Changes In Antibiotic Susceptibility Research Articles

Variable Effects of Exposure to Formulated Microbicides on Antibiotic Susceptibility in Firmicutes and Proteobacteria.

Microbicides are broad-spectrum antimicrobial agents that generally interact with multiple pharmacological targets. While they are widely deployed in disinfectant, antiseptic, and preservative formulations, data relating to their potential to select for microbicide or antibiotic resistance have been generated mainly by testing the compounds in much simpler aqueous solutions. In the current investigation, antibiotic susceptibility was determined for bacteria that had previously exhibited decreased microbicide susceptibility following repeated exposure to microbicides either in formulation with sequestrants and surfactants or in simple aqueous solution. Statistically significant increases in antibiotic susceptibility occurred for 12% of bacteria after exposure to microbicides in formulation and 20% of bacteria after exposure to microbicides in aqueous solutions, while 22% became significantly less susceptible to the antibiotics, regardless of formulation. Of the combinations of a bacterium and an antibiotic for which British Society for Antimicrobial Chemotherapy breakpoints are available, none became resistant. Linear modeling taking into account phylogeny, microbicide, antibiotic, and formulation identified small but significant effects of formulation that varied depending on the bacterium and microbicide. Adaptation to formulated benzalkonium chloride in particular was more likely to increase antibiotic susceptibility than adaptation to the simple aqueous solution. In conclusion, bacterial adaptation through repeated microbicide exposure was associated with both increases and decreases in antibiotic susceptibility. Formulation of the microbicide to which the bacteria had previously adapted had an identifiable effect on antibiotic susceptibility, but it effect was typically small relative to the differences observed among microbicides. Susceptibility changes resulting in resistance were not observed. The safety of certain microbicide applications has been questioned due to the possibility that microbicide exposure could select for microbicide and antibiotic resistance. Evidence that this may happen is based mainly on in vitro experiments where bacteria have been exposed to microbicides in aqueous solution. Microbicides are, however, normally deployed in products formulated with surfactants, sequestrants, and other compounds. While this may influence the frequency and extent of susceptibility changes, few studies reported in the literature have assessed this. In the current investigation, therefore, we have investigated changes in antibiotic susceptibility in bacteria which exhibited decreased microbicide susceptibility following repeated exposure to microbicides in simple aqueous solutions and in formulation. We report that the microbicide formulation had an identifiable effect on antibiotic susceptibility, but it was typically small relative to the differences observed among microbicides. We did not observe susceptibility changes resulting in resistance.

Results from the Survey of Antibiotic Resistance (SOAR) 2002-09 in Turkey.

To investigate changes in antibiotic susceptibility of Streptococcus pneumoniae and Haemophilus influenzae from the Survey of Antibiotic Resistance (SOAR) in community-acquired respiratory tract infections (CA-RTIs) between 2002 and 2009 in Turkey. Previously published SOAR data were used for this analysis. MICs were determined using Etest(®) gradient strips or disc diffusion. Susceptibility against a range of antimicrobial agents was assessed using CLSI breakpoints. A total of 900 S. pneumoniae isolates were analysed: 2002-03 (n = 75), 2004-05 (n = 301) and 2007-09 (n = 524). Four antibiotics were tested consistently throughout and three showed a statistically significant decrease in susceptibility (P < 0.0001): penicillin (74.7% susceptible in 2002-03; 67.8% in 2004-05; and 47.2% in 2007-09); cefaclor (85.3% in 2002-03; 78.7% in 2004-05; and 53.5% in 2007-09) and clarithromycin (85.3% in 2002-03; 82.7% in 2004-05; and 61.9% in 2007-09). Susceptibility to amoxicillin/clavulanic acid did not significantly change (100% in 2002-03; 98.7% in 2004-05; and 97.7% in 2007-09). A total of 930 H. influenzae isolates were analysed: 2002-03 (n = 133), 2004-05 (n = 379) and 2007-09 (n = 418). Four antibiotics were also consistently tested: ampicillin, amoxicillin/clavulanic acid, clarithromycin and cefaclor. All showed >90% susceptibility, but only cefaclor susceptibility significantly reduced (P < 0.0001) over time (99.2% in 2002-03; 96.3% in 2004-05; and 90.4% in 2007-09). In S. pneumoniae from Turkey, there has been a clear statistically significant reduction in susceptibility to key antibiotics since 2002, but not to amoxicillin/clavulanic acid (or amoxicillin). However, susceptibility in H. influenzae remained stable. Continued surveillance is required to monitor future changes in antibiotic susceptibility for CA-RTI bacteria.

