Genotypic validation of extended-spectrum ß-lactamase and virulence factors in multidrug resistance Klebsiella pneumoniae in an Indian hospital.

The emergence of extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae has been increasing rapidly across the world. The presence of virulence factors in ESBL producers further adds to the pathogenicity and severity of infection, which often complicate empirical therapy and sometimes result in treatment failures. In the present study, 227 non-repeated clinical isolates of K. pneumoniae obtained from different clinical specimens from a tertiary care hospital in India were analyzed to detect the genes responsible for ESBL production (blaTEM, blaCTX-M, and blaSHV), virulence (fimH-1, mrkD, entB, irp-1), and capsule production (K1-K2). Phenotypically identified 72 ESBL producing K. pneumoniae isolates were further subjected to PCR based genotypic analysis but only 20 were found to have at least one of the ESBL producing genes. blaTEM was the most predominant gene (100%), followed by blaSHV (90%), and blaCTX-M (85%). Similarly, the most common virulence genes were fimH-1 (70%), entB (65%), markD (55%), irp-1 (25%), K1 (25%), and K2 (20%). REP-PCR profile separated them into five major clusters (I-V), indicating the existing heterogeneity among the isolates. The resistance profile data obtained from the present study can serve as the information base to understand the infection pattern prevailing in the hospital and for physicians to recommend suitable antibiotics for the patients.

Epidemiological trends and molecular dynamics of dengue, chikungunya virus infection, coinfection, and other undifferentiated fever during 2015-2016 in Odisha, India.

Chikungunya virus (CHIKV) infection is spatiotemporally related to dengue virus (DENV) infection and mostly undiagnosed due to similar primary symptoms. In 2013, a high rate (36%) of coinfection of DENV and CHIKV was reported in Odisha. Hence, the hospital-based study was continued to synthesis current epidemiological understanding of their single distribution or coinfection. Suspected DENV patients serum samples were tested for DENV and CHIKV by serology and reverse-transcription polymerase chain reaction. The positive samples were used for analysis of mutation, selection pressure, and phylogenetic relationship. Clinical information was also analyzed. Among 648 (2015 and 2016) suspected DENV patients, 141 (21.7%) were positive for DENV (serotypes 1-3), 22 (3.4%) were positive for CHIKV (ECSA) and 4 (2.8%) were coinfected with both. Sequence analysis showed four consistent mutations (M104V, V112A, K166N, and F169L) in CprM gene of DENV 2 and two consistent mutations (M269V, D284E) in E1 gene of CHIKV. Interestingly, the CHIKV- E1 A226V mutation was absent in the studied population. It was also noticed that the peak incidence of both the infections occurs in August-September in 2015-16. Moreover, Plasmodium species, Salmonella typhi, and Rickettsial typhi infections were also observed in DENV patients. Different etiology was also detected in other undifferentiated fever patients as mixed infections (malaria, S. typhi, and R. typhi ). Hence, this investigation shows the significant reduction of DENV-CHIKV coinfection as compared with previous report, the burden of arboviruses and acute undifferentiated fever in Odisha in 2015-2016, highlighting the importance of epidemiological picture of febrile patients for appropriate patient management.

Extensively drug-resistant and pandrug-resistant Gram-negative bacteria in a tertiary-care hospital in Eastern India: A 4-year retrospective study.

OBJECTIVES: Bacteria resistant to different classes of antimicrobial agents are a major threat to humanity and risk leading the world towards the return of the pre-antimicrobial era. This study was undertaken to detect the incidence of extensively drug-resistant (XDR) and pandrug-resistant (PDR) bacteria in a tertiary-care hospital in Bhubaneswar, Odisha, India. METHODS: Positive bacterial cultures from different clinical samples were identified using a VITEK®2 compact system and the antimicrobial susceptibility profile of different Gram-negative bacteria was analysed. RESULTS: A total of 2489 clinical samples were collected and processed for culture during the period January 2013 to April 2017. Of 1103 pure bacterial cultures, 690 (62.6%) were Gram-negative bacteria. The antimicrobial susceptibility profile of Gram-negative bacterial strains revealed that 41.3% (n=285) were XDR and 8.1% (n=56) were PDR. Rates of colistin and tigecycline resistance were 16% and 51.9%, respectively. CONCLUSION: This situation demands regular surveillance of antimicrobial resistance of Gram-negative bacteria and implementation of an efficient infection control programme.

