Ultrastructural Changes in Clinical and Microbiota ...

Research ArticleUltrastructural Changes in Clinical and Microbiota Isolates ofKlebsiella pneumoniae Carriers of Genes blaSHV blaTEMblaCTX-M or blaKPC When Subject to 120573-Lactam Antibiotics

Dyana Leal Veras1234 Ana Catarina de Souza Lopes3 Grasielle Vaz da Silva125

Gabriel Gazzoni Arauacutejo Gonccedilalves12 Catarina Fernandes de Freitas23

Fernanda Cristina Gomes de Lima23 Maria Ameacutelia Vieira Maciel3

Ana Paula Sampaio Feitosa123 Luiz Carlos Alves125 and Faacutebio Andreacute Brayner123

1Setor deMicroscopia Eletronica Laboratorio de Imunopatologia Keizo Asami (LIKA) Universidade Federal de Pernambuco (UFPE)Avenida Professor Moraes Rego sn Cidade Universitaria 50670-901 Recife PE Brazil2Departamento de Parasitologia Centro de Pesquisas Aggeu Magalhaes (CPqAM)-Fiocruz Avenida Professor Moraes Rego snCaixa Postal 7472 Cidade Universitaria 50670-420 Recife PE Brazil3Departamento de Medicina Tropical Universidade Federal de Pernambuco (UFPE) Avenida Professor Moraes Rego snCidade Universitaria 50670-901 Recife PE Brazil4Departamento de Enfermagem Faculdade de Ciencias Humanas de Olinda (FACHO) Rod PE-015 Jatoba53060-775 Olinda PE Brazil5Instituto de Ciencias Biologicas Universidade de Pernambuco (UPE) Rua Arnobio Marques 310 Santo Amaro50100-130 Recife PE Brazil

Correspondence should be addressed to Dyana Leal Veras dyana lealyahoocombr

Received 5 June 2015 Revised 3 August 2015 Accepted 30 August 2015

Academic Editor Paolo Ruggerone

Copyright copy 2015 Dyana Leal Veras et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aim of this study was to characterize the ultrastructural effects caused by 120573-lactam antibiotics inKlebsiella pneumoniae isolatesThree K pneumoniae clinical isolates were selected for the study with resistance profiles for third-generation cephalosporinsaztreonam andor imipenem and with different resistance genes for extended-spectrum 120573-lactamases (ESBL) or Klebsiellapneumoniae carbapenemase (KPC) Two K pneumoniae isolates obtained from the microbiota which were both resistant toamoxicillin and ampicillin were also analyzed In accordance with the susceptibility profile the clinical isolates were subjectedto subminimum inhibitory concentrations (sub-MICs) of cefotaxime ceftazidime aztreonam and imipenem and the isolates fromthe microbiota to ampicillin and amoxicillin for analysis by means of scanning and transmission electron microscopy The Kpneumoniae isolates showed different morphological and ultrastructural changes after subjection to 120573-lactams tested at differentconcentrations such as cell filamentation loss of cytoplasmic material and deformation of dividing septa Our results demonstratethatK pneumoniae isolates harboring different genes that encode for 120573-lactamases show cell alterations when subjected to different120573-lactam antibiotics thus suggesting that they possess residual activity in vitro despite the phenotypic resistance presented in theisolates analyzed

1 Introduction

Klebsiella pneumoniae is one of the most common Gram-negativemultidrug-resistant (MDR) organisms foundworld-wide and it is responsible for high morbidity and mortalityboth in hospitals and in other healthcare units [1]

120573-Lactam antibiotics are the most commonly prescribedantibacterial agents because of the high efficiency of theirmechanism of action and low toxicity [2] Penicillin-bindingproteins are the lethal targets of 120573-lactams in sensitive bacte-ria and their inhibition leads to degradation of the bacterialcell wall and eventual cell lysis [3] Several studies have

Hindawi Publishing Corporatione Scientific World JournalVolume 2015 Article ID 572128 13 pageshttpdxdoiorg1011552015572128

2 The Scientific World Journal

reported different bacterial morphological changes caused bysubminimum inhibitory concentrations (sub-MICs) of third-generation cephalosporins and carbapenems in sensitiveisolates of several bacterial species [4ndash6] However there isa lack of studies using resistant isolates

In pathogenic bacteria 120573-lactamase production is themost common and important mechanism of resistance to 120573-lactam antibiotics [2]The classical 120573-lactamases are enzymescapable of inactivating penicillins and narrow-spectrumcephalosporins before reaching their target [7]MostK pneu-moniae isolates carry the gene for chromosomal class A SHV-1 120573-lactamaseThis has limited activity against ampicillin anddoes not hydrolyze extended-spectrum 120573-lactam antibiotics[8] The presence of this gene has been previously studiedin K pneumoniae isolates originating from the microbiota ofindividuals without bacterial infection [9]

The introduction into clinical practice of oxyiminoce-phalosporins for treating infections due to Gram-negativebacteria was soon followed by the appearance of extended-spectrum 120573-lactamases (ESBLs) derived from the classical 120573-lactamases SHV-1 TEM-1 andTEM-2These conferred resis-tance to third-generation cephalosporins and monobactamssuch as aztreonam [2] More than 180 variants of the gene119887119897119886SHV and more than 210 of the gene 119887119897119886TEM have alreadybeen reported [10] Another group of ESBLs which has a highprofile of hydrolysis in relation to cefotaxime and is less than40 identical to the SHV and TEM enzymes is the group ofCTX-M enzymesThese are widely found in different speciesof the family Enterobacteriaceae including K pneumoniae[11]

Treatment of infections due to K pneumoniae isolatesproducing ESBLs is limited to the use of a few available anti-biotics Carbapenems are often the last line of effective treat-ment against these infections [12] However production ofthe class A carbapenemase Klebsiella pneumoniae carbapen-emase (KPC) which is an extended-spectrum 120573-lactamasehas been reported to be the primary form of carbapenemresistance in this bacterial species Fifteen variants with dis-semination in different parts of the world have been reported[10]

Production of these 120573-lactamases by clinical isolates ofKpneumoniae has been a major problem in many countriesand this has hampered the therapeutic options available forthe treatment of bacterial infections caused by this species

The increasing prevalence of multidrug resistance isleading towards the threat that a postantibiotic era may beabout to begin characterized by decreased effectiveness ofcommon antibiotics and routine application of complemen-tary therapeutic approaches for treating bacterial infections[13] Therefore new studies need to be developed in whichold and discarded antibiotics should be reinvestigated andreused Even rejected antibiotics possibly should be usedwhen necessary [14]

