Phage therapy to control multidrug-resistant Pseudomonas aeruginosa skin infections: in vitro and ex vivo experiments

The main goal of this study was to evaluate the efficiency of phage therapy against one of the most common multidrug-resistant (MDR) agents of skin infections, Pseudomonas aeruginosa. A phage suspension [10⁸?plaque-forming units (PFU)?mL^?1] was obtained using the clinical strain P. aeruginosa 709 as the host. The ability of the phage to inactivate P. aeruginosa was evaluated in vitro and ex vivo (human skin), using a multiplicity of infection (MOI) of 0.5 to 50. In the presence of the phage, the density of P. aeruginosa 709 [10⁵?colony-forming units (CFU)?mL^?1] in the human skin decreased by 4 logs after 2?h of incubation. The application of a second dose of phage did not increase the efficiency of the therapy. This study indicates that the topical application of phage PA709 efficiently inactivates MDR P. aeruginosa 709. The high efficiency in the inactivation of MDR P. aeruginosa 709, its considerable host range (infection of 30?% of the P. aeruginosa isolates) and its high stability in buffer and ex vivo human skin make this phage very promising for the treatment of P. aeruginosa skin infections. The phage–bacteria interactions were examined in vitro and in ex vivo in order to provide a basis for the selection of the most suitable protocol for subsequent in vivo experiments.     

Metal nanoparticles and nanoparticle composites are effective against Haemophilus influenzae,Streptococcus pneumoniae,and multidrug-resistant bacteria

BackgroundTreatment for lower respiratory tract infection caused by multidrug-resistant organisms (MDRO) are often limited. This study explored the activity of different metal nanoparticles against several respiratory pathogens including MDROs.MethodsClinical isolates of carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Klebsiella pneumoniae (CRKP), Pseudomonas aeruginosa, Haemophilus influenzae, methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus pneumoniae were tested for in vitro susceptibilities to various antibiotics and nanoparticles. Minimum inhibitory concentrations (MICs) of silver-nanoparticle (Ag-NP), selenium-nanoparticle (Se-NP), and three composites solutions ND50, NK99, and TPNT1 (contained 5 ppm Ag-NP, 60 ppm ZnO-nanoparticle, and different concentrations of gold-nanoparticle or ClO₂) were determined by broth microdilution method.ResultsFifty isolates of each bacterial species listed above were tested. Ag-NP showed lower MICs to all species than Se-NP. The MIC₅₀s of Ag-NP for CRAB, CRKP, P. aeruginosa, and H. influenzae were <3.125 ppm, 25 ppm, <3.125 ppm, and <3.125 ppm, respectively, while those for S. pneumoniae and MRSA were >50 ppm and 50 ppm. Among CRAB, CRKP and P. aeruginosa, the MIC₅₀s of ND50, NK99, and TPNT1 for CRAB were the lowest (1/8 dilution, 1/8 dilution, and 1/8 dilution, respectively), and those for CRKP (>1/2 dilution, 1/2 dilution, and 1/2 dilution, respectively) were the highest. Both MRSA and S. pneumoniae showed high MIC₅₀s to ND50, NK99, and TPNT1.ConclusionsMetal nanoparticles had good in vitro activity against Gram-negative bacteria. They might be suitable to be prepared as environmental disinfectants or inhaled agents to inhibit the growth of MDR Gram-negative colonizers in the lower respiratory tracts of patients with chronic lung diseases.     

Multicenter surveillance of antimicrobial susceptibilities and resistance mechanisms among Enterobacterales species and non-fermenting Gram-negative bacteria from different infection sources in Taiwan from 2016 to 2018

