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Rapid diagnosis and treatment are critical for patient recovery in bacterial meningitis. Delayed initiation of antibiotic therapy is associated with increased in-hospital mortality and an unfavorable outcome at discharge. We report a 31-year-old female patient presenting with a history of acute sinusitis and cefuroxime medication for 7 days. On the 7th day, the patient was admitted to the emergency department with nausea, vomiting, confusion, headache, tendency to sleep. On physical examination, the patient presented with unresponsiveness and weak neck stiffness as a sign of meningeal irritation. Cerebrospinal fluid analysis revealed 13,446 leukocytes per mm3 with 95% neutrophils and 5% lymphocytes. CSF biochemical analysis indicated low CSF glucose (2000,00 mg/L albumin. CSF RT-qPCR was positive for Streptococcus pneumoniae. The patient was immediately administered ceftriaxone and vancomycin, and recovered without any neurological sequelae. This study highlights the importance of patient follow-up, accurate prognosis and rapid laboratory testing in patients with failed sinusitis treatment.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant opportunistic pathogen with a wide repertoire of virulence characteristics. Data regarding the molecular profile of MRSA in Northern Cyprus is limited. The current study aimed to examine the virulence profiles of MRSA with a focus on toxin-associated factors. Ninety-one S. aureus isolates collected at a university hospital were included in the study. Identification and antibiotic susceptibility testing were performed with BD Phoenix™ automated system. Methicillin resistance was evaluated by the disc diffusion assay and mecA detection. The presence of nuc was confirmed by conventional PCR. Confirmed MRSA isolates were assessed for the presence of virulence genes hla, eta, etb, etd and tst using molecular methods. Among 91 S. aureus isolates identified as MRSA using the BD Phoenix™ platform, 80.85% (n = 76/91) were confirmed as MRSA using phenotypic and genotypic methods. All confirmed MRSA isolates (n = 76, 100%) were positive for the nuc. MRSA rates were statistically higher in elderly inpatients. The prevalence of toxin-encoding genes was 97.3% (n = 74/76) for hla, 2.63% (n = 2/76) for eta, 1.3% (n = 1/76) for etb, and 2.63% (n = 2/76) for tst. None of the screened isolates harbored the etd gene. These results represent the first report to investigate multiple virulence factors in MRSA isolates in Northern Cyprus.

Aim: SARS-CoV-2 variants of concern (VOCs) carry signature mutations particularly in the spike protein. Most VOCs lineages that carry N501Y substitution have been reported to evade viral diagnostic tests and have impact on vaccine effectiveness. Therefore, monitoring the circulating variants represents a major requirement for a public health response worldwide. We aimed to investigate the prevalence of N501Y bearing SARS-CoV-2 samples in Northern Cyprus. Materials & methods: Reverse transcription quantitative PCR technique was used to identify N501Y mutation from 658 samples. Results: Our results indicate that the proportion of N501Y-bearing lineages increased significantly from January through May 2021 (45.2–75.5%) in the region. Conclusion: These results indicate that VOCs are dominant lineages in the country and highlight an alarming situation which require strict governmental measures to minimize COVID-19 morbidity and mortality.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in December 2019, and shortly after pandemic has been declared by the World Health Organization (WHO) due to its unstoppable global spread. Considerable amount of effort has beenput around the World in order to develop a safe and effective vaccine against SARS-CoV-2. Inactivated and RNA vaccines have already passed phase three studies showing sufficient efficacy and safety, respectively. Nowadays, there is a noticeable dominance of SARS-CoV-2 variants with various mutations over the wild type SARS-CoV-2. However, there is no report showing the efficacy of these vaccines on these variants. This case study describes a thirty-eight-year-old male reported to be infected with SARS-CoV-2 alpha variant following two doses of inactive CoronaVac administration with a protective level of SARS-CoV-2 specific antibodies. The variant analysis of the virus reported to be positive for N501Y mutation.This is the first case in the literature demonstrating that inactive SARS-CoV-2 vaccine might have a lower efficacy on alpha variant.

