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OBJECTIVES: To describe the first nosocomial outbreak of Pseudomonas aeruginosa producing SPM-1 metallo-beta-lactamase (MBL) in southern Brazil. PATIENTS AND METHODS: From January to October 2004, carbapenem-resistant P. aeruginosa (CRPA) were recovered from hospitalized patients. Mortality, site of infection/colonization, patient location and susceptibility profiles were analysed. A sample of CRPA was screened for MBL production, evaluated for the presence of bla(SPM-1), bla(IMP-1) and bla(VIM-2) genes by PCR and submitted for molecular typing by DNA macrorestriction. RESULTS: A total of 135 CRPA (one isolate per patient) were recovered. Two major antibiotic susceptibility profiles comprised 63.7% of the isolates (susceptibility to polymyxin B and aztreonam, and susceptibility only to polymyxin B). Thirty-five CRPA were screened for MBL production (10 isolates from April, June and July, and 25 from September and October) and 27 (77.1%) proved to be positive for MBL production. Twenty-one of the 24 CRPA tested carried the bla(SPM-1) gene. The mortality of patients with CRPA was 48.1% and no variable was associated with death. Molecular typing revealed the presence of a clone with four related subtypes among the bla(SPM-1)-positive CRPA. CONCLUSIONS: The prevalence of MBL production by CRPA is high and horizontal transmission is a major determinant for the spread of SPM-1 CRPA among patients in this institution. As infection control measures failed to control the spread of CRPA, continuous surveillance for MBL production is warranted.
PURPOSE: The emergence of ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA) has created a new therapeutic challenge in ophthalmology. We evaluated ophthalmic antibiotics in vitro and in a rabbit keratitis model to determine effective therapy. DESIGN: Experimental laboratory investigation. METHODS: The susceptibilities of 12 CRPA isolates were determined in vitro for amikacin, ceftazidime, tobramycin, polymyxin B, gentamicin, ticarcillin, and the fluoroquinolones (that is, ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin, and moxifloxacin) using E-tests and National Committee of Clinical Laboratory Standards. A rabbit keratitis model was used to determine the reduction in colony counts of CRPA and ciprofloxacin-susceptible P. aeruginosa (CSPA) isolates following topical treatment with polymyxin B/trimethoprim, tobramycin (14 mg/ml), ceftazidime (50 mg/ml), and ciprofloxacin (3 mg/ml). RESULTS: For 12 CRPA isolates, the susceptibilities and median minimum inhibitory concentrations ([MIC]microg/ml) were as follows: amikacin (92%, 14.0), ceftazidime (75%, 4.0), tobramycin (67%, 1.75), polymyxin B (42%, 7.0), gentamicin (17%, 7.0), ticarcillin (0%, >32.0), and all fluoroquinolones (0%, >32.0). While no antibiotic regimen reduced colony counts in the time frame of the animal model for CRPA, ciprofloxacin alone demonstrated a significant decrease in colony counts for CSPA. Comparing CRPA with CSPA, both tobramycin and ciprofloxacin demonstrated a significant decrease in colony counts for CSPA. CONCLUSION: Our laboratory studies suggest that current antibiotics may be suboptimal in treating CRPA keratitis. Until new antibiotics are available, combination therapy such as fortified tobramycin and ticarcillin, and others may prove effective in aggressive topical long-term therapy.
OBJECTIVE: To investigate an outbreak of carbapenem-resistant Pseudomonas aeruginosa (CRPA) in a urology ward. METHODS: Patients infected or colonized with CRPA were prospectively identified by daily laboratory surveillance. Routine infection-control measures were reinforced, disinfection protocols were revised, and a surveillance program was set up, analyzing cross-transmission in the nursing ward and environment cultures from urology wards and the operating theater. CRPA isolates from clinical and environment samples were studied by pulsed-field gel electrophoresis (PFGE), following XbaI and SpeI restriction. RESULTS: From February 1998 to September 2000, 59 adult urology patients were colonized or infected by CRPA. All patients had been operated on prior to identification of the CRPA isolate and 79% of these procedures were performed in the same cystoscopy room. No patients had received prior carbapenem therapy. No cross-transmission was detected, and environment cultures from the urology ward and theater were negative except for five samples collected in the cystoscopy room. PFGE identified a single clone in the isolates from different patients and the environment samples. CONCLUSIONS: The PFGE analysis indicated that the CRPA outbreak resulted from the contamination of the cystoscopy room via an unsealed drain. The outbreak ended when the drain was sealed.
