Emergence of methicillin resistance predates the clinical use of antibiotics.

The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus-a notorious human pathogen-appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two ß-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development.

Outbreak of Salmonella Typhimurium linked to Swedish pre-washed rocket salad, Sweden, September to November 2022.

In September 2022, the Public Health Agency of Sweden observed an increase in domestic Salmonella Typhimurium cases through the Swedish electronic notification system, and an outbreak strain was identified with whole genome sequencing. Overall, 109 cases with symptom onset between 17 September and 24 November 2022 were reported from 20 of 21 Swedish regions. The median age of cases was 52 years (range 4-87 years) and 62% were female. A case-control study found cases to be associated with consumption of rocket salad (adjusted odds ratio (aOR) = 4.9; 95% confidence interval (CI): 2.4-10, p value < 0.001) and bagged mixed salad (aOR = 4.0; 95% CI: 1.9-8.1, p value < 0.001). Trace-back, supported by Finnish authorities who identified the Swedish outbreak strain in a Finnish cluster during the same time period, identified rocket salad, cultivated, pre-washed and pre-packed in Sweden as the likely source of the outbreak. No microbiological analyses of rocket salad were performed. Our investigation indicates that bagged leafy greens such as rocket salad, regardless of pre-washing procedures in the production chain, may contain Salmonella and cause outbreaks, posing a health risk to consumers. We emphasise the need for primary producers of leafy greens to identify possible contamination points to prevent outbreaks.

Multicentre evaluation of a selective isolation protocol for detection of mcr-positive E. coli and Salmonella spp. in food-producing animals and meat.

This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr-positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre-enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr-1 or mcr-3 and mcr-4 or mcr-5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr-positive colistin-resistant E. coli or Salmonella spp. strains was 86% for CHROMID® Colistin R, 75% for CHROMagarTM COL-APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food-producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food-producing animals and food products in Europe.

Investigation of an international outbreak of multidrug-resistant monophasic Salmonella Typhimurium associated with chocolate products, EU/EEA and United Kingdom, February to April 2022.

An extensive multi-country outbreak of multidrug-resistant monophasic Salmonella Typhimurium infection in 10 countries with 150 reported cases, predominantly affecting young children, has been linked to chocolate products produced by a large multinational company. Extensive withdrawals and recalls of multiple product lines have been undertaken. With Easter approaching, widespread product distribution and the vulnerability of the affected population, early and effective real-time sharing of microbiological and epidemiological information has been of critical importance in effectively managing this serious food-borne incident.

Benzylpenicillin-producing Trichophyton erinacei and methicillin resistant Staphylococcus aureus carrying the mecC gene on European hedgehogs - A pilot-study.

BACKGROUND: A high carriage rate of methicillin-resistant Staphylococcus aureus with the mecC gene (mecC-MRSA) has been described among Wild European hedgehogs (Europeaus erineaus). Due to this frequent occurrence, it has been suggested that hedgehogs could be a natural reservoir for mecC-MRSA. However, the reason why hedgehogs carry mecC-MRSA remains unknown, but it has been hypothesized that mecC-MRSA could have evolved on the skin of hedgehogs due to the co-occurrence with antibiotic producing dermatophytes. The aim of this pilot-study was therefore to investigate if hedgehogs in Sweden carry Trichophyton spp. and to provide evidence that these dermatophytes are able to produce penicillin or similar substances. In addition, the study aimed to identify if dermatophytes co-occurred with mecC-MRSA. METHODS: Samples were collected from hedgehogs (Europeaus erineaus) that were euthanized or died of natural causes. All samples were screened for dermatophytes and mecC-MRSA using selective cultivation methods. Suspected isolates were characterized using PCR-based methods, genome sequencing and bioinformatic analyses. Identification of penicillin was performed by ultra-high-performance liquid chromatography-tandem mass spectrometry. RESULTS: In total 23 hedgehogs were investigated, and it was shown that two carried Trichophyton erinacei producing benzyl-penicillin, and that these hedgehogs also carried mecC-MRSA. The study also showed that 60% of the hedgehogs carried mecC-MRSA. CONCLUSION: The pilot-study demonstrated that Trichophyton erinacei, isolated from Swedish hedgehogs, can produce benzylpenicillin and that these benzylpenicillin-producing T. erinacei co-occurred with mecC-MRSA. The study also reconfirmed the high occurrence of mecC-MRSA among hedgehogs.

