Clinical Isolates Of Group Research Articles

Thymbra capitata essential oil has a significant antimicrobial activity against methicillin-resistant Staphylococcus aureus pre-formed biofilms.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant opportunistic pathogen with a great ability to form biofilms. Herein, the antimicrobial potential of Thymbra capitata essential oil (EO) against MRSA biofilms was investigated. The determination of the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC) of the T. capitata EO was first investigated on a group of clinical isolates from septicaemias, diabetic foot ulcers and osteomyelitis. Biofilms were incubated with the EO at the MLC and its anti-biofilm potential was investigated. A strong antimicrobial activity was observed, with MIC and MLC values between 0·32 and 0·64 mg l-1 . However, the concentration of EO necessary for the eradication of planktonic cells was insufficient to significantly reduce the biofilm biomass of some isolates. Nevertheless, cell culturability and overall cellular metabolism was strongly reduced in all biofilms tested, only when the EO was tested. Contrary to the tested antibiotics, T. capitata EO showed a significant antimicrobial activity against MRSA biofilms, by reducing cellular metabolism and cellular culturability.

Evolution of biofilm-adapted gene expression profiles in lasR-deficient clinical Pseudomonas aeruginosa isolates

The overall success of a pathogenic microbe depends on its ability to efficiently adapt to challenging conditions in the human host. Long-term evolution experiments track and predict adaptive trajectories and have contributed significantly to our understanding of the driving forces of bacterial adaptation. In this study, we conducted a cross-sectional study instead of long-term longitudinal evolution experiments. We analyzed the transcriptional profiles as well as genomic sequence variations of a large number of clinical Pseudomonas aeruginosa isolates that have been recovered from different infected human sites. Convergent changes in gene expression patterns were found in different groups of clinical isolates. The majority of repeatedly observed expression patterns could be attributed to a defective lasR gene, which encodes the major quorum-sensing regulator LasR. Strikingly, the gene expression pattern of the lasR-defective strains appeared to reflect a transcriptional response that evolves in a direction consistent with growth within a biofilm. In a process of genetic assimilation, lasR-deficient P. aeruginosa isolates appear to constitutively express a biofilm-adapted transcriptional profile and no longer require a respective environmental trigger. Our results demonstrate that profiling the functional consequences of pathoadaptive mutations in clinical isolates reveals long-term evolutionary pathways and may explain the success of lasR mutants in the opportunistic pathogen P. aeruginosa in a clinical context.

Hemolytic activity and biofilm-formation among clinical isolates of group B streptococcus causing acute urinary tract infection and asymptomatic bacteriuria

Streptococcus agalactiae, also known as group B Streptococcus, is an aetiological agent of urinary tract infection (UTI) in adults, including cystitis, pyelonephritis and asymptomatic bacteriuria (ABU). Whereas ABU-causing S. agalactiae (ABSA) have been shown to grow and achieve higher culture denstity in human urine compared to uropathogenic S. agalactiae (UPSA) other phenotypic distinctions between S. agalactiae isolated from different forms of UTI are not known. Here, we define the hemolytic activities and biofilm-formation of a collection of clinical isolates of UPSA, ABSA and recurrent S. agalactiae bacteriuria (rSAB) strains to explore these phenotypes in the context of clinical history of isolates. A total of 61 UPSA, 184 ABSA, and 47 rSAB isolates were analyzed for relative hemolytic activity by spot assay on blood agar, which was validated using a erythrocyte lysis suspension assay. Biofilm formation was determined by microtiter plate assay with Lysogeny and Todd-Hewitt broths supplemented with 1% glucose to induce biofilm formation. We also used multiplex PCR to analyze isolates for the presence of genes encoding adhesive pili, which contribute to biofilm formation. Comparing the hemolytic activities of 292 isolates showed, surprisingly, that ABSA strains were significantly more likely to be highly hemolytic compared to other strains. In contrast, there were no differences between the relative abilities of strains from the different clinical history groups to form biofilms. Taken together, these findings demonstrate a propensity of S. agalactiae causing ABU to be highly hemolytic but no link between clinical history of UTI strains and ability to form biofilm.

Determination of Capsular Serotypes, Antibiotic Susceptibility Pattern, and Molecular Mechanism of Erythromycin Resistance among Clinical Isolates of Group B Streptococcus in Isfahan, Iran.

