Case ReportShigellosis Caused by CTX-M Type ESBL ProducingShigella flexneri in Two Siblings of Rural Nepal First CaseReport from the Country

Narayan Prasad Parajuli12 Govardhan Joshi23 Bashu Dev Pardhe2

Jyotsna Shakya2 Anjeela Bhetwal2 Shreena Shakya2 Roshan Pandit2

Sumesh Shreekhanda Shrestha2 and Puspa Raj Khanal2

1Department of Clinical Laboratory Services Manmohan Memorial Medical College and Teaching Hospital Kathmandu Nepal2Department of Laboratory Medicine Manmohan Memorial Institute of Health Sciences Kathmandu Nepal3Kathmandu Center for Genomics and Research Laboratory (KCGRL) Kathmandu Nepal

Correspondence should be addressed to Narayan Prasad Parajuli narayanparajuliiomedunp

Received 22 December 2016 Accepted 5 February 2017 Published 21 February 2017

Academic Editor Larry M Bush

Copyright copy 2017 Narayan Prasad Parajuli et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Shigellosis is an acute infectious disease characterized as severe bloody diarrhea (dysentery) and is accountable for a significantburden of morbidity and mortality especially in children under the age of 5 years Antimicrobial therapy is required in the cases ofsevere dysentery associated with Shigella However emergence of multidrug resistant (MDR) strains of Shigella spp over the lasttwo decades has restricted the use of common therapeutic antimicrobials In MDR strains the third-generation cephalosporinshave been used for the treatment but unfortunately emerging reports of enzyme mediated 120573-lactam resistance among Shigellaisolates from various parts of the world have greatly compromised the therapy of pediatric dysentery In Nepal drug resistantstrains of Shigella spp have been reported butMDR and extended spectrum 120573-lactamase (ESBL) producing strains were previouslyunknown Here we report two Shigella flexneri isolates harboring ESBL genotype-CTX-M associated with acute dysentery in twosiblings which were presented and treated in a tertiary care teaching hospital of Kathmandu Nepal

1 General Background

Shigellosis caused bymembers of the bacterial genus Shigella[1] is an acute diarrheal disease primarily affecting poorcrowded communities that donot have adequate sanitation orclean water [2] Globally Shigella spp are the most commoncause of acute bloody diarrhea (dysentery) and are account-able for a significant burden of morbidity and mortalityassociated with diarrheal disease especially in children underthe age of 5 years [3] Clinically shigellosis may range frommild self-limiting diarrhea to severe dysentery with frequentpassage of blood and mucus high fever cramps tenesmusand dehydration [4] Every year about 125 million new casesof shigellosis occur in Asia alone of which around 14000 arelethal representing the burden of the disease in this region [5]Among various serogroups Shigella flexneri Shigella sonnei

and Shigella boydii are predominant in developing countrieswhile S sonnei is frequently reported from industrializedcountries [6] Antimicrobial therapy is required in the casesof severe dysentery associated with Shigella to reduce theduration of clinical illness minimizing the complications aswell as prevent the dissemination of infectious cases [7 8]However emergence of multidrug resistant (MDR) strains ofShigella spp (ie resistance to more than two first-line oraldrugs such as ampicillin cotrimoxazole and ciprofloxacin)over the last two decades restricted the use of commontherapeutic antimicrobials [9] In MDR strains the third-generation cephalosporins have been used for the treatmentbut unfortunately emerging reports of enzyme mediated120573-lactam resistance among Shigella isolates from variousparts of the world have further compromised the pediatrictherapy [7 10] In Nepal drug resistant strains of Shigella spp

HindawiCase Reports in Infectious DiseasesVolume 2017 Article ID 1862320 5 pageshttpsdoiorg10115520171862320

2 Case Reports in Infectious Diseases

have been reported [11] but MDR and extended spectrum120573-lactamase producing strains were previously unknownHere we report two Shigella flexneri isolates harboring ESBLgenotype-CTX-M associated with acute dysentery in twosiblings which were presented and treated in a tertiary careteaching hospital of Kathmandu Nepal

2 Description of the Cases

A six-month-old infant female was taken to the emergencydepartment of Manmohan Memorial Medical College andTeaching Hospital Kathmandu Nepal at 430 pm on 9 July2016 with complaints of abdominal pain frequent passage ofloose bloody and frothy stool for the last 6 days She did nothave any history of fever nausea and vomiting She belongedto the lower socioeconomic family and living in the earth-quake affected remote area of Dhading district They wereusing drinking water from nearby stream but have no historyof eating anything unusual or unhygienic On examinationshe was conscious but lethargic and appeared pale Herabdomen was soft with increased bowel sound but apparenthepatosplenomegalywas not noted Chest was clinically clearHer body temperature was 98∘F pulse rate was 115minuteand respiratory rate was 30minute The patient was provi-sionally diagnosed as a case of acute diarrheal illness withsuspicion of dysentery and immediate management wasstarted After necessary physical examination blood andstool sampleswere collected aseptically for laboratory investi-gations namely complete blood count blood chemistry testsstool culture and microscopyThe patient was then admittedto the pediatric unit for further treatment Antimicrobialregimen of 1 g24 hours of ceftriaxone and 500mg24 hoursof metronidazole was initiated as empiric therapy for acutediarrheal illness along with zinc tablets (10mg24 hour)probiotic-bifilac (10ml24 hours) and oral rehydration solu-tion (100ml per stool episode)

