Core genome multilocus sequence typing (cgMLST) applicable to the monophyletic Klebsiella oxytoca species complex.

The environmental bacterium Klebsiella oxytoca displays an alarming increase of antibiotic-resistant strains that frequently cause outbreaks in intensive care units. Due to its prevalence in the environment and opportunistic presence in humans, molecular surveillance (including resistance marker screening) and high-resolution cluster analysis are of high relevance. Furthermore, K. oxytoca previously described in studies is rather a species complex (KoSC) than a single species comprising at least six closely related species that are not easily differentiated by standard typing methods. To reach a discriminatory power high enough to identify and resolve clusters within these species, whole genome sequencing is necessary. The resolution is achievable with core genome multilocus sequence typing (cgMLST) extending typing of a few housekeeping genes to thousands of core genome genes. CgMLST is highly standardized and provides a nomenclature enabling cross laboratory reproducibility and data exchange for routine diagnostics. Here, we established a cgMLST scheme not only capable of resolving the KoSC species but also producing reliable and consistent results for published outbreaks. Our cgMLST scheme consists of 2,536 core genome and 2,693 accessory genome targets, with a percentage of good cgMLST targets of 98.31% in 880 KoSC genomes downloaded from the National Center for Biotechnology Information (NCBI). We also validated resistance markers against known resistance gene patterns and successfully linked genetic results to phenotypically confirmed toxic strains carrying the til gene cluster. In conclusion, our novel cgMLST enables highly reproducible typing of four different clinically relevant species of the KoSC and thus facilitates molecular surveillance and cluster investigations.

A head-to-head comparison of three MALDI-TOF mass spectrometry systems with 16S rRNA gene sequencing.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized diagnostics in culture-based microbiology. Commonly used MALDI-TOF MS systems in clinical microbiology laboratories are MALDI Biotyper (Bruker Daltonics) and Vitek MS (bioMérieux), but recently the new EXS2600 (Zybio) has been launched. This study aimed to evaluate the performance of the three devices by comparing the results to 16S rRNA gene sequencing. A set of 356 previously collected difficult-to-identify bacteria was tested in parallel with the three systems. Only the direct smear method and simple formic acid extraction were applied. Valid results were achieved for 98.6%, 94.4%, and 93.3% of all isolates by MALDI Biotyper, EXS2600, and Vitek MS, respectively. Of all valid results, agreement with sequencing data was achieved in 98.9%, 98.5%, and 99.7% by MALDI Biotyper, EXS2600, and Vitek MS, respectively. Considering only the isolates with valid measurements at the single-species level, misidentification rates were 0%, 2.6%, and 1.1% for MALDI Biotyper, EXS2600, and Vitek MS, respectively. Apart from minor performance differences, our data demonstrate that the three systems provide comparable results and are suitable for use in medical diagnostic laboratories.

Real-Time Nanopore Q20+ Sequencing Enables Extremely Fast and Accurate Core Genome MLST Typing and Democratizes Access to High-Resolution Bacterial Pathogen Surveillance.

Next-generation whole-genome sequencing is essential for high-resolution surveillance of bacterial pathogens, for example, during outbreak investigations or for source tracking and escape variant analysis. However, current global sequencing and bioinformatic bottlenecks and a long time to result with standard technologies demand new approaches. In this study, we investigated whether novel nanopore Q20+ long-read chemistry enables standardized and easily accessible high-resolution typing combined with core genome multilocus sequence typing (cgMLST). We set high requirements for discriminatory power by using the slowly evolving bacterium Bordetella pertussis as a model pathogen. Our results show that the increased raw read accuracy enables the description of epidemiological scenarios and phylogenetic linkages at the level of gold-standard short reads. The same was true for our variant analysis of vaccine antigens, resistance genes, and virulence factors, demonstrating that nanopore sequencing is a legitimate competitor in the area of next-generation sequencing (NGS)-based high-resolution bacterial typing. Furthermore, we evaluated the parameters for the fastest possible analysis of the data. By combining the optimized processing pipeline with real-time basecalling, we established a workflow that allows for highly accurate and extremely fast high-resolution typing of bacterial pathogens while sequencing is still in progress. Along with advantages such as low costs and portability, the approach suggested here might democratize modern bacterial typing, enabling more efficient infection control globally.