Pleiotropic consequences of gene knockouts in the phthiocerol dimycocerosate and phenolic glycolipid biosynthetic gene cluster of the opportunistic human pathogen Mycobacterium marinum.

Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) contribute to the pathogenicity of several mycobacteria. Biosynthesis of these virulence factors requires polyketide synthases and other enzymes that represent potential targets for the development of adjuvant antivirulence drugs. We used six isogenic Mycobacterium marinum mutants, each with a different gene knockout in the PDIM/PGL biosynthetic pathway, to probe the pleiotropy of mutations leading to PDIM(-) PGL(-), PDIM(+) PGL(-) or PDIM(-) PGL(+) phenotypes. We evaluated the M. marinum mutants for changes in antibiotic susceptibility, cell envelope permeability, biofilm formation, surface properties, sliding motility and virulence in an amoeba model. The analysis also permitted us to begin exploring the hypothesis that different gene knockouts rendering the same PDIM and/or PGL deficiency phenotypes lead to M. marinum mutants with equivalent pleiotropic profiles. Overall, the results of our study revealed a complex picture of pleiotropic patterns emerging from different gene knockouts, uncovered unexpected phenotypic inequalities between mutants, and provided new insight into the phenotypic consequences of gene knockouts in the PDIM/PGL biosynthetic pathway.

Outline of a bacterial filter-based assay to detect beta-lactamases

We describe a new phenotypic test to detect beta-lactamases. This assay is based on diffusion of beta-lactam/beta-lactamase through a bacterial filter. Beta-lactam hydrolysis on (the other side of) the filter leads to a change in antibiotic susceptibility, which can be measured by disc diffusion tests. We illustrate its ease of use to detect beta-lactamases of different classes.

Development of a protocol for predicting bacterial resistance to microbicides.

Regulations dealing with microbicides in Europe and the United States are evolving and now require data on the risk of the development of resistance in organisms targeted by microbicidal products. There is no standard protocol to assess the risk of the development of resistance to microbicidal formulations. This study aimed to validate the use of changes in microbicide and antibiotic susceptibility as initial markers for predicting microbicide resistance and cross-resistance to antibiotics. Three industrial isolates (Pseudomonas aeruginosa, Burkholderia cepacia, and Klebsiella pneumoniae) and two Salmonella enterica serovar Typhimurium strains (SL1344 and 14028S) were exposed to a shampoo, a mouthwash, eye makeup remover, and the microbicides contained within these formulations (chlorhexidine digluconate [CHG] and benzalkonium chloride [BZC]) under realistic, in-use conditions. Baseline and postexposure data were compared. No significant increases in the MIC or the minimum bactericidal concentration (MBC) were observed for any strain after exposure to the three formulations. Increases as high as 100-fold in the MICs and MBCs of CHG and BZC for SL1344 and 14028S were observed but were unstable. Changes in antibiotic susceptibility were not clinically significant. The use of MICs and MBCs combined with antibiotic susceptibility profiling and stability testing generated reproducible data that allowed for an initial prediction of the development of resistance to microbicides. These approaches measure characteristics that are directly relevant to the concern over resistance and cross-resistance development following the use of microbicides. These are low-cost, high-throughput techniques, allowing manufacturers to provide to regulatory bodies, promptly and efficiently, data supporting an early assessment of the risk of resistance development.

Low prevalence of methicillin resistant Staphylococcus aureus as determined by an automated identification system in two private hospitals in Nairobi, Kenya: a cross sectional study.

