Multicenter comparison of Etest, Vitek2 and BD Phoenix to broth microdilution for beta-lactam susceptibility testing of Streptococcus pneumonia.

PURPOSE: To assess performance of Etest®, Vitek®2 and BD Phoenix™ to determine the susceptibility of Streptococcus pneumoniae strains to penicillin, ampicillin and cefotaxime. METHODS: Sixty unique S. pneumoniae challenge strains were selected to cover a wide range of penicillin, ampicillin and cefotaxime minimal inhibitory concentrations (MICs). Strains were analyzed in four different Belgian laboratories. Etest® benzylpenicillin (BEN), ampicillin/amoxicillin (AMP) and cefotaxime (CTA) (bioMérieux), Vitek®2 AST-ST03 (bioMérieux) and BD Phoenix™ SMIC/ID-11 testing were each performed in two different labs. Results were compared to Sensititre® broth microdilution (BMD) (Thermo Fisher Scientific) results. MIC results were interpreted using EUCAST non-meningitis breakpoints (v 13.0). RESULTS: Essential agreement (EA) was ≥ 90% for all methods compared to BMD, except for Etest® BEN on Oxoid plate (58.3%), Etest® AMP (both on Oxoid (65.8%) and BD BBL plate (84.2%)). Categorical agreement (CA) for penicillin was only ≥ 90% for Vitek®2, for other methods CA ranged between 74 and 84%. CA for AMP was for all methods < 90% (range 75.8-88.3%) and CA for CTA was between 87 and 90% for all methods except for Etest on Oxoid plate (79.2%). CONCLUSIONS: Our study indicates that Vitek®2 and BD Phoenix™ are reliable for providing accurate pneumococcal susceptibility results for BEN, AMP and CTA. Using Etest BEN or AMP on Oxoid plate carries a risk of underestimating the MIC and should be interpreted with caution, especially when the obtained MIC is 1 or 2 doubling dilutions below the S or R clinical breakpoint.

Multicenter interlaboratory study of routine systems for the susceptibility testing of temocillin using a challenge panel of multidrug-resistant strains.

Accurate susceptibility result of temocillin (TMO) is important for treating infections caused by multidrug-resistant Enterobacterales. This multicenter study aimed to investigate the performance of routine temocillin testing assays against Enterobacterales challenging strains. Forty-seven selected clinical isolates were blindly analyzed by 12 Belgian laboratories using VITEK® 2 (n = 5) and BD Phoenix™ (n = 3) automated systems, ETEST® gradient strip (n = 3), and disk (3 brands) diffusion method (DD; n = 6) for temocillin susceptibility using standardized methodology. Results were interpreted using EUCAST 2023 criteria and compared to the broth microdilution (BMD; Sensititre™ panel) method used as gold standard. Methods' reproducibility was assessed by testing 3 reference strains in triplicate. A total of 702 organism-drug results were obtained against 33 TMO-susceptible and 14 TMO-resistant isolates. Excluding Proteae species (P. mirabilis and M. morganii), the essential agreement rates were excellent (91.5-100%) for all MIC-based methods. The highest category agreement was achieved by ETEST® (97.5%) followed by VITEK® 2 (93.2%), disk diffusion (91.6%), and BD Phoenix™ (88.5%). BD Phoenix™ and paper disk diffusion overcalled resistance (11.5% and 6.8% of major discrepancies, respectively), while ROSCO tablets diffusion and VITEK® 2 generated higher very major discrepancies (7.1% and 4.2% respectively). Inter-assay reproducibility was unsatisfactory using recommended E. coli ATCC 25922 strain but was excellent with E. coli ATCC 35218 and K. pneumoniae ATCC 700603 strains. This interlaboratory study suggests that routine testing methods provide accurate and reproducible TMO categorization results except for Proteae species.

Salmonella Durban meningitis: case report and genomics study.

