MLVA Types Research Articles

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.

Multilocus variable number tandem repeat analysis and antimicrobial susceptibility pattern of Staphylococcus epidermidis isolates in Tehran, Iran

Objective: Detection of sources of outbreaks caused by coagulase-negative Staphylococcus relies on molecular epidemiology methods. Little is known about the genetic diversity of the Staphylococcus epidermidis isolates isolated from various sources in Iran. We assessed the molecular epidemiology of S. epidermidis isolates collected from clinical and nonclinical sources from Tehran counties during 2014 to 2016 using MLVA (multilocus variable number tandem repeat analysis). Methods: One hundred and three clinical and nonclinical S. epidermidis isolates were collected from two hospitals in Tehran. Antibiotic susceptibility testing of isolates was evaluated for cefoxitin, tetracycline, erythromycin, clindamycin, mupirocin, vancomycin and linezolid according to Clinical and Laboratory Standards Institute, as well as prevalence of mecA gene was evaluated by PCR method. In addition, genetic relatedness of isolates was assessed by MLVA method. Results: Resistant rate to cefoxitin, tetracycline, erythromycin, clindamycin and mupirocin were 64, 36, 72, 44 and 23% in all isolates. All clinical and nonclinical isolates were susceptible to linezolid and vancomycin. In all, 49.5% of S. epidermidis isolates were multidrug resistant. Prevalence of mecA was 64%. The MLVA profile consists of a series of allele numbers, corresponding to the number of repeats at each variable number tandem repeat locus. The results of MLVA showed 64 types among all 103 isolates. There were 16 MLVA types that were common in two hospitals and 15 MLVA types were existed in various sources of S. epidermidis isolates. The diversity index obtained with MLVA was 0.989 (95% confidence interval [0.984–0.993]) for the 103 S. epidermidis isolates. A range of one to six alleles was identified at variable number tandem repeats loci with Simpson's diversity values between 0.526 and 0.781. Conclusion: Our study demonstrated presence of high molecular diversity among S. epidermidis isolates. In addition, the main conclusion was that some MLVA types can be disseminated over the wards and between the hospitals. In other hand, resistance to various antibiotics in S. epidermidis isolates and prevalence of methicillin-resistant S. epidermidis and multidrug resistant S. epidermidis isolates to be increasing.

Molecular characterization and analysis of Mycoplasma pneumoniae among patients of all ages with community-acquired pneumonia during an epidemic in China

BackgroundAnalysis of the molecular characteristics of isolates is very important for clinical and epidemiological study of community-acquired pneumonia (CAP) caused by Mycoplasma pneumoniae. MethodsBetween 2010 and 2012, an epidemic period, M. pneumoniae was isolated from oropharyngeal swabs of consecutive CAP patients. Minimum inhibitory concentrations of macrolides, 23S rRNA gene sequencing, P1 gene and multilocus variable-number tandem-repeat analysis (MLVA) genotyping was conducted. Results88.3% (181/205) of the isolates were macrolide-resistant M. pneumoniae (MRMP) and all harbored an A2063 G mutation. The strains were clustered into 7 MLVA types, and P1 type 1 and type 2 lineages were co-circulated (86.3% and 13.7%). Compared with adults, no specific MLVA type contributed to higher M. pneumoniae infection in children (p = 0.14). Similar macrolide profile and genotypes of M. pneumoniae was found between outpatients and inpatients. Significant differences in proportion of P1 types and two main MLVA types 4/5/7/2 and 3/5/6/2 were observed between MRMP and macrolide-sensitive M. pneumoniae (MSMP) (p < 0.001). ConclusionsThis study demonstrates a comprehensive profile of M. pneumoniae molecular characterization among CAP patients of all age, and provides more evidences on a correlation between MLVA type 4/5/7/2 and macrolide resistance in the setting of high incidence of MRMP.

Genetic Profiling and Comparison of Human and Animal Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates from Serbia.

