Phenotypic detection of extended-spectrum and AmpC β-lactamases by a new spot-inoculation method and modified three-dimensional extract test: comparison with the conventional three-dimensional extract test

Shahid, Mohammad; Malik, Abida; Agrawal, Manisha; Singhal, Sanjay

doi:10.1093/jac/dkh389

Cited by 52 publications

(45 citation statements)

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“…The isolates which showed a reduced susceptibility to cefoxitin were tested for confirmation by the modified three dimensional test. An indentation or a flattening of the zone of inhibition indicated the AmpC production [7].…”

Section: Methodsmentioning

confidence: 99%

ESBL, MBL and Ampc Lactamases Producing Superbugs – Havoc in the Intensive Care Units of Punjab India

Oberoi¹

2013

JCDR

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Background: An alarming rise in the rates of the antibiotic resistance has now become a serious and an increasingly common public health concern, with severe implications, especially in the intensive care units. A variety of ß-lactamases which include ESBLs, AmpC ß-lactamases and metallo-ß-lactamases, have emerged as the most worrisome mechanism of resistance among the gram negative bacteria, which pose a therapeutic challenge to the health care settings. Materials and Methods:The present study was aimed at knowing the prevalence of various ß-lactamases in the gram negative isolates which were obtained from ICU patients. A total 273 gram negative isolates from 913 clinical samples which were received over a period of one year were processed for their identification and their antimicrobial susceptibility pattern was determined. They were then screened for the ß-lactamase production.Results: Among the 273 isolates, the ß-lactamase production was observed in 193 strains. 96 (35.16%) strains were ESBL producers, followed by 30 (10.98%) metallo ß-lactamase (MBL) producers and 15(5.4%) AmpC producers. The major ESBL and AmpC producer was Escherichia coli, while Klebsiella pneumonia was the predominant MBL producer. The co production of the ESBL/MBL/ AmpC ß-lactamases was observed in 52 (19.04%) strains and it was more common in Escherichia coli. A multidrug resistance to the fluoroquinolones and the aminoglycosides was also observed in the ß-lactamase producing organisms. Conclusion:The high prevalence of the ß-lactamases in the ICU isolates emphasizes the need for a continuous surveillance in the ICUs to detect the resistant strains, strict guidelines for the antibiotic therapy and the implementation of infection control measures to reduce the increasing burden of antibiotic resistance. INTRODUCTIONThe incidence of nosocomial infections in the intensive care units (ICU) is showing a rising trend, mainly because of the severe clinical conditions which are associated with the impaired immunity, increasing the use of invasive diagnostic procedures, lapses in the sterilization and the disinfection techniques and the indiscriminate use of antibiotics. The β-lactam antibiotics are among the most frequently prescribed antibiotics in the ICUs world-wide, which are favoured because of their efficacy, broad spectra and low toxicity. The selective pressures which are generated by the indiscriminate use of the beta-lactam antibiotics have led to the selection of a variety of mutated forms of β-lactamases such as the ESBLs, AmpC β-lactamases and metallo-β-lactamases which have emerged as the most worrisome resistance mechanism which poses a therapeutic challenge to the health care settings [1]. These "newer β-lactamases" are capable of hydrolyzing a wide range of β-lactam antibiotics, notably the extended-spectrum penicillins and the third and fourth generation cephalosporins, which include the carbapenams [2]. The ESBL producing organisms also express the AmpC β-lactamases and they may be co-transferred with the plasmids...

