Wallace Coefficient Research Articles

Assessment of typing methods, virulence genes profile and antimicrobial susceptibility for clinical isolates of Proteus mirabilis

Proteus mirabilis (P. mirabilis) is one of the most important causative pathogens associated with complicated urinary tract infections with a 20% incidence. For epidemiological determinations, several phenotypic and molecular typing methods have been implicated. Sixty P. mirabilis isolated undergo antibiotic susceptibility test by standard Kirby Bauer method. They showed high resistance to nitrofurantoin and trimethoprim/sulfamethoxazole that appear mainly in 3rd age group. The 2nd age group comprised most of the resistant isolates to the tested antibiotics. A total of 73.33% of isolates were classified as multi drug resistance (MDR) and 78.3% of isolates were distributed in several antibiotypes with MAR index over 0.2. Twenty-one isolates were strong biofilm-producers and they were significantly related to MDR. Different virulence factors as protease, urease and hemolysin production are detected. Detection of several virulence genes by PCR; zapA and ureC were harbored by all isolates, followed by rsbA (95%), ureA and flaA (93%), hpmA (91.7%) and mrpA (73.3%). Determination of genetic diversity between isolates was performed by different methods (RAPD, ISSR, ERIC, BOX-AIR and REP-PCR) by using several parameters as typeability and discriminatory power indicating that ERIC-PCR was the best method followed by REP-PCR 1R. Rand’s & Wallace coefficients were used for calculating the congruence among typing methods. Conclusions: The results obtained from both conventional and molecular typing methods indicated that molecular methods are superior to conventional methods in the discrimination of isolates. ERIC-PCR and Rep-PCR provide high discrimination ability among P. mirabilis clinical isolates contributing to epidemiological studies.

Comparison of Three Different Methods in Investigating the Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates

The aim of the study was to evaluate the relationship between carbapenem-resistant Acinetobacter baumannii isolates carrying oxacillinase-type carbapenemase genes with "international high-risk clones" (IC I, II, and III) by different molecular epidemiological methods and to statistically compare the concordance and discrimination power of the methods. Carbapenem-resistant and moderately susceptible A.baumannii isolates from non-repeating blood cultures of 72 patients were included in the study. The presence of "blaOXA-23 , blaOXA-24 , blaOXA-51 ve blaOXA-58 " genes within OXA-type carbapenemases was detected by polymerase chain reaction (PCR) method and confirmed by DNA sequence analysis. Pulsed f ield gel electrophoresis (PFGE), multilocus sequence typing (MLST) and matrix-assisted laser desorption/ ionization time- of-flight mass spectrometry (MALDI-TOF MS) analyses were performed to evaluate the clonal relations of IC I, II and III clones together with clinical isolates. In the statistical comparison of the methods, discrimination power was evaluated by Simpson index of diversity (SID) and concordance by "Wallace coefficient". All of the isolates were found to carry blaOXA-23 and blaOXA-51 genes. As a result of the bioinformatic analysis of the four isolates selected for sequence analysis; blaOXA-23 and blaOXA-51 genes were detected in the selected isolates, and the analysis of two isolates carrying blaOXA-51 gene showed 99% similarity with blaOXA-92 gene. The isolates were clustered into five pulsotypes (A, B, C, D and E) according to ≥ 85% similarity coefficient by PFGE. The isolates and RUH 875, RUH 134, LUH 5875 strains belonging to high-risk clones ICI, ICII and ICIII, respectively, were divided into five main groups [A (n= 58), B (n= 8), C (n= 4), D (n= 4) and E (n= 1)] and 10 subgroups (A1, A2, A4, A5, A6, A9, B1, B4, C3, D1) by PFGE. IC clone III (E1) and seven strains showed singleton PFGE profiles (A3, A7, A8, B2, B3, C1, C2). ICII was found in A5 subtype, ICI in C1 subtype and ICIII in E1 subtype. By PFGE subtype groups, 18 pulsotypes were determined and ST1, ST2, ST81, ST157 and ST604 sequence types were found in 20 isolates randomly selected from pulsotypes according to MLST Pasteur scheme (cpn60, fusA, gltA, pyrG, recA, rplB, rpoB). Principal component analysis (PCA) of the spectra of 72 A. baumannii isolates and ICI, ICII and ICIII clones was performed by MALDI-TOF MS. In PCA analysis, the cluster distance level was defined as 1.5 and the isolates were divided into three clusters. IC clone I, II and III together with 70 clinical isolates were grouped in one cluster, while two clinical isolates (AB083 and AB0115) formed singleton clusters. There was no significant agreement between MALDI-TOF MS; MLST and PFGE data according to Wallace coefficient. It was found that PFGE method gave significant results in terms of discrimination power with SID coefficient, MALDI-TOF MS PCA analysis had the lowest discrimination power value, and the Wallace coefficient result of PFGE and MLST was concordant. In conclusion, MALDI-TOF MS may not function as a gold standard method like PFGE and MLST for epidemiological analysis in A.baumannii species and the epidemiological typing protocols used for MALDI-TOF MS need to be improved and developed.

