Antibodies to <Emphasis Type="Italic">Neospora caninum</Emphasis> and <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> in domestic cats from Brazil

The occurrence of antibodies to Neospora caninum and Toxoplasma gondii was determined in 400 domestic cats (Siamese, Persian, and undetermined breeds) from the Municipality of Araçatuba, Sao Paulo, Brazil, through the indirect fluorescence antibody test (IFAT). Of the 400 cats, 100 were seropositive to T. gondii (25%, titer ≥64) and 98 to N. caninum (24.5%, titer ≥16). The rate of seropositive cats for T. gondii was correlated with age (χ ²=35.7; p<0.001), with a higher number of infected animals at older ages. Of the 219 cats younger than 1-year-old, 13.2% were seropositive for T. gondii, while 39.2% were positive in the 181 older animals. The presence of N. caninum was also correlated with age (÷²=8.8; p<0.01), with 18.7% (41/219) and 31.5% (57/181) of positive animals at ages below and above 12-month, respectively. An association between the occurrences of both protozoa in the felines was also observed (χ ²=19.6; p<0.001).     

The correlation between <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> infection and prenatal depression in pregnant women

Previous studies have demonstrated that latent toxoplasmosis is associated with neuropsychiatric disorders. We evaluated the correlation between Toxoplasma gondii infection and prenatal depression. In this case–control study, we enrolled 116 depressed pregnant women and 244 healthy controls. The Edinburgh Postpartum Depression Scale (EPDS) was used to evaluate the depression symptom severity in study participants. All participants were screened for the anti-Toxoplasma IgG by enzyme-linked immunosorbent assay. Seroprevalence of T. gondii did not significantly differ between the depressed pregnant women and healthy controls (OR?=?1.4; 95 % CI?=?0.9–2.19; P?=?0.142). T. gondii IgG titer was significantly higher in depressed women (18.6?±?10.9 IUs) than those in the control group (13.6?±?8.1 IUs) (z?= ?5.36, P?<?0.001). The T. gondii–positive depressed women showed a positive correlation of T. gondii IgG titer with the EPDS scores (r?=?0.52; P?<?0.01). The mean EPDS score was also significantly higher in the T. gondii–positive depressed women (20.7?±?2.7) compared with the controls (18.36?±?2.7) (P?<?0.001). The results obtained from the current study revealed that T. gondii infection might affect susceptibility to depression and severity of depressive symptoms in pregnant women, particularly in those patients who have high antibody titers. Further study is required to fully elucidate the characteristics and mechanisms of this association.     

Molecular and virulence characterization of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> strains isolated from humans in Portugal

Toxoplasma gondii is an apicomplexan parasite responsible for toxoplasmosis which infects all warm-blooded vertebrates, including mammals and birds. The majority of studies conducted in Europe have revealed that more than 80 % of strains isolated from human infections belong to genotype II, whereas genotypes I and III are responsible for a small number of cases. Atypical and recombinant strains are generally associated with more severe infections. In Portugal, there is a lack of data concerning genetic diversity as the classical typing studies in humans have never been performed. We aimed to determine the Sag2 and microsatellite-based (TUB2, TgM-A, W35, B17, B18) genotypes of T. gondii isolated from humans in Portugal, as well as to study their virulence in mice. We analyzed 48 strains from congenital and acquired toxoplasmosis collected during the last two decades. Sag2-based genotyping of T. gondii was achieved in all 48 strains where 35 (73 %) were classified as type II and 13 (27 %) were type I. The multilocus PCR of five microsatellites allowed the classification of 10 strains (21 %) as recombinant strains that had been previously identified as type II or I by Sag2 typing. Seven out of the 48 strains, including three type I, three recombinant, and one type I, were virulent in mice. This study constitutes the first evidence of recombinant strains circulating in Portugal in humans from congenital infection, highlighting the need for a better evaluation of these strains as their phenotype is still barely understood.     

