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A reusable water bottle was swabbed as part of the citizen science project “Swab and Send,” and a Klebsiella grimontii isolate was recovered on chromogenic agar and designated SS141. Whole‐genome sequencing of SS141 showed it has the potential to be a human pathogen as it contains the biosynthetic gene cluster for the potent cytotoxin, kleboxymycin, and genes for other virulence factors. The genome also contains the antibiotic‐resistant genes, bla OXY‐6‐4, and a variant of fosA, which is likely to explain the observed resistance to ampicillin, amoxicillin, and fosfomycin. We have also shown that SS141 forms biofilms on both polystyrene and polypropylene surfaces, providing a reasonable explanation for its ability to colonize a reusable water bottle. With the increasing use of reusable water bottles as an alternative to disposables and a strong forecast for growth in this industry over the next decade, this study highlights the need for cleanliness comparable to other reusable culinary items., A reusable water bottle was swabbed as part of the citizen science project “Swab and Send,” and a Klebsiella grimontii isolate was recovered on chromogenic agar and designated SS141. Whole‐genome sequencing of SS141 showed it has the potential to be a human pathogen as it contains the biosynthetic gene cluster for the potent cytotoxin, kleboxymycin, and genes for other virulence factors and antibiotic resistance. We have also shown that SS141 is a potent biofilm former, providing a reasonable explanation for its ability to colonize a reusable water bottle.

A re-usable water bottle was swabbed as part of the citizen science project Swab and Send and aKlebsiella grimontiiisolate was recovered on chromogenic agar and designated SS141. Whole genome sequencing of SS141 showed it has the potential to be a human pathogen as it contains the biosynthetic gene cluster for the potent cytotoxin, kleboxymycin, and genes for other virulence factors. The genome also containsblaOXY-6-4 andfosAwhich is likely to explain the observed resistance to ampicillin, amoxicillin and fosfomycin. We have also shown that SS141 is a potent biofilm former, providing a reasonable explanation for its ability to colonise a re-usable water bottle. With the increasing use of re-usable water bottles as an alternative to disposables, and a strong forecast for growth in this industry over the next decade, this study highlights the need for cleanliness comparable to other re-usable culinary items.

During an investigation of CRISPR carriage in clinical, multi-drug resistant, Klebsiella pneumoniae, a novel CRISPR-Cas system (which we have designated Type IV-B) was detected on plasmids from two K. pneumoniae isolates from Egypt (isolated in 2002-2003) and a single K. pneumoniae isolate from the UK (isolated in 2017). Sequence analysis of other genomes available in GenBank revealed that this novel Type IV-B CRISPR-Cas system was present on 28 other plasmids from various Enterobacteriaceae hosts and was never found on the chromosome. Type IV-B is found exclusively on IncHI1B/IncFIB plasmids and is associated with multiple putative transposable elements. Type IV-B has a single repeat-spacer array (CRISPR1) upstream of the cas loci with some spacers matching regions of conjugal transfer genes of IncFIIK/IncFIB(K) plasmids suggesting a role in plasmid incompatibility. Expression of the cas loci was confirmed in available clinical isolates by RT-PCR; indicating the system is active. To our knowledge, this is the first report describing a new subtype within Type IV CRISPR-Cas systems exclusively associated with IncHI1B/IncFIB plasmids.ImportanceHere, we report the identification of a novel subtype of Type IV CRISPR-Cas that is expressed and exclusively carried by IncHI1B/IncFIB plasmids in Enterobacteriaceae, demonstrating unique evolutionarily juxtaposed connections between CRISPR-Cas and mobile genetic elements (MGEs). Type IV-B encodes a variety of spacers showing homology to DNA from various sources, including plasmid specific spacers and is therefore thought to provide specific immunity against plasmids of other incompatible groups (IncFIIK/IncFIB(K)). The relationship between Type IV-B CRISPR-Cas and MGEs that surround and interrupt the system is likely to promote rearrangement and be responsible for the observed variability of this type. Finally, the Type IV-B CRISPR-Cas is likely to co-operate with other cas loci within the bacterial host genome during spacer acquisition.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated cas genes are sequence-specific DNA nuclease systems found in bacteria and archaea. CRISPR/Cas systems use RNA transcripts of previously acquired DNA (spacers) to target invading genetic elements with the same sequence, including plasmids. In this research we studied the relationship between CRISPR/Cas systems and multidrug resistance in Escherichia coli . The presence of Type I-E and Type I-F CRISPR systems was investigated among 82 antimicrobial-susceptible and 96 MDR clinical E. coli isolates by PCR and DNA sequencing. Phylogrouping and MLST were performed to determine relatedness of isolates. RT-PCR was performed to ascertain the expression of associated cas genes. Type I-F CRISPR was associated with the B2 phylogroup and was significantly overrepresented in the susceptible group (22.0%) compared with the MDR group (2.1%). The majority of CRISPR I-F-containing isolates had spacer sequences that matched IncF and IncI plasmids. RT-PCR demonstrated that Type I-F cas genes were expressed and therefore potentially functional. The CRISPR I-F system is more likely to be found in antimicrobial-susceptible E. coli . Given that the Type I-F system is expressed in WT isolates, we suggest that this difference could be due to the CRISPR system potentially interfering with the acquisition of antimicrobial resistance plasmids, maintaining susceptibility in these isolates.

BACKGROUND: Klebsiella pneumoniae outbreaks possessing extended-spectrum β-lactamase- (ESBL) mediated resistance to third-generation cephalosporins have increased significantly in hospital and community settings worldwide. The study objective was to characterize prevalent genetic determinants of TEM, SHV and CTX-M types ESBL activity in K. pneumoniae isolates from Egypt. METHODS: Sixty five ESBL-producing K. pneumoniae strains, isolated from nosocomial and community-acquired infections from 10 Egyptian University hospitals (2000-2003), were confirmed with double disc-synergy method and E-test. blaTEM, blaSHV and blaCTX-m genes were identified by PCR and DNA sequencing. Pulsed-field gel electrophoresis (PFGE) was conducted for genotyping. RESULTS: All isolates displayed ceftazidime and cefotaxime resistance. blaTEM and blaSHV genes were detected in 98% of the isolates' genomes, while 11% carried blaCTX-m. DNA sequencing revealed plasmid-borne SHV-12,-5,-2a (17%), CTX-m-15 (11%), and TEM-1 (10%) prevalence. Among SHV-12 (n=8), one isolate displayed 100% blaSHV-12 amino acid identity, while others had various point mutations: T17G (Leu to Arg, position 6 of the enzyme: n=2); A8T and A10G (Tyr and Ile to Phe and Val, positions 3 and 4, respectively: n=4), and; A703G (Lys to Glu 235: n=1). SHV-5 and SHV-2a variants were identified in three isolates: T17G (n=1); A703G and G705A (Ser and Lys to Gly and Glu: n=1); multiple mutations at A8T, A10G, T17G, A703G and G705A (n=1). Remarkably, 57% of community-acquired isolates carried CTX-m-15. PFGE demonstrated four distinct genetic clusters, grouping strains of different genetic backgrounds. CONCLUSIONS: This is the first study demonstrating the occurrence of SHV-12, SHV-5 and SHV-2a variants in Egypt, indicating the spread of class A ESBL in K. pneumoniae through different mechanisms.