- Volume 58, Issue 11, 2009

The ability to characterize accurately the cause of infection is fundamental to effective treatment. The impact of any antimicrobial agents used to treat infection will, however, always be constrained by both the appropriateness of their use and our ability to determine their effectiveness. Traditional culture-based diagnostic microbiology is, in many cases, unable to provide this information. Molecular microbiological approaches that assess the content of clinical samples in a culture-independent manner promise to change dramatically the types of data that are obtained routinely from clinical samples. We argue that, in addition to the technical advance that these methodologies offer, a conceptual advance in the way that we reflect on the information generated is also required. Through the development of both of these advances, our understanding of infection, as well as the ways in which infections can be treated, may be improved. In the analysis of the microbiological content of certain clinical samples, such as blood, cerebrospinal fluid, brain and bone biopsy, culture-independent approaches have been well documented. Herein, we discuss how extensions to such studies can shape our understanding of infection at the many sites of the human body where a mixed flora, or in more ecological terms, a community of microbes, is present. To do this, we consider the underlying principles that underpin diagnostic systems, describe the ways in which these systems can be applied to community characterization, and discuss the significance of the data generated. We propose that at all locations within the human body where infection is routinely initiated within the context of a community of microbes, the same principles will apply. To consider this further, we take insights from areas such as the gut, oral cavity and skin. The main focus here is understanding respiratory tract infection, and specifically the infections of the cystic fibrosis lung. The impact that the use of culture-independent, molecular analyses will have on the way we approach the treatment of infections is also considered.

Corynebacterium diphtheriae still represents a global medical challenge, particularly due to the significant number of individuals susceptible to diphtheria and the emergence of non-toxigenic strains as the causative agents of invasive infections. In this study, we characterized the clinical and microbiological features of what we believe to be the first case of C. diphtheriae infection of a percutaneous nephrostomy catheter insertion site in an elderly patient with a fatal bladder cancer. Moreover, we demonstrated the potential role of adherence, biofilm formation and fibrin deposition traits in C. diphtheriae from the catheter-related infection. Non-toxigenic C. diphtheriae isolated from the purulent discharge (named strain BR-CAT5003748) was identified by the API Coryne system (code 1 010 324) and a multiplex PCR for detection of dtxR and tox genes. Strain BR-CAT5003748 showed resistance to oxacillin, ceftazidime and ciprofloxacin. In experiments performed in vitro, the catheter isolate was classified as moderately hydrophobic and as moderately adherent to polystyrene surfaces. Glass provided a more effective surface for biofilm formation than polystyrene. Micro-organisms adhered to (>1.5×106 c.f.u.) and multiplied on surfaces of polyurethane catheters. Microcolony formation (a hallmark of biofilm formation) and amorphous accretions were observed by scanning electron microscopy on both external and luminal catheter surfaces. Micro-organisms yielded simultaneous expression of localized adherence-like and aggregative-like (LAL/AAL) adherence patterns to HEp-2 cells. Interestingly, the coagulase tube test resulted in the formation of a thin layer of fibrin embedded in rabbit plasma by the non-toxigenic BR-CAT5003748 strain. In conclusion, C. diphtheriae should be recognized as a potential cause of catheter-related infections in at-risk populations such as elderly and cancer patients. LAL/AAL strains may be associated with virulence traits that enable C. diphtheriae to effectively produce biofilms on catheter surfaces. Biofilm formation and fibrin deposition could have contributed to the persistence of C. diphtheriae at the infected insertion site and the obstruction of the nephrostomy catheter.

The human nasopharynx is a major ecological niche for Haemophilus influenzae colonization. Establishment of infection is critically dependent on the persistence of bacteria in the nasopharynx. Various factors are presumed to mediate this persistence and the influence of biofilm formation has been under scrutiny for a long time. In a prospective population-based study, the nasopharyngeal colonization pattern of 250 children <2 years old was traced to gain further insights into the phenomenon. The association between biofilm formation and persistence was delineated by quantitative biofilm assay, while the true nature of biofilm formers was further evaluated by electron microscopy studies. H. influenzae isolates obtained in this study, when analysed by phenotypic and genotypic means, revealed a clonal distribution of strains within the population. The biofilm formation ability of the isolates was found to be significantly associated with bacterial persistence (P<0.001). The isolates having biofilm formation ability were found to be 7.1 times more likely to persist in the nasopharynx than non-biofilm formers. This study provides direct evidence indicating the intricate relationship between biofilm formation and the persistence of bacteria. Our results emphasize the need to evaluate the potential for biofilm formation before designing preventive and therapeutic strategies.

