Acta Manilana 63 (2015), pp. 39–50Printed in the PhilippinesISSN: 0065–1370

© 2015 UST Research Center for the Natural and Applied Sciences, Manila, Philippines

*To whom correspondence should be addressedgrdedeles@yahoo.com

Antibacterial activity of synthesized zinc oxidenanoparticles against urinary tract infectious pathogens

Queency H. Alcantara, Gina R. Dedeles*, & Christina A. Binag

The Graduate School & Research Center for the Natural and Applied Sciences,University of Santo Tomas, 1015 Manila, Philippines

Inadequacy of effective drugs to control infections is a result of antimicrobial resistance. Withthe constraint in antibiotic usage, there is a delusion that there may not be a cure to infectionsthat were once treatable. Employing metal oxide nanoparticles, zinc oxide in particular, towhich pathogens are unlikely to exhibit resistance could be a promising approach. Microwave-assisted technique involving the reaction of zinc acetate dihydrate and ammonium hydroxidewas used in the synthesis of zinc oxide nanoparticles (ZnO NPs). Sensitivity of ZnO nanoparticlesand bulk against isolated uropathogens was determined through agar-well diffusion and timekill assay. Determinations of minimum inhibitory and minimum bactericidal concentrations werebased on absorbance reading at 600 nm. The ZnO NPs viewed with the transmission electronmicroscope were rod-shaped averaging 23.90±2.66 10.73±1.74 nm. Superiority of ZnO NPsover ZnO in bulk can be observed at all test concentrations against the six urinary tract infections(UTI) bacterial pathogens (p<0.05). Of the isolated pathogens, Pseudomonas mendocinawas the most susceptible with 28.67±0.58 mm zone of inhibition at 10 mg/mL ZnO NP. Minimuminhibitory concentration (MIC) and minimum bactericidal concentration (MBC) varied withthe uropathogens. MICs of ZnO NPs were between 0.6–0.9 mg/mL while MBCs were between0.7–4 mg/mL. Time-kill endpoints of the pathogens at MBC of the nanoparticles were 1 h forP. mendocina and Proteus mirabilis, 3 h for Citrobacter freundii and Escherichia coli, and6 h for Klebsiella pneumoniae ssp. rhinoscleromatis. Growth (CFU/mL) of Serratiamarcescens was down to 90% at 6 h, hence, would have reached 99% at 24 h.

Keywords: urinary tract infection, zinc oxide nanoparticles, zone of inhibition, minimuminhibitory concentration, minimum bactericidal concentration, time-kill endpoint

INTRODUCTION

The development of antimicrobial resistance isthe ultimate obstruction in the treatment ofurinary tract infection (UTI). As one of the mostcommon clinical diseases, UTI carries asignificant healthcare burden. Several differentmicroorganisms can cause UTI, including fungi

and viruses, but bacteria are the major causativeorganisms responsible for more than 95% ofcases [1].

The current undertaking in approachingantimicrobial resistance is the utilization ofnanomaterials to which pathogens are unlikelyto exhibit resistance [2]. Although the interestin the study of nanoparticles (NPs) in relationto microorganisms and biomolecules is

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booming, the application aspect still needs tobe explored. Ravikumar et al. [3] had alreadytested against UTI pathogens five metal oxidenanoparticles (MONPs), namely, aluminumoxide (Al2O3), ferric oxide (Fe2O3), ceriumoxide (CeO), zirconium oxide (ZrO), andmagnesium oxide (MgO), but not zinc oxide(ZnO).

Zinc oxide nanostructure is the leading MONPowing to its unique properties and broadapplications [4]. Specifically, the essentialfactor in considering ZnO as antimicrobialagent is its non-toxicity to human cells [5]. Infact, ZnO, like titanium oxide (TiO2),magnesium oxide (MgO), and calcium oxide(CaO), is listed as “Generally Recognized asSafe” (GRAS) by the U.S. Food and DrugAdministration (21CFR182.8991) [6]. It hasshown different behavior towardmicroorganisms compared to the previouslymentioned metal oxides as well as to siliconoxide (SiO2) [7–9]. At low concentrations, iteffectively prevents or considerably reducesthe growth of pathogenic microbes [4],including both Gram-positive and Gram-negative bacteria [9].

