Double-locus Sequence Typing Research Articles

Core genome multilocus sequence typing of Clostridioides difficile to investigate transmission in the hospital setting

PurposeTraditional epidemiological investigations of healthcare-associated Clostridioides difficile infection (HA-CDI) are often insufficient. This study aimed to evaluate a procedure that includes secondary isolation and genomic typing of single toxigenic colonies using core genome multilocus sequence typing (cgMLST) for the investigation of C. difficile transmission.MethodsWe analyzed retrospectively all toxigenic C. difficile-positive stool samples stored at the Lausanne University Hospital over 6 consecutive months. All isolates were initially typed and classified using a modified double-locus sequence typing (DLST) method. Genome comparison of isolates with the same DLST and clustering were subsequently performed using cgMLST. The electronic administrative records of patients with CDI were investigated for spatiotemporal epidemiological links supporting hospital transmission. A comparative descriptive analysis between genomic and epidemiological data was then performed.ResultsFrom January to June 2021, 86 C. difficile isolates were recovered from thawed samples of 71 patients. Thirteen different DLST types were shared by > 1 patient, and 13 were observed in single patients. A genomic cluster was defined as a set of isolates from different patients with ≤ 3 locus differences, determined by cgMLST. Seven genomic clusters were identified, among which plausible epidemiological links were identified in only 4/7 clusters.ConclusionAmong clusters determined by cgMLST analysis, roughly 40% included unexplained HA-CDI acquisitions, which may be explained by unidentified epidemiological links, asymptomatic colonization, and/or shared common community reservoirs. The use of DLST, followed by whole genome sequencing analysis, is a promising and cost-effective stepwise approach for the investigation of CDI transmission in the hospital setting.

Evolutionary trajectories of beta-lactamase NDM and DLST cluster in Pseudomonas aeruginosa: finding the putative ancestor

ABSTRACT Pseudomonas aeruginosa has different antibiotic resistance pathways, such as broad-spectrum lactamases and metallo-β-lactamases (MBL), penicillin-binding protein (PBP) alteration, and active efflux pumps. Polymerase chain reaction (PCR) and sequencing methods were applied for double-locus sequence typing (DLST) and New Delhi metallo-β-lactamase (NDM) typing. We deduced the evolutionary pathways for DLST and NDM genes of P. aeruginosa using phylogenetic network. Among the analyzed isolates, 62.50% of the P. aeruginosa isolates were phenotypically carbapenem resistance (CARBR) isolates. Characterization of isolates revealed that the prevalence of blaNDM, blaVIM, blaIMP, undetermined carbapenemase, and MexAB-OprM were 27.5%, 2%, 2.5%, 12.5%, and 15%, respectively. The three largest clusters found were DLST t20–105, DLST t32–39, and DLST t32–52. The network phylogenic tree revealed that DLST t26–46 was a hypothetical ancestor for other DLSTs, and NDM-1 was as a hypothetical ancestor for NDMs. The combination of the NDM and DLST phylogenic trees revealed that DLST t32–39 and DLST tN2-N3 with NDM-4 potentially derived from DLST t26–46 along with NDM-1. Similarly, DLST t5–91 with NDM-5 diversified from DLST tN2-N3 with NDM-4. This is the first study in which DLST and NDM evolutionary routes were performed to investigate the origin of P. aeruginosa isolates. Our study showed that the utilization of medical equipment common to two centers, staff members common to two centers, limitations in treatment options, and prescription of unnecessary high levels of meropenem are the main agents that generate new types of resistant bacteria and spread resistance among hospitals.

Molecular typing of Clostridioides difficile from frozen stool samples to investigate cross-transmissions: A proof of concept