High level of multidrug-resistant Escherichia coli in young dairy calves in southern Vietnam.

This study investigated the occurrence of antimicrobial-resistant Escherichia coli in dairy calves in southern Vietnam. Fecal samples were taken directly from the rectum of 84 calves from 41 smallholder dairy farms, when newborn and at 14 days of age for isolation of E. coli. Escherichia coli strains were isolated from 144 of the 168 fecal samples tested. Of the 144 E. coli isolates, 40% were found to be susceptible to all 12 antimicrobial drugs tested and 53% of the E. coli isolates were resistant to at least three antimicrobials. Calves were colonized with antimicrobial-resistant E. coli already on the day of birth. Resistance to tetracycline was most common, followed by resistance to sulfamethoxazole, ampicillin, trimethoprim, and ciprofloxacin. Four isolates carried a gene encoding for extended-spectrum cephalosporinases (ESC), and these genes belonged to blaCTX-M group 1 (2 isolates), blaCTX-M group 9 (1 isolate), and blaCMY-2 (1 isolate). Thirty-three isolates had a plasmid-mediated quinolone resistance (PMQR) phenotype, and 30 of these carried the qnrS gene. These results are of importance for management routines of dairy cattle to prevent the spread of antimicrobial resistance.

Mobile colistin resistance gene mcr-5 in porcine Aeromonas hydrophila.

Objectives: To characterize the mobile colistin resistance gene mcr-5 in Aeromonas hydrophila from backyard pigs in rural areas of China. Methods: Pig faecal samples from 194 households were directly tested for the presence of mcr-5 by PCR assay and the phenotypic antimicrobial susceptibility profiles of the mcr-5-positive isolates were determined using the broth dilution method. The genomic location and transferability of mcr-5 were analysed by S1-PFGE with Southern blotting and DNA hybridization, and natural transformation, respectively. One strain isolated from an mcr-5-positive sample was subjected to WGS and the stability of the mcr-5-harbouring plasmid over successive generations was examined by subculturing. Results: One mcr-5-positive A. hydrophila isolate showing resistance, with a colistin MIC of 4 mg/L, was isolated from a backyard pig faecal sample. mcr-5 was located on a 7915 bp plasmid designated pI064-2, which could naturally transform into a colistin-susceptible A. hydrophila strain of porcine origin and mediated colistin resistance in both the original isolate and its transformants. The plasmid backbone (3790 bp) of pI064-2 showed 81% nucleotide sequence identity to the corresponding region of the ColE2-type plasmid pAsa1 from Aeromonas salmonicida, while similar replication primases are widely distributed among aeromonads, Enterobacteriaceae and Pseudomonas species. Conclusions: To the best of our knowledge, this is the first identification of the novel colistin resistance gene mcr-5 in an A. hydrophila isolate from the faeces of a backyard pig. mcr-5 is expected to be able to disseminate among different bacterial species and genera.

Occurrence and characterization of methicillin-resistant Staphylococcus pseudintermedius in successive parturitions of bitches and their puppies in two kennels in Italy.

BACKGROUND: Multi-drug methicillin-resistant Staphylococcus pseudintermedius (MRSP) detection is rapidly increasing in microbial specimens from pets across Europe. MRSP has also been isolated from bitches and newborns in dog breeding kennels. This study assessed whether MRSP lineage differs between breeding kennels and is maintained over time. Post-partum bitches (at day 3 vaginal and day 3, 9 and 35 milk samples) and their litters (at day 3, 9 and 35 oral and abdominal skin samples) from two Italian breeding kennels (A and B) were sampled and MRSP was subsequently characterized via whole-genome sequencing and antibiotic susceptibility testing. The study was carried out from October 2014 to March 2016 and included successive parturitions from the same animals. RESULTS: The analysis revealed different situations in both investigated kennels. In kennel A, circulating strains were from 7-locus sequence types ST688, ST258 and closely related isolates of ST71, which included most isolates. In kennel B, only a new isolate, ST772, was detected. In addition, most isolates from both kennels had multi-resistant antibiotic profiles. MRSP was only isolated from litters of MRSP-positive bitches, thus suggesting that bitch-litter transmission is likely. CONCLUSIONS: Our data show that MRSP circulation can differ in different settings, that several clonal lineages can circulate together, and that vertical transmission appears common. MRSP colonization did not affect the health conditions of the bitches or of their litters.

Occurrence of blaKPC-2, blaCTX-M, and mcr-1 in Enterobacteriaceae from Well Water in Rural China.

