PFGE Genotyping Research Articles

High Genetic Diversity and Antimicrobial Resistance in Escherichia coli Highlight Arapaima gigas (Pisces: Arapaimidae) as a Reservoir of Quinolone-Resistant Strains in Brazilian Amazon Rivers.

The increasing prevalence of multi-drug resistant (MDR) Escherichia coli in distinct ecological niches, comprising water sources and food-producing animals, such as fish species, has been widely reported. In the present study, quinolone-resistant E. coli isolates from Arapirama gigas, a major fish species in the Brazilian Amazon rivers and fish farms, were characterized regarding their antimicrobial susceptibility, virulence, and genetic diversity. A total of forty (40) specimens of A. gigas, including 20 farmed and 20 wild fish, were included. Thirty-four quinolone-resistant E. coli isolates were phenotypically tested by broth microdilution, while resistance and virulence genes were detected by PCR. Molecular epidemiology and genetic relatedness were analyzed by MLST and PFGE typing. The majority of isolates were classified as MDR and detected harboring blaCTX-M, qnrA and qnrB genes. Enterotoxigenic E. coli pathotype (ETEC) isolates were presented in low prevalence among farmed animals. MLST and PFGE genotyping revealed a wide genetic background, including the detection of internationally spread clones. The obtained data point out A. gigas as a reservoir in Brazilian Amazon aquatic ecosystems and warns of the interference of AMR strains in wildlife and environmental matrices.

Predominance of community-associated, methicillin-susceptible Staphylococcus aureus infections among hospitalized children and adolescents.

Introduction. Staphylococcus aureus infections cause significant morbidity and mortality in children and adolescents.Gap Statement. There is limited data on the characteristics of S. aureus infections requiring hospitalization in childhood.Aim.To investigate the molecular epidemiology and antibiotic resistance of S. aureus clinical isolates from children and adolescents.Methodology.All S. aureus isolates recovered from patients aged <18 years, admitted to a referral hospital, with culture-proven invasive or non-invasive infections during the 4 year period 2015 to 2018 were analysed for antimicrobial resistance, virulence genes, PFGE and multilocus sequence typing (MLST). Cases were assigned to community-associated, community-onset healthcare-associated or hospital-associated infections based on epidemiological case definitions.Results.Among 139 S. aureus infections, 88.5 % (123/139) were caused by methicillin-susceptible isolates (MSSA) and 73.4 % (102/139) were classified as community-associated infections. tst and lukS/lukF-PV genes were more common among MRSA as compared to MSSA isolates (tst, p 0.04; lukS/lukF-PV, p 0.007). Invasive disease was noted in 22/139 patients (15.8 %). Staphylococcal scalded skin syndrome caused by fusidic-resistant MSSA increased over time (22.8 % in 2017-2018 vs 8.3 % in 2015-2016, OR 3.24; 95 % CI 1.10-8.36; P 0.03). By PFGE genotyping, 22 pulsotypes were identified. A total of five sequence types (STs) were identified among 58 isolates analysed by MLST. More than one third of MSSA isolates (40/123, 32.5 %) and 13/23 (56.5 %) of SSSS isolates belonged to pulsotype 1, classified as sequence type 121 (ST121). MRSA isolates were equally distributed to pulsotypes A (ST30), B (ST239), C (ST80), H (ST225). ST121 isolates carried fnbA (40/40), eta/etb genes (29/40), exhibited high resistance to fusidic acid and were increasingly resistant to mupirocin.Conclusion.In our population, community-associated MSSA was the predominant cause of S. aureus infections characterized by polyclonality, increasing resistance to fusidic acid and mupirocin. PFGE type 1 ST121 clone, harboured exfoliative toxin genes and was associated with rising trends of SSSS.

Antibacterial effect of acetic acid during an outbreak of carbapenem-resistant Acinetobacter baumannii in an ICU (II).

