Neuronal, affective, and sensory correlates of targeted helping behavior in male and female Sprague Dawley rats.

Introduction: Empathic behaviors are driven by the ability to understand the emotional states of others along with the motivation to improve it. Evidence points towards forms of empathy, like targeted helping, in many species including rats. There are several variables that may modulate targeted helping, including sex, sensory modalities, and activity of multiple neural substrates. Methods: Using a model of social contact-independent targeted helping, we first tested whether sex differences exist in helping behavior. Next, we explored sex differences in sensory and affective signaling, including direct visualization and an analysis of ultrasonic vocalizations made between animal pairs. Finally, we examined the neural activity in males and females of multiple regions of interest across time. Here, we aim to examine any behavioral differences in our lab's social contact independent targeted helping task between males and females. Results and Discussion: These findings are the first to intimate that, like other prosocial behaviors, males and females may exhibit similar social-independent targeted helping behavior, but the underlying sensory communication in males and females may differ. In addition, this is the first set of experiments that explore the neural correlates of social-independent targeted helping in both males and females. These results lay the groundwork for future studies to explore the similarities and differences that drive targeted helping in both sexes.

Inhibition of Estradiol Signaling in the Basolateral Amygdala Impairs Extinction Memory Recall for Heroin-Conditioned Cues in a Sex-Specific Manner.

INTRODUCTION: Relapse is a major treatment barrier for opioid use disorder. Environmental cues become associated with the rewarding effects of opioids and can precipitate relapse, even after numerous unreinforced cue presentations, due to deficits in extinction memory recall (EMR). Estradiol (E2) modulates EMR of fear-related cues, but it is unknown whether E2 impacts EMR of reward cues and what brain region(s) are responsible for E2s effects. Here, we hypothesize that inhibition of E2 signaling in the basolateral amygdala (BLA) will impair EMR of a heroin-associated cue in both male and female rats. METHODS: We pharmacologically manipulated E2 signaling to characterize the role of E2 in the BLA on heroin-cue EMR. Following heroin self-administration, during which a light/tone cue was co-presented with each heroin infusion, rats underwent cued extinction to extinguish the conditioned association between the light/tone and heroin. During extinction, E2 signaling in the BLA was blocked by an aromatase inhibitor or specific estrogen receptor (ER) antagonists. The next day, subjects underwent a cued test to assess heroin-cue EMR. RESULTS: In both experiments, females took more heroin than males (mg/kg) and had higher operant responding during cued extinction. Inhibition of E2 synthesis in the BLA impaired heroin-cue EMR in both sexes. Notably, E2s actions are mediated by different ER mechanisms, ERα in males but ERß in females. CONCLUSIONS: This study is the first to demonstrate a behavioral role for centrally-produced E2 in the BLA and that E2 also impacts EMR of reward-associated stimuli in both sexes.

Differential Roles of Oxytocin Receptors in the Prefrontal Cortex and Nucleus Accumbens on Cocaine Self-Administration and Reinstatement of Cued Cocaine Seeking in Male Rats.

BACKGROUND: Little is known about the specific roles of cortical and accumbal oxytocin receptors in drug use disorders. To better understand the importance of the endogenous oxytocin system in cocaine relapse behavior, we developed an adeno-associated viral vector-expressing short hairpin (sh) RNAs to selectively degrade the rat oxytocin receptor (OxyR) mRNA in vivo. METHODS: Male (Sprague-Dawley) rats received bilateral infusions of the shRNA for the oxytocin receptor (shOxyR) or an shRNA control virus into the prefrontal cortex (PFC) or the nucleus accumbens core (NAc). Rats self-administered cocaine on an escalating FR ratio for 14 days, lever responding was extinguished, and rats were tested for cued and cocaine-primed reinstatement of drug seeking. RESULTS: OxyR knockdown in the PFC delayed the acquisition of lever pressing on an fixed ratio 1 schedule of reinforcement. All rats eventually acquired the same level of lever pressing and discrimination, and there were no differences in extinction. OxyR knockdown in the NAc had no effect during acquisition. In both the PFC and NAc, the shOxyR decreased cued reinstatement relative to shRNA control virus but was without effect during drug-primed reinstatement. OxyR knockdown in the PFC increased chamber activity during a social interaction task. CONCLUSIONS: This study provides critical new information about how endogenous OxyRs function to affect drug seeking in response to different precipitators of relapse. The tool developed to knockdown OxyRs in rat could provide important new insights that aid development of oxytocin-based therapeutics to reduce return-to-use episodes in people with substance use disorder and other neuropsychiatric disorders.

