Multiple Locus Sequence Typing Research Articles

Genetic diversity of endosymbiotic bacteria Wolbachia infecting two mosquito species of the genus Eretmapodites occurring in sympatry in the Comoros archipelago.

The influence of Wolbachia on mosquito reproduction and vector competence has led to renewed interest in studying the genetic diversity of these bacteria and the phenotypes they induced in mosquito vectors. In this study, we focused on two species of Eretmapodites, namely Eretmapodites quinquevittatus and Eretmapodites subsimplicipes, from three islands in the Comoros archipelago (in the Southwestern Indian Ocean). Using the COI gene, we examined the mitochondrial genetic diversity of 879 Eretmapodites individuals from 54 sites. Additionally, we investigated the presence and genetic diversity of Wolbachia using the wsp marker and the diversity of five housekeeping genes commonly used for genotyping through Multiple Locus Sequence Typing (MLST). Overall, Er. quinquevittatus was the most abundant species in the three surveyed islands and both mosquito species occurred in sympatry in most of the investigated sites. We detected a higher mitochondrial genetic diversity in Er. quinquevittatus with 35 reported haplotypes (N = 615 specimens, Hd = 0.481 and π = 0.002) while 13 haplotypes were found in Er. subsimplicipes (N = 205 specimens, Hd = 0.338 and π = 0.001), this difference is likely due to the bias in sampling size between the two species. We report for the first time the presence of Wolbachia in these two Eretmapodites species. The prevalence of Wolbachia infection varied significantly between species, with a low prevalence recorded in Er. quinquevittatus (0.8%, N = 5/627) while infection was close to fixation in Er. subsimplicipes (87.7%, N = 221/252). Both male and female individuals of the two mosquito species appeared to be infected. The analysis of MLST genes revealed the presence of two Wolbachia strains corresponding to two new strain types (STs) within the supergroups A and B, which have been named wEretA and wEretB. These strains were found as mono-infections and are closely related, phylogenetically, to Wolbachia strains previously reported in Drosophila species. Finally, we demonstrate that maternal transmission of Wolbachia is imperfect in Er. subsimplicipes, which could explain the presence of a minority of uninfected individuals in the field.

Brucella abortus in Kazakhstan, population structure and comparison with worldwide genetic diversity.

Brucella abortus is the main causative agent of brucellosis in cattle, leading to severe economic consequences in agriculture and affecting public health. The zoonotic nature of the infection increases the need to control the spread and dynamics of outbreaks in animals with the incorporation of high resolution genotyping techniques. Based on such methods, B. abortus is currently divided into three clades, A, B, and C. The latter includes subclades C1 and C2. This study presents the results of whole-genome sequencing of 49 B. abortus strains isolated in Kazakhstan between 1947 and 2015 and of 36 B. abortus strains of various geographic origins isolated from 1940 to 2004. In silico Multiple Locus Sequence Typing (MLST) allowed to assign strains from Kazakhstan to subclades C1 and to a much lower extend C2. Whole-genome Single-Nucleotide Polymorphism (wgSNP) analysis of the 46 strains of subclade C1 with strains of worldwide origins showed clustering with strains from neighboring countries, mostly North Caucasia, Western Russia, but also Siberia, China, and Mongolia. One of the three Kazakhstan strains assigned to subclade C2 matched the B. abortus S19 vaccine strain used in cattle, the other two were genetically close to the 104 M vaccine strain. Bayesian phylodynamic analysis dated the introduction of B. abortus subclade C1 into Kazakhstan to the 19th and early 20th centuries. We discuss this observation in view of the history of population migrations from Russia to the Kazakhstan steppes.

