Development of a Multiplex-PCR probe system for the proper identification of <Emphasis Type="Italic">Klebsiella variicola</Emphasis>

Background

Klebsiella variicola was very recently described as a new bacterial species and is very closely related to Klebsiella pneumoniae; in fact, K. variicola isolates were first identified as K. pneumoniae. Therefore, it might be the case that some isolates, which were initially classified as K. pneumoniae, are actually K. variicola. The aim of this study was to devise a multiplex-PCR probe that can differentiate isolates from these sister species.

Result

This work describes the development of a multiplex-PCR method to identify K. variicola. This development was based on sequencing a K. variicola clinical isolate (801) and comparing it to other K. variicola and K. pneumoniae genomes. The phylogenetic analysis showed that K. variicola isolates form a monophyletic group that is well differentiated from K. pneumoniae. Notably, the isolate K. pneumoniae 342 and K. pneumoniae KP5-1 might have been misclassified because in our analysis, both clustered with K. variicola isolates rather than with K. pneumoniae. The multiplex-PCR (M-PCR-1 to 3) probe system could identify K. variicola with high accuracy using the shared unique genes of K. variicola and K. pneumoniae genomes, respectively. M-PCR-1 was used to assay a collection of multidrug-resistant (503) and antimicrobial-sensitive (557) K. pneumoniae clinical isolates. We found K. variicola with a prevalence of 2.1% (23/1,060), of them a 56.5% (13/23) of the isolates were multidrug resistant, and 43.5% (10/23) of the isolates were antimicrobial sensitive. The phylogenetic analysis of rpoB of K. variicola-positive isolates identified by multiplex-PCR support the correct identification and differentiation of K. variicola from K. pneumoniae clinical isolates.

Conclusions

This multiplex-PCR provides the means to reliably identify and genotype K. variicola. This tool could be very helpful for clinical, epidemiological, and population genetics studies of this species. A low but significant prevalence of K. variicola isolates was found, implying that misclassification had occurred previously. We believe that our multiplex-PCR assay could be of paramount importance to understand the population dynamics of K. variicola in both clinical and environmental settings.

     

Extended spectrum beta-lactamase and metallo beta-lactamase production among <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> isolated from different clinical samples in a tertiary care hospital in Kathmandu,Nepal

Background

Extended spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL) production in Klebsiella pneumoniae and Escherichia coli are the commonest modes of drug resistance among these commonly isolated bacteria from clinical specimens. So the main purpose of our study was to determine the burden of ESBL and MBL production in E. coli and K. pneumoniae isolated from clinical samples. Further, the antimicrobial susceptibility patterns of E. coli and K. pneumoniae were also determined.

Methods

A cross-sectional study was conducted at Om Hospital and Research Centre, Kathmandu, Nepal by using the E. coli and K. pneumoniae isolated from different clinical samples (urine, pus, body fluids, sputum, blood) from May 2015 to December 2015. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion technique. Extended spectrum beta-lactamase production was detected by combined disc method using ceftazidime and ceftazidime/clavulanic acid discs and cefotaxime and cefotaxime/clavulanic acid discs. Similarly, metallo beta-lactamase production was detected by combined disc assay using imipenem and imipenem/ethylenediaminetetracetate discs. Bacteria showing resistance to at least three different classes of antibiotics were considered multidrug resistant (MDR).

Results

Of total 1568 different clinical samples processed, 268 (17.1%) samples were culture positive. Among which, E. coli and K. pneumoniae were isolated from 138 (51.5%) and 39 (14.6%) samples respectively. Of the total isolates 61 (34.5%) were ESBL producers and 7 (4%) isolates were found to be MBL producers. High rates of ESBL production (35.9%) was noted among the clinical isolates from outpatients, however no MBL producing strains were isolated from outpatients. Among 138 E. coli and 39 K. pneumoniae, 73 (52.9%) E. coli and 23 (59%) K. pneumoniae were multidrug resistant. The lowest rates of resistance was seen toward imipenem followed by piperacillin/tazobactam, amikacin and cefoperazone/sulbactam.

