The survival of epidemic and sporadic MRSA on human skin mimics is determined by both host and bacterial factors.

Bacterial survival on, and interactions with, human skin may explain the epidemiological success of MRSA strains. We evaluated the bacterial counts for 27 epidemic and 31 sporadic MRSA strains on 3D epidermal models based on N/TERT cells (NEMs) after 1, 2 and 8 days. In addition, the expression of antimicrobial peptides (hBD-2, RNase 7), inflammatory cytokines (IL-1ß, IL-6) and chemokine IL-8 by NEMs was assessed using immunoassays and the expression of 43 S. aureus virulence factors was determined by a multiplex competitive Luminex assay. To explore donor variation, bacterial counts for five epidemic and seven sporadic MRSA strains were determined on 3D primary keratinocyte models (LEMs) from three human donors. Bacterial survival was comparable on NEMs between the two groups, but on LEMs, sporadic strains showed significantly lower survival numbers compared to epidemic strains. Both groups triggered the expression of immune factors. Upon interaction with NEMs, only the epidemic MRSA strains expressed pore-forming toxins, including alpha-hemolysin (Hla), gamma-hemolysin (HlgB), Panton-Valentine leucocidin (LukS) and LukED. Together, these data indicate that the outcome of the interaction between MRSA and human skin mimics, depends on the unique combination of bacterial strain and host factors.

VNTR confirms the heterogeneity of Madurella mycetomatis and is a promising typing tool for this mycetoma causing agent.

The neglected tropical disease mycetoma is a chronic granulomatous inflammatory and infectious disease affecting various body parts. The most common causative agent is the fungus Madurella mycetomatis. In order to study the genetic diversity of this fungus and to monitor any potential outbreaks, a good typing method that can be used in endemic settings is needed. Previous typing methods developed were not discriminative and not easy to perform in resource-limited laboratories. Variable-Number-Tandem-Repeat (VNTR) typing overcomes these difficulties and further enables interlaboratory data comparison. Therefore, in this study we developed a VNTR method for typing M. mycetomatis. Six tandem-repeats were identified in the genome of M. mycetomatis isolate MM55 using an online tandem repeats software. The variation in these repeats was determined by PCR and gel-electrophoresis on DNA obtained from 81 M. mycetomatis isolates obtained from patients. These patients originated from Sudan, Mali, Peru, and India. The 81 isolates were divided into 14 genotypes which separated into two main clusters with seven and five subdivisions, respectively. VNTR typing confirms the heterogeneity of M. mycetomatis strains and can be used to study the epidemiology of M. mycetomatis. The results presented in this article are made fully available to the scientific community on request from the Eumycetoma Working Group. We hope that this open resource approach will bridge scientific community working with mycetoma from all around the world and lead to a deeper understanding of M. mycetomatis.

Campylobacter jejuni translocation across intestinal epithelial cells is facilitated by ganglioside-like lipooligosaccharide structures.

Translocation across intestinal epithelial cells is an established pathogenic feature of the zoonotic bacterial species Campylobacter jejuni. The number of C. jejuni virulence factors known to be involved in translocation is limited. In the present study, we investigated whether sialylation of C. jejuni lipooligosaccharide (LOS) structures, generating human nerve ganglioside mimics, is important for intestinal epithelial translocation. We here show that C. jejuni isolates expressing ganglioside-like LOS bound in larger numbers to the Caco-2 intestinal epithelial cells than C. jejuni isolates lacking such structures. Next, we found that ganglioside-like LOS facilitated endocytosis of bacteria into Caco-2 cells, as visualized by quantitative microscopy using the early and late endosomal markers early endosome-associated protein 1 (EEA1), Rab5, and lysosome-associated membrane protein 1 (LAMP-1). This increased endocytosis was associated with larger numbers of surviving and translocating bacteria. Next, we found that two different intestinal epithelial cell lines (Caco-2 and T84) responded with an elevated secretion of the T-cell attractant CXCL10 to infection by ganglioside-like LOS-expressing C. jejuni isolates. We conclude that C. jejuni translocation across Caco-2 cells is facilitated by ganglioside-like LOS, which is of clinical relevance since C. jejuni ganglioside-like LOS-expressing isolates are linked with severe gastroenteritis and bloody stools in C. jejuni-infected patients.

