- Volume 63, Issue 11, 2014

Infectious gastrointestinal disease is caused by a diverse array of pathogens, and is a challenging syndrome to correctly diagnose and manage. Conventional laboratory diagnostic methods are often time-consuming and frequently suffer from low detection rates. Two commercial multiplex nucleic acid amplification tests [Luminex xTAG Gastrointestinal Pathogen Panel (GPP) and Savyon Diagnostics Gastrointestinal Infection Panel (GIP)] were applied to 1000 stored diarrhoeal clinical stool samples. The Luminex xTAG GPP and Savyon GIP detected Campylobacter in 42/44 and 44/44 culture-positive samples, Salmonella in 4/4 and 3/4 culture-positive samples, Shigella in 1/1 culture-positive sample, Clostridium difficile toxin in 32/35 ELISA-positive samples, and Giardia in 6/6 wet-preparation-microscopy-positive samples, respectively. When the Luminex GPP assay was used concurrently with conventional methods for 472 clinical samples, it detected Campylobacter in 22/22 culture-positive samples, Salmonella in 1/1 culture-positive sample, Clostridium difficile toxin in 14/14 ELISA-positive samples and Giardia in 4/4 wet-preparation-microscopy-positive samples. The pathogen/toxin detection rate for conventional methods in both sample sets was <10 %. The Luminex xTAG GPP detection rate was 24.8 % in the stored samples and 32.6 % in the concurrently tested samples. The Savyon GIP detection rate was 22.5 %. From stored samples, 2.4 % of Luminex xTAG GPP detections and 3.1 % of Savyon GIP detections could not be confirmed using alternative nucleic acid amplification tests. Enhanced detection rates resulted from increased detection of pathogens routinely sought using conventional methods and were also due to ascertainment of micro-organisms that current testing strategies do not diagnose. Use of multiplex nucleic acid amplification tests will allow clinical laboratories to diagnose infectious gastroenteritis in more patients with diarrhoeal disease by increasing the sensitivity of pathogen detection and by reducing the selective bias of current strategies. The clinical and economic impact of these results warrants further investigation.

The aim of this study was to compare the performance of serological versus molecular typing methods to detect capsular polysaccharide (CP) and surface-associated polysaccharide antigen 336 phenotypes of Staphylococcus aureus isolates. Molecular typing of CP types 1, 5 and 8 was carried out using PCR, whereas serological typing of CP1, 2, 5, 8 and antigen 336 was carried out by slide agglutination using specific antisera. By genotyping, 14/31 strains were CP8 positive, 12/31 strains were CP5 and the remaining 6/31 isolates were non-typable (NT). One isolate was positive for both CP5 and CP8 by PCR, but was confirmed as CP8 type serologically. Detection of CP2 and type 336 by PCR was not possible because specific primers were either not available or non-specific. Using serotyping, 14/31 strains were CP8 positive, 11/31 CP5 positive and 2/31 positive for antigen 336. The remaining four S. aureus isolates were serologically NT. However, three of four NT and two 336-positive S. aureus isolates were encapsulated as determined by light microscopy after capsular staining. This discovery was surprising and warrants further investigations on the identification and characterization of additional capsular phenotypes prevalent among S. aureus clinical isolates. It was concluded that serological typing was a better method than molecular typing for use in epidemiological investigations based upon the distribution of surface-associated polysaccharide antigens-based phenotypes.

Detection of latent Mycobacterium tuberculosis is a challenge in the diagnosis of asymptomatic, subclinical tuberculosis. We report the development of an immunofluorescence technique to visualize and enumerate M. tuberculosis in latently infected rabbit lungs where no acid-fast–stained organisms were seen and no cultivable bacilli were obtained by the agar-plating method.