The slow growth of hepatitis A virus (HAV) in cell culture is one of the primary pitfalls in the development of sensitive and rapid methods for the detection and quantification of HAV and associated neutralizing antibodies. Currently, in vitro assays frequently require 8 days or more to detect and quantify the presence of HAV neutralizing antibodies. This study describes a rapid immunoassay that allowed the detection of anti-HAV neutralizing antibodies in only 3 days. This microplate-based enzymic assay may be applicable in virological diagnostics, in evaluating the immunogenicity of HAV vaccines and in quantifying neutralizing antibodies during the course of HAV infection.

Candida albicans is the species most frequently isolated from oral specimens, but the recovery of other Candida species such as Candida dubliniensis is increasing. Differentiation of C. dubliniensis from C. albicans requires special tests and both species are misidentified in some studies. CHROM-Pal (CH-P) is a novel chromogenic medium used in our laboratory for differentiation between C. albicans and C. dubliniensis on the basis of colony colour and morphology, and chlamydospore production. The performance of CH-P and CHROMagar Candida (CAC) was compared for primary isolation and presumptive identification of yeasts from oral specimens from human immunodeficiency virus (HIV)-infected and uninfected individuals. The identification of Candida species on both media was compared with two reference identification methods (API ID 32 C and multiplex PCR). A total of 137/205 oral swabs (66.8 %) plated onto CH-P and CAC media were positive by culture and resulted in the growth of 171 isolates of Candida species on CH-P, whilst only 159 isolates grew on CAC. C. albicans was the most frequently isolated species in both groups of patients, followed by Candida parapsilosis in the HIV-negative group, and by C. dubliniensis in the HIV-infected group. The other Candida species isolated were Candida guilliermondii, Candida glabrata, Candida krusei, Candida tropicalis, Candida famata, Candida rugosa, Candida kefyr, Candida pelliculosa and Candida pulcherrima. The sensitivity and specificity for identifying C. albicans, C. krusei, C. tropicalis and C. dubliniensis on CH-P were over 98.5 %, always equal to or higher than those obtained when CAC was used. CH-P is a simple reliable medium for primary isolation and presumptive identification of yeast isolates from oral samples. The ability of CH-P to discriminate between C. dubliniensis and C. albicans was significantly higher (P <0.05) than that of CAC.

Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53×102 copies μl−1. Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli has spread rapidly worldwide and poses a serious threat to human and animal health. This study collected 51 non-replicate E. coli isolates from 14 different chicken farms in Henan Province in China from December 2007 to August 2008. The prevalence of ESBL-producing E. coli, molecular characterization of the ESBL-related bla genes, including bla TEM, bla SHV and bla CTX-M, and the susceptibilities of these bacteria to various antimicrobial agents were determined. Thirty-one of the 51 isolates were positive for an ESBL phenotype and 29 of these isolates carried one or more bla genes. Twenty-two isolates harboured bla TEM genes and 15 isolates carried bla CTX-M genes (one CTX-M-14, three CTX-M-24 and 11 CTX-M-65). One isolate carried bla TEM -57; the remaining bla TEM isolates carried bla TEM-1 with one silent nucleotide base variation (T18C). We believe that this is the first study to report TEM-57 in E. coli isolates. All isolates harbouring bla CTX-M-24 and bla CTX-M-14 and five of the bla CTX-M-65 isolates also harboured the bla TEM-1 gene. To our knowledge, this study is the first to describe detection of CTX-M-65-producing E. coli isolated from chickens. None of the isolates contained the bla SHV gene. Conjugation experiments demonstrated that bla CTX-M and bla TEM genes could be transferred to E. coli DH5α. The results indicate that ESBL frequency has reached an alarming level in chicken isolates in China, with TEM-1 and CTX-M-65 enzymes being the two predominant β-lactamases detected.

