Decreased detection of ESBL- or pAmpC-producing Escherichia coli in broiler breeders imported into Sweden.

In the spring of 2010, it was discovered that a large proportion of broilers in Sweden were colonized with Escherichia coli producing extended-spectrum beta-lactamases (ESBL) or plasmid mediated AmpC (pAmpC). It was hypothesized that the high prevalence was due to transfer from an upper level in the production pyramid and sampling upwards in the production pyramid was initiated. From 2010 to 2019, all shipments (n = 122) of broiler breeders were screened on arrival to Sweden for the occurrence of ESBL- or pAmpC-producing E. coli using selective methods. Samples of paper linings from shipments of breeders were cultured on MacConkey agar supplemented with cefotaxime (1 mg/L) after pre-enrichment in either MacConkey broth with cefotaxime (1 mg/L), or from late June 2015 in buffered peptone water without antibiotics. ESBL- or pAmpC-producing E. coli was isolated from 43 (35%) of these. Over the years, the proportion of positive imports have decreased and during 2018 and 2019 all imports were negative. In conclusion, the occurrence of ESBL- or pAmpC-producing E. coli in broiler breeders on arrival to Sweden has decreased. Such bacteria have not been detected in any shipments since 2017.

Low occurrence of extended-spectrum cephalosporinase producing Enterobacteriaceae and no detection of methicillin-resistant coagulase-positive staphylococci in healthy dogs in Sweden.

Sweden has a long tradition of monitoring occurrence of antibiotic resistant bacteria in both animals and humans, but there currently is no organised and harmonized monitoring on carriage of Enterobacteriaceae producing extended-spectrum beta-lactamase (ESBL), plasmid-mediated AmpC beta-lactamase (pAmpC), or methicillin-resistant coagulase positive staphylococci e.g. methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) in dogs. The aim of the current study was therefore to determine the prevalence of ESBL/pAmpC producing Enterobacteriaceae and methicillin-resistant coagulase positive staphylococci in healthy dogs in Sweden, and to phenotypically and genotypically characterize any identified isolates. It was shown that 0.9% (95% confident interval 0.3-2.7%) of the dogs (n = 325) carried multi-resistant ESBL-producing Escherichia coli, but that no methicillin-resistant coagulase positive staphylococci could be detected. In conclusion, the occurrence of multi-drug resistant bacteria remains rare among healthy dogs in Sweden. In addition, the ESBL-producing E. coli identified showed genetic characteristics related to those reported from humans.

A link between the newly described colistin resistance gene mcr-9 and clinical Enterobacteriaceae isolates carrying blaSHV-12 from horses in Sweden.

OBJECTIVES: The aim of this study was to investigate the occurrence of the newly described transferable colistin resistance gene mcr-9 in extended-spectrum ß-lactamase (ESBL)-producing clinical Enterobacteriaceae isolates from horses in Sweden. METHODS: A total of 56 whole-genome sequenced ESBL-producing Enterobacteriaceae isolates from horses were subjected to in silico detection of antimicrobial resistance genes and identification of plasmid replicons types. The colistin minimum inhibitory concentration (MIC) for mcr-positive isolates was determined by broth microdilution. Relatedness between Enterobacteriaceae carrying mcr genes was determined by multilocus sequence typing (MLST) and core genome MLST. RESULTS: Thirty ESBL-producing Enterobacteriaceae isolates from horses were positive for the colistin resistance gene mcr-9. These isolates included Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca and Citrobacter freundii and belonged to diverse MLST sequence types within each species. Two of the mcr-9-containing isolates originated from the same horse. All mcr-9-positive isolates had colistin MICs below or equal to the EUCAST epidemiological cut-off value of 2 mg/L and were negative for the two potential regulatory genes qseB-like and qseC-like for mcr-9. Except for one isolate carrying only blaTEM-1B, all of the isolates carried blaSHV-12 and blaTEM-1B, and were all considered multidrug-resistant as they harboured genes encoding resistance to aminoglycosides, chloramphenicol, fosfomycin, macrolides, quinolones, sulfonamides, trimethoprim and tetracyclines. Plasmid replicon types IncHI2 and IncHI2A were detected in all mcr-9-positive isolates. CONCLUSION: The occurrence of mcr-9 was common among clinical ESBL-producing Enterobacteriaceae isolates from horses in Sweden and was linked to the ESBL-encoding gene blaSHV-12 and plasmid replicon types IncHI2 and IncHI2A.

Clonal spread of Escherichia coli resistant to cephalosporins and quinolones in the Nordic broiler production.