ObjectivesTo explore the in vitro antimicrobial susceptibility among clinically important Gram-negative bacteria (GNB) in Taiwan.MethodsFrom 2016 through 2018, a total of 5458 GNB isolates, including Escherichia coli (n = 1545), Klebsiella pneumoniae (n = 1255), Enterobacter species (n = 259), Pseudomonas aeruginosa (n = 1127), Acinetobacter baumannii complex (n = 368), and Stenotrophomonas maltophilia (n = 179), were collected. The susceptibility results were summarized by the breakpoints of minimum inhibitory concentration (MIC) of CLSI 2020, EUCAST 2020 (for colistin), or published articles (for ceftolozane/tazobactam). The resistance genes among multidrug-resistant (MDR) or extensively drug-resistant (XDR)-GNB were investigated by multiplex PCR.ResultsSignificantly higher rates of non-susceptibility (NS) to ertapenem and carbapenemase production, predominantly KPC and OXA-48-like beta-lactamase, were observed in Enterobacterales isolates causing respiratory tract infection than those causing complicated urinary tract or intra-abdominal infection (12.7%/3.44% vs. 5.7%/0.76% or 7.7%/0.97%, respectively). Isolates of Enterobacter species showed higher rates of phenotypic extended-spectrum β-lactamase and NS to ertapenem than E. coli or K. pneumoniae isolates. Although moderate activity (54–83%) was observed against most potential AmpC-producing Enterobacterales isolates, ceftolozane/tazobactam exhibited poor in vitro (44.7–47.4%) activity against phenotypic AmpC Enterobacter cloacae isolates. Additionally, 251 (22.3%) P. aeruginosa isolates exhibited the carbapenem-NS phenotype, and their MDR and XDR rate was 63.3% and 33.5%, respectively. Fifteen (75%) of twenty Burkholderia cenocepacia complex isolates were inhibited by ceftolozane/tazobactam at MICs of ≤4 μg/mL.ConclusionsWith the increase in antibiotic resistance in Taiwan, it is imperative to periodically monitor the susceptibility profiles of clinically important GNB.     

Nosocomial pathogen biofilms on biomaterials: Different growth medium conditions and components of biofilms produced in vitro

Background/Purpose (s)Nosocomial pathogens can develop biofilms on hospital surfaces and medical devices; however, few studies have focused on the evaluation of mono-and dual-species biofilms developed by nosocomial pathogens under different growth conditions.MethodsThis study investigated biofilm development by nosocomial pathogens (Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) on biomaterials in different culture media and their components of the extracellular matrix biofilm.ResultsThe mono-species biofilms showed cell densities from 7.50 to 9.27 Log₁₀ CFU/cm² on natural rubber latex type I (NLTI) and from 7.58 to 8.79 Log₁₀ CFU/cm² on stainless steel (SS). Dual-species biofilms consisted of S. aureus + P. aeruginosa (7.87–8.27 Log₁₀ CFU/cm² in TSBP and TSBME onto SS; p < 0.05), E. coli + P. aeruginosa (8.32–8.86 Log₁₀ CFU/cm² in TSBME onto SS and TSBP onto NLTI; p < 0.05), and S. aureus + E. coli (7.82 Log₁₀ CFU/cm² in TSBME onto SS; p < 0.05). Furthermore, biofilm detachment after proteinase K treatment was 5.54–32.81% compared to 7.95–24.15% after DNase I treatment in the mono-dual species biofilm matrix. Epifluorescence microscopy and scanning electron microscopy (SEM) enabled visualizing the bacteria and extracellular polymeric substances of biofilms on SS and NLTI.ConclusionNosocomial pathogens can develop biofilms on biomaterials. Mono-species biofilms of Gram-negative bacteria showed lower densities than dual-species biofilms in TSBME and TSBP. Additionally, dual-species biofilms showed a higher concentration of proteins and eDNA in the extracellular matrix.     

Influence of Clinical Breakpoint Changes from CLSI 2009 to EUCAST 2011 Antimicrobial Susceptibility Testing Guidelines on Multidrug Resistance Rates of Gram-Negative Rods

Multidrug resistance (MDR) rates of Gram-negative rods were analyzed comparing CLSI 2009 and EUCAST 2011 antibiotic susceptibility testing guidelines. After EUCAST 2011 was applied, the MDR rates increased for Klebsiella pneumoniae (2.2%), Enterobacter cloacae (1.1%), Pseudomonas aeruginosa (0.7%), and Escherichia coli (0.4%). A total of 24% of Enterobacteriaceae MDR isolates and 12% of P. aeruginosa MDR isolates were categorized as MDR due to breakpoint changes.     

