Small Colony Variant Phenotype Research Articles

Manuka honey as a non-antibiotic alternative against Staphylococcus spp. and their small colony variant (SCVs) phenotypes

The antibiotic resistance (ABR) crisis is an urgent global health priority. Staphylococci are among the problematic bacteria contributing to this emergency owing to their recalcitrance to many clinically important antibiotics. Staphylococcal pathogenesis is further complicated by the presence of small colony variants (SCVs), a bacterial subpopulation displaying atypical characteristics including retarded growth, prolific biofilm formation, heightened antibiotic tolerance, and enhanced intracellular persistence. These capabilities severely impede current chemotherapeutics, resulting in chronic infections, poor patient outcomes, and significant economic burden. Tackling ABR requires alternative measures beyond the conventional options that have dominated treatment regimens over the past 8 decades. Non-antibiotic therapies are gaining interest in this arena, including the use of honey, which despite having ancient therapeutic roots has now been reimagined as an alternative treatment beyond just traditional topical use, to include the treatment of an array of difficult-to-treat staphylococcal infections. This literature review focused on Manuka honey (MH) and its efficacy as an anti-staphylococcal treatment. We summarized the studies that have used this product and the technologies employed to study the antibacterial mechanisms that render MH a suitable agent for the management of problematic staphylococcal infections, including those involving staphylococcal SCVs. We also discussed the status of staphylococcal resistance development to MH and other factors that may impact its efficacy as an alternative therapy to help combat ABR.

Klebsiella pneumoniae exhibiting a phenotypic hyper-splitting phenomenon including the formation of small colony variants.

In this study, we characterized a Klebsiella pneumoniae strain in a patient with shrapnel hip injury, which resulted in multiple phenotypic changes, including the formation of a small colony variant (SCV) phenotype. Although already described since the 1960s, there is little knowledge about SCV phenotypes in Enterobacteriaceae. The formation of SCVs has been recognized as a bacterial strategy to evade host immune responses and compromise the efficacy of antimicrobial therapies, leading to persistent and recurrent courses of infections. In this case, 14 isolates with different resisto- and morpho-types were distinguished from the patient's urine and tissue samples. Whole genome sequencing revealed that all isolates were clonally identical belonging to the K. pneumoniae high-risk sequence type 147. Subculturing the SCV colonies consistently resulted in the reappearance of the initial SCV phenotype and three stable normal-sized phenotypes with distinct morphological characteristics. Additionally, an increase in resistance was observed over time in isolates that shared the same colony appearance. Our findings highlight the complexity of bacterial behavior by revealing a case of phenotypic "hyper-splitting" in a K. pneumoniae SCV and its potential clinical significance.

Bigger problems from smaller colonies: emergence of antibiotic-tolerant small colony variants of Mycobacterium avium complex in MAC-pulmonary disease patients.

Mycobacterium avium complex (MAC) is a group of slow-growing mycobacteria that includes Mycobacterium avium and Mycobacterium intracellulare. MAC pulmonary disease (MAC-PD) poses a threat to immunocompromised individuals and those with structural pulmonary diseases worldwide. The standard treatment regimen for MAC-PD includes a macrolide in combination with rifampicin and ethambutol. However, the treatment failure and disease recurrence rates after successful treatment remain high. In the present study, we investigated the unique characteristics of small colony variants (SCVs) isolated from patients with MAC-PD. Furthermore, revertant (RVT) phenotype, emerged from the SCVs after prolonged incubation on 7H10 agar. We observed that SCVs exhibited slower growth rates than wild-type (WT) strains but had higher minimum inhibitory concentrations (MICs) against multiple antibiotics. However, some antibiotics showed low MICs for the WT, SCVs, and RVT phenotypes. Additionally, the genotypes were identical among SCVs, WT, and RVT. Based on the MIC data, we conducted time-kill kinetic experiments using various antibiotic combinations. The response to antibiotics varied among the phenotypes, with RVT being the most susceptible, WT showing intermediate susceptibility, and SCVs displaying the lowest susceptibility. In conclusion, the emergence of the SCVs phenotype represents a survival strategy adopted by MAC to adapt to hostile environments and persist during infection within the host. Additionally, combining the current drugs in the treatment regimen with additional drugs that promote the conversion of SCVs to RVT may offer a promising strategy to improve the clinical outcomes of patients with refractory MAC-PD.

