MLVA Research Articles

Shedding and genetic diversity of Coxiella burnetii in Polish dairy cattle.

Q fever is a worldwide zoonotic disease reported in humans and many animal species including cattle. The aims of this study were to evaluate the prevalence of Coxiella (C.) burnetii shedding in Polish dairy cattle herds and to identify the pathogen’s genotypes and sequence types (STs) using multiple-locus variable number tandem repeat analysis (MLVA) and multispacer sequence typing (MST) methods. The presence of C. burnetii DNA was detected using a commercial real-time PCR kit, targeting the IS1111 element. Overall, 1,439 samples from 279 herds were tested including: 897 individual milk specimens, 101 bulk tank milk samples, 409 genital tract swabs and 32 placentas. Furthermore, 30 consumer milk samples, including 10 from vending machines and 77 dairy products were also analyzed. C. burnetii shedding was confirmed in 31.54% of tested cattle herds as well as in 69.16% of consumer milk and dairy products. Among real-time PCR–positive samples, 49 specimens obtained from 49 cattle herds and 8 samples of purchased dairy products were selected for genotyping. Overall, five previously known MLVA genotypes (I, J, BG, BE, and NM) and three new ones (proposed as PL1, PL2, and PL3) were identified. Two MST sequence types were recorded: ST16 and a novel sequence (ST61). The new genotypes and sequence types need further research particularly into their pathogenicity to humans.

Molecular Epidemiology of Bordetella pertussis.

Although vaccination has been effective, Bordetella pertussis is increasingly causing epidemics, especially in industrialized countries using acellular vaccines (aPs). One factor behind the increased circulation is the molecular changes on the pathogen level. After pertussis vaccinations were introduced, changes in the fimbrial (Fim) serotype of the circulating strains was observed. When bacterial typing methods improved, further changes between the vaccine and circulating strains, especially among the common virulence genes including pertussis toxin (PT) and pertactin (PRN) were noticed. Moreover, development of genome based techniques including pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA) and whole-genome sequencing (WGS) have offered a better resolution to monitor B. pertussis strains. After the introduction of aP vaccines, B. pertussis strains that are deficient to vaccine antigens, especially PRN, have appeared widely. On the other hand, antimicrobial resistance to first line drugs (macrolides) against B. pertussis is still low in many countries and therefore no globally evaluated antimicrobial susceptibility test values have been recommended. In this review, we focus on the molecular changes in the bacteria, which have or may have affected the past and current epidemiology of pertussis.

Brucella melitensis caused abortion in a serologically positive dromedary camel

An abortion case in a dromedary camel caused by a B. melitensis strain belonging to the East-Mediterranean genetic group (GT42 by multiple-locus variable-number tandem-repeat analysis (MLVA8) and GT108 by MLVA11) is reported. Earlier, the dromedary was seronegative for Brucellosis for 6 years. The animal got infected between 112 and 204 days of gestation from an unknown source, developed bacteriaemia and the acute infection resulted in the abortion at 249 days of gestation. The pathological findings in the foetus and the placenta were not pathognomonic for Brucellosis. In the foetus, both pleural and abdominal cavities were filled with bloody fluid and pleurisy was observed. In the placenta, oedema, diffuse mineralisation and focal detachment of the trophoblast were detected without inflammatory changes. The Brucella pathogens were excreted to the environment with the aborted foetus and the placenta. The bacteria disappeared from the blood and the uterus of the dam within a short time after abortion and were isolated only from some of the lymph nodes. The Brucella seropositive camel in its acute phase of the disease did not infect other contact animals. In addition, the transmission of the disease to other dromedaries at the time of abortion was also prevented with appropriate biosecurity measures.