PurposeToxigenic Clostridioides difficile is responsible for up to one third of post antibiotic diarrhea and for more than 95% of pseudomembranous colitis. Nowadays, diagnosis relies on the documentation of the presence of the toxin in stools by specific antigenic or PCR tests. Stool cultures have been mostly abandoned, leading to the absence of isolates for further epidemiological analyses. MethodsAliquots of stool samples, frozen for up to two years, were thawed and inoculated onto commercial C. difficile media. Eighteen stools were recovered from patients hospitalized in the pediatric ward where at that time a chain of transmission was suspected. Eleven stools were recovered from patients hospitalized in a medical ward over a three months period with no suspected transmission event. Up to 16 characteristic colonies were isolates per culture. PCR of toxins genes and molecular typing by Double Locus Sequence Typing (DLST) were performed on these colonies. Whole genome multi locus sequence typing (wgMLST) was performed on selected isolates. ResultsAmong the 29 stool specimens, no growth was observed for four stools and only one colony grew for one stool. Except the latter, all 16 colonies of the 24 stools showed identical toxin genes profiles than the original stool. However, variant DLST genotypes was observed within 20% of investigated stools. The majority of variants were single locus variant due to an IN/DEL of the repeat in one of the two DLST locus.Despite this variation, results of molecular typing overrule the putative transmission chain in the pediatric ward and revealed undetected chains of transmission in the medical ward. These results were confirmed with wgMLST. ConclusionsThe developed protocol allows prospective and retrospective molecular and genomic epidemiological investigation of C. difficile infections for infection control purpose.

Molecular characterization of NDM-1-producing Pseudomonas aeruginosa isolates from hospitalized patients in Iran

BackgroundThe emergence of carbapenem-resistant Pseudomonas aeruginosa is one of the most important challenges in a healthcare setting. The aim of this study is double-locus sequence typing (DLST) typing of blaNDM-1 positive P. aeruginosa isolates.MethodsTwenty-nine blaNDM-1 positive isolates were collected during three years of study from different cities in Iran. Modified hodge test (MHT), double-disk synergy test (DDST) and double-disk potentiation test (DDPT) was performed for detection of carbapenemase and metallo-beta-lactamase (MBL) producing blaNDM-1 positive P. aeruginosa isolates. The antibiotic resistance genes were considered by PCR method. Clonal relationship of blaNDM-1 positive was also characterized using DLST method.ResultsAntibiotic susceptibility pattern showed that all isolates were resistant to imipenem and ertapenem. DDST and DDPT revealed that 15/29 (51.8%) and 26 (89.7%) of blaNDM-1 positive isolates were MBL producing isolates, respectively. The presence of blaOXA-10,blaVIM-2, blaIMP-1 and blaSPM genes were detected in 86.2%, 41.4%, 34.5% and 3.5% isolates, respectively. DLST typing results revealed the main cluster were DLST 25-11 with 13 infected or colonized patients.ConclusionsThe presence of blaNDM-1 gene with other MBLs encoding genes in P. aeruginosa is a potential challenge in the treatment of microorganism infections. DLST showed partial diversity among 29 blaNDM-1 positive isolates.

Molecular Epidemiology of Multi-Drug Resistant Pseudomonas aeruginosa Isolates from Hospitalized Patients in Greece.

Resistant Pseudomonas aeruginosa isolates are one of the major causes of both hospital-acquired infections (HAIs) and community-acquired infections (CAIs). However, management of P. aeruginosa infections is difficult as the bacterium is inherently resistant to many antibiotics. In this study, a collection of 75 P. aeruginosa clinical isolates from two tertiary hospitals from Athens and Alexnadroupolis in Greece was studied to assess antimicrobial sensitivity and molecular epidemiology. All P. aeruginosa isolates were tested for susceptibility to 11 commonly used antibiotics, and the newly introduced Double Locus Sequence Typing (DLST) scheme was implemented to elucidate the predominant clones. The tested P. aeruginosa isolates presented various resistant phenotypes, with Verona Integron-Mediated Metallo-β-lactamase (VIM-2) mechanisms being the majority, and a new phenotype, FEPR-CAZS, being reported for the first time in Greek isolates. DLST revealed two predominant types, 32-39 and 8-37, and provided evidence for intra-hospital transmission of the 32-39 clone in one of the hospitals. The results indicate that DLST can be a valuable tool when local outbreaks demand immediate tracking investigation with limited time and financial resources.

Combining Standard Molecular Typing and Whole Genome Sequencing to Investigate Pseudomonas aeruginosa Epidemiology in Intensive Care Units.