We report on the coexistence of mcr-1 and blaCTX-M in multidrug-resistant, extended-spectrum ß-lactamase-producing Escherichia coli belonging to the sequence type 10 complex isolated from well water in rural China. Raoultella ornithinolytica with blaKPC-2 was also detected in well water from the same area. This study shows that genes coding for resistance to last-resort antibiotics are present in wells in rural China, indicating a potential source of antibiotic resistance.

Community carriage of ESBL-producing Escherichia coli is associated with strains of low pathogenicity: a Swedish nationwide study.

OBJECTIVES: Community carriage of ESBL-producing Escherichia coli (EPE) is common worldwide and there is a need to understand the connection between carriage and infection. We compared the molecular characteristics of EPE among Swedish community carriers with those of EPE causing invasive infections. METHODS: We collected 2134 faecal samples from randomly selected Swedish inhabitants and examined them for the presence of EPE. All participating volunteers answered a questionnaire about putative risk factors for EPE carriage. Suspected EPE isolates (n = 418) from patients with bloodstream infection (BSI) were collected from Swedish laboratories. Isolates were genotypically and phenotypically characterized. RESULTS: Our results show that the EPE population found in carriers generally had lower pathogenicity compared with the isolates from BSIs, since carriers had a lower proportion of E. coli belonging to phylogroup B2, ST131 and ST131 subclone H30-Rx. Isolates from carriers also had lower levels of multiresistance. The Swedish carriage rate of EPE was 4.7% (101/2134) among healthy volunteers. Risk factors associated with carriage were travel to countries in Asia (OR = 3.6, 95% CI = 1.4-9.2) and Africa (OR = 3.6, 95% CI = 1.7-7.7) and a diet without pork (OR = 0.5, 95% CI = 0.3-0.8 for pork eaters). CONCLUSIONS: E. coli host factors previously associated with higher pathogenicity were all more common in BSIs compared with carriers. This indicates that the risk of invasive infection with EPE may be relatively modest in many community carriers and that EPE carriage of high-risk strains should be the focus of attention for prevention.

Limited Dissemination of Extended-Spectrum ß-Lactamase- and Plasmid-Encoded AmpC-Producing Escherichia coli from Food and Farm Animals, Sweden.

Extended-spectrum ß-lactamase (ESBL)- and plasmid-encoded ampC (pAmpC)-producing Enterobacteriaceae might spread from farm animals to humans through food. However, most studies have been limited in number of isolates tested and areas studied. We examined genetic relatedness of 716 isolates from 4,854 samples collected from humans, farm animals, and foods in Sweden to determine whether foods and farm animals might act as reservoirs and dissemination routes for ESBL/pAmpC-producing Escherichia coli. Results showed that clonal spread to humans appears unlikely. However, we found limited dissemination of genes encoding ESBL/pAmpC and plasmids carrying these genes from foods and farm animals to healthy humans and patients. Poultry and chicken meat might be a reservoir and dissemination route to humans. Although we found no evidence of clonal spread of ESBL/pAmpC-producing E. coli from farm animals or foods to humans, ESBL/pAmpC-producing E. coli with identical genes and plasmids were present in farm animals, foods, and humans.

Isolation of coagulase-positive staphylococci from bitches' colostrum and milk and genetic typing of methicillin-resistant Staphylococcus pseudintermedius strains.

BACKGROUND: Among the coagulase-positive, potentially pathogenic staphylococci, Staphylococcus pseudintermedius has been frequently isolated from bitches' milk. This organism colonizes the mammary gland or causes infection, while S. aureus has been only occasionally reported. The objective of this study was to investigate the occurrence and persistence of coagulase-positive staphylococci in the colostrum and milk of postpartum bitches, either treated or untreated with antimicrobials, and to assess the incidence, antibiotic resistance profile and genetic type of the methicillin-resistant strains. On postpartum D1, D7 and D15, drops of secretion were collected from the mammary glands of 27 postpartum bitches, nine of which were treated with antimicrobials. Coagulase-positive staphylococci were identified, antimicrobial susceptibility and the presence of mecA were tested and the genetic profile of methicillin-resistant strains was assessed. RESULTS: Staphylococcus pseudintermedius was the only coagulase-positive staphylococcus isolated, and its presence was detected in 21 out of 27 bitches and in 66 out of 145 swabs. In a single bitch, it caused puerperal mastitis. In untreated bitches, the frequency of isolation was lower in colostrum than in milk. All of the isolates except one were resistant to at least three antimicrobial classes, while 14 out of 66 S. pseudintermedius strains were methicillin-resistant mecA positive (MRSP) and were isolated from eight bitches housed in the same breeding kennel. A significant association was found between antimicrobial treatment and the presence of MRSP. Six of the 12 typed isolates belonged to spa-type t02 carrying SCCmec II/III, and another six were non-typeable with spa carrying SCCmec IV. The t02-SCCmec II/III isolates were sequence type (ST) 71; four NT-SCCmec IV isolates were ST258 and two were ST369. PFGE showed that isolates from the same dog had identical band patterns, while isolates from different dogs had unique band patterns. MRSP strains showed multidrug resistance profiles. CONCLUSIONS: Our results confirm that S. pseudintermedius is the most frequently isolated coagulase-positive staphylococcus from bitches' milk. The isolation of several different strains of MRSP with different genetic characteristics in the same kennel and the fact that two of the strains belonged to a sequence type (ST) described for the first time are noteworthy findings.

