887119897119886SHV 119887119897119886CTX-M-1 119887119897119886CTX-M-9and 119887119897119886CTX-M-825 (Table 1) in a 50120583L volume containing 10xPCR buffer 2mM deoxynucleoside triphosphates 34 pmolof each primer 25mM MgCl

2 1 U Taq DNA polymerase

and 1 120583L of genomic DNA [12] Amplification was carriedout as follows initial denaturation at 94∘C for 10 minutesfollowed by 40 cycles of DNA denaturation at 94∘C for40 seconds primer annealing at 60∘C for 40 seconds andprimer extension at 72∘C for 1 minute and a final elongationstep at 72∘C for 7 minutes The annealing temperature wasoptimal at 55∘C instead of 60∘C for amplification of 119887119897119886VIMAmplicons were then visualized after running in 2 agarosegel at 100V for 30mins A 50ndash1000 bpDNA ladder (USA)wasused as a size marker Finally PCR products were purifiedwith innuPREP PCRpure kit (Analytik Jena Germany) andsubjected to direct sequencing via GATC Company by use ofABI 3730xl DNA sequencer

24 Bioinformatics and Sequences Analysis The obtainedchromatogram sequencing files were inspected and correctedusing the software application Chromas 23 (TechnelysiumHelensvale Australia) and JalView (28)

The sequences obtained from our samples were alignedwith GenBank sequences The phylogenetic tree for eachsequence was obtained by performing neighbor-joining anal-ysis of the alignment of sequences with reference strains(accession numberscountry of origin) that were retrievedfrom GenBank The studied strains were marked by the sign[◼] Meanwhile the reference sequences were marked by thesign [998771]

The BLAST and FASTA programs of the National Centerfor Biotechnology Information (httpblastncbinlmnihgovBlastcgi) were used to search databases for similarnucleotide sequences [13] Multiple sequence alignments ofthe nucleic acidwere carried out using theClustalWprogramThe statistical analysis was performed using SPSS version

International Journal of Microbiology 3

477

615538

231323

184

015 15 46 92 610 0

508446 415

677615

815

100

SusceptibleIntermediateResistant

0102030405060708090

100

()

Imip

enem

Am

ikac

in

Cipr

oflox

acin

Pipe

raci

llin

Cef

oxiti

n

Cef

otax

ime

Cef

oper

azon

e(s

ulba

ctam

)

Figure 1 Antimicrobial susceptibility patterns of all 65 Gram-negative bacilli isolates

2001205942 = chi-square test and119875 valueslt 005were consideredsignificant

3 Results

31 Isolation and Identification The present study was con-ducted on 65 screened isolates of Gram-negative bacilliobtained from 108 various clinical samples such as urineblood pus and sputum where 41 (788) were isolatedfrom urine 12 (666) from blood 14 (482) from respi-ratory secretions and 2 (222) from pus Escherichia coli(461) was the most commonly isolated organism amongGram-negative bacilli followed by Klebsiella pneumoniae(262) Pseudomonas aeruginosa (107) Enterobacter cloa-cae (61) Proteus mirabilis (307) and Acinetobacter bau-manii (46) while Citrobacter freundii and Proteus vulgarisgave 15 The isolation of Gram-negative bacilli isolates wassignificantly higher in patients with trauma (119875 lt 0001)those hospitalized for more than 7 days (119875 lt 0001) andthose with ICU admission (119875 lt 0001) harboring risk factorsfor acquiring Gram-negative bacilli infection

32 The Antibiotic Susceptibility Testing It was shown inFigure 1 as observed that 40 (615) were susceptibleto amikacin 35 (538) were susceptible to ciprofloxacin31 (477) were susceptible to imipenem and 21 (323)were susceptible to cefoperazonesulbactam However allisolates were resistant to cefotaxime (100) 53 (815) ofisolates were resistant to cefoxitin 44 (677) were resis-tant to piperacillin 40 (615) were resistant to cefoper-azonesulbactam 33 (508) were resistant to imipenemfollowed by 24 (446) resistant to amikacin and 27 (415)were resistant to ciprofloxacin

33 PCRAssay Results As shown in Table 6 of 33 imipenem-resistant Gram-negative bacillistrains the results of PCR

amplification products of 120573-lactamases genes showed that667 of isolates carried 119887119897119886VIM at 390 bp (Figure 2(a))727 had 119887119897119886TEM at 800 bp (Figure 2(b)) 360 harbored119887119897119886CTX-M-1 at 688 bp (Figure 2(c)) and 150 showed 119887119897119886SHV(Figure 2(d)) while 182 (Figure 2(e)) 000 (Figure 2(f))harbored 119887119897119886CTX-M-9 119887119897119886CTX-M-825 respectively Sequencingconfirmed presence of these 120573-lactamase genes the GenBanknucleotide sequence accession numbers for the sequencesstudied are detailed Aligning of the obtained sequences withthose of reference strains in GenBank confirmed the correctidentification of 119887119897119886VIM 119887119897119886TEM 119887119897119886SHV and 119887119897119886CTX-M genesby PCR

34 Sequences Analysis and Polymorphism

341 The Analysis of VIM Gene (119887119897119886VIM12) The sequence ofthe purified product of VIM gene (119887119897119886VIM12) was comparedwith homologousGenBank sequences using BLAST programand resulted in significant similarity to many metallo-120573-lactamases genes of different bacterial strains

(1) VIM Gene (blaVIM12) in Escherichia coli Strains The pair-wise sequences alignments of resulting VIM gene (119887119897119886VIM12)in E coli strains isolated from Zagazig University (ZU)Hospitals in comparison with published VIM gene in Ecoli strains from GenBank for example (E coli KC4173771)showed single common SNP (single nucleotide polymor-phism) sites between the different strains The SNPs posi-tion was indicated in position 382 in the Egyptian strains(Figure 3(a))The phylogenetic tree of VIM gene sequence inE coli strains isolated fromZagazigUniversityHospitals andpublished homologous sequences in GenBank showed dif-ferent degrees of dissimilarity between the different strains(Figure 3(b)) It was interesting to detect that both strainsthe most similar and most dissimilar strains were from thesame country Greece indicating biodiversity in the samegeographical location

(2) VIMGene (blaVIM12) in Klebsiella pneumoniae StrainsTheVIM gene isolated from K pneumoniae strains in ZagazigUniversity Hospitals was comparedwith publishedVIM genein K pneumoniae strains from GenBank (eg K pneumoniaeDQ1439131) The results showed 6 different common SNPsbetween the different strains The SNPs positions were indi-cated in 45 150 168 284 309 and 363 (Figure 3(c)) Thephylogenetic tree of VIM gene sequence in K pneumoniaestrains isolated from ZU Hospitals and published homol-ogous sequences in GenBank showed different degrees ofdissimilarity between the different strains with many uniquesequences in the Egyptian strain (Figure 3(d))

(3) VIM Gene (blaVIM12) in Acinetobacter baumanii StrainsThe sequence of purified product of VIM gene (119887119897119886VIM12)from Acinetobacter baumanii strain was compared with theGenBank sequence using BLAST program Interestingly itwas revealed that therewas a single strain present inGenBankwhich is completely different from the studied strains andthis is notmatchingwith our study (eg Staphylococcus phageStB12 complete genome) The sequence alignment showed 9


1 2 3 4 5 6 7 8 9 10

(bp)

1000900800

700

600

500

400

300

200

100

50

390

(a)

1 2 3 4 5 6 7 8 9 10

(bp)

1000900800

700

600

500

400

300

200

100

50

800

(b)1 2 3 4 5 6 7 8 9 10

(bp)

1000900800

700

600

500

400

300

200

100

50

688

(c)

1 2 3 4 5 6 7 8 9 10

(bp)

1000900800700

600

500

400

300

200

100

50

713

(d)1 2 3 4 5 6 7 8 9 10

(bp)

1000900800700

600

500

400

300

200

100

50

561

(e)

1 2 3 4 5 6 7 8 9 10

(bp)

1000900800700

600

500

400

300

200

100

50

326

(f)

Figure 2 Presence and absence of 120573-lactamase genes by PCR amplification in some isolated samples (a)The existence of VIM amplificationfragment (390 bp) (b) The amplification of TEM fragments (800 bp) (c) The amplification of CTX-M-1 (688 bp) (d) The presence of SHV(713 bp) in some samples (e) The presence of CTX-M-9 (561 bp) (f) No amplification was shown with CTX-M-825 primer at 326 bp

different common SNPs between the different strains Thesepositions were indicated in 246 284 306 309 330 363 378382 and 383 (Figure 3(e)) The phylogenetic tree of VIMsequence of A baumanii isolated from ZU Hospitals andpublished ones in GenBank showed dissimilarity betweenEgyptian strains and others (Figure 3(f))

342 Sequence Analysis of TEM Gene (119887119897119886VIM12) Sequencesof the purified product of VIM gene (119887119897119886VIM12) were com-paredwith homologous counterpartGenBank database using

BLASTprogramand resulted in significant similarity tomanymetallo-120573-lactamases genes of different bacterial strains

(1) TEM Gene (blaTEM12) of Escherichia coli The TEM genesequences of E coli strains isolated from ZU Hospitalswere aligned with sequences of published TEM genes in Ecoli strains from GenBank (eg E coli KM5986651) Theresulting alignments showed 4 different common SNP sitesbetween the different strains The SNPs positions were indi-cated commonly in 216 232 385 and 433 (Figure 4(a)) Phy-logenetic tree was constructed from TEM gene (119887119897119886TEM12)


