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Antimicrobial susceptibility testing (AST) has been used to guide therapy of airway infection in persons with cystic fibrosis (CF) for decades. However, evidence that AST adds benefit to treatment outcomes in CF is lacking. In fact, the routine use of AST has potential to exacerbate inappropriate antibiotic use. Several features of airway infection in CF contribute to the limitations of AST in predicting treatment outcomes, providing rationale for abandoning this practice altogether. Other features of CF infection suggest, however, that select use of AST can provide worthwhile guidance to antibiotic selection., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Stenotrophomonas maltophilia is a non-fermenting, Gram-negative bacillus that has emerged as an opportunistic nosocomial pathogen. Its intrinsic multidrug resistance makes treating infections caused by S. maltophilia a great clinical challenge. Clinical management is further complicated by its molecular heterogeneity that is reflected in the uneven distribution of antibiotic resistance and virulence determinants among different strains, the shortcomings of available antimicrobial susceptibility tests and the lack of standardized breakpoints for the handful of antibiotics with in vitro activity against this microorganism. Herein, we provide an update on the most recent literature concerning these issues, emphasizing the impact they have on clinical management of S. maltophilia infections., (Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy 2022.)

The presence of Burkholderia cepacia complex (BCC) strains has resulted in recalls of pharmaceutical products, since these opportunistic pathogens can cause serious infections. Rapid and sensitive diagnostic methods to detect BCC are crucial to determine contamination levels. We evaluated bacterial cultures, real-time PCR (qPCR), droplet digital PCR (ddPCR), and flow cytometry to detect BCC in nuclease-free water, in chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions. Twenty BCC strains were each suspended (1, 10, 100, and 1000 CFU/ml) in autoclaved nuclease-free water, 10 μg/ml CHX, and 50 μg/ml BZK. Five replicates of each strain were tested at each concentration (20 strains × 4 concentrations × 5 replicates = 400 tests) to detect BCC using the aforementioned four methods. We demonstrated the potential of ddPCR and flow cytometry as more sensitive alternatives to culture-based methods to detect BCC in autoclaved nuclease-free water and antiseptics samples.

Median cystic fibrosis (CF) survival has increased dramatically over time due to several factors, including greater availability and use of antimicrobial therapies. During the progression of CF lung disease, however, the emergence of multidrug antimicrobial resistance can limit treatment effectiveness, threatening patient longevity. Current planktonic-based antimicrobial susceptibility testing lacks the ability to predict clinical response to antimicrobial treatment of chronic CF lung infections. There are numerous reasons for these limitations including bacterial phenotypic and genotypic diversity, polymicrobial interactions, and impaired antibiotic efficacy within the CF lung environment. The parallels to other chronic diseases such as non-CF bronchiectasis are discussed as well as research priorities for moving forward., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The Burkholderia cepacia complex (BCC) includes opportunistic pathogenic bacteria that have occasionally been recovered from various pharmaceutical products, including antiseptics and disinfectants. Plausible reasons for the contamination include intrinsic sources, such as inadequate process controls, especially for water or equipment used during product manufacture, or extrinsic sources, such as improper handling and dilution or distribution in contaminated containers. Because the survival of BCC in antiseptics is a concern to the public health and pharmaceutical industry, we determined minimum inhibitory concentrations (MICs) of 36 BCC strains against the antiseptics, following exposure to chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions (1-500 µg/ml for each chemical). Susceptibility to CHX and BZK varied across the BCC strains and was recorded as mean 90.3 and 111.1 µg/ml, respectively, at initial inoculation, which was significantly higher than the 46.4 and 61.1 µg/ml levels measured for BCC incubated in water for 40 days. After determining antiseptic MICs of individual BCC strains, BCC recovery was measured on Tryptic Soy Agar (TSA), Reasoner's Second Agar (R2A) and diluted preparations of these media under their sub-MICs. The survival of BCC was monitored for 14 days (336 h) in sub-MICs diluted to less than their antiseptic susceptible concentration value. Diluted TSA and R2A media exhibited greater efficiency of recovery for most BCC strains from the CHX and BZK solutions than full strength TSA or R2A. For BCC survival in antiseptic solutions, the cell number of BCC decreased rapidly within the first 20 min in both antiseptics, but after this, recovery remained constant in CHX and increased in BZK over the 14 day incubation period. The results indicate that BCC in water can remain viable with low susceptibility to antiseptics for 14 days, which suggests the necessity for improved detection methods and control measures to monitor BCC contamination in pharmaceutical products.

