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Emergence of a New Multidrug Resistant Serotype X Variant in an Epidemic Clone of Shigella flexneri

Emergence of a New Multidrug Resistant Serotype X Variant in an Epidemic Clone of Shigella flexneri

developing countries including Indonesia, Bangladesh, Paki- stan, and China (35–37). These epidemiological observations were unexplained, but it was generally assumed that the sero- type shifts were due to different clones replacing each other over time with each serotype being treated as a clone. In this study we combined epidemiological surveillance and genetic analysis to find a single epidemic clone causing the majority of the S. flexneri infections in China and found that switching of serotypes was occurring frequently within the epidemic clone. Serotype switching was also reflected in the shifts of serotypes over time (Fig. 1). Therefore, we uncovered a novel mecha- nism of epidemic persistence of S. flexneri over long periods through serotype switching to escape infection-induced immu- nity. This finding has major implications for prevention. As immunity to S. flexneri is serotype specific (15), infection-in- duced protection against one serotype will provide protection only against reinfection by the homologous serotype. Frequent serotype switching in S. flexneri may partly explain why the incidence of shigellosis in China and other developing coun- tries remains high despite the fact that safe water supplies, sanitation, hygiene, and the economy have greatly improved in recent years (26, 35). The easy conversion among the S. flexneri serotypes through modification of the O antigen and rapid emergence of new serotypes render the population susceptible to serotypes to which they have not been exposed previously. The emergence of the serotype X variant and frequent se- rotype switching in an epidemic sequence type require updat- ing of the current vaccine development and other prevention and control strategies against shigellosis. Major efforts have been directed to develop vaccines against the predominant S. flexneri serotype 2a. Our mice experiment data show no cross- protection between serotype X variant and serotype 2a (data not shown), and thus, the only

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Molecular Characterization of an Epidemic Clone of Panantibiotic Resistant Pseudomonas aeruginosa

Molecular Characterization of an Epidemic Clone of Panantibiotic Resistant Pseudomonas aeruginosa

We describe the molecular characterization of a multiresistant Pseudomonas aeruginosa clone causing an outbreak in the intensive care unit (ICU) of a tertiary-care university hospital. Analysis included antimicrobial susceptibility profile, O-serotyping, pulsed-field gel electrophoresis, and amplified fragment length polymor- phism. Resistance mechanisms were characterized, including production of naturally occurring and acquired ␤ -lactamases, porin expression, and efflux pump systems. Eighteen patients were colonized or infected with multiresistant P. aeruginosa. Multiresistant P. aeruginosa was panresistant to penicillins, cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones and remained susceptible only to colistin. Sixteen iso- lates (89%) belonged to serotype O:11, pulsed-field gel electrophoresis type A1, and amplified fragment length polymorphism type A. Resistance characterization of this epidemic clone showed an overexpression of the chromosomal cephalosporinase AmpC combined with decreased expression of porin OprD and the absence of metallo- ␤ -lactamase or extended-spectrum beta-lactamase. An upregulation of the MexXY efflux system due to an agrZ mutation in the mexZ repressor was detected. This epidemic clone was restricted to the ICU and was not found elsewhere in hospital. Contamination of the ICU environment and the hands of an ICU nurse with this clone suggests possible hand-borne transmission. Implementation of contact precautions effectively con- trolled transmission of the epidemic clone. This study illustrates the ability of multiresistant P. aeruginosa to cause an outbreak with significant morbidity and mortality and underscores the need to identify clonal outbreaks, which require targeted infection control measures.

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Heterogeneous vancomycin-intermediate susceptibility in a community-associated methicillin-resistant Staphylococcus aureus epidemic clone, in a case of Infective Endocarditis in Argentina

Heterogeneous vancomycin-intermediate susceptibility in a community-associated methicillin-resistant Staphylococcus aureus epidemic clone, in a case of Infective Endocarditis in Argentina

ST59, ST5) or to the new one (ST72) have been detected, particularly in Europe and Asian-Pacific coun- tries [1,19-21]. In the case of the CA-MRSA IE reported here, the molecular typing revealed that although these strains share the same successful lineage ST5 with CA- MRSA (ST5-IV) and -HA-MRSA (ST5-I) epidemic clones disseminated in this country, their PFGE pattern is closer to that of the CA-MRSA clone (one band of difference with I1, additional Figure 3). Moreover, this isolate carries the SCCmec type IV and is non-multire- sistant to antibiotics. Consequently, results identify a PVL-negative variant (PVL - ) of the ST5-IV CA-MRSA epidemic clone detected in Argentina. Importantly, this clone belongs to lineage ST5, which has been demon- strated [5] to develop the VISA phenotype, but so far associated to HA-MRSA clones (ST5-II), unlike our case. These characteristics could confer another selective advantage for its dissemination in Argentina and neigh- boring countries.

