Evaluation and validation of laboratory procedures for the surveillance of ESBL-, AmpC-, and carbapenemase-producing Escherichia coli from fresh meat and caecal samples.

Introduction: Extended-spectrum ß-lactamase- (ESBL) and AmpC- ß-lactamase-producing Enterobacterales are widely distributed and emerging in both human and animal reservoirs worldwide. A growing concern has emerged in Europe following the appearance of carbapenemase-producing Escherichia coli (E. coli) in the primary production of food animals. In 2013, the European Commission (EC) issued the Implementing Decision on the monitoring and reporting of antimicrobial resistance in zoonotic and commensal bacteria. The European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) was tasked with providing two laboratory protocols for samples derived from meat and caecal content, respectively, for the isolation of ESBL- and AmpC-producing E. coli (part 1) and carbapenemase-producing (CP) E. coli (part 2). In this study, we describe the current protocols, including the preparatory work for the development. Methods: Up to nine laboratory procedures were tested using minced meat as the matrix from beef, pork, and chicken as well as six procedures for the caecal content of cattle, pigs, and chicken. Variables included sample volume, pre-enrichment volume, pre-enrichment broth with and without antimicrobial supplementation, and incubation time/temperature. The procedures were evaluated against up to nine E. coli strains harboring different AMR genes and belonging to the three ß-lactamase groups. Results and discussion: The laboratory procedures tested revealed that the most sensitive and specific methodologies were based on a Buffered Peptone Water pre-enrichment of 225 ml to 25 g or 9 ml to 1 g for minced meat and caecal content, respectively, incubated at 37°C overnight, followed by inoculation onto MacConkey agar supplemented with 1 mg/L cefotaxime for detecting ESBL- and AmpC-producing E. coli and Chrom ID SMART (Chrom ID CARBA and OXA) for CP E. coli, incubated overnight at 37 and 44°C, respectively. We provided two isolation protocols for the EU-specific monitoring of ESBL- and AmpC- producing E. coli (part 1) and CP E. coli (part 2) from fresh meat (protocol 1) and caecal (protocol 2) samples, which have been successfully implemented by all EU Member States for the monitoring period 2014-2027 (EU 2020/1729).

Intrinsic tet(L) sub-class in Bacillus velezensis and Bacillus amyloliquefaciens is associated with a reduced susceptibility toward tetracycline.

Annotations of non-pathogenic bacterial genomes commonly reveal putative antibiotic resistance genes and the potential risks associated with such genes is challenging to assess. We have examined a putative tetracycline tet(L) gene (conferring low level tetracycline resistance), present in the majority of all publicly available genomes of the industrially important operational group Bacillus amyloliquefaciens including the species B. amyloliquefaciens, Bacillus siamensis and Bacillus velezensis. The aim was to examine the risk of transfer of the putative tet(L) in operational group B. amyloliquefaciens through phylogenetic and genomic position analysis. These analyses furthermore included tet(L) genes encoded by transferable plasmids and other Gram-positive and -negative bacteria, including Bacillus subtilis. Through phylogenetic analysis, we could group chromosomally and plasmid-encoded tet(L) genes into four phylogenetic clades. The chromosomally encoded putative tet(L) from operational group B. amyloliquefaciens formed a separate phylogenetic clade; was positioned in the same genomic region in the three species; was not flanked by mobile genetic elements and was not found in any other bacterial species suggesting that the gene has been present in a common ancestor before species differentiation and is intrinsic. Therefore the gene is not considered a safety concern, and the risk of transfer to and expression of resistance in other non-related species is considered negligible. We suggest a subgrouping of the tet(L) class into four groups (tet(L)1.1, tet(L)1.2 and tet(L)2.1, tet(L)2.2), corresponding with the phylogenetic grouping and tet(L) from operational group B. amyloliquefaciens referred to as tet(L)2.2. Phylogenetic analysis is a useful tool to correctly differentiate between intrinsic and acquired antibiotic resistance genes.

The Tetracycline Resistance Gene, tet(W) in Bifidobacterium animalis subsp. lactis Follows Phylogeny and Differs From tet(W) in Other Species.