A pseudo-outbreak of MRSA due to laboratory contamination related to MRSA carriage of a laboratory staff member

BackgroundMethicillin resistant Staphylococcus aureus (MRSA) is a major burden for hospitals globally. However, in the Netherlands, the MRSA prevalence is relatively low due to the ‘search and destroy’ policy. Routine multiple-locus variable-number of tandem repeat analysis (MLVA) of MRSA isolates supports outbreak detection. However, whole genome multiple locus sequence typing (wgMLST) is superior to MLVA in identifying (pseudo-)outbreaks with MRSA. The present study describes a pseudo-outbreak of MRSA at the bacteriology laboratory of a large Dutch teaching hospital.MethodsAll staff members of the bacteriology laboratory of the Elisabeth-TweeSteden hospital were screened for MRSA carriage, after a laboratory contamination with MRSA was suspected. Clonal relatedness between the index isolate and the MRSA isolates from laboratory staff members and all previous MRSA isolates from the Elisabeth-TweeSteden hospital with the same MLVA-type as the index case was examined based on wgMLST using whole genome sequencing.ResultsOne of the staff members was identified as the probable source of the laboratory contamination, because of carriage of a MRSA possessing the same MLVA-type as the index case. Eleven other isolates with the same molecular characteristics were found in the database, of which seven were retrospectively suspected of contamination. Clonal relatedness was found between ten isolates, including the isolate found in the staff member and the MRSA found in the index patient with a maximum of eleven alleles difference. All isolates were epidemiologically linked through the laboratory staff member, who had worked on all these cultures.ConclusionsThe present study describes a MRSA pseudo-outbreak over a 2.5-year period due to laboratory contamination caused by a MRSA carrying laboratory staff member involving nine patients. In case of unexpected bacteriological findings, the possibility of a laboratory contamination should be considered.

Caracterização fenotípica e genotípica de isolados de Brucella abortus biovar 4 em bovinos no Brasil

ABSTRACT: The aim of the present study was to characterize (phenotypically and genotypically) two strains of Brucella abortus identified as belonging to biovar 4 isolated from cattle in Brazil. The strains were isolated from cervical bursitis from cattle in the states of Pará and Rio Grande do Sul, respectively. In the phenotypic identification, the isolates were positive in CO2 requirement, produced H2S, were resistant to basic fuchsin (20 µg / mL) and sensitive to thionin (20 µg / mL and 40 µg / mL) and presented M surface antigen, but A surface antigen is absent. The isolates were positive in the PCR for the bcsp31 gene (genus-specific) and in the AMOS-enhanced PCR, both isolates showed a band profile consistent with B. abortus biovar 1, 2 or 4. Moreover, both isolates also showed restriction patterns identical to the reference strain when tested by the omp2b PCR-RFLP. In genotyping using Multiple Locus Variable Number of Tandem Repeat (VNTR) Analysis - MLVA (MLVA16), the isolates showed differences in several loci (Bruce42, Bruce19, Bruce04, Bruce16 and Bruce30); by Multiple Locus Sequence Typing (MLST), they also exhibited differences in sequence type (ST), strain 16/02 ST1 (2-1-1-2-1-3-1-1-1) and strain 128/11 ST (22-1-1 -8-9-3-1-1-1). The extensive typing of B. abortus strains isolated from cattle in Brazil using different approaches confirmed the occurrence of rare B. abortus biovar 4 in the country.

High frequency of colonization by extended-spectrum beta-lactamase-producing Gram-negative bacilli in hemodialysis patients and their household contacts in Colombia: dissemination between the community and the hospital.

Increasing colonization by beta-lactam-resistant Gram-negative bacilli (BR-GNB) represents a risk for infections and bacterial resistance spread, both in hospitals and the community. Hemodialysis patients and their household contacts regularly transit between these environments. This study investigated the clinical and epidemiological characteristics of BR-GNB colonization in hemodialysis patients and their household contacts, as well as the genetic relationship between their isolates. A cross-sectional study was conducted on hemodialysis patients at a hospital-associated dialysis center in Medellín, Colombia and their household contacts. Clinical and epidemiological information was collected. Colonization was assessed from stool or rectal swab samples. Bacterial identification and susceptibility were determined using chromogenic media and Vitek-2. Molecular characterization included beta-lactamase detection by polymerase chain reaction, multiple-locus sequence typing (MLST), pulsed-field gel electrophoresis, and identification of Escherichia coli phylogroups by the Clermont protocol. This study included 36 hemodialysis patients and 90 household contacts. Colonization by BR-GNB occurred in 58.3% of patients and 22.2% of household contacts. The main beta-lactamase detected was CTX-M group-1 (40.5%). In 3 of the 9 homes that had more than 1 colonized individual, a genetic relationship was found. MLST showed a high diversity in E. coli isolates, and the most frequent phylogroups were B1 and B2. These results show a high frequency of colonization and the presence of potentially pathogenic BR-GBN both in hospitals and the community. This highlights the importance of populations who move between those 2 environments, and the need to prevent the spread of bacterial resistance outside hospitals.

