WGS based analysis of acquired antimicrobial resistance in human and non-human Acinetobacter baumannii isolates from a German perspective.

BACKGROUND: Acinetobacter baumannii ability to develop and acquire resistance makes it one of the most critical nosocomial pathogens globally. Whole-genome sequencing (WGS) was applied to identify the acquired or mutational variants of antimicrobial resistance (AMR) genes in 85 German A. baumannii strains utilizing Illumina technology. Additionally, the whole genome of 104 German isolates deposited in the NCBI database was investigated. RESULTS: In-silico analysis of WGS data revealed wide varieties of acquired AMR genes mediating resistance mostly to aminoglycosides, cephalosporins, carbapenems, sulfonamides, tetracyclines and macrolides. In the 189 analyzed genomes, the ant (3″)-IIa conferring resistance to aminoglycosides was the most frequent (55%), followed by blaADC.25 (38.6%) conferring resistance to cephalosporin, blaOXA-23 (29%) and the blaOXA-66 variant of the intrinsic blaOXA-51-likes (26.5%) conferring resistance to carbapenems, the sul2 (26%) conferring resistance to sulfonamides, the tet. B (19.5%) conferring resistance to tetracycline, and mph. E and msr. E (19%) conferring resistance to macrolides. blaTEM variants conferring resistance to cephalosporins were found in 12% of genomes. Thirteen variants of the intrinsic blaOXA-51 carbapenemase gene, blaOXA-510 and blaADC-25 genes were found in isolates obtained from dried milk samples. CONCLUSION: The presence of strains harboring acquired AMR genes in dried milk raises safety concerns and highlights the need for changes in producing dried milk. Acquired resistance genes and chromosomal gene mutation are successful routes for disseminating AMR determinants among A. baumannii. Identification of chromosomal and plasmid-encoded AMR in the genome of A. baumannii may help understand the mechanism behind the genetic mobilization and spread of AMR genes.

Treatment of Yersinia similis with the cationic lipid DOTAP enhances adhesion to and invasion into intestinal epithelial cells - A proof-of-principle study.

The monocationic quaternary surfactant DOTAP has been used for the delivery of nucleic acids and peptides into mammalian cells. This study tested the applicability of DOTAP for the enhancement of adhesion and invasion frequencies of Yersinia (Y.) similis to enable the analysis of the effects of low-pathogenic bacteria on intestinal epithelial cells. Incubation of Y. similis with DOTAP ahead of infection of C2BBe1 intestinal epithelial cells increased invasion and adhesion frequency four- and five-fold, respectively, in plating assays. Proteomic approaches confirmed the increased bacterial load on infected cells: analysis of protein extracts by two-dimensional difference gel electrophoresis (2D-DIGE) revealed higher amounts of bacterial proteins present in the cells infected with DOTAP-treated bacteria. MALDI-TOF mass spectrometry of selected spots from gel-separated protein extracts confirmed the presence of both bacterial and human cell proteins in the samples. Label-free quantitative proteomics analysis identified 1170 human cell proteins and 699 bacterial proteins. Three times more bacterial proteins (279 vs. 93) were detected in C2BBe1 cells infected with DOTAP-treated bacteria compared to infections with untreated bacteria. Infections with DOTAP-treated Y. similis led to a significant upregulation of the stress-inducible ubiquitin-conjugating enzyme UBE2M in C2BBe1 cells. This points towards a stronger impact of the stress and infection responsive transcription factor AP-1 by enhanced bacterial load. DOTAP-treatment of uninfected C2BBe1 cells led to a significant downregulation of the transmembrane trafficking protein TMED10. The application of DOTAP could be helpful for investigating the impact of otherwise low adherent or invasive bacteria on cultivated mammalian cells without utilisation of genetic modifications.

Creating a Primary Care Workforce: Strategies for Leaders, Clinicians, and Nurses.