Background: Herbaspirillum species are gram-negative Betaproteobacteria that inhabit the rhizosphere. We investigated a potential cluster of hospital-based Herbaspirillum species infections., Methods: Cases were defined as Herbaspirillum species isolated from a patient in our comprehensive cancer center between 1 January 2006 and 15 October 2013. Case finding was performed by reviewing isolates initially identified as Burkholderia cepacia susceptible to all antibiotics tested, and 16S ribosomal DNA sequencing of available isolates to confirm their identity. Pulsed-field gel electrophoresis (PFGE) was performed to test genetic relatedness. Facility observations, infection prevention assessments, and environmental sampling were performed to investigate potential sources of Herbaspirillum species., Results: Eight cases of Herbaspirillum species were identified. Isolates from the first 5 clustered cases were initially misidentified as B. cepacia, and available isolates from 4 of these cases were indistinguishable. The 3 subsequent cases were identified by prospective surveillance and had different PFGE patterns. All but 1 case-patient had bloodstream infections, and 6 presented with sepsis. Underlying diagnoses included solid tumors (3), leukemia (3), lymphoma (1), and aplastic anemia (1). Herbaspirillum species infections were hospital-onset in 5 patients and community-onset in 3. All symptomatic patients were treated with intravenous antibiotics, and their infections resolved. No environmental source or common mechanism of acquisition was identified., Conclusions: This is the first report of a hospital-based cluster of Herbaspirillum species infections. Herbaspirillum species are capable of causing bacteremia and sepsis in immunocompromised patients. Herbaspirillum species can be misidentified as Burkholderia cepacia by commercially available microbial identification systems., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Burkholderia cepacia complex (BCC) presence has been the cause of recalls of both sterile and non-sterile pharmaceutical products since these opportunistic pathogens have been implicated to cause infections to susceptible individuals. BCC are ubiquitous in nature, but in pharmaceutical settings the most common source is contaminated water systems. Some strains of BCC, previously described as Pseudomonas cepacia, were not readily detected by standard culture methods. We have explored different strategies to recover and enrich Burkholderia cenocepacia previously cultured in distilled water for 40 days. Enrichment media of varied nutrient concentrations and composition were used, including modified Tryptic Soy Agar or Broth (TSA or TSB), Reasoner's 2nd Agar or Broth (R2A or R2AB), Brain-Heart Infusion Broth (BHIB), Mueller-Hinton Broth (MHB), and Ashdown's (ASH) medium. Of the various broth media tested, cell growth was significantly greater in TSB and R2AB than in BHIB, MHB, or ASH broth. TSB and R2AB were also compared for their recovery efficiency. Generally, there was no significant difference between the numbers of B. cenocepacia grown on 15 differently modified TSA and five modified R2A solid media. Overall, however, diluted TSA and TSB media, and R2A and R2AB showed better recovery efficiency than TSA and TSB for inocula containing small numbers of cells. All strains persisted in distilled water for 40 days. Broth media were more effective than solid media for recovery of B. cenocepacia from distilled water. These results may assist in improving detection assays with recovery and enrichment strategies to maximize recovery of these fastidious organisms.

