Distinct Clonal Types Research Articles

Antibiotic Resistance and Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Infant Formula Milk.

Background: Cronobacter sakazakii is a new emerging foodborne bacterial pathogen associated with severe lethal diseases such as meningitis, necrotizing enterocolitis, and septicemia in infants and neonates. Powdered infant formula milk (PIFM) has been recognized as one of the main transmission vehicles and contaminated sources of this pathogen. This study aimed to investigate the prevalence rate, genotypic and phenotypic antibiotic resistance profile, and clonal relatedness of C. sakazakii strains isolated from 364 PIFM samples collected from Tehran city, Iran. Methods: Culture-based methods, Kirby–Bauer disk diffusion antibiotic resistance testing, conventional Polymerase Chain Reaction (PCR), and Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) assays were used in this study to detect and characterize the C. sakazakii isolates. Results: We isolated 25 C. sakazakii strains from PIFM samples (6.86%). The isolates were highly resistant to amoxicillin-clavulanic acid, amoxicillin, ampicillin, cefoxitin, cefepime, erythromycin, ceftriaxone, ciprofloxacin, and chloramphenicol and susceptible to gentamicin, tetracycline, norfloxacin, and azithromycin antibiotics. The blaCTX-M-1 gene was detected in 96% of the isolates. The isolates were categorized into eight distinct clonal types using the ERIC-PCR method, showing a high genetic diversity among the isolates. However, there was a significant correlation between the genotypic and phenotypic antibiotic resistance properties of the isolates. Conclusions: Novel microbial surveillance systems for detecting multi-drug-resistant C. sakazakii are required to control the contamination of this foodborne pathogen in infant foods.

Emergence of NDM-1-producing Klebsiella pneumoniae in Greece: evidence of a widespread clonal outbreak.

NDM-producing Enterobacteriaceae clinical isolates remain uncommon in the European region. We describe the emergence and broad dissemination of one successful NDM-1-producing Klebsiella pneumoniae clone in Greek hospitals. During a 4 year survey (January 2013-December 2016), 480 single-patient carbapenem non-susceptible K. pneumoniae isolates, phenotypically MBL positive, were consecutively recovered in eight Greek hospitals from different locations and subjected to further investigation. Antimicrobial susceptibility testing, combined-disc test, identification of resistance genes by PCR and sequencing, molecular fingerprinting by PFGE, plasmid profiling, replicon typing, conjugation experiments and MLST were performed. Molecular analysis confirmed the presence of the blaNDM-1 gene in 341 (71%) K. pneumoniae isolates. A substantially increasing trend of NDM-1-producing K. pneumoniae was noticed during the survey (R2 = 0.9724). Most blaNDM-1-carrying isolates contained blaCTX-M-15, blaOXA-1, blaOXA-2 and blaTEM-1 genes. PFGE analysis clustered NDM-1 producers into five distinct clonal types, with five distinct STs related to each PFGE clone. The predominant ST11 PFGE clonal type was detected in all eight participating hospitals, despite adherence to the national infection control programme; it was identical to that observed in the original NDM-1 outbreak in Greece in 2011, as well as in a less-extensive NDM-1 outbreak in Bulgaria in 2015. The remaining four ST clonal types (ST15, ST70, ST258 and ST1883) were sporadically detected. blaNDM-1 was located in IncFII-type plasmids in all five clonal types. This study gives evidence of possibly the largest NDM-1-producing K. pneumoniae outbreak in Europe; it may also reinforce the hypothesis of an NDM-1 clone circulating in the Balkans.

NDM-1 Hazard in the Balkan States: Evidence of the First Outbreak of NDM-1-Producing Klebsiella pneumoniae in Bulgaria.

