- Volume 65, Issue 5, 2016

Pseudomonas aeruginosa uses a type III secretion system to deliver toxic effector proteins directly into host cells and alter host protein functions. Exoenzyme S (ExoS), a type III effector protein, ADP-ribosylates Rab5 GTPase and impairs early phagocytic events in macrophage cells. In this study, we tested the hypothesis that Rin1, a Ras effector protein and Rab5 guanine nucleotide exchange factor, generates an intrinsic Rab5 activity cycle during phagocytosis of live P. aeruginosa; thus, allowing proper phagocytic killing. We found that Rab5 activity was attenuated at a very early time point (2.5 min) of the phagocytic process of live but not of heat-inactivated P. aeruginosa. However, upon overexpressing Rin1 in macrophages, the Rab5 activity sustained for a prolonged time (∼20 min) counteracting the negative effects during phagocytosis of live P. aeruginosa. Ras, also a substrate of the ADP-ribosyltransferase activity of ExoS, remained active during the early events of phagocytosis of live as well as heat-inactivated P. aeruginosa. Further examinations revealed that the Rin1 : Vps9 domain (the Rab5 nucleotide catalytic domain) and the Rin1 : RA domain (the Ras association domain of Rin1) are both required for optimal Rin1 function. Finally, the time-based analysis of the ADP-ribosylation status of Rab5 and Ras obtained from this study was consistent in the context of the regulation of (i) Rab5 activity by Rin1 : Vps9 domain and (ii) Ras interaction with Rin1 via the Rin1 : RA domain. These observations highlight a novel crosstalk between Rin1–Rab5 and Rin1–Ras complexes that offsets the anti-phagocytic effects of ExoS in macrophages.

Understanding how inhaled Mycobacterium tuberculosis achieves dramatic replication and crosses the alveolar barrier to establish systemic latent infection, before adaptive immunity is elicited in humans, is limited by the small infecting inoculum carried in aerosol droplets (1–5 μm diameter) and the inability to identify the time of infection. M. tuberculosis is believed to disseminate via infected macrophages. However, like other invasive bacterial pathogens, M. tuberculosis could also cross the barrier directly using adhesins and toxins. An in vitro alveolar barrier mimicking the gas-exchange regions of the alveolus was devised comprising monolayers of human alveolar epithelial and endothelial cells cultured on opposing sides of a basement membrane. Migration of dissemination-competent strains of M. tuberculosis, and dissemination-attenuated M. tuberculosis and Mycobacterium bovis mutant strains lacking adhesin/toxin ESAT-6 and adhesin HBHA were tested for macrophage-free migration across the barrier. Strains that disseminate similarly in vivo migrated similarly across the in vitro alveolar barrier. Strains lacking ESAT-6 expression/secretion were attenuated, and absence of both ESAT-6 and HBHA increased attenuation of bacterial migration across the barrier. Thus, as reported for other bacteria, M. tuberculosis utilizes adhesins and toxins for macrophage-independent crossing of the alveolar barrier. This in vitro model will allow identification and characterization of molecules/mechanisms employed by M. tuberculosis to establish systemic latent tuberculosis infection during primary infection.

Clinical management of bacterial vaginosis (BV) is difficult owing to inaccurate diagnostic tests, limited drug choices, and a high rate of recurrence. To our knowledge, there has not been a previous study of antimicrobial resistance (AMR) genes in community practice using next-generation sequencing (NGS). A case–control study (1 : 1 age-matched with and without BV) was undertaken in a series of 326 nongravid women of reproductive age with symptoms of BV to determine the prevalence of AMR genes. NGS was used to describe the complete vaginal microbiota and identify bacterial genes associated with resistance to: macrolides and/or lincosamides – ermA, ermB, ermC, erM, ermTR and mefA; tetracyclines, β-lactams, streptomycin, gentamicin and/or tobramycin – acrA, acrB, mecA, tet, tetA, tolC and aac2; 5-nitroimadazoles – nim and nimB; and triazoles – cdr1 and mdr1. An evidence base was created to inform treatment decisions applicable to individual patients. AMR genes were identified in all drug classes: macrolides, 35.2 %; lincosamides, 35.6 %; tetracyclines, 21.8 %; aminoglycosides (streptomycin, gentamicin and tobramycin), 5.2 % each; 5-nitroimidazoles, 0.3 %; and triazoles, 18.7 %. There was more than a fourfold-higher frequency of AMR genes in pathogens from BV than from non-BV patients for macrolides (58.2 versus 12.3 %, respectively), lincosamides (58.9 versus 12.3 %) and tetracyclines (35.6 versus 8.0 %) (Fisher's exact test; all p < 0.001). For each patient with BV, the spectrum of resistance genes was matched to the pathogens present. AMR genes were present in the majority of vaginal microbiomes of patients with symptoms of BV.

