scholarly journals Existence of Multiple ESBL Genes among Phenotypically Confirmed ESBL Producing Klebsiella pneumoniae and Escherichia coli Concurrently Isolated from Clinical, Colonization and Contamination Samples from Neonatal Units at Bugando Medical Center, Mwanza, Tanzania

Antibiotics ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 476
Author(s):  
Vitus Silago ◽  
Dory Kovacs ◽  
Happyness Samson ◽  
Jeremiah Seni ◽  
Louise Matthews ◽  
...  

The proportions and similarities of extended-spectrum β-lactamase (ESBL) producing K. pneumoniae (ESBL-KP) and E. coli (ESBL-EC) carrying multiple ESBL genes is poorly known at our setting. This study investigated the existence of multiple ESBL genes (blaCTX-M, blaTEM, and blaSHV) among ESBL-KP and ESBL-EC concurrently isolated from clinical, colonization, and contamination samples from neonatology units in Mwanza-Tanzania. Twenty and 55 presumptive ESBL-EC and ESBL-KP, respectively, from a previous study archived at −80 °C were successfully recovered for this study. Isolates were screened and confirmed for production of ESBLs by phenotypic methods followed by multiplex PCR assay to determine ESBL genes. All (100%) and 97.3% of presumptive ESBL isolates were phenotypically confirmed by Clinical and Laboratory Standards Institute (CLSI) and modified double-disc synergy methods, respectively. About 93.3% (70/75) of phenotypically confirmed ESBL isolates had at least one ESBL gene, whereby for 62.9% (44/70), all ESBL genes (blaCTX-M, blaTEM, and blaSHV) were detected. Eight pairs of ESBL bacteria show similar patterns of antibiotics susceptibility and ESBL genes. ESBL-KP and ESBL-EC, concurrently isolated from clinical, colonization and contamination samples, harbored multiple ESBL genes. Further, eight pairs of ESBL isolates had similar patterns of antibiotics susceptibility and ESBL genes, suggesting transmission of and/or sharing of mobile genetic elements (MGEs) among ESBL-KP and ESBL-EC.

2016 ◽  
Vol 1 (2) ◽  
pp. 38-42 ◽  
Author(s):  
Khairun Nessa ◽  
Dilruba Ahmed ◽  
Johirul Islam ◽  
FM Lutful Kabir ◽  
M Anowar Hossain

A multiplex PCR assay was evaluated for diagnosis of diarrheagenic Escherichia coli in stool samples of patients with diarrhoea submitted to a diagnostic microbiology laboratory. Two procedures of DNA template preparationproteinase K buffer method and the boiling method were evaluated to examine isolates of E. coli from 150 selected diarrhoeal cases. By proteinase K buffer method, 119 strains (79.3%) of E. coli were characterized to various categories by their genes that included 55.5% enteroaggregative E. coli (EAEC), 18.5% enterotoxigenic E. coli (ETEC), 1.7% enteropathogenic E. coli (EPEC), and 0.8% Shiga toxin-producing E. coli (STEC). Although boiling method was less time consuming (<24 hrs) and less costly (<8.0 US $/ per test) but was less efficient in typing E. coli compared to proteinase K method (41.3% vs. 79.3% ; p<0.001). The sensitivity and specificity of boiling method compared to proteinase K method was 48.7% and 87.1% while the positive and negative predictive value was 93.5% and 30.7%, respectively. The majority of pathogenic E. coli were detected in children (78.0%) under five years age with 53.3% under one year, and 68.7% of the children were male. Children under 5 years age were frequently infected with EAEC (71.6%) compared to ETEC (24.3%), EPEC (2.7%) and STEC (1.4%). The multiplex PCR assay could be effectively used as a rapid diagnostic tool for characterization of diarrheagenic E. coli using a single reaction tube in the clinical laboratory setting.Bangladesh J Med Microbiol 2007; 01 (02): 38-42


2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Yasufumi Matsumura ◽  
Johann D. D. Pitout ◽  
Gisele Peirano ◽  
Rebekah DeVinney ◽  
Taro Noguchi ◽  
...  

