Neelam Taneja

Molecular characterization of multidrug-resistant Shigella species isolated from epidemic and endemic cases of shigellosis in India.

Shigella species represent one of the growing numbers of antimicrobial-resistant bacteria in developing countries. Fluoroquinolone-resistant strains of Shigella dysenteriae type 1 and Shigella flexneri type 2a emerged in India during 2002 and 2003, respectively. Sixty strains of Shigella from different parts of India were analysed for antimicrobial susceptibility, the presence of the qnr plasmid, mutations in the quinolone resistance determining regions (QRDRs), fluoroquinolone accumulation, and the presence of other genes encoding resistance to various antimicrobials. Fluoroquinolone-resistant strains had mutations in gyrA and parC genes and had an active efflux system. They were also resistant to several other antimicrobials but were susceptible to azithromycin and ceftriaxone. The majority of the strains harboured genes encoding resistance to ampicillin (97 %), tetracycline (95 %), streptomycin (95 %) and chloramphenicol (94 %). PFGE analysis revealed clonality among strains of S. dysenteriae types 1 and 5, S. flexneri type 2a and Shigella boydii type 12.

Cephalosporin-resistant Shigella flexneri over 9 years (2001–09) in India

OBJECTIVES To determine the pattern and antimicrobial resistance genes of cephalosporin resistance in Shigella flexneri and Shigella dysenteriae over 9 years. METHODS Isolates of Shigella (S. flexneri, n = 119 and S. dysenteriae, n = 24) were tested for resistance to ceftriaxone and cefepime by disc diffusion, for MIC by Etest and for extended-spectrum β-lactamase (ESBL) and AmpC production. The presence of antimicrobial resistance genes was investigated by PCR using specific primers for bla(TEM), bla(OXA-1), bla(CTX-M-15), bla(SHV) and bla(CMY-2) for all the isolates. RESULTS Twenty (16.8%) S. flexneri isolates were resistant/intermediately susceptible to ceftriaxone/cefepime, while all S. dysenteriae were susceptible. In S. flexneri isolates, the MIC(50) values of ceftriaxone and cefepime were found to be 0.032 and 0.125 mg/L, respectively, while their MIC(90) values were 12 and 8 mg/L, respectively. The MIC(50) and MIC(90) for S. dysenteriae were below 1 mg/L for ceftriaxone; however, for cefepime the MIC(90) was found to be 4 mg/L. Of the 20 resistant/intermediately susceptible S. flexneri isolates, 9 were positive for ESBL production and 4 for AmpC production by phenotypic tests. All 20 isolates were found to be positive for bla(TEM), 10 for bla(CTX-M-15), 8 for bla(OXA) and 7 for bla(CMY-2); none was positive for bla(SHV). CONCLUSIONS We report a high level of cephalosporin resistance with high MICs and ESBL- and AmpC-mediated antibiotic resistance in Shigella from north India.

Outbreaks caused by new variants of Vibrio cholerae O1 El Tor, India.