AM_372945-22_2-group16-_Vim-group16-_H031-316gi1-390

Consensus

152227

264339


Consensus

265340

316390

(a)

AM 372945-22-2-group16-Vim-group16-H03 Ecoli Egypt0000

0000

0000

117376

117339

0037

20

KC4173771

gi Escherichia coli (blaVIM-1) EF0786971 Greece

gi Escherichia coli (blaVIM-1) GU7248711 France

gi Escherichia coli (blaVIM-2) gene KF8566171 Spaingi Escherichia coli blaVIM-1 gene AY7814131 Greece

(b)1111111111

121121121121121121121121121121

361361361361361361361361361361

241241241241241241241241241241

120120120120120120120120120120

240240240240240240240240240240

390390390390390390390390390390

360360360360360360360360360360

Consensus

Consensus

Consensus

Consensus

gi1-390Contig 11-390

gi1-390gi|695214489|ref|NG_0368121|1-390gi|257043832|gb|GQ4228271|1-390gi|257043832|gb|GQ4228271|1-390

gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390

(c)

gi Klebsiella pneumoniae VIM-2 genes |DQ1532171| Koreagi| Klebsiella pneumoniae strain (VIM-1) |GQ4228371| USA

gi Klebsiella pneumoniae plasmid|NG 0362531| USAAM 372945-24 4-group16-Vim-group16-H03 Kpneumonia Egypt

000

0011

00040002

000900090009

DQ1439131

0008 0006 0004 0002 00000010Relative times

gi Klebsiella pneumoniae strain (blaVIM-1) |GQ4228271| USA

(d)

Figure 3 Continued


168168184168168243243

289289305289289364364

288288304288288363363

317315331315315390390

Consensus

Consensus

Contig 11-317

gi|336171074|gb|JF7029191|1-315gi|336171074|gb|JF7029191|1-315gi|151564628|gb|EF6906961|1-390gi|151564622|gb|EF6906951|1-390

Contig 11-317


gi|336171078|gb|JF7029211|1-331gi|336171074|gb|JF7029191|1-315

gi|336171078|gb|JF7029211|1-331gi|336171074|gb|JF7029191|1-315

(e)

gi Acinetobacter baumannii VIM-1 |EF6906951| Greece

AM 372945-23 3-group16-Vim-group16-G03 Abaumanii Egypt0556

0009

0000

00000011

0542

0006

01

JN7005202

gi A baumannii class I integron (blaVIM-1) |EF6906961| Greece

gi Acinetobacter baumannii strain VIM (blaVIM) |JF7029211| India

gi Acinetobacter baumannii strain (blaVIM) |JF7029191| India

(f)

Figure 3Themultiple sequences alignments and trees of VIM gene in different species (a) VIM gene in E coli strains isolated from ZagazigUniversity (ZU) Hospitals in comparison with published VIM gene of E coli strains from GenBank (b) A tree of VIM sequence of E coliisolated from ZU Hospitals and published homologs in GenBank (c) The multiple sequences alignments of VIM gene in K pneumoniaestrains isolated from ZUHospitals in comparison with published VIM gene inK pneumoniae strains fromGenBank (d) Phylogenetic tree ofVIM sequence of K pneumoniae isolated from ZUHospitals and published sequences in GenBank (e)Themultiple sequences alignments ofVIM gene in A baumanii strains isolated from ZUHospitals in comparison with published VIM gene in A baumanii strains from GenBank(f) Phylogenetic tree of VIM sequence of A baumanii isolated from ZU Hospitals and published sequences in GenBank

sequence of Escherichia coli strains isolated from ZU Hos-pitals and published homologous sequences in GenBank(Figure 4(b)) showing similarity degree between Egyptianand Indian strains

(2) TEM Gene (blaTEM12) of Klebsiella pneumonia Multiplesequences alignments of TEM gene inK pneumoniae strainsisolated from ZU Hospitals were compared with other pub-lished TEM genes in K pneumoniae strains from GenBank(eg K pneumoniae KF2683571) (Figure 4(c)) showed 2different common SNPs between different strains The SNPspositions were indicated in 174 and 343 A phylogenetictree of TEM sequence of K pneumoniae isolated fromZagazig University Hospitals and other published ones inGenBank showed the degree of similarity between strainswhere Egyptian strain was dissimilar to that of Iranian andIndian strains (Figure 4(d))

343 The Analysis of SHV Gene (119887119897119886SHV1) in Klebsiellapneumoniae Strains The pairwise sequences alignments ofresulting SHV gene (119887119897119886SHV1) in K pneumoniae strains iso-lated from Zagazig University Hospitals in comparison withpublished SHV gene inK pneumoniae strains fromGenBankusing BLAST program (eg K pneumoniae AF1249841)

showed five common SNPs between the different strainsTheSNPs position was indicated in 454 563 631 635 and 650in Egyptian strains (Figure 5(a)) The phylogenetic tree ofSHV gene in this case showed that Egyptian strain was moresimilar to France strain (Figure 5(b))

344The Analysis of CTX-M-1 Gene (119887119897119886CTX-M-1) Sequencesof the purified product of CTX-M-1 gene (119887119897119886CTX-M-1) werecompared with homologous counterpart GenBank databaseusing BLAST program and resulted in significant similarityto many metallo-120573-lactamases genes of different bacterialstrains

(1) CTX-M-1 Gene (blaCTX-M-1) of Escherichia coli Strains Thesequence of the purified product of CTX-M-1 gene fromEscherichia coli strains was compared with the GenBanksequence using BLASTprogram Interestingly it was revealedthat there were strains present in GenBank which are com-pletely different from the studied strains and this is notmatchingwith our study (egPseudomonas aeruginosaDNAAP0146461) The sequence alignment showed 81 differentcommon SNPs between the different strains The SNPspositions were indicated in 601 604rarr 615 617rarr 626 and628rarr 688 (Figure 6(a)) The phylogenetic tree of CTX-M-1


91115115115

205229229229

319343343343

433457457457

547571571571

204228228228

318342342342

432456456456

546570570570

660684684684

Consensus

Consensus

Consensus

Consensus

Consensus

AM_372945-25_5-group16-_T-group16-_A041-779

gi|347810717|gb|JN1883661|1-800gi|347810716|gb|JN1883651|1-800gi|675594620|gb|KJ9230021|1-800

AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


(a)

TEM Ecoli Egypt

00041

0002700000

00014

00005

KM5986651gi|675594620|gb|KJ9230021| Escherichia coli strain blaTEM (blaTEM-1) India

gi|383282208|gb|JQ8231751| Escherichia beta-lactamase TEM (blaTEM) India

gi|347810716|gb|JN1883651| Escherichia coli strain ARY 4 blaTEM gene India

(b)115102111111111111111

229216225225225225225

343330339339339339339

228215224224

342329338338338338338

456443452452452452452

224224224

AM_372945-26_6-group16-_T-group16-_B041-751

gi|291603834|gb|GU7346961|1-525gi|291603832|gb|GU7346951|1-525gi|291603830|gb|GU7346941|1-525gi|291603822|gb|GU7346901|1-525gi|288190943|gb|GQ4704601|1-525

gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

Consensus

Consensus

Consensus

(c)

AM 372945-26 6-group16-T-group16-B04 Kpneumonia Egypt00022

00033

00000

00000

00000

00000

00000

0001500004

00015

00005

00005

KF2683571gi Klebsiella pneumoniae strain (blaTem1) gene partial cds |GU7346951| Iran

gi Klebsiella pneumoniae beta-lactamase TEM-1 (blaTem) |GQ4704601| Irangi Klebsiella pneumoniae strain (blaTEM1) gene partial cds |GU7346961| Iran

gi Klebsiella pneumoniae strain TEM-1 (blaTem) gene |GU7346941| Iran

gi Klebsiella pneumoniae strain (blaTem1) gene partial cds |GU7346901| Iran

gi Klebsiella pneumoniae strain (blaTEM1) gene partial cds |JN0433841| India

(d)

Figure 4 The multiple sequences alignments and trees of TEM gene (119887119897119886TEM12) in different bacterial species (a) TEM gene in E coli strainsisolated fromZUHospitals in comparisonwith publishedTEMgene ofE coli strains fromGenBank (b)Aphylogenetic tree of TEMgene ofEcoli isolated fromZUHospitals and published homologous ones inGenBank (c)Themultiple sequences alignments of gene inK pneumoniaestrains isolated from ZU Hospitals in comparison with published TEM gene in K pneumoniae strains from GenBank (d) Phylogenetic treeof sequence of K pneumoniae isolated from ZU Hospitals and published homologous ones in GenBank


340

340340340340

340340

453

453453453453

453453

566

566566566566

566566

324

324324

211

211211

98

9898

452

452452452452

452452

210

210210

565

565565565

565565

565

323

323323

678

678678678678

678678

436

436436

Consensus

Consensus

Consensus

AM_372945-30_13-group16-_T-group16-_A041-713gi|295414017|gb|HM0605401|1-470

gi|295414013|gb|HM0605381|1-470gi|313766531|gb|HM7511021|1-713gi|86278447|gb|DQ3557841|1-713

gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

(a)

gi Klebsiella pneumoniae conjugative plasmid beta-lactamase SHV-13 (bla) gene |AF1645771| UK

