Development of a Multiplex Real-Time PCR for the Rapid Detection of the Predominant Beta-Lactamase Genes CTX-M, SHV, TEM and CIT-Type AmpCs in Enterobacteriaceae

Roschanski, Nicole; Fischer, Jennie; Guerra, Beatriz; Roesler, Uwe

doi:10.1371/journal.pone.0100956

Cited by 158 publications

(90 citation statements)

References 24 publications

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“…Subsequently to the antimicrobial susceptibility testing, isolates were further analyzed using real-time PCR for the detection of the predominant beta-lactamase genes bla CTX-M , bla SHV , bla TEM , as well as CIT-type pAmpC genes (43). Therefore, DNA was extracted by a simple boiling method, and the supernatant was used for all PCR analyses (43). Positive controls and no-template controls were used as published previously (43).…”

Section: Methodsmentioning

confidence: 99%

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Extended-Spectrum-Beta-Lactamase- and Plasmid-Encoded Cephamycinase-Producing Enterobacteria in the Broiler Hatchery as a Potential Mode of Pseudo-Vertical Transmission

Projahn

Daehre

Roesler

et al. 2017

Appl Environ Microbiol

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Antimicrobial resistance through extended-spectrum beta-lactamases (ESBLs) and transferable (plasmid-encoded) cephamycinases (pAmpCs) represents an increasing problem in human and veterinary medicine. The presence of ESBL-/ pAmpC-producing commensal enterobacteria in farm animals, such as broiler chickens, is considered one possible source of food contamination and could therefore also be relevant for human colonization. Studies on transmission routes along the broiler production chain showed that 1-day-old hatchlings are already affected. In this study, ESBL-/pAmpC-positive broiler parent flocks and their corresponding eggs, as well as various environmental and air samples from the hatchery, were analyzed. The eggs were investigated concerning ESBL-/pAmpC-producing enterobacteria on the outer eggshell surface (before/after disinfection), the inner eggshell surface, and the egg content. Isolates were analyzed concerning their species, their phylogroup in the case of Escherichia coli strains, the respective resistance genes, and the phenotypical antibiotic resistance. Of the tested eggs, 0.9% (n ϭ 560) were contaminated on their outer shell surface. Further analyses using pulsed-field gel electrophoresis showed a relationship of these strains to those isolated from the corresponding parent flocks, which demonstrates a pseudo-vertical transfer of ESBL-/pAmpC-producing enterobacteria into the hatchery. Resistant enterobacteria were also found in environmental samples from the hatchery, such as dust or surfaces which could pose as a possible contamination source for the hatchlings. All 1-day-old chicks tested negative directly after hatching. The results show a possible entry of ESBL-/pAmpC-producing enterobacteria from the parent flocks into the hatchery; however, the impact of the hatchery on colonization of the hatchlings seems to be low.IMPORTANCE ESBL-/pAmpC-producing enterobacteria occur frequently in broilerfattening farms. Recent studies investigated the prevalence and possible transmission route of these bacteria in the broiler production chain. It seemed very likely that the hatcheries play an important role in transmission and/or contamination events. There are only few data on transmission investigations from a grandparent or parent flock to their offspring. However, reliable data on direct or indirect vertical transmission events in the hatchery are not available. Therefore, we conducted our study and intensively investigated the broiler hatching eggs from ESBL-/pAmpCpositive broiler parent flocks as well as the hatchlings and the environment of the hatchery.KEYWORDS antibiotic resistance, Enterobacteriaceae, broiler chicken, hatchery, ESBL, pAmpC, AmpC

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Section: Methodsmentioning

confidence: 99%

“…Therefore, DNA was extracted by a simple boiling method, and the supernatant was used for all PCR analyses (43). Positive controls and no-template controls were used as published previously (43). Detected ESBL/pAmpC genes for at least one isolate of each sample were verified by sequencing, each with the primers shown in Table 3.…”

Section: Methodsmentioning

confidence: 99%

Extended-Spectrum-Beta-Lactamase- and Plasmid-Encoded Cephamycinase-Producing Enterobacteria in the Broiler Hatchery as a Potential Mode of Pseudo-Vertical Transmission

Projahn

Daehre

Roesler

et al. 2017

Appl Environ Microbiol

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“…The modified Hodge test (MHT) and the modified Carba NP (MCNP) test were used to screen for carbapenemase activity (15,16). All strains were screened using real-time PCR for the presence of the following ␤-lactamase-encoding genes: bla KPC , bla NDM , bla VIM , bla IMP , bla OXA-48 , bla CTX-M , bla TEM , and bla SHV (17)(18)(19). Strains positive by real-time PCR were subjected to standard PCR and sequencing (18,20).…”

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confidence: 99%

First Report of German Cockroaches (Blattella germanica) as Reservoirs of CTX-M-15 Extended-Spectrum-β-Lactamase- and OXA-48 Carbapenemase-Producing Enterobacteriaceae in Batna University Hospital, Algeria