New Delhi MBL (NDM) carbapenemase-producing Klebsiella pneumoniae has become one of the most concerning multidrug-resistant pathogens. The Balkan counties are considered a reservoir for the spread of such strains based on several reports documenting NDM infections after hospitalization in this region. Nevertheless, NDM-producing K. pneumoniae have been only occasionally documented from Balkans. The current study documents the first polyclonal outbreak caused by NDM-1-producing K. pneumoniae in Bulgaria. From July 2015 to April 2016, all 25 single-patient carbapenem-nonsusceptible K. pneumoniae isolates were collected. Phenotypic and molecular screening revealed that 17 produced NDM-1 carbapenemase. All NDM-1 producers harbored blaCTX-M-15, blaCMY-4, blaTEM-1, and blaOXA-2; five also harbored blaOXA-1. In all cases, blaNDM-1 was flanked upstream by ISAba125 element and downstream by bleMBL. Pulsed-field gel electrophoresis (PFGE) clustered NDM-1-positive isolates into four distinct clonal types, A to D. MLST assigned isolates of the dominant clonal type A (n = 14) to sequence type (ST) 11, while isolates of clonal types B, C, and D to ST16, ST15, and ST391, respectively. Of interest, ST11 isolates belonged to the same PFGE type as those of the recently described NDM-1 ST11 clonal outbreak in Greece. Traveling abroad or overseas hospitalization was not reported in any case, suggesting most likely intra- and interhospital dissemination. The study presents the first polyclonal outbreak of NDM-producing K. pneumoniae in the Balkans and underlines the need for larger epidemiological studies in the region to illustrate commonalities in the transmission of NDM clones and possible sources in the community.

An assessment on DNA microarray and sequence-based methods for the characterization of methicillin-susceptible Staphylococcus aureus from Nigeria.

Staphylococcus aureus is an important human pathogen causing nosocomial and community-acquired infections worldwide. In the characterization of this opportunistic pathogen, DNA microarray hybridization technique is used as an alternative to sequence based genotyping to obtain a comprehensive assessment on the virulence, resistance determinants, and population structure. The objective of this study was to characterize a defined collection of S. aureus isolates from Nigeria using the microarray technique, and to assess the extent that it correlates with sequence-based genotyping methods. The clonal diversity and genomic content of 52 methicillin-susceptible Staphylococcus aureus (MSSA) were investigated by spa typing, MLST and DNA microarray hybridization. More than half (55.8%) of these isolates were associated with clonal complexes (CCs) typically associated with methicillin-resistant S. aureus (MRSA) clones i.e., CC1, CC5, CC8, CC30, and CC45. Certain genes linked with virulence (hlgA and clfA) and adherence (ebpS, fnbA, sspA, sspB, and sspP) were detected in all isolates. A number of genes or gene clusters were associated with distinct clonal types. The enterotoxin gene cluster (egc) was linked with CC5, CC25, CC30, CC45, and CC121, enterotoxin H gene (seh) with CC1, exfoliative toxin D gene (etd) with CC25 and CC80, and the epidermal cell differentiation inhibitor B gene (edinB) with CC25, CC80, and CC152. The excellent agreement between data from DNA microarray and MLST in the delineation of Nigerian MSSA isolates indicates that the microarray technique is a useful tool to provide information on antibiotic resistance, clonal diversity and virulence factors associated with infection and disease.

Circulation of a multiresistant, conjugative, IncA/C plasmid within the nosocomial Providencia stuartii population in the Athens area

The objective of the study is to report a multidrug-resistant outbreak of Providencia stuartii that occurred in inpatients in the Athens area in 2012 resulting from a very successful transmissible A/C multidrug-resistant plasmid. Thirteen multidrug-resistant P. stuartii clinical isolates from 5 hospitals were studied. Molecular typing was performed by pulsed-field gel electrophoresis. Antibiotic resistance genes and their genetic surround were detected by PCR and sequencing. Plasmid analysis included conjugation experiments using liquid cultures, sizing by S1 digestion, and incompatibility replicon typing by PCR. Isolates were grouped into 2 distinct clonal types A and B, exhibiting similarity less than 70%. Isolates of type A were recovered from patients hospitalized in 4 different hospitals with no obvious epidemiological linkage, while isolates of type B were recovered from patients treated in a single hospital. Both clonal types harbored a conjugative plasmid of 130bp and IncA/C replicon type carrying 5 β-lactamase genes blaSHV-5, blaVEB-1, blaVIM-1, blaOXA-10, and blaTEM-1 and aminoglycosides resistant determinants. All β-lactamase genes were included in stable structures as IS26, IS1999, and In-e541. The current plasmid seemed to have many common determinants with previously reported plasmids derived from P. stuartii and Proteus mirabilis clinical isolates and exhibited the ability to circulate in nosocomial bacterial populations.

