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Simple quantitative methods based on purified Clostridium difficile spores and a murine transmission model are described for analyzing health care disinfection regimens. Pure Clostridium difficile spores have facilitated practical methods for analyzing the efficacy of Clostridium difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control.

Otitis media is a common reason for hearing loss, especially in children. Otitis media is a multifactorial disease and environmental factors, anatomic dysmorphology and genetic predisposition can all contribute to its pathogenesis. However, the reasons for the variable susceptibility to otitis media are elusive. MCPH1 mutations cause primary microcephaly in humans. So far, no hearing impairment has been reported either in the MCPH1 patients or mouse models with Mcph1 deficiency. In this study, Mcph1-deficient (Mcph1tm1a/tm1a) mice were produced using embryonic stem cells with a targeted mutation by the Sanger Institute's Mouse Genetics Project. Auditory brainstem response measurements revealed that Mcph1tm1a/tm1a mice had mild to moderate hearing impairment with around 70% penetrance. We found otitis media with effusion in the hearing-impaired Mcph1tm1a/tm1a mice by anatomic and histological examinations. Expression of Mcph1 in the epithelial cells of middle ear cavities supported its involvement in the development of otitis media. Other defects of Mcph1tm1a/tm1a mice included small skull sizes, increased micronuclei in red blood cells, increased B cells and ocular abnormalities. These findings not only recapitulated the defects found in other Mcph1-deficient mice or MCPH1 patients, but also revealed an unexpected phenotype, otitis media with hearing impairment, which suggests Mcph1 is a new gene underlying genetic predisposition to otitis media. [ABSTRACT FROM AUTHOR]

Relapsing C. difficile disease in humans is linked to a pathological imbalance within the intestinal microbiota, termed dysbiosis, which remains poorly understood. We show that mice infected with epidemic C. difficile (genotype 027/BI) develop highly contagious, chronic intestinal disease and persistent dysbiosis characterized by a distinct, simplified microbiota containing opportunistic pathogens and altered metabolite production. Chronic C. difficile 027/BI infection was refractory to vancomycin treatment leading to relapsing disease. In contrast, treatment of C. difficile 027/BI infected mice with feces from healthy mice rapidly restored a diverse, healthy microbiota and resolved C. difficile disease and contagiousness. We used this model to identify a simple mixture of six phylogenetically diverse intestinal bacteria, including novel species, which can re-establish a health-associated microbiota and clear C. difficile 027/BI infection from mice. Thus, targeting a dysbiotic microbiota with a defined mixture of phylogenetically diverse bacteria can trigger major shifts in the microbial community structure that displaces C. difficile and, as a result, resolves disease and contagiousness. Further, we demonstrate a rational approach to harness the therapeutic potential of health-associated microbial communities to treat C. difficile disease and potentially other forms of intestinal dysbiosis. [ABSTRACT FROM AUTHOR]

Bicomponent pore-forming leukocidins are a family of potent toxins secreted by Staphylococcus aureus, which target white blood cells preferentially and consist of an S- and an F-component. The S-component recognizes a receptor on the host cell, enabling high-affinity binding to the cell surface, after which the toxins form a pore that penetrates the cell lipid bilayer. Until now, six different leukocidins have been described, some of which are host and cell specific. Here, we identify and characterise a novel S. aureus leukocidin; LukPQ. LukPQ is encoded on a 45 kb prophage (ΦSaeq1) found in six different clonal lineages, almost exclusively in strains cultured from equids. We show that LukPQ is a potent and specific killer of equine neutrophils and identify equine-CXCRA and CXCR2 as its target receptors. Although the S-component (LukP) is highly similar to the S-component of LukED, the species specificity of LukPQ and LukED differs. By forming non-canonical toxin pairs, we identify that the F-component contributes to the observed host tropism of LukPQ, thereby challenging the current paradigm that leukocidin specificity is driven solely by the S-component. [ABSTRACT FROM AUTHOR]

