The use of whole-cell protein profile analysis by SDS-PAGE as an accurate tool to identify species and subspecies of coagulase-negative staphylococci

We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a tool to characterize coagulase-negative staphylococci (CoNS). Of 253 clinical isolates and 10 control strains, five species and four subspecies were analyzed. All the isolates were identified using conventional phenotypic tests and SDS-PAGE. Discrepant results between these methods, as well as less common species and subspecies, were confirmed by sodA and 16S rDNA gene sequencing. Intraspecies similarities, calculated by the Dice coefficient, were significantly higher when compared to interspecies similarities. The conventional method failed to identify eight (3.2%) molecularly defined and SDS-PAGE-determined isolates. Therefore, SDS-PAGE was able to discriminate between all unidentified or misidentified isolates using a phenotypic method. In addition, SDS-PAGE identified all atypical isolates using biochemistry and CoNS at the subspecies level.     

Detection of methicillin resistance in coagulase-negative staphylococci by cefoxitin disc diffusion and oxacillin Etest. A study of consecutive bacteraemia isolates

Coagulase-negative staphylococci (CoNS) are a significant cause of nosocomial bacteraemia and their susceptibility to beta-lactamase-stabile penicillins is unpredictable. To ensure appropriate antibiotic therapy reliable methods for detection of methicillin resistance (MR) are needed. The objectives of this study were to determine the frequency of MR in a set of CoNS from cases of monomicrobial bacteraemia and to evaluate two phenotypic assays for detection of MR, the 10 microg cefoxitin disk test on Iso-Sensitest agar using a semiconfluent inoculum and the oxacillin Etest. MR was determined by a commercial genomic mecA assay. Of 110 CoNS, 75 were mecA positive and 35 mecA negative. Using interpretive zone diameters R < 22 mm and S > or = 27 mm, the cefoxitin disk test had a sensitivity and specificity of 100%. A correct prediction was obtained for 86 isolates, while 23 were indeterminate (> or = 22 mm; <27 mm). Using CLSI's guidelines, sensitivity and specificity of the oxacillin Etest were 100% and 80%, respectively. A correct prediction was obtained for 102 isolates, while 7 mecA negative isolates were classified as resistant. Thus, the cefoxitin disk test and the oxacillin Etest performed with high accuracy and both seem to be suitable for routine use.     

Prevalence of inducible clindamycin resistance among community-associated staphylococcal isolates in central Serbia

The emergence of resistance to most antimicrobial agents in staphylococci indicates the need for new effective agents in the treatment of staphylococcal infections. Clindamycin is considered to be one safe, effective and less costly agent. We analysed 482 staphylococcal isolates. Detection of inducible clindamycin resistance was performed by the D-test, while the presence of methylases genes: erm (A), erm (B) and erm (C), as well as, macrolide efflux gene mef was determined by polymerase chain reaction. Inducible clindamycin resistance phenotype was significantly higher in Staphylococcus aureus (S. aureus) strains then in coagulase-negative staphylococci (CNS). Among analysed S. aureus isolates, the predominance of the erm (C) gene, followed by the erm (A) gene were detected. These results indicate that the D-test should be routinely performed on each staphylococcal isolates.     

Antimicrobial susceptibility and body site distribution of community isolates of coagulase‐negative staphylococci

The primary aim of this study was to determine antimicrobial resistance in coagulase‐negative staphylococci (CoNS) from healthy adults in the community. Healthy adults (n = 114) were swabbed on six body sites; both armpits, both knee pits and both sides of the groin. Species determination was performed using Matrix Assisted Laser Desorption Ionization – Time of Flight (MALDI‐TOF) and susceptibility testing for 11 relevant antimicrobials was performed by the disc diffusion method and minimal inhibitory concentration gradient test. In total, 693 CoNS isolates were identified. Susceptibility testing was done on 386 isolates; one CoNS from each species found on each participant from the different body sites. The prevalence of antimicrobial resistance in the CoNS isolates were; erythromycin (24.6%), fusidic acid (19.9%), tetracycline (11.4%), clindamycin (7.8%), gentamicin (6.2%) and cefoxitin (4.1%). Multidrug resistance was observed in 5.2% of the isolates. Staphylococcus epidermidis and S. hominis were the first and second most prevalent species on all three body sites. We conclude that CoNS isolates from healthy adults in the community have a much lower prevalence of antimicrobial resistance than reported in nosocomial CoNS isolates. Still, we believe that levels of resistance in community CoNS should be monitored as the consumption of antimicrobials in primary care in Norway is increasing.     

