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Pharmaceutical wastewater discharged into open drainage channels around hospitals and pharmaceutical facilities may serve as an important reservoir of multidrug-resistant bacteria and other contaminants of public health concern. This study evaluated the antimicrobial potential of selected medicinal plant extracts against multidrug-resistant bacterial isolates recovered from pharmaceutical-contaminated drainage water in Kano State, Nigeria. Wastewater samples were collected from open drains located outside Aminu Kano Teaching Hospital, Murtala Mohammed Specialist Hospital, Sabon Gari Pharmaceutical Market, and pharmaceutical facilities in Sharada Industrial Estate. Physicochemical parameters of the wastewater were determined using standard methods. Bacteria were isolated, identified by biochemical characterisation and 16S rRNA confirmation, and tested for antibiotic susceptibility using the Kirby–Bauer disc diffusion method. Aqueous and ethanolic extracts of Azadirachta indica, Moringa oleifera, Vernonia amygdalina, Psidium guajava, Allium sativum, and Zingiber officinale were prepared and evaluated against the multidrug-resistant isolates. The wastewater samples showed elevated levels of electrical conductivity (1215–1582 µS/cm), biochemical oxygen demand (48.7–73.5 mg/L), and chemical oxygen demand (108.5–152.3 mg/L), exceeding recommended limits. Escherichia coli was the most prevalent bacterial isolate, accounting for 29.8% of all isolates, followed by Staphylococcus aureus (21.1%) and Pseudomonas aeruginosa (18.5%). Pseudomonas aeruginosa exhibited the highest multidrug resistance rate (71.2%), with marked resistance to ampicillin (92.7%) and tetracycline (75.6%). Among the plant extracts, ethanolic extracts were more active than aqueous extracts. Garlic ethanolic extract showed the strongest antimicrobial activity, producing inhibition zones of 26.8 ± 1.7 mm against Escherichia coli, 29.4 ± 1.6 mm against Staphylococcus aureus, and 23.1 ± 1.4 mm against Pseudomonas aeruginosa. Guava leaf ethanolic extract also demonstrated substantial antibacterial activity. The minimum inhibitory concentration of garlic extract ranged from 6.25 to 25 mg/mL, while the minimum bactericidal concentration ranged from 12.5 to 50 mg/mL. The findings indicate that pharmaceutical-contaminated drainage water in Kano State contains highly resistant bacteria and that selected medicinal plants, particularly Allium sativum and Psidium guajava, possess strong antimicrobial activity against these organisms. The study suggests that these plants may provide low-cost alternative agents for the control of multidrug-resistant bacteria associated with environmental contamination.pharmaceutical wastewater
Keywords: pharmaceutical wastewater; multidrug-resistant bacteria; medicinal plants; antimicrobial activity; Kano State; garlic extract; guava leaf
tubewasinoculatedwithastandardisedbacterialsus- Thephysicochemicalpropertiesofthepharmaceuticalpensionandincubatedat37°Cfor24h. Thelowest contaminatedwaterrevealedseriouspollutionacross concentrationthatshowednovisiblebacterialgrowth all sampling sites. The Sabon Gari Pharmaceutical wasrecordedastheminimuminhibitoryconcentration. Marketdrainrecordedthehighestelectricalconductiv- Todeterminetheminimumbactericidalconcentration, ity(1582±47µS/cm),totaldissolvedsolids(785±26 aliquotsfromtubesshowingnovisiblegrowthwere mg/L),biochemicaloxygendemand(73.5±4.1mg/L), inoculated onto nutrient agar plates and incubated. andchemicaloxygendemand(152.3±6.7mg/L).These Thelowestconcentrationthatproducednobacterial valuesexceededtheWHOpermissiblelimits,indicating growthontheagarwasrecordedastheminimumbachighlevelsofdissolvedpollutantsandorganicmatter. tericidalconcentration.
