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BackgroundKlebsiella 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.MethodsSixty 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.ResultsAll 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.ConclusionsThis 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.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are diverse immune systems found in many prokaryotic genomes that target invading foreign DNA such as bacteriophages and plasmids. There are multiple types of CRISPR with arguably the most enigmatic being Type IV. During an investigation of CRISPR carriage in clinical, multi-drug resistant, Klebsiella pneumoniae, a Type IV-A3 CRISPR-Cas system was detected on plasmids from two K. pneumoniae isolates from Egypt (isolated in 2002-2003) and a single K. pneumoniae isolate from the United Kingdom (isolated in 2017). Sequence analysis of all other genomes available in GenBank revealed that this CRISPR-Cas system was present on 28 other plasmids from various Enterobacteriaceae hosts and was never found on a bacterial chromosome. This system is exclusively located on IncHI1B/IncFIB plasmids and is associated with multiple putative transposable elements. Expression of the cas loci was confirmed in the available clinical isolates by RT-PCR. In all cases, the CRISPR-Cas system has a single CRISPR array (CRISPR1) upstream of the cas loci which has several, conserved, spacers which, amongst things, match regions within conjugal transfer genes of IncFIIK/IncFIB(K) plasmids. Our results reveal a Type IV-A3 CRISPR-Cas system exclusively located on IncHI1B/IncFIB plasmids in Enterobacteriaceae that is likely to be able to target IncFIIK/IncFIB(K) plasmids presumably facilitating intracellular, inter-plasmid competition.

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.