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Section: Methodsmentioning

confidence: 99%

ESBL, MBL and Ampc Lactamases Producing Superbugs – Havoc in the Intensive Care Units of Punjab India

Oberoi¹

2013

JCDR

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“…Since there are no published CLSI criteria for phenotypic screening or confirmatory test for AmpC b-lactmases, a modified three dimensional test was used for the detection of AmpC enzymes in cefoxitin resistant isolates [16].…”

Section: Phenotypic Ampc Detectionmentioning

confidence: 99%

Occurrence of Plasmid-Mediated AmpC β-Lactamases Among Escherichia coli and Klebsiella pneumoniae Clinical Isolates in a Tertiary Care Hospital in Bangalore

Sasirekha

Shivakumar

2011

Indian J Microbiol

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Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC b-lactamases are limited because these organisms are usually resistant to all the b-lactam antibiotics, except for cefepime, cefpirome and the carbapenems. These organisms are a major concern in nosocomial infections and should therefore be monitored in surveillance studies. Hence, this study was aimed out to determine the prevalence of plasmid-mediated AmpC b-lactamases in E. coli and K. pneumoniae from a tertiary care in Bangalore. A total of 63 E. coli and 27 K. pneumoniae were collected from a tertiary care hospital in Bangalore from February 2008 to July 2008. The isolates with decreased susceptibility to cefoxitin were subjected to confirmation test with three dimensional extract tests. Minimum inhibitory concentrations (MICs) were determined by agar dilution method. Conjugation experiments, plasmid profiling and susceptibility testing were carried out to investigate the underlying mechanism of resistance. In our study, 52 (57.7%) isolates showed resistance to cefoxitin, the occurrence of AmpC was found to be 7.7% of the total isolates. Plasmid analysis of the selected isolates showed the presence of a single plasmid of 26 kb in E. coli and 2 Kb in K. pneumoniae. Plasmid-mediated AmpC b-lactamases were found in 11.1% of K. pneumoniae and in 6.3% of E. coli. Curing and conjugation experiments showed that resistance to cephamycins and cephalosporins was plasmid-mediated. Our study has demonstrated the occurrence of plasmid-mediated AmpC in E. coli and K. pneumoniae which illustrates the importance of molecular surveillance in tracking AmpC-producing strains at general hospitals and emphasizes the need for epidemiological monitoring.

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“…1) were performed. To detect AmpC enzymes phenotypically, all cefoxitin-resistant isolates were subjected to the modified three-dimensional extract test (MTDET) as described previously (12).…”

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confidence: 99%

Concurrent Occurrence of bla _ampC Families and bla _CTX-M Genogroups and Association with Mobile Genetic Elements IS Ecp1 , IS 26 , IS CR1 , and sul1 -Type Class 1 Integrons in Escherichia coli and Klebsiella pneumoniae Isolates Originating from India

et al. 2012

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b Cefoxitin-resistant Escherichia coli (n ‫؍‬ 109) and Klebsiella pneumoniae (n ‫؍‬ 16) isolates collected from patients in India in 2009 to 2010 were screened for bla ampC families and mobilizing elements (ISEcp1, IS26, ISCR1, and sul-1-type class 1 integrons) and their association with bla ampC and for the occurrence of class A beta-lactamases (BLs) (CTX-M, TEM, and SHV). The concurrent occurrences of two distinct AmpC families (bla CIT and bla EBC ) and of class A with class C beta-lactamase were observed. All but one of the isolates harboring CTX-M extended-spectrum BLs (ESBLs) were carrying bla CTX-M genogroup 1; the remaining isolate carried bla CTX-M genogroup 9. The mobilizing elements occurred in different combinations in the study isolates.A mpC beta-lactamases (BLs) were originally described as chromosomal, inducible enzymes in members of Enterobacteriaceae; however, the plasmid-mediated AmpC beta-lactamases (pMAmpCs) were first discovered in the late 1980s (6). Since then, they have been detected in different regions around the globe (11,13). pMAmpCs are assumed to be less common than extendedspectrum BLs (ESBLs) in Escherichia coli and Klebsiella isolates, but their importance should be noted, since they impart a broader spectrum of resistance, especially when present along with other classes of ESBLs. The data regarding the worldwide distribution and prevalence of AmpC-mediated resistance are fragmentary compared to those regarding ESBLs. This may be due to the limited number of surveillance studies performed and the fact that laboratories often face difficulties in accurately detecting these resistance mechanisms (5). Reports from molecular studies in India analyzing such a resistance mechanism are fragmentary (2, 4, 11). Notably, none of the researchers from around the globe have reported the simultaneous occurrences of these two bla ampC families on the plasmid isolated from a single bacterial strain. Similarly, reports describing the simultaneous occurrences of two bla CTX-M genogroups are also fragmentary (9).Clinical isolates of E. coli (n ϭ 109) and K. pneumoniae (n ϭ 16) demonstrating resistance to cefoxitin from clinical samples of in-patients undergoing routine microbiological examination were studied during 2009 to 2010 in the Department of Microbiology at the Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, India. Further determinations of antibiotic susceptibility to the antibiotics (as shown in Fig. 1) were performed. To detect AmpC enzymes phenotypically, all cefoxitin-resistant isolates were subjected to the modified three-dimensional extract test (MTDET) as described previously (12).bla ampC genes were detected in all isolates on monoplex PCR (the primers span the universal region of the ampC gene) as described by Féria et al. (3). Further, all 125 isolates were tested by multiplex PCR to detect pMAmpCs by the method described by Pérez-Pérez and Hanson (7). Isolates were screened for mobile genetic elements (ISEcp1, IS26, ISCR1, and sul-1-type clas...

