Corrigendum: Rapid typing of Klebsiella pneumoniae and Pseudomonas aeruginosa by Fourier-transform Infrared spectroscopy informs infection control in veterinary settings.

[This corrects the article DOI: 10.3389/fmicb.2024.1334268.].

Rapid typing of Klebsiella pneumoniae and Pseudomonas aeruginosa by Fourier-transform Infrared spectroscopy informs infection control in veterinary settings.

Introduction: The emergence of multi-drug resistant (MDR) pathogens linked to healthcare-associated infections (HCAIs) is an increasing concern in modern veterinary practice. Thus, rapid bacterial typing for real-time tracking of MDR hospital dissemination is still much needed to inform best infection control practices in a clinically relevant timeframe. To this end, the IR Biotyper using Fourier-Transform InfraRed (FTIR) spectroscopy has the potential to provide fast cluster analysis of potentially related organisms with substantial cost and turnaround time benefits. Materials and methods: A collection of MDR bacterial isolates (n = 199, comprising 92 Klebsiella pneumoniae and 107 Pseudomonas aeruginosa) obtained from companion animal (i.e., dogs, cats and horses) clinical investigations, faecal and environmental screening from four veterinary facilities between 2012 and 2019 was analysed retrospectively by FTIR spectroscopy. Its performance was compared against MLST extracted from whole genomes of a subset of clustering isolates (proportionally to cluster size) for investigation of potential nosocomial transmission between patients and the surrounding hospital environments. Results: Concordance between the FTIR and MLST types was overall high for K. pneumoniae (Adjusted Rand Index [ARI] of 0.958) and poor for P. aeruginosa (ARI of 0.313). FTIR K. pneumoniae clusters (n = 7) accurately segregated into their respective veterinary facility with evidence of intra-hospital spread of K. pneumoniae between patients and environmental surfaces. Notably, K. pneumoniae ST147 intensely circulated at one Small Animal Hospital ICU. Conversely, Pseudomonas aeruginosa FTIR clusters (n = 18) commonly contained isolates of diversified hospital source and heterogeneous genetic background (as also genetically related isolates spread across different clusters); nonetheless, dissemination of some clones, such as P. aeruginosa ST2644 in the equine hospital, was apparent. Importantly, FTIR clustering of clinical, colonisation and/or environmental isolates sharing genomically similar backgrounds was seen for both MDR organisms, highlighting likely cross-contamination events that led to clonal dissemination within settings. Conclusion: FTIR spectroscopy has high discriminatory power for hospital epidemiological surveillance of veterinary K. pneumoniae and could provide sufficient information to support early detection of clonal dissemination, facilitating implementation of appropriate infection control measures. Further work and careful optimisation need to be carried out to improve its performance for typing of P. aeruginosa veterinary isolates.

Intervention with impact: Reduced isolation of methicillin-resistant Staphylococcus pseudintermedius from dogs following the introduction of antimicrobial prescribing legislation in Germany.

BACKGROUND: Legislation was introduced in Germany in 2018, requiring bacterial culture and antimicrobial susceptibility testing before the prescription of fluoroquinolones and third-generation cephalosporins to dogs. We hypothesised that, following this intervention, the number of clinical samples testing positive for methicillin-resistant Staphylococcus pseudintermedius (MRSP) would reduce. METHODS: Reports of S. pseudintermedius isolated from canine clinical samples by three German veterinary diagnostic microbiology laboratories during the 38 months before the introduction of the legislation and the 46 months after were compared. Bacterial identification was performed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and antimicrobial susceptibility testing followed recognised recommendations but with changes during the study period. RESULTS: Among a total of 120,571 S. pseudintermedius isolates, MRSP accounted for 7.1% overall. Following the legislative intervention, monthly submissions yielding S. pseudintermedius increased at all three laboratories. The MRSP percentage was lower in the period after the intervention in two of the three laboratories (p < 0.001); in the third laboratory, there was no change between periods, but a year-on-year reduction in MRSP percentages occurred after the intervention (p = 0.0004). LIMITATIONS: Changing susceptibility testing methods limited the direct comparison of resistance patterns among laboratories. CONCLUSION: The reduction in MRSP in canine clinical samples following the introduction of this legislation suggests a positive impact of compulsory laboratory testing on reducing antimicrobial resistance.