To the Editor: Vibrio cholerae O1, the causative agent of cholera, has 2 biotypes (classical and El Tor), which have traditionally been distinguished by phenotypic tests and by genetic differences in the major toxin-coregulated pilus (TCP) gene, the tcpA allele of the TCP cluster (1), the rstR region (regulatory region for phage lysogeny) of CTX phages (2), the type of cholera toxin (CT) produced, and the infection pattern of the disease they cause. However, 3 variants of the El Tor biotype have been described recently: Matlab (a place in Bangladesh) variants in 2002 (3), which could not be biotyped because they have a mixture of both classical and El Tor (4), Mozambique variant in 2004–2005, which has a typical El Tor genome but a tandem repeat of the classical CTX prophage in the small chromosome (5), and the altered El Tor type (a typical El Tor biotype and an El Tor CTX prophage that produces CT of the classical type) predominant in Bangladesh since 2001 (6). Hybrid vibrios have also been described in other regions of Asia and Africa (7). CT, encoded by the ctxA and ctxB genes, is the principal toxin produced by V. cholerae O1 and O139. Methods for differentiating the biotype-specific CT-B subunit of V. cholerae O1 include sequencing the ctxB gene, performing an ELISA wth a monoclonal antibody specific to the classical or El Tor CT, or by using a mismatch amplification mutation assay (MAMA)–PCR to distinguish between 2 kinds of ctxB genes. This assay detects sequence polymorphisms based on nt position 203 of the ctxB gene (8). In Punjab and Haryana states of northern India, during July–September 2007, 6 clusters of cholera outbreak were identified. A total of 745 case-patients were admitted to local government hospitals; the cholera attack rate was 183/1,000 population. Four deaths were reported (case-fatality rate 0.5%). The number of cases per cluster varied from 15 to 400, and adults were primarily affected (74%); 20% of patients had severe dehydration. V. cholerae O1 Ogawa was confirmed from stool cultures by using standard isolation, biochemical, and serotyping methods. Twenty-six isolates were phenotypically and genotypically characterized according to biotype. Phenotypic characterization included the Voges-Proskauer reaction, polymyxin B (50 U) susceptibility, chick cell agglutination, and sheep erythrocyte hemolysis; all isolates were confirmed as El Tor biotype. Genotypic characterization included PCR assays for ctxA and tcpA (2), rstR (3), and ctxB (8). All strains were toxigenic because each carried the ctxA gene. All strains also carried El Tor–specific tcpA (472 bp) and rstR genes (500 bp). MAMA-PCR showed the ctxB gene of both El Tor and the classical type in 21 (80%) of 26 isolates tested. Similarly, we also tested 20 available isolates from the 2002 outbreak and 4 and 19 sporadic isolates from 2003 and 2004, respectively; all were phenotypically and genotypically confirmed as El Tor and had only ctxB of the El Tor type. Of 53 water samples tested during the 2007 outbreak, 4 grew V. cholerae. Three samples were confirmed to be non-O1, non-O139 strains. Only 1 isolate was V. cholerae O1, which was positive for tcp, ctxA, and ctxB of both classical and El Tor types (Table). Table Phenotypic and genotypic traits of Vibrio cholerae O1 clinical strains isolated from northern India, 2002–2007* During the cholera outbreak of 2002 in Chandigarh (9), only 1 death was reported (case-fatality rate, <0.01%); the attack rate was 20/1,000, 58.6% were children, and only 10% had severe dehydration. Before the most recent outbreak, the affected regions of Panjab and Haryana (Ambala, Nurpur, Kurali, Mohali, Panchkula, and Raili) had been free of cholera outbreaks since 1994, though sporadic cases had been reported. The 4 deaths from cholera in 2007, along with adult preponderance, high attack rate, more severe illness, and 6 different clusters, point towards a change in the disease’s epidemiology. This change may be related to circulation of the hybrid vibrios in this region. In Bangladesh, all strains of V. cholerae O1 examined since 2001 belong to the altered El Tor type (6), which produces CT of the classical type. This altered type has replaced the seventh pandemic strain of the El Tor biotype that produced CT of the El Tor type, which indicates that a cryptic change has occurred in the seventh pandemic El Tor biotype strains of V. cholerae O1. Newly emerged variants from Bangladesh (8) have the genetic makeup of El Tor with ctxB gene of only classical, whereas our strains are unique in having ctxB of both the classical and El Tor biotypes. Our strains appear to be different from the Mozambique variant V. cholerae O1 (10), which has rstR of the classical type, in that our strains have rstR of only the El Tor type. Of 5 Matlab variants analyzed with MAMA-PCR, 3 had classical ctxB and 2 had El Tor type. Our study highlights the different genetic recombinations possible in V. cholerae and the epidemiologic role of these recombinations.

Insights into Newer Antimicrobial Agents Against Gram-negative Bacteria

Currently, drug resistance, especially against cephalosporins and carbapenems, among gram-negative bacteria is an important challenge, which is further enhanced by the limited availability of drugs against these bugs. There are certain antibiotics (colistin, fosfomycin, temocillin, and rifampicin) that have been revived from the past to tackle the menace of superbugs, including members of Enterobacteriaceae, Acinetobacter species, and Pseudomonas species. Very few newer antibiotics have been added to the pool of existing drugs. There are still many antibiotics that are passing through various phases of clinical trials. The initiative of Infectious Disease Society of America to develop 10 novel antibiotics against gram-negative bacilli by 2020 is a step to fill the gap of limited availability of drugs. This review aims to provide insights into the current and newer drugs in pipeline for the treatment of gram-negative bacteria and also discusses the major challenging issues for their management.

Viability kinetics, induction, resuscitation and quantitative real-time polymerase chain reaction analyses of viable but nonculturable Vibrio cholerae O1 in freshwater microcosm

Aim:  To study the induction of a viable but nonculturable (VBNC) state in Vibrio cholerae O1 in freshwater, in response to cold temperatures (4°C) and starvation.

Environmental and epidemiological surveillance of Vibrio cholerae in a cholera-endemic region in India with freshwater environs

Aim:  To conduct epidemiological and ecological surveillance of cholera in freshwater environments.

Detection of Shiga toxin variants among Shiga toxin-forming Escherichia coli isolates from animal stool, meat and human stool samples in India.