Evaluation of whole-genome sequencing for outbreak detection of Verotoxigenic Escherichia coli O157:H7 from the Canadian perspective

BackgroundRapid and accurate identification of Verotoxigenic Escherichia coli (VTEC) O157:H7 is dependent on well-established, standardized and highly discriminatory typing methods. Currently, conventional subtyping tests for foodborne bacterial pathogen surveillance are rapidly being replaced with whole-genome sequencing (WGS) in public health laboratories. The capacity of WGS to revolutionize global foodborne disease surveillance has positioned this tool to become the new gold standard; however, to ensure evidence standards for public health decision making can still be achieved, the performance of WGS must be thoroughly validated against current gold standard methods prior to implementation. Here we aim to verify the performance of WGS in comparison to pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) for eight retrospective outbreaks of VTEC O157:H7 from the Canadian perspective. Since real-time implementation and routine use of WGS in public health laboratories is highly reliant on standardized data analysis tools, we also provide a comparative analysis of two popular methodologies for WGS analyses; an in-house developed single nucleotide variant phylogenomics (SNVPhyl) pipeline and the BioNumerics whole genome multilocus sequence typing (wgMLST) tool. To provide a useful and consistent starting point for examining laboratory-based surveillance data for VTEC O157:H7 in Canada, we also aim to describe the number of genetic differences observed among outbreak-associated isolates.ResultsWGS provided enhanced resolution over traditional subtyping methods, and accurately distinguished outbreak-related isolates from non-outbreak related isolates with high epidemiological concordance. WGS also illuminated potential linkages between sporadic cases of illness and contaminated food, and isolates spanning multiple years. The topologies generated by SNVPhyl and wgMLST were highly congruent with strong statistical support. Few genetic differences were observed among outbreak-related isolates (≤5 SNVs/ < 10 wgMLST alleles) unless the outbreak was suspected to be multi-strain.ConclusionsThis study validates the superiority of WGS and indicates the BioNumerics wgMLST schema is suitable for surveillance and cluster detection of VTEC O157:H7. These findings will provide a useful and consistent starting point for examining WGS data for prospective laboratory-based surveillance of VTEC O157:H7, but however, the data will continue to be interpreted according to context and in combination with epidemiological and food safety evidence to inform public-health decision making in Canada.

Capsular Type, Sequence Type and Microbial Resistance Factors Impact on DNase Activity of Streptococcus agalactiae Strains from Human and Bovine Origin.

Extracellular deoxyribonucleases (DNases) contribute to the spread of pathogenic bacteria through the evasion from host innate immunity. Our main objective was to evaluate the production of extracellular DNases by human and bovine Streptococcus agalactiae clinical strains and perform a correlation of genetic lineages and DNase activity with capsular type, genetic determinants, clinical origin (colonization and infection), and host (human or bovine). DNase activity was evaluated by qualitative and quantitative assays for a collection of 406 human (n = 285) and bovine (n = 121) strains. All (121/121) bovine were isolated from mastitis and revealed to be DNase (+), indicating a putative pathogenic role in this clinical scenario. From the human S. agalactiae strains, 86% (245/285) showed DNase activity, among which all strains belonging to capsular types, namely, Ia, Ib, III-2, and IV. All CC17 strains (n = 58) and 56/96 (58.3%) of the CC19 displayed DNase activity. DNase (–) strains belonged to the CC19 group. However, the subcharacterization of CC19 S. agalactiae strains through multiple-locus variable number tandem repeat analysis (MLVA), antibiotic resistance, mobile elements, and surface proteins did not provide any distinction among DNase producers and non-producers.The production of DNases by all human CC17 strains, about two-fifths of human CC19, and all bovine strains, suggest an important contribution of DNases to hypervirulence.

High frequency of hybrid Escherichia coli strains with combined Intestinal Pathogenic Escherichia coli (IPEC) and Extraintestinal Pathogenic Escherichia coli (ExPEC) virulence factors isolated from human faecal samples