Previous studies regarding UTI have providedprofound understanding on its pathogenesis, butno attempt has been done to employ alternativetreatment strategies, therefore, application ofZnO NPs as antimicrobial agents could be apromising approach to overcome antimicrobialresistance [2, 10].

MATERIALS AND METHODS

Synthesis of ZnO nanoparticles. Generalprocedures for the preparation of nanoparticleswere adapted from Kajbafvala et al. [11] withmodifications. A primary solution ofammonium ions was prepared by adding 25 mLammonium hydroxide into 75 mL distilledwater with stirring at room temperature. Withcontinuous stirring, 10 g of zinc acetatedihydrate powder was gradually added until

completely dissolved. The resulting solutionwas then heated in a microwave oven for 2 minand centrifuged at 3300 rpm for 10 min. Theprecipitate was then washed thrice withacetone, then with deionized water, and ovendried at 60ºC for 18 h. Particle size wasdetermined with TEM. The reported data forshape and size (length and width) were basedon solitary particles depicted in the processedTEM micrograph.

Preparation of ZnO nanosuspension. A100 mg ZnO NPs were suspended in 10 mLglycerol with continuous stirring according tothe procedure of Jalal et al. [12]. The ZnOnanosuspension obtained was then placed in awater bath (90ºC) for 30 min to breakaggregates for uniform dispersal [13] and tolessen its thickness. This was diluted furtherwith glycerol down to the differentconcentrations for use in subsequent assaysagainst uropathogens.

Isolation and identification of UTI pathogens.Forty-four fresh urine samples were obtainedfrom two hospitals in Nueva Ecija Provincewith full accord to standard requisitionprotocol. These samples were collected andsubmitted originally by the patients themselvesas commonly practiced in the country forurinalysis. Combined from both sources, 33samples were from female, and 11 from malepatients with 59% of the total aged 18–30years. Samples were collected in batches andall in each batch were stored at 4ºC for 1 hminimum and 8–12 h maximum before directmicrobiological culture in the laboratory toquantify the approximate total bacterial load ofeach sample.

For each urine sample, 1-L loopful ofundiluted aliquots were plated in triplicate onSheep Blood Agar (SBAP) and MacConkeyAgar (MaC) by multiple-interrupted streakingand T-pattern, respectively. SBAP was used fortotal plate count and MAC for Gram-negative

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putative UTI uropathogens. The culture plateswere incubated at 37ºC for 24 h for the initialcounts on SBAP and for as long as 48 h for thefinal counts when no growth on both media wasobserved. Colony forming units per mL (CFU/mL) equal to or greater than 100,000 on SBAPis indicative of UTI.

All isolated uropathogens were subjected to aseries of biochemical tests and results thereofwere used for identifications (IDs) to genericor species level using ID flowcharts inBergey’s Manual of DeterminativeBacteriology. Confirmation of IDs arrived atwas done using received 16S rRNA sequencesgenerated by Macrogen (Korea) that weremanually aligned using Codon Code Aligner v4.0, and neighbor-joining trees for finalidentifications were constructed (BLASTn;http://blast.ncbi.nlm.nih. gov/Blast.cgi).

Agar well-diffusion assay. The assay was donefollowing the procedures of Guven et al. [14]and Hsouna et al. [15] with some modifications.A 24 h-old dense culture in NA slantrepresenting each of isolated species ofuropathogens was flooded with normal salinesolution (NSS, 0.85% NaCl). The cellsuspension was centrifuged with washing insterile-distilled water to rinse off inadvertentNA nutrients and bits of the agar medium thatwould interfere with the assay. The harvestedbiomass was re-suspended in NSS and theconcentration or titer of bacterial cells in theinocula was adjusted to 108/mL by matching to0.5 McFarland Standard.

Bacterial inocula were swabbed evenly on15 mL Mueller-Hinton Agar (MHA) solidifiedto 4 mm depth in 90-mm Petri dishes. Thebacterial lawn was dried partially in a laminarflow before four wells (5 mm) were punchedout with sterile cork borers. The prepared ZnOnanosuspension and bulk in 30 L volumes weremicropipetted into three wells for testconcentrations 5, 8, and 10 mg/mL with

amounts of equal to 0.15, 0.24, and 0.30 mg,respectively. Plain glycerol (30 L) wasdispensed in the 4th well to serve as control.

