Molecular epidemiology and antimicrobial resistance patterns of carbapenem-resistant Acinetobacter baumannii isolates from patients admitted at ICUs of a teaching hospital in Zunyi, China.

Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a predominant strain of healthcare-associated infections worldwide, particularly in intensive care units (ICUs). Therefore, it is imperative to study the molecular epidemiology of CRAB in the ICUs using multiple molecular typing methods to lay the foundation for the development of infection prevention and control strategies. This study aimed to determine the antimicrobial susceptibility profile, the molecular epidemiology and conduct homology analysis on CRAB strains isolated from ICUs. Methods: The sensitivity to various antimicrobials was determined using the minimum inhibitory concentration (MIC) method, Kirby-Bauer disk diffusion (KBDD), and E-test assays. Resistance genes were identified by polymerase chain reaction (PCR). Molecular typing was performed using multilocus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). Results: Among the 79 isolates collected, they exhibited high resistance to various antimicrobials but showed low resistance to levofloxacin, trimethoprim-sulfamethoxazole, and tetracyclines. Notably, all isolates of A. baumannii were identified as multidrug-resistant A. baumannii (MDR-AB). The bla OXA-51-like, adeJ, and adeG genes were all detected, while the detection rates of bla OXA-23-like (97.5%), adeB (93.67%), bla ADC (93.67%), qacEΔ1-sul1 (84.81%) were higher; most of the Ambler class A and class B genes were not detected. MLST analysis on the 79 isolates identified five sequence types (STs), which belonged to group 3 clonal complexes 369. ST1145Ox was the most frequently observed ST with a count of 56 out of 79 isolates (70.89%). MLST analysis for non-sensitive tigecycline isolates, which were revealed ST1145Ox and ST1417Ox as well. By using the MLVA assay, the 79 isolates could be grouped into a total of 64 distinct MTs with eleven clusters identified in them. Minimum spanning tree analysis defined seven different MLVA complexes (MCs) labeled MC1 to MC6 along with twenty singletons. The locus MLVA-AB_2396 demonstrated the highest Simpson's diversity index value at 0.829 among all loci tested in this study while also having one of the highest variety of tandem repeat species. Conclusion: The molecular diversity and clonal affinities within the genomes of the CRAB strains were clearly evident, with the identification of ST1144Ox, ST1658Ox, and ST1646Oxqaq representing novel findings.

Comparison of Multiple-Locus Variable-Number Tandem Repeat Analysis Profiles of Enteropathogenic Yersinia spp. Obtained from Humans, Domestic Pigs, Wild Boars, Rodents, Pork and Dog Food.

The enteropathogenic Yersinia genus is commonly detected in wildlife including wild boars. Difficulties in its cultivation may hamper subsequent epidemiological studies and outbreak investigations. Multiple-locus variable-number tandem repeat analysis (MLVA) of Yersinia (Y.) enterocolitica and Y. pseudotuberculosis has proven useful in source attribution and epidemiological studies but has hitherto relied on the analysis of isolates. In the present study, MLVA profiles generated from 254 isolates of Y. enterocolitica indicated similarities between human, pig and rodent isolates. Further, MLVA analyses of 13 Y. pseudotuberculosis pure-cultured isolates were compared to MLVA analyses performed directly on the 14 PCR-positive enrichment broths from which the isolates originated, which showed matching MLVA profiles. This indicates that MLVA analysis performed directly on enrichment broths could be a useful method for molecular epidemiological investigations. In addition, 10 out of 32 samples of wild boar minced meat obtained from private hunters and from approved wild-game-handling establishments were PCR-positive for the presence of Y. enterocolitica and may indicate a risk for public health.

A Novel Marine Mammal Coxiella burnetii-Genome Sequencing Identifies a New Genotype with Potential Virulence.