A human origin type II strain of Toxoplasma gondii causing severe encephalitis in mice

Despite its capacity for sexual reproduction and global distribution, Toxoplasma gondii has a highly clonal structure, with the majority of isolates belonging to three distinct clonal types. Congenital toxoplasmosis has been associated with type I and type II strains. We here present the first characterization of a T. gondii strain (BGD1) from South-East Europe, isolated from the umbilical blood of a 24-week-old fetus in Serbia. Genotyping, performed by PCR-RFLP using a set of nested PCR markers (5′SAG2, 3′SAG2, BTUB, SAG3, and GRA6), showed that the BGD1 strain possessed a type II genotype. The cytokine patterns in Swiss-Webster mice inoculated with brain cysts of BGD1 and the prototype type II ME49 strain were similar until 180 days post-infection, with highly elevated IFN-γ, IL-12 and IL-10 by d7 and decreasing thereafter. While both strains induced pneumonia and hepatitis in acute infection (d14), chronic infection (d56) was characterized, in addition to hepatitis, by severe meningoencephalitis, associated with numerous brain cysts. Thus, the BGD1 strain of T. gondii has type II genotypic and immunologic characteristics, but unlike other type II strains of human origin, induces severe encephalitis, making it an alternative to the sheep-derived ME49 strain for experimental models of infection.

CLONAL DIVERSITY OF STREPTOCOCCUS MUTANS CLARKE (1924) IN CARIES-FREE ADULTS

Very few information about the clonal profile of Streptococcus mutans in caries-free adults is available. Microbial samples of saliva, tongue dorsum, and dental biofilm were taken from eight caries-free volunteers (DMFT = 0). Presumptive colonies of mutans streptococci were identified until species level. After confirmation, S. mutans strains were grown in liquid culture media and harvested for whole-cell protein extraction. Multilocus Enzyme Electrophoresis (MLEE) separated protein extracts and the bands’ patterns were assessed for six enzymes. Results showed that two or more distinct clonal types (avg = 4.75) were found cocolonizing all the enrolled subjects, confirming the premise that caries-free subjects are prone to be colonized by more than one strain of S. mutans.

Molecular epidemiology and diversity ofSalmonellaserovar Typhimurium in pigs using phenotypic and genotypic approaches

For epidemiological investigations of the most common and non-host-adapted Salmonella serotypes, such as Typhimurium, highly discriminatory approaches are essential. In the present study, we evaluated three genotyping methods; amplified fragment length polymorphism (AFLP), pulsed-field gel electrophoresis (PFGE) and repetitive palindromic extragenic-PCR (Rep-PCR) using 40 isolates. AFLP showed the highest discriminatory index (0.939), resolution and throughput. To determine clonality of Salmonella Typhimurium isolates and epidemiological relatedness in different commercial pig production units, we employed AFLP in combination with antimicrobial resistance pattern and phage typing. Salmonella serovar Typhimurium isolates (n=196) obtained from a longitudinal study of 18 pig farms over a 3-year period were studied. Using this approach, 16 distinct clonal types were identified. We found two common multidrug- resistant patterns including AmCmStSuTe and AmKmStSuTe. Two commonly multidrug- resistant phage types that are of known public health importance, DT104 and DT193, were also common. AFLP differentiated distinct clones within DT104, a phage type previously reported to be clonal. Fourteen of the clonal types were unique to one of the two production systems, showing diversity between independent commercial pig production systems located in the same geographical area. Clonal types obtained from nursery farms and corresponding finishing units were, however, similar.

Substantially increased faecal carriage of vancomycin-resistant enterococci in a tertiary Greek hospital after a 4 year time interval.

In a tertiary Greek hospital with no documented vancomycin-resistant enterococci (VRE) infections, a cross-sectional study was conducted in order to determine the degree of VRE faecal carriage among adult patients hospitalized in high-risk units. Specimens for the surveillance were collected from separate patients in two periods (January-May 1999 and January-May 2003); 258 specimens were submitted during the first period and 149 during the second period. Three patients (1.2%) were colonized with VRE during the first period, whereas 52 (34.9%) were colonized during the second period. Two VRE isolates of the first period were Enterococcus faecalis and one Enterococcus faecium, whereas those of the second period were E. faecium except for three E. faecalis and two Enterococcus gallinarum. All VRE isolates apart from the two E. gallinarum isolates were positive for the vanA gene. The 48 vancomycin-resistant E. faecium were classified into eight clonal types, one of those predominating with 29 isolates; the remaining included one to nine isolates. The five vancomycin-resistant E. faecalis formed four distinct clonal types. The study reports a substantially higher prevalence of VRE carriage when the surveillance was repeated after a 4 year time interval. Urgent infection control measures are needed to prevent emergence of VRE outbreaks in our hospital setting.

