ERIC PCR and RAPD based fingerprinting of Salmonella Typhi strains isolated over a period of two decades

Salmonella enterica serotype Typhi strains (n = 113) were isolated from typhoid patients over a period of 2 decades, i.e. 1987–2006. RAPD and ERIC PCR methods were used for random whole genome typing of these strains. ERIC PCR was found to be very efficient with the discriminatory index (DI) of 0.9821 with 100% reproducibility. RAPD was satisfactory in discriminating the strains (DI = 0.8978) but with poor reproducibility (40%). However, composite genotypic analysis was still better with DI of 0.9981 but with inherent poor reproducibility due to RAPD. Two major clones were observed to be circulating in the community with few unrelated strains too. The dendrogram constructed based on ERIC PCR banding pattern by involving 89 Typhi strains revealed 71 patterns, indicating that the genome of the bacterium is capable of rapid changes and variations. Thus, the spectrum of biological manifestations of human infection by S. Typhi may be related to its capacity for genetic diversity underlined by its highly plastic hypermutable genome.     

Genetic diversity of clinical Salmonella enterica serovar Typhimurium in a university hospital of south Tunisia, 2000–2013

Typhimurium is one of the main Salmonella serovar responsible for non-typhoidal gastro-enteritis in Tunisia. Here, we aimed to assess the genetic diversity of 88 clinical Salmonella Typhimurium strains recovered during 14 years from 2000 to 2013. Phage typing, CRISPR polymorphisms (CRISPOL), pulsed-field gel electrophoresis (PFGE), multi-locus variable-number tandem repeat analysis (MLVA) and Whole genome sequencing (WGS) were used to study the relatedness and spatio-temporal evolution of Salmonella Typhimurium populations (Typhimurium (n = 81), monophasic (n = 3) and nonmotile (n = 4) variants).Seven-locus MLST from whole genome assemblies showed that all isolates, except one, belonged to ST19. The isolates were divided into 10 definitive phage (DT) types, dominated by DT104-L (39.8%), DT41 (14.8%), DT116 (11.4%) and DT120 (5.7%). Fifty-seven MLVA patterns (DI, 0.978) were obtained compared to 11 different CRISPOL types and 15 PFGE types (DI,0.845). For cgMLST analysis, 20 profiles were found. A total of 3056 SNPs were identified from the whole genome of the 88 Salmonella Typhimurium isolates. These SNPs resolved these isolates into 86 SNP haplotypes. The phylogeny result allocated most Salmonella Typhimurium isolates into four distinct clades and seven subclades. Genetic diversity between the four clades ranged in the order of 249 to 720 nucleotide changes. The prevalent phage type DT104L formed a major clade on the phylogenetic tree. Pairwise SNP differences between the strains of this clade ranged between 0 and 59.SNP-based WGS typing seems to be the most valuable molecular markers for studying the evolutionary relationships of homogeneous serovar Typhimurium isolates.     

Specific and cross-reactive immune response to oral Salmonella Typhi Ty21a and parenteral Vi capsular polysaccharide typhoid vaccines administered concomitantly

BackgroundSince protective efficacy of the current typhoid vaccines—oral whole-cell Salmonella Typhi Ty21a and parenteral Vi-capsular polysaccharide preparation—is not optimal, and no vaccines are available against paratyphoid or non-typhoidal Salmonella (NTS) serotypes, new approaches deserve to be explored. The immunological mechanisms elicited by the two typhoid vaccines are mainly targeted against different structures. We studied whether these vaccines would enhance S. Typhi-specific immune response and cross-reactivity against other Salmonellae, if administered concomitantly.Materials and methodsVolunteers were immunized simultaneously with Ty21a and Vi vaccines (Ty21a + Vi group) or with either of the two singly (Ty21a and Vi groups). All volunteers were investigated for circulating specific and cross-reactive plasmablasts, identified by ELISPOT as IgA, IgG or IgM antibody-secreting cells (ASC) reactive with S. Typhi, S. Paratyphi A/B/C, or selected NTS serotypes (S. Enteritidis, S. Typhimurium).ResultsIn the Ty21a + Vi group, no specific or cross-reactive plasmablasts were detected before vaccination. After vaccination, the number of S. Typhi-specific plasmablasts (878 ASC/10⁶ PBMC, 95%CI 554–1201) proved higher than in the Ty21a (339 ASC/10⁶ PBMC; p < 0.001) and Vi (149 ASC/10⁶ PBMC; p < 0.001) groups. Likewise, cross-reactive responses in the Ty21a + Vi group were higher than in the Ty21a and Vi groups (Ty21a + Vi vs Ty21a: ASC against S. Paratyphi A/B, S. Enteritidis and S. Typhimurium p < 0.05, against S. Paratyphi C p < 0.01; Ty21a + Vi vs Vi: against S. Paratyphi C not significant, others p < 0.0001). A gut-directed homing profile was seen among O antigen-specific and a systemic one among Vi antigen-specific plasmablasts.ConclusionsConcomitant administration of Ty21a and Vi vaccines is well tolerated and induces an additive immune response to the two vaccines. Thus it enhances the magnitude of both typhoid-specific plasmablast responses and those cross-reacting with paratyphoid and most important NTS serotypes. The data encourage concomitant use of Ty21 and Vi vaccines for those at risk.     