The Role of the Respiratory Microbiome and Viral Presence in Lower Respiratory Tract Infection Severity in the First Five Years of Life.

Lower respiratory tract infections (LRTIs) in children are common and, although often mild, a major cause of mortality and hospitalization. Recently, the respiratory microbiome has been associated with both susceptibility and severity of LRTI. In this current study, we combined respiratory microbiome, viral, and clinical data to find associations with the severity of LRTI. Nasopharyngeal aspirates of children aged one month to five years included in the STRAP study (Study to Reduce Antibiotic prescription in childhood Pneumonia), who presented at the emergency department (ED) with fever and cough or dyspnea, were sequenced with nanopore 16S-rRNA gene sequencing and subsequently analyzed with hierarchical clustering to identify respiratory microbiome profiles. Samples were also tested using a panel of 15 respiratory viruses and Mycoplasma pneumoniae, which were analyzed in two groups, according to their reported virulence. The primary outcome was hospitalization, as measure of disease severity. Nasopharyngeal samples were isolated from a total of 167 children. After quality filtering, microbiome results were available for 54 children and virology panels for 158 children. Six distinct genus-dominant microbiome profiles were identified, with Haemophilus-, Moraxella-, and Streptococcus-dominant profiles being the most prevalent. However, these profiles were not found to be significantly associated with hospitalization. At least one virus was detected in 139 (88%) children, of whom 32.4% had co-infections with multiple viruses. Viral co-infections were common for adenovirus, bocavirus, and enterovirus, and uncommon for human metapneumovirus (hMPV) and influenza A virus. The detection of enteroviruses was negatively associated with hospitalization. Virulence groups were not significantly associated with hospitalization. Our data underlines high detection rates and co-infection of viruses in children with respiratory symptoms and confirms the predominant presence of Haemophilus-, Streptococcus-, and Moraxella-dominant profiles in a symptomatic pediatric population at the ED. However, we could not assess significant associations between microbiome profiles and disease severity measures.

Campylobacter jejuni Cas9 Modulates the Transcriptome in Caco-2 Intestinal Epithelial Cells.

The zoonotic human pathogen Campylobacter jejuni is known for its ability to induce DNA-damage and cell death pathology in humans. The molecular mechanism behind this phenomenon involves nuclear translocation by Cas9, a nuclease in C. jejuni (CjeCas9) that is the molecular marker of the Type II CRISPR-Cas system. However, it is unknown via which cellular pathways CjeCas9 drives human intestinal epithelial cells into cell death. Here, we show that CjeCas9 released by C. jejuni during the infection of Caco-2 human intestinal epithelial cells directly modulates Caco-2 transcriptomes during the first four hours of infection. Specifically, our results reveal that CjeCas9 activates DNA damage (p53, ATM (Ataxia Telangiectasia Mutated Protein)), pro-inflammatory (NF-κB (Nuclear factor-κB)) signaling and cell death pathways, driving Caco-2 cells infected by wild-type C. jejuni, but not when infected by a cas9 deletion mutant, towards programmed cell death. This work corroborates our previous finding that CjeCas9 is cytotoxic and highlights on a RNA level the basal cellular pathways that are modulated.

Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota.