Pseudomonas aeruginosa is one of the main pathogens responsible for nosocomial infections, particularly in Intensive Care Units (ICUs). Due to the complexity of P. aeruginosa ecology, only powerful typing methods can efficiently allow its surveillance and the detection during expanding outbreaks. An increase in P. aeruginosa incidence was observed in the ICUs of the Lausanne University Hospital between 2010 and 2014. All clinical and environmental isolates retrieved during this period were typed with Double locus sequence typing (DLST), which detected the presence of three major genotypes: DLST 1–18, DLST 1–21, and DLST 6–7. DLST 1–18 (ST1076) isolates were previously associated with an epidemiologically well-described outbreak in the burn unit. Nevertheless, DLST 1–21 (ST253) and DLST 6–7 (ST17) showed sporadic occurrence with only few cases of possible transmission between patients. Whole genome sequencing (WGS) was used to further investigate the epidemiology of these three major P. aeruginosa genotypes in the ICUs. WGS was able to differentiate between outbreak and non-outbreak isolates and confirm suspected epidemiological links. Additionally, whole-genome single nucleotide polymorphisms (SNPs) results considered isolates as closely related for which no epidemiological links were suspected, expanding the epidemiological investigation to unsuspected links. The combination of a first-line molecular typing tool (DLST) with a more discriminatory method (WGS) proved to be an accurate and cost-efficient typing strategy for the investigation of P. aeruginosa epidemiology in the ICUs.

Molecular typing of methicillin and vancomycin-resistant Staphylococcus aureus isolated from clinical specimens by doublelocus sequence typing (DLST) method

Methicillin-resistant Staphylococcus aureus (MRSA) strains are the essential cause of infections in communities and hospitals. The present study was conducted to determine the molecular typing of MRSA, isolated from hospitalized patients, using the double-locus sequence typing (DLST). In total, 280 S. aureus isolated from clinical specimens by phenotypic (catalase, coagulase, DNase, oxacillin, vancomycin screening agar and antibiotic disk diffusion), and molecular methods (PCR for determining the mecA, vanA and nuc genes). The DLST and sequencing was performed for MRSA containing mecA. Out of 280 specimens, confirmed as Staphylococcus aureus (S. aureus), 123 (43.9%) strains were MRSA. The highest resistance toward the erythromycin (15 μg), followed by ciprofloxacin (5 μg), clindamycin (2 μg), tetracycline (30 μg), gentamicin (10 μg) and rifampicin (5 μg), was 98.3%, 97.5%, 94.3%, 90.2%, 83.7% and 41.4%, respectively. Also, the least resistance (0%) was observed in each of teicoplanin (30 μg), linzolide (30 μg), and vancomycin (30 μg). All (100%) of MRSA strains had the mecA, and none of them have had the vanA. The results of DLST showed that the most common sequence types were BPH 2003 and 0217. The DLST type 18-32 was a significant cluster of MRSA. By sequencing MRSA and comparing the dominant types via the DLST, it is possible to establish the etiology of the disease in a much shorter time, and prevent the complications of the disease. Therefore, the combination of partial sequences of clfB and spa can serve as useful genetic markers for MRSA typing. It concluded that the MRSA in our region was relatively high, but no vancomycin resistance was found. The majority of the MRSA DLST type was 18–32.

Transmissible ST3-IncHI2 Plasmids Are Predominant Carriers of Diverse Complex IS26-Class 1 Integron Arrangements in Multidrug-Resistant Salmonella.

Diverse mobile genetic elements (MGEs) including plasmids, insertion sequences, and integrons play an important role in the occurrence and spread of multidrug resistance (MDR) in bacteria. It was found in previous studies that IS26 and class 1 integrons integrated on plasmids to speed the dissemination of antibiotic-resistance genes in Salmonella. It is aimed to figure out the patterns of specific genetic arrangements between IS26 and class 1 integrons located in plasmids in MDR Salmonella in this study. A total of 74 plasmid-harboring Salmonella isolates were screened for the presence of IS26 by PCR amplification, and 39 were IS26-positive. Among them, 37 isolates were resistant to at least one antibiotic. The thirty-seven antibiotic-resistant isolates were further involved in PCR detection of class 1 integrons and variable regions, and all were positive for class 1 integrons. Six IS26-class 1 integron arrangements with IS26 inserted into the upstream or downstream of class 1 integrons were characterized. Eight combinations of these IS26-class 1 integron arrangements were identified among 31 antibiotic-resistant isolates. Multidrug-resistance plasmids of the IncHI2 incompatibility group were dominant, which all belonged to ST3 by plasmid double locus sequence typing. These 21 IncHI2-positive isolates harbored six complex IS26-class 1 integron arrangement patterns. Conjugation assays and Southern blot hybridizations confirmed that conjugative multidrug-resistance IncHI2 plasmids harbored the different complex IS26-class 1 integron arrangements. The conjugation frequency of IncHI2 plasmids transferring alone was 10−5-10−6, reflecting that different complex IS26-class 1 integron arrangement patterns didn't significantly affect conjugation frequency (P > 0.05). These data suggested that class 1 integrons represent the hot spot for IS26 insertion, forming diverse MDR loci. And ST3-IncHI2 was the major plasmid lineage contributing to the horizontal transfer of composite IS26-class 1 integron MDR elements in Salmonella.

