Pulsed-field Gel Electrophoresis Genotypes Research Articles

Genotyping of German and Austrian Taylorella equigenitalis isolates using repetitive extragenic palindromic (REP) PCR and pulsed-field gel electrophoresis (PFGE)

A total of 124 Taylorella (T.) equigenitalis and five T. asinigenitalis field isolates collected between 2002 and 2014 were available for genotyping using REP- (repetitive extragenic palindromic) PCR and PFGE (pulsed-field gel electrophoresis). The study comprised 79 T. equigenitalis field isolates originating from ten defined breeds of German horses and revealed a spectrum of five REP (rep-E1-E4, rep-E3a) and 15 PFGE (TE-A1-A9, TE-B1-B3, TE-C, TE-E1, and TE-E2) genotypes. T. equigenitalis field isolates (n=40) obtained from Austrian Lipizzaner horses were differentiated into three REP (rep-E1, rep-E3a, and rep-E4) and three PFGE genotypes (TE-A2, TE-A5, and TE-D); those isolated from four Austrian Trotters belonged to the REP/PFGE genotype rep-E2/TE-A1. Interestingly, a T. equigenitalis isolate recovered from a Holsteiner stallion living in South Africa revealed the REP/PFGE genotype rep-E1/TE-A5 which was otherwise exclusively present in the majority of Austrian Lipizzaner horses in our study. The type strain included in this study revealed the genotype REP/PFGE rep-E1/TE-F. Six strains of T. asinigenitalis including the type strain were separated into three REP (rep-A1-A3) and six PFGE genotypes (TA-A1, TA-A2, TA-A3, TA-B, TA-C, TA-D). Overall, the generated REP and PFGE genotypes showed a good correlation, whereas REP-PCR proved to be a suitable method for molecular epidemiological screening of T. equigenitalis and T. asinigenitalis isolates that should be differentiated in detail by genotyping using PFGE.

Prevalence and molecular characterization of Salmonella enterica isolates throughout an integrated broiler supply chain in China.

A total of 1145 samples were collected from chicken breeder farms, hatcheries, broiler farms, a slaughterhouse and retail refrigerated chicken stores in an integrated broiler supply chain in Guangdong Province, China, in 2013. One-hundred and two Salmonella enterica strains were isolated and subjected to serotyping, antimicrobial susceptibility testing, virulence profile determination and molecular subtyping by pulsed field gel electrophoresis (PFGE). The contamination rates in samples from breeder farms, hatcheries, broiler farms, the slaughterhouse and retail stores were 1·46%, 4·31%, 7·00%, 62·86% and 54·67%, respectively. The isolated strains of S. enterica belonged to 10 serotypes; most of them were S. Weltevreden (46·08%, 47/102) and S. Agona (18·63%, 19/102). Isolates were frequently resistant to streptomycin (38·2%), tetracycline (36·3%), sulfisoxazole (35·3%) and gentamicin (34·3%); 31·4% of isolates were multidrug resistant. The isolates were screened for 10 virulence factors. The Salmonella pathogenicity island genes avrA, ssaQ, mgtC, siiD, and sopB and the fimbrial gene bcfC were present in 100% of the strains. PFGE genotyping of the 102 S. enterica isolates yielded 24 PFGE types at an 85% similarity threshold. The PFGE patterns show that the genotypes of S. enterica in the production chain are very diverse, but some strains have 100% similarity in different parts of the production chain, which indicates that some S. enterica persist throughout the broiler supply chain.

Prevalence, antimicrobial resistance profiling and genetic diversity of Campylobacter jejuni and Campylobacter coli isolated from broilers at slaughter in China