Subtyping of Campylobacter coli isolated from raw poultry meat in retail markets using amplified intergenic locus polymorphism - A novel rapid subtyping method

In order to provide more phylogenetic information of Campylobacter coli in large-scale epidemiological investigation, this work was undertaken to develop a novel genotyping method based on amplified intergenic locus polymorphism (AILP), by using pulsed-field gel electrophoresis (PFGE; using SmaI enzymes) as control. Eleven pairs of primers were selected to type C. coli strains for this purpose. A total of 68 C. coli isolates recovered from 51 retail raw chicken and 37 retail raw duck were subtyped. The Simpson's index of diversity (SID) of AILP and PFGE, as well as the adjusted Rand index (AR) and the adjusted Wallace coefficient (AW) between AILP and PFGE, were calculated. The new AILP method differentiated 68 C. coli isolates into 55 different subtypes (SID = 0.993), compared with 46 different profiles obtained from PFGE (SID = 0.980). The SID value of the AILP method was improved with the increasing number of primers, and a combination of 7 loci was selected as the optimal combination. The congruent analysis of the AILP method and PFGE showed moderate congruence between the two methods (AR = 0.462). The AW indicated that if AILP data is the available one can confidently predict the PFGE cluster. The results of this study showed that the AILP method had higher discrimination than PFGE and also allowed for significant reduction in time and cost.

Comparison of Shuttleworth–Wallace and Dual Crop Coefficient Method for Estimating Evapotranspiration of a Tea Field in Southeast China

Determination of evaporation (E) and transpiration (T) in tea fields separately is important in developing precise irrigation scheduling and enhancing water use efficiency. In this study, the Shuttleworth–Wallace (S-W) model was applied to simulate the variations of E and T based on the data from 2015 to 2018 in a tea field in southeast China. The dual crop coefficient (D-K) method recommended by FAO-56 was also applied to calculate E and T, using the same data set to compare with the S-W model. The measured crop coefficient (Kc) ranged from 0.43 to 1.44 with the average value was 0.90 during 1–150 DOY (days of year), and the measured Kc tended to be stable with the average value of 0.83 during 151–365 DOY in 2015. The S-W model estimated ETc with root mean square error (RMSE) and R2 of 0.45 mm d−1 and 0.97, while for the D-K method the values were 0.61 mm d−1 and 0.95. Therefore, both approaches could estimate the E and T separately in tea fields in southeast China, however, the D-K method had a slightly poorer accuracy compared to the S-W model in the estimation of ETc.

Evaluation of Fourier Transform Infrared Spectroscopy as a First-Line Typing Tool for the Identification of Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae Outbreaks in the Hospital Setting.

Early detection of pathogen cross-transmission events and environmental reservoirs is needed to control derived nosocomial outbreaks. Whole-genome sequencing (WGS) is considered the gold standard for outbreak confirmation, but, in most cases, it is time-consuming and has elevated costs. Consequently, the timely incorporation of WGS results to conventional epidemiology (CE) investigations for rapid outbreak detection is scarce. Fourier transform infrared spectroscopy (FTIR) is a rapid technique that establishes similarity among bacteria based on the comparison of infrared light absorption patterns of bacterial polysaccharides and has been used as a typing tool in recent studies. The aim of the present study was to evaluate the performance of the FTIR as a first-line typing tool for the identification of extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL-Kp) outbreaks in the hospital setting in comparison with CE investigations using WGS as the gold standard method. Sixty-three isolates of ESBL-Kp collected from 2018 to 2021 and classified according to CE were typed by both FTIR and WGS. Concordance was measured using the Adjusted Rand index (AR) and the Adjusted Wallace coefficient (AW) for both CE and FTIR clustering considering WGS as the reference method. Both AR and AW were significantly higher for FTIR clustering than CE clustering (0.475 vs. 0.134, p = 0.01, and 0.521 vs. 0.134, p = 0.009, respectively). Accordingly, FTIR inferred more true clustering relationships than CE (38/42 vs. 24/42, p = 0.001). However, a similar proportion of genomic singletons was detected by both FTIR and CE (13/21 vs. 12/21, p = 1). This study demonstrates the utility of the FTIR method as a quick, low-cost, first-line tool for the detection of ESBL-Kp outbreaks, while WGS analyses are being performed for outbreak confirmation and isolate characterization. Thus, clinical microbiology laboratories would benefit from integrating the FTIR method into CE investigations for infection control measures in the hospital setting.