Molecular characterisation of antimicrobial resistance in Pseudomonas aeruginosa and Acinetobacter baumannii during 2014 and 2015 collected across India

Background: Surveillance of antimicrobial resistance (AMR) is of great importance. Pseudomonas aeruginosa and Acinetobacter baumannii are important pathogens and emergence of resistance in these have increased the morbidity and mortality rates. This surveillance study was initiated by the Government of India - Indian Council of Medical Research. The aim of this study is to determine the antimicrobial susceptibility profile and to characterise the enzyme mediated antimicrobial resistance such as extended spectrum beta-lactamases (ESBLs) and carbapenemases among multidrug-resistant (MDR) P. aeruginosa and A. baumannii. Materials and Methods: A multi-centric study was conducted from January 2014 to December 2015 with a total number of 240 MDR P. aeruginosa and 312 MDR A. baumannii isolated from blood, cerebrospinal fluid, respiratory, pus, urine and intra-abdominal infections. Kirby–Bauer disc diffusion was done to determine the antimicrobial susceptibility profile. Further, MDR isolates were characterised by multiplex polymerase chain reaction to determine the resistance genes for ESBLs and carbapenemases. Results: Among the ESBLs, bla_VEB (23%), bla_TEM (5%) and bla_SHV (0.4%) in P. aeruginosa and bla_PER (54%), bla_TEM (16%) and bla_SHV (1%) in A. baumannii were the most prevalent. Likewise, bla_VIM (37%), bla_NDM (14%), bla_GES (8%) and bla_IMP (2%) in P. aeruginosa and bla_OXA-23like (98%), bla_OXA-58like (2%), bla_NDM (22%) and bla_VIM (3%) in A. baumannii were found to be the most prevalent carbapenemases. bla_OXA-51like gene, intrinsic to A. baumannii was present in all the isolates tested. Conclusion: The data shown highlight the wide difference in the molecular mechanisms of AMR profile between P. aeruginosa and A. baumannii. In P. aeruginosa, plasmid-mediated mechanisms are much lesser than the chromosomal mediated mechanisms. In A. baumannii, class D oxacillinases are more common than other mechanisms. Continuous surveillance to monitor the trends in AMR among MDR pathogens is important for implementation of infection control and to guide appropriate empirical antimicrobial therapy.     

Activity of doripenem against extended-spectrum β-lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates

The in vitro activity of doripenem was evaluated against a recent collection of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates (201 ESBL-producing Enterobacteriaceae [153 Escherichia coli and 48 Klebsiella pneumoniae] and 201 P. aeruginosa). Comparator agents included amikacin, tobramycin, ciprofloxacin, cefepime, cefotaxime, ceftazidime piperacillin-tazobactam, imipenem, and meropenem. Both doripenem and meropenem inhibited 100% of the ESBL-producing Enterobacteriaceae at ≤0.5 μg/mL. For these isolates, the MIC₉₀ of doripenem (0.12 μg/mL) was 4-fold lower than that of imipenem (0.5 μg/mL). Against P. aeruginosa, the MIC₉₀ of doripenem and meropenem was 2 μg/mL, 4-fold lower than that of imipenem (8 μg/mL). At an MIC of ≤2 μg/mL, doripenem, meropenem, and imipenem inhibited 90.5%, 89.6%, and 82.1% of P. aeruginosa isolates, respectively. Doripenem maintained activity against imipenem-nonsusceptible isolates of P. aeruginosa; at an MIC of ≤4 μg/mL, it inhibited 15 of the 25 isolates with MICs for imipenem of >4 μg/mL. Doripenem is active against ESBL-producing Enterobacteriaceae and P. aeruginosa isolates. Its activity is similar to that of meropenem and slightly better than that of imipenem. The results of this study suggest that doripenem could be an alternative therapeutic agent for infections caused by these organisms.     

Nosocomial Outbreak of a Non-Cefepime-Susceptible Ceftazidime-Susceptible Pseudomonas aeruginosa Strain Overexpressing MexXY-OprM and Producing an Integron-Borne PSE-1 ?-Lactamase