3 Laboratory Findings

There was reduction in the hemoglobin concentration (Hb95) but a normal (unremarkable) level of total leukocytecount with adequate cellular distribution (52 granulocytesand 48 lymphocytes) Other blood cell parameters andindices were found in the acceptable range The clinicalparameters of blood urea electrolytes and common liverenzymes were found normal C-reactive protein by latexagglutinationwas also negative Urinemicroscopy and chem-ical findings were normalThere were no remarkable changesobserved in abdominal ultrasonography

On stool microscopy numerous leucocytes and erythro-cytes along with cysts and trophozoites of amoeba (Enta-moeba species) were observed Further the stool specimenwas plated onto the Blood and MacConkey agar plates (Hi-Media Laboratories Mumbai India) and incubated aero-bically at 37∘C for 24 hours After incubation numerousnonlactose fermenting colonies were observed on Mac-Conkey agar while pale colonies with no hemolysis were seenon Blood agar These nonlactose fermenting colonies werepicked and processed for biochemical characterization and

Table 1 Antibiogram of Shigella flexneri isolated from two siblings

Antibiotics ResultsAmpicillin ResistantAzithromycin ResistantCotrimoxazole ResistantCeftazidime ResistantCefixime ResistantCiprofloxacin ResistantGentamycin ResistantPiperacillin tazobactam SensitiveAmpicillin Sulbactam ResistantImipenem SensitiveMeropenem Sensitive

antimicrobial susceptibility testing by Kirby Bauer disk diffu-sion method and result of susceptibility test was interpretedaccording to the CLSI guidelines for Enterobacteriaceae [12]

Biochemical results suggested that the isolate belongsto the genus Shigella and further serotyping was carriedout using specific antisera (Denka-Seiken Japan) and it wasconfirmed as Shigella flexneri On the susceptibility testingthe isolate was found resistant to penicillins macrolidescephalosporins fluoroquinolones and trimethoprim-sulph-amethoxazole group of antibiotics (Table 1) Therefore wesuspect the isolate to be presumptive ESBL producer andfurther characterization of ESBL was done by combinationdisk test In this test Ceftazidime (30120583g) disks alone and incombination with clavulanic acid (Ceftazidime + clavulanicacid 3010 120583g) disks were applied onto a plate of MuellerHinton Agar (MHA) which was inoculated with the strainand then incubated in ambient air for 16ndash18 hours at 35plusmn2∘CThe isolate showed the increase of ge5mm zone diameter oncombination disk compared to that of the Ceftazidime diskalone and was considered an ESBL producer [12] which waslater tested for genotypes

Despite continuous fluid replacement and intravenoustherapy of cephalosporin (ceftriaxone) and metronidazoleapparent prognosis in the disease was not observed Onday four the bacteriological report stating the presence ofESBL producing Shigella flexneriwas received and the antimi-crobial regimen was changed to Piperacillin tazobactam(2 gm24 hour) and Metronidazole (450mg24 hour) Fortu-nately after instituting the durataz (Piperacillin + tazobac-tam) the frequency of loose motion was dropped downgradually and clinical signs of abdominal discomfort wereresolved This therapy was continued for the next 5 days andthe patient was completely recovered and discharged Stoolculture on follow-up visit was negative for Shigella confirmingthe complete recovery

While the infant girl was being treated in our hospitalher 25-year-old brother was taken to the hospital on 12th Julywith major complaints of fever bloody diarrhea and abdom-inal cramps for the last three days On examination he wasconscious but appeared pale and was dehydrated (grade II)His abdomen was tender and lower zone nondistendedNo apparent hepatosplenomegaly was observed His body

Case Reports in Infectious Diseases 3

Table 2 Primers for the bla-CTX-M bla-TEM and bla-SHV genes

Gene Primers (51015840-31015840) Amplicon size (bp)