Paraventricular Nucleus of the Hypothalamus Oxytocin and Incubation of Heroin Seeking.

INTRODUCTION: There are numerous pharmacologic treatments for opioid use disorder (OUD), but none that directly target the underlying addictive effects of opioids. Oxytocin, a peptide hormone produced in the paraventricular nucleus (PVN) of the hypothalamus, has been investigated as a potential therapeutic for OUD. Promising preclinical and clinical results have been reported, but the brain region(s) and mechanism(s) by which oxytocin impacts reward processes remain undetermined. METHODS: Here, we assess peripherally administered oxytocin's impacts on cued reinstatement of heroin seeking following forced abstinence and its effects on neuronal activation in the PVN and key projection regions. We also examine how designer receptors exclusively activated by designer drug (DREADD)-mediated activation or inhibition of oxytocinergic PVN neurons alters cued heroin seeking and social interaction. RESULTS: As predicted, peripheral oxytocin administration successfully decreased cued heroin seeking on days 1 and 30 of abstinence. Oxytocin administration also led to increased neuronal activity within the PVN and the central amygdala (CeA). Activation of oxytocinergic PVN neurons with an excitatory (Gq) DREADD did not impact cued reinstatement or social interaction. In contrast, suppression with an inhibitory (Gi) DREADD reduced heroin seeking on abstinence day 30 and decreased time spent interacting with a novel conspecific. DISCUSSION: These findings reinforce oxytocin's therapeutic potential for OUD, the basis for which may be driven in part by increased PVN-CeA circuit activity. Our results also suggest that oxytocin has distinct signaling and/or other mechanisms of action to produce these effects, as inhibition, but not activation, of oxytocinergic PVN neurons did not recapitulate the suppression in heroin seeking.

The role of the anterior insular during targeted helping behavior in male rats.

Empathy, the understanding of the emotional state of others, can be examined across species using the Perception Action Model, where shared affect promotes an action by "Observers" to aid a distressed "Target". The anterior insula (AI) has garnered interest in empathic behavior due to its role integrating sensory and emotional information of self and other. In the following studies, the AI was inhibited pharmacologically and chemogenetically during targeted helping. We demonstrate the insula is active during, and is necessary for the maintenance of, targeted helping. Analysis of ultrasonic vocalizations revealed distress calls from Targets increased when Observers' helping was attenuated due to insula inhibition. Targets' elevated distress was directly correlated to Observers' diminished helping behavior, suggesting emotional transfer between Observer and Target is blunted following Observer AI inhibition. Finally, the AI may selectively blunt targeted helping, as social exploration did not change in a social reward place conditioning task. These studies help further establish the anterior insula as a critical node in the empathic brain during targeted helping, even in the absence of direct social contact.

Chemogenetic inhibition of corticostriatal circuits reduces cued reinstatement of methamphetamine seeking.

Methamphetamine (meth) causes enduring changes within the medial prefrontal cortex (mPFC) and the nucleus accumbens (NA). Projections from the mPFC to the NA have a distinct dorsal-ventral distribution, with the prelimbic (PL) mPFC projecting to the NAcore, and the infralimbic (IL) mPFC projecting to the NAshell. Inhibition of these circuits has opposing effects on cocaine relapse. Inhibition of PL-NAcore reduces cued reinstatement of cocaine seeking and IL-NAshell inhibition reinstates cocaine seeking. Meth, however, exhibits a different profile, as pharmacological inhibition of either the PL or IL decrease cued reinstatement of meth-seeking. The potentially opposing roles of the PL-NAcore and IL-NAshell projections remain to be explored in the context of cued meth seeking. Here we used an intersectional viral vector approach that employs a retrograde delivery of Cre from the NA and Cre-dependent expression of DREADD in the mPFC, in both male and female rats to inhibit or activate these parallel pathways. Inhibition of the PL-NAcore circuit reduced cued reinstatement of meth seeking under short and long-access meth self-administration and after withdrawal with and without extinction. Inhibition of the IL-NAshell also decreased meth cued reinstatement. Activation of the parallel circuits was without an effect. These studies show that inhibition of the PL-NAcore or the IL-NAshell circuits can inhibit reinstated meth seeking. Thus, the neural circuitry mediating cued reinstatement of meth seeking is similar to cocaine in the dorsal, but not ventral, mPFC-NA circuit.

