QUINOLONE- AND ETA-LACTAM- RESISTANCE IN Escherichia coli FROM DANISH AND ITALIAN BROILER FLOCKS

The prevalence of quinolone- and -lactam-resistant E. coli was investigated among healthy broiler flocks in Denmark and Italy. In Denmark, sock samples were collected from 10 parent flocks and 10 offspring flocks, according to the procedure currently used for the surveillance of Salmonella in the EU. Samples were enriched in McConkey broth and streaked on McConkey agar plates added with nalidixic acid (32 g/ml), ciprofloxacin (2 g/ml), ampicillin (32 g/ml), cefotaxime (2 g/ml) or ceftiofur (8 g/ml). The -glucuronidase test was performed for verification of presumptive E. coli. The same methods were used to analyse sock samples collected from 6 Italian broiler flocks. PCR with primers for the CTX-M-type extended-spectrum -lactamases (ESBLs) was performed on cephalosporin-resistant isolates. While resistance to ampicillin and nalidixic acid was widespread in both countries, resistance to ciprofloxacin and cephalosporins was more common among Italian flocks. In Denmark, ciprofloxacin resistance was only detected in 1 parent flock without any history of quinolone usage and none of the flocks was positive for cephalosporin-resistant E. coli. In Italy, resistance to ciprofloxacin was detected in all flocks and resistance to ceftiofur and cefotaxime were detected in 5 flocks. Primers specific for the CTX-M-type ESBLs generated PCR amplicons from isolates from 3 of these flocks. In industrialized countries, the poultry production system is highly standardized, and therefore comparable. However, the use of broad-spectrum antimicrobials is particularly limited in Danish poultry production. Accordingly, the results of this study could reflect the different policies in antimicrobial usage between the two countries

A culture-independent method for studying transfer of IncI1 plasmids from wild-type Escherichia coli in complex microbial communities

IncI1 plasmids play a central role in the transfer of antimicrobial resistance genes among Enterobacteriaceae in animals and humans. Knowledge on the dynamics of IncI1 plasmid transfer is limited, mainly due to lack of culture-independent methods that can quantify donor strain survival and plasmid transfer in complex microbial communities. The aim of this study was to develop a culture-independent method to study the dynamics of IncI1 plasmids transfer by fluorescence-activated cell sorting. We genetically modified three wild-type Escherichia coli of animal (n = 2) and human (n = 1) origin carrying blaCMY-2 or blaCTX-M-1 on two epidemic IncI1 plasmids (pST12 and pST7). Non-coding regions on the chromosome and on the IncI1 plasmid of each strain were tagged with mCherry (red) and GFPmut3 (green) fluorescent proteins, respectively, using lambda recombineering. A gene cassette expressing mCherry and lacIq was inserted into the chromosome, whereas the plasmid was marked with a GFPmut3 cassette with LacIq repressible promoter. Therefore, gfpmut3 was repressed in donor strains but expressed in recipient strains acquiring the plasmids. We demonstrated that genetic engineering of the strains did not affect the growth rate and plasmid transfer-ability in filter and broth matings. A proof-of-concept experiment using the CoMiniGut, an in vitro model of the colon, proved the validity of our method for studying the survival of wild-type E. coli and horizontal transfer of IncI1 plasmids under different pH and oxygen conditions. The dual-labeling method by fluorescent proteins is useful to determine persistence of exogenous E. coli and transfer dynamics of IncI1 plasmids in microbial communities

Fate of CMY-2-encoding plasmids introduced into the human fecal microbiota by exogenous Escherichia coli

The gut is a hot spot for transfer of antibiotic resistance genes from ingested exogenous bacteria to the indigenous microbiota. The objective of this study was to determine the fate of two nearly identical blaCMY-2-harboring plasmids introduced into the human fecal microbiota by two Escherichia coli strains isolated from human and poultry meat, respectively. The chromosome and the CMY-2-encoding plasmid of both strains were labeled with distinct fluorescent markers (mCherry and GFP), allowing Fluorescence Activated Cell Sorting (FACS)-based tracking of the strain and the resident bacteria that have acquired its plasmid. Each strain was introduced into an established in vitro gut model (CoMiniGut) inoculated with individual feces from ten healthy volunteers. Fecal samples collected 2, 6 and 24 h after strain inoculation were analyzed by FACS and plate counts. Although the human strain survived better than the poultry meat strain, both strains transferred their plasmids to the fecal microbiota at concentrations as low as 102 CFU/mL. Strain survival and plasmid transfer varied significantly depending on inoculum concentration and individual fecal microbiota. Identification of transconjugants by 16S rRNA gene sequencing and MALDI-TOF mass spectrometry revealed that the plasmids were predominantly acquired by Enterobacteriaceae such as E. coli and Hafnia alvei. Our experimental data demonstrate that exogenous E. coli of human or animal origin can readily transfer CMY-2-encoding IncI1 plasmids to the human fecal microbiota. Low amounts of exogenous strain are sufficient to ensure plasmid transfer if the strain is able to survive the gastric environment

Transcriptome analysis of extended-spectrum ß-lactamase-producing Escherichia coli and methicillin-resistant Staphylococcus aureus exposed to cefotaxime