Sphingosine-l-phosphate (SIP) is lipid messenger involved in the regulation of embryonic development, immune system functions, and many other physiological processes. However, the mechanisms of SIP transport across cellular membranes remain poorly understood, with several ATP-binding cassette family members and the spinster 2 (Spns2) member of the major facilitator superfamily known to mediate SIP transport in cell culture. Spns2 was also shown to control SIP activities in zebrafish in vivo and to play a critical role in zebrafish cardiovascular development. However, the in vivo roles of Spns2 in mammals and its involvement in the different SIP-dependent physiological processes have not been investigated. In this study, we characterized Spns2-null mouse line carrying the Spns2tmlalKOMP)Wtsi allele (Spns2tmla). The Spns2tmla/tmla animals were viable, indicating a divergence in Spns2 function from its zebrafish ortholog. However, the immunological phenotype of the Spns2tmla/tmla mice closely mimicked the phenotypes of partial SIP deficiency and impaired SIP-dependent lymphocyte trafficking, with a depletion of lymphocytes in circulation, an increase in mature single-positive T cells in the thymus, and a selective reduction in mature B cells in the spleen and bone marrow. Spns2 activity in the nonhematopoietic cells was critical for normal lymphocyte development and localization. Overall, Spns2tmla/tmla resulted in impaired humoral immune responses to immunization. This study thus demonstrated a physiological role for Spns2 in mammalian immune system functions but not in cardiovascular development. Other components of the SIP signaling network are investigated as drug targets for immunosuppressive therapy, but the selective action of Spns2 may present an advantage in this regard. [ABSTRACT FROM AUTHOR]

Clostridium difficile is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of C. difficile that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious C. difficile spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified C. difficile spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure spores and blocking spore-mediated transmission. Complete inactivation of 106 pure C. difficile spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with C. difficile spores (17 to 29 spores/cm²) and block host transmission. Thus, pure C. difficile spores facilitate practical methods for evaluating the efficacy of C. difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control. [ABSTRACT FROM AUTHOR]

• 53 % of 265 Streptococcus uberis isolates were oxacillin resistant. • A total of 101 SNPs were identified in 5 penicillin binding proteins (PBP). • 11 SNPs were associated with oxacillin resistance. • All resistant isolates had E 381 K and Q 554 E substitutions in PBP 2x. • Acquisition of resistance appears to have occurred multiple times. A number of veterinary clinical pathology laboratories in New Zealand have been reporting emergence of increased minimum in inhibitory concentrations for β-lactams in the common clinical bovine mastitis pathogen Streptococcus uberis. The objective of this study was to determine the genetic basis of this increase in MIC for β-lactams amongst S. uberis. Illumina sequencing and determination of oxacillin MIC was performed on 265 clinical isolates. Published sequences of the five penicillin binding proteins pbp1a , pbp1b, pbp2a, pbp2b, and pbp2x were used to identify, extract and align these sequences from the study isolates. Amino acid substitutions resulting from single nucleotide polymorphisms (SNP) within these genes were analysed for associations with elevated (≥ 0.5 mg/L) oxacillin MIC together with a genome wide association study. The population structure of the study isolates was approximated using a phylogenetic tree generated from an alignment of the core genome. A total of 53 % of isolates had MIC ≥ 0.5 mg/L for oxacillin. A total of 101 substitutions within the five pbp were identified, of which 11 were statistically associated with an MIC ≥ 0.5 mg/L. All 140 isolates which exhibited an increased β-lactam MIC had SNPs leading to pbp2x E 381 K and Q 554 E substitutions. The phylogenetic tree indicated that the genotype and phenotype associated with the increased MIC for oxacillin were present in several different lineages suggesting that acquisition of this increased β-lactam MIC had occurred in multiple geographically distinct regions. Reanalysis of the data from the intervention studies from which the isolates were originally drawn found a tendency for the pbp2x E 381 K substitution to be associated with lower cure rates. It is concluded that there is geographically and genetically widespread presence of pbp substitutions associated with reduced susceptibility to β-lactam antimicrobials. Additionally, presence of pbp substitutions tended to be associated with poorer cure rate outcomes following antimicrobial therapy for clinical mastitis. [ABSTRACT FROM AUTHOR]