A total of 651 samples from broiler cecal samples, carcasses, carcass parts collected at the slaughterhouse level in Sichuan Province of China were examined for the occurrence of Campylobacter jejuni and Campylobacter coli. After confirmed by species-specific multiplex PCR, the recovered isolates were examined for resistance to antimicrobials using an agar dilution method and investigated for the mutation of gyrA, tetO gene and V domain of 23S rRNA as well as the presence of class 1 integron and the associated gene cassettes. In addition, the genotype relatedness of the isolates was determined by pulsed-field gel electrophoresis (PFGE) profiling. The prevalence of Campylobacter was 56.1% in cecal samples, 31.0% in carcasses and 17.0% in carcass parts, respectively. Among them, C. jejuni accounted for 24.6% and C. coli occupied 20.0% of the samples. The strains of C. jejuni were most frequently resistant to ciprofloxacin (88.1%), followed by resistance to tetracycline (79.4%) and levofloxacin (78.1%). Most of the C. coli isolates were resistant to ciprofloxacin (100%), tetracycline (98.5%), levofloxacin (98.5%), clindamycin (98.5%) and erythromycin (93.9%). Antimicrobial resistance profiling showed that 93.7% of campylobacters were multidrug resistant (MDR) strains. Moreover, class 1 integrons were detected in 98.6% of MDR campylobacters, among which 98.7% were positive for C. jejuni and 98.5% for C. coli. Three kinds of gene cassettes-associated amplicons were identified and the amplicons profile of 1000–750–500–250 bp was the predominant pattern linked to the aminoglycoside resistance gene of aadA2. The presence of mutation in gyrA, tetO and 23S rRNA between C. jejuni and C. coli varied from 89.7% to 97.3%, 96.6% to 94.1%, and 95.0% to 96.7%, respectively. Finally, the results of PFGE indicated that, 33 PFGE profiles were generated among 78 isolates of C. jejuni and the similarity level ranged from 42.1% to 99.1%. By contrast, 15 PFGE patterns were produced among 68 C. coli isolates sharing a similarity level of 54.0%–99.7%. Comparison of the PFGE and antimicrobial resistance profiles of the isolates reflected the high genetic diversity of Campylobacter tested. A poor correlation among the antimicrobial resistance patterns, resistance determinants and PFGE genotypes was observed in C. jejuni. Our study showed that there were several points of cross-contamination during broiler slaughter, and a high diversity of PFGE types in the Campylobacter isolates with high resistances to ciprofloxacin and tetracycline.

Listeria monocytogenes in ready-to-eat vacuum and modified atmosphere packaged meat and fish products of Estonian origin at retail level

The prevalence, counts and genetic diversity of Listeria monocytogenes in ready-to-eat (RTE) vacuum and modified atmosphere packaged meat and fish products was studied in Estonia. Within two consecutive years 370 RTE food samples were collected at retail level from which 11% were found to be positive for L. monocytogenes. Contamination was higher among RTE fish products (17%) than in RTE meat products (6%). Generally, the counts of L. monocytogenes in positive products remained under ten colony forming units (CFU) per gram of product. Only 1.6% of the RTE meat and fish products contained L. monocytogenes in range of 10–100 CFU/g and 0.3% more than 100 CFU/g at the end of shelf-life. The food category containing highest L. monocytogenes prevalence was RTE lightly salted fish products with the prevalence of 32%. Only one (0.3%) RTE food sample exceeded the 100 CFU/g food safety criterion set out in the EU Regulation 2073/2005. Pulsed-field gel electrophoresis (PFGE) characterization of the isolates showed an overall similarity higher than 70%, and nine clusters based on 100% similarity were revealed. PFGE genotyping revealed that the few predominant pulsotypes were associated with particular food plants.

The worldwide spread of ciprofloxacin-resistant Shigella sonnei among HIV-infected men who have sex with men, Taiwan