Vulvovaginal candidiasis (VVC) is one of the most common causes of vaginitis and affects about 75 % of women of reproductive age. In order to better understand the epidemiology and pathogenesis of this disease, we evaluated genetic relatedness among 62 clinical isolates of Candida albicans sequentially obtained from the anus and vagina of patients with sporadic and recurrent VVC. Evaluation of patients’ demographic and clinical data, direct examination, and colony forming units (c.f.u.) counts of vaginal and anal samples were also performed. The genotypes of strains were determined with ABC genotyping and Randomly Amplified Polymorphic DNA (RAPD). Genotype A was the most prevalent (93.6 %), followed by genotype C (6.4 %), whereas genotype B was not found. We found the maintenance of the same ABC genotype, regardless of the body site of each patient. Most of the vaginal strains suffered microevolution, whereas most of the anal strains were replaced during the period of study. Vaginal and anal isolates of C. albicans obtained simultaneously from the same patient showed the same ABC genotype and high genetic similarity as determined by RAPD. Genotype A seemed to be dominant in both vaginal and anal isolates of patients with VVC. Our results corroborate the hypothesis that there are ‘substrains’ of the C. albicans vaginal clone successfully established, which dominate in an apparently random manner over the course of time. It is suggested that the anal reservoir constitutes a possible source for vaginal infection in most of the cases.

Efflux is the most common mechanism of tetracycline resistance. Class A tetracycline efflux pumps, which often have high prevalence in Enterobacteriaceae, are encoded by tet(A) and tet(A)-1 genes. These genes have two potential start codons, GTG and ATG, located upstream of the genes. The purpose of this study was to determine the start codon(s) of the class A tetracycline resistance (tet) determinants tet(A) and tet(A)-1, and the tetracycline resistance level they mediated. Conjugation, transformation and cloning experiments were performed and the genetic environment of tet(A)-1 was analysed. The start codons in class A tet determinants were investigated by site-directed mutagenesis of ATG and GTG, the putative translation initiation codons. High-level tetracycline resistance was transferred from the clinical strain of Klebsiella pneumoniae 10-148 containing tet(A)-1 plasmid pHS27 to Escherichia coli J53 by conjugation. The transformants harbouring recombinant plasmids that carried tet(A) or tet(A)-1 exhibited tetracycline MICs of 256–512 µg ml−1, with or without tetR(A). Once the ATG was mutated to a non-start codon, the tetracycline MICs were not changed, while the tetracycline MICs decreased from 512 to 64 µg ml−1 following GTG mutation, and to ≤4 µg ml−1 following mutation of both GTG and ATG. It was presumed that class A tet determinants had two start codons, which are the primary start codon GTG and secondary start codon ATG. Accordingly, two putative promoters were predicted. In conclusion, class A tet determinants can confer high-level tetracycline resistance and have two start codons.

Carbapenem-resistant Acinetobacter baumannii (CR-AB) represents a major health-care problem, causing high rates of morbidity and mortality. This study investigated the clonality of CR-AB isolated from diabetic patients from different regions in Saudi Arabia, as well as the relatedness of the β-lactamase genes. A total of 64 non-repetitive CR-AB clinical isolates were collected from 16 different regions in Saudi Arabia from intensive care patients. Isolates were identified phenotypically by the Vitek 2 compact system and genotypically by amplification of the bla OXA-51-like gene. The target sequences were amplified by PCR and the clonal diversity of the isolates was explored by PFGE. Resistance studies revealed that the prevalence of imipenem and meropenem resistance was 92 % and 96 %, respectively, while the vast majority of the isolates were susceptible to tigecycline and colistin. In addition, bla VIM and bla OXA-23 were the most prevalent genes in the isolates under investigation, while ISAba1 was the most dominant insertion sequence. PFGE results showed 13 clusters; clone H was dominant, comprising 20 isolates from four hospitals, followed by clones C and F, comprising 11 isolates each from three and six hospitals, respectively. Moreover, the current study signified the clonal diversity of CR-AB in Saudi Arabia and showed the ability of some clones to infect patients in many different cities.