Background: New therapeutic targets for antibiotic-resistant bacterial pathogens are desperately needed. The bacterial surface polysaccharide poly-β-(1-6)-N-acetyl-glucosamine (PNAG) mediates biofilm formation by some bacterial species, and antibodies to PNAG can confer protective immunity. By analyzing sequenced genomes, we found that potentially multidrug-resistant bacterial species such as Klebsiella pneumoniae, Enterobacter cloacae, Stenotrophomonas maltophilia, and the Burkholderia cepacia complex (BCC) may be able to produce PNAG. Among patients with cystic fibrosis patients, highly antibiotic-resistant bacteria in the BCC have emerged as problematic pathogens, providing an impetus to study the potential of PNAG to be targeted for immunotherapy against pan-resistant bacterial pathogens., Methods: The presence of PNAG on BCC was assessed using a combination of bacterial genetics, microscopy, and immunochemical approaches. Antibodies to PNAG were tested using opsonophagocytic assays and for protective efficacy against lethal peritonitis in mice., Results: PNAG is expressed in vitro and in vivo by the BCC, and cystic fibrosis patients infected by the BCC species B. dolosa mounted a PNAG-specific opsonophagocytic antibody response. Antisera to PNAG mediated opsonophagocytic killing of BCC and were protective against lethal BCC peritonitis even during coinfection with methicillin-resistant Staphylococcus aureus., Conclusions: Our findings raise potential new therapeutic options against PNAG-producing bacteria, including even pan-resistant pathogens.

The therapeutic potential of bacteriophages (phages) in a mouse model of acute Burkholderia cenocepacia pulmonary infection was assessed. Phage treatment was administered by either intranasal inhalation or intraperitoneal injection. Bacterial density, macrophage inflammatory protein 2 (MIP-2), and tumor necrosis factor alpha (TNF-alpha) levels were significantly reduced in lungs of mice treated with intraperitoneal phages (P < .05). No significant differences in lung bacterial density or MIP-2 levels were found between untreated mice and mice treated with intranasal phages, intraperitoneal ultraviolet-inactivated phages, or intraperitoneal lambda phage control mice. Mock-infected mice treated with phage showed no significant increase in lung MIP-2 or TNF-alpha levels compared with mock-infected/mock-treated mice. We have demonstrated the efficacy of phage therapy in an acute B. cenocepacia lung infection model. Systemic phage administration was more effective than inhalational administration, suggesting that circulating phages have better access to bacteria in lungs than do topical phages.

The epidemiology of Burkholderia infection in persons with chronic granulomatous disease is poorly understood. We used species-specific polymerase chain reaction-based assays and genotyping analyses to identify 32 strains representing 9 Burkholderia species among 50 isolates recovered from 18 patients with chronic granulomatous disease. We found that recurrent pulmonary infection with distinct Burkholderia strains is common in chronic granulomatous disease.

The Burkholderia cepacia complex includes 9 genomovars. The relative virulence of each is unknown. Host and pathogen features associated with mortality were evaluated among patients with B. cepacia complex bacteremia. Cases were ascertained through review of blood culture results for the period of May 1996 through May 2002. Isolates were identified to species level with 16S rDNA and recA-based species-specific polymerase chain reaction analyses and recA restriction fragment-length polymorphism. Strain typing was performed with pulsed-field gel electrophoresis. Fifty-three patients with B. cepacia complex bacteremia were identified; only 9 (17%) had cystic fibrosis. Twenty-five patients (47%) died within 14 days of bacteremia. After controlling for comorbid conditions and therapeutic interventions, 2 outbreak-related strains of Burkholderia cenocepacia (genomovar III) were associated with 14-day mortality (odds ratio, 5.5; 95% confidence interval, 1.20-25.02). B. cenocepacia is an emerging nosocomial pathogen. Certain strains are associated with an enhanced capacity for interpatient spread and poor outcome.

Objectives: To determine the susceptibility of Burkholderia multivorans and Burkholderia cenocepacia to bismuth-thiols (BTs), and to examine the synergistic effects of tobramycin and subinhibitory concentrations of BTs against these organisms., Methods: The susceptibilities of 25 clinical isolates each of B. multivorans and B. cenocepacia to six BTs were measured by broth dilution in accordance with NCCLS protocols. Ten strains were selected to evaluate the antimicrobial interaction between BTs and tobramycin. Fractional inhibitory concentration (FIC) and fractional bactericidal concentration (FBC) indices were calculated to assess synergy., Results: B. multivorans and B. cenocepacia showed a wide range of susceptibilities to BTs. Bismuth ethanedithiol (BisEDT) was one of the more potent BTs against these organisms (MIC50 7.8 microM), and was selected for synergy studies. Selected strains were highly resistant to tobramycin. The addition of subinhibitory concentrations of BisEDT (2 microM) reduced the MIC and MBC of tobramycin against all strains, achieving synergy in many instances. The FIC index was in the range 0.28-0.66 and the FBC in the range 0.12-0.85. Most strains became susceptible to tobramycin at clinically achievable concentrations in the presence of non-toxic BisEDT levels., Conclusions: Treatment with subinhibitory BisEDT and tobramycin reduces the MICs and MBCs for B. multivorans and B. cenocepacia. BTs may represent an important adjunctive therapy for resistant Burkholderia cepacia complex.