Retrospective study on clonal relationship of multidrug-resistant Klebsiella spp. indicates closed circulation and initiation of clonal divergence.

PURPOSE: Antibiotic resistance patterns often exhibit geographical variations. Periodic analyses of resistance spectra and phylogenetic trends are important guides for facilitating judicious use of therapeutic interventions. The present study retrospectively analysed the infection trends, resistance patterns, and clonal relationships between isolates of Klebsiella spp. from a tertiary care hospital. METHODOLOGY: Bacterial isolates were collected from January 2013 to June 2014 and their resistance profiles were identified using an automated bacterial identification system. A phylogenetic tree was constructed using housekeeping genes with Molecular Evolutionary Genetic Analysis software. The dN/dS ratio was determined by the Synonymous Non-synonymous Analysis Program while polymorphic sites, and the difference per site was calculated using DNA Sequence Polymorphism software. Statistical Package for Social Science software was used to perform all statistical analyses. KEY FINDINGS: The results of this study indicated the prevalence of community-acquired urinary tract and lower respiratory tract infections caused by Klebsiella spp. among geriatric patients. The occurrence of new allelic profiles, a low dN/dS ratio and the lack of strong evolutionary descent between isolates indicated that mutations play a major role in the evolution of the organism. CONCLUSION: The findings of this study highlight the consequences of antimicrobial agents exerting a silent and strong selective force on the evolution of Klebsiella spp. The expansion of such analyses is of great importance for addressing rapidly emerging antibiotic-resistant opportunistic pathogens.

Molecular characterization of extended spectrum ß-lactamase-producing Enterobacteriaceae strains isolated from a tertiary care hospital.

Infectious diseases caused by ESBL producing Enterobacteriaceae are an emerging problem worldwide which increases the empirical treatment failure, hospital cost, rate of morbidity and mortality. This also leads to the Hospital infection outbreak. Present study was undertaken to determine the frequency of blaTEM, blaCTX-M and blaSHV genes among Enterobacteriaceae. A total of 751 non-repeated clinical isolates of Enterobacteriaceae family were included in this study. Antibiotic susceptibility test was done and minimal inhibitory concentration (MIC) against four antibiotics was carried out. Five hundred fifteen multi drug resistant isolates were tested for ESBL by CLSI confirmatory method. Isolates showing ESBL positive by phenotypic method were screened for blaTEM, blaCTX-M and blaSHV genes by monoplex PCR. Two blaTEM and two blaCTX-M amplified products were selected randomly for sequencing. Sequencing data was submitted to NCBI data base. Of the 515 MDR isolates, 140 showed ESBL production by phenotypic method. All the ESBL producing isolates showed resistant to ceftazidime (100%). IMP, TGC and CL drugs could be preferred for the treatment of ESBL producers as these drugs showed a lower rate of resistance. blaTEM gene was the predominant (96.42%) followed by blaCTX-M (75%) and blaSHV (17.85%). All the three bla genes were occurred in 22 (17.14%) isolates. All the phenotypically confirmed ESBL producers were found contain any one of the three bla genes. It is concluded from the study that the blaTEM was predominantly found in Enterobacteriaceae and blaCTX-M gene also seemed to emerging.

Genetic diversity study of various ß-lactamase-producing multidrug-resistant Escherichia coli isolates from a tertiary care hospital using ERIC-PCR.