Effect of infectious bursal disease (IBD) vaccine on Salmonella Enteritidis infected chickens

BackgroundChickens infected with both infectious bursal disease virus (IBDV) and Salmonella had higher mortality. In this work, we investigated the effect of IBDV vaccine (modified live-virus bursal disease vaccine, Nobilis strain 228E®) on experimentally infected chickens with Salmonella Enteritidis (SE).MethodsFour experimental groups were included in this study, negative control group, 228E®group, 228E® + SE infected group, and SE infected group. Chickens were ocularly administrated 228E® at 12 days of age and orally infected with S. Enteritidis at 13 days of age. Sera, intestinal fluid, blood, cloacal swabs and tissue samples were collected at 1, 2 and 3 weeks post vaccination (PV).ResultsThe recorded mortalities were higher in the 228E® + SE infected group, compared to the SE infected group. The anti-S. Enteritidis serum antibody titer and the intestinal mucosal IgA level were higher in the SE infected group at 2 and 3 weeks PV, compared to 228E® + SE infected group. S. Enteritidis fecal shedding and organ colonization were significantly higher in the 228E® + SE infected group than the SE infected group at 2 and 3 weeks PV. The 228E® + SE group had significantly lower bursa to body weight ratios at 2 and 3 weeks PV, as well as had higher bursal lesion scores than the SE infected group. IBDV vaccine depressed the specific-SE systemic and mucosal antibody responses, but did not affect the specific-SE cellular immune responses.ConclusionChickens administrated IBDV vaccine, followed by S. Enteritidis infection, could cause a significant effect on the bursa of Fabricius, resulting in failure of systemic and mucosal antibody responses to the S. Enteritidis and reduce the elimination and the clearance of S. Enteritidis.     

Performance of a PCR assay for the rapid identification of the Klebsiella pneumoniae ST258 epidemic clone in Latin American clinical isolates

The worldwide dissemination of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae ST258 demands a rapid PCR-based typing method to detect unique genes of the ST258 clone. This study evaluates a PCR developed by Adler et al. (2014) for the detection of ST258 in K. pneumoniae clinical isolates centered on the identification of the pilv-I and prp genes. We tested 143 clinical isolates from Argentina (n = 109), Chile (n = 1), Colombia (n = 1), Costa Rica (n = 2), Ecuador (n = 5), El Salvador (n = 2), Nicaragua (n = 5), Panamá (n = 2), Paraguay (n = 2), Perú (n = 3) and Trinidad and Tobago (n = 11) recovered from 2006 to 2015. bla_KPC, pilv-l and prp genes were detected by PCR and sequenced by standard procedures. ST258 and non-ST258 were defined by PFGE and/or MLST. Isolates were grouped according to PFGE patterns: 58 were compatible with ST258 (group 1) and 85 with non-ST258 (group 2). MLST study was done on an arbitrary selection of isolates. The pilv-l gene was present only in ST258 isolates, regardless of the presence of the bla_KPC gene. Results for the prp gene were variable. Its presence did not define ST258. The pilv-I PCR had a sensitivity and specificity of 100%, respectively, for the detection of ST258 in the isolates under investigation. Given our findings, the pilv-I PCR could replace more time and resource consuming methods, allowing for more rapid detection of the circulating high risk K. pneumoniae clone ST258 in Latin American (LA) countries.     