Investigating Major Recurring Campylobacter jejuni Lineages in Luxembourg Using Four Core or Whole Genome Sequencing Typing Schemes.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis, which has motivated the monitoring of genetic profiles circulating in Luxembourg since 13 years. From our integrated surveillance using a genotyping strategy based on an extended MLST scheme including gyrA and porA markers, an unexpected endemic pattern was discovered in the temporal distribution of genotypes. We aimed to test the hypothesis of stable lineages occurrence by implementing whole genome sequencing (WGS) associated with comprehensive and internationally validated schemes. This pilot study assessed four WGS-based typing schemes to classify a panel of 108 strains previously identified as recurrent or sporadic profiles using this in-house typing system. The strain collection included four common lineages in human infection (N = 67) initially identified from recurrent combination of ST-gyrA-porA alleles also detected in non-human samples: veterinary (N = 19), food (N = 20), and environmental (N = 2) sources. An additional set of 19 strains belonging to sporadic profiles completed the tested panel. All the strains were processed by WGS by using Illumina technologies and by applying stringent criteria for filtering sequencing data; we ensure robustness in our genomic comparison. Four typing schemes were applied to classify the strains: (i) the cgMLST SeqSphere+ scheme of 637 loci, (ii) the cgMLST Oxford scheme of 1,343 loci, (iii) the cgMLST INNUENDO scheme of 678 loci, and (iv) the wgMLST INNUENDO scheme of 2,795 loci. A high concordance between the typing schemes was determined by comparing the calculated adjusted Wallace coefficients. After quality control and analyses with these four typing schemes, 60 strains were confirmed as members of the four recurrent lineages regardless of the method used (N = 32, 12, 7, and 9, respectively). Our results indicate that, regardless of the typing scheme used, epidemic or endemic signals were detected as reflected by lineage B (ST2254-gyrA9-porA1) in 2014 or lineage A (ST19-gyrA8-porA7), respectively. These findings support the clonal expansion of stable genomes in Campylobacter population exhibiting a multi-host profile and accounting for the majority of clinical strains isolated over a decade. Such recurring genotypes suggest persistence in reservoirs, sources or environment, emphasizing the need to investigate their survival strategy in greater depth.

Epidemiology and Risk Factors for Carbapenem-Resistant Klebsiella Pneumoniae and Subsequent MALDI-TOF MS as a Tool to Cluster KPC-2-Producing Klebsiella Pneumoniae, a Retrospective Study.

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) appeared recently and now presents a particularly critical problem to hospitalized patients worldwide. We aim to investigate the epidemiology and the risk factors for CRKP colonization and infections, and to evaluate the application performance of MALDI-TOF MS in clustering CRKP.Results: CRKP colonization and infections incidence was 2.7 (35/1,319,427) per 100,000 patient-days. Inpatients in CRKP group had higher medical expense than CSKP group. Inpatients with underlying conditions, particularly with pulmonary diseases, and with antimicrobial use prior to culture within 30 days, especially with carbapenem use, were risk factors for CRKP acquisition. All CRKP isolates were detected producing KPC-2. The MALDI-TOF MS system and PFGE system provided similar results, with a good concordance between the two methods (adjusted Rand's coefficient, 0.846) and a high probability of MALDI-TOF MS to predict PFGE results (Wallace coefficient, 0.908).Conclusions: Underlying conditions, particularly pulmonary diseases, and antimicrobial use prior to culture within 30 days, especially carbapenem use, are risk factors for CRKP acquisition. BlaKPC−2 is the mainstream gene of CRKP in our geographic area of analysis. As only simple sample preparation is needed and the results can be obtained in a short time, MALDI-TOF MS may be considered a probable alternative to PFGE in clustering KPC-2-producing CRKP.