In this regard some studies have tried to demonstratethe in vitro and in vivo action of therapeutic combinationsof 120573-lactams and aminoglycosides in MDR K pneumoniaeisolates aiming to obtain new options for treating infectionsdue to this bacterial species [15] However no study hasdemonstrated any alterations induced by different 120573-lactam

antibiotics in the bacterial cell structure of MDR K pneumo-niae isolates

K pneumoniae isolates presenting resistance to cefo-taxime when subjected to sub-MICs of this antibiotic wereshown to cause damage to cell surfaces Filamentationthrough a dose-dependent adaptive process was observedcaused by the stressful environment that was induced by thepresence of the antibiotic thereby contributing towards thetherapeutic effects [16]

Unfortunately knowledge about the effects caused by 120573-lactam antibiotics on the bacterial cell structure of MDRK pneumoniae isolates is still scarce These isolates carryimportant resistance genes such as 119887119897119886SHV 119887119897119886TEM 119887119897119886CTX-Mand 119887119897119886KPC Likewise little is known about the effect of theseantibiotics on isolates from the microbiota of individualswithout bacterial infection Therefore the present studyaimed to characterize the morphological and ultrastructuraleffects on pathogenic and nonpathogenic K pneumoniaeisolates that carry genes encoding the classical 120573-lactamasesESBL andKPC caused by120573-lactam antibiotics that are widelyused in clinical medicine

2 Material and Methods

21 Bacterial Isolates For this study five K pneumoniae iso-lates from Recife PE Brazil were selected (Tables 1 and 2)The K211-F and K581-F isolates were obtained from the fecalmicrobiota of children who did not have bacterial infectionThese isolates showed resistance or intermediate resistanceto ampicillin andor amoxicillin but lacked the 119887119897119886SHV gene(Table 1) [9] Three other K pneumoniae clinical isolatesobtained from public hospitals in Recife were used K3CK16R and K652 The susceptibility profile and presence ofthe genes 119887119897119886SHV and 119887119897119886CTX-M had been determined inprevious studies on the K3C and K16R isolates [9 17] whichshowed resistance to cefotaxime ceftazidime and aztreonamaccording to the interpretive criteria of the Clinical andLaboratory Standards Institute (CLSI) of 2013 [18]

The K3C isolate possesses the 119887119897119886SHV-11 gene and the K16Risolate has the 119887119897119886SHV-1 and 119887119897119886CTX-M2 genes [9 17] The K652isolate which was studied for the first time in the presentwork was obtained by means of surveillance testing on rectalswabs from individuals without clinical symptoms of bacte-rial infection who were admitted to a public hospital in 2011This isolate was identified and its susceptibility profile in rela-tion to different antimicrobials was determined using theVITEK 2 automated system (BioMerieux) It was manuallyconfirmed in relation to cefotaxime ceftazidime and imipe-nem (Sigma-Aldrich) by means of broth macrodilution test-ing in accordance with the 2013 CLSI recommendations [18]

22 DNA and PCR Extraction The genomic DNA of K pneu-moniae isolates was extracted using the Wizard GenomicDNA Purification Kit (Promega) in accordance with the man-ufacturerrsquos instructions The presence of the 119887119897119886TEM gene wasinvestigated by means of PCR in the macrobiota isolates andin the three clinical isolates using the T1 primer 51015840-ATAAAA-TTCTTGAAGACGAAA-31015840 and the T2 primer 51015840-GAC-AGTTACCAATGCTTAATC-31015840 [19] The K652 isolate was

The Scientific World Journal 3

Table 1 MICs of K pneumoniae isolates obtained in hospital and sub-MICs used for analysis by electron microscopy

Isolates Cefotaxime Ceftazidime Aztreonam(120583gmLminus1) (120583gmLminus1) (120583gmLminus1)

ESBL MICsa Sub-MICsb MICsa Sub-MICsb MICsa Sub-MICsb

TEMc SEMd TEMc SEMd TEMc SEMd

K3C gt256 64 32 32 128 32 16 32 64 32 16 32K16R gt256 64 32 32 32 16 8 16 16 4 4

Isolates Cefotaxime Ceftazidime Imipenem(120583gmLminus1) (120583gmLminus1) (120583gmLminus1)

KPC MICsa Sub-MICsb MICsa Sub-MICsb MICsa Sub-MICsb


K652 ge256 98 98 ge256 27 27 ge128 16 16aMICs minimum inhibitory concentrations bsub-MICs sub-minimum inhibitory concentrations cTEM transmission electron microscopy and dSEMscanning electron microscopy

Table 2 MICs of K pneumoniae isolates obtained of microbiota and sub-MICs used for analysis by electron microscopy

IsolatesAmpicillin (120583gmLminus1) Amoxicillin (120583gmLminus1)

MICsa Sub-MICsb MICsa Sub-MICsb

TEMc SEMd TEMc SEMd

K581-F 16 05 2 8 05 8 32 05 4 16 05 16K211-F 128 05 16 32 64 16 32 64 gt128 05 4 16 64 128 05 16 128aMICs minimum inhibitory concentrations bsub-MICs sub-minimum inhibitory concentrations cTEM transmission electron microscopy and dSEMscanning electron microscopy

also evaluated bymeans of PCR for the presence of the 119887119897119886SHVand 119887119897119886KPC genes using the primers 51015840-GGTTATGCGTTA-TATTCGCC-31015840 and 51015840-TTAGCGTTGCCAGTGCTC-3 [20]and using KPC1F 51015840-GCTACACCTAGCTCCACCTTC-31015840and KPC1R 51015840-ACAGTGGTTGGTAATCCATGC-31015840 [21]respectively

23 DNA Sequencing Sequencing of the PCR products fromthe 119887119897119886TEM and 119887119897119886KPC geneswas performed using theAppliedBiosystemsHitachi automated sequencer after purificationof the PCR amplicons using the PureLink Micro Kit (Invit-rogen) in accordance with the manufacturerrsquos instructionsThe 119887119897119886TEM gene was sequenced in the K3C and K16R isolatesbecause of phenotypic resistance that was presented in rela-tion to the third-generation cephalosporins that were testedOnly the 119887119897119886KPC gene was sequenced in K652 isolate dueto phenotypic resistance to carbapenems that was presentedThe primers were the same as for PCR plus TEMup21 51015840-TCCCTTTTTTGCGGCATTTTGC-31015840 andTEMdwn280 51015840-CAGTGAGGCACCTATCTC-31015840 [22] for the 119887119897119886TEM geneand KPC F 51015840-GAGCTGAACTCCGCCATC-31015840 and KPC R51015840-TATTTTTCCGAGATGGGTGAC-31015840 [23] for the 119887119897119886KPCgene

The analyses on the DNA sequence and the multiplealignments were performed using the DNAstar software andBLAST [24] The sequences for the 119887119897119886TEM-1 119887119897119886TEM-15 and119887119897119886KPC-2 geneswere deposited in theGenBank database underthe access numbers KF906436 KF906435 and KF906437respectively