SHV F 51015840-GTCAGCGAAAAACACCTTGCC-31015840 383 bpR 51015840-GTCTTATCGGCGATAAACCAG-31015840

TEM F 51015840-GAGACAATAACCCTGGTAAAT-31015840 459 bpR 51015840-AGAAGTAAGTTGGCAGCAGTG-31015840

CTX-M F 51015840-GAAGGTCATCAAGAAGGTGCG-31015840 560 bpR 51015840-GCATTGCCACGCTTTTCATAG-31015840

temperature was 101∘F pulse rate was 120minute and res-piratory rate was 32minute He was passing blood mixedthin stool frequently (9 times in the last twelve hours) Con-sidering the previous case of acute dysentery in his youngersister he was provisionally diagnosed as a case of acute diar-rheal illness (probably dysentery) Necessary clinical and lab-oratory investigations along with appropriate clinical man-agement were carried out with the antimicrobial regimen ofPiperacillin and tazobactam (2 gm24 hour) and 500mg24hour metronidazole as empiric therapy was started as previ-ous ESBL Shigella was documented in his sister Zinc tablets(10mg24 hour) probiotic -bifilac (10ml24 hours) and oralrehydration solution (100ml per stool episode) were alsoincluded in the therapy

On ultrasonography remarkable feature of intestinalintussusception between small bowels was noted Consider-ing it as a surgical emergency immediate exploratory lapara-tomy was done to reduce the intussusceptions Inflamed ter-minal ileum and caecum with ileocolic intussusception wasnoted during the surgery and it was reduced In additioninflamed appendix with mesenteric lymph nodes was notedand appendectomy was done

On laboratory investigations moderate decrease inhemoglobin concentration (107 gm) with normal leuco-cytes and erythrocytes was observed Blood chemistry testsfor urea creatinine and electrolytes were found on the nor-mal range Latex agglutination test for C-reactive protein wasfound strongly positive (++) indicating the systemic inflam-mation Stool microscopy findings and cultural isolates alongwith its susceptibility were exactly similar to that of the pre-vious case (Table 1) The patient was treated with intravenousPiperacillin tazobactam and metronidazole for 7 days anddischarged on complete recovery Similar to the previous casefollow-up culture of stool sample was negative for Shigellaspp

4 Genetic Characterization of ESBL byPolymerase Chain Reaction

Crude plasmid DNA was isolated from bacterial cells byusing plasmid isolation kit (GeNei) by using manufacturerinstructions Primers were obtained from GeNei India andthey were used for identification of TEM SHV and CTX-Mtype ESBLsThe primer sequence is as shown in Table 2 Poly-merase Chain Reaction (PCR) was carried out to detect theplasmid genes for SHV CTX-M and TEM type ESBL as pre-viously described [13] After PCR amplification 25 120583l of each

Figure 1 Electrophoretic bands of CTX-M ESBL after PCR

reaction was separated by electrophoresis in 15 agarose gelfor 30min at 100V in 05 times TBE buffer DNA was stainedwith ethidiumbromide (1120583gml) and the bandswere detectedusingUV-transilluminator [Cleaver Scientific Ltd]We foundCTX-M type ESBL in both of the isolates tested confirmingthe ESBL Shigella in these two siblings (Figure 1)

5 Discussion

Worldwide acute gastrointestinal infections including diar-rhea are among the leading causes of morbidity andmortalityamong children particularly in underdeveloped countries[2] Poor access of safe water inadequate sanitary conditionslower literacy rate and unavailability of health care facilitiesin the remote area are the major factors predisposing diar-rheal illness among these countries [4 13] In our case toothe unavailability of safe drinking water and lower socioe-conomic status of the family might be associated with theseShigella infections in both siblings

In both cases of this report general symptoms of bacillarydysentery including abdominal discomfort pallor dehydra-tion and passage of blood tinged stool containing mucus

4 Case Reports in Infectious Diseases

have helped us to promptly investigate the clinical illnessMicroscopic findings of erythrocytes and numerous nonlac-tose fermenting colonies grown on MacConkey agar furthersimulated the Shigella associated dysentery In addition tothis abdominal ultrasonography was extremely useful in thesecond case for timely detection of intussusception in thesmall intestine Similar cases of intestinal intussusceptionassociated with Shigella spp were reported from India [14]and USA [15 16] among others

Shigellosis or severe bacillary dysentery is disease of pub-lic health importance because it is associated with increasedmortality and morbidity especially among the children ofdeveloping countries [17] It is highly necessary to start aprompt and rational antibiotic regimen to minimize theclinical effects of severe dysentery and its complications[8] Antimicrobial agents including fluoroquinolones andcephalosporins are the mainstay of therapy in severe cases ofshigellosis but emerging reports ofmultidrug resistant strainsfrom various parts of world made them less effective options[5] Furthermore the growing prevalence of ESBL producingstrains among Shigella species is of immense concern [14]

