Improving tuberculosis surveillance by detecting international transmission using publicly available whole genome sequencing data.

IntroductionImproving the surveillance of tuberculosis (TB) is especially important for multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. The large amount of publicly available whole genome sequencing (WGS) data for TB gives us the chance to re-use data and to perform additional analyses at a large scale.AimWe assessed the usefulness of raw WGS data of global MDR/XDR Mycobacterium tuberculosis isolates available from public repositories to improve TB surveillance.MethodsWe extracted raw WGS data and the related metadata of M. tuberculosis isolates available from the Sequence Read Archive. We compared this public dataset with WGS data and metadata of 131 MDR- and XDR M. tuberculosis isolates from Germany in 2012 and 2013.ResultsWe aggregated a dataset that included 1,081 MDR and 250 XDR isolates among which we identified 133 molecular clusters. In 16 clusters, the isolates were from at least two different countries. For example, Cluster 2 included 56 MDR/XDR isolates from Moldova, Georgia and Germany. When comparing the WGS data from Germany with the public dataset, we found that 11 clusters contained at least one isolate from Germany and at least one isolate from another country. We could, therefore, connect TB cases despite missing epidemiological information.ConclusionWe demonstrated the added value of using WGS raw data from public repositories to contribute to TB surveillance. Comparing the German with the public dataset, we identified potential international transmission events. Thus, using this approach might support the interpretation of national surveillance results in an international context.

Assessment of the use and need for an integrated molecular surveillance of tuberculosis: an online survey in Germany.

BACKGROUND: The implementation of an integrated molecular surveillance (IMS) of tuberculosis (TB) is of high priority for TB control. IMS is defined as the systematic inclusion of molecular typing results in the national TB surveillance system. Although not standardized, an IMS of TB is already implemented in several low TB incidence countries. Germany is in the process of implementing a nationwide IMS of TB. This requires close collaboration between national and local health authorities. We conducted an online survey to understand the current use of molecular typing results for TB surveillance among the local public health offices (PHO)s in Germany, and to collect their perception and expectations towards the implementation of a nationwide IMS of TB. METHODS: The online survey was developed using the software Voxco and included 31 questions. The survey was sent to all the 377 local PHOs in Germany in April 2017. Responses were collected until June 2017. RESULTS: A total of 174/377 (46.2%) local PHOs participated in our survey, and 88/377 (23.3%) used molecular typing results in their routine TB surveillance work. The PHOs used molecular typing results especially as support for epidemiological contact tracing (62/88, 70.4%). We found statistically significant differences between answers of PHOs that did not use molecular typing results (n = 86) vs. PHOs that did use molecular typing results (n = 88): the latter perceived the use of molecular typing results as more beneficial for their work compared to the former (65.9% vs. 34.9%, p < 0.05). Moreover, the PHOs using molecular typing results expect for the future more support and coordination from regional and national public health institutes, especially regarding the identification and analysis of molecular clusters. CONCLUSIONS: Our study is a step forward in the broader goal of implementing an IMS of TB in Germany. The local PHOs currently using the molecular typing results highlighted their positive attitude towards the implementation of an IMS, but also their needs of more support. Similar assessments might serve as an example for other countries which are on the way to implement a nationwide IMS of TB.

A hypervariable genomic island identified in clinical and environmental Mycobacterium avium subsp. hominissuis isolates from Germany.

Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic human pathogen widespread in the environment. Genomic islands (GI)s represent a part of the accessory genome of bacteria and influence virulence, drug-resistance or fitness and trigger bacterial evolution. We previously identified a novel GI in four MAH genomes. Here, we further explored this GI in a larger collection of MAH isolates from Germany (n=41), including 20 clinical and 21 environmental isolates. Based on comparative whole genome analysis, we detected this GI in 39/41 (95.1%) isolates. Although all these GIs integrated in the same insertion hotspot, there is high variability in the genetic structure of this GI: eight different types of GI have been identified, designated A-H (sized 6.2-73.3kb). These GIs were arranged as single GI (23/41, 56.1%), combination of two different GIs (14/41, 34.1%) or combination of three different GIs (2/41, 4.9%) in the insertion hotspot. Moreover, two GI types shared more than 80% sequence identity with sequences of M. canettii, responsible for Tuberculosis. A total of 253 different genes were identified in all GIs, among which the previously documented virulence-related genes mmpL10 and mce. The diversity of the GI and the sequence similarity with other mycobacteria suggests cross-species transfer, involving also highly pathogenic species. Shuffling of potential virulence genes such as mmpL10 via this GI may create new pathogens that can cause future outbreaks.

