- Volume 61, Issue 1, 2012

Candida albicans cells have the ability to form biofilms on biotic and abiotic surfaces, such as indwelling medical devices. C. albicans cells can interconvert between budded and hyphal growth forms, herein termed the budded-to-hyphal transition (BHT), which is important for the formation of mature biofilms. Previous work identified 23 small organic molecules that could inhibit the BHT but did not affect C. albicans cell viability or budded cell growth. These BHT inhibitors were proposed to inhibit multiple signalling pathways regulating the BHT, many of which also regulate biofilm formation. However, only three of the BHT inhibitors, buhytrinA, ETYA and CGP-37157, were capable of inhibiting in vitro biofilm formation of wild-type laboratory C. albicans strains. When clinical C. albicans isolates were examined for their ability to form biofilms, only 11 of the 28 clinical isolates tested (39 %) were capable of forming biofilms. Although buhytrinA, ETYA and CGP-37157 could inhibit the BHT of all 28 clinical isolates, they were only able to inhibit biofilm formation of a subset of these clinical isolates, with ETYA having 100 % efficacy. These data indicate that the biofilm-forming capability of laboratory and clinical isolates of C. albicans, as well as the efficacy of BHT inhibitors against these different isolates, can differ dramatically. These differences between laboratory and clinical isolates should be an important aspect to consider when examining potentially new antifungal therapeutics.

Several outbreaks of infections caused by rapidly growing mycobacteria (RGM) were reported in many Brazilian states (2032 notified cases) from 2004 to 2010. Most of the confirmed cases were mainly associated with Mycobacterium massiliense (recently renamed as Mycobacterium abscessus subsp. bolletii) BRA100 clone, recovered from patients who had undergone invasive procedures in which medical instruments had not been properly sterilized and/or disinfected. Since quinolones have been an option for the treatment of general RGM infections and have been suggested for therapeutic schemes for these outbreaks, we evaluated the in vitro activities of all generations of quinolones for clinical and reference RGM by broth microdilution, and analysed the peptide sequences of the quinolone resistance determining regions (QRDRs) of GyrA and GyrB after DNA sequencing followed by amino acid translation. Fifty-four isolates of M. abscessus subsp. bolletii, including clone BRA100, recovered in different states of Brazil, and 19 reference strains of RGM species were characterized. All 54 M. abscessus subsp. bolletii isolates were resistant to all generations of quinolones and showed the same amino acids in the QRDRs, including the Ala-83 in GyrA, and Arg-447 and Asp-464 in GyrB, described as being responsible for an intrinsic low level of resistance to quinolones in mycobacteria. However, other RGM species showed distinct susceptibilities to this class of antimicrobials and patterns of mutations contrary to what has been traditionally defined, suggesting that other mechanisms of resistance, different from gyrA or gyrB mutations, may also be involved in resistance to high levels of quinolones.

A total of 143 group B streptococcus (GBS) isolates collected from mothers at the Maternity Hospital in Kuwait were investigated for their serotypes and antibiotic resistance, and screened by PCR for the carriage of genes for resistance to tetracycline (tetk, tetM, tetL, tetO), erythromycin (ermA, ermB, ermC, ermTR, ermM, mefA, mefE, msrA) and aminoglycosides (aph3, ant4, ant6). All isolates were serotyped using a latex agglutination test. Most of the isolates belonged to serotypes V (38.5 %), III (20.9 %), Ia (7.7 %) and II (11.2 %). Sixteen isolates (11.2 %) were nontypable. All isolates were susceptible to penicillin, ampicillin and cefotaxime (MICs 0.016–0.094 µg ml−1) but were resistant to trimethoprim (92.3 %), tetracycline (89.5 %), minocycline (89.5 %), high-level kanamycin (76.9 %), chloramphenicol (30.0 %), erythromycin (12.6 %), clindamycin (7.0 %), high-level streptomycin (3.5 %) and ciprofloxacin (0.7 %). The tetracycline-resistant isolates contained tetM (94.5 %), tetO (3.9 %), tetL (1.6 %) and tetK (0.8 %). The erythromycin-resistant isolates contained ermB (61.1 %), ermTR (38.9 %), ermA (5.5 %), mefA (5.5 %) and mefE (11 %). All high-level kanamycin-resistant isolates contained aph3. One of the high-level streptomycin-resistant isolates contained ant6. Partial DNA sequencing of aph3 revealed sequences with 99 % similarity to aph3 found in Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis, suggesting that the GBS isolates could have acquired aph3 from other Gram-positive cocci. The high proportion of isolates with resistance to tetracycline, high-level kanamycin and trimethoprim, and the increase in the prevalence of erythromycin resistance, represents an emerging public health concern that needs further surveillance.

