- Volume 60, Issue 7, 2011

We investigated interactions of Aeromonas caviae, Aeromonas veronii biotype sobria and Aeromonas hydrophila strains, isolated from faecal specimens of humans with gastroenteritis, with HT29 intestinal epithelial cells. All strains were found to be cytotoxic to the cells. Bacterial infection caused generation of reactive oxygen species (ROS) and nitric oxide radical (NO·). The maximal levels of ROS and NO· were 14 and 35 times, respectively, greater in cells infected with Aeromonas spp. than in those incubated with non-pathogenic Escherichia coli. The cells incubated with cytolytic enterotoxin isolated from A. veronii biotype sobria induced the highest level of ROS and caused the highest cytotoxicity. We observed that increased accumulation of intracellular ROS leads to a loss of mitochondrial membrane potential (ΔΨm). Analyses of cellular morphology and DNA fragmentation revealed characteristic features of cells undergoing apoptosis. The process was dependent on the activation of caspases, and was completely blocked by the pan-caspase inhibitor z-VAD-fmk. Treatment of infected HT29 cells with three distinct antioxidants prevented intracellular ROS production, mitochondrial damage and apoptosis. The Pearson linear test revealed positive correlations between apoptotic index at 24 h and percentage cytotoxicity, ROS production, NO· production and loss of ΔΨm. This study has provided new insights into the mechanisms contributing to the development of Aeromonas-associated gastroenteritis. The results indicate that bacteria-induced apoptosis of epithelial cells results from mitochondrial depolarization due to oxidative stress.

The implementation of widespread unselected screening for Neisseria gonorrhoeae in England, using nucleic acid amplification tests (NAATs), has raised concerns regarding the potential increase in misdiagnoses. To increase the positive predictive value, confirmatory testing of positive specimens has been recommended; however, in practice this can be difficult to perform. This study examined the role of two different testing strategies for confirming the N. gonorrhoeae status of specimens that had been examined by the ProbeTec Strand Displacement Amplification (SDA) assay (Becton Dickinson). A total of 227 residual clinical specimens in SDA assay collection tubes were sent for confirmatory testing using two different testing approaches: (i) examination using two in-house real-time PCR assays (opa and porA pseudogene) and (ii) examination using the APTIMA Combo 2 (AC2) assay and the APTIMA Monospecific N. gonorrhoeae (AGC) tests (Gen-Probe). Of the 113 SDA-positive specimens (including low positives) examined, 93 % were confirmed as N. gonorrhoeae-positive using either one or both real-time PCR assays. In contrast, only 34 % were confirmed using the AC2 and/or the AGC assays. All 114 SDA-negative specimens were confirmed as negative using all four confirmatory tests. Clearly the AC2 and AGC assays cannot reliably be used to confirm residual specimens in SDA assay transportation buffers, due to the incompatibility of different platform chemistries. Although high rates of confirmation (93 %) can be achieved when examining residual SDA assay specimens using independent real-time PCR assays, establishing well-validated in-house real-time PCR assays for diagnostic use is a large undertaking for many primary laboratories and so such tests may be better confined to specialist laboratory services.

The central nervous system (CNS) is the most common site of dissemination during Aspergillus infection. PCR has the potential to facilitate early diagnosis of CNS aspergillosis, which could assist in reducing disease mortality. In two experiments, neutropenic CD-1 male mice were infected intracranially with 5×106 conidia of Aspergillus fumigatus. At time points up to 120 h after infection, mice were euthanized and samples of blood, brain, spinal cord and cerebrospinal fluid (CSF) were taken. The brain fungal burden was determined by quantitative culture, and fungal DNA was detected by quantitative PCR. Plating for A. fumigatus from the brain confirmed that all mice had burdens of log10 >3 from 4 to 120 h after infection. A. fumigatus DNA was detected in blood (88 %), brain (96 %), CSF (52 %) and spinal cord (92 %) samples. The brain and spinal cord contained the highest concentrations of fungal DNA. Adapting the extraction protocol to maximize yield from small sample volumes (10 µl CSF or 200 µl blood) allowed PCR detection of A. fumigatus in infected mice, suggesting the use of CSF and blood as diagnostic clinical samples for CNS aspergillosis.