The composition and antifungal activity of clove essential oil (EO), obtained from Syzygium aromaticum, were studied. Clove oil was obtained commercially and analysed by GC and GC-MS. The EO analysed showed a high content of eugenol (85.3 %). MICs, determined according to Clinical and Laboratory Standards Institute protocols, and minimum fungicidal concentration were used to evaluate the antifungal activity of the clove oil and its main component, eugenol, against Candida, Aspergillus and dermatophyte clinical and American Type Culture Collection strains. The EO and eugenol showed inhibitory activity against all the tested strains. To clarify its mechanism of action on yeasts and filamentous fungi, flow cytometric and inhibition of ergosterol synthesis studies were performed. Propidium iodide rapidly penetrated the majority of the yeast cells when the cells were treated with concentrations just over the MICs, meaning that the fungicidal effect resulted from an extensive lesion of the cell membrane. Clove oil and eugenol also caused a considerable reduction in the quantity of ergosterol, a specific fungal cell membrane component. Germ tube formation by Candida albicans was completely or almost completely inhibited by oil and eugenol concentrations below the MIC values. The present study indicates that clove oil and eugenol have considerable antifungal activity against clinically relevant fungi, including fluconazole-resistant strains, deserving further investigation for clinical application in the treatment of fungal infections.

The emergence of multidrug-resistant (MDR) and extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae poses a serious antibiotic management problem as resistance genes are easily transferred from one organism to another. Fifty-one strains of K. pneumoniae isolated from sporadic cases in various hospitals throughout Malaysia were analysed by antimicrobial susceptibility testing, PCR detection of ESBL-encoding genes and DNA fingerprinting. Although 27 of the 51 K. pneumoniae strains were MDR (i.e. resistant to three or more classes of antibiotics), the majority of the strains (98 %) were sensitive to imipenem. PCR detection using ESBL gene-specific primers showed that 46 of the K. pneumoniae strains harboured bla SHV, 19 harboured bla CTX-M, 5 harboured bla OXA-1 and 4 harboured bla TEM-1. Class 1 integron-encoded intI1 integrase was detected in 21 of the 51 K. pneumoniae strains and amplification of the integron 5′CS region showed the presence of several known antibiotic resistance gene cassettes of various sizes. Results of conjugation and transformation experiments indicated that some of the ESBL-encoding genes (i.e. bla SHV, bla CTX-M and bla TEM-1) were transmissible and were likely plasmid-encoded. DNA fingerprinting using PFGE and PCR-based methods indicated that the 51 K. pneumoniae strains were genetically diverse and heterogeneous.

Xylitol is a sugar alcohol that inhibits the growth and adherence of Streptococcus pneumoniae. In clinical trials, xylitol has been shown to decrease the occurrence of acute otitis media in day-care children but did not decrease nasopharyngeal carriage of the pneumococci. It has also been shown that xylitol affects the ultrastructure of the pneumococcal capsule. Here, it was hypothesized that xylitol might affect the expression of pneumococcal capsular genes. Capsule gene expression levels were studied in 24 clinical pneumococcal isolates and one ATCC strain (49619) by using a real-time RT-PCR method targeting the mRNA of the second gene of the pneumococcal capsular locus, the cpsB gene. The isolates were exposed to 5 % glucose, 5 % xylitol and control medium (brain heart infusion medium containing 10 % fetal bovine serum) for 2 h. cpsB gene expression levels were measured by using a relative quantification method with calibrator normalization where the 16S rRNA gene of pneumococcus was used as a reference. Exposure to xylitol lowered cpsB gene expression levels significantly compared with those in the control (P=0.035) and glucose (P=0.011) media. This finding supports previous results where exposure to xylitol changed the ultrastructure of the pneumococcal capsule and could explain further the high clinical efficacy of xylitol in preventing otitis media.