Cefepime (FEP) and ceftazidime (CAZ) are broad-spectrum cephalosporins that display similar MICs for wild-type Pseudomonas aeruginosa strains. Recently, P. aeruginosa isolates showing a discordance in susceptibility to CAZ and FEP have been noted at the Hospital de Bellvitge in Barcelona, Spain, and a clustering was suspected. During the study period (March to December 2007), 51 patients, particularly those in an intensive care units (ICUs) (n = 29 [57%]), infected or colonized with at least one P. aeruginosa non-FEP-susceptible and CAZ-susceptible (Fep^ns Caz^s) phenotype strain were detected. Twenty-three (45%) patients were infected, and the respiratory tract was the most frequent site of infection. Changes in the consumption of antimicrobials in the ICUs were observed over time: a progressive reduction in the levels of consumption of carbapenems (247 defined daily doses [DDD]/1,000 patient days to 66 DDD/1,000 patient days; P = 0.008), after restriction of its use in 2006, and an expected increase in the rate of piperacillin-tazobactam use (42 DDD/1,000 patient days in 2004 to 200 DDD/1,000 patient days in 2007; P < 0.001). Throughout the whole study period, only a single clone of a P. aeruginosa Fep^ns Caz^s phenotype strain was identified by pulsed-field gel electrophoresis analysis to be associated with the hyperexpression of MexXY-OprM and the production of an integron-borne PSE-1 ß-lactamase. In conclusion, we identified an epidemic P. aeruginosa clone of an Fep^ns Caz^s phenotype strain involving 51 patients, in particular, ICU patients. The combination of the overexpression of an efflux pump and PSE-1 ß-lactamase production is associated with the multidrug-resistant phenotype. The dominant use of a single class of antibiotics could have provided the selective pressure required for the emergence and spread of this P. aeruginosa strain.Pseudomonas aeruginosa is an opportunistic pathogen that causes a variety of nosocomial infections. Cefepime (FEP) and ceftazidime (CAZ) are broad-spectrum cephalosporins that display similar MICs for wild-type P. aeruginosa strains. Although the rates of susceptibility of these two antipseudomonal cephalosporins appear to be identical for P. aeruginosa isolates in the United States (20), several European studies have recently reported an unusual discordance in the MICs of FEP and CAZ (4, 6, 7), with P. aeruginosa isolates being less susceptible to the former cephalosporin.The most frequent mechanisms of resistance to extended-spectrum cephalosporins in P. aeruginosa are derepression of the chromosomal AmpC ß-lactamase, the impermeability of the outer membrane, and increased efflux (9). FEP, in contrast to CAZ, is efficiently extruded by various efflux pumps, including the MexCD-OprJ and MexXY-OprM efflux pumps (16, 22). In addition, previous studies have noted the selectively reduced susceptibility to FEP among P. aeruginosa strains producing OXA ß-lactamases (2), as opposed to those that produce class A extended-spectrum ß-lactamases (32), which usually confer resistance to CAZ and FEP.In response to an increase in the incidence of carbapenem-resistant and multidrug-resistant P. aeruginosa isolates in the Hospital de Bellvitge, Barcelona, Spain, since 2004, a microbiology surveillance program was introduced. Recently, however, P. aeruginosa isolates showing variable susceptibilities to carbapenems and a discordance in susceptibility between CAZ and FEP have been noted. A clustering of the P. aeruginosa non-FEP-susceptible and CAZ-susceptible (Fep^ns Caz^s) phenotype was suspected in the hospital''s intensive care unit (ICUs). Here we elucidate the clinical and molecular epidemiology of the isolates with this discordant phenotype and the resistance mechanisms involved.     

Silencing acpP gene via antisense oligonucleotide-niosome complex in clinical Pseudomonas aeruginosa isolates

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is one of the major causes of nosocomial infections. In addition to its physiological adaptation capacity, it can develop resistance to disinfectants and antibiotics through various mechanisms. Recently, new eradication methods are gaining attention. Therefore, in this study, an LNA-2′-O-methyl hybrid antisense oligonucleotide targeting the acyl carrier protein P (acpP) gene was introduced into P. aeruginosa isolates. The design was determined through sequence analysis and prediction of the secondary structure of mRNA by software. Niosomes were used for enhancing cellular uptake. The control of the binding and transfection ability of the sequence was determined fluorometrically by labeling with 6-Fam. The effects were determined with broth microdilution method and qPCR studies. Eight different formulations were prepared. Among these, one formulation has shown to have ASO complexation ability whose composition was 312 μl Span 80 + 69.5 mg Cholesterol+ 36.4 mg CTAB+1 ml Chloroform and 5 ml dH₂O. Thus this formulation was determined as the delivery system for the next stages. Significant gene inhibition was detected at the six isolates. Results of this study suggested that niosomes can be used as a delivery system for cellular uptake of ASO and could eliminate bacterial growth.     

