Prevalence and Antibiotics Susceptibility Pattern of ...

Research ArticlePrevalence and Antibiotics Susceptibility Pattern ofSalmonella and Shigella Species among Diarrheal PatientsAttending Nekemte Referral Hospital Oromia Ethiopia

Alemayehu Terfassa1 andMulissa Jida 12

1Department of Biology College of Natural and Computational Sciences Wollega University Nekemte Ethiopia2Environmental Biotechnology Directorate Ethiopian Biotechnology Institute Addis Ababa Ethiopia

Correspondence should be addressed to Mulissa Jida mulaeabagedagmailcom

Received 25 July 2017 Revised 3 December 2017 Accepted 28 December 2017 Published 24 January 2018

Academic Editor Joseph Falkinham

Copyright copy 2018 Alemayehu Terfassa and Mulissa Jida This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The main objective of this study was determining the prevalence and antibiotics resistance pattern of Salmonella and Shigellasp from diarrheal patients attending Nekemte Referral Hospital A total of 422 patients were included in the study and theirsociodemographic and clinical information was collected using questionnaire Stool samples of the patients were collected andprocessed following standard bacteriological protocols Presumptive colonies of Salmonella and Shigella species were identified andsubcultured to their respective genera by standard biochemical tests Antibiotics susceptibility of the isolates was tested using diskdiffusion assayThe prevalences of Salmonella and Shigella sp among the patients were 71 and 21 respectivelyThe antimicrobialsusceptibility test results of the isolates showed that they are highly resistant to amoxicillin (30120583g) In contrast the isolates showedrelatively lower resistance level to ceftriaxone (30120583g) nalidixic acid (30 120583g) norfloxacin (10 120583g) and ciprofloxacin (5 120583g)This studyrevealed 92 prevalence of Salmonella and Shigella sp which were resistant to commonly prescribed antibioticsThus interventionmeasures such as health education provision of safe drinking water improvement of waste disposal systems and surveillance ofantibiotics susceptibility of the pathogens should be done regularly

1 Introduction

Acute gastroenteritis is one of the leading causes of illness anddeath in infants children immune-compromised and agedindividuals throughout the world Of the enteric pathogensSalmonella and Shigella species are of particular concernsas causes of enteric fevers food poisoning and gastroen-teritis [1 2] They are transferred from person to personusually by asymptomatic carriers and via contaminated foodand water They are Gram-negative rods which commonlyinhabit intestinal tracts of humans and many animals [3ndash5]Currently Salmonella and Shigella species are progressivelybecoming resistant to the commonly used antimicrobialagents [6ndash9] The emergence of antibiotic resistance amongtheir species is serious problem in developing countries

Diseases caused by Salmonella and Shigella species arecommon public health problems in many parts of the world

including Ethiopia [10ndash13] Studies conducted in differentcities of Ethiopia indicated that they are widespread amongdiarrheal patients [8 14ndash16] Moreover their increased resis-tance to commonly used antimicrobials has also been noted[13ndash16] On the contrary there is no information on theirprevalence and antimicrobial resistance patterns in NekemteTherefore this study was aimed at determining prevalenceand antimicrobial resistance patterns of Salmonella andShigella species among diarrheal patients attending NekemteReferral Hospital

2 Materials and Methods

21 Study Area Design and Population This study wasconducted in Nekemte Referral Hospital from October 2015to February 2016 Nekemte is located in Western parts ofOromia regional state at geographical location of 9∘461015840N

HindawiInternational Journal of MicrobiologyVolume 2018 Article ID 9214689 6 pageshttpsdoiorg10115520189214689

2 International Journal of Microbiology

latitude and 36∘311015840E longitude with elevation ranging from1960m to 2170m asl The study populations were allpatients with acute diarrhea attending Nekemte ReferralHospital Patients who were under antibiotics treatment inthe last 14 days of sampling period were excluded from thestudy

A cross-sectional study design was carried out to deter-mine the prevalence and antimicrobial resistance patternof Salmonella and Shigella species among diarrheal patientsattendingNekemteReferralHospital Pretested questionnairewas used to gather information concerning sociodemo-graphic data clinical characteristics and hygiene practice ofthe patients