Gut Microbiota Dysbiosis in Suspected Food Protein Induced Proctocolitis-A Prospective Comparative Cohort Trial.

OBJECTIVES: In infants with suspected food protein induced proctocolitis (sFPIP) only a minority of patients are finally diagnosed with the disease following diagnostic dietary intervention (DDI). There is a need for a pathophysiological explanation for the cause of hematochezia in the majority of sFPIP infants. METHODS: We prospectively recruited infants with sFPIP and healthy controls. Fecal samples were collected at inclusion, week 4 (end of DDI in sFPIP), and week 8. For 16S rRNA sequencing (515F/806R) we used Illumina MiSeq sequencing system. Amplicon sequence variants were generated using Qiime2 and DADA2. Qiime diversity alpha and beta group comparisons and linear discriminant analysis effect size analysis was performed. For shotgun metagenomic analysis on species level we used KneadData and MetaPhlAn2. RESULTS: Fourteen sFPIP infants were compared to 55 healthy infants. At inclusion overall microbial composition of sFPIP infants differed significantly from controls (weighted UniFrac; Pairwise PERMANOVA, P = 0.002, pseudo- F = 5.008). On genus level healthy infant microbiota was significantly enriched with Bifidobacterium ( B ) compared to sFPIP patients (linear discriminant analysis [LDA] = 5.5, P < 0.001, 31.3% vs 12.1%). sFPIP stool was significantly enriched by Clostridium sensu stricto 1 over controls (LDA = 5.3, P = 0.003, 3.5% vs 18.3%). DDI caused a significant and sustained increase of Bifidobacterium (LDA = 5.4, P = 0.048, 27.9%) in sFPIP infants. Species level analysis revealed significant reduction of abundance of B longum in sFPIP patients, which after DDI was reversed by B. species other than B longum . CONCLUSIONS: We revealed a gut microbiota dysbiosis phenomenon in sFPIP infants. DDI induces a microbiota composition comparable to that of healthy infants. In most sFPIP infants hematochezia might be triggered by a gut microbiota dysbiosis phenomenon.

Toxin-Producing Klebsiella oxytoca in Healthy Infants: Commensal or Pathobiont?

OBJECTIVES: Klebsiella oxytoca is a gastrointestinal pathobiont with the potential to produce the toxins tilivalline and tilimycin, which cause antibiotic-associated hemorrhagic colitis. Overgrowth of toxigenic K oxytoca has recently been implicated in necrotizing enterocolitis. K oxytoca colonizes 2-9% of healthy adults, however, there is no systematic data on colonization in healthy children. We investigated K oxytoca colonization and its toxigenic properties in healthy infants. METHODS: We sampled stool of healthy infants and determined K oxytoca colonization using stool culture and PCR (pehX). Toxin in stool was measured with HPLC/high-resolution mass spectrometry. K oxytoca isolates were typed using multi-locus sequence typing (MLST) and K oxytoca toxin PCR (npsA/B). Cytotoxin production of isolates was analyzed by MTT assay. RESULTS: K oxytoca was detected in 30 of 61 infants (49%) using stool culture and in 45 of 61 (73%) using PCR (pehX). Toxin marker PCR (npsA/B) was positive in 66% of stool samples positive for K oxytoca PCR. Stool toxin levels were too low for quantitation but traces of tilivalline were detected. Contrarily, 49% of K oxytoca isolates demonstrated toxicity in the MTT assay. MLST revealed 36 distinct sequence types affiliated with all known K oxytoca sequence type clusters (A, B1 and B2). CONCLUSIONS: More than 70% of healthy infants were colonized with K oxytoca. Toxin quantities in stool of colonized healthy infants were below detection level, yet half of the isolates produced toxin in vitro demonstrating their pathobiont potential. The high occurrence of toxigenic K oxytoca in healthy infants has to be considered for future disease association studies.