Many primary care clinics struggle with rapid implementation and systematic expansion of primary care behavioral health (PCBH) services. Often, an uneven course of program development is due to lack of attention to preparing clinic leadership, addressing operational factors, and training primary care providers (PCPs) and nurses. This article offers competency tools for clinic leaders, PCPs, and nurses to use in assessing their status and setting change targets. These tools were developed by researchers working to disseminate evidence-based interventions in primary care clinics that included fully integrated behavioral health consultants and were then used by early adaptors of the PCBH model. By deploying these strategies, both practicing and teaching clinics will take a big step forward in developing the primary care workforce needed for primary care teams, where the behavioral health needs of a patient of any age can be addressed at the time of need.

Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts.

Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.

Phenotypic and genotypic characterization of resistance and virulence in Pseudomonas aeruginosa isolated from poultry farms in Egypt using whole genome sequencing.

Pseudomonas aeruginosa (P. aeruginosa) is an ESKAPE pathogen that can quickly develop resistance to most antibiotics. This bacterium is a zoonotic pathogen that can be found in humans, animals, foods, and environmental samples, making it a One-Health concern. P. aeruginosa threatens the poultry industry in Egypt, leading to significant economic losses. However, the investigation of this bacterium using NGS technology is nearly non-existent in Egypt. In this study, 38 isolates obtained from broiler farms of the Delta region were phenotypically investigated, and their genomes were characterized using whole genome sequencing (WGS). The study found that 100% of the isolates were resistant to fosfomycin and harbored the fosA gene. They were also resistant to trimethoprim/sulfamethoxazole, although only one isolate harbored the sul1 gene. Non-susceptibility (resistant, susceptible with increased dose) of colistin was observed in all isolates. WGS analysis revealed a high level of diversity between isolates, and MLST analysis allocated the 38 P. aeruginosa isolates into 11 distinct sequence types. The most predominant sequence type was ST267, found in 13 isolates, followed by ST1395 in 8 isolates. The isolates were susceptible to almost all tested antibiotics carrying only few different antimicrobial resistance (AMR) genes. Various AMR genes that confer resistance mainly to ß-lactam, aminoglycoside, sulfonamide, and phenicol compounds were identified. Additionally, several virulence associated genes were found without any significant differences in number and distribution among isolates. The majority of the virulence genes was identified in almost all isolates. The fact that P. aeruginosa, which harbors several AMR and virulence-associated factors, is present in poultry farms is alarming and threatens public health. The misuse of antimicrobial compounds in poultry farms plays a significant role in resistance development. Thus, increasing awareness and implementing strict veterinary regulations to guide the use of veterinary antibiotics is required to reduce health and environmental risks. Further studies from a One-Health perspective using WGS are necessary to trace the potential transmission routes of resistance between animals and humans and clarify resistance mechanisms.

First Description of Mergibacter septicus Isolated from a Common Tern (Sterna hirundo) in Germany.

Mergibacter septicus (M. septicus), previously known as Bisgaard Taxon 40, is a recently described species within the Pasteurellaceae family. In this study, we present a M. septicus strain isolated from a common tern (Sterna hirundo) chick that died just after fledging from the Banter See in Wilhelmshaven, Germany. The recovered M. septicus strain underwent microbiological phenotypic characterization, followed by whole genome sequencing on Illumina and Nanopore platforms. Phenotypically, M. septicus 19Y0039 demonstrated resistance to colistin, cephalexin, clindamycin, oxacillin, and penicillin G. The genome analysis revealed a circular 1.8 Mbp chromosome without any extrachromosomal elements, containing 1690 coding DNA sequences. The majority of these coding genes were associated with translation, ribosomal structure and biogenesis, followed by RNA processing and modification, and transcription. Genetic analyses revealed that the German M. septicus strain 19Y0039 is related to the American strain M. septicus A25201T. Through BLAST alignment, twelve putative virulence genes previously identified in the M. septicus type strain A25201T were also found in the German strain. Additionally, 84 putative virulence genes distributed across nine categories, including immune modulation, effector delivery system, nutrition/metabolic factors, regulation, stress survival, adherence, biofilm, exotoxin, and motility, were also identified.