Fourier-Transform InfraRed Spectroscopy Can Quickly Type Gram-Negative Bacilli Responsible for Hospital Outbreaks.

The typing of epidemic bacterial pathogens in hospitals relies on DNA-based, expensive, and time-consuming techniques, that are often limited to retrospective studies. However, the quick identification of epidemic pathogens in the routine of the microbiology laboratories would expedite infection control procedures that limit the contamination of new patients. IR Biotyper (Bruker Daltonics GmbH) is a new typing machine based on Fourier-transform infrared (FTIR) spectroscopy which generates spectra, aiming at typing the micro-organisms within 3 h. This technique discriminates the isolates by exploring the differences of the surface cell polysaccharides. In this work, we evaluated the ability of the FTIR spectroscopy to recognize Gram-negative bacilli clones responsible for hospital outbreaks. Isolates of Pseudomonas aeruginosa (n = 100), Klebsiella pneumoniae (n = 16), Enterobacter cloacae (n = 23), and Acinetobacter baumannii (n = 20) were typed by the reference methods Multi-Locus Sequence Typing (defining sequence types – STs) along with or without pulsed field gel electrophoresis (PFGE) (defining pulsotypes), and by FTIR spectroscopy. The congruence of FTIR spectroscopy clustering was compared to those of MLST and PFGE by Adjusted Rand index and Adjusted Wallace coefficient. We found that FTIR spectroscopy accurately clustered P. aeruginosa, K. pneumoniae, and E. cloacae isolates belonging to the same ST. The performance of the FTIR spectroscopy was slightly lower for A. baumannii. Furthermore, FTIR spectroscopy also correctly clustered P. aeruginosa isolates having a similar pulsotype. Overall, the IR Biotyper can quickly (in less than 3 h) detect the spread of clones of P. aeruginosa, K. pneumoniae, E. cloacae, and A. baumannii. The use of this technique by clinical microbiology laboratories may help to tackle the spread of epidemic clones by the quick implementation of infection control measures.

Comparison of biochemical and genotypic speciation methods for vancomycin-resistant enterococci isolated from urban wastewater treatment plants

Enterococci species in wastewater including Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus and Enterococcus gallinarum isolates (n = 308) with low or high level vancomycin resistance were determined and compared using a phenotypic method (RapID™ STR system), 16S rRNA sequencing, and multi-locus (atpA, groESL, and pheS) sequence analysis (MLSA). Error rates for the RapID™ STR system were E. faecalis (15.9%), E. faecium (21.5%), and E. casseliflavus/E. gallinarum (56.9%) when referenced to the consensus of all methods tested. Comparison of single nucleotide polymorphism (SNP) distances and phylogenetic trees suggested that the groESL locus delineated species more effectively than other loci. The groESL locus was the most reliable loci for the correct identification of Enterococcus spp., including E. casseliflavus and E. gallinarum, with high congruence compared to the consensus (Adjusted Rand Index = 0.954; Adjusted Wallace Co-efficient = 0.941). All of the methods were compared to whole genome sequencing, which acted as a gold standard, for the isolates from this study and those downloaded from NCBI.

Comparison of Shuttleworth-Wallace model and dual crop coefficient method for estimating evapotranspiration of tomato cultivated in a solar greenhouse

The accurate determination of crop evapotranspiration (ETc) is important for improving irrigation water use efficiency and optimizing fruit quality in solar greenhouses. Taking drip-irrigated tomato in a solar greenhouse as an example, this study simulated the variations of ETc by the Shuttleworth–Wallace (S–W) and Dual crop coefficient (Dual-Kc) models at initial, development, mid and late stages in 2015 and 2016 respectively. Continuous measurements of crop ETc with weighing lysimeter, and sap flow system combined with micro-lysimeter were used to validate the performance of two approaches. Results indicated that the total soil evaporation accounted for 25% of ETc over the whole growth period, and decreased gradually with the increasing of canopy coverage ratio. The locally developed basal crop coefficients values during the initial, mid, and late stages were 0.15, 0.94 and 0.65 in 2015, and 0.15, 1.02 and 0.70 in 2016, respectively. The variations of the estimated ETc from S–W and Dual-Kc models were similar to the measured ETc. The S–W model performed well in estimating ETc when LAI was between 0.5 and 2.7, but slightly overestimated and underestimated at initial and mid stages, respectively. Additionally, the performance of the S–W model was also better under different weather conditions as well as after irrigation. However, the Dual-Kc method was superior to the S–W model in estimating daily soil evaporation and daily plant transpiration. The good agreements were found between the predicted ETc using the Dual-Kc method and the measurements for the greenhouse tomato, with the coefficients of regression of 1.03 (R2=0.93) in 2015, and 0.96 (R2=0.93) in 2016. Therefore, the Dual-Kc method was finally recommended for estimating evapotranspiration of tomato cultivated in the solar greenhouse.