The basic studies on detection and titration of the 60-kDa cross-reacting protein antigen (CRPA), which is common to gram-negative rods, by reversed passive gelatin agglutination test with monoclonal and polyclonal antibodies were performed. The gelatin particles sensitized with 500 micrograms/ml of the monoclonal antibody had an ability to react with 200 ng/ml of the purified CRPA for 1.5 h. In contrast, the particles sensitized with 200 micrograms/ml of the polyclonal antibody had an ability to react with 25 ng/ml of the purified CRPA for 1.5 h. In addition, the detection of the 60-kDa CRPA in urinary tract pathogens, which consisted of 60 bacterial strains representing 14 species, was carried out by using the gelatin particles sensitized with 200 micrograms/ml of the polyclonal antibody. The CRPA was detected in all gram-negative rods of urinary tract pathogens, but not in gram-positive cocci. These results suggested that the detection of the 60-kDa CRPA by reversed passive gelatin agglutination test was a rapid and simple method for screening gram-negative bacteriuria.
Expression of the deo operon of Escherichia coli is subjected to double negative control by DeoR and CytR repressors and to the positive control by cAMP-CRP complex. However, sensitivity of the deo operon to catabolite repression is only revealed in bacteria with disrupted synthesis of the CytR protein, since the function of the latter is to prevent CRP activation of the deo operon transcription. In the present work we have studied the influence of crpa specific mutation at the genetic locus of CRP protein on the expression of the deo operon of E. coli. It has been found that the presence of crpa mutation in bacterial genome completely eliminates CytR repression of the deo operon, so that activation of Deo enzymes synthesis by cAMP-CRPa complex becomes possible, even in the presence of CytR. Besides, the modified CRPa protein appears to block the activity of catabolite-sensitive deoPO2 promoter of the deo operon under conditions of cAMP deficiency, which is manifested in the two-fold decrease of deoR derepression in the crpa cells, as compared to the wild type bacteria during the growth on glucose containing medium. It has been supposed that both effects are due to increased affinity of the modified CRPa protein to the specific sites of catabolite sensitive promoters, as compared to the wild type CRP protein.
We report on an outbreak of colistin-resistant Pseudomonas aeruginosa (CRPA) that occurred in a United Kingdom pediatric cystic fibrosis (CF) unit and involved six children over a period of 5 years. All CRPA-positive children had received aerosolized colistin therapy before first isolation of resistant organisms (mean duration, 3.1 years). Four of the 6 had also received courses of intravenous colistin in the year before the first isolation of CRPA. No impact of CRPA acquisition on respiratory function, clinical condition, or radiological parameters could be demonstrated. Four of the 6 children carried isolates of CRPA indistinguishable on genotyping. Two of these 4 children were sisters. The other 2 were on the same ward together at time of first isolation, and subsequently shared overlapping admissions with one of the sisters. While there is no conclusive evidence for the route of transmission, the frequency of overlapping in-patient admissions between 3 of these patients is suggestive of patient-to-patient transfer in the nosocomial setting.CF clinicians should be aware that colistin resistance can occur in P. aeruginosa, and some of these strains are capable of spread within CF units.
Yersinia enterocolitica, a highly antigenic 60-kDa protein, designated cross-reacting protein antigen (CRPA), is a member of the chaperonin-60 family of molecular chaperones. The gene encoding CRPA was cloned, expressed and sequenced. A partial library from Y. enterocolitica 0:3 genomic DNA was constructed in vector pUC19 and was screened by the immunoreactivity to monoclonal antibody 1A4, which has specificity for a species-specific epitope on the CRPA molecule. The crpA gene region consists of a putative two-cistron operon encoding proteins of 549 and 97 amino acids. The operon structure was led by a consensus heat-shock promoter sequence. Homology searches using the derived amino acid sequence have revealed that CRPA is 88.2% identical to GroEL of Escherichia coli. CRPB, another protein encoded by the operon, shows extensive sequence homology, 91.8% identical to GroES of E. coli which is a member of chaperonin-10.
This study was performed to identify risk factors for the nosocomial acquisition of ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA) in a Veterans Administration hospital between January 1994, and March 1995. The study was a retrospective comparison of host factors and in-hospital exposures of patients who acquired nosocomially CRPA and ciprofloxacin-sensitive P. aeruginosa (CSPA). Participants included 42 adult patients with nosocomial CRPA acquisition and 52 adult patients with nosocomial CSPA acquisition. Before pseudomonal acquisition, antecedent ciprofloxacin receipt (50% compared with 8%; odds ratio [OR], 12; p = 0.001), the presence of an indwelling airway (36% compared to 17%; OR, 2.6; p = 0.04), and documented antecedent infection (74% compared to 52%; OR, 2.6; p = 0.03) were significantly associated with acquisition of CRPA. On multivariate analysis, antecedent ciprofloxacin receipt (OR, 16.8; p = 0.0001) and presence of an indwelling airway (OR, 10.5; p = 0.009) remain as significant associations. Furthermore, the test of significance confirmed synergy between these two factors. Antecedent ciprofloxacin therapy and indwelling airway act independently and synergistically to promote CRPA acquisition.