The aim of this study was to characterize a collection of methicillin-resistant Staphylococcus aureus (MRSA) isolates of human and animal origin from Serbia. In total, 36 MRSA isolates—30 obtained from humans and six from companion animals—were investigated by PCR for the presence of antibiotic and biocide resistance determinants and virulence genes (PVL—Panton–Valentine leukocidin, ETs—exfoliative toxins, TSST—toxic shock syndrome toxin, SEs—staphylococcal enterotoxins, and MSCRAMMs—microbial surface components recognizing adhesive matrix molecules and biofilm). Isolates were analyzed by staphylococcal cassette chromosome mec (SCCmec), spa, and dru typing, as well as by multiple locus variable number of tandem repeat analyses (MLVA), multilocus sequence typing (MLST), and subsequently, eBURST. The majority of human MRSA isolates were resistant to gentamicin, erythromycin, clindamycin, and ciprofloxacin. Different antibiotic resistance genes were detected: aac-aphD, ant(6′)-Ia, erm(A), erm(B), erm(C), tet(K), tet(M), fexA, and catpC221. All isolates were susceptible to teicoplanin and linezolid. SCCmec type III was prevalent in human isolates, while SCCmec elements in animals were mostly nontypeable. t037 was the predominant spa type in human and t242 in animal MRSA isolates. The prevalent dru type was dt11c in human and dt10a in animal MRSA isolates. MRSA isolates exhibited 27 different MLVA types. ST239 was predominant in human, while ST5 was prevalent in canine MRSA isolates. PVL was found in two, while tsst-1 was detected in three human isolates. Human-associated clones belonging to ST5, ST45, and ST239 MRSA clones were discovered in companion animals, which suggests anthropozoonotic transmission.

Evaluation of Multilocus Variable-Number Tandem-Repeat (MLVA-8Orsay) for Typing of Carbapenem-Resistant Acinetobacter baumannii Isolated from Patients in Tehran, Iran

Objectives: Acinetobacter baumannii has emerged worldwide as a major nosocomial pathogen. The aim of this study was to evaluate the MLVA-8Orsay scheme suitability for clustering and differentiating A. baumannii strains isolated from two hospitals in Tehran. Methods: A panel of eight variable-number tandem-repeat (VNTR) markers was used for typing analysis (MLVA-8Orsay) of 89 carbapenem-resistant A. baumannii strains isolated from two hospitals in Tehran. Results: Abaum0845 (S-VNTR marker) was excluded from the MLVA scheme because of its great allelic diversity at this locus. The modified MLVA-7 scheme provided excellent discriminatory power and detected 82 MLVA types among the 83 isolates. Additionally, the great diversity associated with MLVA markers did not permit to apply the previously proposed criteria for clustering the isolates. However, six MLVA complexes were defined assuming a 71% genetic similarity cut-off, corresponding to the existence of identically sized alleles for at least five VNTR markers. The blaOXA-23-like gene (93.2%) was the most prevalent gene in resistant isolates collected from the two hospitals. Conclusions: MLVA-7 discriminatory power was found to be excellent. This method was also appeared to be useful and reliable in epidemiological investigations of A. baumannii strains. MLVA-7 analysis revealed that carbapenemase-carrying A. baumannii isolates might be polyclonal and provided evidence for gene transfer among different strains collected from the two hospitals. Overall, the study provided evidence supporting the use of MLVA-7 scheme as an alternative or complementary method for typing of A. baumannii isolates.

Prevalence of Extended-Spectrum β-Lactamases Among Klebsiella pneumoniae Isolated from Intensive Care Unit Patients in a Tertiary Hospital