Orthogonal typing methods identify genetic diversity among Belgian Campylobacter jejuni strains isolated over a decade from poultry and cases of sporadic human illness

Campylobacter jejuni is a zoonotic pathogen commonly associated with human gastroenteritis. Retail poultry meat is a major food-related transmission source of C. jejuni to humans. The present study investigated the genetic diversity, clonal relationship, and strain risk-analysis of 403 representative C. jejuni isolates from chicken broilers (n = 204) and sporadic cases of human diarrhea (n = 199) over a decade (2006–2015) in Belgium, using multilocus sequence typing (MLST), PCR binary typing (P-BIT), and identification of lipooligosaccharide (LOS) biosynthesis locus classes. A total of 123 distinct sequence types (STs), clustered in 28 clonal complexes (CCs) were assigned, including ten novel sequence types that were not previously documented in the international database. Sequence types ST-48, ST-21, ST-50, ST-45, ST-464, ST-2274, ST-572, ST-19, ST-257 and ST-42 were the most prevalent. Clonal complex 21 was the main clonal complex in isolates from humans and chickens. Among observed STs, a total of 35 STs that represent 72.2% (291/403) of the isolates were identified in both chicken and human isolates confirming considerable epidemiological relatedness; these 35 STs also clustered together in the most prevalent CCs. A majority of the isolates harbored sialylated LOS loci associated with potential neuropathic outcomes in humans. Although the concordance between MLST and P-BIT, determined by the adjusted Rand and Wallace coefficients, showed low congruence between both typing methods. The discriminatory power of P-BIT and MLST was similar, with Simpson's diversity indexes of 0.978 and 0.975, respectively. Furthermore, P-BIT could provide additional epidemiological information that would provide further insights regarding the potential association to human health from each strain. In addition, certain clones could be linked to specific clinical symptoms. Indeed, LOS class E was associated with less severe infections. Moreover, ST-572 was significantly associated with clinical infections occurring after travelling abroad. Ultimately, the data generated from this study will help to better understand the molecular epidemiology of C. jejuni infection.

Molecular epidemiology of Neisseria gonorrhoeae strains circulating in Indonesia using multi-locus variable number tandem repeat analysis (MLVA) and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) techniques

BackgroundControl of gonorrhea in resource-limited countries, such as Indonesia, is mostly unsuccessful. Examining Neisseria gonorrhoeae (Ng) transmission networks using strain typing might help prioritizing public health interventions.MethodsIn 2014, urogenital Ng strains were isolated from clients of sexually transmitted infection clinics in three Indonesian cities. Strains were typed using Multiple-Locus Variable Number Tandem Repeat (VNTR) Analysis (MLVA) and Ng Multi-Antigen Sequence Typing (NG-MAST) at the Public Health Service, Amsterdam, the Netherlands, and compared to Dutch strains collected from 2012 to 2015. Minimum spanning trees (MSTs) were constructed using MLVA profiles incorporating demographics and NG-MAST genogroups. A cluster was defined as ≥5 strains differing in ≤1 VNTR locus. The concordance between MLVA and NG-MAST was examined with the adjusted Wallace coefficients (AW).ResultsWe collected a total of 78 Indonesian strains from Yogyakarta (n = 44), Jakarta (n = 25), and Denpasar (n = 9). Seven MLVA clusters and 16 non-clustered strains were identified. No cluster was specific for any geographic area, risk group or age group.Combined with 119 contemporary Dutch strains, 8 MLVA clusters were identified, being four clusters of Indonesian strains, two of Dutch strains, and two of both Indonesian and Dutch strains. Indonesian strains (79.5%) were more often clustered compared to Dutch strains (24.3%).The most prevalent NG-MAST genogroups among Indonesian strains was G1407 (51.3%) and among Dutch strains was G2992 (19.3%). In Indonesian strains, the AW [95% confidence interval] for MLVA to NG-MAST was 0.07 [0.00–0.27] and for NG-MAST to MLVA was 0.03 [0.00–0.12].ConclusionIndonesian Ng strains are more often clustered than Dutch strains, but show no relation with geographical area, risk group, or age group, suggesting a more clonal Ng epidemic in Indonesia. Some similar Ng strains circulate in both Indonesia and the Netherlands.