Dissolvedoxygenvalueswerelowatallsites,ranging from2.4±0.1to3.5±0.3mg/L,suggestingpoorwater
qualityandinadequateaerationasshowninTable1 Allexperimentswerecarriedoutintriplicate,andthe Escherichiacoliwasthemostprevalentbacterialisoresultswereexpressedasmean±standarddeviation. Data was analysed using the Statistical Package for SocialSciencesversion25.Thenormalityofthedata wastestedusingtheShapiro–Wilktest,whilehomogeneityofvariancewasassessedusingLevene’stest. One-wayanalysisofvariancewasthenusedtocomparemeansamongthesamplinglocationsandplant extracts.Duncan39;smultiplerangetestwasusedto separatesignificantlydifferentmeans.Statisticalsignificancewasacceptedatp<0.05.Thepresentreview lateacrossallsites,withameanoccurrenceof29.8%, wasconductedusingacomprehensiveandsystematic followedbyStaphylococcusaureus(21.1%)andPseuevaluationofpublishedscientificliteraturerelatedto domonasaeruginosa(18.5%).Thehighestpercentage metagenomicanalysisofsoilmicrobialcommunities ofE.coliwasobservedintheSabonGaridrain(33.2%), underregenerativeandconventionalagriculturalsysindicatingsignificantfaecalcontamination.Enterococtems.Relevantpeer-reviewedarticleswereretrieved cusfaecaliswasmoreabundantinthereceivingstream fromelectronicdatabasesincludingPubMed,Scopus, (23.7%),suggestingthatcontaminationfromthedrains WebofScience,andGoogleScholarusingkeywords extendsintodownstreamwaterbodies,asshowninTasuchas“soilmetagenomics,”“regenerativeagriculture ble2.
microbiome,” “conventional farming soil microbes,” “16SrRNAsequencing,”“shotgunmetagenomics,”and Theantibioticresistancepatternshowedthatthebac- “soilmicrobialdiversity”(17,18). Studiespublished terialisolateswerehighlyresistanttocommonlyused between2005and2025werepreferentiallyincluded antibiotics. Pseudomonasaeruginosaexhibitedthe tocapturerecentadvancementsinnext-generationsehighestmultidrugresistance(71.2%),followedbyKlebquencingtechnologiesandmicrobialecology. Inclusiellapneumoniae(66.5%)andEscherichiacoli(64.8%). sioncriteriacomprisedstudiesinvestigatingsoilmi- ResistancetoampicillinwashighestinPseudomonas crobialdiversity,functionalgeneprofiling,microbial aeruginosa(92.7%),whiletetracyclineresistanceexabundance,antimicrobialresistancegenes,andecoceeded60%inallisolates.Gentamicinshowedcomparlogicalfunctionsunderregenerativeandconventional ativelylowerresistancevalues,indicatingthatitmay agriculturalpractices.Studiesbasedsolelyonculturestillretainsomeeffectivenessagainsttheorganisms, dependenttechniqueswereexcludedbecauseoftheir asshowninTable3 fromtheplants,asshowninTable5.
Theantimicrobialactivityofgarlicethanolicextract againstPseudomonasaeruginosaincreasedwithconcentration. Theinhibitionzonerosefrom10.2±0.8 mmat25mg/mLto23.1±1.4mmat100mg/mL.This concentration-dependentrelationshipindicatesthat higherconcentrationsoftheextractprovidestronger antibacterialactivity,asshowninTable6. Themini-
Among the aqueous plant extracts, garlic demonstratedthegreatestantimicrobialactivity,producing inhibitionzonesof21.4±1.5mmagainstE.coliand24.8 ±1.4mmagainstStaphylococcusaureus.Guavaleaf muminhibitoryconcentrationandminimumbactericiextractwasthesecondmosteffective,whileMoringa dalconcentrationresultsconfirmedthatgarlicethanooleiferashowedtheweakestantibacterialeffect.Pseulic extract was the most potent plant extract. The domonasaeruginosawasgenerallylesssusceptible lowestMICvalue(6.25mg/mL)wasobservedagainst totheextractsthantheotherorganisms,asshownin Staphylococcusaureus, whilethelowestMBCvalue Table4.
(12.5mg/mL)wasalsorecordedforthesameorganism. Pseudomonasaeruginosarequiredhigherconcentrationsoftheextract,confirmingitsgreaterresistance, asshowninTable7.