Acinetobacter baumannii Global Clone-Specific Resistomes Explored in Clinical Isolates Recovered from Egypt.

Acinetobacter baumannii (A. baumannii) is a highly problematic pathogen with an enormous capacity to acquire or upregulate antibiotic drug resistance determinants. The genomic epidemiology and resistome structure of 46 A. baumannii clinical isolates were studied using whole-genome sequencing. The isolates were chosen based on reduced susceptibility to at least three classes of antimicrobial compounds and were initially identified using MALDI-TOF/MS, followed by polymerase chain reaction amplification of blaOXA-51-like genes. The susceptibility profiles were determined using a broth microdilution assay. Multi-, extensive-, and pan-drug resistance was shown by 34.8%, 63.0%, and 2.2% of the isolates, respectively. These were most susceptible to colistin (95.7%), amikacin, and trimethoprim/sulfamethoxazole (32.6% each), while only 26.1% of isolates were susceptible to tigecycline. In silico multi-locus sequence typing revealed 8 Pasteur and 22 Oxford sequence types (STs) including four novel STs (STOxf 2805, 2806, 2807, and 2808). The majority of the isolates belonged to Global Clone (GC) 2 (76.4%), GC5 (19.6%), GC4 (6.5%), GC9 (4.3%), and GC7 (2.2%) lineages. An extensive resistome potentially conferring resistance to the majority of the tested antimicrobials was identified in silico. Of all known carbapenem resistance genes, blaOXA-23 was carried by most of the isolates (69.6%), followed by ISAba1-amplified blaADC (56.5%), blaNDM-1 and blaGES-11 (21.7% each), and blaGES-35 (2.2%) genes. A significant correlation was found between carbapenem resistance and carO mutations, which were evident in 35 (76.0%) isolates. A lower proportion of carbapenem resistance was noted for strains possessing both blaOXA-23- and blaGES-11. Amikacin resistance was most probably mediated by armA, aac(6')-Ib9, and aph(3')-VI, most commonly coexisting in GC2 isolates. No mutations were found in pmrABC or lpxACD operons in the colistin-resistant isolates. Tigecycline resistance was associated with adeS (N268Y) and baeS (A436T) mutations. While the lineage-specific distribution of some genes (e.g., blaADC and blaOXA-51-like alleles) was evident, some resistance genes, such as blaOXA-23 and sul1, were found in all GCs. The data generated here highlight the contribution of five GCs in A. baumannii infections in Egypt and enable the comprehensive analysis of GC-specific resistomes, thus revealing the dissemination of the carbapenem resistance gene blaOXA-23 in isolates encompassing all GCs.

Zoonotic agents in small ruminants kept on city farms in southern Germany.

Sheep and goats are popular examples of livestock kept on city farms. In these settings, close contacts between humans and animals frequently occur. Although it is widely accepted that small ruminants can carry numerous zoonotic agents, it is unknown which of these agents actually occur in sheep and goats on city farms in Germany. We sampled feces and nasal liquid of 48 animals (28 goats, 20 sheep) distributed in 7 city farms and on one activity playground in southern Germany. We found that 100% of the sampled sheep and 89.3% of the goats carried Shiga toxin-producing Escherichia coli (STEC). The presence of Staphylococcus spp. in 75% of both sheep and goats could be demonstrated. Campylobacter spp. were detected in 25% and 14.3% of the sheep and goats, respectively. Neither Salmonella spp. nor Coxiella burnetii was found. On the basis of these data, we propose a reasonable hygiene scheme to prevent transmission of zoonotic agents during city farm visits.

Effectiveness of an antimicrobial treatment scheme in a confined glanders outbreak.