Typhoidal and non-typhoidal <Emphasis Type="Italic">Salmonella</Emphasis> infections in Africa

Salmonella infections in humans can range from self-limiting gastroenteritis typically associated with non-typhoidal Salmonella (NTS) to typhoidal fever, which can be life-threatening. Salmonellosis causes considerable morbidity and mortality in both humans and animals, and has a significant socioeconomic impact worldwide. In Africa, it is difficult to evaluate the situation of salmonellosis due to the non-availability of facilities capable of performing the tests essential for the diagnosis of typhoidal and non-typhoidal Salmonella infections. This article reviews important work in the literature, including the epidemiology, disease burden, pathogenesis, genomics, diagnosis, treatment, emergence and tracking of multidrug-resistant (MDR) Salmonella infections and intercontinental transmission of Salmonella to Africa. Searches of PubMed and Google Scholar were completed and the retrieved list of relevant publications were further screened. The literature revealed that the most common form of the disease in Africa is gastroenteritis, with bacterial multiplication in intestinal submucosa and diarrhoea caused by the inflammatory response and, perhaps, also by toxins. In addition to the high burden of Salmonella infection in Africa, MDR Salmonella species is on the rise in the continent, which might pose difficulties in the treatment of the disease.     

Incidence of <Emphasis Type="Italic">Cryptosporidium andersoni</Emphasis> in diarrheal patients from southern Assam,India: a molecular approach

The distribution and public health significance of Cryptosporidium species and genotypes in humans and bovine differ across geographical areas. Cryptosporidium species causes a disease known as cryptosporidiosis in humans and animals. To characterize the prevalence of cryptosporidiosis in humans in southern Assam, India, stool samples (n?=?1119) of diarrhea patients were collected from different hospitals and from the community during the period January 2014 to July 2016. Fecal smears were examined microscopically for Cryptosporidium species using modified acid fast staining and were screened to ascertain the presence of Cryptosporidium antigen by enzyme-linked immunosorbent assay (ELISA). The genomic DNA of positive fecal samples were analyzed by nested polymerase chain reaction (PCR), which were subsequently genotyped by PCR-restriction fragment length polymorphism (RFLP), based on small subunit (SSU) 18S rRNA. It was found that the prevalence of Cryptosporidium spp. was high during the monsoon season. The average infection rate of Cryptosporidium spp. was found to be 2.4% (27/1119) microscopically. When subjected to nested PCR using amplification of the 18S rRNA gene, Cryptosporidium was found to be 8.57% (98/1119). Based on the 18S rRNA gene, two Cryptosporidium spp., namely Cryptosporidium andersoni (6.97%: 78/1119) and Cryptosporidium parvum (1.7%: 20/1119), were identified. Cryptosporidium andersoni infections were found to be of either zoonotic or anthroponotic origin. The prevalence was statistically significant (p?=?0.03, R²?=?0.042) considering age, gender, and cast.     

<Emphasis Type="Italic">Anaplasma marginale</Emphasis> and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis>: Rickettsiales pathogens of veterinary and public health significance

Anaplasma marginale and Anaplasma phagocytophilum are the most important tick-borne bacteria of veterinary and public health significance in the family Anaplasmataceae. The objective of current review is to provide knowledge on ecology and epidemiology of A. phagocytophilum and compare major similarities and differences of A. marginale and A. phagocytophilum. Bovine anaplasmosis is globally distributed tick-borne disease of livestock with great economic importance in cattle industry. A. phagocytophilum, a cosmopolitan zoonotic tick transmitted pathogen of wide mammalian hosts. The infection in domestic animals is generally referred as tick-borne fever. Concurrent infections exist in ticks, domestic and wild animals in same geographic area. All age groups are susceptible, but the prevalence increases with age. Movement of susceptible domestic animals from tick free non-endemic regions to disease endemic regions is the major risk factor of bovine anaplasmosis and tick-borne fever. Recreational activities or any other high-risk tick exposure habits as well as blood transfusion are important risk factors of human granulocytic anaplasmosis. After infection, individuals remain life-long carriers. Clinical anaplasmosis is usually diagnosed upon examination of stained blood smears. Generally, detection of serum antibodies followed by molecular diagnosis is usually recommended. There are problems of sensitivity and cross-reactivity with both the Anaplasma species during serological tests. Tetracyclines are the drugs of choice for treatment and elimination of anaplasmosis in animals and humans. Universal vaccine is not available for either A. marginale or A. phagocytophilum, effective against geographically diverse strains. Major control measures for bovine anaplasmosis and tick-borne fever include rearing of tick-resistant breeds, endemic stability, breeding Anaplasma-free herds, identification of regional vectors, domestic/wild reservoirs and control, habitat modification, biological control, chemotherapy, and vaccinations (anaplasmosis and/or tick vaccination). Minimizing the tick exposure activities, identification and control of reservoirs are important control measures for human granulocytic anaplasmosis.     

