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In vitro conjugation kinetics of AmpC, broad spectrum and extended-spectrum beta-lactamase-producing Escherichia coli donors and various Enterobacteriaceae recipients.

ABSTRACT: BACKGROUND:Extended spectrum beta-lactamase (ESBL)-producing enterobacteria pose a major hazard to public health. Due to the possibility of genetic transfer, ESBL genes might spread to pathogenic enterobacterial strains. Thus, information on possible genetic transfer between enterobacteria is of high interest. It was therefore the aim of this in vitro study to screen the capacity of a wide range of Enterobacteriaceae for differences in conjugation at different time points with five ESBL-producing Escherichia coli strains. RESULTS:Conjugation frequencies for five potential E. coli donor strains producing the enzymes CTX-M-1, CTX-M-15, SHV-12, TEM-1, TEM-52 and CMY-2, and six potential recipient strains commonly detected in the gastrointestinal tract of poultry (E. coli, Serratia marcescens subsp. marcescens, Enterobacter cloacae, Salmonella (S.) enterica serovar Typhimurium and Proteus mirabilis) were obtained. Different combinations of donor and recipient strains were co-incubated for between 0 and 22?h and spread on selective agar. Conjugation frequencies were calculated as transconjugants per donor. Some donor and recipient strain combinations did not perform plasmid transfer within 22?h. Hence, the recipient Proteus mirabilis did not accept plasmids from any of the given donors and the E. coli ESBL10716 donor was unable to transfer its plasmid to any recipient. Enterobacter cloacae only accepted the plasmids from the donors E. coli ESBL10708 and E. coli ESBL10716 while E. coli ESBL10708 did not transfer its plasmid to Serratia marcescens subsp. marcescens. E. coli IMT11716 on the other hand did not perform conjugation with the donor E. coli ESBL10689. The remaining mating pairs differed in conjugation frequency, ranging from 10-?5 to 10-?9 transconjugants/donor. The earliest conjugation events were detected after 4?h. However, some mating pairs turned positive only after 22?h of coincubation. CONCLUSION:A suitable mating pair for future in vivo studies to combat transfer of antibiotic resistance to pathogenic bacteria in broiler chicken was determined. The results of this study also suggest that the kinetic of conjugation differs between mating pairs and is independent of species origin. This should be considered when performing conjugation experiments.

SUBMITTER: Saliu EM

PROVIDER: S-EPMC7249311 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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In vitro conjugation kinetics of AmpC, broad spectrum and extended-spectrum beta-lactamase-producing Escherichia coli donors and various Enterobacteriaceae recipients.

Saliu Eva-Maria EM Zentek Jürgen J Vahjen Wilfried W

BMC microbiology 20200525 1

<h4>Background</h4>Extended spectrum beta-lactamase (ESBL)-producing enterobacteria pose a major hazard to public health. Due to the possibility of genetic transfer, ESBL genes might spread to pathogenic enterobacterial strains. Thus, information on possible genetic transfer between enterobacteria is of high interest. It was therefore the aim of this in vitro study to screen the capacity of a wide range of Enterobacteriaceae for differences in conjugation at different time points with five ESBL- ...[more]

PMID: 32450799

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Project description:Extended spectrum beta-lactamases and AmpC-producing Enterobacteriaceae (ESBL/AmpC-E) have become a great concern in both human and veterinary medicine. One setting in which this risk could be particularly prominent is petting zoos, in which humans, especially children, directly and indirectly interact with the animals. Yet, while the zoonotic transmission of various Enterobacteriaceae has been reported previously in petting zoos, reports on ESBL/AmpC-E shedding in this setting is currently lacking, despite the high potential risk. To fill this knowledge gap, we conducted a prospective cross-sectional study to explore the prevalence, molecular epidemiology, and risk for shedding of ESBL/AmpC-E in petting zoos. We performed a prospective cross-sectional study in eight petting zoos. Altogether, we collected 381 fecal and body-surface samples from 228 animals, broth-enriched them, and then plated them onto CHROMagar ESBL-plates for ESBL/AmpC-E isolation. Next, we identified the isolated species and tested their susceptibility to various antibiotics using the Vitek-2 system, determined bacterial relatedness by multilocus sequence typing (MLST), and identified ESBL/AmpC genes by using PCR and sequencing. Finally, we asked petting zoo owners and veterinarians to complete questionnaires, which we then analyzed to evaluate risk factors for ESBL/AmpC-E shedding. We found that ESBL/AmpC-E shedding is an important, currently oversighted risk in petting zoos, as the overall shedding rate was 12% (35 isolates, including 29% ESBL-producers, 34% AmpC-producers, and 37% ESBL and AmpC-producers). The isolated bacteria included Enterobacter cloacae (55%), Escherichia coli (31%), and Citrobacter freundii (14%), with diverse ESBL genes. MLST revealed diverse sequence types (STs), including the highly virulent Enterotoxigenic ST656 and the Uropathogenic ST127 E. coli strains, indicating complex epidemiology with inter-animal bacterial transmission. Shedding was associated with petting permission and antibiotic treatment in the petting zoo (OR = 7.34), which were identified as risk factors for ESBL/AmpC shedding. Our findings highlight petting zoos as a source for antibiotic-resistant ESBL/AmpC-producing bacteria, including highly virulent, disease-associated MDR E. coli strains. As this risk has not been previously described in detail, it calls for the implementation of infection control and active surveillance programs in petting zoos and raises the need for a comprehensive guideline to restrain this emerging concern.