Illumina and nanopore sequencing technologies are powerful tools that can be used to determine the bacterial composition of complex microbial communities. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. We also monitored the progression of nanopore sequencing in the accurate identification of species, using pure, single species cultures, and evaluated the performance of the nanopore EPI2ME 16S data analysis pipeline. Fifty-nine nasal swabs were sequenced using Illumina MiSeq and Oxford Nanopore 16S rRNA gene sequencing technologies. In addition, five pure cultures of relevant bacterial species were sequenced with the nanopore sequencing technology. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. Albacore and Guppy base calling, a workflow in nanopore EPI2ME (Oxford Nanopore Technologies-ONT, Oxford, UK) and an in-house developed bioinformatics script were used to analyze the nanopore data. At genus level, similar bacterial diversity profiles were found, and five main and established genera were identified by both platforms. However, probably due to mismatching of the nanopore sequence primers, the nanopore sequencing platform identified Corynebacterium in much lower abundance compared to Illumina sequencing. Further, when using default settings in the EPI2ME workflow, almost all sequence reads that seem to belong to the bacterial genus Dolosigranulum and a considerable part to the genus Haemophilus were only identified at family level. Nanopore sequencing of single species cultures demonstrated at least 88% accurate identification of the species at genus and species level for 4/5 strains tested, including improvements in accurate sequence read identification when the basecaller Guppy and Albacore, and when flowcell versions R9.4 (Oxford Nanopore Technologies-ONT, Oxford, UK) and R9.2 (Oxford Nanopore Technologies-ONT, Oxford, UK) were compared. In conclusion, the current study shows that the nanopore sequencing platform is comparable with the Illumina platform in detection bacterial genera of the nasal microbiota, but the nanopore platform does have problems in detecting bacteria within the genus Corynebacterium. Although advances are being made, thorough validation of the nanopore platform is still recommendable.

Guide-free Cas9 from pathogenic Campylobacter jejuni bacteria causes severe damage to DNA.

CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, Campylobacter jejuni secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of Streptococcus pyogenes (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications.

Optical fingerprinting in bacterial epidemiology: Raman spectroscopy as a real-time typing method.

Hospital-acquired infections (HAI) increase morbidity and mortality and constitute a high financial burden on health care systems. An effective weapon against HAI is early detection of potential outbreaks and sources of contamination. Such monitoring requires microbial typing with sufficient reproducibility and discriminatory power. Here, a microbial-typing method is presented, based on Raman spectroscopy. This technique provides strain-specific optical fingerprints in a few minutes instead of several hours to days, as is the case with genotyping methods. Although the method is generally applicable, we used 118 Staphylococcus aureus isolates to illustrate that the discriminatory power matches that of established genotyping techniques (numerical index of diversity [D]=0.989) and that concordance with the gold standard (pulsed-field gel electrophoresis) is high (95%). The Raman clustering of isolates was reproducible to the strain level for five independent cultures, despite the various culture times from 18 h to 24 h. Furthermore, this technique was able to classify stored (-80 degrees C) and recent isolates of a methicillin-resistant Staphylococcus aureus-colonized individual during surveillance studies and did so days earlier than established genotyping techniques did. Its high throughput and ease of use make it suitable for use in routine diagnostic laboratory settings. This will set the stage for continuous, automated, real-time epidemiological monitoring of bacterial infections in a hospital, which can then be followed by timely corrective action by infection prevention teams.

Clinical and molecular epidemiologic characteristics of coagulase-negative staphylococcal bloodstream infections in intensive care neonates.

OBJECTIVES: This study aimed to determine clinical characteristics of coagulase-negative staphylococcal (CoNS) sepsis in neonates, to assess the molecular epidemiology and biofilm forming properties of isolated strains, and to assess antibiotic susceptibility of clonal compared with incidentally occurring strains. METHODS: We performed a retrospective study on late-onset CoNS sepsis in infants in the neonatal intensive care unit of a Dutch university hospital in 2003. CoNS isolates were genotyped by restriction fragment end labeling and pulsed-field gel electrophoresis. Resistance profiles, biofilm production, and the presence of mecA and icaA were determined. RESULTS: Twenty-six percent of all 339 infants developed late-onset sepsis, 66% of these with CoNS sepsis. Eighty-six percent of all CoNS sepsis occurred in very low birth weight infants. Sixty-six CoNS strains were isolated. In multivariate analysis, small for gestational age and prolonged hospitalization were associated with CoNS sepsis. Among 3 restriction fragment end labeling clusters, we found 1 large cluster comprising 32% of the isolates. Biofilm producing Staphylococcus epidermidis were more frequently icaA positive than nonbiofilm formers (74% vs. 12%; P < 0.001). In other species, this association was not found. Nearly all isolates were resistant to antibiotics. MecA was present in 87% of the isolates. Multiresistance occurred in 77% of all strains and in 73% of clustered strains. There was significantly less multiresistance among the largest cluster. CONCLUSIONS: Small for gestational age and prolonged hospitalization were associated with CoNS sepsis. The icaA gene is a predictor for biofilm formation in S. epidermidis, but not in other species. Multiresistance is not associated with clonality.