Novel diagnostic ELISA test for discrimination between infections with <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> and <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

Yersiniosis is a foodborne infection caused by Yersinia enterocolitica or Yersinia pseudotuberculosis. Although yersiniosis is most often self-limiting, some patients develop chronic infections, such as reactive arthritis, glomerulonephritis, or myocarditis, which require an antibiotic treatment. Whereas early infections can be diagnosed by direct detection of bacteria, chronic infections can only be identified by serological tests. At this point, a serological method for differentiation between infections with the two Yersinia species is important since antibiotic susceptibility of these bacteria is different. Traditional immunoassays do not distinguish between infections with Y. enterocolitica and Y. pseudotuberculosis. The only test that allows for this differentiation is Mikrogen’s strip test where discrimination between the two types of infection is based on two recombinant bacterial proteins, MyfA and PsaA (specific for Y. enterocolitica and Y. pseudotuberculosis, respectively). Here, we show that Y. enterocolitica and Y. pseudotuberculosis, cultured under the conditions that mimic the natural rout of infection, express surface antigens different from MyfA and PsaA that can also be used in a discrimination test. Further, we describe a new ELISA that is based on the whole bacteria and recombinant MyfA and PsaA as antigens, and that allows the differentiation between infections with Y. enterocolitica and Y. pseudotuberculosis and simultaneous detection of yersiniosis.     

A novel inhibition ELISA for the detection and monitoring of <Emphasis Type="Italic">Penicillium marneffei</Emphasis> antigen in human serum

The thermally dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis marneffei, a disease considered to be an acquired immune deficiency syndrome (AIDS)-defining illness in Southeast Asia and southern China. We have developed an inhibition enzyme-linked immunosorbent assay (inh-ELISA) incorporating the yeast phase specific mannoprotein-binding monoclonal antibody 4D1 for the detection of P. marneffei infection. In our sample set, the test detected antigenemia in all 45 (100 %) patients with P. marneffei, with a mean antigen concentration of 4.32 μg/ml. No cross-reactivity in this assay was found using serum from 44 additional patients with other fungal infections, such as Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans, as well as 44 patients with bacterial infections, such as Mycobacterium tuberculosis and Streptococcus suis. Additionally, no reactivity occurred using serum from 31 human immunodeficiency virus (HIV)-infected patients without a history of fungal infections and 113 healthy controls residing in endemic areas. To investigate the potential of the inh-ELISA for disease monitoring, we followed the reduction in antigenemia in six patients who clinically responded to itraconazole and P. marneffei was no longer isolated from their blood or tissues. In contrast, we correlated increased concentrations of antigenemia in patients with relapsed P. marneffei infection with the progression of their clinical symptoms and the isolation of P. marneffei from their clinical specimens. In summary, the P. marneffei inh-ELISA is a promising new assay for the rapid diagnosis of P. marneffei, as well as a tool for evaluating clinical response and clearance of the fungus during treatment.     

Early-Onset Invasive Infection Due to <Emphasis Type="Italic">Corynespora cassiicola</Emphasis> Associated with Compound Heterozygous <Emphasis Type="Italic">CARD9</Emphasis> Mutations in a Colombian Patient

Purpose

CARD9 deficiency is an inborn error of immunity that predisposes otherwise healthy humans to mucocutaneous and invasive fungal infections, mostly caused by Candida, but also by dermatophytes, Aspergillus, and other fungi. Phaeohyphomycosis are an emerging group of fungal infections caused by dematiaceous fungi (phaeohyphomycetes) and are being increasingly identified in patients with CARD9 deficiency. The Corynespora genus belongs to phaeohyphomycetes and only one adult patient with CARD9 deficiency has been reported to suffer from invasive disease caused by C. cassiicola. We identified a Colombian child with an early-onset, deep, and destructive mucocutaneous infection due to C. cassiicola and we searched for mutations in CARD9.

Methods

We reviewed the medical records and immunological findings in the patient. Microbiologic tests and biopsies were performed. Whole-exome sequencing (WES) was made and Sanger sequencing was used to confirm the CARD9 mutations in the patient and her family. Finally, CARD9 protein expression was evaluated in peripheral blood mononuclear cells (PBMC) by western blotting.