The tetracycline resistance gene tet(W) encodes a ribosomal protection protein that confers a low level of tetracycline resistance in the probiotic bacterium Bifidobacterium animalis subsp. lactis. With the aim of assessing its phylogenetic origin and potential mobility, we have performed phylogenetic and in silico genome analysis of tet(W) and its flanking genes. tet(W) was found in 41 out of 44 examined B. animalis subsp. lactis strains. In 38 strains, tet(W) was flanked by an IS5-like element and an open reading frame encoding a hypothetical protein, which exhibited a similar GC content (51-53%). These genes were positioned in the same genomic context within the examined genomes. Phylogenetically, the B. animalis subsp. lactis tet(W) cluster in a clade separate from tet(W) of other species and genera. This is not the case for tet(W) encoded by other bifidobacteria and other species where tet(W) is often found in association with transferable elements or in different genomic regions. An IS5-like element identical to the one flanking the B. animalis subsp. lactis tet(W) has been found in a human gut related bacterium, but it was not associated with any tet(W) genes. This suggests that the IS5-like element is not associated with genetic mobility. tet(W) and the IS5 element have previously been shown to be co-transcribed, indicating that co-localization may be associated with tet(W) expression. Here, we present a method where phylogenetic and in silico genome analysis can be used to determine whether antibiotic resistance genes should be considered innate (intrinsic) or acquired. We find that B. animalis subsp. lactis encoded tet(W) is part of the ancient resistome and thereby possess a negligible risk of transfer.

Putative antibiotic resistance genes present in extant Bacillus licheniformis and Bacillus paralicheniformis strains are probably intrinsic and part of the ancient resistome.

Whole-genome sequencing and phenotypic testing of 104 strains of Bacillus licheniformis and Bacillus paralicheniformis from a variety of sources and time periods was used to characterize the genetic background and evolution of (putative) antimicrobial resistance mechanisms. Core proteins were identified in draft genomes and a phylogenetic analysis based on single amino acid polymorphisms allowed the species to be separated into two phylogenetically distinct clades with one outlier. Putative antimicrobial resistance genes were identified and mapped. A chromosomal ermD gene was found at the same location in all B. paralichenformis and in 27% of B. licheniformis genomes. Erythromycin resistance correlated very well with the presence of ermD. The putative streptomycin resistance genes, aph and aadK, were found in the chromosome of all strains as adjacent loci. Variations in amino acid sequence did not correlate with streptomycin susceptibility although the species were less susceptible than other Bacillus species. A putative chloramphenicol resistance gene (cat), encoding a novel chloramphenicol acetyltransferase protein was also found in the chromosome of all strains. Strains encoding a truncated CAT protein were sensitive to chloramphenicol. For all four resistance genes, the diversity and genetic context followed the overall phylogenetic relationship. No potentially mobile genetic elements were detected in their vicinity. Moreover, the genes were only distantly related to previously-described cat, aph, aad and erm genes present on mobile genetic elements or in other species. Thus, these genes are suggested to be intrinsic to B. licheniformis and B. paralicheniformis and part of their ancient resistomes. Since there is no evidence supporting horizontal transmission, these genes are not expected to add to the pool of antibiotic resistance elements considered to pose a risk to human or animal health. Whole-genome based phylogenetic and sequence analysis, combined with phenotypic testing, is proposed to be suitable for determining intrinsic resistance and evolutionary relationships.

Antimicrobial Susceptibility Testing and Tentative Epidemiological Cutoff Values for Five Bacillus Species Relevant for Use as Animal Feed Additives or for Plant Protection.

