Sensitive and Specific Loop-Mediated Isothermal Amplification Assays for Detection of Salmonella, CTX-M-1 Group Genes, mph(A), and ermB in Stool and Blood Samples Based on Orange to Green Visible Dye.

Salmonella is a globally prevalent foodborne bacterium, and ceftriaxone and azithromycin have been regarded as drugs of choice for treating Salmonella infections, particularly in children. With the growing incidence of ceftriaxone and azithromycin resistance in Salmonella, there is an urgent requirement for a rapid and dependable gene testing approach to enhance the efficacy of treating Salmonella infections. Utilizing the orange to green visible dye approach, this study developed loop-mediated isothermal amplification (LAMP) assays for the sensitive and specific detection of Salmonella, ceftriaxone and azithromycin resistance genes (including CTX-M-1 group, mph(A), and ermB genes) in stool and blood samples. The specificity and sensitivity of primers during the LAMP assays for detection of Salmonella, CTX-M-1 group, mph(A), and ermB genes were determined in this study. The detection threshold for Salmonella was found to be 1.5 × 103 colony-forming units (CFU)/mL, while it was 1.5 × 102 CFU/mL for CTX-M-1 group genes (including blaCTX-M-3, blaCTX-M-15, and blaCTX-M-55), 1.5 × 102 CFU/mL for mph(A), and 1.5 × 102 CFU/mL for ermB, showing 10-103-fold, 103-fold, and 105-fold increased sensitivity compared with the polymerase chain reaction assay, respectively. Results indicated that the LAMP primers designed for Salmonella, CTX-M-1 group, mph(A), and ermB genes possess high specificity (100%) and sensitivity (over 94%). This novel approach advocates its application in detecting Salmonella, CTX-M-1 group, mph(A), and ermB genes.

Emergence of carriage of CTX-M-15 in faecal Escherichia coli in horses at an equine hospital in the UK; increasing prevalence over a decade (2008-2017).

BACKGROUND: This study investigated changes over time in the epidemiology of extended-spectrum ß-lactamase (ESBL) producing Escherichia coli within a single equine referral hospital in the UK. Faecal samples were collected from hospitalised horses in 2008 and 2017, processed using selective media and standard susceptibility laboratory methods. A novel real-time PCR with high resolution melt analysis was used to distinguish blaCTX-M-1 and blaCTX-M-15 within CTX-M-1 group. RESULTS: In 2008, 457 faecal samples from 103 horses were collected, with ESBL-producing E. coli identified in 131 samples (28.7, 95% CI 24.6-33.1). In 2017, 314 faecal samples were collected from 74 horses with ESBL-producing E. coli identified in 157 samples (50.0, 95% CI 44.5-55.5). There were 135 and 187 non-duplicate ESBL-producing isolates from 2008 and 2017, respectively. In 2008, 12.6% of isolates belonged to CTX-M-1 group, all carrying blaCTX-M-1, whilst in 2017, 94.1% of isolates were CTX-M-1 group positive and of these 39.2 and 60.8% of isolates carried blaCTX-M-1 and blaCTX-M-15, respectively. In addition, the prevalence of doxycycline, gentamicin and 3rd generation cephalosporin resistance increased significantly from 2008 to 2017 while a decreased prevalence of phenotypic resistance to potentiated sulphonamides was observed. CONCLUSIONS: The real-time PCR proved a reliable and high throughput method to distinguish between blaCTX-M-1 and blaCTX-M-15. Furthermore, its use in this study demonstrated the emergence of faecal carriage of CTX-M-15 in hospitalised horses, with an increase in prevalence of ESBL-producing E. coli as well as increased antimicrobial resistance to frequently used antimicrobials.

High prevalence of CTX-M-1 group in ESBL-producing enterobacteriaceae infection in intensive care units in southern Chile.

Enterobacteria-producing extended-spectrum ß-lactamases (ESBL) play an important role in healthcare infections, increasing hospitalization time, morbidity and mortality rates. Among several ESBLs that emerge from these pathogens, CTX-M-type enzymes had the most successful global spread in different epidemiological settings. Latin America presents high prevalence of CTX-M-2 in ESBL-producing enterobacterial infections with local emergence of the CTX-M-1 group. However, this high prevalence of the CTX-M-1 group has not yet been reported in Chile. The aim of this study was to identify ESBLs among enterobacteria isolated from clinical samples of critically ill patients from southern Chile. One-hundred thirty seven ESBL-producing bacteria were isolated from outpatients from all critical patient units from Hernán Henríquez Aravena Hospital. Phenotype characterization was performed by antibiogram, screening of ESBL, and determination of minimum inhibitory concentration (MIC). PCR was used for genetic confirmation of resistance. Molecular typing was performed by ERIC-PCR. ESBL-producing isolates were identified as Klebsiella pneumoniae (n=115), Escherichia coli (n=18), Proteus mirabilis (n=3), and Enterobacter cloacae (n=1), presenting multidrug resistance profiles. PCR amplification showed that the strains were positive for blaSHV (n=111/81%), blaCTX-M-1 (n=116/84.7%), blaTEM (n=100/73%), blaCTX-M-2 (n=28/20.4%), blaCTX-M-9 (0.7%), blaPER-1 (0.7%), and blaGES-10 (0.7%). The multiple production of ESBL was observed in 93% of isolates, suggesting high genetic mobility independent of the clonal relationship. The high frequency of the CTX-M-1 group and a high rate of ESBL co-production are changing the epidemiology of the ESBL profile in Chilean intensive care units. This epidemiology is a constant and increasing challenge, not only in Chile, but worldwide.

High prevalence of CTX-M-1 group in ESBL-producing enterobacteriaceae infection in intensive care units in southern Chile

ABSTRACT Enterobacteria-producing extended-spectrum β-lactamases (ESBL) play an important role in healthcare infections, increasing hospitalization time, morbidity and mortality rates. Among several ESBLs that emerge from these pathogens, CTX-M-type enzymes had the most successful global spread in different epidemiological settings. Latin America presents high prevalence of CTX-M-2 in ESBL-producing enterobacterial infections with local emergence of the CTX-M-1 group. However, this high prevalence of the CTX-M-1 group has not yet been reported in Chile. The aim of this study was to identify ESBLs among enterobacteria isolated from clinical samples of critically ill patients from southern Chile. One-hundred thirty seven ESBL-producing bacteria were isolated from outpatients from all critical patient units from Hernán Henríquez Aravena Hospital. Phenotype characterization was performed by antibiogram, screening of ESBL, and determination of minimum inhibitory concentration (MIC). PCR was used for genetic confirmation of resistance. Molecular typing was performed by ERIC-PCR. ESBL-producing isolates were identified as Klebsiella pneumoniae (n = 115), Escherichia coli (n = 18), Proteus mirabilis (n = 3), and Enterobacter cloacae (n = 1), presenting multidrug resistance profiles. PCR amplification showed that the strains were positive for blaSHV (n = 111/81%), blaCTX-M-1 (n = 116/84.7%), blaTEM (n = 100/73%), blaCTX-M-2 (n = 28/20.4%), blaCTX-M-9 (0.7%), blaPER-1 (0.7%), and blaGES-10 (0.7%). The multiple production of ESBL was observed in 93% of isolates, suggesting high genetic mobility independent of the clonal relationship. The high frequency of the CTX-M-1 group and a high rate of ESBL co-production are changing the epidemiology of the ESBL profile in Chilean intensive care units. This epidemiology is a constant and increasing challenge, not only in Chile, but worldwide.