Acetic acid (AA) has been commonly used in medicine as an antiseptic agent for the past 6000 years. This study evaluated the antibacterial effect of AA during an outbreak in an intensive care unit (ICU) facility in Baja California Sur, México. Thirty-five environmental samples were collected, subsequently, disinfection with AA (4%) was performed, and two days later the same areas were sampled inside the ICU facility. Carbapenem-resistant A. baumannii (CRAB) was detected with loop-mediated isothermal amplification assay (Garciglia-Mercado et al. companion paper), targeting blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaIMP and blaVIM genes. CRAB isolates before and after disinfection were compared by PFGE. Eighteen (54.5%) and five (14.3%) of thirty-five environmental samples were identified as Acinetobacter baumannii before and after disinfection, respectively, showing a significant decrease of 85.7% (p < 0.05) both by Loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). Furthermore, the presence of blaOXA-23-like and blaOXA-58-like genes significantly decreased (p < 0.05) both by LAMP and PCR methods. PFGE genotype showed high similarity among CRAB isolates before and after disinfection, suggesting wide clonal dissemination in the ICU facility. This study demonstrated the novel application of AA with the LAMP assays developed for detecting CRAB. AA promises to be a cheap and efficacious disinfectant alternative to both developed and especially developing countries, preventing the spread of this organism in the environment and to other susceptible patients in health care settings.

Tracing Clostridium perfringens strains from beef processing of slaughter house by pulsed-field gel electrophoresis, and the distribution and toxinotype of isolates in Shaanxi province, China

The purpose of this study was to investigate the distribution and specify the transmission and cross-contamination of Clostridium perfringens (C. perfringens) in the beef slaughtering and butchering process. The prevalence of 21.2% (150/708) yielded 208 isolates of C. perfringens, including 80.8% type A and 19.2% type D, 0.4% (3/708) samples carried both type A and D strains, and 72.5% type D isolates carried both cpe and atyp.cpb2 genes. C. perfringens were identified through the whole slaughtering process but no type F (cpe and cpa isolates) was found. 69 isolates were further analyzed and classified into 28 PFGE genotypes and clade I contained 94.2% isolates and 24 PFGE genotypes, which showed the genetic diversity and epidemic correlation. Our study traced C. perfringens contamination along the handling processes and showed a gradually ascending contamination rate during the whole process, revealing widespread cross-contamination from the feces and hides of slaughtered cattle to the carcass in the slaughtering workshop, so as from tools and personnel to meat of the cutting workshops. Strains from different slaughterhouses (regions) have high homology, and type A is the predominant toxinotype. It is necessary to monitor and control several key points of cross-contamination during slaughtering process to reduce a risk of C. perfringens infection.

Investigation of Carbapenem resistance mechanisms in Klebsiella pneumoniae by using phenotypic tests and a molecular assay.

The aim of this study was to investigate the presence of carbapenemase production and carbapenem resistance mechanisms in 47 carbapenem resistant Klebsiella pneumoniae isolates by phenotypic confirmatory tests and molecular assay. Carbapenem resistance genes KPC, OXA-48 and NDM were investigated with the BD MAX CRE assay kit in the BD MAX real time PCR instrument. Modified Hodge test, MBL gradient strip test, D70C Carbapenemase Detection Set, Temocillin gradient strip test methods were used as phenotypic confirmatory tests. Clonal relationship between study isolates was investigated with pulsed-field gel electrophoresis. Analysis with BD MAX CRE assay revealed OXA-48 positivity in 17 (36%) strains, NDM positivity in 6 (13%) strains and coexistence of OXA-48 + NDM positivity in 8 (17%) strains. In 16 (34%) strains, none of the KPC, OXA-48 and NDM genes were detected. While MHT was the most sensitive phenotypic confirmatory test, D70C disc set had not been considered as a useful tool to assist the search for carbapenemase production. Temocillin gradient test alone could not be considered as sufficient to detect the presence of OXA-48. PFGE analyses revealed that 23 of 31 carbapenemase producing strains were in three major PFGE genotypes (A, B and C). This study revealed that carbapenem resistance observed in K. pneumoniae isolates was mainly due to OXA-48 and NDM genes and the increase of carbapenem resistance among K. pneumoniae strains in our hospital was due to the interhospital spread of especially 3 epidemic clones.