The intestinal flora of healthy broilers can contain Escherichia coli resistant to extended spectrum cephalosporins (ESC) and fluoroquinolones (FQ), representing a possible public health problem. We investigated the clonal epidemiology of E. coli with reduced susceptibility to ESC or FQ in broilers in three Nordic countries interconnected by a common source of breeding animals. Isolates (n = 319 and n = 132 non-wild type for ESC and FQ, respectively) from Norwegian, Swedish and Icelandic production originated mainly from the intestinal flora of broilers at the age of 20-35 days. Genetic relationships were investigated by ten loci multilocus variable number tandem repeat analyses (MLVA) and representative isolates of inter-Nordic clusters were subjected to multilocus sequence typing (MLST). Antimicrobial susceptibility data based on minimum inhibitory concentrations was compiled. Approximately one third of the ESC non-wild type isolates, including isolates from all three countries, clustered together. These isolates belonged to sequence type (ST) 38 and contained blaCMY-2. The FQ non-wild type isolates were more genetically diverse, but related isolates occurred in more than one country. MLST typing showed clusters belonging to ST10, ST355, ST349, ST665 and ST93. Our study demonstrated inter-Nordic distribution of E. coli ST38 with blaCMY-2, suggesting clonal proliferation as a contributing factor for spread of ESC resistance in the broiler production. The international trade in breeding material may explain introduction of resistant E. coli. The reason for their success and the success of certain clonal lineages in broiler production not exposed to antimicrobial selection pressure is currently unknown.

Introduction of quinolone resistant Escherichia coli to Swedish broiler population by imported breeding animals.

During recent years a rapid increase of quinolone resistant Escherichia coli have been noted in the Swedish broiler population, despite the lack of a known selective pressure. The current study wanted to investigate if imported breeding birds could be a source for the quinolone resistant E. coli. The occurrence of quinolone resistant E. coli was investigated, using selective cultivation with nalidixic acid, in grand-parent birds on arrival to Sweden and their progeny. In addition, sampling in hatcheries and empty cleaned poultry houses was performed. Clonality of isolates was investigated using a 10-loci multiple-locus variable number tandem repeat analysis (MLVA). To identify the genetic basis for the resistance isolates were also analysed for occurrence of plasmid-mediated quinolone resistance (PMQR) determinants and characterization of chromosomal mutations. E. coli resistant to nalidixic acid occurred in grandparent birds imported to Sweden for breeding purposes. Four predominant MLVA types were identified in isolates from grandparent birds, parent birds and broilers. However, resistant E. coli with identical MLVA patterns were also present in hatcheries and poultry houses suggesting that the environment plays a role in the occurrence. Nalidixic acid resistance was due to a mutation in the gyrA gene and no PMQR could be identified. The occurrence of identical clones in all levels of the production pyramid points to that quinolone resistant E. coli can be introduced through imported breeding birds and spread by vertical transmission to all levels of the broiler production pyramid.

Vertical transmission of Escherichia coli carrying plasmid-mediated AmpC (pAmpC) through the broiler production pyramid.

OBJECTIVES: In Sweden the prevalence of Enterobacteriaceae with transferable resistance to extended-spectrum cephalosporins (ESCs) is low. However, in broilers ESC-resistant Escherichia coli is common, with a clear dominance of blaCMY-2. Antimicrobials are rarely used in broiler production in Sweden and cephalosporins are never used. Introduction through imported breeding stock and subsequent vertical transmission of the bacteria through the production pyramid could be one explanation for this high prevalence. METHODS: To test this hypothesis, paper linings from imported flocks of grandparent animals were screened for the presence of ESC-resistant E. coli and a positive flock, together with its progeny, was followed longitudinally through the production pyramid using boot swabs. The relationship of isolated ESC-resistant E. coli was investigated using multiple-locus variable number tandem repeat analysis (MLVA). RESULTS: ESC-resistant E. coli carrying blaCMY-2 was isolated from six out of eight imported flocks of grandparent animals. One clone of E. coli carrying blaCMY-2 occurred in all levels of the production pyramid and in flocks of imported grandparent animals. CONCLUSIONS: E. coli carrying blaCMY-2 is frequently present among grandparent animals imported to Sweden for breeding purposes. The occurrence of one clone in all levels of the production pyramid indicates that its introduction through imported breeding stock and vertical transmission through the production pyramid could be one explanation for the high proportion of Swedish broilers colonized with ESC-resistant E. coli.

Antimicrobial susceptibility of porcine Brachyspira hyodysenteriae and Brachyspira pilosicoli isolated in Sweden between 1990 and 2010.