Monoclonal antibodies (mAbs) against the Yersinia enterocolitica immunodominant 60 kDa antigen, termed cross-reacting protein antigen (CRPA), were obtained by fusion of spleen cells from mice immunized with CRPA with murine myeloma cells. The reactivities of the mAbs were examined by Western blotting against extracts of Y. enterocolitica and 23 other species of Gram-positive and Gram-negative bacteria. Cross-reactions were recognized with a wide range of bacteria, but not with Gram-positive cocci. The reactivities were different for each mAb, suggesting that both species-specific and multiple cross-reactive epitopes were present on the CRPA molecule. CRPA was produced under heat-shock conditions in Y. enterocolitica and was shown to correspond immunologically to the GroEL protein in Escherichia coli, a protein involved in the morphogenesis of coliphage. In addition to CRPA, at least nine other major heat-shock proteins were detected by two-dimensional gel electrophoresis of extracts of heat-shocked Y. enterocolitica.
UNLABELLED: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) represents a critical-priority pathogen capable of causing life-threatening, multidrug-resistant infections. We performed susceptibility testing, whole-genome sequencing, and bioinformatic analyses on 137 CRPA isolates from a Guangdong hospital. We found that the major specimen types were respiratory specimens (57/137, 41.6%) and bronchoalveolar lavage (42/137, 30.7%). All isolates were carbapenem-resistant but had low resistance to polymyxin B (0.7%, 1/137). IncP-6-positive isolates exhibited ≥2- to 32-fold higher resistance to 9/12 antibiotics (P < 0.05), with no difference to imipenem and meropenem. Fifty-four sequence types and 11 O-serogroups were identified, with ST1971 (6.6%) and O11 (29.9%) being predominant. Temporal and spatial patterns suggest persistent co-occurrence of clade 1 and clade 2 isolates, indicating potential nosocomial outbreak and clonal transmission. IMPORTANCE: The prevalence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) has increased rapidly in recent years, yet few genetic and epidemiological studies on CRPA isolates have been performed. We performed susceptibility testing, whole-genome sequencing, and bioinformatic analyses on hospital isolates to investigate their resistance profiles and molecular epidemiology. These findings may offer new insights for developing effective global strategies to control CRPA and reduce untreatable infections in clinical settings.
OBJECTIVES: Carbapenem-resistant Pseudomonas aeruginosa (CRPA), especially KPC-producing P. aeruginosa, is rapidly expanding and posing a serious public health threat. Here, we aim to characterise the epidemiology of KPC-3-producing P. aeruginosa in a tertiary hospital over a 10-year period and elucidate the mechanism of ceftazidime-avibactam (CZA) resistance driven by blaKPC-3 to blaKPC-267 mutations in CRPA, along with conducting a global phylogeographic analysis of KPC-3-producing P. aeruginosa. METHODS: 11 non-duplicate KPC-3-producing CRPA isolates collected over a 10-year period were characterized by antimicrobial susceptibility testing and whole-genome sequencing (WGS). The genetic context and transferability of blaKPC-3/267 and the mechanism of KPC-267-mediated CZA resistance were investigated. Global phylogenomic analysis was performed to characterize the geographic distribution and population structure of blaKPC-3-carrying P. aeruginosa. RESULTS: All 11 KPC-3-producing CRPA strains in this study belonged to ST1076 and exhibited multidrug resistance. The blaKPC-267-positive CZA-resistant strain SRMPA3523 was isolated from patient 1 after blaKPC-3-positive P. aeruginosa SRMPA1139 and SRMPA1630 were treated with CZA. WGS indicated that blaKPC-3/267 was located on the Tn6296 transposon contained in the transferable IncP-2 plasmid. KPC-267 mediates resistance to CZA by reducing the inhibitory effect of avibactam and increasing affinity for ceftazidime. Global analysis indicated that blaKPC-3-carrying P. aeruginosa were predominantly in China, America, and Colombia, with ST1076 and ST111 as dominant clones. CONCLUSIONS: This study characterised the global phylogeography of blaKPC-3-carrying P. aeruginosa and identified KPC-267 as a KPC-3-derived variant associated with CZA resistance. This finding highlighted the risk of developing CZA resistance in KPC-producing P. aeruginosa strains under therapeutic pressure.