Isolation and characterization of Staphylococcus argenteus strains from retail foods and slaughterhouses in Japan

Staphylococcus argenteus has been recently established as a novel species of Staphylococcus aureus complex. It is known to cause various human diseases, such as skin and soft-tissue infections, sepsis, and staphylococcal food poisoning, although the source of infection has not been clearly described. In food poisoning cases, the source of bacterial contamination in food is unknown. This study examined the prevalence of S. argenteus among retail fresh food and poultry slaughterhouses in Japan. Among 642 food samples examined, successful isolation of S. argenteus was achieved in 21 of 151 (13.9%) chicken samples. No isolations from pork, beef, fish, or vegetables in retail markets were confirmed. Multiple-locus sequence typing revealed that the 21 isolates were classified into four sequence types (ST) that were divided into 14 subtypes using spa-typing. All food isolates were susceptible to methicillin and did not show positivity for the Panton-Valentine leukocidin gene. When bacteria were isolated from two poultry slaughterhouses in the same region, 14 S. argenteus strains were successfully isolated from only one slaughterhouse. Thirteen of 14 strains were isolated from a poultry carcass and slaughterhouse environments during a certain sampling period and were all classified as ST5961 with identical spa-type. Also, the number of single nucleotide variants (SNVs) detected on the core genomes of the same 13 strains were between 0 and 17, suggesting a long-term inhabitation of an S. argenteus strain inside the facility. Furthermore, one isolate from chicken meat was also genetically linked with the same lineage of slaughterhouse isolates, with ≤15 SNVs being detected. Additionally, one slaughterhouse isolate from chiller water and three chicken isolates were classified into the same cluster by phylogenetic analysis, although the number of pairwise SNVs ranged from 62 to 128. To the best of the authors' knowledge, this is the first study that demonstrated S. argenteus in a food processing facility and the possible bacterial contamination on food during food processing.

Molecular Epidemiological Analysis of ST11-K64 Extensively Drug-Resistant Klebsiella pneumoniae Infections Outbreak in Intensive Care and Neurosurgery Units Based on Whole-Genome Sequencing.

Klebsiella pneumoniae (Kp) is the primary causative bacteria for nosocomial infections and hospital outbreaks. In particular, extensively drug-resistant K. pneumoniae (XDRKp) causes severe clinical infections in hospitalized patients. Here, we used pulsed-field gel electrophoresis (PFGE), drug susceptibility tests, and the whole-genome sequencing (WGS) technology to examine genetic relatedness and phenotypic traits of the strains isolated during an outbreak period. Based on PFGE, a distinct clones cluster comprised of eight XDRKp was observed. These strains were confirmed as ST11-K64 via multiple-locus sequence typing database of Kp. The strains also had genes related to the regulation of biofilm biosynthesis (type 1 & 3 fimbriae, type IV pili biosynthesis, RcsAB, and type VI secretion system) and multiple drug resistance (β-lactamase and aminoglycoside antibiotic resistance). WGS data based on core-single nucleotide polymorphisms and epidemiological investigation showed that the neurosurgery unit was likely the source of the outbreak, the strain was likely to have been transmitted to the ICU through patients. In addition, the two highly probable transmission routes were in the ICU (exposure through shared hospital beds) and the neurosurgery units (all cases were treated by the same rehabilitation physician and were most likely infected during the physical therapy). Notably, the bed mattress had played a crucial transmission role of this outbreak, served as a pathogen reservoir.