A high intermittent dose regimen (group A: 10 mg kg−1 on day 1, 5 mg kg−1 on days 3 and 6) was compared with standard dosing (group B: 3 mg kg−1 per day for 14 days) of liposomal amphotericin B (LAB) for empirical treatment of persistent febrile neutropenia. A total cumulative dose of 1275 mg (group A) and 2800 mg (group B) was administered. Infusion-related adverse drug events, mainly rigors/chills, occurred more frequently with group A (11/45, 24 % infusions) than with group B (12/201, 6 % infusions) (P=0.002), which extended the mean infusion time by 20 min (P=0.001). Creatinine levels were similar in the two regimens: the A : B ratio of the area under the curve for creatinine (AUCCREATININE) for days 2–7 was 1.09 (P=0.27) and for days 2–14 was 1.05 (P=0.51). Rises in creatinine were mild (clinical toxicity criteria 1) in all patients with elevations. Hypokalaemia tended to be less severe in group A with a lower proportion of hypokalaemic days [57/143 (39 %) vs 80/137 (58 %), P=0.21], a higher AUCPOTASSIUM (A : B ratio of 1.06, P=0.12), a lower proportion of patients with hypokalaemia at the end of study (10 vs 61 %, P=0.01) and fewer potassium-supplemented days [12/210 (6 %) vs 41/210 (19.5 %), P<0.1]. There were mildly elevated median levels of serum bilirubin, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, which were similar for the two regimens and were usually associated with other co-existing co-morbid conditions. The AUC for these enzymes was also similar in the two groups. No patient had discontinuation of the study drug due to toxicity. Composite success was identical for each regimen (11/15 patients, 73 %). Three of the fifteen patients in group B and none in group A developed invasive fungal infections (IFIs). β-d-Glucan levels were similar in both groups for patients without an IFI [AUCGLUCAN of 362 and 683 (P=0.36) for groups A and B, respectively]. The rate of defervescence was similar for each regimen (P=0.75). This feasibility study suggests that a short intermittent high-dose course of 10/5/5 mg LAB kg−1 on days 1, 3 and 6 may be as safe and effective as a standard 14 day course of 3 mg kg−1 per day, with drug-acquisition cost savings and reduced drug exposure. A larger study is indicated for confirmation of this.

Shigella sonnei infection in industrialized countries is often associated with foreign travel. A total of 195 S. sonnei isolates in Japan, isolated from cases associated with foreign travel, were analysed by biotyping and molecular typing using PFGE and multilocus variable-number tandem-repeat analysis (MLVA); their antimicrobial susceptibilities were also evaluated. The isolates were from 26 countries, most of which were Asian. Molecular typing revealed a correlation among the genotypes, biotypes and their geographical areas of origin. The isolates were classified into two biotypes, a and g. Biotype g isolates (n=178) were further divided into distinct clusters mainly on the basis of their geographical areas of origin by both PFGE and MLVA. Isolates from South Asian countries constituted one of the distinct clusters. Biotype g isolates from countries other than South Asia constituted other distinct clusters. Most of the isolates from other countries and continents, excluding the South Asian countries, were included in one major cluster by PFGE analysis. However, by MLVA, they were further divided into minor subclusters mainly on the basis of their countries of origin. MLVA was also demonstrated to be useful in molecular epidemiological analysis, even when only seven loci were applied, resulting in a high resolution with Simpson's index of diversity (D) of 0.993. A core drug-resistance pattern of streptomycin, sulfisoxazole, tetracycline and trimethoprim–sulfamethoxazole was observed in 108 isolates, irrespective of their geographical areas of origin, but the frequency of resistance to nalidixic acid was high among the South Asian and East Asian isolates. Two isolates from China and India were resistant to cefotaxime and harboured the bla CTX-M-14 and bla CTX-M-15 genes, respectively; these isolates were also resistant to nalidixic acid, which is a matter of concern in terms of shigellosis treatment. Use of a combination of methods was found to be effective for epidemiological investigation in the case of S. sonnei infection.

Since 1996, Malta has experienced an upsurge of invasive meningococcal disease (IMD) following an almost 30 year period with a negligible number of annually reported cases. We reviewed the 233 IMD cases notified during a 14 year period (1994–2007), and analysed epidemiological and laboratory surveillance data. The crude incidence per 100 000 inhabitants peaked in 2000 at 8.1 [95 % confidence interval (CI) 5.7–11.6] and again in 2006 at 8.9 (95 % CI 6.4–12.4), thereby placing Malta amongst the countries with the highest incidence of the disease in Europe. Of the total cases, 137 (59 %) were confirmed and 30 (13 %) were classified as probable. However, 66 cases (28 %) had no laboratory evidence of the disease and were classified as possible. Information on the serogroup was available for 114 cases. Serogroup B formed the largest proportion (76 %, n=87) followed by serogroup C (16 %, n=18). B : 4 : P1.19,15 strains (n=46) predominated throughout the study period since their first identification in 1998. With 28 deaths attributed to IMD, the overall case fatality rate was 12 %. Apart from stressing the importance of maintaining high vigilance for IMD, our findings underscore the importance of enhancing laboratory surveillance of the disease, including characterization of the meningococci. Until vaccines against a broad range of serogroup B meningococci become available for universal use, the main methods of control remain the early treatment of cases and the prevention of secondary cases.