24 Transmission ElectronMicroscopy (TEM) To analyze theaction of antibiotics on K pneumoniae isolates these isolateswere subjected to different sub-MICs of ampicillin amox-icillin ceftazidime cefotaxime aztreonam and imipenem(Sigma-Aldrich) at 37∘C for 6 hours according to the originand susceptibility profile of each isolate using clinically rele-vant concentrations (Tables 1 and 2) [25 26] In all the pro-cesses a control for the isolate was included under the sameconditions and at the same dilutions without the presence ofthe antibiotic After growth the bacterial cells were centri-fuged and fixed using 25 glutaraldehyde and 4 parafor-maldehyde (Sigma-Aldrich) The cells were postfixed in 1osmium tetroxide and then contrasted in 5 uranyl acetate(Electron Microscopy Science) Dehydration was carried outusing acetone (Sigma-Aldrich) followed by infiltration andembedment of the material in epon 812 resin (ElectronMicroscopy Science) The samples were viewed under atransmission electron microscope (Zeiss EM109)

25Scanning Electron Microscopy (SEM) The K pneumoniaeisolates were subjected to antimicrobial agents using the con-centrations and criteria described above (Tables 1 and 2) Aftergrowth the bacterial cells were gently centrifuged and fixedusing 25 glutaraldehyde (Sigma-Aldrich) Postfixation wasperformed using 1 osmium tetroxide (ElectronMicroscopyScience) followed by dehydration using ethanol (Sigma-Aldrich) After dehydration the material was dried in prepa-ration for metallization and viewing of the bacterial cellsunder a scanning electron microscope (JEOL JSM-5600 LV)


3 Results

31 Antimicrobial Susceptibility The susceptibility profiles ofthe K3C K16R K211-F and K581-F isolates had previouslybeen determined in other studies [9 17] and are describedin Tables 1 and 2 The K pneumoniae K652 isolate showedresistance to all the antibiotics tested using the VITEK 2automated system (ampicillin ampicillinsulbactam aztreo-nam cephalosporins cefepime cefotaxime cefoxitin cef-tazidime ciprofloxacin ertapenem gentamicin imipenemmeropenem and piperacillintazobactam) except for ami-kacin and tigecycline The macrodilution broth test con-firmed the resistance of this isolate to cefotaxime ceftazi-dime and imipenem with MICs of 256 120583gmLminus1

32 Presence of bla119878119867119881

bla119879119864119872

and bla119870119875119862

Genes The PCRanalyses identified the presence of 119887119897119886TEM gene in the threeclinical isolates of K pneumoniae and in the K211-F isolatefrom the microbiota Additionally the 119887119897119886SHV and 119887119897119886KPCgenes were also identified by means of PCR in the Kpneumoniae isolate K652

33 Sequencing of the bla119879119864119872

and bla119870119875119862

Genes The 119887119897119886TEM-1gene was identified in the K pneumoniae isolate K16R whichalso had the 119887119897119886CTX-M2 gene [17] The 119887119897119886TEM gene found inthe K3C isolate showed a nucleotide change in relation tothe 119887119897119886TEM-1 gene at positions 512 GrarrA and 914 GrarrAleading to replacement of Glu-104rarr Lys and Gly-238rarr SerThe 119887119897119886KPC-2 gene was identified in the K pneumoniae isolateK652

34 Ultrastructural and Morphological Analyses The cellsof the control K pneumoniae isolates which were ana-lyzed without having been subjected to 120573-lactam antibioticsshowed morphology that had been preserved with an intactcell wall and cytoplasmic contents of electron-dense appear-ance on analysis bymeans of TEMThepresence of ribosomesand genetic material distributed in the bacterial cytoplasmcould be seenThe cells undergoing a division process showedformation of septa without irregularities (Figures 1(a) 2(a)and 3(a)) The SEM analysis showed conserved rod-shapedbacterial morphology and an average cell length of 15 to 3 120583m(Figures 4(a) 4(b) 5(a) and 6(a))

35 K16R Isolate Although the K16R isolate presented resis-tance to cefotaxime ceftazidime and aztreonam and hadthe 119887119897119886SHV-1 119887119897119886TEM-1 and 119887119897119886CTX-M2 genes subjection ofthis isolate to sub-MICs of these antimicrobials causedcellular events that differed from those of the control cellsAfter subjection to cefotaxime at concentrations of 32 and64 120583gmLminus1 cells with increased periplasmic space overtheir entire extent with greater amounts of space at theirends were observed thus suggesting that the cytoplasm haddecreased in volume or retracted (Figure 2(b)) No significantmorphological changes to cell size were observed in the SEManalysis (Figure 5(b))

Subjection of the K16R isolate to ceftazidime at concen-trations of 8 and 16 120583gmLminus1 allowed identification of mor-phological and cellular organizational changes by means of

TEM and SEM in which several elongated cells were viewedThrough TEM it could be seen that this event occurredbecause of unfinished cell division and this was identifiedthrough the presence of several consecutive division septathat were incomplete Some cells also showed membranecell spaces inside the bacterial cytoplasm and cytoplasmiccontent identical to the rest of the cell with ribosomes andgenetic material equally distributed (Figures 2(c)ndash2(e)) Thissuggests that cell division did not happen thus enablingformation of membrane compartments within the bacterialcells Structural disorganization of the continuity of thecytoplasmic membrane of some cells and decreased amountsof bacterial cytoplasm could also be observedThrough SEMit was possible to identify cells that were more than 15 120583m inlength (Figure 5(c))

After the K16R isolate was subjected to application of4 120583gmLminus1 of aztreonam some cells presented large electron-lucent spaces in the bacterial cytoplasm with decreasedcytoplasmic mass (Figures 2(f)ndash2(h)) No morphological orcell size changes were identified through SEM (Figure 5(d))

36 K3C Isolate The K pneumoniae isolate K3C also hasphenotypic resistance to cefotaxime ceftazidime and aztre-onam and has the 119887119897119886SHV-11 and 119887119897119886TEM-15 genes After thisisolate was subjected to 32 and 64 120583gmLminus1 of cefotaximesome cells showed a decrease in cytoplasmic material withformation of membrane compartments within the cellswhich presented internal material containing ribosomesThese cells showed cytoplasmic membrane disorganizationSeveral cells also contained in their cytoplasm a large numberof nonmembrane electron-lucent structures near the cellmembrane at both tested concentrations (Figures 1(b) and1(c)) No morphological change was identified through SEM(Figure 6(b))

Various filamentous cells were seen through TEM andSEM after the K3C isolate was subjected to 16 and 32 120583gmLminus1of ceftazidime Through TEM it could be seen that cellelongationwas followed by a decrease in cytoplasmicmaterialin the middle of the dividing cell or at the ends of cells thathad already separatedThe presence of a large electron-lucentspace between the cytoplasmic membrane and cell wall ofthese cells highlighted the presence of cell compartmentsseparated bymembranes that contained cytoplasmicmaterial(Figure 1(d)) Through SEM cells of up to 18 120583m in lengthcould be seen (Figure 6(c))