Molecular characterization of a carbon dioxide-dependent Proteus mirabilis small-colony variant isolated from a clinical specimen

IntroductionCarbon dioxide-dependent Proteus mirabilis has been isolated from clinical specimens. It is not clear whether mutations in carbonic anhydrase are responsible for the carbon dioxide dependence of P. mirabilis. The pathogenicity of carbon dioxide-dependent P. mirabilis also remains unclear. The purpose of this study was to determine the cause carbon dioxide dependence of P. mirabilis and its pathogenicity. MethodsThe DNA sequence of can encoding carbonic anhydrase of a carbon dioxide-dependent P. mirabilis small colony variant (SCV) isolate was analyzed. To confirm that impaired carbonic anhydrase activity is responsible for the formation of the carbon dioxide-dependent SCV phenotype of P. mirabilis, we performed complementation experiments using plasmids with intact can. Additionally, mouse infection experiments were performed to confirm the change in virulence due to the mutation of carbonic anhydrase. ResultsWe found that the can gene of the carbon dioxide-dependent P. mirabilis SCV isolate showed had a frameshift mutation with a deletion of 1 bp (c. 173delC). The can of P. mirabilis encodes carbonic anhydrase was also found to function in Escherichia coli. The cause of the carbon dioxide-dependent SCV phenotype of P. mirabilis was an abnormality in carbonic anhydrase. Nevertheless, no changes were observed in virulence due to the mutation of carbonic anhydrase in mouse infection experiments. ConclusionsThe can gene is essential for the growth of P. mirabilis in ambient air. The mechanisms underlying this fitness advantage in terms of infection warrant further investigation.

The first genomic characterization of a stable, hemin-dependent small colony variant strain of Staphylococcus epidermidis isolated from a prosthetic-joint infection

Phenotype switching from a wild type (WT) to a slow-growing subpopulation, referred to as small colony variants (SCVs), supports an infectious lifestyle of Staphylococcus epidermidis, the leading cause of medical device-related infections. Specific mechanisms underlying formation of SCVs and involved in the shaping of their pathogenic potential are of particular interest for stable strains as they have been only rarely cultured from clinical specimens. As the SCV phenotype stability implies the existence of genetic changes, the whole genome sequence of a stable, hemin-dependent S. epidermidis SCV strain (named 49SCV) involved in a late prosthetic joint infection was analyzed. The strain was isolated in a monoculture without a corresponding WT clone, therefore, its genome was compared against five reference S. epidermidis strains (ATCC12228, ATCC14990, NBRC113846, O47, and RP62A), both at the level of the genome structure and coding sequences. According to the Multilocus Sequence Typing analysis, the 49SCV strain represented the sequence type 2 (ST2) regarded as the most prominent infection-causing lineage with a worldwide dissemination. Genomic features unique to 49SCV included the absence of the Staphylococcal Cassette Chromosome (SCC), ~12 kb deletion with the loss of genes involved in the arginine deiminase pathway, and frameshift-generating mutations within the poly(A) and poly(T) homopolymeric tracts. Indels were identified in loci associated with adherence, metabolism, stress response, virulence, and cell wall synthesis. Of note, deletion in the poly(A) of the hemA gene has been considered a possible trigger factor for the phenotype transition and hemin auxotrophy in the strain. To our knowledge, the study represents the first genomic characterization of a clinical, stable and hemin-dependent S. epidermidis SCV strain. We propose that previously unreported indels in the homopolymeric tracts can constitute a background of the SCV phenotype due to a resulting truncation of the corresponding proteins and their possible biological dysfunction. Streamline of genetic content evidenced by the loss of the SCC and a large genomic deletion can represent a possible strategy associated both with the SCV phenotype and its adaptation to chronicity.

