Rebekah Henry

Colistin Resistance in Acinetobacter baumannii Is Mediated by Complete Loss of Lipopolysaccharide Production

ABSTRACT Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.

Colistin-resistant, lipopolysaccharide-deficient Acinetobacter baumannii responds to lipopolysaccharide loss through increased expression of genes involved in the synthesis and transport of lipoproteins, phospholipids and poly-β-1,6-N-acetylglucosamine.

ABSTRACT We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis (Moffatt JH, et al., Antimicrob. Agents Chemother. 54:4971–4977). Consequently, strains harboring these mutations are unable to produce the major Gram-negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC 19606 to that of an isogenic, LPS-deficient, lpxA mutant strain. The analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, upregulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-β-1,6-N-acetylglucosamine (PNAG) also were upregulated, and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited the reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein AssC in culture supernatants of the A. baumannii wild-type strain but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface.

Major Surface Protein LipL32 Is Not Required for Either Acute or Chronic Infection with Leptospira interrogans

ABSTRACT Leptospira interrogans is responsible for leptospirosis, a zoonosis of worldwide distribution. LipL32 is the major outer membrane protein of pathogenic leptospires, accounting for up to 75% of total outer membrane protein. In recent times LipL32 has become the focus of intense study because of its surface location, dominance in the host immune response, and conservation among pathogenic species. In this study, an lipL32 mutant was constructed in L. interrogans using transposon mutagenesis. The lipL32 mutant had normal morphology and growth rate compared to the wild type and was equally adherent to extracellular matrix. Protein composition of the cell membranes was found to be largely unaffected by the loss of LipL32, with no obvious compensatory increase in other proteins. Microarray studies found no obvious stress response or upregulation of genes that may compensate for the loss of LipL32 but did suggest an association between LipL32 and the synthesis of heme and vitamin B12. When hamsters were inoculated by systemic and mucosal routes, the mutant caused acute severe disease manifestations that were indistinguishable from wild-type L. interrogans infection. In the rat model of chronic infection, the LipL32 mutant colonized the renal tubules as efficiently as the wild-type strain. In conclusion, this study showed that LipL32 does not play a role in either the acute or chronic models of infection. Considering the abundance and conservation of LipL32 among all pathogenic Leptospira spp. and its absence in saprophytic Leptospira, this finding is remarkable. The role of this protein in leptospiral biology and pathogenesis thus remains elusive.

Genome-wide transposon mutagenesis in pathogenic Leptospira species.

ABSTRACT Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans.

Leptospira interrogans requires heme oxygenase for disease pathogenesis

We recently characterised the Leptospira interrogans heme oxygenase (hemO) gene and showed that HemO was required for growth with hemoglobin as the sole iron source. Here we investigated the role of HemO in pathogenesis. Hamsters inoculated with the hemO mutant showed 83% survival, compared with 33% for a control mutant (intergenic transposon insertion). Lung pathology was consistent with survival data, showing that HemO contributes significantly to pathogenesis and heme is a major in vivo iron source for L. interrogans. This is only the second defined, attenuated mutant in pathogenic Leptospira and the first to define function of the mutated gene.

Mutations affecting Leptospira interrogans lipopolysaccharide attenuate virulence

Leptospira interrogans is the causative agent of leptospirosis. Lipopolysaccharide (LPS) is the major outer membrane component of L. interrogans. It is the dominant antigen recognized during infection and the basis for serological classification. The structure of LPS and its role in pathogenesis are unknown. We describe two defined mutants of L. interrogans serovar Manilae with transposon insertions in the LPS locus. Mutant M895 was disrupted in gene la1641 encoding a protein with no known homologues. M1352 was disrupted in a gene unique to serovar Manilae also encoding a protein of unknown function. M895 produced truncated LPS while M1352 showed little or no change in LPS molecular mass. Both mutants showed altered agglutination titres against rabbit antiserum and against a panel of LPS‐specific monoclonal antibodies. The mutants were severely attenuated in virulence via the intraperitoneal route of infection, and were cleared from the host animal by 3 days after infection. M895 was also highly attenuated via the mucosal infection route. Resistance to complement in human serum was unaltered for both mutants. While complementation of mutants was not possible, the attenuation of two independently derived LPS mutants demonstrates for the first time that LPS plays an essential role leptospiral virulence.