Bacillus megaterium (n = 29), Bacillus velezensis (n = 26), Bacillus amyloliquefaciens (n = 6), Bacillus paralicheniformis (n = 28), and Bacillus licheniformis (n = 35) strains from different sources, origins, and time periods were tested for the MICs for nine antimicrobial agents by the CLSI-recommended method (Mueller-Hinton broth, 35°C, for 18 to 20 h), as well as with a modified CLSI method (Iso-Sensitest [IST] broth, 37°C [35°C for B. megaterium], 24 h). This allows a proposal of species-specific epidemiological cutoff values (ECOFFs) for the interpretation of antimicrobial resistance in these species. MICs determined by the modified CLSI method were 2- to 16-fold higher than with the CLSI-recommended method for several antimicrobials. The MIC distributions differed between species for five of the nine antimicrobials. Consequently, use of the modified CLSI method and interpretation of resistance by use of species-specific ECOFFs is recommended. The genome sequences of all strains were determined and used for screening for resistance genes against the ResFinder database and for multilocus sequence typing. A putative chloramphenicol acetyltransferase (cat) gene was found in one B. megaterium strain with an elevated chloramphenicol MIC compared to the other B. megaterium strains. In B. velezensis and B. amyloliquefaciens, a putative tetracycline efflux gene, tet(L), was found in all strains (n = 27) with reduced tetracycline susceptibility but was absent in susceptible strains. All B. paralicheniformis and 23% of B. licheniformis strains had elevated MICs for erythromycin and harbored ermD The presence of these resistance genes follows taxonomy suggesting they may be intrinsic rather than horizontally acquired. Reduced susceptibility to chloramphenicol, streptomycin, and clindamycin could not be explained in all species.IMPORTANCE When commercializing bacterial strains, like Bacillus spp., for feed applications or plant bioprotection, it is required that the strains are free of acquired antimicrobial resistance genes that could potentially spread to pathogenic bacteria, thereby adding to the pool of resistance genes that may cause treatment failures in humans or animals. Conversely, if antimicrobial resistance is intrinsic to a bacterial species, the risk of spreading horizontally to other bacteria is considered very low. Reliable susceptibility test methods and interpretation criteria at the species level are needed to accurately assess antimicrobial resistance levels. In the present study, tentative ECOFFs for five Bacillus species were determined, and the results showed that the variation in MICs followed the respective species. Moreover, putative resistance genes, which were detected by whole-genome sequencing and suggested to be intrinsic rather that acquired, could explain the resistance phenotypes in most cases.

A sampling and metagenomic sequencing-based methodology for monitoring antimicrobial resistance in swine herds.

OBJECTIVES: Reliable methods for monitoring antimicrobial resistance (AMR) in livestock and other reservoirs are essential to understand the trends, transmission and importance of agricultural resistance. Quantification of AMR is mostly done using culture-based techniques, but metagenomic read mapping shows promise for quantitative resistance monitoring. METHODS: We evaluated the ability of: (i) MIC determination for Escherichia coli; (ii) cfu counting of E. coli; (iii) cfu counting of aerobic bacteria; and (iv) metagenomic shotgun sequencing to predict expected tetracycline resistance based on known antimicrobial consumption in 10 Danish integrated slaughter pig herds. In addition, we evaluated whether fresh or manure floor samples constitute suitable proxies for intestinal sampling, using cfu counting, qPCR and metagenomic shotgun sequencing. RESULTS: Metagenomic read-mapping outperformed cultivation-based techniques in terms of predicting expected tetracycline resistance based on antimicrobial consumption. Our metagenomic approach had sufficient resolution to detect antimicrobial-induced changes to individual resistance gene abundances. Pen floor manure samples were found to represent rectal samples well when analysed using metagenomics, as they contain the same DNA with the exception of a few contaminating taxa that proliferate in the extraintestinal environment. CONCLUSIONS: We present a workflow, from sampling to interpretation, showing how resistance monitoring can be carried out in swine herds using a metagenomic approach. We propose metagenomic sequencing should be part of routine livestock resistance monitoring programmes and potentially of integrated One Health monitoring in all reservoirs.

Evidence for Human Adaptation and Foodborne Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus.

We investigated the evolution and epidemiology of a novel livestock-associated methicillin-resistant Staphylococcus aureus strain, which colonizes and infects urban-dwelling Danes even without a Danish animal reservoir. Genetic evidence suggests both poultry and human adaptation, with poultry meat implicated as a probable source.

Heavy metal and disinfectant resistance genes among livestock-associated methicillin-resistant Staphylococcus aureus isolates.

Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has emerged in animal production worldwide. Most LA-MRSA in Europe belong to the clonal complex (CC) 398. The reason for the LA-MRSA emergence is not fully understood. Besides antimicrobial agents used for therapy, other substances with antimicrobial activity applied in animal feed, including metal-containing compounds might contribute to their selection. Some of these genes have been found in various novel SCCmec cassettes. The aim of this study was to assess the occurrence of metal-resistance genes among a LA-S. aureus collection [n=554, including 542 MRSA and 12 methicillin-susceptible S. aureus (MSSA)] isolated from livestock and food thereof. Most LA-MRSA isolates (76%) carried at least one metal-resistance gene. Among the LA-MRSA CC398 isolates (n=456), 4.8%, 0.2%, 24.3% and 71.5% were positive for arsA (arsenic compounds), cadD (cadmium), copB (copper) and czrC (zinc/cadmium) resistance genes, respectively. In contrast, among the LA-MRSA non-CC398 isolates (n=86), 1.2%, 18.6% and 16.3% were positive for the cadD, copB and czrC genes, respectively, and none were positive for arsA. Of the LA-MRSA CC398 isolates, 72% carried one metal-resistance gene, and the remaining harboured two or more in different combinations. Differences between LA-MRSA CC398 and non-CC398 were statistically significant for arsA and czrC. The czrC gene was almost exclusively found (98%) in the presence of SCCmec V in both CC398 and non-CC398 LA-MRSA isolates from different sources. Regarding the LA-MSSA isolates (n=12), some (n=4) were also positive for metal-resistance genes. This study shows that genes potentially conferring metal-resistance are frequently present in LA-MRSA.

World Health Organization Ranking of Antimicrobials According to Their Importance in Human Medicine: A Critical Step for Developing Risk Management Strategies to Control Antimicrobial Resistance From Food Animal Production.

Antimicrobial use in food animals selects for antimicrobial resistance in bacteria, which can spread to people. Reducing use of antimicrobials-particularly those deemed to be critically important for human medicine-in food production animals continues to be an important step for preserving the benefits of these antimicrobials for people. The World Health Organization ranking of antimicrobials according to their relative importance in human medicine was recently updated. Antimicrobials considered the highest priority among the critically important antimicrobials were quinolones, third- and fourth-generation cephalosporins, macrolides and ketolides, and glycopeptides. The updated ranking allows stakeholders in the agriculture sector and regulatory agencies to focus risk management efforts on drugs used in food animals that are the most important to human medicine. In particular, the current large-scale use of fluoroquinolones, macrolides, and third-generation cephalosporins and any potential use of glycopeptides and carbapenems need to be addressed urgently.

Host-Specific Patterns of Genetic Diversity among IncI1-Iγ and IncK Plasmids Encoding CMY-2 ß-Lactamase in Escherichia coli Isolates from Humans, Poultry Meat, Poultry, and Dogs in Denmark.

UNLABELLED: CMY-2 is the most common plasmid-mediated AmpC ß-lactamase in Escherichia coli isolates of human and animal origin. The aim of this study was to elucidate the epidemiology of CMY-2-producing E. coli in Denmark. Strain and plasmid relatedness was studied in 93 CMY-2-producing clinical and commensal E. coli isolates collected from 2006 to 2012 from humans, retail poultry meat, broilers, and dogs. Multilocus sequence typing (MLST), antimicrobial susceptibility testing, and conjugation were performed in conjunction with plasmid replicon typing, plasmid multilocus sequence typing (pMLST), restriction fragment length polymorphism (RFLP), and sequencing of selected blaCMY-2-harboring plasmids. MLST revealed high strain diversity, with few E. coli lineages occurring in multiple host species and sample types. blaCMY-2 was detected on plasmids in 83 (89%) isolates. Most (75%) of the plasmids were conjugative and did not (96%) cotransfer resistance to antimicrobials other than cephalosporins. The main replicon types identified were IncI1-Iγ (55%) and IncK (39%). Isolates from different host species mainly carried distinct plasmid subtypes. Seven of the 18 human isolates harbored IncI1-Iγ/sequence type 2 (ST2), IncI1-Iγ/ST12, or IncK plasmids highly similar to those found among animal isolates, even though highly related human and animal plasmids differed by nonsynonymous single nucleotide polymorphisms (SNPs) or insertion sequence elements. This study clearly demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this ß-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids that are generally distributed according to host-specific patterns. These baseline data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources. IMPORTANCE: CMY-2 is the most common plasmid-mediated AmpC ß-lactamase in Escherichia coli This ß-lactamase is poorly inhibited by clavulanic acid and confers resistance to cephamycins, third-generation cephalosporins, and aztreonam. Furthermore, resistance to carbapenems has been reported in E. coli as a result of production of plasmid-encoded CMY-2 ß-lactamase in combination with decreased outer membrane permeability. The gene encoding CMY-2 generally resides on transferable plasmids belonging to different incompatibility groups. The prevalence of CMY-2-mediated cephalosporin resistance in E. coli varies significantly depending on the geographical region and host. This study demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this ß-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids, which are generally distributed according to host-specific patterns. These data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources.