Penicillin-resistant,ampicillin-susceptible Enterococcus faecalis of hospital origin: pbp4 gene polymorphism and genetic diversity

Despite the spread of penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF) isolates in diverse countries, the mechanisms leading to this unusual resistance phenotype have not yet been investigated. The aim of this study was to evaluate whether polymorphism in the pbp4 gene is associated with penicillin resistance in PRASEF isolates and to determine their genetic diversity. E. faecalis isolates were recovered from different clinical specimens of hospitalized patients from February 2006 to June 2010. The β-lactam minimal inhibitory concentrations (MICs) were determined by E-test®. The PCR-amplified pbp4 gene was sequenced with an automated sequencer. The genetic diversities of the isolates were established by PFGE (pulsed-field gel electrophoresis) and MLST (multilocus sequencing typing). Seventeen non-producing β-lactamase PRASEF and 10 penicillin-susceptible, ampicillin-susceptible E. faecalis (PSASEF) strains were analyzed. A single-amino-acid substitution (Asp-573 → Glu) in the penicillin-binding domain was significantly found in all PRASEF isolates by sequencing of the pbp4 gene but not in the penicillin-susceptible isolates. In contrast to the PSASEF isolates, a majority of the PRASEFs had similar PFGE profiles. Six representative PRASEF isolates were resolved by MLST into ST9 and ST524 and belong to the globally dispersed clonal complex 9 (CC9). In conclusion, it appears quite likely that the amino acid alteration (Asp-573 → Glu) found in the PBP4 of the Brazilian PRASEF isolates may account for their reduced susceptibility to penicillin, although other resistance mechanisms remain to be investigated.     

Serogroup,virulence, and molecular traits of Vibrio parahaemolyticus isolated from clinical and cockle sources in northeastern Thailand

Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n = 74) and cockles (Anadara granosa) (n = 74) in Thailand were analyzed by serotyping, determination of virulence and related marker genes present, response to antimicrobial agents, and genetic relatedness. Serological analysis revealed 31 different serotypes, 10 of which occurred among both clinical and cockle samples. The clinical isolates commonly included the pandemic serogroup O3:K6, while a few of the cockle isolates exhibited likely pandemic serovariants such as O3:KUT and O4:KUT, but not O3:K6. The pandemic (orf8 gene-positive) strains were more frequently found among clinical isolates (78.4%) than cockle isolates (28.4%) (p < 0.001). Likewise, the virulence and related marker genes were more commonly detected among clinical than cockle isolates; i.e., tdh gene (93.2% versus 29.7%), vcrD2 (97.3% versus 23.0%), vopB2 (89.2% versus 13.5%), vopT (98.6% versus 36.5%) (all p < 0.001) and trh (10.8% versus 1.4%) (p < 0.05). Pulsed-field gel electrophoresis of NotI-digested genomic DNA of 41 randomly selected V. parahaemolyticus isolates representing different serotypes produced 33 pulsotypes that formed 5 different clusters (clonal complexes) (A-E) in a dendrogram. Vibrio parahaemolyticus O3:K6 and likely related pandemic serotypes were especially common among the numerous clinical isolates in cluster C, suggesting a close clonal link among many of these isolates. Most clinical and cockle isolates were resistant to ampicillin. This study indicates that O3:K6 and its likely serovariants based on the PFGE clusters, are causative agents. Seafoods such as cockles potentially serve as a source of virulent V. parahaemolyticus, but further work is required to identify possible additional sources.     

Molecular epidemiology of Corynebacterium pseudotuberculosis isolated from horses in California

Corynebacterium pseudotuberculosis biovar Equi is an important pathogen of horses. It is increasing in frequency in the United States, and is responsible for various clinical forms of infection, including external abscesses, internal abscesses of the abdominal or thoracic cavities, and ulcerative lymphangitis. The host/pathogen factors dictating the form or severity of infection are currently unknown. Our recent investigations have shown that genotyping C. pseudotuberculosis isolates using enterobacterial repetitive intergenic consensus (ERIC)-PCR is useful for understanding the evolutionary genetics of the species as well for molecular epidemiology studies. The aims of the present study were to assess (i) the genetic diversity of C. pseudotuberculosis strains isolated from horses in California, United States and (ii) the epidemiologic relationships among isolates. One hundred and seven C. pseudotuberculosis biovar Equi isolates from ninety-five horses, and two C. pseudotuberculosis biovar Ovis strains, C. pseudotuberculosis ATCC 19410^T type strain and C. pseudotuberculosis 1002 vaccine strain, were fingerprinted using the ERIC 1 + 2-PCR. C. pseudotuberculosis isolated from horses showed a high genetic diversity, clustering in twenty-seven genotypes with a diversity index of 0.91. Minimal spanning tree showed four major clonal complexes with a pattern of temporal clustering. Strains isolated from the same horse showed identical ERIC 1 + 2-PCR genotype, with the exception of two strains isolated from the same animal that showed distinct genotypes, suggesting a co-infection. We found no strong genetic signals related to clinical form (including internal versus external infections). However, temporal clustering of genotypes was observed.     