Isolate-Based Surveillance of Bordetella pertussis, Austria, 2018-2020.

Pertussis is a vaccine-preventable disease, and its recent resurgence might be attributable to the emergence of strains that differ genetically from the vaccine strain. We describe a novel pertussis isolate-based surveillance system and a core genome multilocus sequence typing scheme to assess Bordetella pertussis genetic variability and investigate the increased incidence of pertussis in Austria. During 2018-2020, we obtained 123 B. pertussis isolates and typed them with the new scheme (2,983 targets and preliminary cluster threshold of <6 alleles). B. pertussis isolates in Austria differed genetically from the vaccine strain, both in their core genomes and in their vaccine antigen genes; 31.7% of the isolates were pertactin-deficient. We detected 8 clusters, 1 of them with pertactin-deficient isolates and possibly part of a local outbreak. National expansion of the isolate-based surveillance system is needed to implement pertussis-control strategies.

An epidemic CC1-MRSA-IV clone yields false-negative test results in molecular MRSA identification assays: a note of caution, Austria, Germany, Ireland, 2020.

We investigated why a clinical meticillin-resistant Staphylococcus aureus (MRSA) isolate yielded false-negative results with some commercial PCR tests for MRSA detection. We found that an epidemic European CC1-MRSA-IV clone generally exhibits this behaviour. The failure of the assays was attributable to a large insertion in the orfX/SCCmec integration site. To ensure the reliability of molecular MRSA tests, it is vital to monitor emergence of new SCCmec types and junction sites.

Primary resistance of Helicobacter pylori is still low in Southern Austria.

OBJECTIVES: We determined primary and secondary resistance rates of H. pylori in different regions of Austria and potential bacterial and host factors associated with resistance. METHODS: In a prospective multicentre study H. pylori was cultivated from biopsies and susceptibility testing was performed according to EUCAST. Resistance to clarithromycin and levofloxacin was determined by sequencing of the resistance-determining regions of 23S rRNA and gyrA genes. cagA, vacA and babA2 genotypes were determined. RESULTS: A total of 1266 patients were included. 178 isolates were cultured: 128 from patients without prior eradication therapy, 50 from patients after failed eradication. Primary resistance to clarithromycin, levofloxacin and metronidazole were 17.2%, 9.4% and 10.2%, respectively. Secondary resistance to clarithromycin, levofloxacin and metronidazole were 64%, 18% and 44%, respectively. Prior eradication was associated with a higher risk of clarithromycin as well as metronidazole resistance (OR=8.1; 95% CI 3.8-17.1 and OR 5.7; 95% CI 2.5-13, respectively). CONCLUSION: Primary resistance to both clarithromycin and levofloxacin was markedly lower in Southern Austria than recently reported.

Accuracy of commercial kits and published primer pairs for the detection of periodontopathogens.

OBJECTIVES: Despite the input of microbiome research, a group of 20 bacteria continues to be the focus of periodontal diagnostics and therapy. The aim of this study was to compare three commercial kits and laboratory-developed primer pairs for effectiveness in detecting such periodontopathogens. MATERIALS AND METHODS: Fourteen bacterial mock communities, consisting of 16 randomly assembled bacterial strains, were used as reference standard for testing kits and primers. Extracted DNA from mock communities was analyzed by PCR in-house with specific primers and forwarded for analysis to the manufacturer's laboratory of each of the following kits: ParoCheck®Kit 20, micro-IDent®plus11, and Carpegen® Perio Diagnostik. RESULTS: The kits accurately detected Fusobacterium nucleatum, Prevotella intermedia/Prevotella nigrescens, Parvimonas micra, Aggregatibacter actinomycetemcomitans, Campylobacter rectus/showae, Streptococcus mitis, Streptococcus mutans, and Veillonella parvula. The in-house primers for F.nucleatum were highly specific to subtypes of the respective periopathogen. Other primers repeatedly detected oral pathogens not present in the mock communities, indicating reduced specificity. CONCLUSIONS: The commercial kits used in this study are reliable tools to support periodontal diagnostics. Whereas the detection profile of the kits is fixed at a general specificity level, the design of primers can be adjusted to differentiate between highly specific strains. In-house primers are more error-prone. Bacterial mock communities can be established as a reference standard for any similar testing. CLINICAL RELEVANCE: The tested kits render good results with selected bacterial species. Primers appear to be less useful for routine clinical diagnostics and of limited applicability in research. Basic information about the periodontopathogens identified in this study supports clinical decision-making.