Sequence-Based Characterization of Tn5801-Like Genomic Islands in Tetracycline-Resistant Staphylococcus pseudintermedius and Other Gram-positive Bacteria from Humans and Animals.

Antibiotic resistance in pathogens is often associated with mobile genetic elements, such as genomic islands (GI) including integrative and conjugative elements (ICEs). These can transfer resistance genes within and between bacteria from humans and/or animals. The aim of this study was to investigate whether Tn5801-like GIs carrying the tetracycline resistance gene, tet(M), are common in Staphylococcus pseudintermedius from pets, and to do an overall sequences-based characterization of Tn5801-like GIs detected in Gram-positive bacteria from humans and animals. A total of 27 tetracycline-resistant S. pseudintermedius isolates from Danish pets (1998-2005) were screened for tet(M) by PCR. Selected isolates (13) were screened for GI- or ICE-specific genes (int Tn5801 or xis Tn916 ) and their tet(M) gene was sequenced (Sanger-method). Long-range PCR mappings and whole-genome-sequencing (Illumina) were performed for selected S. pseudintermedius-isolates (seven and three isolates, respectively) as well as for human S. aureus isolates (seven and one isolates, respectively) and one porcine Enterococcus faecium isolate known to carry Tn5801-like GIs. All 27 S. pseudintermedius were positive for tet(M). Out of 13 selected isolates, seven contained Tn5801-like GIs and six contained Tn916-like ICEs. Two different Tn5801-like GI types were detected among S. pseudintermedius (Tn5801 and GI6287) - both showed high similarity compared to GenBank sequences from human pathogens. Two distinct Tn5801-like GI types were detected among the porcine E. faecium and human S. aureus isolates (Tn6014 and GI6288). Tn5801-like GIs were detected in GenBank-sequences from Gram-positive bacteria of human, animal or food origin worldwide. Known Tn5801-like GIs were divided into seven types. The results showed that Tn5801-like GIs appear to be relatively common in tetracycline-resistant S. pseudintermedius in Denmark. Almost identical Tn5801-like GIs were identified in different Gram-positive species of pet and human origin, suggesting that horizontal transfer of these elements has occurred between S. pseudintermedius from pets and human pathogens, including S. aureus.

Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat, Denmark 2015.

The plasmid-mediated colistin resistance gene, mcr-1, was detected in an Escherichia coli isolate from a Danish patient with bloodstream infection and in five E. coli isolates from imported chicken meat. One isolate from chicken meat belonged to the epidemic spreading sequence type ST131. In addition to IncI2, an incX4 replicon was found to be linked to mcr-1. This report follows a recent detection of mcr-1 in E. coli from animals, food and humans in China.

Pharmacodynamic modelling of in vitro activity of tetracycline against a representative, naturally occurring population of porcine Escherichia coli.

BACKGROUND: The complex relationship between drug concentrations and bacterial growth rates require not only the minimum inhibitory concentration but also other parameters to capture the dynamic nature of the relationship. To analyse this relationship between tetracycline concentration and growth of Escherichia coli representative of those found in the Danish pig population, we compared the growth of 50 randomly selected strains. The observed net growth rates were used to describe the in vitro pharmacodynamic relationship between drug concentration and net growth rate based on E max model with three parameters: maximum net growth rate (amax); concentration for a half-maximal response (E max); and the Hill coefficient (γ). RESULTS: The net growth rate in the absence of antibiotic did not differ between susceptible and resistant isolates (P = 0.97). The net growth rate decreased with increasing tetracycline concentrations, and this decline was greater in susceptible strains than resistant strains. The lag phase, defined as the time needed for the strain to reach an OD600 value of 0.01, increased exponentially with increasing tetracycline concentration. The pharmacodynamic parameters confirmed that the αmax between susceptible and resistant strains in the absence of a drug was not different. EC 50 increased linearly with MIC on a log-log scale, and γ was different between susceptible and resistant strains. CONCLUSIONS: The in vitro model parameters described the inhibition effect of tetracycline on E. coli when strains were exposed to a wide range of tetracycline concentrations. These parameters, along with in vivo pharmacokinetic data, may be useful in mathematical models to predict in vivo competitive growth of many different strains and for development of optimal dosing regimens for preventing selection of resistance.

