Rapid Emergence of Third-Generation Cephalosporin Resistance in Shigella Sp. Isolated in Andaman and Nicobar Islands,India

Abstract

Shigellosis is a major cause of diarrheal diseases among children in Andaman & Nicobar Islands, India, which have a population of 350,000 people, including settlers from mainland India and 6 indigenous tribes. From the last one-and-half decade, we have been monitoring the species distribution and emergence of antibiotic resistance among the isolates of Shigella. The circulating Shigella strains have been found rapidly acquiring resistance to a wide spectrum of antibiotics. The recent data indicate that a significant proportion of Shigella isolates have been resistant to newer generation of cephalosporins, which are used as an alternative of quinolones to treat the patients with shigellosis. In this communication, we report the antibiotic-resistant pattern of Shigella isolates that are recently isolated from these islands. From January 2008 to December 2009, 311 stool samples were processed and 44 (14%) Shigella isolates were recovered. Out of these 44 Shigella isolates, 6 (14%) were found to be resistant to all the three third-generation cephalosporins tested. The minimum inhibitory concentrations of the resistant isolates were all above the breakpoint for reduced susceptibility as per the Clinical and Laboratory Standards Institute guidelines. All of the cephalosporin-resistant Shigella strains were confirmed to produce extended-spectrum β-lactamases. By analyzing trends in the resistance patterns of the various Shigella species, we found that Shigella dysenteriae (40%) is currently more resistant, followed by Shigella flexneri (14%), than the other Shigella species in these islands of India, especially to the third-generation cephalosporins. The acquisition of resistance by enteric pathogens to the increasing number of antibacterial drugs is becoming a grave concern, particularly in developing countries where shigellosis is of a common occurrence.

Infections caused by Shigella sp. are a major cause of diarrhea in children. More than 100 million diarrheal episodes occur per year among children in the age group of 0–4 years in developing countries.⁹ Because antimicrobial susceptibility patterns of shigellae may vary greatly between geographical areas and over time, monitoring resistance patterns is needed to guide selection of appropriate empiric treatment.^1,7 The emergence of multiple-drug-resistant strains of Shigella sp. especially during the last two decades has made the treatment for shigellosis more difficult. Currently, ciprofloxacin (or other fluoroquinolones) is recommended as the drug of choice by World Health Organization for the therapy of Shigella infections in both adults and children.¹⁷ In addition, ceftriaxone, pivmecillinam (amdinocillin pivoxil), and azithromycin are considered as alternative drugs.¹⁷ Shigellae expressing extended-spectrum β-lactamases (ESBLs) have emerged globally, and this has limited the treatment strategies available for shigellosis.¹⁰

Andaman and Nicobar Islands, an archipelago of >500 islands situated in the Bay of Bengal about 1,200 km southeast of peninsular India (92–94°E; 6–14°N), is an Union Territory of India. More than 350,000 people, including six aboriginal tribes and settlers from mainland India, live in these islands. Healthcare is provided almost entirely by the Government. G.B. Pant Hospital, located at Port Blair (capital of the territory), is the only referral hospital in the islands. Hospital-based surveillance in the islands has identified shigellosis as a major cause of acute diarrhea in children.^5,6 The proportion of different species and serotypes among Shigella isolates and their drug resistance patterns has been showing considerable variation over the years.^5,15 Extended-spectrum cephalosporins and quinolones are generally used for the treatment of shigellosis in India. However, these antibiotics were ineffective in many cases, and the development of resistance to extended-spectrum cephalosporins was strongly suspected.¹¹ Resistance to first-generation cephalosporins was observed for the first time in these islands in the year 2006 and to third-generation cephalosporins in late 2008, when an isolate from a 3-year-old boy hospitalized with acute dysentery was found to be resistant to cefixime, ceftriaxone, and cefotaxime. In this communication, we report the status of third-generation cephalosporin resistance in Shigella isolates in these islands.