BackgroundStaphylococcus aureus (S.aureus) is a major cause of both healthcare and community acquired infections. In developing countries, manual phenotypic tests are the mainstay for the identification of staphylococci with the tube and slide coagulase tests being relied upon as confirmatory tests for S. aureus. The subjectivity associated with interpretation of these tests may result in misidentification of coagulase negative staphylococci as S.aureus. Given that antibiotic resistance is more prevalent in CONS, this may result in over estimation of methicillin resistant S.aureus (MRSA) prevalence.MethodsA review of susceptibility data from all non-duplicate S.aureus isolates generated between March 2011 and May 2013 by the Vitek-2 (bioMérieux) automated system was performed by the authors. The data was generated routinely from processed clinical specimens submitted to the microbiology laboratories for culture and sensitivity at the Aga Khan University Hospital and Gertrude’s children’s hospital both situated in Nairobi.ResultsAntimicrobial susceptibility data from a total of 731 non-duplicate S.aureus isolates was reviewed. Majority (79.2%) of the isolates were from pus swabs. Only 24 isolates were both cefoxitin and oxacillin resistant while 3 were resistant to oxacillin but susceptible to cefoxitin giving an overall MRSA prevalence of 3.7% (27/731). None of the isolates were resistant to mupirocin, linezolid, tigecycline, teicoplanin or vancomycin.ConclusionThe prevalence of MRSA in this study is much lower than what has been reported in most African countries. The significant change in antibiotic susceptibility compared to what has previously been reported in our hospital is most likely a consequence of the transition to an automated platform rather than a trend towards lower resistance rates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0669-y) contains supplementary material, which is available to authorized users.

Bacteriology and changes in antibiotic susceptibility in adults with community-acquired perforated appendicitis.

This study evaluated bacterial etiology and antibiotic susceptibility in patients diagnosed with community-acquired perforated appendicitis over a 12-year-period. We retrospectively reviewed records of adult patients diagnosed with perforated appendicitis at an 800-bed teaching hospital between January 2000 and December 2011. In total, 415 culture-positive perforated appendicitis cases were analyzed. Escherichia coli was the most common pathogen (277/415, 66.7%), followed by Streptococcus species (61/415, 14.7%). The susceptibility of E. coli to ampicillin, piperacillin/tazobactam, ceftriaxone, cefepime, amikacin, gentamicin, and imipenem was 35.1%, 97.1%, 97.0%, 98.2%, 98.9%, 81.8%, and 100%, respectively. The overall susceptibility of E. coli to quinolones (ciprofloxacin or levofloxacin) was 78.7%. During the study period, univariate logistic regression analysis showed a significant decrease in E. coli susceptibility to quinolones (OR = 0.91, 95% CI 0.84–0.99, P = 0.040). We therefore do not recommend quinolones as empirical therapy for community-acquired perforated appendicitis.

SiRNA-mediated gene silencing of MexB from the MexA-MexB-OprM efflux pump in Pseudomonas aeruginosa

To gain insights into the effect of MexB gene under the short interfering RNA (siRNA), we synthesized 21 bp siRNA duplexes against the MexB gene. RT-PCR was performed to determine whether the siRNA inhibited the expression of MexB mRNA. Changes in antibiotic susceptibility in response to siRNA were measured by the E-test method. The efficacy of siRNAs was determined in a murine model of chronic P. aeruginosa lung infection. MexB-siRNAs inhibited both mRNA expression and the activity of P. aeruginosa in vitro. In vivo, siRNA was effective in reducing the bacterial load in the model of chronic lung infection and the P. aeruginosa-induced pathological changes. MexB-siRNA treatment enhanced the production of inflammatory cytokines in the early infection stage (P ＜ 0.05). Our results suggest that targeting of MexB with siRNA appears to be a novel strategy for treating P. aeruginosa infections. [BMB Reports 2014; 47(4): 203-208]