Previous studies on bacterial response to antibiotics mainly focused on susceptible strains. Here we characterized the transcriptional responses of distinct cephalosporin-resistant bacteria of public health relevance to cefotaxime (CTX), a cephalosporin widely used in clinical practice. Adaptation to therapeutic concentrations of CTX (30 µg/ml) was investigated by RNA sequencing in mid-exponential phase cultures of a methicillin-resistant Staphylococcus aureus (MRSA) and two genetically diverse E. coli producing CTX-M-15 or CMY-2 β-lactamase following genome sequencing and annotation for each strain. MRSA showed the most notable adaptive changes in the transcriptome after exposure to CTX, mainly associated with cell envelope functions. This reprogramming coincided with a transient reduction in cell growth, which also occurred in the CMY-2-producing E. coli but not in the CTX-M-15-producing strain. Re-establishment of growth in the CMY-2 producer proceeded without any notable adaptive transcriptional response, while limited reprogramming of gene transcription was observed in the CTX-M-15 producer. Our data show that the transcriptional response of CTX-resistant bacteria to CTX depends on the bacterial species, level of resistance and resistance determinant involved. Gene products induced in the presence of CTX may play an essential role for bacterial survival during therapy and merit further investigation as possible targets for potentiating CTX

Characterization of plasmids encoding extended-spectrum \u3b2-lactamases (ESBLs) and QnrS1 in Avian Pathogenic Escherichia coli (APEC) isolated from commercial poultry flocks in Italy.

Avian pathogenic Escherichia coli (APEC) cause infections with high morbidity and mortality in poultry flocks. The aim of this study was to characterize the mobilizable pool mediating resistance to cephalosporins and fluoroquinolones in APEC collected in Italy between 2008 and 2012. Non-repetitive APEC from turkeys (n=109), broilers (n=98) and layers (n=22) were examined. Isolates resistant to third-generation cephalosporins were screened for presence of blaTEM, blaSHV, blaCTX-M and blaCMY-2 and for chromosomal ampC promoter mutations, while all isolates were tested by PCR for all known plasmid-mediated quinolone resistance (PMQR) genes. ESBL/AmpC or PMQR-harboring plasmids were typed by traditional typing methods. Twenty-eight (12%) isolates displayed resistance to third-generation cephalosporins either mediated by mutations leading to chromosomal ampC overproduction (n=10) or by the following plasmid/gene combinations: IncI1/ST26/blaSHV-12 (n=1), IncI1/ST3/blaCTX-M-1 (n=7), IncI1/ST26/blaCTX-M-1 (n=1), IncI1/ST36/blaCTX-M-1 (n=2), IncI1/STnew/blaCTX-M-1 (n=1), IncN/blaCTX-M-1 (n=1), IncI1/ST26/blaCTX-M-2 (n=1), IncFII/blaCTX-M-14 (n=1), IncK/blaCTX-M-14 (n=1), IncI1/ST26/blaCMY-2 (n=1) and IncK/blaCMY-2 (n=1). Plasmids measured approximately 40 to 200 kb and mainly exhibited different RFLP profiles. Sixty (26%) and 21 (9%) isolates displayed resistance to nalidixic acid and ciprofloxacin, respectively. qnrS1 was detected in two isolates on IncX2 and a non-typeable plasmid of ca. 30 and 40 kb, respectively. The APEC population in Italian poultry harbours diverse ESBL-encoding genes and plasmids, often in association with fluroquinolone resistance. Interestingly, IncI1/ST26 plasmids were associated with four \u3b2-lactamases (SHV-12, CTX-M-1, CTX-M-2 and CMY-2), suggesting that this plasmid lineage is well adapted in APEC isolated from Italian poultry production. These findings underline the need to develop new strategies for prevention and therapy of multidrug-resistant APEC infections

ST131 fimH22 Escherichia coli isolate with a blaCMY-2/IncI1/ST12 plasmid obtained from a patient with bloodstream infection: highly similar to E. coli isolates of broiler origin.

OBJECTIVES: This study compares the genome of an ST131 CMY-2-producing Escherichia coli isolate from a Danish patient with other ST131 CMY-2-producing E. coli isolates of both human and animal&nbsp;origin. METHODS: In 2016, an ST131 CMY-2-producing E. coli isolate (ESBL20160056) was obtained from a patient with a bloodstream infection. The genome of the ESBL20160056 isolate was compared with genomes from six ST131 CMY-2-producing E. coli isolates obtained from broiler meat imported to Denmark, 15 ST131 CMY-2-producing E. coli isolates obtained from Enterobase (http://enterobase.warwick.ac.uk) and two ST131 CMY-2-producing E. coli from European collaborators. The plasmid from ESBL20160056 was sequenced using a MinION Mk1B (Oxford Nanopore&nbsp;Technologies). RESULTS: The E. coli isolate from the Danish patient clustered together with 13 other fimH22 ST131 CMY-2-producing E. coli isolates in a distinct clade. The clade consisted of genomes from six E. coli isolates from humans collected in Denmark, Spain, Cambodia and the USA, six E. coli isolates obtained from broiler meat samples imported to Denmark from France, the Netherlands and Germany, and two E. coli isolates obtained from broilers in Belgium and Luxembourg. The 101.5 kb plasmid with blaCMY-2 from ESBL20160056 had an IncI1 replicon and belonged to ST12 using the plasmid MLST scheme. In total, 10 of the 14 ST131 E. coli isolates belonging to the fimH22 clade carried an IncI1 ST12 plasmid with&nbsp;blaCMY-2. CONCLUSIONS: From our data, it seems plausible that the ST131 fimH22 CMY-2-producing E. coli isolate obtained from the Danish patient could have a zoonotic broiler&nbsp;origin.</p