AM_372945-30 13-group16-T-group16-A04 Ecloacae Egypt

gi Klebsiella pneumoniae beta-lactamase SHV-14 (bla) gene |AF2266221| UK

00000

00022

00087

00022

00000

00000

000000004300000

0002200022

00022

00022

00000

0001

AF1249841

gi Klebsiella pneumoniae extended spectrum beta-lactamase SHV-1 (blaSHV) gene |HM0605401| Iran

gi Klebsiella pneumoniae strain K16R SHV-1 beta-lactamase (blaSHV) gene |HM7511021| Brazil

gi Klebsiella pneumoniae strain extended spectrum beta-lactamase SHV-11 (blaSHV) |GQ4704281| Iran

gi Klebsiella pneumoniae strain extended spectrum beta-lactamase SHV-1 (blaSHV) |HM0605381| Iran

gi Klebsiella pneumoniae plasmid blaSHV gene for beta-lactamase SHV |AB6757231| Japan

gi Klebsiella pneumoniae blaSHV type 1 gene complete cds|DQ3557841| Brazil

gi Klebsiella pneumoniae blaSHV gene for beta-lactamase class A |Y110691| France

(b)

Figure 5Themultiple sequences alignments and trees of SHV gene in different species (a) SHV gene inK pneumoniae strains isolated fromZUHospitals in comparison with published SHV gene of K pneumoniae strains from GenBank (b) Phylogenetic tree of SHV sequence of Kpneumoniae isolated from ZU Hospitals and published homologous ones in GenBank

sequence of E coli isolated fromZagazig University Hospitalsand published homologous ones in GenBank showed dissim-ilarity degree between Egyptian and other universal strains(Figure 6(b))

(2) CTX-M-1 Gene (blaCTX-M-1) of Klebsiella pneumoniaeStrains The alignment in this case showed 39 differentcommon SNPs between the different strains The SNPspositions were indicated in positions 1 rarr 39 (Figure 6(c))A phylogenetic tree of CTX-M-1 gene sequence in K pneu-moniae strains isolated from ZU Hospitals and publishedhomologous sequences in GenBank showed different degrees

of dissimilarity between the different strains with manyunique sequences in Egyptian strain (Figure 6(d))

(3) CTX-M-1 Gene (blaCTX-M-1) of Pseudomonas aeruginosaStrainsTheCTX-M-1 gene isolated fromP aeruginosa strainsin Zagazig University Hospitals was compared using BLASTprogram with published CTX-M-1 gene in P aeruginosastrains from GenBank (eg Paeruginosa KC5712551) Theresults showed 4 different common SNPs between the differ-ent strainsThe SNPs positions were indicated in 117 206 228and 283 (Figure 6(e)) A phylogenetic tree showed similaritydegree between Egyptian and Russian strains (Figure 6(f))


601601601601

629688688688

Consensus

CTX11-629gi|695214033|ref|NG_0367291|1-688gi|224696954|emb|FM2133711|1-688gi|402795953|dbj|AB7015731|1-688

(a)

CTX1 Ecoli Egypt05168

00000

00000

0000005168

01

AP0146461

gi |NG 0367291| Escherichia coli blaCTX-M-3 gene for beta-lactamase USA

gi Escherichia coli DNA ISEcp1-blaCTX-M-15 |AB7015731| Japan

gi Escherichia coli blaCTX-M-3 gene for beta-lactamase |FM2133711| Poland

(b)227

2911

340142

3391417474

452254187187

7575

Consensus

Consensus

gi|288190909|gb|GQ4704431|1-496gi|288190907|gb|GQ4704421|1-429gi|288190905|gb|GQ4704411|1-429

AM_372945-30_15-group16-_g1-group16-_F041-497


AM_372945-30_15-group16-_g1-group16-_F041-497

(c)

gi Klebsiella pneumoniae strain 285L extended spectrum beta-lactamase CTX-M-15 |GQ4704351|Iran

CTX1 Klebsiella pneumoniae Egyptgi Klebsiella pneumoniae strain 212L extended spectrum beta-lactamase CTX-M-15 |GQ4704311| Irangi Klebsiella pneumoniae CTX-M-15|GQ8655681| India

00000

0000000000

07585

07515

00070

02

KJ4514101

gi Klebsiella pneumoniae (blaCTX-M-15) gene |EU5567551|Hungary

(d)56

113113

167224224

279336336

391448448

168225225

280337337

Consensus

Consensus

Consensus

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

(e)

gi|584111314|gb|KF8761331| Pseudomonas aeruginosa strain B-757P Russia

gi|584111314|gb|KF8761331| Pseudomonas aeruginosa strain B-757P Russia(2)CTX1 Pseudomonas aeruginosa Egypt00024

00000

0000000024

00005

KC5712551

(f)

Figure 6 The multiple sequences alignments and trees of CTX-M-1 gene in different species (a) CTX-M-1 gene in E coli strains isolatedfrom ZU Hospitals in comparison with published CTX-M-1 gene of E coli strains from GenBank (b) A tree of CTX-M-1 sequence of E coliisolated from ZUHospitals and published homologs in GenBank (c)Themultiple sequences alignments of CTX-M-1 gene in K pneumoniaestrains isolated from ZU Hospitals in comparison with published CTX-M-1 gene in K pneumoniae strains from GenBank (d) Phylogenetictree of CTX-M-1 sequence of K pneumoniae isolated from ZU Hospitals and published homologous sequences in GenBank (e) Multiplesequences alignments of CTX-M-1 gene in P aeruginosa strains isolated from ZU Hospitals in comparison with published CTX-M-1 gene inP aeruginosa strains from GenBank (f) Phylogenetic tree of CTX-M-1 sequence of P aeruginosa isolated from ZU Hospitals and publishedhomologs in GenBank


11111

113113113113113

225225225225225

Consensus

Consensus

Consensus

112112112112112

224224224224224

336336336336336

gi|221149276|gb|EU4189151|1-561gi|295917909|gb|GU7328351|1-561gi|668347703|dbj|AB9765961|1-561gi|299483205|gb|HM5697351|1-561

AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552

(a)

00000

00000

00037

00037

00005

JN6768431

AM 372945-35 21-group16- g9-group16- C05 (830bp) Ecoli Egypt

gi Escherichia coli (blaCTX-M-9b) gene complete cds |EU4189151|Australia

gi Escherichia coli strain K-340 (blaCTX-M-9) gene complete cds |HM5697351|Russia

gi Escherichia coli DNA blaCTX-M-14 gene |AB9765961|Japan

(b)

Figure 7Themultiple sequences alignments and tree of CTX-M-9 gene in different species (a) CTX-M-9 gene in E coli strains isolated fromZU Hospitals in comparison with published CTX-M-9 gene of E coli strains from GenBank (b) Phylogenetic tree of CTX-M-9 sequence ofE coli isolated from ZU Hospitals and published homologous ones in GenBank

345 The Analysis of CTX-M-9 Gene (119887119897119886CTX-M-9) inEscherichia coli Strains The CTX-M-9 gene isolated fromE coli strains in Zagazig University Hospitals was comparedwith published CTX-M-1 gene in P aeruginosa strains fromGenBank using BLAST program (eg E coli JN6768431)The multiple sequences alignments showed 2 differentcommon SNPs in positions 74 and 272 (Figure 7(a))The phylogenetic tree of CTX-M-9 gene sequence in Ecoli strains isolated from ZU Hospitals and publishedhomologous sequences in GenBank showed differentdegrees of dissimilarity between the different strains andmany unique sequences in the Egyptian strain similar tothat of Russia and Australia and dissimilar to that of Japan(Figure 7(b))

4 Discussion

The Gram-negative bacilli are among the most importantcauses of serious nosocomial and community-onset bacte-rial infections in humans and antimicrobial resistance hasbecome a global threat to effective health care delivery [14]However carbapenem-resistant Gram-negative bacilli havebeen increasingly reported worldwide [4] Various acquiredcarbapenemases have been identified in the last years belong-ing to either acquired metallo-beta-lactamases (IMP VIM

Table 2 The distribution of Gram-negative bacilli isolates in eachclinical specimen

Clinical specimens Gram-negative bacilli isolatesType Number Number ()Urine 52 41 788Blood 18 12 666Sputum 29 10 345Pus 9 2 222Total 108 65 (602)1205942= 2128 119875 lt 0001 (statistically significant)

SPM GIM NDM andDIM types) or class A (KPC andGES)and class D 120573-lactamase OXA-48 [15]

In the present study prevalence of 120573-lactamases-producing isolates was found in Table 2 Different studiescarried out by other workers in various parts of the worldshow quite variable results In a study carried out thefrequency of beta-lactamases-producing isolates was urine(61) followed by blood cultures (38) wound swabs (13)and tracheal aspirates (5) (119875 lt 0001) [16] And this issimilar to our study By contrast Shanthi and Sekar [17] inIndia reported that Gram-negative isolates were obtainedfrom the respiratory tract (418) followed by urinary tract


Table 3 API 20E identification of Gram-negative bacilli isolatesamong positive clinical specimens

Organism Total = 65Number ()

Escherichia coli 30 461Klebsiella pneumoniae 17 262Enterobacter cloacae 4 61Pseudomonas aeruginosa 7 107Proteus mirabilis 2 307Acinetobacter baumanii 3 46Citrobacter freundii 1 15Proteus vulgaris 1 15119875 lt 0001 (statistically significant)

(255) wound (20) and blood (127) Also pus wasthe most common specimen accounting for 21 followedby tracheal aspirate (17) sputum (16) urine (11) andblood (7) [18]

Various risk factors of 120573-lactamases have been implicatedin selection and spread producing strains from variousclinical samples