Complex Interactions Between Sex and Stress on Heroin Seeking.

Rationale: Stress plays a dual role in substance use disorders as a precursor to drug intake and a relapse precipitant. With heroin use at epidemic proportions in the United States, understanding interactions between stress disorders and opioid use disorder is vital and will aid in treatment of these frequently comorbid conditions. Objectives: Here, we combine assays of stress and contingent heroin self-administration (SA) to study behavioral adaptations in response to stress and heroin associated cues in male and female rats. Methods: Rats underwent acute restraint stress paired with an odor stimulus and heroin SA for subsequent analysis of stress and heroin cue reactivity. Lofexidine was administered during heroin SA and reinstatement testing to evaluate its therapeutic potential. Rats also underwent tests on the elevated plus maze, locomotor activity in a novel environment, and object recognition memory following stress and/or heroin. Results: A history of stress and heroin resulted in disrupted behavior on multiple levels. Stress rats avoided the stress conditioned stimulus and reinstated heroin seeking in response to it, with males reinstating to a greater extent than females. Lofexidine decreased heroin intake, reinstatement, and motor activity. Previous heroin exposure increased time spent in the closed arms of an elevated plus maze, activity in a round novel field, and resulted in object recognition memory deficits. Discussion: These studies report that a history of stress and heroin results in maladaptive coping strategies and suggests a need for future studies seeking to understand circuits recruited in this pathology and eventually help develop therapeutic approaches.

Behavioral and accumbens synaptic plasticity induced by cues associated with restraint stress.

Exposure to acute stress can increase vulnerability to develop or express many psychiatric disorders, including post-traumatic stress disorder. We hypothesized that stress-induced psychiatric vulnerability is associated with enduring neuroplasticity in the nucleus accumbens core because stress exposure can alter drug addiction-related behaviors that are associated with accumbens synaptic plasticity. We used a single 2-h stress session and 3 weeks later exposed male and female rats to stress-conditioned odors in a modified defensive burying task, and quantified both active and avoidant coping strategies. We measured corticosterone, dendritic spine and astrocyte morphology in accumbens, and examined reward sensitivity using a sucrose two-bottle choice and operant sucrose self-administration. Exposure to stress odor increased burying (active coping) and immobility (avoidant coping) in the defensive burying task in female and male rats. Systemic corticosterone was transiently increased by both ongoing acute restraint stress and stress-conditioned odors. Three weeks after administering acute restraint stress, we observed increased dendritic spine density and head diameter, and decreased synaptic association with astroglia and the astroglial glutamate transporter, GLT-1. Exposure to conditioned stress further increased head diameter without affecting spine density or astroglial morphology, and this increase by conditioned stress was correlated with burying behavior. Finally, we found that stress-exposed females have a preference for sweet solutions and higher motivation to seek sucrose than stressed male rats. We conclude that acute stress produced enduring plasticity in accumbens postsynapses and associated astroglia. Moreover, conditioned stress odors induced active behavioral coping strategies that were correlated with dendritic spine morphology.

Long-term impact of acute restraint stress on heroin self-administration, reinstatement, and stress reactivity.

