Monitoring of Water Quality, Antibiotic Residues, and Antibiotic-Resistant Escherichiacoli in the Kshipra River in India over a 3-Year Period.

The emergence of antibiotic resistance is a major global and environmental health issue, yet the presence of antibiotic residues and resistance in the water and sediment of a river subjected to excessive anthropogenic activities and their relationship with water quality of the river are not well studied. The objectives of the present study were a) to investigate the occurrence of antibiotic residues and antibiotic-resistant Escherichiacoli (E.coli) in the water and sediment of the Kshipra river in India at seven selected sites during different seasons of the years 2014, 2015, and 2016 and b) to investigate the association between antibiotic residues and antibiotic-resistant E.coli in water and sediment and measured water quality parameters of the river. Antibiotic residues and resistant E.coli were present in the water and sediment and were associated with the measured water quality parameters. Sulfamethoxazole was the most frequently detected antibiotic in water at the highest concentration of 4.66 µg/L and was positively correlated with the water quality parameters. Significant (p < 0.05) seasonal and spatial variations of antibiotic-resistant E.coli in water and sediment were found. The resistance of E.coli to antibiotics (e.g., sulfamethiazole, norfloxacin, ciprofloxacine, cefotaxime, co-trimoxazole, ceftazidime, meropenem, ampicillin, amikacin, metronidazole, tetracycline, and tigecycline) had varying associations with the measured water and sediment quality parameters. Based on the results of this study, it is suggested that regular monitoring and surveillance of water quality, including antibiotic residues and antibiotic resistance, of all rivers should be taken up as a key priority, in national and Global Action Plans as these can have implications for the buildup of antibiotic resistance.

Molecular characterization of carbapenem-resistant Acinetobacter baumannii strains from a tertiary care center in South India / Caracterización molecular de cepas de Acinetobacter baumannii carbapenem-resistante en un centro de atención terciaria en el sur de la India

Objectives: Carbapenem resistantAcinetobacter baumannii is an important therapeutic and infection control challenge worldwide. In this study, we investigated the prevalence and distribution of molecular mechanisms of resistance among carbapenem resistant A. baumannii species at a tertiary care setting in South India. Materials and Methods: A total of 89 non-duplicate clinical isolates of carbapenem-resistantA. baumannii were collected from critical care units of St. John's Medical College Hospital, Bengaluru, India. Polymerase chain reaction (PCR) was performed to detect blaOXA type carbapenemase blaOXA-51-like, blaOXA-23-like, blaOXA-24-like and bla OXA-58-like, MBL genes blaNDM, blaIMP, and blaVIM genes. Molecular typing of carbapenem-resistant A. baumannii strains was performed by using Rep-PCR. Results: Eighty-seven of the isolates were found to carry the blaOXA-51 gene and 81 (91%) isolates were found to have blaOXA-51-like gene and blaOXA-23, gene. The bla OXA-24 like gene was detected in two isolates of which one also carried blaOXA-51 like and one isolate carried blaVIM coding gene. The prevalence of blaNDM, blaIMP, bla VIM genes was 12(13%),14 (16%) and 57(64%) respectively. Cluster analyses revealed a 90% similarity and were divided into 5 clusters. Most of the isolates containing carbapenemases coding genes grouped under cluster A, C and UC. Considerable heterogeneity was observed within UC cluster indicating circulation of multiple strains of A. baumannii within our institution. Conclusions: Carbapenemase coding blaOXA-23, blaOXA-24 and blaOXA-51 -like were more common than blaVIM and blaNDM. The presence of blaNDM with other genes coding for carbapenemases indicate the ability of the strains to acquire novel genes despite having its share of the blaOXA like carbapenemase.