Background: The increased prevalence of extended-spectrum β-lactamases (ESBLs)-producing virulent strains possibly leads to serious outbreaks in hospitals. Objectives: The aims of the study were to detect ESBLs in Klebsiella pneumoniae isolated from patients in intensive care units (ICUs) of a hospital and to investigate their genetic relationships. Methods: A total number of 85 K. pneumoniae isolates were detected in the specimens of ICU patients in a tertiary hospital in the southwest of Tehran, Iran. Antibiotic susceptibility was determined by the disk diffusion method. The rmpA and wcaG virulence genes were detected by polymerase chain reaction (PCR). The ESBL-positive isolates were typed using multilocus variable-number tandem repeat analysis (MLVA) method. Results: Of the 85 K. pneumoniae isolates, 48 (56.5%) produced ESBLs. There were statistically significant differences in the prevalence of virulence genes based on ESBL production. The typing of the 48 ESBL-positive isolates showed a significant genetic relatedness with the identification of nine MLVA types. Conclusions: This study showed a relatively high prevalence of ESBL in K. pneumoniae isolated from ICU patients and a low diversity between these isolates. The selection of ESBL-producing clones was mainly responsible for the spread of K. pneumoniae in the ICUs.

Genetic diversity of Mycoplasma hyopneumoniae in Mendoza province

In Argentina, enzootic pneumonia (EP) is highly prevalent and different genetic types of Mycoplasma hyopneumoniae have been identified. However, there is a lack of information about prevalence and other epidemiological aspects of EP in Mendoza province. A multiple Locus variable-number tandem repeat analysis (MLVA) targeting P97 R1, P97 R1A and P146 R3 loci was used to assess the genetic diversity of M. hyopneumoniae from clinical specimens recovered from pigs from five herds located in different districts of Mendoza province. M. hyopneumoniae could be typed from 27 bronchoalveolar lavages (BAL) specimens, and eight different MLVA types were identified. This is the first report about diversity of M. hyopneumoniae in Mendoza. Results obtained in this work allow drawing a better picture of the genetic diversity of this pathogen in Argentina.

First detection and characterization of macrolide-resistant Mycoplasma pneumoniae strains in Cuba

ObjectivesMycoplasma pneumoniae is a common cause of community-acquired pneumonia in humans. Treatment of infections can be complicated by the occurrence of macrolide resistant strains. The study was conducted to evaluate the presence of resistant strains in Cuba and to determine the corresponding genotypes. MethodsDNA of M. pneumoniae isolates and positive respiratory tract specimens collected in the years 2012 and 2017 were tested for resistance-associated mutations of 23S rRNA. In addition, strain types (P1 and MLVA) were determined. ResultsMacrolide resistance mutations were confirmed in 5 out of 27 strains (18.5%). Whereas both P1 subtypes 1 and 2 as well variants V2a and V2c were identified, only two MLVA types (4/5/7/2 and 3/5/6/2) could be found. ConclusionsDuring both sampling years, circulation of macrolide resistant strains was demonstrated. No association of resistance with a particular P1/MLVA type was found. Future longitudinal sampling to monitor prevalence of macrolide resistance of M. pneumoniae is recommended to verify the resistance pattern of this important pathogen of human respiratory tract infections.

Genomic epidemiology of erythromycin-resistant Bordetella pertussis in China

ABSTRACT Macrolides such as erythromycin are the empirical treatment of Bordetella pertussis infections. China has experienced an increase in erythromycin-resistant B. pertussis isolates since they were first reported in 2013. Here, we undertook a genomic study on Chinese B. pertussis isolates from 2012 to 2015 to elucidate the origins and phylogenetic relationships of erythromycin-resistant B. pertussis isolates in China. A total of 167 Chinese B. pertussis isolates were used for antibiotic sensitivity testing and multiple locus variable-number tandem repeat (VNTR) analysis (MLVA). All except four isolates were erythromycin-resistant and of the four erythromycin-sensitive isolates, three were non-ptxP1. MLVA types (MT), MT55, MT104 and MT195 were the predominant types. Fifty of those isolates were used for whole genome sequencing and phylogenetic analysis. Genome sequencing and phylogenetic analysis revealed three independent erythromycin-resistant lineages and all resistant isolates carried a mutation in the 23S rRNA gene. A novel fhaB3 allele was found uniquely in Chinese ptxP1 isolates and these Chinese ptxP1-ptxA1-fhaB3 had a 5-fold higher mutation rate than the global ptxP1-ptxA1 B. pertussis population. Our results suggest that the evolution of Chinese B. pertussis is likely to be driven by selection pressure from both vaccination and antibiotics. The emergence of the new non-vaccine fhaB3 allele in Chinese B. pertussis population may be a result of selection from vaccination, whereas the expansion of ptxP1-fhaB3 lineages was most likely to be the result of selection pressure from antibiotics. Further monitoring of B. pertussis in China is required to better understand the evolution of the pathogen.

