PCR-based Open Reading Frame Typing Research Articles

Assessing the discriminability of PCR-based open reading frame typing versus single-nucleotide polymorphism analysis via draft whole-genome sequencing of methicillin-resistant Staphylococcus aureus in nosocomial transmission analysis

Phylogenetic analysis based on single-nucleotide polymorphism (SNP)-based through whole-genome sequencing is recognized as the standard method for probing nosocomial transmission. However, the application of WGS is constrained by the high cost of equipment and the need for diverse analysis tools, which limits its widespread use in clinical laboratory settings. In Japan, the prevalent use of PCR-based open reading frame typing (POT) for tracing methicillin-resistant Staphylococcus aureus (MRSA) transmission routes is attributed to its simplicity and ease of use. Although POT's discriminatory power is considered insufficient for nosocomial transmission analysis, conclusive data supporting this notion is lacking. This study assessed the discriminatory capabilities of SNP analysis and POT across 64 clinical MRSA strains. All 21 MRSA strains of ST5/SCCmec IIa, having more than 16 SNPs, demonstrated distinct clones. Conversely, two strains shared the same POT number and were identified as group A. Among the 12 MRSA strains of ST8/SCCmec IVl with over nine SNPs, five fell into POT group B, and five into POT group C. All four MRSA strains of ST8/SCCmec IVa were classified into POT group D, although they included strains with more than 30 SNPs. Among the 27 MRSA strains of ST1/SCCmec IVa, 14 were classified into POT group E. However, except for two clusters (each comprising two or three strains), all had SNP counts >10 (Fig. 1-D). SNP analysis of MRSA in CC1/SCCmec IV showed that several strains had the same number of SNPs in POT number (106-183-37), even among bacteria with >100 SNPs, indicating POT's limited use in detailed nosocomial transmission analysis.

Increase in the frequency of community-acquired methicillin-resistant Staphylococcus aureus clones among inpatients of acute care hospitals in the Kyoto and Shiga regions, Japan

BackgroundSpecific epidemic clones of hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) are responsible for the worldwide spread of MRSA. However, in recent years, the isolation of community-acquired MRSA (CA-MRSA) clones has been increasing. We investigated the latest molecular epidemiology trends of HA-MRSA and CA-MRSA clones in the Kyoto and Shiga regions, Japan. Materials and methodsAll nonduplicate MRSA isolates obtained from the clinical specimens of inpatients at four acute care hospitals in the Kyoto and Shiga regions between 2014 and 2019 were typed using the PCR-based open reading frame typing (POT) method. CA-MRSA and HA-MRSA were classified according to the POT1 values. We performed whole-genome sequencing analysis for representative isolates displaying common POT types. ResultsA total of 2413 isolates were included in the study, comprising 1730 nosocomial and 683 nonnosocomial isolates. The rates of HA-MRSA decreased from 50.2% in 2014 to 19.0% in 2019, while those of CA-MRSA increased from 44.7% to 76.4% (p < 0.001). Isolates belonging to the most common 10 POT types (CA-MRSA, n = 6; HA-MRSA, n = 4) accounted for 42% of the isolates studied and were obtained from 3 or more hospitals. Whole-genome sequencing analysis showed that the common CA-MRSA isolates with POT types 106-137-80, 106-9-80, 106-9-2, and 106-137-2, those with POT types 106-183-37 and 106-129-5, and HA-MRSA isolates with POT types 93-191-103, 93-157-127, 93-137-103, and 93-223-111 belonged to ST8-SCCmecIV, ST1-SCCmecIV, and ST764-SCCmecII, respectively. ConclusionA recent clonal shift from HA-MRSA to CA-MRSA occurred, and specific regional clones were prevalent among inpatients in the Kyoto and Shiga regions.