Characterisation of Pseudomonas aeruginosa related to bovine mastitis

Pseudomonas aeruginosa is one of the causative pathogens of bovine mastitis. Most P. aeruginosa strains possess the type III secretion system (TTSS), which may increase somatic cell counts (SCCs) in milk from mastitis-affected cows. Moreover, most of P. aeruginosa cells can form biofilms, thereby reducing antibiotic efficacy. In this study, the presence and effect of TTSS-related genotypes on increase of SCCs among 122 P. aeruginosa isolates obtained from raw milk samples from mastitis-affected cows and their antibiotic susceptibility at planktonic and biofilm status were investigated. Based on the presence of TTSS-related genes a total of 82.7% of the isolates were found to harbour exoU and/or exoS genes, including the invasive (exoU-/exoS+, 69.4%), cytotoxic (exoU+/exoS-, 8.3%) and cytotoxic/invasive strains (exoU+/ exoS+, 5.0%). Milk containing exoS-positive isolates had higher SCCs than those containing exoS-negative isolates. The majority of isolates showed gentamicin, amikacin, meropenem and ciprofloxacin susceptibility at planktonic status. However, the susceptibility was decreased at the biofilm status. Based on minimum biofilm eradication concentration (MBEC)/minimum inhibitory concentration (MIC) ratios, the range of change in antibiotic susceptibility varied widely depending on the antibiotics (from ≥ 3.1-fold to ≥ 475.0-fold). In conclusion, most P. aeruginosa isolates studied here had a genotype related to increase in SCCs. The efficiency of antibiotic therapy against P. aeruginosa-related bovine mastitis could be improved by analysing both the MBEC and the MIC of isolates.

English

2-Phenoxyethanol (2-PE) is a biocide used as a preservative in pharmaceutical, cosmetic and perfumery formulations. It is known for its rapid bactericidal effect against a wide range of bacteria. In the present study, clinical samples of multi drug resistant (MDR)Pseudomonas aeruginosa&nbsp;and&nbsp;Escherichia coli&nbsp;were grown in an increasing sub-minimal inhibitory concentration of 2-PE to observe if adaptation could be obtained and consequently retrieval of antibiotic susceptibility to clinically used antibiotics. Minimal inhibitory concentration (MIC) of 2-PE was determined for both MDR&nbsp;E. coli&nbsp;&nbsp; and&nbsp;&nbsp;P. aeruginosa. Adaptation to the biocide was attempted by repeated sub- culturing of the isolates in increasing sub-minimal-inhibitory concentration of the biocide.&nbsp;E. coli&nbsp;was able to grow in the presence of sub-MIC of 2-PE for two passages only and a slight rise in MIC was observed. MDR&nbsp;P. aeruginosa&nbsp;rapidly obtained an adaptive resistance towards the biocide with an increase in MIC from 5.4 to 13.7g%. Antibiotic sensitivities for both, MDR and MDR-2-PE adapted&nbsp;E. coli&nbsp;and&nbsp;P. aeruginosa&nbsp;were detected. MDR2-PE resistant&nbsp;P. aeruginosa&nbsp;showed significant improvement in antibiotic sensitivity while no change was &nbsp; &nbsp; observed with&nbsp;E. coli. Our results suggest an intriguing species specific link between biocide resistance and changes in antibiotic susceptibilities, findings that could be utilized in controlling the spread of antibiotic resistance and ambitiously improving the activity of some existing antibiotics. &nbsp; Key words:&nbsp;Pseudomonas aeruginosa,&nbsp;2-phenoxyethanol, antibiotic resistance.