RATIONALE: There is a robust relationship between anxiety disorders, including post-traumatic stress disorder (PTSD) and substance abuse. In fact, 30-50% of people seeking treatment for substance abuse have a comorbid diagnosis for PTSD. Heroin use is at epic proportions in the USA and is commonly used by people with co-occurring PTSD symptoms and substance use disorder. OBJECTIVES: Here, we combined animal assays of acute restraint stress and contingent heroin self-administration (SA) to study comorbidity between stress disorders and opioid use disorder and identify shifts in anxiety-like behaviors following stress and/or heroin in response to a stress-conditioned cue. Our objective for this approach was to determine the long-term impact of acute restraint stress and heroin self-administration on stress reactivity and basic reward processes. METHODS: We used 2-h acute restraint stress paired with an odor stimulus to condition a stress cue (CS) for testing of subsequent stress reactivity in a burying task and reinstatement and extinction to heroin seeking. Rats were also tested for social place preference for measures of social reward and anxiety-like behaviors. RESULTS: Stress rats exhibited multiple levels of disrupted behavior including enhanced acquisition of heroin intake and reinstatement in response to the stress CS, as well as delayed extinction in response to the stress CS. All rats developed a social place preference, but stress rats spent more time in nose-to-nose contact with the unfamiliar rat while heroin rats spent time exploring the chamber. In the burying task, stress shortened latencies to bury the CS and increased burying and immobility in male and female rats relative to sham counterparts. CONCLUSIONS: Acute restraint stress results in anxiety-like behaviors and a stress-associated cue is sufficient to reinstate extinguished heroin seeking. This project has the potential to elucidate the complex relationship between stress/anxiety disorders, including some PTSD-like characteristics, and the onset, maintenance, and relapse to heroin seeking.

Global Scale Dissemination of ST93: A Divergent Staphylococcus aureus Epidemic Lineage That Has Recently Emerged From Remote Northern Australia.

Background: In Australia, community-associated methicillin-resistant Staphylococcus aureus (MRSA) lineage sequence type (ST) 93 has rapidly risen to dominance since being described in the early 1990s. We examined 459 ST93 genome sequences from Australia, New Zealand, Samoa, and Europe to investigate the evolutionary history of ST93, its emergence in Australia and subsequent spread overseas. Results: Comparisons with other S. aureus genomes indicate that ST93 is an early diverging and recombinant lineage, comprising of segments from the ST59/ST121 lineage and from a divergent but currently unsampled Staphylococcal population. However, within extant ST93 strains limited genetic diversity was apparent with the most recent common ancestor dated to 1977 (95% highest posterior density 1973-1981). An epidemic ST93 population arose from a methicillin-susceptible progenitor in remote Northern Australia, which has a proportionally large Indigenous population, with documented overcrowded housing and a high burden of skin infection. Methicillin-resistance was acquired three times in these regions, with a clade harboring a staphylococcal cassette chromosome mec (SCCmec) IVa expanding and spreading to Australia's east coast by 2000. We observed sporadic and non-sustained introductions of ST93-MRSA-IVa to the United Kingdom. In contrast, in New Zealand, ST93-MRSA-IVa was sustainably transmitted with clonal expansion within the Pacific Islander population, who experience similar disadvantages as Australian Indigenous populations. Conclusion: ST93 has a highly recombinant genome including portions derived from an early diverging S. aureus population. Our findings highlight the need to understand host population factors in the emergence and spread of antimicrobial resistant community pathogens.

Robust Prediction of Resistance to Trimethoprim in Staphylococcus aureus.

The rise of antibiotic resistance threatens modern medicine; to combat it new diagnostic methods are required. Sequencing the whole genome of a pathogen offers the potential to accurately determine which antibiotics will be effective to treat a patient. A key limitation of this approach is that it cannot classify rare or previously unseen mutations. Here we demonstrate that alchemical free energy methods, a well-established class of methods from computational chemistry, can successfully predict whether mutations in Staphylococcus aureus dihydrofolate reductase confer resistance to trimethoprim. We also show that the method is quantitatively accurate by calculating how much the most common resistance-conferring mutation, F99Y, reduces the binding free energy of trimethoprim and comparing predicted and experimentally measured minimum inhibitory concentrations for seven different mutations. Finally, by considering up to 32 free energy calculations for each mutation, we estimate its specificity and sensitivity.

Novel multiresistance cfr plasmids in linezolid-resistant methicillin-resistant Staphylococcus epidermidis and vancomycin-resistant Enterococcus faecium (VRE) from a hospital outbreak: co-location of cfr and optrA in VRE.