Combinatorial efficacy of Manuka honey and antibiotics in the in vitro control of staphylococci and their small colony variants

IntroductionStaphylococci are among the list of problematic bacteria contributing to the global antibiotic resistance (ABR) crisis. Their ability to adopt the small colony variant (SCV) phenotype, induced by prolonged antibiotic chemotherapy, complicates staphylococcal infection control options. Novel and alternative approaches are needed to tackle staphylococcal infections and curb ABR. Manuka honey (MH), a non-antibiotic alternative is recognized for its unique antibacterial activity based on its methylglyoxal (MGO) component.MethodsIn this study, MH (MGO 830+) was tested in combination with gentamicin (GEN), rifampicin (RIF), or vancomycin (VA) against staphylococcal wildtype (WT) and SCVs. To our knowledge, there are no current studies in the literature documenting the effects of MH on staphylococcal SCVs. While Staphylococcus aureus is well-studied for its international ABR burden, limited data exists demonstrating the effects of MH on S. epidermidis and S. lugdunensis whose pathogenic relevance and contribution to ABR is also rising.Results & discussionThe three staphylococci were most susceptible to RIF (0.06-0.24 μg/ml), then GEN (0.12-0.49 μg/ml), and lastly VA (0.49-0.96 μg/ml). The MICs of MH were 7%, 7-8%, and 6-7% (w/v), respectively. Fractional inhibitory concentration (FIC) evaluations showed that the combined MH + antibiotic effect was either additive (FICI 1-2), or partially synergistic (FICI >0.5-1). While all three antibiotics induced SCVs in vitro, stable SCVs were observed in GEN treatments only. The addition of MH to these GEN-SCV-induction analyses resulted in complete suppression of SCVs (p<0.001) in all three staphylococci, suggesting that MH’s antibacterial properties interfered with GEN’s SCV induction mechanisms. Moreover, the addition of MH to growth cultures of recovered stable SCVs resulted in the inhibition of SCV growth by at least 99%, indicating MH’s ability to prevent subsequent SCV growth. These in vitro analyses demonstrated MH’s broad-spectrum capabilities not only in improving WT staphylococci susceptibility to the three antibiotics, but also mitigated the development and subsequent growth of their SCV phenotypes. MH in combination with antibiotics has the potential to not only resensitize staphylococci to antibiotics and consequently require less antibiotic usage, but in instances where prolonged chemotherapy is employed, the development and growth of SCVs would be hampered, providing a better clinical outcome, all of which mitigate ABR.

P242 Small colony variants of Staphylococcus aureus often exhibit a mucoid phenotype in the airways of people with cystic fibrosis

In a recent multicenter study, we determined the prevalence (cross-sectional) and the persistence of mucoid Staphylococcus aureus in people with cystic fibrosis (pwCF) in a one-year prospective study: from 41 of 451 S. aureus-positive pwCF from 13 centers (9.1%) mucoid S. aureus was cultured (unpublished observation). SCVs have been shown to be associated with more severe lung disease. It is not known whether mucoid isolates also exhibit the small colony variant (SCV) phenotype. Therefore, we analyzed the study isolates according to the SCV phenotype.

Genomic Modification of TonB and Emergence of Small-Colony Phenotype in VIM- and NDM-Producing Escherichia coli following Cefiderocol Exposure In Vitro.

Knowledge on resistance mechanisms toward cefiderocol, a novel siderophore-conjugated cephalosporin antibiotic, is still limited. Although the presence of New-Delhi metallo-β-lactamase has been demonstrated to facilitate the resistance development toward cefiderocol via siderophore receptor mutations in Enterobacter cloacae and Klebsiella pneumoniae, the impact of metallo-β-lactamases on facilitating such mutations in Escherichia coli is not yet elucidated. Our study aimed to study the effect of the presence of various β-lactamases, such as NDM-5, VIM-1, KPC-2, and OXA-48, on the development of cefiderocol resistance in E. coli. To this end, we performed liquid mating to transfer these β-lactamases onto a defined K-12 E. coli background (J53) and exposed these transconjugants to increasing cefiderocol concentrations in a serial passage experiment. Cefiderocol-resistant isolates were genotyped by whole-genome sequencing to investigate the underlying resistance mechanism. Cefiderocol-resistant isolates emerged only in isolates producing VIM-1 and NDM-5 metallo-β-lactamase, but not in those producing the serine β-lactamases KPC-2 and OXA-48. We observed two distinct morphological changes of the J53 E. coli strain exhibiting reduced colony size after insertions of transposable elements in the tonB gene leading to alterations in the TonB binding site and morphological changes consistent with the small-colony variant (SCV) phenotype due to mutations in the hemB and hemH genes. Passaging experiments suggested that these phenotypes were highly plastic. The SCV phenotype is attributed to immune evasion and decreased susceptibility toward antibiotics. The emergence of SCV following cefiderocol exposure may have clinical implications for bacterial clearance and warrants further investigation.

