Staphylococcus aureus bacteremia, Australia

Staphylococcus aureus bacteremia (SAB) is common and increasing worldwide. A retrospective review was undertaken to quantify the number of cases, their place of acquisition, and the proportions caused by methicillin-resistant.S. aureus (MRSA) in 17 hospitals in Australia. Of 3,192 episodes, 1,571 (49%) were community onset. MRSA caused 40% of hospital-onset episodes and 12% of community-onset episodes. The median rate of SAB was 1.48/1,000 admissions (range 0.61-3.24; median rate for hospital-onset SAB was 0.7/1,000 and for community onset 0.8/1,000 admissions). Using these rates, we estimate that approximate to 6,900 episodes of SAB occur annually in Australia (35/100,000 population). SAB is common, and a substantial proportion of cases may be preventable. The epidemiology is evolving, with > 10% of community-onset SAB now caused by MRSA. This is an emerging infectious disease concern and is likely to impact on empiric antimicrobial drug prescribing in suspected cases of SAB.

Antimicrobial resistance in Staphylococcus aureus in Australian teaching hospitals, 1989-1999

An annual survey of antimicrobial resistance in clinical isolates of Staphylococcus aureus was conducted in 21 Australian teaching hospital microbiology laboratories in eight major cities from 1989 to 1999. A total of 19,000 isolates were tested for susceptibility to 18 antimicrobials, with 3795 being methicillin-resistant (MRSA). Resistance to ciprofloxacin in MRSA increased from 4.9% to 75.9%. The proportion of MRSA resistant to erythromycin decreased significantly (99.0%-88.9%), as did that to trimethoprim (98.4%-82.4%) and to tetracycline (96.5%-80.1%). The proportion of MRSA isolated increased in Sydney, Melbourne, Canberra, Adelaide, Perth, and Darwin, but not in Brisbane. The proportion in Hobart peaked in 1994. MRSA in Perth were predominantly non-multiresistant (nmMRSA) throughout the survey (i.e., resistant to less than three of eight indicator antibiotics) due mainly to local strains that originated in the community. The proportion of nmMRSA increased to modest levels in the other cities. In eastern cities, this was due to the appearance of strains closely related to nmMRSA seen in other countries of the southwestern Pacific.

Antimicrobial resistance and plasmid detection in strains of the Bacteroides fragilis group

Resistant populations of the Bacteroides fragilis group bacteria (two reference ones and two isolated from human and Callithrix penicillata marmoset) were obtained by the gradient plate technique, to clindamycin, penicillin G, metronidazole and mercuric chloride. All the four tested strains were originaly susceptible to the four antimicrobial drugs at the breakpoint used in this study. MICs determination for the four cultures gave constant values for each antimicrobial, on the several steps by the gradient plate technique. The intestinal human B. fragilis strains showed three DNA bands, that could be representative of only two plasmids in the closed covalently circular (CCC) form with molecular weights of approximately 25 and 2.5 Md. The results do not permit an association between the presence of plasmid in the human strain with the susceptibility to the studied drugs. The four strains were ß-lactamase negative in the two methods used, and no particular chromosomal genetic resistance marker was demonstred. The resistance (MIC) observed, after contact with penicillin G and mercuric chloride, were two-fold in the four tested strains

Epidemiology and antimicrobial resistance of B. fragilis group organisms isolated from clinical specimen and human intestinal microbiota

Epidemiological aspects and the antimicrobial susceptibility profile of the Bacteroides fragilis group isolated from clinical and human intestinal specimens were examined in this study. B. fragilis group strains were isolated from 46 (37%) of 124 clinical specimens and the source of the samples was: Blood culture (3), intraabdominal infection (27), brain abscess (2), soft tissue infection (17), respiratory sinus (3), pleural aspirate (9), breast abscess (3), surgical infected wound (22), pelvic inflammatory disease (22), chronic otitis media (9) and miscellaneous (7). Intraabdominal and soft tissue infections were responsible for more than half of the clinical isolates. Susceptibility to penicillin, cefoxitin, tetracycline, metronidazole, chloramphenicol and clindamycin was examined. All isolates were susceptible to metronidazole and chloramphenicol. For clindamycin and cefoxitin the resistance rates observed were 21.7% and 10.9% respectively. Susceptibility profiles varied among the different species tested. A total of 37 species of B. fragilis group isolated from intestinal microbiota of individuals who had no antimicrobial therapy for at least 1 month before the sampling was also examined. All strains were also susceptible to chloramphenicol and motronidazole and the resistance rates to clindamycin and cefoxitin were 19.4% and 5.4% respectively. A few institutions, in Brazil, have monitored the antimicrobial susceptibility of B. fragilis group strains isolated from anaerobic infections. The resistance rates to cefoxitin and clindamycin and the variation in susceptibility patterns among the species isolated in this study emphasize the need for monitoring of susceptibility patterns of B. fragilis group organisms isolated, especially at our University Hospitals.