1206. Epidemiological interaction of MRSA, ESBL E.coli and C. difficile between ICU/Step-down Unit of University-affiliated Community Hospital and LTACs in Japan: Utility of Molecular Epidemiology Analysis by PCR-based ORF Typing (POT method)

Abstract Background Epidemiological interaction of drug resistant organisms between ICU and surrounding health facilities including LTACs have been of interest in the era of antimicrobial resistance (AMR) and aging society. In addition, conventional molecular epidemiology methods such as pulsed-field gel electrophoresis, and multilocus sequence typing are time-consuming and difficult to perform in a standard microbiology laboratory. Methods Patients admitted to a university-affiliated community hospital in Japan was recruited if their cultures collected clinically during admission grew the following target pathogens: Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin Resistant Enterococci (VRE), ESBL Gram negative rods (GNR), carbapenem-resistant Enterobacteriaceae (CRE), multi-drug resistant Pseudomonas (MDRP) and Clostridioides difficile (C. diff). Their clinical information including admission to ICU/step-down unit vs regular wards and disposition was analyzed with chi-square test for binary outcomes. For the isolates of MRSA, ESBL E. coli, and C. diff, further molecular epidemiology analysis was made, using PCR-based open-reading frame (ORF) Typing (POT method), a more resource-friendly and time-efficient technology originally developed in Japan. Results Among total 9639 hospital admissions in 2018-2019, the above-mentioned target pathogens grew from 233 patients (2.4%). There were 96 MRSA isolates (41.2%, out of 233), followed by 93 ESBL GNR (39.9%), 21 C. diff (9.0%), 13 CRE (5.6%), 10 MDRP (4.3%), and no VRE, respectively. Patients admitted to ICU/step-down unit were more likely to grow the target pathogens (117/1605 vs 116/7801, RR 4.90, 95% CI 3.81-6.30, p&amp;lt; 0.001), and to be discharged to LTACs than patients admitted to regular wards (70/113 vs 32/116, RR 2.17, 95% CI 1.56-3.02). Molecular epidemiology analysis with POT method identified 5, 3 and 3 common strains across 20, 11, and 7 patients with MRSA, ESBL E.coli, C. diff, respectively. Conclusion The study suggests the pathogens of higher priority were more prevalent in ICU/step-down unit and that the discharge from ICU/step-down unit to LTACs could impact on further transmission in the surrounding region. POT method can be utilized for more efficient molecular epidemiology analysis in the era of AMR. Disclosures All Authors: No reported disclosures.

A 12-year epidemiological study of Acinetobacter baumannii from blood culture isolates in a single tertiary-care hospital using polymerase chain reaction (PCR)-based open reading frame typing.

Acinetobacter baumannii is a causative agent of healthcare-associated infections, and the introduction and spread of A. baumannii that has acquired drug resistance within a hospital are serious healthcare problems. We investigated the transition of epidemic clones and the occurrence of outbreaks by molecular epidemiological analysis to understand the long-term behavior of A. baumannii within a single facility. A. baumannii isolates collected from blood-culture-positive patients between January 2009 and December 2020 were subjected to PCR-based open reading frame typing (POT) for species identification, clonal typing, and homology searches. Of the strains isolated from blood cultures, 49 were identified as A. baumannii and analyzed with POT. The POT#1=122 clones had different antimicrobial resistance profiles to the other POT clones, and strains belonging to this clone were dominant during outbreaks of multidrug-resistant Acinetobacter. Although the clonal diversity of A. baumannii decreased and its antimicrobial resistance increased during the outbreaks, clonal diversity and the in-hospital antibiogram improved at the end of the outbreaks. The POT#1=122 clone was not eliminated from the hospital during the study period. POT is a simple and suitable method for molecular epidemiological monitoring and can show the introduction, outbreak, and subsequent transition of an epidemic clone of A. baumannii.

Clinical characteristics and molecular analysis of USA300 and ST 764 methicillin-resistant Staphylococcus aureus isolates from outpatients in Japan by PCR-Based open reading frame typing

IntroductionUSA300 is the most common community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strain. Sequence type (ST) 764 MRSA is a new local variant of the ST 5 lineage. The objective of this study was to determine the clinical characteristics of USA300 and ST 764 infections among outpatients in Japan. MethodsWe obtained MRSA isolates from 132 outpatients who visited our hospital from January 2016 to December 2017 and compared USA300 infection group to ST 764 infection group. Molecular analysis, including that of various toxins and other virulence factors, of the MRSA isolates were performed. In particular, we investigated the relationships among PCR-based open reading frame typing (POT) scores, MRSA clones, and virulence factors. ResultsTwenty-seven USA300 isolates (20.5%) and 16 ST 764 isolates (12.1%) were identified. Although USA300 and ST 764 had lower rates of risk factors, their infection rates were higher. USA300-infected patients had higher rates of deep skin and soft tissue infections compared with the non-USA300 CA-MRSA-infected patients. Notably, the USA300 and ST 764 isolates had unique POT scores. ConclusionsOur results indicated that USA300 MRSA was spreading in an area 120 km west of Tokyo, Japan. We observed multiple cases of ST 764 MRSA infection, raising concerns about the antimicrobial resistance of ST 764, as it limits the choices of antibiotics to treat infection. The POT score can predict the presence of toxins and virulence factors, as well as the clone identity of MRSA with high accuracy.