Severe forefoot infection complicated by Fusobacterium russii.

We present the first case of a complicated foot infection caused by Fusobacterium russii in Austria. F. russii is highly associated with mammals such as cats and dogs. Our case underlines the difficulties in isolation and identification of anaerobes and the pitfalls in antimicrobial treatment of polymicrobial infections.

Contaminated handwashing sinks as the source of a clonal outbreak of KPC-2-producing Klebsiella oxytoca on a hematology ward.

We investigated sinks as possible sources of a prolonged Klebsiella pneumonia carbapenemase (KPC)-producing Klebsiella oxytoca outbreak. Seven carbapenem-resistant K. oxytoca isolates were identified in sink drains in 4 patient rooms and in the medication room. Investigations for resistance genes and genetic relatedness of patient and environmental isolates revealed that all the isolates harbored the blaKPC-2 and blaTEM-1 genes and were genetically indistinguishable. We describe here a clonal outbreak caused by KPC-2-producing K. oxytoca, and handwashing sinks were a possible reservoir.

Prevalence of emm types and antimicrobial susceptibility of Streptococcus dysgalactiae subsp. equisimilis in Austria.

OBJECTIVES: An increase of severe infections caused by Streptococcus dysgalactiae subsp. equisimilis (SDSE) similar to infections caused by Streptococcus pyogenes has been reported over the last years. Little is known about infections with SDSE in Austria. Therefore, we investigated a collection of 113 SDSE invasive and non-invasive isolates from different infection sites and type of infections as well as patients' characteristics. METHODS: The isolates were phenotypically identified and emm typed using the enlarged emm database from the Centers for Disease Control and Prevention. Additionally, 13 antimicrobial agents were tested using EUCAST guidelines and virulence genes were investigated. RESULTS: Severe SDSE infections were most common in elderly men with underlying diseases especially diabetes mellitus. With VitekMS identification of SDSE isolates was successful to the species level only. Emm typing revealed 24 different emm types, one new type and one new subtype. StG485, stG6, stC74a, stG643, and stG480 were the predominant types in this study, stC74a and stG652 in invasive infections and stG643, stC74a and stG485 in non-invasive infections. Resistance was observed to tetracycline (62%), macrolides (13%) with one M phenotype, and clindamycin (12%) presenting 6 constitutive MLS(B) phenotypes and 8 inducible MLS(B) phenotypes. Levofloxacin resistance was detected only in one isolate. All isolates tested for virulence genes were positive for scpA, ska, saga and slo. Superantigenic genes were negative except speG(dys) (positive 17/34; 50%). CONCLUSION: This paper presents the first report of SDSE infections in Austria. Severe SDSE infections were found mainly in elderly men with underlying diseases. SDSE isolates demonstrated substantial emm type diversity without association with infections site or invasiveness. Analysis of virulence genes showed no significant difference between invasive and non-invasive infections.

Detection of central venous catheter-related bloodstream infections in haematooncological patients.