Biocide Susceptibility of Staphylococcus aureus CC398 and CC30 Isolates from Pigs and Identification of the Biocide Resistance Genes, qacG and qacC.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA), in particular clonal complex (CC) 398, is increasingly found in livestock. Recently, MRSA CC30 was identified in Danish pigs. We determined the susceptibility of porcine S. aureus isolates of CC398 and CC30 to disinfectants used in pig farming (benzalkonium chloride, hydrogen peroxide, formaldehyde, sodium hypochlorite, and caustic soda). Furthermore, efflux pump activity, antimicrobial resistance profiles, hemolysis properties, and the presence of toxic shock syndrome toxin-1 (TSST-1) and Panton-Valentine Leukocidin (PVL)-encoding virulence factors were investigated. METHODS: Susceptibilities to biocides and antimicrobial agents of 79 porcine S. aureus isolates were determined by the microdilution method. Isolates comprised 21 methicillin-sensitive S. aureus (MSSA) and 40 MRSA isolates belonging to CC398 and 13 MSSA and 5 MRSA isolates belonging to CC30. The presence of quaternary ammonium compound (QAC) resistance efflux pumps was analyzed using an ethidium bromide accumulation assay. The presence of qac resistance genes in active efflux pump positive isolates was determined by whole-genome sequencing data. All isolates were screened for lukPV and tst genes with PCR, and hemolytic activities were determined using an agar plate assay. RESULTS: S. aureus isolates did not show reduced susceptibility to the biocides tested. However, the QAC resistance gene, qacG, was detected in three MRSA CC30 isolates and the qacC in one MRSA CC30 isolate. CC30 isolates were generally more susceptible to non-beta-lactam antibiotics than CC398. Isolates generally had low hemolytic activity and none encoded PVL or TSST-1. CONCLUSION: The presence of qac genes in European porcine S. aureus isolates and in livestock-associated MRSA CC30 is for the first time described in this study. This finding is concerning as it ultimately may compromise disinfection with QACs and thereby contribute to the selection and spread of MRSA CC30.

Antibiotic resistance and resistance genes in Escherichia coli from poultry farms, southwest Nigeria.

INTRODUCTION: This study investigated the mechanisms of resistance in 36 E. coli isolated from waste, litter, soil and water samples collected from poultry farms in Southwestern Nigeria. METHODOLOGY: Minimum inhibitory concentration (MIC) distributions of the isolates were determined using the methods of the Clinical and Laboratory Standard Institute and resistance genes detected by PCR. RESULTS: A total of 30 isolates (94%) showed resistance to more than one antimicrobial. Percentage resistance was: tetracycline 81%, sulphamethoxazole 67%, streptomycin 56%, trimethoprim 47 %, ciprofloxacin 42%, ampicillin 36%, spectinomycin 28%, nalidixic acid 25%, chloramphenicol 22%, neomycin 14%, gentamicin 8%, amoxicillin-clavulanate, ceftiofur, cefotaxime, colistin, florfenicol and apramycin 0%. Resistance genes found among the isolates include bla-TEM (85%), sul2 (67%), sul3 (17%), aadA (65%), strA (70%), strB (61%), catA1 (25%), cmlA1 (13%), tetA (21%) and tetB (17%). Class 1 and 2 integrons were found in five (14%) and six (17%) isolates, respectively, while one isolate was positive for both classes of integrons. Seven out of eight isolates with resistance to ciprofloxacin and MIC ≤ 32 mg/L to nalidixic acid contained qnrS genes. CONCLUSIONS: Our findings provided additional evidence that the poultry production environment in Nigeria represents an important reservoir of antibiotic resistance genes such as qnrS that may spread from livestock production farms to human populations via manure and water.

Detection and linkage to mobile genetic elements of tetracycline resistance gene tet(M) in Escherichia coli isolates from pigs.