Conclusions

High rate of ESBL production was found in the E. coli and K. pneumoniae isolated from outpatients suggesting the dissemination of ESBL producing isolates in community. This is very serious issue and can’t be neglected. Regular monitoring of rates of ESBL and MBL production along with multidrug resistance among clinical isolates is very necessary.

     

Hypocrealean fungi associated with populations of <Emphasis Type="Italic">Ips typographus</Emphasis> in West Carpathians and selection of local <Emphasis Type="Italic">Beauveria</Emphasis> strains for effective bark beetle control

In Slovakia, a diversity of entomopathogenic fungi (Ascomycota, Hypocreales) associated with outbreaks of Ips typographus was studied in 81 localities and as many as 113 in vitro cultures of five entomopathogenic species were isolated from infected individuals: Beauveria bassiana (87 isolates), B. pseudobassiana (14 isolates), B. caledonica (6 isolates), Lecanicillium lecanii (4 isolates) and Isaria farinosa (2 isolates). B. pseudobassiana is recorded in natural populations of I. typographus for the first time. Biological properties of selected Beauveria isolates, including colony growth, biomass production, conidia yield and pathogenicity to I. typographus adults, were studied in a series of laboratory bioassays and much intra- and interspecific variability was detected. B. bassiana isolates produced biomass or conidia at significantly higher rate than B. pseudobassiana and B. caledonica isolates. Two B. bassiana isolates were selected as the most virulent to bark beetle adults, demonstrating a mean LC₅₀ ranging from 0.72 to 2.05?×?10⁶ conidia ml^?1, and were qualified as promising candidates for biocontrol of I. typographus. Their virulence was significantly higher than that of the mycoinsecticides Boverol®, which was used as a reference strain in the virulence bioassays.     

Virulence plasmid stability in environmentally occurring <Emphasis Type="Italic">Bacillus anthracis</Emphasis> from North East Turkey

The Bacillus anthracis virulence plasmid pXO2, which encodes for a polypeptide capsule, can be lost during long term laboratory storage. To determine if pXO2 is lost in nature we screened B. anthracis isolates obtained from B. anthracis spores from contaminated animal burial sites in Turkey for their ability to express a capsule upon primary culture. A total of 672 B. anthracis colonies were examined of which ten produced a mixed mucoid (capsule +ve)/non-mucoid (capsule ?ve) phenotype and a further one colony yielded non-mucoid colonies upon repeated culture. Screening by PCR using pXO2 specific primers revealed that seven of these isolates had eliminated the plasmid. Of the four colonies which were positive by PCR, one regained the ability to express a capsule upon repeated culture suggesting that the defect was reversible. This is an important observation as capsule expression is a principal marker of virulence and in the absence of PCR serves as a key diagnostic marker. The results of this preliminary study suggest that pXO2 is lost in nature and that further studies are need to determine the mechanisms by which this occurs.     

Clonal Transmission of ESBL-Producing <Emphasis Type="Italic">Klebsiella</Emphasis> spp. at a University Hospital in Brazil

The present study was designed to determine the prevalence and extended-spectrum β-lactamase (ESBL) types in clinical isolates of Klebsiella spp. at a university hospital located in the Brazilian southern region (Ribeirão Preto, São Paulo) as well as their antibiotic susceptibility and genetic profiles. This study included 147 non-repeat Klebsiella spp. isolates collected from January to June 2000, of which 23 K. pneumoniae and 8 K. oxytoca were selected as ESBL producers by using the Oxoid combination disk method and Etest ESBL strip. β-lactamases were characterized by IEF, PCR and sequencing assays using primers for ESBL genes. Antibiotic susceptibility was evaluated by MicroScan system. Dissemination of two major clones of ESBL-producing Klebsiella spp. occurred in the hospital. According to the results obtained in this study there was a clonal spread of CTX-M-producing K. oxytoca in five clinics and dissemination of ESBL-producing K. pneumoniae in the nursery and pediatrics wards.     

Genotypic and metabolic approaches towards the segregation of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> strains producing different antibiotic resistant enzymes

Introduction

Genotype and metabolomic variation are important for bacterial survival and adaptation to environmental changes.

Objectives

In this study, we compared the relationship among Klebsiella pneumoniae strains based on their genotypic and metabolic profiles. In addition, we also evaluated the association of the relationship with beta-lactamase production.

