Debdutta Bhattacharya

Rapid emergence of third-generation cephalosporin resistance in Shigella sp. isolated in Andaman and Nicobar Islands, India.

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.

Green and ecofriendly synthesis of silver nanoparticles: Characterization, biocompatibility studies and gel formulation for treatment of infections in burns.

The current study summarizes a unique green process for the synthesis of silver nanoparticles (AgNPs) by simple treatment of silver nitrate with aqueous extract of Ammania baccifera. Phytosynthesized AgNPs were characterized by various advanced analytical methods and studied for its use against infections associated with burns. Formation of AgNPs was observed by visual color change from colorless to dark brown and confirmed by UV-visible characteristic peak at 436 nm. Zeta potential, particle size and polydispersity index of nano-silver were found to be -33.1 ± 1.12, 112.6 ± 6.8 nm and 0.3 ± 0.06 respectively. XRD spectra revealed crystalline nature of AgNPs whereas TEM confirmed the presence of mixed morphology of AgNPs. The overall approach designated in the present research investigation for the synthesis of AgNPs is based on all 12 principles of green chemistry, in which no man-made chemical other than the silver nitrate was used. Synthesized nano-silver colloidal dispersion was initially tested for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against a panel of organisms involved in infections associated with burns (Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA) and methicillin resistant S. aureus (MRSA)). MIC and MBC were found to be in range of 0.992 to 7.93 and 7.93 to 31.75 μg/mL respectively. MBC was used for formulation of AgNP gel and tested for its efficacy using agar well diffusion method against PA, SA and MRSA. Comparative bactericidal efficacy of formulated gel (0.03% w/w) and marked formulation Silverex™ ionic (silver nitrate gel 0.2% w/w) showed equal zone of inhibition against all pathogenic bacteria. Formulated AgNP gel consisting of 95% lesser concentration of silver compared to marketed formulation was found to be equally effective against all organisms. Hence, the formulated AgNP gel could serve as a better alternative with least toxicity towards the treatment presently available for infections in burns.

Emergence of an Unusual Genotype of Rotavirus in Andaman and Nicobar Islands, India

Rotavirus is the most common cause of severe diarrhoea worldwide, affecting over 125 million young children every year in developing countries. The present study is a part of ongoing childhood diarrhoeal surveillance to determine the strain diversity of rotaviruses prevalent in Andaman and Nicobar Islands. From October 2010 to February 2012, 296 stool samples from children (age group 6–60 months) with gastroenteritis were obtained from different referral hospitals/primary health centres and community health centres in Andaman and Nicobar Islands. A total of 47 samples were found positive for GARV. Among these, 21 (44.7%) samples belong to G2P[4], 12 (25.5%) samples were G1P[8], 10 (21.2%) samples belong to G9P[4], 2 (4.3%) samples belong to G1P[4] and 2 (4.3%) samples had a mixed genotype. Rotavirus G2 genotype remains the most common genotype in these islands. The prevalence of G9 rotavirus reported in the present study is higher than that reported from mainland India. The results emphasize the role of the unusual serotype G9 as an epidemiologically important genotype and the need to include G9 specificity in a rotavirus vaccine.

Prevalence of the plasmid-mediated quinolone resistance determinants among clinical isolates of Shigella sp. in Andaman & Nicobar Islands, India.

Aims:  This study was carried out to find the prevalence of various plasmid‐mediated quinolone‐resistant (PMQR) determinants among the quinolone‐resistant clinical isolates of Shigella sp. from paediatric patients in Andaman & Nicobar Islands.

Changing patterns and widening of antibiotic resistance in Shigella spp. over a decade (2000-2011), Andaman Islands, India.

This study is a part of the surveillance study on childhood diarrhoea in the Andaman and Nicobar Islands; here we report the drug resistance pattern of recent isolates of Shigella spp. (2006-2011) obtained as part of that study and compare it with that of Shigella isolates obtained earlier during 2000-2005. During 2006-2011, stool samples from paediatric diarrhoea patients were collected and processed for isolation and identification of Shigella spp. Susceptibility to 22 antimicrobial drugs was tested and minimum inhibitory concentrations were determined for third-generation cephalosporins, quinolones, amoxicillin-clavulanic acid combinations and gentamicin. A wide spectrum of antibiotic resistance was observed in the Shigella strains obtained during 2006-2011. The proportions of resistant strains showed an increase from 2000-2005 to 2006-2011 in 20/22 antibiotics tested. The number of drug resistance patterns increased from 13 in 2000-2005 to 43 in 2006-2011. Resistance to newer generation fluoroquinolones, third-generation cephalosporins and augmentin, which was not observed during 2000-2005, appeared during 2006-2011. The frequency of resistance in Shigella isolates has increased substantially between 2000-2006 and 2006-2011, with a wide spectrum of resistance. At present, the option for antimicrobial therapy in shigellosis in Andaman is limited to a small number of drugs.