Ciprofloxacin-resistant shigellosis outbreaks among men who have sex with men (MSM) have not been reported in Asia. During 3 March to 6 May 2015, the Notifiable Disease Surveillance System detected nine non-imported Shigella sonnei infections among human immunodeficiency virus (HIV) -infected Taiwanese MSM. We conducted a molecular epidemiological investigation using a 1 : 5 matched case–control study and laboratory characterizations for the isolates. Of the nine patients, four reported engagement in oral–anal sex before illness onset. Shigellosis was associated with a syphilis report within 12 months (adjusted odds ratio (aOR) 8.6; 95% CI 1.05–70.3) and no HIV outpatient follow-up within 12 months (aOR 22.3; 95% CI 2.5–201). Shigella sonnei isolates from the nine patients were all ciprofloxacin-resistant and the resistance was associated with S83L and D87G mutations in gyrA and S80I mutation in parC. The nine outbreak isolates were discriminated into two closely related pulsed-field gel electrophoresis (PFGE) genotypes and seven 8-locus multilocus variable-number tandem repeat analysis (MLVA8) types that suggest multiple sources of infections for the outbreak and possible under-recognition of infection among Taiwanese MSM. The outbreak isolates were characterized to be variants of the intercontinentally transmitted SS18.1 clone, which falls into the globally prevalent phylogenetic sub-lineage IIIb. Inter-database pattern similarity searching indicated that the two PFGE genotypes had emerged in the USA and Japan. The epidemiological characteristics of this outbreak suggest roles of risky sexual behaviours or networks in S. sonnei transmission. We urge enhanced surveillance and risk-reduction interventions regionally against the interplay of HIV and shigellosis among MSM.

Prevalence and Characterization of Integrons in Multidrug Resistant Acinetobacter baumannii in Eastern China: A Multiple-Hospital Study.

Objective: The aim of this multiple-hospital study was to investigate the prevalence of integrons in multidrug-resistant Acinetobacter baumannii (MDRAB) in Eastern China, and characterize the integron-integrase genes, so as to provide evidence for the management and appropriate antibiotic use of MDRAB infections. Methods: A total of 425 clinical isolates of A. baumannii were collected from 16 tertiary hospitals in 11 cities of four provinces (Fujian, Jiangsu, Zhejiang and Shandong) from January 2009 to June 2012. The susceptibility of A. baumannii isolates to ampicillin/sulbactam, piperacillin/tazobactam, ceftazidime, ceftriaxone, cefepime, aztreonam, meropenem, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, sulfamethoxazole/trimenthoprim, minocycline and imipenem was tested, and integrons and their gene cassettes were characterized in these isolates using PCR assay. In addition, integron-positive A. baumannii isolates were genotyped using pulsed-field gel electrophoresis (PFGE) assay, and intI1 gene cassette was sequenced. Results: intI1 gene was carried in 69.6% of total A. baumannii isolates, while intI2 and intI3 genes were not detected. The prevalence of resistance to ampicillin/sulbactam, piperacillin/tazobactam, ceftazidime, ceftriaxone, cefepime, aztreonam, imipenem, meropenem, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin and sulfamethoxazole/trimenthoprim was significantly higher in integron-positive A. baumannii isolates than in negative isolates (all p values <0.05), while no significant difference was observed in the prevalence of minocycline resistance (p > 0.05). PFGE assay revealed 27 PFGE genotypes and 4 predominant genotypes, P1, P4, P7 and P19. The PFGE genotype P1 contained 13 extensive-drug resistant and 89 non-extensive-drug resistant A. baumannii isolates, while the genotype P4 contained 34 extensive-drug resistant and 67 non-extensive-drug resistant isolates, appearing a significant antimicrobial resistance pattern (both p values <0.05). Sequencing analysis revealed two gene cassette assays of aacA4-catB8-aadA1 and dfrXII-orfF-aadA2 in MDRAB isolates. Conclusions: The results of this study demonstrate a high prevalence of class 1 integrons in MDRAB in Eastern China, and a greater prevalence of antimicrobial resistance in intI1 gene-positive MDRAB isolates than in negative isolates. Four predominant PFGE genotypes are identified in intI1 gene-positive MDRAB isolates, in which P4 is an epidemic PFGE genotype in Fujian Province, and it has a high proportion of extensive drug resistant A. baumannii. The gene cassette dfrXII-orfF-aadA2 is reported, for the first time, in A. baumannii strains isolated from Fujian Province, Eastern China.

Methicillin-Resistant Staphylococcus aureus from Brazilian Dairy Farms and Identification of Novel Sequence Types.