Patterns of multidrug resistance among methicillin-resistant hospital isolates of coagulase-positive and coagulase-negative staphylococci collected in the international multicenter study RESIST in 1997 and 1998

The primary purpose of the multicenter international study "RESIST" was to obtain an update on the degree of multidrug resistance among methicillin-resistant staphylococci collected from a geographically diverse sample. A total of 3,307 staphylococcal isolates were recovered from single patients and primarily from clinical specimens that were collected at 20 collaborating regional health centers located in several countries in Europe, Asia, and Latin America during a 3- to 4-month period each in 1997 and 1998. All strains were deposited at the Laboratory of Molecular Genetics at ITQB/UNL in Oeiras, Portugal, for quality control and for testing by microbiological and molecular typing techniques; the Laboratory of Microbiology at The Rockefeller University serving as organizational center. The majority of strains, 3,100, were methicillin-resistant, of which 1,749 were coagulase positive (methicillin-resistant Staphylococcus aureus, MRSA), and 1,351 were coagulase negative (methicillin-resistant coagulase negative staphylococci, MRCNS). The overall frequency of drug resistance traits among the 1,749 MRSA strains was high (over 70% and up to and over 90% of the strains) to ciprofloxacin, erythromycin, clindamycin, gentamicin, and tetracycline, and was somewhat less frequent to sulfamethoxazole-trimethoprim (45%), chloramphenicol (30%), and rifampin (38%). None of the 3,307 staphylococcal isolates showed reduced susceptibility to vancomycin except for a single methicillin-resistant coagulase-negative isolate. The great majority of staphylococci were also susceptible to the new antimicrobial Synercid. In contrast, resistance to teicoplanin was significant among methicillin-resistant strains of coagulase-negative staphylococci, particularly among Staphylococcus haemolyticus. MRSA isolates showed marked geographic variation in their patterns of multiresistance, most likely reflecting the properties of unique multiresistant MRSA clones dominant in the hospitals that provided the MRSA isolates from the various geographic areas. The multiresistance patterns of MRSA strains and strains of methicillin-resistant coagulase-negative staphylococci originating at the same country source also showed striking differences, suggesting that resistance to antimicrobial agents emerged under different antibiotic pressures in these bacterial species.     

Antimicrobic susceptibility and plasmid profile analysis as identity tests for multiple blood isolates of coagulase-negative staphylococci.

We compared a disk diffusion antimicrobic susceptibility panel with plasmid DNA profiles as tests for identity of 106 isolates of coagulase-negative staphylococci cultured from the blood of 45 patients on multiple occasions. The antimicrobic panel included penicillin, oxacillin, clindamycin, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, tobramycin, kanamycin, and gentamicin. Nineteen patterns of antimicrobic susceptibility were found. The most common pattern was present in 25% of the isolates, and at least one isolate from 31% of the patients had this pattern. Forty-seven distinct plasmid DNA profiles were found. The most common plasmid profile was present in 8.5% of the isolates, and at least one isolate from 15% of the patients had this profile. Twenty-eight patients had multiple isolates that were identical by plasmid profile analysis. Twenty-seven (96%) of these patients had isolates that were also identical by antimicrobic susceptibility. Nineteen patients had multiple isolates that were different by plasmid profile analysis. In 18 (95%) of these patients, the isolates were also different by antimicrobic susceptibility. Although plasmid DNA profile analysis is a more discriminating tool, these data confirm that a selected disk diffusion antimicrobic susceptibility panel may be used to screen multiple blood isolates of coagulase-negative staphylococci for identity or differences.     