Results

The patient was affected by a large, indurated, foul-smelling, and verrucous ulcerated lesion on the left side of the face with extensive necrosis and crusting, due to a C. cassiicola infectious disease. WES led to the identification of compound heterozygous mutations in the patient consisting of the previously reported p.Q289* nonsense (c.865C > T, exon 6) mutation, and a novel deletion (c.23_29del; p.Asp8Alafs10*) leading to a frameshift and a premature stop codon in exon 2. CARD9 protein expression was absent in peripheral blood mononuclear cells from the patient.

Conclusion

We describe here compound heterozygous loss-of-expression mutations in CARD9 leading to severe deep and destructive mucocutaneous phaeohyphomycosis due to C. cassiicola in a Colombian child.

     

Prevalence and molecular diversity of invasive <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> in a German tertiary care medical centre

Prevalence of invasive ß-haemolytic streptococci (BHS) at a tertiary care hospital and molecular diversity of S. pyogenes and S. dysgalactiae was studied. Between 2012 and 2016, all blood culture sets (n?=?55,839), CSF (n?=?8413) and soft tissue (n = 20,926) samples were analysed for BHS positivity using HYBASE software. Molecular profiles of 99 S. pyogenes and S. dysgalactiae were identified by sequencing of M protein genes (emm types) and multiplex PCR typing of 20 other virulence determinants. Streptococci contributed to 6.2% of blood, 10.7% of CSF and 14.5% of soft tissue isolates, being among the most common invasive isolates. The overall rates of invasive S. pyogenes, S. agalactiae, S. dysgalactiae and S. pneumoniae were 2.4, 4.4, 2.1, and 5.3%. Whereas S. pneumoniae was 1.5% more common in CSF samples, BHS isolates were 2-fold and 11-fold higher in bacteraemia and invasive soft tissue infections. Genetic BHS typing revealed wide molecular diversity of invasive and noninvasive group A and group G BHS, whereas one emm-type (stG62647.0) and no other virulence determinants except scpA were detected in invasive group C BHS. BHS were important invasive pathogens, outpacing S. pneumoniae in bacteraemia and invasive soft tissue infections. The incidence of S. dysgalactiae infections was comparable to that of S. pyogenes even with less diversity of molecular virulence. The results of this study emphasise the need for awareness of BHS invasiveness in humans and the need to develop BHS prevention strategies.     

Clinical characteristics of infections caused by <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis> P1 genotypes in children

Mycoplasma pneumoniae (M. pneumoniae) isolates can be classified into two major genetic groups, P1 type 1 (MP1) and P1 type 2 (MP2), based on the DNA sequence of the P1 adhesion protein gene. The aim of our study was to determine if M. pneumoniae P1 genotype is associated with disease manifestation and severity of acute M. pneumoniae infection. We compared epidemiological and clinical data of children infected with either MP1 or MP2. In addition, we separately analysed data of patients presenting with individual manifestations of M. pneumoniae infection. Data of 356 patients infected with MP1 were compared with those of 126 patients infected with MP2. MP2-infected children presented with higher median baseline C-reactive protein levels and were admitted to the hospital more often. The distribution of P1 genotype varied among groups of patients with different manifestations of M. pneumoniae infection. MP2 was more common than MP1 among patients with neurological and cardiovascular manifestations, whereas MP1 was more prevalent in other manifestations. The results from our large cohort indicate that the two P1 subtypes may have different pathogenic potential and that infections with MP2 strains could be more virulent than those with MP1 strains.     