The aim of this study was to investigate the phenotypic and genotypic diversity and anti-microbial resistance among staphylococci of dairy herds that originated from Paraiba State, north-eastern Brazil, a region where such studies are rare. Milk samples (n = 552) were collected from 15 dairy farms. Isolates were evaluated for anti-microbial susceptibility by Kirby-Bauer disc diffusion method. Confirmation of methicillin-resistant Staphylococcus aureus (MRSA) was performed using multiplex PCR targeting mecA and nuc genes in addition to phenotypic assay based on PBP-2a latex agglutination. Clonal relatedness of isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) genotyping. Staphylococci were detected in 269 (49%) of the samples. Among these, 65 (24%) were S. aureus. The remaining 204 isolates were either coagulase-negative staphylococci (n = 188; 70%) or coagulase positive other than S. aureus (n = 16; 6%). Staphylococci were cultured in seven (35%) of the 20 hand swab samples, from which five isolates were S. aureus. The isolates were most commonly resistant against penicillin (43%), ampicillin (38%) and oxacillin (27%). The gene mecA was detected in 21 S. aureus from milk and in one isolate from a milker's hand. None of the isolates were resistant to vancomycin. PFGE findings showed high clonal diversity among the isolates. Based on MLST, we identified a total of 11 different sequence types (STs 1, 5, 6, 83, 97, 126, 1583, 1622, 1623, 1624 and 1625) with four novel STs (ST1622-ST1625). The findings show that MRSA is prevalent in milk from semi-extensive dairy cows in north-eastern Brazil, and further investigation on its extent in various types of milk production systems and the farm-to-table continuum is warranted.

Development of live attenuated Streptococcus agalactiae vaccine for tilapia via continuous passage in vitro.

Fish Streptococcus agalactiae (S. agalactiae) seriously harms the world's aquaculture industry and causes huge economic losses. This study aimed to develop a potential live attenuated vaccine of S. agalactiae. Pre-screened vaccine candidate strain S. agalactiae HN016 was used as starting material to generate an attenuated strain S. agalactiae YM001 by continuous passage in vitro. The biological characteristics, virulence, and stability of YM001 were detected, and the protective efficacy of YM001 immunization in tilapia was also determined. Our results indicated that the growth, staining, characteristics of pulsed-field gel electrophoresis (PFGE) genotype, and virulence of YM001 were changed significantly as compared to the parental strain HN016. High doses of YM001 by intraperitoneal (IP) injection (1.0 × 10(9) CFU/fish) and oral gavage (1.0 × 10(10) CFU/fish) respectively did not cause any mortality and morbidity in tilapia. The relative percent survivals (RPSs) of fishes immunized with YM001 (1.0 × 10(8) CFU/fish, one time) via injection, immersion, and oral administration were 96.88, 67.22, and 71.81%, respectively, at 15 days, and 93.61, 60.56, and 53.16%, respectively, at 30 days. In all tests with 1-3 times of immunization in tilapia, the dosages at 1 × 10(8) and 1 × 10(9) CFU/fish displayed the similar best results, whereas the immunoprotection of the dosages at 1 × 10(6) and 1 × 10(7) CFU/fish declined significantly (P < 0.01), and 1 × 10(5) CFU/fish hardly displayed any protective effect. In addition, the efficacy of 2-3 times of immunization was significantly higher than that of single immunization (P < 0.01) while no significant difference in the efficacy between twice and thrice of immunization was seen (P > 0.05). The level of protective antibody elicited by oral immunization was significantly higher compared to that of the control group (P < 0.01), and the antibody reached their maximum levels 14-21 days after the immunization but decreased significantly after 28 days of vaccination. YM001 bacteria were isolated from the brain, liver, kidney, and spleen tissues of fish after oral immunization and the bacteria existed for the longest time in the spleen (up to 15 days). Taken together, this study obtained a safe, stable, and highly immunogenic attenuated S. agalactiae strain YM001; oral immunization of tilapia with this strain produced a good immune protection.

Resistance-Gene Cassettes Associated With Salmonella enterica Genotypes.