Fungaemia caused by Malassezia spp. in hospitalized patients requires prompt and appropriate therapy, but standard methods for the definition of the in vitro antifungal susceptibility have not been established yet. In this study, the in vitro susceptibility of Malassezia furfur from bloodstream infections (BSIs) to amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC), posaconazole (POS) and voriconazole (VRC) was assessed using the broth microdilution (BMD) method of the Clinical and Laboratory Standards Institute (CLSI) with different media such as modified Sabouraud dextrose broth (SDB), RPMI and Christensen’s urea broth (CUB). Optimal broth media that allow sufficient growth of M. furfur, and produce reliable and reproducible MICs using the CLSI BMD protocol were assessed. Thirty-six M. furfur isolates collected from BSIs of patients before and during AMB therapy, and receiving FLC prophylaxis, were tested. A good growth of M. furfur was observed in RPMI, CUB and SDB at 32 °C for 48 and 72 h. No statistically significant differences were detected between the MIC values registered after 48 and 72 h incubation. ITC, POS and VRC displayed lower MICs than FLC and AMB. These last two antifungal drugs showed higher and lower MICs, respectively, when the isolates were tested in SDB. SDB is the only medium in which it is possible to detect isolates with high FLC MICs in patients receiving FLC prophylaxis. A large number of isolates showed high AMB MIC values regardless of the media used. In conclusion, SDB might be suitable to determine triazole susceptibility. However, the media, the drug formulation or the breakpoints herein applied might not be useful for assessing the AMB susceptibility of M. furfur from BSIs.

Arbidol (ARB) is an antiviral drug that has broad-spectrum activity against a number of viral infections. To date, there are no specific data regarding its effects against a herpesvirus. Here, the in vitro antiviral effect of ARB and structurally related derivatives were evaluated in HaCat cells on different steps of herpes simplex virus type 1 replication: adsorption, entry and post-entry. The simplified pyrrolidine analogue, 9a2, showed the best antiviral activity in vitro by reducing the plaque numbers by about 50 % instead of 42 % obtained with ARB at the same concentration. Furthermore, we have reported that all tested compounds evaluated for their immunomodulatory activity showed the ability to reduce the viral proteins VP16 and ICP27 and to modify the virus-induced cytokine expression, allowing the host cell a more efficient antiviral response.

This study examined invasive Neisseria meningitidis recovered from invasive meningococcal disease (IMD) cases in Western Canada between 2009 and 2013. A total of 161 isolates from individual IMD cases were analysed for serogroup, serotype, serosubtype, PorA genotype, multi-locus sequence type and nucleotide sequence of their 4CMenB vaccine antigen genes. Sixty-nine isolates were serogroup B (MenB), 47 were serogroup Y (MenY), 22 were serogroup C (MenC), 19 were serogroup W (MenW), three were serogroup E and one was non-encapsulated. MenC, MenY and MenW were mainly clonal, represented primarily by clonal complex (cc) 11, cc23 or cc167, and cc22, respectively. In contrast, MenB were composed of eight different ccs together with 11 isolates not assigned to any known cc. Antigenic analysis and PorA genotyping confirmed the heterogeneity of MenB isolates, while such results supported the clonal nature of most MenC, MenY and MenW isolates. Thirty-four (21.1 %) isolates had at least one gene that encoded one matching vaccine protein component of the 4CMenB vaccine (i.e. PorA P1.4; fHbp variant 1.1; NHBA peptide 2; and NadA-1, -2, or -3). An additional 18 isolates had genes that encoded variant 1 or subfamily B factor H binding proteins of this same vaccine.

