Antimicrobial resistance-Do we share more than companionship with our dogs?

AIMS: To investigate and compare antimicrobial resistance genes (ARGs) in faeces from cohabiting dogs and owners. METHODS AND RESULTS: DNA from faecal samples from 35 dogs and 35 owners was screened for the presence of 34 clinically relevant ARGs using high throughput qPCR. In total, 24 and 25 different ARGs were present in the dog and owner groups, respectively. The households had a mean of 9.9 ARGs present, with dogs and owners sharing on average 3.3 ARGs. ARGs were shared significantly more in households with dogs over 6 years old (3.5, interquartile range 2.75-5.0) than in households with younger dogs (2.5, interquartile range 2.0-3.0) (p = 0.02). Dogs possessed significantly more mecA and aminoglycoside resistance genes than owners. CONCLUSIONS: Dogs and owners can act as reservoirs for a broad range of ARGs belonging to several antimicrobial resistance classes. A modest proportion of the same resistance genes were present in both dogs and owners simultaneously, indicating that ARG transmission between the dog and human gut is of minor concern in the absence of antimicrobial selection. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insight into the common dog and human gut resistomes, contributing to an improved knowledge base in risk assessments regarding ARG transmission between dogs and humans.

Aliivibrio salmonicida requires O-antigen for virulence in Atlantic salmon (Salmo salar L.).

Aliivibrio salmonicida is the causative agent of cold-water vibriosis, a hemorrhagic septicemia of salmonid fish. The bacterium has been shown to rapidly enter the fish bloodstream, and proliferation in blood is seen after a period of latency. Although the pathogenesis of the disease is largely unknown, shedding of high quantities of outer-membrane complex VS-P1, consisting of LPS and a protein moiety, has been suggested to act as decoy and contribute to immunomodulation. To investigate the role of LPS in the pathogenesis, we constructed O-antigen deficient mutants by knocking out the gene encoding O-antigen ligase waaL. As this gene exists in two copies in the Al. salmonicida genome, we constructed single and double in-frame deletion mutants to explore potential effects of copy number variation. Our results demonstrate that the LPS structure of Al. salmonicida is essential for virulence in Atlantic salmon. As the loss of O-antigen did not influence invasive properties of the bacterium, the role of LPS in virulence applies to later stages of the pathogenesis. One copy of waaL was sufficient for O-antigen ligation and virulence in experimental models. However, as a non-significant decrease in mortality was observed after immersion challenge with a waaL single mutant, it is tempting to suggest that multiple copies of the gene are beneficial to the bacterium at lower challenge doses. The loss of O-antigen was not found to affect serum survival in vitro, but quantification of bacteria in blood following immersion challenge suggested a role in in vivo survival. Furthermore, fish challenged with the waaL double mutant induced a more transient immune response than fish challenged with the wild type strain. Whether the reduction in virulence following the loss of waaL is caused by altered immunomodulative properties or impaired survival remains unclear. However, our data demonstrate that LPS is crucial for development of disease.

A unique role of flagellar function in Aliivibrio salmonicida pathogenicity not related to bacterial motility in aquatic environments.

Aliivibrio salmonicida is the causative agent of cold-water vibriosis, a septicemia of farmed salmonid fish. The mechanisms of disease are not well described, and few virulence factors have been identified. However, a requirement for motility in the pathogenesis has been reported. Al. salmonicida is motile by the means of lophotrichous polar flagella, consisting of multiple flagellin subunits that are expressed simultaneously. Here we show that flagellin subunit FlaA, but not FlaD, is of major importance for motility in Al. salmonicida. Deletion of flaA resulted in 62% reduction in motility, as well as a reduction in the fraction of flagellated cells and number of flagella per cell. Similarly, deletion of the gene encoding motor protein motA gave rise to an aflagellate phenotype and cessation of motility. Surprisingly, we found that Al. salmonicida does not require motility for invasion of Atlantic salmon. Nevertheless, in-frame deletion mutants defective of motA and flaA were less virulent in Atlantic salmon challenged by immersion, whereas an effect on virulence after i.p. challenge was only seen for the latter. Our results indicate a complex requirement for motility and/or flagellation in the pathogenesis of cold-water vibriosis, but the mechanisms involved remain unknown. We hypothesize that the differences in virulence observed after immersion and i.p. challenge are related to the immune response of the host.

The autoinducer synthases LuxI and AinS are responsible for temperature-dependent AHL production in the fish pathogen Aliivibrio salmonicida.