Hydrogen production under salt stress conditions by a freshwater Rhodopseudomonas palustris strain.

Hydrogen represents a possible alternative energy carrier to face the growing request for energy and the shortage of fossil fuels. Photofermentation for the production of H2 constitutes a promising way for integrating the production of energy with waste treatments. Many wastes are characterized by high salinity, and polluted seawater can as well be considered as a substrate. Moreover, the application of seawater for bacterial culturing is considered cost-effective. The aims of this study were to assess the capability of the metabolically versatile freshwater Rhodopseudomonas palustris 42OL of producing hydrogen on salt-containing substrates and to investigate its salt stress response strategy, never described before. R. palustris 42OL was able to produce hydrogen in media containing up to 3 % added salt concentration and to grow in media containing up to 4.5 % salinity without the addition of exogenous osmoprotectants. While the hydrogen production performances in absence of sea salts were higher than in their presence, there was no significant difference in performances between 1 and 2 % of added sea salts. Nitrogenase expression levels indicated that the enzyme was not directly inhibited during salt stress, but a regulation of its expression may have occurred in response to salt concentration increase. During cell growth and hydrogen production in the presence of salts, trehalose was accumulated as a compatible solute; it protected the enzymatic functionality against salt stress, thus allowing hydrogen production. The possibility of producing hydrogen on salt-containing substrates widens the range of wastes that can be efficiently used in production processes.

Molecular typing of toxic shock syndrome toxin-1- and Enterotoxin A-producing methicillin-sensitive Staphylococcus aureus isolates from an outbreak in a neonatal intensive care unit.

Outbreaks of Staphylococcus aureus are common in neonatal intensive care units (NICUs). Usually they are documented for methicillin-resistant strains, while reports involving methicillin-susceptible S. aureus (MSSA) strains are rare. In this study we report the epidemiological and molecular investigation of an MSSA outbreak in a NICU among preterm neonates. Infection control measures and interventions were commissioned by the Local Public Health Authority and supported by the Robert Koch Institute. To support epidemiological investigations molecular typing was done by spa-typing and Multilocus sequence typing; the relatedness of collected isolates was further elucidated by DNA SmaI-macrorestriction, microarray analysis and bacterial whole genome sequencing. A total of 213 neonates, 123 healthcare workers and 205 neonate parents were analyzed in the period November 2011 to November 2012. The outbreak strain was characterized as a MSSA spa-type t021, able to produce toxic shock syndrome toxin-1 and Enterotoxin A. We identified seventeen neonates (of which two died from toxic shock syndrome), four healthcare workers and three parents putatively involved in the outbreak. Whole-genome sequencing permitted to exclude unrelated cases from the outbreak and to discuss the role of healthcare workers as a reservoir of S. aureus on the NICU. Genome comparisons also indicated the presence of the respective clone on the ward months before the first colonized/infected neonates were detected.

Molecular typing of clinical Cryptococcus neoformans isolates collected in Germany from 2004 to 2010.

Cryptococcosis is a fungal infection mostly caused by Cryptococcus neoformans. We identified agents of cryptococcosis diagnosed in Germany from 2004 to 2010. We used multi-locus sequence typing (MLST) to understand the molecular epidemiology of cryptococcosis. Sero- and mating types of individual patient isolates were determined by PCR. MLST was performed using the seven-locus scheme. Allele and nucleotide diversity was calculated for each locus of C. neoformans var. grubii and C. neoformans var. neoformans. Phylogenetic relations were assessed by dendrograms. Clinical data were compared between infections caused by the two variants. We studied 101 isolates. Eight were identified as hybrids (8%). All non-hybrids were of the α mating type. Among 78 C. neoformans var. grubii (77%), 16 sequence types (STs) were identified including three novel STs. They clustered in four groups, previously isolated in Asia, Europe or worldwide. Among 15 C. neoformans var. neoformans (15%), 10 STs were identified, without clustering. These isolates showed higher allele, and nucleotide diversity compared with C. neoformans var. grubii. C. neoformans var. neoformans was more likely to cause soft-tissue infections (3/9, 33 vs. 1/63, 2%, p = 0.005) and to affect non-AIDS patients (7/14, 50 vs. 15/76, 20%, p = 0.036). C. neoformans var. grubii is the predominant agent of cryptococcosis in Germany. MLST suggests that a part of these cases are acquired abroad by immigrants or tourists. C. neoformans var. neoformans isolates represent a greater genetic diversity and are associated with more variable clinical presentations.