Aggregation of children in schools has been established to be a key driver of transmission of infectious diseases. Mathematical models of transmission used to predict the impact of control measures, such as vaccination and testing, commonly depend on self-reported contact data. However, the link between self-reported social contacts and pathogen transmission has not been well described. To address this, we used Staphylococcus aureus as a model organism to track transmission within two secondary schools in England and test for associations between self-reported social contacts, test positivity and the bacterial strain collected from the same students. Students filled out a social contact survey and their S. aureus colonization status was ascertained through self-administered swabs from which isolates were sequenced. Isolates from the local community were also sequenced to assess the representativeness of school isolates. A low frequency of genome-linked transmission precluded a formal analysis of links between genomic and social networks, suggesting that S. aureus transmission within schools is too rare to make it a viable tool for this purpose. Whilst we found no evidence that schools are an important route of transmission, increased colonization rates found within schools imply that school-age children may be an important source of community transmission.

Methicillin-resistant Staphylococcus aureus (MRSA) clonal-complex 398 (CC398) is the dominant livestock-associated (LA) MRSA lineage in European livestock and an increasing cause of difficult-to-treat human disease. LA-CC398 MRSA evolved from a diverse human-associated methicillin-sensitive population, and this transition from humans to livestock was associated with three mobile genetic elements (MGEs). In this study, we apply transposon-directed insertion site sequencing (TraDIS), a high-throughput transposon mutagenesis approach, to investigate genetic signatures that contribute to LA-CC398 causing disease in humans. We identified 26 genes associated with LA-CC398 survival in human blood and 47 genes in porcine blood. We carried out phylogenetic reconstruction on 1,180 CC398 isolates to investigate the genetic context of all identified genes. We found that all genes associated with survival in human blood were part of the CC398 core genome, while 2/47 genes essential for survival in porcine blood were located on MGEs. Gene SAPIG0966 was located on the previously identified Tn 916 transposon carrying a tetracycline resistance gene, which has been shown to be stably inherited within LA-CC398. Gene SAPIG1525 was carried on a phage element, which in part, matched phiSa2wa_st1, a previously identified bacteriophage carrying the Panton-Valentine leucocidin (PVL) virulence factor. Gene deletion mutants constructed in two LA-CC398 strains confirmed that the SAPIG0966 carrying Tn 916 and SAPIG1525 were important for CC398 survival in porcine blood. Our study shows that MGEs that carry antimicrobial resistance and virulence genes could have a secondary function in bacterial survival in blood and may be important for host adaptation. IMPORTANCE CC398 is the dominant type of methicillin-resistant Staphylococcus aureus (MRSA) in European livestock and a growing cause of human infections. Previous studies have suggested MRSA CC398 evolved from human-associated methicillin-sensitive Staphylococcus aureus and is capable of rapidly readapting to human hosts while maintaining antibiotic resistance. Using high-throughput transposon mutagenesis, our study identified 26 and 47 genes important for MRSA CC398 survival in human and porcine blood, respectively. Two of the genes important for MRSA CC398 survival in porcine blood were located on mobile genetic elements (MGEs) carrying resistance or virulence genes. Our study shows that these MGEs carrying antimicrobial resistance and virulence genes could have a secondary function in bacterial survival in blood and may be important for blood infection and host adaptation.