Population dynamics and antigenic drift of Bordetella pertussis following whole cell vaccine replacement, Barcelona, Spain, 1986–2015

ABSTRACT Among the factors associated with the resurgence of whooping cough, special emphasis has been given to pathogen adaptation after the introduction of the acellular vaccine (ACV). To assess the impact of the vaccine transition strategy from whole-cell vaccine (WCV) to ACV on population dynamics of Bordetella pertussis in Barcelona (Spain), we studied 339 isolates collected from 1986 to 2015 by PFGE and multi-locus variable-number tandem repeat analysis (MLVA). Additionally, allelic variants for the pertussis toxin and its promoter, pertactin, type 3 fimbriae and fimbrial serotyping were assessed to determine its antigenic drift. A shift was observed in the B. pertussis population as well as in its antigenic profile concurrently with the introduction of ACV in Barcelona. Four out of the five most prevalent PFGE profiles were replaced by new profiles following the ACV introduction. MLVA type 27 was the dominant genotype, and its frequency increased from 25% to 79.3% after WCV replacement. Antigen typing demonstrated the emergence of prn2, ptxP3, fim3-2 and a shift from the fimbriae 3 to the fimbriae 2 serotypes after the ACV introduction. Our findings support the presence of population and antigenic dynamic changes in B. pertussis likely driven by the introduction of ACV.

Genetic Profiling of the Causative Agents of Natural-Focal infections, Circulating in the Stavropol Territory

Objective of the study was genetic typing of the strains and nucleic acid isolates of causative agents of natural focal infections, both bacterial and viral etiology, accumulation of data on genetic features of regional strains cir­culating in the Stavropol Territory . Material and methods. To study the genetic spectrum of causative agents of natural- focal infections, analysis of the strains and nucleic acids isolates detected in the samples of field and clinical material was carried out. Indication of causative agents of natural focal infections in the samples was carried out by PCR. For genetic typing of the strains and DNA/RNA isolates of natural focal infections agents, MLVA and sequencing of genome regions with subsequent phylogenetic analysis were used. The analysis of territorial distribution of the causative agent genetic variants and mapping was performed in ArcGIS 10.1. Results and conclusions. MLVA-25 typing of 20 strains of Francisella. tularensis, MLVA-10 typing of 4 Coxiella burnetii isolates, species identification of 20 isolates of Rickettsia sp., sub-species genetic typing of 40 RNA isolates of CCHF virus and 8 RNA isolates of hantaviruses circulating in the Stavropol Territory in 2016-2017 were performed. The studied strains of F. tularensis belong to eight MLVA genotypes. They are mainly confined to specific areas. The isolates of C. burnetii have the same MLVA type. Rickettsia, belonging to 5 species: R. massiliae R. raoultii, R. sibirica, R. aeschlimannii, R. slovaca, RNA-isolates of hantavirus of the «Tula» genotype and variants of the CCHF virus of the Europe-1 and Europe-3 genotypes were identified. The obtained data can be used in the epidemiological investigation of possible cases of infectious diseases to determine the source and pathways of infection.

Genomic Study of a Clostridium difficile Multidrug Resistant Outbreak-Related Clone Reveals Novel Determinants of Resistance.