Objective Natural plant products are considered as a source of novel and effective compounds for the treatment of leishmaniasis. In this study, the in vitro activities of essential oils obtained from Origanum dubium (OD), Origanum majorana (OM), Salvia fruticosa (SF) and Laurus nobilis (LN) plants in Northern Cyprus were investigated against Leishmania tropica. Methods Leishmania tropica strain (MHOM/TR/2012/CBCL-LT) was obtained. RPMI-1640 was added to 96-well plates in 100 μL aliquots, 100 μg/mL essential oil was added to the first well of each row and serial 2-fold dilutions were performed. A promastigote suspension was pipetted into all wells, and the plates were incubated. The promastigotes were enumerated using a haemocytometer. Results OD essential oil was effective at killing all promastigotes at a minimum inhibitor height (MIC)=0.2 μg/mL and had high activity at the lowest concentrations. Both SF and LN oils had MIC=1.56 μg/mL and LD50=0.78 μg/mL. SF was observed to impair promastigote morphology at the lowest concentrations, while LN did not exert any effect at concentrations

Objective: Pseudomonas aeruginosa is among the most common nosocomial opportunistic pathogens capable of acquiring multi-drug resistance. Recent data on antibiotic resistance in Northern Cyprus is limited. We aimed evaluate antibiotic susceptibility profiles of P. aeruginosa strains isolated from clinical samples and contribute to the update of antibiotic use policies in the country. Methods: Total of 504 P. aeruginosa strains sent to Microbiology Laboratory from various clinics from September 2016 to October 2019 were retrospectively analyzed. Clinical samples were cultured on %5 sheep blood agar and eosin-methylene blue agar. BD Phoenix™ 100 (Becton Dickinson Co., Sparks, Maryland, USA) automated bacterial identification system and conventional antimicrobial susceptibility testing was used. Antimicrobial susceptibility was determined in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The distribution of P. aeruginosa isolates according to hospital services, sample types, admission types, age group, and antibiotic resistance rates were investigated. Results: P. aeruginosa was mostly isolated from urine (37.3%), deep tracheal aspirate (17.9%) and abscess/wound material (17.9%) samples. Strains were most commonly obtained from the intensive care clinics (35.9%). Infections were observed mainly in >65 age group (49.6%) and in-patients (70.8%) group. Antibiotic resistance rates were detected as 47.7% for aztreonam, 32% for cefepime, 26.3% for ceftazidime, 26.4% for imipenem, 25.6% for ciprofloxacin, 24.6% for meropenem, 19.7% for piperacillin-tazobactam, 13.5% for gentamicin, 12.2% for colistin and 5.3% for amikacin. Multi-drug resistant P. aeruginosa rate was 19.2%. When compared with 2010-2014, a substantial increase in rates of cefepime, ceftazidime, ciprofloxacin, piperacillin-tazobactam and carbapenem resistance was detected within the last three years. Conclusions: In this study, the highest resistance rates were detected against aztreonam and cefepime, and the highest susceptibility was observed for colistin and amikacin. These results may be used for empirical treatments, establishment of rational antibiotic use policies and prevention of emerging antibiotic resistance problem in Cyprus.

Highlights • Of the 1001 influenza positive samples, 3 outpatients and 1 inpatient were co-infected with SARS-CoV-2 in Wuhan in January, 2020. • Clinical and laboratory features indicate the 3 outpatients showed mild symptoms, and the inpatient experienced malignant tumor and had worse outcomes from COVID-19. • In January, when the testing capacity is insufficient, co-infection of SARS-CoV-2 with influenza viruses in winter influenza activity peak season may contribute the expansion of SARS-CoV-2 in the local population.