BACKGROUND: Quorum sensing (QS) is a cell-to-cell communication system used by bacteria to regulate activities such as virulence, bioluminescence and biofilm formation. The most common QS signals in Gram-negative bacteria are N-acyl-homoserine lactones (AHLs). Aliivibrio salmonicida is the etiological agent of cold water vibriosis in Atlantic salmon, a disease which occurs mainly during seasons when the seawater is below 12°C. In this work we have constructed several mutants of A. salmonicida LFI1238 in order to study the LuxI/LuxR and AinS/AinR QS systems with respect to AHL production and biofilm formation. RESULTS: Using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) we found that LuxI in A. salmonicida LFI1238 is responsible for producing seven of the different AHLs, whereas AinS is responsible for producing only one. The production of these various AHLs is dependent on both cell density and growth temperature. The AHLs were efficiently produced when wild type LFI1238 was grown at 6 or 12°C, however at 16°C AHL production decreased dramatically, and LFI1238 produced less than 5% of the maximum concentrations observed at 6°C. LitR, the master regulator of QS, was found to be a positive regulator of AinS-dependent AHL production, and to a lesser extent LuxI-dependent AHL production. This implies a connection between the two systems, and both systems were found to be involved in regulation of biofilm formation. Finally, inactivation of either luxR1 or luxR2 in the lux operon significantly reduced production of LuxI-produced AHLs. CONCLUSION: LuxI and AinS are the autoinducer synthases responsible for the eight AHLs in A. salmonicida. AHL production is highly dependent on growth temperature, and a significant decrease was observed when the bacterium was grown at a temperature above its limit for disease outbreak. Numerous AHLs could offer the opportunity for fine-tuning responses to changes in the environment.

LitR is a repressor of syp genes and has a temperature-sensitive regulatory effect on biofilm formation and colony morphology in Vibrio (Aliivibrio) salmonicida.

Vibrio (Aliivibrio) salmonicida is the etiological agent of cold water vibriosis, a disease in farmed Atlantic salmon (Salmo salar) that is kept under control due to an effective vaccine. A seawater temperature below 12°C is normally required for disease development. Quorum sensing (QS) is a cell density-regulated communication system that bacteria use to coordinate activities involved in colonization and pathogenesis, and we have previously shown that inactivation of the QS master regulator LitR attenuates the V. salmonicida strain LFI1238 in a fish model. We show here that strain LFI1238 and a panel of naturally occurring V. salmonicida strains are poor biofilm producers. Inactivation of litR in the LFI1238 strain enhances medium- and temperature-dependent adhesion, rugose colony morphology, and biofilm formation. Chemical treatment and electron microscopy of the biofilm identified an extracellular matrix consisting mainly of a fibrous network, proteins, and polysaccharides. Further, by microarray analysis of planktonic and biofilm cells, we identified a number of genes regulated by LitR and, among these, were homologues of the Vibrio fischeri symbiosis polysaccharide (syp) genes. The syp genes were regulated by LitR in both planktonic and biofilm lifestyle analyses. Disruption of syp genes in the V. salmonicida ΔlitR mutant alleviated adhesion, rugose colony morphology, and biofilm formation. Hence, LitR is a repressor of syp transcription that is necessary for expression of the phenotypes examined. The regulatory effect of LitR on colony morphology and biofilm formation is temperature sensitive and weak or absent at temperatures above the bacterium's upper threshold for pathogenicity.

The Home Environment Is a Reservoir for Methicillin-Resistant Coagulase-Negative Staphylococci and Mammaliicocci.

Coagulase-negative staphylococci (CoNS) and mammaliicocci are opportunistic human and animal pathogens, often resistant to multiple antimicrobials, including methicillin. Methicillin-resistant CoNS (MRCoNS) have traditionally been linked to hospitals and healthcare facilities, where they are significant contributors to nosocomial infections. However, screenings of non-hospital environments have linked MRCoNS and methicillin-resistant mammaliicocci (MRM) to other ecological niches. The aim of this study was to explore the home environment as a reservoir for MRCoNS and MRM. A total of 33 households, including households with a dog with a methicillin-resistant staphylococcal infection, households with healthy dogs or cats and households without pets, were screened for MRCoNS and MRM by sampling one human, one pet (if present) and the environment. Samples were analyzed by a selective culture-based method, and bacterial species were identified by MALDI-TOF MS and tested for antibiotic susceptibility by the agar disk diffusion method. Following whole-genome sequencing, a large diversity of SCCmec elements and sequence types was revealed, which did not indicate any clonal dissemination of specific strains. Virulome and mobilome analyses indicated a high degree of species specificity. Altogether, this study documents that the home environment is a reservoir for a variety of MRCoNS and MRM regardless of the type of household.

LitR of Vibrio salmonicida is a salinity-sensitive quorum-sensing regulator of phenotypes involved in host interactions and virulence.

Vibrio (Aliivibrio) salmonicida is the causal agent of cold-water vibriosis, a fatal bacterial septicemia primarily of farmed salmonid fish. The molecular mechanisms of invasion, colonization, and growth of V. salmonicida in the host are still largely unknown, and few virulence factors have been identified. Quorum sensing (QS) is a cell-to-cell communication system known to regulate virulence and other activities in several bacterial species. The genome of V. salmonicida LFI1238 encodes products presumably involved in several QS systems. In this study, the gene encoding LitR, a homolog of the master regulator of QS in V. fischeri, was deleted. Compared to the parental strain, the litR mutant showed increased motility, adhesion, cell-to-cell aggregation, and biofilm formation. Furthermore, the litR mutant produced less cryptic bioluminescence, whereas production of acylhomoserine lactones was unaffected. Our results also indicate a salinity-sensitive regulation of LitR. Finally, reduced mortality was observed in Atlantic salmon infected with the litR mutant, implying that the fish were more susceptible to infection with the wild type than with the mutant strain. We hypothesize that LitR inhibits biofilm formation and favors planktonic growth, with the latter being more adapted for pathogenesis in the fish host.