Exploring diagnostic methods for drug-resistant tuberculosis: A comprehensive overview.

Despite available global efforts and funding, Tuberculosis (TB) continues to affect a considerable number of patients worldwide. Policy makers and stakeholders set clear goals to reduce TB incidence and mortality, but the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) complicate the reach of these goals. Drug-resistance TB needs to be diagnosed rapidly and accurately to effectively treat patients, prevent the transmission of MDR-TB, minimise mortality, reduce treatment costs and avoid unnecessary hospitalisations. In this narrative review, we provide a comprehensive overview of laboratory methods for detecting drug resistance in MTB, focusing on phenotypic, molecular and other drug susceptibility testing (DST) techniques. We found a large variety of methods used, with the BACTEC MGIT 960 being the most common phenotypic DST and the Xpert MTB/RIF being the most common molecular DST. We emphasise the importance of integrating phenotypic and molecular DST to address issues like resistance to new drugs, heteroresistance, mixed infections and low-level resistance mutations. Notably, most of the analysed studies adhered to the outdated definition of XDR-TB and did not consider the pre-XDR definition, thus posing challenges in aligning diagnostic methods with the current landscape of TB resistance.

Livestock-associated methicillin-resistant Staphylococcus aureus responsible for human colonization and infection in an area of Italy with high density of pig farming.

BACKGROUND: Livestock-Associated MRSA (LA-MRSA) belonging to ST398 lineage, common among pigs and other animals, emerged in Central and Northern Europe, becoming a new risk factor for MRSA among farm workers. Strains belonging to ST398 can be responsible for human colonization and infection, mainly in areas with high livestock-farming. The aim of this study was to investigate the occurrence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) human colonization and infections in an area of the Lombardy Region (Italy), the Italian region with the highest density of pig farming. METHODS: In the period March-April 2010, 879 nasal swabs were taken from subjects at admission to a local hospital serving an area of the Lombardy Region devoted to agriculture and farming. In the period March 2010-February 2011, all MRSA strains from community-acquired infection (CAI) observed in the same hospital, were collected. Molecular characterization of the isolates included SCCmec typing, spa typing and multilocus sequence typing (MLST). RESULTS: Out of 879 nasal swabs examined, 9 (1%) yielded MRSA. Five strains were assigned to sequence type (ST)398 (spa t899, 3 isolates; t108 and t2922, 1 isolate each) and were therefore categorized as LA-MRSA. The other 4 isolates were likely of hospital origin. No strains were positive for Panton-Valentine Leukocidin genes. Twenty MRSA isolates were detected from CAI, 17 were from skin and soft-tissue infections and 3 from other infections. An MRSA isolate from otitis externa was t899/ST398 and PVL-negative, hence categorized as LA-MRSA. Four isolates were assigned to t127/ST1. Eight strains were PVL-positive community acquired (CA)-MRSA and belonged to different clones, the most frequent being ST8. CONCLUSIONS: In an area of Italy with high density of pig farming, LA-MRSA is able to colonize the population and rarely to produce infections. Typical CA-MRSA is more common than LA-MRSA among CAI.

Staphylococcus aureus in a northern Italian region: phenotypic and molecular characterization.

BACKGROUND: Staphylococcus aureus is a leading cause of community-acquired infections and healthcare-associated infections. Epidemiological data are useful for understanding the dynamics of the diffusion of this pathogen, and to plan control activities and monitor their efficacy. METHODS: S. aureus isolates were collected in 13 public hospital laboratories of Emilia-Romagna (northern Italy region) during February-March 2009; phenotypic and molecular characterizations of these isolates were performed. RESULTS: The study sample included 267 isolates, 57 from blood, 81 from respiratory tract, and 129 from wounds; 106 (40%) were methicillin-resistant S. aureus (MRSA). MRSA showed a limited number of circulating clones with 2 predominant spa types--t008 and t041--accounting for 36% and 27% of MRSA isolates, respectively. The t041 type had a higher prevalence of antimicrobial resistance compared to other spa types and accounted for most of the retrieved hetero-vancomycin-intermediate S. aureus (h-VISA), while t008 was more frequently detected in non-hospital isolates. A higher degree of genetic diversity was observed in methicillin-susceptible S. aureus (MSSA), with no predominant clones and low prevalence of antimicrobial resistance. The occurrence of community-acquired MRSA infection appears to be rare in Emilia-Romagna. CONCLUSIONS: In contrast to previous studies reporting Italian data, t008 was the most frequent spa type among MRSA isolates in Emilia-Romagna. The prevalence of antimicrobial resistance of different MRSA spa types could influence their ability to cause infections with hospital onset. The presence of only 2 major MRSA clones circulating in Emilia-Romagna increases the chances that a regional strategy aimed at MRSA prevention will be effective.