BACKGROUND: Bacterial meningitis caused by non-typhoid Salmonella can be a fatal condition which is more common in low and middle-income countries. CASE PRESENTATION: We report the case of a Salmonella meningitis in a Belgian six-month old male infant. The first clinical examination was reassuring, but after a few hours, his general state deteriorated. A blood test and a lumbar puncture were therefore performed. The cerebrospinal fluid analysis was compatible with a bacterial meningitis which was later identified by the NRC (National Reference Center) as Salmonella enterica serovar Durban. CONCLUSIONS: In this paper, we present the clinical presentation, genomic typing, and probable sources of infection for an unusually rare serovar of Salmonella. Through an extended genomic analysis, we established its relationship to historical cases with links to Guinea.

A Bioinformatics Whole-Genome Sequencing Workflow for Clinical Mycobacterium tuberculosis Complex Isolate Analysis, Validated Using a Reference Collection Extensively Characterized with Conventional Methods and In Silico Approaches.

The use of whole-genome sequencing (WGS) for routine typing of bacterial isolates has increased substantially in recent years. For Mycobacterium tuberculosis (MTB), in particular, WGS has the benefit of drastically reducing the time required to generate results compared to most conventional phenotypic methods. Consequently, a multitude of solutions for analyzing WGS MTB data have been developed, but their successful integration in clinical and national reference laboratories is hindered by the requirement for their validation, for which a consensus framework is still largely absent. We developed a bioinformatics workflow for (Illumina) WGS-based routine typing of MTB complex (MTBC) member isolates allowing complete characterization, including (sub)species confirmation and identification (16S, csb/RD, hsp65), single nucleotide polymorphism (SNP)-based antimicrobial resistance (AMR) prediction, and pathogen typing (spoligotyping, SNP barcoding, and core genome multilocus sequence typing). Workflow performance was validated on a per-assay basis using a collection of 238 in-house-sequenced MTBC isolates, extensively characterized with conventional molecular biology-based approaches supplemented with public data. For SNP-based AMR prediction, results from molecular genotyping methods were supplemented with in silico modified data sets, allowing us to greatly increase the set of evaluated mutations. The workflow demonstrated very high performance with performance metrics of >99% for all assays, except for spoligotyping, where sensitivity dropped to ∼90%. The validation framework for our WGS-based bioinformatics workflow can aid in the standardization of bioinformatics tools by the MTB community and other SNP-based applications regardless of the targeted pathogen(s). The bioinformatics workflow is available for academic and nonprofit use through the Galaxy instance of our institute at https://galaxy.sciensano.be.

Genomic epidemiology of persistently circulating MDR Shigella sonnei strains associated with men who have sex with men (MSM) in Belgium (2013-19).

OBJECTIVES: Shigella sonnei resistant to first-line antibiotics azithromycin and ciprofloxacin are on the rise globally. The aim of this study was to describe the epidemiology of MDR S. sonnei in Belgium and to identify origins and circulating clusters through WGS. METHODS: We undertook demographic, temporal and geographical analysis of 930 S. sonnei isolates submitted to the Belgian National Reference Centre for Salmonella and Shigella between 2017 and 2019. Phylogenetic analysis of WGS data, genotyping and identification of genetic markers of antimicrobial resistance was performed on 372 Belgian isolates submitted between 2013 and 2019. RESULTS: S. sonnei was identified in 75% (930/1253) of Belgian Shigella isolates submitted between 2017 and 2019. Overall, 7% (69/930) of isolates were resistant to ciprofloxacin alone, 6% (57/930) showed reduced susceptibility to azithromycin alone, and 24% (223/930) exhibited both. Men were at higher risk of carrying a double resistant S. sonnei strain, compared with women (risk ratio = 8.6, 95% CI = 5.4-13.9). Phylogenetic analysis revealed four independent Belgian clusters of persistently circulating MDR strains, associated with men who have sex with men (MSM) and of the same genotypes as previously described international MSM-related clades. Belgian isolates carried various incompatibility (Inc)-type plasmids, the SpA plasmid and ESBL genes. CONCLUSIONS: In Belgium, S. sonnei isolates from men are much more likely to be resistant to important first-line antibiotics than isolates from women. Multiple co-circulating MDR S. sonnei clusters of different genotypes were identified in the MSM community. Further studies on risk groups are needed for targeted prevention, improved clinical and public health management and antimicrobial stewardship in Belgium.

