Cefotaxime/sulbactam plus gentamicin as a potential carbapenem- and amikacin-sparing first-line combination for neonatal sepsis in high ESBL prevalence settings.

BACKGROUND: Infection with ESBL-producing Enterobacteriaceae infection is ubiquitous in some neonatal ICUs and increasing levels of antibiotic resistance are a cause for urgent concern. Delineation of bacterial and viral sepsis can be challenging, often leading to patients receiving empirical antibiotics without or whilst waiting for a definitive causal diagnosis. Empirical therapy is often dependent on broad-spectrum 'Watch' antibiotics, contributing to further resistance. METHODS: ESBL-producing Enterobacteriaceae clinical isolates found to have caused neonatal sepsis and meningitis underwent a detailed in vitro screening including susceptibility testing, chequerboard combination analysis and hollow-fibre infection model dynamic analyses using combinations of cefotaxime, ampicillin and gentamicin in combination with ß-lactamase inhibitors. RESULTS: Additivity or synergy was found for all antibiotic combinations against seven Escherichia coli and three Klebsiella pneumoniae clinical isolates. Cefotaxime or ampicillin plus sulbactam combined with gentamicin was able to consistently inhibit the growth of ESBL-producing isolates at typical neonatal doses, and the combination cleared the hollow-fibre infection model system of organisms resistant to each agent alone. The combination of cefotaxime/sulbactam and gentamicin was consistently bactericidal at clinically achievable concentrations (Cmax of 180, 60 and 20 mg/L for cefotaxime, sulbactam and gentamicin, respectively). CONCLUSIONS: The addition of sulbactam to cefotaxime or ampicillin to the typical first-line empirical therapy could obviate the need for carbapenems and amikacin in settings with high ESBL-infection prevalence.

Implications of identifying the recently defined members of the Staphylococcus aureus complex S. argenteus and S. schweitzeri: a position paper of members of the ESCMID Study Group for Staphylococci and Staphylococcal Diseases (ESGS).

BACKGROUND: Staphylococcus argenteus and Staphylococcus schweitzeri, previously known as divergent Staphylococcus aureus clonal lineages, have been recently established as novel, difficult-to-delimit, coagulase-positive species within the S. aureus complex. Methicillin-resistant strains of S. argenteus are known from Australia and the UK. Knowledge of their epidemiology, medical significance and transmission risk is limited and partly contradictory, hampering definitive recommendations. There is mounting evidence that the pathogenicity of S. argenteus is similar to that of 'classical' S. aureus, while as yet no S. schweitzeri infections have been reported. AIM: To provide decision support on whether and how to distinguish and report both species. SOURCES: PubMed, searched for S. argenteus and S. schweitzeri. CONTENT: This position paper reviews the main characteristics of both species and draws conclusions for microbiological diagnostics and surveillance as well as infection prevention and control measures. IMPLICATIONS: We propose not distinguishing within the S. aureus complex for routine reporting purposes until there is evidence that pathogenicity or clinical outcome differ markedly between the different species. Primarily for research purposes, suitably equipped laboratories are encouraged to differentiate between S. argenteus and S. schweitzeri. Caution is urged if these novel species are explicitly reported. In such cases, a specific comment should be added (i.e. 'member of the S.aureus complex') to prevent confusion with less- or non-pathogenic staphylococci. Prioritizing aspects of patient safety, methicillin-resistant isolates should be handled as recommended for methicillin-resistant Staphylococcus aureus (MRSA). In these cases, the clinician responsible should be directly contacted and informed by the diagnosing microbiological laboratory, as they would be for MRSA. Research is warranted to clarify the epidemiology, clinical impact and implications for infection control of such isolates.

Genomic insights into the emergence and spread of international clones of healthcare-, community- and livestock-associated meticillin-resistant Staphylococcus aureus: Blurring of the traditional definitions.

