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Extended spectrum beta-lactamases (ESBL) are frequently found in Enterobacterales isolates from Western Africa. However, information on the molecular epidemiology of regional ESBL-positive Enterobacterales strains is scarce. In order to provide epidemiological information, ESBL-positive Escherichia coli isolates from stool samples of European soldiers with diarrhea deployed to a field camp in Mali were subjected to whole-genome sequencing (Illumina MiSeq and Oxford Nanopore MinION) and antimicrobial susceptibility testing. With two exemptions, sequence-based analysis suggested an absence of transmission events between soldiers as indicated by a high genetic diversity of isolates and sequence types, confirming previous rep-PCR results. Third-generation cephalosporin resistance was associated with the presence of blaCTX-M-15 genes with (n = 14) and without (n = 5) co-occurring blaTEM-1b genes. Between 0 and 6 virulence and resistance plasmids per isolate were recorded. The detected resistance plasmids could be categorized into five types, which, in turn, share different sequence-identical segments, representing particular antimicrobial resistance gene-associated mobile genetic elements (MGEs). Phenotypic resistance rates within the 19 assessed isolates that showed distinguishable colony morphologies were 94.7% (18/19) against ampicillin-sulbactam and trimethoprim/sulfamethoxazole, 68.4% (13/19) against moxifloxacin, 31.6% (6/19) against ciprofloxacin, 42.1% (8/19) against gentamicin, 31.6% (6/19) against tobramycin, and 21.1% (4/19) against piperacillin-tazobactam and fosfomycin. Virulence-associated genes mediating infectious gastroenteritis were rarely detected. The gene aggR, which is characteristic for enteroaggregative E. coli, was only detected in one single isolate. In summary, we found a variety of different strains and clonal lineages of ESBL-carrying E. coli. Transmission either between soldiers or from common contaminated sources was demonstrated in two cases and played only a minor role in this military field camp, while there were indications that resistance gene bearing MGEs had been exchanged between antimicrobial resistance gene-(ARG-)carrying plasmids.

Background The main natural reservoir for Campylobacter jejuni is the intestinal tract of birds, especially in the context of human nutrition chickens and turkeys play the main role. There, C. jejuni multiplies optimally at 42°C, the avian body temperature, while after infection of humans by peroral intake in the human intestinal tract only 37°C prevail. Proteome profiling by label-free mass spectrometry (DIA-MS) was performed to examine the processes which enable C. jejuni strain 81-176 to adapt to 37°C in comparison to 42°C. In total, four states were compared with each other: incubation for 12 h at 37°C, for 24 h at 37°C, for 12 h at 42°C and 24 h at 42°C. Results It was shown that the proteome profile changes not only according to the different incubation temperature but also with the length of the incubation period, i.e. significant differences in protein expression were evident when comparing 37°C and 42°C as well as 12 h and 24 h of incubation. Altogether, the expression of 957 proteins was quantifiable. Out of these, 37.1% - 47.3% were considered to be significantly differentially expressed, that means these proteins showed at least a 1.5-fold change in either direction (i.e log2 FC ≥ 0.585 or log2 FC ≤ -0.585) and an FDR-adjusted p-value of less than 0.05. The significantly differentially expressed proteins could be arranged in 4 different clusters and 16 functional catogories. Conclusions The C. jejuni proteome at 42°C is better adopted to high replication rates than that at 37°C, which was in particular indicated by the up-expression of proteins belonging to the functional categories "replication", "DNA synthesis and repair factors", "lipid and carbohydrate biosynthesis" and "vitamin synthesis, metabolism, cofactor biosynthesis". The relative up-expression of proteins with chaperone function at 37°C in comparison to 42°C after 12 h incubation indicates a temporary lower-temperature adaptation response. Additionally the up-expression of factors for DNA uptake at 37°C compared to 42°C indicate a higher competence for the acquisition of extraneous DNA at human body temperature.

Despite recent advances in prophylactic vaccination, SARS-CoV-2 infections continue to cause significant morbidity. A better understanding of immune response differences between vaccinated individuals with and without later SARS-CoV-2 breakthrough infection is urgently needed. CoV-ADAPT is a prospective long-term study comparing humoral (anti-spike-RBD-IgG, neutralization capacity, avidity) and cellular (spike-induced T-cell interferon-γ release) immune responses in individuals vaccinated against SARS-CoV-2 at four different time points (three before and one after third vaccination). In this cohort study, 62 fully vaccinated individuals presented with SARS‑CoV-2 breakthrough infections vs 151 without infection 3-7 months following third vaccination. Breakthrough infections significantly increased anti-spike-RBD-IgG (p

BackgroundKlebsiella pneumoniae, which is frequently associated with hospital- and community-acquired infections, contains multidrug-resistant (MDR), hypervirulent (hv), non-MDR/non-hv as well as convergent representatives. It is known that mostly international high-risk clonal lineages including sequence types (ST) 11, 147, 258, and 307 drive their global spread. ST395, which was first reported in the context of a carbapenemase-associated outbreak in France in 2010, is a less well-characterized, yet emerging clonal lineage.MethodsWe computationally analyzed a large collection ofK. pneumoniaeST395 genomes (n = 297) both sequenced in this study and reported previously. By applying multiple bioinformatics tools, we investigated the core-genome phylogeny and evolution of ST395 as well as distribution of accessory genome elements associated with antibiotic resistance and virulence features.ResultsClustering of the core-SNP alignment revealed four major clades with eight smaller subclades. The subclades likely evolved through large chromosomal recombination, which involved differentK. pneumoniaedonors and affected,inter alia, capsule and lipopolysaccharide antigen biosynthesis regions. Most genomes contained acquired resistance genes to extended-spectrum cephalosporins, carbapenems, and other antibiotic classes carried by multiple plasmid types, and many were positive for hypervirulence markers, including the siderophore aerobactin. The detection of “hybrid” resistance and virulence plasmids suggests the occurrence of the convergent ST395 pathotype.ConclusionsTo the best of our knowledge, this is the first study that investigated a large international collection ofK. pneumoniaeST395 genomes and elucidated phylogenetics and detailed genomic characteristics of this emerging high-risk clonal lineage.