BackgroundClassification of pathogenic Escherichia coli (E. coli) has traditionally relied on detecting specific virulence associated genes (VAGs) or combinations thereof. For E. coli isolated from faecal samples, the presence of specific genes associated with different intestinal pathogenic pathovars will determine their classification and further course of action. However, the E. coli genome is not a static entity, and hybrid strains are emerging that cross the pathovar definitions. Hybrid strains may show gene contents previously associated with several distinct pathovars making the correct diagnostic classification difficult. We extended the analysis of routinely submitted faecal isolates to include known virulence associated genes that are usually not examined in faecal isolates to detect the frequency of possible hybrid strains.MethodsFrom September 2012 to February 2013, 168 faecal isolates of E. coli routinely submitted to the Norwegian Institute of Public Health (NIPH) from clinical microbiological laboratories throughout Norway were analysed for 33 VAGs using multiplex-PCR, including factors associated with extraintestinal pathogenic E. coli (ExPEC) strains. The strains were further typed by Multiple Locus Variable-Number Tandem-Repeat Analysis (MLVA), and the phylogenetic grouping was determined. One isolate from the study was selected for whole genome sequencing (WGS) with a combination of Oxford Nanopore’s MinION and Illumina’s MiSeq.ResultsThe analysis showed a surprisingly high number of strains carrying ExPEC associated VAGs and strains carrying a combination of both intestinal pathogenic E. coli (IPEC) and ExPEC VAGs. In particular, 93.5% (101/108) of isolates classified as belonging to an IPEC pathovar additionally carried ExPEC VAGs. WGS analysis of a selected hybrid strain revealed that it could, with present classification criteria, be classified as belonging to all of the Enteropathogenic Escherichia coli (EPEC), Uropathogenic Escherichia coli (UPEC), Neonatal meningitis Escherichia coli (NMEC) and Avian pathogenic Escherichia coli (APEC) pathovars.ConclusionHybrid ExPEC/IPEC E. coli strains were found at a very high frequency in faecal samples and were in fact the predominant species present. A sequenced hybrid isolate was confirmed to be a cross-pathovar strain possessing recognised hallmarks of several pathovars, and a genome heavily influenced by horizontal gene transfer.

Molecular Investigation of the Transmission Pattern of Brucella suis 3 From Inner Mongolia, China.

Brucellosis is an endemic disease in China affecting both humans and livestock. The aim of the present study was to analyze two Brucella strains isolated from sheep spleens from Ulanqab in Inner Mongolia, China using classical and molecular typing techniques. The two strains were identified as Brucella suis biovar 3 and were closely related to isolates previously obtained from two different hosts (human and swine) in Guangxi Province. Our results suggest that B. suis can be directly or indirectly transferred from swine to sheep, which act as reservoirs for B. suis infection and later transmitted to humans. Multiple locus variable-number tandem repeat analysis (MLVA) is a useful tool for tracing the geographical origin of brucellosis infections and elucidating its transmission patterns.

Virulence genes and genetic diversity assessment of Shiga toxin-producing Escherichia coli O91 strains from cattle, beef and poultry products

Shiga toxin-producing Escherichia coli (STEC) O91 has ranked in the top five of the non-O157 serogroups most frequently associated with human cases. In order to gain insight into the genetic diversity of O91 Latin American STEC strains, we analyzed their virulence properties and carried out a subtyping assay. A panel of 21 virulence genetic markers associated with human and animal infections was evaluated and the relatedness among strains was determined by a multiple-locus variable-number tandem repeats analysis (MLVA) comprising 9 VNTR loci. Twenty-two STEC O91 isolated from cattle and meat food and belonging to 5 serotypes (O91:H21, O91:H8, O91:H14, O91:H28, O91:H40) were studied. Eight virulence profiles were obtained for the O91 STEC strains: 4 for O91:H21 plus one for O91:H8, O91:H14, O91:H28 and O91:H40. All strains contained ehxA and lpfA0113 genes and only both stx1-positive strains lacked saa, which encodes the STEC autoagglutinating adhesin. Other genes involved in adhesion were detected: ehaA (91%), elfA and espP (86%), ecpA (82%) and, hcpA (77%). The gene encoding the cytolethal distending toxin type-V (CDT-V) was found only in O91:H8 and O91:H21, being present in the majority (89%) of strains of this last serotype. MLVA typing divided the total number of strains into 12 genotypes, and 9 of them were unique to a single strain. No association was observed between the virulence profiles and the source of the strains. Although they lack the eae gene, most of the strains have the genetic potential to adhere to host cells through other structures and possess cdt-V, which has been found in STEC strains involved in serious diseases. The MLVA showed clonal relatedness among strains isolated from cattle belonged to a same dairy farm and suggested that the same clone remains circulating throughout the year and, on the other hand, the need to increase the number of VNTR loci which could allow a higher discrimination among O91:H21 isolates.

Investigation of the Relationships Between Clinical and Environmental Isolates of Aspergillus fumigatus by Multiple-locus Variable Number Tandem Repeat Analysis During Major Demolition Work in a French Hospital.