Prevalence and mechanisms of macrolide resistance among Staphylococcus epidermidis isolates from neutropenic patients in Tunisia

The prevalence of macrolide-lincosamide-streptogramin (MLS) resistance phenotypes was determined among erythromycin-resistant Staphylococcus epidermidis isolates collected at the Bone Marrow Transplant Centre, Tunisia during 2002. The erm(A), erm(B), erm(C), msrA, mefA and icaA genes were detected by PCR. The vga, vgb and vat genes were amplified from pristinamycin-resistant isolates. The icaA gene was detected in 76.5% of 34 isolates examined in detail. The erm(C) (53%) and erm(A) (32%) genes predominated because of clonal dissemination, followed by msrA (15%). Gene distribution was related to the methicillin resistance pattern. The vga gene was present in combination with erm(A) in three isolates.     

Analysis of methicillin resistance among Staphylococcus aureus blood isolates in an emergency department

The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has become of great concern in both hospital and community settings. To evaluate the prevalence and risk factors for methicillin resistance among Staphylococcus aureus, blood isolates in our Emergency Department (ED) were collected. All patients with S. aureus bacteremia (SAB) who presented to the ED from January 2000 to August 2005 were included, and a retrospective study was performed. A total of 231 patients with SAB were enrolled (median age, 59 yr; M:F, 125:106). Among these patients, methicillin-resistant strains accounted for 27.3% (63 patients). Catheter-related infection was the most frequent primary site of SAB (39.0%), followed by skin and soft tissue infection (16.5%). In multivariate analysis, recent surgery (OR, 3.41; 95% CI, 1.48-7.85), recent hospitalization (2.17; 1.06-4.62), and older age (> or =61 yr) (2.39; 1.25-4.57) were independently associated with the acquisition of methicillin-resistant strains. When antimicrobial therapy is considered for the treatment of a patient with suspected SAB, clinicians should consider obtaining cultures and modifying empirical therapy to provide MRSA coverage for patients with risk factors: older age, recent hospitalization, and recent surgery.     

Study of clinical profile and antibiotic response in typhoid fever

The objective of the present study is to evaluate the clinical profile and pattern of various drugs used in the treatment of typhoid fever. A retrospective analysis of adult patients suffering from typhoid fever was done at Kasturba Medical College hospital, Attavar during the year 1999-2001. Diagnosis of patients was based on clinical features, widal test and blood culture. The sensitivity pattern of isolates from blood culture was recorded. The mode of presentation, clinical course, treatment history, laboratory investigations reports, antibiotic administered, response to therapy and the complications were recorded. Total number of 44 cases of typhoid fever were studied. Out of these 21(47.7%) were males and 23(52.3%) were females. Average age of presentation was 23.9 years. Average duration of hospital stay was 10.8 days. Fever was present in all patients. Resistance of S. typhi to amoxicillin, chloramphenicol, ampicillin and co-trimoxazole were significantly high. Ciprofloxacin also showed resistance in 18.1% of cases. Sensitivity to cephalosporin was 100% in our study. Ciprofloxacin was the most commonly used antibiotic in our study (23 patients). Chloramphenicol alone was used in 2 patients and in 3 patients it was given after 6 days of ciprofloxacin treatment. Third generation cephalosporins (ceftriaxone) alone were used in 16 patients. Indiscriminate use of drugs in typhoid fever should be discouraged. Appropriate antibiotic as indicated by sensitivity tests should be employed to prevent the development of resistant strains of S. typhi.     

A kalilo-like linear plasmid in Louisiana field isolates of the pseudohomothallic fungus Neurospora tetrasperma

Two Louisiana strains of Neurospora tetrasperma contain a linear plasmid (LA-kalDNA) with a restriction map identical to the Hawaiian Neurospora intermedia senescence plasmid, kalDNA, but with termini 100 nucleotide pairs shorter. One of these strains also bore a circular plasmid similar to the Hawaiian circular plasmid Hanalei-2. One species probably acquired both plasmids from the other by horizontal transfer, at a time sufficiently distant for sequence divergence to take place. Many LA-kalDNA-bearing derivative strains senesced, but this plasmid does not guarantee senescence. Furthermore, LA-kalDNA does not insert into mtDNA. One senescent strain showed no LA-kalDNA. The plasmids are effectively transmitted via the pseudohomothallic sexual cycle. Single mating-type derivatives transmit plasmids maternally.     