Background: Clostridium difficile infection (CDI) is prevalent in healthcare settings. The emergence of hypervirulent and antibiotic resistant strains has led to an increase in CDI incidence and frequent outbreaks. While the main virulence factors are the TcdA and TcdB toxins, antibiotic resistance is thought to play a key role in the infection by and dissemination of C. difficile.Methods: A CDI outbreak involving 12 patients was detected in a tertiary care hospital, in Lisbon, which extended from January to July, with a peak in February, in 2016. The C. difficile isolates, obtained from anaerobic culture of stool samples, were subjected to antimicrobial susceptibility testing with Etest®strips against 11 antibiotics, determination of toxin genes profile, PCR-ribotyping, multilocus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS).Results: Of the 12 CDI cases detected, 11 isolates from 11 patients were characterized. All isolates were tcdA-/tcdB+ and belonged to ribotype 017, and showed high level resistance to clindamycin, erythromycin, gentamicin, imipenem, moxifloxacin, rifampicin and tetracycline. The isolates belonged to four genetically related MLVA types, with six isolates forming a clonal cluster. Three outbreak isolates, each from a different MLVA type, were selected for WGS. Bioinformatics analysis showed the presence of several antibiotic resistance determinants, including the Thr82Ile substitution in gyrA, conferring moxifloxacin resistance, the substitutions His502Asn and Arg505Lys in rpoB for rifampicin resistance, the tetM gene, associated with tetracycline resistance, and two genes encoding putative aminoglycoside-modifying enzymes, aadE and aac(6′)-aph(2″). Furthermore, a not previously described 61.3 kb putative mobile element was identified, presenting a mosaic structure and containing the genes ermG, mefA/msrD and vat, associated with macrolide, lincosamide and streptogramins resistance. A substitution found in a class B penicillin-binding protein, Cys721Ser, is thought to contribute to imipenem resistance.Conclusion: We describe an epidemic, tcdA-/tcdB+, multidrug resistant clone of C. difficile from ribotype 017 associated with a hospital outbreak, providing further evidence that the lack of TcdA does not impair the infectious potential of these strains. We identified several determinants of antimicrobial resistance, including new ones located in mobile elements, highlighting the importance of horizontal gene transfer in the pathogenicity and epidemiological success of C. difficile.

Diversity of Yersinia enterocolitica isolated from pigs in a French slaughterhouse over 2years.

The pig is one of the main reservoirs of Yersinia enterocolitica strains pathogenic to humans. A description of the Y. enterocolitica population in this reservoir, and accurate discriminatory techniques for typing isolates are needed for prevention, outbreak investigation, and surveillance. This study investigates the genetic diversity of pathogenic Y. enterocolitica isolates obtained from pig tonsils in a French pig slaughterhouse in 2009 (S1) and 2010 (S2). The use of Pulsed‐Field Gel Electrophoresis (PFGE) and MLVA as typing techniques was also compared and evaluated. First, a total of 167 isolates (12 of biotype 3 recovered during S1, and 155 of biotype 4 recovered during S1 and S2) were typed by PFGE using the XbaI enzyme. MLVA was then tested on all the biotype 3 isolates in addition to 70 selected biotype 4 isolates recovered over the 2 years. PFGE generated two specific XbaI‐PFGE profiles for biotype 3 isolates. Nine XbaI profiles were obtained for biotype 4, with a higher diversity (ID = 0.599) than biotype 3 (ID = 0.167). Two out of the nine XbaI profiles were reported during both surveys and at different months. MLVA improved the differentiation between isolates; the index of diversity reached 0.621 and 0.958, respectively, for biotype 3 (three MLVA types) and biotype 4 (32 MLVA types). The MLVA types for biotype 4 differed over the two surveys, but some isolates with different MLVA types were genetically closely related. This study provides an initial evaluation of the genetic diversity of Y. enterocolitica strains isolated from pigs in France. We show that some PFGE profiles are maintained in the pig production sector, and, through MLVA, that part of the Y. enterocolitica population remained genetically close over the two years. MLVA proved its effectiveness as a tool for investigating pathogenic Y. enterocolitica strains isolated from pigs.

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.

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.

Colonization of Pigs Experimentally Infected with a Monophasic Variant of Salmonella Typhimurium.

