- Volume 72, Issue 1, 2023

Introduction. Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a serious threat to global public health. Colistin is regarded as the last-resort antibiotic for CRKP infections, but colistin resistance among CRKP is increasingly being reported, making clinical treatment for CRKP infections more difficult.

Hypothesis/Gap Statement. The molecular mechanisms of colistin resistance in Klebsiella spp. under the pressure of colistin have not been fully investigated.

Aim. We aimed to investigate the phenotypic and genetic variation in two colistin-susceptible Klebsiella spp. strains under selective pressure of colistin.

Methodology. One hundred microlitres of overnight cultures of the CRKP clinical strain CRKP12-130 and of ATCC 700603 was spread on five Mueller-Hinton Agar (MHA) plates with colistin concentrations of 2, 4, 8, 16 and 32 µg ml−1, and growth of colonies was observed for five consecutive days. Colonies collected from plates were passaged daily for 10 days on MHA plates without colistin and susceptibility testing of colistin was performed by broth microdilution. Thirty-four colistin-resistant strains randomly selected were submitted to whole genome sequencing (WGS). Transcriptional levels of genes involved in colistin resistance (mgrB, phoP, phoQ, pmrA, pmrB, pmrD, pmrE and pmrK) were measured by quantitative real-time PCR.

Results. A total of 114 and 119 colistin-resistant colonies were obtained from CRKP12-130 and ATCC 700603 in this study, among which 16 and 18 colonies were submitted to WGS, respectively. Among these 34 sequenced isolates, mutation in phoQ (13/16, 81.25 %) was the main genetic factor mediating colistin resistance in strains from CRKP12-130, while for strains from ATCC 700603, mutation associated with mgrB (8/18, 44.44 %) was found to be the commonest. Mutation of mgrB led to a significant increase in the MIC for colistin (from 64 to >128 µg ml−1), and a novel mutation C28R in mgrB was first reported in this study.

Conclusion. Colistin-resistant Klebsiella spp. could be easily selected under pressure of different concentrations of colistin. Mutations of mgrB, phoP, phoQ and pmrB genes were the main mechanisms leading to chromosomally mediated colistin resistance in Klebsiella spp.

Introduction. Proteus mirabilis is part of the family Enterobacteriaceae , and is naturally resistant to various antimicrobial drugs. In recent years, outbreaks of severe nosocomial infections caused by carbapenem-resistant P. mirabilis (CR-PMI) have been frequently reported. Few studies exist on the whole-genome molecular characteristics of this bacterium in China and elsewhere, which stimulated the implementation of this study.

Hypothesis. CR-PMI strains contained the multiple drug resistance genes and exhibited a high resistance rate to commonly used antimicrobial drugs.

Aim. Our goals here were to identify resistance mechanisms and homology of CR-PMI strains and provide a theoretical basis for clinical treatment and controlling nosocomial infections.

Methodology. Bacterial species identification was carried out using matrix-assisted laser desorption/ionization time of flight MS (MALDI-TOF-MS). Antimicrobial susceptibility was determined using the VITEK 2 system and Kirby-Bauer (K-B) disc-diffusion method. Whole-genome sequencing (WGS) was conducted by the Illumina platform NovaSeq sequencer. Antibiotic resistance genes (ARGs) were identified using the NCBI database with Abricate. Plasmid replicon types were identified using PlasmidFinder, available at the Center for Genomic Epidemiology.

Results. Five CR-PMI strains collected in our hospital from July 2019 to September 2021 were resistant to almost all antimicrobial agents except aztreonam (ATM), amikacin (AMK) and cefotetan (CTT). All CR-PMI strains contained the carbapenem resistance gene New Delhi metallo-β-lactamase 1 (bla NDM-1), and two strains harboured extended-spectrum β-lactamase (ESBL) genes bla PER-4 and bla CTX-M-65. The five CR-PMI strains contained 27, 18, 30, 25 and 24 drug-resistance genes, respectively. Most antimicrobial resistance genes were detected for aminoglycosides (n=14), followed by cephalosporins (n=7). The phylogenetic tree was divided into five evolutionary groups, and the five CR-PMI strains were in the four evolutionary groups B–E.

Conclusion Overall, CR-PMI strains exhibited a high resistance rate to commonly used antimicrobial drugs, and contained the carbapenem resistance gene bla NDM-1. The CR-PMI strains showed a polyclonal trend in different wards at different times. Most importantly, all strains identified contained important antimicrobial resistance genes, which may lead to severe drug resistance transmission and fatal multiple resistant bacterial infections.

Introduction. The prompt and specific diagnosis of Listeria monocytogenes meningoencephalitis (LMM) is challenging. Next-generation sequencing (NGS) of cerebrospinal fluid (CSF) is an emerging technique for diagnosing infrequent causative pathogens.

Hypothesis/Gap statement. We hypothesized that NGS of CSF is an effective approach for diagnosing LMM.

Aim. To evaluate the effectiveness of NGS, we present five cases of LMM diagnosed using NGS of the CSF.