The cellular events induced by aztreonam in this iso-late at concentrations of 16 and 32 120583gmLminus1 were discreetSome cells showed large numbers of nonmembrane electron-lucent spaces in the bacterial cytoplasm similar to thosefound after treatmentwith cefotaxime (Figure 1(e))Howevermost of the cells showed conservation of morphology withpreservation of the cytoplasmic membrane and cell wallNo morphological changes were observed through SEM(Figure 6(d))

37 K652 Isolate The K652 isolate of K pneumoniae has the119887119897119886KPC-2 gene and shows high phenotypic resistance to carba-penems and third-generation cephalosporins Subjection of


C

w

(a)

lowast

C

(b)

C lowast

lowast

lowast

(c)

C

(d)

lowast

lowast

lowastC

(e)

Figure 1 (andashe) Transmission electron micrographs of isolate K3C from K pneumoniae (a) bacterial cell without treatment (control)mdashpreserved morphology cytoplasmic membrane (arrow) cell wall (w) and cytoplasm (C) intact (b-c) Cells subjected to CTX (64120583gmLminus1)mdashdecreased cytoplasmic material (C) presence of membrane compartments (stars) electron-lucent spaces (asterisks) and disorganization ofthe membrane and cell wall (d) Cell subjected to CAZ (32 120583gmLminus1)mdashelongated aspect morphology with loss of cytoplasmic material (C)and the presence of small membrane compartments (stars) (e) Cell subjected to ATM (32120583gmLminus1)mdashnormal morphology in the presence ofelectron-lucent spaces (asterisks) in the cytoplasm (C) Bars = 05 120583m CAZ ceftazidime CTX cefotaxime and ATM aztreonam

this isolate to both ceftazidime at 27120583gmLminus1 and imipenemat 16 120583gmLminus1 caused cell filamentation as seen in analysesusing SEM Cells of more than 50mm in length could be seen(Figures 4(e)ndash4(i)) The number of elongated cells seemed tobe greater after subjection to imipenem than to ceftazidime(Figures 3(d)ndash3(h)) The TEM analysis on the K652 isolate

subjected to these antibiotics showed that most of the cellsshowed formation of consecutive division septa withoutcompleting the division process Many cells also showedelongation and disorganization of the cytoplasmicmembraneand cell wall at the site of cell division thus providing aresilient appearance to these cellular components


C

w

CC

C

C

C

C

C

(a)

(c) (d)

(h)

(f)(e)

(g)

(b)

Figure 2 (andashh) Transmission electronmicrographs of isolate K16R fromK pneumoniae (a) Untreated bacterial cellmdashpreservedmorphologycytoplasmic membrane (arrow) cell wall (w) and cytoplasm (C) intact (b) Cell subjected to CTX (64120583gmLminus1)mdashpresence of large electron-lucent space due to increased periplasmic space (stars) and reduced cytoplasmic material (C) (cndashe) Cells subjected to CAZ (16 120583gmLminus1)(c-d) Filamentous cells and the presence of membrane compartments containing cytoplasmic material (star) (e) Elongated cell showingloss of cytoplasmic material (star) (fndashh) Cell subjected to ATM (4120583gmLminus1)mdashloss of cytoplasmic material (C) and presence of membranecompartments (stars) Bars = 05120583m CAZ ceftazidime CTX cefotaxime and ATM aztreonam


C

w

(d)

(e)(f)

(a) (c)(b)

(h)(g)

Figure 3 (andashh) Transmission electron micrographs of isolate K652 of K pneumoniae (a) Untreated bacterial cellmdashcell wall with preservedmorphology (w) and cytoplasm (C) intact besides regular septation (arrowheads) (b-c) Cell subjected to CTX (98 120583gmLminus1)mdashpresenceof cells not grown and rounded (stars) at the ends of cells with normal morphology (dndashf) Cells subjected to CAZ (27 120583gmLminus1)mdashconsecutive formation of septa (arrowheads) providing elongation of bacterial morphology Observe disorganization of membrane and cellwall with elastic aspect (g-h) Cell subjected to IMP (16120583gmLminus1)mdashmorphology of elongated appearance with formation of consecutive septa(arrowheads) Bars = 05 120583m CAZ ceftazidime CTX cefotaxime and IMP imipenem


(a) (b)

(c) (d)

(e) (f) (g)

(h) (i)

Figure 4 (andashi) Scanning electron micrographs of isolate K652 (a-b) Control cells with preserved morphology (c-d) Cells subjected to CTX(98 120583gmLminus1) Observe spherical cells that are not grown being formed from cells with preserved morphology (endashg) Cells subjected to CAZ(27 120583gmLminus1) Observe elongated cells forming bacterial filaments (h-i) Cells subjected to IMP (16120583gmLminus1) Observe elongated cells due tounfinished successive divisions Bars = 2120583m CAZ ceftazidime CTX cefotaxime and IMP imipenem


(a) (b)

(c) (d)

Figure 5 (andashd) Scanning electron micrographs of isolate K16R (a) Control cells with preserved morphology (b) Cells subjected to CTX(32 120583gmLminus1) (c) Cells subjected to CAZ (16120583gmLminus1) (d) Cells subjected to ATM (4120583gmLminus1) Bars = 2 120583m CAZ ceftazidime CTXcefotaxime and ATM aztreonam

Through SEM various rod-shaped bacilli that gave riseto nondeveloped cells in the form of cocci could be observedafter the K652 isolate was subjected to 98120583gmLminus1 of cefo-taxime (Figures 4(c) and 4(d)) The TEM confirmed thatsome spherical cells were being formed from rod-shaped cellsthrough changes to the density of the bacterial cytoplasm(Figures 3(b) and 3(c))

38 K581-F and K211-F Isolates The results from the TEMand SEM analyses on the K pneumoniae isolates from themicrobiota after subjection to amoxicillin and ampicillinwere quite similar to those from the isolates obtained fromthe hospital environment when the latter were subjected tocefotaxime and aztreonam

The K581-F isolate showed phenotypic resistance toamoxicillin and intermediate resistance to ampicillin whenexposed to sub-MICs of these antibiotics (Table 2) This wasdemonstrated by the enlarged electron-lucent spaces at theends of the cells in comparison with the control as seenthrough TEM thus suggesting that there was a decrease inthe cytoplasmic content Large quantities of electron-lucentspaces and nonmembrane electron-dense structures werealso identified in the bacterial cytoplasm of several cells(Figures 7(b) and 7(c)) These cellular events became morepronounced with higher concentrations of each drug thusdemonstrating that there was a dose-dependent correlation