Outbreak of Central American born Shigella sonnei in two youth camps in Belgium in the summer of 2019.

In 2019, an outbreak of Shigella sonnei occurred during two youth camps in Belgium. The clustering of isolates from both camps was confirmed by next-generation sequencing, as well as a secondary infection of a technician. The outbreak strain clustered with internationally isolated strains from patients with recent travel history to Central America. This report exemplifies enhanced surveillance and international collaboration between public health institutes by enabling to link local outbreaks to region-specific sublineages circulating abroad.

Strong increase of true and false positive mycobacterial cultures sent to the National Reference Centre in Belgium, 2007 to 2016.

IntroductionIn 2007, a new federal legislation in Belgium prohibited non-biosafety level 3 laboratories to process culture tubes suspected of containing mycobacteria.AimTo present mycobacterial surveillance/diagnosis data from the Belgian National Reference Centre for mycobacteria (NRC) from 2007 to 2016.MethodsThis retrospective observational study investigated the numbers of analyses at the NRC and false positive cultures (interpreted as containing mycobacteria at referring clinical laboratories, but with no mycobacterial DNA detected by PCR in the NRC). We reviewed mycobacterial species identified and assessed trends over time of proportions of nontuberculous mycobacteria (NTM) vs Mycobacterium tuberculosis complex (MTBc), and false positive cultures vs NTM.ResultsFrom 2007 to 2016, analyses requests to the NRC doubled from 12.6 to 25.3 per 100,000 inhabitants. A small but significant increase occurred in NTM vs MTBc proportions, from 57.9% (587/1,014) to 60.3% (867/1,437) (p < 0.001). Although NTM infection notification is not mandatory in Belgium, we annually received up to 8.6 NTM per 100,000 inhabitants. M. avium predominated (ca 20% of NTM cultures), but M. intracellulare culture numbers rose significantly, from 13.0% (74/587) of NTM cultures in 2007 to 21.0% (178/867) in 2016 (RR: 1.05; 95% CI: 1.03-1.07). The number of false positive cultures also increased, reaching 43.3% (1,097/2,534) of all samples in 2016.ConclusionWe recommend inclusion of NTM in sentinel programmes. The large increase of false positive cultures is hypothesised to result from processing issues prior to arrival at the NRC, highlighting the importance of sample decontamination/transport and equipment calibration in peripheral laboratories.

Aloe Emodin Reduces Phthiodiolone Dimycocerosate Potentiating Vancomycin Susceptibility on Mycobacteria.

Treatment of tuberculosis still represent a major public health issue. The emergence of multi-and extensively-drug resistant (MDR and XDR) Mycobacterium tuberculosis clinical strains further pinpoint the urgent need for new anti-tuberculous drugs. We previously showed that vancomycin can target mycobacteria lacking cell wall integrity, especially those lacking related phthiocerol and phthiodolone dimycocerosates, PDIM A and PDIM B, respectively. As aloe emodin was previously hypothesized to be able to target the synthesis of mycobacterial cell wall lipids, we tested its ability to potentiate glycopeptides antimycobacterial activity. The aloe emodin with the vancomycin induced a combination effect beyond simple addition, close to synergism, at a concentration lower to reported IC50 cytotoxic value, on M. bovis BCG and on H37Rv M. tuberculosis. Interestingly, out of six MDR and pre-XDR clinical strains, one showed a strong synergic susceptibility to the drug combination. Mycobacterial cell wall lipid analyses highlighted a selective reduction of PDIM B by aloe emodin.

Salmonella enterica serovar Typhi Producing CTX-M-15 Extended Spectrum ß-Lactamase in the Democratic Republic of the Congo.