Introduction. We recently revealed that a significant proportion of clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates are susceptible to pencillins and clavulanic acid (potentiated penicillins), including widely available combinations such as co-amoxiclav. These isolates also showed increased susceptibility to oxacillin on Iso-Sensitest Agar (ISA). Hypothesis/Gap Statement. The increased susceptibility to oxacillin displayed on ISA by these MRSA isolates may be used to distinguish them from the resistant ones. Aim. We aimed to develop a method to simultaneously screen a S. aureus clinical isolate for its susceptibility to methicillin and potentiated penicillins. Methodology. A double-disc diffusion method using 10 µg cefoxitin and 1 µg oxacillin discs on ISA was developed and tested against a panel of 120 whole genome-sequenced MRSA isolates. The sensitivity of the method was compared with that of previously published genotypic and phenotypic methods. In addition, double-disc diffusion was performed for all isolates on Müller-Hinton agar (MHA) following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) protocol. Results. All isolates (120/120) were reconfirmed to be phenotypically MRSA, as indicated by the result of cefoxitin disc diffusion testing. All isolates (40/40) that had a pencillins and clavulanic acid (Pen-Clav)-resistant genotype were not inhibited by oxacillin, while 77/80 (96.3 %) isolates that had a Pen-Clav-susceptible genotype were inhibited by oxacillin on ISA. The results also showed that the EUCAST method using MHA correctly identified all isolates as MRSA but failed to distinguish the Pen-Clav-susceptible isolates from the Pen-Clav-resistant isolates. Conclusions. This double-disc diffusion method using ISA could be used to accurately screen for clinical MRSA isolates and determine their susceptibility to Pen-Clav simultaneously, rapidly identifying MRSA infections that might be suitable for treatment with potentiated penicillins.

Otitis media is a common reason for hearing loss, especially in children. Otitis media is a multifactorial disease and environmental factors, anatomic dysmorphology and genetic predisposition can all contribute to its pathogenesis. However, the reasons for the variable susceptibility to otitis media are elusive. MCPH1 mutations cause primary microcephaly in humans. So far, no hearing impairment has been reported either in the MCPH1 patients or mouse models with Mcph1 deficiency. In this study, Mcph1-deficient (Mcph1(tm1a) (/tm1a) ) mice were produced using embryonic stem cells with a targeted mutation by the Sanger Institute's Mouse Genetics Project. Auditory brainstem response measurements revealed that Mcph1(tm1a) (/tm1a) mice had mild to moderate hearing impairment with around 70% penetrance. We found otitis media with effusion in the hearing-impaired Mcph1(tm1a) (/tm1a) mice by anatomic and histological examinations. Expression of Mcph1 in the epithelial cells of middle ear cavities supported its involvement in the development of otitis media. Other defects of Mcph1(tm1a) (/tm1a) mice included small skull sizes, increased micronuclei in red blood cells, increased B cells and ocular abnormalities. These findings not only recapitulated the defects found in other Mcph1-deficient mice or MCPH1 patients, but also revealed an unexpected phenotype, otitis media with hearing impairment, which suggests Mcph1 is a new gene underlying genetic predisposition to otitis media.

Clostridium difficile persists in hospitals by exploiting an infection cycle that is dependent on humans shedding highly resistant and infectious spores. Here we show that human virulent C. difficile can asymptomatically colonize the intestines of immunocompetent mice, establishing a carrier state that persists for many months. C. difficile carrier mice consistently shed low levels of spores but, surprisingly, do not transmit infection to cohabiting mice. However, antibiotic treatment of carriers triggers a highly contagious supershedder state, characterized by a dramatic reduction in the intestinal microbiota species diversity, C. difficile overgrowth, and excretion of high levels of spores. Stopping antibiotic treatment normally leads to recovery of the intestinal microbiota species diversity and suppresses C. difficile levels, although some mice persist in the supershedding state for extended periods. Spore-mediated transmission to immunocompetent mice treated with antibiotics results in self-limiting mucosal inflammation of the large intestine. In contrast, transmission to mice whose innate immune responses are compromised (Myd88(-/-)) leads to a severe intestinal disease that is often fatal. Thus, mice can be used to investigate distinct stages of the C. difficile infection cycle and can serve as a valuable surrogate for studying the spore-mediated transmission and interactions between C. difficile and the host and its microbiota, and the results obtained should guide infection control measures.