Multidrug-resistant Klebsiella quasipneumoniae subsp. similipneumoniae carrying blaNDM-blaCTX-M15 isolated from flies in Rio de Janeiro, Brazil

ObjectivesFlies have been implicated in the dispersal of medically important bacteria including members of the genus Klebsiella between different environmental compartments. The aim of this study was to retrieve and characterize antibiotic-resistant bacteria from flies collected near to hospitals. MethodsFlies were collected in the vicinity of medical facilities and examined for bacteria demonstrating phenotypic resistance to ceftriaxone, followed by determination of phenotypic and genotypic resistance profiles. In addition, whole genome sequencing followed by phylogenetic analysis and resistance genotyping were performed with the multidrug-resistant (MDR) strain Lemef23, identified as Klebsiella quasipneumoniae subsp. similipneumoniae. ResultsThe strain Lemef23, classified by multiple locus sequence typing as novel ST 3397, harboured numerous resistance genes. The blaNDM was located on a Tn3000 element, a common genetic platform for the carriage of this gene in Brazil. Inference of phylogenetic orthology of strain Lemef23 and other clinical isolates suggested an anthropogenic origin. ConclusionsThe findings of this study support the role of flies as vectors of MDR bacteria of clinical importance and provide the first record of blaNDM-1 and blaCTXM-15 in a Brazilian isolate of K. quasipneumoniae subsp. similipneumoniae, demonstrating the value of surveying insects as reservoirs of antibiotic resistance.

Genotypic Characteristics and Correlation of Epidemiology of Staphylococcus aureus in Healthy Pigs, Diseased Pigs, and Environment.

China is one of the largest producers of pigs and pork in the world. However, large-scale studies on pig-associated Staphylococcus aureus in relation to healthy pigs, diseased pigs and environment are scarce. The objective of the present study was to characterize and compare S. aureus isolates from healthy pigs, diseased pigs and environment through antimicrobial susceptibility testing, multiple locus sequence typing, spa typing, and antimicrobial resistance gene screening. Results showed all isolates were susceptible to linezolid and vancomycin. However, 66.7% (104/156) isolates were multidrug-resistant by displaying resistance to three or more antibiotics and high rates of resistance to penicillin, tetracycline, clindamycin, and clarithromycin were observed. Of the 20 multilocus sequence types (STs) identified among the isolates, ST9, ST188, and ST7 were most commonly isolated from healthy pigs and environment, while ST1 was most commonly isolated from diseased pigs. In total, 17 spa types were represented among the isolates, while t4792 was most commonly isolated from diseased pigs and t899, t189 were most commonly isolated from healthy pigs and environment. In conclusion, the genotypic and epidemiology characteristics observed among the isolates suggest pigs and pork could be important players in S. aureus dissemination.

Cross-sectional evaluation of multiple epidemiological cycles of Leptospira species in peri-urban wildlife in California.

To perform a cross-sectional survey to estimate prevalence of and potential risk factors for Leptospira spp infection and exposure in peri-urban wildlife throughout California. 723 animals representing 12 wildlife species. Blood and urine samples were obtained from wildlife in California from 2007 to 2017. Live animals were captured in humane traps, anesthetized, and released. Carcasses were donated by wildlife services and necropsied for urine, blood, and kidney tissue samples. Samples were tested for antibodies against 6 serovars of Leptospira spp with a microscopic agglutination test and for pathogenic Leptospira spp DNA with a real-time PCR assay targeting the LipL32 gene. Potential risk factors for Leptospira spp exposure were assessed by logistic regression. Genetic relatedness of Leptospira spp were assessed with DNA sequencing of the rrs2 gene and multiple locus sequence analysis. Statewide Leptospira spp seroprevalence was 39.1%, and prevalence of positive PCR assay results for Leptospira spp DNA was 23.0%. Risk factors for Leptospira spp exposure included being an adult, being from northern California, and being a western gray squirrel, coyote, striped skunk, raccoon, gray fox, or mountain lion. Antibodies against serovar Pomona predominated in most species, followed by serovar Copenhageni. Complete rrs2 sequences were identified as Leptospira interrogans and multiple locus sequence type analysis revealed sequence type 140. Pathogenic Leptospira spp appeared to be common and widespread among peri-urban wildlife in California. Our data highlight the potential for exposure to infectious disease for both humans and domestic animals at the urban-wildland interface.

