OXA-48 has rapidly disseminated worldwide and become one of the most common carbapenemases in many countries with more than 45 variants reported with, in some cases, significant differences in their ...hydrolysis profiles. The R214 residue, located in the ß5-ß6 loop, is crucial for the carbapenemase activity, as it stabilizes carbapenems in the active site and maintains the shape of the active site through interactions with D159. In this study, we have characterized a novel variant of OXA-48, OXA-933 with a single D159N change. To evaluate the importance of this residue, point mutations were generated (D159A, D159G, D159K, and D159W), kinetic parameters of OXA-933, OXA-48 D159G, and OXA-48 D159K were determined and compared to those of OXA-48 and OXA-244. The
gene was borne on Tn
, a 2,696-bp composite transposon made of two IS
elements surrounded by 9 bp target site duplications and inserted into a non-self-transmissible plasmid pOXA-933 of 7,872 bp in size. Minimal inhibitory concentration values of
expressing the
gene or of its point mutant derivatives were lower for carbapenems (except for D159G) as compared to those expressing the
gene. Steady-state kinetic parameters revealed lower catalytic efficiencies for expanded spectrum cephalosporins and carbapenems. A detailed structural analysis confirmed the crucial role of D159 in shaping the active site of OXA-48 enzymes by interacting with R214. Our work further illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutations at positions outside the β5-β6 loop, but interacting with key residues of it.
To genetically characterize VIM-producing Enterobacter cloacae complex (ECC) isolates recovered in France from 2015 to 2018.
WGS, species determination, MLST, clonal relationship and genetic ...characterization were performed on 149 VIM-producing ECC isolates.
Among VIM-producing Enterobacterales, the prevalence of ECC increased drastically from 6% in 2012 to 52% in 2018. The most prevalent species were Enterobacter hormaechei subsp. hoffmannii (40.9%), E. hormaechei subsp. steigerwaltii (21.5%), E. hormaechei subsp. xiangfangensis (14.8%) and ECC clade S (17.4%). Major STs were ST-873 (17.5%), ST-66 (12.1%), ST-78 (9.4%), ST-419 (8.1%), ST-145 (4.7%), ST-50 (4.0%), ST-118 (4.0%) and ST-168 (4.0%). Finally, six different integrons were identified, with some being specific to a given blaVIM variant (In916 with blaVIM-1-aacA4'-aphA15-aadA1-catB2 and In416 with blaVIM-4-aacA7-dfrA1b-aadA1b-smr2 genes).
This study demonstrated the genetic diversity among VIM-producing ECC isolates, indicating that their spread is not linked to a single clone.
Fast and accurate diagnostic tests to identify carbapenemase-producing Enterobacteriaceae (CPE) are mandatory for proper antimicrobial therapy and implementing infection control measures. Here, we ...have developed a rapid Carbapenem Inactivation Method (rCIM) for CPE detection.
The rCIM consists of the incubation of a potential carbapenemase producer with meropenem discs and use of the resulting supernatant to challenge a susceptible indicator strain. Growth of the indicator strain is monitored using a nephelometer. The performances of the rCIM were compared with the CIM and Carba NP tests using a collection of 113 well-characterized carbapenem-resistant enterobacterial isolates, including 85 carbapenemase producers and 28 non-carbapenemase producers. In addition, rCIM was compared with the Carba NP test and PCR sequencing in a prospective analysis of 101 carbapenem-resistant enterobacterial isolates addressed to the French National Reference Center for Antimicrobial Resistance in July 2017.
The rCIM correctly identified 84/85 carbapenemase producers and 28/28 non-carbapenemase producers, yielding a sensitivity of 99% and a specificity of 100%, slightly higher than the CIM and Carba NP test. In the prospective validation study, the rCIM showed a sensitivity and specificity of 97% and 95%, respectively. Two cephalosporinase-hyperproducing Enterobacter cloacae gave false-positive results, whereas an IMI-17-producing Enterobacter asburiae gave a false-negative result. The result was, however, positive when the isolate was grown on selective antibiotic-containing media.
The rCIM is a rapid (less than 3 h), cheap and accurate test for the detection of CPEs, which can be implemented in low-resource settings, making it a useful tool for microbiology laboratories.
In Enterobacterales, the most common carbapenemases are Ambler's class A (KPC-like), class B (NDM-, VIM- or IMP-like) or class D (OXA-48-like) enzymes. This study describes the characterization of ...twenty-four OXA-23 or OXA-58 producing-Proteus mirabilis isolates recovered from human and veterinary samples from France and Belgium. Twenty-two P. mirabilis isolates producing either OXA-23 (n = 21) or OXA-58 (n = 1), collected between 2013 and 2018, as well as 2 reference strains isolated in 1996 and 2015 were fully sequenced. Phylogenetic analysis revealed that 22 of the 24 isolates, including the isolate from 1996, belonged to a single lineage that has disseminated in humans and animals over a long period of time. The bla
gene was located on the chromosome and was part of a composite transposon, Tn6703, bracketed by two copies of IS15∆II. Sequencing using Pacbio long read technology of OXA-23-producing P. mirabilis VAC allowed the assembly of a 55.5-kb structure encompassing the bla
gene in that isolate. By contrast to the bla
genes, the bla
gene of P. mirabilis CNR20130297 was identified on a 6-kb plasmid. The acquisition of the bla
gene on this plasmid involved XerC-XerD recombinases. Our results suggest that a major clone of OXA-23-producing P. mirabilis is circulating in France and Belgium since 1996.