Antibiogram and plasmid profiling of carbapenemase and extended spectrum Beta-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae in Abeokuta,South western,Nigeria

Background

The increased reports of ESBL dissemination from various centres in south western, Nigeria and the recent emergence of carbapenem resistant bacteria prompted the conception of this study.

Objectives

To demonstrate the relationship between high molecular weight plasmids and the expression of antibiotic multi-resistance including ESBL and carbapenemase.

Methods

We investigated 97 isolates of selected organisms consisting of 67 E. coli and 30 Klebseilla spp for the presence of plasmids expressing ESBL including carbapenem-hydrolysing enzymes. Beta-lactamase was determined using acidometric method, while ESBL and carbapenemase activity was determined using the double-disk diffusion test as well as the Modified Hodge test (MHT). Plasmid profiles of ESBL and carbapenemase positive isolates were determined according to standard protocols.

Results

An ESBL prevalence rate of 21.6% and carbapenem- resistance rate of 9.3% was recorded. Antibiotic susceptibility profile of ESBL isolates showed 100.0% resistance against Amoxicillin, Cotrimoxazole and Erythromycin. Moderate susceptibility was recorded against the Quinolone class of antibiotics; Meropenem remained the most active antibiotic against ESBL isolates with 62.5% against E. coli and 60% against K. pneumoniae. The plasmid profiles of our study isolates ranged from 11.8kbp to 35.5kbp.

Conclusion

Due to the relationship between high molecular weight plasmids and multi-drug resistance, we hereby recommend regular molecular surveillance of this form in our study setting.     

Studies on prevalence of biofilm associated genes and primary observation on sasX gene in clinical isolates of coagulase negative staphylococci (CoNS)

Coagulase negative staphylococci (CoNS) are nosocomial pathogens that cause indwelling medical device associated infections due to its biofilm forming potential and multiple antibiotic resistance. The current study focused on species identification, antibiotic resistance profile and molecular basis of biofilm formation and attachment of CoNS isolated from clinical samples. Along with this, molecular screening for mecA and newly identified surface colonization protein encoded by sasX gene was also conducted. S. epidermidis (n = 19, 47%) was identified as the most prevalent CoNS species and very interestingly two biofilm forming, mecA positive S. epidermidis isolates were found to carry all the biofilm associated genes screened in this study, which indicates its potential to form the strong biofilm. Another novel observation of the study is the detection of sasX gene in one biofilm positive S. epidermidis isolate. The study also identified one doxycycline resistant mecA positive, multidrug resistant S. haemolyticus isolate. In conclusion, the study signifies the existence of multiple biofilm related genes, multidrug resistance and the presence of sasX gene among clinical isolates of CoNS.     

Variability of resistance plasmids in coagulase-negative staphylococci and their importance as a reservoir of antimicrobial resistance

Coagulase-negative staphylococci (CoNS) are an important cause of human and animal diseases. Treatment of these diseases is complicated by their common antimicrobial resistance, caused by overuse of antibiotics in hospital and veterinary environment. Therefore, they are assumed to serve as a reservoir of resistance genes often located on plasmids. In this study, we analyzed plasmid content in 62 strains belonging to 10 CoNS species of human and veterinary origin. In 48 (77%) strains analyzed, 107 different plasmids were detected, and only some of them showed similarities with plasmids found previously. In total, seven different antimicrobial-resistance genes carried by plasmids were identified. Five of the CoNS staphylococci carried plasmids identical with either those of other CoNS species tested, or a well characterized Staphylococcus aureus strain COL, suggesting plasmid dissemination through horizontal transfer. To demonstrate the possibility of horizontal transfer, we performed electroporation of four resistance plasmids among Staphylococcus epidermidis, Staphylococcus petrasii, and coagulase-positive S. aureus strains. Plasmids were transferred unchanged, were stably maintained in recipient strains, and expressed resistance genes. Our work demonstrates a great variability of plasmids in human and veterinary staphylococcal strains and their ability to maintain and express resistance plasmids from other staphylococcal species.     