Genotyping is needed to explore the link between clinical cases from colonization of invasive aspergillosis (IA) and major building construction. Attempts to correlate Aspergillus fumigatus strains from clinical infection or colonization with those found in the environment remain controversial due to the lack of a large prospective study. Our aim in this study was to compare the genetic diversity of clinical and environmental A. fumigatus isolates during a demolition period. Fungal contamination was monitored daily for 11 months in 2015. Environmental surveillance was undertaken indoors and outdoors at 8 locations with automatic agar samplers. IA infection cases were investigated according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group criteria. Isolates were identified by amplification and sequencing of the β- tubulin gene. They were genotyped by multiple-locus variable number tandem repeat analysis (MLVA). The phylogenetic relationships between isolates were assessed by generating a minimum spanning tree. Based on 3885 samples, 394 A. fumigatus isolates (383 environmental and 11 clinical) were identified and genotyped using MLVA. Clinical isolates were collected from patients diagnosed as having probable IA (n = 2), possible IA (n = 1), or bronchial colonization (n = 6). MLVA generated 234 genotypes. Seven clinical isolates shared genotypes identical to environmental isolates. Among the diversity of genotypes described, similar genotypes were found in clinical and environmental isolates, indicating that A. fumigatus infection and colonization may originate from hospital environments.

Investigation and source-tracing of an anthrax outbreak in Gansu Province, China.

Anthrax is an endemic disease in China. Cases are reported every year, especially in the northwestern areas. In August 2016, an outbreak of 21 cutaneous anthrax cases was reported in Min County, Gansu Province, China. In this study, the general characteristics of the anthrax outbreak are described. Two molecular typing methods, canonical single-nucleotide polymorphism (canSNP) and multiple-locus variable-number tandem repeat analysis with 15 markers (MLVA15), were used to investigate the possible source of transmission and to identify the genetic relationship among the strains/samples isolated in this outbreak as well as previous isolates. In this outbreak, all patients were infected through contact with diseased livestock or contaminated animal products. Livestock had been introduced into the local area shortly before the outbreak from Gannan Prefecture (in Gansu Province), Sichuan and Qinghai Provinces. In the molecular typing analysis, there were two canSNP subgroups found in Gansu, A.Br.001/002 and A.Br.Ames, and five MLVA15 genotypes were observed. The strains collected from the anthrax outbreak in Min County in 2016 belonged to the A.Br.001/002 canSNP subgroup and the MLVA15-28 and MLVA15-30 genotype. Strains previously isolated from Sichuan, Inner Mongolia and Maqu County (in Gannan Prefecture, Gansu Province) were clustered with these outbreak-related strains/samples according to the MLVA15-30 genotype. The MLVA15-28 genotype was found in strains isolated from Gansu and Xinjiang in previous studies. Combining the epidemiological investigation and molecular typing results, we conclude that the patients in this outbreak were infected by a local pathogen present in the adjoining area of Gansu, Sichuan and Qinghai Provinces.

Comparison of the molecular characteristics of Mycoplasma pneumoniae from children across different regions of China

Previous molecular characterization of Mycoplasma pneumoniae in China focused only on one or two cities. In this study, we characterized 835 samples from patients suspected to be infected with M. pneumoniae; these samples were collected in 2016 from pediatric patients from different regions of China. Multiple locus variable number tandem repeat analysis (MLVA), P1-restriction fragment length polymorphism (RFLP) analysis, and sequencing of the domain V of 23S rRNA were performed to compare genotype distribution across different locations. Two-hundred-and-thirteen samples tested positive for M. pneumoniae by PCR. P1 types were identified in 154 samples: 78.6% were type I and 21.4% were type II. Type I was the most prevalent genotype in five locations, except Nanjing where type II was the most common type (p < 0.01). Five distinct MLVA types were identified in the 172 samples. Genotype M4-5-7-2 was predominant at all locations, except Nanjing where type 3-5-6-2 was the most common (p < 0.01). Macrolide resistance-associated mutations were identified in 186 (76.3%) samples. The resistance rate differed with the location. This study showed that genotypes and macrolide resistance rate differed across China. The most prevalent genotype in China remains M4-5-7-2/P1-1. The resistance rate decreased, along with changes to the M4-5-7-2 genotype.