Carbapenems such as imipenem and meropenem are first-line agents for the treatment of serious nosocomial infections caused by multidrug-resistant clinical isolates of bacteria belonging to the family Enterobacteriaceae. However, resistance to carbapenems has increased dramatically among members of the family Enterobacteriaceae isolated from a teaching hospital in Shanghai, China. In the present study, we investigated the prevalence and molecular characteristics of carbapenem-resistant clinical isolates of Enterobacteriaceae. None of the 77 clinical isolates collected from 2002 to 2009 were susceptible to ertapenem and only 6.5 % and 1.3 % of isolates were susceptible to imipenem and meropenem, respectively. Colistin and tigecycline were found to be the most active agents against carbapenem-resistant Enterobacteriaceae isolates, inhibiting 90 % of isolates at a concentration of 1 µg ml−1 and 4 µg ml−1, respectively. The results of PFGE analysis suggested that many of the KPC-2-producing isolates of Citrobacter freundii and Klebsiella pneumoniae were clonally related. Most of these isolates were isolated from the same ward, namely the neurosurgical ward, suggesting horizontal transfer of the KPC-2-encoding gene in these isolates. Of the 77 isolates, 84.4 % were found, by PCR, to be capable of carbapenemase production. SDS-PAGE analysis revealed that 75.3 % (58/77) of the isolates had lost at least one porin protein. Our results suggested that the prompt detection of carbapenemase-producing strains is critical for the containment of nosocomial transmission. As no novel antimicrobials have been identified for use in the treatment of these pan-drug-resistant isolates, further studies should focus on the rational use of available antibiotics, the implementation of active antibiotic resistance surveillance and the strict implementation of infection control measures to avoid the rapid spread or outbreak of carbapenemase-producing Enterobacteriaceae in health-care facilities.

Acinetobacter species have emerged as opportunistic nosocomial pathogens in intensive care units. Epidemic spread and outbreaks of multidrug-resistant or carbapenem-resistant Acinetobacter baumannii infections have been described worldwide. Species distribution, antimicrobial resistance and genotypes were investigated for Acinetobacter species isolates collected from a single institution in Korea over 7 years. Two hundred and eighty-seven Acinetobacter species isolates were collected from patients with bloodstream infections in one Korean hospital from 2003 to 2010. Most of them belonged to the Acinetobacter calcoaceticus–baumannii complex (94.4 %). The most frequently isolated species was A. baumannii (44.2 %), followed by Acinetobacter nosocomialis (formerly Acinetobacter genomic species 13TU) (34.1 %). The proportion of A. baumannii increased significantly from 2008 to 2010 (40.4 to 50.0 %). From 2008, imipenem and meropenem resistance rates increased significantly compared with 2003–2007 (12.9 % and 20.5 %, respectively, to 41.4 % and 41.5 %, respectively). An increased carbapenem resistance rate between the two periods was identified more clearly amongst A. baumannii isolates. Polymyxin-resistant A. baumannii isolates emerged in 2008–2010, despite the availability of few isolates. The increase of carbapenem resistance in A. baumannii might be due to the substitution of main clones. Although ST92 and ST69 were the most prevalent clones amongst A. baumannii in 2003–2007 (47.8 % and 15.9 %, respectively), ST75 and ST138 had increased in 2008–2010 (39.7 % and 25.9 %, respectively). Although ST92 showed moderate resistance to carbapenems, most ST75 and ST138 isolates were resistant to carbapenems. All ST75 and ST138 isolates, but only one ST92 isolate, contained the bla OXA-23-like gene. Increased carbapenem resistance in Acinetobacter species and A. baumannii isolates might be due to the expansion of specific carbapenem-resistant clones.