The rmtB gene, one of the 16S rRNA methylase genes whose products confer high-level resistance to aminoglycosides, is most prevalent among Enterobacteriaceae strains. In this study, eight non-duplicate rmtB-carrying avian Escherichia coli strains from a farm in China were isolated and characterized, and further examined by phylogenetic grouping, conjugation experiments and PCR-based replicon typing. In addition, the genetic environment of rmtB was investigated by cloning and sequencing. Six rmtB-carrying E. coli were identified as phylogroup A, sequence type (ST) 156 (A-ST156), with two assigned to D-ST117; however, all of them carried the same IncI1 ST136 plasmid. The genetic environment of the rmtB gene in these eight plasmids was the same, as shown by PCR mapping. A multidrug-resistant region carrying bla TEM-1, rmtB, a class 1 integron cassette array (intI1-dfrA12-orfF-aadA2-qacEΔ1-sul1) and aacC2 was characterized on the conjugative IncI1 ST136 plasmid. Co-location of the rmtB gene with a class 1 integron cassette array and aacC2 on the conjugative plasmid will facilitate its maintenance and dissemination.

Multilocus sequence typing (MLST) is considered a reliable method for providing insight into the Streptococcus suis population structure, clonal relationships and the potential of particular clones to cause disease. Indeed, MLST has revealed the presence of several clonal complexes (CCs) within the Streptococcus suis population. However, the method is costly, time-consuming and difficult to use for screening large numbers of isolates. In this study, a multiplex PCR assay was developed to identify Streptococcus suis CCs that are relevant to human infections. The multiplex PCR assay was capable of simultaneously distinguishing CC1, CC25, CC28, CC104, CC221/234 and CC233/379, which are related to human infections in Thailand, in a single reaction. The multiplex PCR assay is useful for low-cost screening of large numbers of isolates with rapid analytical capacity and could be utilized in most laboratories.

Multiple studies have shown that the antibacterial dressing Acticoat can inhibit growth of bacteria but is unable to completely clear a wound of infection, which could leave patients vulnerable to sepsis. Agar inoculated with four different Staphylococcus aureus strains and overlain with Acticoat showed growth inhibition beneath and within a 1 mm perimeter of the dressing after 24 h. When lifted from inoculated agar and briefly blotted onto fresh agar plates, Acticoat transferred viable bacteria. Scanning electron microscopy of the surface of Acticoat that overlaid meticillin-resistant S. aureus for 24, 48 and 72 h showed dense clusters of apparently undamaged bacteria distributed across the mesh. The number of bacteria growing on inoculated pig skin, underneath and on the surface of Acticoat, was lower than on controls for the first 8 h, but after 24 h the number of bacteria on the skin was 2.3-fold greater than the untreated controls. In contrast, after 24 h the number of bacteria surviving on the surface of the Acticoat was 11.9 % of controls. Acticoat moistened with 10 % glycerol plus antimicrobial peptides (AMPs) mel12–26 or bac8c (50 μg ml− 1) reduced the numbers of bacteria on the dressing and on the skin underneath to below 10 % and 0.01 % of the controls, respectively. When lysozyme (1 mg ml− 1) was added to Acticoat wetted with glycerol and the AMP bac8c, the dressing was able to prevent the survival of bacteria on densely inoculated pig skin and on the surface of Acticoat for up to 24 h. In effect, biocompatible solvents and AMPs significantly enhance the bactericidal efficacy of Acticoat.