The catheterized urinary tract provides ideal conditions for the development of biofilm populations. Catheter-associated urinary tract infections (CAUTIs) are recalcitrant to existing antimicrobial treatments; therefore, established biofilms are not eradicated completely after treatment and surviving biofilm cells will carry on the infection. Cis-2-decenoic acid (CDA), an unsaturated fatty acid, is capable of inhibiting biofilm formation by Pseudomonas aeruginosa and of inducing the dispersion of established biofilms by multiple types of micro-organisms. Here, the ability of CDA to induce dispersal in pre-established single- and dual-species biofilms formed by Escherichia coli and Klebsiella pneumoniae was measured by using both semi-batch and continuous cultures bioassays. Removal of the biofilms by combined CDA and antibiotics (ciprofloxacin or ampicillin) was evaluated using microtitre plate assays (crystal violet staining). The c.f.u. counts were determined to assess the potential of combined CDA treatments to kill and eradicate pre-established biofilms formed on catheters. The effects of combined CDA treatments on biofilm surface area and bacteria viability were evaluated using fluorescence microscopy, digital image analysis and live/dead staining. To investigate the ability of CDA to prevent biofilm formation, single and mixed cultures were grown in the presence and absence of CDA. Treatment of pre-established biofilms with only 310 nM CDA resulted in at least threefold increase in the number of planktonic cells in all cultures tested. Whilst none of the antibiotics alone exerted a significant effect on c.f.u. counts and percentage of surface area covered by the biofilms, combined CDA treatments led to at least a 78 % reduction in biofilm biomass in all cases. Moreover, most of the biofilm cells remaining on the surface were killed by antibiotics. The addition of 310 nM CDA significantly prevented biofilm formation by the tested micro-organisms, even within mixed cultures, indicating the ability of CDA to inhibit biofilm formation by other types of bacteria in addition to Pseudomonas aeruginosa. These findings suggested that the biofilm-preventive characteristics of CDA make it a noble candidate for inhibition of biofilm-associated infections such as CAUTIs, which paves the way toward developing new strategies to control biofilms in clinical as well as industrial settings.

A series of clinical isolates of drug-resistant (DR) Acinetobacter baumannii with diverse drug susceptibility was detected from eight patients in the emergency intensive care unit of Tokai University Hospital. The initial isolate was obtained in March 2010 (A. baumannii Tokai strain 1); subsequently, seven isolates were obtained from patients (A. baumannii Tokai strains 2–8) and one isolate was obtained from an air-fluidized bed used by five of the patients during the 3 months from August to November 2011. The isolates were classified into three types of antimicrobial drug resistance patterns (RRR, SRR and SSR) according to their susceptibility (S) or resistance (R) to imipenem, amikacin and ciprofloxacin, respectively. Genotyping of these isolates by multilocus sequence typing revealed one sequence type, ST208, whilst that by a DiversiLab analysis revealed two subtypes. All the isolates were positive for bla OXA-51-like and bla OXA-66, as assessed by PCR and DNA sequencing. A. baumannii Tokai strains 1–8 and 10 (RRR, SRR and SSR) had quinolone resistance-associated mutations in gyrA/parC, as revealed by DNA sequencing. The ISAba1 upstream of bla OXA-51-like and aminoglycoside resistance-associated gene, armA, were detected in A. baumannii Tokai strains 1–7 and 10 (RRR and SRR) as assessed by PCR. Among the genes encoding resistance–nodulation–division family pumps (adeB, adeG and adeJ) and outer-membrane porins (oprD and carO), overexpression of adeB and adeJ and suppression of oprD and carO were seen in isolates of A. baumannii Tokai strain 2 (RRR), as assessed by real-time PCR. Thus, the molecular characterization of a series of isolates of DR A. baumannii revealed the outbreak of ST208 and diverse antimicrobial drug susceptibilities, which almost correlated with differential gene alterations responsible for each type of drug resistance.