BACKGROUND: Catheter-related bloodstream infections (CRBSIs) are currently detected in patients with clinically suspicion. The aim of our study was to evaluate whether CRBSIs could be anticipated and detected in a subclinical stage by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) using universal hybridization probes or acridine orange leucocyte cytospin (AOLC) tests in haematooncological patients with central venous catheters (CVCs) in situ. MATERIALS AND METHODS: Peptide nucleic acid fluorescence in situ hybridization and AOLC tests using blood samples from one CVC lumen/port chamber in haematooncological patients were continuously performed. These results were compared to those obtained from routinely performed CRBSI diagnostic tests. RESULTS: One hundred and eighty-two patients with 342 catheter periods were investigated. Seventeen CRBSI cases were detected in 6466 CVC days by routine measures resulting in a CRBSI rate of 2.6/1000 catheter days. Two of 17 showed positive PNA FISH tests, and five positive AOLC test results before the diagnosis were established with routine measures. The screening revealed further seven patients with positive universal PNA FISH tests and 10 positive AOLC tests without symptoms indicative for infection and were therefore considered not to have CRBSI. CONCLUSIONS: Sampling of only one CVC lumen/port chamber screening for CRBSI in haematooncological patients seems not to be a useful tool for anticipative diagnosis of CRBSI. Reasons for false-negative results might include origin of CRBSIs from the other CVC lumina not sampled for screening, and false-positive results might origin from catheter colonization without subsequent spread of micro-organisms into the peripheral bloodstream.

The Toxin-Producing Pathobiont Klebsiella oxytoca Is Not Associated with Flares of Inflammatory Bowel Diseases.

BACKGROUND: Alterations in the intestinal microbiota are thought to be involved in the pathogenesis of inflammatory bowel diseases (IBD). Klebsiella oxytoca is an intestinal pathobiont that can produce a cytotoxin (tillivaline). AIM: We aimed to elucidate the pathogenetic relevance of toxin-producing K. oxytoca in patients with IBD flares and investigated the clonal relationship of K. oxytoca isolates from IBD patients using multilocus sequence typing (MLST). METHODS: Fecal samples of 235 adult IBD patients were collected from January 2008 to May 2009 and were tested for K. oxytoca, C. difficile toxin, and other pathogens by standard microbiological methods. Clinical data and disease activity scores were collected. K. oxytoca isolates were tested for toxin production using cell culture assays. A total of 45 K. oxytoca isolates from IBD patients, healthy, asymptomatic carriers and from patients with antibiotic-associated hemorrhagic colitis in part from our strain collection were tested for their clonal relationship using MLST. RESULTS: The prevalence of K. oxytoca in IBD overall was 4.7%. Eleven K. oxytoca isolates were detected. Two of 11 isolates were tested positive for toxin production. There was no significant difference in the distribution of K. oxytoca isolates between the groups (active vs. remission in UC and CD). MLST yielded 33 sequence types. K. oxytoca isolates from IBD did not cluster separately from isolates from asymptomatic carriers. CONCLUSIONS: Our data demonstrate that toxin (tilivalline)-producing K. oxytoca is not associated with IBD flares.

Genotypes of Klebsiella oxytoca isolates from patients with nosocomial pneumonia are distinct from those of isolates from patients with antibiotic-associated hemorrhagic colitis.

Klebsiella oxytoca acts as a pathobiont in the dysbiotic human intestinal microbiota, causing antibiotic-associated hemorrhagic colitis (AAHC), but it also infects other organs, resulting in pneumonia and urinary tract and skin infections. The virulence of K. oxytoca is still poorly understood. The production of a specific cytotoxin has been linked to AAHC pathogenesis. To investigate the clonal relationships of K. oxytoca with regard to clinical origin and virulence attributes, we established a multilocus sequence typing (MLST) method and analyzed 74 clinical K. oxytoca isolates from asymptomatic carriers and patients with AAHC, respiratory infections, and other infections. The isolates were phenotypically characterized, typed, and compared phylogenetically based on the sequences of seven housekeeping genes. MLST analysis yielded 60 sequence types, 12 of which were represented by more than one isolate. The phylogenetic tree distinguished clusters of K. oxytoca isolates between patients with AAHC and those with respiratory infections. Toxin-positive and -negative strains were observed within one sequence type. Our findings indicate that AAHC isolates share a genetic background. Interestingly, K. oxytoca isolates from nosocomial pneumonia showed a different genetic clustering, suggesting that these strains do not originate from the intestines or that they are specialized for respiratory tract colonization. Our results further indicate a polyphyletic origin and possible horizontal transfer of the genes involved in K. oxytoca cytotoxin production. This work provides evidence that K. oxytoca isolates colonizing the two main clinically relevant habitats (lower gastrointestinal [GI] tract and respiratory tract) of the human host are genetically distinct. Applications of this MLST analysis should help clarify the sources of nosocomial infections.