Isolation and identification of Leptospira in patients with fever of unknown origin in Guizhou province

Objective: To understand the etiological characteristics of the patients with fever of unknown origin in Guizhou province through the isolation and identification of Leptospira interrogans and provide evidence for the control, prevention and treatment of human leptospirosis. Methods: Blood and urine samples were collected from patients with fever symptoms in Qiandongnan, an epidemic area, in Guizhou. The suspected Leptospira strains were primarily identified using pathogenic Leptospira specific G1/G2-PCR, and subsequently identified by using Leptospira serogroups specific PCR. The Leptospira strains were then genotyped with multiple locus sequence typing. MLST data based cluster analysis on the isolates and Leptospira reference strains of common serogroups were analyzed by using software NTsys 2.10e. Results: Three suspected strains of Leptospira were isolated from human blood samples, the isolation rate was 8.６%, which were designated as strain 17BX002, 17BX003 and 17AJX008. Strain 17BX002 was further identified as serogroup grippotyphosa by using Leptospira serogroup specific PCR, while the other two strains were negative (excluded as iterohaemorrhagiae, sejroe, canicola, autumnalis, grippotyphosa and hebdomadis). MLST genotyping showed that strain 17BX002 was typed as ST106, most closely clustered with Leptospira grippotyphosa, while strain 17BX003 and 17AJX008 were typed as ST96, the same as serogroup badaviae. Conclusion: There are leptospirosis cases in epidemic area of Guizhou in high incidence season, grippotyphosa and bataviae are the newly discovered serogroups of Leptospira in Guizhou.

Molecular Characterization and Antibiotic Profile of Clostridium difficile Isolated from Bacteremia

Introduction: Clostridium difficile infection always manifests as diarrhea associated with antibiotic use. Nevertheless, extraintestinal infections caused by C. difficile are reported. The current report was on a case of C. difficile bacteremia in a 51-year-old male patient with appendiceal perforation and abscess as well as confirmed cirrhosis. Case Presentation: The patient was admitted to the infectious diseases department with repeated abdominal pain associated with fever. His anaerobic blood culture was positive for C. difficile two days after transarterial chemoembolization. The same toxin strain was isolated from stool four days later. Both of the two isolates were confirmed positive for toxin A (tcd A) and toxin B (tcd B) genes by polymerase chain reaction, and identified as ST3 by multiple locus sequence typing. The two strains showed the same susceptibility to the tested antibiotics. The patient was treated with vancomycin intravenously, and got remitted shortly after that. The patient was discharged with good general health conditions and followed up. Conclusions: A patient with C. difficile bacteremia presenting with appendiceal perforation and abscess as well as confirmed cirrhosis was described, and the microbiological and molecular biological analysis suggested that C. difficile strains isolated from blood came from gut. It is necessary that clinicians detect C. difficile and/or toxins in patients with long terms antibiotic therapy, in case of transformation of intestinal flora, which could cause the infection outside the intestinal tract.

Sequential emergence of colistin and rifampicin resistance in an OXA-72- producing outbreak strain of Acinetobacter baumannii

ObjectivesThis study reported a hospital outbreak due to an extensively drug-resistant (XDR) OXA-72-producing strain of Acinetobacter baumannii (A. baumannii). Methods and ResultsThe isolates were found to be genotypically indistinguishable by whole-genome multiple locus sequence typing, and to belong to the international clonal complex CC2. One of these isolates sequentially developed a high resistance to colistin and rifampicin under treatment, as a result of mutations in genes pmrB and rpoB, respectively. The blaOXA-72 gene was localised on a 10-kb transferable plasmid, named pAB-STR-1, whose sequence is nearly identical to that of another plasmid previously found in Lithuanian strains, pAB120. ConclusionThis report highlighted the need to carefully monitor the emergence of colistin and rifampicin resistance in patients treated for infections with multidrug-resistant A. baumannii.