This report represents the first case of ESBL producingShigella strain associated with the clinical cases of shigellosisfrom the Himalayan country Nepal Previously ESBL pro-ducing clinical strains of Shigella spp has been reported fromdeveloping countries like India [5 8 14] Pakistan [18] Bang-ladesh [19 20] Turkey [21] and others In the past twodecades both the isolation frequencies and the types of ESBLshave gradually increased CTX-M SHV andTEM type ESBLsare being increasingly reported in the Shigella species aroundthe globe [8] The ESBLs are detected most commonly inKlebsiella pneumoniae and Escherichia coli but have beennoted in other members of the Enterobacteriaceae familyas well [22] Macrolides such as azithromycin have beendescribed as an alternative regime for empirical therapy forcases of severe dysentery particularly in children [5] but inour cases simultaneous resistance of fluoroquinolones andmacrolides in the isolated strains of Shigella further limitedtherapeutic options Frequent isolation of these multidrugresistant (MDR) and ESBL producing strains demands thehigher antimicrobial regimens to be instituted Howevertherapy with the intravenous broad spectrum drugs hasgreater economic as well as therapeutic constraints particu-larly in developing countries like Nepal where no any antimi-crobial guidelines for specific infection are available

CTX-M types ESBLs are plasmid-mediated 120573-lactamaseshaving higher hydrolytic effect against cefotaxime CTX-M-15 has been reported as common genotype of ESBL amongShigella isolates [23] CTX-M type ESBL detected in both ourcases could be the same genotype but we cannot analyze thefurther sequence The molecular detection of various genesis the gold standard technique for identifying ESBL genes butroutine screening with molecular tools is not practical in ourcountry Regular screening of all isolates strains for beta lacta-mase production and rational antibiotic prescription is highlynecessary to curtail the spread of resistance determinants toother organisms of vicinity

6 Conclusions

Detection of ESBL in Shigella has created the undeniableproblem constricting the therapeutic choices for acute dysen-tery Alongside the mobile resistance determinants maytransfer to wild strains of Shigella causing further dissemi-nation of drug resistance Timely diagnosis and appropriateantimicrobial regimen selection are vital in the managementof invasive infections caused by Shigella strains

Consent

Written informed consent was obtained from the mother ofthe two siblings for necessary clinical information and casepublication

Competing Interests

The authors have no competing interests to declare in thiswork

Authorsrsquo Contributions

Narayan Prasad Parajuli identified the case performed nec-essary laboratory investigations followed up the treatmentand drafted the manuscript Govardhan Joshi Bashu DevPardhe Anjeela Bhetwal Shreena Shakya Roshan Panditand Sumesh Shreekhanda Shrestha performed moleculartests and helped in drafting the manuscript Jyotsna Shakyaand Puspa Raj Khanal guided the procedure and helped indrafting of the manuscript All authors contributed towarddrafting and revising the paper gave final approval of theversion to be published and agreed to be accountable for allaspects of the work

References

[1] H L DuPont ldquoShigella species (bacillary dysentery)rdquo in Man-dell Douglas and Bennettrsquos Principles and Practice of InfectiousDiseases G L Mandell J E Bennett and R Dolin Eds pp2905ndash2910 Churchill Livingstone Elsevier Philadelphia PaUSA 2010

[2] Centers forDisease Control and Prevention Shigella-ShigellosisCenters for Disease Control and Prevention Atlanta Ga USA2016 httpwwwcdcgovshigellageneral-informationhtml

[3] K L Kotloff J P Nataro W C Blackwelder et al ldquoBurden andaetiology of diarrhoeal disease in infants and young childrenin developing countries (the Global Enteric Multicenter StudyGEMS) a prospective case-control studyrdquoThe Lancet vol 382no 9888 pp 209ndash222 2013

[4] S K Niyogi ldquoShigellosisrdquo Journal of Microbiology vol 43 no 2pp 133ndash143 2005

[5] S Ghosh G P Pazhani G Chowdhury et al ldquoGenetic charac-teristics and changing antimicrobial resistance among Shigellaspp isolated from hospitalized diarrhoeal patients in KolkataIndiardquo Journal ofMedicalMicrobiology vol 60 no 10 pp 1460ndash1466 2011

[6] C NThompson P T Duy and S Baker ldquoThe rising dominanceof Shigella sonnei an intercontinental shift in the etiology of

Case Reports in Infectious Diseases 3

Table 2 Primers for the bla-CTX-M bla-TEM and bla-SHV genes

Gene Primers (51015840-31015840) Amplicon size (bp)