MLVA subtyping of Listeria monocytogenes isolates from meat products and meat processing plants

Listeria monocytogenes is widely distributed in meat products and the meat-processing industry thus posing a risk to consumers. The aim of this study was to evaluate the suitability of the multilocus variable-number tandem-repeat analysis (MLVA) for use as a L. monocytogenes subtyping technique for surveillance and routine control in meat products and meat processing plants. A collection of 113 isolates (including control strains and isolates from meat products and meat processing plants) were subject to MLVA analysis using two different platforms for fragment sizing: 1.) ABI 3730xl DNA analyzer (Life Technologies) as the reference method and 2.) The QIAxcel Advanced System (Qiagen). Although discrepancies in fragment sizing were observed it was possible to standardize results in order to assign the same allele for a given fragment independently of the platform used for fragment sizing. A total of 27 different MLVA profiles were obtained considering all the isolates (N=113), 24 of them corresponding to the meat industry isolates (N=106). MLVA and multilocus sequence typing (MLST) results were compared and yielded Simpson's diversity indices of 0.907 and 0.872, respectively. The congruence between both typing methods was measured with the adjusted Wallace coefficient (AW). Using MLVA as the primary method, AW=0.946 suggested that MLVA can predict the sequence type with high accuracy. Given its discriminatory power and high throughput, MLVA could be considered a rapid, reliable, and high-throughput alternative to existing subtyping methods for surveillance and control of L. monocytogenes in the meat-processing industry.

C-source metabolic profilings of foodborne Shiga-toxin producing E. coli match serogroup differentiations and highlight functional adaptations

The tropism of pathogenic STEC for foodstuffs and cattle reservoir is related to functional specializations. An investigation of C-source utilization patterns among and between STEC serogroups was performed using omnilog phenotypic microarrays (OM). OM functional groupings were compared with STEC phylogroups, seropathotypes, EFSA's molecular risk assessment groups and serogroups. OM INT reduction activities of 37 STEC strains growing on 190 C-substrates were compared. Each strain had its own specific C-utilization profile but 23% of the substrates was used by all strains, 47% by none, and 30% was variably metabolized. Galactose, mannose, N-acetyl-glucosamine (GlcNAc), and N-acetyl neuraminic acid (Neu5Ac) found in the mucus layer of the bovine small intestine were metabolized by all strains. The 56 most informative substrates divided the C-utilization patterns (CP) into three clusters with: (A) harboring all O157 and O145 strains; (B) all O26 strains, and (C) strains of the other serogroups. Significant correlations between INT reduction values of pair of strains per CP group supported these differentiations. CP of group A and B strains were respectively defective in the use of galactonic acid-γ-lactone and rhamnose. Most CP group C strains grew with l-lyxose. Adjusted Wallace coefficients analyses of the datasets indicated high probabilities for the prediction of the use of glycolic acid, β-hydroxybutyric acid, l-lyxose and d-galactonic acid-γ-lactone and 5-keto-d-gluconic acid by a serogroup. The use of a C-substrate could be predicted from the classification of a strain into a phylogroup or seropathotype. Significantly lower numbers of C-substrates were used by seropathotype A strains like O157 ones. Improvements of STEC identification keys were proposed using the most discriminant C-substrates found in this study.

Characterization of Multidrug Resistant E. faecalis Strains from Pigs of Local Origin by ADSRRS-Fingerprinting and MALDI -TOF MS; Evaluation of the Compatibility of Methods Employed for Multidrug Resistance Analysis.