Trends in antibiotic susceptibility and incidence of late-onset Klebsiella pneumoniae neonatal sepsis over a six-year period in a neonatal intensive care unit in Karachi, Pakistan

The incidence, change in antibiotic susceptibility, and risk factors associated with mortality of late-onset Klebsiella pneumoniae sepsis during 2006-2011, in a neonatal intensive care unit (NICU) of a developing country, were analyzed. The medical records of neonates with a discharge diagnosis of sepsis due to late-onset K. pneumoniae were retrieved. Demographic features, gestational age, date and year of admission, antibiotic susceptibility of isolates, and discharge status were recorded. The late-onset K. pneumoniae incidence per 1000 NICU admissions and risk factors for mortality due to late-onset K. pneumoniae sepsis are reported. During the period 2006-2011, 104 of 2768 neonates developed late-onset K. pneumoniae sepsis. The overall incidence of late-onset K. pneumoniae sepsis was 3.7% (37/1000 NICU admissions), with the highest annual incidence being 53/1000 in 2010. Most cases were males (n = 64; 62%) and most were premature and very low birth weight (n = 68; 65%). More than 80% of isolates were resistant to ampicillin + clavulanic acid, gentamicin, aztreonam, and cephalosporins. An increasing trend of resistance to amikacin, fluoroquinolones, piperacillin/tazobactam, and imipenem was observed. In 2011, three-quarters (72%; n=13) of late-onset K. pneumoniae were CR K. pneumoniae. Seventeen (16%) neonates died. Being male (p = 0.06, adjusted odds ratio (AOR) 9.2, 95% confidence interval (CI) 1.3-66.9), having an extremely low birth weight (p = 0.01, AOR 6.1, 95% CI 0.8-44.4), having severe thrombocytopenia (p = 0.07, AOR 3.9, 95% CI 1.2-13.0), and failure to achieve microbiological clearance (p < 0.001, AOR 19.6, 95% CI 4.0-98.0) were significantly associated with mortality due to late-onset K. pneumoniae sepsis. There has been a rise in carbapenem-resistant strains of late-onset K. pneumoniae, associated with an increased mortality and limited antibacterial choices. Antimicrobial stewardship and rigorous infection control measures seem to be the only way to limit the spread of these strains.

Trends in Incidence and Susceptibility among Methicillin-Resistant Staphylococcus aureus Isolated from Intranasal Cultures Associated with Rhinosinusitis

Reports regarding the incidence and antibiotic susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) in rhinosinusitis (RS) are limited. This study was designed to identify epidemiology and trends of MRSA incidence and antimicrobial resistance in the sinonasal cavities. This is a retrospective case series. All intranasal/sinus cultures obtained by otolaryngologists at Stanford over a 20-year period (1990-2010) were retrospectively reviewed by mining the microbiology database. Nested searches were then made for all S. aureus and MRSA cultures. Patterns of incidence and changes in antibiotic susceptibilities were tabulated and statistical analysis was performed. Our search retrieved 10,387 positive intranasal culture samples, with S. aureus found in 800 (7.7%), and MRSA comprising 110 (1.06%) of this subset. Between the years of 1990 and 1999, only 2/112 (1.7%) of S. aureus-positive nasal cultures were positive for MRSA, with a sharp rise in incidence to 86/606 (14.2%) from 2000 to 2005, and to 22/82, 26.8% from 2006 to 2010. On a percent basis, using logistic regression modeling, this represents a statistically significant increasing trend (p < 0.0001) for MRSA sinusitis. However, over the 20-year interval studied, the patterns of antibiotic resistance among MRSA remained unaltered, especially with regard to trimethoprim-sulfamethoxazole and vancomycin. S. aureus and MRSA isolates from intranasal cultures, which were essentially absent before the year 2000, became significantly more common earlier this decade. These data show the increased role of MRSA in sinusitis. MRSA antibiotic susceptibilities have remained, however, largely stable during this time period.

