Resistance to carbapenems: mechanisms and detection methods

In the last two decades, carbapenems have been implemented for the treatment of infections due to last-generation cephalosporin-resistant Gram-negative infections. This massive use has contributed to the selection of carbapenem-resistant bugs that usually produce carbapenemase enzymes. Nowadays, the spread of such untreatable organisms has reached dramatic levels representing one of the most global public health problems. This issue has led to the realization of the need for more rapid diagnosis. This talk presents a summary of the carbapenem resistance mechanisms. In particular, the main classes of carbapenemases will be discussed, emphasizing on their genetic background that support global spread. The currently available phenotypic and molecular methods that can be implemented by diagnostic and research laboratories will be also debated

Complete Genome Sequence of the First Colistin-Resistant Raoultella electrica Strain.

We present the full genome sequence of a colistin-resistant Raoultella electrica strain (MIC, >4 μg/mL) that was isolated from the stool of a healthy person living in India. The sequence consists of a chromosome and three plasmids (5,455,992-bp and 98,913-bp, 4,232-bp, and 3,961-bp, respectively). No previously described colistin resistance mechanisms were detected

Genome stability during serial sub-culturing in hyperepidemic multidrug-resistant Klebsiella pneumoniae and Escherichia coli.

BACKGROUND Core-genome single-nucleotide variant (cgSNV) analysis represents a powerful tool for epidemiological investigations of multidrug-resistant (MDR) bacteria. However, cgSNV thresholds to confirm whether isolates are the same clone are not formally defined. METHODS We implemented hybrid whole-genome sequencing to study the genomic changes of 4 MDR isolates belonging to hyperepidemic sequence types (STs) during 20 propagation steps (T20) on MacConkey and CHROMID ESBL plates. The following strains were analyzed: K. pneumoniae AE-2247421 (OXA-48/NDM-1-producing, ST101), K. pneumoniae MCL-2017-2 (CTX-M-15-producing, ST307), E. coli Ec-042 (OXA-181-producing, ST410), and E. coli Ec-050 (NDM-5-producing, ST167). The genome assembly at T5 and T20 was compared to that at time point zero (T0) and to two reference genomes. RESULTS At T20, AE-2247421 lost the IncL blaOXA-48-carrying plasmid when grown on CHROMID ESBL plates, while a large fragment encompassing blaNDM-1 was lost from its IncC plasmid when grown on both plates. In contrast, no structural changes were noted for the other 3 strains. With regard to the cgSNVs, the following results were obtained at T5 and T20 (ranges considering the different agar plates and reference genomes): AE-2247421 (1-8 and 2-12 cgSNVs), MCL-2017-2 (both 1-2 cgSNVs), Ec-042 (both 0 cgSNVs), and Ec-050 (0-6 and 0-9 cgSNVs). CONCLUSIONS We showed that structural changes and accumulation of cgSNVs can occur in few propagation steps under laboratory conditions. These changes might also arise in the clinical context in a short time, especially under antibiotics treatment. This phenomenon should be carefully considered because it might affect the final interpretation of epidemiological genomic analyses

Complete Genome Sequence of Entomomonas sp. Strain E2T0, Isolated from the Darkling Beetle Zophobas morio Larvae.

Here, we present the complete genome sequence of Entomomonas sp. E2T0, a strain isolated from larvae of the darkling beetle Zophobas morio. The isolate was fully resistant to aztreonam and possessed a novel class D β-lactamase gene. The 3,325,929-bp genome consists of a chromosome and a 9,996-bp plasmid

Detection of SHV β-lactamases in Gram-negative bacilli using fluorescein-labeled antibodies

<p>Abstract</p> <p>Background</p> <p>β-lactam resistance in Gram-negative bacteria is a significant clinical problem in the community, long-term care facilities, and hospitals. In these organisms, β-lactam resistance most commonly results from the production of β-lactamases. In Gram-negative bacilli, TEM-, SHV-, and CTX-M-type β-lactamases predominate. Therefore, new and accurate detection methods for these β-lactamase producing isolates are needed.</p> <p>Results</p> <p><it>E. coli </it>DH10B cells producing SHV-1 β-lactamase and a clinical isolate of <it>K. pneumoniae </it>producing SHV-5 β-lactamase were rendered membrane permeable, fixed and adhered to poly-L-lysine coated slides, and stained with purified polyclonal anti-SHV antibodies that were fluorescein labeled. <it>E. coli </it>DH10B cells without a <it>bla</it>_SHVgene were used as a negative control. The procedure generated a fluorescence signal from those slides containing cells expressing SHV β-lactamase that was sufficient for direct imaging.</p> <p>Conclusion</p> <p>We developed a rapid and accurate method of visualizing the SHV family of enzymes in clinical samples containing Gram-negative bacilli using a fluorescein-labeled polyclonal antibody.</p

Extended-spectrum cephalosporin-resistant Escherichia coli in community, specialized outpatient clinic and hospital settings in Switzerland