Angiotensin-converting enzyme 2 (ACE2) is a membrane-bound zinc metallopeptidase that generates the vasodilatory peptide angiotensin 1–7 and thus performs a protective role in heart disease. It is considered an important therapeutic target in controlling the COVID-19 outbreak, since SARS-CoV-2 enters permissive cells via an ACE2-mediated mechanism. The present in silico study attempted to repurpose existing drugs for use as prospective viral-entry inhibitors targeting human ACE2. Initially, a clinically approved drug library of 7,173 ligands was screened against the receptor using molecular docking, followed by energy minimization and rescoring of docked ligands. Finally, potential binders were inspected to ensure molecules with different scaffolds were engaged in favorable contacts with both the metal cofactor and the critical residues lining the receptor’s active site. The results of the calculations suggest that lividomycin, burixafor, quisinostat, fluprofylline, pemetrexed, spirofylline, edotecarin, and diniprofylline emerge as promising repositionable drug candidates for stabilizing the closed (substrate/inhibitor-bound) conformation of ACE2, thereby shifting the relative positions of the receptor’s critical exterior residues recognized by SARS-CoV-2. This study is among the rare ones in the relevant scientific literature to search for potential ACE2 inhibitors. In practical terms, the drugs, unmodified as they are, may be introduced into the therapeutic armamentarium of the ongoing fight against COVID-19 now, or their scaffolds may serve as rich skeletons for designing novel ACE2 inhibitors in the near future., Graphical abstract Image 1, Highlights • A drug library of 7173 ligands was screened against ACE2 using molecular docking. • The predicted ACE2–ligand complexes were optimized via molecular mechanics. • On closer examination, eight promising repositionable drug candidates were proposed. • The drugs, unmodified as they are, may serve as entry inhibitors of SARS-CoV-2. • Their scaffolds may serve as rich skeletons for designing novel ACE2 inhibitors.

Aim: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated over 8 000 000 cases of the coronavirus disease 2019 (COVID-19) worldwide and has led to the implementation of strict measures in all countries. The aim of this study was to investigate the knowledge, attitude and implementation status of personal protective measures (PPMs) by ordinary citizens in Northern Cyprus and their access to personal protective equipment (PPE) during the COVID-19 outbreak.Subject and Methods: This was a cross-sectional study based on internet-based survey. A total of 406 participants were recruited between May 1 and 4, 2020 via authors’ networks to complete a questionnaire. Participants were asked to indicate how often they implemented the five PPMs recommended by the World Health Organization (WHO), their daily frequency of hand hygiene events, their COVID-19 knowledge as well as the availability of PPE in their hometown.Results: The prevalence of the five PPMs ranged from 65.0% to 95.6%, with the highest being hand hygiene and the lowest being avoiding touching the eyes, nose, and mouth. Hand hygiene events were recorded to be more than 10 per day in 47.3% of the participants. Gloves (86.2%), surgical masks (52.2%), and cloth face masks (47.3%) were the most commonly used PPE in public areas. The majority of the responders were aware of the incubation time (90.4%) and indirect route of viral transmission (89.7%), whereas knowledge of droplet (72.2%) and aerosol transmission (43.6%) were lower. Knowledge was gained mainly through social media and TV.Conclusion: Overall, citizens implemented protective measures effectively and were strictly coherent to government-induced curfew and self-isolation measurements with a high public awareness.

There are limited data regarding the potential effect of erbium, chromium: yttrium&ndash, scandium&ndash, gallium&ndash, garnet (Er,Cr:YSGG) laser-activated irrigation (LAI) on the microleakage qualities of calcium silicate-based cements. The objective of the present study was to assess the effect of LAI on the microleakage qualities of MTA Repair HP (MTA-HP) and NeoMTA Plus (Neo) used in root-end filling and to compare the antimicrobial effectiveness of MTA- HP. Two experimental sets were conducted: antimicrobial activity (agar diffusion test/at 24, 48 h) and microleakage (glucose leakage model/at 1st, 10th, 20th days). Antimicrobial activities of MTA-HP, Neo, Biodentine, ProRoot and MTA Angelus were evaluated, and inhibition zones were observed not only against a range of Gram-positive and Gram-negative bacteria but also against yeast at 48h. For microleakage evaluation, fifty teeth were prepared to simulate the clinical situation where the root-tips (apex) are open, and randomly divided into two experimental groups (n = 20/group) according to the cement type (MTA-HP and Neo), and two control (n = 5/group) groups. Each experimental group was further divided into two subgroups (n = 10/group) with respect to LAI: MTA-HP, L-HP, Neo, L-Neo. A statistical difference was only detected between Neo and L-HP groups on day 1. Subsequently, MTA-HP exhibited superior microleakage quality compared to Neo in the short-term. Er,Cr:YSGG laser-activated irrigation could be used as a reliable technique without creating adverse effects on the sealing abilities of MTA Repair HP and NeoMTA Plus.