Emergence of Small Colony Variants Is an Adaptive Strategy Used by Pseudomonas aeruginosa to Mitigate the Effects of Redox Imbalance.

The ability to generate a subpopulation of small colony variants (SCVs) is a conserved feature of Pseudomonas aeruginosa and could represent a key adaptive strategy to colonize and persist in multiple niches. However, very little is known about the role of the SCV phenotype, the conditions that promote its emergence, and its possible involvement in an adaptive strategy. In the present work, we investigated the in vitro selective conditions promoting the emergence of SCVs from the prototypical strain PA14, which readily forms SCVs in nonagitated standing cultures. We found that O2 limitation, which causes a redox imbalance, is the main factor selecting for the SCV phenotype, which promotes survival of the population via formation of a biofilm at the air-liquid interface to access the electron acceptor. When this selective pressure is relieved by aeration or supplementation of an alternative electron acceptor, SCVs are barely detectable. We also observed that SCV emergence contributes to redox rebalancing, suggesting that it is involved in an adaptive strategy. We conclude that selection for the SCV phenotype is an adaptive solution adopted by P. aeruginosa to access poorly available O2. IMPORTANCE The bacterium Pseudomonas aeruginosa is an opportunistic pathogen that thrives in many environments. It poses a significant health concern, notably because it is a causative agent of nosocomial infections and the most prevalent pathogen found in the lungs of people with cystic fibrosis. In infected hosts, its persistence is often related to the emergence of an alternative phenotype known as small colony variant (SCV). Identification of conditions selecting for the SCV phenotype contributes to knowledge regarding adaptive mechanisms exploited by P. aeruginosa to survive in multiple niches and persist during infections. Hindering this adaptation strategy could help control persistent P. aeruginosa infections.

Iron restriction induces the small-colony variant phenotype in Staphylococcus aureus.

Pathogens such as Staphylococcus aureus must overcome host-induced selective pressures, including limited iron availability. To cope with the harsh conditions of the host environment, S. aureus can adapt its physiology in multiple ways. One of these adaptations is the fermenting small-colony variant (SCV) phenotype, which is known to be inherently tolerant to certain classes of antibiotics and heme toxicity. We hypothesized that SCVs might also behave uniquely in response to iron starvation since one of the major cellular uses of iron is the respiration machinery. In this study, a respiring strain of S. aureus and fermenting SCV strains were treated with different concentrations of the iron chelator, 2,2' dipyridyl (DIP). Our data demonstrate that a major impact of iron starvation in S. aureus is the repression of respiration and the induction of the SCV phenotype. We demonstrate that the SCV phenotype transiently induced by iron starvation mimics the aminoglycoside recalcitrance exhibited by genetic SCVs. Furthermore, prolonged growth in iron starvation promotes increased emergence of stable aminoglycoside-resistant SCVs relative to the naturally occurring subpopulation of SCVs within an S. aureus community. These findings may have relevance to physiological and evolutionary processes occurring within bacterial populations infecting iron-limited host environments.

Priority changes between biofilm exopolysaccharides synthesis and rhamnolipids production are mediated by ac-di-GMP-specific phosphodiesterase NbdA inPseudomonas aeruginosa.