Theethanolicextractsweremoreeffectivethanthe aqueousextractsagainstallbacterialisolates.Garlic ethanolicextractshowedthehighestantimicrobialactivity,withinhibitionzonesof26.8±1.7mmagainst The comparison between medicinal plant extracts E.coli,29.4±1.6mmagainstStaphylococcusaureus, andconventionalantibioticsshowedthatgarlicand and23.1±1.4mmagainstPseudomonasaeruginosa. guava ethanolic extracts produced larger inhibition Guavaleafethanolicextractalsodemonstratedstrong zonesthanciprofloxacinandgentamicinagainstthe antibacterialactivity. Theenhancedperformanceof multidrug-resistantbacteria.Garlicethanolicextract ethanolicextractssuggeststhatethanolwasmoreeffirecordedthehighestinhibitionzoneagainstStaphylocientinextractingtheactiveantimicrobialcompounds coccusaureus(29.4±1.6mm),whereasciprofloxacin produced only 20.4 ± 1.3 mm. These findings suggestthatselectedmedicinalplants,especiallygarlic andguavaleaf,mayprovideeffectivealternativetreatmentsformultidrug-resistantbacterialinfections,as showninTable8.
Thephysicochemicalpropertiesofthedrainagewater indicatedsubstantialenvironmentalcontaminationat allthesamplingsites.Electricalconductivity,totaldissolvedsolids,biochemicaloxygendemand,andchemicaloxygendemandexceededrecommendedlimits, particularlyintheSabonGariPharmaceuticalMarket drain.Suchelevatedvaluessuggestthatthedrainage channelscontainedlargequantitiesofdissolvedsalts, organicmatter,suspendedsolids,andpharmaceutical residues. Akpor and Muchie (2011) similarly reportedthatuntreatedwastewaterfromurbanenvironmentsisoftencharacterisedbyhighbiochemicaloxygendemandandchemicaloxygendemandbecause ofthepresenceofdecomposableorganicsubstances. Kayode-Afolayanetal.(2022)furthernotedthatpharmaceuticaleffluentsfrequentlydegradewaterquality by increasing organic and chemical pollution loads. Thelowdissolvedoxygenconcentrationsrecordedin thepresentstudyindicatepoorecologicalqualityof the drainage water. Dissolved oxygen values below 5mg/Laregenerallyconsideredunsuitableformany aquaticorganismsbecausesuchconditionsreflectintensemicrobialdecompositionandhighoxygendemand. SimilarobservationswerereportedbyAkpor andMuchie(2011),whofoundthatpollutedwastewatersystemsoftenexhibitlowdissolvedoxygendue toexcessiveorganicloading(Okpojietal.,2025).The lowoxygenvaluesobservedinthedrains,therefore, suggest that the receiving streams may be exposed tosignificantenvironmentalstress. Escherichiacoli was the most abundant bacterial isolate recovered from the wastewater. The predominance of this organismsuggestsconsiderablefaecalcontaminationof thedrainagesystems,probablyresultingfromhospital sewage, domestic waste, and poor sanitary conditions around the sampling sites. Similar findings havebeenreportedinwastewaterstudieswhereEscherichiacoliwasidentifiedasthemajorindicatororganismoffaecalpollution(Nibamureke Barnhoorn, 2025). The high occurrence of Escherichia coli also raisesconcernbecausetheorganismcantransferresistancegenestootherbacteriaintheenvironment.The occurrenceofStaphylococcusaureus,Pseudomonas aeruginosa,Klebsiellapneumoniae,andEnterococcus faecalisinthedrainagewaterisofconsiderablepublic healthimportance.Thesebacteriaarecommonopportunisticpathogensassociatedwithhospitalenvironmentsandhavebeenimplicatedinsevereinfections, including septicemia, wound infections, respiratory tractinfections,andurinarytractinfections.Kayode- Afolayanetal.