22 Sampling and Sample Size Determination The study par-ticipants were selected via systematic random samplingtechnique from OPD of the hospital A minimum samplesize of 384 was calculated using single population proportionformula assuming 95 confidence interval 50 prevalenceand 5marginal error as a result 422 patients were includedin the study [17]

23 Stool Sample Collection and Handling Stool specimenswere collected in sterile container and transported to Micro-biology Laboratory Oromia Regional Health LaboratoryNekemte within 2 hrs of collection All plates and test tubeswere incubated at 37∘C for 24 hrs

24 Isolation of Salmonella and Shigella Species About 1 gof the stool sample was transferred to Selenite F broth andincubated at 37∘C for 24 hrs Simultaneously a loop full ofstool sample suspension was streaked on SSA and XLDAand then incubated at 37∘C for 24 hrs Furthermore culturenegative specimens on SSA and XLDA media were subcul-tured from Selenite F broth enrichment broth to SSA andXLDA plates to improve recovery of the isolates Growth ofSalmonella and Shigella species were detected by their typicalcolonies characteristic on SSA and on XLDA [18] All isolateswere purified by repeated subculturing streaked on nutrientagar slant and preserved in refrigerator set at 4∘C untilfurther analysis

25 Morphological and Biochemical Characterization ofthe Isolates

251 Morphological Characterization Purified single colonyfrom SSAXLDA was taken with loop spread on the centerof clean slide and subject to Gram staining Finally thestained smear was observed under microscope (oil immer-sion objective) and then morphology cell arrangement andGram reaction of the isolates were determined

252 Motility Test To determine whether the tested organ-ism was motile or not motility media were inoculated witha straight inoculating needle through a single stab about1-2 cm down into the medium and incubated at 37∘C for24 hrs Based on their appearance onmotilitymedium the testorganisms were categorized as motile and nonmotile

253 Biochemical Characterization of the Isolates Colonieswith typical characteristics of Salmonella and Shigella speciesfrom primary media were further cultured on Kligler IronAgar (KIA) medium by stabbing the butt and streaking theslant and then incubated at 37∘C for 24 hrs to determine theirglucose and lactose fermentation abilities and productionof hydrogen sulfide [19] The KIA tubes were examined forspecific growth and appearance of Salmonella and Shigellaspecies Colonies of the isolates from primary media werefurther examined by stabbing the butt and streaking onsurface of the slant of Lysine Iron Agar (LIA) and incubatingat 37∘C for 24 hrsThe growth of positive isolate was detectedby their specific growth and appearance on LIA tubes [19]Pure colonies of the isolates were tested for citrate utilizationability by stabbing the butt and streaking the slant of SimmonsCitrate Agar and incubating at 37∘C for 24 hrs [19] Suspectedcolonies of Salmonella and Shigella species were transferredfrom growth of KIA to nutrient broth with sterile loop andincubated for few hrs Indole productions were tested byadding 5 drop of Kovacrsquos reagent into the test tubes Ureaseproduction ability of the suspected colony of Salmonella andShigella was tested by inoculating heavily over the entire sur-face of the slant urease medium the caps were loosened andincubated for an overnight at 37∘C [19]

254 Antibiotics Susceptibility Test Antibiotics resistancepattern of the isolates was determined using disc diffusionmethods [20] A single pure colony of each isolates wasinoculated into the nutrient broth and incubated at 37∘C for24 hrs and their turbidity was adjusted with 05 McFarlandThe culture was inoculated on Muller Hinton agar usingsterilized cotton swab Discs impregnated with appropriateconcentration of antibiotics were placed on inoculated plateAmoxicillin (30 120583g) ceftriaxone (30 120583g) chloramphenicol(30 120583g) ciprofloxacin (5120583g) gentamicin (10 120583g) nalidixicacid (30 120583g) and norfloxacin (10 120583g) disks were used for thetest E coli (ATCC 25922) was used as reference strain toensure quality of the data Zones of inhibition were measuredto the nearest mm and results were interpreted according tothe CLSI [20]

