الملخص
The mismatch amplification assay is a modified version of polymerase chain reaction (PCR) that permits specific amplification of gene sequences with single base pair change. The basis of the technique relies on primer designing. The single nucleotide mismatch at the 3' proximity of the reverse oligonucleotide primer makes Taq DNA polymerase unable to carry out extension process. Thus, the primers produce a PCR fragment in the wild type, whereas it is not possible to yield a product with a mutation at the site covered by the mismatch positions on the mismatch amplification mutation assay (MAMA) primer from any gene. The technique offers several advantages over other molecular methods, such as PCR-restriction fragment length polymorphism (RFLP) and oligonucleotide hybridization, which is routinely used in the detection of known point mutations. Since multiple point mutations in the quinolone resistance determining region play a major role in high-level fluoroquinolone resistance in Gram-negative bacteria, the MAMA-PCR technique is preferred for detecting these mutations over PCR-RFLP and sequencing technology.
الملخص
To understand antimicrobial resistance (AMR) patterns and mechanisms of horizontal gene transfer in human-associated environments is essential to AMR surveillance. Gram-negative bacteria (1122 isolates) from food-animal environments were characterized for antimicrobial susceptibility and AMR genes. Seventy five per cent of the isolates (837 of 1122) were resistant to at least one of the antibiotics tested. Resistance to more than three groups of antimicrobials (multidrug resistance) was observed in 43 isolates with most often encountered (12 of 43) resistance to ?-lactams, tetracycline, quinolones and nitrofurantoin. The profile of frequently reported plasmid-mediated resistance gene in these isolates was determined. The mobility of these elements as plasmids or phages was examined. The blaCTX-M gene was present in the plasmid of 61 per cent and packed in induced phage fractions in 72 per cent of the isolates and blaTEMin 69 per cent phage fractions compared to 15 per cent presence in the plasmid.
الملخص
Background & objectives: Infections caused by extended-spectrum ?-lactamase (ESBL)-producing Escherichia coli carrying blaCTX-M genes have been spreading globally, but there are geographical variations in the type of blaCTX-Mgenes prevalent and there are scanty data from India. This study was conducted to determine the CTX-M type ESBLs in E. coli isolates obtained from clinical specimens from patients with extra-intestinal infections attending a tertiary care hospital in south India. Methods: ESBL-producing E. coli isolated from patients with extra-intestinal infections were subjected to PCR using CTX-M group-specific primers. From a representative isolate, full-length CTX-M-15 gene was amplified and sequenced. An internal fragment of this gene was sequenced in 10 representative isolates. Results: Of the 300 isolates of E. coli tested, 88 per cent carried CTX-M genes and blaCTX-M-15was the most dominant gene present in 90 per cent of the positive isolates. Most (91%) of the isolates positive for blaCTX-M were sensitive to meropenem. Interpretation & conclusions: Our findings showed blaCTX-M-15 to be the dominant gene. Based on the data on antimicrobial susceptibility, cefoperazone-sulbactum could be an antimicrobial of choice.
الملخص
Background & objectives: The resistance to antibiotics in pathogenic bacteria has increased at an alarming rate in recent years due to the indiscriminate use of antibiotics in healthcare, livestock and aquaculture. In this context, it is necessary to monitor the antibiotic resistance patterns of bacteria isolated from the environmental samples. This study was conducted to determine the phenotypic and genotypic profile of antimicrobial resistance in Gram-negative bacteria isolated from environmental samples. Methods: Two hundred and fifty samples were collected from different sources, viz. fish and fishery products (99), livestock wastes (81) and aquaculture systems (70), in and around Mangaluru, India. Isolation, identification and antimicrobial profiling were carried out as per standard protocols. The isolates were screened for the presence of resistance genes using PCR. Results: A total of 519 Gram-negative bacteria comprising Escherichia coli (116), Salmonella spp. (14), Vibrio spp. (258), Pseudomonas spp. (56), Citrobacter spp. (26) and Proteus spp. (49) were isolated and characterized from 250 samples obtained from different sources. A total of 12 antibiotics were checked for their effectiveness against the isolates. While 31.6 per cent of the isolates were sensitive to all the antibiotics used, 68.4 per cent of the isolates showed resistance to at least one of the antibiotics used. One-third of the isolates showed multidrug resistance. Maximum resistance was observed for ampicillin (43.4%), followed by nitrofurantoin (20.8%). Least resistance was seen for carbapenems and chloramphenicol. PCR profiling of the resistant isolates confirmed the presence of resistance genes corresponding to their antibiotic profile. Interpretation & conclusions: This study results showed high rate of occurrence of antimicrobial resistance and their determinants in Gram-negative bacteria isolated from different environmental sources.
الملخص
Background & objectives: Infection from fluoroquinolone-resistant extra-intestinal Escherichia coli is a global concern. In this study, isolation and characterization of fluoroquinolone-resistant extra-intestinal E. coli isolates obtained from hospital samples were undertaken to detect plasmid-mediated quinolone resistance (PMQR) genes. Methods: Forty three isolates of E. coli obtained from patients with extra-intestinal infections were subjected to antibiogram to detect fluoroquinolone resistance. The mechanism of fluoroquinolone resistance was determined by the detection of PMQR genes and mutations in quinolone resistance determining region (QRDR). Results: Of the 43 isolates, 36 were resistant to nalidixic acid (83.72%) and 28 to ciprofloxacin (65.11%). Eight E. coli isolates showed total resistance to both the antimicrobials without any minimum inhibitory concentration. The detection of PMQR genes with qnr primers showed the presence of qnrA in two, qnrB in six and qnrS in 21 isolates. The gene coding for quinolone efflux pump (qepA) was not detected in any of the isolates tested. The presence of some unexpressed PMQR genes in fluoroquinolone sensitive isolates was also observed. Interpretation & conclusions: The detection of silent PMQR genes as observed in the present study presents a risk of the transfer of the silent resistance genes to other microorganisms if present in conjugative plasmids, thus posing a therapeutic challenge to the physicians. Hence, frequent monitoring is to be done for all resistance determinants.