BACKGROUND: In Escherichia coli the genes involved in the acquisition of tetracycline resistance are mainly tet(A) and tet(B). In addition, tet(M) is the most common tetracycline resistance determinant in enterococci and it is associated with conjugative transposons and plasmids. Although tet(M) has been identified in E. coli, to our knowledge, there are no previous reports studying the linkage of the tet(M) gene in E. coli to different mobile genetic elements. The aim of this study was to determine the occurrence of tet(A), tet(B), and tet(M) genes in doxycycline-resistant E. coli isolates from pigs, as well as the detection of mobile genetic elements linked to tet(M) in E. coli and its possible transfer from enterococci. RESULTS: tet(A) was the most frequently detected gene (87.9%) in doxycycline-resistant isolates. tet(M) was found in 13.1% E. coli isolates. The tet(M) gene was detected in relation with conjugative transposons in 10 out of 36 enterococci isolates analyzed but not in any of E. coli isolates positive for tet(M). Southern blot showed that in E. coli and in most of the enterococci isolates the tet(M) gene was carried on a plasmid. According to the phylogenetic analysis, E. coli contained a new tet(M) allele grouping separately. Mating experiments revealed that tet(M) was carried on a mobile element successfully transferred between enterococci and between enterococci and E. coli. CONCLUSIONS: The detection of tet(M) in E. coli isolates from pigs was higher than expected. In our study, tet(M) detected in E. coli seems not to have been transferred from enterococci, although it can not be ruled out that the horizontal transfer of this gene occurred from other intestinal tract bacteria.

Characterization of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli obtained from Danish pigs, pig farmers and their families from farms with high or no consumption of third- or fourth-generation cephalosporins.

OBJECTIVES: To compare and characterize extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli from pigsties, pig farmers and their families on farms with previous high or no use of third- or fourth-generation cephalosporins. METHODS: Twenty farms with no third- or fourth-generation cephalosporin use and 19 herds with previous frequent use were included. The ESBL-producing isolates detected in humans and pigs were characterized by ESBL genotype, PFGE, susceptibility to non-ß-lactam antibiotics and phylotype, and selected isolates were characterized by multilocus sequence typing (MLST). Furthermore, transferability of bla(CTX-M-)1 from both human and pig isolates was studied and plasmid incompatibility groups were defined. The volunteers answered a questionnaire including epidemiological risk factors for carriage of ESBL-producing E. coli. RESULTS: ESBL-producing E. coli was detected in pigs on 79% of the farms with high consumption of cephalosporins compared with 20% of the pigs on farms with no consumption. ESBL-producing E. coli was detected in 19 of the 195 human participants and all but one had contact with pigs. The genes found in both humans and pigs at the same farms were blaCTX-M-1 (eight farms), bla(CTX-M-14) (one farm) and bla(SHV-12) (one farm). At four farms ESBL-producing E. coli isolates with the same CTX-M enzyme, phylotype, PFGE type and MLST type were detected in both pigs and farmers. The majority of the plasmids with bla(CTX-M-1) were transferable by conjugation and belonged to incompatibility group IncI1, IncF, or IncN. CONCLUSIONS: The present study shows an increased frequency of ESBL-producing E. coli on farms with high consumption of third- or fourth-generation cephalosporins and indicates transfer of either ESBL-producing E. coli or plasmids between pigs and farmers.

Spread of extended spectrum cephalosporinase-producing Escherichia coli clones and plasmids from parent animals to broilers and to broiler meat in a production without use of cephalosporins.

OBJECTIVES: This study investigated the occurrence of extended spectrum cephalosporinase (ESC)-producing Escherichia coli in a broiler production with no cephalosporin use and a low use of antimicrobials in general. Furthermore, it investigated whether the current consumption of aminopenicillins selects for ESC-producing E. coli and whether certain clones or plasmids spread from imported parent flocks to the meat. MATERIALS AND METHODS: ESC-producing E. coli was isolated using MacConkey broth with 1 mg/L of ceftriaxone. ESC genes were identified using polymerase chain reaction and sequencing. Isolates with blaCMY-2 were subtyped by pulsed-field gel electrophoresis (PFGE), phylotyping, and antimicrobial susceptibility testing. Selected isolates were used as donors in filter-mating experiments, multilocus sequence typing (MLST), and plasmid replicons were typed. Aminopenicillin use at the farm (not flock) level was obtained from VetStat, a database for mandatory registration of veterinary prescriptions in Denmark. RESULTS: ESC-producing E. coli occurred in 93% (27/29) of broiler parent farms in 2011, 27% (53/197) of broiler flocks in 2010, and 3.3% (4/121) of Danish retail broiler meat in 2009 and 8.6% (16/187) in 2010. The ESC producing E. coli contained blaCMY-2, blaSHV-2 or blaCTX-M-1. Isolates with blaCMY-2 represented 35 PFGE groups. One group dominated (39 isolates) and included isolates with indistinguishable PFGE patterns from parents, broilers, and meat. Most blaCMY-2 isolates were susceptible to non-ß-lactams, and blaCMY-2 was mostly present on horizontally transferable incI1 or incK plasmids. Phylogroup D was most common and E. coli MLST types previously found in humans were observed. Broiler farms with registered aminopenicillin use had significantly higher occurrence of ESC E. coli. CONCLUSIONS: ESC-producing E. coli from flocks of imported broiler parents spread clonally and horizontally to broiler meat (including potentially human pathogenic types) even in a country with no cephalosporin use. Use of aminopenicillins may influence the persistence of ESC-producing E. coli in the broiler production, but other factors should be investigated.