Three-year epidemiological analysis of Clostridioides difficile infection and the value of hospital-infection control measures in a tertiary-care teaching hospital in Japan using PCR-based open-reading frame typing

IntroductionClostridioides difficile is an important causative pathogen in antibiotic-associated colitis and nosocomial infections. This study aimed to assess immunochromatographic test results for C. difficile infection and the utility of PCR-based open-reading frame typing (POT) for potentially controlling the intra-ward transmission of C. difficile. MethodsWe conducted a molecular epidemiological analysis using POT to investigate 102 inpatients who tested positive for the C. difficile toxin using immunochromatography in a tertiary-care teaching hospital in Japan between 2016 and 2018; isolates from the patients were obtained and cultured. ResultsThe number of POT numbers detected in 2016, 2017, and 2018 were 27 (among 34 patients), 20 (among 31 patients), and 28 (among 37 patients), respectively. During this three-year period, there were seven cases whose bacterial strains with the same POT number was identified in the same ward within 6 months. The intra-ward transmission rate was the highest in 2017 (16.1%). Intra-ward transmission was identified at a higher rate in patients whose sample cultures tested toxin-positive than in patients whose sample cultures tested toxin- and glutamate-dehydrogenase-positive via immunochromatography (16% vs. 3%, p < 0.05). ConclusionsWe conclude that the use of immunochromatographic tests for C. difficile diagnosis and epidemiological analyses via POT may be helpful for evaluating intra-ward transmission of C. difficile.

Systematic research to overcome newly emerged multidrug-resistant bacteria.

In the 1980s, I found that the chromosomal β-lactamase of Klebsiella pneumoniae LEN-1 showed a very high similarity to the R-plasmid-mediated penicillinase TEM-1 on the amino acid sequence level, and this strongly suggested the origination of TEM-1 from the chromosomal penicillinases of K. pneumoniae or related bacteria. Moreover, the chromosomal K1 β-lactamase (KOXY) of Klebsiella oxytoca was found to belong to the class A β-lactamases that include LEN-1 and TEM-1, although KOXY can hydrolyze cefoperazone (CPZ) like the chromosomal AmpC-type cephalosporinases of various Enterobacteriaceae that can hydrolyze several cephalosporins including CPZ. Furthermore, my collaborators and I found plural novel serine-type β-lactamases, such as MOX-1, SHV-24, TEM-91, CTX-M-64, CMY-9, CMY-19, GES-3, GES-4, and TLA-3, mediated by plasmids. Besides these serine-type β-lactamases, we also first identified exogenously acquired metallo-β-lactamases (MBLs), IMP-1 and SMB-1, in imipenem-resistant Serratia marcescens, and the IMP-1-producing S. marcescens TN9106 became the index case for carbapenemase-producing Enterobacteriaceae. I developed the sodium mercaptoacetic acid (SMA)-disk test for the simple identification of MBL-producing bacteria. We were also the first to identify a variety of plasmid-mediated 16S ribosomal RNA methyltransferases, RmtA, RmtB, RmtC, and NpmA, from various Gram-negative bacteria that showed very high levels of resistance to a wide range of aminoglycosides. Furthermore, we first found plasmid-mediated quinolone efflux pump (QepA) and fosfomycin-inactivating enzymes (FosA3 and FosK). We also first characterized penicillin reduced susceptible Streptococcus agalactiae, macrolide-resistant Mycoplasma pneumoniae, as well as Campylobacter jejuni, and Helicobacter pylori, together with carbapenem-resistant Haemophilus influenzae. We constructed a PCR-based open reading frame typing method for rapid identification of Acinetobacter baumannii international clones.

Molecular epidemiology of clinically high-risk Pseudomonas aeruginosa strains: Practical overview.