In accordance with distribution of Gram-negative bacilli(461) Escherichia coli and (262) Klebsiella pneumoniaeisolates followed by (107) P aeruginosa were identifiedas the commonest isolates among Gram-negative bacilli(Table 3) and this coincidedwith that concluded by Sahu et al[19]who found that 58were identified asEscherichia coli fol-lowed by 277 Klebsiella pneumoniae and 15 Pseudomonasaeruginosa in Udaipur Rajasthan In a study by Vipin etal [20] 52 (5842) isolates of Escherichia coli were foundto be the most common organisms in Allahabad followedby Klebsiella pneumoniae (2022) Pseudomonas aerugi-nosa (1235) Proteus vulgaris (337) Proteus mirabilis(224) and Enterobacter cloacae (224) Also in IndiaSankarankutty and Kaup [21] documented the same resultin that 5842 isolates of Escherichia coli were found to bethe most common organisms followed by Klebsiella pneu-moniae (2022) Pseudomonas aeruginosa (1235) Proteusvulgaris (337) Proteus mirabilis (224) and Enterobacteraerogenes (224)

But our study disagreed with that published by Aboderinet al [22] who reported that Pseudomonas aeruginosarecorded the highest prevalence followed by Klebsiella pneu-moniae and ESBL producers whereas frequency among Ecoli isolates was much lower than Klebsiella pneumoniaeHence prevalence of pathogens often varies dramaticallybetween communities according to geography hospitals inthe same community and among different patient popula-tions in the same hospital

In this study risk factors associated with isolation ofGram-negative bacilli isolates were shown in Table 4 Andthis was matched with that reported by Kumar et al [23] whoexhibitedmajor risk factors such as prolonged hospitalizationgt 8 days previous antibiotic use trauma and mechanicalventilation which may contribute to the mortality

In Turkey Aktas et al [24] reported risk factors foracquisition including prolonged hospitalization an ICU stayventilator usage previous use of carbapenem antibiotics andthe presence of underlying diseases and this is compatiblewith our research But in Brazil Tuon et al [25] documentedthat there was statistical significance in isolation of Klebsiellapneumoniae isolates according to age (119875 = 0005) andmechanical ventilation (119875 = 0003) while trauma (119875 = 087)and ICU stay (119875 = 025) had a statistical significance asmajorrisk factors

The importance of these risk factors lies in the epidemi-ological implications at the hospital level because the resultssuggest a probable nosocomial transmission of the infection

Resistance pattern among nosocomial bacterialpathogens may vary widely from country to country atany time and within the same country over time [26]

In our study all the isolates were resistant to cefotaxime(100) and displayed unusually high level of imipenem-resistance (508) isolates with MICs ranging from 156 to250 120583gmL (Table 5) (119875 lt 0001) In Egypt this was parallelto that reported by Mohamed and Raafat [27] who reported(522) imipenem-resistance among isolates (100) cefo-taxime resistance (55) susceptible-ciprofloxacin and (70)susceptibility to amikacin In theMiddle East the occurrenceof imipenem-resistant Gram-negative bacilli is alarminglyelevated Another similar study showed that all 84 Klebsiellapneumoniae isolates exhibited resistance to imipenem withMICs ranging from 4 to gt32 120583gmL in a Greek hospital [28]

Another dissimilar study was shown in Saudi Arabiawhere the susceptibility rate of Gram-negative organismsisolated from a tertiary care hospital to imipenem wasreported to be as low as 10 [29] Galani et al [30] reportedthat Klebsiella pneumoniae and Escherichia coli isolates werefound to be susceptible to imipenem in routine suscep-tibility disk diffusion tests Other authors observed thatall beta-lactamases producers of Escherichia coli Klebsiellapneumoniae and Pseudomonas aeruginosa were suscepti-ble to imipenem showing coresistance to other antibioticsof aminoglycosides fluoroquinolones and others [17] Theextensive use of carbapenems in some locations has likelycreated a selective antibiotic pressure which in turn hasresulted in an increased prevalence of carbapenem-resistantGram-negative isolates

The carbapenem resistance due to production of 120573-lactamases has a potential for rapid dissemination since it isoften plasmid-mediated [2] Consequently rapid detection of120573-lactamases is necessary to initiate effective infection con-trol measures to prevent their uncontrolled spread in clinicalsettings In Egyptian hospitals the 120573-lactamases presence wasconfirmed by PCR amplification

In our study the percentage of 119887119897119886VIM 119887119897119886TEM 119887119897119886SHVand 119887119897119886CTX-M genes among Gram-negative bacilli isolateswas shown in Table 6 As regards 119887119897119886VIM gene detectedin our results it is similar to that 119887119897119886VIM gene encodingMBL among the isolates of P aeruginosa (613) in Tehranhospitals [31] On the contrary 119887119897119886VIM genes were notdetected among the studied Gram-negative isolates in otherTehran hospitals [32] TEM SHV and CTX-M genes are themost common plasmid-mediated lactamases often found in


Table 4 Risk factors associated with isolation of Gram-negative bacilli isolates

Risk factor Gram-negativebacilli isolates

Total number ofsamples Relative risk (95 CI)lowast 119875 value significance

Agelt40 26 45 107 (078ndash147) 066gt40 39 63

SexMale 29 49 109 (08ndash147) 059Female 36 59

TraumaYes 48 59 234 (157ndash351) lt0001No 17 49

Hospitalization length other than ICUlt7 days 15 51 298 (133ndash461) lt0001gt7 days 50 57

ICU admissionYes 28 46 102 (045ndash247) lt0001No 37 62

Urinary catheterYes 18 32 091 (064ndash129) 058No 47 76

Ventilator supportYes 10 18 083 (051ndash135) 044No 20 30

Central venous catheterYes 4 9 067 (031ndash143) 026No 26 39lowast = 95 confidence interval

Table 5 Antimicrobial susceptibility patterns by disc diffusion method

AntibioticAntimicrobial susceptibility patterns of all Gram-negative bacilli isolates

Susceptible Intermediate ResistantNumber () Number () Number ()

Imipenem 31 477 1 15 33 508Amikacin 40 lowast615 1 15 24 446Ciprofloxacin 35 538 3 46 27 415Piperacillin 15 231 6 lowast92 44 677Cefoperazonesulbactam 21 323 4 61 40 615Cefoxitin 12 184 0 000 53 815Cefotaxime 0 000 0 000 65 lowast10000lowast119875 lt 0001 (statistically significant)

Enterobacteriaceae and P aeruginosa [33] In Iran Eftekharet al [34] showed 693 119887119897119886TEM and 3137 119887119897119886CTX-M-1 genesamongGram-negative isolates and this agreedwith our studyAlso presence of 119887119897119886CTX-M19 119886SHV and 119887119897119886TEM genes amongtested rods of the Enterobacteriaceae family was revealed byOjdana et al [35] Another study reported by Cuzon et al[36] harbored similar results regarding carbapenem-resistantGram-negative isolates carrying TEM SHV CTX-M-1 andCTX-M-9 genes

In Switzerland Zurfluh et al [37] mentioned that theisolates of Enterobacteriaceae were further screened for blagenes encoding SHV TEM CTX-M group 1 CTX-M group2 or CTX-M group 9 enzymes with (23) (713) (392)(201) and (156) respectively

Other authors noted different observations in that clinicalisolates of Escherichia coli and Pseudomonas aeruginosacarried 119887119897119886CTX-M-types which was the most common (958)followed by 119887119897119886TEM (292) 119887119897119886SHV (73) and 119887119897119886VIM


Table 6 The percentage of blaVIM blaTEM blaSHV and blaCTX-M genes among Gram-negative bacilli isolates

Isolates 120573-lactamases genes

Organism Number VIM TEM SHV CTX-M-1 CTX-M-9 CTX-M-825Number () Number () Number () Number () Number () Number ()

Escherichia coli 15 10 666 13 870 0 00 3 200 4 266 0 000Klebsiella pneumonia 10 8 800 9 900 5 50 6 600 1 100 0 000Enterobacter cloaca 2 1 500 1 500 0 00 0 000 0 000 0 000Pseudomonas aeruginosa 4 2 500 1 250 0 00 3 750 1 250 0 000Proteus mirabilis 1 0 000 0 000 0 00 0 000 0 000 0 000Acinetobacter baumanii 1 1 100 0 000 0 00 0 000 0 000 0 000Total 33 22 667 24 727 5 150 12 360 6 182 0 000

(125) in Nepal [38] These observations contribute to theknowledge of the epidemiology of VIM TEM SHV andCTX-M-producing Gram-negative isolates that have nowbecome endemic in major hospitals in Egypt Continuousmonitoring proper infection control and surveillance andprevention practices will limit the further spread of theseinfections within these hospitals and clinical settings

Multiple sequences analysis is used in such biologicalstudies to extract important phylogenetic and evolutionaryinformation using different scoringmatrices (BLOSUM62 forBLAST BLOSUM50 for SEARCH and FASTA) [39]

5 Conclusion

There is a high prevalence of120573-lactamase genes in our clinicalisolates that are responsible for such resistance Hence itis essential to report 120573-lactamases production along withroutine sensitivity reports which will help the clinician inprescribing proper antibiotics Also the sequence analysis ofamplified genes showed differences between multiple SNPsin the same gene among different local isolates and withinternationally published sequences In the end it has beenfelt that there is a need to formulate strategies to detect andprevent the emergence of 120573-lactamases producing strains forthe effective treatment of infections which are caused bythem

Conflict of Interests

The authors have declared no conflict of interests and theyhave no financial conflicts

Acknowledgments

The authors would like to express their sincere thanksand deepest gratitude to Professor Dr Ahmed Mansour AlZohairy Assistant Professor of genetics Faculty of Agri-culture Zagazig University for his help in achieving andplanning bioinformatics of sequences