Acinetobacter baumannii is an emerging multidrug-resistant pathogen and very little information is available regarding its imipenem resistance in Latin American countries such as Bolivia. This study investigated the antimicrobial resistance profile of 46 clinical strains from different hospitals in Cochabamba, Bolivia, from March 2008 to July 2009, and the presence of carbapenemases as a mechanism of resistance to imipenem. Isolates were obtained from 46 patients (one isolate per patient; 30 males,16 females) with an age range of 1 day to 84 years, and were collected from different sample types, the majority from respiratory tract infections (17) and wounds (13). Resistance to imipenem was detected in 15 isolates collected from different hospitals of the city. These isolates grouped into the same genotype, named A, and were resistant to all antibiotics tested including imipenem, with susceptibility only to colistin. Experiments to detect carbapenemases revealed the presence of the OXA-58 carbapenemase. Further analysis revealed the location of the bla OXA-58 gene on a 40 kb plasmid. To our knowledge, this is the first report of carbapenem resistance in A. baumannii isolates from Bolivia that is conferred by the OXA-58 carbapenemase. The presence of this gene in a multidrug-resistant clone and its location within a plasmid is of great concern with regard to the spread of carbapenem-resistant A. baumannii in the hospital environment in Bolivia.

The aim of this study was to assess risk factors for primary Helicobacter pylori antibiotic resistance by an extended anamnesis. In total, 519 H. pylori strains from untreated symptomatic adults who answered a questionnaire were evaluated. Strain susceptibility was assessed by a breakpoint susceptibility test. Primary resistance rates were 29.5 % for metronidazole, 17.9 % for clarithromycin, 7.3 % for metronidazole+clarithromycin, 4.0 % for tetracycline and 10.8 % for ciprofloxacin. On multivariate analysis, younger (≤65 years) age was an independent predictor for metronidazole resistance. To our knowledge, for the first time, being a member of the health-care profession was revealed as a risk factor for H. pylori resistance to metronidazole and both metronidazole and clarithromycin. Respiratory and urinary tract infections were independent predictors of clarithromycin and ciprofloxacin resistance, respectively. The presence of co-infections was an independent risk factor for clarithromycin, metronidazole and ciprofloxacin resistance. Surprisingly, female sex was the only predictor for tetracycline resistance. The antibiotic resistance rates were not associated with disease type, place of residence, birthplace, educational level, non-steroidal anti-inflammatory drug or proton pump inhibitor use, smoking or dietary factors, such as consumption of coffee, yogurt, green tea, raw garlic, raw onion, honey or meat. There was a trend for higher metronidazole resistance in strains from diabetic patients. In conclusion, the extended anamnesis of H. pylori-positive patients should include data on patient age, sex, whether they are in the health-care profession, co-infections and possibly diabetes to improve the choice of empiric therapy. Tailored treatment based on the extended anamnesis is suggested, and susceptibility testing of the strains is recommended for patients at risk for antibiotic resistance, especially to clarithromycin, fluoroquinolones or both metronidazole and clarithromycin.

In this study of the diversity of AmpC β-lactamase in clinical isolates of Enterobacter spp., a strain was found carrying the plasmid-mediated AmpC β-lactamase ACT-1 gene on its chromosome. The strain was identified as Enterobacter hormaechei using phylogenetic analysis of 16S rRNA and hsp60 genes. In addition, the species was confirmed by DNA–DNA hybridization. The genetic environment of the bla ACT-1 gene was characterized, including the ampR and ampG genes, using a two-step PCR. The amino acid sequences of AmpR at serine 35, arginine 86, glycine 102, aspartic acid 135 and tyrosine 264 were conserved. Measurement of the transcription level of the bla ACT-1 gene using real-time quantitative PCR showed that it increased 1.98-fold following cefoxitin induction. These results suggest that the plasmid-mediated bla ACT-1 gene originated from the chromosome of E. hormaechei.