First report of Nocardia asiatica olecranon bursitis in an immunocompetent traveler returning to Austria.

Nocardia spp. are rarely isolated in extrapulmonary clinical specimens. We describe the first case of olecranon bursitis caused by Nocardia asiatica. The patient, a traveler returning from Thailand, was successfully treated with linezolid.

Resistance patterns of Escherichia coli isolated from sewage sludge in comparison with those isolated from human patients in 2000 and 2009.

For some time now, antibiotic-resistant bacterial strains have been found in the human population, in foods, in livestock and wild animals, as well as in surface waters. The entry of antibiotics and resistant bacterial strains into the environment plays an important role in the spread of antibiotic resistance. The goal of the present study was to monitor the entry of antibiotic resistances into the environment through the contamination of wastewater. To assess the extent of transmission of antibiotic resistances from human sources into the environment, the resistance patterns of Escherichia coli strains isolated from human patients have been compared to those found in strains isolated from sewage sludge. Our results may indicate if resistances to particular antibiotics are more prone than others to spread into the environment. To monitor the increase of specific resistances over time, samples taken in the years 2000 and 2009 were analysed. Our study shows that for some antibiotics a parallel development of resistance patterns has taken place in both patient and environmental samples over time. For other sets of antibiotics, independent developments have occurred in the samples. A clear increase of multi-resistant E. coli strains over time was observed in samples from both sources.

Establishing breakpoints for amoxicillin/clavulanate and ampicillin/sulbactam for rapid antimicrobial susceptibility testing directly from positive blood culture bottles.

Introduction. In 2018, EUCAST released guidelines on rapid antimicrobial susceptibility testing (RAST) directly from positive blood culture bottles for selected bacterial species and antimicrobial agents, but not for the commonly used agents amoxicillin/clavulanate (AMC) and ampicillin/sulbactam (SAM).Hypothesis/Gap statement. This work addresses the Enterobacterales RAST capability gap for betalactam/betalactamase inhibitor combinations.Aim. We aimed to determine RAST breakpoints for AMC and SAM for Escherichia coli and Klebsiella pneumoniae after 4 and 6 h of incubation directly from positive blood cultures.Methodology. Blood culture bottles were spiked with clinical isolates of E. coli (n=89) and K. pneumoniae (n=81). RAST was performed according to EUCAST guidelines and zones were read after 4 and 6 h. Breakpoints were defined to avoid very major errors.Results. The proportion of readable zone diameters after 4 h of incubation were 90.8 % in E. coli and 85.8 % in K. pneumoniae isolates. After 6 h of incubation all zone diameters could be read. The proposed breakpoints for E. coli after 6 h of incubation were ≥16 mm S (susceptible), 14-15 mm ATU (area of technical uncertainty) and <14 mm R (resistant) for AMC; ≥15 mm S, 12-14 mm ATU and <12 mm R for SAM; for K. pneumoniae these were ≥16 mm S, 14-15 mm ATU and <14 mm R for AMC; ≥13 mm S, 12 mm ATU, <12 mm R for SAM. Applying our newly set breakpoints, major errors were infrequent (2.6 %).Conclusion. We propose novel AMC and SAM breakpoints for RAST directly from positive blood cultures for reading after 4 and 6 h of incubation.

Complete Genome Sequence of Klebsiella oxytoca Strain AHC-6, Isolated from a Patient during Acute Antibiotic-Associated Hemorrhagic Colitis.

Klebsiella oxytoca is a ubiquitous bacterium that is increasingly associated with inflammatory diseases. Here, we report the hybrid assembled genome for cytotoxic K. oxytoca strain AHC-6. The genome comprises a total of 5.7 Mbp, with a GC content of 55.2% and 5,258 coding sequences after assembly and annotation.

Corrigendum: Variation in accessory genes within the Klebsiella oxytoca species complex delineates monophyletic members and simplifies coherent genotyping.

[This corrects the article DOI: 10.3389/fmicb.2021.692453.].