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Molecular Epidemiology of Multidrug Resistant Acinetobacter baumannii in a Tertiary Care Hospital in Naples, Italy, Shows the Emergence of a Novel Epidemic Clone

Molecular Epidemiology of Multidrug Resistant Acinetobacter baumannii in a Tertiary Care Hospital in Naples, Italy, Shows the Emergence of a Novel Epidemic Clone

the isolation rate of A. baumannii was significantly associated with length of stay in the ward, being higher in the CR-ICU than in the PO-ICU (10, 15). Based on a previous study of an A. baumannii outbreak occurring in the same institution be- tween June 2003 and June 2004 (25), we could assume that the epidemic described herein was caused by the spread of a single epidemic clone. However, the present report revealed the emergence of two distinct A. baumannii epidemic clones that were isolated in two consecutive temporal clusters in the same wards of the hospital. Indeed, the identity of 3LST, ST, and resistance profiles/genes and the near-identity of PFGE pro- files indicate that these two sets of isolates each represent a clone. The first epidemic clone showed identical PFGE profiles of the A. baumannii strains responsible for two epidemics oc- curring in Naples in the Federico II and V. Monaldi hospitals during 2002 and during 2003 and 2004, respectively (25, 26), and was assigned to 3LST group 1 and ST2, which corre- sponded to the previously characterized European clone II (9, 10). The second epidemic clone, which was first isolated in the CR-ICU in May 2006 and replaced the previous clone in both wards from March 2007 onward, showed a distinct genotype, assigned to novel 3LST group 6 and ST78, which has never been isolated before and is described for the first time herein. This is consistent with previous studies showing that carbap- enem-resistant A. baumannii epidemics in southern Europe are caused by genotypes belonging to 3LST groups 1 and 2, corresponding to the European clones II and I, respectively, but also by additional genotypes of 3LST groups 4 and 5 (10, 11, 15, 22). Our data are also in agreement with a recent report showing that four distinct clones are responsible for a cluster of carbapenem-resistant A. baumannii infections in the ICU of a Greek hospital (21). Also, the isolation of A. baumannii ST78/B strains in the ICUs of two other hospitals in Naples during 2007 suggests that the spread of the novel A. baumannii epidemic clone described herein might have been caused by interhospital transfer of colonized patients in the city. In agree- ment with previous studies, the respiratory tract was the most frequent site of isolation for both clones (9, 15, 25, 26). How- ever, ST78/B strains caused a higher but not statistically sig- nificant proportion of bacteremias than did the other clone in patients from the V. Monaldi hospital, thus suggesting that the novel epidemic clone may possess some inherent properties for developing invasive disease. Moreover, no statistically signifi- cant differences in mean age and female/male ratio were ob- served between patients of the V. Monaldi hospital infected by ST2/A strains and those infected by ST78/B strains of A. bau- mannii.

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tcdC Genotypes Associated with Severe TcdC Truncation in an Epidemic Clone and Other Strains of Clostridium difficile

tcdC Genotypes Associated with Severe TcdC Truncation in an Epidemic Clone and Other Strains of Clostridium difficile