Genetic diversity and antimicrobial resistance of enterococcal isolates from Southern region of Brazil

In the present study, a total of 455 enterococcal isolates, recovered from patients living in the city of Porto Alegre, State of Rio Grande do Sul, Brazil, during the period from July 1996 to June 1997, were identified to the species level by conventional biochemical and microbiological tests, and assayed for their susceptibilities to antimicrobial agents. The genetic diversity of antimicrobial resistant strains was evaluated by pulsed-field gel electrophoresis (PFGE) analysis of SmaI restricted chromosomal DNA. The most frequent species was Enterococcus faecalis (92.8%). Other species identified were: E. faecium (2.9%), E. gallinarum (1.5%), E. avium (1.1%), E. hirae (0.7%), E. casseliflavus (0.4%), E. durans (0.4%) and E. raffinosus (0.2%). The overall prevalence of isolates with high-level resistance (HLR) to aminoglycosides was 37.8%. HLR to gentamicin was found in 24.8%. No strains with acquired resistance to vancomycin were found. PFGE analysis showed the predominance of clonal group A, comprising strains isolated from different clinical specimens obtained from patients in three hospitals. These results suggest intra and inter-hospital dissemination of one predominant clonal group of E. faecalis isolates with HLR to gentamicin in the hospitals included in this study.

Epidemiological characterization, antimicrobial resistance and virulence mechanisms of human and animal streptococci

Dissertação para obtenção do Grau de Doutor em Biologia

Prevalence of enterotoxin-encoding genes and antimicrobial resistance in coagulase-negative and coagulase-positive Staphylococcus isolates from black pudding

INTRODUCTION: Staphylococcal species are pathogens that are responsible for outbreaks of foodborne diseases. The aim of this study was to investigate the prevalence of enterotoxin-genes and the antimicrobial resistance profile in staphylococcus coagulase-negative (CoNS) and coagulasepositive (CoPS) isolates from black pudding in southern Brazil. METHODS: Two hundred typical and atypical colonies from Baird-Parker agar were inoculated on mannitol salt agar. Eighty-two mannitol-positive staphylococci were submitted to conventional biochemical tests and antimicrobial susceptibility profiling. The presence of coagulase (coa) and enterotoxin (se) genes was investigated by polymerase chain reaction. RESULTS: The isolates were divided into 2 groups: 75.6% (62/82) were CoNS and 24.4% (20/82) were CoPS. The biochemical tests identified 9 species, of which Staphylococcus saprophyticus (37.8%) and Staphylococcus carnosus (15.9%) were the most prevalent. Antimicrobial susceptibility tests showed resistance phenotypes to antibiotics widely administered in humans, such as gentamicin, tetracycline, chloramphenicol, and erythromycin. The coa gene was detected in 19.5% (16/82) of the strains and 4 polymorphic DNA fragments were observed. Five CoNS isolates carrying the coa gene were submitted for 16S rRNA sequencing and 3 showed similarity with CoNS. Forty strains were positive for at least 1 enterotoxin-encoding gene, the genes most frequently detected were sea (28.6%) and seb (27.5%). CONCLUSIONS: The presence of antimicrobial resistant and enterotoxin-encoding genes in staphylococci isolates from black pudding indicated that this fermented food may represent a potential health risk, since staphylococci present in food could cause foodborne diseases or be a possible route for the transfer of antimicrobial resistance to humans.

Antimicrobial resistance profiles and genetic characterisation of macrolide resistant isolates of Streptococcus agalactiae

In this study, 100 clinical isolates of Streptococcus agalactiae recovered from genitourinary tract specimens of non-pregnant individuals living in Rio de Janeiro were submitted for antimicrobial susceptibility testing, detection of macrolide resistance genes and evaluation of the genetic diversity of erythromycin-resistant isolates. By agar diffusion method, all isolates were susceptible to ceftazidime, penicillin and vancomycin. Isolates were resistant to levofloxacin (1%), clindamycin (5%), erythromycin (11%) and tetracycline (83%) and were intermediated to erythromycin (4%) and tetracycline (6%). Erythromycin-resistant and intermediated isolates presented the following phenotypes: M (n = 3), constitutive macrolide-lincosamide-streptogramin B (MLS B, n = 5) and inductive MLS B (n = 7). Determinants of macrolide resistance genes, erm and mef, were detected in isolates presenting MLS B and M phenotypes, respectively. Randomly amplified polymorphic DNA profiles of erythromycin-resistant isolates were clustered into two major groups of similarity.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

Use of pyrosequencing to identify streptococci and to detect mutations causing antimicrobial resistance