A prolonged outbreak of carbapenem-resistant Acinetobacter baumannii in a German university medical centre in 2006 was investigated; the investigation included a descriptive epidemiological analysis, a case–control study, environmental sampling, molecular typing of A. baumannii isolates using PFGE and repetitive-sequence-based PCR (rep-PCR) typing, and detection of OXA-type carbapenemases by multiplex PCR. Thirty-two patients acquired the outbreak strain in five intensive care units (ICUs) and two regular wards at a tertiary care hospital within 10 months. The outbreak strain was resistant to penicillins, cephalosporins, ciprofloxacin, gentamicin, tobramycin, imipenem and meropenem, and carried the bla OXA-23-like gene. Based on PFGE and rep-PCR typing, it was shown to be related to the pan-European A. baumannii clone II. The most likely mode of transmission was cross-transmission from colonized or infected patients via the hands of health-care workers, with the severity of disease and intensity of care (therapeutic intervention scoring system 28 score >median) being independently associated with acquisition of the outbreak strain (odds ratio 6.67, 95 % confidence interval 1.55–36.56). Control of the outbreak was achieved by enforcement of standard precautions, education of personnel, screening of ICU patients for carbapenem-resistant A. baumannii and cohorting of patients. This is believed to be the first report of an outbreak of A. baumannii carrying the carbapenemase OXA-23 in Germany.

Most cases of Streptococcus suis infection in humans are caused by serotype 2 strains, and only a few cases caused by other serotypes have been reported. Among 177 human isolates of S. suis in Thailand, 12 (6.8 %) were identified as being of serotype 14, and an occurrence of sporadic S. suis serotype 14 infection was noted during 2006–2008, particularly in northern Thailand. Clinical presentations of the 12 patients (median age 62.9 years) included meningitis (58.3 %), septic arthritis (25 %) and sepsis (16.7 %). These clinical features were similar to those previously reported for S. suis infections, except that there were no fatal cases. All of the 12 serotype 14 strains belonged to the multilocus sequence types (ST) 105 (n=11) and the novel ST127 (n=1). Molecular typing by PFGE revealed four different pulsotypes, including an identical pattern for nine ST105 strains and three closely related patterns for two ST105 strains and one ST127 strain. Our PFGE data suggested clonal dissemination of ST105 strains in Thailand. Because serotype 14 is becoming a more common cause of S. suis infections in humans, diagnostic tests for serotype 14 should be performed in South-East Asian countries.

The genus Roseomonas comprises groups of slow-growing, Gram-negative coccobacilli, which only infrequently cause infection in humans. When identified, they are associated with immunocompromised adults, often causing bacteraemia. Due to their rarity, members of this genus can be overlooked or misidentified using automated laboratory identification systems. We report on an immunocompetent adolescent patient who developed septic arthritis due to Roseomonas gilardii following surgery for a sports injury. The isolate was initially misidentified as Bordetella bronchiseptica using the Vitek 2 system, but confirmed as R. gilardii based on 16S rRNA gene sequencing. To the best of our knowledge, this is the first case of a healthy paediatric patient with septic arthritis due to R. gilardii.

Keratitis with endophthalmitis of the right eye occurred in a 78-year-female following a complicated cataract surgery. Prompt intravitreal vancomycin and ceftazidime with topical fortified tobramycin and cefazolin treatments were started. The corneal, aqueous and vitreous cultures grew a Burkholderia cepacia complex (Bcc) strain on the fourth day. Restriction fragment length polymorphism analysis of the recA amplicon revealed B. cepacia genomovar I. The organism was found to be susceptible to ceftazidime, ciprofloxacin and ofloxacin. Topical ciprofloxacin was given immediately. At day 10, the pain relieved and the clinical condition of the patient improved with resolution of the purulent discharge, severe circumcorneal congestion and chemosis. The size of the corneal abscess and anterior chamber exudation decreased. The Bcc should be included among the bacterial species that may cause keratitis following intraocular surgeries.

We report a case of monomicrobial necrotizing fasciitis caused by hypermucoviscous Klebsiella pneumoniae in an immunocompromised white male after travel to China. The K. pneumoniae isolate belonged to the K2 serotype, and carried the virulence factors RmpA and aerobactin. To the best of our knowledge this is the first report of necrotizing fasciitis caused by hypermucoviscous K. pneumoniae resembling the highly virulent K. pneumoniae isolates associated with liver abscess syndrome in Asia.