The monophasic variant of Salmonella Typhimurium is highly prevalent in human and in pork. However, little is known about colonization dynamics and serology in pigs. We orally inoculated 24 seven-week-old piglets with 109 CFU/pig of a porcine strain of monophasic Salmonella Typhimurium in an experimental trial. Three groups of eight piglets were orally inoculated and monitored for 21, 49, or 84 days post-inoculation until necropsied. From 3 days post-inoculation to necropsy, individual feces were sampled twice weekly and blood once weekly. At necropsy, the tonsils, mesenteric lymph nodes, and the contents of the duodenum, jejunum, ileum, and cecum were collected from each pig. We determined the number of CFU/g in all the samples and measured also Salmonella antibodies in OD% in all blood samples. At different times during the trial, we tested by MLVA (Multilocus Variable Number Tandem Repeat Analysis) the genomic stability of the strain after passing through the intestinal tract. Salmonella was continuously excreted by pigs, ranging from 1.4 to 5.8 log10 CFU/g. At necropsy, Salmonella was present in all samples, but the tonsils were particularly infected. Salmonella antibodies were detected in five pigs 7 days post-inoculation. At 49 days post-inoculation, all the pigs were seropositive. We observed new MLVA types for 3.3% of the isolates tested over the trial. Our study allowed us to show the serovar's ability to persist in pigs after infection up to 84 days post-inoculation. We demonstrated that Salmonella seroconversion appeared earlier than in naturally infected pigs and that the strain's genome can evolve after passing through the digestive tract of pigs.

Diversity Determination of CTX-M1 Producing Klebsiella pneumoniae Using Multilocus Variable-Number Tandem Repeat Analysis, Semnan, Iran

Background: CTX-M is the most prevalent and rapidly growing type of the extended-spectrum β-lactamase (ESBL) family and CTX-M1 is the most common type of blaCTX-M. Objectives: The current study aimed at investigating the genetic diversity of CTX-M-1-producing Klebsiella pneumoniae circulating in Semnan, Iran evaluated by multilocus variable-number tandem repeat analysis (MLVA). Methods: A total of 110 isolates of K. pneumoniae were collected from different clinical samples. The antibiotic suceptibility and double disk synergy test were determined according to CLSI (the clinical and laboratory standards institute) guidelines. The polymerase chain reaction (PCR) method was performed to detect CTX-M-1. The eight loci for MLVA genotyping were selected along with the primers previously described. Results: Imipenem, with 84.7% susceptibility, was the most effective antibiotic against K. pneumoniae. Seventy (63.63%) isolates had ESBL positive results and 42 (60 %) of them were positive for CTX-M-1 gene. Totally, 28 MLVA genotypes were discriminated, evaluation of diversity indexes (DIs) for eight loci showed that six different alleles were the most polymorphic and the most DI was 0.807. Conclusions: The findings of the current study demonstrated heterogeneity among CTX-M-1-producing K. pneumonia strains. The presence of CTX-M-1 in different MLVA types demonstrated that a certain clone is not responsible for spreading the isolates.

Antimicrobial susceptibility and MLVA analysis of S. Typhimurium strains isolated from human and poultry samples in Tunisia.

Salmonella enterica infections are a significant public health concern worldwide, being Salmonella Typhimurium one of the most prevalent serovars. Human salmonellosis is typically associated with the consumption of contaminated foods, such as poultry, eggs and processed meat. The extensive use of antimicrobials in humans and animals has led to an increase in multidrug resistance among Salmonella strains, becoming multidrug-resistant (MDR) strains a major public health concern. This study was designed to investigate the antimicrobial susceptibility and the genotypic diversity of Salmonella Typhimurium strains isolated in Tunisia from human and poultry sources from 2009 to 2015. Fortyfive strains were analyzed by disk-diffusion test to determine the antimicrobial susceptibility. The presence of antimicrobial resistance genes was tested by PCR, and genotyping was performed using multiple-locus variable-number tandem repeats analysis (MLVA). About 50% of the strains were resistant to at least 3 antibiotics (multidrug-resistant strains, MDR). The most frequent resistance profile in clinical strains was AMP-TIC-TET-MIN-SXT (n = 7) and TET-MIN in poultry origin strains (n = 7). The MLVA typing grouped the strains in 2 main clusters. Cluster I was mostly formed by human isolates, whereas in cluster II both human and poultry isolates were grouped. Simpson's diversity index was 0.870 and 0.989 for antimicrobial resistance profiles and MLVA, respectively. Multiresistance is common in Salmonella Typhimurium isolated from human and poultry sources in Tunisia. The genotyping results suggest that some strains isolated from both sources may descend from a common subtype.