BACKGROUND: Linezolid is often the drug of last resort to treat infections caused by Gram-positive cocci. Linezolid resistance can be mutational (23S rRNA or L-protein) or, less commonly, acquired [predominantly cfr, conferring resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A compounds (PhLOPSA) or optrA, encoding oxazolidinone and phenicol resistance]. OBJECTIVES: To investigate the clonality and genetic basis of linezolid resistance in 13 linezolid-resistant (LZDR) methicillin-resistant Staphylococcus epidermidis (MRSE) isolates recovered during a 2013/14 outbreak in an ICU in an Irish hospital and an LZDR vancomycin-resistant Enterococcus faecium (VRE) isolate from an LZDR-MRSE-positive patient. METHODS: All isolates underwent PhLOPSA susceptibility testing, 23S rRNA sequencing, DNA microarray profiling and WGS. RESULTS: All isolates exhibited the PhLOPSA phenotype. The VRE harboured cfr and optrA on a novel 73 kb plasmid (pEF12-0805) also encoding erm(A), erm(B), lnu(B), lnu(E), aphA3 and aadE. One MRSE (M13/0451, from the same patient as the VRE) harboured cfr on a novel 8.5 kb plasmid (pSEM13-0451). The remaining 12 MRSE lacked cfr but exhibited linezolid resistance-associated mutations and were closely related to (1-52 SNPs) but distinct from M13/0451 (202-223 SNPs). CONCLUSIONS: Using WGS, novel and distinct cfr and cfr/optrA plasmids were identified in an MRSE and VRE isolate, respectively, as well as a cfr-negative LZDR-MRSE ICU outbreak and a distinct cfr-positive LZDR-MRSE from the same ICU. To our knowledge, this is the first report of cfr and optrA on a single VRE plasmid. Ongoing surveillance of linezolid resistance is essential to maintain its therapeutic efficacy.

Molecular Epidemiology of Staphylococcus aureus Skin and Soft Tissue Infections in the Lao People's Democratic Republic.

This is the first report of the molecular epidemiology of Staphylococcus aureus from skin and soft tissue infections (SSTI) in Laos. We selected a random sample of 96 S. aureus SSTI isolates received by the Microbiology Laboratory, Mahosot Hospital, Vientiane, between July 2012 and June 2014, including representation from seven referral hospitals. Isolates underwent susceptibility testing by Clinical and Laboratory Standards Institute methods, spa typing and DNA microarray analysis, with whole genome sequencing for rare lineages. Median patient age was 19.5 years (interquartile range 2-48.5 years); 52% (50) were female. Forty-three spa types, representing 17 lineages, were identified. Fifty-eight percent (56) of all isolates encoded Panton-Valentine leukocidin (PVL), representing six lineages: half of these patients had abscesses and three had positive blood cultures. The dominant lineage was CC121 (39; 41%); all but one isolate encoded PVL and 49% (19) were from children under five. Staphyococcus argenteus was identified in six (6%) patients; mostly adults > 50 years and with diabetes. Six isolates (6%) belonged to rare lineage ST2885; two possibly indicate cross-infection in a neonatal unit. One isolate from a previously undescribed lineage, ST1541, was identified. Antibiotic resistance was uncommon except for penicillin (93; 97%) and tetracycline (48; 50%). Seven (7%) isolates were methicillin-resistant S. aureus (MRSA), belonging to ST239-MRSA-III, CC59-MRSA-V(T) Taiwan Clone, ST2250-MRSA-IV, ST2885-MRSA-V and CC398-MRSA-V. Globally widespread CC5 and CC30 were absent. There are parallels in S. aureus molecular epidemiology between Laos and neighboring countries and these data highlight the prominence of PVL and suggest infiltration of MRSA clones of epidemic potential from surrounding countries.

Methicillin-resistant Staphylococcus aureus emerged long before the introduction of methicillin into clinical practice.