Sputum containing zinc enhances carbapenem resistance,biofilm formation and virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa chronic lung infections are the leading cause of mortality in cystic fibrosis patients, a serious problem which is notably due to the numerous P. aeruginosa virulence factors, to its ability to form biofilms and to resist the effects of most antibiotics. Production of virulence factors and biofilm formation by P. aeruginosa is highly coordinated through complex regulatory systems. We recently found that CzcRS, the zinc and cadmium-specific two-component system is not only involved in metal resistance, but also in virulence and carbapenem antibiotic resistance in P. aeruginosa. Interestingly, zinc has been shown to be enriched in the lung secretions of cystic fibrosis patients. In this study, we investigated whether zinc might favor P. aeruginosa pathogenicity using an artificial sputum medium to mimic the cystic fibrosis lung environment. Our results show that zinc supplementation triggers a dual P. aeruginosa response: (i) it exacerbates pathogenicity by a CzcRS two-component system-dependent mechanism and (ii) it stimulates biofilm formation by a CzcRS-independent mechanism. Furthermore, P. aeruginosa cells embedded in these biofilms exhibited increased resistance to carbapenems. We identified a novel Zn-sensitive regulatory circuit controlling the expression of the OprD porin and modifying the carbapenem resistance profile. Altogether our data demonstrated that zinc levels in the sputum of cystic fibrosis patients might aggravate P. aeruginosa infection. Targeting zinc levels in sputum would be a valuable strategy to curb the increasing burden of P. aeruginosa infections in cystic fibrosis patients.     

Association between histo-blood group antigens and Pseudomonas aeruginosa-associated diarrheal diseases

BackgroundPseudomonas aeruginosa is not a common enteric pathogen. The association between human histo-blood group antigens (HBGAs) and P. aeruginosa enteric infection has not yet been studied.MethodsWe collected stool samples from healthy children under 2 years of age for P. aeruginosa gut colonization rate. Saliva samples were collected from patients with P. aeruginosa-associated diarrheal diseases and normal healthy children. Genomic DNA was extracted from saliva samples for ABO blood group typing and FUT2 genotyping. Lewis phenotype was detected using ELISA assay.ResultsA total of 85 patients with P. aeruginosa-associated diarrheal diseases and 105 healthy children were enrolled for collecting saliva specimens. The stool colonization rate was 5/101 (5%) in healthy children, 4/58 (6.9%) in infants, and 1/43 (2.3%) in children 1–2 years old, respectively. Blood group A was more frequent in patients with P. aeruginosa-associated diarrheal diseases 24/77 (31.2%) than in healthy children 18/102 (17.6%) (P = 0.035). All patients and healthy children were secretor positive. The distribution of weak-secretor genotype Se³⁸⁵/Se³⁸⁵ was 23/84 (27.4%) in patients with P. aeruginosa-associated diarrheal diseases and 17/104 (16.3%) in healthy children, respectively (P = 0.06). Patients with P. aeruginosa-associated diarrheal diseases had a higher percentage of Le^a+b+ phenotype 25/81 (30.9%) than healthy children 17/105 (16.2%) (P = 0.018). There was no association between ABO or secretor or Lewis status with the clinical severity of P. aeruginosa-associated diarrheal diseases.ConclusionInfants had a higher gut P. aeruginosa colonization rate than children. Children with blood group A and Le^a+b+ phenotype are prone to P. aeruginosa-associated diarrheal diseases.     

Persistence and genetic adaptation of Pseudomonas aeruginosa in patients with chronic obstructive pulmonary disease

ObjectivesIt is unclear whether recurrent sputum culture with Pseudomonas aeruginosa from patients with chronic obstructive pulmonary disease (COPD) is caused by intermittent airway carriage by different P. aeruginosa lineages or persistent carriage by the same lineage, and whether lineages genetically adapt during carriage.MethodsWhole-genome sequencing was performed for P. aeruginosa isolates sampled longitudinally from sputum cultures in patients with COPD who were enrolled in an ongoing randomized controlled trial (clinicaltrials.gov: NCT03262142).ResultsA total of 153 P. aeruginosa isolates were sequenced for 23 patients during 365 days of follow-up. Recurrent presence of P. aeruginosa was seen in 19 patients (83%) and was caused by persistence of the same clonal lineage in all but one patient. We identified 38 genes mutated in parallel in two or more lineages, suggesting positive selection for adaptive mutations. Mutational enrichment analysis revealed genes important in antibiotic resistance and chronic infections to be more frequently mutated.DiscussionRecurrent P. aeruginosa was common and carried for a prolonged time after initial detection in the airways of patients with COPD. Recurrence was caused by persistence of the same clonal lineage and was associated with genetic adaptation. Trial data on possible clinical benefits of attempting antibiotic eradication of P. aeruginosa in COPD are warranted.     