Changes in Antibiotic Susceptibility ofHelicobacter pyloriin the Course of Eight Years in the Zaanstreek Region in The Netherlands

Background. Failure of anti-Helicobacter therapy is the result noncompliance and resistance to the prescribed antibiotics. Aim. Antibiotic susceptibility of H. pylori was determined in native Dutch patients and patients of Turkish descent. Methods. In a period of eight years a total of 925 strains of H. pylori were cultured. Bacterial susceptibility was successfully determined in 746 (80.6%) of these isolates. Three hundred and nine strains (33%) originated from patients of Turkish descent. Results. In total clarithromycin resistance was found in 146 (20.5%) strains, metronidazole resistance in 147 (19.9%) strains. Amoxicillin resistance was found to be present in five strains. There is a slight but nonsignificant decrease in the percentage of clarithromycin-resistant strains in the consecutive period of eight years from 20% to 18%. No changes were seen in the consecutive years in metronidazole resistance. The number of clarithromycin-resistant strains decreased in Turkish patients, not in native Dutch patients. Conclusion. Resistance did not change significantly in consecutive years. But clinicians should take not only the antibiotic history into account but also ethnicity before prescribing metronidazole or clarithromycin.

Comparison of Antibiotic Susceptibility of Old and Current Serratia

We explored changes in antibiotic susceptibility of Serratia marcescens in the last 50 years by comparing isolates collected between 1945 and 1950, and current isolates. Isolates were divided into three groups: environmental, clinical and 'old'. Susceptibility was determined by microdilution. Class 1 integrons were determined by PCR. Statistical analysis was conducted using the Kruskal-Wallis (K-W) tests with Bonferroni correction for multiplicity. Antimicrobials showing differences in the K-W test were analyzed by Mann-Whitney U test. Differences were considered significant when p < 0.05. All isolates were sensitive to ceftazidime, cefotaxime, kanamycin, gentamicin, ofloxacin and ciprofloxacin, and resistant to rifampicin, penicillin, ampicillin, amoxicillin, tetracycline, amoxicillin-clavulanic acid, cefazolin, cefamandole, polymyxin B/colistin, fusidic acid, lincosamides, streptogramins, daptomycin, linezolid and cefuroxime. Old isolates exhibited reduced susceptibility to streptomycin. Cefotaxime and streptomycin showed significant differences in the K-W test. None of the strains studied presented ESBL. Resistance to antimicrobials was not drastically different in Serratia when old and current strains were compared. Despite the multiple molecular mechanisms involved in bacterial resistance, withdrawing the antibiotics tends to restore the original phenotypes. Results from this report essentially confirm the conclusion obtained through metagenomic analysis that resistance to antibiotics already existed in ancient times.

Unchanged Antibiotic Susceptibility in Escherichia coli and Pseudomonas aeruginosa after Long-Term in vitro Exposure to Antineoplastic Drugs

Background: Certain antineoplastic drugs inhibit bacterial growth. Whether these drugs also cause genetic changes in bacteria that lead to increased antibiotic resistance is not yet documented. Given the massive and repeated antibiotic treatment most cancer patients undergo, this question is important. We have examined the possible effects of in vitro long-term antineoplastic exposure on antibiotic resistance. Methods: Using the disc diffusion method, two bacterial strains (Escherichia coli, ATCC 25922, and Pseudomonas aeruginosa, ATCC 27583) were exposed to methotrexate, fluorouracil, vincristine, doxorubicin and cytarabine during 50 overnight cycles. The bacterial strains were susceptibility-tested to several antibiotics before and after repeated exposure to antineoplastics. Results: No changes in antibiotic susceptibility were seen in the two bacterial strains after long-term exposure to any of the antineoplastic drugs tested. Conclusion: Long-term in vitro antineoplastic exposure did not change the antibiotic susceptibility in the E. coli or P. aeruginosa strains.

Chronological change of antibiotic use and antibiotic resistance in Escherichia coli causing urinary tract infections