Although the K211-F isolate showed higher MICs foramoxicillin and ampicillin than that of the K581-F isolate the


(a) (b) (c)

(d)

Figure 6 (andashd) Scanning electron micrographs of isolate K3C (a) Control cells with preserved morphology (b) Cells subjected to CTX(32 120583gmLminus1) (c) Cells subjected to CAZ (32120583gmLminus1) (d) Cells subjected to ATM (32120583gmLminus1) Bars = 2 120583m CAZ ceftazidime CTXcefotaxime and ATM aztreonam

TEM analysis after the isolates were subjected to sub-MICs ofthese antibiotics showed that the results from the two isolateswere similar Large quantities of electron-lucent spaces andnonmembrane electron-dense structures were identified inthe bacterial cytoplasm of several cells of the K211-F iso-late even at the lowest concentration tested (05120583gmLminus1)(Figure 7(a)) Several cells with irregular formation of septaduring bacterial division but without loss of cytoplasmiccontent even at higher concentrations were simultaneouslyobserved (Figure 7) The SEM analysis did not show anysignificant changes to any of the antibiotic concentrationstested in relation to the K211-F and K581-F isolates

4 Discussion

Production of 120573-lactamases is one of the main mechanismsof resistance among isolates of the Enterobacteriaceae familyESBL andKPC in particular are often found inK pneumoniaeclinical isolates thus conferring a phenotype that is resistantto the main 120573-lactams used in clinical medicine

The 119887119897119886TEM-15 119887119897119886CTX-M2 and 119887119897119886KPC-2 genes foundrespectively in the K3C K16R and K652 isolates encodeenzymes of ESBL and carbapenemase type These genes havepreviously been reported in different bacterial species [27ndash29] The changes of Glurarr Lys at position 104 and GlyrarrSer at position 238 of the 119887119897119886TEM-15 gene are known to favorincreased substrate fixation and an enzyme-active site thusgiving rise to an enzyme with increased affinity and catalyticefficiency for oxyiminocephalosporins [29] ESBL CTX-Mbelonging to the CTX-M2 phylogenetic group is one ofthe CTX-M-type enzymes most found in Enterobacteriaceaeisolates in Brazil [28] with high hydrolysis power comparedwith third-generation cephalosporins especially cefotaxime

KPC 120573-lactamases efficiently hydrolyze penicillinscephalosporins carbapenems and aztreonam and areinhibited by clavulanic acid and tazobactam [28] The K652isolate presents the resistance gene 119887119897119886KPC-2 This gene wasfirst detected in Brazil in K pneumoniae isolates obtainedfrom patients hospitalized in Recife PE [27] Since thenother studies have demonstrated the presence of this gene in


(a)

(c) (b)

lowast

Figure 7 (andashc) Transmission electron micrographs of isolate K211-F and K581-F of K pneumoniae (a) Isolate K211-F submitted to05 120583gmLminus1 ampicillin Observe irregular formation of septa in a dividing cell (arrow) (b) Isolate K581-F subjected to 05 120583gmLminus1 ofampicillin Cells present electron-lucent (fine arrow) and electron-dense spaces with different sizes and shape throughout the bacterialcytoplasm (arrowheads) in addition to reduction of cytoplasmic contents (star) (c) Isolate K581-F subjected to 4120583gmLminus1 of amoxicillinThe cells exhibit altered morphologies with cell elongation (large arrow) and undefined forms (asterisk) with the presence of electron-lucentstructures with shape and varying sizes scattered throughout the cell cytoplasm (fine arrow) Bars = 05120583m

K pneumoniae isolates obtained from several other states inBrazil [27 28]

Cases of infections by isolates producing these enzymesgive rise to considerable concern regarding the most appro-priate antibiotic therapy This is because bacterial growthoften does not change and cell death due to subjection to120573-lactams does not occur In this context the present studywas designed to demonstrate the in vitro effect of imipenempenicillin third-generation cephalosporins and aztreonamon MDR K pneumoniae and microbiota isolates obtainedfrom individuals under different clinical conditions Thisvariability made it possible to analyze the effects of 120573-lactamantibiotics in isolates with distinct genetic and phenotypicresistance profiles which would have the capacity to evolvedifferently in their host organisms

Subjection of the K3C K16R and K652 isolates to differ-ent concentrations of ceftazidime and of the K652 isolate toimipenem caused cell filamentation in all the isolates ana-lyzed with formation of several consecutive septa and disor-ganization of the cell membrane and cell wall in several cellsThis induction of cell filamentation was previously reportedin other studies using sensitive and resistant isolates fromdifferent bacterial species including Serratia marcescens Kpneumoniae and P aeruginosa subjected to third-generationcephalosporins carbapenems and monobactams Thesestudies showed that inactivation of different penicillin-binding proteins may be associated with inability to completethe cell division process after replication of the bacterial mass[4 5 16 30]

In our study the presence of bacterial filaments andcell disorganization caused by K pneumoniae isolates aftersubjection to ceftazidime and imipenem makes us believethat these antibiotics have some residual activity despite thepresence of genes that code for ESBL and KPC Howevereven though the 120573-lactam antibiotics tested here were capa-ble of causing changes to the K pneumoniae isolates usedin this study these antibiotics were clearly incapable ofinterferingwith the survival of isolates with a resistant pheno-type

Because of the great need for effective therapeutic optionsfor treating MDR bacterial isolates new therapeutic com-binations have been tested Hirsch et al conducted a studythat showed that meropenem and amikacin in combinationwere able to inhibit in vitro and in vivo growth of resistant Kpneumoniae isolates that produced KPC-2 and KPC-3 [15] Inthe present study it was found that imipenemand ceftazidimealone were able to promote in vitro cellular changes thatincluded loss of cytoplasmic material filamentation anddisorganization of the cell membrane and cell wall at thedivision site Understanding the effect of antibiotics aloneon MDR K pneumoniae isolates is extremely importantbecause this enables understanding of how the develop-ment of therapeutic combinations can effectively contributetowards treating the infections caused by these isolates In ourstudy resistant K pneumoniae isolates subjected to clinicallyrelevant concentrations 120573-lactam antibiotics seemed to haveresponses similar to those of sensitive cells at low concentra-tions of these antibiotics


Deloney and Schiller [31] used Helicobacter pylori-sen-sitive isolates subjected to sub-MICs of 120573-lactam antibio-tics including ampicillin and aztreonam and showed thatthe bacterial cells became filamentous when subjected toaztreonam and spherical when subjected to other 120573-lactamsOur data showed that aztreonam was unable to induce cellfilamentation in the clinical isolates tested However theappearance of electron-lucent spaces suggested that loss orretraction of cytoplasmic contents occurred in the sameway that occurred when microbial isolates were subjected toampicillin and amoxicillin