High Heterogeneity of Escherichia coli Sequence Types Harbouring ESBL/AmpC Genes on IncI1 Plasmids in the Colombian Poultry Chain.

BackgroundEscherichia coli producing ESBL/AmpC enzymes are unwanted in animal production chains as they may pose a risk to human and animal health. Molecular characterization of plasmids and strains carrying genes that encode these enzymes is essential to understand their local and global spread.ObjectivesTo investigate the diversity of genes, plasmids and strains in ESBL/AmpC-producing E. coli from the Colombian poultry chain isolated within the Colombian Integrated Program for Antimicrobial Resistance Surveillance (Coipars).MethodsA total of 541 non-clinical E. coli strains from epidemiologically independent samples and randomly isolated between 2008 and 2013 within the Coipars program were tested for antimicrobial susceptibility. Poultry isolates resistant to cefotaxime (MIC ≥ 4 mg/L) were screened for ESBL/AmpC genes including blaCTX-M, blaSHV, blaTEM, blaCMY and blaOXA. Plasmid and strain characterization was performed for a selection of the ESBL/AmpC-producing isolates. Plasmids were purified and transformed into E. coli DH10B cells or transferred by conjugation to E. coli W3110. When applicable, PCR Based Replicon Typing (PBRT), plasmid Multi Locus Sequence Typing (pMLST), plasmid Double Locus Sequence Typing (pDLST) and/or plasmid Replicon Sequence Typing (pRST) was performed on resulting transformants and conjugants. Multi Locus Sequence Typing (MLST) was used for strain characterization.ResultsIn total, 132 of 541 isolates were resistant to cefotaxime and 122 were found to carry ESBL/AmpC genes. Ninety-two harboured blaCMY-2 (75%), fourteen blaSHV-12 (11%), three blaSHV-5 (2%), five blaCTX-M-2 (4%), one blaCTX-M-15 (1%), one blaCTX-M-8 (1%), four a combination of blaCMY-2 and blaSHV-12 (4%) and two a combination of blaCMY-2 and blaSHV-5 (2%). A selection of 39 ESBL/AmpC-producing isolates was characterized at the plasmid and strain level. ESBL/AmpC genes from 36 isolates were transferable by transformation or conjugation of which 22 were located on IncI1 plasmids. These IncI1 plasmids harboured predominantly blaCMY-2 (16/22), and to a lesser extend blaSHV-12 (5/22) and blaCTX-M-8 (1/22). Other plasmid families associated with ESBL/AmpC-genes were IncK (4/33), IncHI2 (3/33), IncA/C (2/33), IncΒ/O (1/33) and a non-typeable replicon (1/33). Subtyping of IncI1 and IncHI2 demonstrated IncI1/ST12 was predominantly associated with blaCMY-2 (12/16) and IncHI2/ST7 with blaCTX-M-2 (2/3). Finally, 31 different STs were detected among the 39 selected isolates.ConclusionsResistance to extended spectrum cephalosporins in E. coli from Colombian poultry is mainly caused by blaCMY-2 and blaSHV-12. The high diversity of strain Sequence Types and the dissemination of homogeneous IncI1/ST12 plasmids suggest that spread of the resistance is mainly mediated by horizontal gene transfer.

Molecular Characterization and Phylogenetic Analysis of Pseudomonas aeruginosa Isolates Recovered from Greek Aquatic Habitats Implementing the Double-Locus Sequence Typing Scheme.