SHV F 51015840-GTCAGCGAAAAACACCTTGCC-31015840 383 bpR 51015840-GTCTTATCGGCGATAAACCAG-31015840

TEM F 51015840-GAGACAATAACCCTGGTAAAT-31015840 459 bpR 51015840-AGAAGTAAGTTGGCAGCAGTG-31015840

CTX-M F 51015840-GAAGGTCATCAAGAAGGTGCG-31015840 560 bpR 51015840-GCATTGCCACGCTTTTCATAG-31015840

temperature was 101∘F pulse rate was 120minute and res-piratory rate was 32minute He was passing blood mixedthin stool frequently (9 times in the last twelve hours) Con-sidering the previous case of acute dysentery in his youngersister he was provisionally diagnosed as a case of acute diar-rheal illness (probably dysentery) Necessary clinical and lab-oratory investigations along with appropriate clinical man-agement were carried out with the antimicrobial regimen ofPiperacillin and tazobactam (2 gm24 hour) and 500mg24hour metronidazole as empiric therapy was started as previ-ous ESBL Shigella was documented in his sister Zinc tablets(10mg24 hour) probiotic -bifilac (10ml24 hours) and oralrehydration solution (100ml per stool episode) were alsoincluded in the therapy

On ultrasonography remarkable feature of intestinalintussusception between small bowels was noted Consider-ing it as a surgical emergency immediate exploratory lapara-tomy was done to reduce the intussusceptions Inflamed ter-minal ileum and caecum with ileocolic intussusception wasnoted during the surgery and it was reduced In additioninflamed appendix with mesenteric lymph nodes was notedand appendectomy was done

On laboratory investigations moderate decrease inhemoglobin concentration (107 gm) with normal leuco-cytes and erythrocytes was observed Blood chemistry testsfor urea creatinine and electrolytes were found on the nor-mal range Latex agglutination test for C-reactive protein wasfound strongly positive (++) indicating the systemic inflam-mation Stool microscopy findings and cultural isolates alongwith its susceptibility were exactly similar to that of the pre-vious case (Table 1) The patient was treated with intravenousPiperacillin tazobactam and metronidazole for 7 days anddischarged on complete recovery Similar to the previous casefollow-up culture of stool sample was negative for Shigellaspp

4 Genetic Characterization of ESBL byPolymerase Chain Reaction

Crude plasmid DNA was isolated from bacterial cells byusing plasmid isolation kit (GeNei) by using manufacturerinstructions Primers were obtained from GeNei India andthey were used for identification of TEM SHV and CTX-Mtype ESBLsThe primer sequence is as shown in Table 2 Poly-merase Chain Reaction (PCR) was carried out to detect theplasmid genes for SHV CTX-M and TEM type ESBL as pre-viously described [13] After PCR amplification 25 120583l of each

Figure 1 Electrophoretic bands of CTX-M ESBL after PCR

reaction was separated by electrophoresis in 15 agarose gelfor 30min at 100V in 05 times TBE buffer DNA was stainedwith ethidiumbromide (1120583gml) and the bandswere detectedusingUV-transilluminator [Cleaver Scientific Ltd]We foundCTX-M type ESBL in both of the isolates tested confirmingthe ESBL Shigella in these two siblings (Figure 1)

5 Discussion

Worldwide acute gastrointestinal infections including diar-rhea are among the leading causes of morbidity andmortalityamong children particularly in underdeveloped countries[2] Poor access of safe water inadequate sanitary conditionslower literacy rate and unavailability of health care facilitiesin the remote area are the major factors predisposing diar-rheal illness among these countries [4 13] In our case toothe unavailability of safe drinking water and lower socioe-conomic status of the family might be associated with theseShigella infections in both siblings

In both cases of this report general symptoms of bacillarydysentery including abdominal discomfort pallor dehydra-tion and passage of blood tinged stool containing mucus

4 Case Reports in Infectious Diseases

have helped us to promptly investigate the clinical illnessMicroscopic findings of erythrocytes and numerous nonlac-tose fermenting colonies grown on MacConkey agar furthersimulated the Shigella associated dysentery In addition tothis abdominal ultrasonography was extremely useful in thesecond case for timely detection of intussusception in thesmall intestine Similar cases of intestinal intussusceptionassociated with Shigella spp were reported from India [14]and USA [15 16] among others

Shigellosis or severe bacillary dysentery is disease of pub-lic health importance because it is associated with increasedmortality and morbidity especially among the children ofdeveloping countries [17] It is highly necessary to start aprompt and rational antibiotic regimen to minimize theclinical effects of severe dysentery and its complications[8] Antimicrobial agents including fluoroquinolones andcephalosporins are the mainstay of therapy in severe cases ofshigellosis but emerging reports ofmultidrug resistant strainsfrom various parts of world made them less effective options[5] Furthermore the growing prevalence of ESBL producingstrains among Shigella species is of immense concern [14]