Homologous and heterologous SARS-CoV-2 vaccinations yield different spike protein-directed humoral and cellular immune responses. This study aimed to explore their currently unknown interdependencies.COV-ADAPT is a prospective, observational cohort study of 417 healthcare workers who received vaccination with homologous ChAdOx1 nCoV-19, homologous BNT162b2 or with heterologous ChAdOx1 nCoV-19/BNT162b2. We assessed humoral (anti-spike-RBD-IgG, neutralizing antibodies, and avidity) and cellular (spike-induced T-cell interferon-γ release) immune responses in blood samples up to 2 weeks before (T1) and 2-12 weeks following secondary immunization (T2).Initial vaccination with ChAdOx1 nCoV-19 resulted in lower anti-spike-RBD-IgG compared with BNT162b2 (70 ± 114 vs. 226 ± 279 BAU/ml, p .01) at T1. Booster vaccination with BNT162b2 proved superior to ChAdOx1 nCoV-19 at T2 (anti-spike-RBD-IgG: ChAdOx1 nCoV-19/BNT162b2 2387 ± 1627 and homologous BNT162b2 3202 ± 2184 vs. homologous ChAdOx1 nCoV-19 413 ± 461 BAU/ml, both p .001; spike-induced T-cell interferon-γ release: ChAdOx1 nCoV-19/BNT162b2 5069 ± 6733 and homologous BNT162b2 4880 ± 7570 vs. homologous ChAdOx1 nCoV-19 1152 ± 2243 mIU/ml, both p .001). No significant differences were detected between BNT162b2-boostered groups at T2. For ChAdOx1 nCoV-19, no booster effect on T-cell activation could be observed. We found associations between anti-spike-RBD-IgG levels (ChAdOx1 nCoV-19/BNT162b2 and homologous BNT162b2) and T-cell responses (homologous ChAdOx1 nCoV-19 and ChAdOx1 nCoV-19/BNT162b2) from T1 to T2. Additionally, anti-spike-RBD-IgG and T-cell response were linked at both time points (all groups combined). All regimes yielded neutralizing antibodies and increased antibody avidity at T2.Interdependencies between humoral and cellular immune responses differ between common SARS-CoV-2 vaccination regimes. T-cell activation is unlikely to compensate for poor humoral responses.

In recent years, Arcobacter butzleri has gained clinical significance as an emerging diarrheagenic pathogen associated with poultry and water reservoirs. The full clinical significance of Arcobacter remains rather speculative due to variable virulence and antibiotic susceptibility of individual strains. The aims of the present study were (i) to identify antibiotic resistance genes (ARGs) in the genome sequences of two multidrug-resistant A. butzleri isolates, (ii) to use multilocus-sequence typing (MLST) to generate a guiding phylogeny of A. butzleri isolates collected in Kumasi, Ghana, (iii) to examine the distribution of ARGs in the test cohort, and (iv) to assess the strain’s virulence and possible antibiotic treatment options for arcobacteriosis based on the genome sequences and the ARG distribution. A total of 48 A. butzleri isolates obtained from poultry were included in the analysis. These isolates were genotyped by MLST and the antibiotic susceptibilities of isolates to ampicillin, ciprofloxacin, tetracycline, gentamicin, and erythromycin were tested by disk diffusion. Whole genome sequence data of two multidrug-resistant (MDR) A. butzleri isolates were obtained by a combination of single-molecule real-time (SMRT) and Illumina sequencing technology. A total of 14 ARGs were identified in the two generated genome sequences. For all 48 isolates, the frequency of these 14 ARGs was investigated by PCR or amplicon sequencing. With 44 different sequence types found among 48 isolates, strains were phylogenetically heterogeneous. Four of 48 isolates showed an ARG constellation indicating a multidrug-resistant phenotype. The virulence genes in the two A. butzleri genomes showed that the species might be characterized by a somewhat lower virulence as Campylobacter species. The phenotypic susceptibility data combined with the distribution of the particular ARGs especially oxa-464 and the T81I point mutation of the quinolone resistance determining region (QRDR) in a significant percentage of isolates indicated that macrolides and tetracycline can be recommended for calculated antibiotic treatment of arcobacteriosis in Ghana, but not ampicillin and quinolones.

Worldwide, farm animals, in particular poultry, are an important reservoir for Campylobacter spp. However, information on Campylobacter colonization in farm animals in Africa is scarce. Hence, this cross-sectional study determined antibiotic-resistant Campylobacter from both commercial and smallholder farm animals in the Asante Akim North Municipality of Ghana. Fecal samples from poultry and livestock kept by commercial and smallholder farms were collected and analyzed using standard microbiological methods. The overall Campylobacter frequency was 20.3% (n/N = 322/1,585), and frequencies detected were similarly high in isolates from commercial (21.0%, n/N = 169/805) and smallholder (19.6%, n/N = 153/780) farms. Species isolated were C. coli (67.7%, n/N = 218/322) and C. jejuni (32.3%, n/N = 104/322). However, the frequency of C. coli was 2.1 (95% CI: 1.8–2.5) times higher than what was found for C. jejuni. Campylobacter frequencies in the rainy season was 22.2% (n/N = 258/1,160) and 15.1% (n/N = 64/425) in the dry season (prevalence ratio = 1.48, 95% CI: 1.2–1.9). About 1.7% (n/N = 6/322) of the Campylobacter isolates, all from smallholder farms, were susceptible to all antibiotics tested. Multidrug resistance was observed for 4.7% (n/N = 15/322) of the Campylobacter isolates, of which 93.3% (n/N = 14/15) occurred in isolates from commercial farms. This study highlights the need for the implementation of control programs, in commercial farming but also at the smallholder farm level, to formulate clear guidelines aimed at decreasing Campylobacter contamination of meat products and reducing the use of antibiotics in the farming sector.

T cells orchestrate adaptive and innate immune responses against pathogens and transformed cells. However, T cells are also the main adaptive effector cells that mediate allergic and autoimmune reactions. Within the last few years, it has become abundantly clear that activation, differentiation, effector function, and environmental adaptation of T cells is closely linked to their energy metabolism. Beyond the provision of energy equivalents, metabolic pathways in T cells generate building blocks required for clonal expansion. Furthermore, metabolic intermediates directly serve as a source for epigenetic gene regulation by histone and DNA modification mechanisms. To date, several antibiotics were demonstrated to modulate the metabolism of T cells especially by altering mitochondrial function. Here, we set out to systematically review current evidence about how beta-lactam antibiotics, macrolides, fluoroquinolones, tetracyclines, oxazolidinones, nitroimidazoles, and amphenicols alter the metabolism and effector functions of CD4+ T helper cell populations and CD8+ T cells in vitro and in vivo. Based on this evidence, we have developed an overview on how the use of these antibiotics may be beneficial or detrimental in T cell-mediated physiological and pathogenic immune responses, such as allergic and autoimmune diseases, by altering the metabolism of different T cell populations.