Impact of single-room contact precautions on hospital-acquisition and transmission of multidrug-resistant Escherichia coli: a prospective multicentre cohort study in haematological and oncological wards

ObjectivesColonization and infection with third-generation cephalosporin-resistant Escherichia coli (3GCR-EC) are frequent in haematological and oncological patients. In this high-risk setting, German guidelines recommend single-room contact precautions (SCP) for patients with 3GCR-EC that are non-susceptible to fluoroquinolones (F3GCR-EC). However, this recommendation is controversial, as evidence is limited. MethodsWe performed a prospective, multicentre cohort study at four haematology and oncology departments assessing the impact of SCP on hospital-acquired colonization or bloodstream infection (BSI) with F3GCR-EC. Two sites performed SCP for F3GCR-EC patients including single rooms, gloves and gowns (SCP sites), and two did not (NCP sites). Active screening for 3GCR-EC was performed and isolates were characterized with molecular typing methods including whole genome sequencing and core genome multiple locus sequence typing to assess patient-to-patient transmission. Potential confounders were assessed by competing-risk regression analysis. ResultsWithin 12 months, 1386 patients at NCP sites and 1582 patients at SCP sites were included. Hospital-acquisition of F3GCR-EC was observed in 22/1386 (1.59%) and 16/1582 (1.01%) patients, respectively (p 0.191). There were 3/1386 (0.22%) patients with BSI caused by F3GCR-EC at NCP sites and 4/1582 (0.25%) at SCP sites (p 1.000). Patient-to-patient transmission occurred in three cases at NCP and SCP sites each (p 1.000). The number of patients needed to screen in order to prevent one patient-to-patient transmission of F3GCR-EC was determined to be 3729. ConclusionsUse of SCP had no significant impact on hospital-acquisition or patient-to-patient transmission of F3GCR-EC in this high-risk setting.

Multilocus sequence typing for phylogenetic view and vip gene diversity of Bacillus thuringiensis strains of the Assam soil of North East India.

An agriculturally important insecticidal bacterium, Bacillus thuringiensis have been isolated from the soil samples of various part of Assam including the Kaziranga National Park. Previously, the isolates were characterized based on morphology, 16S rDNA sequencing, and the presence of the various classes' crystal protein gene(s). In the present study, the phylogenetic analysis of a few selected isolates was performed by an unambiguous and quick method called the multiple locus sequence typing (MLST). A known B. thuringiensis strain kurstaki 4D4 have been used as a reference strain for MLST. A total of four the MLST locus of housekeeping genes, recF, sucC, gdpD and yhfL were selected. A total of 14 unique sequence types (STs) was identified. A total number of alleles identified for the locus gdpD and sucC was 12, followed by locus yhfL was 11, however, only 6 alleles were detected for the locus recF. The phylogenetic analysis using MEGA 7.0.26 showed three major lineages. Approximately, 87% of the isolates belonged to the STs corresponding to B. thuringiensis, whereas two isolates, BA07 and BA39, were clustered to B. cereus. The isolates were also screened for the diversity of vegetative insecticidal protein (vip) genes. In all, 8 isolates showed the presence of vip1, followed by 7 isolates having vip2 and 6 isolates for vip3 genes. The expression of Vip3A proteins was analyzed by western blot analyses and expression of the Vip3A protein was observed in the isolate BA20. Thus, the phylogenetic relationship and diversity of Bt isolates from Assam soil was established based on MLST, in addition, found isolates having vip genes, which could be used for crop improvement.

DST659 genotype of Candida albicans showing positive association between biofilm formation and dominance in Taiwan.