The evolution of meticillin-resistant Staphylococcus aureus (MRSA) from meticillin-susceptible S. aureus has been a result of the accumulation of genetic elements under selection pressure from antibiotics. The traditional classification of MRSA into healthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA) is no longer relevant as there is significant overlap of identical clones between these groups, with an increasing recognition of human infection caused by livestock-associated MRSA (LA-MRSA). Genomic studies have enabled us to model the epidemiology of MRSA along these lines. In this review, we discuss the clinical relevance of genomic studies, particularly whole-genome sequencing, in the investigation of outbreaks. We also discuss the blurring of each of the three epidemiological groups (HA-MRSA, CA-MRSA and LA-MRSA), demonstrating the limited relevance of this classification.

What Is the Origin of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 Isolates from Humans without Livestock Contact? An Epidemiological and Genetic Analysis.

Fifteen percent of all methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) human carriers detected in The Netherlands had not been in direct contact with pigs or veal calves. To ensure low MRSA prevalence, it is important to investigate the likely origin of this MRSA of unknown origin (MUO). Recently, it was shown that CC398 strains originating from humans and animals differ in the presence of specific mobile genetic elements (MGEs). We hypothesized that determining these specific MGEs in MUO isolates and comparing them with a set of CC398 isolates of various known origin might provide clues to their origin. MUO CC398 isolates were compared to MRSA CC398 isolates obtained from humans with known risk factors, a MRSA CC398 outbreak isolate, livestock associated (LA) MRSA CC398 isolates from pigs, horses, chickens, and veal calves, and five methicillin-susceptible Staphylococcus aureus (MSSA) CC398 isolates of known human origin. All strains were spa typed, and the presence or absence of, scn, chp, φ3 int, φ6 int, φ7 int, rep7, rep27, and cadDX was determined by PCRs. The MRSA CC398 in humans, MUO, or MRSA of known origin (MKO) resembled MRSA CC398 as found in pigs and not MSSA CC398 as found in humans. The distinct human MSSA CC398 spa type, t571, was not present among our MRSA CC398 strains; MRSA CC398 was tetracycline resistant and carried no φ3 bacteriophage with scn and chp. We showed by simple PCR means that human MUO CC398 carriers carried MRSA from livestock origin, suggestive of indirect transmission. Although the exact transmission route remains unknown, direct human-to-human transmission remains a possibility as well.

The alpha-1 adrenergic antagonist doxazosin for treatment of cocaine dependence: A pilot study.

BACKGROUND: Medications decreasing central noradrenergic activity have been associated with attenuation of cocaine effects. AIMS: This pilot study examined the efficacy of doxazosin versus placebo for reducing cocaine use in treatment-seeking cocaine dependent persons. METHODS: We screened 108 cocaine dependent subjects and assigned 35 participants to receive either doxazosin (8mg/day) or placebo for 13 weeks. Participants were titrated on the study medication according to two different schedules. During the initial phase of the study, patients were titrated onto the study medication over an 8-week period (DOX-slow). After reviewing data from our human laboratory study, a second phase was initiated, wherein titration was accelerated to a 4-week period (DOX-fast). All participants received weekly cognitive behavioral therapy. Urine toxicology was performed thrice weekly. RESULTS: Baseline subject characteristics were comparable. Thirty subjects entered the study: 8 subjects in DOX-slow, 9 subjects in DOX-fast, and 13 subjects in placebo. Total number of cocaine-negative urines was significantly increased in the DOX-fast group; and percentage of total cocaine-negative urines by group were 10% for DOX-slow group, 35% for DOX-fast group, and 14% for placebo (χ(2)=36.3, df=2, p<0.0001). The percentage of participants achieving two or more consecutive weeks of abstinence by group was 0% for DOX-slow group, 44% for DOX-fast group, and 7% for placebo (χ(2)=7.35, df=2, p<0.023). CONCLUSIONS: This pilot study suggests the potential efficacy of doxazosin when rapidly titrated in reducing cocaine use.

Prevalence of and risk factors for MRSA carriage in companion animals: a survey of dogs, cats and horses.