Worldwide, farm animals, in particular poultry, are an important reservoir for

Erysipelothrix rhusiopathiae is a facultative anaerobic, environmentally stable, Gram-positive rod that causes swine and avian erysipelas as a zoonotic pathogen. In humans, the main manifestations described are circumscribed erysipeloid, generalized erysipeloid, and endocarditis. Here, we report a 46-year-old female patient who presented to the physician because of redness and marked functio laesa of the hand, in terms of a pain-related restricted range of motion, and was treated surgically. E. rhusopathiae was detected in tissue biopsy. The source of infection was considered to be a pond in which both swine and, later, her dog bathed. The genome of the isolate was completely sequenced and especially the presumptive virulence associated factors as well as the presumptive antimicrobial resistance genes, in particular a predicted homologue to the multiple sugar metabolism regulator (MsmR), several predicted two-component signal transduction systems, three predicted hemolysins, two predicted neuraminidases, three predicted hyaluronate lyases, the surface protective antigen SpaA, a subset of predicted enzymes that potentially confer resistance to reactive oxygen species (ROS), several predicted phospholipases that could play a role in the escape from phagolysosomes into host cell cytoplasm as well as a predicted vancomycin resistance locus (vex23-vncRS) and three predicted MATE efflux transporters were investigated in more detail.

Background: Humoral and cellular immune responses to SARS-CoV-2 vaccines wane with time. In the COV‑ADAPT cohort, we recently studied both immunological components and their interdependencies following different vaccine combinations before (T1) and up to three months after second immunization (T2). This follow-up investigated the stability of long-term immune responses and aimed to identify predictive markers. Methods: We assessed humoral (anti-spike-RBD-IgG, neutralization capacity, avidity) and cellular (spike-induced T-cell interferon‑γ release) immune responses three-seven months after secondary vaccination (T3) in blood samples of 318 healthcare workers with previous homologous ChAdOx1 nCoV-19 (ChAdOx1), homologous BNT162b2 or heterologous ChAdOx1/BNT162b2 vaccinations. Results: At T3, homologous ChAdOx1 vaccination resulted in significantly lower anti-spike-RBD-IgG (152±151 BAU/ml) as compared to heterologous ChAdOx1/BNT162b2 (388±300 BAU/ml) and homologous BNT162b2 (435±327 BAU/ml). In all groups, anti-spike-RBD-IgG (T3) exceeded antibody levels before second vaccination (T1). T-cell interferon-γ release following heterologous ChAdOx1/BNT162b2 vaccination was significantly higher at T3 (1062±2083 mIU/ml) vs. T1 (680±1691 mIU/ml), yet did not differ significantly between the three groups at T3. Associations between humoral and cellular responses were found at T3 (all groups combined). Additionally, the early cellular response (at T1) was significantly associated with late (T3) humoral (ChAdOx1/BNT162b2, BNT162b2/BNT162b2) and cellular responses (all groups). In contrast to T2, neutralization capacity at T3 was significantly higher for ChAdOx1/BNT162b2 and BNT162b2/BNT162b2 vs. ChAdOx1/ChAdOx1. Conclusions: We identified (i) long-term interdependencies between the humoral and the cellular immune system, (ii) observed distinct waning dynamics following different vaccination regimes, and (iii) uncovered early T-cell responses as a useful predictor of long-term immune responses.

IntroductionTo efficiently monitor the COVID-19 pandemic for surveillance purposes, reliable serological rapid diagnostic tests (RDTs) are desirable for settings where well-established high-throughput bench-top solutions are not available. Here, we have evaluated such an RDT.MethodsWe have assessed the Xiamen AmonMed Biotechnology COVID-19 IgM/IgG test kit (Colloidal gold) and the EUROIMMUN benchtop assay with serum samples from patients with polymerase chain reaction (PCR)-confirmed COVID-19 disease. Samples from patients with Epstein-Barr-virus (EBV) infection and blood donors were used for specificity testing.ResultsFor the colloid gold rapid test and the EUROIMMUN assay, the study indicated overall sensitivity of 15.2% and 67.4%, respectively, while specificity of 99.0% and 97.9% with the blood donor sera, as well as 100% and 96.8% with the EBV-patients, were observed, respectively. An association of the time period between positive PCR results and serum acquisition with serological test positivity could be observed for the immunologlobulin G subclass of the EUROIMMUN assay only.ConclusionsIn spite of acceptable specificity of the assessed RDT, the detected poor sensitivity leaves room for improvement. The test results remain difficult to interpret and therefore the RDT can currently not be recommended for routine diagnostic or surveillance use.

Background: Homologous and heterologous SARS-CoV-2 vaccinations yield different spike protein-directed humoral and cellular immune responses. This study aimed to explore their currently unknown interdependencies. Methods: COV-ADAPT is a prospective, observational cohort study of 417 healthcare workers who received vaccination with homologous ChAdOx1 nCoV-19, homologous BNT162b2 or with heterologous ChAdOx1 nCoV-19/BNT162b2. We assessed humoral (anti-spike-RBD-IgG, neutralizing antibodies, avidity) and cellular (spike-induced T cell interferon‑γ release) immune responses in blood samples up to 2 weeks before (T1) and 2 to 12 weeks following secondary immunization (T2). Results: Initial vaccination with ChAdOx1 nCoV-19 resulted in lower anti-spike-RBD-IgG compared to BNT162b2 (70±114 vs. 226±279 BAU/ml, pConclusions: Interdependencies between humoral and cellular immune responses differ between common SARS-CoV-2 vaccination regimes. T cell activation is unlikely to compensate for poor humoral responses.