26 Data Analysis and Interpretation The data was editedand analyzed using SPSS version 23 Multivariate logisticregression test adjusted odds ratio (AOR) and 95 CI (119875 lt005 significance level) were used to assess the level of asso-ciation among prevalence of the pathogens and associatedrisk factors

3 Results

31 Sociodemographic and Clinical Characteristics of thePatients In this study 422 patients with acute diarrhea andattending Nekemte Referral Hospital from October 2015 toFebruary 2016 were considered Of these 528 were maleand the mean age of the study participants was 207 years(Table 1) The majority of study participants (832) wereurban dwellers and 621 of them have the habit of washingtheir hands regularly after using toilet Most (832) of the

International Journal of Microbiology 3

Table 1 Sociodemographic clinical characteristics and prevalence of Salmonella and Shigella sp among diarrheal patients attendingNekemteReferral Hospital October 2015 to February 2016

Variables Frequency Percent () Number of Salmonella or Shigella species positive patients ()Sex

M 223 528 17 (76)F 199 199 22 (111)

Age Category (years)1ndash19 225 533 18 (8)19ndash29 81 192 6 (74)30ndash44 69 164 11 (159)45ndash59 35 83 4 (114)gt60 12 28 0

Educational statusIlliterate 137 325 12 (88)Grade 1ndash4 70 166 4 (53)Grade 5ndash8 71 168 4 (56)Grade 9ndash12 81 192 11 (136)Certificate dip 57 135 7 (123)1st degree amp above 6 14 1 (167)

Hand washing after toiletGood 262 621 29 (111)Poor 160 379 10 (63)

Source of drinking waterTap 351 832 31 (88)River 70 166 8 (114)well 1 02 0 (00)

Residence areasUrban 320 911 31 (88)Rural 63 89 8 (113)

Clinical characteristicsAbdominal pain 419 993 38 (91)Vomiting 272 645 28 (103)Fever 310 735 38 (123)Headache 222 526 34 (153)Joint pain 182 431 27 (148)

Prevalence of Salmonella amp Shigella spShigella sp 9 21 -Salmonella sp 30 71 -Total Prevalence 39 92 -

Note Salmonella and Shigella positive values as No () sp species

diarrheal patients sources of drinking water was tap whereas166 of them depend on river Abdominal pain fevervomiting and headache were the clinical symptoms observedamong the study population Abdominal pain was found tobe the most common (993) clinical symptom followed byfever and vomiting (Table 1)

32 Prevalence of Salmonella and Shigella Species Out of thetotal stool specimens 92 were positive for Salmonella andShigella species (Table 1)Of these 71 and 21were positivefor Salmonella and Shigella species respectively 777 of

Shigella isolates were obtained from male patients Of allisolates 461 were isolated from patients with age lessthan 19 years In contrast neither Salmonella nor Shigellaspecies were found in study participants with age of 60years and above Our results also indicated that there wereno statistically significant associations (119875 gt 005) betweengender age groups and prevalence of Salmonella and Shigellaspecies whereas poor hand washing habit after toilet (AOR41 119875 0002) vomiting (AOR 100 119875 0040) and headache(AOR 47 119875 0013) had statistically significant (119875 lt 005)association with the rate of isolation of the pathogens (datanot shown)


Table 2 Antibiotics susceptibility pattern of Salmonella and Shigella species from diarrheal patients attending Nekemte Referral HospitalOctober 2015 to February 2016

Antimicrobial Salmonella species N () Shigella species N ()S I R S I R

Ceftriaxone 28 (9333) 1 (333) 1 (333) 9 (100) 0 (0) 0 (0)Norfloxacin 29 (9667) 0 (0) 1 (333) 9 (100) 0 (0) 0 (0)Ciprofloxacin 28 (9333) 0 (0) 2 (667) 8 (8889) 1 (1111) 0 (0)Nalidixic acid 22 (7333) 7 (2333) 1 (333) 8 (8889) 0 (0) 1 (1111)Chloramphenicol 21 (70) 7 (233) 2 (667) 7 (7778) 1 (1111) 1 (1111)Gentamicin 21 (70) 6 (20) 3 (10) 7 (7778) 1 (1111) 1 (1111)Amoxicillin 0 (0) 3 (10) 27 (90) 2 (2222) 0 (0) 7 (7778)Parenthesis S = sensitive I = intermediate R = resistance