In recent years, numerous outbreaks of multidrug-resistant Pseudomonas aeruginosa have been reported across the world. Once an outbreak occurs, besides routinely testing isolates for susceptibility to antimicrobials, it is required to check their virulence genotypes and clonality profiles. Replacing pulsed-field gel electrophoresis DNA fingerprinting are faster, easier-to-use, and less expensive polymerase chain reaction (PCR)-based methods for characterizing hospital isolates. P. aeruginosa possesses a mosaic genome structure and a highly conserved core genome displaying low sequence diversity and a highly variable accessory genome that communicates with other Pseudomonas species via horizontal gene transfer. Multiple-locus variable-number tandem-repeat analysis and multilocus sequence typing methods allow for phylogenetic analysis of isolates by PCR amplification of target genes with the support of Internet-based services. The target genes located in the core genome regions usually contain low-frequency mutations, allowing the resulting phylogenetic trees to infer evolutionary processes. The multiplex PCR-based open reading frame typing (POT) method, integron PCR, and exoenzyme genotyping can determine a genotype by PCR amplifying a specific insertion gene in the accessory genome region using a single or a multiple primer set. Thus, analyzing P. aeruginosa isolates for their clonality, virulence factors, and resistance characteristics is achievable by combining the clonality evaluation of the core genome based on multiple-locus targeting methods with other methods that can identify specific virulence and antimicrobial genes. Software packages such as eBURST, R, and Dendroscope, which are powerful tools for phylogenetic analyses, enable researchers and clinicians to visualize clonality associations in clinical isolates.

Tracing the dissemination of the international clones of multidrug-resistant Acinetobacter baumannii among cancer patients in Egypt using the PCR-based open reading frame typing (POT) method.

The aim of this study was to perform an epidemiological surveillance of multidrug-resistant (MDR) Acinetobacter baumannii genetic lineages among cancer patients in Egypt using the PCR-based open reading frame typing (POT) method. A total of 160 MDR A. baumannii isolates were collected between January 2015 and December 2017 at a tertiary-care centre in Egypt. VITEK®2 system was used for preliminary species identification and antimicrobial susceptibility testing. The POT method was applied for confirmation of species identification and molecular epidemiological typing of the isolates. MDR A. baumannii isolates were classified into 15 POT types, including POT 122 (n=69), POT 69 (n=22) and other miscellaneous POT types (MPOTs) including POT 0, 8, 10, 12, 14, 40, 44, 52, 56, 93, 104, 106 and 108 (n=69). POT 122 isolates infected or colonised 61% of patients hospitalised in surgical wards and 54% of patients diagnosed with solid tumours and 51% were adults; whereas MPOT isolates infected or colonised 51% of patients hospitalised in paediatric wards and 49% of patients diagnosed with haematological malignancies and 51% were paediatric patients (P=0.007, 0.001 and 0.004, respectively). MPOT isolates were recovered from 46% of the collected blood specimens, whilst POT 122 isolates were recovered from 61% of the collected respiratory specimens (P=0.05). The current study demonstrates that the easy, low-cost POT method is convenient for rapid delineation of A. baumannii clonal diversity in a tertiary-care hospital in Egypt.

Spread of seb-Positive Methicillin-Resistant Staphylococcus aureus SCCmec Type II-ST764 Among Elderly Japanese in Nonacute Care Settings.

We investigated the prevalence and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) among 356 residents of nine long-term care facilities (LTCFs) in Japan during 2015 and 2017. In total, 800 specimens were tested and 39 MRSA isolates were recovered from 31 (8.71%) residents. PCR-based open reading frame typing (POT) and pulsed-field gel electrophoresis typing were performed for the 39 MRSA isolates; five of them showing identical pulsotypes, and POT scores were excluded in further analysis. Staphylococcal cassette chromosome mec (SCCmec) typing, multilocus sequence typing, and toxin gene detection were performed for one representative MRSA isolate per resident. Among the 34 unrelated MRSA isolates, 15 (44.1%) and 19 (55.9%) were of SCCmec types II and IV, respectively, and belonged to seven sequence types (STs). Among the 15 SCCmec II isolates, 11 (73.3%), 3, and 1 belonged to ST764 (clonal complex [CC]5), ST5 (CC5), and ST630 (CC8), respectively. Among the 19 SCCmec IV isolates, 13 (68.4%), 3, 2, and 1 belonged to ST1 (CC1), ST474 (CC1), ST8 (CC8), and ST380 (CC8), respectively. Among the 14 CC5 lineage-SCCmec II isolates, one ST5 isolate and 7 of the 11 ST764 isolates (63.6%) carried seb gene, and 14 (87.5%) of 16 CC1 lineage-SCCmec IV isolates had sea gene (p < 0.05). The results indicate that the seb-positive SCCmec type II-ST764 clone has spread in Japanese LTCF environments. As LTCF residents have multiple comorbidities and increased susceptibility to infections, it is necessary to monitor MRSA colonization in LTCFs through periodic screening to prevent dissemination.