Spigaglia and Mastrantonio have described three tcdC ge- notypes from nonepidemic C. difficile isolates. Genotype tcdC-A is characterized by a nonsense mutation (C184T) that is predicted to result in severe truncation of the TcdC protein from 232 to 61 amino acid residues. In addition, this genotype contains a 39-bp deletion from nucleotides 341 to 379. Geno- type tcdC-A was identified in C. difficile isolates of toxinotypes V and VI. In the same study, genotypes tcdC-B and tcdC-C were identified. These tcdC alleles were characterized by 18-bp deletions at nucleotides 330 to 347 (23). Of note, genotype tcdC-C was derived from a C. difficile isolate of an asymptom- atic patient. On the other hand, similar 18-bp deletions have been detected by fragment size discrimination and sequencing of partial tcdC amplicons (G. Killgore, Centers for Disease Control and Prevention, unpublished data) among BI/NAP1 isolates and are proposed to result in a loss of toxin regulation accounting for the more severe disease attributed to the epi- demic clone (12, 24). Six epidemic clone isolates from Canada * Corresponding author. Mailing address: University of Pittsburgh

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Identification of a Novel Methicillin Resistant Staphylococcus aureus Epidemic Clone in Córdoba, Argentina, Involved in Nosocomial Infections

Identification of a Novel Methicillin Resistant Staphylococcus aureus Epidemic Clone in Córdoba, Argentina, Involved in Nosocomial Infections

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are increasingly a main health concern worldwide for hospitalized patients. In addition, the prevalence of community-acquired infection has risen continuously during the last few years. Some MRSA clones spread easier than others within the hospital environment and therefore are frequently implicated in outbreaks. Thus, the spread of a unique epidemic multiresistant clone, the so-called South American clone, is the main cause of nosocomial infections produced by this bacterium in Brazil and in some regions of Argentina, Chile, and Uruguay. In the present work we describe the identification of a novel clone of MRSA that is involved in nosocomial infections and that shows a prevalence as high as that for the South American clone. A total of 53 consecutive single-patient MRSA isolates were recovered during a 3-month period (May to July 1999) from six different hospitals (955 beds) in Córdoba. The isolates were initially typed according the antibiotic resistance and phage susceptibility patterns, followed by genotyping using pulsed-field gel electrophoresis (PFGE). PFGE analysis of the 53 MRSA isolates revealed six major types (A to F) and 25 subtypes. The B-type DNA pattern was indistinguishable from that of the South American epidemic clone observed in 34% of the isolates. A novel highly prevalent clone, showing the A-type DNA pattern and representing 38% of the isolates, was also identified. Moreover, the most frequent subtype of the A clonal family triggered an outbreak in a hospital 2 months later, further confirming its epidemic feature.

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Endemic, Epidemic Clone of Salmonella enterica Serovar Typhi Harboring a Single Multidrug Resistant Plasmid in Vietnam between 1995 and 2002

Endemic, Epidemic Clone of Salmonella enterica Serovar Typhi Harboring a Single Multidrug Resistant Plasmid in Vietnam between 1995 and 2002

If the distinction between phage types E1 and E3 is not reliable, and taking into account a proportional part of the Vi-untypeable strains, all harboring the 3a ribotype, most MDR serovar Typhi isolates (four-fifths) exhibited genetic ho- mogeneity. This means that the endemic, epidemic MDR ty- phoid fever in Vietnam was due for the most part to a single clone spreading from the south to the center and the north from 1995 to 2002. This is supported by results from the BBPE- Unit collection (Institut Pasteur, Paris): among ribotype 3a strains, phage types E1 and E3 represent 49.2 and 0%, respec- tively; of 29 3a/E1 serovar Typhi isolates with travel history, 20 were associated with travel in Asia (India, Pakistan, and Laos). Moreover, serovar Typhi type E3 is less susceptible to Vi bacteriophages than type E1. Thus, clone 3a-E1, found in Viet- nam as well as in Asian countries, could have evolved in Viet- nam into a new clone, 3a-E3, which was never seen in our collection before. Therefore, one clonal expansion (one ri- botype) which evolved from one phage type to another and underwent limited chromosomal rearrangements (pointed out by PFGE fingerprints) is a valid hypothesis.