Assay for MIC and MBC. The method ofVargas-Reus et al. [16] was followed with somemodifications. The concentrations of ZnOnanosuspension in Mueller-Hinton Broth(MHB) tested for minimum inhibitoryconcentration (MIC) and minimum bactericidalconcentration (MBC) against eachrepresentative isolate of uropathogens are asfollows: 4.0–1.0 mg/mL and 0.9–0.3 mg/mL.Aliquot 0.9-mL volumes of each weredispensed in Eppendorf tubes and mixed with0.1 mL fresh cell suspension of uropathogensprepared as above in NSS and calibrated tocontain 108 cells/mL. Final 0.2-mL volumeswere pipetted into three wells (96-wellMicrotiter Plate) per test concentration anduropathogen isolate. Inoculated MHB withoutZnO NPs was placed in “control” wells. Useof Microtiter plates was necessitated by thesmall volumes of assayed cultures.

The optical density (OD) of each mixture wasat 600 nm were read using a Microplate Reader(Corona Electric SH-1000) before and after24 h incubation at 37ºC. A significant increasein OD indicated growth of pathogen, and theconverse, either bacteriostatic (MIC) orbacteriocidal (MBC) effect of testconcentration of ZnO NPs on the uropathogen.Sample aliquots from the wells includingcontrols were streaked on MHA plates whichwere then incubated at 37ºC for 24–48 h todetermine the final MIC and MBC for eachisolate of test uropathogens.

Time kill assay. Procedures for time kill assaywere adapted from Ojo et al. [17] withmodifications. The MBC concentration of ZnONPs for each test uropathogen was prepared intubes with 10 mL MHB. Control tubescontained MHB free of ZnO NPs. The bacterialinoculum (1 mL) was then added, and the

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the species in the Philippines. Ergin & Mutlu[24] recently isolated it with 0.05% distributionrate in a hospital. Though referred to primarilyas an environmental bacterium, Sarris &Scoulica [25] considered it as an opportunistichuman pathogen with type VI secretion system(T6SS) which is said to contribute in itspathogenesis by acting as a nano-syringe.

The isolates of S. marcescens were non-pigmented like most clinical strains [26], i.e.,did not produce in culture the blood-redpigment prodigiosin believed to contribute topathogenicity but the exact role of which is stillunknown. Proteus mirabilis is dimorphic —swarming or motile. Isolates in this study wereof the motile type. The gene FlGn codes forthe initiation of the flagellar filament assemblywhich enables the cells of the motile type toascend the urinary tract and cause UTI [27].

All test uropathogens except Pseudomonas (inPseudomonadaceae) are in familyEnterobacteriaceae, a group known forinherent resistance to most current organicantibiotics. This resistance is due to R-(Resistance-) genes, plasmid-encodedExtended Spectrum -Lactamases (9 ESBLs),and a host of sophisticated operon-mediatedefflux pumps [28–30].

Figure 2. Occurrence percentages of six species of UTIpathogenic bacteria in 30 isolations from urinesamples used in the study.

P. mendocina3%

S. marcescens10%

P. mirabilis7%

K. pneumoniae10%

C. freundii3%

E. coli67%

Table 1. Biochemical featuresa and initial identities of 30 isolates of UTI bacterial pathogens based on Bergey’s Manual of Determinative Bacteriology

Characteristics Escherichia

coli (n=20)

Citrobacter freundii

(n=1)

Klebsiella pneumoniae

(n=3)

Proteus mirabilis

(n=2)

Serratia marcescens

(n=3)

Pseudomonas sp.

(n=1) Growth on MaC LF LF LF NLF NLF NLF Gram staining – – – – – – Oxidase test – – – – – + Indole test + – – – – – Methyl Red test + + + + – – Vogues-Proskauer – – – – + – Citrate test – + + – + – Lys decarboxylase + – + + + + Nitrate test + + + + + + Urease test – – + + – – TSI slant test A A A K A K Butt A A A A A K Gas + – + – + – H2S Prod. – + – + + – Motility + + – + + –

aPositive (+); Negative (–); Alkaline (K); Acid (A); Lactose fermentative (LF); Non-lactose fermentative NLF).