We report a typhoid fever case with a Salmonella enterica serovar Typhi isolate showing extended spectrum ß-lactamase (ESBL) production in the Democratic Republic of the Congo. Whole genome sequencing revealed that the strain carried a plasmid-mediated CTX-M-15 ESBL gene and did not belong to the dominant H58 Salmonella Typhi clade.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria.

Species identification and drug susceptibility testing (DST) of mycobacteria are important yet complex processes traditionally reserved for reference laboratories. Recent technical improvements in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has started to facilitate routine mycobacterial identifications in clinical laboratories. In this paper, we investigate the possibility of performing phenotypic MALDI-based DST in mycobacteriology using the recently described MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). We randomly selected 72 clinical Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) strains, subjected them to MBT-ASTRA methodology, and compared its results to current gold-standard methods. Drug susceptibility was tested for rifampin, isoniazid, linezolid, and ethambutol (M. tuberculosis, n = 39), and clarithromycin and rifabutin (NTM, n = 33). Combined species identification was performed using the Biotyper Mycobacteria Library 4.0. Mycobacterium-specific MBT-ASTRA parameters were derived (calculation window, m/z 5,000 to 13,000, area under the curve [AUC] of >0.015, relative growth [RG] of <0.5; see the text for details). Using these settings, MBT-ASTRA analyses returned 175/177 M. tuberculosis and 65/66 NTM drug resistance profiles which corresponded to standard testing results. Turnaround times were not significantly different in M. tuberculosis testing, but the MBT-ASTRA method delivered on average a week faster than routine DST in NTM. Databases searches returned 90.4% correct species-level identifications, which increased to 98.6% when score thresholds were lowered to 1.65. In conclusion, the MBT-ASTRA technology holds promise to facilitate and fasten mycobacterial DST and to combine it directly with high-confidence species-level identifications. Given the ease of interpretation, its application in NTM typing might be the first in finding its way to current diagnostic workflows. However, further validations and automation are required before routine implementation can be envisioned.

Viral interference of the bacterial RNA metabolism machinery.

In a recent publication, we reported a unique interaction between a protein encoded by the giant myovirus phiKZ and the Pseudomonas aeruginosa RNA degradosome. Crystallography, site-directed mutagenesis and interactomics approaches revealed this 'degradosome interacting protein' or Dip, to adopt an 'open-claw' dimeric structure that presents acidic patches on its outer surface which hijack 2 conserved RNA binding sites on the scaffold domain of the RNase E component of the RNA degradosome. This interaction prevents substrate RNAs from being bound and degraded by the RNA degradosome during the virus infection cycle. In this commentary, we provide a perspective into the biological role of Dip, its structural analysis and its mysterious evolutionary origin, and we suggest some therapeutic and biotechnological applications of this distinctive viral protein.

Development of a Luminex xTAG® assay for cost-effective multiplex detection of ß-lactamases in Gram-negative bacteria.

OBJECTIVES: The objective of this study was to design and validate a genotyping method for multiplex identification of ESBLs and carbapenemases in Gram-negative bacilli. This assay had to be (i) superior to traditional (multiplex) PCR/sequencing-based tests in turn-around time, gene coverage and the ability to detect multiple variants of the same allele, and (ii) significantly more cost-effective than commercial microarrays and WGS. The targeted ß-lactamases include ESBLs (CTX-M families and subtypes, ESBL and non-ESBL SHV- and TEM-likes, OXA-1/2/7-likes, PER, VEB, GES), plasmid-mediated cephalosporinases (CMY, MOX, FOX, ACC, DHA, MIR/ACT) and carbapenemases (OXA-48, NDM, KPC, VIM, IMP). METHODS: A modular multiplex oligonucleotide ligation-PCR procedure was used, with read-out on a Luminex MAGPIX(®) platform. We designed 46 xTAG(®)-compatible probes targeting ß-lactamase alleles and allele variants, and one probe targeting a conserved 16S rRNA region serving as a DNA extraction control. The assay was optimized using a collection of 48 reference strains and further validated using 105 foodborne ESBL-producing Escherichia coli isolates. RESULTS: The specificity and selectivity of the test are 100% and 99.4%, respectively. Multiple variants of the same allele were successfully discriminated, as exemplified by five E. coli strains encoding both blaTEM-1 and blaTEM-52 genes. The turn-around time from single colony to result is 5 h and total consumable costs remained <€5 per sample. CONCLUSIONS: We designed and validated the first Luminex-compatible genotyping assay that reliably and rapidly identifies a broad range of ESBL, pAmpC and carbapenemase producers in culture.