Virulence and Antimicrobial Characteristics of Escherichia Coli Isolated from Diseased Chickens in China and Algeria

To reveal and compare the prevalence of pathotypes and virulence genes, as well as antimicrobial resistance and genotyping of poultry E. coli isolates from China and Algeria. Pathotype and seven virulence genes were tested by PCR, susceptibility to antimicrobials was evaluated using broth microdilution method, and genotyping was analyzed by PFGE. Six isolates were identified as pathogenic E. coli. Virulence gene testing showed that the frequency of ompT, iss, fimC, iroN, hlyF and iutA was high in the isolates from Shandong, Shanxi, Jiangsu and Xinjiang province, while stx2 was detected in only two isolates from Shandong, and stx2, iss, fimC, hlyF and iutA could not be detected in Tibet isolates. Importantly, nearly all isolates from Algeria carried seven virulence genes. Drug resistance testing of 141 strains showed that 98.2% (109/111) of the isolates from China and all isolates (30/30) from Algeria resist to more than three classes of antimicrobials. The PFGE genotyping of 157 isolates yielded 134 types, demonstrated a high level of genetic diversity among these isolates. Thus, the poultry E. coli from both China and Algeria exhibited either high frequencies of antimicrobial resistance or high rates of virulence genes carrying.

Pulsed-field gel electrophoresis fingerprinting of Campylobacter jejuni and Campylobacter coli strains isolated from clinical specimens, Iran.

The aim of this study was to determine the clonal correlation of Campylobacter strains isolated from diarrheal children under 5years of age in Iran using the PFGE method and to determine the antimicrobial susceptibility and virulence gene content of strains. Of 750 patients with bacterial diarrhea, 33 (4%) Campylobacter spp., including 31 C. jejuni (94%) and 2 C. coli (6%), were isolated during 18-month period in Tehran, Iran. Except for one strain, remaining Campylobacter strains were positive for the flaA gene. A complete set of cytolethal distending toxin (CDT) encoding genes (cdtABC) were detected in 52% of the C. jejuni strains, while the 2 C. coli isolates under study only harbored cdtA and cdtB of the CDT cluster. All strains were resistant to at least three antibiotic classes. Resistance to ampicillin among C. coli and C. jejuni strains was 100% and 84%, respectively, and 80% of all strains were susceptible to gentamicin. PFGE genotyping generated 19 pulsotypes with two major clusters, displaying the maximum and minimum similarity of 100% and 26%, respectively. The C. coli strains showed clearly distinct pulsotypes and each fell within separate clusters. A very homogeneous Campylobacter population was detected among Iranian patients with 33 % of strains showing identical banding patterns and no clear correlation was observed between antibiotic resistance profiles and PFGE patterns of the isolates.

Molecular Identification of Multidrug-Resistant Campylobacter Species From Diarrheal Patients and Poultry Meat in Shanghai, China.

Emerging resistance to the antimicrobial agents of choice for treatment of thermophilic Campylobacter infections is becoming a serious threat to public health. In this study, 548 Campylobacter (372 C. jejuni and 176 C. coli) isolates from diarrheal patients and poultry meat were subjected for antibiotic susceptibility analysis to ciprofloxacin, tetracycline, gentamicin, erythromycin and clindamycin. Among them, 151 Campylobacter (32 C. jejuni and 119 C. coli) were identified as multidrug resistant isolates. PFGE analysis was performed on the 151 multidrug resistant isolates to determine their genetic relatedness, and 103 PFGE genotypes were determined. Some isolates from both human and chicken belonged to identical genotypes, indicating these clones might be able to spread between human and chicken. Antibiotic resistant genes of the 151 isolates were identified. The numbers of isolates carried tet (O), aadE, ermB, and aadE-sat4-aphA were 148 (98%), 89 (58.9%), 31 (20.5%), and 10 (6.6%), respectively. Almost all (n = 150, 99.3%) had gyrA mutation at codon 86. And the 23s rRNA A2075G point mutation was found in 56 (37.1%) isolates. Gene mutations at the cmeR-cmeABC intergenic region may lead to the activation of CmeABC multidrug efflux pump, and in this study novel sequence types of the intergenic region were identified in both C. jejuni and C. coli. This study determined the genetic prerequisites for antibiotic resistance of multidrug resistant Campylobacter isolates from diarrheal patients and poultry meat in Shanghai, China.

Emergence of different Acinetobacter baumannii clones in a Croatian hospital and correlation with antibiotic susceptibility.