We characterized a collection of IMI-like-producing Enterobacter spp. isolates (n = 112) in France. The main clone corresponded to IMI-1-producing sequence type 820 E. cloacae subspecies cloacae that ...was involved in an outbreak. Clinicians should be aware of potential antimicrobial resistance among these bacteria.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gram-negative bacteria, especially Enterobacterales, have emerged as major players in antimicrobial resistance worldwide. Resistance may affect all major classes of anti-gram-negative agents, ...becoming multidrug resistant or even pan-drug resistant. Currently, β-lactamase-mediated resistance does not spare even the most powerful β-lactams (carbapenems), whose activity is challenged by carbapenemases. The dissemination of carbapenemases-encoding genes among Enterobacterales is a matter of concern, given the importance of carbapenems to treat nosocomial infections. Based on their amino acid sequences, carbapenemases are grouped into three major classes. Classes A and D use an active-site serine to catalyze hydrolysis, while class B (MBLs) require one or two zinc ions for their activity. The most important and clinically relevant carbapenemases are KPC, IMP/VIM/NDM, and OXA-48. However, several carbapenemases belonging to the different classes are less frequently detected. They correspond to class A (SME-, Nmc-A/IMI-, SFC-, GES-, BIC-like…), to class B (GIM, TMB, LMB…), class C (CMY-10 and ACT-28), and to class D (OXA-372). This review will address the genetic diversity, biochemical properties, and detection methods of minor acquired carbapenemases in Enterobacterales.
KPC-producing Pseudomonas aeruginosa are increasingly isolated in the Americas and in the Caribbean islands. Here, we determined the whole-plasmid sequence of two plasmids carrying the blaKPC-2 gene ...from multidrug-resistant P. aeruginosa clinical isolates from Colombia.
The two plasmids, pCOL-1 and pPA-2, were transferred to Escherichia coli recipient strain TOP10 and completely sequenced using high-throughput pyrosequencing for pCOL-1 and classical Sanger sequencing for pPA-2.
Both plasmids could be transferred to E. coli by transformation and displayed no other resistance marker besides KPC. Plasmid pCOL-1 was 31 529 bp in size, contained 31 open reading frames (ORFs) and belonged to the IncP-6 replicon group. It exhibited genes involved in replication, mobilization and partitioning, but none involved in conjugation. Plasmid pPA-2 was 7995 bp in size and contained seven ORFs. It exhibited a replicase gene of IncU, but was lacking genes involved in mobilization, partitioning and conjugation. Only 2072 bp matched Tn4401, including the blaKPC-2 gene, part of ISKpn6 and a 73 bp segment located upstream of the blaKPC-2 gene, containing the P1 promoter. Sequence identity was interrupted by a Tn3 transposon, itself interrupted by an IS26 element inserted within the β-lactamase blaTEM-1 gene.
Here we present the genetic features of the very first plasmids carrying the blaKPC-2 gene from P. aeruginosa. The emergence of the blaKPC-2 gene on unrelated plasmids, differing in size and in incompatibility group, and harbouring different genetic structures containing the blaKPC-2 genes in P. aeruginosa isolates suggests that this resistance trait may follow a dissemination scheme in P. aeruginosa similar to that seen in Enterobacteriaceae.
The differential expression of VIM-1 in
WEB-2 and
ssp.
WEB-1 clinical isolates from a rectal swab of a hospitalized patient in France was investigated.
WEB-2 was resistant to all β-lactams except ...carbapenems. It produced ESBL SHV-12, but the Carba NP test failed to detect any carbapenemase activity despite the production of VIM-1. Conversely,
WEB-1, previously recovered from the same patient, was positive for the detection of carbapenemase activity. The
gene was located on a plasmid and embedded within class 1 integron. Both plasmids were of the same IncA incompatibility group and conferred the same resistance pattern when electroporated in
TOP10 or
CIP7933. Quantitative RT-PCR experiments indicated a weaker replication of pWEB-2 in
as compared to
. An isogenic mutant of
WEB-2 selected after sequential passages with increased concentrations of imipenem possessed higher MICs for carbapenems and cephalosporins including cefiderocol, higher levels of the
gene transcripts, and detectable carbapenemase activity using the Carba NP test. Assessment of read coverage demonstrated that a duplication of the region surrounding
gene occurred in the
mutant with detectable carbapenemase activity. The lack of detection of the VIM-1 carbapenemase activity in
WEB-2 isolate was likely due to a weak replication of the IncA plasmid harboring the
gene. Imipenem as selective pressure led to a duplication of this gene on the plasmid and to the restoration of a significant carbapenem-hydrolyzing phenotype.
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