BACKGROUND: The anaerobic spirochetes Brachyspira hyodysenteriae and Brachyspira pilosicoli cause diarrheal diseases in pigs. Their fastidious nature has hampered standardization of methods for antimicrobial susceptibility testing. For monitoring of antimicrobial susceptibility wild type cutoff values are needed to define where the wild type distribution of MICs ends and no approved cutoffs are available for Brachyspira spp. In this study antimicrobial susceptibility data for both species (in total 906 isolates) were compiled and analyzed and wild type cut off values for B. hyodysenteriae proposed. METHODS: The MICs of tiamulin, valnemulin, tylosin, tylvalosin, doxycycline and lincomycin were determined by broth dilution in brain heart infusion broth supplemented with 10% fetal calf serum. RESULTS: The compiled MICs from the broth dilution tests of the B. hyodysenteriae type strain, B78T (ATCC® 27164T), showed that the method yields reproducible results. In an international perspective the frequencies of isolates with decreased antimicrobial susceptibility were low among both B. hyodysenteriae and B. pilosicoli. However, in B. pilosicoli a constant level of 10-15% isolates with tiamulin MICs >4 µg/ml was detected between 2002 and 2010 and in B. hyodysenteriae a gradual increase in tiamulin MICs was seen between 1990 and 2003 although this increase has ceased during the last years. The wild type cutoff values proposed for B. hyodysenteriae are: tiamulin >0.25 µg/ml, valnemulin >0.125 µg/ml, tylosin >16 µg/ml, tylvalosin >1 µg/ml, lincomycin >1 µg/ml and doxycycline >0.5 µg/ml. CONCLUSIONS: The broth dilution method used in this study has over the years generated tightly grouped MIC populations for the field isolates and reproducible results for the control strain B78T and is therefore a suitable antimicrobial susceptibility test method for monitoring of Brachyspira spp. Here we propose wild type cutoff values for six antimicrobial agents for B. hyodysenteriae tested by broth dilution based on MIC distributions and the current knowledge on mechanisms of resistance in this species. There are few studies on antimicrobial resistance mechanisms and MIC distributions in B. pilosicoli but to some extent the cutoff values proposed for B. hyodysenteriae may be applicable also for monitoring of antimicrobial susceptibility in B. pilosicoli.

Methicillin-resistant Staphylococcus pseudintermedius in Sweden.

Staphylococcus pseudintermedius is an opportunistic pathogen that is one of the most frequent causes of infections in dogs. In Europe, there are increasing reports of methicillin-resistant S. pseudintermedius (MRSP), and in Sweden, MRSP has also been more frequently isolated during recent years. However, there is limited knowledge regarding the epidemiology and genetic relationship among the Swedish isolates. This study therefore investigated the genetic relationship of MRSP isolated from companion animals in Sweden. In the study, MRSP isolates taken in the period January 2008-June 2010 from a total of 226 dogs and cats were characterized by spa typing, pulsed-field gel electrophoresis, and antimicrobial susceptibility testing. In addition, the geographical distribution of the isolates based on year of isolation and genetic typing was determined using a geographical information system. One multiresistant clonal lineage dominated among Swedish MRSP isolates, corresponding to the European winning lineage ST71-J-t02-SCCmec II-III. Furthermore, the geographical dissemination of MRSP corresponded to areas with high dog densities, centered on the three major cities in Sweden where the largest animal hospitals are situated.

The first nosocomial outbreak of methicillin-resistant Staphylococcus aureus in horses in Sweden.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) in animals is a rare finding in Sweden. In horses, MRSA was first detected in a screening survey in 2007. In 2008, six clinical cases occurred in an equine hospital, indicating an outbreak. METHOD: All MRSA isolates detected, 11 spa-type t011 and one t064 (n = 12), in infected horses (n = 10) and screening of horses (n = 2) in Sweden from December 2007 to March 2010 were retrospectively analysed with pulsed-field gel electrophoresis (PFGE) using Cfr9I and ApaI restriction enzymes, to study relationship between the isolates. Medical records of infected horses and outbreak investigation notes were scrutinised to monitor the clinical outcome and other aspects of the outbreak. RESULTS: Eight of the 10 infected horses were linked to one equine hospital and two to another hospital in the same region. The six horses infected with MRSA in 2008 underwent surgery during the period 22 May-7 July in one of the hospitals. Four more infections linked to the two hospitals were notified between 2009 and March 2010.Nine of the 11 spa-type t011 isolates had identical Cfr9I and ApaI PFGE pattern. All six infected horses from 2008 presented with this MRSA. Two t011 isolates differed in one and two bands, respectively, in PFGE.Nine horses suffered from surgical site infections (SSI). No antimicrobials were used following the MRSA diagnosis and the infections cleared. The time from surgery to MRSA diagnosis differed greatly between the horses (range 15-52 days). CONCLUSIONS: Association in time and space of six horses infected with an identical MRSA strain of spa-type t011 confirmed an outbreak. Two isolates found in 2009 and 2010 in the outbreak hospital were closely related to the outbreak strain, indicating one circulating strain. Both spa-type t011 and t064 have been reported in horses in Europe prior to these findings. The observation that the infections cleared although antimicrobials were not used is encouraging for future prudent use of antimicrobials. The time from surgery to bacteriological diagnosis was not acceptable in most cases, as contagious spread was a risk. Sampling when symptoms of infection are noticed and accurate analysis are thus important.