Characterization Of Clinical Isolates Research Articles

Comparison of mutations in human parainfluenza viruses during passage in primary human bronchial/tracheal epithelial air-liquid interface cultures and cell lines.

Adaptation of viruses to cultured cells can increase the risk of misinterpretation in virological characterization of clinical isolates. In human parainfluenza virus (HPIV) 3, it has been reported that the human airway epithelial and lung organoid models are preferable for the study of viral characteristics of clinical strains without mutations. Therefore, we analyzed clinical isolates of all four HPIVs for the occurrence of mutations after five laboratory passages in human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) or conventional culture. We found a high risk of hemagglutinin-neuraminidase mutagenesis in all four HPIVs in conventional cultured cells. In addition, in HPIV1 and HPIV2, mutations of the large protein were also more frequent in conventional cultured cells than in HBTEC-ALI culture. HBTEC-ALI culture was useful for maintaining the original sequence and characteristics of clinical isolates in all four HPIVs. The present study contributes to the understanding of HPIV pathogenesis and antiviral strategies.

Characteristics of Clinical Isolates of Streptococcus mutans

Dental caries is an infectious disease which remains a significant health problem all over the world. The purpose of the study was to characterise a collection of 60 clinical isolates of S. mutans from adults’ and children’s dental plaque (natural biofilm). The paper describes the process of isolation, identification, analysis of biofilm formation and collection testing for the presence of 13 two-component systems (TCS) identified earlier in reference strain ATCC 700610 (UA159). In the case of S. mutans strains, plaque formation is specifically influenced by binary systems. All isolated strains of S. mutans form biofilm at high levels and possess a set of 26 genes forming TSC binary systems, which have an important role in plaque formation.

Correlation of multi-drug resistance integron genes, blaESBL carriage of Extended-spectrum β-lactamas producing clinical isolates from equine with respiratory disorders

The present study was aimed to identify phenotypical and genotypic antimicrobial characterization of clinical isolates obtained from equine with respiratory disorders in Egypt, as well as the correlation between the β-lactamases and integron genes in clinical isolates. Thirty two clinical isolates were identified as K.pneumoneae (15 isolates); P.aeruginosa (8 isolates); S.zooepidemicus (3 isolates) and S.equi (6 isolates). All isolates showed resistance to more than 3 classes of antibiotic and were considered as multidrug resistance isolates (MRD isolates), K. pneumoneae harbor ctx-M genes and shv-1 gene in highest rate (53.3% and 60% respectively). P.aeruginosa isolates harbor ctx-M genes and shv-1 gene (20% and 33.3% respectively) while S.equi and S.zooepidemicus harbor only ctx-M genes (33.3% and 20% respectively), tem-1 gene was observed in S. equi (66.7%) followed by K,pneumoneae (33.3%) and absent in P.aeruginosa. Significant correlation or association of intI genes with ctx-M genes in K. pneumoneae, P.aeruginosa and streptococcus species was observed, while it was significant with shv-1 in K.pneumoneae and streptococcus species. The significance was at p<0.05. It was concluded that the detection of integrons in most of the bacterial isolates promoters the phenomenon of in depth resistance because of these mobile genetic elements therefore the detection of those mobile gene cassettes in equine may be a keystone of multidrug resistance. Companion animals predominantly contain bla-ctx-M clusters, which might be animal origin, causing quick changes in drug-resistance epidemiology. ctx-M-1 and shv-1 also are commonly found among human pathogens signifying a standard clonal heredity and a possible ESBL dissemination source in farm and livestock.

Genotypic Characterization of Clinical Isolates of Staphylococcus aureus from Pakistan.