Based on multiple locus sequence typing, we previously found that DST659 and DST693 were dominant genotypes of Candida albicans among the bloodstream isolates at Chang-Gung Memorial Hospital at Linkou. Biofilm-forming activity, which is critical for C. albicans virulence, probably contributed to the dominance of antifungal sensitive isolates in hospital. Both in vitro membrane weighting and in vivo zebrafish egg infection assays were used to evaluate the biofilm-forming activity of DST659 and DST693 genotypes. Medical records of the patients infected by these two genotypes were retrospectively reviewed. High biofilm-forming activity of DST659 isolates was demonstrated in vitro and further proved with the zebrafish egg infection model, which showed a positive correlation between the biofilm-forming extent on chorion and the in vitro biofilm activity. Moreover, significantly less embryos survived when infected with DST659 isolates than those with DST693 (1.25%vs. 11.43%), and the high-biofilm subset of DST659 showed a greater reduction in survival of embryos at 48h post-infection than the low-biofilm subset (0vs. 1.92%). Patients infected with DST659 seemed to survive slightly worse than those infected with DST693, although the difference was insignificant. It is noteworthy that DST659-infected patients were associated with a higher incidence in renal insufficiency as compared to those with DST693, the low biofilm genotype. We suggest that a strong biofilm activity of DST659 contributed to a high mortality rate in zebrafish hosts and poor renal function in patients, as well as gaining the dominance in the northern Taiwan.

Antimicrobial resistance and virulence gene profiling of Salmonella Agona isolated from patients with diarrhea

Objective To investigate the antimicrobial resistance, molecular phenotypes, virulence gene profiles of Salmonella Agona (S.Agona) isolated from patients with acute diarrhea, and to better understand its epidemic trend, prevention and treatment. Methods Clinical data and stool samples of patients with acute diarrhea during April to October in 2013 and 2014 from the Second Hospital of Tianjin Medical University were collected. Enrichment culture, biochemical identification and serotyping analysis were used to isolate and identify S. Agona strains. The isolated strains were further analyzed with antibiotics susceptibility test, pulsed field gel electrophoresis (PFGE), multiple locus sequence typing (MLST), Quinolone resistance determining region (QRDR). Plasmid-mediated quinolone resistance (PMQR) and β-lactamases genes (TEM, SHV, OXA, and CTX-M) were amplified by polymerase chain reaction (PCR) and sequencing. The representative genes carried by Salmonella pathogenicity islands (SPI) 1—6, 9—12 and virulence plasmids were amplified by PCR. And the clinical characteristics of S. Agona infection were analyzed. Results Among 119 non-repetitive (non-typhoidal salmonella, NTS) isolates during the two years, eight isolates (6.7%) of S. Agona were identified. The resistance rate of S. Agona strains to streptomycin was 100.0%, those to ampicillin and gentamicin were 62.5%, to levofloxacin, ciprofloxacin and nalicixic acid were 25.0%, to chloramphenicol, amoxillin/clavulanic acid and piperacillin tazobactam were 12.5%. The strains were susceptible to other drugs. All 8 isolates had the identical ST13 genotype. PFGE showed 5 clones, and 4 out of 5 isolates had the exact same patterns of PFGE and drug susceptibility. Two (fluoroquinolones, FQ) resistant strains carried gyrA mutation leading to amino acid substitutions at position 87 in GyrA, and no PMQR genes was detected, while one of which was sensitive to ciprofloxacin by K-B method. All five ampicillin-resistant isolates were positive for TEM-1b gene and one isolate of them was resistant to β-lactam/β-lactamase inhibitor complex. The representative genes carried by SPI 1—6, 9, 11, 12 (hilA, sseL, mgtC, siiE, sopB, pagN, bapA, pagC and sspH2) were 100.0% positive, while the genes carried by SPI10 (sefA) virulence plasmids (spvB, prot6E) were negative. Two patients with FQ resistant strains infection were clinically diagnosed with bacillary dysentery, and the remaining six cases with FQ susceptible strains infection were clinically diagnosed with acute gastroenteritis. Conclusions FQs-resistant and multi-drug resistant S. Agona isolates have emerged in clinical settings. These isolates carry a variety of virulence genes. Resistance to FQ of S. Agona may cause more severe illness. ST13 might be the dominant genotype of S. Agona in China, and we should try to prevent the infection outbreak of S. Agona. Key words: Salmonella Agona; Antimicrobial resistance; Multiple locus sequence typing; Pulsed field gel electrophoresis; Infection outbreak; Salmonella pathogenicity islands

ST37 Klebsiella pneumoniae: development of carbapenem resistance in vivo during antimicrobial therapy in neonates.