Table 3 Multidrug resistance pattern of Salmonella and Shigellaspecies from diarrheal patients attending Nekemte Referral Hospi-tal October 2015 to February 2016

Bacterial Isolates Number isolate Antibiotics resistedShigella species 3 (3333 ) GEN AM

Salmonella species

1 (333) C AM1 (333) CIP AM

1 (333) C CIP CRO NA GENNOR

Parenthesis GEN = gentamicin AM = amoxicillin C = chloramphenicolCIP = ciprofloxacin CRO = ceftriaxone NA = nalidixic acid NOR =norfloxacin

33 Antibiotic Susceptibility Pattern of the Isolates Of allSalmonella isolates 90 were resistant to amoxicillin (30 120583g)whereas only 10 67 67 33 33 and 33 of theisolates were found to be resistant to gentamicin (10 120583g)ciprofloxacin (5120583g) chloramphenicol (30 120583g) ceftriaxone(30 120583g) norfloxacin (10 120583g) and nalidixic acid (30120583g)respectively Of all 778 of Shigella isolates were resistant toamoxicillin whereas 1111 of themwere found to be resistantto ciprofloxacin chloramphenicol gentamicin and nalidixicacid Shigella isolateswere also found to be of high susceptibil-ity to ceftriaxone and norfloxacin followed by ciprofloxacinnalidixic acid chloramphenicol and gentamicin (Table 2)Multiple drug resistances were observed among Salmonellaand Shigella isolates obtained from the participants of thisstudy Amazingly one Salmonella isolate showed resistanceto six antibiotics while only 10 of the isolates exhibitedmultidrug resistance to different antibiotics tested in thestudy Our results demonstrated that 3333 of Shigellaisolates exhibited multidrug resistance to gentamicin andamoxicillin (Table 3)

4 Discussion

According to this study the predominant bacterial pathogenswere Salmonella (711) indicating that it is commonhuman health problem at the study area The prevalence ofSalmonella in this study is in agreement with the result (78)of study done in Bahir Dar [14] and higher than the result of

studies done in Gonder (108) [8] and Addis Ababa 395[21] Ethiopia Similarly isolation rate of Salmonella speciesobtained in this study is higher than the results of studiesin other countries 33 in Nigeria [11] and 15 in Turkey[12] In contrast our result is lower than studies done inGonder (1739) [22] Harar (115) [13] Butajira (105)[23] and Jimma (108) [16] Ethiopia The variation couldbe due to differences in sociodemographic characteristicsgeographical location and study time

The isolation rate of Shigella species obtained in this studyis lower than the earlier studies done in Ethiopia 95 inBahirDar [14] 91 inAddisAbaba [21] 69 inMekelle [15]67 inHarar [13] 457 inGonder [8] 45 in Butajira [23]and 29 in Ambo [10] Comparable results were reported bystudies carried out elsewhere 29 in Nigeria Abeokuta [11]and 3 inTurkey [12] In contrast the isolation rate of Shigellaspecies in this study is higher than the results of studies donein Ambo (13) [24] and Jimma (11) [16]

Our results showed that 621 of the study participantswash their hands after using latrine Good hand washingpattern after using latrine has statically significant associationwith prevalence of Salmonella and Shigella species Howeverthere were no statistically significant association betweenthe percentage of patients with abdominal pain joint painand prevalence of the pathogens But there were statisticallysignificant associations between headache vomiting andprevalence of Salmonella and Shigella species The infectionrate (4615) within age group below 19 yearsmight be due todifference in immunity status of the study group

According to this study the resistance rate of Salmonellawas 90 to amoxicillin which is in agreement with reportsfromother areas of Ethiopia [8 13 22 25]Thepossible reasoncould be due to long time service and wide use of this drug inthe country and frequent exposure of Salmonella species to itSalmonella species showed that less resistance to gentamicinin this study is lower than result (25) study done in Gonder[8] In contrast the result of this study is higher than resultsreported from other areas in Ethiopia [13 21 23]