The recently described double-locus sequence typing (DLST) scheme implemented to deeply characterize the genetic profiles of 52 resistant environmental Pseudomonas aeruginosa isolates deriving from aquatic habitats of Greece. DLST scheme was able not only to assign an already known allelic profile to the majority of the isolates but also to recognize two new ones (ms217-190, ms217-191) with high discriminatory power. A third locus (oprD) was also used for the molecular typing, which has been found to be fundamental for the phylogenetic analysis of environmental isolates given the resulted increased discrimination between the isolates. Additionally, the circulation of acquired resistant mechanisms in the aquatic habitats according to their genetic profiles was proved to be more extent. Hereby, we suggest that the combination of the DLST to oprD typing can discriminate phenotypically and genetically related environmental P. aeruginosa isolates providing reliable phylogenetic analysis at a local level.

New genotyping method discovers sustained nosocomial Pseudomonas aeruginosa outbreak in an intensive care burn unit

Pseudomonas aeruginosa is a leading cause of healthcare-associated infections in the intensive care unit (ICU). To investigate an unexplained increase in the incidence of P.aeruginosa recovered from clinical samples in the ICU over a two-year period. After unsuccessful epidemiological investigation by conventional tools, P.aeruginosa clinical isolates of all patients hospitalized between January 2010 and July 2012 were typed by a novel double-locus sequence typing (DLST) method and compared to environmental isolates recovered during the investigation period. In total, 509 clinical isolates from 218 patients and 91 environmental isolates were typed. Thirty-five different genotypic clusters were found in 154 out of 218 patients (71%). The largest cluster, DLST 1-18, included 23 patients who were mostly hospitalized during overlapping periods in the burn unit. Genotype DLST 1-18 was also recovered from floor traps, shower trolleys and the shower mattress in the hydrotherapy rooms, suggesting environmental contamination of the burn unit as the source of the outbreak. After implementation of appropriate infection control measures, this genotype was recovered only once in a clinical sample from a burned patient and twice in the environment, but never thereafter during a 12-month follow-up period. The use of a novel DLST method allowed the genotyping of a large number of clinical and environmental isolates, leading to the identification of the environmental source of a large unrecognized outbreak in the burn unit. Eradication of the outbreak was confirmed after implementation of a continuous epidemiological surveillance of P.aeruginosa clones in the ICU.

Hand soap contamination by Pseudomonas aeruginosa in a tertiary care hospital: no evidence of impact on patients

During an environmental investigation of Pseudomonas aeruginosa in intensive care units, the liquid hand soap was found to be highly contaminated (up to 8 × 10(5)cfu/g) with this pathogen. It had been used over the previous five months and was probably contaminated during manufacturing. To evaluate the burden of this contamination on patients by conducting an epidemiological investigation using molecular typing combined with whole genome sequencing (WGS). P. aeruginosa isolates from clinical specimens were analysed by double locus sequence typing (DLST) and compared with isolates recovered from the soap. Medical charts of patients infected with a genotype identical to those found in the soap were reviewed. WGS was performed on soap and patient isolates sharing the same genotype. P. aeruginosa isolates (N = 776) were available in 358/382 patients (93.7%). Only three patients (0.8%) were infected with a genotype found in the soap. Epidemiological investigations showed that the first patient was not exposed to the soap, the second could have been exposed, and the third was indeed exposed. WGS showed a high number of core single nucleotide polymorphism differences between patients and soap isolates. No close genetic association was observed between soap and patient isolates, ruling out the hypothesis of transmission. Despite a highly contaminated soap, the combined investigation with DLST and WGS ruled out any impact on patients. Hand hygiene performed with alcohol-based solution for >15 years was probably the main reason. However, such contamination represents a putative reservoir of pathogens that should be avoided in the hospital setting.

Current Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in Elderly French People: Troublesome Clones on the Horizon.

Objective: In 2015, we conducted at 44 healthcare facilities (HCFs) and 21 nursing homes (NHs) a 3-month bloodstream infection (BSI) survey, and a 1-day prevalence study to determine the rate of carriage of methicillin-resistant Staphylococcus aureus (MRSA) in 891 patients and 470 residents. We investigated the molecular characteristics of the BSI-associated and colonizing MRSA isolates, and assessed cross-transmission using double-locus sequence typing and pulsed-field gel electrophoresis protocol.Results: The incidence of MRSA-BSI was 0.040/1000 patient-days (19 cases). The prevalence of MRSA carriage was 4.2% in patients (n = 39) and 8.7% in residents (n = 41) (p < 0.001). BSI-associated and colonizing isolates were similar: none were PVL-positive; 86.9% belonged to clonal complexes 5 and 8; 93.9% were resistant to fluoroquinolones. The qacA/B gene was carried by 15.8% of the BSI-associated isolates [3/3 BSI cases in intensive care units (ICUs)], and 7.7% of the colonizing isolates in HCFs. Probable resident-to-resident transmission was identified in four NHs.Conclusion: Despite generally reassuring results, we identified two key concerns. First, a worryingly high prevalence of the qacA/B gene in MRSA isolates. Antisepsis measures being crucial to prevent healthcare-associated infections, our findings raise questions about the potential risk associated with chlorhexidine use in qacA/B+ MRSA carriers, particularly in ICUs. Second, NHs are a weak link in MRSA control. MRSA spread was not controlled at several NHs; because of their frequent contact with the community, conditions are favorable for these NHs to serve as reservoirs of USA300 clone for local HCFs.