To investigate the mechanism leading to in vivo carbapenem resistance development in Klebsiella pneumoniae. Carbapenemase was detected using the modified carbapenem inactivation method. β-lactamases resistant genes were identified by PCR and sequencing. Clonal relatedness was evaluated by random amplified polymorphic DNA and multiple locus sequence typing. The relationship between sequence typing and resistant genes was analyzed by using the chi-squared test. All ST37 carbapenem-resistant isolates were blaOXA-1 positive and all ST37 carbapenem-sensitive isolates were blaOXA-1 negative at Stage I. A significant relationship between carbapenem resistance and blaOXA-1 was observed. The blaOXA-1 -positive rate was significantly higher in ST37 K. pneumoniae than others. This is the first study about the development of carbapenem resistance in vivo potentially mediated by blaOXA-1 in ST37 K. pneumoniae among neonates.

Molecular Epidemiology and Antibiotic Resistance Profiles of Methicillin-Resistant Staphylococcus aureus Strains in a Tertiary Hospital in China.

Analysis of the genotypic characteristics and antimicrobial susceptibility patterns of methicillin-resistant Staphylococcus aureus (MRSA) is essential for the control and treatment of diseases caused by this important pathogen. In this study, MRSA isolates obtained from a tertiary caret hospital in China were subjected to spa typing, SCCmec typing, multiple locus sequence typing (MLST), and PCR targeting of the genes encoding Panton-Valentine leukocidin (PVL). The disk diffusion method was used to test the antimicrobial susceptibility of the isolates to 10 non-beta-lactam antibiotics. Among the 120 MRSA isolates studied, 18 spa types and 15 ST types were identified. The spa t311 type was the most common (a total of 60 isolates; 50%) among the study strains, and nearly all the t311 strains belonged to ST5, which is the most common ST type that was previously reported from China among the t002 isolates. ST5-II/t311 was the major prevalent clone (55, 45.8%), which was followed by ST5-II/t002 (12, 10.0%) and ST59-IV/t437 (11, 9.2%). PVL-encoding genes were found in 6.7% of the isolates. Although the ST5-II/t311 and ST5-II/t002 clones are different spa types, they shared the same resistance profile (clindamycin, erythromycin, and ciprofloxacin). Most isolates of the ST239-III/t037 clone were resistant to clindamycin, erythromycin, ciprofloxacin, gentamicin, tetracycline, and trimethoprim/sulfamethoxazole. By contrast, the MRSA isolates of the ST239-III/t030 clone were more resistant to rifampin, but they were susceptible to trimethoprim/sulfamethoxazole. Our data emphasize the need for ongoing epidemiologic surveillance.

Leptospirosis in Central America: Techniques for Diagnosis and Molecular Characterization

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the genus Leptospira. In Central America, Leptospira is endemic, and almost all countries experience frequent outbreaks. Diagnosis of leptospirosis is primarily based on serology. More technologically advanced methods, such as polymerase chain reaction (PCR), are used in only a few laboratories, although it can identify genes specific to pathogenic species. New techniques, such as isothermal reactions and molecular typing could also contribute to disease surveillance in humans, animals, and the environment. Advanced techniques, including molecular characterization methods, have been used only rarely up until now. In Nicaragua, we have used phenotypic analysis to characterize pathogenic Leptospira species and multiple locus sequence typing (MLST) to facilitate the identification of a completely new serovar in Costa Rica. Improved technology and expertise for molecular typing of Leptospira are needed in order to improve surveillance and provide the basis for epidemiologic studies. Current MLST characterization schemes include representative strains known to be circulating in Central America, and a more widespread implementation of this technique could enrich information about the epidemiology of Leptospira and enable identification of novel strains emerging in the region.