Objetivos: El Acinetobacter baumannii resistente a Carbapenem es un reto importante en todo el mundo para su tratamiento y para el control de infecciones hospitalarias. Nosotros estudiamos la prevalencia y los mecanismos de resistencia en aislados de un centro de atención terciario, en el sur de la India Materiales y Métodos: Se estudiaron 89 aislados clínicos de A. baumannii recolectados en unidades de cuidado crítico del Hospital St. John's Medical College en Bengaluru, India. Se realizó amplificación por PCR (Reacción en Cadena de Polimerasa) y luego tipificación molecular con la técnica Rep-PCR (PCR de elementos repetitivos palindromicos) para detectar los genes de carbapenemasa blaOXA, blaOXA-51, blaOXA-23, blaOXA-24, blaOXA-58, MBL, blaNDM, blaIMP y blaVIM. Resultados: Se encontraron 87 aislados que llevaban el gen blaOXA-51 y de ellos en 81 (91%) se encontró blaOXA-51 y blaOXA-23. El blaOXA-24 se detectó en dos aislados de los cuales uno de ellos llevaba blaOXA-51 y otro blaVIM. Los genes blaNDM, blaIMP y blaVIM se encontraron en 12 (13%),14 (16%) y 57(64%) de los aislados, respectivamente. El análisis de agrupamiento reveló un 90% de similitud entre los aislados y que podían asignarse a 5 agrupamientos. La mayoría de aislados llevaban genes de carbapenemasas de los grupos A, C y UC. Se observó mucha heterogeneidad dentro del agrupamiento UC indicando que existe circulación de múltiples cepas de A. baumannii dentro de nuestra institución. Conclusiones: Las carbapenemasas que codifican para blaOXA-23, blaOXA-24 y blaOXA-51 son más comunes que blaVIM y blaNDM en nuestra institución. La presencia de NDM con otros genes codificando para carbapenemasas indica la capacidad que tienen este tipo de aislados para adquirir nuevos genes a pesar de contar con blaOXA.

Detection of virulence genes in ESBL producing, quinolone resistant commensal Escherichia coli from rural Indian children.

INTRODUCTION: Extended-spectrum ß-lactamase producing commensal Escherichia coli are considered as a reservoir of antibiotic resistance genes that may be transmitted in the community. This study aimed to determine the genes coding for ESBLs, plasmid mediated quinolone resistance and virulence markers in commensal E. coli isolated from healthy school children. METHODOLOGY: ESBL producing E. coli isolates (n = 47) were obtained from 529 fecal samples of healthy school children from a rural area in central India. Multiplex PCR was used to detect the genes coding for cephalosporin and quinolone resistance, for virulence fluA, fluB, stx1, stx2, eae, bfp, lt, stII, virF, ipaH, daaE, aafII and phylogenetic groups. RESULTS: Of the 47 ESBL producing E. coli, 41 were positive for CTXM-15, 23 for TEM-1, 8 for OXA-1and a single for SHV-12. For plasmid-mediated quinolone resistance, all the 47 isolates carried the aac(6')-ib-cr gene, and amongst them18 were qnrS positive. Virulence gene, fluA was detected in 32,whereas eae in 14, daaE in 7 and fluB in 1. In 10 isolates, fluA and eae and in 7, fluA and daaE co-existed. Of the 47 E. coli isolates, 18 were grouped into the phylogenetic group B2, 17 in D and 12 in A. The proportion of isolates positive for fluA gene in the phylogenetic group B2 (18/18), was significantly higher than in group A (7/12) and D (6/17). CONCLUSION: Commensal E. coli in healthy children in rural India may serve as reservoirs of resistance towards cephalosporins and fluoroquinolones and virulence coding genes for urinary tract and diarrheal infections.

Frequency and factors associated with carriage of multi-drug resistant commensal Escherichia coli among women attending antenatal clinics in central India.