On the other hand Salmonella isolates showed lower(333) resistance rate to ceftriaxone and nalidixic acid thanresults obtained by earlier study done in Hawassa which were75 and 25 respectively [25] Similarly resistance levelof Salmonella isolates to chloramphenicol gentamicin and


nalidixic acid is lower than results of studies conducted inHarar [13] Addis Ababa [21] and Gonder [8] Resistance rate(333) of Salmonella isolates to ceftriaxone observed in thisstudy is slightly higher than earlier studies done in other areasof the country [8 21 23]The proportion (10) of multidrug-resistant Salmonella isolates found in this study is lower thanthe previous reports [14]

Shigella species isolated in this study were highly suscep-tible to both ceftriaxone and norfloxacin (100) followed byciprofloxacin and nalidixic acidThese findings are supportedby the results of previous study conducted in other areas ofEthiopia [8 21 23] The resistance rate Shigella isolates toamoxicillin was 7778 which is in agreement with the studydone in Gonder (882) [8] and slightly lower than resultreported from Addis Ababa (914) [21] Harar (100) [13]and Hawassa (100) [25] On the other hand low resistantrate of Shigella isolates to chloramphenicol and gentamicin(1111) observed in this study is contradictory to the resultsof earlier studies [8 13 14 23 25] and supported by lowerresistance level to gentamicin reported by other studies [11 1421 23 26]More than 33 of Shigella isolates weremultidrug-resistant which is higher than the previous study [8] inGonder Ethiopia

5 Conclusion

Our results revealed 92 prevalence of Salmonella thanShigella species among diarrheal patients in the studyarea Antimicrobial susceptibility test results showed thatSalmonella and Shigella species isolated during this studyare highly resistant to amoxicillin Salmonella and Shigellaisolates were highly sensitive to ceftriaxone and norfloxacinBased on the results of the current study we stronglyrecommend continuous surveillance on the prevalence andantibiotic resistance pattern of Salmonella and Shigella speciesamong relevant patients in hospitals and other health centersin the study area which should be basis for empirical therapySince Salmonella and Shigella species were resistant to mostcommon drugs care should be taken in selecting antimicro-bials in treating disease caused by themMoreover improvingenvironmental and personal hygiene and providing safepotable water and intensive health education together withmore careful use of antimicrobials could conserve antimicro-bial efficacy and significantly reduce diarrheal illness

In this study the pathogens were not identified to thespecies level using serological analysis and hence biochemicaltests should be substantiated by serological and molecularidentification for better taxonomy of the pathogens to speciesand strain level This study was conducted only on patientspresented with diarrheal case and visited Nekemte ReferralHospital and did not include patients from health centersand private clinics However the study verified that furtherepidemiological study should be conducted on patientsattending health centers and private clinics

Additional Points

Availability of Data and Materials Data supporting thisresearch article are available from the corresponding authoror first author on reasonable request

Conflicts of Interest

There are no financial or other relationships that might leadto conflicts of interest in this study The authors of thismanuscript declare that they have no conflicts of interest

Authorsrsquo Contributions

Mulissa Jida was involved in concept design supervisedthe data collection and critical revision of the article andprovided final approval of the article Alemayehu Terfassawas involved in concept design and data collection draftedthe manuscript performed laboratory analysis and providedanalysis and interpretation of the data All authors read andapproved the final manuscript

Acknowledgments

The authors are so grateful to Oromia Regional HealthLaboratory Nekemte for providing laboratorymaterials bio-chemical test reagents and working bench Nekemte ReferralHospital for assistance during stool sample collection andDepartment of Biology Wollega University for supportingthis research work

References

[1] C Yildiz C Ozturk and G Emekdas ldquoResearch of the E coli0157H7 strains cases of the Gastro-enteritsrdquo Infeksiyon Dergisivol 19 pp 189ndash192 2005

[2] M A Raji S F H Jiwa M U Minga and P S GwakisaldquoEscherichia coli 0157 H7 reservoir transmission diagnosisand the African situation A reviewrdquo East African MedicalJournal vol 80 no 5 pp 271ndash276 2003