Human papillomavirus (HPV) and Chlamydia trachomatis are pathogens with oncogenic potential associated with persistent infections. Epidemiological data on C. trachomatis infection status, C. trachomatis/HPV co-infection and the relationship between HPV genotypes in Italian women are only preliminary. The aim of the present study was to characterize the relationship between HPV genotypes and C. trachomatis in an extending cohort of asymptomatic immunocompetent women from an area of north-east Italy. A retrospective study was conducted using Luminex technology on cervical swabs from asymptomatic immunocompetent women, comprising 921 attending the prevention centre for the Cervical Cancer Program and 6214 who had been referred to the Sexually Transmitted Infections Center, with clinical indications of HPV and C. trachomatis infections. A quantitative real-time PCR was performed to assess chronic C. trachomatis infection by heat-shock protein 60 (Hsp60) gene expression. The overall prevalence of the investigated pathogens was 39 % (359/921) for HPV and 4 % (251/6214) for C. trachomatis. The Hsp60 gene was detected in 57 % of the women infected with C. trachomatis. HPV co-infection was present in 58 % of C. trachomatis-infected women. A high prevalence of co-infection was found in women with chronic C. trachomatis infection (68 %, P = 0.0002), especially in women ≤ 25 years (72 %) where HPV multiple infections were found in 78 % (P = 0.022). HPV genotype distribution showed that uncommon low-risk genotypes were associated with C. trachomatis. These results indicate a high frequency of co-detection of multiple HPV genotypes in chronically infected young women and suggest that the expression of the C. trachomatis Hsp60 gene may favour HPV infection.

Chlamydia pecorum infection is a threat to the health of free-ranging koalas (Phascolarctos cinereus) in Australia. Utilizing an extensive sample archive we determined the prevalence of C. pecorum in koalas within six regions of Victoria, Australia. The ompA genotypes of the detected C. pecorum were characterized to better understand the epidemiology of this pathogen in Victorian koalas. Despite many studies in northern Australia (i.e. Queensland and New South Wales), prior Chlamydia studies in Victorian koalas are limited. We detected C. pecorum in 125/820 (15 %) urogenital swabs, but in only one ocular swab. Nucleotide sequencing of the molecular marker C. pecorum ompA revealed that the majority (90/114) of C. pecorum samples typed were genotype B. This genotype has not been reported in northern koalas. In general, Chlamydia infection in Victorian koalas is associated with milder clinical signs compared with infection in koalas in northern populations. Although disease pathogenesis is likely to be multifactorial, the high prevalence of genotype B in Victoria may suggest it is less pathogenic. All but three koalas had C. pecorum genotypes unique to southern koala populations (i.e. Victoria and South Australia). These included a novel C. pecorum ompA genotype and two genotypes associated with livestock. Regression analysis determined that significant factors for the presence of C. pecorum infection were sex and geographical location. The presence of ‘wet bottom’ in males and the presence of reproductive tract pathology in females were significantly associated with C. pecorum infection, suggesting variation in clinical disease manifestations between sexes.

Our aim was to identify long-term β-lactam resistance trends in local Klebsiella pneumoniae isolates, which are a common cause of sepsis in Western Australia. We studied three collections of K. pneumoniae isolates from Western Australia between 1977 and 2015 comprising contemporary blood culture (n = 98), multiresistant (n = 21) and historical (n = 50) isolates. Antimicrobial resistance was determined by Clinical and Laboratory Standards Institute agar dilution methods. PCR DNA sequencing identified β-lactamase variants and porin mutations contributing to β-lactam resistance. Isolates were genotyped by PFGE, multilocus sequence typing and a variable number tandem repeat method. From 1989 onwards, we detected the SHV-2a extended-spectrum β-lactamase (ESBL) in ceftriaxone-resistant isolates, and in ceftazidime- and aztreonam-resistant isolates from 1993. Ceftriaxone, ceftazidime and aztreonam resistance persisted, with bla CTX-M types becoming the dominant ESBLs by 2010. CTX-M-15 was encountered in both multiresistant and blood culture isolates. Meropenem resistance was detected for the first time in 2011 in a locally isolated bla IMP-4-positive K. pneumoniae. We found sequence types ST23 and ST86 that occurred in multiple isolates from invasive infections. ST86 was the most common and maintained a high degree (90 %) of similarity by PFGE since 1977. Ceftazidime-resistant K. pneumoniae sequence types have caused invasive infections in Western Australia since 1993. Invasive isolates producing CTX-M-14 and CTX-M-15 appeared in Western Australia during the last decade, before the appearance of carbapenemases. The diversity of β-lactam resistance and β-lactamase resistance mechanisms in Western Australian K. pneumoniae has increased since ESBLs were first described locally.