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Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum ?-lactamases (ESBL).

ABSTRACT: The current global spread of multi-resistant Gram-negatives, particularly extended spectrum ?-lactamases expressing bacteria, increases the likelihood of inappropriate empiric treatment of critically ill patients with subsequently increased mortality. From a clinical perspective, fast detection of resistant pathogens would allow a pre-emptive correction of an initially inappropriate treatment. Here we present diagnostic amplification-sequencing approach as proof of principal based on the fast molecular detection and correct discrimination of CTX-M-?-lactamases, the most frequent ESBL family. The workflow consists of the isolation of total mRNA and CTX-M-specific reverse transcription (RT), amplification and pyrosequencing. Due to the high variability of the CTX-M-?-lactamase-genes, degenerated primers for RT, qRT as well as for pyrosequencing, were used and the suitability and discriminatory performance of two conserved positions within the CTX-M genes were analyzed, using one protocol for all isolates and positions, respectively. Using this approach, no information regarding the expected CTX-M variant is needed since all sequences are covered by these degenerated primers. The presented workflow can be conducted within eight hours and has the potential to be expanded to other ?-lactamase families.

SUBMITTER: Stein C

PROVIDER: S-EPMC3818264 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Project description:CTX-M-type extended-spectrum beta-lactamases (ESBLs) have become increasingly common worldwide, with the notable exception of the United States, where TEM- and SHV-type ESBLs have appeared to predominate. We have noted the emergence of ESBLs in our health care system (the University Health System in San Antonio, TX), especially in Escherichia coli isolates, that preferentially hydrolyze cefotaxime rather than ceftazidime, suggesting the possibility of CTX-M-type enzymes. Microbiology laboratory records were reviewed to identify ESBL-producing isolates and to compare the diameters of ceftazidime disk diffusion zones of inhibition to cefotaxime zone diameters. All isolates had been initially detected and confirmed using the procedures recommended by the Clinical and Laboratory Standards Institute. A total of 94 stored ESBL-producing isolates recovered between January 2000 and June 2006 (predominately from blood and normally sterile fluids) were retrieved for further study and screened using PCR primers specific for the presence of CTX-M, TEM, and SHV ESBLs. Only small numbers of retained ESBL-producing isolates were available for study in 2000 and 2002. The percentages of available ESBL-producing organisms in the following years were found to produce CTX-M enzymes: 2000, 25%; 2001, 10%; 2002, 0%; 2003, 60%; 2004, 69%; 2005, 89%; and 2006, 70%. The most common CTX-M-type ESBL was CTX-M-15, followed by CTX-M-16, CTX-M-8, and CTX-M-14. Comparing the disk diffusion zone diameters of cefotaxime and ceftazidime was helpful with the initial recognition of CTX-M-producing E. coli, which had an average cefotaxime zone diameter 7 mm smaller than the ceftazidime zone. However, comparing ceftazidime and cefotaxime zones for CTX-M-producing Klebsiella spp. was not helpful with initial recognition. CTX-M enzymes were also identified in Proteus mirabilis, Enterobacter spp., and Morganella morganii. Based on pulsed-field gel electrophoresis typing of the E. coli isolates, the CTX-M-producing isolates did not represent the spread of a single clone in the institution or in the community. In conclusion, CTX-M-type ESBLs are now the most common ESBL type isolated from patients in our health care system and may also be present but unrecognized in other U.S. locales.

Project description:The presence of antimicrobial-resistant bacteria and resistance genes in aquatic environments is a serious public health concern. This study focused on Escherichia coli possessing bla CTX-M genes in wastewater inflows. Twelve crude inflow water samples from wastewater treatment plant (WWTP) A and two samples each from three other WWTPs were collected in 2017 and 2018. A total of 73 E. coli isolates with 31 different sequence types (STs) harboring distinctive bla CTX-M gene repertoires were detected. In WWTP A influents, bla CTX-M-14 (14 isolates) was dominant, followed by bla CTX-M-15 (12 isolates) and bla CTX-M-27 (10 isolates). The chimeric bla CTX-M-64 and bla CTX-M-123 genes were each identified in one of the E. coli isolates from the same WWTP A inflow port. The bla CTX-M-27 gene was associated with five of seven B2-ST131 isolates, including three isolates of the B2-O25b-ST131-H30R/non-Rx lineage. One of the remaining two isolates belonged to the B2-O25b-ST131-H30R/Rx lineage harboring the bla CTX-M-15 gene. As for the B2-O25b-ST131-H30R/non-Rx lineage, two isolates with bla CTX-M-27 were recovered from each of the WWTP B and D influents, and one isolate with bla CTX-M-174 was also recovered from WWTP B influent. Whole-genome sequencing of chimeric bla CTX-M-harboring E. coli isolates revealed that the bla CTX-M-64 gene was integrated into the chromosome of ST10 E. coli B22 via ISEcp1-mediated transposition of a 9,467-bp sequence. The bla CTX-M-123-carrying IncI1 plasmid pB64 was 109,169?bp in length with pST108. The overall findings suggest that wastewater may act as a probable reservoir of clinically significant clonal lineages mediating antimicrobial resistance genes and chimeric genes that have not yet been identified from human isolates of domestic origin in Japan.IMPORTANCE Global spread of CTX-M-type extended-spectrum ?-lactamase (ESBL)-producing Enterobacteriaceae is a critical concern in both clinical and community settings. This dominance of CTX-M-type ESBL producers may be largely due to the successful international spread of epidemic clones, as represented by the extraintestinal pathogenic Escherichia coli (ExPEC) ST131. Our findings highlight the worrisome presence of diverse E. coli clones associated with humans, including ExPEC lineages harboring the most common bla CTX-M variants in untreated wastewater samples. Moreover, the chimeric genes bla CTX-M-64 and bla CTX-M-123, which have not yet been identified from human isolates of domestic origin in Japan, were identified. Exposure to untreated wastewater through combined sewer overflow caused by heavy rains derived from abnormal weather change could pose a risk for human health due to ingesting those antimicrobial-resistant bacteria.

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Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum ?-lactamases (ESBL).

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