Vibrio salmonicida pathogenesis analyzed by experimental challenge of Atlantic salmon (Salmo salar).

Cold-water vibriosis (CV) is a bacterial septicemia of farmed salmonid fish and cod caused by the Gram-negative bacterium Vibrio (Aliivibrio) salmonicida. To study the pathogenesis of this marine pathogen, Atlantic salmon was experimentally infected by immersion challenge with wild type V. salmonicida and the bacterial distribution in different organs was investigated at different time points. V. salmonicida was identified in the blood as early as 2 h after challenge demonstrating a rapid establishment of bacteremia without an initial period of colonization of the host. Two days after immersion challenge, only a few V. salmonicida were identified in the intestines, but the amount increased with time. In prolonged CV cases, V. salmonicida was the dominating bacterium of the gut microbiota causing a release of the pathogen to the water. We hypothesize that V. salmonicida uses the blood volume for proliferation during the infection of the fish and the salmonid intestine as a reservoir that favors survival and transmission. In addition, a motility-deficient V. salmonicida strain led us to investigate the impact of motility in the CV pathogenesis by comparing the virulence properties of the mutant with the wild type LFI1238 strain in both i.p. and immersion challenge experiments. V. salmonicida was shown to be highly dependent on motility to gain access to the fish host. After invasion, motility was no longer required for virulence, but the absence of normal flagellation delayed the disease development.

Transmission of Methicillin-Resistant Staphylococcus spp. from Infected Dogs to the Home Environment and Owners.

Dogs with methicillin-resistant Staphylococcus spp. (MRS) infections often undergo treatment in their homes, interacting with their owners and surroundings. This close contact between dogs and owners may facilitate the interspecies transmission of MRS. Therefore, this study aimed to investigate the transmission of MRS from infected dogs to their owners and home environments. Seven households with dogs that had been diagnosed with methicillin-resistant S. pseudintermedius (MRSP) and one household with a dog with methicillin-resistant S. epidermidis (MRSE) participated in the study. Dogs, owners, and the home environments were screened for the presence of clinical MRS. A selection of 36 staphylococcal isolates were whole-genome sequenced and screened for resistance genes and virulence genes. Clinical MRS were primarily identified from the dogs and their immediate surroundings, but these were also detected in locations that were out of reach for the dogs, indicating indirect transmission. Two of eight owners carried clinical MRS in their nostrils, while one owner carried methicillin-susceptible S. pseudintermedius (MSSP). All clinical MRS were multi-resistant, and several possessed resistance genes that were not expressed phenotypically. Clinical MRSP persisted in the home environment for a prolonged period, despite infection recovery and one dog being euthanized. Regardless of the stable presence of MRSP in the surroundings, the owners in these homes remained negative, but tested positive for MSSP on three occasions.

Prevalence of Salmonella serovars isolated from reptiles in Norwegian zoos.

BACKGROUND: Reptiles are known to be asymptomatic carriers of Salmonella spp. in their gastrointestinal mucosa and a variety of Salmonella serovars including exotic serovars mainly associated with reptiles as well as human pathogenic serovars have been isolated. There are many case reports of reptile-associated Salmonella infections worldwide, including one case in Norway in 2000. In August 2017, there was a legislative change in Norway that allowed more permissive reptile ownership and legalized the keeping of 19 different reptile species by private persons. There has been a concern that this new legislation will lead to an increase in reptile-associated salmonellosis in Norway, however knowledge is lacking on the occurrence of Salmonella spp. in Norwegian reptiles. The aim of this study was therefore to investigate the prevalence of Salmonella spp. in captive reptile species in Norway, identify the serovars and evaluate their zoonotic potential. Thus, cloacal swabs were taken from 53 snakes, 15 lizards and 35 chelonians from three Norwegian zoos, and assessed for the presence of Salmonella spp. by culture, biochemical testing and serotyping. RESULTS: In total, 43% of the reptiles were shedding Salmonella spp., with a prevalence of 62%, 67% and 3% in snakes, lizards and chelonians, respectively. A total of 26 different serovars were found, including Salmonella enterica spp. enterica (40%) and S. enterica spp. arizonae (4%), both of which are considered to have a high zoonotic potential. S. enterica spp. diarizonae, salamae and houtenae were also identified, however these serovars are considered to have a lower zoonotic potential. CONCLUSIONS: The current study demonstrates that captive Norwegian reptiles are carriers of potentially zoonotic Salmonella spp. Given the increasing popularity of reptiles as pets and the legislative change, reptile-associated salmonellosis could become an increasingly important public health concern in Norway. Adequate public information about the risk of Salmonella infection as well as preventive measures to avoid Salmonella transmission from reptiles to humans is needed. The risk of Salmonella infection is considered low when recommended precautions are taken and good hygiene exhibited.