Previous studies have identified specific Burkholderia cepacia complex strains that are common to multiple persons with cystic fibrosis (CF). Such so-called epidemic strains have an apparent enhanced capacity for inter-patient spread and reside primarily in Burkholderia cenocepacia (formerly B. cepacia complex genomovar III). We sought to identify strains from B. cepacia complex species other than B. cenocepacia that are similarly shared by multiple CF patients. We performed genotype analysis of 360 recent sputum culture isolates from 360 persons residing in 29 cities by using repetitive extragenic palendromic polymerase chain reaction (rep-PCR) and pulsed field gel electrophoresis. The results indicate that sharing of a common Burkholderia multivorans strain occurs relatively infrequently; however, several small clusters of patients infected with the same strain were identified. A cluster of seven patients infected with the same B. cepacia (genomovar I) strain was found. We also identified a large group of 28 patients receiving care in the same treatment center and infected with the same Burkholderia dolosa strain. These observations suggest that B. cepacia complex strains in species other than B. cenocepacia may be spread among CF patients.

Persons with cystic fibrosis (CF) are susceptible to chronic pulmonary infection due to certain Burkholderia species, but it is not clear whether this typically involves persistent infection with the same strain or sequential infection with distinct strains. We analyzed 1095 Burkholderia isolates recovered from serial sputum cultures from 379 patients with CF receiving care in 112 CF treatment centers in the United States. Genotyping was performed by random amplified polymorphic DNA typing or pulsed-field gel electrophoresis. Overall, a change in infecting strain was found in 24 (6.9%) of 347 patients infected with Burkholderia cepacia complex and in 3 (9%) of 32 patients infected with Burkholderia gladioli. Several patients were likely coinfected, at least transiently, with >1 B. cepacia complex strain. The potential for strain replacement during chronic infection may confound studies of the relationship between strain and clinical outcome and must be considered in designing effective infection-control practices.

Burkholderia cepacia complex infections contribute significantly to mortality and morbidity in persons with cystic fibrosis (CF). The aim of this study was to evaluate the use of a novel typing method, multilocus restriction typing (MLRT), for investigation of the global epidemiology of B. cepacia complex genomovar III, the species most commonly encountered in CF. In the MLRT method, variation at several loci is indexed by restriction analysis of polymerase chain reaction-amplified genes. Data obtained by MLRT and pulsed-field gel electrophoresis analysis of a large number of B. cepacia genomovar III isolates (including isolates belonging to epidemic lineages and environmental isolates) show a strong correlation. MLRT extends the utility of isolate genotyping by allowing comparisons of isolates collected in studies of larger scale (both temporal and spatial). The portability of MLRT data will facilitate comparison of data obtained in different laboratories. In addition, data obtained with MLRT can be used in studies of bacterial population structure.

The recently described genus Pandoraea consists of five named species and four unnamed genomospecies, several of which have been identified in clinical specimens including respiratory secretions from persons with cystic fibrosis. We investigated whether it is possible to distinguish species of the genus Pandoraea by means of restriction fragment length polymorphism (RFLP) analysis and direct sequencing of the gyrB gene. Sixty-seven Pandoraea isolates were included. Species-specific RFLP patterns were obtained following digestion of the PCR-amplified gyrB gene with MspI. Specificity of RFLP groupings was confirmed by direct sequencing of several representative isolates. Our results indicate that RFLP analysis and sequencing of the gyrB gene are useful for the identification of Pandoraea species. We also found that further taxonomic studies within the beta-Proteobacteria using the gyrB gene would benefit from the development of additional primers allowing more efficient amplification of the gyrB gene. Our data also indicate that the taxonomic status of Pandoraea genomospecies 2 should be reinvestigated.