Approaches to characterize extended spectrum beta-lactamase/beta-lactamase producing Escherichia coli in healthy organized vis-a-vis backyard farmed pigs in India

The study was undertaken to investigate the occurrence and to characterize the ESBL/beta-lactamase producing-Escherichia coli in healthy pigs of organized and backyard farms in West Bengal, India. Total 200 rectal swabs were collected randomly from healthy pigs maintained in four organized farms and 10 backyard farms (n = 100 each) and 76 isolates were identified as E. coli from organized (48/100, 48%) and backyard pigs (28/100, 28%). Twelve E. coli isolates (6%) in the present study were detected to possess any of the ESBL/beta-lactamase genes studied. ESBL/beta-lactamase producers were isolated with significantly more frequency from backyard pigs than the organized farm pigs (p = 0.026). Six of ESBL/beta-lactamase producing isolates were phenotypically confirmed as CTX-M producers and ten of them were confirmed as TEM/SHV producers. PCR and sequencing of the amplified product from representative isolates revealed the presence of bla_CTX-M-9, bla_SHV-12 and bla_TEM-1. No unique combination of the studied beta lactamase genes for organized and backyard farm pig isolates was noted. The ESBL isolates belonged to O13, O55, O133, O153, O157, O158, O166, rough and OUT serogroups. The association of heat labile toxin (elt) (p < 0.0005) with organized farm isolates and heat stable toxin (estA) (p = 0.0143) with backyard piggery sector was significantly higher. The ESBL/beta-lactamase producers from organized farm (Ak/Ex) and indigenous pigs (Ak/Ex/Te; Ak/CoT/G) showed a characteristic phenotypical antibiotic resistance pattern. Two pairs of isolates from organized and backyard farm pigs showed clonal relationship indicating a possible transmission between the farms which were situated adjacently. Thus the present study revealed backyard farm pigs as major source of ESBL/beta-lactamase producing-E. coli associated with STa and characteristic antibiotic resistance pattern in India.     

Salmonella Enteritidis with double deletion in phoP fliC and a competitive exclusion culture elicit substantial additive protective effects against Salmonella exposure in newly hatched chicks

A live Salmonella Enteritidis vaccine (SE 147N ΔphoP fliC), able to express both a homologous intestinal colonisation-inhibition effect and a systemic invasion-inhibition effect, was tested for its potential to generate a postulated additive protective effect in case of combined application with a competitive exclusion (CE) culture against Salmonella exposure in very young chicks. Both, SE 147N ΔphoP fliC and the CE culture alone were highly protective against systemic and intestinal colonisation of the challenge strain in case of moderate Salmonella exposure, consequently, additive protective effects in combined use could not be detected. However, in case of high Salmonella Enteritidis challenge with 10⁶ cfu/bird at day 3 of life the combination of the ΔphoP fliC vaccine and the CE culture resulted in a protective effect much more pronounced than either of the single preparations and most substantial compared to untreated control birds. The term additive protective effects reflects the recognition that exclusion effects by gut flora cultures and inhibition effects by Salmonella vaccines are caused by different mechanisms.     

Polyphenols,vitamin C and antioxidant activity in wines from Rosa canina L. and Rosa rugosa Thunb.