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Phenotypic detection of extended-spectrum and AmpC β-lactamases by a new spot-inoculation method and modified three-dimensional extract test: comparison with the conventional three-dimensional extract test

Abstract: The method described here is simple and cost-effective. Furthermore, up to 16 isolates may be tested on a single culture plate, thus it is a less labour-intensive and more economic technique than other reported phenotypic methods.

Cited by 52 publications

References 9 publications

ESBL, MBL and Ampc Lactamases Producing Superbugs – Havoc in the Intensive Care Units of Punjab India

ESBL, MBL and Ampc Lactamases Producing Superbugs – Havoc in the Intensive Care Units of Punjab India

Occurrence of Plasmid-Mediated AmpC β-Lactamases Among Escherichia coli and Klebsiella pneumoniae Clinical Isolates in a Tertiary Care Hospital in Bangalore

Concurrent Occurrence of bla _ampC Families and bla _CTX-M Genogroups and Association with Mobile Genetic Elements IS Ecp1 , IS 26 , IS CR1 , and sul1 -Type Class 1 Integrons in Escherichia coli and Klebsiella pneumoniae Isolates Originating from India

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Phenotypic detection of extended-spectrum and AmpC β-lactamases by a new spot-inoculation method and modified three-dimensional extract test: comparison with the conventional three-dimensional extract test

Abstract: The method described here is simple and cost-effective. Furthermore, up to 16 isolates may be tested on a single culture plate, thus it is a less labour-intensive and more economic technique than other reported phenotypic methods.

Cited by 52 publications

References 9 publications

ESBL, MBL and Ampc Lactamases Producing Superbugs – Havoc in the Intensive Care Units of Punjab India

ESBL, MBL and Ampc Lactamases Producing Superbugs – Havoc in the Intensive Care Units of Punjab India

Occurrence of Plasmid-Mediated AmpC β-Lactamases Among Escherichia coli and Klebsiella pneumoniae Clinical Isolates in a Tertiary Care Hospital in Bangalore

Concurrent Occurrence of bla ampC Families and bla CTX-M Genogroups and Association with Mobile Genetic Elements IS Ecp1 , IS 26 , IS CR1 , and sul1 -Type Class 1 Integrons in Escherichia coli and Klebsiella pneumoniae Isolates Originating from India

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Concurrent Occurrence of bla _ampC Families and bla _CTX-M Genogroups and Association with Mobile Genetic Elements IS Ecp1 , IS 26 , IS CR1 , and sul1 -Type Class 1 Integrons in Escherichia coli and Klebsiella pneumoniae Isolates Originating from India