Methods

A total of 53 K. pneumoniae strains isolated in 2013–2014 from a tertiary teaching hospital in Malaysia were subjected to antimicrobial susceptibility testing (AST) via disk diffusion method and beta-lactamase production confirmation. The bacterial strains were also typed genotypically and metabolically via REP-PCR and ¹H-NMR spectroscopy respectively. The concordance of the matrices derived based on genotypic and metabolic characterization was measured based on Spearman’s rank correlation.

Results

Spearman’s correlation rank showed that there is a weak but significant negative correlation between the genetic fingerprints and metabolic profiles of K. pneumoniae. Specifically, K. pneumoniae strains were clustered into five major clusters based on REP-PCR where most of the carbapenem resistant K. pneumoniae (CRKP) strains made up the major cluster. In contrast, metabolic patterns of the three groups (i.e. CRKP, extended spectrum beta-lactamase producing K. pneumoniae (ESBL), susceptible) of K. pneumoniae were clearly differentiated on PLS-DA score plots derived from ¹H-NMR spectroscopy.

Conclusion

Overall, this study showed that metabolomic profiling using ¹H-NMR spectroscopy is able to discriminate K. pneumoniae strains based on their beta-lactamase production status.

     

Incidence of metallo-beta-lactamase-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> isolates from hospital setting in Pakistan

The aim of this study was monitoring and surveillance in different wards of the PIMS hospital, Islamabad, to understand emerging challenges of antibiotic resistance in particular association with most virulent serotypes of Klebsiella pneumoniae. The study was conducted during March 2015 to September 2015. The study showed that rate of isolation of K. pneumoniae was 37% (103 positives out of a total of 277 clinical samples) and 7.7% (8) were phenotypically and genotypically confirmed to be metallo-β-lactamase resistant (carbapenem resistant) and all of them were multidrug resistant (MDR). These carbapenem-resistant isolates were isolated from blood, endotracheal tubes, and pus. Molecular screening for the presence of integrons indicated that distribution of class I integrons (87.5% of carbapenem-resistant K. pneumoniae isolates) was higher than class II integrons (1.25%) among given isolates. The study indicated that exposure of metallo-beta-lactamase-producing strains through hospitalizations increases the chances of spread of MDR pathogens. There is an urgent need for effective surveillance and monitoring strategies to control the spread of extremely resistant K. pneumoniae implicated in nosocomial infections leading to the increased health burden and enforcement of policy guideline on appropriate antibiotics usage.     

An evaluation of multidrug-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates in urinary tract infections from Aguascalientes,Mexico: cross-sectional study

Background

Uropathogenic Escherichia coli (UPEC) are one of the main bacteria causing urinary tract infections (UTIs). The rates of UPEC with high resistance towards antibiotics and multidrug-resistant bacteria have increased dramatically in recent years and could difficult the treatment.

Methods

The aim of the study was to determine multidrug-resistant bacteria, antibiotic resistance profile, virulence traits, and genetic background of 110 E. coli isolated from community (79 isolates) and hospital-acquired (31 isolates) urinary tract infections. The plasmid-mediated quinolone resistance genes presence was also investigated. A subset of 18 isolates with a quinolone-resistance phenotype was examined for common virulence genes encoded in diarrheagenic and extra-intestinal pathogenic E. coli by a specific E. coli microarray.

Results

Female children were the group most affected by UTIs, which were mainly community-acquired. Resistance to trimethoprim–sulfamethoxazole, ampicillin, and ampicillin–sulbactam was most prevalent. A frequent occurrence of resistance toward ciprofloxacin (47.3%), levofloxacin (43.6%) and cephalosporins (27.6%) was observed. In addition, 63% of the strains were multidrug-resistant (MDR). Almost all the fluoroquinolone (FQ)-resistant strains showed MDR-phenotype. Isolates from male patients were associated to FQ-resistant and MDR-phenotype. Moreover, hospital-acquired infections were correlated to third generation cephalosporin and nitrofurantoin resistance and the presence of kpsMTII gene. Overall, fimH (71.8%) and fyuA (68.2%), had the highest prevalence as virulence genes among isolates. However, the profile of virulence genes displayed a great diversity, which included the presence of genes related to diarrheagenic E. coli. Out of 110 isolates, 25 isolates (22.7%) were positive to qnrA, 23 (20.9%) to qnrB, 7 (6.4%) to qnrS1, 7 (6.4%) to aac(6′)lb-cr, 5 (4.5%) to qnrD, and 1 (0.9%) to qnrC genes. A total of 12.7% of the isolates harbored bla_CTX-M genes, with bla_CTX-M-15 being the most prevalent.