Overuse of antibiotics can cause the emergence of resistant bacterial strains. This study retrospectively investigated recent trends in Escherichia coli causing urinary tract infections (UTIs), focusing on antibiotic use and antibiotic susceptibilities. Patients diagnosed with UTIs caused by E. coli in Akashi Municipal Hospital between April 2004 and March 2010 were enrolled in the study. A total of 858 UTI cases were examined. Antibiotics used in our hospital during that period and the antibiotic susceptibilities of E. coli in UTI cases were assessed. We analyzed the data on a yearly basis, with the year being defined as the period from April to the following March (e.g., in this study the period from April 2004 to March 2005 represents 2004). The first 3years (2004-2006) were compared to the last 3years (2007-2009). The use of piperacillin, cephazolin, amikacin, oral cefotiam, and levofloxacin decreased significantly and the use of imipenem, gentamicin (GM), cefcapene, and oral minocycline (MINO) increased significantly in the last 3years compared to the previous 3years. The susceptibilities of MINO in complicated cystitis significantly increased and those of GM in uncomplicated pyelonephritis significantly decreased in these 3years (2007-2009) compared to the previous 3years (2004-2006) (P<0.05). Additionally, extended-spectrum β-lactamase (ESBL)-producing E. coli tended to be isolated more often; this was statistically significant in the last 3years (2007-2009) compared to the previous 3years (2004-2006) (P<0.05). In conclusion, we found changes in our pattern of antibiotic use associated with changes in antibiotic susceptibilities and an increase in ESBL-producing E. coli isolated from our UTI cases. Monitoring of antibiotic use and emergence of resistant strains should be continued.

Phenotypic diversity of Escherichia coli O157:H7 strains associated with the plasmid O157.

Escherichia coli O157:H7, a food-borne pathogen, causes hemorrhagic colitis and the hemolytic-uremic syndrome. A putative virulence factor of E. coli O157:H7 is a 60-MDa plasmid (pO157) found in 99% of all clinical isolates and many bovine-derived strains. The well characterized E. coli O157:H7 Sakai strain (Sakai) and its pO157-cured derivative (Sakai-Cu) were compared for phenotypic differences. Sakai-Cu had enhanced survival in synthetic gastric fluid, did not colonize cattle as well as wild-type Sakai, and had unchanged growth rates and tolerance to salt and heat. These results are consistent with our previous findings with another E. coli O157:H7 disease outbreak isolate ATCC 43894 and its pO157-cured (43894-Cu). However, despite the essentially sequence identical pO157 in these strains, Sakai-Cu had changes in antibiotic susceptibility and motility that did not occur in the 43894-Cu strain. This unexpected result was systematically analyzed using phenotypic microarrays testing 1,920 conditions with Sakai, 43894, and the plasmid-cured mutants. The influence of the pO157 differed between strains on a wide number of growth/survival conditions. Relative expression of genes related to acid resistance (gadA, gadX, and rpoS) and flagella production (fliC and flhD) were tested using quantitative real-time PCR and gadA and rpoS expression differed between Sakai-Cu and 43894-Cu. The strain-specific differences in phenotype that resulted from the loss of essentially DNA-sequence identical pO157 were likely due to the chromosomal genetic diversity between strains. The O157:H7 serotype diversity was further highlighted by phenotypic microarray comparisons of the two outbreak strains with a genotype 6 bovine E. coli O157:H7 isolate, rarely associated with human disease.

Changes in antibiotic susceptibility and ribotypes in Clostridium difficile isolates from southern Scotland, 1979–2004

An increase in the incidence of clinical cases of Clostridium difficile infection has been reported in recent years, but few studies have examined changes in molecular epidemiology and antibiotic resistance over a long period of time. A collection of 179 isolates of C. difficile obtained from symptomatic adult patients in southern Scotland between 1979 and 2004 was used to determine changes in the prevalence of epidemiological types and antibiotic susceptibilities to common antibiotics. PCR ribotyping and MIC determination were performed on all isolates. A total of 56 different ribotypes were identified, among which ribotype 002 was the commonest type overall (14 .0%), followed by ribotypes 014 (7.3 %), 012 (5 .0%), 015 (5.0 %), 020 (5 .0%) and 001 (4.5 %). Ribotype 078 was also identified. The 10 commonest ribotypes comprised 55 % of the total isolates. Ribotype 001 increased in prevalence from 1.5 to 12.2 % over the study years, whereas the prevalence of ribotype 012 decreased from 8.7 to 2 .0%. Resistance to clindamycin, erythromycin and ceftriaxone was found in 95.5, 14.0 and 13.4 % of isolates, respectively. Resistance to vancomycin or metronidazole was not detected. Thirty-two (17.9 %) and 14 (7.8 %) isolates were resistant to two and three or more antibiotics, respectively. Ribotype 001 displayed maximum resistance, with 50 % of isolates resistant to erythromycin, moxifloxacin and ceftriaxone, and 100 % resistant to clindamycin. Over the 26 years of the study, antibiotic resistance and ribotype prevalence have changed, and antibiotic pressures may have been the major driver of this change.