Two new mechanisms of macrolide resistance in clinical strains of Streptococcus pneumoniae from Eastern Europe and North America.

Resistance to macrolides in pneumococci is generally mediated by methylation of 23S rRNA via erm(B) methylase which can confer a macrolide (M)-, lincosamide (L)-, and streptogramin B (S(B))-resistant (MLS(B)) phenotype or by drug efflux via mef(A) which confers resistance to 14- and 15-membered macrolides only. We studied 20 strains with unusual ML or MS(B) phenotypes which did not harbor erm(B) or mef(A). The strains had been isolated from patients in Eastern Europe and North America from 1992 to 1998. These isolates were found to contain mutations in genes for either 23S rRNA or ribosomal proteins. Three strains from the United States with an ML phenotype, each representing a different clone, were characterized as having an A2059G (Escherichia coli numbering) change in three of the four 23S rRNA alleles. Susceptibility to macrolides and lincosamides decreased as the number of alleles in isogenic strains containing A2059G increased. Sixteen MS(B) strains from Eastern Europe were found to contain a 3-amino-acid substitution ((69)GTG(71) to TPS) in a highly conserved region of the ribosomal protein L4 ((63)KPWRQKGTGRAR(74)). These strains formed several distinct clonal types. The single MS(B) strain from Canada contained a 6-amino-acid L4 insertion ((69)GTGREKGTGRAR), which impacted growth rate and also conferred a 500-fold increase in MIC on the ketolide telithromycin. These macrolide resistance mechanisms from clinical isolates are similar to those recently described for laboratory-derived mutants.

Molecular population and virulence factor analysis of Staphylococcus aureus from bovine intramammary infection.

Staphylococcus aureus isolates from cows in Ireland (n = 102) and the USA (n = 42) were characterized by RAPD-PCR and analysed for the production of a number of putative virulence factors. Of these strains 63 representative isolates were screened for the corresponding virulence factor genes by PCR or Southern hybridization or both. The isolates were divided into 12 distinct clonal types on the basis of their RAPD fingerprint profiles. Of the isolates, 107 (74.3%) tested positive for clumping factor in a slide agglutination test, all 24 RAPD type 7 isolates being negative for clumping factor. PCR analysis of region R, a repeat region of the clfA gene, revealed eight region-R sizes. There was a strong association between RAPD type and the clfA region-R genotype among Irish isolates. Of the RAPD type 7 isolates, 21 (87.5%) coproduced toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin C (SEC). Over 90% of isolates demonstrated haemolytic activity on sheep or rabbit red blood cells and all isolates harboured the gamma-haemolysin (hlg) locus. Of the Irish isolates, all those of RAPD type 7 were sensitive to penicillin G, whereas 86% of RAPD types 4 and 5 strains were resistant. Furthermore, RAPD types 5 and 7 were more likely to be associated with clinical mastitis whereas RAPD type 4 isolates were more often associated with a latent infection. The current study identifies some of the putative virulence factors produced by the predominant clonal types of bovine Staph. aureus that may be considered as components of a vaccine.

Testing the efficacy of a molecular surveillance network: methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF) genotypes in six hospitals in the metropolitan New York City area. The BARG Initiative Pilot Study Group. Bacterial Antibiotic Resistance Group.

Molecular fingerprinting techniques are rapidly becoming indispensable tools for hospital epidemiology. On the other hand, the relative complexity and unfamiliarity of these techniques to most hospital diagnostic laboratories limit their usefulness. In an attempt to provide a solution for this dilemma, we tested the feasibility and efficacy of a cooperative venture in which molecular typing of isolates recovered from patients in six hospitals was performed at two microbiology research laboratories with expertise in these techniques. In a small preliminary study, 30 methicillin-resistant Staphylococcus aureus (MRSA) and 30 vancomycin-resistant Enterococcus faecium (VREF) isolates were collected over a 3-week period from six hospitals in the metropolitan New York area and transported to the Laboratory of Microbiology at The Rockefeller University during the summer months of 1994. Nineteen of the 27 confirmed MRSA isolates were closely related strains carrying the same mecA and the same Tn554 polymorphs in a pulsed-field gel electrophoresis (PFGE) background represented by closely related subtypes of a single pattern, indicating the wide distribution of this MRSA clone among the participating hospitals. Typing of the same 27 MRSA isolates was also performed at the Tuberculosis Center of the Public Health Research Institute and identical results were obtained. The 29 confirmed VREF isolates were highly heterogeneous and belonged to as many as 23 distinct clonal types as defined by PFGE patterns and probing with vanA. Characterization of the 60 isolates by these methods was completed in one month of full-time effort by a single experienced laboratory assistant guided by a doctoral-level expert in molecular fingerprinting techniques. The collection of samples for both MRSA and VREF was not intended to address epidemiological questions but to determine the feasibility of a multicenter study. On the basis of our preliminary findings we are encouraged that a larger cooperative effort is possible and with the correct sampling method we believe that epidemiological and surveillance studies could be accomplished that would provide a tracking system to assist hospitals, clinics, and chronic care facilities in controlling the spread of multidrug-resistant pathogens.