Genome-Based Epidemiological Analysis of 13 Acinetobacter Strains Isolated from Blood Cultures of Hospitalized Patients from a University Hospital in Japan.

The genus Acinetobacter comprises many species that can cause infectious diseases. Despite their importance as nosocomial pathogens, the clinical distributions of individual species or clones are not well understood. In this study, we aimed to characterize 13 Acinetobacter strains isolated from blood cultures from Osaka City University Hospital. We conducted whole-genome sequencing to reveal their genetic background. We also performed PCR-based open reading frame typing (POT) and compared the results with those of multilocus sequence typing (MLST) to confirm its reliability as a genotyping method. Although biochemical analysis suggested that most isolates were A. baumannii, genomic analysis revealed that the collection of Acinetobacter isolates comprised six different species, with non-baumannii Acinetobacter species representing the majority. All strains possessed an inherent ADC-type β-lactamase gene, whereas the distribution of OXA-type enzymes was limited to A. baumannii, A. pittii, and A. colistiniresistens. While MLST properly discriminated four A. baumannii strains as different clones, POT failed to distinguish three of the four A. baumannii strains from each other, highlighting a potential pitfall that may be encountered when applying POT to non-epidemiological A. baumannii strains.

Efficacy of PCR-based open reading frame typing assay for outbreak investigation of metallo-β-lactamase-producing Pseudomonas aeruginosa in hematology unit.

We investigated the efficacy of the PCR-based open reading frame typing (POT) assay for outbreak investigation of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa (MBL-PA). A total of 53 P. aeruginosa isolates were detected between January 2010 and December 2012 on a hematology ward, of which 6 were identified as MBL-PA with the blaIMP-1 gene. The POT assay revealed the same genotype (207-41) in 3 of 6 MBL-PA, suggesting an outbreak caused by a single strain. Environmental investigation of bathroom samples revealed the same POT genotype (207-41) as those of the clinical isolates and no other MBL-PA strains. Genetic relatedness of the MBL-PA isolates was confirmed by the DiversiLab repetitive-sequence-based PCR typing system, suggesting the POT type 207-41 as a genetically identical clone. The POT assay can be successfully applied to MBL-PA genotyping.

Microbiological and molecular epidemiological analyses of community-associated methicillin-resistant Staphylococcus aureus at a tertiary care hospital in Japan.

Molecular characterization of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is generally conducted referred to staphylococcal cassette chromosome mec (SCCmec) type IV or V. CA-MRSA is now a cause of concern since such strains have been isolated not only from individuals in a community but also from patients in healthcare settings. The aim of this study was to analyze microbiological and molecular epidemiological features of CA-MRSA strains at a Japanese tertiary care hospital using PCR based-open reading frame typing (POT). This technique allows for molecular classification into CA-MRSA (POT-CA) and hospital-associated (HA-) MRSA (POT-HA) with clonal discrimination. Clinical MRSA isolates obtained from consecutive patients between October 1, 2012 and September 30, 2013 at the hospital were analyzed in combination with the clinical definition for CA-MRSA by the Centers for Disease Control and Prevention and POT. Of 219 isolates (76 clonal groups), 64 (29.3%) were clinical-HA/POT-CA isolates (22 clonal groups). Some clones of them accumulated in this hospital and might be involved in nosocomial transmission. Virulent factors of the isolates were analyzed, and only one (1.6%) Panton-Valentine leukocidin gene positive isolate but no arginine catabolic mobile element genes positive isolate were found in clinical-HA/POT-CA. Additionally, clinical-HA/POT-CA isolates showed higher antimicrobial susceptibility than clinical-HA/POT-HA, especially to minocycline, doxycycline, and amikacin. The most frequent genotype of molecular CA-MRSA was multi-locus sequence type 5-SCCmecIV, previously not detected in Japan. Although CA-MRSA at this hospital showed low virulence and higher antimicrobial susceptibility, the risk of nosocomial infection from them should be recognized, requiring stricter infection control measures.