Staphylococcus epidermidis, Clonality and Accessory Gene Regulator Diversity in Clinical Isolates

Background: Quorum-sensing systems are considered as important mechanisms for pathogenesis and bacterial communication. The accessory gene regulator (agr) is one of the quorum-sensing systems in staphylococci, which is generally conserved. It is believed that there is a correlation between agr groups and infection. Multiple-locus VNTR analysis is a method for bacterial typing, previously applied for several different species of bacteria. This study aimed at determining the diversity of Staphylococcus epidermidis accessory gene regulator and clonality in clinical isolates from intensive care unit patients, Tehran, Iran. Methods: A total of 59 Staphylococcus epidermidis isolates were obtained from intensive care unit patients. The MLVA was performed for S. epidermidis isolates, using seven VNTR loci, including SE2395, SE0331, SE 828, SE1632, SE0175, Se2, and Se4. Specific primers were used for agr diversity determination. Results: The agr type I was observed in 29 (49%), while each of the agr type II and agr type III were observed in 10 (17%). Furthermore, 10 (17%) isolates were untypeable with using primers. In total, 49 MLVA genotypes were discriminated. Isolates were classified to six clonal complexes. Of the 59 isolate, 33 were included in clonal complex 1 (CC1), the largest of which harbored 15 (45.4%) agr type I. Conclusions: Agr type I was observed in the majority of the isolates. The MLVA results of this study suggest that there was a clone with 33 samples, comprised of 56% of isolates; smaller clones each comprised of two to seven isolates, and four isolates in the form of singleton. It seems that big clone isolates were settled in the intensive care unit (ICU), and singleton isolates entered the ward by visitors or medical personnel and caused infection.

Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) and Tandem Repeat Sequence Typing (TRST), helpful tools for subtyping Staphylococcus lugdunensis

Staphylococcus lugdunensis is an emergent virulent coagulase-negative Staphylococcus that is increasingly responsible for severe infections. In an attempt to generate informative sequence data for subtyping S. lugdunensis, we selected and sequenced seven polymorphic variable number of tandem repeats (VNTRs) to develop two new methods: a classic length-based multiple-locus VNTR analysis (MLVA) method and a tandem repeat sequence typing (TRST) method. We assessed their performances compared to two existing methods, multilocus sequence typing (MLST) and multivirulence-locus sequence typing (MVLST) for 128 isolates from diverse clinical settings and geographical origins. The clustering achieved by the four methods was highly congruent, with MLVA discriminating within clonal complexes as defined by MLST. Indeed, MLVA was highly discriminant compared to MLST and MVLST in terms of number of genotypes as well as diversity indexes. Sequencing of the seven VNTRs showed that they were stable, and analysis of sequence polymorphisms provided superior discriminatory power. The typeability, reproducibility, and epidemiological concordance of these new methods were excellent. Of note, no link between clustering and clinical settings was identified. This study demonstrates that MLVA and TRST provide valuable information for molecular epidemiological study of S. lugdunensis, and represent promising tools to distinguish between strains of homogenous lineages in this clonal species.

Salmonella spp. transmission in a vertically integrated poultry operation: Clustering and diversity analysis using phenotyping (serotyping, phage typing) and genotyping (MLVA).

The transmission of Salmonella enterica within a vertically integrated poultry operation was investigated longitudinally over an 18-month period (2013–2014). Thirty six percent of all samples collected (1503 of 4219) were positive for salmonellae with seven Salmonella enterica subsp. enterica serovars, and one Salmonella enterica subsp. salamae serovar detected. Both Salmonella enterica subsp. enterica serovars Infantis and Typhimurium were detected in all locations sampled. Salmonella Typhimurium was the most frequently detected serovar (63% of serotyped samples) with 8 phage types (PT) and 41 multiple-locus variable-number tandem-repeats analysis (MLVA) profiles identified. The most frequently identified phage types were PT135a and DT135. A total of 62 PT/MLVA combinations were observed. MLVA profiles 03-14-10-09-525 and 03-15-11-11-525 were the most frequently identified and 83% of the isolates shared at least one MLVA profile with an isolate from another phage type. The use of phage typing and MLVA profiling, on their own or in combination, were insufficient to understand the complexity of the epidemiological relationships between locations within this production system. Despite the high level of apparent diversity, cluster analysis was unable to differentiate the transmission pathways of all S. Typhimurium variants detected within the integrated enterprise. Using additional epidemiological information, the parent breeder rearing site was identified as the most likely point of introduction of two S. Typhimurium isolates into the production system with subsequent dissemination to the broiler flocks via the hatchery. This complexity is unable to be resolved in the absence of intensive sampling programs at all generations of the production system.