BACKGROUND: The spread of drug-resistant bacterial pathogens poses a major threat to global health. It is widely recognised that the widespread use of antibiotics has generated selective pressures that have driven the emergence of resistant strains. Methicillin-resistant Staphylococcus aureus (MRSA) was first observed in 1960, less than one year after the introduction of this second generation beta-lactam antibiotic into clinical practice. Epidemiological evidence has always suggested that resistance arose around this period, when the mecA gene encoding methicillin resistance carried on an SCCmec element, was horizontally transferred to an intrinsically sensitive strain of S. aureus. RESULTS: Whole genome sequencing a collection of the first MRSA isolates allows us to reconstruct the evolutionary history of the archetypal MRSA. We apply Bayesian phylogenetic reconstruction to infer the time point at which this early MRSA lineage arose and when SCCmec was acquired. MRSA emerged in the mid-1940s, following the acquisition of an ancestral type I SCCmec element, some 14 years before the first therapeutic use of methicillin. CONCLUSIONS: Methicillin use was not the original driving factor in the evolution of MRSA as previously thought. Rather it was the widespread use of first generation beta-lactams such as penicillin in the years prior to the introduction of methicillin, which selected for S. aureus strains carrying the mecA determinant. Crucially this highlights how new drugs, introduced to circumvent known resistance mechanisms, can be rendered ineffective by unrecognised adaptations in the bacterial population due to the historic selective landscape created by the widespread use of other antibiotics.

Detection of diverse genotypes of Methicillin-resistant Staphylococcus aureus from hospital personnel and the environment in Armenia.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a public health concern internationally. Studies examining a range of cohorts have been reported from various regions of the world, but little is known about the molecular epidemiology of MRSA in Armenia. METHODS: Between May and September 2013, twenty isolates of methicillin-resistant Staphylococcus aureus (MRSA; mecA positive) were recovered from hospital personnel (n = 10; 9 females, 1 male) and environmental sites (n = 10) in the maternity ward of one of the teaching hospitals in Armenia. RESULTS: Multi-locus sequence type clonal complex (MLST-CC) assignments inferred from spa typing data revealed the majority belonged to 3 pandemic lineages of MRSA including: t008-CC8-SCCmecV (n = 10; 7 from personnel); t021-CC30-SCCmecIV (n = 5; all environmental); and t1523-CC45 (n = 2; 1 from personnel), one harboured SCCmecV the other was SCCmec non-typable. The remainder identified as belonging to genotype t364-CC182, both of which harboured a novel SCCmec cassette with kdp, rif5, ccrB2 and ccrC detected by PCR (both from personnel); and t325-CC88-SCCmecIV (n = 1; environmental). All MRSA were negative for the Panton-Valentine Leukocidin (PVL) locus and three CC8 strains were positive for the arginine catabolic element (ACME). CONCLUSIONS: In this small study, we report for the first time of the occurrence of diverse MRSA genotypes belonging to both pandemic and more sporadic international clones in Armenia harbouring the smaller SCCmec types and/or ACME, both of which have been associated with strain fitness. Further surveillance is warranted to better understand the prevalence, clinical and molecular epidemiology of MRSA throughout Armenia.

Re-emergence of methicillin susceptibility in a resistant lineage of Staphylococcus aureus.

Objectives: MRSA is a leading cause of hospital-associated infection. Acquired resistance is encoded by the mecA gene or its homologue mecC , but little is known about the evolutionary dynamics involved in gain and loss of resistance. The objective of this study was to obtain an expanded understanding of Staphylococcus aureus methicillin resistance microevolution in vivo , by focusing on a single lineage. Methods: We compared the whole-genome sequences of 231 isolates from a single epidemic lineage [clonal complex 30 (CC30) and spa -type t018] of S. aureus that caused an epidemic in the UK. Results: We show that resistance to methicillin in this single lineage was gained on at least two separate occasions, one of which led to a clonal expansion around 1995 presumably caused by a selective advantage. Resistance was, however, subsequently lost in vivo by nine strains isolated between 2008 and 2012. We describe the genetic mechanisms involved in this loss of resistance and the imperfect relationship between genotypic and phenotypic resistance. Conclusions: The recent re-emergence of methicillin susceptibility in this epidemic lineage suggests a significant fitness cost of resistance and reduced selective advantage following the introduction in the mid-2000s of MRSA hospital control measures throughout the UK.