This report represents the first case of ESBL producingShigella strain associated with the clinical cases of shigellosisfrom the Himalayan country Nepal Previously ESBL pro-ducing clinical strains of Shigella spp has been reported fromdeveloping countries like India [5 8 14] Pakistan [18] Bang-ladesh [19 20] Turkey [21] and others In the past twodecades both the isolation frequencies and the types of ESBLshave gradually increased CTX-M SHV andTEM type ESBLsare being increasingly reported in the Shigella species aroundthe globe [8] The ESBLs are detected most commonly inKlebsiella pneumoniae and Escherichia coli but have beennoted in other members of the Enterobacteriaceae familyas well [22] Macrolides such as azithromycin have beendescribed as an alternative regime for empirical therapy forcases of severe dysentery particularly in children [5] but inour cases simultaneous resistance of fluoroquinolones andmacrolides in the isolated strains of Shigella further limitedtherapeutic options Frequent isolation of these multidrugresistant (MDR) and ESBL producing strains demands thehigher antimicrobial regimens to be instituted Howevertherapy with the intravenous broad spectrum drugs hasgreater economic as well as therapeutic constraints particu-larly in developing countries like Nepal where no any antimi-crobial guidelines for specific infection are available

CTX-M types ESBLs are plasmid-mediated 120573-lactamaseshaving higher hydrolytic effect against cefotaxime CTX-M-15 has been reported as common genotype of ESBL amongShigella isolates [23] CTX-M type ESBL detected in both ourcases could be the same genotype but we cannot analyze thefurther sequence The molecular detection of various genesis the gold standard technique for identifying ESBL genes butroutine screening with molecular tools is not practical in ourcountry Regular screening of all isolates strains for beta lacta-mase production and rational antibiotic prescription is highlynecessary to curtail the spread of resistance determinants toother organisms of vicinity

6 Conclusions

Detection of ESBL in Shigella has created the undeniableproblem constricting the therapeutic choices for acute dysen-tery Alongside the mobile resistance determinants maytransfer to wild strains of Shigella causing further dissemi-nation of drug resistance Timely diagnosis and appropriateantimicrobial regimen selection are vital in the managementof invasive infections caused by Shigella strains

Consent

Written informed consent was obtained from the mother ofthe two siblings for necessary clinical information and casepublication

Competing Interests

The authors have no competing interests to declare in thiswork

Authorsrsquo Contributions

Narayan Prasad Parajuli identified the case performed nec-essary laboratory investigations followed up the treatmentand drafted the manuscript Govardhan Joshi Bashu DevPardhe Anjeela Bhetwal Shreena Shakya Roshan Panditand Sumesh Shreekhanda Shrestha performed moleculartests and helped in drafting the manuscript Jyotsna Shakyaand Puspa Raj Khanal guided the procedure and helped indrafting of the manuscript All authors contributed towarddrafting and revising the paper gave final approval of theversion to be published and agreed to be accountable for allaspects of the work

References

[1] H L DuPont ldquoShigella species (bacillary dysentery)rdquo in Man-dell Douglas and Bennettrsquos Principles and Practice of InfectiousDiseases G L Mandell J E Bennett and R Dolin Eds pp2905ndash2910 Churchill Livingstone Elsevier Philadelphia PaUSA 2010

[2] Centers forDisease Control and Prevention Shigella-ShigellosisCenters for Disease Control and Prevention Atlanta Ga USA2016 httpwwwcdcgovshigellageneral-informationhtml

[3] K L Kotloff J P Nataro W C Blackwelder et al ldquoBurden andaetiology of diarrhoeal disease in infants and young childrenin developing countries (the Global Enteric Multicenter StudyGEMS) a prospective case-control studyrdquoThe Lancet vol 382no 9888 pp 209ndash222 2013

[4] S K Niyogi ldquoShigellosisrdquo Journal of Microbiology vol 43 no 2pp 133ndash143 2005

[5] S Ghosh G P Pazhani G Chowdhury et al ldquoGenetic charac-teristics and changing antimicrobial resistance among Shigellaspp isolated from hospitalized diarrhoeal patients in KolkataIndiardquo Journal ofMedicalMicrobiology vol 60 no 10 pp 1460ndash1466 2011

[6] C NThompson P T Duy and S Baker ldquoThe rising dominanceof Shigella sonnei an intercontinental shift in the etiology of

4 Case Reports in Infectious Diseases

have helped us to promptly investigate the clinical illnessMicroscopic findings of erythrocytes and numerous nonlac-tose fermenting colonies grown on MacConkey agar furthersimulated the Shigella associated dysentery In addition tothis abdominal ultrasonography was extremely useful in thesecond case for timely detection of intussusception in thesmall intestine Similar cases of intestinal intussusceptionassociated with Shigella spp were reported from India [14]and USA [15 16] among others