The present study studied the dynamics of SARS-CoV-2 in Northern-Cyprus (NC) by using real data and a designed mathematical model. The model consisted of two equilibrium points, which were disease-free and epidemic. The stability of the equilibrium points was determined by the magnitude of the basic reproduction number (𝑹𝟎). If 𝑹𝟎 < 1, the disease eventually disappears, if 𝑹𝟎 ≥ 1, the presence of an epidemic is stated. 𝑹𝟎 has been calculated patient zero, with a range of 2.38 to 0.65. Currently, the 𝑹𝟎 for NC was found to be 0.65, indicating that NC is free from the SARS-CoV-2 epidemic.

The public health burden of the novel coronavirus disease 2019 (COVID-19) is expected to increase and urgent strict measures by decision-makers is critical for the containment of the novel coronavirus (SARS-CoV-2) outbreak worldwide.This study aimed to give a real-time analysis of COVID-19 presence in northern Cyprus.All official SARS-CoV-2 positive cases were tracked and reported in terms of the origin, nationality, and transmission routes. Preventive measures taken after the first reported case were analyzed for their effectiveness as control strategies.The index case of SARS-CoV-2 in northern Cyprus was identified as a female German tourist. First local case had travel history from the United Kingdom after which local transmission occurred. Rapid and strict containment measures have currently delayed a peak in observed cases.Rapid implementation of social-distancing measures, good hygiene measures and travel/gathering bans in northern Cyprus has been effective in controlling the outbreak.الحالة الراهنة لكوفيد-19 في شمال قبرص.نظيف سلطانوجلو، بوكت بادال، كايا سوار، تامر سانليداج.من المتوقع أن يزداد العبء الصحي العام لمرض فيروس كورونا المستجد 2019 (كوفيد-19)، ومن المهم أن يتخذ صانعو القرارات تدابير صارمة وعاجلة لاحتواء فاشية الفيروس التاجي المستجد (فيروس كورونا سارس- 2) في جميع أنحاء العالم.هدفت هذه الدراسة إلى تقديم تحليل آني لوجود فيروس كوفيد-19 في شمال قبرص.تُتُبعت جميع الحالات الإيجابية لفيروس كورونا سارس-2 الرسمية وأُبلغ عنها من حيث المنشأ والجنسية وطرق انتقال العدوى للمصابين. وحُللت التدابير الوقائية المتخذة بعد الإبلاغ عن الحالة الأولى من حيث فعاليتها كاستراتيجيات للمكافحة.حُددت الحالة الدالة لمتلازمة «فيروس كورونا سارس-2» في شمال قبرص وكانت لسائحة ألمانية. وكان لأول حالة محلية تاريخ سفر إلى المملكة المتحدة وبعدها حدثت العدوى محليًا. وقد أدت تدابير الاحتواء الصارمة والسريعة في الوقت الآني إلى تأخير حدوث الذروة في الحالات المُلاحَظة.إن التنفيذ السريع لتدابير التباعد الاجتماعي (البدني) وتدابير النظافة الصحية الجيدة وحظر السفر والتجمع في شمال قبرص كان فعا لً في مكافحة فاشية المرض.Situation actuelle de la COVID-19 dans la partie nord de Chypre.La charge de morbidité de la maladie à nouveau coronavirus 2019 (COVID-19) devrait augmenter. De ce fait, des mesures strictes et urgentes prises par les décideurs sont essentielles pour endiguer la flambée épidémique de nouveau coronavirus (SARS-CoV-2) dans le monde.La présente étude visait à fournir une analyse en temps réel de la présence de la COVID-19 dans le nord de Chypre.Tous les cas positifs officiels au SARS-CoV-2 ont été suivis et signalés en termes d’origine, de nationalité et de voies de transmission. Les mesures préventives prises après le premier cas notifié ont été analysées pour établir leur efficacité en tant que stratégies de lutte.Le cas indicateur de SARS-CoV-2 dans la partie nord de Chypre a été identifié comme étant une touriste allemande. Le premier cas local avait des antécédents de voyage en provenance du Royaume-Uni, ce qui a ensuite entraîné une transmission locale. Des mesures de confinement rapides et strictes ont actuellement retardé un pic dans le nombre des cas observés.La mise en oeuvre rapide de mesures de distanciation sociale, de bonnes mesures d’hygiène et l’interdiction de voyages/rassemblements dans la partie nord de Chypre ont été efficaces pour endiguer la flambée.