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Temperature-Dependent Phage Resistance in Listeria monocytogenes Epidemic Clone II Strains

Temperature-Dependent Phage Resistance in Listeria monocytogenes Epidemic Clone II Strains

companies, and were located in different, non-adjoining states. Putative serotypic designations were determined by means of multiplex polymerase chain reaction, with serotypic designations being 1/2a (or 3a), 1/2b (or 3b), 1/2c (or 3c), and the serotype 4b complex (4b, 4d, 4e) (7). A complete description of L. monocytogenes (including serotypes and genomic fingerprints) and other Listeria spp. isolated from these plants will be described elsewhere. In addition, a panel of strains representing different serotypes was used to determine the host range of the listeriaphage. These strains, along with representatives of other Listeria species, are part of our laboratory’s Listeria strain collection and are listed in Table 2. Six of these strains were used as indicators to screen environmental samples for the presence of phage. These six strains were L. monocytogenes F2365 (serotype 4b, epidemic clone I), H7550 (serotype 4b, epidemic clone II), 4b1 (serotype 4b, sporadic), F6854 (serotype 1/2a), G3978 (serotype 1/2b), and WSLC1001 (serotype 1/2c) (Table 2). Listeria strains were routinely grown in Brain Heart Infusion (BHI; Difco, Sparks, MD) broth or BHI agar (BHI broth supplemented with 1.5% agar, Difco) at 37 ° C and preserved at -80 ° C in BHI with 20% glycerol.

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Subtyping Listeria monocytogenes isolates genetically related to the Swiss epidemic clone

Subtyping Listeria monocytogenes isolates genetically related to the Swiss epidemic clone

Listeria monocytogenes is a facultative human and animal pathogen responsible mainly for abortion, stillbirth, meningi- tis, encephalitis, and septicemia. L. monocytogenes is wide- spread in the environment and often contaminates food, which is considered to be the main source of infection. Listeriosis occurs usually as sporadic cases (2 to 15 cases per million people per year [11]). However, listeriosis epidemics have been repeatedly described in the past years (11, 13) and have caused great concern in the medical community and the food industry. Several typing methods have been applied to the epidemiolog- ical surveillance and tracing of L. monocytogenes. Multilocus enzyme electrophoresis (MEE) has shown that several of the recent major epidemics over the world were caused by a group of genetically closely related serovar 4b strains of L. monocy- togenes (18), designated electrophoretic type 1 (ET1) clone in the present study and in several previous studies (5, 17, 18, 23). Other groups have confirmed these results by using different typing methods (9, 13). This clone has also been shown to be responsible for a significant proportion of the sporadic human and animal listeriosis cases (5, 17, 23).

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Vancomycin-resistant Enterococcus faecium sequence type 796 - rapid international dissemination of a new epidemic clone

Vancomycin-resistant Enterococcus faecium sequence type 796 - rapid international dissemination of a new epidemic clone

Results: A novel clone of vanB E. faecium (ST796) was first detected at one Australian hospital in late 2011, then in two New Zealand hospitals linked by inter-hospital transfers from separate Melbourne hospitals. ST796 also appeared in hospitals in South Australia and New South Wales and was responsible for at least one major colonization outbreak in a Neonatal Intensive Care Unit without identifiable links between centers. No exceptional AMR was detected in the isolates. While WGS analysis showed very limited diversity at the core genome, consistent with recent emergence of the clone, clustering by institution was observed.

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Widespread Dissemination in The Netherlands of the Epidemic Berlin Methicillin Resistant Staphylococcus aureus Clone with Low Level Resistance to Oxacillin

Widespread Dissemination in The Netherlands of the Epidemic Berlin Methicillin Resistant Staphylococcus aureus Clone with Low Level Resistance to Oxacillin

From 2000 through 2002, a total of 2,012 cases (one isolate per case) of MRSA were identified in the Netherlands and sent to the RIVM for further typing (34, 35). Approximately 80% of the strains were isolated from patients, and the remainder were from hospital staff. Of these isolates, 419 (21%) represented a predominant clone based on BT and PFGE: phage type Z-252 and PFGE type 16 (PFT-16), respectively (Table 1). Based on the PFGE results, the strain appeared to be closely related to the Berlin epidemic clone or USA600 strain (W. Witte and F. Tenover, personal communication) (Fig. 1). The proportion of PFT-16 MRSA isolates steadily increased from 3% in 2000 to 33% in 2002, based on data from the national MRSA surveil- lance program carried out by the RIVM (Fig. 2). Before the year 2000, virtually no PFT-16 MRSA isolates were observed in the Netherlands. Furthermore, 394 of 419 (94%) of the PFT-16 MRSA isolates showed low-level resistance to oxacil- lin, with MICs varying between 4 and 32 mg/liter (median, 8 mg/liter) (Table 1). Other predominant epidemic MRSA PFTs observed in the Netherlands during the study period were PFT-4, PFT-18, and PFT-129 (all 5%) and PFT-28 (4%). All four MRSA subtypes displayed oxacillin MICs of ⱖ256 mg/ liter and therefore could, in contrast to MRSA PFT-16, easily be recognized as MRSA.