The transmission of viral and non-viral infectious pathogens continues to be the most serious of the potential adverse effects of allogenic tissue transplantations. EU Directive 2006/17/EC stipulates that cadaveric blood specimens for serology testing in the context of post-mortem tissue donation must be taken not later than 24 h post-mortem. An expanded time slot would significantly improve the availability of tissue donations, but there are no significant data on the stability of infectious serology assays for anti-human immunodeficiency virus (HIV), anti-hepatitis C virus (HCV), hepatitis B virus (HBV) surface antigen (HBsAg) and anti-HBC core antigen (HBc) in samples collected more than 24 h post-mortem. In this prospective study, serum samples of 30 deceased persons were taken upon admission to the Institute of Forensic Medicine (University Hospital Hamburg-Eppendorf, Germany) and at 12, 24, 36 and 48 h post-mortem. All samples were measured twice, first using the Abbott AxSYM system, and then after ~9 months of storage at −70 °C using the BEP III System with Siemens and Ortho reagents. For HIV, six deceased persons with a pre-mortem HIV history were included. All samples (at committal and at 12, 24, 36, 48 h) were reactive. Indeterminate or false-negative results did not occur. For HCV, 17 deceased persons with a pre-mortem HCV history were included; 16 samples were reactive up to 48 h and one was reactive at 36 h post-mortem (48 h sample was not available). Indeterminate or false-negative results did not occur. For HBV, nine deceased persons were included: five samples were initially positive for HBsAg and remained positive up to 48 h, and eight of the samples were reactive for anti-HBc up to 48 h and one up to 36 h post-mortem (48 h sample was not available). Indeterminate or false-negative results did not occur. These data suggest that infectious serological testing may be extended for blood samples of potential tissue donors collected up to 48 h post-mortem to detect antibodies or antigens for HIV, HBV and HCV.

The standard use of a single universal broad-range PCR in direct 16S rDNA sequencing from polybacterial samples leaves the minor constituents at risk of remaining undetected because all bacterial DNA will be competing for the same reagents. In this article we introduce a set of three broad-range group-specific 16S rDNA PCRs that together cover the clinically relevant bacteria and apply them in the investigation of 25 polybacterial clinical samples. Mixed DNA chromatograms from samples containing more than one species per primer group were analysed using RipSeq Mixed (iSentio, Norway), a web-based application for the interpretation of chromatograms containing up to three different species. The group-specific PCRs reduced complexity in the resulting DNA chromatograms and made the assay more sensitive in situations with unequal species concentrations. Together this allowed for identification of a significantly higher number of bacterial species than did standard direct sequencing with a single universal primer pair and RipSeq analysis (95 vs 51). The method could improve microbiological diagnostics for important groups of patients and can be established in any laboratory with experience in direct 16S rDNA sequencing.

Routine laboratory diagnosis of Pneumocystis jirovecii is currently achieved by PCR in almost all laboratories with sufficient equipment due to its high sensitivity and specificity compared to staining methods. A current issue that limits the reliability and sensitivity of PCR is the degree of inhibition caused by inhibitory substances in respiratory samples. The present study aimed to analyse the degree and frequency of inhibition in real-time PCR detecting P. jirovecii in respiratory specimens submitted to a Pneumocystis pneumonia (PcP) diagnosis laboratory in Ege University Medical School, Turkey. Between July 2009 and December 2010, 76 respiratory specimens [63 bronchoalveolar lavage (BAL) fluid, 10 sputum samples, two tracheal aspiration fluid and one thoracentesis fluid] obtained from 69 PcP-suspected patients were investigated for the presence of P. jirovecii using real-time PCR targeting the cdc2 gene. Of these samples, 42 of the specimens were stained and examined by microscopy according to the request of the clinicians. PCR was positive in 15 specimens in the initial run. Of the remaining 61 samples, 41 of them were negative with positive internal inhibition controls (i.e. true-negative group). The frequency of inhibition in the initial run was 26.31 % (20/76) as determined by spiked negative controls. All of the inhibited samples were resolved after 1 : 2, 1 : 5, 1 : 10 and 1 : 20 dilutions. P. jirovecii was detected by PCR in two inhibited specimens after retesting with diluted samples which were also positive by microscopy. The incidence of P. jirovecii in respiratory specimens was 22.36 % (17/76) as determined by real-time PCR and 7.14 % (3/42) by microscopy. Overall, the incidence of P. jirovecii in respiratory samples was 23.68 % (18/76) as detected by both methods. In conclusion, inclusion of spiked positive controls in each sample and retesting with diluted samples to resolve inhibition increased the reliability of the real-time PCR assay in terms of determining false-negative results and influencing the treatment of the patient. Furthermore, results of the present study determined for the first time the frequency and degree of inhibition in a real-time PCR detecting P. jirovecii in respiratory specimens during routine diagnosis of PcP.