The epidemiology of salmonellosis is complex because of the diversity and different serotypes of Salmonella enterica (S. enterica) that occur in different reservoirs and geographic incidences. To determine the genotype distribution and resistance-gene content of 2 classes of integron among S. enterica isolates. Thirty-six S. enterica species were isolated and tested for their serological distribution and the resistance-gene contents of 2 classes of integron, as well as for their genetic diversity, using the pulsed-field gel electrophoresis (PFGE) genotyping method. Serogroups E (36.1%) and D (30.5%) were dominant among the isolates. All of the isolates in serogroup D belonged to the serovar enteritidis. The aadA1 gene was found within all resistance-gene cassettes. We observed 4 common and 26 single pulsotypes among the isolates, which indicated a high degree of genetic diversity among the isolates. Using the PulseNet International standard protocol, it was found that these isolates were different from those reported previously in Iran. The presence of a few common and new pulsotypes among the isolates suggests the emergence and spread of new clones of S. enterica in Iran.

Resistance of Stenotrophomonas maltophilia to Fluoroquinolones: Prevalence in a University Hospital and Possible Mechanisms.

Objective: The purpose of this study was to investigate the clinical distribution and genotyping of Stenotrophomonas maltophilia, its resistance to antimicrobial agents, and the possible mechanisms of this drug resistance. Methods: S. maltophilia isolates were collected from clinical specimens in a university hospital in Northwestern China during the period between 2010 and 2012, and were identified to the species level with a fully automated microbiological system. Antimicrobial susceptibility testing was performed for S. maltophilia with the Kirby-Bauer disc diffusion method. The minimal inhibitory concentrations (MICs) of norfloxacin, ofloxacin, chloramphenicol, minocycline, ceftazidime, levofloxacin and ciprofloxacin against S. maltophilia were assessed using the agar dilution method, and changes in the MIC of norfloxacin, ciprofloxacin and ofloxacin were observed after the addition of reserpine, an efflux pump inhibitor. Fluoroquinolone resistance genes were detected in S. maltophilia using a polymerase chain reaction (PCR) assay, and the expression of efflux pump smeD and smeF genes was determined using a quantitative fluorescent (QF)-PCR assay. Pulsed-field gel electrophoresis (PFGE) was employed to genotype identified S. maltophilia isolates. Results: A total of 426 S. maltophilia strains were isolated from the university hospital from 2010 to 2012, consisting of 10.1% of total non-fermentative bacteria. The prevalence of norfloxacin, ciprofloxacin and ofloxacin resistance was 32.4%, 21.9% and 13.2% in the 114 S. maltophilia isolates collected from 2012, respectively. Following reserpine treatment, 19 S. maltophilia isolates positive for efflux pump were identified, and high expression of smeD and smeF genes was detected in two resistant isolates. gyrA, parC, smeD, smeE and smeF genes were detected in all 114 S. maltophilia isolates, while smqnr gene was found in 25.4% of total isolates. Glu-Lys mutation (GAA-AAA) was detected at the 151th amino acid of the gyrA gene, while Gly-Arg mutation (GGC-CGC) was found at the 37th amino acid of the parC gene. However, no significant difference was observed in the prevalence of gyrA or parC mutation between fluoroquinolone-resistant and -susceptible isolates (p> 0.05). The smqnr gene showed 92% to 99% heterogenicity among the 14 S. maltophilia clinical isolates. PFGE of 29 smqnr gene-positive S. maltophilia clinical isolates revealed 25 PFGE genotypes and 28 subgenotypes. Conclusions: Monitoring the clinical distribution and antimicrobial resistance of S. maltophilia is of great significance for the clinical therapy of bacterial infections. Reserpine is effective to inhibit the active efflux of norfloxacin, ciprofloxacin and ofloxacin on S. maltophilia and reduce MIC of fluoroquinolones against the bacteria. The expression of efflux pump smeD and smeF genes correlates with the resistance of S. maltophilia to fluoroquinolones.

The association of serotype and pulsed-field gel electrophoresis genotype in isolates of Streptococcus pneumoniae isolated in Israel

The relationship between Streptococcus pneumoniae isolates causing invasive infections in children admitted to a single center in central Israel was examined by pulsed-field gel electrophoresis (PFGE) and serotyping. Although there was a close correlation between serotype and PFGE clone, the genetic diversity varied by serotype, with some genotypes comprising multiple serotypes. Additionally, clones C and D were associated with higher penicillin minimum inhibitory concentrations. Serotyping alone may be insufficient for epidemiological mapping of pneumococcal isolates in the era of pneumococcal conjugate polysaccharide vaccines.