Dengue: a newly emerging viral infection in Andaman and Nicobar Islands, India

Prior to 2009 dengue fever had not been reported in the Andaman and Nicobar archipelago. In 2009, a few patients with dengue fever-like illness were reported, some of whom tested positive for dengue antibodies. In 2010, 516 suspected cases were reported, including some with dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS); 80 (15·5%) were positive for dengue antibodies. DENV RNA was detected in five patients and PCR-based typing showed that three of these belonged to serotype 1 and two to serotype 2. This was confirmed by sequence typing. Two clones of dengue virus, one belonging to serotype 1 and the other to serotype 2 appeared to be circulating in Andaman. Emergence of severe diseases such as DHF and DSS might be due to recent introduction of a more virulent strain or because of the enhancing effect of sub-neutralizing levels of antibodies developed due to prior infections. There is a need to revise the vector-borne disease surveillance system in the islands.

Isolation of NDM-1-producing multidrug-resistant Pseudomonas putida from a paediatric case of acute gastroenteritis, India

Pseudomonas putida is an uncommon opportunistic pathogen, usually susceptible to antimicrobial agents. Data concerning resistance to antimicrobial agents in clinical P. putida isolates are limited. To the best of our knowledge we report for the first time the isolation of NDM-1-producing multidrug-resistant P. putida from a case of acute gastroenteritis. The isolate showed resistance to a wide range of antimicrobials, including fluoroquinolones, third-generation cephalosporins and carbapenems. The isolate also exhibited multiple mutations in the quinolone resistance determining region and showed the presence of qepA, blaTEM, blaOXA1 and blaOXA7 genes. The present study highlights the importance of looking for the relatively rare aetiological agents in clinical samples that do not yield common pathogens.

Emergence of Multidrug-Resistant Vibrio cholerae O1 Biotype El Tor in Port Blair, India