The purpose of this study was to determine the concentration of biologically active compounds (polyphenols and l-ascorbic acid) in Rosa canina L. and Rosa rugosa Thunb. wines. The antioxidant capacity and antimutagenicity of the wines were also investigated. Aged and young wines contained phenolics levels of 2786–3456 and 3389–3990 mg/L GAE, respectively. The final concentrations of ascorbic acid were 1200 for Rosa rugosa Thunb. and 600 mg/L for Rosa canina L. R. rugosa and R. canina wines revealed high antioxidant activity in different assays (with ABTS, DPPH, and DMPD radicals). Expressed in terms of Trolox equivalent antioxidant capacity (TEAC), the activity ranged from 8 to 13.5 mM. Significant differences were found between the tested wines terms of their reactivity against the ABTS and DMPD radicals. The wines inhibited in vitro N-methyl-N′-nitro-nitrosoguanidine (MNNG) and the number of induced His⁺ revertants increased in a dose-dependent manner by 16–48% in Salmonella Typhimurium TA98 and 12–52% in Salmonella Typhimurium TA100. Wines from dog rose (Rosa canina L.) showed a greater ability to reduce mutations.     

Zebrafish as a novel model for non-typhoidal Salmonella pathogenesis,transmission and vaccine efficacy

Salmonella-induced gastroenteritis causes massive morbidity and mortality in both adults and children of developing countries. However, it is difficult to study the mode of infection and vaccine efficacy due to inadequacies of current animal models. For this reason, we have explored using zebrafish as an improved model for non-typhoidal Salmonella (NTS) infection, including Salmonella enterica Typhimurium, Salmonella enterica Enteritidis and Salmonella enterica Weltevreden. In this study, we found that after infection of zebrafish with NTS, severe diarrhea like symptoms were observed and NTS significantly colonized the zebrafish intestine without any manipulation of the normal intestinal microbiota of the fish. Furthermore, these strains can colonize for longer than 72 h and induce severe inflammation in the intestine, which may induce fish death. We also found that infected fish can transmit the pathogen into naïve fish. Moreover, we have established that zebrafish is an excellent model for vaccine study. Successive triple bath vaccination with heat-killed single serotype S. Typhimurium and S. Enteritidis immunogen induced protective efficacy against a high dose (10⁸ CFU/ml) of infection with these pathogens. This study provides a natural infection model for the study of NTS infection, transmission and vaccine efficacy.     

Quantitative PCR analysis of genes expressed during biofilm development of methicillin resistant Staphylococcus aureus (MRSA)

Staphylococcus aureus biofilm associated infections remains a major clinical concern in patients with indwelling devices. Quantitative real-time PCR (qPCR) can be used to investigate the pathogenic role of such biofilms. We describe qPCRs for 12 adhesion and biofilm-related genes of four S. aureus isolates which were applied during in vitro biofilm development. An endogenous control (16S rRNA) was used for signal normalization. We compared the qPCR results with structural analysis using scanning electron microscopy (SEM). The SEM studies showed different cellular products surrounding the aggregated cells at different times of biofilm formation. Using qPCR, we found that expression levels of the gene encoding fibronectin binding protein A and B and clumping factor B (fnbA/B and clfB), which involves in primary adherence of S. aureus, were significantly increased at 24 h and decreased slightly and variably at 48 h when all 4 isolates were considered. The elastin binding protein (ebps) RNA expression level was significantly enhanced more than 6-fold at 24 and 48 h compared to 12 h. Similar results were obtained for the intercellular adhesion biofilm required genes type C (icaC). In addition, qPCR revealed a fluctuation in expression levels at different time points of biofilm growth of other genes, indicating that different parameter modes of growth processes are operating at different times.     

Genetic diversity of Mycobacterium avium subsp. hominissuis strains isolated from humans,pigs, and human living environment

Mycobacterium avium subsp. hominissuis (MAH) strains are genetically diverse and cause infections in pigs and humans. To elucidate the geographical and host-dependent variations in the genetic diversity of MAH, we performed variable numbers of tandem repeat (VNTR) analysis targeting 19 loci for MAH samples from humans (n = 146), bathroom environments (n = 37), and pigs (n = 75) in Japan; these data were then compared with previously reported VNTR data from other countries. The minimum spanning tree (MST) and the multi-dimensional scaling (MDS) analyses based on the VNTR data indicated a high degree of genetic relatedness between isolates from humans and bathrooms in Japan, but a low degree of similarity with the isolates from France and Finland. Moreover, the comparison showed a higher similarity of isolates from Japanese pigs with those from French humans and pigs and Finnish humans and pigs than with other isolates from humans and bathrooms in Japan. The singularity of the Japanese MAH was characterized as the prevalence of hsp65 sequevar code 15 and ISMav6 for the human and bathroom isolates; however, none of the isolates obtained from the pigs belonged to the code 15 or possessed ISMav6. The genetic diversity of MAH and its regional variations imply a possible regional or local specific source of infection and route of transmission of MAH for humans.     