Comparison of multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) for genetic typing of Staphylococcus aureus.

We compared multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) for typing of Staphylococcus aureus and show that the methods yield similar results, although with differences in resolving power and reproducibility. Epidemiological conditions should determine which is the optimal typing method to be used.

5 years of experience implementing a methicillin-resistant Staphylococcus aureus search and destroy policy at the largest university medical center in the Netherlands.

OBJECTIVE: To evaluate the effectiveness of a rigorous search and destroy policy for controlling methicillin-resistant Staphylococcus aureus (MRSA) infection or colonization. DESIGN: Hospital-based observational follow-up study. SETTING: Erasmus University Medical Center Rotterdam, a 1,200-bed tertiary care center in Rotterdam, the Netherlands. METHODS: Outbreak control was accomplished by the use of active surveillance cultures for persons at risk, by the preemptive isolation of patients at risk, and by the strict isolation of known MRSA carriers and the eradication of MRSA carriage. For unexpected cases of MRSA colonization or infection, patients placed in strict isolation or contact isolation and healthcare workers (HCWs) were screened. We collected data from 2000-2004. RESULTS: During the 5-year study period, 51,907 MRSA screening cultures were performed for 21,598 persons at risk (8,403 patients and 13,195 HCWs). By screening, it was determined that 123 (1.5%) of 8,403 patients and 31 (0.2%) of 13,195 HCWs were MRSA carriers. From the performance of clinical cultures, it was determined that 54 additional patients were MRSA carriers, resulting in a total of 177 patients carrying MRSA. Of the 177 patients carrying MRSA, 144 (81%) were primary patients, and 33 (19%) secondary patients. The average number of nosocomial transmissions was 6.7 per year. The cumulative incidence of MRSA colonization among this group of patients was 0.10 cases per 100 admissions. Of 156 cases of MRSA colonization, 44 (28%) were acquired in a foreign healthcare institution, and 45 (29%) were acquired in other Dutch hospitals, 22 (47%) of which were acquired in a single hospital in our region. There were 16 cases (10%) that occurred in a nursing home and another 16 cases (10%) that fulfilled our definition of community-acquired MRSA colonization; there were 4 cases (3%) categorized as "other" and 31 cases (20%) for which the source of MRSA acquisition remained unknown. The basic reproduction rate was 10-fold less for patients isolated on admission, compared with those who were not. During the 5-year study period, 5 episodes of MRSA bacteremia occurred in which 4 patients died, an incidence rate of 0.28 cases of infection per 100,000 patient-days per year. CONCLUSION: Our results show that, during a rigorous search and destroy policy, a low incidence of MRSA in our medical center was continuously observed and that this policy most likely contributed to a very low nosocomial transmission rate.

Candida krusei transmission among hematology patients resolved by adapted antifungal prophylaxis and infection control measures.

A sudden increase in neutropenic hematology patients with Candida krusei colonization and bacteremia prompted a longitudinal epidemiological investigation. We identified 39 patients; 13 developed candidemia, and three died; 25 patients carried the same genotype. We intervened by changing antifungal prophylaxis and implementing strict infection control measures. The incidence dropped immediately.