Source tracing of the Yersinia pestis strains isolated from Heqing county, Yunnan province in 2017

Objective: To understand the genotype of the Yersinia (Y.) pestis strains isolated from Heqing county, Yunnan province in 2017 and provide evidence for the prevention and control of plague in this area. Methods: Ten Y. pestis strains isolated from Heqing were typed by the detections of different region (DFR) and clustered regularly interspaced short palindromic repeats (CRISPRs) as well as multiple-locus variable-number tandem repeat analysis (MLVA). And the results were compared with those of the 93 Y. pestis strains from the adjacent plague foci of Heqing obtained from the established database for clustering analysis. Results: The results showed that Heqing strains had the same type of DFR (Genomovar 05) and CRISPRs (Cluster Ca7, Type 22) with isolates from the plague focus in Lijiang. Heqing strains and Lijiang strains were in the same cluster in MST and only VNTR loci N2117 and M23 of Heqing strains were different from that of Lijiang strains. Conclusion: The Y. pestis strains isolated from Heqing in 2017 were highly homogenous with the strains isolated from wild rodents in plague focus in Lijiang, and Heqing plague might be the result of further southward spread of Lijiang plague.

Genotyping Mycoplasma hyopneumoniae isolates based on multi-locus sequence typing, multiple-locus variable-number tandem repeat analysis and analysing gene p146

Mycoplasma hyopneumoniae is a swine pathogen bacterium, causing significant economic losses worldwide. Epidemiological investigations based on molecular typing methods support the prevention and eradication strategies for the control of M. hyopneumoniae, through tracing the spreading of the pathogen. The present study describes the genotyping of 44 M. hyopneumoniae strains isolated from Hungarian, Czech and Slovakian porcine lung samples by multi-locus sequence typing (MLST), multiple-locus variable-number tandem repeat analysis (MLVA) and analysing gene p146, and the evaluation of the used methods. The resolution of the three-gene (adk, rpoB, tpiA) and the seven-gene (efp, metG, pgiB, recA, adk, rpoB, tpiA) based MLST systems was identical with 27 sequence types. MLVA utilising loci P97-RR1 and Locus1 extended with the serine repeat numbers of gene p146 showed the highest resolution power among the studied methods differentiating 40 genotypes. The independent analysis of gene p146 revealed 31 different types among the isolates.High variability of M. hyopneumoniae strains was detected by the used typing methods. The results confirmed that utilization of the minimal MLST is suitable for phylogenetic analyses of M. hyopneumoniae strains. The MLVA method extended with the evaluation of serine repeat numbers of gene p146 is adequate for the resolution of genetic relationships within MLST groups. Examination of the p146 gene is suitable to complement both MLST and MLVA methods in order to refine closer genetic relationships.

Phenotypic Characterization and Genotypic Subtyping of Salmonella enterica Serovars Enteritidis and Gallinarum Isolated from Human and Poultry-Related Samples

ABSTRACTThis study aimed to assess the serotypes, antibiotic susceptibility, and molecular subtyping of Salmonella enterica isolated from food products and human patients with gastroenteritis. A total of 59 isolates were investigated, and the results revealed a predominance of S. Enteritidis with 57.63% (34/59) S. Gallinarum held second with 15.62% (5/32) of total food borne. While, isolates from humans showed 18.51% (5/27) of S. Typhimurium. High level of resistance to nalidixic acid was noted among food strains and 35.29% of human isolates were found to be multidrug resistant. Eight representative isolates were subtyping using three molecular approaches, ribotyping, Multilocus sequence typing (MLST) and Multiple-locus variable number tandem repeat analysis (MLVA). MLST showed three sequence types corresponding to two clonal complexes, (ST-78, CC-4) for S. Gallinarum, (ST-11, CC-4) for S. Enteritidis and (S-367, CC-37) for S. Cerro. While, MLVA generated six different profiles targeting nine loci for S. Enteritidis and S. Gallinarum.