Rapid identification and resistance determination of pathogens in clinical specimens is vital for accurate treatment and monitoring of infectious diseases. Antimicrobial drug resistance is increasing globally and healthcare settings are facing this cost-intensive and even life-threatening problem. The incidence of resistant pathogens in Finland has remained relatively steady and manageable at least for the time being. DNA sequencing is the gold standard method for genotyping, mutation analysis, and identification of bacteria. Due to significant cost decrease in recent years, this technique is available to many research and clinical laboratories. Pyrosequencing technique, a rapid real-time DNA sequencing method especially suitable for analyzing fairly short stretches of DNA, was used in this study. Due to its robustness and versatility, pyrosequencing was applied in this study for identification of streptococci and detection of certain mutations causing antimicrobial resistance in different bacteria. Certain streptococcal species such as S. pneumoniae and S. pyogenes are significantly important clinical pathogens. S. pneumoniae causes e.g. pneumonia and otitis media and is one of the most important community-acquired pathogens. S. pyogenes, also known as group A streptococcus, causes e.g. angina and erysipelas. In contrast, the socalled alpha-haemolytic streptococci, such as S. mitis and S. oralis, belong to the normal microbiota, which are regarded to be non-pathogenic and are nearly impossible to identify by phenotypic methods. In this thesis, a pyrosequencing method was developed for identification of streptococcal species based on the 16S rRNA sequences. Almost all streptococcal species could be differentiated from one another by the developed method, including S. pneumoniae from its close relatives S. mitis and S. oralis . New resistance genes and their variants are constantly discovered and reported. In this study, new methods for detecting certain mutations causing macrolide resistance or extended spectrum beta-lactamase (ESBL) phenotype were developed. These resistance detection approaches are not only suitable for surveillance of mechanisms causing antimicrobial resistance but also for routine analysis of clinical samples particularly in epidemic settings. In conclusion, pyrosequencing was found to be an accurate, versatile, cost-effective, and rapid DNA sequencing method that is especially suitable for mutation analysis of short DNA fragments and identification of certain bacteria.

Antimicrobial Resistance in Campylobacter jejuni and Campylobacter coli

Antimicrobial Resistance in Campylobacter jejuni and Campylobacter coli Campylobacters are a common cause of bacterial gastroenteritis worldwide, with Campylobacter jejuni and C. coli being the most common species isolated in human infections. If antimicrobial treatment is required, the drugs of choice at the moment are the macrolides and fluoroquinolones. In this thesis, the in vitro resistance profiles of the C. jejuni and C. coli strains were evaluated with emphasis on multidrug resistance. The aim was also to evaluate the different resistance mechanisms against the macrolides. Further, the disk diffusion method was compared to agar dilution method and its repeatability was evaluated, since it has been widely used for the susceptibility testing of campylobacters. The results of the present study showed that resistance to the fluoroquinolones is common in strains isolated from Finnish patients, but resistance to the macrolides is still rare. Multidrug resistance was associated with resistance to both ciprofloxacin and erythromycin. Among the available per oral drugs, least resistance was observed to coamoxiclav There was no resistance to the carbapenems. Sitafloxacin and tigecycline were in vitro highly effective towards Campylobacter species. A point mutation A2059G of the 23S rRNA gene was the main mechanism behind the macrolide resistance, whereas the efflux pumps did not seem to play an important role when a strain had A2059G mutation. A five amino acids insertion, which has not been described previously, in the ribosomal protein L22 of one highly-resistant C. jejuni strain without mutation in the 23S rRNA gene was also detected. Concerning the disk diffusion method, there was variation in the repeatability In conclusion, macrolides still appear to be the first-choice alternative for suspected Campylobacter enteritis. The in vitro susceptibilities found suggest that co-amoxiclav might be a candidate for clinical trials on campylobacteriosis, but in life-threatening situations, a carbapenem may be the drug of choice. More studies are needed on whether the disk diffusion test method could be improved or whether all susceptibilities of campylobacters should be done using a MIC based method.

Antimicrobial resistance of Staphylococcus spp. from small ruminant mastitis in Brazil

The study aimed to determine the antimicrobial resistance patterns and to identify molecular resistance markers in Staphylococcus spp. (n=210) isolated from small ruminant mastitis in Brazil. The antimicrobial resistance patterns were evaluated by the disk diffusion test and by detection of the presence of mecA, blaZ, ermA, ermB, ermC and msrA genes by PCR. The efflux pump test was performed using ethidium bromide and biofilm production was determined by Congo red agar test along with PCR for detection of the icaD gene. The isolates were most resistant to amoxicillin (50.0%), streptomycin (42.8%), tetracycline (40.4%), lincomycin (39.0%) and erythromycin (33.8%). Pan-susceptibility to all tested drugs was observed in 71 (33.8%) isolates and 41 Staphylococcus isolates were positive for the efflux pump. Although phenotypic resistance to oxacillin was observed in 12.8% of the isolates, none harbored the mecA gene. However, 45.7% of the isolates harbored blaZ indicating that beta-lactamase production was the main mechanism associated with staphylococci resistance to beta-lactams in the present study. The other determinants of resistance to antimicrobial agents ermA, ermB, ermC, and msrA were observed in 1.4%, 10.4%, 16.2%, and 0.9% of the isolates, respectively. In addition, the icaD gen was detected in 32.9% of the isolates. Seventy three isolates (54 from goats and 19 from sheep) were negative for all resistance genes tested and 69 isolates presented two or more resistance genes. Association among blaZ, ermA, ermB, ermC and efflux pump were observed in 17 isolates, 14 of which originated from goats and three from sheep. The data obtained in this study show the resistance of the isolates to beta-lactamics, which may be associated with the use of antimicrobial drugs without veterinary control.