Livestock-Associated Methicillin Resistant Staphylococcus aureus (LA-MRSA) Clonal Complex (CC) 398 Isolated from UK Animals belong to European Lineages.

In recent years, there has been an increase in the number of livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) clonal complex (CC) 398 recovered from S. aureus isolated animals in the UK. To determine possible origins of 12 LA-MRSA CC398 isolates collected after screening more than a thousand S. aureus animal isolates from the UK between 2013 and 2015, whole genome sequences (WGS) of CC398 European, including UK, and non-European isolates from diverse animal hosts were compared. Phylogenetic reconstruction applied to WGS data to assess genetic relatedness of all 89 isolates, clustered the 12 UK CC398 LA-MRSA within the European sub-lineages, although on different nodes; implicating multiple independent incursions into the UK, as opposed to a single introduction followed by clonal expansion. Three UK isolates from healthy pigs and one from turkey clustered within the cassette chromosome recombinases ccr C S. aureus protein A (spa)-type t011 European sub-lineage and three UK isolates from horses within the ccrA2B2 t011 European sub-lineage. The remaining UK isolates, mostly from pigs, clustered within the t034 European lineage. Presence of virulence, antimicrobial (AMR), heavy metal (HMR), and disinfectant (DR) resistance genes were determined using an in-house pipeline. Most, including UK isolates, harbored resistance genes to ≥3 antimicrobial classes in addition to ß-lactams. HMR genes were detected in most European ccrC positive isolates, with >80% harboring czrC, encoding zinc and cadmium resistance; in contrast ~60% ccrC isolates within non-European lineages and 6% ccrA2B2 isolates showed this characteristic. The UK turkey MRSA isolate did not harbor φAVß avian prophage genes (SAAV_2008 and SAAV_2009) present in US MSSA isolates from turkey and pigs. Absence of some of the major human-associated MRSA toxigenic and virulence genes in the UK LA-MRSA animal isolates was not unexpected. Therefore, we can conclude that the 12 UK LA-MRSA isolates collected in the past 2 years most likely represent separate incursions into the UK from other European countries. The presence of zinc and cadmium resistance in all nine food animal isolates (pig and poultry), which was absent from the 3 horse isolates may suggest heavy metal use/exposure has a possible role in selection of some MRSA.

Identification of commonly expressed exoproteins and proteolytic cleavage events by proteomic mining of clinically relevant UK isolates of Staphylococcus aureus.

The range of exoproteins and core exoproteome of 14 Staphylococcus aureus isolates representing major lineages associated with asymptomatic carriage and clinical disease in the UK was identified by MS proteomics using a combined database incorporating sequences derived from 39 S. aureus genomes. In all, 632 different proteins were identified and, of these, only 52 (8 %) were found in all 14 isolates whereas 144 (23 %) were found in just a single isolate. Comparison of the observed mass of each protein (based on migration by SDS-PAGE) with its predicted mass (based on amino acid sequence) suggested that 95 % of the proteins identified were not subject to any major post-translational modification. Migration of 5 % of the proteins was not as expected: 1 % of the proteins migrated at a mass greater than predicted, while 4 % appeared to have undergone proteolytic cleavage; these included SsaA2, Aur, SspP, Ebh as well as BlaR1, MecR1, FsH, OatA and LtaS. Intriguingly, a truncated SasG was produced by a single CC8 USA300-like strain. The analysis provided evidence of the marked heterogeneity in protein expression by S. aureus in broth, while yielding a core but narrow common exoproteome.

First report of lukM-positive livestock-associated methicillin-resistant Staphylococcus aureus CC30 from fattening pigs in Northern Ireland.

The increasing number of reports of livestock-associated meticillin-resistant Staphylococcus aureus (LA-MRSA) world-wide attests to the public health concern surrounding this pathogen in animal husbandry and in-contact humans. In Europe, LA-MRSA CC398 is predominant and generally regarded as being of low virulence for animals. Herein we report the recovery of a lineage of LA-MRSA, belonging to CC30, from three pigs in Northern Ireland and which encodes a marker of virulence (lukM and lukF-P83) restricted to animal-associated clones of S. aureus.