We investigated the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) carriage in a convenience sample of purposely selected populations of dogs, cats and horses in the Greater London area. Swabs from carriage sites were pooled, enriched and processed by standard bacteriological methods. The presence of nuc and mecA was confirmed for MRSA. Risk factors were investigated among veterinary treatment group animals using exact logistic regression analysis. Twenty-six (1.53%) MRSA carriers were identified in the 1692 animals (15/704 dogs, 8/540 cats, 3/152 horses). Animals presenting for veterinary treatment more frequently carried MRSA than healthy animals (OR 7.27, 95% CI 2.18-24.31, P<0.001). Concurrent carriage of non-MRSA coagulase-positive staphylococci was associated with MRSA carriage (OR 0.088, 95% CI 0.016-0.31, P<0.001); none of the other 13 putative risk factors was significant. MRSA carriage was rare in the selected companion animal populations. The absence of typical risk factors indicates that companion animals act as contaminated vectors rather than as true reservoirs.

Meticillin-resistant Staphylococcus aureus carriage in UK veterinary staff and owners of infected pets: new risk groups.

Meticillin-resistant Staphylococcus aureus (MRSA) nasal carriage on admission to hospital remains one of the most important risk factors for subsequent infection. Identification of high risk groups for MRSA carriage is vital for the success of infection control programmes. Veterinary staff may be one such risk group but little is known about pet owners and the role of contact with infected pets. As part of a UK-wide case-control study investigating risk factors for MRSA infection in dogs and cats between 2005 and 2008, 608 veterinary staff and pet owners in contact with 106 MRSA and 91 meticillin-susceptible S. aureus (MSSA)-infected pets were screened for S. aureus nasal carriage. Laboratory isolation and characterisation included salt broth enrichment, standard and automated microbiological tests, demonstration of the S. aureus-specific thermonuclease gene (nuc) and of mecA, and polymerase chain reaction-based lineage characterisation. MRSA carriage was 12.3% in veterinarians attending MRSA-infected animals and 7.5% in their owners. In the MSSA control group, MRSA carriage was 4.8% in veterinary staff and 0% in owners. Veterinary staff carried MRSA more frequently than owners (odds ratio: 2.33; 95% confidence interval: 1.10-4.93). All MRSA from humans and all but one animal MRSA were CC22 or CC30, typical for hospital MRSA in the UK. This study indicates for the first time an occupational risk for MRSA carriage in small animal general practitioners. Veterinary staff and owners of MRSA-infected pets are high risk groups for MRSA carriage despite not having direct hospital links. Strategies to break the cycle of MRSA infection must take these potential new reservoirs into account.

Lack of transmission of methicillin-resistant Staphylococcus aureus (MRSA) between apparently healthy dogs in a rescue kennel.

Although it is widely accepted that methicillin-resistant Staphylococcus aureus (MRSA) can be transmitted between humans and animals in both directions, little is known about the dynamics of animal-to-animal transfer. This study aimed to investigate aspects of dog-to-dog MRSA transfer in a rescue facility in the South-East of England during an MRSA outbreak. One hundred and twenty-nine apparently healthy dogs, mostly housed in pairs, were swabbed at nasal, oral, axillary and perianal sites. Swabs were enriched in selective broth and staphylococci identified using standard biological methods. MRSA isolates were confirmed by demonstration of the thermonuclease gene (nuc) and mecA. After initial swabbing, a dog excluded from the study design but housed at the same facility was discovered to have a wound infection due to MRSA. MRSA carriage was identified in 10/129 dogs (7.8%) and all isolates were of the same lineage as the one isolated from the infected dog. All carrier dogs lived in shared kennels and their 16 kennel partners sampled negative on two occasions. Concurrently with successful antimicrobial treatment of the infected patient, MRSA carriage resolved spontaneously in all dogs within two weeks. In conclusion, MRSA did not transmit readily between apparently healthy dogs, MRSA carriage was not supported for long periods in a regularly cleaned environment and exposure alone may not lead to MRSA acquisition by dogs without the presence of additional risk factors.

Genetic diversity in CC398 methicillin-resistant Staphylococcus aureus isolates of different geographical origin.