References

[1] J Breslow ldquoIllinois lsquoNightmare Bacteriarsquo Outbreak RaisesAlarmsrdquo April 2014 httpwwwpbsorg

[2] HM ZowawiHH Balkhy T RWalsh andD L Paterson ldquo120573-Lactamase production in key gram-negative pathogen isolatesfrom theArabian PeninsulardquoClinicalMicrobiology Reviews vol26 no 3 pp 361ndash380 2013

[3] J N Kattan M V Villegas and J P Quinn ldquoNew developmentsin carbapenemsrdquoClinicalMicrobiology and Infection vol 14 no12 pp 1102ndash1111 2008

[4] K M Papp-Wallace A Endimiani M A Taracila and R ABonomo ldquoCarbapenems past present and futurerdquo Antimicro-bial Agents and Chemotherapy vol 55 no 11 pp 4943ndash49602011

[5] H Lee K S Ko J-H Song and K R Peck ldquoAntimicrobialactivity of doripenem and other carbapenems against gram-negative pathogens fromKoreardquoMicrobial Drug Resistance vol17 no 1 pp 37ndash45 2011

[6] M A Toleman and T R Walsh ldquoEvolution of the ISCR3 groupof ISCR elementsrdquoAntimicrobial Agents and Chemotherapy vol52 no 10 pp 3789ndash3791 2008

[7] S A Jemima and S Verghese ldquoMultiplex PCR for 119887119897119886CTX-Mand 119887119897119886SHV in the extended spectrum-B-lactamase (ESBL) pro-ducing gram-negative isolatesrdquo The Indian Journal of MedicalResearch vol 128 no 3 pp 313ndash317 2008

[8] R C Picao L Poirel A C Gales and P Nordmann ldquoDiver-sity of 120573-lactamases produced by ceftazidime-resistant Pseu-domonas aeruginosa isolates causing bloodstream infections inBrazilrdquo Antimicrobial Agents and Chemotherapy vol 53 no 9pp 3908ndash3913 2009

[9] E SMoland J A Black J OuradaMD ReisbigNDHansonandK SThomson ldquoOccurrence of newer120573-lactamases inKleb-siella pneumoniae isolates from24USHospitalsrdquoAntimicrobialAgents and Chemotherapy vol 46 no 12 pp 3837ndash3842 2002

[10] L K Dianna and H G Peter ldquoPseudomonasrdquo in Manual ofClinical Microbiology R M Patrick J B Ellen H J James A PMiccael and H Y Robert Eds vol 1 pp 719ndash728 ASM PressWashington DC USA 8th edition 2003

[11] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing Twenty-SecondInformational Supplement CLSI M100-S22 Clinical and Labo-ratory Standards Institute Wayne Pa USA 2012

[12] C Dallenne A da Costa D Decre C Favier and G ArletldquoDevelopment of a set of multiplex PCR assays for the detection


of genes encoding important 120573-lactamases in Enterobacteri-aceaerdquo Journal of Antimicrobial Chemotherapy vol 65 no 3Article ID dkp498 pp 490ndash495 2010

[13] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[14] J D D Pitout ldquoMultiresistant enterobacteriaceae new threat ofan old problemrdquo Expert Review of Anti-InfectiveTherapy vol 6no 5 pp 657ndash669 2008

[15] J Manuel R Martinez P Nordmann N Fortineau and LPoirel ldquoVIM-19 a metallo-120573-lactamase with increased car-bapenemase activity from Escherichia coli and Klebsiella pneu-moniaerdquo Antimicrobial Agents and Chemotherapy vol 54 no 1pp 471ndash476 2010

[16] J-M Rodriguez-Martinez P Nordmann N Fortineau andL Poirel ldquoVIM-19 a metallo-120573-lactamase with increased car-bapenemase activity from Escherichia coli and Klebsiella pneu-moniaerdquo Antimicrobial Agents and Chemotherapy vol 54 no 1pp 471ndash476 2010

[17] M Shanthi and U Sekar ldquoExtended spectrum beta lactamaseproducing Escherichia coli and Klebsiella pneumoniae riskfactors for infection and impact of resistance on outcomesrdquoJournal of the Association of Physicians of India vol 58 no 1pp 41ndash44 2010

[18] K K BenachinmardiM Padmavathy JMalini and B V Nave-neeth ldquoPrevalence of non-fermenting Gram-negative bacilliand their in vitro susceptibility pattern at a tertiary care teachinghospitalrdquo Journal of the Scientific Society vol 41 no 3 pp 162ndash166 2014

[19] S K Sahu A S Dalal and G Bansal ldquoDetection of extended-spectrum 120573-lactamases in clinical isolates of E coli and Kleb-siella species fromUdaipurRajasthanrdquoBiomedical Research vol22 no 3 pp 367ndash373 2011

[20] K Vipin K M Rohit C Avantika and G Pramila ldquoIncidenceof 120573-lactamase producing gram-negative clinical isolates andtheir antibiotic susceptibility pattern a case study inAllahabadrdquoInternational Journal of Research in Pure and Applied Microbiol-ogy vol 1 no 3 pp 36ndash39 2011

[21] J Sankarankutty and S Kaup ldquoMicrobiological profile andantibiogram of uropathogens from a tertiary care centre inTumkur Indiardquo Journal of Microbiology and BiotechnologyResearch vol 4 no 2 pp 46ndash51 2014

[22] O A Aboderin L-R Abdu BWOdetoyin and A LamikanraldquoAntimicrobial resistance in Escherichia coli strains from uri-nary tract infectionsrdquo Journal of the National Medical Associa-tion vol 101 no 12 pp 1268ndash1273 2009

[23] S H Kumar A S De SM Baveja andMA Gore ldquoPrevalenceand risk factors ofMetallo120573-lactamase producingPseudomonasaeruginosa and Acinetobacter species in burns and surgicalwards in a tertiary care hospitalrdquo Journal of Laboratory Physi-cians vol 4 no 1 pp 39ndash42 2012

[24] Z Aktas D Satana C Kayacan et al ldquoCarbapenem resistancein Turkey repeat report on OXA-48 in Klebsiella pneumoniaeand first report on IMP-1 beta-lactamase in Escherichia colirdquoAfrican Journal ofMicrobiology Research vol 6 no 17 pp 3874ndash3878 2012

[25] F F Tuon J L Rocha P Toledo et al ldquoRisk factors for KPC-producingKlebsiella pneumoniae bacteremiardquo Brazilian Journalof Infectious Diseases vol 16 no 5 pp 416ndash419 2012

[26] K Prashanth and S Badrinath ldquoIn vitro susceptibility patternof Acinetobacter species to commonly used cephalosporins

quinolones and aminoglycosidesrdquo Indian Journal of MedicalMicrobiology vol 22 no 2 pp 97ndash103 2004

[27] N M Mohamed and D Raafat ldquoPhenotypic and genotypicdetection of metallo-beta-lactamases in imipenem-resistantAcinetobacter baumannii isolated from a tertiary hospital inAlexandria Egyptrdquo Research Journal of Microbiology vol 6 no10 pp 750ndash760 2011

[28] A Poulou E Voulgari G Vrioni et al ldquoImported Klebsiellapneumoniae carbapenemase-producing K pneumoniae clonesin a Greek hospital Impact of infection control measures forrestraining their disseminationrdquo Journal of Clinical Microbiol-ogy vol 50 no 8 pp 2618ndash2623 2012

[29] S M Al Johani J Akhter H Balkhy A El-Saed M Younanand Z Memish ldquoPrevalence of antimicrobial resistance amonggram-negative isolates in an adult intensive care unit at a tertiarycare center in Saudi Arabiardquo Annals of Saudi Medicine vol 30no 5 pp 364ndash369 2010

[30] I Galani P D Rekatsina D Hatzaki D Plachouras M SouliandH Giamarellou ldquoEvaluation of different laboratory tests forthe detection of metallo-120573-lactamase production in Enterobac-teriaceaerdquo Journal of Antimicrobial Chemotherapy vol 61 no 3pp 548ndash553 2008

[31] F Shahcheraghi V S Nikbin and M M Feizabadi ldquoIdentifi-cation and genetic characterization of metallo-beta-lactamase-producing strains of Pseudomonas aeruginosa in Tehran IranrdquoNew Microbiologica vol 33 no 3 pp 243ndash248 2010

[32] F Shahcheraghi M Abbasalipour M M Feizabadi G HEbrahimipour and N Akbari ldquoIsolation and genetic character-ization of metallo-120573-lactamase and carbapenamase producingstrains of Acinetobacter baumannii from patients at tehranhospitalsrdquo Iranian Journal of Microbiology vol 3 no 2 pp 68ndash74 2011

[33] A A Shah F Hasan S Ahmed and A Hameed ldquoCharac-teristics epidemiology and clinical importance of emergingstrains of Gram-negative bacilli producing extended-spectrum120573-lactamasesrdquoResearch inMicrobiology vol 155 no 6 pp 409ndash421 2004

[34] F EftekharM RastegarM Golalipoor andNMansoursamaeildquoDetection of extended spectrum Β-lactamases in urinaryisolates of Klebsiella pneumoniae in relation to 119861119897119886SHV 119861119897119886TEMand 119861119897119886CTX-M gene carriagerdquo Iranian Journal of Public Healthvol 41 no 3 pp 127ndash132 2012

[35] D Ojdana P Sacha P Wieczorek et al ldquoThe occurrence of119887119897119886CTX-M 119887119897119886SHV and 119887119897119886TEM genes in extended-spectrum 120573-lactamase-positive strains ofKlebsiella pneumoniae Escherichiacoli and Proteus mirabilis in Polandrdquo International Journal ofAntibiotics vol 2014 Article ID 935842 7 pages 2014