Influence of biofilm formation in the susceptibility of Pseudomonas aeruginosa from Brazilian patients with cystic fibrosis

Ferreira AG, Leão RS, Carvalho‐Assef APD, Folescu TW, Barth AL, Marques EA. Influence of biofilm formation in the susceptibility of Pseudomonas aeruginosa from Brazilian patients with cystic fibrosis. APMIS 2010; 118: 606–12. Biofilms play a key role in the occurrence of lung infections by Pseudomonas aeruginosa in patients with cystic fibrosis (CF). In this study, we examined 40 isolates of P. aeruginosa from CF patients according to their capacity to form biofilm. We also compared their in vitro response to antimicrobials according to different modes of growth (planktonic vs biofilm) and performed molecular typing. All isolates proved capable of forming biofilm. However, there was no difference in biofilm development according to the mucoid and nonmucoid phenotypes and among isolates obtained at different periods of the chronic infection. All isolates tested for antimicrobial susceptibility in the biofilm state (BIC) were consistently more resistant to antibiotics than the same isolate tested in the planktonic state. The molecular typing indicates a considerable clonal diversity among isolates. We identified five patients harboring the same strain over different periods. These strains, however, displayed different levels of biofilm formation and BIC values for antibiotics tested. The results of the present study demonstrate that there is a marked difference in the susceptibility profile according to the mode of growth of CF P. aeruginosa, as cells tested in the biofilm state proved consistently more resistant to antibiotics.     

In vitro activity of the novel siderophore cephalosporin,cefiderocol, in Gram-negative pathogens in Europe by site of infection

ObjectivesWe assessed the activity of the novel siderophore cephalosporin, cefiderocol and selected other antibacterial agents against Gram-negative bacterial isolates in Europe.MethodsIsolates were obtained between 2013 and 2018 from European countries participating in the SIDERO-WT and SIDERO-Proteeae multinational surveillance studies. Isolates were categorised by infection site, focusing on bloodstream infections, hospital-acquired/ventilator-associated bacterial pneumonia (HABP/VABP), complicated intra-abdominal infections and complicated urinary tract infections. Cefiderocol activity was compared with ceftazidime–avibactam, ceftolozane–tazobactam, colistin and meropenem using standard susceptibility testing methods. European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were used to interpret susceptibility data.ResultsIsolates (n = 20 911) were collected from 145 sites in 24 countries in Europe, the highest proportion (34%) being from patients with HABP/VABP. Enterobacterales (66.6% of isolates) were more frequent than glucose non-fermenting species (33.4%) overall, with some differences between infection sites. Across all infection sites, the MIC₅₀/MIC₉₀ for cefiderocol was ≤0.5/≤2 mg/L for Enterobacter spp., ≤0.25/<2 mg/L for Klebsiella spp., 0.12/2 mg/L for Acinetobacter spp., ≤0.25/1 mg/L for Pseudomonas aeruginosa and ≤0.12/≤0.5 mg/L for Stenotrophomonas maltophilia. Across all infection sites, cefiderocol MICs were ≤2 mg/L for ≥96% of Enterobacter spp., ≥95% of Klebsiella spp., ≥90% of Acinetobacter spp. and ≥99% of Pseudomonas aeruginosa and Stenotrophomonas maltophilia isolates. Cefiderocol maintained high activity in carbapenem-resistant isolates, and the difference in activity between carbapenem-resistant (percentage susceptibility at EUCAST breakpoint: E. coli 77.8%, Klebsiella spp. 69.2%, Pseudomonas aeruginosa 97.5%, Acinetobacter spp. 90.7%, Stenotrophomonas maltophilia 99.6%) and carbapenem-susceptible (percentage susceptibility at EUCAST breakpoint: E. coli 99.4%, Klebsiella spp. 98.0%, Pseudomonas aeruginosa 99.7%, Acinetobacter spp. 94.9%) isolates was lower for cefiderocol than other agents.ConclusionsCefiderocol had excellent activity against all Gram-negative species, independent of key infection site and carbapenem MIC. Cefiderocol is a useful addition to the therapeutic options available for these difficult-to-treat infections.     