Patients included in the study were the diarrhea cases admitted to G.B. Pant Hospital, Port Blair, and primary health centers in Andaman and Nicobar Islands during January 2008 to December 2009. Written consents were obtained from the patient or guardian. No death caused by shigellosis was reported during the study period. Stool samples were collected on arrival to the hospital/primary health centre (PHC) before the administration of antimicrobials. The stool samples were cultured using standard procedures for isolation of enteric bacterial pathogens. Shigella species were identified biochemically by standard methods and serologically typed by slide agglutination with specific antisera (Denka Seiken Co., Ltd.). Antibiotic susceptibility was tested by disk diffusion method, according to Clinical and Laboratory Standards Institute guidelines³ using disks (Hi-Media) of ampicillin (AMP, 10 μg), carbenicillin (CAR, 100 μg), imipenem (IMP, 30 μg), amoxicillin-clavulanate (AMC, 20/10 μg), cefuroxime (CXM, 5 μg), cephalothin (CEF, 30 μg), cefixime (CFM, 30 μg), ceftriaxone (CRO, 30 μg), cefotaxime (CTX, 30 μg), ceftazidime (CAZ, 30 μg), tetracycline (TET, 30 μg), co-trimoxazole (CoT, 20 μg), nalidixic acid (NAL, 30 μg), ciprofloxacin (CIP, 30 μg), norfloxacin (NOR, 10 μg), ofloxacin (OFX, 5 μg), gatifloxacin (GAT, 5 μg), gentamicin (GEN, 10 μg), amikacin (AMK, 30 μg), nitrofurantoin (NIT, 300 μg), azithromycin (AZM, 30 μg), and chloramphenicol (CHL, 30 μg). ESBL production was detected using the double-disk synergy method with ceftazidime-clavulanic acid (CAC, 30/10 μg) and ceftriaxone-clavulanic acid (CAC, 30/10 μg). Control strains Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were included in each test. The minimum inhibitory concentrations (MICs) of third-generation cephalosporins (ceftriaxone, ceftazidime, and cefotaxime) and amoxicillin-clavulanate was determined by E-test (AB Biodisk). All the isolates resistant to third-generation cephalosporins were screened for the presence of various β-lactam genes (bla_TEM, bla_SHV, bla_OXA-1, bla_OXA-7, and bla_CTX-M-3) following standard techniques.¹³ Genomic DNA of each isolate was used as a template to generate random amplified polymorphic DNA, enterobacteriaceae repetitive intergenic consensus, repetitive extragenic palindrome, and BOX-sequence-based PCR fingerprints following the standard techniques.^12,15

Three hundred eleven stool samples were processed during January 2008 to December 2009, and 44 (14%) Shigella isolates were recovered. The species distribution of the isolates is as follows: Shigella flexneri 29 (66%) followed by Shigella sonnei 10 (23%) and Shigella dysenteriae 5 (11%). No Shigella boydii was isolated during the study period. Out of these 44 Shigella isolates, 6 (14%) were found to be resistant to all the three third-generation cephalosporins tested in disk diffusion tests. All these cephalosporin-resistant strains were also resistant to fluoroquinolones up to fourth generation. The MICs of the resistant isolates ranged from 30 to >256 mg/L for ceftriaxone, 5 to >256 mg/L for cefotaxime, 5 to >256 mg/L for ceftazidime, and 5 to >256 mg/L for amoxicillin-clavulanate (Table 1). The MICs for these strains were above the breakpoint for reduced susceptibility as per the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2007). All the cephalosporin-resistant Shigella strains were found to produce ESBL as the zone diameters around ceftazidime-clavulanic acid and ceftriaxone-clavulanic acid disk were at least 5 mm more than that around the ceftazidime and ceftriaxone disk.

Table 1.