In this study, we compared pulsed-field gel electrophoretic (PFGE), multilocus sequence typing (MLST), Staphylococcal cassette chromosome mec (SCCmec), spa typing, and virulence gene profiles of 19 Panton–Valentine leucocidin (PVL)-positive, multidrug-, and methicillin-resistant clinical Staphylococcus aureus (MRSA) isolates obtained from a hospital intensive care unit in Pakistan. The isolates exhibited 10 pulsotypes, contained eight adhesin genes (bbp, clfA, clfB, cna, fnbA, fnbB, map-eap, and spa), 10 toxin genes (hla, hlb, hld, hlg, pvl, sed, see, seg, seh, and tst), and two other virulence genes (cfb, v8) that were commonly present in all isolates. The spa-typing indicated seven known spa types (t030, t064, t138, t314, t987, t1509, and t5414) and three novel spa types. MLST analysis indicated eight ST types (ST8, ST15, ST30, ST239, ST291, ST503, ST772, and ST1413). All isolates belonged to the agr group 1. Most of the isolates possessed SCCmec type III, but some isolates had it in combination with types SCCmec IV and V. The presence of multidrug-resistant MRSA isolates in Pakistan indicates poor hygienic conditions, overuse of antibiotics, and a lack of rational antibiotic therapy that have led to the evolution and development of hypervirulent MRSA clones. The study warrants development of a robust epidemiological screening program and adoption of effective measures to stop their spread in hospitals and the community.

Secretome characterization of clinical isolates from the Mycobacterium abscessus complex provides insight into antigenic differences

BackgroundMycobacterium abscessus (MAB) is a widely disseminated pathogenic non-tuberculous mycobacterium (NTM). Like with the M. tuberculosis complex (MTBC), excreted / secreted (ES) proteins play an essential role for its virulence and survival inside the host. Here, we used a robust bioinformatics pipeline to predict the secretome of the M. abscessus ATCC 19977 reference strain and 15 clinical isolates belonging to all three MAB subspecies, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense.ResultsWe found that ~ 18% of the proteins encoded in the MAB genomes were predicted as secreted and that the three MAB subspecies shared > 85% of the predicted secretomes. MAB isolates with a rough (R) colony morphotype showed larger predicted secretomes than isolates with a smooth (S) morphotype. Additionally, proteins exclusive to the secretomes of MAB R variants had higher antigenic densities than those exclusive to S variants, independent of the subspecies. For all investigated isolates, ES proteins had a significantly higher antigenic density than non-ES proteins. We identified 337 MAB ES proteins with homologues in previously investigated M. tuberculosis secretomes. Among these, 222 have previous experimental support of secretion, and some proteins showed homology with protein drug targets reported in the DrugBank database. The predicted MAB secretomes showed a higher abundance of proteins related to quorum-sensing and Mce domains as compared to MTBC indicating the importance of these pathways for MAB pathogenicity and virulence. Comparison of the predicted secretome of M. abscessus ATCC 19977 with the list of essential genes revealed that 99 secreted proteins corresponded to essential proteins required for in vitro growth.ConclusionsThis study represents the first systematic prediction and in silico characterization of the MAB secretome. Our study demonstrates that bioinformatics strategies can help to broadly explore mycobacterial secretomes including those of clinical isolates and to tailor subsequent, complex and time-consuming experimental approaches accordingly. This approach can support systematic investigation exploring candidate proteins for new vaccines and diagnostic markers to distinguish between colonization and infection. All predicted secretomes were deposited in the Secret-AAR web-server (http://microbiomics.ibt.unam.mx/tools/aar/index.php).

Vancomycin-resistant Enterococcus faecium in Algeria: phenotypic and genotypic characterization of clinical isolates.

vancomycin-resistant Enterococcus faecium (VREfm) is a major public health problem worldwide. The aim of our study was to determine the microbiological, epidemiological and molecular characteristics of VREfm isolated in north-central, eastern and western Algeria. a collection of 48 VREfm isolated from September 2010 to April 2017 in several Algerian hospitals were studied. Minimum inhibitory concentrations (MICs) were determined by E-test method according to CLSI guidelines. the detection of van genotype of all strains was performed by PCR. Clonal relationship of five VREfm targeted by region were characterized using multilocus sequence typing (MLST). All isolates have multidrug-resistance (MDR) and were resistant to at least five classes of antibiotics; however, all were susceptible to tigecycline and daptomycin with MIC50 at 0.094 µg/mL and 2 µg/mL respectively. All strains belonged to vanA genotype and have high level of resistance to vancomycin and teicoplanin. MLST revealed two sequence types (STs): ST80 (from the four regions of Algeria) and ST789, both belonging to the former hospital-adapted clonal complex CC17. the alarming dissemination of MDR E. faecium vanA and the ST80 in several regions of Algeria suggest a clonal spread of VREfm strains, which urgently require implementation of adequate infection control measures.