Dissemination of blaNDM-5-carrying IncX3-type plasmid among non-clonal Escherichia coli strains colonising a dog with a skin infection caused by a carbapenem-resistant Klebsiella pneumoniae, United Kingdom.

A successful outcome of a post-surgical wound infection management by a carbapenem-resistant Klebsiella pneumoniae is described in a dog. Four multidrug-resistant and carbapenem-resistant Escherichia coli strains belonging to ST410 (n = 1) and ST648 (n = 3) were isolated from faecal samples and nasal swabs of this dog at admission to a veterinary hospital in the United Kingdom, and one month after discharge. Whole-genome sequencing analysis suggests dissemination of a 46,161-bp IncX3 blaNDM-5-carrying plasmid among E. coli strains from the different lineages. In this study, the E. coli ST648 strains were virtually identical to each other (5 SNPs difference) indicating dissemination and persistence of this clone over time and across different anatomical sites in the same dog maybe due to the prolonged antimicrobial therapy. The carbapenemase carrying plasmid also showed homology with other publicly available plasmid sequences from Asian countries. These results suggests that plasmids may be a major vehicle in mediating the dissemination of carbapenem-resistance. Further studies investigating the selection and flow of plasmids carrying important resistance genes amongst companion animals are needed as it may further contaminate other environments posing a threat to public health.

Longitudinal study of ESBL/AmpC-producing Enterobacterales strains sharing between cohabiting healthy companion animals and humans in Portugal and in the United Kingdom.

Extended-spectrum beta-lactamase (ESBL)- and plasmid-mediated cephalosporinase (AmpC)-producing Enterobacterales (ESBL/AmpC-E) are an increasing healthcare problem in both human and veterinary medicine. The aim of this study was to investigate the possible sharing of ESBL/AmpC-E strains between healthy companion animals and humans of the same household in Portugal (PT) and the United Kingdom (UK). In a prospective longitudinal study, between 2018 and 2020, faecal samples were collected from healthy dogs (n=90), cats (n=20) and their cohabiting humans (n=119) belonging to 41 PT and 44 UK households. Samples were screened for the presence of ESBL/AmpC-E and carbapenemase-producing bacteria. Clonal relatedness between animal and human strains was established by using REP-PCR fingerprinting method, followed by whole-genome sequencing (WGS) of selected strains. ESBL/AmpC-E strains were detected in companion animals (PT=12.7%, n=8/63; UK=8.5%, n=4/47) and humans (PT=20.7%, n=12/58; UK=6.6%, n=4/61) in at least one timepoint. REP-PCR identified paired multidrug-resistant ESBL/AmpC-producing Escherichia coli strains from companion animals and owners in two Portuguese households (4.8%) and one UK household (2.3%). WGS analysis of nine E. coli strains from these three households confirmed that interhost sharing occurred only between the two animal-human pairs from Portugal. Three shared strains were identified: one CTX-M-15-producing E. coli strain in a cat-human pair (O15-H33-ST93) and two CTX-M-15- and CTX-M-55/CMY-2-producing E. coli strains, in a dog-human pair (O8:H9-ST410 and O11:H25-ST457, respectively) at different timepoints. These E. coli clonal lineages are human pandemic, highlighting the role of companion animals living in close contact with humans in the dissemination and persistence of antimicrobial resistance in the household environment.

mcr-1 colistin resistance gene sharing between Escherichia coli from cohabiting dogs and humans, Lisbon, Portugal, 2018 to 2020.