Introduction: Acute tonsillopharyngitis is the most important infectious cause of hospital admission in pediatric age groups. The aim of this study was to investigate the prevalence of group A beta-hemolytic streptococci in patients prediagnosed with acute tonsillopharyngitis in a university hospital in Northern Cyprus and determine the effectiveness of Streptococcus rapid antigen test (SRAT) in diagnosis. Materials and Methods: Patients with acute tonsillopharyngitis prediagnosis and admitted to our hospital between September 1, 2015 and December 31, 2017 were retrospectively screened from physical and electronic records. Patients with a throat culture and/or SRAT result were included in the study. Culture method was used as the golden standard. Statistical analysis was performed with SPSS version 13.0 (SPSS Inc., Chicago, IL, USA) programme and p

Pathogens constantly interact with their hosts and the environment, and therefore have evolved unique virulence mechanisms to target and breach host defense barriers and manipulate host immune response to establish an infection. Advances in technologies that allow genome mining, gene editing such as CRISPR/Cas9, genomic, epigenomic and transcriptomic studies such as dual RNA-seq, coupled with bioinformatics, have accelerated the field of host–pathogen interactions within a broad range of infection models. Underpinning of the molecular changes that accompany invasion of eukaryotic cells with pathogenic microorganisms at the intersection of host, pathogen and their local environment has provided a better understanding of infectious disease mechanisms and antimicrobial strategies. The recent evolution of physiologically relevant three-dimensional (3-D) tissue/organ models and microfluidic organ-on-chip devices also provided a window to a more predictive framework of infectious disease processes. These approaches combined hold the potential to highly impact discovery of novel drug targets and vaccine candidates of the future. Here, we review three of the available and emerging technologies—dual RNA-seq, CRISPR/Cas9 screening and organs-on-chips, applicable to the high throughput study and deciphering of interaction networks between pathogens and their hosts that are critical for the development of novel therapeutics.

Bioinspired organ-level in vitro platforms that recapitulate human organ physiology and organ-specific responses have emerged as effective technologies for infectious disease research, drug discovery, and personalized medicine. A major challenge in tissue engineering for infectious diseases has been the reconstruction of the dynamic 3D microenvironment reflecting the architectural and functional complexity of the human body in order to more accurately model the initiation and progression of host–microbe interactions. By bridging the gap between in vitro experimental models and human pathophysiology and providing alternatives for animal models, organ-on-chip microfluidic devices have so far been implemented in multiple research areas, contributing to major advances in the field. Given the emergence of the recent pandemic, plug-and-play organ chips may hold the key for tackling an unmet clinical need in the development of effective therapeutic strategies. In this review, latest studies harnessing organ-on-chip platforms to unravel host–pathogen interactions are presented to highlight the prospects for the microfluidic technology in infectious diseases research.