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Genomic Portrait of the Evolution and Epidemic Spread of a Recently Emerged Multidrug Resistant Shigella flexneri Clone in China

Genomic Portrait of the Evolution and Epidemic Spread of a Recently Emerged Multidrug Resistant Shigella flexneri Clone in China

Shigella flexneri is the major cause of shigellosis in developing countries. A new S. flexneri serotype, Xv, appeared in 2000 and replaced serotype 2a as the most prevalent serotype in China. Serotype Xv is a variant of serotype X, with phosphoethanolamine modification of its O antigen mediated by a plasmid that contained the opt gene. Serotype Xv isolates belong to sequence type 91 (ST91). In this study, whole-genome sequencing of 59 S. flexneri isolates of 14 serotypes (serotypes 1 to 4, Y, Yv, X, and Xv) indi- cated that ST91 arose around 1993 by acquiring multidrug resistance (MDR) and spread across China within a decade. A com- parative analysis of the chromosome and opt-carrying plasmid pSFXv_2 revealed independent origins of 3 serotype Xv clusters in China, with different divergence times. Using 18 cluster-dividing single-nucleotide polymorphisms (SNPs), SNP typing di- vided 380 isolates from 3 provinces (Henan, Gansu, and Anhui) into 5 SNP genotypes (SGs). One SG predominated in each prov- ince, but substantial interregional spread of SGs was also evident. These findings suggest that MDR is the key selective pressure for the emergence of the S. flexneri epidemic clone and that Shigella epidemics in China were caused by a combination of local expansion and interregional spread of serotype Xv.

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To Enhance Type 4 Clone Detection in Clone          Testing

To Enhance Type 4 Clone Detection in Clone Testing

A code clone means similar or duplicate code in a source code or code that is created either by replication or some modifications .These cloned code needs high maintenance cost of software and also cause the code bloating. This is because when changes are performed on one clone, then the same action is performed on respected clone, this will increase the maintenance. These clones can also increase risk of faults in system. Past research conclude that around 7% to 23% of the source code in a software system contains code clone. There are number of techniques and tools to detect the code clones, but it is not effective to remove the clones. Because code clones are needed for software to function properly. So we can apply the principal of refactoring or modularity to improve the reusability and maintainability of software from clone code.

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Clone-Chunk Algorithm for Non-Contiguous Clone Detection

Clone-Chunk Algorithm for Non-Contiguous Clone Detection

ABSTRACT: A research [1] says “as a program evolves, it becomes more complex, and extra resources are needed to preserve and simplify its structure”. Basically, the main reason for the occurrence of clones is copy-paste practice since it is easier to write an already developed code and make some changes in it rather than writing it from scratch, if the implementation is almost similar. Software maintenance is the longest and crucial stage of the software lifecycle, since in order to fulfil the requirements of the user, the system needs to be updated and bugs should be fixed timely. [2] So software evolution is the critical phase of the software maintenance. [3,4] found that due to maintenance more than half time is consumed almost 40% of effort is done on enhancements and extensions in the evolution phase. So, if the cloned codes can be identified correctly than the cost and effort of software development can be reduced to a much lower extent. This paper extends the clone chunk extraction algorithm given in [9] by using the ANTLR grammar thereby giving the view in chain view. Along with this the algorithm provides various statistics so that the programmer can decide whether he want to check the code for cloning or not.

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A novel sub-epidemic modeling framework for short-term forecasting epidemic waves

A novel sub-epidemic modeling framework for short-term forecasting epidemic waves

The myriad of interrelated, and often unobserved, factors that influence the propagation of pathogens at different spatial and temporal scales create major challenges for pre- dicting the transmission dynamics of infectious disease [1]. The factors influencing infectious disease transmission in- clude the mode of transmission (e.g., close contact, airborne, via vector, sexual route), the individual-level network that captures the dynamics of disease-relevant interactions (which are often influenced by cultural factors) [2], the nat- ural history of the disease, variations in the risk behavior of individuals, reactive public health interventions, the behavior changes in response to an epidemic, and the background immunity of the population shaped by genetic factors and prior exposure to the disease or vaccination campaigns [3– 6]. Our ability to generate accurate epidemic forecasts is challenged by the sparse data on the individual- and group- level heterogeneity that affect the dynamics of infectious dis- ease transmission [7–9].