Shigellosis or severe bacillary dysentery is disease of pub-lic health importance because it is associated with increasedmortality and morbidity especially among the children ofdeveloping countries [17] It is highly necessary to start aprompt and rational antibiotic regimen to minimize theclinical effects of severe dysentery and its complications[8] Antimicrobial agents including fluoroquinolones andcephalosporins are the mainstay of therapy in severe cases ofshigellosis but emerging reports ofmultidrug resistant strainsfrom various parts of world made them less effective options[5] Furthermore the growing prevalence of ESBL producingstrains among Shigella species is of immense concern [14]

This report represents the first case of ESBL producingShigella strain associated with the clinical cases of shigellosisfrom the Himalayan country Nepal Previously ESBL pro-ducing clinical strains of Shigella spp has been reported fromdeveloping countries like India [5 8 14] Pakistan [18] Bang-ladesh [19 20] Turkey [21] and others In the past twodecades both the isolation frequencies and the types of ESBLshave gradually increased CTX-M SHV andTEM type ESBLsare being increasingly reported in the Shigella species aroundthe globe [8] The ESBLs are detected most commonly inKlebsiella pneumoniae and Escherichia coli but have beennoted in other members of the Enterobacteriaceae familyas well [22] Macrolides such as azithromycin have beendescribed as an alternative regime for empirical therapy forcases of severe dysentery particularly in children [5] but inour cases simultaneous resistance of fluoroquinolones andmacrolides in the isolated strains of Shigella further limitedtherapeutic options Frequent isolation of these multidrugresistant (MDR) and ESBL producing strains demands thehigher antimicrobial regimens to be instituted Howevertherapy with the intravenous broad spectrum drugs hasgreater economic as well as therapeutic constraints particu-larly in developing countries like Nepal where no any antimi-crobial guidelines for specific infection are available

CTX-M types ESBLs are plasmid-mediated 120573-lactamaseshaving higher hydrolytic effect against cefotaxime CTX-M-15 has been reported as common genotype of ESBL amongShigella isolates [23] CTX-M type ESBL detected in both ourcases could be the same genotype but we cannot analyze thefurther sequence The molecular detection of various genesis the gold standard technique for identifying ESBL genes butroutine screening with molecular tools is not practical in ourcountry Regular screening of all isolates strains for beta lacta-mase production and rational antibiotic prescription is highlynecessary to curtail the spread of resistance determinants toother organisms of vicinity

6 Conclusions

Detection of ESBL in Shigella has created the undeniableproblem constricting the therapeutic choices for acute dysen-tery Alongside the mobile resistance determinants maytransfer to wild strains of Shigella causing further dissemi-nation of drug resistance Timely diagnosis and appropriateantimicrobial regimen selection are vital in the managementof invasive infections caused by Shigella strains

Consent

Written informed consent was obtained from the mother ofthe two siblings for necessary clinical information and casepublication

Competing Interests

The authors have no competing interests to declare in thiswork

Authorsrsquo Contributions

Narayan Prasad Parajuli identified the case performed nec-essary laboratory investigations followed up the treatmentand drafted the manuscript Govardhan Joshi Bashu DevPardhe Anjeela Bhetwal Shreena Shakya Roshan Panditand Sumesh Shreekhanda Shrestha performed moleculartests and helped in drafting the manuscript Jyotsna Shakyaand Puspa Raj Khanal guided the procedure and helped indrafting of the manuscript All authors contributed towarddrafting and revising the paper gave final approval of theversion to be published and agreed to be accountable for allaspects of the work

References

[1] H L DuPont ldquoShigella species (bacillary dysentery)rdquo in Man-dell Douglas and Bennettrsquos Principles and Practice of InfectiousDiseases G L Mandell J E Bennett and R Dolin Eds pp2905ndash2910 Churchill Livingstone Elsevier Philadelphia PaUSA 2010

[2] Centers forDisease Control and Prevention Shigella-ShigellosisCenters for Disease Control and Prevention Atlanta Ga USA2016 httpwwwcdcgovshigellageneral-informationhtml

[3] K L Kotloff J P Nataro W C Blackwelder et al ldquoBurden andaetiology of diarrhoeal disease in infants and young childrenin developing countries (the Global Enteric Multicenter StudyGEMS) a prospective case-control studyrdquoThe Lancet vol 382no 9888 pp 209ndash222 2013

[4] S K Niyogi ldquoShigellosisrdquo Journal of Microbiology vol 43 no 2pp 133ndash143 2005

[5] S Ghosh G P Pazhani G Chowdhury et al ldquoGenetic charac-teristics and changing antimicrobial resistance among Shigellaspp isolated from hospitalized diarrhoeal patients in KolkataIndiardquo Journal ofMedicalMicrobiology vol 60 no 10 pp 1460ndash1466 2011