(2022)observedthathospitalwastewaterfrequentlycontainssuchpathogenicmicroorganismsbecauseofcontaminationfromclinicalactivities and pharmaceutical discharges. Among the bacterialisolates,Pseudomonasaeruginosaexhibitedthe highestmultidrugresistance. Theorganismshowed particularlyhighresistancetoampicillinandtetracycline,indicatingthatithasdevelopedstrongadaptive mechanismsagainstconventionalantibiotics. Pseudomonasaeruginosaisnaturallyresistanttoseveral antibioticsbecauseofitsabilitytoproducebiofilms,effluxpumps,and-lactamaseenzymes.Theseresistance mechanismsreducethepenetrationandeffectiveness ofantibiotics,makingtheorganismdifficulttoeradicate.SimilarobservationsweremadebyJaishankaret al.(2014),whoexplainedthatpersistentcontaminants andenvironmentalstresscanstimulatebacterialresistance.KlebsiellapneumoniaeandEscherichiacolialso demonstrated considerable resistance to the tested antibiotics. Theirhighresistancemaybeassociated withplasmid-mediatedresistanceandthepresence ofextended-spectrum-lactamasegenes.Accordingto Pateletal.(2019),pharmaceuticalresiduespresentin wastewatercreateselectivepressurethatencourages thesurvivalandmultiplicationofresistantmicroorganisms. Li et al. (2024) also emphasised that wastewatercontaminatedbyantibioticsprovidesidealconditionsforthespreadofmultidrug-resistantbacteria. Thehighfrequencyofmultidrugresistanceobserved inthisstudymaythereforebeattributedtothecontinuousexposureofbacteriatoresidualantibioticsin thepharmaceutical-contaminateddrains.Whensusceptiblebacteriaarerepeatedlyexposedtolowconcentrationsofantibiotics,theyareeliminatedwhile resistantorganismssurviveanddominate.Overtime, theseresistantbacteriabecomeestablishedwithinthe microbialcommunityandmaytransfertheirresistance genestootherorganisms.Obinnaetal.(2023)similarly concluded that untreated pharmaceutical wastewaterisamajorenvironmentalsourceofantimicrobial resistance.Themedicinalplantextractsexaminedin thisstudyshowedvaryingdegreesofantibacterialactivityagainstthemultidrug-resistantbacteria.Ingeneral,theethanolicextractsproducedlargerinhibition zonesthantheaqueousextracts.Thisfindingmaybe explainedbythegreaterabilityofethanoltoextractactivephytochemicalsfromplantmaterials.Flavonoids, alkaloids,tannins,terpenoids,phenolics,andessential oilsaregenerallymoresolubleinethanolthaninwater. Consequently,ethanolicextractsareoftenmoreconcentratedandmoreeffectivethanaqueouspreparations.Amongalltheplantstested,garlicexhibitedthe strongestantibacterialactivity.Theethanolicextractof garlicproducedthehighestinhibitionzonesagainstEscherichiacoli,Staphylococcusaureus,Pseudomonas aeruginosa,andKlebsiellapneumoniae. Thestrong activityofgarlicmaybeattributedtothepresenceofallicin,ajoene,diallylsulfide,andothersulfur-containing compounds.Thesesubstancesarecapableofdisruptingbacterialcellwalls,alteringmembranepermeability,inhibitingproteinsynthesis,andsuppressingessentialmetabolicpathways.Theeffectivenessofgarlic against both Gram-positive and Gram-negative bacteria agrees with previous findings reported by Patel et al. (2019). The inhibitory effect of garlic was particularlypronouncedagainstStaphylococcusaureus.ThisobservationmaybeduetothegreatersusceptibilityofGram-positivebacteriatoplant-derived compoundsbecausetheypossesssimplercellwalls thanGram-negativebacteria.Nevertheless,garlicalso showedconsiderableactivityagainstPseudomonas aeruginosa,despitethehighresistanceofthisorganism to conventional antibiotics. This suggests that garlicactsthroughmechanismsthatdifferfromthose ofsyntheticantimicrobialagents. Guavaleafextract also demonstrated substantial antibacterial activity andrankedsecondonlytogarlic. Theeffectiveness of guava leaves may be linked to their high concentrationsoftannins,flavonoids,quercetin,andother