BackgroundThe emergence of colistin resistance is a One Health antimicrobial resistance challenge worldwide. The close contact between companion animals and humans creates opportunities for transmission and dissemination of colistin-resistant bacteria.AimTo detect potential animal reservoirs of colistin-resistant Escherichia coli and investigate the possible sharing of these bacteria between dogs, cats and their cohabiting humans in the community in Lisbon, Portugal.MethodsA prospective longitudinal study was performed from 2018 to 2020. Faecal samples from dogs and cats either healthy or diagnosed with a skin and soft tissue or urinary tract infection, and their cohabiting humans were screened for the presence of colistin-resistant E. coli. All isolates were tested by broth microdilution against colistin and 12 other antimicrobials. Colistin-resistant isolates were screened for 30 resistance genes, including plasmid-mediated colistin resistance genes (mcr-1 to mcr-9), and typed by multilocus sequence typing. Genetic relatedness between animal and human isolates was analysed by whole genome sequencing.ResultsColistin-resistant E. coli strains harbouring the mcr-1 gene were recovered from faecal samples of companion animals (8/102; 7.8%) and humans (4/125; 3.2%). No difference between control and infection group was detected. Indistinguishable multidrug-resistant E. coli ST744 strains harbouring the mcr-1 gene were found in humans and their dogs in two households.ConclusionsThe identification of identical E. coli strains containing the plasmid-mediated mcr-1 gene in companion animals and humans in daily close contact is of concern. These results demonstrate the importance of the animal-human unit as possible disseminators of clinically important resistance genes in the community setting.

Investigation of In Vitro Susceptibility and Resistance Mechanisms in Skin Pathogens: Perspectives for Fluoroquinolone Therapy in Canine Pyoderma.

Fluoroquinolones (FQ) are commonly used in dogs with bacterial skin infections. Their use as first choice, along with the increased incidence of FQ-resistance, represents a risk to animal and public health. Our study determined minimum inhibitory (MIC) and bactericidal (MBC) concentrations of five FQs in Staphylococcus aureus, Staphylococcus pseudintermedius, and Escherichia coli, together with FQ-resistance mechanisms. MICs, efflux pump (EP) overexpression and MBCs were measured in 249 skin infection isolates following CLSI guidelines (CLSI VET01-A4, CLSI M26-A). Chromosomal and plasmid-mediated resistance genes were investigated after DNA extraction and sequencing. FQ-resistance was detected in 10% of methicillin-susceptible (MS), 90% of methicillin-resistant (MR) staphylococci and in 36% of E. coli. Bactericidal effect was observed except in 50% of MRSA/P for ciprofloxacin and in 20% of MRSPs for enrofloxacin. Highest MICs were associated with double mutation in gyrA (Ser83Leu + Asp87Asn), efflux pumps and three PMQR genes in E. coli, and grlA (Ser80Phe + Glu84Lys) in S. aureus. EP overexpression was high among E. coli (96%), low in S. aureus (1%) and absent in S. pseudintermedius. Pradofloxacin and moxifloxacin showed low MICs with bactericidal effect. Since in vitro FQ resistance was associated with MR, FQ use should be prudently guided by susceptibility testing.

The nose is not enough: Multi-site sampling is best for MRSP detection in dogs and households.

BACKGROUND: Following recovery from meticillin-resistant Staphylococcus pseudintermedius (MRSP) infection of any type, dogs may continue to carry MRSP asymptomatically on skin and mucosae, contributing to the spread of this multidrug-resistant, veterinary hospital-associated pathogen with zoonotic potential to others and into the environment. OBJECTIVES: This study determined which canine anatomic and household environmental sites are most sensitive for sampling to identify carriage and contamination. METHODS AND MATERIALS: Fifty-one dogs and 22 households, MRSP-positive on at least one tested site, were sampled on 132 and 40 occasions over time, respectively. Dogs were swabbed at six sites (mouth, nose, conjunctiva, skin, prepuce/vulva, perianal area); household environments were sampled using contact plates (mannitol salt agar [MSA] and MSA + 6 mg/L oxacillin [MS+]) on five sites. MRSP was isolated after enrichment, grown on MSA/MS+ and was confirmed by PCR. Generalized estimating equations were used for calculation of sensitivity (95% confidence interval) for each site/combination. RESULTS: Each anatomical and environmental site yielded MRSP at least once. MRSP was isolated from only a single site in 27.3% of dogs, with the buccal mucosa showing the highest sensitivity (63.8%). Multi-site sampling of a minimum of four canine anatomical or four environmental sites, respectively, was needed to achieve >95% sensitivity. CONCLUSIONS AND CLINICAL RELEVANCE: The canine buccal mucosa should be included in MRSP sampling protocols, ideally in addition to at least three other anatomical sites. Likewise, environment sampling should be of multiple household sites in cases where it is used as a part of clinical case management.