ABSTRACT Escherichia coli sequence type 131 (ST131) is a pandemic clonal lineage that is responsible for the global increase in fluoroquinolone resistance and extended-spectrum-β-lactamase (ESBL) producers. The members of ST131 clade C, especially subclades C2 and C1-M27, are associated with ESBLs. We developed a multiplex conventional PCR assay with the ability to detect all ST131 clades (A, B, and C), as well as C subclades (C1-M27, C1-nM27 [C1-non-M27], and C2). To validate the assay, we used 80 ST131 global isolates that had been fully sequenced. We then used the assay to define the prevalence of each clade in two Japanese collections consisting of 460 ESBL-producing E. coli ST131 (2001-12) and 329 E. coli isolates from extraintestinal sites (ExPEC) (2014). The assay correctly identified the different clades in all 80 global isolates: clades A (n = 12), B (n = 12), and C, including subclades C1-M27 (n = 16), C1-nM27 (n = 20), C2 (n = 17), and other C (n = 3). The assay also detected all 565 ST131 isolates in both collections without any false positives. Isolates from clades A (n = 54), B (n = 23), and C (n = 483) corresponded to the O serotypes and the fimH types of O16-H41, O25b-H22, and O25b-H30, respectively. Of the 483 clade C isolates, C1-M27 was the most common subclade (36%), followed by C1-nM27 (32%) and C2 (15%). The C1-M27 subclade with bla CTX-M-27 became especially prominent after 2009. Our novel multiplex PCR assay revealed the predominance of the C1-M27 subclade in recent Japanese ESBL-producing E. coli isolates and is a promising tool for epidemiological studies of ST131.


2013 ◽  
Vol 5 (2) ◽  
pp. 59-66 ◽  
Author(s):  
Sushmita Roy ◽  
SM Shamsuzzaman ◽  
Kazi Z Mamun

Objective: Multiplex PCR assay was used for diagnosis of diarrheagenic Escherichia coli (DEC) in stool samples of children (under 5 years) with acute diarrhea.  Methods: Samples were collected from January 2011 to December 2011, from Dhaka Medical College Hospital and Dhaka Shishu Hospital. Multiplex PCR with five specific primer pairs to detect enteropathogenic E. coli (eae, bfp), enterotoxigenic E. coli (lt, st) and enteroaggregative E. coli (aat) were used. However, enteroinvasive E. coli, enterohemorrhagicE. coli and diffusely adhererentE. coli were not sought. Result: In total, 135 (67.5%) E. coli were isolated from 200 stool samples. The prevalence of DEC was 68 (34%). Among DEC, most frequently isolated pathotype was EPEC 40 (58.82%), followed by ETEC 24 (35.29%) and EAggEC 18 (26.47%). Among the EPEC, 5 (12.5%) were typical EPEC. Among the 68 DEC positive cases, 22 samples contained more than one pathogenic gene in various combinations. Among the combination of DEC, EPEC+ETEC combination was 6 (27.27%) followed by ETEC+EAggEC 4 (18.18%), EPEC+EAggEC and ETEC+EPEC+EAggEC were both in 3 (13.6%). Conclusion:This study shows that DEC is a common cause of childhood diarrhea in Dhaka city of Bangladesh. By using multiplex PCR assay, DEC can be diagnosed in one PCR reaction that makes a conclusive diagnosis of diarrhea. DOI: http://dx.doi.org/10.3126/ajms.v5i2.8576 Asian Journal of Medical Science, Volume-5(2) 2014: 59-66


2007 ◽  
Vol 70 (7) ◽  
pp. 1663-1669 ◽  
Author(s):  
PINA M. FRATAMICO ◽  
LORI K. BAGI