BACKGROUND: Glanders is a contagious and fatal zoonotic disease of solipeds caused by the Gram-negative bacterium Burkholderia (B.) mallei. Although regulations call for culling of diseased animals, certain situations e.g. wild life conservation, highly valuable breeding stock, could benefit from effective treatment schemes and post-exposure prophylaxis. RESULTS: Twenty three culture positive glanderous horses were successfully treated during a confined outbreak by applying a treatment protocol of 12 weeks duration based on the parenteral administration of enrofloxacin and trimethoprim plus sulfadiazine, followed by the oral administration of doxycycline. Induction of immunosupression in six randomly chosen horses after completion of treatment did not lead to recrudescence of disease. CONCLUSION: This study demonstrates that long term treatment of glanderous horses with a combination of various antibiotics seems to eliminate the agent from the organism. However, more studies are needed to test the effectiveness of this treatment regime on B. mallei strains from different endemic regions. Due to its cost and duration, this treatment can only be an option in certain situations and should not replace the current "testing and culling" policy, in conjunction with adequate compensation to prevent spreading of disease.

WGS-Based Phenotyping and Molecular Characterization of the Resistome, Virulome and Plasmid Replicons in Klebsiella pneumoniae Isolates from Powdered Milk Produced in Germany.

The emergence of Klebsiella pneumoniae (K. pneumoniae) in German healthcare is worrying. It is not well-investigated in the veterinary world and food chains. In the current study, antibiotic susceptibility profiles of 24 K. pneumoniae strains isolated from powdered milk samples produced in Germany were investigated by a microdilution test. Next-generation sequencing (NGS) was applied to identify genomic determinants for antimicrobial resistance (AMR), virulence-associated genes and plasmids replicons. All isolates were susceptible to the majority (14/18) of tested antibiotics. Resistance to colistin, fosfomycin, chloramphenicol and piperacillin was found. The ambler class A ß-lactamase, blaSHV variants were identified in all isolates, of which blaSHV-187 was most prevalent and found in 50% of isolates. Single-nucleotide-variants of oqxA and oqxB conferring resistance to phenicol/quinolone were found in all isolates, and the oqxB17 was the most prevalent found in 46% of isolates. 67% of isolates harbored fosA genes; however, only one was fosfomycin-resistant. Two isolates harbored genes conferring resistance to colistin, despite being susceptible. The majority of identified virulome genes were iron uptake siderophores. Two enterobactins (entB, fepC), six adherence-related genes belonging to E. coli common pilus (ECP) and one secretion system (ompA gene) were found in all isolates. In contrast, yersiniabactin was found in two isolates. One ST23 strain was susceptible to all tested antibiotics, and harbored determinants discriminatory for hypervirulent strains, e.g., aerobactin, salmochelin, yersiniabactin, enterobactin and regulator of mucoid phenotype A genes that are highly associated with hypervirulent K. pneumoniae. The IncF plasmid family was found in all strains, while almost half of the isolates harbored Col440I-type plasmids and nine isolates harbored various Inc-type plasmids. The presence of K. pneumoniae carrying different resistomes and major virulent specific virulomes in powdered milk samples is alarming. This could threaten public health, particularly of neonates and infants consuming dried milk.

High frequency of chlamydial co-infections in clinically healthy sheep flocks.

BACKGROUND: The epidemiological situation of ovine chlamydial infections in continental Europe, especially Germany is poorly characterised. Using the German state of Thuringia as a model example, the chlamydial sero- and antigen prevalence was estimated in thirty-two randomly selected sheep flocks with an average abortion rate lower than 1%. Seven vaccinated flocks were reviewed separately. RESULTS: A wide range of samples from 32 flocks were examined. Assumption of a seroprevalence of 10% (CI 95%) at flock level, revealed that 94% of the tested flocks were serologically positive with ongoing infection (i.e. animals with seroconversion) in nearly half (47%) of the flocks. On the basis of an estimated 25% antigen prevalence (CI 95%), PCR and DNA microarray testing, together with sequencing revealed the presence of chlamydiae in 78% of the flocks. The species most frequently found was Chlamydophila (C.) abortus (50%) followed by C. pecorum (47%) and C. psittaci genotype A (25%). Mixed infections occurred in 25% of the tested flocks. Samples obtained from the vaccinated flocks revealed the presence of C. abortus field samples in 4/7 flocks. C. pecorum was isolated from 2/7 flocks and the presence of seroconversion was determined in 3/7 flocks. CONCLUSIONS: The results imply that chlamydial infections occur frequently in German sheep flocks, even in the absence of elevated abortion rates. The fact that C. pecorum and the potentially zoonotic C. psittaci were found alongside the classical abortifacient agent C. abortus, raise questions about the significance of this reservoir for animal and human health and underline the necessity for regular monitoring. Further studies are needed to identify the possible role of C. psittaci infections in sheep.