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Antibiosis on uropathogens

Agar well-diffusion. Most noticeable off handwas the clear overall superiority of ZnO NPover ZnO in bulk at all test concentrationsagainst the six UTI bacterial pathogens(p<0.05), except on C. freundii at 5 mg/mLwhere both formed no zone of inhibition (ZOI),as did all control wells with glycerol only(p>0.05). Near equipotency by NP and bulkwas evident only at 8 mg/mL of the former and10 mg/mL of the latter with K. pneumonia, E.coli, P. mirabilis, and S. marcescens.

The ZOI for both NP and bulk particles (BP)increased, hence, the susceptibility ofpathogens, with concentrations. However,whereas such increases in ZOI with NP werecontinuous for all concentrations andpathogens, those with BP were limitedapparently to P. mendocina at 5–8 mg/mL andE. coli at 8–10 mg/mL (Table 2). To both ZnONP and BP the most susceptible among theuropathogens was P. mendocina, and to the BP,S. marcescens was the most resistant.

For ZnO NP alone, ZOI differed for P.mendocina, E. coli, and C. freundii at 5 mg/L(0–12 mm) and 8 mg/mL (6–16 mm) (p<0.05)but not for P. mirabilis, K. pneumonia, and S.marcescens, 6–11 mm and 7–12 mm also atthe same concentration levels, respectively(p>0.05). All test uropathogens, however, had

ZOI 3-13 mm higher at 10 than at 8 mg/mL(p<0.05). And these may be ranked as followsfrom most susceptible to most resistantrelative to the sum of ZOI for all testconcentrations — P. mendocina, P. mirabilis,K. pneumoniae, S. marcescens = E. coli > C.freundii.

For ZnO BP, increases in ZOI for the mostsusceptible pathogen, P. mendocina, differedfrom 5 to 8 and on to 10 mg/L (p<0.05). Thesum of ZOI for 8 and 10 mg/mL would makeE. coli second in susceptibility. For the otherfour pathogens at 10 mg/mL, K. pneumoniae,P. mirabilis and C. freundii were equallysusceptibility (10–11 mm ZOI), and S.marcescens was resistant.

In Table 3 are the equivalent qualitativeexpressions of quantitative results (ZOImeasures) in Table 2. These are as given inQuinto & Santos [31] as follows: (1) >19 mm,very active; (2) 13–19 mm, active; (3) 10–12mm, partially active; (4) <10 mm, inactive.Corresponding equivalent rankings, given the1-mm variance from a lower rank to the nexthigher rank and vice versa, may not be in fullagreement with the statistical analyses of thedata in Table 3. Nevertheless, the tabulationhighlighted again the overall superiority of ZnONP over ZnO in bulk form in antibiosis on testbacterial uropathogens.

Table 2. Inhibition zones (mm) in MHA agar well (5 mm)-diffusion assay of ZnO, NP, and ZnO bulk against UTI pathogens (n=3)a

Uropathogens ZnO NP (mg/mL) ZnO Bulk (mg/mL) 5 8 10 5 8 10

Pseudomonas mendocina 11.670.58 15.670.58 28.670.58 8.660.58 11.670.58 24.670.58 Klebsiella pneumoniae ssp.

rhinoscleromatidis 9.330.58 10.001.00 14.001.00 0 0 9.670.58

Escherichia coli 6.330.58 8.330.58 11.330.58 0 6.330.58 8.660.58 Proteus mirabilis 11.330.58 11.670.58 16.001.00 0 0 10.670.58 Serratia marcescens 6.330.58 7.000.00 12.000.00 0 0 6.670.58 Citrobacter freundii 0 6.330.58 12.001.00 0 0 10.330.58