BackgroundHomologous and heterologous SARS-CoV-2-vaccinations yield different spike protein-directed humoral and cellular immune responses. However, their interdependencies remain elusive.MethodsCOV-ADAPT is a prospective, observational cohort study of 417 healthcare workers who received homologous vaccination with Astra (ChAdOx1-S; AstraZeneca) or BNT (BNT162b2; Biontech/Pfizer) or heterologous vaccination with Astra/BNT. We assessed the humoral (anti-spike-RBD-IgG, neutralizing antibodies, antibody avidity) and cellular (spike-induced T cell interferon-γ release) immune response in blood samples up to 2 weeks before (T1) and 2 to 12 weeks following secondary immunization (T2).FindingsInitial vaccination with Astra resulted in lower anti-spike-RBD-IgG responses compared to BNT (70±114 vs. 226±279 BAU/ml, pγ release: Astra/BNT 5069±6733 and BNT/BNT 4880±7570 vs. Astra/Astra 1152±2243 mIU/ml, both pInterpretationInterdependencies between humoral and cellular immune responses differ between common SARS-CoV-2 vaccination regimes. T cell activation is unlikely to compensate for poor humoral responses.FundingDeutsche Forschungsgemeinschaft (DFG), ER723/3-1Research in contextEvidence before this studyWe searched Pubmed for papers published between 01/01/2019 and 14/05/2021 with the search terms “covid-19” combined with “vaccination” and “heterologous”, excluding “BCG”. Of the 41 papers found, none addressed the combination of ChAdOx1-S by AstraZeneca (Astra) and BNT162b2 by Biontech/Pfizer (BNT). After our study was initiated, the CombiVacS trial reported a significant booster effect when BNT was given after initial vaccination with Astra.1 The investigators of the CoCo trial subsequently published data on heterologous immunization in comparison to homologous Astra in a small population (n=87), with the heterologous immunization scheme showing a superior humoral and cellular immune response.2 Further studies investigated heterologous vaccinations with Astra and BNT as well as homologous Astra and BNT regimes and also found superior humoral and cellular immune responses in the heterologous regimes compared to homologous Astra, and comparable or slightly superior immune responses when compared to homologous BNT vaccination.3–6 The body of research covering the effects of heterologous immunization regimes has recently been aggregated in a systematic review.7Added value of this studyTo our knowledge, this is the first study that evaluates the interdependencies of cellular and humoral immune responses following heterologous vaccination with Astra/BNT in a large group of individuals. Our data show strong correlations between humoral and cellular immune responses with the prime-boost combination Astra/BNT. The findings suggest that individuals with a robust initial response developed strong humoral and cellular immune responses after booster immunization.Implications of all the available evidenceOur study and the available data suggest that due to its superior capacity to elicit a humoral and cellular immune response, mRNA-based vaccines such as BNT should be chosen for booster vaccination rather than Astra. This seems to be particularly important in individuals whose immune response was poor after initial vaccination with Astra.We demonstrate here an association between humoral and cellular immune responses following vaccination. Our findings suggest that distinct differences between common COVID-19 vaccination regimes should be taken into account in population-based vaccine programs. The present data indicate that a poor humoral immune response is unlikely to be mitigated by a strong cellular immune response.

The intriguing recent discovery of Campylobacter coli strains, especially of clade 1, that (i) possess mosaic C. coli / C. jejuni alleles, (ii) demonstrate mixed multilocus sequence types (MLSTs) and (iii) have undergone genome-wide introgression has led to the speculation that these two species may be involved in an accelerated rate of horizontal gene transfer that is progressively leading to the merging of both species in a process coined ‘despeciation’. In an MLST-based neighbour-joining tree of a number of C. coli and C. jejuni isolates of different clades, three prominent Campylobacter isolates formed a seemingly separate cluster besides the previously described C. coli and C. jejuni clades. In the light of the suspected, ongoing genetic introgression between the C. coli and C. jejuni species, this cluster of Campylobacter isolates is proposed to present one of the hybrid clonal complexes in the despeciation process of the genus. Specific DNA methylation as well as restriction modification systems are known to be involved in selective uptake of external DNA and their role in such genetic introgression remains to be further investigated. In this study, the phylogeny and DNA methylation of these putative C. coli / C. jejuni hybrid strains were explored, their genomic mosaic structure caused by C. jejuni introgression was demonstrated and basic phenotypic assays were used to characterize these isolates. The genomes of the three hybrid Campylobacter strains were sequenced using PacBio SMRT sequencing, followed by methylome analysis by Restriction-Modification Finder and genome analysis by Parsnp, Smash++ and blast. Additionally, the strains were phenotypically characterized with respect to growth behaviour, motility, eukaryotic cell invasion and adhesion, autoagglutination, biofilm formation, and water survival ability. Our analyses show that the three hybrid Campylobacter strains are clade 1 C . coli strains, which have acquired between 8.1 and 9.1 % of their genome from C. jejuni . The C. jejuni genomic segments acquired are distributed over the entire genome and do not form a coherent cluster. Most of the genes originating from C. jejuni are involved in chemotaxis and motility, membrane transport, cell signalling, or the resistance to toxic compounds such as bile acids. Interspecies gene transfer from C. jejuni has contributed 8.1–9.1% to the genome of three C. coli isolates and initiated the despeciation between C. jejuni and C. coli . Based on their functional annotation, the genes originating from C. jejuni enable the adaptation of the three strains to an intra-intestinal habitat. The transfer of a fused type II restriction-modification system that recognizes the CAYNNNNNCTC/GAGNNNNNRTG motif seems to be the key for the recombination of the C. jejuni genetic material with C. coli genomes.

Flagellar motility is important for the pathogenesis of many intestinal pathogens, allowing bacteria to move to their preferred ecological niche. Clostridioides difficile is currently the major cause for bacterial health care-associated intestinal infections in the western world. Most clinical strains produce peritrichous flagella and are motile in soft-agar. However, little knowledge exists on the C. difficile swimming behaviour and its regulation at the level of individual cells. We report here on the swimming strategy of C. difficile at the single cell level and its dependency on environmental parameters. A comprehensive analysis of motility parameters from several thousand bacteria was achieved with the aid of a recently developed bacterial tracking programme. C. difficile motility was found to be strongly dependent on the matrix elasticity of the medium. Long run phases of all four motile C. difficile clades were only observed in the presence of high molecular weight molecules such as polyvinylpyrrolidone (PVP) and mucin, which suggests an adaptation of the motility apparatus to the mucin-rich intestinal environment. Increasing mucin or PVP concentrations lead to longer and straighter runs with increased travelled distance per run and fewer turnarounds that result in a higher net displacement of the bacteria. The observed C. difficile swimming pattern under these conditions is characterised by bidirectional, alternating back and forth run phases, interrupted by a short stop without an apparent reorientation or tumbling phase. This motility type was not described before for peritrichous bacteria and is more similar to some previously described polar monotrichous bacteria.

This study was performed as a head-to-head comparison of the performance characteristics of (1) two SARS-CoV-2-specific rapid antigen assays with real-time PCR as gold standard as well as (2) a fully automated high-throughput transcription-mediated amplification (TMA) assay and real-time PCR in a latent class analysis-based test comparison without a gold standard with several hundred samples in a low prevalence “real world” setting. Recorded sensitivity and specificity of the NADAL and the LumiraDx antigen assays and the Hologic Aptima SARS-CoV-2 TMA assay were 0.1429 (0.0194, 0.5835), 0.7644 (0.7016, 0.8174), and 0.7157 (0, 1) as well as 0.4545 (0.2022, 0.7326), 0.9954 (0.9817, 0.9988), and 0.9997 (not estimable), respectively. Agreement kappa between the positive results of the two antigen-based assays was 0.060 (0.002, 0.167) and 0.659 (0.492, 0.825) for TMA and real-time PCR. Samples with low viral load as indicated by cycle threshold (Ct) values &gt, 30 were generally missed by both antigen assays, while 1:10 pooling suggested higher sensitivity of TMA compared to real-time PCR. In conclusion, both sensitivity and specificity speak in favor of the use of the LumiraDx rather than the NADAL antigen assay, while TMA results are comparably as accurate as PCR, when applied in a low prevalence setting.