Staphylococcus aureus lineages associated with a free-ranging population of the fruit bat Pteropus livingstonii retained over 25 years in captivity.

Conservation of endangered species has become increasingly complex, and costly interventions to protect wildlife require a robust scientific evidence base. This includes consideration of the role of the microbiome in preserving animal health. Captivity introduces stressors not encountered in the wild including environmental factors and exposure to exotic species, humans and antimicrobial drugs. These stressors may perturb the microbiomes of wild animals, with negative consequences for their health and welfare and hence the success of the conservation project, and ultimately the risk of release of non-native organisms into native ecosystems. We compared the genomes of Staphylococcus aureus colonising critically endangered Livingstone's fruit bats (Pteropus livingstonii) which have been in a captive breeding programme for 25 years, with those from bats in the endemic founder population free ranging in the Comoros Republic. Using whole genome sequencing, we compared 47 isolates from captive bats with 37 isolates from those free ranging in the Comoros Republic. Our findings demonstrate unexpected resilience in the bacteria carried, with the captive bats largely retaining the same two distinctive lineages carried at the time of capture. In addition, we found evidence of genomic changes which suggest specific adaptations to the bat host.

Hostage to history - questioning the duration of systemic antimicrobial therapy for the treatment of canine superficial bacterial folliculitis.

Current guidelines for the use of systemic antimicrobials for the treatment of superficial bacterial folliculitis in dogs include the recommendation that the disease be treated for a minimum of 3 weeks and for at least 1 week beyond clinical resolution. With increasing antimicrobial resistance being noted for bacteria involved in this condition, as well as the increased use of evidence-based medicine, this dogma needs to be reevaluated.

Effect of topical antimicrobial therapy and household cleaning on meticillin-resistant Staphylococcus pseudintermedius carriage in dogs.

BACKGROUND: Meticillin-resistant Staphylococcus pseudintermedius (MRSP) is a multidrug-resistant canine pathogen with a low zoonotic potential. This study investigated MRSP carriage and clearance through topical antimicrobial therapy and household cleaning in dogs recovered from MRSP infection. METHODS: Dogs were swabbed for MRSP carriage; household contamination was assessed using contact plates. Carrier dogs were allocated randomly to receive topical fusidic acid and chlorhexidine/miconazole treatment combined with owners implementing a household hygiene protocol (H&T) or implementation of hygiene alone (H) over three weeks. Carriage-negative dogs were monitored monthly. The relatedness of isolates over time was investigated by pulsed-field gel electrophoresis (PFGE). RESULTS: At inclusion, MRSP carriage was confirmed in 31/46 (67.4%) index dogs and 16/24 (66.7%) contact dogs, and contamination was found in 18/40 (45%) environments. In dogs completing all cycles, interventions cleared carriage in 5/9 (55.6%) dogs in group H&T and 2/6 (33.3%) in group H. Environmental contamination was infrequent but associated with carrier dogs (p = 0.047). Monthly monitoring of initially negative dogs showed intermittent carriage in 9/14 dogs. PFGE-concordance was found among all 34 MRSP isolated from eight index dogs over time. CONCLUSION: MRSP carriage was common in dogs after recovery from infection. Topical antimicrobial therapy temporarily eliminated carriage but recurrence was frequent. Management efforts must include the prevention of recurrent infections and hygiene.