BACKGROUND: Commensal Escherichia coli are a prominent reservoir of genes coding for antibiotic resistance and also responsible for endogenous infections in pregnant women. We studied the factors in pregnant women associated with carriage of multi-drug resistant (MDR) E. coli and genetic determinants of antibiotic resistance in them. METHODS: Women attending to Obstetric and Gynaecology department outpatient clinics for routine antenatal check-up were administered a questionnaire. Peri-anal swabs were collected for culture isolation and identification of E.coil. Antibiotic sensitivity was done using the Kirby-Bauer disc diffusion method as recommended by the CLSI guidelines. MICs for quinolones and third generation cephalosporins were done using the agar dilution method. Genes coding for production of beta lactamses and for the quinolone resistance determinant were screened by polymerase chain reaction. Rep-PCR was done on MDR isolates for detecting possible genetic similarity. Multiple logistic regression models were used to determine the independent factors associated with carriage of MDR isolates. RESULTS: A total of 710 isolates of E. coli from 710 women (mean age 26 years) were included in the study. Resistance to at least one antibiotic tested was detected in 94% of the E. coli isolates. A total of 109 isolates were ESBL producing and 35 isolates were MDR. In the MDR isolates MIC(50) and MIC(90) for quinolones and third generation cephalosporins were high for those isolates that carried bla(TEM) gene (26 isolates) and bla(CTX-M) gene (24 isolates). Both bla(TEM) and bla(CTX-M) genes were detected in 19 isolates. The commonest Plasmid Mediated Quinolone Resistance (PMQR) gene identified was aac(6')-Ib-cr (n = 23/25). All isolates carrying the PMQR genes were also positive for bla(CTX-M) and bla(TEM) gene. Mutations in gyr A and par C genes were present in all 35 MDR isolates. The statistically significant risk factors for carriage of MDR E. coli were graduate or post-graduate education, a self-employed status, a family size of more than 10 members, antibiotic usage in last four weeks, and history of hospitalization in the last four weeks. CONCLUSIONS: The presence of genes coding for extended spectrum of beta lactamases and plasmid mediated quinolone resistance in commensal E. coli is disconcerting. The study provides strong basis good antibiotic stewardship.

Identification of extended-spectrum ß-lactamase and quinolone resistance genes in Escherichia coli isolated from hospital wastewater from central India.

OBJECTIVES: To investigate the presence of antibiotic resistance genes (ARGs), related to commonly used ß-lactams and quinolones, in Escherichia coli present in hospital wastewater in central India. METHODS: Cefotaxime- and ciprofloxacin-resistant E. coli isolates from hospital-associated wastewater samples were collected from two tertiary care hospitals in the Ujjain district of India during 2008-09. The presence of bla(CTX-M), bla(TEM,) bla(SHV), qnrA, qnrB, qnrS, aac(6')-Ib-cr and qepA genes was detected by PCR and sequencing. RESULTS: Twenty-five E. coli isolates were extended-spectrum ß-lactamase (ESBL) positive. bla(CTX-M-15) and bla(TEM-1) genes were identified in 21 and 16 ESBL-positive isolates by PCR, respectively. Amongst 30 fluoroquinolone-resistant E. coli isolates, aac(6')-Ib-cr was detected in 27 isolates, qnrA in 1 isolate, qnrB in 2 isolates and qepA in 3 isolates. CONCLUSIONS: bla(CTX-M-15,) bla(TEM-1), aac(6')-Ib-cr, qnrA, qnrB and qepA genes are present in E. coli occurring in hospital wastewater in central India.

Efficacy of loop mediated isothermal amplification (LAMP) assay for the laboratory identification of Mycobacterium tuberculosis isolates in a resource limited setting.

Current methods of TB diagnosis are time consuming and less suited for developing countries. The LAMP (loop mediated isothermal amplification) is a rapid method more suitable for diagnosis in resource limited settings and has been proposed as a viable test requiring further evaluation for use as a laboratory method as well. We evaluated two LAMP assays, using culture lysates of clinical sputum samples (from Southern India) and compared it to a proprietary multiplex PCR reverse-hybridization line probe assay ('GenoType MTBC' from HAIN Lifescience GmbH, Germany). The LAMP procedure was modified to suit the local conditions. The Mycobacterium tuberculosis specific LAMP assay ('MTB LAMP') showed sensitivity and specificity, of 44.7% and 94.4% respectively in a 60 min format, 85.7% and 93.9% respectively in a 90 min format and 91.7%, and 90.9% respectively in a 120 min format. The Mycobacteria universal LAMP assay ('Muniv LAMP') showed a sensitivity of 99.1%. The LAMP was shown to be a rapid and accessible assay for the laboratory identification of M. tuberculosis isolates. Initial denaturation of template was shown to be essential for amplification in unpurified/dilute samples and longer incubation was shown to increase the sensitivity. The need for modification of protocols to yield better efficacy in this scenario needs to be addressed in subsequent studies.