Background:Documented streptococcal resistance to erythromycin has recently been raised. The aim of this study is to identify the molecular mechanism of erythromycin resistance among group B Streptococcus (GBS) strains and to correlate with the clinical origin of strains.Materials and Methods:A total number of 134 colonizing (n = 36), invasive (n = 36), noninvasive (n = 46), and asymptomatic (n = 16) GBS isolates were characterized by the detection of dltS gene, capsular serotyping, antibiotic susceptibility profiles using disc diffusion method, and screening of the ermB, ermTR, and mefA resistance genes.Results:The distribution of capsular serotypes was as follow: serotype III (24.6%), Ia (21.6%), V (17.9%), Ib (14.9%), II (8.9%), IV (8.9%), VI (1.5%), and VII (1.5%). From 134 GBS isolates, 51 (38%) isolates were resistant to erythromycin. The constitutive macrolide lincosamide streptogrmin B (MLSB) was the most common resistance phenotype (62.7%), followed by inducible MLSB (27.4%) and M phenotype (9.8%). Erythromycin resistance rate was higher among asymptomatic GBS strains (13/16, 81.2%). Serotype III was the most prevalent type among resistant isolates (41.1%). The ermB gene highly distributed among resistant strains (64.7%), followed by ermTR (21.5%) and mefA (9.8%). The ermB gene was related to constitutive MLSB phenotype (84.3%, P < 0.05) and serotypes III (61.9%), Ib (87.5%), and V (83.3%). All M phenotype strains harbored mefA gene and were in association with serotype Ia (90%).Conclusion:The current study suggests that ribosomal modification with erm genes is the main mechanism of erythromycin resistance. Because of relatively high prevalence of erythromycin resistance, double disc test highly recommended for GBS disease treatment and intrapartum prophylaxis among penicillin intolerant patients in our region.

Influence of environmental and genotypic factors on biofilm formation by clinical isolates of group B streptococci

Group B Streptococcus (GBS) colonizes the gastrointestinal and urogenital tracts of approximately 30% of women, and it can cause sepsis and meningitis in neonates. GBS has been shown to form biofilms in vitro, but the effects of environmental and genotypic factors upon GBS biofilm formation are unclear. The aim of the present study was to optimize culture conditions for enhanced GBS biofilm production. Furthermore, this study also investigated the influences of strain lineage, pilus profile, and isolation source on GBS biofilm formation. The results demonstrate that the fed-batch mode and acidic pH strongly enhanced GBS biofilm formation in vitro. These findings suggest that the fed-batch mode may be suitable for both screening and fundamental studies of GBS biofilm formation. Moreover, this study demonstrated a correlation between the hyper virulent clonal complex 17 and a strong biofilm phenotype.

Susceptibility of Clinical Isolates of Burkholderia pseudomallei to a Lipid A Biosynthesis Inhibitor

.Burkholderia pseudomallei is the causative agent of melioidosis, a serious infection associated with high mortality and relapse. Current antimicrobial therapy using ceftazidime (CAZ) is often ineffective. Inhibitors of LpxC, the enzyme responsible for lipid A biosynthesis, have potential antimicrobial activity against several Gram-negative bacteria in vivo, but their activity against B. pseudomallei is unclear. Herein, we investigated the susceptibility of B. pseudomallei clinical isolates to LpxC-4, an LpxC inhibitor, and LpxC-4 in combination with CAZ. Time-kill assays for bactericidal activity were conducted for B. pseudomallei K96243, revealing growth inhibition and bactericidal effect at LpxC-4 concentrations of 2 μg/mL and 4 μg/mL, respectively. No significant synergistic effect was observed with the combination of LpxC-4 and CAZ. LpxC-4 susceptibility was tested on three groups of clinical isolates:1) CAZ- and trimethoprim–sulfamethoxazole (SXT)–susceptible (N = 71), 2) CAZ-resistant (N = 14), and 3) SXT-resistant (N = 23) isolates, by broth microdilution. The minimum concentration of LpxC-4 required to inhibit the growth of 90% of organisms was 2 μg/mL for all isolates. The median minimum inhibitory concentration of both the CAZ/SXT-susceptible and CAZ-resistant groups was 1 μg/mL (interquartile range [IQR] = 1–2 μg/mL), compared with 2 μg/mL (IQR = 2–4 μg/mL) for the SXT-resistant group. Cell morphology was observed after drug exposure by immunofluorescent staining, and a change from rod-shaped to cell wall–defective spherical cells was observed in surviving bacteria. LpxC-4 is a potent bactericidal agent against B. pseudomallei and warrants further testing as a new antibiotic to treat melioidosis.

M type of Clinical Isolates of Group A Streptococcus in Pleven (Bulgaria) and vaccine development

M type of Clinical Isolates of Group A Streptococcus in Pleven (Bulgaria) and vaccine development

Classification of group B streptococci with reduced β-lactam susceptibility (GBS-RBS) based on the amino acid substitutions in PBPs.