The obligate intracellular bacterial pathogen Coxiella burnetii has been identified in a few species of marine mammals, some of which are showing population declines. It has been hypothesized that C. burnetii in marine mammals is a distinct genotype that varies significantly from the typical terrestrial genotypes. It appears to lack an IS1111. Isolates originating from Australian marine animals have a distinctly non-Australian profile of multiple-locus variable-number tandem-repeat analysis (MLVA). Extracted Coxiella DNA of Australian fur seal placental origin was sequenced using the Novaseq platform. Illumina 150 bp paired-end reads were filtered and trimmed with Trimgalore. The microbial community present in the sequenced genome was evaluated with Kraken and Bracken software using the NCBI database. A phylogenetic analysis was performed using 1131 core genes. Core genes were identified using Panaroo and inputted into Iqtree to determine the maximum-likelihood tree. A second phylogenetic tree was created using Rickettsiella grylii and using seven housekeeping genes. Results were compared with the C. burnetii Nine Mile RSA439 virulent genome. This new Australian marine mammal isolate of Coxiella (PG457) appears to be a novel genotype that lacks IS1111 and has a distinct MLVA signature (ms26, ms27, ms28, ms30, and ms31). The presence of genes for multiple virulence factors appears to give this genotype sufficient pathogenicity for it to be considered a possible causative agent of abortion in Australian fur seals as well as a potential zoonotic risk.

Genotyping of Coxiella burnetii from Cattle by Multispacer Sequence Typing and Multiple Locus Variable Number of Tandem Repeat Analysis in the Republic of Korea.

Genotyping of Coxiella burnetii using multispacer sequence typing (MST) and multiple locus variable number tandem repeat analysis (MLVA) was conducted from infected animals for the first time in the Republic of Korea. C. burnetii was detected by real-time PCR, and followed by MST and MLVA genotyping. The result showed that detected C. burnetii all had the same MLVA genotype, 6-13-2-7-9-10 for markers MS23-MS24-MS27-MS28-MS33-MS34, respectively, and genotype group 61 for MST. The same genotypes were previously identified in Poland. Importantly, this MLVA type was detected in humans in France, suggesting that the Korean strain can also potentially cause Q fever in humans. MST and MLVA were very useful tools for analyzing the molecular epidemiology of C. burnetii and helpful for interpreting the epidemiological relationship between isolates from domestic and international resources.

The role of co-infections in M. hyopneumoniae outbreaks among heavy fattening pigs: a field study.

Little is known about how co-infections and genotype dynamics affect Mycoplasma hyopneumoniae infection in fattening pigs. This study was aimed at assessing the role of co-infections in M. hyopneumoniae outbreaks, their influence on the presence of M. hyopneumoniae genotypes and their impact on consequent lung lesions. Tracheobronchial swabs (TBS) from 300 finishers were collected from 10 farms at the onset of enzootic pneumonia outbreaks and 1 month later, sampling of 3 groups per farm: Group A showed clinical signs first, Group B was housed near Group A, and Group C was located in a different building. Pigs' lungs were scored at the slaughterhouse. TBS were tested for the main pathogens involved in respiratory diseases, and samples positive for M. hyopneumoniae were genotyped by multiple-locus variable-number tandem repeat analysis (MLVA). Pigs in Group A showed the highest prevalence and load of M. hyopneumoniae. A positive association was detected between M. hyopneumoniae and Mycoplasma hyorhinis, whereas Actinobacillus pleuropneumoniae was more frequent when the M. hyopneumoniae load was higher. Nevertheless, co-infection had no effect on lung lesion scores. The presence of multiple MLVA types (mixed infections) increased in time only in pigs from Group C and was positively associated with porcine reproductive and respiratory syndrome virus infection. Lung lesions were more severe in pigs with at least one TBS positive for M. hyopneumoniae and in pigs with a history of mixed infections. The central role of M. hyopneumoniae and relevance of mixed infections suggest that increased biosecurity might be beneficial for lung lesion sequelae.

A Suggested Diagnostic Approach for Sporadic Anthrax in Cattle to Protect Public Health.

The repeated occurrence of anthrax in grazing animals should be a reminder of a widespread presence of Bacillus anthracis spores in the environment. Its rapid diagnosis is critical to protect public health. Here, we report a case of anthrax in cattle that was investigated using conventional and molecular methods. In 2015, six cows suddenly died within three days and the number of dead animals increased to a total of 12 within two weeks. At necropsy, anthrax was suspected. Therefore, spleen tissue samples were collected (from 6/12 animals) and laboratory tests (microscopy, cultivation, and real-time PCR) performed. The results of tissue staining for microscopy and cultivation were in congruence, while B. anthracis real-time PCR outperformed both. Spleen tissues from all six animals were real-time PCR-positive, while B. anthracis was successfully cultivated and detected by microscopy from the spleen of only three animals. Additionally, the ear tissue from another (1/12) cow tested positive by real-time PCR, supporting the suitability of ear clippings for molecular confirmation of B. anthracis. Genotyping of the isolates using multiple-locus variable-number tandem repeat analysis (MLVA) revealed a common source of infection as all three typed isolates had an indistinguishable MLVA genotype, which has not been observed previously in Europe. The results indicate that molecular testing should be selected as the first-line tool for confirming anthrax outbreaks in animals to ensure timely protection of public health.