During routine diagnostic laboratory work, the clinical microbiologist observed an increase of Acinetobacter baumannii isolates with three different carbapenem susceptibility patterns: susceptible, intermediate and resistant. Isolates belonging to the same carbapenem susceptibility phenotype exhibited identical susceptibility/resistance patterns to non-β-lactam antibiotics. This prompted us to analyse the mechanisms of carbapenem-resistance and the molecular epidemiology of the isolates. A total of 59 A. baumannii isolates were analysed and grouped according to their susceptibility to imipenem: group 1 were susceptible (N=24), group 2 were intermediate (N=8) and group 3 were resistant (N=27) to imipenem. PCR and sequencing was used to detect resistance genes. Genotyping of the isolates was performed by PFGE and MLST. Out of 27 resistant isolates, 20 harboured blaOXA-40-like and 7 blaOXA-23-like genes. ISAba1 was found upstream of blaOXA-51 and blaOXA-23 genes. PFGE genotyping demonstrated the existence of three major A. baumannii clones in GH Pula and determination of sequence groups showed that the isolates belonged to international clones commonly associated with multidrug-resistance. MLST (performed on six isolates) showed diverse population structure of isolates belonging to the same cluster, including ST 195, ST 231, ST 775 and ST 1095. A previous study conducted in 2009-2010 showed that reduced susceptibility to carbapenems in GH Pula was only associated with upregulation of the intrinsic OXA-51 β-lactamase. In this study a shift to isolates with acquired oxacillinases, belonging to two major clones was reported.

A single Legionella pneumophila genotype in the freshwater system in a ship experiencing three separate outbreaks of legionellosis in 6 years.

BackgroundRecurrent legionella outbreaks at one and the same location are common. We have identified a single Legionella pneumophila genotype associated with recurrent Legionella outbreaks over 6 years.MethodsField emergency surveys following Legionella outbreaks were performed on a vessel in 2008, 2009 and 2013. Water samples from both the distribution and technical parts of the potable water system were analyzed with respect to L. pneumophila [Real-Time PCR, cultivation, serotyping and genotyping (PFGE)] and free-living amoebae, (FLA).ResultsLegionella pneumophila serogroup 1 was present in the ship's potable water system during every outbreak. Genotyping of the 2008 survey material showed two separate PFGE genotypes while those in 2009 and 2013 demonstrated the presence of only one of the two genotypes. FLA with intracellular L. pneumophila of the same genotype were also detected. Analyses of the freshwater system on a ship following three separate Legionella outbreaks, for L. pneumophila and FLAs, revealed a single L. pneumophila genotype and FLA (Hartmanella).ConclusionsIt is reasonable to assume that the L. pneumophila genotype detected in the freshwater system was the causal agent in the outbreaks onboard. Persistence of an apparently low-pathogenic L. pneumophila genotype and FLA in a potable water system represent a potential risk for recurrent outbreaks.

The curli biogenesis genes expression level is unassociated with Enterobacter cloacae hsp60 clusters and PFGE genotypes

The objective of this study was to determine the correlation between Enterobacter cloacae complex subspecies and clusters involved in UTI infections and specific pulsotypes, and to assess the contribution of major curli biogenesis genes (csgD, csgA) expression level to pathogenesis of clusters and genotypes.Based on the PFGE analysis, 37 different profiles were observed among which 8 profiles were common types. Real time PCR of csgD and csgA genes of 50 E. cloacae complex in relation to PFGE and hsp60 genotypes showed that all the genetic clusters are not equally involved in pathogenesis of urinary tract infections. It was elucidated in this study that isolates with common PFGE genotypes belonged to identical hsp60 clusters, and the foremost clusters (VI, III, and V) mainly comprised within PFGE common types. In our study, no significant correlation was detected between the specific hsp60 clusters or PFGE genotypes and the expression level of csgD and csgA genes (P-value > 0.05).This is the first study describing that unequivalent contribution of E. cloacae genotypes and clusters in pathogenesis of UTI, is not owing to varied curli biogenesis expression potential. The PFGE genotyping showed more discriminatory power than hsp60 genotyping for epidemiological studies and source tracking purpose.

Clonal diversity and epidemiological characteristics of Staphylococcus aureus: high prevalence of oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) associated with clinical isolates in Brazil.