Monitoring of abdominal<Emphasis Type="Italic"> Staphylococcus aureus</Emphasis> infection using magnetic resonance imaging: a murine animal model for hepatic and renal abscesses

To establish a routine workflow for in vivo magnetic resonance imaging (MRI) of mice infected with bacterial biosafety level 2 pathogens and to generate a mouse model for systemic infection with Staphylococcus aureus suitable for monitoring by MRI. A self-contained acrylic glass animal bed complying with biosafety level 2 requirements was constructed. After intravenous infection with 10⁵ colony-forming units (CFU) (n?=?3), 10⁶ CFU (n?=?11) or 10⁷ CFU (n?=?6) of S. aureus strain Newman, female Balb/c mice were whole-body scanned by 7T MRI. Abdominal infections such as abscesses were visualized using a standard T2-weighted scan. Infection monitoring was performed for each animal by measurements at 1, 3, and 7 days after infection. Intravenous pathogen application led to a dose-dependent decrease in survival probability (p?=?0.03). In the group with the highest infectious dose the 7-day survival rate was 33 %. An intermediate S. aureus dose showed a survival rate of 80 %, whereas at the lowest infection dose, none of the animals died. All animals with the highest infection dose exhibited hepatic abscesses 4 days after inoculation, 80 % developed renal abscesses on the 3rd day. Mice obtaining the intermediate S. aureus load reached a plateau at day 4 with 72 % liver and 60 % renal abscess probability. No abscesses were observed in other abdominal organs at any time point. The implemented experimental setup provides a suitable and reliable in vivo MRI method to study murine abdominal infection models using BSL-2 pathogen. Systemic Staphylococcus aureus infection leads to a dose-dependent development of hepatic and renal abscesses.     

Genomic analysis reveals the presence of a class D beta-lactamase with broad substrate specificity in animal bite associated <Emphasis Type="Italic">Capnocytophaga</Emphasis> species

Capnocytophga canimorsus and Capnocytophga cynodegmi can be transmitted from cats and dogs to humans, and can cause a wide range of infections including wound infections, sepsis, or endocarditis. We and others recently discovered two new Capnocytophaga species, C. canis and C. stomatis, mainly associated with wound infections. The first-line treatment of animal bite related infections is penicillin, and in case of allergy, doxycycline and trimethoprim/sulfamethoxazole. However, there is a lack of antibiotic susceptibility patterns for animal bite associated Capnocytophaga species. Thus, we ?set out to study the antibiotic profiles against animal bite associated Capnocytophaga species isolated from wound and blood cultures after cat and dog bites and coupled the findings to whole genome sequencing data. A total of 24 strains were included in the study. Phenotypic analysis of antibiotic resistance was performed with E-tests. The web-based tool ‘Resfinder’ was used to identify resistance genes in the whole genome dataset. Two strains of C. cynodegmi and two strains of the recently discovered C. stomatis were resistant to penicillin (MIC?> 24 mg?/L) and cephalosporins (MIC?>?24 mg/?L), and three out of these strains also exhibited resistance to imipenem (MIC?=?32 mg/?L). Genomic analysis revealed that these strains carried a class D beta-lactamase gene, which has not previously been found in Capnocytophaga spp. A class D beta lactamase with broad substrate specificity was found in animal bite associated Capnocytophaga species, which could have important implications when treating wound infections after cat and dog bites. It also suggests that pet animal bacteria can harbour resistance genes with relevance for human infections.     

Use of Dense Granule Antigen GRA6 in an Immunoglobulin G Avidity Test To Exclude Acute Toxoplasma gondii Infection during Pregnancy