[3] M Abdullahi ldquoIncidence and antimicrobial susceptibility pat-tern of Salmonella species in children attending some hospitalsin kano metropolis kano statendashNigeriardquo Bajopas vol 3 pp202ndash206 2010

[4] H Yan L L Alam M J Shinoda and S Miyoshi ldquoPrevalenceand antimicrobial resistance of Salmonella in retail foods innorthern Chinardquo FoodMicrobiology vol 143 pp 230ndash234 2010

[5] C A Arias and B E Murray ldquoAntibiotic-resistant bugs in the21st centurymdasha clinical super-challengerdquo e New EnglandJournal of Medicine vol 360 no 5 pp 439ndash443 2009

[6] R Sharma C L Sharma and B Kapoor ldquoAntibacterial resis-tance current problems and possible solutionsrdquo Indian Journalof Medical Sciences vol 59 no 3 pp 120ndash129 2005

[7] B Gu Y Cao S Pan et al ldquoComparison of the prevalenceand changing resistance to nalidixic acid and ciprofloxacin ofShigella between Europe-America and Asia-Africa from 1998 to2009rdquo International Journal of Antimicrobial Agents vol 40 no1 pp 9ndash17 2012

[8] T A Demissie T Moges M Feleke M Dagnachew and AGetnet ldquoPrevalence and antimicrobial susceptibility patterns ofShigella and Salmonella species among patients with diar-rhea attending Gondar tOwn Health Institutions NorthwestEthiopiardquo Science Journal of Public Health vol 2 no 5 pp 469ndash475 2014

[9] K Huruy A Kassu A Mulu et al ldquoHigh level of antimicrobialresistance in Shigella species isolated from diarrhoeal patients


in University of Gondar Teaching Hospital Gondar EthiopiardquoPharmacologyonline vol 2 pp 328ndash340 2008

[10] S Kefyalew G Kebede and A Keneni ldquoPrevalence of Shigellarelated diarrhea in Ambo town and antibiotic susceptibility ofthe isolated strainsrdquoGreener Journal of Epidemiology and PublicHealth vol 3 no 1 pp 001ndash006 2015

[11] O A Akingbade P O Okerentugba O B Awoderu and A AShobayo ldquoPrevalence of Samonella sp and Shigella sp amongchildren with diarrhoea in Abeokuta Ogun State NigeriardquoAcadem Arena vol 6 no 9 pp 13ndash16 2014

[12] T T Kara H Ozdemir F Kurt et al ldquoPrevalence of Salmonellaand Shigella spp and antibiotic resistance status in acutechildhood gastroenteritis Ankara Turkeyrdquo J Pediatr Inf vol9 pp 102ndash107 2015

[13] A A Reda B Seyoum J Yimam G Andualem S Fisehaand J M Vandeweerd ldquoAntibiotic susceptibility patterns ofSalmonella and Shigella isolates in Harer Eastern EthiopiardquoJournal of Infectious Diseases and Immunity vol 3 no 8 pp134ndash139 2011

[14] M A Admassu G Yemane M Kibret B Abera E Nibretand M Adal ldquoPrevalence and antibiogram of Shigella andSalmonella spp from under five children with acute diarrhea inBahir Dar Townrdquo Ethiopian Journal of Science and Technologyvol 8 no 1 pp 27ndash35 2015

[15] A Gebrekidan T A Dejene G Kahsay and A G WasihunldquoPrevalence and antimicrobial susceptibility patterns of Shigellaamong acute diarrheal outpatients in Mekelle hospital North-ern Ethiopiardquo BMC Research Notes vol 8 no 611 2015

[16] T Lamboro T Ketema and K Bacha ldquoPrevalence andantimicrobial resistance in Salmonella and Shigella species iso-lated from outpatients Jimma University Specialized HospitalSouthwest Ethiopiardquo Canadian Journal of Infectious Diseases ampMedical Microbiology vol 2016 Article ID 4210760 8 pages2016

