- Volume 60, Issue 3, 2011

This study examined Klebsiella mobilis isolates cultured from clinical specimens for virulence-associated properties and antibiotic resistance. The strains produced a number of siderophores, including enterobactin, aerobactin and yersiniabactin. All isolates were able to adhere to and invade epithelial cells. They had cytotoxic activity, which caused destruction of human laryngeal epithelial HEp-2 cells and evoked lysis of murine macrophage J774 cells. Analyses of HEp-2 and J774 cellular morphology and DNA fragmentation in the cells showed features typical of cells undergoing apoptosis. Some K. mobilis strains harboured class 1 integrons carrying the aadA1 gene encoding an aminoglycoside adenyltransferase.

Pandoraea species have emerged as opportunistic pathogens among cystic fibrosis (CF) and non-CF patients. Pandoraea pulmonicola is the predominant Pandoraea species among Irish CF patients. The objective of this study was to investigate the pathogenicity and potential mechanisms of virulence of Irish P. pulmonicola isolates and strains from other Pandoraea species. Three patients from whom the P. pulmonicola isolates were isolated have since died. The in vivo virulence of these and other Pandoraea strains was examined by determining the ability to kill Galleria mellonella larvae. The P. pulmonicola strains generally were the most virulent of the species tested, with three showing a comparable or greater level of virulence in vivo relative to another CF pathogen, Burkholderia cenocepacia, whilst strains from two other species, Pandoraea apista and Pandoraea pnomenusa, were considerably less virulent. For all Pandoraea species, whole cells were required for larval killing, as cell-free supernatants had little effect on larval survival. Overall, invasive Pandoraea strains showed comparable invasion of two independent lung epithelial cell lines, irrespective of whether they had a CF phenotype. Pandoraea strains were also capable of translocation across polarized lung epithelial cell monolayers. Although protease secretion was a common characteristic across the genus, it is unlikely to be involved in pathogenesis. In conclusion, whilst multiple mechanisms of pathogenicity may exist across the genus Pandoraea, it appears that lung cell invasion and translocation contribute to the virulence of P. pulmonicola strains.

Pseudomonas aeruginosa, an opportunistic pathogen, is the third most common pathogen associated with nosocomial urinary tract infections (UTIs). The virulence of this organism is due to its ability to produce quorum-sensing (QS) signal molecules and form biofilms. These biofilms are usually resistant to conventional antibiotics and host immune responses. Recently, beneficial effects of macrolides, especially azithromycin (AZM), have been shown in patients suffering from chronic infections caused by P. aeruginosa. These were due to anti-inflammatory and modulatory effects of AZM on the expression of virulence factors of this pathogen. The present study was designed to evaluate the potential of AZM to inhibit QS signal molecules and its ability to attenuate the virulence of P. aeruginosa in an experimental UTI model. Sub-MIC concentrations of AZM significantly inhibited the production of QS signals, swimming, swarming and twitching motilities, and biofilm formation in vitro. The therapeutic evaluation of AZM in this experimental UTI model showed complete clearance of the organisms from the mouse kidneys. The results of this study highlight the potential effectiveness of AZM in attenuating the virulence of P. aeruginosa in a UTI model.

A total of 1204 meticillin-resistant Staphylococcus aureus (MRSA) screens (3340 individual swabs) were tested to evaluate a staphylococcal cassette chromosome mec (SCCmec) real-time PCR. In total, 148 (12.3 %) of the screens were MRSA-positive, where 146 (12.1 %) were MRSA-positive by the SCCmec real-time PCR assay. In contrast, 128 (10.6 %) screens were MRSA-positive by culture. One hundred and twenty-six (10.5 %) of the screens were positive by both culture and PCR. Twenty of the 1204 screens (1.66 %) were negative by culture but positive by PCR; these samples were sequenced. In 14 of the cases, a homology search confirmed the sequence as SCCmec, indicating that these samples could be considered true positives. Two of the 1204 (0.2 %) screens were positive by culture and negative by PCR. The mean turnaround time (TAT) for PCR-negative swabs was 6 h 12 min and for PCR-positive swabs was 6 h 48 min. In comparison, for culture-negative swabs the mean TAT was 29 h 30 min and for culture-positive swabs was 69 h. The cost per swab for routine culture was £0.41 (€0.48) and that of the real-time PCR assay was £2.35 (€2.75). This optimized, in-house, inexpensive, real-time PCR test maintained a very high sensitivity and specificity when evaluated under real-time laboratory conditions. The TAT of this real-time PCR assay was substantially lower than that of chromogenic culture. It was also maintained throughout the entire process, which can be taken as an indirect measure of test performance. This study showed that implementation of a molecular test can be achieved with limited resources in a standard microbiology laboratory.