Loucif

Gacemi-Kirane

Cherak

et al. 2016

Antimicrob Agents Chemother

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Here we report the isolation of extended-spectrum ␤-lactamase (ESBL)-and carbapenemase-producing Enterobacteriaceae from German cockroaches caught in the burn unit of Batna University Hospital in Algeria. Nine of 12 isolates harbored the bla CTX-M-15 ESBL gene. One Enterobacter cloacae isolate belonging to sequence type 528 coexpressed the bla OXA-48 , bla CTX-M-15 , and bla TEM genes. Our findings indicate that cockroaches may be one of the most dangerous reservoirs for ESBL and carbapenemase producers in hospitals.T he emergence of multidrug-resistant Enterobacteriaceae (MDRE) and their dissemination in hospitals have become a major health concern worldwide (1). The production of ␤-lactamases such as extended-spectrum ␤-lactamase (ESBL) and carbapenemase remains the main mechanism of resistance in Enterobacteriaceae (2). Algeria is now feaures among the Mediterranean countries known to be affected by the spread of CTX-M-15 type ESBL and the emergence of the phantom menace OXA-48 type carbapenemase (3, 4). Indeed, the transmission and dissemination of MDRE in hospitals are usually related to hand contact (5) and poorly sterilized instruments (6). However, cockroaches which colonize these environments may also act as potential mechanical vectors of antibiotic-resistant bacteria (7). Cockroaches are among the most prevalent pests in hospitals, where they are attracted by moisture, food, and suitable temperatures (8, 9). The medical significance of cockroaches has been largely overlooked, but it has been proven that they can harbor a number of pathogenic microorganisms with different levels of antibiotic resistance (10). Blattella germanica is the most abundant cockroach species (11), and the external surfaces and excrement of these cockroaches can contaminate food, human habitats, and hospital equipment; therefore, these insects can present a threat to human health (12).The aim of our study was to screen for broad-spectrum-cephalosporin-and carbapenem-resistant Enterobacteriaceae in B. germanica cockroaches collected from the burn unit of Batna University Hospital in Algeria and then to investigate the molecular support of ESBL and carbapenemase production.In March 2015, 10 cockroaches were randomly captured from different parts of the burn unit, including the kitchen and the guard, treatment, and patient rooms, directly in sterile containers. Samples were immobilized by freezing at 0°C for 10 min (8). Each cockroach was soaked in 5 ml of Tween 80 solution at 0.05% and vortexed vigorously for 2 min. The resulting wash was used as an external-body-homogenate sample. To remove the external body contamination, cockroaches were immersed in bleach for 2 min, in sterile physiological saline for 2 min, and then in 70% ethanol for 5 min (12). Then, each sample was washed with sterile physiological saline. Subsequently, the insect was soaked in a sterile bottle containing 5 ml of Tween 80 solution at 0.05% and then crushed inside using a sterile pestle. The triturate was then vortexed vigorously for 2 min. The resulting s...

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“…ESBL/AmpC E. coli were selected using MacConkey agar (Oxoid, CM 0115, Wesel, Germany) supplemented with 1 mg/L cefotaxime, followed by species confirmation using MALDI-TOF identification (MALDI Microflex LT and Biotyper database, Bruker Daltonics, Bremen, Germany). The presence of the β-lactamase genes bla CTX , bla TEM , bla SHV and the CIT-type AmpCs (e.g., CMY-2) was confirmed by real-time PCR as described by Roschanski et al (2014).…”

Section: Methodsmentioning

confidence: 98%

The Efficacy of Isolated Bacteriophages from Pig Farms against ESBL/AmpC-Producing Escherichia coli from Pig and Turkey Farms

et al. 2017

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Extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases are plasmid (but also chromosomally) encoded enzymes found in Enterobacteriaceae, determining resistance to a variety of important antibiotics including penicillins, cephalosporins, and monobactams. In recent decades, the prevalence of ESBL/AmpC-producing bacteria has increased rapidly across the world. Here, we evaluate the potential use of bacteriophages in terms of a reduction of antibiotic-resistant bacteria in healthy animals. The aim of our studies was to isolate bacteriophages capable of destroying ESBL/AmpC-producing Escherichia coli isolated from livestock habitats. The efficacy of isolated phages against ESBL/AmpC E. coli strains varies, but creation of a phage cocktail with broad activity spectrum is possible. This may indicate that the role of phages may not be limited to phage therapy, but bacterial viruses may also be applied against spread of bacteria with antibiotic resistance genes in the environment. We also addressed the hypothesis, that phages, effective for therapeutic purposes may be isolated from distant places and even from different environments other than the actual location of the targeted bacteria. This may be beneficial for practical purposes, as the construction of effective phage preparations does not require access to disease outbreaks.

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Development of a Multiplex Real-Time PCR for the Rapid Detection of the Predominant Beta-Lactamase Genes CTX-M, SHV, TEM and CIT-Type AmpCs in Enterobacteriaceae

Cited by 158 publications

References 24 publications

Extended-Spectrum-Beta-Lactamase- and Plasmid-Encoded Cephamycinase-Producing Enterobacteria in the Broiler Hatchery as a Potential Mode of Pseudo-Vertical Transmission

Extended-Spectrum-Beta-Lactamase- and Plasmid-Encoded Cephamycinase-Producing Enterobacteria in the Broiler Hatchery as a Potential Mode of Pseudo-Vertical Transmission

First Report of German Cockroaches (Blattella germanica) as Reservoirs of CTX-M-15 Extended-Spectrum-β-Lactamase- and OXA-48 Carbapenemase-Producing Enterobacteriaceae in Batna University Hospital, Algeria

The Efficacy of Isolated Bacteriophages from Pig Farms against ESBL/AmpC-Producing Escherichia coli from Pig and Turkey Farms

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