Cluster of infections caused by methicillin-resistant Staphylococcus pseudintermedius in humans in a tertiary hospital.

The dog-associated Staphylococcus pseudintermedius is a rare pathogen in humans. Here we describe a cluster of infections caused by the methicillin-resistant S. pseudintermedius clone ST71-J-t02-II-III. It involved four elderly patients at a tertiary hospital. Three patients had wound infections, and the strain had a tendency to cause bullous skin lesions.

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.

Frequent occurrence of extended-spectrum beta-lactamase- and transferable ampc beta-lactamase-producing Escherichia coli on domestic chicken meat in Sweden.

Forty-four percent of Swedish chicken meat fillets were contaminated with extended-spectrum or transferable AmpC beta-lactamase-producing Escherichia coli strains. Isolates from Swedish chicken meat and broilers were closely related to isolates from chicken meat imported into Sweden; these results indicate a common source of the contamination.

An investigation of household dogs as the source in a case of human bacteraemia caused by Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is a commensal and an opportunistic pathogen in dogs, and is also an opportunistic pathogen in humans. Here we report about a case of bacteraemia with a fatal outcome in a 77-year-old co-morbid male likely caused by a S. pseudintermedius and the investigation into the possible transmission from the two dogs in the patient's household. The two dogs carried the same S. pseudintermedius strain, but this dog strain was unrelated to the strain from the patient. In contrast to the patient strain, the dog strain showed reduced susceptibility to several antibiotics and both dogs had received antibiotic treatment prior to sampling. So, it is conceivable that these treatments can have eliminated the patient's strain between the transmission event and the dog sampling. It is also worth noting that the patient strain was positive for the expA gene, which encodes an exfoliative toxin closely related to the S. aureus exfoliative toxin B. This toxin has been linked to canine pyoderma, but its effect on humans remains unknown. Transmission of S. pseudintermedius was confirmed in the household between the dogs. However, we could not verify that the dogs were the source for the S. pseudintermedius in the patient.

Dissemination of carbapenemase-producing Enterobacterales through wastewater and gulls at a wastewater treatment plant in Sweden.

Here we report the detection of carbapenemase-producing Enterobacterales (CPE) isolated from Swedish wastewater and gull faeces. CPE have not been detected in samples from animals in Sweden preceding this report. Sampling of wastewater treatment plant (WWTP) inlet and outlet, sedimentation basins, surface seawater from key aquatic bird habitats and freshly deposited gull faeces was done on six separate occasions during May to September 2021. Following broth enrichment, selective screening of putative CPE was performed on mSuperCarba™ (CHROMagar). Species identification was done with MALDI-TOF. Antimicrobial susceptibility testing was performed according to EUCAST. In total, seventeen CPE were verified by genome sequencing carrying blaGES-5, blaIMI-3, blaOXA-181 or blaOXA-244. The blaGES-5 was carried on IncP plasmids in four different species; Escherichia coli ST10 isolated from WWTP outlet, Raoultella ornithinolytica isolated from WWTP inlet, outlet and sedimentation basins as well as gull faeces collected at the WWTP and Klebsiella spp. isolates from WWTP inlet and outlet. The genetic environment surrounding blaGES-5 was similar in two Citrobacter freundii causing human infections. The blaIMI-3 was carried on IncFII(Yp) plasmids in four Enterobacter ludwigii, isolated from WWTP outlet and gull faeces collected at a recreational city park 2 km from the WWTP. The blaOXA-181 was located on a COLKP3 plasmid found in an E. coli, while blaOXA-244 was chromosomally located in an E. coli ST10, both isolated from WWTP inlet. Phylogenetic analysis of R. ornithinolytica and E. ludwigii isolates indicate that the gulls carried strains related to those identified in the WWTP samples. The results thus add to the increasing evidence of WWTPs as anthropogenic reservoirs for mobile genetic elements with antibiotic-resistance functionality. Such environments could profoundly impact the dissemination and spread of such genetic elements via for example aquatic birds, thereby warranting further study and surveillance.