Recent Developments in Phenotypic and Molecular Diagnostic Methods for Antimicrobial Resistance Detection in Staphylococcus aureus: A Narrative Review.

Staphylococcus aureus is an opportunistic pathogen responsible for a wide range of infections in humans, such as skin and soft tissue infections, pneumonia, food poisoning or sepsis. Historically, S. aureus was able to rapidly adapt to anti-staphylococcal antibiotics and become resistant to several classes of antibiotics. Today, methicillin-resistant S. aureus (MRSA) is a multidrug-resistant pathogen and is one of the most common bacteria responsible for hospital-acquired infections and outbreaks, in community settings as well. The rapid and accurate diagnosis of antimicrobial resistance in S. aureus is crucial to the early initiation of directed antibiotic therapy and to improve clinical outcomes for patients. In this narrative review, I provide an overview of recent phenotypic and molecular diagnostic methods for antimicrobial resistance detection in S. aureus, with a particular focus on MRSA detection. I consider methods for resistance detection in both clinical samples and isolated S. aureus cultures, along with a brief discussion of the advantages and the challenges of implementing such methods in routine diagnostics.

A high-resolution record of Holocene primary productivity and water-column mixing from the varved sediments of Lake Zabinskie, Poland.

Eutrophication and anoxia are increasing in lakes worldwide. However, our understanding of variations of primary productivity and anoxia in lakes over thousands of years is limited. Long-term records are needed to understand the natural variability of lake ecosystems and to improve our understanding of drivers of productivity and anoxia. In this study, we used the varved sediment record of Lake Zabinskie, Poland to answer the following research questions: 1) How have primary production and water column oxygen concentrations varied during the past 10,800 years?; 2) what role did natural and anthropogenic forces have in driving changes in primary production or lake mixing regime? Recently developed hyperspectral imaging (HSI) techniques were used to quantify sedimentary chloropigments-a and bacteriopheopigments-a (Bphe-a) at sub-annual resolution. These data, combined with elemental data from micro X-ray fluorescence (µ-XRF) and pigment assemblage data from high-performance liquid chromatography (HPLC) measurements, were used to reconstruct paleolimnological conditions. Bphe-a was used as an indicator of anoxia, and its presence suggests that an extensive anoxic zone was present nearly continuously from 10.8 to 2.8 ka BP. Anoxic conditions, driven by thermal stratification, were promoted by closed forest cover during that time, which limited wind-driven mixing of the water column. After 2.8 ka BP, water column oxygenation occurred more frequently, particularly during periods of increased human agricultural activity and forest opening. Pronounced anoxia was again present continuously from ~610 to 1470 CE, concurrent with a period of reforestation. After ~1610 CE, deforestation caused increases in erosion rates, algal production, and water column oxygenation. Pigment assemblages indicate that the algal community during the past 150 years was different from any other time during the Holocene. This study demonstrates a clear link between lake biogeochemical processes and forest cover and shows the potential of HSI to produce extremely high-resolution records of past productivity and redox conditions from varved lake sediments.

Metabolic phenotype of clinical and environmental Mycobacterium avium subsp. hominissuis isolates.

BACKGROUND: Mycobacterium avium subsp. hominissuis (MAH) is an emerging opportunistic human pathogen. It can cause pulmonary infections, lymphadenitis and disseminated infections in immuno-compromised patients. In addition, MAH is widespread in the environment, since it has been isolated from water, soil or dust. In recent years, knowledge on MAH at the molecular level has increased substantially. In contrast, knowledge of the MAH metabolic phenotypes remains limited. METHODS: In this study, for the first time we analyzed the metabolic substrate utilization of ten MAH isolates, five from a clinical source and five from an environmental source. We used BIOLOG Phenotype MicroarrayTM technology for the analysis. This technology permits the rapid and global analysis of metabolic phenotypes. RESULTS: The ten MAH isolates tested showed different metabolic patterns pointing to high intra-species diversity. Our MAH isolates preferred to use fatty acids such as Tween, caproic, butyric and propionic acid as a carbon source, and L-cysteine as a nitrogen source. Environmental MAH isolates resulted in being more metabolically active than clinical isolates, since the former metabolized more strongly butyric acid (p = 0.0209) and propionic acid (p = 0.00307). DISCUSSION: Our study provides new insight into the metabolism of MAH. Understanding how bacteria utilize substrates during infection might help the developing of strategies to fight such infections.