Phylogenetic Relatedness and Genome Structure of Yersinia ruckeri Revealed by Whole Genome Sequencing and a Comparative Analysis.

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), a serious infection that affects global aquaculture with high economic impact. The present study used whole genome sequences to perform a comparative analysis on 10 Y. ruckeri strains and to explore their genetic relatedness to other members of the genus. Y. ruckeri, Yersinia entomophaga, and Yersinia nurmii formed a species complex that constitutes the most basal lineage of the genus. The results showed that the taxonomy of Y. ruckeri strains is better defined by using a core genome alignment and phylogenetic analysis. The distribution of accessory genes in all Yersinia species revealed the presence of 303 distinctive genes in Y. ruckeri. Of these, 169 genes were distributed in 17 genomic islands potentially involved in the pathogenesis of ERM via (1) encoding virulence factors such as Afp18, Yrp1, phage proteins and (2) improving the metabolic capabilities by enhancing utilization and metabolism of iron, amino acids (specifically, arginine and histidine), and carbohydrates. The genome of Y. ruckeri is highly conserved regarding gene structure, gene layout and functional categorization of genes. It contains various components of mobile genetic elements but lacks the CRISPR-Cas system and possesses a stable set of virulence genes possibly playing a critical role in pathogenicity. Distinct virulence plasmids were exclusively restricted to a specific clonal group of Y. ruckeri (CG4), possibly indicating a selective advantage. Phylogenetic analysis of Y. ruckeri genomes revealed the co-presence of multiple genetically distant lineages of Y. ruckeri strains circulating in Germany. Our results also suggest a possible dissemination of a specific group of strains in the United States, Peru, Germany, and Denmark. In conclusion, this study provides new insights into the taxonomy and evolution of Y. ruckeri and contributes to a better understanding of the pathogenicity of ERM in aquaculture. The genomic analysis presented here offers a framework for the development of more efficient control strategies for this pathogen.

WGS-Based Analysis of Carbapenem-Resistant Acinetobacter baumannii in Vietnam and Molecular Characterization of Antimicrobial Determinants and MLST in Southeast Asia.

Carbapenem-resistant Acinetobacter baumannii (A. baumannii, CRAb) is an emerging global threat for healthcare systems, particularly in Southeast Asia. Next-generation sequencing (NGS) technology was employed to map genes associated with antimicrobial resistance (AMR) and to identify multilocus sequence types (MLST). Eleven strains isolated from humans in Vietnam were sequenced, and their AMR genes and MLST were compared to published genomes of strains originating from Southeast Asia, i.e., Thailand (n = 49), Myanmar (n = 38), Malaysia (n = 11), Singapore (n = 4) and Taiwan (n = 1). Ten out of eleven Vietnamese strains were CRAb and were susceptible only to colistin. All strains harbored ant(3")-IIa, armA, aph(6)-Id and aph(3") genes conferring resistance to aminoglycosides, and blaOXA-51 variants and blaADC-25 conferring resistance to ß-lactams. More than half of the strains harbored genes that confer resistance to tetracyclines, sulfonamides and macrolides. The strains showed high diversity, where six were assigned to sequence type (ST)/2, and two were allocated to two new STs (ST/1411-1412). MLST analyses of 108 strains from Southeast Asia identified 19 sequence types (ST), and ST/2 was the most prevalent found in 62 strains. A broad range of AMR genes was identified mediating resistance to ß-lactams, including cephalosporins and carbapenems (e.g., blaOXA-51-like, blaOXA-23, blaADC-25, blaADC-73, blaTEM-1, blaNDM-1), aminoglycosides (e.g., ant(3")-IIa, aph(3")-Ib, aph(6)-Id, armA and aph(3')-Ia), phenicoles (e.g., catB8), tetracyclines (e.g., tet.B and tet.39), sulfonamides (e.g., sul.1 and sul.2), macrolides and lincosamide (e.g., mph.E, msr.E and abaF). MLST and core genome MLST (cgMLST) showed an extreme diversity among the strains. Several strains isolated from different countries clustered together by cgMLST; however, different clusters shared the same ST. Developing an action plan on AMR, increasing awareness and prohibiting the selling of antibiotics without prescription must be mandatory for this region. Such efforts are critical for enforcing targeted policies on the rational use of carbapenem compounds and controlling AMR dissemination and emergence in general.