The synthesis of biofilm exopolysaccharides and rhamnolipids (RLs) are two interrelated processes in Pseudomonas aeruginosa, but how bacteria coordinate these two processes remains unclear. We collected a P.aeruginosa KT1115 with rugose small colony variant (RSCV) phenotype from soil, and used it to study the dynamic regulation mechanism of biofilm polysaccharide and RLs synthesis. The results showed that the overproduction of biofilm exopolysaccharides at biofilm stage ultimately contributed the surge of RLs production at RLs stage. This phenomenon was further verified by comparing PAO1 with its engineered RSCV mutant, PAO1ΔwspF. Further genomic, transcriptomic analyses and gene deletion revealed that downregulation of c-di-GMP level was the key to switch biofilm exopolysaccharides accumulation to RLs surge, by transcriptionally upregulating a c-di-GMP phosphodiesterase NbdA. Overall, this study demonstrates the importance of c-di-GMP in coordinating biofilm exopolysaccharides and RLs synthesis, and provides an inspiration for enhancing RLs production through regulating c-di-GMP level.

Pseudomonas aeruginosa Strains from Both Clinical and Environmental Origins Readily Adopt a Stable Small-Colony-Variant Phenotype Resulting from Single Mutations in c-di-GMP Pathways.

A subpopulation of small-colony variants (SCVs) is a frequently observed feature of Pseudomonas aeruginosa isolates obtained from colonized cystic fibrosis lungs. Since most SCVs have until now been isolated from clinical samples, it remains unclear how widespread the ability of P. aeruginosa strains to develop this phenotype is and what the genetic mechanism(s) behind the emergence of SCVs are according to the origin of the isolate. In the present work, we investigated the ability of 22 P. aeruginosa isolates from various environmental origins to spontaneously adopt an SCV-like smaller alternative morphotype distinguishable from that of the ancestral parent strain under laboratory culture conditions. We found that all the P. aeruginosa strains tested could adopt an SCV phenotype, regardless of their origin. Whole-genome sequencing of SCVs obtained from clinical and environmental sources revealed single mutations exclusively in two distinct c-di-GMP signaling pathways, the Wsp and YfiBNR pathways. We conclude that the ability to switch to an SCV phenotype is a conserved feature of P. aeruginosa and results from the acquisition of a stable genetic mutation, regardless of the origin of the strain. IMPORTANCE P. aeruginosa is an opportunistic pathogen that thrives in many environments. It poses a significant health concern, notably because this bacterium is the most prevalent pathogen found in the lungs of people with cystic fibrosis. In infected hosts, its persistence is considered related to the emergence of an alternative small-colony-variant (SCV) phenotype. By reporting the distribution of P. aeruginosa SCVs in various nonclinical environments and the involvement of c-di-GMP in SCV emergence from both clinical and environmental strains, this work contributes to understanding a conserved adaptation mechanism used by P. aeruginosa to adapt readily in all environments. Hindering this adaptation strategy could help control persistent infection by P. aeruginosa.

Molecular Mechanisms of Staphylococcus and Pseudomonas Interactions in Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder that is characterized by recurrent and chronic infections of the lung predominantly by the opportunistic pathogens, Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa. While S. aureus is the main colonizing bacteria of the CF lungs during infancy and early childhood, its incidence declines thereafter and infections by P. aeruginosa become more prominent with increasing age. The competitive and cooperative interactions exhibited by these two pathogens influence their survival, antibiotic susceptibility, persistence and, consequently the disease progression. For instance, P. aeruginosa secretes small respiratory inhibitors like hydrogen cyanide, pyocyanin and quinoline N-oxides that block the electron transport pathway and suppress the growth of S. aureus. However, S. aureus survives this respiratory attack by adapting to respiration-defective small colony variant (SCV) phenotype. SCVs cause persistent and recurrent infections and are also resistant to antibiotics, especially aminoglycosides, antifolate antibiotics, and to host antimicrobial peptides such as LL-37, human β-defensin (HBD) 2 and HBD3; and lactoferricin B. The interaction between P. aeruginosa and S. aureus is multifaceted. In mucoid P. aeruginosa strains, siderophores and rhamnolipids are downregulated thus enhancing the survival of S. aureus. Conversely, protein A from S. aureus inhibits P. aeruginosa biofilm formation while protecting both P. aeruginosa and S. aureus from phagocytosis by neutrophils. This review attempts to summarize the current understanding of the molecular mechanisms that drive the competitive and cooperative interactions between S. aureus and P. aeruginosa in the CF lungs that could influence the disease outcome.