Emergence of β-lactamases and extended-spectrum β-lactamases (ESBLs) producing Salmonella in retail raw chicken in China.

β-Lactamases and extended-spectrum β-lactamases (ESBLs) producing pathogenic bacteria were widely studied previously in China, but were seldom focused on foodborne Salmonella. In this study, an investigation concerning β-lactamases and ESBLs producing Salmonella recovered from retail raw chickens was performed. Sixty of 699 foodborne Salmonella isolates were detected as β-lactamases and ESBLs-producing ones that covered 12 Salmonella serotypes and exhibited different pulsed-field gel electrophoresis genotypes. Forty-four of 60 β-lactamases and ESBLs-producing strains were simultaneously resistant to ampicillin, amoxicillin/clavulanic acid, ceftiofur, ceftriaxone, and cefoxitin. The most commonly detected β-lactamases and ESBLs-encoding gene was bla(TEM-1) (n = 44), followed by bla(OXA-1) (n = 38), bla(CMY-2) (n = 29), bla(PSE-1-like) (n = 1), bla(CTX-M-3) (n = 16), and bla(CTX-M-15) (n = 1), respectively. Fourteen, 24, 21, and 1 isolates were detected simultaneously positive for 1, 2, 3, and 4 of the detected β-lactamases and ESBLs-encoding genes, respectively. A Salmonella strain simultaneously co-carrying bla(TEM-1), bla(OXA-1), bla(CMY-2), and bla(CTX-M-3) was first reported in the present study. Amino acid substitution of Trp244Cys/His247Leu was detected in PSE-1, Val218Asp in CMY-2, and Asp242Gly in CTX-M-15 enzymes, respectively. A difference was found among the amino acid sequences of the detected OXA-1, CMY-2, CTX-M, PSE-1, and TEM-1. The results demonstrated that β-lactamases and ESBLs were emerging and prevalent in foodborne Salmonella.

Emergence of an outbreak-associated Clostridium difficile variant with increased virulence.

The prevalence of Clostridium difficile infections has increased due to the emergence of epidemic variants from diverse genetic lineages. Here we describe the emergence of a novel variant during an outbreak in a Costa Rican hospital that was associated with severe clinical presentations. This C. difficile variant elicited higher white blood cell counts and caused disease in younger patients than did other strains isolated during the outbreak. Furthermore, it had a recurrence rate, a 30-day attributable disease rate, and disease severity as great as those of the epidemic strain NAP1. Pulsed-field gel electrophoresis genotyping indicated that the outbreak strains belong to a previously undescribed variant, designated NAPCR1. Whole-genome sequencing and ribotyping indicated that the NAPCR1 variant belongs to C. difficile ribotype 012 and sequence type 54, as does the reference strain 630. NAPCR1 strains are resistant to fluoroquinolones due to a mutation in gyrA, and they possess an 18-bp deletion in tcdC that is characteristic of the epidemic, evolutionarily distinct, C. difficile NAP1 variant. NAPCR1 genomes contain 10% more predicted genes than strain 630, most of which are of hypothetical function and are present on phages and other mobile genetic elements. The increased virulence of NAPCR1 was confirmed by mortality rates in the hamster model and strong inflammatory responses induced by bacteria-free supernatants in the murine ligated loop model. However, NAPCR1 strains do not synthesize toxin A and toxin B at levels comparable to those in NAP1 strains. Our results suggest that the pathogenic potential of this emerging C. difficile variant is due to the acquisition of hypothetical functions associated with laterally acquired DNA.

Multilocus sequence typing of Leuconostoc gelidum subsp. gasicomitatum, a Psychrotrophic lactic acid bacterium causing spoilage of packaged perishable foods.