Molecular Characterization of German Acinetobacter baumannii Isolates and Multilocus Sequence Typing (MLST) Analysis Based on WGS Reveals Novel STs.

Acinetobacter baumannii (A. baumannii) is a major cause of severe nosocomial infections worldwide. The emergence of infections associated with A. baumannii poses a significant health risk in Germany. A. baumannii is part of the ACB complex and is difficult to distinguish from other species phenotypically, necessitating its reliable identification. The current study analyzed 89 A. baumannii strains from human and non-human origins by matrix-assisted laser desorption/ionization (MALDI-TOF) and PCR detection of intrinsic blaOXA-51-like carbapenemase, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and ISAba 1 genes. Whole-genome sequencing (WGS) was applied for species confirmation and strain type determination. Combining the molecular detection of the intrinsic blaOXA-51-like carbapenemase gene together with MALDI-TOF with a score value of >2.300 proved to be a suitable tool for A. baumannii identification. WGS data for all of the sequenced strains confirmed the identity of all A. baumannii strains. The Pasteur scheme successfully assigned 79.7% of the strains into distinct STs, while the Oxford scheme succeeded in allocating only 42.7% of isolates. Multilocus sequence typing (MLST) analysis based on the Pasteur scheme identified 16 STs. ST/241 was the most prevalent in samples from non-human origin, whereas ST/2 was predominant in human samples. Furthermore, eight isolates of non-human origin were allocated to seven new STs (ST/1410, ST/1414, ST/1416, ST/1417, ST/1418, ST/1419, and ST/1421). Ten isolates from non-human origin could not be typed since new alleles were observed in the loci Pas_cpn60, Pas_rpoB, and Pas_gltA. MLST analysis based on the Pasteur scheme was more appropriate than the Oxford scheme for the current group of A. baumannii.

Spatio-Temporal Distribution of Acinetobacter baumannii in Germany-A Comprehensive Systematic Review of Studies on Resistance Development in Humans (2000-2018).

Acinetobacter (A.) baumannii has gained global notoriety as a significant nosocomial pathogen because it is frequently associated with multi-drug resistance and hospital-based outbreaks. There is a substantial difference in the incidence of A. baumannii infections between different countries and within Germany. However, its continuous spread within Germany is a matter of concern. A systematic literature search and analysis of the literature published between 2000 and 2018 on A. baumannii in humans was performed. Forty-four studies out of 216 articles met the criteria for inclusion, and were selected and reviewed. The number of published articles is increasing over time gradually. Case reports and outbreak investigations are representing the main body of publications. North Rhine-Westphalia, Hesse and Baden-Wuerttemberg were states with frequent reports. Hospitals in Cologne and Frankfurt were often mentioned as specialized institutions. Multiresistant strains carrying diverse resistance genes were isolated in 13 of the 16 German states. The oxacillinase blaOXA-23-like, intrinsic blaOXA-51-like, blaOXA-58 variant, blaNDM-1, blaGES-11, blaCTX-M and blaTEM are the most predominant resistance traits found in German A. baumannii isolates. Five clonal lineages IC-2, IC-7, IC-1, IC-4 and IC-6 and six sequence types ST22, ST53, ST195, ST218, ST944/ST78 and ST348/ST2 have been reported. Due to multidrug resistance, colistin, tigecycline, aminoglycosides, fosfomycin, ceftazidime/avibactam and ceftolozan/tazobactam were often reported to be the only effective antibiotics left to treat quadruple multi-resistant Gram-negative (4MRGN) A. baumannii. Dissemination and infection rates of A. baumannii are on the rise nationwide. Hence, several aspects of resistance development and pathogenesis are not fully understood yet. Increased awareness, extensive study of mechanisms of resistance and development of alternative strategies for treatment are required. One-Health genomic surveillance is needed to understand the dynamics of spread, to identify the main reservoirs and routes of transmission and to develop targeted intervention strategies.