All clinical isolates of group B Streptococcus (GBS; Streptococcus agalactiae) are considered uniformly susceptible to β-lactams, including penicillins. However, GBS with reduced penicillin susceptibility (PRGBS) were first identified by our group in Japan and have also been reported from North America. PRGBS are non-susceptible to penicillin because of acquisition of amino acid substitutions near the conserved active-site motifs in PBP2X. In particular, V405A and Q557E are considered the key amino acid substitutions responsible for penicillin non-susceptibility. We revealed that in addition to the substitutions in PBP2X, an amino acid substitution in PBP1A confers high-level cephalosporin resistance in GBS. As the number of publications on GBS with reduced β-lactam susceptibility (GBS-RBS), especially PRGBS, and concomitantly the need for a systematic classification of GBS-RBS is increasing, we propose here a classification of GBS-RBS based on the amino acid substitutions in their PBPs.

Bacteriological and clinical evaluations of imipenem/cilastatin sodium in neonates and premature infants

The antimicrobial activity of imipenem against group B streptococci was investigated. The clinical efficacy of imipenem/cilastatin sodium (IPM/CS) was determined in neonates and premature infants. The results are summarized below. 1. The distribution of MIC values of IPM against 55 clinical isolates of group B streptococci isolated from the vagina of pregnant women peaked at 0.024 micrograms/ml. MIC values of IPM against all clinical isolates tested were 0.05 microgram/ml or less. 2. IPM/CS was used for treatment of bacterial infections in 9 neonates and premature infants. Among these patients, clinical responses were excellent in 5 patients and good in 4 patients. No adverse reaction was observed. Abnormal laboratory test values were observed in 4 patients, eosinophilia in 2 patients and increased platelets in 2. 3. In 7 neonates and premature infants, quantitative cultures for aerobic and anaerobic fecal flora were performed. Fecal flora change was not significantly different than those observed during treatment with latamoxef or cefmenoxime. 4. Ten neonates and premature infants were investigated for effects of IPM/CS on the blood coagulation system. Prolongations of prothrombin time (PT) and activated partial thromboplastin time (APTT) were observed in 1 patient. Abnormal prothrombin (PIVKA II) was detected in the same patient. There was no tendency for inhibition of platelet function.

Characterization of protease IV expression in Pseudomonas aeruginosa clinical isolates

Expression of protease IV by Pseudomonas aeruginosa during ocular infections contributes significantly to tissue damage. However, several P. aeruginosa strains isolated from ocular infections or inflammatory events produce very low levels of protease IV. The aim of the present study was to characterize, genetically and phenotypically, the presence and expression of the protease IV gene in a group of clinical isolates that cause adverse ocular events of varying degrees, and to elucidate the possible control mechanisms of expression associated with this virulence factor. Protease IV gene sequences from seven clinical isolates of P. aeruginosa were determined and compared to P. aeruginosa strains PAO1 and PA103-29. Production and enzyme activity of protease IV were measured in test strains and compared to that of quorum-sensing gene (lasRI) mutants and the expression of other virulence factors. Protease IV gene sequence similarities between the isolates were 97.5-99.5 %. The strains were classified into two distinct phylogenetic groups that correlated with the presence of exo-enzymes from type three secretion systems (TTSS). Protease IV concentrations produced by PAOΔlasRI mutants and the two clinical isolates with a lasRI gene deficiency were restored to levels comparable to strain PAO1 following complementation of the quorum-sensing gene deficiencies. The protease IV gene is highly conserved in P. aeruginosa clinical isolates that cause a range of adverse ocular events. Observed variations within the gene sequence appear to correlate with presence of specific TTSS genes. Protease IV expression was shown to be regulated by the Las quorum-sensing system.

In vitro and in vivo antibacterial activities of garenoxacin against group G Streptococcus dysgalactiae subsp. equisimilis

In this study, garenoxacin showed potent in vitro activity against clinical isolates of group G Streptococcus dysgalactiae subsp. equisimilis [minimum inhibitory concentration for 90% of the organisms (MIC 90) = 0.125 μg/mL] and was superior to levofloxacin (MIC 90 = 1 μg/mL) and moxifloxacin (MIC 90 = 0.25 μg/mL). In experimental pneumonia caused by group G S. dysgalactiae subsp. equisimilis in mice, the effective dose for 50% survival (ED 50) of garenoxacin following single oral administration was 1.87 mg/kg, >10.7-fold and 4.6-fold less than the ED 50 values of levofloxacin (>20 mg/kg) and moxifloxacin (8.54 mg/kg), respectively. The area under the free serum concentration–time curve from 0–24 h ( fAUC 0–24)/MIC ratio of garenoxacin in serum following oral administration of 20 mg/kg was 73.2, which was 8.7–11.4-fold and 1.4-fold greater than that of levofloxacin (6.44–8.46) and moxifloxacin (51.4), respectively. These results suggest that garenoxacin has potential for the treatment of infectious diseases caused by S. dysgalactiae subsp. equisimilis.