Microbiological, clinical and molecular findings of non-typhoidal Salmonella bloodstream infections associated with malaria, Oriental Province, Democratic Republic of the Congo.

BACKGROUND: In sub-Saharan Africa, non-typhoidal Salmonella (NTS) can cause bloodstream infections, referred to as invasive non-typhoidal Salmonella disease (iNTS disease); it can occur in outbreaks and is often preceded by malaria. Data from Central Africa is limited. METHODS: Clinical, microbiological and molecular findings of NTS recovered in a blood culture surveillance project (2009-2014) were analyzed. RESULTS: In March-July 2012 there was an epidemic increase in malaria infections in the Oriental Province of the Democratic Republic of the Congo (DRC). In one referral hospital, overall hospital admissions in June 2012 were 2.6 times higher as compared to the same period in the years before and after (336 versus an average of 128 respectively); numbers of malaria cases and blood transfusions were nearly three- and five-fold higher respectively (317 versus 112 and 250 versus 55). Case fatality rates (in-hospital deaths versus all admissions) peaked at 14.6 %. Salmonella Typhimurium and Salmonella Enteritidis together accounted for 88.9 % of pathogens isolated from blood cultures collected during an outreach visit to the affected districts in June 2012. Children infected with Salmonella Enteritidis (33 patient files available) tended to be co-infected with Plasmodium falciparum more often than children infected with Salmonella Typhimurium (40 patients files available) (81.8 % versus 62.5 %). Through the microbiological surveillance project (May 2009-May 2014) 113 unique NTS isolates were collected (28.5 % (113/396) of pathogens); most (95.3 %) were recovered from children < 15 years. Salmonella Typhimurium (n = 54) and Salmonella Enteritidis (n = 56) accounted for 47.8 % and of 49.6 % NTS isolates respectively. Multilocus variable-number tandem-repeat analysis (MLVA) revealed more heterogeneity for Salmonella Typhimurium than for Salmonella Enteritidis. Most (82/96, 85.4 %) NTS isolates that were available for antibiotic susceptibility testing were multidrug resistant. All isolates were susceptible to ceftriaxone and azithromycin. CONCLUSION: During the peak of an epidemic increase in malaria in the DRC in 2012, a high proportion of multidrug resistant Salmonella Typhimurium and Salmonella Enteritidis were isolated from blood cultures. Overall, the two serovars showed subtle differences in clinical presentation and genetic diversity.

Expression of a novel P22 ORFan gene reveals the phage carrier state in Salmonella typhimurium.

We discovered a novel interaction between phage P22 and its host Salmonella Typhimurium LT2 that is characterized by a phage mediated and targeted derepression of the host dgo operon. Upon further investigation, this interaction was found to be instigated by an ORFan gene (designated pid for phage P22 encoded instigator of dgo expression) located on a previously unannotated moron locus in the late region of the P22 genome, and encoding an 86 amino acid protein of 9.3 kDa. Surprisingly, the Pid/dgo interaction was not observed during strict lytic or lysogenic proliferation of P22, and expression of pid was instead found to arise in cells that upon infection stably maintained an unintegrated phage chromosome that segregated asymmetrically upon subsequent cell divisions. Interestingly, among the emerging siblings, the feature of pid expression remained tightly linked to the cell inheriting this phage carrier state and became quenched in the other. As such, this study is the first to reveal molecular and genetic markers authenticating pseudolysogenic development, thereby exposing a novel mechanism, timing, and populational distribution in the realm of phage-host interactions.