Development of a novel, high resolution melting analysis based genotyping method for Cryptosporidium parvum.

This study employed the post-real-time PCR application, high resolution melting (HRM) analysis, in order to differentiate between characterised clinical and reference Cryptosporidium parvum samples obtained from Cork University Hospital (Cork, Ireland) and the Cryptosporidium Reference Unit (Swansea, Wales). A sample set composed of 18 distinct C. parvum gp60-subtypes of the IIa gp60-subtype family (an allele family accounting for over 80% of all cryptosporidiosis cases in Ireland) was employed. HRM analysis-based interrogation of the gp60, MM5 and MS9-Mallon tandem repeat loci was found to completely differentiate between 10 of the 18 studied gp60-subtypes. The remaining eight gp60-subtypes were differentiated into three distinct groupings, with the designations within these groupings resolved to two to three potential gp60-subtypes. The current study aimed to develop a novel, reproducible, real-time PCR based multi-locus genotyping method to distinguish between C. parvum gp60-subtypes. These preliminary results support the further expansion of the multi-locus panel in order to increase the discriminatory capabilities of this novel method.

Comparison of conventional molecular and whole-genome sequencing methods for subtyping Salmonella enterica serovar Enteritidis strains from Tunisia.

We sought to determine the relative value of conventional molecular methods and whole-genome sequencing (WGS) for subtyping Salmonella enterica serovar Enteritidis recovered from 2000 to 2015 in Tunisia and to investigate the genetic diversity of this serotype. A total of 175 Salmonella Enteritidis isolates were recovered from human, animal, and foodborne outbreak samples. Pulsed-field gel electrophoresis (PFGE), multiple locus variable-number tandem repeat analysis (MLVA), and whole-genome sequencing were performed. Eight pulsotypes were detected for all isolates with PFGE (DI = 0.518). Forty-five Salmonella Enteritidis isolates were selected for the MLVA and WGS techniques. Eighteen MLVA profiles were identified and classified into two major clusters (DI = 0.889). Core genome multilocus typing (cgMLST) analysis revealed 16 profiles (DI = 0.785). Whole-genome analysis indicated 660 single-nucleotide polymorphism (SNP) divergences dividing these isolates into 43 haplotypes (DI = 0.997). The phylogenetic tree supported the classification of Salmonella Enteritidis isolates into two distinct lineages subdivided into five clades and seven subclades. Pairwise SNP differences between the isolates ranged between 302 and 350. We observed about 311 SNP differences between the two foodborne outbreaks, while only less or equal to 4 SNP differences within each outbreak. SNP-based WGS typing showed an excellent discriminatory power comparing with the conventional methods such as PFGE and MLVA. Besides, we demonstrate the added value of WGS as a complementary subtyping method to discriminate outbreak from non-outbreak isolates belonging to common subtypes. It is important to continue the survey of Salmonella Enteritidis lineages in Tunisia using WGS.

[Characteristics on molecular epidemiology of Brucella melitensis in Jiangxi province].

Objective: To understand the molecular characteristics and correlation among isolated strains of Brucella melitensis (BM) so as to improve the strategies on prevention and control of the disease in Jiangxi province. Methods: A total of 25 strains of BM isolated from human in 17 counties of Jiangxi province were analyzed by multiple locus variable-number tandem repeat analysis (MLVA) method. Results: A total of 25 strains of BM were classified into 24 independent genotypes with similarities between 67.00% and 100.00% and Simpson index between 0.000 and 0.773. There were 3 genotypes in MLVA8, including 60.00% (15/25) as 42 genotype, 32.00% (8/25) as 43 genotype, and 8.00% (2/25) as 63 genotype, respectively. There were 7 genotypes in MLVA11 identified, with 116 genotype and 125 genotype the main genotypes, accounting for 56.00% (14/25) of all the identified strains. Conclusions: Genes from all the 25 strains of BM that isolated from human being were with high genetic diversities, and various, genotypes. However, no obvious epidemiological correlation was noticed among these strains, indicating the complexity of the source of infection on Brucella in Jiangxi province.