BACKGROUND & OBJECTIVES: The prevalence of multidrug-resistant (MDR) Escherichia coli isolates producing ß-lactamase enzyme is a growing problem across the globe. Strain typing is an epidemiologically important tool not only for detecting the cross transmission of nosocomial pathogens but also for determining the source of infection. The present study was conducted to understand the clonal relationship among various ß-lactamase-producing MDR E. coli isolates using enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR). METHODS: A total of 41 MDR E. coli isolates were randomly collected from various clinical samples and processed. Isolated organisms were tested for antibiotics resistance pattern. Phenotypic detection of metallo ß-lactamases (MBL) was carried out by the imipenem-ethylenediaminetetraacetic acid disc diffusion/double-disc synergy test. AmpC enzyme production was tested by a modified three-dimensional extract test. RESULTS: Almost all isolates were found sensitive to colistin. A high percentage of drug resistance was observed in these isolates against ceftazidime (100%), cefotaxime (100%), cefepime (100%), ofloxacin (97.56%), amoxicillin/clavulanic acid (97.56%) and norfloxacin (85.36%). Of the 41 isolates, ESBL producers were found to be predominant, i.e., 22 (53.65%), followed by AmpC (6, 14.63%) and MBL (5, 12.19%). INTERPRETATION & CONCLUSIONS: At 60 per cent similarity cut-off value, the dendrogram analysis showed that there were a total of 14 unique clusters of ERIC (CL-1 - CL-14) within the 41 E. coli isolates, which revealed the genetic diversity existing between them.

Prevalence of TEM, SHV, and CTX-M genes of extended-spectrum ß-lactamase-producing Escherichia coli strains isolated from urinary tract infections in adults.

Urinary tract infections (UTIs) are one of the major sources of widespread infectious diseases in the community as well as in the hospitals which increase the cause of morbidity and mortality. Prevalence of extended-spectrum-ß-lactamase (ESBL)-producing uropathogenic E. coli isolates has been found to be increased rapidly across the world. The present study was undertaken to find out the frequency of bla TEM, bla CTX-M, and bla SHV genes among E. coli isolates from UTI and detect their sensitivity pattern. A total of 112 non-repeated E. coli isolates obtained from urine samples of UTI diagnosed patients were included in this study. Antibiotic susceptibility test was done by disc diffusion method. Seventy seven (68.75%) isolates were MDR and tested for ESBL. ESBL-positive isolates were screened for bla TEM, bla CTX-M, and bla SHV genes by monoplex PCR (polymerase chain reaction). Among 46 ESBL-producing E. coli isolates, 8.69% harboured all the three bla genes. The bla TEM was the predominant (93.47%) gene followed by bla CTX-M (82.6%) and bla SHV (4.34%). It can be concluded that the prevalence of MDR (multidrug resistance) ESBL-producing E. coli appears to be high and the highest identified gene was bla TEM. The knowledge of resistance pattern can help physician's select suitable empirical antibiotic regimens, so that antibiotics showing high-resistance pattern can be avoided.

Functional Genome Screening to Elucidate the Colistin Resistance Mechanism.

Antibiogram profile of 1590 clinical bacterial isolates based on thirteen different antimicrobial compounds showed that 1.6% of the bacterial isolates are multidrug resistant. Distribution pattern based on 16S rRNA sequence analysis showed that Pseudomonas aeruginosa constituted the largest group (83.6%) followed by Burkholderia pseudomallei sp. A191 (5.17%), Staphylococcus sp. A261 (3.45%). Among the various antibiotics used, colistin appeared to be the most effective against the Gram negative bacteria. Burkholderia pseudomallei sp. A191 and Pseudomonas aeruginosa sp. A111 showed resistance to 1500 µg/ml and 750 µg/ml of colistin respectively which constitutes 7.7% of the bacterial population. A functional genomics strategy was employed to discover the molecular support for colistin resistance in Burkholderia pseudomallei sp. A191. A pUC plasmid-based genomic expression library was constructed with an estimated library size of 2.1 × 10(7)bp. Five colistin resistant clones were obtained after functional screening of the library. Analysis of DNA sequence of five colistin resistant clones showed homology to two component regularity systems (TCRS) encoding for a histidine kinase (mrgS) and its regulatory component (mrgR). Cross complementation assay showed that mutations in mrgS were sufficient enough to confer colistin resistant phenotype in a sensitive strain.

High rates of co-infection of Dengue and Chikungunya virus in Odisha and Maharashtra, India during 2013.