Biological characteristics of coxsackievirus A6 clinical isolates

Objective To analyze the biological characteristics of clinical isolates of coxsackievirus A6 (CVA6), a pathogen of hand, foot and mouth disease (HFMD), and to provide reference for vaccine development. Methods CVA6 strains were isolated from 21 stool and throat swab specimens of patients with HFMD in Yunnan Province and then identified. Their growth characteristics, plaque morphology and virulence to suckling mice were analyzed. Results Five CVA6 strains, named CVA6-129, CVA6-113, CVA6-57, CVA6-94 and CVA6-162, were isolated and all belonged to D3 subtype. Only the CVA6-129 strain could proliferate rapidly in Vero and KMB17 cells and the proliferation peaked 30 h after inoculation. The infectious titer of the CVA6-129 strain was 7.54 lgCCID50 (50% cell culture infective dose)/ml in KMB17 cells. Different morphologies of plaques were formed by the CVA6-129 strain in Vero and KMB17 cells at the same time points, which were small and round with clear edges in Vero cells, and large and irregular with blurry edges in KMB17 cells. Suckling mice were susceptible to CVA6 via intramuscular and intraperitoneal injection. The most common symptoms in infected suckling mice were reduced mobility, hind limb paralysis and quadriplegia. CVA6 infection could result in death in severe cases. Conclusions This study isolated five CVA6 strains from a number of clinical samples of suspected HFMD cases, of which the CVA6-129 strain showed potential as a vaccine candidate. Key words: Coxsackievirus A6 (CVA6); Hand, foot and mouth disease (HFMD); Vaccine; Biological characteristics

Mycobacterial inactivation protein extraction protocol for matrix-assisted laser desorption ionization time-of-flight characterization of clinical isolates.

Rapid identification of mycobacteria has been made possible with matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) in recent years. Working with high concentrations of mycobacteria in a PC-3 containment facility makes MALDI-TOF cumbersome and costly. Therefore removing the inactivated isolate's protein extract from the PC-3 facility is needed for efficient identification in a routine PC-2 laboratory. This work describes a novel chemical and mechanical disruption protein extraction method, which provides reliable MALDI-TOF results from solid and liquid media, while ensuring laboratory safety. When compared to sequencing results, 93.9% of the clinical isolates were identified in LJ media and 89% of the clinical isolates were identified in MGIT media. The MIPE protocol produces a high quality protein extract with improved isolate identification without compromising result turn-around-times or laboratory safety.

The role of Aggregatibacter actinomycetemcomitans and its leukotoxin in periodontitis

ABSTRACTAggregatibacter actinomycetemcomitans is a Gram-negative periodontitis-associated bacterium that expresses a toxin that selectively affects leukocytes. Leukotoxin is encoded by an operon belonging to the core genome of this bacterial species. Variations in the expression of the leukotoxin have been reported, and a specific clonal type (JP2) of this bacterium with enhanced leukotoxin expression has been identified. Experimental studies have shown that leukotoxin, besides to kill leukocytes, also activates a substantial pro-inflammatory response in these cells. Characterization of clinical isolates from individuals with different geographic origin, age and periodontal status linked carriership of highly leukotoxic A. actinomycetemcomitans to disease. These highly virulent bacteria are of serotype b and have several common phenotypic and genetic features. Based on a great genetic diversity, A. actinomycetemcomitans can be distributed into a high number of different genotypes with various virulence capacities. It can be concluded that carriership of highly leukotoxic bacteria is associated with a significantly increased risk for initiation of aggressive forms of periodontitis.