Staphylococcus aureus of sequence type 398 has emerged in Europe, North America and Asia, and has typically been associated with livestock and their human contacts. We analysed two Panton-Valentine leukocidin (PVL)-negative t034-ST398 isolates from humans in contact with pigs and two t034-ST398 PVL-positive isolates from two unrelated, adopted Chinese children, using multistrain microarrays to determine genomic variability between the two sets of isolates. The ST398 isolates clearly belong to the same lineage when compared to other clonal lineages. However, the four isolates cluster into two distinct groups corresponding to differences in epidemiology based on mobile genetic elements and resistance patterns, suggesting that the two groups are epidemiologically distinct.

Transcriptional signature following inhibition of early-stage cell wall biosynthesis in Staphylococcus aureus.

To facilitate mode of action studies on antibacterial inhibitors of early-stage cell wall biosynthesis (CWB), we determined the transcriptional response of Staphylococcus aureus to depletion/inhibition of enzymes in this pathway by DNA microarray analysis. We identified a transcriptional signature distinct from that previously observed following exposure to inhibitors of late-stage CWB.

Molecular characterisation of the dominant UK methicillin-resistant Staphylococcus aureus strains, EMRSA-15 and EMRSA-16.

Epidemic methicillin-resistant Staphylococcus aureus types 15 and 16 (EMRSA-15 and EMRSA-16) are the dominant types of MRSA found in UK hospitals, but accurate designation of strains has been difficult. Restriction fragment length polymorphism (RFLP) profiles of seven core virulence genes were used to classify unambiguously isolates of MRSA from St George's Hospital into two groups corresponding to EMRSA-15 and EMRSA-16. Variants of both EMRSA-15 and EMRSA-16 isolates occurred that had lost virulence genes encoded on mobile genetic elements. EMRSA-16 isolates had core gene profiles identical to a cluster of previously characterised MSSA (methicillin-sensitive S. aureus) isolates from St George's Hospital, suggesting that they have arisen from this source, or that loss of the accessory genetic element encoding methicillin resistance is frequent. EMRSA-15 and EMRSA-16 strains were distinct from other MRSA strains previously identified in UK hospitals, and always carried a mobile genetic element encoding multiple superantigens. These results contribute to the understanding of the types of MRSA found in UK hospitals, how they vary and how they arose.

Genetic variation among hospital isolates of methicillin-sensitive Staphylococcus aureus: evidence for horizontal transfer of virulence genes.

Staphylococcus aureus strains often carry in their genomes virulence genes that are not found in all strains and that may be carried on discrete genetic elements. Strains also differ in that they carry one of four classes of an accessory gene regulator (agr) locus, an operon that regulates virulence factor expression and that has been proposed to be a therapeutic target. To look at their distribution among hospital strains, we investigated 38 methicillin-sensitive S. aureus isolates, classifying the isolates by agr class and screening them for the presence and restriction fragment length polymorphisms (RFLPs) of 12 core and 14 accessory virulence genes. Twenty-three (61%) were agr class I, 10 (26%) were agr class II, and 5 (13%) were agr class III. None were agr class IV. The S. aureus strains had distinguishable RFLP profiles, although clusters of isolates with clearly related core gene profiles were found among our strains, including all five agr class III strains, two sets of six strains within agr class I, and six strains within agr class II. Within these clusters there was evidence of horizontal acquisition and/or loss of multiple accessory virulence genes. Furthermore, two isolates from the same patient were identical except for the presence of the sea gene, indicating that movement of mobile elements may occur in vivo. Several strong correlations with the carriage of virulence genes between strains were seen, including a positive correlation between tst and agr class III and negative correlations between tst and lukE-splB and between lukE-splB and seg-sei. This suggests that the core genome or the presence of accessory genetic elements within a strain may influence acquisition and loss of other elements encoding virulence genes.

Interactive regulatory pathways control virulence determinant production and stability in response to environmental conditions in Staphylococcus aureus.