BackgroundStaphylococcus aureus is the major cause of global and nosocomial infections with a significant impact in hospitals worldwide. Our objective was to investigate clinical and molecular characteristics of S. aureus isolates causing infections in patients admitted to hospitals from Recife city, Brazil, and investigate the prevalence of oxacillin-susceptible mecA-positive S. aureus (OS-MRSA) in the region, as well as genetically characterize the isolates and compare with epidemic clones.ResultsWe characterized 89 isolates in total, 31 clinical methicillin-resistant S. aureus (MRSA) and 58 methicillin-sensitive (MSSA) isolates by PFGE, MLST, spa typing and SCCmec genotyping. Isolates belonging to international MRSA clones were present: Brazilian epidemic clone (BEC) (61 % of MRSA isolates), Paediatric (36 %), New York/Japan (3 %). Some MSSA isolates were related to MRSA clones: USA400-related (10 % of MSSA isolates), Berlin clone (2 %), Paediatric (14 %), New York/Japan (2 %) and Southwest Pacific clone (17 %). MLST revealed new sequence types (ST’s): ST2381, ST2382, and ST2383 and new spa types: 10548 and 10550. Among isolates phenotypically identified as MSSA by antimicrobial susceptibility assays, we verified 30 oxacillin-susceptible isolates, which exhibited the mecA gene, without mec complex amplification and were thus classified as OS-MRSA. We observed clonal spread of MRSA and MSSA, including OS-MRSA, within several areas of the main hospital investigated and closely related isolates between hospitals analyzed.ConclusionsThe results of this study suggest a possible spread of the strains in hospital environment that could be responsible for nosocomial infections. We documented the presence of several MRSA clones, as well as new MLST and spa types, that were responsible for severe infections in hospitalized patients. The finding of OS-MRSA isolates could have implications for therapy, because testing for mecA and PBP2a is not a routine procedure performed by clinical microbiology laboratories in Brazil and, as consequence, these isolates could be misclassified as MSSA. Our data alert to the necessity to develop more effective strategies for epidemiological control of S. aureus in order to avoid an increase of hospital infections provoked by this pathogen. We reinforce the use of genetic methods, in addition to phenotypic tests, for a precise identification of MRSA.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0733-4) contains supplementary material, which is available to authorized users.

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus Isolated from Australian Veterinarians.

This work investigated the molecular epidemiology and antimicrobial resistance of methicillin-resistant Staphylococcus aureus (MRSA) isolated from veterinarians in Australia in 2009. The collection (n = 44) was subjected to extensive molecular typing (MLST, spa, SCCmec, dru, PFGE, virulence and antimicrobial resistance genotyping) and antimicrobial resistance phenotyping by disk diffusion. MRSA was isolated from Australian veterinarians representing various occupational emphases. The isolate collection was dominated by MRSA strains belonging to clonal complex (CC) 8 and multilocus sequence type (ST) 22. CC8 MRSA (ST8-IV [2B], spa t064; and ST612-IV [2B], spa variable,) were strongly associated with equine practice veterinarians (OR = 17.5, 95% CI = 3.3–92.5, P < 0.001) and were often resistant to gentamicin and rifampicin. ST22-IV [2B], spa variable, were strongly associated with companion animal practice veterinarians (OR = 52.5, 95% CI = 5.2–532.7, P < 0.001) and were resistant to ciprofloxacin. A single pig practice veterinarian carried ST398-V [5C2], spa t1451. Equine practice and companion animal practice veterinarians frequently carried multiresistant-CC8 and ST22 MRSA, respectively, whereas only a single swine specialist carried MRSA ST398. The presence of these strains in veterinarians may be associated with specific antimicrobial administration practices in each animal species.

Salmonellosis Surveillance and Epidemiological Trend in Taiwan

Nontyphoidal Salmonella is a prevalent foodborne pathogen in Taiwan. A national Salmonella reference laboratory was established in 2004 in Centers for Disease Control, Ministry of Health and Welfare, for performing serotyping, PFGE genotyping, and antimicrobial susceptibility testing for isolates collected from hospitals across the country to investigate the epidemiological trend and to build a Salmonella fingerprint database for disease surveillance. In 2004-2013, a total of 20,370 Salmonella isolates were characterized. The isolates belonged to 100 serovars and Enteritidis (accounting for 28.1%), Typhimurium (23.8%), Stanley (7.8%), Newport (6.8%), and Albany (3.7%) were the first 5 most commonly isolated serovars. The profile of serovar distribution suggested that human salmonellosis in Taiwan was caused by Salmonella from numerous host reservoirs. A total of 3,087 PFGE patterns were identified in the 20,370 isolates. A database with the PFGE patterns is an important information platform for the use in predicting Salmonella serotypes, tracing back animal reservoirs of Salmonella strains (multidrug resistant Salmonella strains in particular), performing real-time disease surveillance, and investigating domestic and international foodborne salmonellosis outbreaks. Source management is the most effective control measure for foodborne diseases. For the control of salmonellosis, the Centers for Disease Control, Food and Drug Administration, and Bureau of Animal and Plant Inspection and Quarantine have to work together on the disease surveillance and elimination of Salmonella contamination in food and agricultural products.