Emm gene diversity, superantigen gene profiles and presence of SlaA among clinical isolates of group A, C and G streptococci from western Norway

In order to investigate molecular characteristics of beta-hemolytic streptococcal isolates from western Norway, we analysed the entire emm gene sequences, obtained superantigen gene profiles and determined the prevalence of the gene encoding streptococcal phospholipase A2 (SlaA) of 165 non-invasive and 34 contemporary invasive group A, C and G streptococci (GAS, GCS and GGS). Among the 25 GAS and 26 GCS/GGS emm subtypes identified, only emm3.1 was significantly associated with invasive disease. M protein size variation within GAS and GCS/GGS emm types was frequently identified. Two non-invasive and one invasive GGS possessed emm genes that translated to truncated M proteins as a result of frameshift mutations. Results suggestive of recombinations between emm or emm-like gene segments were found in isolates of emm4 and stG485 types. One non-invasive GGS possessed speC, speG, speH, speI and smeZ, and another non-invasive GGS harboured SlaA. speA and SlaA were over-represented among invasive GAS, probably because they were associated with emm3. speG dys was identified in 83% of invasive and 63% of non-invasive GCS/GGS and correlated with certain emm subtypes. Our results indicate the invasive potential of isolates belonging to emm3, and show substantial emm gene diversity and possible lateral gene transfers in our streptococcal population.

Pathogenic potential of Aeromonas hydrophila isolated from surface waters in Kolkata, India

Members of the genus Aeromonas (family Aeromonadaceae) are medically important, Gram-negative, rod-shaped micro-organisms and are ubiquitous in aquatic environments. Aeromonas species are increasingly recognized as enteric pathogens; they possess several virulence factors associated with human disease, and represent a serious public health concern. In the present study, putative virulence traits of Aeromonas hydrophila isolates collected from different natural surface waters of Kolkata, India, were compared with a group of clinical isolates from the same geographical area using tissue culture and PCR assays. Enteropathogenic potential was investigated in the mouse model. Of the 21 environmental isolates tested, the majority showed cytotoxicity to HeLa cells (81 %), haemolysin production (71 %) and serum resistance properties (90 %), and they all exhibited multi-drug resistance. Some of the isolates induced fluid accumulation (FA ratio>or=100), damage to the gut and an inflammatory reaction in the mouse intestine; these effects were comparable to those of clinical strains of A. hydrophila and toxigenic Vibrio cholerae. Interestingly, two of the isolates evoked a cell vacuolation effect in HeLa cells, and were also able to induce FA. These findings demonstrate the presence of potentially pathogenic and multi-drug-resistant A. hydrophila in the surface waters, thereby indicating a significant risk to public health. Continuous monitoring of surface waters is important to identify potential water-borne pathogens and to reduce the health risk caused by the genus Aeromonas.

Biofilm formation in clinical isolates of group B streptococci from north India

Many bacterial species are capable of living as biofilm, thought to be the predominant growth mode for bacteria in natural environments. Increasing evidence implicates biofilm as the cause of various human infections. In this study, biofilm formation was demonstrated in group B streptococci (GBS) isolated from different sources in the north Indian community at various pH ranges as well as sugar concentrations and correlated it with different serotypes and surface gene ( c alpha) profiles. The capability to form biofilm was better demonstrated in strains from asymptomatic carriers (pregnant women) compared to symptomatic patients. Quantitatively bacterial adherence with host cells was greater in isolates that produced biofilm under neutral conditions. This study assessed the biofilm formation in clinical isolates of GBS, which is a step towards understanding its role in pathogenesis.