A shigellosis outbreak associated with a sports festival at a kindergarten in Kitakyushu City, Japan.

INTRODUCTION: Shigellosis cases have decreased gradually in Japan in recent years, but indigenous shigellosis outbreaks sometimes occur in childcare facilities. From national surveillance data, we identified a shigellosis outbreak involving a kindergarten. METHODS: After detecting Shigella sonnei in Kitakyushu City, we conducted active case finding and epidemiological investigation in Kindergarten Z, including stool specimen collection and interviews. The stool specimens were cultured, and isolated strains were subjected to pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat analysis (MLVA). RESULTS: Between September 1 and December 31, 2014, we identified 19 cases: 14 confirmed, 2 suspected, and 3 asymptomatic. Of the 19 cases, 16 were epidemiologically associated with Kindergarten Z (10 pupils, 5 family members, and 1 teacher). On October 19, a pupil with gastrointestinal illness participated in the kindergarten's sports festival, in which the pupils were split into "red" and "white" teams; the pupil in question belonged to the red team. Attack rates of the red and white teams were 8% (7/82) and 0% (0/108), respectively (relative risk, 10.5; 95% confidence interval, 1.3-82.1). PFGE patterns were identical or similar for the isolates in all 17 cases; 7 isolates were identical, and the others had one locus difference on MLVA. CONCLUSIONS: We concluded that contact during the sports festival could have been responsible for spread of the shigellosis outbreak at the kindergarten, although the infection source was not determined. It is vital to inform guardians immediately after detection of shigellosis cases that symptomatic pupils should not participate in activities such as sports festivals.

Genetic Diversity of Australian Bacillus anthracis Isolates Revealed by Multiple-Locus Variable-Number Tandem Repeat Analysis.

Outbreaks of anthrax occur sporadically in Australia and most commonly in the "anthrax belt", a region which extends from southern Queensland through the centre of New South Wales and into northern Victoria. Little is known about the epidemiological links between Bacillus anthracis isolates taken from different outbreaks and the diversity of strains within Australia. We used multiple-locus variable-number tandem repeat analysis employing 25 markers (MLVA25) to genotype 99 B. anthracis isolates from an archival collection of Australian isolates. MLVA25 genotyping revealed eight unique genotypes which clustered within the previously defined A3 genotype of B. anthracis. Genotyping of B. anthracis strains from outbreaks of disease in Victoria identified the presence of multiple genotypes associated with these outbreaks. The geographical distribution of genotypes within Australia suggests that a single genotype was introduced into the eastern states of Australia, followed by the spread and localised differentiation of the pathogen (MLVA25 genotypes MG1-MG6) throughout the anthrax belt. In contrast, unexplained occurrences of disease in areas outside of this anthrax belt which are associated with different genotypes, (MLVA25 genotypes MG7 and MG8) indicate separate introductions of B. anthracis into Australia.

First Detection and Characterization of Macrolide-Resistant Mycoplasma pneumoniae from People with Community-Acquired Pneumonia in Iran.

Objectives: Increasing macrolide resistance of Mycoplasma pneumoniae strains is becoming a public health concern worldwide. Nevertheless, no comprehensive genomic background of circulating isolates is available in our region. We aimed to study the genetic diversity of this microorganism using the multiple-locus variable-number tandem-repeat analysis method and to investigate the relationships between MLVA types and macrolide susceptibility profiles of the isolates. Materials and Methods: A total of 270 patients attending Tehran general university hospitals were included in this study. One throat swab was taken from each patient. M. pneumoniae was identified using culture and PCR assay. Macrolide resistance was determined using the broth microdilution method. The MLVA was performed by amplification of four variable-number tandem-repeat loci. Results: Of 270 specimens, M. pneumoniae was detected in 25.2% (n = 68) and 21.8% (n = 59) samples using PCR and culture, respectively. Approximately 56.9% of isolates were resistant to macrolides. Fifty-one of 59 M. pneumoniae isolates were divided into 6 distinct MLVA types. Conclusion: The macrolide-resistant M. pneumoniae (MRMP) rate in this study was relatively high and most of the MRMP isolates were assigned into the type 4/5/7/2. Since a significant association between MLVA type 4/5/7/2 and macrolide resistance of M. pneumoniae isolates was observed, further monitoring of genetic diversity of MRMP isolates might facilitate better understanding of epidemiology of this microorganism. Besides surveillance of the antibiotic susceptibility might be helpful to make necessary reconsiderations on guidelines for treatment of M. pneumoniae infection.