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Mortality results in Jiang et al. [13] stronglysuggested that NPs effectivity varies with testorganism, oxide composition andconcentration, and experimental condition.Thus, Heinlaan et al. [32] found no significantdifference in toxicity of NPs and BPs ZnO andTiO2 to Vibrio fischeri and two crustaceans, asdid Adams et al. [9] for NPs of both oxidesand SiO2 to B. subtilis and E. coli, and alsoFranklin et al. [33] for ZnO NP to thefreshwater microalga Pseudokirchneriellasubcapitata. In Collins et al. [34], in situincorporation in soil of NPs CuO and ZnOnegatively affected diversity of bacterial taxain Flavobacteriales and Sphingomonadales butthose in Rhizobiales proved more resilient. Itwas the opposite in Ge et al. [35]; impact ofNP ZnO was positive on taxa in theSphingomanadales and Streptomycetacetalesand negative on those in molecular nitrogen-fixing Rhizobiales and methane-oxidizingMethylobacteriaceae.

The glycerol-ZnO NPs was colloidal but themixture (0.03 mL) had dried up already whenthe agar-well assay plates were examined forZOI measurement. The ZnO NPs and BPs,although not examined directly, must haveaggregated in the agar wells within the 24-h

period of incubation at 37ºC. That ZOI formedwithin this time meant rapid expression oftoxicity. In Jiang et al. [13], results wereobtained after only 2 h in tubes with 1 g/L NaCl.Nevertheless, gradual drying of glycerol in theincubator in this study could have affected theextent of growth suppression of theuropathogens with different growth rates. ZnONPs similarly aggregated during the dryingprotocol in TEM (Fig. 1A). The clusters, alsoin Fig. 1A (not circled), were not much largerbut with greater surface areas than solitary NPs.Aggregation occurred also in other studies, thesizes equal for NPs [9] and larger for NPs thanBPs [13].

Glycerol could have affected also the extentsof growth suppression of the uropathogens inthe agar-well setups. A sugar alcohol, glycerolis used as sole carbon and energy source forbiomass buildup at least by S. marcescens [36]and C. freundii [37]. Such growth induction byglycerol could probably account for delayednegative effect, hence, both pathogens ranked“most resistant” to both ZnO NPs and BPs. ZOIscores of “0” or “inactive” at 5 and 8 mg/mLimproved quantitatively and qualitatively onlyat 10 mg/mL (Table 2 and Table 3).

Table 3. Relative bioactivitya of ZnO NP and ZnO bulk equivalent to average ZOI values (excluding standard deviation)

for uropathogens in Table 2.

Uropathogens ZnO NP (mg/mL) ZnO Bulk (mg/mL) 5 8 10 5 8 10

Pseudomonas mendocina Partially active Active Very

active Inactive Partially active

Very active

Klebsiella pneumoniae ssp. rhinoscleromatidis Inactive Partially

active Active 0 (Inactive)

0 (Inactive)

Partially active

Escherichia coli Inactive Inactive Partially active

0 (Inactive) Inactive Inactiveb

Proteus mirabilis Partially active

Partially active Active 0

(Inactive) 0

(Inactive) Partially active

Serratia marcescens Inactive Inactive Partially active

0 (Inactive)

0 (Inactive) Inactive

Citrobacter freundii 0 (Inactive) Inactive Partially

active 0

(Inactive) 0

(Inactive) Partially active

aAfter Quinto & Santos (2005); bConsidered “partially active” in text above

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MIC and MBC. As a rule, turbidity of mediumis indicative of bacterial growth readily visibleto the naked eye. However, this dictum is notapplicable to this study since the ZnOnanoparticles also contributed to the turbidityof the medium. Moreover, only 0.2 mL totalvolume of NP suspension and inoculated MHB(1:9, v/v) was in the wells of microtiter platesused in the assay. To solve this problem, opticaldensity (OD) at 600 nm was measured. OD600is typically used to measure bacterial cells. Nosignificant difference between the first andsecond OD readings is suggestive of inhibitionor bactericidal activity. Results are in Table 4.

Bactericidal activity was verified by theabsence of growth when streaked onto NAplates. The least concentration without growthwas considered the MBC while the least

concentration with growth but no significantdifference between the initial and final readingswas considered the MIC (Fig. 3).