[36] G Cuzon T Naas P Bogaerts Y Glupczynski and P Nord-mann ldquoEvaluation of a DNAmicroarray for the rapid detectionof extended-spectrum 120573-lactamases (TEM SHV and CTX-M)plasmid-mediated cephalosporinases (CMY-2-like DHA FOXACC-1 ACTMIR and CMY-1-likeMOX) and carbapenemases(KPCOXA-48VIM IMPandNDM)rdquo Journal of AntimicrobialChemotherapy vol 67 no 8 Article ID dks156 pp 1865ndash18692012

[37] K Zurfluh H HachlerM Nuesch-Inderbinen and R StephanldquoCharacteristics of extended-spectrum 120573-lactamase- andcarbapenemase-producing Enterobacteriaceae isolates fromrivers and lakes in Switzerlandrdquo Applied and EnvironmentalMicrobiology vol 79 no 9 pp 3021ndash3026 2013

[38] R H Pokhrel B Thapa R Kafle P K Shah and C Tribud-dharat ldquoCo-existence of beta-lactamases in clinical isolates of


Escherichia coli from Kathmandu Nepalrdquo BMC Research Notesvol 7 article 694 2014

[39] H Alsayed Gaber M A Ahmed B M Rania M Mohammedand M A Osama ldquoMathematical modeling and classificationof viruses from herpesvirus familyrdquo International Journal ofComputer Applications vol 87 no 12 pp 5ndash13 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

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Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


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Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Primer name primer sequences and expected amplicon size of amplified DNA products

PCR name 120573-lactamase targeted Primer name Sequence (51015840-31015840) Amplicon size (bp)

VIM VIM variants including VIM-1 andVIM-2

VIM-for GATGGTGTTTGGTCGCATA 390VIM-rev CGAATGCGCAGCACCAG

TEM TEM variants including TEM-1 andTEM-2

TSO-T-for CATTTCCGTGTCGCCCTTATTC 800TSO-T-rev CGTTCATCCATAGTTGCCTGAC

SHV SHV variants including SHV-1 TSO-S-for AGCCGCTTGAGCAAATTAAAC 713TSO-S-rev ATCCCGCAGATAAATCACCAC

CTX-Mgroup 1

Variants of CTX-M group 1 includingCTX-M-1 CTX-M-3 and CTX-M-15

CTXMGp 1-for TTAGGAARTGTGCCGCTGYAb688

CTXMGp 12 rev CGATATCGTTGGTGGTRCCATb

CTX-Mgroup 9

Variants of CTX-M-9 includingCTX-M-9 and CTX-M-14

CTX-9-F TCAAGCCTGCCGATCTGGT 561CTX-9-R TGATTCTCGCCGCTGAAG

CTX-Mgroup 825

CTX-M-8 CTX-M-25 CTX-M-26 andCTX-M-39 to CTX-M-41

CTX-825-F AACRCRCAGACGCTCTACb326

CTX-825-R TCGAGCCGGAASGTGTYATb

bY = T or C R = A or G S = G or C

some genes encoding beta-lactamase enzymes responsible forsuch resistance in Zagazig University Hospitals in Egypt

2 Material and Methods

21 Bacterial Isolates Clinical isolates of Gram-negativebacilli including Escherichia coli (119899 = 30) Klebsiella pneu-moniae (119899 = 17) Pseudomonas aeruginosa (119899 = 7) Proteusmirabilis (119899 = 2) Citrobacter freundii (119899 = 1) Acinetobacterbaumanii (119899 = 3) and Enterobacter cloacae (119899 = 4) werecollected fromblood urine pus and sputum specimens fromhospitalized patients in ZagazigUniversityHospitals in Egyptfrom January 2013 toMarch 2014These clinical samples wereprocessed by plating on blood agar and MacConkey agar[9] A growth temperature of 44∘C was used sometimes toconfirm the identity of these isolates and the identified strainswere stored in glycerol (20 VV) at 70∘C and subculturedseveral times to be viable All isolates were identified bystandard biochemical tests [10] and confirmed by API 20E(BioMerieux Marcy lrsquoEtoile France)

22 Antibiotic Susceptibility Testing The susceptibility testingof studied isolates was performed by disc diffusion method(modified Kirby-Bauer method) using Muller-Hinton agar(Becton Dickinson MA USA) and interpreted accordingto the Clinical Laboratory Standard Institute (CLSI) guide-lines [11] The antibiotic disks used imipenem (IPM 10 120583g)amikacin (AK 30 120583g) ciprofloxacin (CIP 5 120583g) piperacillin(PRL 100 120583g) cefoperazonesulbactam (CES 10 + 5 120583g)cefoxitin (FOX 30 120583g) and cefotaxime (CTX 30120583g) whichwere placed 15mm away from the central disc and the plateswere incubated for about 18ndash24 hrs at 37∘C

23 Molecular Detection of 120573-Lactamase Genes by PCR Fordetection of 120573-lactamase genes responsible for imipenem-resistance rapid genomic DNA was prepared from aboutfive colonies heated in 100mL distilled water (95∘C for10min) followed by a centrifugation step of cell suspension at

12000 rpm for 5min then supernatant was taken as a sourceof template DNA PCR amplification was carried out by usingDNA thermal cycler (Biometra Singapore) using a specificprimer for 119887119897119886VIM 119887119897119886TEM 119887119897119886SHV 119887119897119886CTX-M-1 119887119897119886CTX-M-9and 119887119897119886CTX-M-825 (Table 1) in a 50120583L volume containing 10xPCR buffer 2mM deoxynucleoside triphosphates 34 pmolof each primer 25mM MgCl

2 1 U Taq DNA polymerase

and 1 120583L of genomic DNA [12] Amplification was carriedout as follows initial denaturation at 94∘C for 10 minutesfollowed by 40 cycles of DNA denaturation at 94∘C for40 seconds primer annealing at 60∘C for 40 seconds andprimer extension at 72∘C for 1 minute and a final elongationstep at 72∘C for 7 minutes The annealing temperature wasoptimal at 55∘C instead of 60∘C for amplification of 119887119897119886VIMAmplicons were then visualized after running in 2 agarosegel at 100V for 30mins A 50ndash1000 bpDNA ladder (USA)wasused as a size marker Finally PCR products were purifiedwith innuPREP PCRpure kit (Analytik Jena Germany) andsubjected to direct sequencing via GATC Company by use ofABI 3730xl DNA sequencer

24 Bioinformatics and Sequences Analysis The obtainedchromatogram sequencing files were inspected and correctedusing the software application Chromas 23 (TechnelysiumHelensvale Australia) and JalView (28)

The sequences obtained from our samples were alignedwith GenBank sequences The phylogenetic tree for eachsequence was obtained by performing neighbor-joining anal-ysis of the alignment of sequences with reference strains(accession numberscountry of origin) that were retrievedfrom GenBank The studied strains were marked by the sign[◼] Meanwhile the reference sequences were marked by thesign [998771]

The BLAST and FASTA programs of the National Centerfor Biotechnology Information (httpblastncbinlmnihgovBlastcgi) were used to search databases for similarnucleotide sequences [13] Multiple sequence alignments ofthe nucleic acidwere carried out using theClustalWprogramThe statistical analysis was performed using SPSS version


477

615538

231323

184

015 15 46 92 610 0

508446 415

677615

815

100


0102030405060708090

100

()

Imip

enem

Am

ikac

in

Cipr

oflox

acin

Pipe

raci

llin

Cef

oxiti

n

Cef

otax

ime

Cef

oper

azon

e(s

ulba

ctam

)



3 Results











1 2 3 4 5 6 7 8 9 10

(bp)

1000900800

700

600

500

400

300

200

100

50

390

(a)

1 2 3 4 5 6 7 8 9 10

(bp)

1000900800

700

600

500

400

300

200

100

50

800

(b)1 2 3 4 5 6 7 8 9 10

(bp)

1000900800

700

600

500

400

300

200

100

50

688

(c)

1 2 3 4 5 6 7 8 9 10

(bp)

1000900800700

600

500

400

300

200

100

50

713

(d)1 2 3 4 5 6 7 8 9 10

(bp)

1000900800700

600

500

400

300

200

100

50

561

(e)

1 2 3 4 5 6 7 8 9 10

(bp)

1000900800700

600

500

400

300

200

100

50

326

(f)








Consensus

152227

264339


Consensus

265340

316390

(a)


0000

0000

117376

117339

0037

20

KC4173771




(b)1111111111

121121121121121121121121121121

361361361361361361361361361361

241241241241241241241241241241

120120120120120120120120120120

240240240240240240240240240240

390390390390390390390390390390

360360360360360360360360360360

Consensus

Consensus

Consensus

Consensus



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390

(c)



000

0011

00040002

000900090009

DQ1439131

0008 0006 0004 0002 00000010Relative times


(d)

Figure 3 Continued


168168184168168243243

289289305289289364364

288288304288288363363

317315331315315390390

Consensus

Consensus

Contig 11-317


Contig 11-317


gi|336171078|gb|JF7029211|1-331gi|336171074|gb|JF7029191|1-315

gi|336171078|gb|JF7029211|1-331gi|336171074|gb|JF7029191|1-315

(e)



0009

0000

00000011

0542

0006

01

JN7005202




(f)









91115115115

205229229229

319343343343

433457457457

547571571571

204228228228

318342342342

432456456456

546570570570

660684684684

Consensus

Consensus

Consensus

Consensus

Consensus

AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


(a)