Methodology. Between August 2017 and 30 September 2020, we used NGS of the CSF to detect pathogens in patients with clinically suspected central nervous system infections. The clinical characteristics, laboratory tests, imaging findings and NGS results are reviewed.

Results. Five patients were diagnosed with LMM using NGS of the CSF within 2 to 4 days, although the clinical manifestations, medical history and imaging findings varied strikingly. NGS of CSF showed sequence reads corresponding to L. monocytogenes species ranging from 118 to 1997 bp, genomic coverage of 0.29–5.96 %, relative abundance of 14.83–32.16 % and sequencing depth of 1.12 to 1.35. The prompt diagnosis resulted in targeted and effective treatment with the appropriate antibiotics, although two patients with the most severe cerebral parenchymal lesions showed little improvement.

Conclusion. Our results demonstrate the power of NGS of CSF for the prompt diagnosis of LMM. NGS of CSF is an important complementary tool for identifying L. monocytogenes .

Introduction. In 2009, the World Health Organization (WHO) established the Global Invasive Bacterial Vaccine Preventable Disease (IB-VPD) Surveillance Network (GISN) to monitor the global burden and aetiology of bacterial meningitis, pneumonia and sepsis caused by Haemophilus influenzae (Hi), Neisseria meningitidis (Nm) and Streptococcus pneumoniae (Sp).

Hypothesis/Gap Statement. The GISN established an external quality assessment (EQA) programme for the characterization of Hi, Nm and Sp by culture and diagnostic PCR.

Aim. To assess the performance of sentinel site laboratories (SSLs), national laboratories (NLs) and regional reference laboratories (RRLs) between 2014 and 2019 in the EQA programme.

Methodology. Test samples consisted of bacterial smears for Gram-staining, viable isolates for identification and serotyping or serogrouping (ST/SG), plus simulated cerebrospinal fluid (CSF) samples for species detection and ST/SG by PCR. SSLs and NLs were only required to analyse the slides for Gram staining and identify the species of the live isolates. RRLs, and any SLs and NLs that had the additional laboratory capacity, were also required to ST/SG the viable isolates and analyse the simulated CSF samples.

Results. Across the period, 69–112 SS/NL labs and eight or nine RRLs participated in the EQA exercise. Most participants correctly identified Nm and Sp in Gram-stained smears but were less successful with Hi and other species. SSLs/NLs identified the Hi, Nm and Sp cultures well and also submitted up to 56 % of Hi, 62 % of Nm and 33 % of Sp optional ST/SG results each year. There was an increasing trend in the proportion of correct results submitted over the 6 years for Nm and Sp. Some SSLs/NLs also performed the optional detection and ST/SG of the three organisms by PCR in simulated CSF from 2015 onwards; 89–100 % of the CSF samples were correctly identified and 76–93 % of Hi-, 90–100 % of Nm- and 75–100 % of Sp-positive samples were also correctly ST/SG across the distributions. The RRLs performed all parts of the EQA to a very high standard, with very few errors across all aspects of the EQA.

Conclusion. The EQA has been an important tool in maintaining high standards of laboratory testing and building of laboratory capacity in the GISN.

Introduction. Rapid identification (ID) and antimicrobial susceptibility testing (AST) of bloodstream infections (BSI) pathogens are fundamental to switch from empirical to targeted antibiotic therapy improving patients outcome and reducing antimicrobial resistance spreading.

Hypothesis. The adoption of a rapid microbiological protocol (RP) based on Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS) and Light Scattering Technology (LST) for rapid diagnosis of BSI could positively impact on patients' antimicrobial management.

Aim. The study aim was to evaluate a RP for BSI microbiological diagnosis in terms of accuracy, turnaround time (TAT) and potential therapeutic impact.

Methodology. A prospective observational study was conducted: monomicrobial bacterial blood cultures of septic patients were analysed in parallel by RP and standard protocol (SP). In RP the combination of MALDI-TOF MS and LST was used for rapid ID and AST assessments, respectively. To determine the potential impact of RP on antimicrobial therapy management, clinicians were interviewed on therapeutic decisions based on RP and SP results. RP accuracy, TAT and impact were evaluated in comparison to SP results.

Results. A total of 97 patients were enrolled. ID and AST concordance between RP and SP were 96.9 and 94.7 %, respectively. RP technical and real-life TAT were lower than SP (6.4 h vs. 18.4 h; 9.5 vs. 27.1 h). The agreement between RP- and SP-based therapeutic decisions was 90.7 (90 % CI 84.4–95.1). RP results could produce 24/97 correct antibiotic changes with 18/97 possible de-escalations and 25/97 prompt applications of infection control precautions.

Conclusion. With the application of RP in BSI management, about one-fourth of patients may safely benefit from early targeted antibiotic therapy and infection control policies with one working day in advance in comparison to conventional methods. This protocol is feasible for clinical use in microbiology laboratories and potentially helpful for Antimicrobial Stewardship.