Genotyping, local prevalence and international dissemination of β-lactamase-producing Kingella kingae strains

β-lactamase production has been sporadically reported in the emerging Kingella kingae pathogen but the phenomenon has not been studied in-depth. We investigated the prevalence of β-lactamase production among K. kingae isolates from different geographical origins and genetically characterized β-lactamase-producing strains. Seven hundred and seventy-eight isolates from Iceland, the USA, France, Israel, Spain and Canada were screened for β-lactamase production and, if positive, were characterized by PFGE and MLST genotyping, as well as rtxA, por, blaTEM and 16S rRNA sequencing. β-lactamase was identified in invasive strains from Iceland (n = 4/14, 28.6%), the USA (n = 3/15, 20.0%) and Israel (n = 2/190, 1.1%) and in carriage strains in the USA (n = 5/17, 29.4%) and Israel (n = 66/429, 15.4%). No French, Spanish or Canadian isolates were β-lactamase producers. Among β-lactamase producers, a perfect congruency between the different typing methods was observed. Surprisingly, all US and Icelandic β-lactamase-producing isolates were almost indistinguishable, belonged to the major international invasive PFGE clone K/MLST ST-6, but differed from the four genetically unrelated Israeli β-lactamase-producing clones. Representative strains of different genotypes produced the TEM-1 enzyme. K. kingae β-lactamase producers exhibit a clear clonal distribution and have dissimilar invasive potential. The presence of the enzyme in isolates belonging to the major worldwide invasive clone K/ST-6 highlights the possible spread of β-lactam resistance, and emphasizes the importance of routine testing of all K. kingae clinical isolates for β-lactamase production.

Predictive factors for mortality in patients with methicillin-resistant Staphylococcus aureus bloodstream infection: impact on outcome of host, microorganism and therapy

AbstractMortality related to methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection (BSI) remains high, despite changes in the epidemiology. To analyze the current predictive factors for mortality we conducted a prospective study in a large cohort of patients with MRSA-BSI from 21 Spanish hospitals. Epidemiology, clinical data, therapy and outcome were recorded. All MRSAstrains were analysed, including susceptibility to antibiotics and molecular characterization. Vancomycin MICs (V-MIC) were tested by the E-test and microdilution methods. Time until death was the dependent variable in a Cox regression analysis. Overall, 579 episodes were included. Acquisition was nosocomial in 59% and vascular catheter was the most frequent source (38%). A dominant PFGE genotype was found in 368 (67%) isolates, which belonged to Clonal Complex (CC)5 and carried SCCmecIV and agr2. Microdilution V-MIC50 and V-MIC90 were 0.7 and 1.0 mg/L, respectively. Initial therapy was appropriate in 66% of episodes. Overall mortality was observed in 179 (32%) episodes. The Cox-regression analysis identified age >70 years (HR 1.88), previous fatal disease (HR 2.16), Pitt score >1 (HR 3.45), high-risk source (HR 1.85) and inappropriate initial treatment (HR 1.39) as independent predictive factors for mortality. CC5 and CC22 (HR 0.52 and 0.45) were associated with significantly lower mortality rates than CC8. V-MIC ≥ 1.5 did not have a significant impact on mortality, regardless of the method used to assess it.

Emergence of Vibrio cholerae O1 classical biotype in 2012 in Iran

Cholera outbreaks annually occur in many parts of Iran. The aim of this study was to investigate the biotype and genotype diversity of V. cholerae isolates from recent outbreak (2012) in Iran and to characterize the ctxB allelic sequence of isolates. The ctxB sequence of all isolates was analyzed and compared with the reference ctxB sequences for El Tor and classical biotypes in GenBank database. The PFGE genotype specification of isolates was determined and genetic relatedness among isolates and also with those previously reported from Iran was assessed. Ten out of eleven isolates were identified as El Tor biotype and one single isolate belonged to classical biotype. All isolates except three possessed tcpA, ctxA, ctxB and wbeT genes. All the ctxB(+) isolates in this study (classical and El Tor biotypes) possessed the ctxB sequence of classical biotype allele providing evidences of El Tor variants. Eight out of 11 isolates (73%) showed identical pulsotypes (P1). Each of the remaining three isolates showed distinct pulsotypes (P2-P4) with more than three band differences. Pulsotype P2 was corresponded to an isolate (9%) with classical biotype. The result demonstrated diversity in virulence gene content of strains with identical PFGE patterns and also provided evidences of the import of a V. cholerae strain with classical biotype from out of our country as no classical biotype strain has been previously (1998-2011) reported from Iran. Emergence of V. cholerae with Classical biotype after at least one decade in Iran is alarming due to fear of expansion of V. cholerae strains with high virulence potential and signifies the need to monitor and analyze all new cases in countries with cholera outbreaks or even sporadic cases as this could conceivably affect the neighbouring countries and may expose the world to the seventh pandemic with Classical biotype strains.