Nontypeable Haemophilus influenzae (NTHi) is a common airway commensal and opportunistic pathogen that persists within biofilm communities in vivo. Biofilm studies so far are mainly based on assays on plastic surfaces. The aim of this work was to investigate the capacity of clinical NTHi strains to form biofilm structures on polarized Calu-3 human airway epithelial cells and primary normal human bronchial epithelial cells and to characterize the biofilm architecture. Formation of adherent NTHi biofilms post colonization of host cells at multiple time-points was evaluated using confocal laser scanning microscopy and electron microscopy. NTHi biofilms were analyzed in terms of biofilm height and presence of extracellular matrix components, and their apoptotic effects on epithelial cells were measured by TUNEL assay. Strain Fi176 was observed to form robust biofilms on airway epithelia over time, while disrupting the integrity of Calu-3 monolayer by 72 h of co-culture. NTHi biofilms were observed to induce apoptotic DNA fragmentation in host cells at 24 h post infection. Biofilm formation on cell monolayers by Fi176ΔpilA strain was markedly reduced compared to WT strain. Biofilm inhibition and disruption assays by crystal violet staining indicated that DNA and proteins are part of NTHi biofilms in vitro. Our findings highlight critical stages of NTHi pathogenesis following host colonization and provide useful biofilm models for future antimicrobial drug discovery investigations.

The globally dominant, invasive M1T1 strain of group A Streptococcus (GAS) harbors polymorphisms in the promoter region of an operon that contains the genes encoding streptolysin O (SLO) and NAD+-glycohydrolase (NADase), resulting in high-level expression of these toxins. While both toxins have been shown experimentally to contribute to pathogenesis, many GAS isolates lack detectable NADase activity. DNA sequencing of such strains has revealed that reduced or absent enzymatic activity can be associated with a variety of point mutations in nga, the gene encoding NADase; a commonly observed polymorphism associated with near-complete abrogation of activity is a substitution of aspartic acid for glycine at position 330 (G330D). However, nga has not been observed to contain early termination codons or mutations that would result in a truncated protein, even when the gene product contains missense mutations that abrogate enzymatic activity. It has been suggested that NADase that lacks NAD-glycohydrolase activity retains an as-yet-unidentified inherent cytotoxicity to mammalian cells and thus is still a potent virulence factor. We now show that expression of NADase, either enzymatically active or inactive, augments SLO-mediated toxicity for keratinocytes. In culture supernatants, SLO and NADase are mutually interdependent for protein stability. We demonstrate that the two proteins interact in solution and that both the translocation domain and catalytic domain of NADase are required for maximal binding between the two toxins. We conclude that binding of NADase to SLO stabilizes both toxins, thereby enhancing GAS virulence., IMPORTANCE The global increase in invasive GAS infections in the 1980s was associated with the emergence of an M1T1 clone that harbors a 36-kb pathogenicity island, which codes for increased expression of toxins SLO and NADase. Polymorphisms in NADase that render it catalytically inactive can be detected in clinical isolates, including invasive strains. However, such isolates continue to produce full-length NADase. The rationale for this observation is not completely understood. This study characterizes the binding interaction between NADase and SLO and reports that the expression of each toxin is crucial for maximal expression and stability of the other. By this mechanism, the presence of both toxins increases toxicity to keratinocytes and is predicted to enhance GAS survival in the human host. These observations provide an explanation for conservation of full-length NADase expression even when it lacks enzymatic activity and suggest a critical role for binding of NADase to SLO in GAS pathogenesis.

The ability to adhere and adapt to the human respiratory tract mucosa plays a pivotal role in the pathogenic lifestyle of nontypeable Haemophilus influenzae (NTHi). However, the temporal events associated with a successful colonization have not been fully characterized. In this study, by reconstituting the ciliated human bronchial epithelium in vitro, we monitored the global transcriptional changes in NTHi and infected mucosal epithelium simultaneously for up to 72 h by dual RNA sequencing. The initial stage of colonization was characterized by the binding of NTHi to ciliated cells. Temporal profiling of host mRNA signatures revealed significant dysregulation of the target cell cytoskeleton elicited by bacterial infection, with a profound effect on the intermediate filament network and junctional complexes. In response to environmental stimuli of the host epithelium, NTHi downregulated its central metabolism and increased the expression of transporters, indicating a change in the metabolic regime due to the availability of host substrates. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms, including the transport of exogenous glutathione and activation of the toxin-antitoxin system. The results of this analysis were validated by those of confocal microscopy, Western blotting, Bio-plex, and real-time quantitative reverse transcription-PCR (qRT-PCR). Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs (sRNAs) regulated during human host infection in Haemophilus species. Our data, by providing a robust and comprehensive representation of the cross talk between the host and invading pathogen, provides important insights into NTHi pathogenesis and the development of efficacious preventive strategies.Simultaneous monitoring of infection-linked transcriptome alterations in an invading pathogen and its target host cells represents a key strategy for identifying regulatory responses that drive pathogenesis. In this study, we report the progressive events of NTHi colonization in a highly differentiated model of ciliated bronchial epithelium. Genome-wide transcriptome maps of NTHi during infection provided mechanistic insights into bacterial adaptive responses to the host niche, with modulation of the central metabolism as an important signature of the evolving milieu. Our data indicate that infected epithelia respond by substantial alteration of the cytoskeletal network and cytokine repertoire, revealing a dynamic cross talk that is responsible for the onset of inflammation. This work significantly enhances our understanding of the means by which NTHi promotes infection on human mucosae and reveals novel strategies exploited by this important pathogen to cause invasive disease.