Dengue viral (DENV) infection is endemic in different parts of India and because of similar primary signs and symptoms, Chikungunya virus (CHIKV) is mostly undiagnosed. Hence, we investigated 204 suspected Dengue cases in a hospital based cross-sectional study in Odisha, India in 2013. It was observed that 50 samples were positive for DENV only, 28 were positive for CHIKV only and interestingly, 28 patients were co-infected with both DENV and CHIKV. Additionally, a total of 18 confirmed Dengue samples from Maharashtra, India were screened for CHIKV and out of those, 15 were co-infected. All CHIKV strains were of East Central South African (ECSA) type and serotype 2 (genotype IV) was predominant in the DENV samples. Additionally, Dengue serotype 1 and 3 were also detected during this time. Further, sequence analysis of E1 gene of CHIKV strains revealed that two substitution mutations (M269V and D284E) were observed in almost 50% strains and they were from co-infected patients. Similarly, sequence analysis of C-prM gene showed the presence of five substitution mutations, (G70S, L72F, N90S, S93N and I150L) in all serotype 1 and two consistent mutations (A101V and V112A) in serotype 2 Dengue samples. Together, it appears that a significantly high number of dengue patients (43, 44.8%) were co-infected with DENV and CHIKV during this study. This emphasizes the need of a routine diagnosis of CHIKV along with DENV for febrile patients. This will be useful in early and proper recognition of infecting pathogen to study the correlation of clinical symptoms with single or co-infection which will ultimately help to implement proper patient care in future.

Phylogenetic study of metallo-ß-lactamase producing multidrug resistant Pseudomonas aeruginosa isolates from burn patients.

The present study was carried out to understand the clonal relationship using enterobacteriaceae repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) among metallo-ß-lactamase (MBL) producing multidrug resistant Pseudomonas aeruginosa isolates from burn victims and their susceptibility to commonly used anti-pseudomonal agents. In the present study 94 non-duplicate P. aeruginosa strains from the wound samples of burn patients were included. Identification of the isolates was done by biochemical methods and antibiotic sensitivity was done by disc diffusion method following CLSI (Clinical Laboratory Standard Institute) guidelines. By using imipenem (IPM)-EDTA disk diffusion/double disc synergy method carbapenem resistant organisms were tested for MBL. To define the clonal relationship ERIC-PCR was used. Of the 94 isolates, 18 (19.14%) were found resistant to IPM and MBL production was shown 11 (11.70%) by the IPM-EDTA disc diffusion method. From dendrogram of the ERIC-PCR profile four major clusters were obtained (A, B, C and D). Cluster B contained the majority of the isolates (6 strains 1, 4, 8, 9, 10 and 11). This study using ERIC-PCR of randomly collected isolates exhibits high genetic diversity which rules out cross contamination frequency.

Surveillance of multidrug resistant uropathogenic bacteria in hospitalized patients in Indian.

OBJECTIVE: To record surveillance, antibiotic resistance of uropathogens of hospitalized patients over a period of 18 months. METHODS: Urine samples from wards and cabins were used for isolating urinary tract infection (UTI)-causing bacteria that were cultured on suitable selective media and identified by biochemical tests; and their antibiograms were ascertained by Kirby-Bauer's disc diffusion method, in each 6-month interval of the study period, using 18 antibiotics of five different classes. RESULTS: From wards and cabins, 1 245 samples were collected, from which 996 strains of bacteria belonging to 11 species were isolated, during April 2011 to September 2012. Two Gram-positive, Staphylococcus aureus (S. aureus) and Enterococcus faecalis (E. faecalis), and nine Gram-negative bacteria, Acinetobacter baumannii, Citrobacter sp., Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris and Pseudomonas aeruginosa were isolated. Both S. aureus and E. faecalis were vancomycin resistant, and resistant-strains of all pathogens increased in each 6-month period of study. Particularly, all Gram-negatives were resistant to nitrofurantoin and co-trimoxazole, the most preferred antibiotics of empiric therapy for UTI. CONCLUSIONS: Antibiograms of 11 UTI-causing bacteria recorded in this study indicated moderately higher numbers of strains resistant to each antibiotic studied, generating the fear of precipitating fervent episodes in public health particularly with bacteria, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and S. aureus. Moreover, vancomycin resistance in strains of S. aureus and E. faecalis is a matter of concern.