Molecular epidemiology and genetic diversity of Mycobacterium tuberculosis complex in the Cross River State,Nigeria

This study provides with a first insight on Mycobacterium tuberculosis complex epidemiology and genetic diversity in the Cross River State, Nigeria. Starting with 137 smear positive patients recruited over a period of 12 months (June 2008 to May 2009), we obtained 97 pure mycobacterial isolates out of which 81 (83.5%) were identified as M. tuberculosis complex. Genotyping revealed a total of 27 spoligotypes patterns with 10 clusters (n = 64% or 79% of clustered isolates, 2–32 isolates/cluster), with patients in the age group range 25–34 years being significantly associated with shared-type pattern SIT61 (p = 0.019). Comparison with SITVIT2 database showed that with the exception of a single cluster (SIT727/H1), all other clusters observed were representative of West Africa; the two main lineages involved were LAM10-CAM (n = 42/81% or 51.8%) of M. tuberculosis and AFRI_2 sublineage of Mycobacterium africanum (n = 27/81% or 33.3%). Subsequent 12-loci MIRU typing resulted in a total of 13 SIT/MIT clusters (n = 52 isolates, 2–9 isolates per cluster), with a resulting recent n ? 1 transmission rate of 48.1%. Available drug-susceptibility testing (DST) results for 58/81 clinical isolates revealed 6/58% or 10.4% cases of multiple drug-resistance (MDR); 5/6 MDR cases were caused by strains belonging to LAM10-CAM lineage (a specific cluster SIT61/MIT266 in 4/6 cases, and an orphan spoligotype pattern in 1/6 case). Additionally, MIT266 was associated with streptomycin resistance (p = 0.016). All the six MDRTB isolates were concomitantly resistance to streptomycin and ethambutol; however, 4/6 MDR strains with identical MIRU patterns were characterized by consecutive strain numbers hence the possibility of laboratory cross contamination could not be excluded in 3/4 serial cases. The present preliminary study underlines the usefulness of spoligotyping and 12-loci MIRU–VNTRs to establish a baseline of circulating genotypic lineages of M. tuberculosis complex in Nigeria.     

Genetic basis of antimicrobial resistance and clonal dynamics of carbapenem-resistant Acinetobacter baumannii sequence type 191 in a Korean hospital

This study investigated the genetic basis of antimicrobial resistance and the epidemiological characteristics of 125 carbapenem-resistant Acinetobacter baumannii (CRAB) isolates collected from 2011 to 2012 in a Korean hospital. All CRAB isolates showed an extensively drug-resistant phenotype, but were susceptible to tigecycline. The bla_OXA − 23 and armA genes were mainly responsible for resistance to carbapenems and aminoglycosides, respectively. Four colistin-resistant CRAB isolates with different pulsotypes were identified. All four colistin-resistant isolates had a deletion at nucleotide 776 in lpxA, while one also had an insertion at nucleotide 732 in lpxA. All CRAB isolates belonged to three sequence types (STs): ST191 (n = 118), ST208 (n = 6), and ST436 (n = 1), but were classified into 33 arbitrary pulsotypes. Of the CRAB ST191 isolates, two main arbitrary pulsotypes 5 (n = 20) and 18 (n = 17) emerged sequentially, but were not clonally related to CRAB isolates collected from 2009 to 2010 in the same hospital. Furthermore, of the two main pulsotypes identified among CRAB ST191 isolates from 2009 to 2010, one was clonally related to sporadic CRAB ST191 isolates from 2011 to 2012, but the other was not related to any CRAB isolate from 2011 to 2012. In conclusion, this study shows the clonal dynamics of CRAB ST191 isolates in a Korean hospital during the last four years.     