Our results demonstrate that K pneumoniae isolatesharboring different genes that encode for 120573-lactamases canundergo ultrastructural changes when subjected to sub-MICsof 120573-lactam drugs thus suggesting that this antimicrobialshave residual activity in vitro despite the phenotypic resis-tance presented in the isolates analyzed

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study is financially supported by Programa Estrategicode Apoio a Pesquisa em Saude-PAPES IV (Process no4078262012-2) Fundacao Oswaldo Cruz (FIOCRUZ) Theauthors thank the Programa de Desenvolvimento Tecnologicoem Insumos para Saude (PDTIS) of Fundacao Oswaldo Cruz(FIOCRUZ) and the Nucleo de Plataformas Tecnologicas ofCentro de Pesquisas AggeuMagalhaes (CpqAM)FIOCRUZespecially MSc Viviane Carvalho and Dr Cassia Docena forthe technical assistance

References

[1] C Mammina C Bonura A Aleo et al ldquoSequence type 101(ST101

) as the predominant carbapenem-non-susceptible Kleb-siella pneumoniae clone in an acute general hospital in ItalyrdquoInternational Journal of Antimicrobial Agents vol 39 no 6 pp543ndash545 2012

[2] S Haeligggman S Lofdahl A Paauw J Verhoef and S BrisseldquoDiversity and evolution of the class A chromosomal beta-lactamase gene in Klebsiella pneumoniaerdquo Antimicrobial Agentsand Chemotherapy vol 48 no 7 pp 2400ndash2408 2004

[3] M E Stefanova J Tomberg M Olesky J-V Holtje W GGutheil and R A Nicholas ldquoNeisseria gonorrhoeae penicillin-binding protein 3 exhibits exceptionally highy carboxypepti-dase and 120573-lactam binding activitiesrdquo Biochemistry vol 42 no49 pp 14614ndash14625 2003

[4] J Buijs A S M Dofferhoff J W Mouton J H Wagenvoortand J van der Meer ldquoConcentration-dependency of 120573-lactam-induced filament formation inGram-negative bacteriardquoClinicalMicrobiology and Infection vol 14 no 4 pp 344ndash349 2008

[5] T Horii M Kobayashi K Sato S Ichiyama and M Ohta ldquoAnin-vitro study of carbapenem-induced morphological changesand endotoxin release in clinical isolates of Gram-negativebacillirdquo Journal of Antimicrobial Chemotherapy vol 41 no 4pp 435ndash442 1998

[6] J J Jackson andH Kropp ldquo120573-lactam antibiotic-induced releaseof free endotoxin in vitro comparison of penicillin-bindingprotein (PBP) 2-specific imipenem and PBP 3-specific cef-tazidimerdquo Journal of Infectious Diseases vol 165 no 6 pp 1033ndash1041 1992

[7] F Robin J Delmas C Chanal D Sirot J Sirot and R BonnetldquoTEM-109 (CMT-5) a natural complex mutant of TEM-1 120573-lactamase combining the amino acid substitutions of TEM-6and TEM-33 (IRT-5)rdquo Antimicrobial Agents and Chemotherapyvol 49 no 11 pp 4443ndash4447 2005

[8] E C Nelson H Segal and B G Elisha ldquoOuter membrane pro-tein alterations and 119887119897119886TEM-1 variants their role in 120573-lactamresistance in Klebsiella pneumoniaerdquo Journal of AntimicrobialChemotherapy vol 52 no 6 pp 899ndash903 2003

[9] D L Veras L C Alves F A Brayner et al ldquoPrevalence of theblaSHV gene in Klebsiella pneumoniae Isolates obtained fromhospital and community infections and from the microbiota ofhealthy individuals in Recife Brazilrdquo Current Microbiology vol62 no 5 pp 1610ndash1616 2011

[10] G Jacoby and K Bush ldquo120573-Lactamase Classification and AminoAcid Sequences for TEM SHV and OXA Extended-Spectrumand Inhibitor Resistant Enzymesrdquo 2013 httpwwwlaheyorgstudies

[11] M Saladin V T B Cao T Lambert et al ldquoDiversity of CTX-M120573-lactamases and their promoter regions from Enterobacteri-aceae isolated in three Parisian hospitalsrdquo FEMS MicrobiologyLetters vol 209 no 2 pp 161ndash168 2002

[12] C Lascols G Peirano M Hackel K B Laupland and J D DPitout ldquoSurveillance and molecular epidemiology of Klebsiellapneumoniae isolates that produce carbapenemases first reportof OXA-48-like enzymes in North Americardquo AntimicrobialAgents and Chemotherapy vol 57 no 1 pp 130ndash136 2013

[13] B F Cress J A Englaender W He D Kasper R J Linhardtand M A G Koffas ldquoMasquerading microbial pathogenscapsular polysaccharides mimic host-tissue moleculesrdquo FEMSMicrobiology Reviews vol 38 no 4 pp 660ndash697 2014

[14] K Bush P Courvalin G Dantas et al ldquoTackling antibioticresistancerdquoNature Reviews Microbiology vol 9 no 12 pp 894ndash896 2011

[15] E B Hirsch B Guo K-T Chang et al ldquoAssessment of anti-microbial combinations for Klebsiella pneumoniae carbapene-mase-producing K pneumoniaerdquo Journal of Infectious Diseasesvol 207 no 5 pp 786ndash793 2013

[16] H Rajeshwari S Nagveni A Oli D Parashar and K R Chan-drakanth ldquoMorphological changes of Klebsiella pneumoniae inresponse to Cefotaxime a scanning electronmicroscope studyrdquoWorld Journal of Microbiology and Biotechnology vol 25 no 12pp 2263ndash2266 2009

[17] A C S Lopes D L Veras A M S Lima R D C A Melo andJ Ayala ldquo 119887119897119886CTX-M-2 and 119887119897119886CTX-M-28 extended-spectrum120573-lactamase genes and class 1 integrons in clinical isolatesof Klebsiella pneumoniae from Brazilrdquo Memorias do InstitutoOswaldo Cruz vol 105 no 2 pp 163ndash167 2010

[18] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial susceptibility testing fifteenthinformational supplementrdquoDocumentM100-S15 CLSIWaynePa USA 2010

[19] C Mabilat and S Goussard ldquoPCR detection and identificationof genes for extended-spectrum 120573-lactamasesrdquo in DiagnosticMolecular Microbiology Principles and Applications D H Pers-ing T F Smith F C Tenover and T J White Eds pp 553ndash559American Society for Microbiology Washington DC USA1993


[20] J K Rasheed C Jay B Metchock et al ldquoEvolution of extended-spectrum 120573-lactam resistance (SHV-8) in a strain of Escherichiacoli during multiple episodes of bacteremiardquo AntimicrobialAgents and Chemotherapy vol 41 no 3 pp 647ndash653 1997