The usefulness of a specific immunoglobulin G (IgG) avidity enzyme-linked immunosorbent assay (ELISA) based on recombinant GRA6 antigen for distinguishing between acute and chronic Toxoplasma infection was investigated. Two sets of serum samples obtained from pregnant women with acute, chronic, or no Toxoplasma infection collected in France and Iran were used. Among the French subjects, 19 of 20 (95%) women who experienced seroconversion during the past 4 months before sampling displayed low-avidity IgG antibodies against GRA6, while all 17 (100%) women with chronic infection had high-avidity antibodies. When the Euroimmun IgG avidity ELISA was used, 15 of 19 (78.9%) recently infected women had low-avidity antibodies, and 20 of 22 (90.9%) women with chronic infection displayed high-avidity antibodies. The results suggested better performance of the GRA6 avidity ELISA than the Euroimmun avidity ELISA for exclusion of a recent infection occurring less than 4 months previously. Similarly, all 35 Iranian women with acute Toxoplasma infection had low-avidity antibodies against GRA6, whereas all 34 women with chronic infection displayed IgG antibodies of high avidity, indicating the value of GRA6 avidity testing for ruling out a recent infection. Avidity tests based on lysed whole-cell Toxoplasma gondii antigen are currently used to exclude recently acquired infections; however, the use of recombinant antigen(s) might improve the diagnostic performance of avidity tests and facilitate the development of more standardized assays.Congenital toxoplasmosis may occur when maternal infection is acquired during pregnancy and results in severe fetopathy or miscarriage (28, 38). While the rate of fetal infection with Toxoplasma gondii is extremely low in preconception infections, the transmission rate increases and the severity of fetal infection decreases as gestational age progresses (7, 18). Fortunately, prenatal treatment of the infection is effective for reducing both the incidence of clinical manifestations in infected newborns (8) and the maternal-fetal transmission rate (6, 29, 37). It is, therefore, essential to estimate the gestational age of the primary Toxoplasma infection as precisely as possible for the proper clinical management of pregnant women.Diagnosis of acute toxoplasmosis depends mainly on serological testing, as the infection is asymptomatic in 93% to 97% of pregnant women (10, 27). In countries where a prenatal screening of T. gondii infection is performed, detection of seroconversion or a significant rise in the specific immunoglobulin G (IgG) titer establishes a recently acquired infection. In most countries, however, a single serum sample from a pregnant woman is available to determine whether the infection occurred during gestation. IgM antibodies have been traditionally known as the markers of acute infection; however, persistent specific IgM has been found for up to several years after primary infection (15, 24, 39). Measurement of specific IgG avidity was shown to be an effective confirmatory test to help discriminate between recently acquired and distant infections (4, 16, 20, 22, 30, 31). In fact, it was shown that the combination of a sensitive test for T. gondii IgM and a specific IgG avidity assay is the best tool for determining the time of infection (34). The functional affinity (avidity) of specific IgG antibodies is low in the beginning of infection and usually increases with time by antigen-driven B-cell selection. However, low-avidity antibodies may persist for several months after acute infection, indicating that the avidity test is best used to rule out a recent infection in individuals with high-avidity results (5, 20, 30). Some studies reported the presence of very rare borderline- or high-avidity antibodies in acute infection with T. gondii (11, 20-22). The discordant results of avidity assays are attributed to the presence of antiparasitic treatment and immunodeficiency status; however, the complex nature of lysed whole-cell T. gondii antigen might cause such results (4, 35). Recombinant antigens might improve the performance of avidity assays for distinguishing between acute and chronic infection (1, 32, 33). Furthermore, preparation of recombinant antigens with more constant quality, compared to T. gondii antigen prepared from parasites grown either in cell culture or in mice peritoneal cavities, would facilitate development of better standardized assays (2).In this study, we developed an IgG avidity test based on recombinant GRA6 antigen and evaluated its value for differentiation between recently acquired and distant Toxoplasma infections in pregnant women. The potential usefulness of the GRA6 avidity test was compared with that of the Euroimmun IgG avidity enzyme-linked immunosorbent assay (ELISA; Euroimmun, Lübeck, Germany) for exclusion of a recent Toxoplasma infection that occurred less than 4 months before.     

Prevalence,genotypes, and risk factors of <Emphasis Type="Italic">Enterocytozoon bieneusi</Emphasis> in Asiatic black bear (<Emphasis Type="Italic">Ursus thibetanus</Emphasis>) in Yunnan Province,Southwestern China

Microsporidiosis is an important zoonotic disease, even leading to severe diarrhea. However, no information about prevalence and genotypes of Enterocytozoon bieneusi infection in Asiatic black bears in southwestern China is available. The objectives of the present study were to investigate the prevalence of E. bieneusi and to characterize their genotypes using the nested PCR amplification of the internal transcribed spacer region of the ribosomal RNA gene cluster and multilocus sequence typing (MLST). The overall prevalence of E. bieneusi was 19.75% (80/405) and the rate of E. bieneusi in Xishuangbanna (33.33%) was significantly higher than that in any other regions (Honghe, 17.65%; Dehong, 13.04%; Kunming, 0; P?=?0.01). Sequence analysis revealed that 4 known genotypes (D, n?=?2; SC02, n?=?10; SC01, n?=?5; and CHB1, n?=?4) and 13 novel genotypes (designed MJ1–MJ13) were identified. When 17, 5, 14, and 34 sequences at loci MS1, MS3, MS4, and MS7 via MLST analyses, representing 4, 4, 5, and 10 genotypes, respectively, were completed, one multilocus genotype (MLG novel-ABB1) was identified. This is the first report of E. bieneusi in Asiatic black bear in Yunnan province, Southwestern China. The results indicated the potential zoonotic risk of this parasite through the Asiatic black bear in this region and provided foundation data for preventing and controlling E. bieneusi infection of many other animals and humans in these regions.     