The predominance and clustering of Clostridioides (Clostridium) difficile PCR ribotype 001 isolates in three hospitals in Eastern Slovakia, 2017.

This study aimed to implement a toxigenic culture as an optional third diagnostic step for glutamate dehydrogenase (GDH)-positive and toxin A/B-negative diarrheal stool samples into a diagnostic algorithm for Clostridioides (Clostridium) difficile infection (CDI), and to characterise C. difficile isolates for epidemiological purposes. During the 5-month study, 481 diarrhoeal stool samples from three Slovak hospitals were investigated and 66 non-duplicated GDH-positive stool samples were found. Of them, 36 were also toxin A/B-positive. Twenty-three GDH-positive and toxin A/B-negative stool samples were shown subsequently to be positive following toxigenic culture (TC). Molecular characterisation of C. difficile isolates showed the predominance of PCR ribotype (RT) 001 (n = 37, 56.1%) and the occurrence of RT 176 (n = 3, 4.5%). C. difficile RT 001 isolates clustered to eight clonal complexes (CCs) using multiple-locus variable-number tandem repeats analysis (MLVA). Interestingly, one third of RT 001 isolates clustering in these CCs were cultured from toxin A/B-negative stool samples. Our observations highlight the need of use multiple step diagnostic algorithm in CDI diagnosis in order to detect all CDI cases and to avoid the spread of C. difficile in healthcare settings.

Genetic Analysis Method for Staphylococcus chromogenes Associated with Goat Mastitis.

Mastitis in goats is mainly caused by coagulase-negative Staphylococcus (CNS). The identification methods for this group are based on evaluating the expression of phenotypic characteristics such as the ability to metabolize various substrates; however, this is disadvantageous as these methods are dependent on gene expression. In recent years, genotyping methods such as the Multiple Locus Variable-Number Tandem Repeat Analysis (MLVA) and gene identification have been useful for epidemiological study of several bacterial species. To develop a genotyping method, the genome sequence of Staphylococcus chromogenes MU970 was analysed. The analysis showed nine virulence genes described in Staphylococcus aureus. The MLVA was developed using four loci identified in the genome of S. chromogenes MU970. This genotyping method was examined in 23 strains of CNS isolated from goat mastitis. The rate of discrimination for MLVA was 0.8893, and the highest rates of discrimination per the index of Simpson and Hunter-Gaston were 0.926 and 0.968 for the locus 346_06, respectively. The virulence genes were present in all strains of S. chromogenes but not in other CNS. The genotyping method presented in this paper is a viable and easy method for typifying CNS isolates from mastitis cases in different regions and is an ideal mean of tracking this disease.

Reemergence of human plague in Yunnan, China in 2016.

The third plague pandemic originated from Yunnan Province, China in the middle of the 19th century. The last human plague epidemic in Yunnan occurred from 1986–2005. On June 6, 2016, a case of human plague was reported in the Xishuangbanna Prefecture, Yunnan. The patient suffered from primary septicemic plague after exposure to a dead house rat (Rattus flavipectus), which has been identified as the main plague reservoir in the local epizootic area. Moreover, a retrospective investigation identified another bubonic plague case in this area. Based on these data, human plague reemerged after a silent period of ten years. In this study, three molecular typing methods, including a clustered regularly interspaced short palindromic repeats (CRISPR) analysis, different region analysis (DFR), and multiple-locus variable number of tandem repeats analysis (MLVA), were used to illustrate the molecular characteristics of Yersinia pestis (Y. pestis) strains isolated in Yunnan. The DFR profiles of the strains isolated in Yunnan in 2016 were the same as the strains that had previously been isolated in this Rattus flavipectus plague focus. The c3 spacer present in the previously isolated strains was absent in the spacer arrays of the Ypc CRISPR loci of the strains isolated in 2016. The MLVA analysis using MLVA (14+12) showed that the strains isolated from the human plague case and host animal plague infection in 2016 in Yunnan displayed different molecular patterns than the strains that had previously been isolated from Yunnan and adjacent provinces.