[Relationship Between the VNTR Loci Polymorphism and MALDI-TOF MS Protein Profile Diversity in Mycobacterium tuberculosis Complex].

OBJECTIVE: To study the relationship between the copy numbers of repetitive units at variable number tandem repeat (VNTR) loci of Mycobacterium tuberculosis complex (MTBC) with its diversity of protein profiles. METHODS: The MTBC strains were subjected to genotyping using multiple locus variable number tandem repeat analysis (MLVA). Also, the principal component analysis (PCA) was performed for bacterial protein profiles of MTBC using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The relationship between the polymorphism of VNTR loci and PCA clustering was analyzed. RESULTS: A total of 157 MTBC strains were collected. 146 MTBC strains (MS identification score values ≥1.700) were performed PCA and three clusters, clusterⅠ(61 strains), clusterⅡ(26 strains) and cluster Ⅲ(59 strains), were generated. Polymorphic diversities were observed in 24 VNTR loci, among them, 7 were highly various, 7 were moderately, and 10 were low various. The polymorphism of Mtub39, QUB26 and QUB4156 loci were correlated with the results of MALDI-TOF MS clustering (P=0.000, P=0.035, P=0.017). CONCLUSION: The polymorphism of Mtub39, QUB26 and QUB4156 loci in MTBC was correlated with the difference of MALDI-TOF MS protein profiles, suggesting that these loci may play a role in regulating the composition of protein profiles of MTBC strains.

Phenotypic and genotypic characteristics of Brucella isolates from the Republic of Kazakhstan.

The purposes of this study were to determine phenotypic and genotypic characteristics of Brucella isolates from the Republic of Kazakhstan and to determine their biotype. The focus was laid on culture-morphological, biochemical, and biological properties of 59 Brucella isolates from primary cultures. Material was isolated from blood and tissue of serum-positive killed, dead diseased, or aborted domestic cattle from different regions of Kazakhstan where brucellosis is a common problem. Multiple-locus variable number tandem repeat analysis (MLVA) of all strains, isolated in different regions, has shown that Brucella isolates from the epizootic form two clusters. Based on the comparison with strains available in the MLVA database, B. abortus 0015/B is alike the B. abortus strains isolated from Italy and Portugal. B. melitensis 0016/B isolated from the Almaty region fits the third cluster and is alike the B. melitensis strains isolated from humans in Turkey, China, and Portugal. More than 90% of the overall B. abortus samples were isolated from the northern regions of the East and West Kazakhstan, while B. melitensis strains were registered in the southeast Kazakhstan. The most frequently recorded B. abortus biovar is biovar 3. The most frequently recorded B. melitensis biovars are biovars 1 and 3. SIGNIFICANCE AND IMPACT OF STUDY: These results contribute to a better understanding of the geographic pattern of Brucella infection in Kazakh cattle also important for developing the specific control measures. The results of current research can be used for creating a gene bank of Brucella strains circulating in Kazakhstan for producing diagnostic and therapeutic agents. The research material will be used to solve the problems of genetic characterization of Brucella species and to establish the phylogenetic relationships of strains.

Molecular epidemiology of Leptospira interrogans in Rattus norvegicus in Hanoi, Vietnam.