Rapid detection of vancomycin-resistant enterococci (VRE) infection is very important for control and prevention of nosocomial spread of these bacteria. A multiplex PCR method for rapid screening of VRE has recently been developed. We performed a prospective study of VRE screening tests to compare the performance of PCR to that of a chromogenic agar-based culture method. From January to December 2009, a total of 8815 rectal swab specimens were tested simultaneously for VRE by VRE selective culture and by PCR. The specimens were inoculated onto ChromID VRE agar containing 8 µg vancomycin ml−1 and examined after 24 and 48 h of incubation. Identification and antibiotic susceptibility tests were performed using the automated VITEK-2 system and a supplementary E-test and disk diffusion test. Detection of the vanA and vanB genes was performed with the Seeplex VRE detection kit. Specimens were inoculated in enterococcosel broth for 16–24 h before PCR for enrichment of VRE. VRE were isolated from 741 of the 8815 specimens by chromogenic agar-based culture (8.4 %). vanA and vanB genotypes were detected in 758 (8.6 %) and 3 (0.03 %) specimens, respectively, by multiplex PCR. Sensitivity, specificity, positive predictive value and negative predictive value of PCR for detection of VRE were 98.2 %, 99.6 %, 95.7 %, and 99.8 %. No VRE were isolated from vanB-positive specimens. The overall performance of PCR is comparable to that of a chromogenic agar-based culture method for screening of VRE, so PCR could be an alternative or supportive method for effective control of nosocomial VRE infection.

Cryptococcus neoformans and Cryptococcus gattii are aetiological agents of cryptococcosis, a major opportunistic systemic mycosis of increasing global importance. This study reports the antifungal susceptibility profiles of clinical and environmental isolates of C. neoformans var. grubii, genotype VNI/AFLP1 (n = 246), and C. gattii serotype B, genotype VGI/AFLP4 (n = 62), originating from patients and environmental sources in north-western India. All of the C. neoformans var. grubii and C. gattii isolates were mating type α. Using the broth microdilution method, both species were found to be susceptible to the antifungals tested except for two clinical C. neoformans var. grubii isolates that were resistant to 5-flucytosine (MIC >64 µg ml−1). Data on the geometric mean of MICs revealed that C. gattii was significantly less susceptible than C. neoformans var. grubii to fluconazole, itraconazole and voriconazole (P<0.0001). In addition, the MIC90 of C. gattii was twofold higher than that of C. neoformans var. grubii for fluconazole, itraconazole and voriconazole. However, no statistically significant difference in susceptibility of the two Cryptococcus species was observed against amphotericin B and 5-flucytosine. Furthermore, the environmental C. neoformans var. grubii isolates were significantly less susceptible to fluconazole, itraconazole and 5-flucytosine (P<0.0001) than the clinical isolates. A continued surveillance of antifungal susceptibility of clinical and environmental strains of C. neoformans and C. gattii is desirable to monitor the emergence of any resistant strains in order to ensure more successful therapy of cryptococcosis.

Cationic antimicrobial agents may prevent device-associated infections caused by Staphylococcus epidermidis and Staphylococcus aureus. This study reports that the cationic antimicrobial polymer poly(2-(dimethylamino ethyl)methacrylate) (pDMAEMA) was more effective at antagonizing growth of clinical isolates of S. epidermidis than of S. aureus. Importantly, mature S. epidermidis biofilms were significantly inactivated by pDMAEMA. The S. aureus isolates tested were generally more hydrophobic than the S. epidermidis isolates and had a less negative charge, although a number of individual S. aureus and S. epidermidis clinical isolates had similar surface hydrophobicity and charge values. Fluorescence spectroscopy and flow cytometry revealed that fluorescently labelled pDMAEMA interacted strongly with S. epidermidis compared with S. aureus. S. aureus ΔdltA and ΔmprF mutants were less hydrophobic and therefore more susceptible to pDMAEMA than wild-type S. aureus. Although the different susceptibility of S. epidermidis and S. aureus isolates to pDMAEMA is complex, influenced in part by surface hydrophobicity and charge, these findings nevertheless reveal the potential of pDMAEMA to treat S. epidermidis infections.