A functional menadione biosynthesis pathway is required for capsule production by Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that utilises a wide array of pathogenic and immune evasion strategies to cause disease. One immune evasion strategy, common to many bacterial pathogens, is the ability of S. aureus to produce a capsule that protects the bacteria from several aspects of the human immune system. To identify novel regulators of capsule production by S. aureus, we applied a genome wide association study (GWAS) to a collection of 300 bacteraemia isolates that represent the two major MRSA clones in UK and Irish hospitals: CC22 and CC30. One of the loci associated with capsule production, the menD gene, encodes an enzyme critical to the biosynthesis of menadione. Mutations in this gene that result in menadione auxotrophy induce the slow growing small-colony variant (SCV) form of S. aureus often associated with chronic infections due to their increased resistance to antibiotics and ability to survive inside phagocytes. Utilising such an SCV, we functionally verified this association between menD and capsule production. Although the clinical isolates with polymorphisms in the menD gene in our collections had no apparent growth defects, they were more resistant to gentamicin when compared to those with the wild-type menD gene. Our work suggests that menadione is involved in the production of the S. aureus capsule, and that amongst clinical isolates polymorphisms exist in the menD gene that confer the characteristic increased gentamicin resistance, but not the major growth defect associated with SCV phenotype.

The MpsB protein contributes to both the toxicity and immune evasion capacity of Staphylococcus aureus.

Understanding the role specific bacterial factors play in the development of severe disease in humans is critical if new approaches to tackle such infections are to be developed. In this study we focus on genes we have found to be associated with patient outcome following bacteraemia caused by the major human pathogen Staphylococcus aureus . By examining the contribution these genes make to the ability of the bacteria to survive exposure to the antibacterial factors found in serum, we identify three novel serum resistance-associated genes, mdeA, mpsB and yycH. Detailed analysis of an MpsB mutant supports its previous association with the slow growing small colony variant (SCV) phenotype of S. aureus , and we demonstrate that the effect this mutation has on membrane potential prevents the activation of the Agr quorum sensing system, and as a consequence the mutant bacteria do not produce cytolytic toxins. Given the importance of both toxin production and immune evasion for the ability of S. aureus to cause disease, we believe that these findings explain the role of the mpsB gene as a mortality-associated locus during human disease.

Methicillin-resistant Staphylococcus aureus of the clonal lineage ST5-SCCmecII-t2460 was associated with high mortality in a Wuhan hospital

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that can cause serious infectious diseases. An emerging MRSA strain, ST5-SCCmecII spa-type-t2460 (SMRSA), has spread rapidly since its recent emergence in China, but little information is available about this lineage. In this study, 91 MRSA isolates were collected from patients treated in the Zhongnan Hospital, Wuhan University, from 2018 to 2019, and investigated for their molecular characteristics, antibiotic resistance profiles, and clinical characteristics. The predominant lineage, SMRSA, accounted for 37.4% (34/91) of the isolates, followed by ST239-SCCmecIII-t030 (19.8%, 18/91) and ST59-SCCmecIV-t437 (8.8%, 8/91). In contrast to the latter two non-SMRSA (nSMRSA) lineages, which are among the main MRSA found in Chinese settings, SMRSA exhibited small colony variant (SCV) phenotype and had extremely high resistance rates to erythromycin (100.0%), clindamycin (100.0%), levofloxacin (100.0%), tetracycline (97.1%), moxifloxacin (97.1%), and ciprofloxacin (100%), but was more susceptible to rifampicin (resistance rate 3%). The levels of white blood cells (WBC) and procalcitonin (PCT) and the 30-day mortality in patients infected with SMRSA were (12.54 ± 6.61) × 109/L, 0.66 ng/mL, and 52.9%, respectively, which were much higher than those in patients infected with nSMRSA. In addition, patients infected with SMRSA were more frequently admitted to the intensive care unit (ICU) and submitted to invasive procedures than those infected with nSMRSA. In conclusion, SMRSA showed SCV phenotype and exhibited multiple antibiotic-resistance profiles. In this study, SMRSA was associated with serious infections and poor prognosis. Compared with ST239, ST59, or other nSMRSA strains, patients infected with SMRSA strains have higher 30-day mortality, increased levels of inflammatory biomarkers, and more frequent ICU hospitalization and invasive procedures.