[21] 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

[22] C H Jones A Ruzin M Tuckman M A Visalli P J Petersenand P A Bradford ldquoPyrosequencing using the single-nucleo-tide polymorphism protocol for rapid determination of TEM-and SHV-type extended-spectrum 120573-lactamases in clinical iso-lates and identification of the novel 120573-lactamase genes 119887119897119886SHV-48119887119897119886SHV-105 and 119887119897119886TEM-155rdquoAntimicrobial Agents andChemother-apy vol 53 no 3 pp 977ndash986 2009

[23] H Yigit A M Queenan G J Anderson et al ldquoNovel carbape-nem-hydrolyzing-lactamase KPC-1 from a carbapenem-resist-ant strain of Klebsiella pneumoniaerdquo Antimicrobial Agents andChemotherapy vol 45 no 4 pp 1151ndash1161 2001

[24] BLAST Basic Local Alignment Search Tool httpblastncbinlmnihgovBlastcgi

[25] G Adamis M G Papaioannou E J Giamarellos-BourboulisP Gargalianos J Kosmidis and H Giamarellou ldquoPharmaco-kinetic interactions of ceftazidime imipenem and aztreonamwith amikacin in healthy volunteersrdquo International Journal ofAntimicrobial Agents vol 23 no 2 pp 144ndash149 2004

[26] M J Ahsman E D Wildschut D Tibboel and R A MathotldquoPharmacokinetics of cefotaxime and desacetylcefotaxime ininfants during extracorporeal membrane oxygenationrdquoAntimi-crobial Agents and Chemotherapy vol 54 no 5 pp 1734ndash17412010

[27] J Monteiro A F Santos M D Asensi G Peirano and A CGales ldquoFirst report of KPC-2-producing Klebsiella pneumoniaestrains in Brazilrdquo Antimicrobial Agents and Chemotherapy vol53 no 1 pp 333ndash334 2009

[28] G Peirano L M Seki V L Val Passos M C F G Pinto L RGuerra and M D Asensi ldquoCarbapenem-hydrolysing 120573-lacta-mase KPC-2 inKlebsiella pneumoniae isolated in Rio de JaneiroBrazilrdquo Journal of Antimicrobial Chemotherapy vol 63 no 2 pp265ndash268 2009

[29] D Sirot C Recule E B Chaibi et al ldquoA complex mutant ofTEM-1 120573-lactamase with mutations encountered in both IRT-4 and extended-spectrum TEM-15 produced by an Escherichiacoli clinical isolaterdquo Antimicrobial Agents and Chemotherapyvol 41 no 6 pp 1322ndash1325 1997

[30] A-GGunkel UHechler andHHMartin ldquoState of penicillin-binding proteins and requirements for their bactericidal inter-action with 120573-lactam antibiotics in Serratia marcescens highlyresistant to extended-spectrum 120573-lactamsrdquo Journal of GeneralMicrobiology vol 137 no 2 pp 243ndash252 1991

[31] C R Deloney and N L Schiller ldquoCompetition of various 120573-lactam antibiotics for the major penicillin-binding proteins ofHelicobacter pylori antibacterial activity and effects on bacterialmorphologyrdquo Antimicrobial Agents and Chemotherapy vol 43no 11 pp 2702ndash2709 1999


reported different bacterial morphological changes caused bysubminimum inhibitory concentrations (sub-MICs) of third-generation cephalosporins and carbapenems in sensitiveisolates of several bacterial species [4ndash6] However there isa lack of studies using resistant isolates

In pathogenic bacteria 120573-lactamase production is themost common and important mechanism of resistance to 120573-lactam antibiotics [2]The classical 120573-lactamases are enzymescapable of inactivating penicillins and narrow-spectrumcephalosporins before reaching their target [7]MostK pneu-moniae isolates carry the gene for chromosomal class A SHV-1 120573-lactamaseThis has limited activity against ampicillin anddoes not hydrolyze extended-spectrum 120573-lactam antibiotics[8] The presence of this gene has been previously studiedin K pneumoniae isolates originating from the microbiota ofindividuals without bacterial infection [9]

The introduction into clinical practice of oxyiminoce-phalosporins for treating infections due to Gram-negativebacteria was soon followed by the appearance of extended-spectrum 120573-lactamases (ESBLs) derived from the classical 120573-lactamases SHV-1 TEM-1 andTEM-2These conferred resis-tance to third-generation cephalosporins and monobactamssuch as aztreonam [2] More than 180 variants of the gene119887119897119886SHV and more than 210 of the gene 119887119897119886TEM have alreadybeen reported [10] Another group of ESBLs which has a highprofile of hydrolysis in relation to cefotaxime and is less than40 identical to the SHV and TEM enzymes is the group ofCTX-M enzymesThese are widely found in different speciesof the family Enterobacteriaceae including K pneumoniae[11]

Treatment of infections due to K pneumoniae isolatesproducing ESBLs is limited to the use of a few available anti-biotics Carbapenems are often the last line of effective treat-ment against these infections [12] However production ofthe class A carbapenemase Klebsiella pneumoniae carbapen-emase (KPC) which is an extended-spectrum 120573-lactamasehas been reported to be the primary form of carbapenemresistance in this bacterial species Fifteen variants with dis-semination in different parts of the world have been reported[10]

Production of these 120573-lactamases by clinical isolates ofKpneumoniae has been a major problem in many countriesand this has hampered the therapeutic options available forthe treatment of bacterial infections caused by this species

The increasing prevalence of multidrug resistance isleading towards the threat that a postantibiotic era may beabout to begin characterized by decreased effectiveness ofcommon antibiotics and routine application of complemen-tary therapeutic approaches for treating bacterial infections[13] Therefore new studies need to be developed in whichold and discarded antibiotics should be reinvestigated andreused Even rejected antibiotics possibly should be usedwhen necessary [14]

In this regard some studies have tried to demonstratethe in vitro and in vivo action of therapeutic combinationsof 120573-lactams and aminoglycosides in MDR K pneumoniaeisolates aiming to obtain new options for treating infectionsdue to this bacterial species [15] However no study hasdemonstrated any alterations induced by different 120573-lactam

antibiotics in the bacterial cell structure of MDR K pneumo-niae isolates

K pneumoniae isolates presenting resistance to cefo-taxime when subjected to sub-MICs of this antibiotic wereshown to cause damage to cell surfaces Filamentationthrough a dose-dependent adaptive process was observedcaused by the stressful environment that was induced by thepresence of the antibiotic thereby contributing towards thetherapeutic effects [16]

Unfortunately knowledge about the effects caused by 120573-lactam antibiotics on the bacterial cell structure of MDRK pneumoniae isolates is still scarce These isolates carryimportant resistance genes such as 119887119897119886SHV 119887119897119886TEM 119887119897119886CTX-Mand 119887119897119886KPC Likewise little is known about the effect of theseantibiotics on isolates from the microbiota of individualswithout bacterial infection Therefore the present studyaimed to characterize the morphological and ultrastructuraleffects on pathogenic and nonpathogenic K pneumoniaeisolates that carry genes encoding the classical 120573-lactamasesESBL andKPC caused by120573-lactam antibiotics that are widelyused in clinical medicine