In vitro synergistic effect of colistin and ampicillin/sulbactam with several antibiotics against clinical strains of multi-drug resistant Acinetobacter baumannii

PurposeNowadays, Acinetobacter baumannii is resistant to almost all available antibiotics. The evaluation of synergistic effects between the antibiotics against this pathogen is among the efforts to counteract its antimicrobial resistance. This study aimed to evaluate possible synergistic effect of colistin and ampicillin/sulbactam (separately) with several antibiotics against clinical isolates of multi-drug resistant (MDR) A. baumannii.MethodsAcinetobacter baumannii strains were isolated from biological samples of hospitalized patients with any type of nosocomial infection related to this pathogen. Only MDR strains (resistance to at least three classes of antibiotics including cephalosporins, fluoroquinolones, and aminoglycosides) were included in the study. After determining the minimum inhibitory concentration (MIC) of antibiotics against the isolates by broth microdilution test, the checkerboard method was used for evaluation of any possible synergistic effect of both colistin and ampicillin/sulbactam with several other antibiotics.ResultsTwenty isolates underwent synergy test for colistin and 20 isolates for ampicillin/sulbacatam. Doxycycline (55%), azithromycin (35%), and co-trimoxazole (35%) had the most frequency of synergistic effect with colistin. On the other hand, amikacin and gentamicin (55%), doxycycline (50%), co-trimoxazole (45%), azithromycin (40%), and cefepime (40%) had the most frequency of synergistic effect with ampicillin/sulbactam. No antagonistic effect was observed for both antibiotics.ConclusionColistin and ampicillin/sulbactam have substantial synergistic effect with several antibiotics especially doxycycline, co-trimoxazole, azithromycin, and amikacin (with ampicillin/sulbactam) against MDR strains of Acinetobacter baumannii.     

Revisiting colistin susceptibility testing: will adding calcium to Mueller–Hinton agar improve the detection of colistin resistance?

ObjectiveThe aim was to investigate whether adding calcium to Mueller–Hinton agar for gradient MIC or disc diffusion tests could improve separation between colistin-susceptible and -resistant populations of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. and if this method could provide a reliable screening test for colistin resistance in routine laboratories.MethodsAn isolate collection of 57 E. coli, K. pneumoniae, P. aeruginosa and Acinetobacter spp. was tested. Ca²⁺ in concentrations from 2.5 to 40 mM was added to the Mueller–Hinton agar plates used for gradient MIC and disc diffusion tests. Broth microdilution (ISO 20776-1) MIC determination was used as reference. Escherichia coli and K. pneumoniae were investigated for colistin resistance genes.ResultsResults were similar for gradient tests and disc diffusion for all species. Correlation between phenotypic expression of resistance and resistance genes was not absolute. Addition of Ca²⁺ to Mueller–Hinton agar improved separation between colistin-susceptible and -resistant isolates for E. coli. For K. pneumoniae, separation was improved for isolates with mcr genes, but not for isolates harbouring other colistin resistance mechanisms. To further increase the concentrations of Ca²⁺ did not improve the separation between susceptible and resistant isolates of E. coli and K. pneumoniae. For P. aeruginosa and Acinetobacter species, addition of Ca²⁺ did not improve separation between susceptible and resistant populations.DiscussionThe results from this study show that addition of Ca²⁺ to the Mueller–Hinton agar does not sufficiently improve detection of colistin resistance by gradient MIC or disc diffusion tests for use in a routine laboratory.     

A procedure for systematic identification of bacteriophage–host interactions of P. aeruginosa phages

Immediately after bacteriophage infection, phage early proteins establish optimal conditions for phage infection, often through a direct interaction with host-cell proteins. We implemented a yeast two-hybrid approach for Pseudomonas aeruginosa phages as a first step in the analysis of these – often uncharacterized – proteins. A 24-fold redundant prey library of P. aeruginosa PAO1 (7.32 × 10⁶ independent clones), was screened against early proteins (gp1 to 9) of ?KMV, a P. aeruginosa-infecting member of the Podoviridae; interactions were verified using an independent in vitro assay. None resembles previously known bacteriophage–host interactions, as the three identified target malate synthase G, a regulator of a secretion system and a regulator of nitrogen assimilation. Although at least two-bacteriophage infections are non-essential to ?KMV infection, their disruption has an influence on infection efficiency. This methodology allows systematic analysis of phage proteins and is applicable as an interaction analysis tool for P. aeruginosa.     