Forty carbapenem-resistant Klebsiella pneumoniae isolates were recovered from 28 patients from various sites in an intensive care unit in Zhejiang Provincial People’s Hospital, China, over a 6 month period. PFGE analysis indicated that the 40 strains were all closely related. The MICs of carbapenems varied from 16 to >256 µg ml−1. Conjugation studies with Escherichia coli resulted in the transfer of reduced carbapenem susceptibility from the original isolates. All K. pneumoniae and E. coli transconjugants produced K. pneumoniae carbapenemase 2 (KPC-2), and most of them produced TEM, SHV and CTX-M. Additionally, 27 isolates and 27 E. coli transconjugants carried the qnr gene (25 were qnrB2 and 2 were qnrS1). K. pneumoniae harboured several plasmids, and bla KPC-2 was located on a 55 kb plasmid. SDS-PAGE and ompK35/36 gene sequence analysis of OMPs suggested that porins in K. pneumoniae are expressed normally. The MICs of the carbapenems did not change in the presence of CCCP. Thus, production of KPC-2 appears to play an important role in resistance to carbapenems, although other mechanisms may be involved. The bla KPC-2 gene is associated with several antibiotic-resistance genes, such as bla TEM, bla SHV, bla CTX-M and qnr.

Three clinical Klebsiella pneumoniae strains, KpARG74, KpARG220 and KpARG185, isolated from a hospital in Algeria, carried the novel β-lactamases SHV-98, SHV-99 and SHV-100, respectively, and co-expressed TEM-1 and either CTX-M-3 or CTX-M-15. In contrast, transformed cells possessing the genes for these novel β-lactamases, i.e. EcDH5α-SHV-98, EcDH5α-SHV-99 and EcDH5α-SHV-100, respectively, carried unique sequence features of bla SHV gene variants, enabling oxyimino-cephalosporin susceptibility and confirming that none of the transformants exhibited extended-spectrum β-lactamase (ESBL) properties. SHV-100 is apparently functional, despite differing from the SHV-1 sequence by duplication of 13 amino acids. The SHV-99 enzyme differed from the parental SHV-1 by the amino acid substitution Asp104→Gly, which is an important position in the development of the ESBL phenotype in TEM β-lactamases. This is the first time, to our knowledge, that this mutation has been reported in clinically occurring isolates. Thus, kinetic characterization of the SHV-99 enzyme was performed. The SHV-99 enzyme showed higher affinity (K m of 196 µM), catalytic activity (k cat of 0.5 s−1) and catalytic efficiency (k cat/K m of 0.003 µM−1 s−1) than SHV-1 β-lactamase against aztreonam. These results showed that the neutral glycine at residue 104 increased the affinity of the enzyme to aztreonam, but was unable to develop the ESBL phenotype in SHV enzymes. As the emergence of new threatening combinations of resistance determinants among nosocomial pathogens is further possible, this study has highlighted the need to reverse the spread of initial mutations.