Project description:BACKGROUND:We investigated the virulence factors, genes, antibiotic resistance patterns, and genotypes (VRE and ESBL/AmpC) production in Enterococci and Enterobacteriaceae strains isolated from fecal samples of humans, dogs, and cats. METHODS:A total of 100 fecal samples from 50 humans, 25 dogs, and 25 cats were used in the study. MICs of nine antimicrobials were determined using the broth microdilution method. Polymerase chain reaction was used for the detection of genes responsible for antibiotic resistance (VRE and ESBL/AmpC) and virulence genes both in Enterococcus species, such as cytolysin (cylA, cylB, cylM), aggregation substance (agg), gelatinase (gelE), enterococcal surface protein (esp), cell wall adhesins (efaAfs and efaAfm), and in Enterobacteriaceae, such as cytolysin (hemolysin) and gelatinase production (afa, cdt, cnf1, hlyA, iutA, papC, sfa). RESULTS:Enterococcus faecium was the most prevalent species in humans and cats, whereas Enterococcus faecalis was the species isolated in the remaining samples. A total of 200 Enterobacteriaceae strains were also detected, mainly from humans, and Escherichia coli was the most frequently isolated species in all types of samples. In the Enterococcus spp, the highest percentages of resistance for ampicillin, amoxicillin/clavulanate, erythromycin, tetracycline, ciprofloxacin, teicoplanin, and vancomycin were detected in cat isolates (41.6%, 52.8%, 38.9%, 23.6%, 62.5%, 20.8%, and 23.6% respectively), and in E. coli, a higher rate of resistance to cefotaxime and ceftazidime emerged in cat and dog samples, if compared with humans (75.4% and 66.0%, 80.0% and 71.4%, and 32.0% and 27.2%, respectively). Regarding the total number of enterococci, 5% and 3.4% of the strains were vancomycin and teicoplanin resistant, and the vancomycin resistance (van A) gene has been detected in all samples by PCR amplification. All the Enterobacteriaceae strains were confirmed as ESBL producers by PCR and sequencing, and the most frequent ESBL genes in E. coli strains from humans and pet samples were blaCTX-M-1 and blaCTX-M-15. CONCLUSIONS:Our results provide evidence that one or more virulence factors were present in both genera, underlining again the ability of pet strains to act as pathogens.

Project description:Plasmid-mediated AmpC beta-lactamase-producing (pAmpC) Enterobacteriaceae are increasing worldwide, difficult to identify and often confounded with extended-spectrum beta-lactamase (ESBL) producers. The low prevalence precludes routine universal admission screening. Therefore, we evaluated potential risk factors for carriage of pAmpC-producing Enterobacteriaceae that would allow targeted screening to improve yield and reduce cost.We performed a case control study at a tertiary care center from 1/2006 to 12/2010. Cases were adult patients in whom pAmpC-producing Enterobacteriaceae were isolated; controls were chosen among carriers of ESBL-producing Enterobacteriaceae. Both infected and colonized patients were included.Over five years, we identified 40 pAmpC producers in 39 patients among 16,247 screened consecutive isolates of Enterobacteriaceae. The pAmpC prevalence was low (0.25%), but more than 30% of pAmpC carriers received incorrect empirical antibiotic treatment. When compared with 39 ESBL controls, pAmpC carriage was associated with clinically confirmed infections in 74% (versus 51%) (p=0.035), mainly of the urinary tract, previous antibiotic exposure in 63% (versus 36%) (p=0.035) and carriage of a nasogastric tube in 23% (versus 0%) (p=0.002). In the multivariate regression analysis only clinically confirmed infections remained significantly associated with pAmpC carriage (OR 1.44 (95%CI 1.15-2.57)). No other clinical and blood test-associated risk factor allowed discrimination of pAmpC-carrying patients from ESBL controls. The type of acquisition - nosocomial versus community-acquired - was also non-informative for resistance type, as 46% of pAmpC- and 44% of ESBL-producing Enterobacteriaceae were community-acquired.This study could not identify a clinical profile that would allow targeted screening for pAmpC-producing Enterobacteriaceae when compared to ESBL carriers. Because empiric antimicrobial therapy was inappropriate in more than 30%, rapid identification of pAmpC carriers is needed. New microbiological methods are therefore required to simplify rapid and reliable detection of pAmpC carriers.

Dataset Information

In vitro conjugation kinetics of AmpC, broad spectrum and extended-spectrum beta-lactamase-producing Escherichia coli donors and various Enterobacteriaceae recipients.

Publications

In vitro conjugation kinetics of AmpC, broad spectrum and extended-spectrum beta-lactamase-producing Escherichia coli donors and various Enterobacteriaceae recipients.

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