Conclusions

Urinary tract infection due to E. coli may be difficult to treat empirically due to high resistance to commonly used antibiotics. Continuous surveillance of multidrug resistant organisms and patterns of drug resistance are needed in order to prevent treatment failure and reduce selective pressure. These findings may help choosing more suitable treatments of UTI patients in this region of Mexico.

     

Molecular testing of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> contaminating tissue allografts recovered from deceased donors

Microbiological screening of tissue allografts is crucial to prevent the transmission of bacterial and fungal infections to transplant recipients. Klebsiella was the most prevalent and resistant contaminating microorganism observed in our setting in the Iranian Tissue Bank. This study was conducted to determine the presence of extended-spectrum β-lactamase (ESBL) genes, antimicrobial resistance patterns of Klebsiella pneumoniae isolates, and their clonal relationships in allograft materials. K. pneumoniae contaminating bone and other tissue allografts recovered from deceased donors were identified and ESBL isolates were detected using a phenotypic confirmatory method. Antimicrobial susceptibility testing was carried out using the disk diffusion method. Distribution of ESBL genes and molecular typing were performed using polymerase chain reaction (PCR) and Repetitive-element (rep-PCR) methods. Of 3828 donated tissues, 51 (1.3%) were found contaminated by K. pneumoniae isolates. Compared to tissue allografts from brain-dead, heart-beating tissue donors, allografts from donors with circulatory cessation were associated with a higher risk of K. pneumoniae contamination [odds ratio (OR), 1.2 (CI 95% 0.9–2.3) (P value < 0.001)]. Half of the isolates produced ESBL, and the rate of susceptibility to cephalosporins was 51%. Among isolates, 22 (43.1%) harbored CTX-M, 31 (60.8%) SHV, and 9 (17.6%) harbored TEM types. The rep-dendrogram indicated that clones having identical or related strains with a similar antibiotype were isolated in the same period. This study provides evidence that a single clone of K. pneumoniae contaminated tissue allografts recovered from many different donors. A single clone found on tissues from several donors suggests contamination of tissues from a single source such as the tissue recovery process and environment. Genomic DNA testing and clonality of contaminating bacteria using molecular methods can focus the epidemiologic investigation on the tissue allograft recovery process including a search for contamination of the tissue recovery room environment, recovery staff, recovery equipment, reagents, solutions and supplies.     

Genomic characterisation,detection of genes encoding virulence factors and evaluation of antibiotic resistance of <Emphasis Type="Italic">Trueperella pyogenes</Emphasis> isolated from cattle with clinical metritis

Trueperella pyogenes is one of the most important microorganisms causing metritis in post-partum cattle. Co-infection with other bacterial species such as Escherichia coli or Fusobacterium necrofurom increases the severity of the disease and the persistence of bacteria in utero. The aim of this study was to investigate the frequency of T. pyogenes strains, and their virulence and antimicrobial resistant profiles in metritis cases. The study was carried out on 200 samples obtained from metritis discharges of postpartum cattle on 18 farms around Tehran, Iran. Sixty-five T. pyogenes isolates (32.5%) were identified, of which 16 isolates were detected as pure cultures and the other 49 isolates from cultures most commonly mixed with E. coli or F. necrofurom. In terms of diversity in biochemical characteristic of T. pyogenes strains, 8 different biotypes were identified among the isolates. Single or multi antimicrobial resistance was observed in 48 isolates (73.9%), which was mostly against trimethoprim sulfamethoxazole, azithromycin, erythromycin and streptomycin. The tetracycline resistance gene tetW and macrolide resistance genes ermB and ermX were detected in 30, 18 and 25 isolates, respectively. In the screening of genes encoding virulence factors, fimA and plo genes were identified in all tested isolates. Genes encoding nanP, nanH, fimC, fimG, fimE and cbpA were detected in 50, 54, 45, 40, 50 and 37 of isolates, respectively. Thirteen different genotypes were observed in these T. pyogenes isolates. A significant association between clonal types and virulence factor genes, biochemical profile, CAMP test result, severity of the disease and sampling time was detected.     