The aim of this study was to characterize multidrug resistant E. faecalis strains from pigs of local origin and to analyse the relationship between resistance and genotypic and proteomic profiles by amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI -TOF MS). From the total pool of Enterococcus spp. isolated from 90 pigs, we selected 36 multidrug resistant E. faecalis strains, which represented three different phenotypic resistance profiles. Phenotypic resistance to tetracycline, macrolides, phenicols, and lincomycin and high-level resistance to aminoglycosides were confirmed by the occurrence of at least one corresponding resistance gene in each strain. Based on the analysis of the genotypic and phenotypic resistance of the strains tested, five distinct resistance profiles were generated. As a complement of this analysis, profiles of virulence genes were determined and these profiles corresponded to the phenotypic resistance profiles. The demonstration of resistance to a wide panel of antimicrobials by the strains tested in this study indicates the need of typing to determine the spread of resistance also at the local level. It seems that in the case of E. faecalis, type and scope of resistance strongly determines the genotypic pattern obtained with the ADSRRS-fingerprinting method. The ADSRRS-fingerprinting analysis showed consistency of the genetic profiles with the resistance profiles, while analysis of data with the use of the MALDI- TOF MS method did not demonstrate direct reproduction of the clustering pattern obtained with this method. Our observations were confirmed by statistical analysis (Simpson’s index of diversity, Rand and Wallace coefficients). Even though the MALDI -TOF MS method showed slightly higher discrimination power than ADSRRS-fingerprinting, only the latter method allowed reproduction of the clustering pattern of isolates based on phenotypic resistance and analysis of resistance and virulence genes (Wallace coefficient 1.0). This feature seems to be the most useful for epidemiological purposes and short-term analysis.

Discriminative power of Campylobacter phenotypic and genotypic typing methods

The aim of this study was to compare different typing methods, individually and combined, for use in the monitoring of Campylobacter in food. Campylobacter jejuni (n=94) and Campylobacter coli (n=52) isolated from different broiler meat carcasses were characterized using multilocus sequence typing (MLST), flagellin gene A restriction fragment length polymorphism typing (flaA-RFLP), antimicrobial resistance profiling (AMRp), the presence/absence of 5 putative virulence genes; and, exclusively for C. jejuni, the determination of lipooligosaccharide (LOS) class. Discriminatory power was calculated by the Simpson's index of diversity (SID) and the congruence was measured by the adjusted Rand index and adjusted Wallace coefficient. MLST was individually the most discriminative typing method for both C. jejuni (SID=0.981) and C. coli (SID=0.957). The most discriminative combination with a SID of 0.992 for both C. jejuni and C. coli was obtained by combining MLST with flaA-RFLP. The combination of MLST with flaA-RFLP is an easy and feasible typing method for short-term monitoring of Campylobacter in broiler meat carcass.

A Novel Molecular Strategy for Surveillance of Multidrug Resistant Tuberculosis in High Burden Settings.

BackgroundIn South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR.MethodsA total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discriminatory Indexes (HGI) and Wallace coefficients were compared between the methods.ResultsOverall clustering rates were high by both IS6110-RFLP (52.8%) and MIRU-VNTR (45.8%), indicative of on-going transmission. Both 24-loci MIRU-VNTR and IS6110-RFLP had similar HGI (0.972 and 0.973, respectively), with close numbers of unique profiles (87 vs. 70), clustered isolates (129 vs. 146), and cluster sizes (2 to 26 vs. 2 to 25 isolates). Spoligotyping alone was the least discriminatory (80.1% clustering, HGI 0.903), with 28 unique types. However, the discriminatory power of spoligotyping was improved when combined with pncA sequencing using the SpoNC approach (61.8% clustering, HGI 0.958). A high proportion of MDR-TB isolates had mutations in pncA (68%, n = 145), and pncA mutations were significantly associated with clustering (p = 0.007 and p = 0.0013 by 24-loci MIRU-VNTR and IS6110-RFLP, respectively), suggesting high rates of resistance to pyrazinamide among all MDR-TB cases and particularly among clustered cases.ConclusionWe conclude that SpoNC provides good discrimination for MDR-TB surveillance and early identification of outbreaks in South Africa, with 24-loci MIRU-VNTR applied for pncA wild-type strains as needed.