Genotypic and Phenotypic Characterization of Clinical Isolates of Staphylococcus Aureus for Biofilm Formation Ability

Genotypic and Phenotypic Characterization of Clinical Isolates of Staphylococcus Aureus for Biofilm Formation Ability

Population Structure of Salmonella enterica Serovar 4,[5],12:b:− Strains and Likely Sources of Human Infection

Salmonella enterica serovar 4,[5],12:b:- is a monophasic serovar not able to express the second-phase flagellar antigen (H2 antigen). In Germany, the serovar is occasionally isolated from poultry, reptiles, fish, food, and humans. In this study, a selection of 67 epidemiologically unrelated Salmonella enterica serovar 4,[5],12:b:- strains isolated in Germany between 2000 and 2011 from the environment, animal, food, and humans was investigated by phenotypic and genotypic methods to better understand the population structure and to identify potential sources of human infections. Strains of this monophasic serovar were highly diverse. Within the 67 strains analyzed, we identified 52 different pulsed-field gel electrophoresis XbaI profiles, 12 different multilocus sequence types (STs), and 18 different pathogenicity array types. The relatedness of strains based on the pathogenicity gene repertoire (102 markers tested) was in good agreement with grouping by MLST. S. enterica serovar 4,[5],12:b:- is distributed across multiple unrelated eBurst groups and consequently is highly polyphyletic. Two sequence types (ST88 and ST127) were linked to S. enterica serovar Paratyphi B (d-tartrate positive), two single-locus variants of ST1583 were linked to S. enterica serovar Abony, and one sequence type (ST1484) was associated with S. enterica serovar Mygdal, a recently defined, new serovar. From the characterization of clinical isolates and those of nonhuman origin, it can be concluded that the potential sources of sporadic human infections with S. enterica serovar 4,[5],12:b:- most likely are mushrooms, shellfish/fish, and poultry.

Antibacterial Susceptibility Patterns and Characterization of Clinical Isolates of <i>Elizabethkingia meningoseptica</i> in Henan Province, China

This study aimed to provide insight into antimicrobial susceptibility and homology of Elizabethkingia meningoseptica in a hospital environment. Samples from environmental surfaces and the hands of medical staff were screened for E. meningoseptica and antimicrobial susceptibility testing was performed; Pulsed-Field Gel Electrophoresis (PFGE) was employed to subtype E. meningoseptica strains; The resistant genes were detected by PCR. In total, six isolates of E. meningoseptica were collected from 280 samples. Antimicrobial susceptibility testing revealed that all of the six strains displayed multiresistance, showing resistance to more than three different classes of antibiotics. The strains were separated into five different PFGE patterns. The sulII gene was detected in four of the strains. Our data show that multiresistant E. meningoseptica strains exist in the hospital environment and susceptibility testing revealed that vancomycin was the most effective antibiotic. These results have practical significance for treatment of E. meningoseptica infection.

Molecular characterization of Cryptosporidium isolates from humans in Equatorial Guinea

The aim of the study was to perform a molecular characterization of clinical isolates of Cryptosporidium species from Equatorial Guinea. Standard laboratory methods were used to identify 35 cryptosporidiosis cases among 185 patients. PCR-RFLP successfully identified 34 Cryptosporidium species from these 35 cases, comprising C. parvum (52.9%), C. hominis (44.1%) and C. meleagridis (2.9%); over 90% of the species were isolated from HIV-positive patients. This is the first report of the molecular characterization of Cryptosporidium species isolated from humans in Equatorial Guinea and shows that zoonotic and anthroponotic transmission is present in this country.

Antimicrobial Susceptibility Patterns and Characterization of Clinical Isolates of Staphylococcus aureus in Immunocompromised Cancer Patients Undergoing Chemotherapy in Pakistan

Cancer patients undergoing chemotherapy are highly prone to infections because of suppression of their immune system. in the present study, the activity of fluoro-quinolones, cephalosporins, glycopeptides and oxazolidinones was evaluated in Staphylococcus aureus strains by broth dilution method according to Clinical and laboratory Standards institute (CLSI), USA guidelines. The frequency of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains was 67% and 33% respectively. the MIC50 and MIC90 of isolates were ciprofloxacin 8 and 32 μg/ml for MRSA, 8 and 16 μg/ml for MSSA, levofloxacin 8 and 16 μg/ml for MRSA and MSSA, gatifloxacin 4 and 16 μg/ml for MRSA and 4 and 8 μg/ml for MSSA. MIC50 and MIC90 of vancomycin and linzolid were 1 and 2 μg/ml, 2 and 4 μg/ml for both MRSA and MSSA strains respectively. This study shows a high prevalence of and resistance in MRSA. However, vancomycin and linezolid were highly active and could be used for treating MRSA infections.

