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

Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes

Background and aim: Plasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between ampli-cons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.3, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing. © 2018, European Centre for Disease Prevention and Control (ECDC). All rights reserved

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

Occurrence of diverse Extended-spectrum beta-lactamase (ESBL)- and AmpC beta-lactamase-encoding genes in Avian Pathogenic Escherichia coli from poultry in Italy

Avian pathogenic Escherichìa coli (APEC) cause infections with high morbidity and mortality in poultry flocks. Beta-lactams like orai penicillins, ampicillin and amoxicillin, are among the most used antimicrobials in poultry but they can be inactivated by enzymes including extendedspectrum |3-lactamases (ESBLs)- and AmpC P-lactamases. Information on occurrence and mechanisms of beta-lactam resistance in APEC is limited, although highly relevant for animai welfare and economy within the poultry industry. This study aimed at investigating the prevalence and diversity of ESBL- and AmpC P-lactamase-encoding genes in APEC from chickens, turkeys and layer hens collected from industriai poultry farms across Italy between 2008 and 2012. A total of 226 non-repetitive clinical E. coli isolates from turkeys (n=104), broilers (n=99) and layer hens (n=23) was screened for ESBL/AmpC production by phenotypic tests according to Clinical and Laboratory Standards Institute (CESI) guidelines. Isolates resistant to at least one thirdgeneration cephalosporin were tested for presence of TEM-, SHV-, CTX-Mand CMY-2-encoding genes and mutations in the chromosomal AmpC promoter by PCR and sequencing. A total of 31 (14%) isolates from ali sources displayed resistance to at least one third-generation cephalosporin and 26 (84%) of those isolates were confìrmed as ESBLs producers. The ESBL-encoding genes è/aSHV-12, è/aCTX-M-1, blaCTX-M-2 and è/aCTX-M-14 were detected in one, twelve, one and two isolates, respectively. The AmpC P-lactamase-encoding gene è/aCMY-2 was detected in two isolates, and ten isolates presented mutations in the chromosomal AmpC promoter compatible with AmpC overproduction. è/aTEM-1 was detected in fifteen isolates associateci with 6/aCTX-M-l (n=8), WaCTX-M-14 (n=l), WaCMY-2 (n=2), and AmpC promoter mutations (n=I). In three isolates, the molecular bases for ESBL phenotype were not elucidated and are currently under investigation. This is the first study characterizing ESBL/AmpC-encoding genes in APEC in poultry in Italy. A broad diversity of ESBL/AmpC-encoding genes was detected, including variants not previously reported in Italian poultry (6/aCTX-M-14). This suggests that APEC acquired such genes on multiple occasions, and emphasizes the need of studies identifying the causes for occurrence of ESBL/AmpC-encoding genes in poultry flocks

Resistenza a chinoloni ed antibiotici beta-lattamici in ceppi di Escherichia coli isolati da broilers in Danimarca ed in Italia

The prevalence of quinolone- and β-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 μg/ml), ciprofloxacin (2 μg/ml), ampicillin (32 μg/ml), cefotaxime (2 μg/ml) or ceftiofur (8 μg/ml). The β-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 β-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