A comparison was made of the relative efficiencies of three enrichment media, RapidChek Escherichia coli O157:H7 enrichment broth (REB), R&F broth (RFB), and modified E. coli broth containing novobiocin (mEC+n), and four selective plating media for detection of cold- and freeze-stressed E. coli O157:H7 in raw ground beef. Ground beef (25 g) was inoculated with E. coli O157:H7 at ≤0.5 and ≤2 CFU/g, and samples were then enriched immediately or were stored at 4°C for 72 h or at −20°C for 2 weeks and then enriched. After 8 or 20 h of enrichment, the cultures were plated onto R&F E. coli O157: H7 chromogenic plating medium, cefixime-tellurite sorbitol MacConkey agar, CHROMagar O157, and Rainbow agar O157 and tested using the RapidChek E. coli O157 lateral flow immunoassay and a multiplex PCR assay targeting the E. coli O157: H7 eae, stx1, and stx2 genes. Recovery of E. coli O157:H7 on the four agar media was 4.0 to 7.9 log CFU/ml with the REB enrichment, 1.4 to 7.4 log CFU/ml with RFB, 1.7 to 6.7 log CFU/ml with mEC+n incubated at 42°C, and 1.3 to 3.3 log CFU/ml from mEC+n incubated at 35°C. The percentages of positive ground beef samples containing nonstressed, cold-stressed, and freeze-stressed E. coli O157:H7 as obtained by plating, the immunoassay, and the PCR assay were 97, 88, and 97%, respectively, with REB, 92, 81, and 78%, respectively, with RFB, 97, 58, and 53%, respectively, with mEC+n incubated at 42°C, and 22, 31, and 25%, respectively, with mEC+n incubated at 35°C. Logistic regression analyses of the data indicated significant main effects of treatment, type of medium, enrichment time, inoculum concentration, and detection method. In particular, a positive result was 1.1 times more likely to occur after 20 h of enrichment than after 8 h, 25 times more likely with RFB and REB than with mEC+nat35°C, 3.7 times more likely with an initial inoculum of ≤2.0 CFU/g than with ≤0.5 CFU/g, 2.5 to 3 times more likely using freeze-stressed or nonstressed bacteria than with cold-stressed bacteria, and 2.5 times more likely by plating than by the immunoassay or the PCR assay. REB had better overall performance for enrichment of cold- and freeze-stressed E. coli O157:H7 present in ground beef than did the other media examined.


2004 ◽  
Vol 132 (1) ◽  
pp. 61-65 ◽  
Author(s):  
R. HORVÁTH ◽  
M. DENDIS ◽  
J. SCHLEGELOVÁ ◽  
F. RŮŽIČKA ◽  
J. BENEDÍK

The original method for molecular typing of E. coli strains was developed using the polymorphism in chromosomal sequences of bacterial interspersed mosaic elements (BIMEs) detected by multiplex PCR and analysed by AFLP assay. The applicability of the method in the epidemiology of E. coli was tested on a group of 524 strains of human and veterinary origin. In the studied group 18 different genotypes were detected. Significant differences were found in the frequencies of the genotypes among various groups of strains, suggesting the method could be a promising tool in the epidemiology of E. coli.


2021 ◽  
Author(s):  
Abed Zahedi bialvaei ◽  
Alireza Dolatyar Dehkharghani ◽  
Farhad Asgari ◽  
Firouzeh Shamloo ◽  
Parisa Eslami ◽  
...  

Abstract Background Timely detection of carbapenemases is essential for developing strategies to control the spread of infections by carbapenem-resistant isolates. The purpose of our study was to determine the epidemiology of carbapenemase genes among carbapenem resistant isolates of Acinetobacter baumannii, Klebsiella pneumoniae and Escherichia coli and to compare efficacy of modified Hodge Test (MHT), Carba NP and modified carbapenem inactivation method (mCIM) tests. Methods A total of 122 carbapenem-resistant clinical isolates including 77 K. pneumoniae, 39 A. baumannii, and six E. coli were collected from hospitalized patients. Three phenotypic methods, including MHT, Carba NP test and mCIM were used for investigation of carbapenemase production. In addition, polymerase chain reaction (PCR) was performed to detect carbapenemase encoding genes. Results The sensitivity and specificity of the MHT were 75.0% and 100% respectively. In addition, CarbaNP displayed 80.8% sensitivity and 100% specificity, whereas the sensitivity and specificity were 90.4% and 100% for the mCIM test, respectively. Among carbapenem-resistant isolates, 70, 84 and 87 isolates exhibited positive results according to MHT, CarbaNP test and mCIM, respectively. PCR indicated the presence of one or more carbapenemase genes in 119 of carbapenem-resistant isolates, with blaKPC and blaVIM being the most commonly encountered. Co-production of ‘KPC and VIM’, ‘KPC and IMP’ and ‘KPC and OXA-48’ was detected in nine, seven and three isolates, respectively. Conclusion Our results confirm that the mCIM test is a useful tool for the reliable detection of carbapenemases-activity in enterobacterial isolates, especially in clinical microbiological laboratories with limited resources.