TEM Ecoli Egypt

00041

0002700000

00014

00005




(b)115102111111111111111

229216225225225225225

343330339339339339339

228215224224

342329338338338338338

456443452452452452452

224224224

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

Consensus

Consensus

Consensus

(c)


00033

00000

00000

00000

00000

00000

0001500004

00015

00005

00005






(d)



340

340340340340

340340

453

453453453453

453453

566

566566566566

566566

324

324324

211

211211

98

9898

452

452452452452

452452

210

210210

565

565565565

565565

565

323

323323

678

678678678678

678678

436

436436

Consensus

Consensus

Consensus



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

(a)




00000

00022

00087

00022

00000

00000

000000004300000

0002200022

00022

00022

00000

0001

AF1249841








(b)







601601601601

629688688688

Consensus


(a)


00000

00000

0000005168

01

AP0146461




(b)227

2911

340142

3391417474

452254187187

7575

Consensus

Consensus


AM_372945-30_15-group16-_g1-group16-_F041-497


AM_372945-30_15-group16-_g1-group16-_F041-497

(c)



00000

0000000000

07585

07515

00070

02

KJ4514101


(d)56

113113

167224224

279336336

391448448

168225225

280337337

Consensus

Consensus

Consensus

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

(e)



00000

0000000024

00005

KC5712551

(f)



11111

113113113113113

225225225225225

Consensus

Consensus

Consensus

112112112112112

224224224224224

336336336336336


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552

(a)

00000

00000

00037

00037

00005

JN6768431





(b)



4 Discussion


























Agelt40 26 45 107 (078ndash147) 066gt40 39 63






















5 Conclusion




Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


477

615538

231323

184

015 15 46 92 610 0

508446 415

677615

815

100


0102030405060708090

100

()

Imip

enem

Am

ikac

in

Cipr

oflox

acin

Pipe

raci

llin

Cef

oxiti

n

Cef

otax

ime

Cef

oper

azon

e(s

ulba

ctam

)



3 Results











1 2 3 4 5 6 7 8 9 10

(bp)

1000900800

700

600

500

400

300

200

100

50

390

(a)

1 2 3 4 5 6 7 8 9 10

(bp)

1000900800

700

600

500

400

300

200

100

50

800

(b)1 2 3 4 5 6 7 8 9 10

(bp)

1000900800

700

600

500

400

300

200

100

50

688

(c)

1 2 3 4 5 6 7 8 9 10

(bp)

1000900800700

600

500

400

300

200

100

50

713

(d)1 2 3 4 5 6 7 8 9 10

(bp)

1000900800700

600

500

400

300

200

100

50

561

(e)

1 2 3 4 5 6 7 8 9 10

(bp)

1000900800700

600

500

400

300

200

100

50

326

(f)








Consensus

152227

264339


Consensus

265340

316390

(a)


0000

0000

117376

117339

0037

20

KC4173771




(b)1111111111

121121121121121121121121121121

361361361361361361361361361361

241241241241241241241241241241

120120120120120120120120120120

240240240240240240240240240240

390390390390390390390390390390

360360360360360360360360360360

Consensus

Consensus

Consensus

Consensus



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390

(c)



000

0011

00040002

000900090009

DQ1439131

0008 0006 0004 0002 00000010Relative times


(d)

Figure 3 Continued


168168184168168243243

289289305289289364364

288288304288288363363

317315331315315390390

Consensus

Consensus

Contig 11-317


Contig 11-317


gi|336171078|gb|JF7029211|1-331gi|336171074|gb|JF7029191|1-315

gi|336171078|gb|JF7029211|1-331gi|336171074|gb|JF7029191|1-315

(e)



0009

0000

00000011

0542

0006

01

JN7005202




(f)









91115115115

205229229229

319343343343

433457457457

547571571571

204228228228

318342342342

432456456456

546570570570

660684684684

Consensus

Consensus

Consensus

Consensus

Consensus

AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


(a)

TEM Ecoli Egypt

00041

0002700000

00014

00005




(b)115102111111111111111

229216225225225225225

343330339339339339339

228215224224

342329338338338338338

456443452452452452452

224224224

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

Consensus

Consensus

Consensus

(c)


00033

00000

00000

00000

00000

00000

0001500004

00015

00005

00005






(d)



340

340340340340

340340

453

453453453453

453453

566

566566566566

566566

324

324324

211

211211

98

9898

452

452452452452

452452

210

210210

565

565565565

565565

565

323

323323

678

678678678678

678678

436

436436

Consensus

Consensus

Consensus



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

(a)




00000

00022

00087

00022

00000

00000

000000004300000

0002200022

00022

00022

00000

0001

AF1249841








(b)







601601601601

629688688688

Consensus


(a)


00000

00000

0000005168

01

AP0146461




(b)227

2911

340142

3391417474

452254187187

7575

Consensus

Consensus


AM_372945-30_15-group16-_g1-group16-_F041-497


AM_372945-30_15-group16-_g1-group16-_F041-497

(c)



00000

0000000000

07585

07515

00070

02

KJ4514101


(d)56

113113

167224224

279336336

391448448

168225225

280337337

Consensus

Consensus

Consensus

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

(e)



00000

0000000024

00005

KC5712551

(f)



11111

113113113113113

225225225225225

Consensus

Consensus

Consensus

112112112112112

224224224224224

336336336336336


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552

(a)

00000

00000

00037

00037

00005

JN6768431





(b)



4 Discussion


























Agelt40 26 45 107 (078ndash147) 066gt40 39 63






















5 Conclusion




Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4 5 6 7 8 9 10

(bp)

1000900800

700

600

500

400

300

200

100

50

390

(a)

1 2 3 4 5 6 7 8 9 10

(bp)

1000900800

700

600

500

400

300

200

100

50

800

(b)1 2 3 4 5 6 7 8 9 10

(bp)

1000900800

700

600

500

400

300

200

100

50

688

(c)

1 2 3 4 5 6 7 8 9 10

(bp)

1000900800700

600

500

400

300

200

100

50

713

(d)1 2 3 4 5 6 7 8 9 10

(bp)

1000900800700

600

500

400

300

200

100

50

561

(e)

1 2 3 4 5 6 7 8 9 10

(bp)

1000900800700

600

500

400

300

200

100

50

326

(f)








Consensus

152227

264339


Consensus

265340

316390

(a)


0000

0000

117376

117339

0037

20

KC4173771




(b)1111111111

121121121121121121121121121121

361361361361361361361361361361

241241241241241241241241241241

120120120120120120120120120120

240240240240240240240240240240

390390390390390390390390390390

360360360360360360360360360360

Consensus

Consensus

Consensus

Consensus



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390

(c)



000

0011

00040002

000900090009

DQ1439131

0008 0006 0004 0002 00000010Relative times


(d)

Figure 3 Continued


168168184168168243243

289289305289289364364

288288304288288363363

317315331315315390390

Consensus

Consensus

Contig 11-317


Contig 11-317


gi|336171078|gb|JF7029211|1-331gi|336171074|gb|JF7029191|1-315

gi|336171078|gb|JF7029211|1-331gi|336171074|gb|JF7029191|1-315

(e)



0009

0000

00000011

0542

0006

01

JN7005202




(f)









91115115115

205229229229

319343343343

433457457457

547571571571

204228228228

318342342342

432456456456

546570570570

660684684684

Consensus

Consensus

Consensus

Consensus

Consensus

AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


(a)

TEM Ecoli Egypt

00041

0002700000

00014

00005




(b)115102111111111111111

229216225225225225225

343330339339339339339

228215224224

342329338338338338338

456443452452452452452

224224224

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

Consensus

Consensus

Consensus

(c)


00033

00000

00000

00000

00000

00000

0001500004

00015

00005

00005






(d)



340

340340340340

340340

453

453453453453

453453

566

566566566566

566566

324

324324

211

211211

98

9898

452

452452452452

452452

210

210210

565

565565565

565565

565

323

323323

678

678678678678

678678

436

436436

Consensus

Consensus

Consensus



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

(a)




00000

00022

00087

00022

00000

00000

000000004300000

0002200022

00022

00022

00000

0001

AF1249841








(b)







601601601601

629688688688

Consensus


(a)


00000

00000

0000005168

01

AP0146461




(b)227

2911

340142

3391417474

452254187187

7575

Consensus

Consensus


AM_372945-30_15-group16-_g1-group16-_F041-497


AM_372945-30_15-group16-_g1-group16-_F041-497

(c)



00000

0000000000

07585

07515

00070

02

KJ4514101


(d)56

113113

167224224

279336336

391448448

168225225

280337337

Consensus

Consensus

Consensus

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

(e)



00000

0000000024

00005

KC5712551

(f)



11111

113113113113113

225225225225225

Consensus

Consensus

Consensus

112112112112112

224224224224224

336336336336336


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552

(a)

00000

00000

00037

00037

00005

JN6768431





(b)



4 Discussion


























Agelt40 26 45 107 (078ndash147) 066gt40 39 63






















5 Conclusion




Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Consensus

152227

264339


Consensus

265340

316390

(a)


0000

0000

117376

117339

0037

20

KC4173771




(b)1111111111

121121121121121121121121121121

361361361361361361361361361361

241241241241241241241241241241

120120120120120120120120120120

240240240240240240240240240240

390390390390390390390390390390

360360360360360360360360360360

Consensus

Consensus

Consensus

Consensus



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390



gi|73747120|gb|DQ1532171|1-390gi|73747126|gb|DQ1532181|1-390

gi|695211273|ref|NG_0360511|1-390gi|695211797|ref|NG_0362531|1-390

(c)



000

0011

00040002

000900090009

DQ1439131

0008 0006 0004 0002 00000010Relative times


(d)