The clones of common lime (Tilia×vulgaris Hayne) planted in England during the seventeenth and eighteenth centuries.

Most avenues of limes planted in England and Wales during the late seventeenth and early eighteenth century contain two distinct clonal types of common lime (Tilia×vulgaris Hayne). They differ in many morphological characters. Clonal group A has a fluted trunk with epicormic bosses or sprouts, a conical crown, scarlet, shining, ellipsoid overwintering buds, bright green leaves with obliquely asymmetric bases, long bracts narrowed to both ends and inflorescences normally of seven flowers. Clonal group B has a cylindrical trunk which is almost without epicormic buds or shoots, a hemispherical crown, crimson, pruinose, ovoid and sub-acute buds, more or less cordate, dull green leaves, bracts narrowed to the stalk but broadly rounded at the distal end, and inflorescences with 3-5 flowers. Clonal group A includes the Dutch clones 'Pallida' and 'Koningslinde'. Clonal group B is closely related to, but not identical with, the Dutch clone 'Svartelinde'. Both clonal groups were probably originally imported from the Netherlands and were possibly derived from ancient village trees, which in the Netherlands are usually T. ×vulgaris.

A statistical approach to the ecology of Porphyromonas gingivalis.

Previous studies have suggested that infections with Porphyromonas gingivalis, associated with periodontal disease, may consist of one clonal type. It has also been shown that each individual patient carries a unique clonal type of P. gingivalis, as assessed by DNA fingerprinting. This issue was further examined by random collection of multiple isolates of P. gingivalis from multiple sites in several patients, and characterization of these isolates by DNA fingerprinting. Although most patients appeared to be infected exclusively by one clonal type of P. gingivalis, at least one patient was found to harbor two distinct clonal types. This indicates that the simultaneous presence of different clonal types of P. gingivalis can occur. A statistical method was developed for retrospective analysis of these data for estimation of whether the remainder of these patients were actually infected with single or multiple clonal types of P. gingivalis. With this statistical method, a confidence interval was calculated for estimation of the true proportion of a single observed clonal type in the potential population of P. gingivalis that might be recovered from an infected patient. Statistically, the sampling of small numbers of sites per patient or isolates per site leads to a wide confidence interval for the estimated true proportion of the observed clonal type in the infecting P. gingivalis population. For example, when five sites in an oral cavity yield indistinguishable P. gingivalis isolates, then the true proportion of this clonal type in the total P. gingivalis population in the infected oral cavity is estimated to be in the interval between 55% and 100% (at a 95% confidence level).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of genomic clonal types of Actinobacillus actinomycetemcomitans by restriction endonuclease analysis

To evaluate its utility in discriminating different strains, restriction endonuclease analysis was applied to 12 strains of Actinobacillus actinomycetemcomitans (3 serotype a, 5 serotype b, and 4 serotype c strains). DNA isolated from each strain was digested by 12 different restriction endonucleases, and the electrophoretic banding patterns of the resulting DNA fragments were compared. The DNA fragment patterns produced by SalI, XhoI, and XbaI for the 12 A. actinomycetemcomitans strains were simple (less than 30 bands) and allowed us to recognize easily 10 distinct genomic clonal types. The three serotype a strains exhibited distinctly different clonal types from one another, the five serotype b strains exhibited an additional four distinct clonal types, and the four serotype c strains showed another three different clonal types. The other endonucleases tested were less useful in typing A. actinomycetemcomitans. We conclude that restriction endonuclease analysis is a powerful tool for typing and discerning genetic heterogeneity and homogeneity among A. actinomycetemcomitans strains. It should, therefore, be very useful for epidemiologic studies.