Molecular epidemiology of carbapenem non-susceptible Acinetobacter nosocomialis in a medical center in Taiwan

The mechanism by which carbapenem non-susceptible Acinetobacter nosocomialis (CNSAN) is disseminated is rarely described in the literature. In this study, we delineated the molecular epidemiology of CNSAN isolated from patients in a medical center in Taiwan. Fifty-four non-duplicate bloodstream isolates of CNSAN were collected at the Taipei Veterans General Hospital between 2001 and 2007. Pulsed-field gel electrophoresis (PFGE) was performed to determine their clonal relationship. Carbapenem-resistance genes and associated genetic structures were detected by polymerase chain reaction (PCR) mapping. Southern hybridization was performed to determine the plasmid location of carbapenem-resistance genes. Transmissibility of these genes to Acinetobacter baumannii was demonstrated by conjugation tests. The overall carbapenem non-susceptibility rate among A. nosocomialis isolates during the study period was 21.6% (54/250). PFGE revealed three major pulsotypes: H (n = 23), I (n = 10), and K (n = 8). The most common carbapenem-resistance gene was bla_OXA-58 (43/54, 79.6%), containing an upstream insertion sequence IS1006 and a truncated ISAba3 (IS1006-ΔISAba3-like-bla_OXA-58). All isolates belonging to the pulsotypes H, I, and K carried plasmid located IS1006-ΔISAba3-like-bla_OXA-58. A common plasmid carrying ISAba1-bla_OXA-82 was found in six isolates, which belonged to five pulsotypes. A type 1 integron that carried bla_IMP-1 was detected in different plasmids of seven isolates, which belonged to five pulsotypes. Plasmids carrying these carbapenem-resistant determinants were transmissible from A. nosocomialis to A. baumannii via conjugation. In this medical center, CNSAN mainly emerged through clonal dissemination; propagation of plasmids and integrons carrying carbapenem-resistant determinants played a minor role. This study showed that plasmids carrying carbapenem-resistant determinants are transmissible from A. nosocomialis to A. baumannii.     

Novel class 1 Integrons and sequence types in VIM-2 and VIM-11-producing clinical strains of Enterobacter cloacae

All VIM-producing Enterobacteriaceae (six Enterobacter cloacae) submitted to the Argentinian Reference Laboratory in Antimicrobial Resistance in the period 2008–13 were characterized. The isolates were referred from 6 nosocomial institutions located in 5 different cities across the country. All isolates showed carbapenem disk diffusion inhibition zones ≤ 22 mm and synergism between a carbapenem disk and EDTA/SMA. The six isolates were PCR positive for bla_VIM. Imipenem MICs were ≤ 1 to 8 μg/ml. Typing by PFGE and MLST distinguished six pulsotypes and sequence types with bla_VIM located on novel class 1 integron arrays: ECL-1: ST182, In883; ECL-2, ST90, In885; ECL-3, ST88, In346 with bla_VIM-11; ECL-4, ST184, In900; ECL-5, ST749-new, In900; ECL-6, ST91 and uncharacterized In. Only ECL-2 was able to transfer bla_VIM-2 to E. coli J53 by biparental conjugation. bla_VIM was located in plasmids of 53–82 Kb and in the chromosome (ECL-1 and ECL-5). The diversity of clones, class 1 integrons, plasmids and location of bla_VIM, reveals the plasticity of the genetic elements described and highlights the importance of surveillance programs as tools to identify the transmission of these highly resistant metallo-β-lactamase-producing Enterobacteriaceae.     

Extensive intra-host genetic diversity uncovered in Cryptosporidium parvum using Next Generation Sequencing

The theory about the Cryptosporidium life cycle predicts genetic diversity of sporozoites within the host. Nevertheless, the Cryptosporidium intra-host genetic diversity is difficult to study using conventional Sanger sequencing or electrophoretic resolution of amplicons, due to the methods’ inability to resolve mixtures of templates. We analysed the within-isolate genetic diversity of two Cryptosporidium parvum isolates sharing common descent, by combining the use of Next Generation Sequencing and cloning of PCR amplicons with database searches. The analysis focused on the single-copy 70 kDa heat shock protein (HSP70) and the 60 kDa surface glycoprotein (gp60) genes, which allowed any diversity to be ascribed to the presence of a heterogeneous population of sporozoites. The results indicated an unprecedented intra-host genetic diversity, with two HSP70 and 10 gp60 alleles in these isolates, in spite of the initial resolution of one allele per locus using Sanger sequencing. At both loci, the predominant alleles were those initially identified by Sanger sequencing. A significant (p < 0.01) overrepresentation of gp60 alleles previously reported in New Zealand was observed. These results further our understanding of the genetic structure of C. parvum populations, and expose the limitations of the use of non-axenic isolates as operational taxonomic units of genetic studies of cryptosporidiosis.