Serological assays can contribute to the estimation of population proportions with previous immunologically relevant contact with the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) virus. In this study, we compared five commercially available diagnostic assays for the diagnostic identification of SARS-CoV-2-specific antibodies. Depending on the assessed immunoglobulin subclass, recorded sensitivity ranged from 17.0% to 81.9% with best results for immunoglobulin G. Specificity with blood donor sera ranged from 90.2% to 100%, with sera from EBV patients it ranged from 84.3% to 100%. Agreement from fair to nearly perfect was recorded depending on the immunoglobulin class between the assays, the with best results being found for immunoglobulin G. Only for this immunoglobulin class was the association between later sample acquisition times (about three weeks after first positive PCR results) and positive serological results in COVID-19 patients confirmed. In conclusion, acceptable and comparable reliability for the assessed immunoglobulin G-specific assays could be shown, while there is still room for improvement regarding the reliability of the assays targeting the other immunoglobulin classes.

SUMMARYBackgroundThe global pandemic of COVID-19 (coronavirus disease 2019) is caused by the novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), with different prevalence rates across countries and regions. Dynamic testing strategies are mandatory to establish efficient mitigation strategies against the disease; to be cost effective, they should adapt to regional prevalences. Seroprevalence surveys that detect individuals who have mounted an immune response against COVID-19 will help to determine the total number of infections within a community and improve the epidemiological calculations of attack and case fatality rates of the virus. They will also inform about the percentage of a population that might be immune against re-infections.MethodsWe developed a sensitive and specific cell-based assay to detect conformational SARS-CoV-2 spike (SARS-2-S) S1 antibodies in human serum, and have cross-evaluated this assay against two FDA-approved SARS-CoV-2 antibody assays. We performed pseudovirus neutralization assays to determine whether sera that were rated antibody-positive in our assay were able to specifically neutralize SARS-2-S. We pooled up to 24 sera and assessed the group testing performance of our cell-based assay. Group testing was further optimized by Monte Carlo like simulations and prospectively evaluated.FindingsHighly significant correlations could be established between our cell-based assay and commercial antibody tests for SARS-CoV-2. SARS-2-S S1 antibody-positive sera neutralized SARS-2-S but not SARS-S, and were sensitively and specifically detected in pools of 24 samples. Monte Carlo like simulations demonstrated that a simple two-step pooling scheme with fixed pool sizes performed at least equally as well as Dorfman’s optimal testing across a wide range of antibody prevalences.InterpretationWe demonstrate that a cell-based assay for SARS-2-S S1 antibodies qualifies for group testing of neutralizing anti-SARS-2-S antibodies. The assay can be combined with an easily implemented algorithm which greatly expands the screening capacity to detect anti-SARS-2-S antibodies across a wide range of antibody prevalences. It will thus improve population serological testing in many countries.FundingThis work was supported by the Bundesministerium für Bildung und Forschung within the network project RAPID (risk assessment in pre-pandemic respiratory infectious diseases [grant number 01KI1723D, S.P.]).

The aim of this study was to assess whether epidemics of sexually transmitted infections caused by poorly transmittable agents corresponded to self-reported sexual activity in a distinct population. To exemplify this, a model was used to investigate whether HIV infection incidences corresponded to the extent of sexual activity as assessed by a questionnaire-based study. The model suggested between 97 and 486 sexual contacts per German individual during a sexually active lifetime based on the annual HIV incidence of 680 among the heterosexual population reported by the German National Health Authority. This is in line with the estimated 296 sexual contacts during one&rsquo, s lifetime, which was indicated by questionnaire respondents. The model confirms the correspondence of self-reported sexual activity with HIV incidence as reported by the German National Health Authority. Accordingly, HIV incidence- and prevalence-based modeling of sexual activity in a population provides crude estimations in situations where a range of uncertainty is acceptable. The model&rsquo, s veracity is limited by a number of assumptions necessitated by the paucity of data. Nevertheless, the model may be suitable in settings where severe reporting bias has to be expected for legal or socio-cultural reasons.

Young children are frequently colonized with Clostridioides (C.) difficile. Depending on their resistance patterns, antibiotic treatment can facilitate gastrointestinal spreading in colonized individuals, potentially leading to transmission to others. C. difficile was isolated from stool samples from infants born in two hospitals in Göttingen and Darmstadt, Germany. All isolates were subjected to phenotypic antimicrobial resistance testing, PCR-based screening for toxin genes and mass spectrometry-based exclusion of ribotypes 027 and 176. Within an initial cohort of 324 neonates with a longitudinal survey of C. difficile, 137 strains were isolated from 48 individuals. Antimicrobial resistance was recorded against metronidazole in one (0.7%), erythromycin in 16 (11.7%) and moxifloxacin in 2 (1.5%) of the strains, whereas no resistance was observed against vancomycin (0.0%) or rifampicin (0.0%). Newly observed resistance against erythromycin in children with detection of previously completely sensitive isolates was reported for C. difficile isolates from 2 out of 48 children. In 20 children (42%), non-toxigenic strains were detected, and from 27 children (56%), toxigenic strains were isolated, while both toxigenic and non-toxigenic strains were recorded for 1 child (2%). Ribotypes 027 or 176 were not observed. In conclusion, the German C. difficile strains isolated from the children showed mild to moderate resistance with predominance of macrolide resistance, a substance class which is frequently applied in children. The observed switches to the dominance of macrolide-resistant isolates suggests likely selection of resistant C. difficile strains already in children.

Background: Antibody mass testing to identify SARS-CoV-2 seropositive people represents one of the major strategies to combat emergence of COVID-19 Postulating

Objectives The identification and characterization of clinical Clostridioides difficile isolates with reduced fidaxomicin susceptibility. Methods Agar dilution assays were used to determine fidaxomicin MICs. Genome sequence data were obtained by single-molecule real-time (SMRT) sequencing in addition to amplicon sequencing of rpoB and rpoC alleles. Allelic exchange was used to introduce the identified mutation into C. difficile 630Δerm. Replication rates, toxin A/B production and spore formation were determined from the strain with reduced fidaxomicin susceptibility. Results Out of 50 clinical C. difficile isolates, isolate Goe-91 revealed markedly reduced fidaxomicin susceptibility (MIC >64 mg/L). A V1143D mutation was identified in rpoB of Goe-91. When introduced into C. difficile 630Δerm, this mutation decreased fidaxomicin susceptibility (MIC >64 mg/L), but was also associated with a reduced replication rate, low toxin A/B production and markedly reduced spore formation. In contrast, Goe-91, although also reduced in toxin production, showed normal growth rates and only moderately reduced spore formation capacities. This indicates that the rpoBV1143D allele-associated fitness defect is less pronounced in the clinical isolate. Conclusions To the best of our knowledge, this is the first description of a pathogenic clinical C. difficile isolate with markedly reduced fidaxomicin susceptibility. The lower-than-expected fitness burden of the resistance-mediating rpoBV1143D allele might be an indication for compensatory mechanisms that take place during in vivo selection of mutants.