2 Material and Methods

21 Bacterial Isolates For this study five K pneumoniae iso-lates from Recife PE Brazil were selected (Tables 1 and 2)The K211-F and K581-F isolates were obtained from the fecalmicrobiota of children who did not have bacterial infectionThese isolates showed resistance or intermediate resistanceto ampicillin andor amoxicillin but lacked the 119887119897119886SHV gene(Table 1) [9] Three other K pneumoniae clinical isolatesobtained from public hospitals in Recife were used K3CK16R and K652 The susceptibility profile and presence ofthe genes 119887119897119886SHV and 119887119897119886CTX-M had been determined inprevious studies on the K3C and K16R isolates [9 17] whichshowed resistance to cefotaxime ceftazidime and aztreonamaccording to the interpretive criteria of the Clinical andLaboratory Standards Institute (CLSI) of 2013 [18]

The K3C isolate possesses the 119887119897119886SHV-11 gene and the K16Risolate has the 119887119897119886SHV-1 and 119887119897119886CTX-M2 genes [9 17] The K652isolate which was studied for the first time in the presentwork was obtained by means of surveillance testing on rectalswabs from individuals without clinical symptoms of bacte-rial infection who were admitted to a public hospital in 2011This isolate was identified and its susceptibility profile in rela-tion to different antimicrobials was determined using theVITEK 2 automated system (BioMerieux) It was manuallyconfirmed in relation to cefotaxime ceftazidime and imipe-nem (Sigma-Aldrich) by means of broth macrodilution test-ing in accordance with the 2013 CLSI recommendations [18]

22 DNA and PCR Extraction The genomic DNA of K pneu-moniae isolates was extracted using the Wizard GenomicDNA Purification Kit (Promega) in accordance with the man-ufacturerrsquos instructions The presence of the 119887119897119886TEM gene wasinvestigated by means of PCR in the macrobiota isolates andin the three clinical isolates using the T1 primer 51015840-ATAAAA-TTCTTGAAGACGAAA-31015840 and the T2 primer 51015840-GAC-AGTTACCAATGCTTAATC-31015840 [19] The K652 isolate was






K3C gt256 64 32 32 128 32 16 32 64 32 16 32K16R gt256 64 32 32 32 16 8 16 16 4 4








TEMc SEMd TEMc SEMd








3 Results


32 Presence of bla119878119867119881

bla119879119864119872

and bla119870119875119862



and bla119870119875119862












C

w

(a)

lowast

C

(b)

C lowast

lowast

lowast

(c)

C

(d)

lowast

lowast

lowastC

(e)





C

w

CC

C

C

C

C

C

(a)

(c) (d)

(h)

(f)(e)

(g)

(b)



C

w

(d)

(e)(f)

(a) (c)(b)

(h)(g)



(a) (b)

(c) (d)

(e) (f) (g)

(h) (i)



(a) (b)

(c) (d)







(a) (b) (c)

(d)



4 Discussion





(a)

(c) (b)

lowast












Acknowledgments


References







































K3C gt256 64 32 32 128 32 16 32 64 32 16 32K16R gt256 64 32 32 32 16 8 16 16 4 4








TEMc SEMd TEMc SEMd








3 Results


32 Presence of bla119878119867119881

bla119879119864119872

and bla119870119875119862



and bla119870119875119862












C

w

(a)

lowast

C

(b)

C lowast

lowast

lowast

(c)

C

(d)

lowast

lowast

lowastC

(e)





C

w

CC

C

C

C

C

C

(a)

(c) (d)

(h)

(f)(e)

(g)

(b)



C

w

(d)

(e)(f)

(a) (c)(b)

(h)(g)



(a) (b)

(c) (d)

(e) (f) (g)

(h) (i)



(a) (b)

(c) (d)







(a) (b) (c)

(d)



4 Discussion





(a)

(c) (b)

lowast












Acknowledgments


References



































3 Results


32 Presence of bla119878119867119881

bla119879119864119872

and bla119870119875119862



and bla119870119875119862












C

w

(a)

lowast

C

(b)

C lowast

lowast

lowast

(c)

C

(d)

lowast

lowast

lowastC

(e)





C

w

CC

C

C

C

C

C

(a)

(c) (d)

(h)

(f)(e)

(g)

(b)



C

w

(d)

(e)(f)

(a) (c)(b)

(h)(g)



(a) (b)

(c) (d)

(e) (f) (g)

(h) (i)



(a) (b)

(c) (d)







(a) (b) (c)

(d)



4 Discussion





(a)

(c) (b)

lowast












Acknowledgments


References



































C

w

(a)

lowast

C

(b)

C lowast

lowast

lowast

(c)

C

(d)

lowast

lowast

lowastC

(e)





C

w

CC

C

C

C

C

C

(a)

(c) (d)

(h)

(f)(e)

(g)

(b)



C

w

(d)

(e)(f)

(a) (c)(b)

(h)(g)



(a) (b)

(c) (d)

(e) (f) (g)

(h) (i)



(a) (b)

(c) (d)







(a) (b) (c)

(d)



4 Discussion





(a)

(c) (b)

lowast












Acknowledgments


References



































C

w

CC

C

C

C

C

C

(a)

(c) (d)

(h)

(f)(e)

(g)

(b)



C

w

(d)

(e)(f)

(a) (c)(b)

(h)(g)



(a) (b)

(c) (d)

(e) (f) (g)

(h) (i)



(a) (b)

(c) (d)







(a) (b) (c)

(d)



4 Discussion





(a)

(c) (b)

lowast












Acknowledgments


References



































C

w

(d)

(e)(f)

(a) (c)(b)

(h)(g)



(a) (b)

(c) (d)

(e) (f) (g)

(h) (i)



(a) (b)

(c) (d)







(a) (b) (c)

(d)



4 Discussion





(a)

(c) (b)

lowast












Acknowledgments


References



































(a) (b)

(c) (d)

(e) (f) (g)

(h) (i)



(a) (b)

(c) (d)







(a) (b) (c)

(d)



4 Discussion





(a)

(c) (b)

lowast












Acknowledgments


References



































(a) (b)

(c) (d)







(a) (b) (c)

(d)



4 Discussion





(a)

(c) (b)

lowast












Acknowledgments


References



































(a) (b) (c)

(d)



4 Discussion





(a)

(c) (b)

lowast












Acknowledgments


References



































(a)

(c) (b)

lowast












Acknowledgments


References







































Acknowledgments


References


































Ultrastructural Changes in Clinical and Microbiota ...

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