Evaluation of double locus (clfB and spa) sequence typing for studying molecular epidemiology of methicillin-resistant Staphylococcus aureus in Taiwan.

Pulsed-field gel electrophoresis (PFGE) is the "gold standard" for epidemiological investigation of methicillin-resistant Staphylococcus aureus (MRSA), but several DNA sequence-based methods have been developed in MRSA typing because of the unambiguous results. Ninety-one MRSA isolates were collected from the blood cultures of different patients from July 2008 to December 2008 in central Taiwan. The molecular characteristics of each isolate, including double locus sequence typing (DLST; spa and clfB typing), Staphylococcus cassette chromosome mec (SCCmec), and PFGE were determined for comparison. Five major clfB types (types A-E), 18 spa types, 33 DLST genotypes, five SCCmec types, 17 pulsotypes have been observed. Three major DLST genotypes (A1-t002, C0-t037, and B1-t437) and two major pulsotypes (6 and 8) were identified. Most clfB type A isolates (97.1%) were SCCmec type II and all clfB type C isolates (100%) were SCCmec type III. Most clfB type B isolates (88.9%) were SCCmec type IV (59.3%) and VT (29.6%). All (100%) clfB subtypes A1, A2, and C isolates and 70.4% of clfB type B isolates belonged to healthcare-associated-MRSA. The average congruence was 57.7% between DLST and PFGE, and 96.6% between clfB and SCCmec type. The index of discrimination of SCCmec, clfB, spa, PFGE, and DLST was 0.72, 0.79, 0.80, 0.81, and 0.87, respectively. ClfB type has high congruence with SCCmec type. The DLST method in this study yielded a higher discriminatory power than PFGE in local investigation of molecular epidemiology of MRSA and a promising alternative to PFGE.

Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.

Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.

Comparison of double-locus sequence typing (DLST) and multilocus sequence typing (MLST) for the investigation of Pseudomonas aeruginosa populations

Reliable molecular typing methods are necessary to investigate the epidemiology of bacterial pathogens. Reference methods such as multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) are costly and time consuming. Here, we compared our newly developed double-locus sequence typing (DLST) method for Pseudomonas aeruginosa to MLST and PFGE on a collection of 281 isolates. DLST was as discriminatory as MLST and was able to recognize “high-risk” epidemic clones. Both methods were highly congruent. Not surprisingly, a higher discriminatory power was observed with PFGE. In conclusion, being a simple method (single-strand sequencing of only 2 loci), DLST is valuable as a first-line typing tool for epidemiological investigations of P. aeruginosa. Coupled to a more discriminant method like PFGE or whole genome sequencing, it might represent an efficient typing strategy to investigate or prevent outbreaks.

Double-Locus Sequence Typing UsingporAandpeb1Afor Epidemiological Studies ofCampylobacter jejuni

Campylobacter jejuni is the leading cause of foodborne bacterial gastroenteritis worldwide. Bacterial typing schemes play an important role in epidemiological investigations to trace the source and route of transmission of the infectious agent by identifying outbreak and differentiating among sporadic infections. In this study, a double-locus sequence typing (DLST) scheme for C. jejuni based on concatenated partial sequences of porA and peb1A genes is proposed. The DLST scheme was validated using 50 clinical and environmental C. jejuni strains isolated from human (C5, H, H15-H19), chicken (CH1-CH15), water (W2-W17), and ovine samples (OV1-OV6). The scheme was found to be highly discriminatory (discrimination index [DI]=0.964) and epidemiologically concordant based on C. jejuni strains studied. The DLST showed discriminatory power above 0.95 and excellent congruence to multilocus sequence typing and can be recommended as a rapid and low-cost typing scheme for epidemiological investigation of C. jejuni. It is suggested that the DLST scheme is suitable for identification of outbreak strains and differentiation of the sporadic infection strains.