Two new DNA FISH-probes for Campylobacter fetus were designed, in silico checked for cross-reactions and successfully evaluated in a multi-centric approach with 41 Campylobacter fetus isolates including isolates of all three know subspecies: Campylobacter fetus ssp. fetus, Campylobacter fetus ssp. venerealis, and Campylobacter fetus ssp. testudinum and 40 strains of five non-target Campylobacter species.

Human immunodeficiency virus (HIV) is the causative agent of the Acquired Immunodeficiency Syndrome (AIDS). The pandemic is believed to have originated within the Northern Congo basin covering large parts of the Democratic Republic of Congo, the Republic of Congo, the Central African Republic, Cameroon and Gabon. Although over decades, HIV-1 has spread throughout the World leaving no country unaffected, sub-Saharan Africa remains the region with more than 80% of all infected individuals. The HIV-2 epidemic has largely remained restricted to West Africa along the Upper Guinean forests. Co-incident with these regions of highest HIV distribution is a part of the malaria belt and therefore, co-infections are common. In this review we carve out the consequences of HIV transmission prevention and synchronous malaria prophylaxis during occupational or leisure travelling activities within this World region. In particular, we elaborate on considering pre-existing drug resistances of both, the malaria parasites and the immunodeficiency viruses, when determining a combination for prophylactic and, if necessary, post-expositional measures with a focus on the compatibility of both medications.

Clostridioides difficile, a Gram-positive spore-forming bacterium, is the leading cause of nosocomial diarrhea worldwide and therefore a substantial burden to the healthcare system. During the past decade, hypervirulent PCR-ribotypes (RT) e.g., RT027 or RT176 emerged rapidly all over the world, associated with both, increased severity and mortality rates. It is thus of great importance to identify epidemic strains such as RT027 and RT176 as fast as possible. While commonly used diagnostic methods, e.g., multilocus sequence typing (MLST) or PCR-ribotyping, are time-consuming, proteotyping offers a fast, inexpensive, and reliable alternative solution. In this study, we established a MALDI-TOF-based typing scheme for C. difficile. A total of 109 ribotyped strains representative for five MLST clades were analyzed by MALDI-TOF. MLST, based on whole genome sequences, and PCR-ribotyping were used as reference methods. Isoforms of MS-detectable biomarkers, typically ribosomal proteins, were related with the deduced amino acid sequences and added to the C. difficile proteotyping scheme. In total, we were able to associate nine biomarkers with their encoding genes and include them in our proteotyping scheme. The discriminatory capacity of the C. difficile proteotyping scheme was mainly based on isoforms of L28-M (2 main isoforms), L35-M (4 main isoforms), and S20-M (2 main isoforms) giving rise to at least 16 proteotyping-derived types. In our test population, five of these 16 proteotyping-derived types were detected. These five proteotyping-derived types did not correspond exactly to the included five MLST-based C. difficile clades, nevertheless the subtyping depth of both methods was equivalent. Most importantly, proteotyping-derived clade B contained only isolates of the hypervirulent RT027 and RT176. Proteotyping is a stable and easy-to-perform intraspecies typing method and a promising alternative to currently used molecular techniques. It is possible to distinguish the group of RT027 and RT176 isolates from non-RT027/non-RT176 isolates using proteotyping, providing a valuable diagnostic tool.

Diagnostic testing in the infectious disease laboratory facilitates decision-making by physicians at the bedside as well as epidemiological assessments and surveillance at study level. Problems may arise if test results are uncritically considered as being the same as the unknown true value. To allow a better understanding, the influence of external factors on the interpretation of test results is introduced with the example of prevalence, followed by the presentation of strengths and weaknesses of important techniques in the infectious disease laboratory like microscopy, cultural diagnostics, serology, mass spectrometry, nucleic acid amplification and hypothesis-free metagenomic sequencing with focus on basic, high-technology and potential future approaches. Special problems like multiplex testing as well as uncertainty of test evaluations, if no gold standard is available, are also stressed with a final glimpse on emerging future technologies for the infectious disease laboratory. In the conclusions, suitability for point-of-care-testing and field laboratory applications is summarized. The aim is to illustrate the limitations of diagnostic accuracy to both clinicians and study planners and to stress the importance of close cooperation with experts in laboratory disciplines so as to avoid potentially critical misunderstandings due to inappropriate interpretation of diagnostic test results.

Salmonella and Campylobacter are important gastroenteric pathogens. Arcobacter butzleri is an emerging enteric pathogen. Data on the frequencies of these poultry-associated pathogens on meat products sold in sub-Saharan Africa are scarce. This study aimed to analyze the frequency of Salmonella, Campylobacter, and Arcobacter antibiotic resistance and underlying mechanisms of resistance to fluoroquinolones in locally produced and imported poultry sold in urban Ghana. Chicken meat was collected and cultured on standard media. Bacterial strains were identified by biochemical methods and by mass spectrometry. Antibiotic susceptibility was tested by disk diffusion. Ciprofloxacin-resistant strains were assessed for molecular mechanisms of resistance. Among 200 samples, comprising 34% (n = 68) from the Ghanaian poultry industry and 66% (n = 132) from imports, 9% (n = 17) contained Salmonella, 11% (n = 22) Campylobacter, and 26.5% (n = 53) A. butzleri. Higher overall contamination frequencies were found in local meat. Most common Salmonella serovars identified were Kentucky (n/N = 5/16; 31%) and Poona (n/N = 4/16; 25%). Campylobacter were C. coli (n/N = 10/19; 53%) and C. jejuni (n/N = 9/19; 47%). Resistance to fluoroquinolones was high with 63% (n = 10), 75% (n = 15), and 52% (n = 25) in Salmonella, Campylobacter, and Arcobacter, respectively. A link between Salmonella Kentucky [sequence type (ST) 198] and a ciprofloxacin minimum inhibitory concentration of 16 μg/mL was found. Salmonella Poona-ST308 revealed transferable qnrB2 fluoroquinolone resistance genes. Markedly high frequencies of resistant Salmonella, Campylobacter, and Arcobacter predominant in locally produced meat represent a probable transmission reservoir for human infections. These findings highlight the need for implementation of surveillance systems that focus on food hygiene, use of antibiotics in animal husbandry, and continuous monitoring of the quality of meat products from imports.