Canine pyoderma: mecA persists autogenous bacterin formulation from meticillin-resistant Staphylococcus pseudintermedius (MRSP) and S. aureus (MRSA).

OBJECTIVE: Autogenous Staphylococcus pseudintermedius bacterins can reduce prescribing of antimicrobials in the management of canine recurrent pyoderma. However, increasing prevalence of meticillin-resistant, mecA-positive S. pseudintermedius (MRSP) raises concern over dispersal of mecA through bacterin therapy. We investigated the presence and integrity of mecA in bacterin formulations after manufacturing. MATERIAL AND METHODS: Twenty clinical isolates (12 MRSP, 7 MR-S. aureus, 1 meticillin-susceptible SP) were investigated. Pellets from overnight growth were washed 3 times with 0.5 % phenol saline, followed by addition of 0.1 ml 10 % formal-saline to 10 ml phenol-saline. Sterility was confirmed, and DNA extracted using both a standard genomic extraction kit and one recommended for formalin-fixed tissue samples (FFPE). The presence of mecA was determined after PCR and its integrity examined in 5 randomly selected samples after sequencing. RESULTS: In all bacterins from meticillin-resistant isolates, mecA was detected following FFPE extraction; products aligned fully to a reported mecA sequence. After standard DNA extraction, mecA was seen in 16/19 samples. CONCLUSION: Persistence of mecA in MRSP bacterins suggests that dispersal of this important resistance mediator through therapy may be possible. While the ability of skin bacteria to uptake naked DNA remains unclear, it seems prudent to only formulate autogenous bacterins from mecA-negative S. pseudintermedius to avoid unnecessary spread of mecA.

Malassezia otitis unresponsive to primary care: outcome in 59 dogs.

BACKGROUND: Otitis externa (OE) is a common disorder in dogs. Infection by the commensal yeast, Malassezia pachydermatis, may result in chronic disease that does not respond to standard primary care. Chronic infectious OE may be associated with otitis media (OM). HYPOTHESIS/OBJECTIVE: To report medical management, clinical outcomes and frequency of middle ear involvement, in dogs with Malassezia otitis unresponsive to primary care. ANIMALS: Fifty-nine dogs from one referral veterinary hospital from January 2007 to September 2018. METHODS AND MATERIALS: Retrospective analysis of medical records of dogs referred with chronic otitis and treated for Malassezia otitis at a referral veterinary hospital. RESULTS: Chronic Malassezia OE was treated successfully in 91% of ears, in 87% of these cases with one ear flush intervention. Median time-to-resolution was 27 days after ear flush intervention. Neither duration of otitis, presence of neutrophils in aural discharge nor administration of oral itraconazole affected clinical outcome. Malassezia OM occurred concurrently in 17% of ears. CONCLUSIONS AND CLINICAL RELEVANCE: These findings assist clinicians and carers of affected dogs in decision-making, by documenting that most cases of canine Malassezia otitis that have not resolved with standard primary care, can be treated successfully with a well-staged and intense medical treatment plan. Malassezia OM should be suspected to occur concurrently in around a fifth of affected ears.

Fatal exudative dermatitis in island populations of red squirrels (Sciurus vulgaris): spillover of a virulent Staphylococcus aureus clone (ST49) from reservoir hosts.