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Diagnostic Potential of Monoclonal Antibodies Specific to the Unique O-Antigen of Multidrug-Resistant Epidemic Escherichia coli Clone ST131-O25b:H4

Diagnostic Potential of Monoclonal Antibodies Specific to the Unique O-Antigen of Multidrug-Resistant Epidemic Escherichia coli Clone ST131-O25b:H4

The Escherichia coli lineage sequence type 131 (ST131)-O25b:H4 is a globally spread multidrug-resistant clone responsible for a great proportion of extraintestinal infections. Driven by the significant medical needs associated with this successful pathogenic lineage, we generated murine monoclonal antibodies (MAbs) against its lipopolysaccharide (LPS) O25b antigen in order to de- velop quick diagnostic tests. Murine monoclonal antibodies were generated by immunizing mice with whole killed nonencapsu- lated ST131-O25b E. coli cells and screening hybridoma supernatants for binding to purified LPS molecules obtained from an E. coli ST131-O25b clinical isolate. The MAbs selected for further study bound to the surface of live E. coli O25b strains irrespective of the capsular type expressed, while they did not bind to bacteria or purified LPS from other serotypes, including the related classical O25 antigen (O25a). Using these specific MAbs, we developed a latex bead-based agglutination assay that has greater specificity and is quicker and simpler than the currently available typing methods. The high specificities of these MAbs can be explained by the novel structure of the O25b repeating unit elucidated in this article. Based on comparative analysis by nuclear magnetic resonance (NMR) and mass spectrometry, the N-acetyl-fucose in the O25a O-antigen had been replaced by O-acetyl- rhamnose in the O25b repeating unit. The genetic determinants responsible for this structural variation were identified by align- ing the corresponding genetic loci and were confirmed by trans-complementation of a rough mutant by the subserotype-specific fragments of the rfb operons.

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Epidemic of Klebsiella pneumoniae ST11 Clone Coproducing KPC 2 and 16S rRNA Methylase RmtB in a Chinese University Hospital

Epidemic of Klebsiella pneumoniae ST11 Clone Coproducing KPC 2 and 16S rRNA Methylase RmtB in a Chinese University Hospital

predominant clone of KPC-KP across the whole world and has caused outbreaks in many countries [5,6,22]. Fur- thermore, ST258 has extended to the recently reported colistin-resistant KPC-KP [18,27]. In the present study, ST11 KPC-KP also seemed to show dissemination advan- tage over other clones and be good colonizers to capture and accumulate resistance determinants. Of particular concern is that increasing prevalence of RPKP coincides with decreasing prevalence of RNKP in our hospital, which might suggest a bigger dissemination advantage of ST11 RPKP over ST11 RNKP, even in the absence of se- lective pressure. Another particular concern is the fact that 3 RPKP isolates (all belonged to ST11-PTA) were imported from other local hospitals, suggesting the pres- ence of inter-hospital dissemination of this extremely multi-drug resistant pathogen. This indicates a dangerous possibility that ST11 RPKP clone might spread widely out- side our hospital in the future.

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Tracking Methicillin Resistant Staphylococcus aureus Clones during a 5 Year Period (1998 to 2002) in a Spanish Hospital

Tracking Methicillin Resistant Staphylococcus aureus Clones during a 5 Year Period (1998 to 2002) in a Spanish Hospital