Rapid increase in occurrence of carbapenem-resistant Enterobacteriaceae in healthy rural residents in Shandong Province, China, from 2015 to 2017.

OBJECTIVES: The global increase in carbapenem-resistant Enterobacteriaceae (CRE) is a growing health concern. Infections caused by CRE are associated with increased mortality and length of hospital stay, emphasising the health and economic burden posed by these pathogens. Although CRE can inhabit the human gut asymptomatically, colonisation with CRE is associated with an increased risk of CRE infection and mortality. In this study, we investigated the occurrence and characteristics of CRE in faecal samples from healthy persons in 12 villages in Shandong Province, China. METHODS: Screening for CRE in faecal samples was performed by selective cultivation. Minimum inhibitory concentrations (MICs) of meropenem were determined by the agar dilution method. Multilocus sequence typing (MLST) and carbapenemase gene carriage of the isolates were determined by whole-genome sequencing. Genetic relatedness of Escherichia coli isolates was determined by core genome MLST. RESULTS: CRE carriage increased from 2.4% in 2015 to 13.4% in 2017. Most CRE isolates (93.0%) were E. coli and all carried NDM-type carbapenemases. Sequence types (STs) among the E. coli isolates were diverse. The single most common ST was the highly epidemic strain ST167, which was only observed in 2017. CONCLUSION: We report a rapid increase in occurrence of CRE (from 2.4% to 13.4%) among faecal samples collected from healthy rural residents of Shandong Province from 2015 to 2017. Colonisation with CRE is known to increase the risk of CRE infection, and the worrying deterioration of the epidemiological situation in the region reported here indicates a need for further monitoring and possible interventions.

Detection of an IMI-2 carbapenemase-producing Enterobacter asburiae at a Swedish feed mill.

Occurrence of multidrug resistant Enterobacteriaceae in livestock is of concern as they can spread to humans. A potential introduction route for these bacteria to livestock could be animal feed. We therefore wanted to identify if Escherichia spp., Enterobacter spp., Klebsiella spp., or Raoutella spp. with transferable resistance to extended spectrum cephalosporins, carbapenems or colistin could be detected in the environment at feed mills in Sweden. A second aim was to compare detected isolates to previous described isolates from humans and animals in Sweden to establish relatedness which could indicate a potential transmission between sectors and feed mills as a source for antibiotic resistant bacteria. However, no isolates with transferable resistance to extended-cephalosporins or colistin could be identified, but one isolate belonging to the Enterobacter cloacae complex was shown to be carbapenem-resistant and showing carbapenemase-activity. Based on sequencing by both short-read Illumina and long-read Oxford Nanopore MinIon technologies it was shown that this isolate was an E. asburiae carrying a bla IMI-2 gene on a 216 Kbp plasmid, designated pSB89A/IMI-2, and contained the plasmid replicons IncFII, IncFIB, and a third replicon showing highest similarity to the IncFII(Yp). In addition, the plasmid contained genes for various functions such as plasmid segregation and stability, plasmid transfer and arsenical transport, but no additional antibiotic resistance genes. This isolate and the pSB89A/IMI-2 was compared to three human clinical isolates positive for bla IMI-2 available from the Swedish antibiotic monitoring program Swedres. It was shown that one of the human isolates carried a plasmid similar with regards to gene content to the pSB89A/IMI-2 except for the plasmid transfer system, but that the order of genes was different. The pSB89A/IMI-2 did however share the same transfer system as the bla IMI-2 carrying plasmids from the other two human isolates. The pSB89A/IMI-2 was also compared to previously published plasmids carrying bla IMI-2, but no identical plasmids could be identified. However, most shared part of the plasmid transfer system and DNA replication genes, and the bla IMI-2 gene was located next the transcription regulator imiR. The IS3-family insertion element downstream of imiR in the pSB89A was also related to the IS elements in other bla IMI-carrying plasmids.