Figure 3 Continued


168168184168168243243

289289305289289364364

288288304288288363363

317315331315315390390

Consensus

Consensus

Contig 11-317


Contig 11-317


gi|336171078|gb|JF7029211|1-331gi|336171074|gb|JF7029191|1-315

gi|336171078|gb|JF7029211|1-331gi|336171074|gb|JF7029191|1-315

(e)



0009

0000

00000011

0542

0006

01

JN7005202




(f)









91115115115

205229229229

319343343343

433457457457

547571571571

204228228228

318342342342

432456456456

546570570570

660684684684

Consensus

Consensus

Consensus

Consensus

Consensus

AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


(a)

TEM Ecoli Egypt

00041

0002700000

00014

00005




(b)115102111111111111111

229216225225225225225

343330339339339339339

228215224224

342329338338338338338

456443452452452452452

224224224

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

Consensus

Consensus

Consensus

(c)


00033

00000

00000

00000

00000

00000

0001500004

00015

00005

00005






(d)



340

340340340340

340340

453

453453453453

453453

566

566566566566

566566

324

324324

211

211211

98

9898

452

452452452452

452452

210

210210

565

565565565

565565

565

323

323323

678

678678678678

678678

436

436436

Consensus

Consensus

Consensus



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

(a)




00000

00022

00087

00022

00000

00000

000000004300000

0002200022

00022

00022

00000

0001

AF1249841








(b)







601601601601

629688688688

Consensus


(a)


00000

00000

0000005168

01

AP0146461




(b)227

2911

340142

3391417474

452254187187

7575

Consensus

Consensus


AM_372945-30_15-group16-_g1-group16-_F041-497


AM_372945-30_15-group16-_g1-group16-_F041-497

(c)



00000

0000000000

07585

07515

00070

02

KJ4514101


(d)56

113113

167224224

279336336

391448448

168225225

280337337

Consensus

Consensus

Consensus

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

(e)



00000

0000000024

00005

KC5712551

(f)



11111

113113113113113

225225225225225

Consensus

Consensus

Consensus

112112112112112

224224224224224

336336336336336


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552

(a)

00000

00000

00037

00037

00005

JN6768431





(b)



4 Discussion


























Agelt40 26 45 107 (078ndash147) 066gt40 39 63






















5 Conclusion




Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


168168184168168243243

289289305289289364364

288288304288288363363

317315331315315390390

Consensus

Consensus

Contig 11-317


Contig 11-317


gi|336171078|gb|JF7029211|1-331gi|336171074|gb|JF7029191|1-315

gi|336171078|gb|JF7029211|1-331gi|336171074|gb|JF7029191|1-315

(e)



0009

0000

00000011

0542

0006

01

JN7005202




(f)









91115115115

205229229229

319343343343

433457457457

547571571571

204228228228

318342342342

432456456456

546570570570

660684684684

Consensus

Consensus

Consensus

Consensus

Consensus

AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


(a)

TEM Ecoli Egypt

00041

0002700000

00014

00005




(b)115102111111111111111

229216225225225225225

343330339339339339339

228215224224

342329338338338338338

456443452452452452452

224224224

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

Consensus

Consensus

Consensus

(c)


00033

00000

00000

00000

00000

00000

0001500004

00015

00005

00005






(d)



340

340340340340

340340

453

453453453453

453453

566

566566566566

566566

324

324324

211

211211

98

9898

452

452452452452

452452

210

210210

565

565565565

565565

565

323

323323

678

678678678678

678678

436

436436

Consensus

Consensus

Consensus



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

(a)




00000

00022

00087

00022

00000

00000

000000004300000

0002200022

00022

00022

00000

0001

AF1249841








(b)







601601601601

629688688688

Consensus


(a)


00000

00000

0000005168

01

AP0146461




(b)227

2911

340142

3391417474

452254187187

7575

Consensus

Consensus


AM_372945-30_15-group16-_g1-group16-_F041-497


AM_372945-30_15-group16-_g1-group16-_F041-497

(c)



00000

0000000000

07585

07515

00070

02

KJ4514101


(d)56

113113

167224224

279336336

391448448

168225225

280337337

Consensus

Consensus

Consensus

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

(e)



00000

0000000024

00005

KC5712551

(f)



11111

113113113113113

225225225225225

Consensus

Consensus

Consensus

112112112112112

224224224224224

336336336336336


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552

(a)

00000

00000

00037

00037

00005

JN6768431





(b)



4 Discussion


























Agelt40 26 45 107 (078ndash147) 066gt40 39 63






















5 Conclusion




Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


91115115115

205229229229

319343343343

433457457457

547571571571

204228228228

318342342342

432456456456

546570570570

660684684684

Consensus

Consensus

Consensus

Consensus

Consensus

AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


AM_372945-25_5-group16-_T-group16-_A041-779


(a)

TEM Ecoli Egypt

00041

0002700000

00014

00005




(b)115102111111111111111

229216225225225225225

343330339339339339339

228215224224

342329338338338338338

456443452452452452452

224224224

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

AM_372945-26_6-group16-_T-group16-_B041-751


gi|354805824|gb|JN0433841|1-516

Consensus

Consensus

Consensus

(c)


00033

00000

00000

00000

00000

00000

0001500004

00015

00005

00005






(d)



340

340340340340

340340

453

453453453453

453453

566

566566566566

566566

324

324324

211

211211

98

9898

452

452452452452

452452

210

210210

565

565565565

565565

565

323

323323

678

678678678678

678678

436

436436

Consensus

Consensus

Consensus



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

(a)




00000

00022

00087

00022

00000

00000

000000004300000

0002200022

00022

00022

00000

0001

AF1249841








(b)







601601601601

629688688688

Consensus


(a)


00000

00000

0000005168

01

AP0146461




(b)227

2911

340142

3391417474

452254187187

7575

Consensus

Consensus


AM_372945-30_15-group16-_g1-group16-_F041-497


AM_372945-30_15-group16-_g1-group16-_F041-497

(c)



00000

0000000000

07585

07515

00070

02

KJ4514101


(d)56

113113

167224224

279336336

391448448

168225225

280337337

Consensus

Consensus

Consensus

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

(e)



00000

0000000024

00005

KC5712551

(f)



11111

113113113113113

225225225225225

Consensus

Consensus

Consensus

112112112112112

224224224224224

336336336336336


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552

(a)

00000

00000

00037

00037

00005

JN6768431





(b)



4 Discussion


























Agelt40 26 45 107 (078ndash147) 066gt40 39 63






















5 Conclusion




Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


340

340340340340

340340

453

453453453453

453453

566

566566566566

566566

324

324324

211

211211

98

9898

452

452452452452

452452

210

210210

565

565565565

565565

565

323

323323

678

678678678678

678678

436

436436

Consensus

Consensus

Consensus



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470



gi|5457153|gb|AF1645771|1-713gi|10443723|gb|AF2266221|1-713

gi|1841439|emb|Y110691|1-713gi|283490011|gb|GQ4704281|1-470

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

gi|370981682|dbj|AB6757231|1-706

(a)




00000

00022

00087

00022

00000

00000

000000004300000

0002200022

00022

00022

00000

0001

AF1249841








(b)







601601601601

629688688688

Consensus


(a)


00000

00000

0000005168

01

AP0146461




(b)227

2911

340142

3391417474

452254187187

7575

Consensus

Consensus


AM_372945-30_15-group16-_g1-group16-_F041-497


AM_372945-30_15-group16-_g1-group16-_F041-497

(c)



00000

0000000000

07585

07515

00070

02

KJ4514101


(d)56

113113

167224224

279336336

391448448

168225225

280337337

Consensus

Consensus

Consensus

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

(e)



00000

0000000024

00005

KC5712551

(f)



11111

113113113113113

225225225225225

Consensus

Consensus

Consensus

112112112112112

224224224224224

336336336336336


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552

(a)

00000

00000

00037

00037

00005

JN6768431





(b)



4 Discussion


























Agelt40 26 45 107 (078ndash147) 066gt40 39 63






















5 Conclusion




Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


601601601601

629688688688

Consensus


(a)


00000

00000

0000005168

01

AP0146461




(b)227

2911

340142

3391417474

452254187187

7575

Consensus

Consensus


AM_372945-30_15-group16-_g1-group16-_F041-497


AM_372945-30_15-group16-_g1-group16-_F041-497

(c)



00000

0000000000

07585

07515

00070

02

KJ4514101


(d)56

113113

167224224

279336336

391448448

168225225

280337337

Consensus

Consensus

Consensus

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

gi|584111314|gb|KF8761331|1-607gi|584111414|gb|KF8771341|_1-607

AM_372945-31_16-group16-_g1-group16-_G041-633

(e)



00000

0000000024

00005

KC5712551

(f)



11111

113113113113113

225225225225225

Consensus

Consensus

Consensus

112112112112112

224224224224224

336336336336336


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552

(a)

00000

00000

00037

00037

00005

JN6768431





(b)



4 Discussion


























Agelt40 26 45 107 (078ndash147) 066gt40 39 63






















5 Conclusion




Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


11111

113113113113113

225225225225225

Consensus

Consensus

Consensus

112112112112112

224224224224224

336336336336336


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552


AM_372945-35_21-group16-_g9-group16-_C051-552

(a)

00000

00000

00037

00037

00005

JN6768431





(b)



4 Discussion


























Agelt40 26 45 107 (078ndash147) 066gt40 39 63






















5 Conclusion




Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















Agelt40 26 45 107 (078ndash147) 066gt40 39 63






















5 Conclusion




Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








5 Conclusion




Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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