Sepsis due to linezolid resistant Staphylococcus cohnii and Staphylococcus kloosii: first reports of linezolid resistance in coagulase negative staphylococci from India

Linezolid, a viable alternative to vancomycin against methicillin resistant staphylococcal isolates, has been in use for a decade around the globe. However, resistance against staphylococci remains extremely rare and unreported from most of the Asian countries. Herein, we report two cases of linezolid resistant, coagulase negative staphylococcal sepsis for the first time from India. The first case was an 18-year-old burn patient, who, after a major graft surgery, landed in sepsis, and linezolid resistant Staphylococcus cohnii with an minimum inhibitory concentration (MIC) of >256 μg/ml by both broth microdilution and Etest, was isolated from multiple blood cultures. The second patient was a 60-year-old male with an intracranial bleed and sepsis, from whose blood cultures, linezolid resistant Staphylococcus kloosii was repeatedly isolated. Linezolid MIC was >32 μg/ml by broth microdilution and >16 μg/ml by Etest.     

Molecular analysis and antimicrobial resistance of Salmonella isolates recovered from raw meat marketed in the area of "Grand Tunis", Tunisia

A total of 315 samples of chicken (60), beef (144), minced meat (56), lamb meat (33), merguez (10) and fish (12) were collected from various local outlet stores in the area of "Grand Tunis", Tunisia between 2006 and 2008. Salmonella was recovered from 80 samples with the highest occurrence in chicken (48.3%) followed by beef (29.8%), minced meat (10.7%) and lamb (6.0%). No Salmonella were isolated from 12 fish and 10 merguez samples (typical Tunisian sausages). Nine serovars were identified among the isolates with the predominance of Salmonella Typhimurium (n=25) followed by Salmonella Kentucky (n=14), Salmonella Suberu (n=12) and Salmonella Zanzibar (n=11). Isolated Salmonella were characterized by serotyping, pulsed-field gel electrophoresis (PFGE) analysis, plasmid content and antimicrobial resistance profiling. Sixteen (20.0%) Salmonella isolates displayed resistance to ampicillin (13 isolates), streptomycin (five isolates), cefoperazone (two isolates), furazolodine (two isolates), with seven of these isolates displaying multiple resistance to at least two of these antimicriobal agents. PFGE analysis showed homogenous restriction patterns in each serovar. Compiled serotyping, PFGE analysis, plasmid profiling and antimicrobial resistance data provided additional discrimination.     

Molecular analysis of isoleucyl-tRNA synthetase mutations in clinical isolates of methicillin-resistant Staphylococcus aureus with low-level mupirocin resistance

Emergence and spread of low-level mupirocin resistance in staphylococci have been increasingly reported in recent years. The aim of this study was to characterize missense mutations within the chromosomal isoleucyl-tRNA synthetase gene (ileS) among clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) with low-level mupirocin resistance. A total of 20 isolates of MRSA with low-level mupirocin resistance (minimal inhibitory concentration, 16-64 microg/mL) were collected from 79 patients in intensive care units for six months. The isolates were analyzed for isoleucyl-tRNA synthetase (IleS) mutations that might affect the binding of mupirocin to the three-dimensional structure of the S. aureus IleS enzyme. All isolates with low-level mupirocin resistance contained the known V588F mutation affecting the Rossman fold, and some of them additionally had previously unidentified mutations such as P187F, K226T, F227L, Q612H, or V767D. Interestingly, Q612H was a novel mutation that was involved in stabilizing the conformation of the catalytic loop containing the KMSKS motif. In conclusion, this study confirms that molecular heterogeneity in ileS gene is common among clinical MRSA isolates with low-level mupirocin resistance, and further study on clinical mutants is needed to understand the structural basis of low-level mupirocin resistance.     