phenoliccompounds.Thesephytochemicalscandenaturebacterialproteins,damagecellmembranes,and inhibitnucleicacidsynthesis.Similarantibacterialactivityofguavahasbeenreportedinstudiesofmedicinalplantsusedagainstresistantpathogens(Obinnaet al.,2023).Neem,ginger,bitterleaf,andmoringaalso showed measurable antibacterial activity, although theirinhibitionzoneswerelowerthanthoseofgarlic andguava. Neemcontainsazadirachtinandnimbin, whilegingercontainsgingerolandshogaol.Bitterleaf andmoringaarealsorichinphytochemicalscapable ofinhibitingmicrobialgrowth.Theloweractivityobservedintheseplantsmayberelatedtodifferences intheconcentration,stability,orsolubilityoftheiractiveconstituents. Theincreaseinantibacterialactivitywithincreasingconcentrationofgarlicextractindicatesaconcentration-dependenteffect.TheinhibitionzoneagainstPseudomonasaeruginosaincreased progressivelyfromthelowesttothehighestconcentrationtested. Thispatternconfirmsthatthephytochemicalsingarlicexertstrongerantibacterialactivityathigherdoses.Similarconcentration-dependent relationshipshavebeenreportedforseveralmedicinalplantextracts(Pateletal., 2019). Theminimum inhibitory concentration and minimum bactericidal concentrationvaluesobtainedinthepresentstudyfurtherconfirmthepotencyoftheplantextracts.Garlic requiredthelowestconcentrationstoinhibitandkill thebacterialisolates,whileguavaalsoproducedrelativelylowinhibitoryconcentrations.Incontrast,Pseudomonasaeruginosarequiredhigherconcentrations oftheextractsthantheotherorganisms,reflectingits greaterresistance.Animportantfindingofthisstudy isthatthemosteffectivemedicinalplantextractsproducedlargerinhibitionzonesthanconventionalantibioticssuchasciprofloxacinandgentamicin. This suggeststhatmedicinalplantsmayprovideusefulalternativeswheresyntheticantibioticsareineffective. Inlow-resourcesettingswhereaccesstoantibiotics islimitedandantimicrobialresistanceiswidespread, medicinalplantsmaythereforeserveasaffordableand sustainabletreatmentoptions(Obinnaetal.,2023;Pateletal.,2019).
Thepresentstudydemonstratedthatwastewaterdischargedintodrainagechannelsoutsidehospitals,pharmaceuticalmarkets,andpharmaceuticalfactoriesin KanoStateisheavilypollutedandcontainssignificant numbersofmultidrug-resistantbacteria.Theelevated valuesofelectricalconductivity,biochemicaloxygen demand,chemicaloxygendemand,andtotaldissolved solidsindicatepoorwaterqualityandconfirmthatthe drainsreceivesubstantialquantitiesofuntreatedpharmaceutical and organic wastes. The predominance ofEscherichiacoli,Pseudomonasaeruginosa,Staphylococcusaureus,andKlebsiellapneumoniaefurther showsthatthesedrainagesystemsfunctionasimportant reservoirs of pathogenic microorganisms. The highresistanceofthesebacteriatocommonlyused antibiotics supports previous findings that pharmaceuticalresiduesinwastewatercreateselectivepressure that promotes the development and spread of antimicrobialresistance. Thestudyalsoestablished thatmedicinalplantspossessconsiderablepotential asalternativeantimicrobialagentsagainstmultidrugresistantbacteria.Ethanolicextractsweremoreeffectivethanaqueousextracts,whilegarlicandguavaleaf extractsexhibitedthestrongestantibacterialactivity. Theabilityoftheseplantextractstoproducelargerinhibitionzonesthanconventionalantibioticsindicates that they may provide affordable, locally available, andenvironmentallysustainablealternativesforthe treatmentofresistantbacterialinfections.Therefore, stricterregulationofpharmaceuticalwastewaterdischarge,improvedwastewatertreatment,andfurther investigationofmedicinalplantsarerecommendedto reducethespreadofmultidrug-resistantbacteriaand protectpublichealth.