The transcriptomic response of Acinetobacter baumannii to colistin and doripenem alone and in combination in an in vitro pharmacokinetics/pharmacodynamics model

OBJECTIVES Colistin remains a last-line treatment for MDR Acinetobacter baumannii and combined use of colistin and carbapenems has shown synergistic effects against MDR strains. In order to understand the bacterial responses to these antibiotics, we analysed the transcriptome of A. baumannii following exposure to each. METHODS RNA sequencing was employed to determine changes in the transcriptome following treatment with colistin and doripenem, both alone and in combination, using an in vitro pharmacokinetics (PK)/pharmacodynamics model to mimic the PK of both antibiotics in patients. RESULTS After treatment with colistin (continuous infusion at 2 mg/L), >400 differentially regulated genes were identified, including many associated with outer membrane biogenesis, fatty acid metabolism and phospholipid trafficking. No genes were differentially expressed following treatment with doripenem (Cmax 25 mg/L, t1/2 1.5 h) for 15 min, but 45 genes were identified as differentially expressed after 1 h of growth under this condition. Treatment of A. baumannii with both colistin and doripenem together for 1 h resulted in >450 genes being identified as differentially expressed. More than 70% of these gene expression changes were also observed following colistin treatment alone. CONCLUSIONS These data suggest that colistin causes gross damage to the outer membrane, facilitates lipid exchange between the inner and outer membrane and alters the normal asymmetric outer membrane composition. The transcriptional response to colistin was highly similar to that observed for an LPS-deficient strain, indicating that many of the observed changes are responses to outer membrane instability resulting from LPS loss.

Into the deep: Evaluation of SourceTracker for assessment of faecal contamination of coastal waters

Faecal contamination of recreational waters is an increasing global health concern. Tracing the source of the contaminant is a vital step towards mitigation and disease prevention. Total 16S rRNA amplicon data for a specific environment (faeces, water, soil) and computational tools such as the Markov-Chain Monte Carlo based SourceTracker can be applied to microbial source tracking (MST) and attribution studies. The current study applied artificial and in-laboratory derived bacterial communities to define the potential and limitations associated with the use of SourceTracker, prior to its application for faecal source tracking at three recreational beaches near Port Phillip Bay (Victoria, Australia). The results demonstrated that at minimum multiple model runs of the SourceTracker modelling tool (i.e. technical replicates) were required to identify potential false positive predictions. The calculation of relative standard deviations (RSDs) for each attributed source improved overall predictive confidence in the results. In general, default parameter settings provided high sensitivity, specificity, accuracy and precision. Application of SourceTracker to recreational beach samples identified treated effluent as major source of human-derived faecal contamination, present in 69% of samples. Site-specific sources, such as raw sewage, stormwater and bacterial populations associated with the Yarra River estuary were also identified. Rainfall and associated sand resuspension at each location correlated with observed human faecal indicators. The results of the optimised SourceTracker analysis suggests that local sources of contamination have the greatest effect on recreational coastal water quality.

Environmental monitoring of waterborne Campylobacter: evaluation of the Australian standard and a hybrid extraction-free MPN-PCR method.

Campylobacter is the leading agent of diarrheal disease worldwide. This study evaluates a novel culture-PCR hybrid (MPN-PCR) assay for the rapid enumeration of Campylobacter spp. from estuarine and wastewater systems. To first evaluate the current, culture-based, Australian standard, an inter-laboratory study was conducted on 69 subsampled water samples. The proposed Most-Probable Number (MPN)-PCR method was then evaluated, by analysing 147 estuarine samples collected over a 2 year period. Data for 14 different biological, hydrological and climatic parameters were also collated to identify pathogen-environment relationships and assess the potential for method specific bias. The results demonstrated that the intra-laboratory performance of the MPN-PCR was superior to that of AS/NZS (σ = 0.7912, P < 0.001; κ = 0.701, P < 0.001) with an overall diagnostic accuracy of ~94%. Furthermore, the analysis of both MPN-PCR and AS/NZS identified the potential for the introduction of method specific bias during assessment of the effects of environmental parameters on Campylobacter spp. numbers.

Source tracking using microbial community fingerprints: Method comparison with hydrodynamic modelling

Urban estuaries around the world are experiencing contamination from diffuse and point sources, which increases risks to public health. To mitigate and manage risks posed by elevated levels of contamination in urban waterways, it is critical to identify the primary water sources of contamination within catchments. Source tracking using microbial community fingerprints is one tool that can be used to identify sources. However, results derived from this approach have not yet been evaluated using independent datasets. As such, the key objectives of this investigation were: (1) to identify the major sources of water responsible for bacterial loadings within an urban estuary using microbial source tracking (MST) using microbial communities; and (2) to evaluate this method using a 3-dimensional hydrodynamic model. The Yarra River estuary, which flows through the city of Melbourne in South-East Australia was the focus of this study. We found that the water sources contributing to the bacterial community in the Yarra River estuary varied temporally depending on the estuarys hydrodynamic conditions. The water source apportionment determined using microbial community MST correlated to those determined using a 3-dimensional hydrodynamic model of the transport and mixing of a tracer in the estuary. While there were some discrepancies between the two methods, this investigation demonstrated that MST using bacterial community fingerprints can identify the primary water sources of microorganisms in an estuarine environment. As such, with further optimization and improvements, microbial community MST has the potential to become a powerful tool that could be practically applied in the mitigation of contaminated aquatic systems.