Cholera is a major disease in the developing world. The World Health Organization reported in 2006 that 236,896 cases of cholera occurred in 52 countries, a 79% increase over 2005.1 During the past decade, the dominance of the O1 Ogawa serotype of Vibrio cholerae and a quiescent period during the O139 era was observed.2 El Tor V. cholerae have replaced the classical biotype over the past few decades.3–5 Cycles of serotype shifting at intervals of 2–8 years have been reported.6 During the monsoon season, sporadic and small clusters of cases of cholera occur almost every year in Port Blair, India (Bhattgacharya DS and others, unpublished data). Two outbreaks of cholera have been reported from Andaman and Nicobar Islands. The first outbreak, which was caused by V. cholerae O1 Ogawa, occurred in Nancowry Islands in 2002.7 The second outbreak, which was caused by V. cholerae O1 Inaba, occurred in Port Blair and its suburbs in 2006.8 We report the emergence of multidrug-resistant V. cholerae O1 cholera in the Andaman Islands. This study was approved by the institutional ethical committee. During May–June 2010, there was an increase in diarrhea cases in Port Blair, the capital city of the Andaman and Nicobar Islands. Fecal samples were collected from persons with suspected cholera who were admitted to the G.B. Pant Hospital in Port Blair, the only referral hospital in the territory, and a private childcare hospital, and processed according to standard procedures for isolation and identification of V. cholerae. Written consent was obtained from the patients or guardians before collection of samples. The V. cholerae strains were serotyped by using polyvalent and monovalent antisera (Denka Seiken Co., Ltd., Tokyo, Japan). Susceptibility to different antimicrobial drugs was tested by using the disk diffusion technique according to Clinical and Laboratory Standards Institute (CLSI) (Wayne, PA) guidelines9 and commercially available antimicrobial drug disks (Hi-Media, Mumbai, India). The drugs tested were ampicillin (10 μg), carbenicillin (100 μg), imipenem (30 μg), amoxicillin-clavulanic acid (20/10 μg), cefixime (30 μg), cefuroxime (5 μg), cephalothin (30 μg), ceftriaxone (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), tetracycline (30 μg), co-trimoxazole (20 μg), nalidixic acid (30 μg), ciprofloxacin (30 μg), norfloxacin (10 μg), ofloxacin (5 μg), gatifloxacin (5 μg), gentamicin (10 μg), amikacin (30 μg), nitrofurantoin (300 μg), azithromycin (30 μg), and chloramphenicol (30 μg). Escherichia coli strain ATCC 25922 was used as the quality control strain. The minimum inhibitory concentrations (MICs) of azithromycin, tetracycline, and ciprofloxacin were determined for all strains by using the Etest (AB Biodisk, Solna, Sweden) following CLSI procedures and interpretative standards for V. cholerae. Because there is no reference zone size for V. cholerae resistance to azithromycin, we considered a zone of inhibition ≥ 18 mm as the cut-off value to determine susceptibility, as followed in other studies on V. cholerae.10 All Vibrio cholerae O1 isolates were screened for virulence genes ctxA, tcpA (El Tor/Classical), toxR, toxS, toxRS, VPI, toxT, ace, zot, and tcpP by using a polymerase chain reaction–based detection technique.11 Random amplified polymorphic DNA fingerprinting was performed for all isolates by using an arbitrary primer M16 (5′-AAAGAAGGACTCAGCGACTGCG-3′).12 Reference strains of V. cholerae O139, V. cholerae O1 serotype Ogawa, and biotype El Tor were used as controls.13 A total of 62 stool samples were collected from patients with suspected cholera who came to or were admitted to the two hospitals in Port Blair. All patients were residents of South Andaman Island. Vibrio cholerae was isolated from 19 patients. Six (31.6%) isolates were V. cholerae Inaba, one (5.2%) was V. cholerae Ogawa, and 12 (63.2%) were non-agglutinating vibrios. The first confirmed case was detected on June 2, 2010, and the last confirmed case was detected on June 23, 2010. The last case-patient apparently had contracted the infection on another island, Little Andaman, and had symptoms develop while he was traveling to Port Blair. The isolate obtained from this patient was V. cholerae O1 Ogawa. No deaths caused by cholera were reported during the study period. None of the patients had any recent history of travel to mainland India or other islands, except the patient who contracted the infection on Little Andaman Island. Although the outbreak that occurred in the islands in 2002 was caused by V. cholerae O1 Ogawa resistant to ampicillin, nalidixic acid and co-trimoxazole, the outbreak of 2006 was caused by V. cholerae O1 Inaba that was resistant to nitrofurantoin, in addition to the above three drugs. All V. cholerae isolates obtained during June 2010 also were resistant to ampicillin, nalidixic acid, co-trimoxazole, nitrofurantoin, tetracycline, cephalexin, and carbenicillin. Although four of the six V. cholerae Inaba isolates obtained during June 2010 were resistant to gentamicin, ciprofloxacin, amikacin, and azithromycin, only one isolate of V. cholerae Ogawa was resistant to amikacin and azithromycin. All isolates showed intermediate resistance to norfloxacin and ofloxacin by the disk diffusion method, with a MIC ranging from 0.125 to 1 μg/mL, respectively. The MICs of tetracycline and ciprofloxacin for strains resistant to these two drugs ranged from 16 to 32 μg/mL. The MICs of azithromycin for strains resistant to this drug ranged from 8 to 64 μg/mL (Table 1). Table 1 Antimicrobial drugs test results for Vibrio cholerae strains, Port Blair, India* A multiplex polymerase chain reaction showed that all V. cholerae O1 isolates had the virulence genes ctxA, tcpA (El Tor), toxR, toxS, toxRS, VPI, toxT, ace, zot, and tcpP. Random amplified polymorphic DNA RAPD analysis with primer M16 generated identical fingerprints for all the V. cholerae Inaba isolates, which were similar to fingerprints of V. cholerae O1 strains isolated during the outbreak of cholera in Port Blair in 2006. The fingerprinting profile of the sole V. cholerae Ogawa isolate, DS-597, was similar to that of V. cholerae O1 Ogawa strains isolated during 2002 outbreak. In the present study, all isolates showed multidrug resistance for 7–11 drugs. Three drug resistance patterns were observed among the seven V. cholerae isolates. Although multidrug resistance in V. cholerae isolates has been reported from elsewhere in India and neighboring countries,2,14–16 it has not been reported from Andaman and Nicobar Islands. It is likely that V. cholerae O1 Inaba has been circulating in the environment of South Andaman, probably in some non-pathogenic/non-cultivable form since the outbreak in 2006. The re-emergence after a quiescent period, when apparently no cholera occurred, might be caused by acquisition of virulence genes by non-pathogenic V. cholerae17,18 or by an increase in contamination of the environment by V. cholerae to a level adequate for successful transmission of infection. During the quiescent period, survival of V. cholerae in water bodies might have enabled dissipation of drug resistance to different serotypes or strains.14 During the last two outbreaks in 2002 and 2006 caused by V. cholerae O1 Ogawa and Inaba, respectively, all strains isolated were sensitive to tetracycline, gentamicin, amikacin, azithromycin, and cephalexin. Many of the V. cholerae strains isolated during the recent outbreak were resistant to these drugs. Ciprofloxacin and azithromycin resistance has already emerged in V. cholerae.19 Resistance to quinolones is generally associated with amino acid substitutions in portions of GyrA and ParC proteins, which are caused by mutations in the quinolone resistance–determining region.20 The presence of an integron, an integrative and conjugative element, and active efflux also adds to the factors conferring resistance to a wide range of antimicrobial drugs. Azithromycin resistance can be mediated by various mechanisms, including overexpression of efflux pumps, production of methylases, and mutations in the drug target, the 23S ribosomal RNA gene (A2059G). The mechanism of this high-level resistance could be novel or a combination of known mechanisms. However, the possibility of the strain being introduced into the environment of the islands by persons with undetected cholera who traveled to the islands from mainland India cannot be ruled out.14 Emergence of resistance to multiple drugs has been reported in other diarrheal pathogens in these islands.21 This finding is not unique because many investigations conducted in different areas have demonstrated an increase in the antimicrobial resistance spectrum among epidemically significant V. cholerae over time.20 Resistance to commonly used antimicrobial drugs is becoming a major public health concern because it complicates treatment and may result in longer hospital stays for patients. Spread of antimicrobial drug resistance has been recognized by the World Health Organization as an extremely serious problem. Vibrio cholerae possesses a number of mechanisms to evade the effects of antimicrobial drugs and a stage may come when the commonly used antimicrobial drugs are no longer effective.20 However, we are not yet stranded because strains are still sensitive to some of the newer quinolones and cephalosporins. Nonetheless, the expanding spectrum of drug resistance among these V. cholerae isolates is a cause for serious concern.

