- Volume 69, Issue 12, 2020

Introduction. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of hospital-acquired infections. Over the past two decades MRSA has become ‘epidemic’ in many hospitals worldwide. However, little is known about the genetic background of S. aureus recovered from hospital personnel in China.

Hypothesis/Gap Statement. The diversity of S. aureus genotypes warrants further surveillance and genomic studies to better understand the relatedness of these bacteria to those recovered from patients and the community.

Aim. The aim of this study was to determine the genetic diversity of MRSA and methicillin-susceptible S. aureus (MSSA) recovered from hospital personnel in Tianjin, North China.

Methodology. Three hundred and sixty-eight hand or nasal swabs were collected from 276 hospital personnel in 4 tertiary hospitals in Tianjin, North China between November 2017 and March 2019. In total, 535 Gram-positive bacteria were isolated, of which 59 were identified as S. aureus . Staphylococcal cassette chromosome mec (SCCmec) typing, multi-locus sequence typing (MLST) and spa typing were performed to determine the molecular characteristics of S. aureus .

Results. Thirty-one out of 276 (11 %) hospital personnel were S. aureus carriers, whereas 11/276 (4 %) carried MRSA. Fifty out of 59 (85 %) S. aureus isolates were resistant or intermediately resistant to erythromycin. The dominant genotypes of MRSA recovered from hospital personnel were ST398-t034-SCCmecIV/V and ST630-t084/t2196, whereas the major genotypes of MSSA included ST15-t078/t084/t346/t796/t8862/t8945/t11653 and ST398-t189/t034/t078/t084/t14014.

Conclusion. Although the predominant genotypes of MRSA recovered from hospital personnel in this study were different from the main genotypes that have previously been reported to cause infections in Tianjin and in other geographical areas of China, the MRSA ST398-t034 genotype has previously been reported to be associated with livestock globally. The dominant MSSA genotypes recovered from hospital personnel were consistent with the those previously reported to have been recovered from the clinic.

Introduction. Resistance against macrolide antibiotics in Mycoplasma pneumoniae is becoming non-negligible in terms of both appropriate therapy and diagnostic stewardship. Molecular methods have attractive features for the identification of Mycoplasma pneumoniae as well as its resistance-associated mutations of 23S ribosomal RNA (rRNA).

Hypothesis/Gap Statement. The automated molecular diagnostic sytem can identify macrolide-resistant M. pneumoniae .

Aim. To assess the performance of an automated molecular diagnostic system, GENECUBE Mycoplasma, in the detection of macrolide resistance-associated mutations.

Methodology. To evaluate whether the system can distinguish mutant from wild-type 23S rRNA, synthetic oligonucleotides mimicking known mutations (high-level macrolide resistance, mutation in positions 2063 and 2064; low-level macrolide resistance, mutation in position 2067) were assayed. To evaluate clinical oropharyngeal samples, purified nucleic acids were obtained from M. pneumoniae -positive samples by using the GENECUBE system from nine hospitals. After confirmation by re-evaluation of M. pneumoniae positivity, Sanger-based sequencing of 23S rRNA and mutant typing using GENECUBE Mycoplasma were performed.

Results. The system reproducibly identified all synthetic oligonucleotides associated with high-level macrolide resistance. Detection errors were only observed for A2067G (in 2 of the 10 measurements). The point mutation in 23S rRNA was detected in 67 (26.9 %) of 249 confirmed M. pneumoniae -positive clinical samples. The mutations at positions 2063, 2064 and 2617 were observed in 65 (97.0 %), 2 (3.0 %) and 0 (0.0 %) of the 67 samples, respectively. The mutations at positions 2063 and 2064 were A2063G and A2064G, respectively. The results from mutant typing using GENECUBE Mycoplasma were in full agreement with the results from sequence-based typing.

Conclusion. GENECUBE Mycoplasma is a reliable test for the identification of clinically significant macrolide-resistant M. pneumoniae .