BACKGROUND: Case-control studies analyzing antibiotic exposure as a risk factor for antimicrobial resistance usually assume single-drug resistance in the bacteria under study, even though resistance to multiple antimicrobials may be present. Since antibiotic selection pressures differ depending on the susceptibility profile of the antimicrobial-resistant bacteria, an accurate assessment of whether exposure to an individual antimicrobial is a risk factor for the emergence of resistance should distinguish between single-drug-resistant and multidrug-resistant bacteria. OBJECTIVE: To determine whether the exposures to individual antibiotics that were identified as independent risk factors in case-control studies differed depending on whether single-drug-resistant or multidrug-resistant bacteria were evaluated. DESIGN: Two retrospective case-control studies were performed with data on patients harboring Pseudomonas aeruginosa strains resistant only to ciprofloxacin (CRPA) and patients harboring P. aeruginosa strains resistant to ciprofloxacin and other antibiotics (multidrug-resistant P. aeruginosa [MDR-PA]). These 2 groups were compared with patients not harboring P. aeruginosa. SETTING: Two tertiary care hospitals. RESULTS: A total of 41 patients harboring CRPA and 151 patients harboring MDR-PA were identified and matched to 192 control subjects. By conditional logistic regression, independent risk factors associated with presence of CRPA were nonambulatory status (OR, 5.6 [95% confidence interval {CI}, 1.4-23]; P=.02) and prior ciprofloxacin exposure (OR, 5.0 [95% CI, 1.2-21]; P=.03). Independent risk factors for presence of MDR-PA were a Charlson score greater than 2 (OR, 3.3 [95% CI 1.8-6.0]; P<.001) and exposure to quinolones (OR, 2.8 [95% CI, 1.2-5.0]; P=.001), third- and fourth-generation cephalosporins (OR, 3.5 [95% CI, 1.7-7.1]; P<.001), imipenem (OR, 3.8 [95% CI, 1.2-12.1]; P=.02), and/or aminoglycosides (OR, 2.3 [95% CI, 1.04-5.1]; P=.04). CONCLUSION: There were substantial differences in exposure to individual antimicrobials between patients harboring CRPA and patients harboring MDR-PA. Future case-control studies addressing risk factors for single-drug-resistant bacteria should consider the complete susceptibility profile of the bacteria under investigation.
BACKGROUND: The prevalence of resistance to imipenem and ceftazidime among Pseudomonas aeruginosa isolates is increasing worldwide. OBJECTIVE: Risk factors for nosocomial recovery (defined as the finding of culture-positive isolates after hospital admission) of imipenem-resistant P. aeruginosa (IRPA) and ceftazidime-resistant P. aeruginosa (CRPA) were determined. DESIGN: Two separate case-control studies were conducted. Control subjects were matched to case patients (ratio, 2:1) on the basis of admission to the same ward at the same time as the case patient. Variables investigated included demographic characteristics, comorbid conditions, and the classes of antimicrobials used. SETTING: The study was conducted in a 400-bed general teaching hospital in Campinas, Brazil that has 14,500 admissions per year. Case patients and control subjects were selected from persons who were admitted to the hospital during 1992-2002. RESULTS: IRPA and CRPA isolates were obtained from 108 and 55 patients, respectively. Statistically significant risk factors for acquisition of IRPA were previous admission to another hospital (odds ratio [OR], 4.21 [95% confidence interval {CI}, 1.40-12.66]; P=.01), hemodialysis (OR, 7.79 [95% CI, 1.59-38.16]; P=.01), and therapy with imipenem (OR, 18.51 [95% CI, 6.30-54.43]; P<.001), amikacin (OR, 3.22 [95% CI, 1.40-7.41]; P=.005), and/or vancomycin (OR, 2.48 [95% CI, 1.08-5.64]; P=.03). Risk factors for recovery of CRPA were previous admission to another hospital (OR, 18.69 [95% CI, 2.00-174.28]; P=.01) and amikacin use (OR, 3.69 [95% CI, 1.32-10.35]; P=.01). CONCLUSION: Our study suggests a definite role for several classes of antimicrobials as risk factors for recovery of IRPA but not for recovery of CRPA. Limiting the use of only imipenem and ceftazidime may not be a wise strategy to contain the spread of resistant P. aeruginosa strains.
A monoclonal antibody against the Yersinia enterocolitica 60-kilodalton (kDa) antigen, designated cross-reacting protein antigen (CRPA), was obtained by cell fusion. The CRPA common to gram-negative bacteria was purified from Y. enterocolitica by the affinity chromatography with the monoclonal antibody (IgG1) thus obtained. The purified CRPA showed a single band of 60 kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and reacted with rabbit antisera against Y. enterocolitica, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, and Shigella sonnei in Western blot analysis. The monoclonal antibody, however, reacted with a 60 kDa peptide from Y. enterocolitica, but not with the antigens from other gram-negative bacteria such as V. cholerae, E. coli, S. sonnei, Salmonella enteritidis, Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and P. aeruginosa. The results suggested that both species-specific and cross-reactive epitopes were present on a CRPA molecule.