Genetic characterization of Hawaiian isolates of <Emphasis Type="Italic">Plasmodium relictum</Emphasis>reveals mixed-genotype infections

Background

The relatively recent introduction of a highly efficient mosquito vector and an avian pathogen (Plasmodium relictum) to an isolated island ecosystem with naïve, highly susceptible avian hosts provides a unique opportunity to investigate evolution of virulence in a natural system. Mixed infections can significantly contribute to the uncertainty in host-pathogen dynamics with direct impacts on virulence. Toward further understanding of how host-parasite and parasite-parasite relationships may impact virulence, this study characterizes within-host diversity of malaria parasite populations based on genetic analysis of the trap (thrombospondin-related anonymous protein) gene in isolates originating from Hawaii, Maui and Kauai Islands.

Methods

A total of 397 clones were produced by nested PCR amplification and cloning of a 1664 bp fragment of the trap gene from two malarial isolates, K1 (Kauai) and KV115 (Hawaii) that have been used for experimental studies, and from additional isolates from wild birds on Kauai, Maui and Hawaii Islands. Diversity of clones was evaluated initially by RFLP-based screening, followed by complete sequencing of 33 selected clones.

Results

RFLP analysis of trap revealed a minimum of 28 distinct RFLP haplotypes among the 397 clones from 18 birds. Multiple trap haplotypes were detected in every bird evaluated, with an average of 5.9 haplotypes per bird. Overall diversity did not differ between the experimental isolates, however, a greater number of unique haplotypes were detected in K1 than in KV115. We detected high levels of clonal diversity with clear delineation between isolates K1 and KV115 in a haplotype network. The patterns of within-host haplotype clustering are consistent with the possibility of a clonal genetic structure and rapid within-host mutation after infection.

Conclusion

Avian malaria (P. relictum) and Avipoxvirus are the significant infectious diseases currently affecting the native Hawaiian avifauna. This study shows that clonal diversity of Hawaiian isolates of P. relictum is much higher than previously recognized. Mixed infections can significantly contribute to the uncertainty in host-pathogen dynamics with direct implications for host demographics, disease management strategies, and evolution of virulence. The results of this study indicate a widespread presence of multiple-genotype malaria infections with high clonal diversity in native birds of Hawaii, which when coupled with concurrent infection with Avipoxvirus, may significantly influence evolution of virulence.

Reviewers

This article was reviewed by Joseph Schall (nominated by Laura Landweber), Daniel Jeffares (nominated by Anthony Poole) and Susan Perkins (nominated by Eugene Koonin).

     

Genetic differentiation of <Emphasis Type="Italic">Puccinia triticina</Emphasis> Erikss. in Russia

A collection of Puccinia triticina isolates was characterized for polymorphism of microsatellite loci and estimated for their differentiation by geographic origin. The collection included 20 isolates from the Ural region, 31 from West Siberia, 53 from Central Europe, 32 from the Northwest region, 32 from the Volga region, and 40 from the North Caucasus (24 from Dagestan and 16 from Krasnodar and Stavropol). The studied isolates were represented by 65 virulence phenotypes. In the polymorphism analysis of 18 microsatellite loci, 69 genotypes were determined. The index values of genetic distances (F _st, R _st, KB _c) between populations for microsatellite loci indicated differentiation of P. triticina isolates according to geographical origin, and they were clustered into three groups: (1) Asian, (2) European, and (3) North Caucasian. The North Caucasian isolates from Krasnodar and Stavropol regions were closer in similarity to European isolates than the Dagestan ones. Current analysis confirmed the assumption made earlier on the basis of the virulence analysis about the existence of several geographic fungi populations in Russia.     