The antibiotic susceptibility of Acinetobacter calcoaceticus–Acinetobacter baumannii complex strains recovered from the intensive care unit (ICU) of West China Hospital, Sichuan, PR China, from 2006 to 2009 was investigated. The identification of A. baumannii and analysis of carbapenemase-encoding genes and their relationship with ISAba1 were performed by PCR. Furthermore, a DiversiLab repetitive extragenic palindromic sequence-based PCR (rep-PCR) microbial typing system and a multilocus sequence typing (MLST) scheme were applied to assess the genetic relationship of the isolates. The results showed that the antibiotic susceptibility of the A. calcoaceticus–A. baumannii complex isolates changed and imipenem resistance increased rapidly between 2006 and 2009. The bla OXA-51-like and ISAba1-associated bla OXA-23 genes were prevalent in the imipenem-resistant A. baumannii isolates. However, the bla OXA-58-like gene was found in only one isolate and no metallo-β-lactamase genes were detected. The representative multidrug-resistant A. baumannii isolates were identified as one cluster by rep-PCR fingerprinting and belonged to the clonal complex 92 (CC92) according to MLST. These findings indicate a situation of increasing resistance and wide distribution of class D β-lactamase genes, especially the acquired ISAba1-associated bla OXA-23 gene, in A. baumannii isolates in the ICU of West China Hospital, probably caused by expansion of the CC92 clone.

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are members of the species Human enterovirus A, and are both major and independent aetiological agents of hand-foot-and-mouth disease. The human enterovirus (HEV) 5′ untranslated region (UTR) is fundamentally important for efficient virus replication and for virulence, whilst the VP1 region correlates well with antigenic typing by neutralization, and can be used for virus identification and evolutionary studies. A comparison was undertaken of the 5′UTR and VP1 nucleotide sequences of five EV71 clinical isolates and 10 CVA16 clinical isolates from one laboratory with the 5′UTR and VP1 sequences of 104 EV71 strains and 45 CVA16 strains available in GenBank. The genetic relationships were analysed using standard phylogenetic methods. The EV71 phylogenetic analysis showed that the VP1 sequences were clustered into three genogroups, A, B and C, with genogroups B and C further divided into five subgenogroups, B1–B5 and C1–C5, respectively. All EV71 strains were clustered similarly in the 5′UTR and VP1 trees, except for one Taiwanese strain, which demonstrated different clustering in the two trees, suggesting a recombination event in the phylogeny. The CVA16 phylogenetic analysis showed that the VP1 sequences were clustered into two genogroups, A and B, with genogroup B further divided into B1 (B1a and B1b), B2 and a possible B3; and that a similar pattern and grouping of all strains were displayed in the 5′UTR tree. This study demonstrated that comparing the two regions provides evidence of epidemiological linkage of HEV-A strains, and that mutation in the two regions plays a vital role in the evolution of these viruses. The combination of molecular typing and phylogenetic sequence analysis will be beneficial in both individual patient diagnosis and public health measures.

A high carriage rate of multidrug-resistant Escherichia coli (MDREC) was observed in elderly residents in long-term care facilities. A double-blinded, placebo-controlled trial was carried out to determine whether the probiotic product E. coli strain Nissle 1917 (Mutaflor) would compete with MDREC in the bowel and thereby reduce the prevalence of the multiresistant bacteria in faeces and urine. Sixty-nine patients excreting norfloxacin-resistant E. coli were randomized to probiotic or placebo groups and administered capsules twice daily. The daily dose of probiotic was 5×109–5×1010 bacteria. Faecal and urine samples were cultured at baseline and during and after the treatment period. A reduction in baseline carriage was not influenced by probiotic administration. The probiotic strain was detected in faecal specimens collected during the treatment period of only two out of 12 probiotic group subjects that were tested. Genotyping of norfloxacin-resistant E. coli isolates showed that 32 strains were prevalent among the patients. Thus, E. coli Nissle 1917 does not have the capacity to compete effectively with MDREC in the bowel of elderly patients.

Acinetobacter junii is a rare human pathogen associated with bacteraemia in neonates and paediatric oncology patients. We present a case of A. junii causing bacteraemia in an adult transplant patient with leukaemia. The correct identification of Acinetobacter species can highlight the clinical significance of the different species of this genus.

Extra-intestinal manifestations of Clostridium difficile infection are uncommon. Most cases are associated with gastrointestinal disease and often occur as a mixed infection with other gut flora. We report a case of breast abscess following monomicrobial C. difficile bacteraemia in a female with background chronic hepatitis C infection and alcoholic liver disease. No evidence of colitis was found. Our case shows that C. difficile is indeed capable of spreading from the gastrointestinal tract.

Daptomycin is licensed for the management of Staphylococcus aureus infections in adults, including those caused by meticillin-resistant S. aureus (MRSA). Few data exist on paediatric use or dose guidance in neonates. We report the case of a neonate with MRSA bacteraemia successfully managed with daptomycin. Dose requirements were substantially higher than those recommended for adults.

Seven epidemiologically unrelated cases of invasive Haemophilus influenzae type a (Hia) disease were identified in First Nations communities of Northwestern Ontario, Canada, in 2004–2008. In all cases, Hia was isolated from blood. The clinical presentation in most of the cases was moderately severe and all patients responded to antibiotic therapy. Laboratory analysis of Hia isolates from Northwestern Ontario indicated striking similarities in their phenotypic and genotypic characteristics. The findings are discussed in the context of current epidemiology of invasive Hia disease. Our data along with some published studies by others suggest an increased susceptibility to this infection among North American indigenous populations.