Leptospirosis is a zoonotic disease that is caused by pathogenic spirochaetes of Leptospira spp. and it has become a public health concern in urban localities in the tropics. Rats are important reservoir animals for the transmission of leptospirosis in urban areas. Leptospirosis is considered endemic in Vietnam. However, information on the causative Leptospira genotypes and serotypes in the country is limited. We investigated the carrier status of Leptospira spp. in rats captured in Hanoi by culturing and DNA detection. Isolates were characterized using a serological method and multiple-locus variable-number tandem repeat analysis (MLVA). We captured 144 rats (1 Rattus argentiventer, 135 R. norvegicus, and 8 R. rattus) and obtained 17 L. interrogans, determined by rrs sequencing, from R. norvegicus (12.6%). Sixteen of the isolates were serogroup Bataviae. Five of the 16 isolates exhibited an MLVA type identical to that of the serovar Bataviae reference strain Van Tienen, while there were nine repeats for the other 11 isolates at VNTR31 compared with the reference strain. The remaining isolate grew poorly, and we were unable to determine its serogroup. However, it had an MLVA type matching those of serogroup Pomona strains isolated from R. norvegicus in Japan. Three different flaB sequences were detected in 23 out of 81 R. norvegicus kidney tissue samples (28.4%) using nested PCR followed by DNA sequencing. Two of the sequences were identical with those of serogroups Bataviae and Pomona, and no strain with another sequence was detected in the present study. The present study reveals a high prevalence rate of L. interrogans among R. norvegicus in Hanoi, Vietnam, indicating a potential risk of rat-borne leptospirosis in the area. The present study also demonstrates that a fastidious L. interrogans strain circulates among rats and that molecular detection is crucial in facilitating the accurate determination of reservoir animals.

[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.

Persistence of the same genetic type of Mycoplasma hyopneumoniae in a closed herd for at least two years / Persistencia del mismo tipo genético de Mycoplasma hyopneumoniae por al menos 2 anos en una piara sin reposición externa de animales

Two cross-sectional studies were carried out in 2013 and 2015 monitoring for Mycoplasma hyopneumoniae presence in a swine farm. In these studies, the genetic diversity of M. hyopneumoniae was assessed in clinical specimens using a Multiple Locus Variable-number tandem repeat Analysis (MLVA) targeting P97 R1, P146 R3 and H4 loci. The samples from August 2015 showed the MLVA profile prevalent in June 2013, therefore it can be concluded that a same genetic type of M. hyopneumoniae can persist for at least two years in a closed herd. In addition, the nested PCR reactions implemented in this study showed to be useful for MLVA typing in non-invasive clinical samples.

Dos estudios transversales fueron realizados en los anos 2013 y 2015 monitorizando la presencia de Mycoplasma hyopneumoniae en una piara. En esos estudios la diversidad genética de M. hyopneumoniae fue evaluada a partir de muestras clínicas utilizando un análisis multilocus de regiones repetidas en tándem (MLVA) de los loci P97 R1, P146 R3 y H4. Las muestras colectadas en agosto del 2015 mostraron el perfil de MLVA prevalente en junio del 2013, por lo tanto, se puede concluir que el mismo tipo genético de M. hyopneumoniae puede persistir por al menos 2 años en una piara sin reposición externa de animales. Además, las reacciones de PCR anidadas implementadas en este estudio mostraron ser útiles para la tipificación por MLVA a partir de muestras clínicas no invasivas.

The development of a screening protocol for Salmonella spp. and enteropathogenic Yersinia spp. in samples from wild boar (Sus scrofa) also generating MLVA-data for Y. enterocolitica and Y. pseudotuberculosis.

Salmonellosis and yersiniosis are notifiable human diseases that are commonly associated with contaminated food. Domestic pigs as well as wild boars and other wild-life have been identified as reservoirs of these bacteria. Methods for cultivation and molecular epidemiological investigations of Salmonella spp. are well established, however, cultivation of enteropathogenic Yersinia spp. is time- consuming and the commonly used method for molecular epidemiological investigations, pulsed-field gel electrophoresis, lack in discriminatory power. The aim of this study was to develop and evaluate a screening protocol well suited for wildlife samples and other highly contaminated samples. The method is based on PCR-screening followed by Multiple Loci Variant number tandem repeat Analysis (MLVA) on enrichment broth to obtain molecular epidemiological data for enteropathogenic Yersinia spp. without the need for pure isolates. The performance of the protocol was evaluated using wild boar samples (n=354) including tonsils, faeces and lymph nodes from 90 Swedish wild boars. The new protocol performed as well as or better than the established ISO-standards for detection and cultivation of Y. enterocolitica and Salmonella spp., however for cultivation of Y. pseudotuberculosis, further development is needed. The selection for motility seems beneficial for the enrichment of Salmonella spp. and Y. enterocolitica. Further, the selective enrichment prior to PCR-analysis eliminates inhibitory factors present in the original sample. In total, ten isolates of Y. enterocolitica of various bio-serotypes were obtained, and the MLVA-profile of these isolates were consistent with the profiles from the corresponding enrichment broth. Further, 22 isolates of Salmonella spp. comprising six different serovars were obtained with S. Fulica, S. Hadar and a monophasic S. Typhimurium being the most common. In conclusion, the presented screening protocol offers a rapid and efficient way to obtain prevalence data from a large sample set as well as MLVA-data within a short time frame. These results can hence improve the knowledge on the epidemiology and distribution of these pathogens and their importance to public health.