2021 ◽  
Vol 71 (1) ◽  
Author(s):  
Abed Zahedi Bialvaei ◽  
Alireza Dolatyar Dehkharghani ◽  
Farhad Asgari ◽  
Firouzeh Shamloo ◽  
Parisa Eslami ◽  
...  

Abstract Purpose Timely detection of carbapenemases is essential for developing strategies to control the spread of infections by carbapenem-resistant isolates. The purpose of this study was to determine the epidemiology of carbapenemase genes among carbapenem-resistant isolates of Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli. In addition, the efficacy of the modified Hodge test (MHT), Carba NP test, and modified carbapenem inactivation method (mCIM) were compared. Methods A total of 122 carbapenem-resistant clinical isolates including 77 K. pneumoniae, 39 A. baumannii, and six E. coli were collected from hospitalized patients. Three phenotypic methods, including the MHT, Carba NP test, and mCIM were used for investigation of carbapenemase production. In addition, polymerase chain reaction (PCR) was performed to detect carbapenemase-encoding genes. Result The sensitivity and specificity of the MHT were 75.0% and 100%, respectively. In addition, Carba NP displayed 80.8% sensitivity and 100% specificity, whereas the sensitivity and specificity were 90.4% and 100% for the mCIM test, respectively. Among carbapenem-resistant isolates, 70, 84, and 87 isolates exhibited positive results according to the MHT, Carba NP test, and mCIM, respectively. PCR indicated the presence of one or more carbapenemase genes in 119 of carbapenem-resistant isolates, with blaKPC and blaVIM being the most commonly encountered. Co-production of ‘KPC and OXA-48’, ‘KPC and VIM’, and ‘KPC and IMP’ was detected in three, nine, and seven isolates, respectively. Conclusion Our results confirm that the mCIM test is a useful tool for the reliable detection of carbapenemase activity in enterobacterial isolates, especially in clinical microbiological laboratories with limited resources.


2019 ◽  
Vol 57 (5) ◽  
Author(s):  
James R. Johnson ◽  
Brian D. Johnston ◽  
Stephen B. Porter ◽  
Connie Clabots ◽  
Tricia L. Bender ◽  
...  

ABSTRACT Escherichia coli sequence type 1193 (ST1193) is an emerging multidrug-resistant pathogen. We performed longitudinal and cross-sectional surveillance for ST1193 among clinical and fecal E. coli isolates from Minneapolis Veterans Affairs Medical Center (VAMC) patients and their household members, other Minnesota centers, and national VAMCs and compared these ST1193 isolates with archival human and canine ST1193 isolates from Australia (2008). We also developed and extensively validated a novel multiplex PCR assay for ST1193 and its characteristic fimH64 (type 1 fimbrial adhesin) allele. We found that ST1193-H64 (where “H64” refers to a phylogenetic subdivision within ST1193 that is characterized by the fimH64 allele), which was uniformly fluoroquinolone resistant, appeared to emerge in the United States in a geographically staggered fashion beginning around 2011. Its prevalence among clinical and fecal E. coli isolates at the Minneapolis VAMC rose rapidly beginning in 2013, peaked in early 2017, and then plateaued or declined. In comparison with other ST14 complex (STc14) isolates, ST1193-H64 isolates were more extensively multidrug resistant, whereas their virulence genotypes were less extensive but included (uniquely) K1 capsule and fimH64. Pulsed-field gel electrophoresis separated ST1193-H64 isolates from other STc14 isolates and showed genetic commonality between archival Australian versus recent U.S. isolates, fecal versus clinical isolates, and human versus canine isolates. Three main ST1193 pulsotypes differed significantly in resistance profiles and capsular types; emergent pulsotype 2123 was associated with trimethoprim-sulfamethoxazole resistance and K1 (versus K5) capsule. These findings clarify ST1193-H64’s temporal prevalence trends as a fluoroquinolone-resistant pathogen and commensal; document clonal subsets with distinctive geographic, temporal, resistance, and virulence gene associations; and establish a new laboratory tool for rapid and simple detection of ST1193-H64.