Campylobacter jejuni and Campylobacter coli are among the leading causes of gastroenteritis in humans worldwide, particularly in Africa. Poultry remains a major source of Campylobacter species and a vector of transmission to humans. This pilot study was aimed at isolating and determining the antibiotic susceptibility profiles of Campylobacter spp. from fresh poultry droppings collected from poultry farms in Lagos State, Nigeria. Susceptibility was assessed using the CLSI standards. Standard microbiological methods were used in isolation, identification, and characterization of Campylobacter spp. Isolates were subjected to antibiotic susceptibility testing by the disk diffusion method. Of the 150 poultry droppings analyzed, 8 (5.3%) harbored Campylobacter spp. All isolates proved to be C. coli since they were all negative for the hip gene. A percentage of 100% showed resistance to nalidixic acid, chloramphenicol, cloxacillin, and streptomycin. While 87.5% were susceptible to amoxicillin and amoxicillin/clavulanic acid, 62.5% were susceptible to tetracycline. Surprisingly, 62.5% of C. coli had decreased (intermediate) susceptibility to erythromycin. Although there was a low prevalence of C. coli from poultry in this study, the presence of antibiotic resistant strains circulating the food chain could result in treatment failures and difficulty in case management if involved in infections of humans.

Campylobacter fetus is a causative agent of intestinal illness and, occasionally, severe systemic infections and meningitis. C. fetus currently comprises three subspecies: C. fetus subspecies fetus (Cff), C. fetus subspecies venerealis (Cfv), and C. fetus subspecies testudinum (Cft). Cff and Cfv are primarily associated with mammals whereas Cft is associated with reptiles. To offer an alternative to laborious sequence-based techniques such as multilocus sequence typing (MLST) and polymerase chain reaction (PCR)-ribotyping for this species, the purpose of the study was to develop a typing scheme based on proteotyping. In total, 41 representative C. fetus strains were analyzed by intact cell mass spectrometry and compared to MLST results. Biomarkers detected in the mass spectrum of C. fetus subsp. fetus reference strain LMG 6442 (NCTC 10842) as well as corresponding isoforms were associated with the respective amino acid sequences and added to the C. fetus proteotyping scheme. In combination, the 9 identified biomarkers allow the differentiation of Cft subspecies strains from Cff and Cfv subspecies strains. Biomarkers to distinguish between Cff and Cfv were not found. The results of the study show the potential of proteotyping to differentiate different subspecies, but also the limitations of the method.

Salmonella and Campylobacter are important gastroenteric pathogens. Arcobacter butzleri is an emerging enteric pathogen. Data on the frequencies of these poultry-associated pathogens on meat products sold in sub-Saharan Africa are scarce. This study aimed to analyze the frequency of Salmonella, Campylobacter, and Arcobacter antibiotic resistance and underlying mechanisms of resistance to fluoroquinolones in locally produced and imported poultry sold in urban Ghana. Chicken meat was collected and cultured on standard media. Bacterial strains were identified by biochemical methods and by mass spectrometry. Antibiotic susceptibility was tested by disk diffusion. Ciprofloxacin-resistant strains were assessed for molecular mechanisms of resistance. Among 200 samples, comprising 34% (n = 68) from the Ghanaian poultry industry and 66% (n = 132) from imports, 9% (n = 17) contained Salmonella, 11% (n = 22) Campylobacter, and 26.5% (n = 53) A. butzleri. Higher overall contamination frequencies were found in local meat. Most common Salmonella serovars identified were Kentucky (n/N = 5/16; 31%) and Poona (n/N = 4/16; 25%). Campylobacter were C. coli (n/N = 10/19; 53%) and C. jejuni (n/N = 9/19; 47%). Resistance to fluoroquinolones was high with 63% (n = 10), 75% (n = 15), and 52% (n = 25) in Salmonella, Campylobacter, and Arcobacter, respectively. A link between Salmonella Kentucky [sequence type (ST) 198] and a ciprofloxacin minimum inhibitory concentration of 16 μg/mL was found. Salmonella Poona-ST308 revealed transferable qnrB2 fluoroquinolone resistance genes. Markedly high frequencies of resistant Salmonella, Campylobacter, and Arcobacter predominant in locally produced meat represent a probable transmission reservoir for human infections. These findings highlight the need for implementation of surveillance systems that focus on food hygiene, use of antibiotics in animal husbandry, and continuous monitoring of the quality of meat products from imports.

Campylobacter jejuni and Campylobacter coli are among the leading causes of gastroenteritis in humans worldwide, particularly in Africa. Poultry remains a major source of Campylobacter species and a vector of transmission to humans. This pilot study was aimed at isolating and determining the antibiotic susceptibility profiles of Campylobacter spp. from fresh poultry droppings collected from poultry farms in Lagos State, Nigeria. Susceptibility was assessed using the CLSI standards. Standard microbiological methods were used in isolation, identification, and characterization of Campylobacter spp. Isolates were subjected to antibiotic susceptibility testing by the disk diffusion method. Of the 150 poultry droppings analyzed, 8 (5.3%) harbored Campylobacter spp. All isolates proved to be C. coli since they were all negative for the hip gene. A percentage of 100% showed resistance to nalidixic acid, chloramphenicol, cloxacillin, and streptomycin. While 87.5% were susceptible to amoxicillin and amoxicillin/clavulanic acid, 62.5% were susceptible to tetracycline. Surprisingly, 62.5% of C. coli had decreased (intermediate) susceptibility to erythromycin. Although there was a low prevalence of C. coli from poultry in this study, the presence of antibiotic resistant strains circulating the food chain could result in treatment failures and difficulty in case management if involved in infections of humans. Open-Access-Publikationsfonds 2019 peerReviewed