Pan-Proteomic Analysis and Elucidation of Protein Abundance among the Closely Related Brucella Species, Brucella abortus and Brucella melitensis.

Brucellosis is a zoonotic infection caused by bacteria of the genus Brucella. The species, B. abortus and B. melitensis, major causative agents of human brucellosis, share remarkably similar genomes, but they differ in their natural hosts, phenotype, antigenic, immunogenic, proteomic and metabolomic properties. In the present study, label-free quantitative proteomic analysis was applied to investigate protein expression level differences. Type strains and field strains were each cultured six times, cells were harvested at a midlogarithmic growth phase and proteins were extracted. Following trypsin digestion, the peptides were desalted, separated by reverse-phase nanoLC, ionized using electrospray ionization and transferred into an linear trap quadrapole (LTQ) Orbitrap Velos mass spectrometer to record full scan MS spectra (m/z 300-1700) and tandem mass spectrometry (MS/MS) spectra of the 20 most intense ions. Database matching with the reference proteomes resulted in the identification of 826 proteins. The Cluster of Gene Ontologies of the identified proteins revealed differences in bimolecular transport and protein synthesis mechanisms between these two strains. Among several other proteins, antifreeze proteins, Omp10, superoxide dismutase and 30S ribosomal protein S14 were predicted as potential virulence factors among the proteins differentially expressed. All mass spectrometry data are available via ProteomeXchange with identifier PXD006348.

Phenotypic and WGS-derived antimicrobial resistance profiles of clinical and non-clinical Acinetobacter baumannii isolates from Germany and Vietnam.

OBJECTIVES: This study aimed to combine in vitro phenotyping analysis and whole-genome-sequencing (WGS) to characterise the phenotype and genetic determinants associated with intrinsic resistance in 100 clinical and non-clinical Acinetobacter baumannii strains originating from Germany and Vietnam. Moreover, it aimed to assess whether powdered milk as a food source functions as a potential reservoir of antibiotic resistance and possesses similar antimicrobial resistance (AMR) genes as in clinical strains isolated from Germany. METHODS: Antimicrobial susceptibility testing was performed using the broth microdilution method and the minimum inhibitory concentration (MIC) was determined for 18 antibiotics. The WGS data from all isolates were mapped to intrinsic genes known to be associated with phenotypic AMR. RESULTS: The highest resistance frequency was observed for chloramphenicol (100%), followed by fosfomycin (96%) and cefotaxime (95%). The lowest resistant rates were observed for colistin (3%), trimethoprim/sulfamethoxazole (17%), tigecycline (19%), and amikacin (19%). Thirty-five percent of tested strains displayed resistance to at least one of the carbapenems. Resistance to fluoroquinolones, aminoglycosides, tigecycline, penicillins, trimethoprim/sulfamethoxazole, and fourth-generation cephalosporins was determined only in human strains. About one-quarter of isolates (24%) was multidrug-resistant (MDR) and all were of human origin. Among them, 16 isolates were extensively drug resistant (XDR) and 10 from those 16 isolates showed resistance to all tested antibiotics except colistin. In silico detection of intrinsic AMR genes revealed the presence of 36 ß-lactamases and 24 non-ß-lactamase resistance genes. Two colistin-resistant and 10 ertapenem-resistant strains were isolated from powdered milk produced in Germany. Thirty-eight AMR genes associated with resistance to antibiotics were found in isolates recovered from milk powder. Several resistance mechanisms towards many classes of antibiotics existed in A. baumannii including ß-lactamases, multidrug efflux pumps and aminoglycoside-modifying enzymes. CONCLUSION: The use of WGS for routine public health surveillance is a reliable method for the rapid detection of emerging AMR in A. baumannii isolates. Milk powder poses a risk to contain MDR Acinetobacter strains or resistance genes in Germany.