2000 ◽  
Vol 38 (5) ◽  
pp. 1791-1796 ◽  
Author(s):  
Philip E. Coudron ◽  
Ellen S. Moland ◽  
Kenneth S. Thomson

AmpC beta-lactamases are cephalosporinases that confer resistance to a wide variety of β-lactam drugs and that may thereby create serious therapeutic problems. Although reported with increasing frequency, the true rate of occurrence of AmpC beta-lactamases inEscherichia coli, Klebsiella pneumoniae, andProteus mirabilis remains unknown. We tested a total of 1,286 consecutive, nonrepeat isolates of these three species and found that, overall, 45 (3.5%) yielded a cefoxitin zone diameter less than 18 mm (screen positive) and that 16 (1.2%) demonstrated AmpC bands by isoelectric focusing. Based on the species, of 683 E. coli, 371 K. pneumoniae, and 232 P. mirabilisisolates tested, 13 (1.9%), 28 (7.6%), and 4 (1.7%), respectively, demonstrated decreased zone diameters and 11 (1.6%), 4 (1.1%), and 1 (0.4%), respectively, demonstrated AmpC bands. Cefoxitin resistance was transferred for all but 8 (E. coli) of the 16 AmpC producers. We also describe a three-dimensional extract test, which was used to detect phenotypically isolates that harbor AmpC beta-lactamase. Of the 45 cefoxitin-resistant isolates, the three-dimensional extract test accurately identified all 16 AmpC producers and 28 of 29 (97%) isolates as non-AmpC producers. Interestingly, most (86%) isolates in the latter group were K. pneumoniae isolates. These data confirm that, at our institution, E. coli, K. pneumoniae, and P. mirabilis harbor plasmid-mediated AmpC enzymes.


2018 ◽  
Vol 12 (03) ◽  
pp. 164-170 ◽  
Author(s):  
George Farah Araj ◽  
Aline Z Avedissian ◽  
Lina Y Itani ◽  
Jowana A Obeid

Introduction: It is not yet clear which antimicrobial agents should be used to treat the ominously increasing infections with carbapenem-resistant (CR) bacteria. We therefore investigated the activity of different antimicrobial agents against CR Escherichia coli and Klebsiella pneumoniae in Lebanon. Methodology: This retrospective study assessed the minimum inhibitory concentrations (MICs) of three carbapenems (by Etest), as well as the in vitro activity of eight other antimicrobials (by disk diffusion) against CR E. coli (n = 300) and K. pneumoniae (n = 232) isolates recovered at a major University Medical Center in Lebanon. Results: Higher percentages of isolates showing carbapenem MICs of ≤ 8 µg/mL were noted among the CR E. coli compared to the CR K. pneumoniae for ertapenem (48% vs 27%), imipenem (74 % vs 58%) and meropenem (82% vs 63%). Among the eight other antimicrobials, activity was generally higher when the MICs for the three carbapenems were ≤ 8 µg/mL. Regardless of the MIC level of the three carbapenems, very low susceptibility rates (≤ 33%) were noted for ciprofloxacin, trimethoprim-sulfamethoxazole and aztreonam against both E. coli and K. pneumoniae isolates. With Amikacin, higher susceptibility rates were seen against E. coli isolates (81%-97%) than against K. pneumoniae isolates (55%-86%), also reflecting higher activity than gentamicin (44%-54%). The best activity (66%-100%) was observed for tigecycline, colistin and fosfomycin against both CR species. Conclusions: Based on the in vitro findings in this study, the combination of a carbapenem showing an MIC of ≤ 8 µg/mL together with an active colistin, tigecycline, or fosfomycin, would offer a promising treatment option for patients infected with CR E. coli or K. pneumoniae.


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