To study the prevalence and distribution of various variants in the stx gene of Shiga toxin–producing Escherichia coli (STEC) isolated from diverse environmental sources (animal stool, meat) and human illness, from a large geographic area in India, and to understand the association between variants, serotype distribution and human disease.

Effectiveness of Indigenous Ready-to-Use Therapeutic Food in Community-based Management of Uncomplicated Severe Acute Malnutrition: a Randomized Controlled Trial from India

A randomized controlled trial was conducted in Chandigarh, India (2011), to determine the effectiveness of indigenous ready-to-use therapeutic food (RUTF) in community-based management of uncomplicated severe acute malnutrition (SAM). Intervention was through outpatient therapeutic program site (OTP). Study and control group children (6 months-5 years) were followed up weekly for 12 weeks, in OTP and at home. All children received supplementary nutrition through anganwadis under integrated child development scheme. Study children, in addition, received therapeutic dose of RUTF in OTP. Primary outcome, 115% of baseline weight, was attained in 6 of 13 (46.2%) and 1 of 13 (7.7%) children among study and control group, respectively [odds ratio: 10.28, 95% confidence interval (CI): 1.02-103.95]. Compared with control group, addition of RUTF in study group resulted in average additional increase in weight by 13 g/kg of baseline weight/week/child (95% CI: 2-23). Indigenous RUTF was effective in community-based management of uncomplicated SAM.

Comparative efficacy evaluation of disinfectants routinely used in hospital practice: India.

Aim: The aim of this study was to evaluate and compare practically achieved disinfection efficacy of some locally available disinfectants on surfaces and infectious microbiological hospital waste. Materials and Methods: Seven disinfectants were tested at concentrations recommended by manufacturers on rough and smooth surfaces that were contaminated experimentally by locally circulating isolates of methicillin-resistant Staphylococcus aureus, multidrug-resistant Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter aerogenes, Pseudomonas aeruginosa strains, standard isolate of Salmonella typhi and Candida albicans. Reduction in microbial counts before and after surface disinfection was expressed as log reduction. A very heavy microbial waste load was simulated by immersing culture plates with heavy microbial growth in disinfectants. Daily, a sample of disinfectant was taken and subjected to in-use test. Results: The highest average log reduction of test microbes on the rough surface was given by DesNet (5.05) and Bacillocid special (5.02). A comparable average log reduction of test microbes on a smooth steel surface was noted (5.68, 5.67, 5.50) for Lysol, Bacillocid sp. and DesNet, respectively. In the discard jars, Bacillocid special worked satisfactorily for 4 days, DesNet for 3 days and Hi-giene Germitol for 1 day. The remainder of the disinfectants failed in the in-use test on Day 1. Phenolics, although widely used in our settings, may not be as good surface disinfectants as newer formulations like DesNet and Bacillocid special. Conclusions: Newer quaternary ammonium compounds and aldehyde formulations were found to be the best disinfectants for disinfection of heavy contamination.

Dipstick Test for Rapid Diagnosis of Shigella dysenteriae 1 in Bacterial Cultures and Its Potential Use on Stool Samples

Background We describe a test for rapid detection of S. dysenteriae 1 in bacterial cultures and in stools, at the bedside of patients. Methodology/Principal Findings The test is based on the detection of S. dysenteriae 1 lipopolysaccharide (LPS) using serotype 1-specific monoclonal antibodies coupled to gold particles and displayed on a one-step immunochromatographic dipstick. A concentration as low as 15 ng/ml of LPS was detected in distilled water and in reconstituted stools in 10 minutes. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 1.6×106 CFU/ml and 4.9×106 CFU/ml of S. dysenteriae 1, respectively. Optimal conditions to read the test have been determined to limit the risk of ambiguous results due to appearance of a faint yellow test band in some negative samples. The specificity was 100% when tested with a battery of Shigella and unrelated strains in culture. When tested on 328 clinical samples in India, Vietnam, Senegal and France by laboratory technicians and in Democratic Republic of Congo by a field technician, the specificity (312/316) was 98.7% (95% CI:96.6–99.6%) and the sensitivity (11/12) was 91.7% (95% CI:59.8–99.6%). Stool cultures and the immunochromatographic test showed concordant results in 98.4 % of cases (323/328) in comparative studies. Positive and negative predictive values were 73.3% (95% CI:44.8–91.1%) and 99.7% (95% CI:98–100%). Conclusion The initial findings presented here for a simple dipstick-based test to diagnose S. dysenteriae 1 demonstrates its promising potential to become a powerful tool for case management and epidemiological surveys.