Reduction in fecal microbiota diversity and short-chain fatty acid producers in Methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> infected individuals as revealed by PacBio single molecule,real-time sequencing technology

Methicillin-resistant Staphylococcus aureus (MRSA) may cause potentially lethal infections. Increasing evidence suggests that the gut microbiota is associated with human health. Yet, whether patients with MRSA infections carry specific signatures in their fecal microbiota composition has not been determined. Thus, this study aimed to compare the fecal microbiota profile of MRSA-positive patients (n=15) with individuals without MRSA infection (n=15) by using the PacBio single molecule, real-time (SMRT) DNA sequencing system and real-time quantitative polymerase chain reaction (qPCR). Mann-Whitney tests and unweighted UniFrac principal coordinate analysis (PCoA) showed that the profile of fecal microbiota was apparently different between the two populations. Both the community richness and diversity were reduced in the MRSA-positive group (p<0.050). The genera Acinetobacter and Enterococcus were highly enriched in the MRSA-positive group, whereas less short-chain fatty acid (SCFA)-producing bacteria, including Butyricimonas, Faecalibacterium, Roseburia, Ruminococcus, Megamonas and Phascolarctobacterium, were detected in the MRSA-positive group. At species level, the species Acinetobacter baumannii and Bacteroides thetaiotaomicron were prevalent in the MRSA-positive group, whereas opposite trends were observed in 17 other species, such as Faecalibacterium prausnitzii, Lactobacillus rogosae, Megamonas rupellensis and Phascolarctobacterium faecium. Positive correlations were observed between Acinetobacter baumannii and erythrocyte sedimentation rate (ESR) (R=0.554, p=0.001), as well as hypersensitive C reactive protein (hsCRP) (R=0.406, p=0.026). Faecalibacterium prausnitzii was negatively associated with ESR (R=-0.545, p=0.002), hsCRP (R=-0.401, p=0.028) and total bile acids (TBA) (R=-0.364, p=0.048). In conclusion, the fecal microbiota structure was different between MRSA-positive and -negative patients. The increase in potential pathogens with the reduction of beneficial populations, such as SCFA-producing bacteria, in MRSA-positive patients may affect prognosis.     

Serodiagnostic evaluation of recombinant CdtB of <Emphasis Type="Italic">S</Emphasis>. Typhi as a potential candidate for acute typhoid

Typhoid fever caused by human restricted Salmonella typhi presents a considerable health burden on developing South-Asian nations like India. The suboptimal sensitivity and specificity associated with culture-based isolation of etiological agent and the extensively used surface antigen-based serological assays often lead to misdiagnosis and inappropriate antimicrobial treatment. The increasing reports of the emergence of resistant strains and undefined disease burden signify the critical need for an inexpensive, reliable, easy-to-use, and highly sensitive diagnostic test for typhoid fever. Utilizing S. typhi-specific and immunogenic antigens in sero-diagnostic assays could lead to precise diagnosis of acute typhoid and prompt treatment. In this study, we report cloning, expression, and purification of recombinant Cytolethal distending toxin subunit B (CdtB) of S. typhi, which is reported to be highly specific, immunogenic, and expressed only upon S. typhi infection. We further evaluated the purified recombinant CdtB for its diagnostic potential in an IgM-based indirect ELISA format using 33 human samples. Twenty-one serum samples from blood culture confirmed cases (n?=?21) of typhoid and 12 samples from healthy controls (n?=?12) were tested. The assay showed sensitivity of 100% and specificity of 83.3% respectively with positive and negative predictive values of 91.3 and 100% respectively. Efficient detection of specific IgM antibodies indicates that CdtB could be highly valuable in sero-diagnosis of acute typhoid and rapid screening of clinical samples.     

Methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> with <Emphasis Type="Italic">mecC</Emphasis>: a description of 45 human cases in southern Sweden

In 2011, a novel mecA gene homologue, mecC, was reported in isolates from both humans and dairy cattle. The epidemiology of mecC methicillin-resistant Staphylococcus aureus (MRSA) in humans is not yet well known. In this retrospective study, we present the epidemiology of human clinical cases with mecC MRSA detected in the southern part of Sweden during the period 2005–2014. A total of 45 patients with an isolate positive for mecC MRSA were included in the study. Twenty-six isolates were found before 2012 and were retrospectively tested for mecC. Nineteen isolates were detected in 2012–2014 through routine testing. Culture results, resistance patterns, Panton–Valentine leukocidin (PVL) genes, and spa types were collected from the Clinical Microbiology Laboratory. Epidemiological data were received from the database at the Regional Centre for Communicable Disease Control and the patient’s medical files. The majority of the patients with mecC MRSA were of Swedish origin, had underlying diseases, and lived in rural areas. The median age was 60 years. Of the mecC MRSA, 76 % belonged to spa types t373 and t843. The median minimum inhibitory concentration (MIC) value for oxacillin was 16 mg/L (1–64 mg/L) and only one isolate was resistant to other classes of antibiotics. The most common type of infection was skin and soft tissue infections, most often in an existing skin lesion. The patients with mecC MRSA were colonized for a short time and gave rise to few secondary cases. mecC MRSA in our region appears to have a domestic origin and mainly affects patients with underlying diseases or patients with an existing skin lesion. Our data indicate that it could be a poor colonizer.     

Cranberry proanthocyanidins have anti-biofilm properties against <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Background

Bacteria within a biofilm are phenotypically more resistant to antibiotics, desiccation, and the host immune system, making it an important virulence factor for many microbes. Cranberry juice has long been used to prevent infections of the urinary tract, which are often related to biofilm formation. Recent studies have found that the A-type proanthocyanidins from cranberries have anti-biofilm properties against Escherichia coli.

Methods

Using crystal violet biofilm staining, resazurin metabolism assays, and confocal imaging, we examined the ability of A-type proanthocyanidins (PACs) to disrupt the biofilm formation of Pseudomonas aeruginosa. We used mass spectrometry to analyze the proteomic effects of PAC treatment. We also performed synergy assays and in vitro and in vivo infections to determine whether PACs, alone and in combination with gentamicin, could contribute to the killing of P. aeruginosa and the survival of cell lines and G. mellonella.

Results

Cranberry PACs reduced P. aeruginosa swarming motility. Cranberry PACs significantly disrupted the biofilm formation of P. aeruginosa. Proteomics analysis revealed significantly different proteins expressed following PAC treatment. In addition, we found that PACs potentiated the antibiotic activity of gentamicin in an in vivo model of infection using G. mellonella.

Conclusions

Results suggest that A-type proanthocyanidins may be a useful therapeutic against the biofilm-mediated infections caused by P. aeruginosa and should be further tested.

     

Multidrug-resistant <Emphasis Type="Italic">Mycoplasma genitalium</Emphasis> infections in Europe

In Japan and Australia, multidrug-resistant Mycoplasma genitalium infections are reported with increasing frequency. Although macrolide-resistant M. genitalium strains are common in Europe and North America, fluoroquinolone-resistant strains are still exceptional. However, an increase of multidrug-resistant M. genitalium in Europe and America is to be expected. The aim of this paper is to increase awareness on the rising number of multidrug-resistant M. genitalium strains. Here, one of the first cases of infection with a genetically proven multidrug-resistant M. genitalium strain in Europe is described. The patient was a native Dutch 47-year-old male patient with urethritis. Mycoplasma genitalium was detected, but treatment failed with azithromycin, doxycycline and moxifloxacin. A urogenital sample was used to determine the sequence of the 23S rRNA, gyrA, gyrB and parC genes. The sample contained an A2059G single nucleotide polymorphism (SNP) in the 23S rRNA gene and an SNP in the parC gene, resulting in an amino acid change of Ser83 → Ile, explaining both azithromycin and moxifloxacin treatment failure. The SNPs associated with resistance were probably generated de novo, as a link with high-prevalence areas was not established. It is, thus, predictable that there is going to be an increase of multidrug-resistant M. genitalium strains in Europe. As treatment options for multidrug-resistant M. genitalium are limited, the treatment of M. genitalium infections needs to be carefully considered in order to limit the rapid increase of resistance to macrolides and fluoroquinolones.