Phylogenetic,toxigenic and virulence profiles of <Emphasis Type="Italic">Alternaria</Emphasis> species causing leaf blight of tomato in Egypt

Species of Alternaria are serious plant pathogens, causing major losses on a wide range of crops. Leaf blight symptoms were observed on tomato leaves, and samples were collected from various regions. Isolation was done from symptomatic tomato leaves, and 15 representatives were selected from a collection of 65 isolates of Alternaria species. The virulence of Alternaria isolates was investigated on detached leaves (DL) and whole plants of tomato cv. Super strain B. A phylogenetic analysis was performed based on three partial gene regions, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the RNA polymerase second largest subunit (RPB2) and the Alternaria major allergen gene (Alt a 1). The potentiality of Alternaria isolates to produce toxins was also investigated on the basis of thin-layer chromatography (TLC). Our investigations revealed that Alternaria isolates showed different levels of virulence either on tomato plants or DL. Based on the phylogeny of three genes, Alternaria isolates encompassed two species of small-spored morphospecies: A. alternata (14 isolates) and A. arborescens (single isolate). The produced toxins varied among Alternaria isolates with tenuazonic acid (TeA) being the most abundant mycotoxin produced by most isolates. This study highlighted on other Alternaria species in Egypt that might represent a serious concern for tomato producers as causal agents of leaf blight over other species, i.e. A. solani.     

Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> virulence genes in <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis> from Vellore,India

Streptococcus dysgalactiae subsp. equisimilis (SDSE), belonging to the group C and G streptococci, are human pathogens reported to cause clinical manifestations similar to infections caused by Streptococcus pyogenes. To scrutinize the distribution of gene coding for S. pyogenes virulence factors in SDSE, 255 isolates were collected from humans infected with SDSE in Vellore, a region in southern India, with high incidence of SDSE infections. Initial evaluation indicated SDSE isolates comprising of 82.35% group G and 17.64% group C. A multiplex PCR system was used to detect 21 gene encoding virulence-associated factors of S. pyogenes, like superantigens, DNases, proteinases, and other immune modulatory toxins. As validated by DNA sequencing of the PCR products, sequences homologous to speC, speG, speH, speI, speL, ssa and smeZ of the family of superantigen coding genes and for DNases like sdaD and sdc were detected in the SDSE collection. Furthermore, there was high abundance (48.12% in group G and 86.6% in group C SDSE) of scpA, the gene coding for C5a peptidase in these isolates. Higher abundance of S. pyogenes virulence factor genes was observed in SDSE of Lancefield group C as compared to group G, even though the incidence rates in former were lower. This study not only substantiates detection of S. pyogenes virulence factor genes in whole genome sequenced SDSE but also makes significant contribution towards the understanding of SDSE and its increasing virulence potential.     

<Emphasis Type="Italic">Aeromonas</Emphasis> and <Emphasis Type="Italic">Plesiomonas</Emphasis> species from scarlet ibis (<Emphasis Type="Italic">Eudocimus ruber</Emphasis>) and their environment: monitoring antimicrobial susceptibility and virulence

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.     

Colistin resistance in <Emphasis Type="Italic">Enterobacter</Emphasis> spp. isolates in Korea

We investigated the colistin resistance rate among 356 Enterobacter spp. clinical isolates from eight hospitals in Korea. Antibiotic susceptibility testing was performed by broth microdilution. While 51 of 213 (23.9%) Enterobacter cloacae isolates were colistin-resistant, only six of 143 (4.2%) E. aerogenes isolates showed resistance. We also identified the skip well phenotype in eight E. cloacae and three E. aerogenes isolates. Multilocus sequence typing for E. cloacae and randomly amplified polymorphic DNA analysis and enterobacterial repetitive intergenic consensus PCR for E. aerogenes revealed that clonal spreading of colistin-resistant and skip well Enterobacter spp. isolates had not occurred. In vitro time-kill assays were performed with three colistin-resistant, three skip well, and two colistin-susceptible isolates of E. cloacae and E. aerogenes. Inconsistent results were observed among isolates with skip well phenotypes; while some were eradicated by 2 mg/L colistin, others were not. This suggests that skip well isolates have differentiated into different categories. As the high rates of colistin resistance in E. cloacae detected are of clinical concern, continuous monitoring is warranted. In addition, the clinical implications and mechanisms of the skip well phenotype should be investigated to ensure the appropriate use of colistin against Enterobacter infections.     