Phenotypic and Genotypic Characteristics of Clinical Isolates of Pseudomonas aeruginosa: Rate of Occurrence and Distribution of Different Serotypes, Antimicrobial Susceptibility Profiles, and Molecular Typing

Background: In the current investigation, 167 clinical isolates of Pseudomonas aeruginosa obtained from a Northwest Ohio hospital were tested for phenotypic diversity using serotyping and antibiotic susceptibility testing. The genetic diversity of the selected strains was determined by DNA fingerprinting. Methods: The isolates were serotyped by slide agglutination. The susceptibility was measured by disk diffusion. To analyze genetic variability, 96 strains (serotypes O:1, O:2, O:6, and O:11) were studied by polymerase chain reaction (PCR)-based single primer DNA fingerprinting.

Genome Content Determination in Methicillin-Resistant Staphylococcus Aureus

Staphylococcus aureus is a major pathogen responsible for both nosocomial and community-acquired infections. While the first S. aureus isolates displaying resistance to methicillin were reported in the early 1960s, endemic strains of methicillin-resistant S. aureus (MRSA) carrying multiple resistance determinants only became a worldwide nosocomial problem in the early 1980s, carrying a threefold attributable cost and a threefold excess length of hospital stay when compared with methicillin-susceptible S. aureus bacteremia. Recent efforts in the field of high-throughput sequencing resulted in the release of several MRSA genome sequences enabling the development of massively parallel tools to study clinical isolates of MRSA at the organism scale. Microarrays covering whole genomes and high-throughput sequencing devices are the two main techniques currently utilizable for whole-genome characterization. These tools not only provide information for the development of genotyping assays but also allow evaluation of potential virulence of the strains, by enumerating genetic-encoded resistance markers and toxin content. This appears particularly attractive for understanding the epidemiology of MRSA and the relationship between genome content on one side and virulence potential or epidemicity on the other side. In addition, sequence information is mandatory for the development of molecular tests allowing the rapid identification, genotyping and characterization of clinical isolates.

Molecular characterization of clinical isolates of M non-typable group A streptococci from invasive disease cases

Currently there are 93 validated M serotypes of Streptococcus pyogenes, Lancefield group A streptococcus (GAS), and >130 emm genotypes. A marked increase in the number of non-typable GAS isolates (2 % in 2000, 4 % in 2001 and 9 % in 2002) from invasive disease cases referred to the authors' reference laboratory was noted during 2000-2002. A total of 217 (92 %) were from blood cultures, 14 (6 %) from deep abscesses and five (2 %) from aspirates. The clinical manifestations included bacteraemia, septicaemia, cellulitis, meningitis, necrotizing fasciitis and toxic-shock syndrome. In order to establish whether this increase was due to the emergence of novel types or the unavailability of M-typing sera, these isolates were subjected to emm sequencing. A total of 144 isolates (61 %) belonged to M types for which sera were no longer available; 112 (48 %) belonged to higher M types, including emm83.1 (9 %), emm94 (8 %) emm87 (6 %) and emm89 (6 %); and 32 (13 %) belonged to lower M types that were not commonly isolated in the UK, and included M25, M43, M49, M64, M73 and M74. Sixty-six (28 %) of the isolates belonged to newly designated emm types. Other isolates belonged to the novel emm types st2147, STNS1033 and st854, recently registered in the Centers for Disease Control (CDC) database by other laboratories. One novel emm type, st2161, was isolated from an injecting drug user. There were differences in the type distribution of these isolates according to geographic location. However, 90 % of emm93, one of seven predominant emm types identified amongst the collection of M non-typable (MNT) isolates, were isolated from the London region.

Follow‐up of epidemiological data of cryptococcosis in Austria, Germany and Switzerland with special focus on the characterization of clinical isolates