Haem toxicity provides a competitive advantage to the clinically relevant Staphylococcus aureus small colony variant phenotype.

Microorganisms encounter toxicities inside the host. Many pathogens exist as subpopulations to maximize survivability. Subpopulations of Staphylococcus aureus include antibiotic-tolerant small colony variants (SCVs). These mutants often emerge following antibiotic treatment but can be present in infections prior to antibiotic exposure. We hypothesize that haem toxicity in the host selects for respiration-deficient S. aureus SCVs in the absence of antibiotics. We demonstrate that some but not all respiration-deficient SCV phenotypes are more protective than the haem detoxification system against transient haem exposure, indicating that haem toxicity in the host may contribute to the dominance of menaquinone-deficient and haem-deficient SCVs prior to antibiotic treatment.

Inactivation of genes in oxidative respiration and iron acquisition pathways in pediatric clinical isolates of Small colony variant Enterobacteriaceae

Isolation of bacterial small colony variants (SCVs) from clinical specimens is not uncommon and can fundamentally change the outcome of the associated infections. Bacterial SCVs often emerge with their normal colony phenotype (NCV) co-isolates in the same sample. The basis of SCV emergence in vivo is not well understood in Gram-negative bacteria. In this study, we interrogated the causal genetic lesions of SCV growth in three pairs of NCV and SCV co-isolates of Escherichia coli, Citrobacter freundii, and Enterobacter hormaechei. We confirmed SCV emergence was attributed to limited genomic mutations: 4 single nucleotide variants in the E. coli SCV, 5 in C. freundii, and 8 in E. hormaechei. In addition, a 10.2 kb chromosomal segment containing 11 genes was deleted in the E. hormaechei SCV isolate. Each SCV had at least one coding change in a gene associated with bacterial oxidative respiration and another involved in iron capture. Chemical and genetic rescue confirmed defects in heme biosynthesis for E. coli and C. freundii and lipoic acid biosynthesis in E. hormaachei were responsible for the SCV phenotype. Prototrophic growth in all 3 SCV Enterobacteriaceae species was unaffected under anaerobic culture conditions in vitro, illustrating how SCVs may persist in vivo.

An ex vivo cystic fibrosis model recapitulates key clinical aspects of chronic Staphylococcus aureus infection.

Staphylococcus aureus is the most prevalent organism isolated from the airways of people with cystic fibrosis (CF), predominantly early in life. Yet its role in the pathology of lung disease is poorly understood. In mice, and many experiments using cell lines, the bacterium invades cells or interstitium, and forms abscesses. This is at odds with the limited available clinical data: interstitial bacteria are rare in CF biopsies and abscesses are highly unusual. Bacteria instead appear to localize in mucus plugs in the lumens of bronchioles. We show that, in an established ex vivo model of CF infection comprising porcine bronchiolar tissue and synthetic mucus, S. aureus demonstrates clinically significant characteristics including colonization of the airway lumen, with preferential localization as multicellular aggregates in mucus, initiation of a small colony variant phenotype and increased antibiotic tolerance of tissue-associated aggregates. Tissue invasion and abscesses were not observed. Our results may inform ongoing debates relating to clinical responses to S. aureus in people with CF.

A novel hemA mutation is responsible for a small-colony-variant phenotype in Escherichia coli.

We identified a small colony variant (SCV) of an amoxicillin/clavulanic acid-resistant derivative of a clinical isolate of Escherichia coli from Malawi, which was selected for in vitro in a subinhibitory concentration of gentamicin. The SCV was auxotrophic for hemin and had impaired biofilm formation compared to the ancestral isolates. A single novel nucleotide polymorphism (SNP) in hemA, which encodes a glutamyl-tRNA reductase that catalyses the initial step of porphyrin biosynthesis leading to the production of haem, was responsible for the SCV phenotype. We showed the SNP in hemA resulted in a significant fitness cost to the isolate, which persisted even in the presence of hemin. However, the phenotype quickly reverted during sequential sub-culturing in liquid growth media. As hemA is not found in mammalian cells, and disruption of the gene results in a significant fitness cost, it represents a potential target for novel drug development specifically for the treatment of catheter-associated urinary tract infections caused by E. coli.