Compared with previous studies on ZnO NP,variations were even among test strains. For thechoice test species E. coli in particular, MICat 600 g/mL in this study was higher inincreasing amounts compared to 500, 400, and>0.5 g/mL as determined respectively viaredox by resazurin incorporation [5],turbidimetry [6] and microdilution. Similarly,MBC for E. coli in this study is at 2000 g/mLwhich is also higher in increasing amounts than0.016 and 800 g/mL obtained respectively byEmami-Karvani & Chehrazi [38] who used agardilution and by Wang et al. [39]. But it is notsurprising for the values for antibiosis to differeither way. The methods, preparation protocols,

Table 4. MIC-MBC of ZnO NPs on uropathogens by spectrophotometry (OD600) before and after 24 h incubation in MHB (1:9 v/v) (n=3).

Pathogens ZnO NPs Concentration (mg/mL) Control 4 3 2 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3

Pseudomonas mendocina

IAbs 0.03 0.45 0.42 0.36 0.16 0.17 0.17 0.15 0.09 0.06 0.05 0.05

FAbs 0.05* 0.46 0.46 0.37 0.17 0.17 0.17 0.15 0.11 0.08* 0.06* 0.06*

Klebsiella pneumoniae ssp. rhinoscleromatis

IAbs 0.05 0.47 0.40 0.30 0.16 0.15 0.14 0.14 0.09 0.06 0.04 0.04

FAbs 0.28*

0.47 0.40 0.30 0.16 0.15 0.14 0.14 0.11* 0.08* 0.07* 0.07*

Escherichia Coli

IAbs 0.04 0.49 0.46 0.31 0.17 0.17 0.14 0.16 0.09 0.05 0.03 0.03

FAbs 0.35* 0.47 0.45 031 0.20 0.20 0.16 0.17 0.10 0.08* 0.07* 0.07*

Proteus mirabilis

IAbs 0.04 0.43 0.41 0.32 0.16 0.16 0.14 0.14 0.07 0.05 0.03 0.03

FAbs 0.30*

0.46 0.44 0.35 0.21 0.20 0.19 0.18 0.11* 0.09* 0.08* 0.07*

Serratia marcescens

IAbs 0.05 0.46 0.46 0.35 0.19 0.17 0.10 0.11 0.05 0.03 0.01 0.01

FAbs 0.49* 0.47 0.47 0.38 0.28 0.26 0.20* 0.18* 0.14* 0.11* 0.09* 0.12*

Citrobacter freundii

IAbs 0.04 0.42 0.42 0.33 0.16 0.15 0.13 0.13 0.07 0.02 0.01 0.01

FAbs 0.27*

0.41 0.40 0.32 0.15 0.15 0.12 0.19* 0.15* 0.12* 0.12* 0.05*

*significantly different at 0.05 level of confidence = growth increase; MBC; MIC; IAbs initial absorbance reading; FAbs final absorbance reading

Antibacterial activity of synthesized zinc oxide nanoparticles

47

exposure conditions and other technicalities inMIC and MBC determination contrast witheach other. Moreover, strains of the samespecies can differ in resistance orsusceptibility to a given antimicrobial. Aclinical strain from UTI was used in this studywhile in others, focus was on food pathogen E.coli 0157:H7 (EHEC) [10] and referencestrains from culture collections, e.g., K88 fromthe Persian Type Culture Collection (PTCC)[50] and a strain from MTCC (Microbial TypeCulture Collection) in India [5].

There is no previous report on the response ofK. pneumoniae ssp. rhinoscleromatis andPseudomonas mendocina to any metal oxideNP. MIC/MBC values for these twouropathogens with ZnO NP in this study were700/2000 g/mL and 600/700 g/mL,respectively. The MIC for the former compared