Accurate identification of Nocardia species remains a challenge due to the complexities of taxonomy and insufficient discriminatory power of traditional techniques. We report the development of a molecular technique that utilizes real-time PCR-based high-resolution melting (HRM) analysis for differentiation of the most common Nocardia species. Based on a novel fusA-tuf intergenic region sequence, Nocardia farcinica , Nocardia cyriacigeorgica and Nocardia beijingensis were clearly distinguished from one another by HRM analysis. The limit of detection of the HRM assay for purified Nocardia spp. DNA was at least 10 fg. No false positives were observed for specificity testing of 20 non-target clinical samples. In comparison to established matrix-assisted laser desorption/ionization-time of flight MS, the HRM assay improved the identification of N. beijingensis . Additionally, all the products of PCR were verified by direct sequencing. In conclusion, the developed molecular assay allows simultaneous detection and differentiation of N. farcinica , N. cyriacigeorgica and N. beijingensis with high sensitivity and specificity.

Introduction. Although new parechovirus A (PeVA) types, including parechovirus A3 (PeVA3) and PeVA4, have been reported in this century, there have not yet been any seroepidemiological studies on PeVA over a period of several decades.

Hypothesis/Gap Statement. The authors hypothesize that PeVA3 and PeVA4 emerged recently.

Aims. The aim was to clarify changes in the seroprevalence of PeVA1, PeVA3 and PeVA4.

Methodology. Neutralizing antibodies (NT Abs) were measured among residents in Yamagata, Japan in 1976, 1983, 1985, 1990, 1999 and 2017.

Results. The total NT Ab-positive rate for PeVA1 was between 90.7 and 100 % for all years analysed, with that for PeVA3 increasing from 39.6 % in 1976 to 69.6 % in 2017, and that for PeVA4 decreasing from 93.9 % in 1976 to 49.1 % in 2017. The distribution of NT Ab titres for PeVA1, PeVA3 and PeVA4 among those aged less than 20 years old was as follows: those ≥1 : 32 for PeVA1 were between 68.0–89.2 % for all years analysed; those ≥1 : 32 for PeVA3 was 15.4 % in 1976, 44.3–54.9 % in 1983–1990 and 64.8–68.0 % in 1999–2017; and those ≥1 : 32 for PeVA4 were between 49.1–67.2 % in 1976–1990, 41.3 % in 1999 and 23.8 % in 2017.

Conclusions. Our findings in this seroepidemiological study over four decades suggested that PeVA1 has been stably endemic, while PeVA3 appeared around 1970s and has spread since then as an emerging disease, and occasional PeVA4 infections were common in 1970s and 1980s but have been decreasing for several decades in our community.

Introduction. Rapid identification of the causative agent of sepsis is crucial for patient outcomes.

Aim. The Sepsityper sample preparation method enables direct microbial identification of positive blood culture samples via matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS).

Hypothesis/Gap statement. The implementation of the Sepsityper method in the routine practice could represent a fundamental tool to achieve a prompt identification of the causative agent of bloodstream infections, and therefore accelerate the adoption of the proper antibiotic treatment.

Methodology. In this study, the novel rapid workflow of the MALDI Biotypr Sepsityper kit (Bruker Daltonik GmbH, Germany) was evaluated using routine samples from a 2-year period (n=6918), and dedicated optimized protocols for the microbial groups that were more difficult to identify were developed. Moreover, the use of the residual bacterial pellet to perform susceptibility testing using different methods (commercial broth microdilution, disc diffusion, gradient diffusion) was investigated.

Results. The rapid Sepsityper protocol allowed the identification of 5470/6338 (86.3 %) monomicrobial samples at species level, with very good performance for all of the clinically most significant pathogens (2510/2592 enterobacteria, 631/669 Staphylococcus aureus and 223/246 enterococci were identified). Streptococcus pneumoniae , Bacteroides fragilis and yeasts were the most troublesome to identify, but the application of specific optimized protocols significantly improved their rate of identification (from 14.7–71.5 %, 47.8–89.7 % and 37.1–89.5 %, respectively). Specificity was 100 % (no identification was made for the false-positive samples). Further, the residual pellet proved to be suitable to investigate susceptibility to antimicrobials, enabling us to simplify the workflow and shorten the time to report.

Conclusion. The Rapid Sepsityper workflow proved to be a reliable sample preparation method for identification and susceptibility testing directly from positive blood cultures, providing novel approaches for accelerated diagnostics of bloodstream infections.