Distribution and pathogenic relationship of virulence associated genes among Vibrio alginolyticus from the mariculture systems

Vibrio alginolyticus has been confirmed as an important pathogen for aquatic animals. However, the pathogenic mechanism of V. alginolyticus is not completely understood. A total of 31 isolates of V. alginolyticus from sea water, fish and shrimp on the mariculture systems were fingerprinted by pulsed-field gel electrophoresis. The pathogenicity of these isolates was tested by challenge and the 21 genes associated with the virulence of Vibrio cholerae or Vibrio parahaemolyticus were examined in V. alginolyticus using PCR. The results showed that the 31 V. alginolyticus isolates belonged to 26 PFGE genotypes and the isolates from different source had different genotypes. Nine of the 31 isolates were confirmed as pathogenic strains by challenge. Moreover, 12 vibrio virulence genes were detected in this study. Of the detected genes, VCtoxR, VCtoxS, hlyA, VPtoxR and tlh were found in both pathogenic and non-pathogenic isolates. However, the other 7 virulence genes, ctxB, zot, tagA, stn, sto, tdh and trh, were only present in pathogenic isolates. Analysis of the relationship between virulence associated genes and pathogenicity of V. alginolyticus provides a possible explanation that the pathogenic mechanism of V. alginolyticus might be similar to that of V. parahaemolyticus instead of V. cholerae.

Characterization of Yersinia enterocolitica Biotype 1A Strains Isolated from Swine Slaughterhouses and Markets

Yersinia enterocolitica is an important foodborne pathogen that causes illness in humans and animals. Y. enterocolitica is also the most heterogeneous species of the genus and is divided into distinct serotypes and over six biotypes. Y. enterocolitica biotype 1A strains are classically considered as nonpathogenic; however, some biotype 1A isolates have been considered as causative of gastrointestinal disease, yielding symptoms indistinguishable from those produced by pathogenic biotypes. Even after decades of isolation of clinical strains, the pathogenic mechanisms of these isolates are still not fully understood. In the present study, 122 Yersinia enterocolitica biotype 1A strains isolated from swine slaughterhouses and meat markets in Sao Paulo, Brazil, were characterized according to the presence of the virulence genes ail, virF, and ystA. A total of 94 strains were positive to at least one virulence gene (77.05%), and 67 were positive to all of them (54.92%). Twenty-two strains were submitted to PFGE genotyping resulting in 22 distinct pulsotypes, varying from 50% to 84% of genetic similarity. Any clustering tendency among pulsotypes related to origin, isolation site, or even virulence profile was not observed. The present study reports an important contamination of the environment in swine slaughterhouses, meat markets, and pork, by potentially virulent Y. enterocolitica biotype 1A.

PFGE and AFLP genotyping of Staphylococcus aureus subsp. anaerobius isolated from goats with Morel’s disease

Staphylococcus aureus subsp. anaerobius is the etiological agent of the Morel’s disease in sheep and goats. The disease presents with subcutaneous abscesses, located mainly in the superficial lymph nodes. Forty-one isolates of S. aureus subsp. anaerobius were collected from two outbreaks of the Morel’s disease in Poland in years 2006–2008. Analysis of DNA SmaI digests by PFGE showed that 35 of 41 isolates belonged to the same PFGE type, identical to the type strain of S. aureus subsp. anaerobius ATCC 35844, confirming high level of clonality of the species. The DNA patterns of the remaining identical 6 isolates, different from the reference strain only by two bands, were found closely related. Genotyping performed with AFLP technique revealed two clonal groups including 16 and 25 isolates, respectively. The study indicated that AFLP technique might be a better discriminatory tool for genetic analysis of S. aureus subsp. anaerobius isolates, when compared to PFGE.