closed to 500 g/mL in Ansari et al. [40] but notto either 5.0 g/mL in Wahab et al. [41] or>5.0 g/mL in Yousef & Danial [42] for whatare presumed here to be strains of the type ssp.pneumoniae. The MIC for the non-fluorescentP. mendocina was distant to >0.5 g/mL forits fluorescent congener P. aeruginosalikewise in Yousef & Danial [42]. For pathogensP. mirabilis, C. freundii, and S. marcescens thecorresponding MIC/MBC reported bySangeetha et al. [43] are as follows — 1.0/7.8,1.8/10.2 and 1.4/8.4 mM. These compare with700/1000, 800/3000, and 900/4000 g/mL,respectively for clinical strains in this study.Notable to account for the variations, inaddition to strain differences, was the methodof synthesis of ZnO NPs. Wahab et al. [41]used the non-hydrolytic solution route.Sangeetha et al. [43] method was biologicalwith the use of Aloe vera leaf broth. The oneby Yousef & Danial [42] was not specified, andthe NPS used by Ansari et al. [40] weresurface-modified for biomedical application.These methods and others yield NPS withdifferent morphologies which are knownconstitute a major determinant in interactionwith target bacterial cells [13].

Time kill endpoints. The rate at which abacterial isolate of known cell density is killedis a major consideration in the development ofantibacterial agent. This would demonstrate theefficacy of the material at a given time. Andwith its probable mode of action known, thelink between concentration and its desiredeffect can be established.

Bactericidal effect is characterized by a 3-logdecrease in CFU and can be determined at 6 h.A 90% kill at this time equates to 99% kill at24 h. In this study bactericidal activity of ZnONPs at pre-determined MBC concentration inMHB (Table 5) was monitored at 0 (pre-incubation), 1, 3, and 6 h. Confluent growth andtoo-numerous-to-count (TNTC) colonies insubsequent plate counts in MHA (10–4 dilution)

Figure 3. Presence-absence of growth in NA plates after24–48 h incubation. Numbers in sectors correspondto assayed ZnO NP concentrations in Table 4.

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have no numerical values and, for graphicalpresentation, these were assumed to be 400 and300 colonies, respectively.

CFU averages for each of the six uropathogenwere plotted against time. Time kill curves forall were combined in one graph for clearercomparison (Fig. 4). Growth variations werenoticeable even at pre-incubation time (time0). Four (K. pneumoniae ssp.rhinoscleromatis, P. mirabilis, S. marcescens,and C. freundii) already had confluent or TNTCcounts, while P. mendocina and E. coli had only124 and 133 colonies, respectively. However,time kill endpoints do not depend on the initialcounts of colonies. Thus, P. mirabilis withTNTC at the beginning gave shorter time killendpoint (1 h) compared to E. coli (3 h). Slow-grower P. mendocina also scored kill endpointat 3 h. Both S. marcescens and K. pneumoniaespp. rhinoscleromatis proved resilient. Growthat 3 h remained confluent in the former and wasdown to 17 colonies in the latter. Kill endpointfor the subspecies was reached at 6 h, at whichtime when the monitoring was ended, however,growth of S. marcescens was still present inMHA plate, though this was down to only 16colonies. Nevertheless, complete eradicationof S. marcescens is expected thereafter within24 h from 0 time since percent kill at 6 h was

more than 90% of the previous hour. Growth incontrol plates for all uropathogens (not shown)remained consistent either as confluent or TNTCthroughout the monitoring period.

Overall, time kill endpoints for the uropathogensunder the conditions in this study increased inthe order P. mirabilis = P. mendocina > E. coli= C. freundii > K. pneumoniae ssp.rhinoscleromatis > S. marcescens. Perhaps notsurprising, this trend corresponds well withincreases in MBC levels for the pathogens inthe same order, with the exception of E. coli: P.mendocina > P. mirabilis > K. pneumoniae ssp.> E. coli = C. freundii = S. marcescens (Table4). Growth rate is inversely proportional to levelof susceptibility to NPs as well as to antibiotics.This implies that slow-growers are moresusceptible than fast growers and vice versa.The results suggest this to be true in reversefor fast-grower E. coli (doubling time =~20 min) and probably also for the others.However, direct growth rate-time kill endpointrelationship was not studied.

CONCLUSION

ZnO NP has proved to have more efficientantibacterial agent against its bulk form. Atconcentrations much lower than the requiredhuman need, it exhibited an active activityagainst UTI pathogens. Thus, ZnO NP can be apotential antibacterial agent, granted thatclinical trials are made.

ACKNOWLEDGMENT

The authors would like to express gratitude tothe Department of Science and Technology –Science Education Institute for funding thisresearch.

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