Identification and comparative analysis of a genomic island in Mycobacterium avium subsp. hominissuis.

Mycobacterium avium subsp. hominissuis (MAH) is an environmental bacterium causing opportunistic infections. The objective of this study was to identify flexible genome regions in MAH isolated from different sources. By comparing five complete and draft MAH genomes we identified a genomic island conferring additional flexibility to the MAH genomes. The island was absent in one of the five strains and had sizes between 16.37 and 84.85kb in the four other strains. The genes present in the islands differed among strains and included phage- and plasmid-derived genes, integrase genes, hypothetical genes, and virulence-associated genes like mmpL or mce genes.

Second international diagnostic accuracy study for the serological detection of West Nile virus infection.

BACKGROUND: In recent decades, sporadic cases and outbreaks in humans of West Nile virus (WNV) infection have increased. Serological diagnosis of WNV infection can be performed by enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA) neutralization test (NT) and by hemagglutination-inhibition assay. The aim of this study is to collect updated information regarding the performance accuracy of WNV serological diagnostics. METHODOLOGY/PRINCIPAL FINDINGS: In 2011, the European Network for the Diagnostics of Imported Viral Diseases-Collaborative Laboratory Response Network (ENIVD-CLRN) organized the second external quality assurance (EQA) study for the serological diagnosis of WNV infection. A serum panel of 13 samples (included sera reactive against WNV, plus specificity and negative controls) was sent to 48 laboratories involved in WNV diagnostics. Forty-seven of 48 laboratories from 30 countries participated in the study. Eight laboratories achieved 100% of concurrent and correct results. The main obstacle in other laboratories to achieving similar performances was the cross-reactivity of antibodies amongst heterologous flaviviruses. No differences were observed in performances of in-house and commercial test used by the laboratories. IFA was significantly more specific compared to ELISA in detecting IgG antibodies. The overall analytical sensitivity and specificity of diagnostic tests for IgM detection were 50% and 95%, respectively. In comparison, the overall sensitivity and specificity of diagnostic tests for IgG detection were 86% and 69%, respectively. CONCLUSIONS/SIGNIFICANCE: This EQA study demonstrates that there is still need to improve serological tests for WNV diagnosis. The low sensitivity of IgM detection suggests that there is a risk of overlooking WNV acute infections, whereas the low specificity for IgG detection demonstrates a high level of cross-reactivity with heterologous flaviviruses.

Diagnosis of west nile virus human infections: overview and proposal of diagnostic protocols considering the results of external quality assessment studies.

West Nile virus, genus Flavivirus, is transmitted between birds and occasionally other animals by ornithophilic mosquitoes. This virus also infects humans causing asymptomatic infections in about 85% of cases and <1% of clinical cases progress to severe neuroinvasive disease. The virus also presents a threat since most infections remain unapparent. However, the virus contained in blood and organs from asymptomatically infected donors can be transmitted to recipients of these infectious tissues. This paper reviews the presently available methods to achieve the laboratory diagnosis of West Nile virus infections in humans, discussing the most prominent advantages and disadvantages of each in light of the results obtained during four different External Quality Assessment studies carried out by the European Network for 'Imported' Viral Diseases (ENIVD).

Mechanisms of antibiotic resistance in Staphylococcus aureus.

Staphylococcus aureus can exemplify better than any other human pathogen the adaptive evolution of bacteria in the antibiotic era, as it has demonstrated a unique ability to quickly respond to each new antibiotic with the development of a resistance mechanism, starting with penicillin and methicillin, until the most recent, linezolid and daptomycin. Resistance mechanisms include enzymatic inactivation of the antibiotic (penicillinase and aminoglycoside-modification enzymes), alteration of the target with decreased affinity for the antibiotic (notable examples being penicillin-binding protein 2a of methicillin-resistant S. aureus and D-Ala-D-Lac of peptidoglycan precursors of vancomycin-resistant strains), trapping of the antibiotic (for vancomycin and possibly daptomycin) and efflux pumps (fluoroquinolones and tetracycline). Complex genetic arrays (staphylococcal chromosomal cassette mec elements or the vanA operon) have been acquired by S. aureus through horizontal gene transfer, while resistance to other antibiotics, including some of the most recent ones (e.g., fluoroquinolones, linezolid and daptomycin) have developed through spontaneous mutations and positive selection. Detection of the resistance mechanisms and their genetic basis is an important support to antibiotic susceptibility surveillance in S. aureus.