Comparison of RAPD-PCR and PFGE analysis for the typing of Streptococcus thermophilus strains isolated from traditional Turkish yogurts

The dairy industry is constantly developing new starters with specific traits. Therefore, strains from natural sources have huge potential for exploration and characterization. The aim of this work was to identify the most efficient, useful, and reliable option for typing 55 Streptococcus thermophilus isolates from traditional Turkish yogurts and four industrial strains. Then, we statistically evaluated whether our conclusions could be extended to the true population. For this purpose, the results of the rapid amplified polymorphic DNA polymerase chain reaction (RAPD-PCR; using the OPI-02 MOD, M13, and XD9 primers) and pulsed-field gel electrophoresis (PFGE; using the SmaI and ApaI enzymes) typing methods were compared. The discriminatory power, typeability, and reproducibility were analyzed. Additionally, the congruence between typing methods was quantified using the adjusted Rand index (AR), Wallace index (W), and expected Wallace coefficient. Both methods revealed high genetic diversity of the S. thermophilus strains, even in the same yogurt sample. The numerical combination of results for these primers or restriction enzymes increased the congruence between the methods and provided more complete information on the strains. The comparison of these two options showed that using SmaI with ApaI was more advisable and useful than using OPI-02 MOD with M13. Additionally, the first combination represented the best tool to discriminate S. thermophilus strains (Simpson’s index of diversity [DI] of 0.999 [0.997–1.000]). This finding was statistically supported. The RAPD (OPI-02 MOD) typing result showed an ability to confidently predict the PFGE (SmaI, ApaI) type (AR = 0.782 [0.618–0.949], W = 0.946 [0.865–1.000]). This result had some statistical support and might represent an important application in the dairy industry for screening strains.

Clustered Regularly Interspaced Short Palindromic Repeats Are emm Type-Specific in Highly Prevalent Group A Streptococci.

Clustered regularly interspaced short palindromic repeats (CRISPR) are the bacterial adaptive immune system against foreign nucleic acids. Given the variable nature of CRISPR, it could be a good marker for molecular epidemiology. Group A streptococcus is one of the major human pathogens. It has two CRISPR loci, including CRISPR01 and CRISPR02. The aim of this study was to analyze the distribution of CRISPR-associated gene cassettes (cas) and CRISPR arrays in highly prevalent emm types. The cas cassette and CRISPR array in two CRISPR loci were analyzed in a total of 332 strains, including emm1, emm3, emm4, emm12, and emm28 strains. The CRISPR type was defined by the spacer content of each CRISPR array. All strains had at least one cas cassette or CRISPR array. More than 90% of the spacers were found in one emm type, specifically. Comparing the consistency between emm and CRISPR types by Simpson’s index of diversity and the adjusted Wallace coefficient, CRISPR01 type was concordant to emm type, and CRISPR02 showed unidirectional congruence to emm type, suggesting that at least for the majority of isolates causing infection in high income countries, the emm type can be inferred from CRISPR analysis, which can further discriminate isolates sharing the same emm type.

Typing of Campylobacter jejuni Isolated from Turkey by Genotypic Methods, Antimicrobial Susceptibility, and Virulence Gene Patterns: A Retrospective Study.

In this retrospective study, typing ability, discriminatory power, and concordance between typing results obtained on 123 Campylobacter jejuni turkey isolates, collected in 1998, within 14 different farms, applying multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), antibiotic resistance profile, and virulence gene pattern, were assessed and compared. Overall, 33 sequence types, 28 pulsotypes, 10 resistotypes, and 5 pathotypes were identified. MLST and PFGE showed the better discriminatory ability (i.e., Simpson's diversity index >0.90) as well as unidirectional (i.e., Wallace and adjusted Wallace coefficients >0.86) and bidirectional (i.e., adjusted Rand coefficient >0.60) concordance. Moreover, both methods showed a good unidirectional and bidirectional concordance with the resistotype. On the contrary, the congruence of both genotyping methods and resistotype with the pathotype seemed due to chance alone. A clonal relationship was identified among 66.7% of the isolates. Furthermore, 59.7% of the investigated isolates were resistant to two or more antimicrobials and 92% to tetracycline. All the isolates harbored cadF and pldA genes, whereas a flaA gene product and a cdtB gene product were amplified from 85.4% and 79.7% of the isolates, respectively, using the primers designed by Bang et al. (2003). The results of this study clarify the level of genetic diversity among the C. jejuni originating from turkeys. MLST level of correlation with PFGE, resistotype, and pathotype is assessed. This result supports the selection of type and number of typing methods to use in epidemiological studies. Finally, the identification of clonal complexes (i.e., groups of profiles differing by no more than one gene from at least one other profile of the group using the entire Campylobacter MLST database) shared between turkey and human isolates suggests that turkeys could be a possible source of Campylobacter infection.