Oligonucleotide primers complementary to conserved regions of the 16S and 23S ribosomal RNA genes were used to amplify the 16S-23S intergenic spacer region of bacterial pathogens. The amplification patterns produced were compared for their potential use in molecular epidemiologic analysis. This method, polymerase chain reaction (PCR) ribotyping, was applied to isolates of Staphylococcus aureus, Enterococcus faecium, Escherichia coli, and Enterobacter species. Length polymorphisms in the amplified DNA distinguished unrelated strains of all bacteria. The banding patterns of 3 S. aureus isolates from the blood of 1 patient on 3 consecutive days were identical. Plasmid analysis, biotyping, and antibiograms were also obtained on the Enterobacter isolates. All three of these methods showed considerable variability after in vitro passage of bacteria, but PCR ribotypes remained stable. Results demonstrate the utility of the conserved primers for PCR ribotyping, a widely applicable method for the molecular epidemiology of genetically diverse bacteria.

Eighty-three isolates of Pseudomonas capacia were recovered from respiratory secretions from 12 chronically colonized cystic fibrosis patients and examined by ribotype analysis. In 9 patients, the ribotype of the cultured P. cepacia remained unchanged throughout the entire period of observation, indicating chronic pulmonary colonization with a single strain. In each of the remaining 3 patients, two genetically distinct strains were detected among serial P. cepacia isolates. No significant change in clinical condition was correlated with the change in identity of the colonizing strain. In control experiments, the stability of strain ribotype was demonstrated among isolated that had been subcultured 100 times in vitro and among isolates recovered from chronically colonized mice. These data demonstrate the utility of ribotype analysis and indicate that most chronically colonized cystic fibrosis patients harbor a single strain of P. cepacia for prolonged periods.

The in-vitro activities of trospectomycin, cefpodoxime, cefamandole, cefonicid, and cefuroxime against beta-lactamase-negative and -positive invasive clinical isolates of Haemophilus influenzae type b were determined by the agar dilution method. Trospectomycin and cefpodoxime inhibited 90% of the strains at concentrations of 5 and 0.06 mg/l, respectively, and no differences between the susceptibilities of the beta-lactamase-negative and -positive strains were noted. The activity of cefpodoxime was minimally affected by increased inoculum size, but significant inoculum effects were noted with cefamandole, cefonicid, and cefuroxime with beta-lactamase positive strains.

Restriction fragment length polymorphism (RFLP) patterns, plasmid profiles, and antimicrobial susceptibility patterns of paired sequential Escherichia coli isolates from antibiotic-treated and untreated elderly women were analyzed. Isolates from 26 of 27 subjects who were treated successfully with antibiotics but became reinfected differed by RFLP analysis, whereas 10 of 12 subjects who failed treatment and 11 of 14 untreated subjects had paired isolates with identical RFLP banding patterns. Only 40 of the 53 pairs of isolates could be analyzed by plasmid profiles; 36 of these 40 were concordant with RFLP analysis. Antimicrobial susceptibility patterns showed poor concordance with RFLP analysis. This study demonstrates the utility of RFLP analysis and indicates that isolation of E. coli from elderly women after sterilization of the urinary tract usually results from introduction of a new strain; elderly women who fail antibiotic therapy or receive no therapy may remain persistently infected with the same E. coli strain.

We investigated the use of ribosomal RNA (rRNA) as a probe for molecular epidemiology of bacterial pathogens. The chromosomal DNA of Escherichia coli, Pseudomonas cepacia, and nontypable Haemophilus influenzae was digested with EcoRI. Agarose gel electrophoresis, Southern blotting, and hybridization by 32P-labeled rRNA revealed eight to 13 bands. The P. cepacia and H. influenzae banding patterns, observed by using an E. coli rRNA probe, were identical to those produced with homologous rRNA probes. Polymorphism of several hybridization bands distinguished all E. coli isolates, nine of 10 H. influenzae isolates, and seven of eight P. cepacia isolates. Two to four bands were common to all P. cepacia and E. coli isolates. The banding patterns of H. influenzae isolates cultured from the trachea and blood of an infant and from the mother's cervix were identical. These data demonstrate that this method is a widely applicable system for determining the molecular epidemiology of genetically diverse gram-negative organisms.