[17] W W Daniel Biostatistics A Foundation for Analysis in eHealth Sciences John Wiley and Sons New York USA 7thedition 1999

[18] WHO Basic Laboratory Procedure in Clinical Bacteriology 56-69 World Health Organization Geneva 2nd edition 2003

[19] M Cheesbrough Medical Laboratory Manual for TropicalCountries vol 2 38-39 138 UK Cambridge Press 2nd edition2006

[20] CLSI Performance Standard for Antimicrobial SusceptibilityTesting Wayne PA 19087 2012

[21] Y Mamuye G Metaferia A Birhanu K Desta and S FantawldquoIsolation and antibiotic susceptibility patterns of Shigella andSalmonella among under 5 children with acute diarrhoea across-sectional study at selected public health facilities in addisababa ethiopiardquo Clinical Microbiology Open Access vol 4 no186 2015

[22] L Garedew Z Hagos Z Addis R Tesfaye and B ZegeyeldquoPrevalence and antimicrobial susceptibility patterns ofSalmonella isolates in association with hygienic status frombutcher shops in Gondar town Ethiopiardquo Antimicrobial Resis-tance and Infection Control vol 4 no 1 article 21 2015

[23] G Mengistu G Mulugeta T Lema and A Aseffa ldquoPrevalenceand antimicrobial susceptibility patterns of Salmonellaserovarsand Shigella speciesrdquo Journal of Microbial amp Biochemical Tech-nology vol 6 Supplemet 2 Article ID Article 6 2014

[24] W Tosisa Prevalence And Antibiotic Resistance of EntericBacterial Pathogens Isolated from Childhood Diarrhea in AmboTown Public Health Institutions Addis Ababa University 2015

[25] M Getamesay B Getenet and Z Ahmed ldquoPrevalence ofShigella Salmonella and Cmpylobacter species and their sus-ceptibility patters among under five children with diarrhea inHawassa Town South Ethiopiardquo Ethiopian Journal of HealthSciences vol 24 no 2 pp 101ndash108 2014

[26] G Yismaw C Negeri and A Kassu ldquoA five-year antimicro-bial resistance pattern observed in Shigella species isolatedfrom stool samples in Gondar University Hospital northwestEthiopiardquoe Ethiopian Journal of Health Development vol 20no 3 pp 194ndash198 2006

Hindawiwwwhindawicom

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MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

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Submit your manuscripts atwwwhindawicom


latitude and 36∘311015840E longitude with elevation ranging from1960m to 2170m asl The study populations were allpatients with acute diarrhea attending Nekemte ReferralHospital Patients who were under antibiotics treatment inthe last 14 days of sampling period were excluded from thestudy

A cross-sectional study design was carried out to deter-mine the prevalence and antimicrobial resistance patternof Salmonella and Shigella species among diarrheal patientsattendingNekemteReferralHospital Pretested questionnairewas used to gather information concerning sociodemo-graphic data clinical characteristics and hygiene practice ofthe patients

22 Sampling and Sample Size Determination The study par-ticipants were selected via systematic random samplingtechnique from OPD of the hospital A minimum samplesize of 384 was calculated using single population proportionformula assuming 95 confidence interval 50 prevalenceand 5marginal error as a result 422 patients were includedin the study [17]

23 Stool Sample Collection and Handling Stool specimenswere collected in sterile container and transported to Micro-biology Laboratory Oromia Regional Health LaboratoryNekemte within 2 hrs of collection All plates and test tubeswere incubated at 37∘C for 24 hrs

24 Isolation of Salmonella and Shigella Species About 1 gof the stool sample was transferred to Selenite F broth andincubated at 37∘C for 24 hrs Simultaneously a loop full ofstool sample suspension was streaked on SSA and XLDAand then incubated at 37∘C for 24 hrs Furthermore culturenegative specimens on SSA and XLDA media were subcul-tured from Selenite F broth enrichment broth to SSA andXLDA plates to improve recovery of the isolates Growth ofSalmonella and Shigella species were detected by their typicalcolonies characteristic on SSA and on XLDA [18] All isolateswere purified by repeated subculturing streaked on nutrientagar slant and preserved in refrigerator set at 4∘C untilfurther analysis