Details of the Shigella Isolates with Third-Generation Cephalosporin Resistance

						MIC (mg/L)
Strain no.	Patient (age/sex)	Date of isolation	Geographical location	Shigella sp.	Resistant antibiotics	CRO	CTX	CAZ	AMC	No. of genetic type
BC 24	2Y8M/F	28/08/2008	Middle Andaman	Shigella flexneri 2a	GEN, CHL, TET, CIP, NOR, NAL, AMP, GAT, OFX, AZT, CAR, CEF, CFM, CXM, CRO, CTX, CAZ, AMC	30	5	5	>256	A
BC188	9M/M	22/09/2009	South Andaman	S. flexneri 2a	GEN, TET, CIP, NOR, NAL, AMP, GAT, OFX, AZT, CAR, CoT, AMK, CRO, CXM, CEF, CTX, CAZ, CFM, AMC	>256	>256	>256	>256	A
DS 500	3Y/M	14/05/2008	South Andaman	S. flexneri 4a	CHL, TET, CIP, NOR, NAL, AMP, GAT, OFX, AZT, CAR, CoT, CRO, CXM, CEF, CTX, CAZ, CFM, AMC	>256	>256	>256	>256	A
DS 529	1Y4M/M	13/01/2009	Middle Andaman	S. flexneri 2a	GEN, TET, CIP, NOR, NAL, GAT, OFX, AZT, CAR, AMK, CRO, CXM, CEF, CTX, CAZ, CFM, AMC	>256	>256	>256	>256	A
BC 33	1Y6M/M	10/9/2008	South Andaman	Shigella dysenteriae 2	GEN, CHL, TET, CIP, NOR, NAL, AMP, GAT, OFX, AZT, CAR, CoT, AMK, CRO, CXM, CEF, CTX, CAZ, CFM, AMC	30	>256	>256	30	B
DS 505	6M/F	30/05/2008	South Andaman	S. dysenteriae 2	GEN, TET, CIP, NOR, NAL, AMP, GAT, OFX, AZM, CAR, CoT, AMK, NIT, CRO, CXM, CEF, CTX, CAZ, CFM, AMC	30	>256	>256	5	B

A, one type of banding/genetic pattern shown by DNA fingerprinting (REP, ERIC, BOX, and RAPD PCR).

B, another type of banding/genetic pattern shown by DNA fingerprinting (REP, ERIC, BOX, and RAPD PCR).

AMP, ampicillin; CAR, carbenicillin; AMC, amoxicillin-clavulanate; CXM, cefuroxime; CEF, cephalothin; CFM, cefixime; CRO, ceftriaxone; CTX, cefotaxime; CAZ, ceftazidime; TET, tetracycline; CoT, co-trimoxazole; NAL, nalidixic acid; CIP, ciprofloxacin; NOR, norfloxacin; OFX, ofloxacin; GAT, gatifloxacin; GEN, gentamicin; AMK, amikacin; NIT, nitrofurantoin; CHL, chloramphenicol; ERIC, enterobacteriaceae repetitive intergenic consensus; RAPD, random amplified polymorphic DNA; REP, repetitive extragenic palindrome; AZM, azithromycin.

All the isolates harbored the β-lactam-resistant genes bla_TEM (GenBank accession nos. HQ203207–HQ203208), bla_OXA-1, and bla_CTX-M-3 (GenBank accession nos. HQ203199–HQ203200)_. The genes bla_SHV and bla_OXA-1 were not found in any of the resistant isolates.

All S. flexneri isolates showed similar patterns in repetitive extragenic palindrome, enterobacteriaceae repetitive intergenic consensus, and BOX and random amplified polymorphic DNA PCR assays, and so did the isolates belonging to S. dysenteriae (fingerprints not shown).

Third-generation cephalosporin resistance among Shigella sp. was reported for the first time from France.⁴ Since then, many cephalosporin-resistant strains of Shigella sp. have been reported from the developing countries in Asia.^8,14 Currently, in India third-generation cephalosporins are used as an alternative in patients who do not respond to fluoroquinolone treatment. The emergence of resistance to these cephalosporins further reduces the choice of drugs for the treatment of shigellosis. Two (40%) out of the five isolates of S. dysenteriae were resistant to third-generation cephalosporins, whereas only 4 (14%) out of the 29 S. flexneri isolates were resistant. Although third-generation cephalosporin resistance appears to be more common among S. dysenteriae than among S. flexneri, the numbers are too small to make a definite conclusions.

Shigella dysenteriae attracts special attention for its epidemic potential and its association with most serious dysentery cases, high case-fatality rate, and various complications.² If any outbreak of dysentery occurs in these islands¹⁶ in the coming years, clinicians are left with a very limited choice of drugs for antibacterial therapy. The acquisition of resistance by enteric pathogens to the increasing number of antibacterial drugs is becoming a grave concern, particularly in developing countries where shigellosis is of common occurrence. The options for effective and inexpensive antibacterial therapy for shigellosis are shrinking. A network of laboratories for real-time monitoring of antibiotic resistance among enteric pathogens and timely dissemination of such information to the clinicians for modification of treatment strategy is the need of the hour.

Footnotes

Acknowledgments

The authors are thankful to the Indian Council of Medical Research (Project no. Tribal-26/2006/ECD-II) for providing financial grant for the study.

Ethics Approval

The study was cleared by institutional ethics committee.

Disclosure Statement

The authors do not have any commercial or other associations that may pose a conflict of interest.

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