Fast and simple epidemiological typing of Pseudomonas aeruginosa using the double-locus sequence typing (DLST) method

Although the molecular typing of Pseudomonas aeruginosa is important to understand the local epidemiology of this opportunistic pathogen, it remains challenging. Our aim was to develop a simple typing method based on the sequencing of two highly variable loci. Single-strand sequencing of three highly variable loci (ms172, ms217, and oprD) was performed on a collection of 282 isolates recovered between 1994 and 2007 (from patients and the environment). As expected, the resolution of each locus alone [number of types (NT) = 35-64; index of discrimination (ID) = 0.816-0.964] was lower than the combination of two loci (NT = 78-97; ID = 0.966-0.971). As each pairwise combination of loci gave similar results, we selected the most robust combination with ms172 [reverse; R] and ms217 [R] to constitute the double-locus sequence typing (DLST) scheme for P. aeruginosa. This combination gave: (i) a complete genotype for 276/282 isolates (typability of 98%), (ii) 86 different types, and (iii) an ID of 0.968. Analysis of multiple isolates from the same patients or taps showed that DLST genotypes are generally stable over a period of several months. The high typability, discriminatory power, and ease of use of the proposed DLST scheme makes it a method of choice for local epidemiological analyses of P. aeruginosa. Moreover, the possibility to give unambiguous definition of types allowed to develop an Internet database ( http://www.dlst.org ) accessible by all.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Switzerland: sampling only invasive isolates does not allow a representative description of the local diversity of clones

We conducted a molecular study of MRSA isolated in Swiss hospitals, including the first five consecutive isolates recovered from blood cultures and the first ten isolates recovered from other sites in newly identified carriers. Among 73 MRSA isolates, 44 different double locus sequence typing (DLST) types and 32 spa types were observed. Most isolates belonged to the New York/Japan, the UK-EMRSA-15, the South German and the Berlin clones. In a country with a low to moderate MRSA incidence, inclusion of non-invasive isolates allowed a more accurate description of the diversity.

Role of IncHI2 plasmids harbouring blaVIM-1, blaCTX-M-9, aac(6′)-Ib and qnrA genes in the spread of multiresistant Enterobacter cloacae and Klebsiella pneumoniae strains in different units at Hospital Vall d’Hebron, Barcelona, Spain

Seven Enterobacter cloacae isolates and seven Klebsiella pneumoniae isolates harbouring a phenotype compatible with the production of a metallo-β-lactamase were recovered between 2009 and 2011 in three Intensive Care Units of Hospital Vall d’Hebron (Barcelona, Spain). The presence of blaVIM, blaIMP, blaNDM, blaCTX-M, aac(6′)-Ib, qnrA, qnrB and qnrS genes was screened by polymerase chain reaction (PCR) and sequencing. Clonal relatedness of the isolates was assessed by pulsed-field gel electrophoresis (PFGE) and, in the case of K. pneumoniae isolates, by multilocus sequence typing (MLST). PCR-based replicon typing, Southern hybridisation, plasmid double-locus sequence typing and MOB relaxase classification methods were used to identify and characterise the plasmids carrying the resistance genes. Transferability of the identified plasmids was tested by conjugation assays. All 14 isolates were found to carry blaVIM-1, blaCTX-M-9 (except one isolate), aac(6′)-Ib and qnrA genes. Clonality assessment demonstrated that E. cloacae isolates were distributed in three clonal clusters, whereas all of the K. pneumoniae isolates belonged to one unique clone, identified as sequence type ST252. All studied isolates harboured a large conjugative IncHI2 MOBH11 plasmid carrying all of the detected resistance genes. Plasmid DNA analysis showed that all of them belonged to the ST1 IncHI2 plasmid cluster and shared the same relaxase partial sequence. In conclusion, the present study describes the dissemination within a hospital of multiresistant E. cloacae and K. pneumoniae isolates producing VIM-1. A complex clonal epidemiology of the E. cloacae isolates was observed and plasmid DNA analysis strongly supports horizontal exchanges of the same IncHI2 plasmid between different strains and species.