Invasive Salmonella Infections at Multiple Surveillance Sites in the Democratic Republic of the Congo, 2011-2014.

BACKGROUND: This study reports the microbiological landscape of Salmonella Typhi and invasive nontyphoidal Salmonella (iNTS) in the Democratic Republic of the Congo (DRC). METHODS: Blood cultures obtained from hospital-admitted patients suspected of bloodstream infection (BSI) in 4 of 11 provinces in DRC (Kinshasa, Bas-Congo, Equateur, and Orientale) were processed. Sampling had started in 2007; the results for the period 2011-2014 are reported. RESULTS: Salmonella Typhi and iNTS were cultured from 194 (1.4%) and 840 (5.9%), respectively, of 14,110 BSI episodes and ranked first among BSI pathogens in adults (65/300 [21.7%]) and children (783/1901 [41.2%]), respectively. A total of 948 of 1034 (91.7%) isolates were available for analysis (164 Salmonella Typhi and 784 iNTS). Salmonella Typhimurium and Salmonella Enteritidis represented 386 (49.2%) and 391 (49.9%), respectively, of iNTS isolates, fluctuating over time and geography and increasing during the rainy season. Adults accounted for <5% of iNTS BSI episodes. Children <5 years accounted for 20.3% of Salmonella Typhi BSI episodes. Among Salmonella Typhi, rates of multidrug resistance and decreased ciprofloxacin susceptibility (DCS) were 37.8% and 37.2%, respectively, and 18.3% displayed combined multidrug resistance and DCS; rates of azithromycin and ceftriaxone resistance were 0.6% and absent, respectively. Among NTS isolates, ≥80% (79.7% of Salmonella Enteritidis and 90.2% of Salmonella Typhimurium isolates) showed multidrug resistance, and <2.5% showed DCS. Combined extended-spectrum ß-lactamase production (blaTEM-1 gene) and azithromycin resistance was noted in 12.7% of Salmonella Typhimurium isolates, appearing in Bas-Congo from 2013 onward. CONCLUSIONS: Salmonella Typhi and NTS are major causes of BSI in DRC; their antimicrobial resistance is increasing.

Trends in serotype distribution and antimicrobial susceptibility in Salmonella enterica isolates from humans in Belgium, 2009 to 2013.

The Belgian National Reference Centre for Salmonella received 16,544 human isolates of Salmonella enterica between January 2009 and December 2013. Although 377 different serotypes were identified, the landscape is dominated by S. enterica serovars Typhimurium (55%) and Enteritidis (19%) in a ratio which is inverse to European Union averages. With outbreaks of Salmonella serotypes Ohio, Stanley, and Paratyphi B variant Java as prime examples, 20 serotypes displayed significant fluctuations in this 5-year period. Typhoid strains account for 1.2% of Belgian salmonellosis cases. Large-scale antibiotic susceptibility analyses (n = 4,561; panel of 12 antibiotics) showed declining resistance levels in S. Enteritis and Typhimurium isolates for 8 and 3 tested agents, respectively. Despite low overall resistance to ciprofloxacin (4.4%) and cefotaxime (1.6%), we identified clonal lineages of Salmonella serotypes Kentucky and Infantis displaying rising resistance against these clinically important drugs. Quinolone resistance is mainly mediated by serotype-specific mutations in GyrA residues Ser83 and Asp87 (92.2% not wild type), while an additional ParC_Ser80Ile mutation leads to ciprofloxacin resistance in 95.5% S. Kentucky isolates, which exceeds European averages. Plasmid-mediated quinolone resistance (PMQR) alleles qnrA1 (n = 1), qnrS (n = 9), qnrD1 (n = 4), and qnrB (n = 4) were found in only 3.0% of 533 isolates resistant to nalidixic acid. In cefotaxime-resistant isolates, we identified a broad range of Ambler class A and C ß-lactamase genes (e.g., bla(SHV-12), blaTEM-52, bla(CTX-M-14), and bla(CTX-M-15)) commonly associated with members of the family Enterobacteriaceae. In conclusion, resistance to fluoroquinolones and cefotaxime remains rare in human S. enterica, but clonal resistant serotypes arise, and continued (inter)national surveillance is mandatory to understand the origin and routes of dissemination thereof.

Development of giant bacteriophage ϕKZ is independent of the host transcription apparatus.

UNLABELLED: Pseudomonas aeruginosa bacteriophage ϕKZ is the type representative of the giant phage genus, which is characterized by unusually large virions and genomes. By unraveling the transcriptional map of the ∼ 280-kb ϕKZ genome to single-nucleotide resolution, we combine 369 ϕKZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the ϕKZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to ϕKZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the ϕKZ genome is performed by the consecutive action of two ϕKZ-encoded, noncanonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampin-resistant transcriptional machinery is conserved within the diverse giant phage genus. IMPORTANCE: The data presented in this paper offer, for the first time, insight into the complex transcriptional scheme of giant bacteriophages. We show that Pseudomonas aeruginosa giant phage ϕKZ is able to infect and lyse its host cell and produce phage progeny in the absence of functional bacterial transcriptional machinery. This unique property can be attributed to two phage-encoded putative RNAP enzymes, which contain very distant homologues of bacterial ß and ß'-like RNAP subunits.

Functional elucidation of antibacterial phage ORFans targeting Pseudomonas aeruginosa.

Immediately after infection, virulent bacteriophages hijack the molecular machinery of their bacterial host to create an optimal climate for phage propagation. For the vast majority of known phages, it is completely unknown which bacterial functions are inhibited or coopted. Early expressed phage genome regions are rarely identified, and often filled with small genes with no homology in databases (so-called ORFans). In this work, we first analysed the temporal transcription pattern of the N4-like Pseudomonas-infecting phages and selected 26 unknown, early phage ORFans. By expressing their encoded proteins individually in the host bacterium Pseudomonas aeruginosa, we identified and further characterized six antibacterial early phage proteins using time-lapse microscopy, radioactive labelling and pull-down experiments. Yeast two-hybrid analysis gaveclues to their possible role in phage infection. Specifically, we show that the inhibitory proteins may interact with transcriptional regulator PA0120, the replicative DNA helicase DnaB, the riboflavin metabolism key enzyme RibB, the ATPase PA0657and the spermidine acetyltransferase PA4114. The dependency of phage infection on spermidine was shown in a final experiment. In the future, knowledge of how phages shut down their hosts as well ass novel phage-host interaction partners could be very valuable in the identification of novel antibacterial targets.

Systematic identification of hypothetical bacteriophage proteins targeting key protein complexes of Pseudomonas aeruginosa.

Addressing the functionality of predicted genes remains an enormous challenge in the postgenomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism, and cell division during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the α subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins. (Data are available via ProteomeXchange with identifier PXD001199.).

Design and Validation of RT-PCR Assays to Differentiate Salmonella Vaccine Strains from Wild-Type Field Isolates.

The timely differentiation of the AviPro Salmonella VAC T and VAC E strains from the wild-type Salmonella enterica ser. Typhimurium and ser. Enteritidis isolates is crucial for effectively monitoring veterinary isolates. Currently, the distinction between field and vaccine strains has been conducted routinely via phenotypic antimicrobial resistance testing since the vaccines were first introduced more than 20 years ago, and the differentiation based on the antimicrobial resistance profile is still a valid and well-established method. However, an alternative method was sought for those laboratories that prefer a PCR-based method for logistic and/or operational reasons. In this study, we developed two triplex Real-Time PCR reactions that targeted conserved and specific mutations and, therefore, enabled the reliable differentiation of field and vaccine strains. To validate the effectiveness of both assays, we extensively tested them on a dataset consisting of 405 bacterial strains. The results demonstrated a 100% sensitivity and specificity for distinguishing both Salmonella enterica ser. Typhimurium and Enteritidis, although a confirmed culture is required.