烧伤病房金黄色葡萄球菌分离株的分子分型及其耐药性

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) infection had been a global problem up to 1980s, and it has become a leading pathogen giving rise to nosocomial infections now. OBJECTIVE: To determine the molecular types and drug susceptibilities of Staphylococcus aureus prevailed in burn ward, and to provide a basis for preventing and controlling MRSA intections. METHODS: A total of 53 Staphylococcus aureus strains were collected from the burn ward in the Urumqi General Hospital of Lanzhou Military Region of Chinese PLA. These MRSA strains were identified by PCR and cefoxitin disc diffusion test, and all MRSA strains were typed by spa, SCCmec and MLST typing. In the meanwhile, antibiotic susceptibilities of 17 kinds of drugs, such as oxacillin, to Staphylococcus aureus were also determined, and drug resistance of different types of Staphylococcus aureus especially MRSA, was analyzed. RESULTS AND CONCLUSION:Among 53 Staphylococcus aureus strains, 43 were identified as MRSA, containing determined for amplification of meoA (n=41) and positive for cefoxitin disc diffusion test (n=2). Three SCCmec types, four spa types, and three ST types were found. The major predominant clone was ST239-MRSA-III-t030 (90.7%), with highest resistant to oxacillin and other nine antibiotics. In conclusion, the higher MRSA isolation rate from the burn ward, and ST239-MRSA-III-t030, as the predominant clone, presents with an outbreak in the burn ward and stronger resistance to many different families of antibiotics.       

Genetics of glycopeptide resistance in gram-positive pathogens

Glycopeptide resistance in enterococci, first detected in 1986, has since spread worldwide and represents a major health problem. Importantly, it has been recently acquired by Staphylococcus aureus. We shall review the genetics which accounts for this successful dissemination and discuss the possible origins of the resistance genes.     

Topical application of plasmid DNA to mouse and human skin

Gene expression following direct injection of naked plasmid DNA into the skin has been demonstrated in the past. Topical application of plasmid DNA represents an attractive route of gene delivery. If successful, it would have great prospects in skin gene therapy since it is painless and easy to apply. In this study, we analyzed the expression of plasmid DNA in vivo and in vitro following topical application of plasmid DNA in various liposomal spray formulations. Therefore, different concentrations of plasmid DNA expressing enhanced green fluorescent protein (pEGFP-N1) were sprayed onto mouse or human skin once daily for three consecutive days and compared with direct injection. Gene expression was assessed 24 h after the final topical application of various liposomal DNA formulations. The results showed that EGFP mRNA and protein were detectable by RT-PCR and Western blot, respectively. However, epicutaneously applied EGFP plasmid DNA did not lead to microscopically detectable EGFP protein, when assessed by confocal laser microscopy or fluorescence-activated cell sorting in contrast to about 4% of fluorescent keratinocytes following intradermal injection. In an in vivo mouse model, the application of pEGFP-N1 DNA led to the generation of GFP-specific antibodies. These results indicate that topical spray application of pEGFP-N1 liposomal DNA formulations is a suitable method for plasmid DNA delivery to the skin, yielding limited gene expression. This spray method may thus be useful for DNA vaccination. To increase its attractiveness for skin gene therapy, the improvement of topical formulations with enhanced DNA absorption is desirable.     

Epidemiological and genetic characterization of methicillin-resistant Staphylococcus aureus isolates from the ear discharge of outpatients with chronic otitis media

The origin of methicillin-resistant Staphylococcus aureus (MRSA) strains from otolaryngology outpatients has not been evaluated yet in Korea. We analyzed epidemiologic and genetic characteristics of MRSA isolates from the ear discharge of 64 outpatients with chronic otitis media in a Korean University Hospital during 2004. MRSA strains were grouped as either from the initial visit (n=33) or the follow-up visit (n=31) based on the timing of isolation. Healthcare-associated risk factors were frequently present among patients of the initial visit group, especially prior visit to primary clinic (79%) and antibiotic use (73%). SCCmec typing and multilocus sequence typing results showed that two genotypes, ST5-MRSA-II and ST239-MRSAIII, were prevalent in both the initial visit (73% vs. 24%) and the follow-up visit (55% vs. 42%). Pulsed-field gel electrophoresis identified eight types, including two major types shared by both groups. We conclude that majority of MRSA strains from ear discharge of chronic otitis media belonged to nosocomial clones that might be circulating in the community. This is the first report of the genetic analysis of MRSA strains from otolaryngology practices in Korea.