Stenotrophomonas maltophilia is an important multidrug-resistant nosocomial pathogen associated with high mortality. Our aim was to examine antimicrobial susceptibility, biofilm production and clonal relatedness of clinical isolates of S. maltophilia. S. maltophilia isolates were collected between 2006 and 2013 from two tertiary care hospitals in Mexico. Antimicrobial susceptibility was evaluated by the broth microdilution method. PCR was used to determine the presence of β-lactamase genes L1 and L2. Biofilm formation was assessed with crystal violet staining. Clonal relatedness was determined by PFGE. Among the 119 collected S. maltophilia isolates, 73 (61.3 %) were from the respiratory tract. Resistance levels exceeded 75 % for imipenem, meropenem, ampicillin, aztreonam, gentamicin and tobramycin. Resistance to trimethoprim-sulfamethoxazole was 32.8 %. L1 and L2 genes were detected in 77.1 % (91/118) and 66.9 % (79/118) of isolates, respectively. All S. maltophilia strains were able to produce biofilms. Strains were classified as weak (47.9 %, 57/119), moderate (38.7 %, 46/119), or strong (13.4 %, 16/119) biofilm producers. A total of 89 distinct PFGE types were identified and 21.6 % (22/102) of the isolates were distributed in nine clusters. This is the first study in Mexico to reveal characteristics of clinical isolates of S. maltophilia. Clonal diversity data indicate low cross-transmission of S. maltophilia in a hospital setting. The high antibiotic resistance underscores the need for continuous surveillance of S. maltophilia in hospital settings in Mexico.

From 2007 to 2009, we collected a total of 83 bacteraemic isolates of Escherichia coli with reduced susceptibility or resistance to third-generation cephalosporins (TGCs). Isolates were genotyped by PFGE and multilocus sequence typing (MLST). The PFGE patterns revealed two highly correlated clusters (cluster E: nine isolates; cluster G: 22 isolates) associated with this prolonged clonal spreading. Compared with cluster E isolates, cluster G isolates were significantly more likely to harbour aac(6')-Ib-cr (P<0.05), and most of these isolates were isolated during a later year than cluster E isolates (P<0.05). By MLST analysis, 94 % of cluster E and G isolates (29/31) were ST68. Although no time or space clustering could be identified by the conventional hospital-acquired infection monitoring system, E. coli cases caused by cluster E and G isolates were significantly associated with having stayed in our hospital’s respiratory care ward (P<0.05). Isolates obtained from patients who had stayed in the respiratory care ward had a significantly higher rate of aac(6')-Ib-cr and bla CTX-M-14 positivity, and were more likely to belong to ST68/S68-like (all P<0.05). To our knowledge, this is the first report of prolonged clonal spreading caused by E. coli ST68 associated with a stay in a long-term care facility. Using epidemiological investigations and PFGE and MLST analyses, we have identified long-term clonal spreading caused by E. coli ST68, with extra antimicrobial-resistance genes possibly acquired during the prolonged spreading period.

Hypervirulent Clostridium difficile strains, most notably BI/NAP1/027, have been increasingly emerging in Western countries as local epidemics. We performed a prospective multicentre observational study from December 2011 to May 2012 to identify recent incidences of toxigenic culture-confirmed hospital-onset C. difficile infections (CDI) and their associated clinical characteristics in South Korea. Patients suspected of having been suffering from CDI more than 48 h after admission and aged ≥20 years were prospectively enrolled and provided loose stool specimens. Toxigenic C. difficile culture (anaerobic culture+toxin A/B/binary gene PCR) and PCR ribotyping were performed in one central laboratory. We enrolled 98 toxigenic culture-confirmed CDI-infected patients and 250 toxigenic culture-negative participants from three hospitals. The incidence of toxigenic culture-confirmed hospital-onset CDI cases was 2.7 per 10 000 patient-days. The percentage of severe CDI cases was relatively low at only 3.1 %. UK ribotype 018 was the predominant type (48.1 %). There were no hypervirulent BI/NAP1/027 isolates identified. The independent risk factors for toxigenic culture-confirmed hospital-onset CDI were invasive procedure (odds ratio (OR) 7.3, P = 0.003) and past CDI history within 3 months (OR 28.5, P = 0.003). In conclusion, the incidence and severity of CDI in our study were not higher than reported in Western countries.