Leuconostoc gelidum subsp. gasicomitatum is a psychrotrophic lactic acid bacterium (LAB) that causes spoilage of a variety of modified-atmosphere-packaged (MAP) cold-stored food products. During the past 10 years, this spoilage organism has been increasingly reported in MAP meat and vegetable products in northern Europe. In the present study, the population structure within 252 L. gelidum subsp. gasicomitatum strains was determined based on a novel multilocus sequence-typing (MLST) scheme employing seven housekeeping genes. These strains had been isolated from meat and vegetable sources over a time span of 15 years, and all 68 previously detected pulsed-field gel electrophoresis (PFGE) genotypes were represented. A total of 46 sequence types (STs) were identified, with a majority of the strains (>60%) belonging to three major STs, which were grouped into three clonal complexes (CCs) and 17 singletons by Global Optimal eBURST (goeBURST). The results by Bayesian analysis of population structure (BAPS) mostly correlated with the grouping by goeBURST. Admixture analysis by BAPS indicated a very low level of exchange of genetic material between the subpopulations. Niche specificity was observed within the subpopulations: CC1 and BAPS cluster 1 consisted mostly of strains from a variety of MAP meats, whereas vegetable strains grouped together with strains from MAP poultry within CC2 and BAPS cluster 2. The MLST scheme presented in this study provides a shareable and continuously growing sequence database enabling global comparison of strains associated with spoilage cases. This will further advance our understanding of the microbial ecology of this industrially important LAB.

The Epidemiology and Resistance Mechanisms of Acinetobacter baumannii Isolates from the Respiratory Department ICU of a Hospital in China

Acinetobacter baumannii is an important nosocomial pathogen able to cause severe infections in an intensive care unit (ICU). However, there is a lack of analysis regarding the epidemiology and resistance of A. baumannii in respiratory department ICUs. In this study, clinical isolates were collected from the respiratory department ICU of Southwest Hospital from January 2009 to December 2010, and the social and demographic information of the patients from whom the isolates were taken was obtained from the Southwest Hospital information system. The minimal inhibitory concentration (MIC) of the isolates was determined by the agar dilution method. The carbapenemase-encoding resistance genes of these isolates were amplified using PCR. The clonal relationship of isolates was analyzed by pulsed-field gel electrophoresis (PFGE). Forty-six isolates were collected from the respiratory department ICU, and the antibiotics minocycline and quinolone had higher drug sensitivity against these isolates. The OXA-51, OXA-23, and IMP-4 genes were present at rates of 100% (46/46), 67.4% (31/46), and 41.3% (19/46), respectively. Forty-six isolates had 12 different PFGE genotypes. The results above suggested that the hospital environment and patients contributed to nosocomial infections, and the spread of resistance genes in the hospital was common.

Spread of carbapenemase-producing enterobacteria in a southwest hospital in China.

BackgroundThe rapid emergence and dissemination of carbapenem resistance in Enterobacteriaceae complicates the treatment of infections caused by these organisms.MethodsWe collected clinical isolates with meropenem inhibition zones of ≤ 22 mm from January 1, 2009, through December 31, 2010. We attempted to amplify the NDM-1 gene from these isolates and conducted the modified Hodge test (MHT). The minimal inhibitory concentration (MIC) of the MHT-positive strains was determined by the agar disk dilution method. The carbapenemase-encoding resistance genes of these strains were examined using polymerase chain reaction (PCR) analysis and a sequencing strategy to characterize these enzymes. The clonal relationship among isolates was analyzed by pulsed-field gel electrophoresis (PFGE).ResultsAmong the 158 Enterobacteriaceae isolates that were collected, there were no NDM-1-positive strains and 26 MHT-positive strains. Among the latter, 18 strains were IMP-4-positive, and 1 was KPC-2-positive. In addition, 15 of the IMP-4-positive Klebsiella pneumoniae strains belonged to 4 PFGE genotypes, with 8 strains having the same genotype.ConclusionThese results suggest that nosocomial infections are one of the main reasons for the spread of these resistant strains.