Outbreak strains of Acinetobacter baumannii are highly clonal, and cross-infection investigations can be difficult. We sought targets based on AbaR resistance islands and on other genes found in some, but not all, sequenced isolates of A. baumannii among a set of clinical isolates ( n = 70) that included multiple representatives of a number of pulsed-field gel electrophoresis (PFGE)-defined types. These included representatives that varied in their profiles at two variable-number tandem repeat (VNTR) loci, which can provide discrimination within a PFGE cluster. Detection, or not, of each element sought provided some degree of discrimination among the set, with the presence or absence of genes coding for a phage terminase (ACICU_02185), a sialic acid synthase (ACICU_00080), a polysaccharide biosynthesis protein (AB57_0094), aphA1 , bla TEM , and integron-associated orfX (Kyoto Encyclopedia of Genes and Genomes [KEGG] no. K03830) proving the most helpful in discriminating between closely related isolates in our panel. The results support VNTR data in describing distinct populations of highly similar isolates. Such analysis, in combination with other typing methods, can inform epidemiological investigations and provide additional characterization of isolates. Most genotypes carrying bla OXA-23-like were PCR positive for a yeeA-bla OXA-23 fragment found in an AbaR4-type island, suggesting that this is widespread.

The ability to adhere and adapt to the human respiratory tract mucosa plays a pivotal role in the pathogenic lifestyle of nontypeable Haemophilus influenzae (NTHi). However, the temporal events associated with a successful colonization have not been fully characterized. In this study, by reconstituting the ciliated human bronchial epithelium in vitro, we monitored the global transcriptional changes in NTHi and infected mucosal epithelium simultaneously for up to 72 h by dual RNA sequencing. The initial stage of colonization was characterized by the binding of NTHi to ciliated cells. Temporal profiling of host mRNA signatures revealed significant dysregulation of the target cell cytoskeleton elicited by bacterial infection, with a profound effect on the intermediate filament network and junctional complexes. In response to environmental stimuli of the host epithelium, NTHi downregulated its central metabolism and increased the expression of transporters, indicating a change in the metabolic regime due to the availability of host substrates. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms, including the transport of exogenous glutathione and activation of the toxin-antitoxin system. The results of this analysis were validated by those of confocal microscopy, Western blotting, Bio-plex, and real-time quantitative reverse transcription-PCR (qRT-PCR). Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs (sRNAs) regulated during human host infection in Haemophilus species. Our data, by providing a robust and comprehensive representation of the cross talk between the host and invading pathogen, provides important insights into NTHi pathogenesis and the development of efficacious preventive strategies., IMPORTANCE Simultaneous monitoring of infection-linked transcriptome alterations in an invading pathogen and its target host cells represents a key strategy for identifying regulatory responses that drive pathogenesis. In this study, we report the progressive events of NTHi colonization in a highly differentiated model of ciliated bronchial epithelium. Genome-wide transcriptome maps of NTHi during infection provided mechanistic insights into bacterial adaptive responses to the host niche, with modulation of the central metabolism as an important signature of the evolving milieu. Our data indicate that infected epithelia respond by substantial alteration of the cytoskeletal network and cytokine repertoire, revealing a dynamic cross talk that is responsible for the onset of inflammation. This work significantly enhances our understanding of the means by which NTHi promotes infection on human mucosae and reveals novel strategies exploited by this important pathogen to cause invasive disease.