[6] C NThompson P T Duy and S Baker ldquoThe rising dominanceof Shigella sonnei an intercontinental shift in the etiology of

Case Reports in Infectious Diseases 5

bacillary dysenteryrdquo PLoS Neglected Tropical Diseases vol 9 no6 Article ID e0003708 2015

[7] A H Sabra G F Araj M M Kattar et al ldquoMolecular char-acterization of ESBL-producing Shigella sonnei isolates frompatients with bacilliary dysentery in Lebanonrdquo Journal of Infec-tion in Developing Countries vol 3 no 4 pp 300ndash305 2009

[8] P Aggarwal B Uppal R Ghosh et al ldquoMulti drug resistanceand extended spectrum beta lactamases in clinical isolates ofShigella a study from New Delhi Indiardquo Travel Medicine andInfectious Disease vol 14 no 4 pp 407ndash413 2015

[9] J Mandal V Sangeetha Nivedithadivya A Das and S C Par-ija ldquoCharacterization of extended-spectrum 120573-lactamasepro-ducing clinical isolates of Shigella flexnerirdquo Journal of HealthPopulation and Nutrition vol 31 no 3 pp 405ndash408 2013

[10] S Varghese and A Aggarwal ldquoExtended spectrum beta-lac-tamase production in Shigella isolatesmdasha matter of concernrdquoIndian Journal of Medical Microbiology vol 29 no 1 pp 76ndash782011

[11] P Kansakar S Malla and G R Ghimire ldquoShigella isolates ofNepal changes in the incidence of shigella subgroups andtrends of antimicrobial susceptibility patternrdquoKathmandu Uni-versity Medical Journal vol 5 no 17 pp 32ndash37 2007

[12] ldquoPerformance Standards for Antimicrobial Disk SusceptibilityTestsrdquo M02-A11 Clinical and Laboratory Standards InstituteWayne Pa USA 2012

[13] A V Sangeetha S C Parija J Mandal and S KrishnamurthyldquoClinical andmicrobiological profiles of Shigellosis in childrenrdquoJournal of Health Population and Nutrition vol 32 no 4 pp580ndash586 2014

[14] S Sreenivasan A Kali and J Pradeep ldquoMultidrug resistantshigella flexneri infection simulating intestinal intussuscep-tionrdquo Journal of Laboratory Physicians vol 8 no 1 pp 55ndash572016

[15] C M Nylund L A Denson and J M Noel ldquoBacterial enteritisas a risk factor for childhood intussusception A RetrospectiveCohort Studyrdquo Journal of Pediatrics vol 156 no 5 pp 761ndash7652010

[16] DW Lett andTDMarsh ldquoShigellosis in a newbornrdquoAmericanJournal of Perinatology vol 10 no 1 pp 58ndash59 1993

[17] S Gupta B Mishra S Muralidharan and H Srinivasa ldquoCeftri-axone resistant Shigella flexneri an emerging problemrdquo Indianjournal of medical sciences vol 64 no 12 pp 553ndash556 2010

[18] A S Sattar S A Abbasi J Usman F Faqir F Kaleem and FHanif ldquoExtended-spectrum 120573-lactamase production in Shigellaflexnerirdquo Journal of the College of Physicians and SurgeonsPakistan vol 20 no 11 pp 768ndash769 2010

[19] H Rashid and M Rahman ldquoPossible transfer of plasmid medi-ated third generation cephalosporin resistance between Esche-richia coli and Shigella sonnei in the human gutrdquo InfectionGenetics and Evolution vol 30 pp 15ndash18 2015

[20] M Rahman S Shoma H Rashid A K Siddique G B Nairand D A Sack ldquoExtended-spectrum 120573-lactamase-mediatedthird-generation cephalosporin resistance in Shigella isolates inBangladeshrdquo Journal of Antimicrobial Chemotherapy vol 54 no4 pp 846ndash847 2004

[21] Z C Acikgoz Z Gulay M Bicmen S Gocer and S Gamber-zade ldquoCTX-M-3 extended-spectrum 120573-lactamase in a Shigellasonnei clinical isolate first report from Turkeyrdquo ScandinavianJournal of Infectious Diseases vol 35 no 8 pp 503ndash505 2003

[22] M Shanthi and U Sekar ldquoExtended spectrum beta lactamaseproducing escherichia coli and klebsiella pneumoniae risk

factors for infection and impact of resistance on outcomesrdquoJournal of Association of Physicians of India vol 58 pp 41ndash442010

[23] N Taneja A Mewara A Kumar G Verma and M SharmaldquoCephalosporin-resistant Shigella flexneri over 9 years (2001-09)in IndiardquoThe Journal of Antimicrobial Chemotherapy vol 67 no6 pp 1347ndash1353 2012

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom