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The human gut microbiome is an important reservoir of antimicrobial resistance genes (ARGs), collec-tively termed the 'resistome'. To date, few studies have examined the dynamics of the human gut resis-tome in healthy individuals. Previously, the authors observed high rates of ARG acquisition and significant abundance shifts during international travel. In order to provide insight into commonly occurring dynam-ics, this study investigated longitudinal fluctuations in prevalent ARGs (cfxA, tetM and ermB) in the resis-tomes of non-travelling healthy volunteers. In addition, this study assessed the prevalence of acquirable ARGs (blaCTX-M, qnrB, qnrS, vanA and vanB) over time. Faecal samples from 23 participants were collected at baseline and after 2 and 4 weeks. DNA was isolated, and ARG quantification was performed by quan-titative polymerase chain reaction adjusting for the total amount of bacterial 16S rDNA. vanA and qnrS were not detected in any of the samples, while the prevalence rates of vanB of non-enterococcal origin and qnrB were 73.9% and 5.7%, respectively. The ss-lactamase encoding blaCTX-M was detected in 17.4% of healthy participants. The results were compared with previous data from 122 travellers. ARG acquisitions observed in travellers were rare in non-travelling individuals during 4 weeks of follow-up, supporting the hypothesis of ARG acquisition during international travel. However, median -1.04-to 1.04-fold abun-dance changes were observed for 100% of cfxA, tetM and ermB, which did not differ from those found in travellers. Thus, common abundance shifts in prevalent ARGs of the gut resistome were found to occur independent of travel behaviour.(c) 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, several variants of concern (VOC) have emerged and became the dominant strain. These are considered VOC because of traits like increased transmissibility, increased severity, and immune-evasion. Understanding their viral load dynamics, with the use of longitudinal follow-up data, can help to understand transmission and could inform policy makers on for example infection prevention guidelines. However, longitudinal follow-up studies are scarce. In this study, we were able to monitor the viral load dynamics of the Delta, Omicron BA.1 and BA.2 variants via an unique dataset that was obtained as a consequence of the implemented infection prevention guidelines for healthcare workers at our institution, based on longitudinal follow-up of viral load. We found that the dynamics are different between Delta, Omicron BA.1, and Omicron BA.2 variants, the latter having the highest viral load on day 5 (5.7 log10 copies/mL compared to 4.3 log10 copies/mL for Delta and 4.8 log10 copies/mL for BA.1) and day 7 (4.4 log10 copies/mL compared to 3.3 log10 copies/mL for Delta and 3.8 log10 copies/mL for BA.1). However, the infection duration does not appear to be different between these variants. Still, considerable viral loads even after the suggested quarantine period were observed, in particular for the Omicron BA.2 sub variant. This highlights the need for a tailored approach per variant as results of previously circulating variants do not always match. This is in particular important for healthcare workers as they can transmit SARS-CoV-2 to vulnerable patients.

Breakthrough SARS-CoV-2 infections have been reported in fully vaccinated individuals, in spite of the high efficacy of the currently available vaccines, proven in trials and real-world studies. Several variants of concern (VOC) have been proffered to be associated with breakthrough infections following immunization. In this study, we investigated 378 breakthrough infections recorded between January and July 2021 and compared the distribution of SARS-CoV-2 genotypes identified in 225 fully vaccinated individuals to the frequency of circulating community lineages in the region of South Limburg (The Netherlands) in a week-by-week comparison. Although the proportion of breakthrough infections was relatively low and stable when the Alpha variant was predominant, the rapid emergence of the Delta variant lead to a strong increase in breakthrough infections, with a higher relative proportion of individuals vaccinated with Oxford-AstraZeneca or J&J/Janssen being infected compared to those immunized with mRNA-based vaccines. A significant difference in median age was observed when comparing fully vaccinated individuals with severe symptoms (83 years) to asymptomatic cases (46.5 years) or individuals with mild-to-moderate symptoms (42 years). There was no association between SARS-CoV-2 genotype or vaccine type and disease symptoms. Interestingly, symptomatic individuals harbored significantly higher SARS-CoV-2 loads than asymptomatic vaccinated individuals and breakthrough infections caused by the Delta variant are associated with increased viral loads compared to those caused by the Alpha variant. Altogether, these results indicate that the emergence of the Delta variant might have lowered the efficiency of particular vaccine types to prevent SARS-CoV-2 infections and that, although rare, the elderly are particularly at risk of becoming severely infected as the consequence of a breakthrough infection.

A DNA fragment from Bordetella pertussis, encoding the fim2 fimbrial subunit gene with adjacent sequences, was used as a probe for the detection of homologous sequences in chromosomal DNA of Bordetella avium. A 1.8 kb Sa1I-PstI fragment from the genome of B. avium, which hybridized with the probe, was isolated and sequenced. No fimbrial subunit gene was located on the B. avium DNA fragment. Two regions could be distinguished in the sequence of the fragment. Region 1, which was 80% identical to the sequence upstream of the fim2 gene of B. pertussis and region 2, which had no identity with any known sequence. A 491 bp EagI DNA fragment (probe A) within region 1 and a 650 bp Eag I DNA fragment (probe B) within region 2 were used as DNA probes on restriction endonuclease digests of chromosomal DNA from various bacterial species. This hybridization experiment showed that the region 2 sequence was specific for B. avium. A polymerase chain reaction (PCR) with specific primers within region 2 resulted in the amplification of a 500 bp DNA fragment with B. avium DNA only. This PCR is a useful method for the rapid detection of B. avium and appeared useful to discriminate B. avium from other Bordetella species and also from Alcaligenes faecalis.

With DNA probes derived from the fimbrial subunit genes fim2 and fim3 of Bordetella pertussis, two homologous subunit genes of Bordetella bronchiseptica were identified and cloned. The nucleotide sequences of these genes were determined. Comparison of these nucleotide sequences with the B. pertussis fimbrial fim2 and fim3 subunit genes showed a pronounced homology. Therefore, the B. bronchiseptica genes were also designated fim2 and fim3. Expression of the two B. bronchiseptica genes was demonstrated by Western blotting (immunoblotting) with polyclonal antiserum directed against the Fim2 and Fim3 fimbrial subunit proteins of B. pertussis and by enzyme-linked immunosorbent assay with monoclonal antibodies. After growth of B. bronchiseptica in the presence of MgSO4, no expression of both fimbrial subunit genes was observed. While no fimbriae were expressed, expression of flagella was observed under these circumstances. A longer C-stretch (up to 19 cytosine residues) than the one in front of the fim2 and fim3 genes of B. pertussis is present in front of the B. bronchiseptica fimbrial genes. In adherence experiments, fimbriated (Bvg+) as well as flagellated (Bvg-) B. bronchiseptica bacteria were able to adhere to HeLa cells, whereas nonflagellated B. pertussis did not. This suggests that fimbriae as well as other factors (possibly flagella) contribute to adherence of B. bronchiseptica to eukaryotic cells.

Invasion, defined as adhesion to, followed by entrance into HeLa cells by Bordetella bronchiseptica was determined by (i) specific staining of intracellular bacteria and (ii) counting of viable intracellular bacteria after killing extracellular bacteria with colistin. It was demonstrated for the first time that B. bronchiseptica, like Bordetella pertussis and Bordetella parapertussis , is able to invade HeLa cells. Comparison of the invasiveness of Bvg+ and Bvg-B. bronchiseptica showed that B. bronchiseptica, in contrast to B. pertussis, invaded HeLa cells in both phases. The number of viable intracellular bacteria isolated after invasion of bacteria in the Bvg- (flagellated) phase was ten-fold lower than when Bvg+ (fimbriated) bacteria were used. Strains which are deficient in the production of either FimX or Fim2 fimbriae were as invasive as wild-type B. bronchiseptica , which indicates that not these fimbriae but probably filamentous haemagglutinin (FHA) is the major adhesin of B. bronchiseptica.

Adhesion of bacteria to epithelial cells of the host is the first and probably the most important step in the pathogenesis of bacterial infections. In humans, adhesion of enterotoxigenic E.coli (ETEC) to the mucosa of the small intestines is mediated by serologically distinct and host-specific fimbrial antigens on the bacterial surface. The first fimbrial antigen to be identified in human ETEC strains was colonization factor antigen I (CFA/I)1. CFA/I is a single fimbrial antigen found on ETEC producing only heat-stable (ST) enterotoxin and on ETEC producing both heat-labile and heat-stable enterotoxin (LT, ST) of at least 15 serogroups. Later CFA/11 was detected2 and was subsequently shown to consist of three distinct coli surface associated (CS) antigens, designated CS1, CS2 and CS33. Nearly all CFA/II-positive E.coli produce CS3, but those of serotype 06.H16 or 06. H- also express either CS1 or CS2 depending on their biotype: biotype A strains produce CS1 whereas those of biotypes B, C and F express CS23. Production of CS1 and CS3 has been reported in only one other ETEC strain. This strain was of serotype 0139.H28 and differs from other 0139.H28 strains that are CS3 only producers4.

Production of the plasmid-coded fimbrial antigen CFA/I of Escherichia coli requires both CFA/I region 1 and CFA/I region 2, which are separated by about 40 kb on the wildtype plasmid. The nucleotide sequence of region 2 was determined and contains an open reading frame (cfa d), encoding a protein of 265 amino acids. The protein has no signal sequence and upon sequence analysis appeared to be a DNA-binding protein. A plasmid was constituted, with a promoterless beta-galactosidase gene preceded by the promoter of region 1. Introduction of a plasmid, carrying the cfa d gene, into a strain containing this construct enhanced expression of beta-galactosidase by at least five-fold indicating that the cfa d protein was enhancing expression from the promoter of region 1. The cfa d gene sequence differed at 28 positions from the Rns gene, which encodes a protein that is a positive regulator of the expression of CS1 or CS2 fimbriae. It was shown that the cfa d gene and the Rns gene can functionally substitute each other in regulating fimbrial synthesis.