Q fever: Evidence of a massive yet undetected cross-border outbreak, with ongoing risk of extra mortality, in a Dutch-German border region.

BACKGROUND: Following outbreaks in other parts of the Netherlands, the Dutch border region of South Limburg experienced a large-scale outbreak of human Q fever related to a single dairy goat farm in 2009, with surprisingly few cases reported from neighbouring German counties. Late chronic Q fever, with recent spikes of newly detected cases, is an ongoing public health concern in the Netherlands. We aimed to assess the scope and scale of any undetected cross-border transmission to neighbouring German counties, where individuals unknowingly exposed may carry extra risk of overlooked diagnosis. METHODS: (A) Seroprevalence rates in the Dutch area were estimated fitting an exponential gradient to the geographical distribution of notified acute human Q fever cases, using seroprevalence in a sample of farm township inhabitants as baseline. (B) Seroprevalence rates in 122 neighbouring German postcode areas were estimated from a sample of blood donors living in these areas and attending the regional blood donation centre in January/February 2010 (n = 3,460). (C) Using multivariate linear regression, including goat and sheep densities, veterinary Q fever notifications and blood donor sampling densities as covariates, we assessed whether seroprevalence rates across the entire border region were associated with distance from the farm. RESULTS: (A) Seroprevalence in the outbreak farm's township was 16.1%. Overall seroprevalence in the Dutch area was 3.6%. (B) Overall seroprevalence in the German area was 0.9%. Estimated mean seroprevalence rates (per 100,000 population) declined with increasing distance from the outbreak farm (0-19 km = 2,302, 20-39 km = 1,122, 40-59 km = 432 and ≥60 km = 0). Decline was linear in multivariate regression using log-transformed seroprevalence rates (0-19 km = 2.9 [95% confidence interval (CI) = 2.6 to 3.2], 20 to 39 km = 1.9 [95% CI = 1.0 to 2.8], 40-59 km = 0.6 [95% CI = -0.2 to 1.3] and ≥60 km = 0.0 [95% CI = -0.3 to 0.3]). CONCLUSIONS: Our findings were suggestive of widespread cross-border transmission, with thousands of undetected infections, arguing for intensified cross-border collaboration and surveillance and screening of individuals susceptible to chronic Q fever in the affected area.

Prevalence-dependent use of serological tests for diagnosing glanders in horses.

BACKGROUND: The internationally mandatory complement fixation test (CFT) for testing of equine sera for the absence of glanders has repeatedly led to discrepant results. Not only do "false positive" sera pose a problem for the diagnostician and the animal health authorities but they can also result in significant financial losses for the animal owners.Due to the very low prevalence of glanders in the horse population it is of major importance to use tests with a high specificity to overcome unreliable predictive values. We have compared formalin-fixed B. mallei whole cell antigen and a well characterised mouse monoclonal antibody with regard to their specificity and sensitivity for glanders serodiagnosis using CFT, an indirect (i) and a competitive (c) ELISA platform. RESULTS: Our results show that the CFT is still a very reliable technique in horse populations with very low glanders prevalence. The cELISA has a high sensitivity and specificity comparable to that of the CFT. The cELISA offers the possibility for automatisation, can be applied to non-complement fixing sera and used for various host species. CONCLUSION: The CFT is still the method of choice for testing horses for the absence of glanders.