25 Morphological and Biochemical Characterization ofthe Isolates

251 Morphological Characterization Purified single colonyfrom SSAXLDA was taken with loop spread on the centerof clean slide and subject to Gram staining Finally thestained smear was observed under microscope (oil immer-sion objective) and then morphology cell arrangement andGram reaction of the isolates were determined

252 Motility Test To determine whether the tested organ-ism was motile or not motility media were inoculated witha straight inoculating needle through a single stab about1-2 cm down into the medium and incubated at 37∘C for24 hrs Based on their appearance onmotilitymedium the testorganisms were categorized as motile and nonmotile

253 Biochemical Characterization of the Isolates Colonieswith typical characteristics of Salmonella and Shigella speciesfrom primary media were further cultured on Kligler IronAgar (KIA) medium by stabbing the butt and streaking theslant and then incubated at 37∘C for 24 hrs to determine theirglucose and lactose fermentation abilities and productionof hydrogen sulfide [19] The KIA tubes were examined forspecific growth and appearance of Salmonella and Shigellaspecies Colonies of the isolates from primary media werefurther examined by stabbing the butt and streaking onsurface of the slant of Lysine Iron Agar (LIA) and incubatingat 37∘C for 24 hrsThe growth of positive isolate was detectedby their specific growth and appearance on LIA tubes [19]Pure colonies of the isolates were tested for citrate utilizationability by stabbing the butt and streaking the slant of SimmonsCitrate Agar and incubating at 37∘C for 24 hrs [19] Suspectedcolonies of Salmonella and Shigella species were transferredfrom growth of KIA to nutrient broth with sterile loop andincubated for few hrs Indole productions were tested byadding 5 drop of Kovacrsquos reagent into the test tubes Ureaseproduction ability of the suspected colony of Salmonella andShigella was tested by inoculating heavily over the entire sur-face of the slant urease medium the caps were loosened andincubated for an overnight at 37∘C [19]

254 Antibiotics Susceptibility Test Antibiotics resistancepattern of the isolates was determined using disc diffusionmethods [20] A single pure colony of each isolates wasinoculated into the nutrient broth and incubated at 37∘C for24 hrs and their turbidity was adjusted with 05 McFarlandThe culture was inoculated on Muller Hinton agar usingsterilized cotton swab Discs impregnated with appropriateconcentration of antibiotics were placed on inoculated plateAmoxicillin (30 120583g) ceftriaxone (30 120583g) chloramphenicol(30 120583g) ciprofloxacin (5120583g) gentamicin (10 120583g) nalidixicacid (30 120583g) and norfloxacin (10 120583g) disks were used for thetest E coli (ATCC 25922) was used as reference strain toensure quality of the data Zones of inhibition were measuredto the nearest mm and results were interpreted according tothe CLSI [20]

26 Data Analysis and Interpretation The data was editedand analyzed using SPSS version 23 Multivariate logisticregression test adjusted odds ratio (AOR) and 95 CI (119875 lt005 significance level) were used to assess the level of asso-ciation among prevalence of the pathogens and associatedrisk factors

3 Results

31 Sociodemographic and Clinical Characteristics of thePatients In this study 422 patients with acute diarrhea andattending Nekemte Referral Hospital from October 2015 toFebruary 2016 were considered Of these 528 were maleand the mean age of the study participants was 207 years(Table 1) The majority of study participants (832) wereurban dwellers and 621 of them have the habit of washingtheir hands regularly after using toilet Most (832) of the




M 223 528 17 (76)F 199 199 22 (111)


















Salmonella species

1 (333) C AM1 (333) CIP AM




4 Discussion










5 Conclusion



Additional Points






Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





M 223 528 17 (76)F 199 199 22 (111)


















Salmonella species

1 (333) C AM1 (333) CIP AM




4 Discussion










5 Conclusion



Additional Points






Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018








Salmonella species

1 (333) C AM1 (333) CIP AM




4 Discussion










5 Conclusion



Additional Points






Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





5 Conclusion



Additional Points






Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Prevalence and Antibiotics Susceptibility Pattern of ...

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