The present survey in Austria, Germany and Switzerland continued the survey of cryptococcosis set up by the European Confederation of Medical Mycology (ECMM) in 1997. From 2000 to 2003 77 cases have been reported. An HIV infection is still the most important risk factor (68%). Young HIV+ women from ASIA contributed to the increase of cryptococcosis in females. A total of 129 clinical isolates of both surveys were genotyped by PCR fingerprinting to study the prevalence of different genotypes. The prevalence of Cryptococcus neoformans var. grubii (serotype A) with the genotypes VNA1 and VNA2 was higher in Germany and Austria (74.5%) than in Switzerland (52%), while in Switzerland the Cr. neoformans hybrids AD (26%) and Cr. neoformans var. neoformans (serotype D) (22%) were more prevalent compared with Germany and Austria (8 and 17.5% respectively). Cryptococcus gattii isolates were studied by FT-IR spectroscopy. DNA in the ITS region was sequenced to get further information about Cr. neoformans serotype AD strains and about the geographical origin of the Cr. gattii isolates. The ITS sequence of the serotype AD isolates of the genotypes VNAD1, VNAD2 and VNAD4 is usually identical to serotype A or serotype D respectively. In the three isolates of the genotype VNAD3 a genotype-specific sequence pattern was detected. Two autochthonous infections due to Cr. gattii could indicate that the genotype VGIV with the ITS type 'Asia 2' might be endemic in Europe.

The Effect of Temperature and Glucose Concentration on the Growth of Microorganisms Commonly Associated with CAPD Peritonitis

Editar: CAPD peritonitis, despite recent advances, re mains a major source of patient morbidity. While many factors contribute to this problem (1,2), the actual growth characteristics of clinically relevant microorganisms might be expected to playa role. To that end, we have studied the in vitra growth characteristics of clinical isolates of Staphylacaccus epidermidis, E. cali, and Pseudamanas aeruginasa in 1.5%, 2.5%, and 4.25% dextrose dialysate fluid. Colonies of the organism were grown on 5% sheep blood agar plates (BAP:BBL, Becton Dickinson Microbiology Systems, Rutherford, NJ) overnight at 35°C. Sufficient quantities of the colonies were suspended in 10 mL normal saline to produce suspensions containing approximately 105 colony-forming units per mL (CFU/mL). The suspensions were inoculated into 200-mL aliquots of the dialysate solutions (Dianeal PD-1, Baxter Healthcare Corporation, Deerfield, IL). The dialysate solutions contained 132 mEq/ L sodium. 3.5 mEq/L calcium, 1.5 mEq/L magnesium, 102 mEq/L chloride and 35 mEq/L of lactate, and 1.5%, 2.5%, or 4.25% dextrose. Each organism and dialysate combination was incubated at 4°C, room temperature (21°C), or 35°C. Twenty microliter volumes at 0, 2, 4, 6, and 24 hours were plated in triplicate onto BAP and incubated at 35°C. The resultant colonies were counted and the mean±SD CFU/mL was determined for each time, temperature, and organism. At 4°C and 21°C regardless of glucose concentration, growth of all organisms appeared to be stagnant with no changes in concentration up to 24 hours. At 35°C, all three organisms exhibited a reduction in colony counts by 24 hours in each dialysate. Figure I represents the observed reduction in colony counts by 24 hours for all organisms and dialysates. Thus changes in medium characteristics (medium osmolalities between 350 mOsm/kg and 510 mOsm/ kg) appear to have little effect on microorganism growth. The ability to recover organisms from inoculated bags for up to 24 hours suggests that early cultures may not be the critical step in microorganism identification. Perhaps the presence of hostproduced inflammatory responses is the factor which significantly alters recovery of organisms in clinical specimens stored outside the body for several hours (3).

Legionella micdadei (Pittsburgh pneumonia agent): direct fluoresent-antibody examination of infected human lung tissue and characterization of clinical isolates.

Legionella micdadei (Pittsburgh pneumonia agent) was identified by direct fluorescent-antibody (DFA) examination of lung tissue in six of seven persons diagnosed previously as having L. micdadei pneumonia only by histopathology and in four persons who also had positive cultures of the organism. No cross-reactions occurred with monospecific DFA conjugates prepared against Legionella pneumophila serogroups 1 to 6, Legionella bozemanii, Legionella dumoffii, and Legionella gormanii. One person had L. pneumophila serogroup 6 identified by DFA examination of lung tissue and subsequent culture of stored pulmonary secretions. Characterization of the four strains of L. micdadei revealed specific DFA reactions, bacteriological behavior, and cellular fatty acid composition that allow identification of the organism. DFA testing appears to be a sensitive method for identifying L. micdadei prescent in human lung tissue or cultured on artificial media.