Fatal exudative dermatitis (FED) is a significant cause of death of red squirrels (Sciurus vulgaris) on the island of Jersey in the Channel Islands where it is associated with a virulent clone of Staphylococcus aureus, ST49. S. aureus ST49 has been found in other hosts such as small mammals, pigs and humans, but the dynamics of carriage and disease of this clone, or any other lineage in red squirrels, is currently unknown. We used whole-genome sequencing to characterize 228 isolates from healthy red squirrels on Jersey, the Isle of Arran (Scotland) and Brownsea Island (England), from red squirrels showing signs of FED on Jersey and the Isle of Wight (England) and a small number of isolates from other hosts. S. aureus was frequently carried by red squirrels on the Isle of Arran with strains typically associated with small ruminants predominating. For the Brownsea carriage, S. aureus was less frequent and involved strains associated with birds, small ruminants and humans, while for the Jersey carriage S. aureus was rare but ST49 predominated in diseased squirrels. By combining our data with publicly available sequences, we show that the S. aureus carriage in red squirrels largely reflects frequent but facile acquisitions of strains carried by other hosts sharing their habitat ('spillover'), possibly including, in the case of ST188, humans. Genome-wide association analysis of the ruminant lineage ST133 revealed variants in a small number of mostly bacterial-cell-membrane-associated genes that were statistically associated with squirrel isolates from the Isle of Arran, raising the possibility of specific adaptation to red squirrels in this lineage. In contrast there is little evidence that ST49 is a common carriage isolate of red squirrels and infection from reservoir hosts such as bank voles or rats, is likely to be driving the emergence of FED in red squirrels.

Non-neoplastic anal sac disorders in UK dogs: Epidemiology and management aspects of a research-neglected syndrome.

BACKGROUND: Non-neoplastic anal sac disorders (ASD) are frequent presentations for dogs in primary-care practice but evidence-based information on disease occurrence and risk is sparse. This study estimates prevalence, breed associations and other risk factors as well as reporting on clinical management. METHODS: A cohort study of dogs attending VetCompass practices between 1 January 2013 and 31 December 2013. Risk factor analysis used multivariable logistic regression methods. RESULTS: Of 104,212 dogs attending 110 UK practices, the 1-year period prevalence of ASD was 4.40% (95% CI: 4.22-4.57). Compared to crossbreds, six breeds showed increased odds of ASD (Cavalier King Charles spaniel, King Charles spaniel, Cockapoo, Shih-tzu, Bichon Frise and Cocker spaniel), and six breeds showed reduced odds (Labrador Retriever, Border collie, Staffordshire Bull Terrier, Lurcher, German Shepherd Dog and Boxer). Brachycephalic types had 2.6 times the odds for ASD compared to dolichocephalic types. Medication prescribed for ASD included antimicrobials (n = 480, 20.24%) and analgesics (n = 284, 11.97%). Anal sacculectomy was performed in under 1% of cases. CONCLUSIONS: High prevalence, strong breed predispositions and evidence of severity suggested from the antimicrobial and analgesic therapies combined with current substantial knowledge gaps identify ASD as a key research-neglected syndrome in dogs.

Genes on the Move: In Vitro Transduction of Antimicrobial Resistance Genes between Human and Canine Staphylococcal Pathogens.

Transmission of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) between people and pets, and their co-carriage, are well-described. Potential exchange of antimicrobial resistance (AMR) genes amongst these staphylococci was investigated in vitro through endogenous bacteriophage-mediated transduction. Bacteriophages were UV-induced from seven donor isolates of canine (MRSP) and human (MRSA) origin, containing tet(M), tet(K), fusB or fusC, and lysates filtered. Twenty-seven tetracycline- and fusidic acid- (FA-) susceptible recipients were used in 122 donor-recipient combinations (22 tetracycline, 100 FA) across 415 assays (115 tetracycline, 300 FA). Bacteriophage lysates were incubated with recipients and presumed transductants quantified on antimicrobial-supplemented agar plates. Tetracycline resistance transduction from MRSP and MRSA to methicillin-susceptible S. pseudintermedius (MSSP) was confirmed by PCR in 15/115 assays. No FA-resistance transfer occurred, confirmed by negative fusB/fusC PCR, but colonies resulting from FA assays had high MICs (≥32 mg/L) and showed mutations in fusA, two at a novel position (F88L), nine at H457[Y/N/L]. Horizontal gene transfer of tetracycline-resistance confirms that resistance genes can be shared between coagulase-positive staphylococci from different hosts. Cross-species AMR transmission highlights the importance of good antimicrobial stewardship across humans and veterinary species to support One Health.