Tribes in Karnataka: Status of health research.

The south Indian State of Karnataka, once part of several kingdoms and princely states of repute in the Deccan peninsula, is rich in its historic, cultural and anthropological heritage. The State is the home to 42,48,987 tribal people, of whom 50,870 belong to the primitive group. Although these people represent only 6.95 per cent of the population of the State, there are as many as 50 different tribes notified by the Government of India, living in Karnataka, of which 14 tribes including two primitive ones, are primarily natives of this State. Extreme poverty and neglect over generations have left them in poor state of health and nutrition. Unfortunately, despite efforts from the Government and non-Governmental organizations alike, literature that is available to assess the state of health of these tribes of the region remains scanty. It is however, interesting to note that most of these tribes who had been original natives of the forests of the Western Ghats have been privy to an enormous amount of knowledge about various medicinal plants and their use in traditional/folklore medicine and these practices have been the subject matter of various scientific studies. This article is an attempt to list and map the various tribes of the State of Karnataka and review the studies carried out on the health of these ethnic groups, and the information obtained about the traditional health practices from these people.

Shewanella algae in acute gastroenteritis

Shewanella algae is an emerging bacteria rarely implicated as a human pathogen. Previously reported cases of S. algae have mainly been associated with direct contact with seawater. Here we report the isolation of S. algae as the sole etiological agent from a patient suffering from acute gastroenteritis with bloody diarrhoea. The bacterium was identified by automated identification system and 16S rRNA gene sequence analysis. Our report highlights the importance of looking for the relatively rare aetiological agents in clinical samples that does not yield common pathogens. It also underscores the usefulness of automated systems in identification of rare pathogens.