Recently, dupA was reported as a new virulence factor in Helicobacter pylori, but its association with gastroduodenal disorders and its mode of action are still unclear. Here, an association of the dupA status with different disease groups was determined and a biological explanation for the observed associations was tested. In total, 216 H. pylori isolates were obtained from 232 presumed H. pylori-infected patients. A positive association was observed between the occurrence of duodenal ulcer (DU) and the presence of dupA [odds ratio (OR) 24.2; 95 % confidence interval (CI) 10.6–54.8]. In addition, an inverse association between the occurrence of gastric cancer (GC) [OR 0.16; 95 % CI 0.05–0.47] and gastric ulcer (GU) [OR 0.34; 95 % CI 0.16–0.68] with the presence of dupA was observed. A putative explanation for the observed associations might be a more corpus-located infection (pan-gastritis) by the dupA-positive strains due to their increased acid resistance. Indeed, a strong association between dupA-positive H. pylori isolated from gastritis patients and in vitro acid resistance was observed (P<0.05). The observed higher acid resistance of the dupA-positive strains suggests that these strains are adapted to a stomach with high gastric acid output. This may in part explain the observed associations, as an increased gastric acid output is thought to be typical for an antrum-predominant H. pylori infection and, whilst this is associated with an increased risk of DU formation, it also decreases the risk for the genesis of GUs and GC.

Normal immunocompetent mice are not susceptible to non-adapted filoviruses. There are therefore two strategies available to establish a murine model of filovirus infection: adaptation of the virus to the host or the use of genetically modified mice that are susceptible to the virus. A number of knockout (KO) strains of mice with defects in either their adaptive or innate immunity are susceptible to non-adapted filoviruses. In this study, A129 α/β −/− interferon receptor-deficient KO mice, strain A129 IFN-α/β −/−, were used to determine the lethality of a range of filoviruses, including Lake Victoria marburgvirus (MARV), Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Reston ebolavirus (REBOV) and Côte d’Ivoire ebolavirus (CIEBOV), administered by using intraperitoneal (IP) or aerosol routes of infection. One hundred percent mortality was observed in all groups of KO mice that were administered with a range of challenge doses of MARV and ZEBOV by either IP or aerosol routes. Mean time to death for both routes was dose-dependent and ranged from 5.4 to 7.4 days in the IP injection challenge, and from 10.2 to 13 days in the aerosol challenge. The lethal dose (50 % tissue culture infective dose, TCID50) of ZEBOV for KO mice was <1 TCID50 ml−1 when administered by either the IP or aerosol route of infection; for MARV the lethal dose was <1 TCID50 ml−1 by the IP route of infection and <10 TCID50 ml−1 by the aerosol route. In contrast, there was no mortality after infection with SEBOV or REBOV by either IP or aerosol routes of infection; all the mice lost weight (~15 % loss of group mean body weight with SEBOV and ~7 % with REBOV) but recovered to their original weights by day 14 post-challenge. There was no mortality in mice administered with CIEBOV via the IP route of infection and no clinical signs of infection were observed. The progression of disease was faster following infection with ZEBOV than with MARV but ultimately both viruses caused widespread infection with high titres of the infectious viruses in multiple organs. Histopathological observations were consistent with other animal models and showed widespread organ damage. This study suggests that MARV and ZEBOV are more virulent when administered via the IP route rather than by aerosol infection, although both are highly virulent by either route. The KO mouse may provide a useful model to test potential antiviral therapeutics against wild-type filoviruses.