Molecular clonality and antimicrobial resistance in Salmonella enterica serovars Enteritidis and Infantis from broilers in three Northern regions of Iran.

BACKGROUND: Multidrug-resistant Salmonella strains are frequently encountered problems worldwide with considerable increased occurrences in recent years. The aim of this study was to investigate the occurrence and frequency of antimicrobial resistance and associated resistance genes in Salmonella isolates from broiler farms in different regions of Iran covering a time period of four years. RESULTS: From 2007 to 2011, 36 Salmonella strains were isolated from broiler farms located in three northern provinces of Iran. The isolates were serotyped, antimicrobial susceptibility tested, and characterized for antimicrobial resistance genes associated to the phenotype. Pulsed-field gel electrophoresis (PFGE) was applied for comparison of genetic relatedness.Two serovars were detected among the isolates; Salmonella enterica serovar Infantis (75%) and S. Enteritidis (25%). Thirty-four (94%) of the isolates exhibited resistance to nalidixic acid and ciprofloxacin caused by a single mutation in the quinolone resistance-determining region (QRDR) of gyrA. For all strains this mutation occurred in the codon of Asp87 leading to a Asp87-Tyr, Asp87-Gly or Asp87-Asn substitutions. All S. Infantis (n = 27) were resistant to tetracycline, spectinomycin, streptomycin, and sulfamethoxazole and harbored the associated resistance genes; tetA, dfrA14, aadA1, and sulI together with class 1 integrons. The isolates revealed highly similar PFGE patterns indicating clonal relatedness across different geographical locations. CONCLUSION: The data provided fundamental information applicable when launching future control programs for broilers in Iran with the aim to conserve the effectiveness of important antimicrobials for treatment in humans.

Voluntary ban on cephalosporin use in Danish pig production has effectively reduced extended-spectrum cephalosporinase-producing Escherichia coli in slaughter pigs.

OBJECTIVES: To measure the effect of a voluntary ban on cephalosporin usage in the Danish pig production on the prevalence of extended-spectrum cephalosporinase (ESC)-producing Escherichia coli in pigs and pork. METHODS: Data on cephalosporin consumption were obtained from the VetStat database. For detection of ESC-producing E. coli, three sampling types were included: at slaughter, caecal samples were collected from pigs in 2009 and 2010 (June) before and in two periods (2010 and 2011) after a voluntary ban on cephalosporins was effected (July 2010); at farm level, pools of five stool samples from different pigsties were collected in 2010 and in 2011; and samples from pork were collected randomly at retail stores and outlets from 2009 to 2011. ESC-producing E. coli was isolated after selective enrichment in MacConkey broth with 1 mg/L ceftriaxone. ESC genes were detected using PCR, microtube array and sequencing. RESULTS: From July 2010 the consumption of cephalosporins approximated zero. The occurrence of ESC-producing E. coli in pigs at slaughter was not significantly different (P=0.7) between 2009 [10.8% (85/786)] and 2010 [11.8% (48/407)], but in 2011 the occurrence [3.6% (28/777)] decreased significantly (P<0.001). A significant decrease (P=0.002) in occurrence of ESC-producing E. coli at pig farm level from 2010 [11% (11/99)] to 2011 (0/78) was also observed. The bla(CTX-M-1) gene was most often detected (63%), but bla(CTX-M-14) and bla(CTX-M-15) were also found. Occurrence in pork was between 1.3% and 0.9%. CONCLUSIONS: The discontinuation of an already low use of cephalosporins in pig production has significantly reduced the occurrence of ESC-producing E. coli.