Objectives Resistance to extended-spectrum cephalosporins (ESCs) in Escherichia coli can be due to the production of ESBLs, plasmid-mediated AmpCs (pAmpCs) or chromosomal AmpCs (cAmpCs). Information regarding type and prevalence of β-lactamases, clonal relations and plasmids associated with the bla genes for ESC-R E. coli (ESC-R-Ec) detected in Switzerland is lacking. Moreover, data focusing on patients referred to the specialized outpatient clinics (SOCs) are needed. Methods We analysed 611 unique E. coli isolated during September-December 2011. ESC-R-Ec were studied with microarrays, PCR/DNA sequencing for blaESBLs, blapAmpCs, promoter region of blacAmpC, IS elements, plasmid incompatibility group, and also implementing transformation, aIEF, rep-PCR and MLST. Results The highest resistance rates were observed in the SOCs, whereas those in the hospital and community were lower (e.g. quinolone resistance of 22.6%, 17.2% and 9.0%, respectively; P = 0.003 for SOCs versus community). The prevalence of ESC-R-Ec in the three settings was 5.3% (n = 11), 7.8% (n = 22) and 5.7% (n = 7), respectively. Thirty isolates produced CTX-M ESBLs (14 were CTX-M-15), 5 produced CMY-2 pAmpC and 5 hyper-expressed cAmpCs due to promoter mutations. Fourteen isolates were of sequence type 131 (ST131; 10 with CTX-M-15). blaCTX-M and blaCMY-2 were associated with an intact or truncated ISEcp1 and were mainly carried by IncF, IncFII and IncI1plasmids. Conclusions ST131 producing CTX-M-15 is the predominant clone. The prevalence of ESC-R-Ec (overall 6.5%) is low, but an unusual relatively high frequency of AmpC producers (25%) was noted. The presence of ESC-R-Ec in the SOCs and their potential ability to be exchanged between hospital and community should be taken into serious consideratio

Pseudomonas aeruginosa bloodstream infections: risk factors and treatment outcome related to expression of the PER-1 extended-spectrum beta-lactamase

BACKGROUND: Bloodstream infection (BSI) due to Pseudomonas aeruginosa (Pa) has relevant clinical impact especially in relation to drug resistance determinants. The PER-1 extended-spectrum beta-lactamase (ESBL) is a common enzyme conferring high-level resistance to anti-pseudomonal cephalosporins. Risk factors and treatment outcome of BSI episodes caused by PER-1-positive Pa (PER-1-Pa) strains were compared to those caused by ESBL-negative Pa isolates (ESBL-N-Pa). METHODS: Twenty-six BSI cases due to ceftazidime-resistant Pa strains have been investigated. MIC values of anti-pseudomonal drugs were determined by the Etest method (AB Biodisk, Solna, Sweden). The double-disk synergy test was used to detect ESBL production. PCR amplification and DNA sequencing were used to characterize ESBL types. Clinical records of BSI-patients were examined retrospectively. Demographic data, underlying diseases (McCabe-Jackson classification and Charlson weighted index), risk factors, antimicrobial therapy, and treatment outcome were evaluated in cases due to ESBL-positive and cases due to ESBL-N-Pa isolates. Unpaired Student's t-test, Mann-Whitney U-test, Fisher's exact test and the χ(2 )test were used for statistical analysis. RESULTS: Nine Pa isolates expressed the PER-1 ESBL; the remaining 17 isolates did not produce ESBLs. Severe sepsis (P = 0.03), bladder and intravascular catheters (both P = 0.01), immunosuppressive therapy (P = 0.04), and mechanical ventilation (P = 0.03) were significantly associated with BSI due to PER-1-Pa. Empirical treatment (P = 0.02) and treatment after ID/AST (P < 0.01) were rarely adequate in PER-1-Pa cases. With regard to treatment outcome, 77.8% BSI cases due to PER-1-Pa vs. 28.6% cases due to ESBL-N-Pa isolates failed to respond (P < 0.03). All cases due to PER-1-Pa that were treated with carbapenems (alone or in combination with amikacin) failed to respond. In contrast, 7/8 cases due to ESBL-N-Pa given carbapenems were responders. CONCLUSION: Therapeutic failure and increased hospital costs are associated with BSI episodes caused by PER-1-Pa strains. Thus, recognition and prompt reporting of ESBL-production appears a critical factor for the management of patients with serious P. aeruginosa infections

Detection of blaCTX-M and blaDHA genes in stool samples of healthy people: comparison of culture- and shotgun metagenomic-based approaches.

We implemented culture- and shotgun metagenomic sequencing (SMS)-based methods to assess the gut colonization with extended-spectrum cephalosporin-resistant Enterobacterales (ESC-R-Ent) in 42 volunteers. Both methods were performed using native and pre-enriched (broth supplemented with cefuroxime) stools. Native culture screening on CHROMID® ESBL plates resulted in 17 positive samples, whereas the pre-enriched culture (gold-standard) identified 23 carriers. Overall, 26 ESC-R-Ent strains (24 Escherichia coli) were identified: 25 CTX-M and 3 DHA-1 producers (2 co-producing CTX-Ms). Using the SMS on native stool ("native SMS") with thresholds ≥60% for both identity and coverage, only 7 of the 23 pre-enriched culture-positive samples resulted positive for blaCTX-M/blaDHA genes (native SMS reads mapping to blaCTX-M/blaDHAs identified in gold-standard: sensitivity, 59.0%; specificity 100%). Moreover, an average of 31.5 and 24.6 antimicrobial resistance genes (ARGs) were detected in the 23 pre-enriched culture-positive and the 19 negative samples, respectively. When the pre-enriched SMS was implemented, more blaCTX-M/blaDHA genes were detected than in the native assay, including in stools that were pre-enriched culture-negative (pre-enriched SMS reads mapping to blaCTX-M/blaDHAs identified in gold-standard: sensitivity, 78.3%; specificity 75.0%). In addition, the pre-enriched SMS identified on average 38.6 ARGs/sample, whereas for the corresponding native SMS it was 29.4 ARGs/sample. Notably, stools resulting false-negative by using the native SMS had lower concentrations of ESC-R-Ent (average: ~105 vs. ~107 CFU/g) and E. coli classified reads (average: 193,959 vs. 1.45 million) than those of native SMS positive samples. Finally, the detection of blaCTX-M/blaDHA genes was compared with two well-established bioinformatic tools. In conclusion, only the pre-enriched SMS assured detection of most carriers of ESC-R-Ent. However, its performance was not comparable to the pre-enriched culture-based approach