Dissemination of IMP-6-producing Pseudomonas aeruginosa ST244 in multiple cities in China

Pseudomonas aeruginosa is an important opportunistic pathogen responsible for nosocomial infections and is currently reported to be a worldwide nosocomial menace. The aim of this study was to investigate the epidemiological traits and the distribution of metallo-β-lactamases (MBLs)-producing P. aeruginosa clinical isolates in ten cities in China between January 2010 and May 2012. Antimicrobial susceptibility was determined by disc diffusion assay and the minimum inhibitory concentrations (MICs) of imipenem and meropenem were also determined by the Etest according to Clinical and Laboratory Standards Institute (CLSI) guidelines. In addition, polymerase chain reaction (PCR) and DNA sequencing were applied to detect bla _MBL genes, and their epidemiological relationships were investigated by multilocus sequence typing (MLST). Of 368 P. aeruginosa isolates, MLST analysis identified 138 sequence types (STs), including 122 known and 16 novel STs, and the most frequently detected clone was ST244, followed by ST235. Besides, our study revealed that 25 isolates carried the bla _IMP-6 gene and three isolates carried the bla _VIM-2 gene, and a probe specific for both genes could be hybridised to an ~1,125-kb fragment in all isolates. Interestingly, all of the bla _IMP-6-producing isolates shared an identical ST, ST244, and exhibited a higher level of resistance to several antibiotics. Overall, these observations suggest that P. aeruginosa ST244 carrying the chromosomally located bla _IMP-6 gene is widely disseminated in multiple cites in China.     

Nosocomial outbreak of VIM-2 metallo-β-lactamase-producing Pseudomonas aeruginosa associated with retrograde urography

Pseudomonas aeruginosa is well adapted to the hospital setting and can cause a wide array of nosocomial infections that occasionally culminate in recalcitrant outbreaks. In the present study, we describe the first nosocomial outbreak of infection caused by bla_VIM-2-positive P. aeruginosa in Germany. In November and December 2007, highly resistant P. aeruginosa isolates were recovered from the urine of 11 patients in the Department of Urology of a University Hospital. Bacterial isolates were typed by multilocus sequence typing and screened for known metallo-β-lactamase (MBL) genes by PCR. Environmental sources of transmission were tested for bacterial contamination using surveillance cultures. Furthermore, a matched case–control study was performed in search of medical procedures significantly associated with case status. Typing of recovered isolates confirmed VIM-2 MBL-producing P. aeruginosa of sequence type 175 in all cases. Surveillance cultures did not lead to the identification of an environmental source of the outbreak strain. Case–control analysis revealed retrograde urography as the only exposure significantly associated with case status. The analyses suggest the transmission of a single clone of VIM-2 MBL-producing P. aeruginosa leading to the infection of 11 patients within 47 days. Events in temporal proximity to retrograde urographies appear to have facilitated infection in the majority of cases. Department-specific infection control measures, including reinforced hygiene procedures during retrograde urography, quickly terminated the outbreak.     

Impact of multidrug resistance on Pseudomonas aeruginosa ventilator-associated pneumonia outcome: predictors of early and crude mortality

The prevalence of multidrug-resistant (MDR) Pseudomonas aeruginosa has increased over the past decade and a significant rise in these isolates in ventilator-associated pneumonia (VAP) has been observed. However, the impact of MDR on VAP outcome has not been analysed in depth. We investigated the risk factors for early and crude mortality in a retrospective study of microbiologically and clinically documented VAP. Ninety-one VAP episodes in 83 patients were included, 31 caused by susceptible P. aeruginosa and 60 by MDR strains, of which 42 (70 %) were extensively drug-resistant (XDR) P. aeruginosa. Thirteen episodes concomitantly presented P. aeruginosa bacteraemia, in seven of which the origin was the respiratory tract. Whereas susceptible P. aeruginosa episodes were more likely than MDR episodes to receive adequate empirical (68 % vs. 30 %; p?<?0.001) and definitive antimicrobial therapy (96 % vs. 50 %; p?<?0.001), susceptible P. aeruginosa VAP presented a trend towards early mortality (29 % vs. 15 %; p?=?0.06). A logistic regression model with early mortality as the dependent variable identified multiorgan dysfunction syndrome (MODS) [odds ratio (OR) 10.4; 95 % confidence interval (CI) 1.7–63.5; p?=?0.01] and inadequate antibiotic therapy (OR 4.27; 95 % CI 0.98–18.4; p?=?0.052) as independent risk factors for early mortality. A similar analysis identified MODS (OR 4.31; 95 % CI 1.14–16.2; p?=?0.03) as the only independent predictor of crude mortality. The severity of acute illness clinical presentation was the main predictor of mortality. Despite adequate antibiotic therapy, susceptible P. aeruginosa seems to cause major early mortality. Although adequate therapy is essential to treat VAP, the severity of acute illness is a more important factor than drug resistance.