In this study, 90 non-replicate imipenem-resistant Pseudomonas aeruginosa (IRPA) Malaysian isolates collected between October 2005 and March 2008 were subjected to a screening test for detection of the integron and the gene cassette. Class 1 integrons were detected in 54 IRPA clinical isolates, whilst three isolates contained class 2 integrons. Analysis of the gene cassettes associated with the class 1 integrons showed the detection of accC1 in isolates carrying bla IMP-7 and aacA7 in isolates carrying bla VIM-2. aadA6 was detected in two isolates carrying bla IMP-4. Using random amplification of polymorphic DNA analysis, 14 PCR fingerprint patterns were generated from the 32 isolates carrying metallo-β-lactamase (MBL) genes (35.5 %), whilst 20 patterns were generated from the 58 non-MBL gene isolates (64.4 %). Based on the differences in the fingerprinting patterns, two clusters (A and B) were identified among the MBL-producing isolates. Cluster A comprised 18 isolates (56 %) carrying the bla VIM gene, whereas cluster B comprised 14 (44 %) isolates carrying the bla IMP gene. The non-MBL isolates were divided into clusters C and D. Cluster C comprised 22 non-MBL isolates harbouring class 1 integrons, whilst cluster D consisted of three isolates carrying class 2 integrons. These findings suggest that the class 1 integron is widespread among P. aeruginosa isolated in Malaysia and that characterization of cassette arrays of integrons will be a useful epidemiological tool to study the evolution of multidrug resistance and the dissemination of antibiotic resistance genes.

Staphylococcus aureus drug resistance to antibiotics is a serious situation that has drawn greater attention to immunotherapy and prophylaxis. Peptidoglycan (PGN) is a common and conserved component of the cell wall of Gram-positive bacteria such as S. aureus. However, PGN, as a thymus-independent antigen, cannot be considered a vaccine candidate because of its very weak immunogenicity. In this study we have attempted to enhance the immunogenicity of PGN by identifying a PGN peptide mimic sequence that would act as a thymus-dependent antigen. Several peptide sequences were obtained from a phage display peptide library using a mAb against S. aureus PGN, and a 12-mer linear single peptide (Sp-31) and a four-branch multiple antigen peptide (MAP) (MAP-P31) were synthesized. Both Sp-31 and MAP-P31 were shown to bind directly to anti-PGN mAb and a polyclonal antibody against S. aureus. These peptides could also inhibit the binding of PGN to a mAb against PGN. Furthermore, MAP-P31 was able to provoke an effective secondary antibody response in mice to PGN and to cell-wall fragments isolated from S. aureus, Escherichia coli, Staphylococcus epidermidis and Pseudomonas aeruginosa by sonication. In addition, the MAP-P31 antiserum showed a potent bactericidal or bacteriostatic activity against S. aureus in the presence and absence of complement in vitro. Importantly, immunization with MAP-P31 significantly prolonged the survival and enhanced bacterial clearance in BALB/c mice challenged with live S. aureus. In addition, the serum IgG-type antibodies against PGN persisted in mice, demonstrating that MAP-P31, as a peptide mimicking epitopes on PGN, provokes an effective secondary or memory antibody response, which can only be induced by a thymus-dependent antigen and which protects against infection with S. aureus. These results suggest that MAP-31 may be a novel vaccine candidate against S. aureus.

This study used a previously described multiplex PCR-based reverse line blot (mPCR/RLB) assay to assess the prevalence and distribution of 14 urogenital pathogens or putative pathogens, namely Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Trichomonas vaginalis, Gardnerella vaginalis, Ureaplasma parvum, Ureaplasma urealyticum, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, herpes simplex virus types 1 and 2, and human adenovirus. First-voided urine specimens and endocervical and self-collected vaginal swabs from each of 216 women attending three sexual health clinics in Sydney, Australia, were tested and the results were compared with those of reference methods for each organism. One hundred and sixty-eight women (77.7 %) had at least one and 105 (48.6 %) had more than one target organism, most commonly G. vaginalis and Ureaplasma spp. The prevalence of each of the four known sexually transmissible pathogens was <5 %. Of the 216 women, 111 (51.4 %) reported at least one symptom consistent with genital or urethral infection, including discharge, pain or discomfort. Only G. vaginalis was detected more frequently in women with symptoms (P = 0.05). The specificity of the mPCR/RLB assay compared with that of the reference methods for each organism and for all specimen types was 100 %. The mean sensitivities of the mPCR/RLB assay compared with those of the reference methods for self-collected vaginal swabs, cervical swabs and first-voided urine specimens for all organisms were 99.3, 98.1 and 84.6 %, respectively; however, these differences were not significant. There were no differences in sensitivities between specimen types for C. trachomatis, N. gonorrhoeae, T. vaginalis and H. influenzae, although all were found infrequently. Overall, the mPCR/RLB platform was found to be an accurate testing platform in a sexual health clinic setting.