Clostridioides difficile, a Gram-positive spore-forming bacterium, is the leading cause of nosocomial diarrhea worldwide and therefore a substantial burden to the healthcare system. During the past decade, hypervirulent PCR-ribotypes (RT) e.g., RT027 or RT176 emerged rapidly all over the world, associated with both, increased severity and mortality rates. It is thus of great importance to identify epidemic strains such as RT027 and RT176 as fast as possible. While commonly used diagnostic methods, e.g., multilocus sequence typing (MLST) or PCR-ribotyping, are time-consuming, proteotyping offers a fast, inexpensive, and reliable alternative solution. In this study, we established a MALDI-TOF-based typing scheme for C. difficile. A total of 109 ribotyped strains representative for five MLST clades were analyzed by MALDI-TOF. MLST, based on whole genome sequences, and PCR-ribotyping were used as reference methods. Isoforms of MS-detectable biomarkers, typically ribosomal proteins, were related with the deduced amino acid sequences and added to the C. difficile proteotyping scheme. In total, we were able to associate nine biomarkers with their encoding genes and include them in our proteotyping scheme. The discriminatory capacity of the C. difficile proteotyping scheme was mainly based on isoforms of L28-M (2 main isoforms), L35-M (4 main isoforms), and S20-M (2 main isoforms) giving rise to at least 16 proteotyping-derived types. In our test population, five of these 16 proteotyping-derived types were detected. These five proteotyping-derived types did not correspond exactly to the included five MLST-based C. difficile clades, nevertheless the subtyping depth of both methods was equivalent. Most importantly, proteotyping-derived clade B contained only isolates of the hypervirulent RT027 and RT176. Proteotyping is a stable and easy-to-perform intraspecies typing method and a promising alternative to currently used molecular techniques. It is possible to distinguish the group of RT027 and RT176 isolates from non-RT027/non-RT176 isolates using proteotyping, providing a valuable diagnostic tool.

Background Campylobacter jejuni is one of the most common bacterial causes of food-borne enteritis worldwide. Chemotaxis in C. jejuni is known to be critical for the successful colonization of the host and key for the adaptation of the microbial species to different host environments. In C. jejuni, chemotaxis is regulated by a complex interplay of 13 or even more different chemoreceptors, also known as transducer-like proteins (Tlps). Recently, a novel chemoreceptor gene, tlp12, was described and found to be present in 29.5% of the investigated C. jejuni strains. Results In this study, we present a functional analysis of Tlp12 with the aid of a tlp12 knockout mutant of the C. jejuni strain A17. Substrate specificity was investigated by capillary chemotaxis assays and revealed that Tlp12 plays an important role in chemotaxis towards glutamate and pyruvate. Moreover, the Δtlp12 mutant shows increased swarming motility in soft agar assays, an enhanced invasion rate into Caco-2 cells and an increased autoagglutination rate. The growth rate was slightly reduced in the Δtlp12 mutant. The identified phenotypes were in partial restored by complementation with the wild type gene. Tlp12-harboring C. jejuni strains display a strong association with chicken, whose excreta are known to contain high glutamate levels. Conclusions TLP12 is a chemoreceptor for glutamate and pyruvate recognition. Deletion of tlp12 has an influence on distinct physiological features, such as growth rate, swarming motility, autoagglutination and invasiveness. Electronic supplementary material The online version of this article (10.1186/s12866-018-1254-0) contains supplementary material, which is available to authorized users.

PURPOSE: Bile acids are crucial components of the intestinal antimicrobial defense and represent a significant stress factor for enteric pathogens. Adaptation processes of Campylobacter jejuni to this hostile environment are analyzed in this study by a proteomic approach. EXPERIMENTAL DESIGN: Proteome profiling by label-free mass spectrometry (SWATH-MS) has been used to characterize the adaptation of C. jejuni to sublethal concentrations of seven bile acids. RESULTS: The bile acids with the lowest inhibitory concentration (IC50 ), deoxycholic and chenodeoxycholic acid, induce the most significant proteome changes. Overall a downregulation of all basic biosynthetic pathways and a general decrease in the transcription machinery are found. Concurrently, an induction of factors involved in detoxification of reactive oxygen species, protein folding, and bile acid exporting efflux pumps is detected. Exposure to deoxycholic and chenodeoxycholic acid results in an increased expression of components of the more energy-efficient aerobic respiration pathway, while the anaerobic branches of the electron transport chain are down-expressed. CONCLUSIONS AND CLINICAL RELEVANCE: The results show that C. jejuni has a differentiated system of adaptation to bile acid stresses. The findings enhance the understanding of the pathogenesis of campylobacteriosis, especially for survival of C. jejuni in the human intestine, and may provide clues to future medical treatment. peerReviewed

Haemophilus influenzae is a key pathogen of upper respiratory tract infections. Its reliable discrimination from nonpathogenic Haemophilus spp. is necessary because merely colonizing bacteria are frequent at primarily unsterile sites. Due to close phylogenetic relationship, it is not easy to discriminate H. influenzae from the colonizer Haemophilus haemolyticus. The frequency of H. haemolyticus isolations depends on factors like sampling site, patient condition, and geographic region. Biochemical discrimination has been shown to be nonreliable. Multiplex PCR including marker genes like sodC, fucK, and hpd or sequencing of the 16S rRNA gene, the P6 gene, or multilocus-sequence-typing is more promising. For the diagnostic routine, such techniques are too expensive and laborious. If available, matrix-assisted laser-desorption–ionization time-of-flight mass spectrometry is a routine-compatible option and should be used in the first line. However, the used database should contain well-defined reference spectra, and the spectral difference between H. influenzae and H. haemolyticus is small. Fluorescence in-situ hybridization is an option for less well-equipped laboratories, but the available protocol will not lead to conclusive results in all instances. It can be used as a second line approach. Occasional ambiguous results have to be resolved by alternative molecular methods like 16S rRNA gene sequencing.

Modern molecular diagnostic approaches in the diagnostic microbiological laboratory like real-time quantitative polymerase chain reaction (qPCR) have led to a considerable increase of diagnostic sensitivity. They usually outperform the diagnostic sensitivity of culture-based approaches. Culture-based diagnostics were found to be insufficiently sensitive for the assessment of the composition of biofilms in chronic wounds and poorly standardized for screenings for enteric colonization with multi-drug resistant bacteria. However, the increased sensitivity of qPCR causes interpretative challenges regarding the attribution of etiological relevance to individual pathogen species in case of multiple detections of facultative pathogenic microorganisms in primarily non-sterile sample materials. This is particularly the case in high-endemicity settings, where continuous exposition to respective microorganisms leads to immunological adaptation and semi-resistance while considerable disease would result in case of exposition of a non-adapted population. While biofilms in chronic wounds show higher pathogenic potential in case of multi-species composition, detection of multiple pathogens in respiratory samples is much more difficult to interpret and asymptomatic enteric colonization with facultative pathogenic microorganisms is frequently observed in high endemicity settings. For respiratory samples and stool samples, cycle-threshold-value-based semi-quantitative interpretation of qPCR results has been suggested. Etiological relevance is assumed if cycle-threshold values are low, suggesting high pathogen loads. Although the procedure is challenged by lacking standardization and methodical issues, first evaluations have led to promising results. Future studies should aim at generally acceptable quantitative cut-off values to allow discrimination of asymptomatic colonization from clinically relevant infection.