Ninety-one consecutive pneumococcal isolates (primarily from sputum), recovered in Chongqing Southwest Hospital during a 12 month period in 2009–2010 from individuals of all ages with suspected cases of pneumococcal disease, were subjected to PCR-serotyping, Quellung reaction serotyping, antimicrobial-susceptibility testing and multilocus sequence typing (MLST). Although 20 different serotypes were observed, most isolates (69, 75.8 %) were of serotypes included in the pneumococcal 13-valent conjugate vaccine (PCV13), including 33 of the 46 (71.7 %) isolates recovered from individuals less than 5 years of age. The prevalent serotypes were 19F (34 %), 19A (9.9 %), 6B (9.9 %), 23F (7.7 %), 14 (6.6 %) and 6A (4.4 %). PCR-determined serotypes were in agreement with Quellung testing, with the exception of two serotype 33C isolates. Most or all isolates within each PCV13 serotype were represented by one genotype, with the globally disseminated MLST sequence types (STs) ST271, ST320, ST90 and ST81 each accounting for the highly resistant isolates within serotypes 19F, 19A, 6B and 23F, respectively. Sixty-six (72.5 %) isolates were resistant to combinations of β-lactam antibiotics (BLAs). A total of 63 of these 66 (95.5 %) BLA-resistant isolates were of serotypes included in PCV13; however, 3 serogroup 15 isolates were also BLA-resistant. Most isolates (88/91 = 96.7 %) were resistant to erythromycin and clindamycin. The majority of isolates were also resistant to tetracycline (76, 84 %) and to cotrimoxazole (67, 74 %). This work revealed that the majority of antimicrobial-resistant isolates (50/91 = 54.9 %) recovered in this Chinese hospital were represented by four global clones. Serotypes for these as well as more obscure strains were readily determined by using PCR.

Clostridium difficile is the most common cause of antibiotic-associated diarrhoea worldwide. Over the past 10 years, the incidence and severity of disease have increased in North America and Europe due to the emergence of a hypervirulent clone designated PCR ribotype 027. In this study, we sought to identify phenotypic differences among a collection of 26 presumed PCR ribotype 027 strains from the US and the UK isolated between 1988 and 2008 and also re-evaluated the PCR ribotype. We demonstrated that some of the strains typed as BI by restriction endonuclease analysis, and presumed to be PCR ribotype 027, were in fact other PCR ribotypes such as 176, 198 and 244 due to slight variation in banding pattern compared to the 027 strains. The reassigned 176, 198 and 244 ribotype strains were isolated in the US between 2001 and 2004 and appeared to have evolved recently from the 027 lineage. In addition, the UK strains were more motile and more resistant to most of the antibiotics compared to the US counterparts. We conclude that there should be a heightened awareness of newly identified PCR ribotypes such as 176, 198 and 244, and that they may be as problematic as the notorious 027 strains.

Microsporum ferrugineum, an uncommon causative agent of dermatophytosis, has restricted endemicity. Iranian strains suspected to be M. ferrugineum from two patients with tinea were analysed using the rDNA internal transcribed spacer (ITS) region and the partial β-tubulin (BT2) and translation elongation factor 1-α (TEF1) genes. Strains were compared to reference strains to differentiate M. ferrugineum from its relatives Microsporum canis and Microsporum audouinii. Inter-species differences for TEF1 and BT2 were found to be higher than for the ITS region, which is the current molecular standard for species identification in dermatophytes. Intra-species variation was zero for each of the markers. In silico analysis showed that the restriction enzymes BanI and BshNI were together sufficient to differentiate the three species based on TEF1, whereas a two-step digestion was needed with BT2 or the ITS region. The prevalence of M. ferrugineum in clinical samples in Iran appeared to be higher than suspected on the basis of routine phenotypic identification.

Pseudomonas aeruginosa is one of the primary pathogens in the cystic fibrosis (CF) lung and a significant cause of morbidity and mortality. Reports of the spread of epidemic or transmissible strains of P. aeruginosa within and across CF centres in Europe have raised concern regarding the possibility of clonal spread among and within CF centres in Ireland. P. aeruginosa isolates (313 isolates from 142 sputum samples and 53 throat swabs) from 68 CF patients were examined using PFGE to explore the diversity of P. aeruginosa isolates among CF patients in a Dublin paediatric hospital. Only 57 different P. aeruginosa genotypes were identified among the 313 isolates. Forty-three of the genotypes were observed only in individual patients (distinct genotypes) while 13 cluster strains (present in two to four patients) were observed. Typing of P. aeruginosa isolates identified one indistinguishable clonal isolate of P. aeruginosa present in 13 CF patients (13/68; 19.1 %) which displayed higher levels of antibiotic resistance than those displayed by P. aeruginosa isolates of distinct genotype.