213: Antibiotic resistance and penicillin tolerance in clinical isolates of group B streptococci

213: Antibiotic resistance and penicillin tolerance in clinical isolates of group B streptococci

In vitro and in vivo activities of DW-224a, a novel fluoroquinolone antibiotic agent

The objective of the present study was to assess the in vitro and in vivo activities of DW-224a in order to eventually use it as an antibiotic. DW-224a was compared with DW286, ciprofloxacin and trovafloxacin. MICs of DW-224a, DW286, ciprofloxacin and trovafloxacin were determined against several groups of clinical isolates. In addition, intraperitoneal infection was induced with various organisms in mice. Test compounds were administered once orally to mice immediately after infection. The 50% protective dose (PD50) was calculated from the survival rates on day 7 after infection. Against Gram-positive bacteria, the in vitro activity of DW-224a was stronger than those of ciprofloxacin and trovafloxacin, but slightly weaker than that of DW286. Against Gram-negative bacteria, the activity of DW-224a was similar to those of trovafloxacin and DW286, but weaker than that of ciprofloxacin. In experimental systemic infections in mice with various organisms, like DW286, DW-224a demonstrated potent activity against Gram-positive bacteria and somewhat less activity against Gram-negative bacteria. DW-224a has a broad spectrum of antimicrobial activity, which is especially potent against Gram-positive bacteria.

Determination of group B streptococcal genes encoding putative adherence factors in GBS clinical strains

Group B streptococcus (GBS) is part of the vaginal flora of many women and is an important cause of invasive bacterial disease in newborns. In spite of extensive research on this organism in recent years, little is known about the specific virulence factors responsible for increased bacterial pathogenicity. The goal of the present work was to assess the presence of putative adherence genes, located on GBS pathogenicity islands in various regions of the genome, in a group of clinical isolates. The putative adherence genes, originally found by subtractive hybridization and designated as sspB1 and sspB2, encoded proteins homologous to the broad family of adherence and aggregation proteins commonly found in Gram-positive bacteria. The clinical GBS strains consisted of 118 isolates were analyzed for the presence of sspB1 and sspB2 employing PCR and DNA hybridization. The occurrence of the genes under study in the genome correlated with severity of the case; for example, sspB1 and sspB2 variants were most common for invasive GBS strains and strains isolated from urine. There was no correlation between the presence of the sspB genes in the genome and specific serotype.

Persistent Macrolide Resistance among Group A Streptococci: The Lack of Accomplishment after 4 Decades

After isolation of erythromycin from a culture of Streptomyces erythreus in 1952 and the soon-to-follow introduction of erythromycin to clinical practice, erythromycin resistance required <2 decades to become apparent among clinical isolates of group A streptococci (GAS; Streptococcus pyogenes). A 1968 report in 1968 by Sanders et al. [1] about an erythromycinresistant GAS (M type 12) isolate recovered from a patient's upper respiratory tract concluded with the admonition, Therefore, determination of the in-vitro susceptibility of group A streptococci to these drugs appears advisable whenever their use is contemplated, and imperative when either the infection threatens life or there is an increased risk that late sequelae of such infections will develop. Now, decades later, Richter et al. [2] similarly admonish physicians in their survey of macrolide resistance in the United States: In vitro testing of S. pyogenes for macrolide susceptibility is warranted for patients who are allergic to 4-lactams (p. 606).

Combined contributions of streptolysin O and streptolysin S to virulence of serotype M5 Streptococcus pyogenes strain Manfredo.

Streptolysin O (SLO) and streptolysin S (SLS) are potent cytolytic toxins produced by almost all clinical isolates of group A streptococci (GAS). Allele-replacement mutagenesis was used to construct nonpolar (in-frame) deletion mutations in the slo and sagB genes of the serotype M5 GAS strain Manfredo, producing isogenic single and double SLO- and SLS-defective mutants. In contrast to recent reports on SLS-defective insertion mutants (I. Biswas, P. Germon, K. McDade, and J. Scott, Infect. Immun. 69:7029-7038, 2001; Z. Li, D. Sledjeski, B. Kreikemeyer, A.Podbielski, and M. Boyle, J. Bacteriol. 181:6019-6027, 1999), none of the mutants described here had notable pleiotropic effects on the expression of other virulence factors examined. Comparison of isogenic parent and mutant strains in various virulence models revealed no differences in their abilities to multiply in human blood or in their 50% lethal doses (LD(50)s) upon intraperitoneal infection of BALB/c mice. A single log unit difference in the LD(50)s of the parent and SLS-defective mutant strains was observed upon infection by the subcutaneous (s.c.) route. Comparisons over a range of infective doses showed that both SLO and SLS contributed to the early stages of infection and to the induction of necrotic lesions in the murine s.c. model. Individually, each toxin made an incremental contribution to virulence that was not apparent at higher infective doses, although the absence of both toxins reduced virulence over the entire dose range examined. Interestingly, in some cases, the contribution of SLO to virulence was clear only from an analysis of the double-mutant strain, highlighting the value of not confining virulence studies to mutant strains defective in the expression of only single virulence factors.

Clinical microbiological case: a firm, right infraclavicular mass in an adult woman with connective tissue disease

Clinical microbiological case: a firm, right infraclavicular mass in an adult woman with connective tissue disease