In addition to the 5 major PFGE types, 16 sporadic types were identified. Since they all together only represent 4.5% of all MRSA isolates, we have not considered them relevant to our main objective of tracking the temporal shifts in the prev- alence of major clones. Consequently, even though the SCCmec type was obtained for all of them, they were not included in the MLST analysis for this study, although they will be further investigated. However, one sporadic isolate was characterized by MLST; it is the isolate nearest one to the ST36 branch in the PFGE-based tree, showing only seven bands of differences from the B1 subtype. This isolate was identified as a ST30-MRSA-IV clone (CC30). This pattern resemblance is in accordance with the high relatedness of STs from ST30 to ST36, since they constitute SLVs and SNVs, differing in the pta allele. These results enforce the robustness of the cutoff criteria for assigning PFGE patterns. It is remark- able that the isolate representing subtype B9, the one with the largest number of band difference from B1 (six bands), repre- sented ST36, while the sporadic PFGE type J, the nearest sporadic to type B, with a seven-band difference from B1, represented ST30. Hence, our results show an interesting pos- itive correlation between PFGE criteria and genetic related- ness. S. aureus isolates with ST30 are common in the United Kingdom (10). There are 80 ST30 isolates deposited in the MLST database, most of which are methicillin-sensitive S. au- reus (83%), with ST30-MRSA isolates in the minority (17%). In several countries (10, 30), a few ST30-MRSA-IV isolates have been found. But there are several reports where ST30- MRSA-IV represents the predominant clone (1, 35, 36, 37). In fact, ST30 is currently considered a particularly successful pathogen, although it does not always carry SCCmec type IV; in particular, community-acquired ST30 presents novel combi- nations of the SCCmec type (40). In our study, we detected only one isolate belonging to the ST30-MRSA-IV clone, which is susceptible to all non-␤-lactam antibiotics, including tobra- mycin. However, it did not spread in our hospital, where mul- tiresistant MRSA clones are dominant.

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Infectious cDNA Clone of the Epidemic West Nile Virus from New York City

Infectious cDNA Clone of the Epidemic West Nile Virus from New York City

West Nile virus (WNV) is found in many regions, including Africa, the Middle East, Europe, Russia, India, Indonesia, and most recently North America (9). Phylogenetic analysis of WNV strains has revealed two distinct lineages (I and II). Lineage I strains are frequently involved in human and equine outbreaks, and lineage II strains are mostly maintained in enzootic cycles (4, 30, 35, 36, 59). Sequence analysis showed that the strain in North America is closely related to other human epidemic strains isolated from Israel, Romania, Russia, and France, all of which belong to lineage I (35, 36). WNV has caused significant human, equine, and avian disease since its appearance in North America in 1999 (2, 28, 36), and the virus has quickly spread from the Northeast to the eastern seaboard and to the Midwest (3). There were 61 human cases (7 deaths) in New York City in 1999 (13); 21 human cases (4 deaths) in New York, New Jersey, and Connecticut in 2000 (42); and 48 human cases (5 deaths) in New York, Florida, New Jersey, Connecticut, Maryland, Massachusetts, Georgia, and Louisi- ana in 2001 (14).

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A Full Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus Host Interactions and Vaccine Development

A Full Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus Host Interactions and Vaccine Development

demonstrate that this mutation increases the fitness of clone- derived virus in Vero cells, we introduced it into the ZIKV-1 cDNA plasmid and generated the ZIKV-NS3m plasmid. The rep- lication of ZIKV-NS3m virus recovered after cDNA plasmid FIG 2 Quasispecies diversity and growth kinetics of ZIKV-wt and ZIKV-ICD viruses in different cell lines. (A) Summary of genome-wide deep-sequencing analysis of ZIKV-wt and ZIKV-ICD RNA. *, total number of nucleotides identified in quality-filtered reads; **, total number of mismatches in quality-filtered reads compared to the consensus sequence of the Paraiba_01/2015 strain. The frequencies of mismatches in ZIKV-wt and ZIKV-ICD were compared using the chi-square test. (B and C) Mutational profiles of ZIKV-wt (B) and ZIKV-ICD (C) RNAs. (D) Growth kinetics of ZIKV-wt and ZIKV-ICD in Vero, C6/36, human foreskin fibroblast, human neuroblastoma SH-SY5Y, mouse testis-derived Sertoli 15P-1, human trophoblast HTR-8/Neo, and human placenta-derived BeWo, JEG-3, and JAR cells. Each cell line was infected at an MOI of 0.01 PFU/cell in duplicate. Titers were determined by plaque assay in Vero cells and are presented as mean values ⫾ standard deviations. Differences in growth kinetics were compared using 2-way ANOVA.

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