Dominance of Multidrug-Resistant Denmark14-32 (ST230) Clone AmongStreptococcus pneumoniaeSerotype 19A Isolates Causing Pneumococcal Disease in Bulgaria from 1992 to 2013

A pneumococcal conjugate vaccine (PCV10) was introduced in Bulgarian national immunization program since April 2010. Clonal composition based on pulsed-field gel electrophoresis and multilocus sequence typing genotyping of 52 serotype 19A Streptococcus pneumoniae isolates was analyzed. These were invasive and respiratory isolates collected between 1992 and 2013 from both children (78.8% <5 years) and adults with pneumococcal infections. Multidrug resistance was found in 82.7% of all 19A isolates. The most prevalent genotype (63.5%) among serotype 19A pneumococcal strains was the multidrug-resistant clonal complex CC230, which is a capsular switched variant of the Denmark(14)-32 (ST230) global clone. The most frequent sequence type (ST) was ST230 (48.1%) and together with four other closely related STs (15.4%), belonging to ST1611, ST276, ST7466, and ST2013, which were single- and double-locus variants; they were included in the main CC230. The disappearance of highly drug-resistant ST663 clone and emergence of new clones as CC320 and CC199 was also observed among the rest 19A isolates. A comparison of clonal composition between invasive and noninvasive isolates did not show a great genetic diversity among both kinds of isolates. Continuous surveillance of serotype 19A population following the introduction of PCV10 is essential to evaluate the impact of the vaccine on the epidemiology of this serotype.

A preliminary molecular epidemiologic study using analysis of variable number of tandem repeats of Acinetobacter baumannii OXA-23 producing strains isolated from hospitals in Rio de Janeiro State, Brazil

The Gram-negative multidrug-resistant (MDR) bacterium Acinetobacter baumannii constitutes a serious cause of nosocomial infections in Brazilian hospitals. A panel of 36 strains, belonging to five pre-determined pulsed-field gel electrophoresis (PFGE) genotypes from four different hospitals in the City of Rio de Janeiro, Rio de Janeiro State, Brazil, was submitted to simple agarose gel electrophoresis to determine the variation of nine variable number tandem repeats (VNTR) band profiles. Based on published data VNTR were classified in two categories, long (L) and short (S) repeats with different evolutionary implications. The results demonstrated the superior discrimination of VNTR over PFGE. The VNTR Abaum_3002 marker was the least discriminatory with only one allele, while VNTR10 and Abaum_2240 each presented four alleles. The use of a combination of the nine VNTR resulted in a refined genotyping tool that provides valuable epidemiological information.

Distribution of Capnocytophaga canimorsus in dogs and cats with genetic characterization of isolates

Capnocytophaga canimorsus, which is often found in the oral cavities of dogs and cats, is sometimes transmitted to humans, causing severe infection. To elucidate the risk of C. canimorsus in humans and animals, this study was undertaken to characterize this bacterium epidemiologically and genetically. We examined the distribution of C. canimorsus in dogs and cats, and analyzed the correlation between the presence of bacteria and individual factors statistically. We also compared C. canimorsus isolates genetically using 16S rRNA gene sequence analysis and pulsed-field gel electrophoresis (PFGE). C. canimorsus was detected in 76 of 109 dogs (69.7%) and 57 of 104 cats (54.8%). A relation between C. canimorsus presence and some individual factors was detected both in dogs and cats, but the predictive factors of carrying the bacterium differed between dogs and cats. 16S rRNA gene sequences from C. canimorsus isolates in this study were combined with previously published sequences to assess their intra-specific phylogeny. Results show that C. canimorsus is classifiable into two main groups (I and II) with differing γ-glutamyl aminopeptidase activity. Strains from human patients belonged unevenly to group I, possibility suggesting that group I can be transmitted to humans and group II is indigenous only to the oral cavities of dogs and cats. PFGE genotyping showed high discriminatory power, and the dendrogram accorded with genetic segregation between isolates of group I and II. Sma I-digest PFGE developed for this study is useful as a molecular typing method for additional epidemiological and phylogenetic studies of C. canimorsus.

Genotyping of Staphylococcus aureus strains isolated from healthy persistent carriers

The paper presents results on the relatedness of Staphylococcus aureus strains colonizing the upper respiratory tract isolated from healthy persistent carriers. Genotyping was carried out using two methods--multiple-locus variable-number tandem-repeat fingerprinting (MLVF) and pulsed-field gel electrophoresis (PFGE). By comparison of the results obtained by both methods, good correlations between MLVF and PFGE genotyping of strains isolated from the asymptomatic carriers were observed. Further studies are needed to evaluate methods useful for genotyping of S. aureus strains circulating in the community.