Isolation and characterization of chromium(VI)-reducing bacteria from tannery effluents and solid wastes

In the present investigation, five novel Cr(VI) reducing bacteria were isolated from tannery effluents and solid wastes and identified as Kosakonia cowanii MKPF2, Klebsiella pneumonia MKPF5, Acinetobacter gerneri MKPF7, Klebsiella variicola MKPF8 and Serratia marcescens MKPF12 by 16S rDNA gene sequence analysis. The maximum tolerance concentration of Cr(VI) as K₂Cr₂O₇ of the bacterial isolates was varying up to 2000 mg/L. Among the investigated bacterial isolates, A. gerneri MKPF7 was best in terms of reduction rate. The optimum temperatures for growth and Cr(VI) reduction by the bacterial isolates were 35 and 40 °C, respectively except A. gerneri MKPF7 which grew and reduced Cr(VI) optimally at 40 °C. The optimum pH for growth and Cr(VI) reduction by K. cowanii MKPF2, A. gerneri MKPF7 and S. marcescens MKPF12 was 7.0 whereas the optimum pH for growth and Cr(VI) reduction by K. pneumoniae MKPF5 and K. variicola MKPF8 were 7.0, 8.0 and 6.0, 7.0, respectively. All the bacterial isolates showed maximum tolerance against Ni²⁺ and Zn²⁺ whereas minimum tolerance was observed against Hg²⁺ and Cd²⁺. The bacteria isolated in the present study thus can be used as eco-friendly biological expedients for the remediation and detoxification of Cr(VI) from the contaminated environments.     

Characterization of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> isolates from Austrian companion animals and horses

Background

The aim of the present study was to investigate the genetic relatedness and the antimicrobial resistance profiles of a collection of Austrian Streptococcus pneumoniae isolates from companion animals and horses. A total of 12 non-repetitive isolates presumptively identified as S. pneumoniae were obtained during routinely diagnostic activities between March 2009 and January 2017.

Results

Isolates were confirmed as S. pneumoniae by bile solubility and optochin susceptibility testing, matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) mass spectrometry and sequence analysis of a part recA and the 16S rRNA genes. Isolates were further characterized by pneumolysin polymerase chain reaction (PCR) and genotyped by multilocus sequence typing (MLST). Antimicrobial susceptibility testing was performed and resistance genes were detected by specific PCR assays. All isolates were serotyped. Four sequence types (ST) (ST36, ST3546, ST6934 and ST6937) and four serotypes (3, 19A, 19F and 23F) were detected. Two isolates from twelve displayed a multidrug-resistance pheno- and genotype.

Conclusions

This study represents the first comprehensive investigation on characteristics of S. pneumoniae isolates recovered from Austrian companion animals and horses. The obtained results indicate that common human sero- (23F) and sequence type (ST36) implicated in causing invasive pneumococcal disease (IPD) may circulate in dogs. Isolates obtained from other examined animals seem to be host-adapted.

     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> as a Probiotic Potential from Kouzeh Cheese (Traditional Iranian Cheese) and Its Antimicrobial Activity

The aim of this study is to isolate and identify Lactobacillus plantarum isolates from traditional cheese, Kouzeh, and evaluate their antimicrobial activity against some food pathogens. In total, 56 lactic acid bacteria were isolated by morphological and biochemical methods, 12 of which were identified as Lactobacillus plantarum by biochemical method and 11 were confirmed by molecular method. For analyzing the antimicrobial activity of these isolates properly, diffusion method was performed. The isolates were identified by 318 bp band dedicated for L. plantarum. The isolated L. plantarum represented an inhibitory activity against four of the pathogenic bacteria and showed different inhibition halos against each other. The larger halos were observed against Staphylococcus aureus and Staphylococcus epidermidis (15 ± 0.3 and 14.8 ± 0.7 mm, respectively). The inhibition halo of Escherichia coli was smaller than that of other pathogen and some L. plantarum did not show any inhibitory activity against E. coli, which were resistant to antimicrobial compounds produced by L. plantarum. The isolated L. plantarum isolates with the antimicrobial activity in this study had strong probiotic properties. These results indicated the nutritional value of Kouzeh cheese and usage of the isolated isolates as probiotic strains.