Background Non-typeable Haemophilus influenzae (NTHi) is a Gram negative microorganism residing in the human nasopharyngeal mucosa and occasionally causing infections of both middle ear and lower respiratory airways. A broadly protective vaccine against NTHi has been a long-unmet medical need, as the high genetic variability of this bacterium has posed great challenges. Results In this study, we developed a robust serum bactericidal assay (SBA) to optimize the selection of protective antigens against NTHi. SBA takes advantage of the complement-mediated lysis of bacterial cells and is a key in vitro method for measuring the functional activity of antibodies. As a proof of concept, we assessed the bactericidal activity of antibodies directed against antigens known to elicit a protective response, including protein D used as carrier protein in the Synflorix pneumococcal polysaccharide conjugate vaccine. Prior to SBA screening, the accessibility of antigens to antibodies and the capacity of the latter to induce C3 complement deposition was verified by flow cytometry. Using baby rabbit serum as a source of complement, the proposed assay not only confirmed the bactericidal activity of the antibodies against the selected vaccine candidates, but also showed a significant reproducibility. Conclusions Considering the rapidity and cost-effectiveness of this novel SBA protocol, we conclude that it is likely to become an important tool to prove the capability of antibodies directed against recombinant antigens to induce NTHi in vitro killing and to both select new protective vaccine candidates, and predict vaccine efficacy. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0420-x) contains supplementary material, which is available to authorized users.

Variable number tandem repeat (VNTR) analysis at nine loci of isolates of Pseudomonas aeruginosa submitted to the national reference laboratory from UK hospitals, from over 2000 patients, between June 2010 and June 2012 revealed four widely found types that collectively were received from approximately a fifth of patients, including from those with cystic fibrosis. These types were also prevalent among related submissions from the clinical environment and were received from up to 54 (out of 143) hospitals. Multi-locus sequence typing and bla OXA-50-like sequencing confirmed the clonal relationship within each cluster, and representatives from multiple centres clustered within about 70 % by pulsed-field gel electrophoresis. Illumina sequencing of 12 isolates of cluster A of VNTR profile 8, 3, 4, 5, 2, 3, 5, 2, x (where the repeat number at the last, most discriminatory locus is variable) revealed a large number of variably present targets in the accessory genome and seven of these were sought by PCR among a larger set of isolates. Representatives from patients within a single centre mostly had distinct accessory gene profiles, suggesting that these patients acquired the strain independently, while those with clear epidemiological links shared the same profile. Profiles also varied between representatives from different centres. Epidemiological investigations of widely found types such as these require the use of finer-typing methods, which increasingly will be informed by next generation sequencing.

Pseudomonas infections are an important cause of morbidity and mortality in immunocompromised patients. We present here data for the spread of Pseudomonas fluorescens caused by a contaminated drinking water dispenser in a bone marrow transplant unit. Over a 1-month period we observed a sharp increase in the isolation of P. fluorescens from weekly pharyngeal surveillance swabs. Environmental samples were taken from a variety of water sources throughout the unit. These samples were cultured on cetrimide agar medium, and isolates were epidemiologically characterized by antibiotic susceptibility patterns and molecular typing methods. Nine patients became colonized with P. fluorescens , and six out of the nine developed febrile neutropenia. P. fluorescens was cultured after the filtration of 100 ml of drinking water from one of two stand-alone chiller units supplying cooled bottled water to the bone marrow transplant unit. All other environmental samples were negative. There were no further cases of P. fluorescens colonization after the contaminated dispenser was removed. Molecular typing showed that all P. fluorescens isolates were identical by both random amplification of polymorphic DNA PCR and pulsed-field gel electrophoresis. We recommend that such bottled water supplies not be used in high-risk areas or be subject to regular microbiological monitoring.