Molecular typing characterization of food-borne methicillin-resistant Staphylococcus aureus in China

Objective: To analyses the antimicrobial resistance and molecular characterization of 21 MRSA isolates cultured from retail foods from different provinces in China, and evaluate the molecular typing methods. Methods: Twenty-one MRSA isolates were obtained from national foodborne pathogen surveillance network in 2012 (Chinese salad, n=3; milk, n=1; cake, n=2; rice, n=1; cold noodle, n=1; spiced beef, n=1; dumpling, n=1; packed meal, n=1; salad, n=1; raw pork, n=9). The antimicrobial resistance of 21 strains to 12 antimicrobial agents was tested by broth dilution method. Polymerase chain reaction (PCR) and DNA sequencing were performed to obtain the genetic types of MLST (ST) and spa typing. The clonal complex (CC) was assigned by eBURST soft and the MLVA type (MT) and MLVA complex (MC) were identified via the database of the MLVA website (http://www.mlva.net). SmaI pulsed-field gel electrophoresis (SmaⅠ-PFGE) was also carried out to obtain the PFGE patterns of 21 strains. The genetic diversity and discriminatory power of typing were calculated by the Simpson's index of diversity (diversity index, DI) to find out the best genotyping method for MRSA. Results: All MRSA isolates showed multi-drug resistance(MDR), and were resistant to oxacillin, benzylpenicillin, clindamycin and erythromycin, and 71.4% (15/21), 47.6% (10/21), 42.9% (9/21) and 28.6% (6/21) of the MRSA isolates were resistant to tetracycline, ciprofloxacin, trimethoprim/sulfamethoxazole and gentamicin, respectively. Moreover, one strain was found to be resistant to all three antimicrobials of levofloxacin, moxifloxacin and rifampicin. Great diversity was found in these food-associated MRSA (6 STs, 7 spa types, and 9 MTs). PFGE patterns were more diverse than those of other three molecular typing methods (19 pulse types). The index of diversity (DI) of PFGE, MLVA, spa typing and MLST was 0.99, 0.80, 0.73, and 0.61, respectively. Among the MRSA isolates, CC9-ST9-t899-MT929-MC2236 (PFGE Cluster Ⅴ) was the most prevalent clone, which were all cultured from raw pork (9 isolates). Besides, two MRSA were identified as CC59-ST338-t437-MT621-MC621 (PFGE Cluster Ⅳ). Different clone had their own resistance spectrum profiles. Conclusion: The food-borne MRSA isolates were all MDR in this study. Different clones had their own resistance spectrum profiles. MLVA represented a promising tool for molecular epidemiology tracing of MRSA in foodborne disease events.

Retrospective genome-wide comparisons of Salmonella enterica serovar Enteritidis from suspected outbreaks in Singapore

The number of salmonellosis cases in Singapore has increased over the years. Salmonella enterica serovar Enteritidis has always been the most predominant serovar in the last five years. The National Public Health Laboratory assisted outbreak investigations by performing multilocus variable number tandem repeat analysis (MLVA) on isolates that were collected at the time of the investigations. Isolates were defined as belonging to a particular cluster if they had identical MLVA patterns. Whilst MLVA has been instrumental in outbreak investigations, it may not be useful when outbreaks are caused by an endemic MLVA type. In this study, we analysed 67 isolates from 12 suspected outbreaks with known epidemiological links to explore the use of next-generation sequencing (NGS) for defining outbreaks. We found that NGS can confidently group isolates into their respective outbreaks. The isolates from each suspected outbreak were closely related and differed by a maximum of 3 single nucleotide polymorphisms (SNPs). They were also clearly separated from isolates that belonged to different suspected outbreaks. This study provides an important insight and further evidence on the value of NGS for routine surveillance and outbreak detection of S. Enteritidis.