Emm type distribution and antibiotic susceptibility of non-invasive group a beta-hemolytic streptococci in Perth, Western Australia

Aims To characterise the emm type distribution and antibiotic susceptibility of non-invasive group A streptococci (GAS) in Perth, Western Australia. Methods We collected consecutive, non-invasive GAS isolates received during a 3 month period in 2008. Isolates were categorised as sampled from either skin (impetigo, cellulitis), throat (pharyngitis) or genital (perianal cellulitis, vaginal discharge, perinatal screening) areas. Antibiotic susceptibility to penicillin, erythromycin, clindamycin and tetracycline was determined by disc diffusion and/or Etest methods. Emm typing was performed by sequencing of the N-terminal hypervariable region of the emm gene. Pulse field gel electrophoresis (PFGE) was performed on selected isolates. Results A total of 186 isolates were obtained from patients with a mean age of 24 (range 0.3–78). Most (138;74%) resided in suburbs within the Perth metropolitan area. The isolates were obtained from the skin (58%), throat (29%) and genital (13%) areas. Forty-five different emm types were represented. A significant difference existed between the proportion of genital isolates compared to skin isolates that were emm 4 (28% vs 9%, p = 0.019). All isolates were susceptible to penicillin (mean minimum inhibitory concentration 0.012, range 0.004–0.023). Rates of erythromycin, clindamycin and tetracycline non-susceptibility were 2%, 2% and 14%, respectively. There were significant differences between the proportion of emm 4 versus non-emm 4 isolates (0% vs 17%, p = 0.017) and emm 87 versus non-emm 87 isolates (69% vs 11%) that were tetracycline non-susceptible. PFGE demonstrated this to due to a dominant clone within these emm types. Conclusions A wide diversity of emm types cause non-invasive GAS disease in Western Australia, yet they remain uniform in their susceptibility to penicillin. Resistance to second line therapeutic antibiotics is rare. Ongoing surveillance will be necessary to monitor changes in antibiotic susceptibility, assist in vaccine design and monitor the potential impact of such vaccines on emm type distribution.

Changes in antibiotic susceptibility in staphylococci habituated to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia)

To investigate the effect of sub-lethal challenge with tea tree oil (TTO) on the antibiotic resistance profiles of staphylococci. Isolates of methicillin-resistant/-sensitive Staphylococcus aureus (MRSA and MSSA) and coagulase-negative staphylococci (CONS) were habituated to sub-lethal concentrations of TTO (72 h). Following habituation, the minimum inhibitory concentrations (MIC) of antibiotics and TTO were determined. Habituated MRSA/MSSA cultures had higher (P < 0.05) MIC values than control cultures for the examined antibiotics. Habituated MRSA/MSSA cultures also displayed decreased susceptibility to TTO. Although the MIC of habituated MRSA/MSSA for the examined antibiotics reverted to control values after subsequent culture in the absence of TTO, the increased MIC against TTO were maintained. When compared with control cultures, habituated CoNS cultures had higher (P < 0.05) MIC values against three-fifths of the antibiotics examined; no changes in TTO MIC were observed. TTO habituation 'stress-hardens' MRSA and MSSA, evidenced by transient decreased antibiotic susceptibility and stable decreased TTO susceptibility. Although TTO habituation did not decrease susceptibility of CoNS to TTO, such cultures showed transient decreased antibiotic susceptibility. Application of TTO at sub-lethal concentrations may reduce the efficacy of topical antibiotics used with TTO in combination therapies.