Molecular epidemiology of Shiga toxin-producing O113:H21 isolates from cattle and meat.

The serotype O113:H21 is considered one of the relevant non-O157 STEC serotypes associated with severe human infections. Due to the increased detection of O113 strains and their relationship with clinical cases, which emphasizes the importance of this serogroup as an emerging pathogen, our aim was to determine the characteristics of STEC O113:H21 strains circulating in bovine cattle and retail meat from Argentina. For this purpose, we determined the presence and combinations of various virulence genes (and their variants) related to adhesion and toxicity in a collection of 34 isolates. Their genetic relatedness using multiple-locus variable-number tandem repeat analysis (MLVA) was also studied. Subtyping of stx genes indicated that O113:H21 strains circulating in Argentina mainly present stx2a alone or together with stx2c or, less frequent, with stx2d , all of which are subtypes associated with human disease. We found plasmid markers, such as saa, ehxA and subA, in a higher proportion than previous studies, and five variants of saa, two of which were novel ones. In relation to MLVA subtyping, we detected a limited diversity among the isolates considering that several loci were not discriminative and, that in some farms, the same clone seemed to remain circulating throughout the year. The O113:H21 strains studied harbour several toxin and adhesion genes (saa, espP, fimCD, ehaA, iha, hcpA, elfA, lpfO113, ecpA, subA, cdt-V) and Stx subtypes associated with human disease. Results also highlighted that subtyping of stx and saa is useful to discriminate O113:H21 strains that share virulence genes. In conclusion, this study shows that a number of O113:H21 strains that occur in foods and bovines could be pathogenic for humans. This situation calls for further attention in the prevention and control of foodborne disease caused by these strains.

Genetic diversity, anti-microbial resistance, plasmid profile and frequency of the Vi antigen in Salmonella Dublin strains isolated in Brazil.

Salmonella Dublin is strongly adapted to cattle causing enteritis and/or systemic disease with high rates of mortality. However, it can be sporadically isolated from humans, usually causing serious disease, especially in patients with underlying chronic diseases. The aim of this study was to molecularly type S. Dublin strains isolated from humans and animals in Brazil to verify the diversity of these strains as well as to ascertain possible differences between strains isolated from humans and animals. Moreover, the presence of the capsular antigen Vi and the plasmid profile was characterized in addition to the anti-microbial resistance against 15 drugs. For this reason, 113 S. Dublin strains isolated between 1983 and 2016 from humans (83) and animals (30) in Brazil were typed by PFGE and MLVA. The presence of the capsular antigen Vi was verified by PCR, and the phenotypic expression of the capsular antigen was determined serologically. Also, a plasmid analysis for each strain was carried out. The strains studied were divided into 35 different PFGE types and 89 MLVA-types with a similarity of ≥80% and ≥17.5%, respectively. The plasmid sizes found ranged from 2 to >150 kb and none of the strains studied presented the capsular antigen Vi. Resistance or intermediate resistance was found in 23 strains (20.3%) that were resistant to ampicillin, ciprofloxacin, chloramphenicol, imipenem, nalidixic acid, piperacillin, streptomycin and/or tetracycline. The majority of the S. Dublin strains studied and isolated over a 33-year period may descend from a common subtype that has been contaminating humans and animals in Brazil and able to cause invasive disease even in the absence of the capsular antigen. The higher diversity of resistance phenotypes in human isolates, as compared with animal strains, may be a reflection of the different anti-microbial treatments used to control S. Dublin infections in humans in Brazil.