The accessory gene regulator (agr) and staphylococcal accessory regulator (sar) loci are important regulators of toxin production in Staphylococcus aureus. In this study we examined how environmental conditions degree of aeration and salt concentration - affect the transcription and translation of mRNAs for alpha-haemolysin (Hla) and serine protease (Ssp) via these pathways and influence the stability of these proteins. Using Northern analysis, we have confirmed earlier observations that sarA is involved in the upregulation of RNAIII, the effector molecule encoded by the agr locus. However, this effect was abolished in highly aerated cultures. While sarA does appear to have an up-regulatory effect on hla transcription that is independent of agr, we propose that the PC1839 (sarA) mutant produces less alpha-haemolysin activity mainly as a result of post-translational inactivation by proteases. The most obvious phenotypic feature of PC1839 (sarA) is the upregulation of proteases. In this study we show that ssp is repressed by SarA at the transcriptional level. Western analysis using an anti-alpha-haemolysin antibody identified a major breakdown product that is only present in the supernatant of strains that are overexpressing serine protease. We have also confirmed that agr exerts a significant regulatory influence on hla at the level of translation, as well as transcription. Finally, the addition of salt upregulates ssp transcription and dramatically downregulates transcription of hla, and is an example of an environmental parameter that affects toxin production independently of agr and sarA. How environmental signals are transduced to control alpha-haemolysin and serine protease production, activity and stability at multiple levels are discussed.

Evidence for Clostridium perfringens enterotoxin (CPE) inducing a mitogenic and cytokine response in vitro and a cytokine response in vivo.

We investigated some immunogenic properties of Clostridium perfringens enterotoxin (CPE) in vitro using murine J774A macrophages (MPhi) and in vivo using Swiss Webster (SW) mice. CPE was a potent mitogen in vitro, where cell proliferation increased with CPE concentration. CPE was nonmitogenic when MPhi were concurrently incubated with CPE and interferon gamma (IFN-gamma). MPhi incubated in the presence of CPE induced the synthesis of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma), but not interleukin-2 (IL-2). In vivo, CPE induced a pro-inflammatory cytokine response with striking production of IFN-gamma, IL-1, and IL-6. Regardless of route of CPE entry, serum cytokine levels generally peaked within 1 h of administration and were maintained for 4-8 h. Although CPE engenders an intense immune response during toxicosis, the toxin does not appear to be a superantigen. Death from CPE-induced shock appears to result from various interrelating immunological mechanisms.

The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus.

Tst, the gene for toxic shock syndrome toxin-1 (TSST-1), is part of a 15.2 kb genetic element in Staphylococcus aureus that is absent in TSST-1-negative strains. The prototype, in RN4282, is flanked by a 17 nucleotide direct repeat and contains genes for a second possible superantigen toxin, a Dichelobacter nodosus VapE homologue and a putative integrase. It is readily transferred to a recA recipient, and it always inserts into a unique chromosomal copy of the 17 nucleotide sequence in the same orientation. It is excised and circularized by staphylococcal phages phi13 and 80alpha and replicates during the growth of the latter, which transduces it at very high frequency. Because of its site and orientation specificity and because it lacks other identifiable phage-like genes, we consider it to be a pathogenicity island (PI) rather than a transposon or a defective phage. The tst element in RN4282, near tyrB, is designated SaPI1. That in RN3984 in the trp region is only partially homologous to SaPI1 and is excised by phage 80 but not by 80alpha. It is designated SaPI2. These PIs are the first in any gram-positive species and the first for which mobility has been demonstrated. Their mobility may be responsible for the spread of TSST-1 production among S. aureus strains.

Chronic sequelae of foodborne disease.

In the past decade the complexity of foodborne pathogens, as well as their adaptability and ability to cause acute illness, and in some cases chronic (secondary) complications, have been newly appreciated. This overview examines long-term consequences of foodborne infections and intoxications to emphasize the need for more research and education.

Chronic health effects of microbial foodborne disease.

The acute effects of foodborne disease are sometimes not the end of the illness. Several significant foodborne pathogens are capable of triggering chronic disease, and even permanent tissue or organ destruction, probably via immune mechanisms. Arthritis, septic and reactive, inflammatory bowel disease, haemolytic uraemic syndrome, Guillain-Barré syndrome, and possible several autoimmune disorders can be triggered by foodborne pathogens or their toxins. Research is needed to more fully understand the mechanisms by which the immune system is inappropriately activated by these common foodborne disease-causing agents.