Aeruginosa Isolates

Genotypic and phenotypic variation within a collection of 17 P. aeruginosa isolates from the airways of CF patients, from a hospital environment, and from a plant rhizosphere was inves- tigated. The evolutionary genetic relationships of P. aeruginosa were examined by comparative sequence analysis of a 714-bp region of the housekeeping gene mdh from each of our iso- lates. Phylogenetic analysis of the mdh sequences did not re- veal distinct and separate branching of clinical and environ- mental isolates. Clinical and environmental isolates clustered with each other on the mdh tree. In addition, analysis of a second chromosomal locus, groEL, also demonstrated a close genetic relationship among strains and underscored the con- served nature of the core genome. These findings correlate with those of previous studies showing that clinical and envi- ronmental isolates do not form distinct lineages. In our study, of the four pairs of serial isolates from CF patients examined, we found that two P. aeruginosa isolates (CF177 and CF195) had identical mdh sequences and virulence profiles as deter- FIG. 1. Neighbor-joining tree constructed by the Jukes-Cantor

aeruginosa isolates which we obtained from our cystic fibrosis patients, it is important to select multiple isolates from sputum cultures for antimicrobial susceptibility testing so as t[r]

Transmissible strains of Pseudomonas aeruginosa have been described for cystic fibrosis (CF) and may be associated with a worse prognosis. Using a comprehensive strain biobank spanning 3 decades, we sought to determine the prevalence and stability of chronic P. aeruginosa infection in an adult population. P. aeruginosa isolates from sputum samples collected at initial enroll- ment in our adult clinic and at the most recent clinic visit were examined by a combination of pulsed-field gel electrophoresis and multilocus sequence typing and compared against a collection of established transmissible and local non-CF bronchiectasis (nCFB) isolates. A total of 372 isolates from 107 patients, spanning 674 patient-years, including 66 patients with matched isolates from initial and final encounters, were screened. A novel clone with increased antibacterial resistance, termed the prairie epi- demic strain (PES), was found in 29% (31/107 patients) of chronically infected patients referred from multiple prairie-based CF centers. This isolate was not found in those diagnosed with CF as adults or in a control population with nCFB. While 90% (60/66 patients) of patients had stable infection over a mean of 10.8 years, five patients experienced strain displacement of unique iso- lates, with PES occurring within 2 years of transitioning to adult care. PES has been present in our cohort since at least 1987, is unique to CF, generally establishes chronic infection during childhood, and has been found in patients at the time of transition of patients from multiple prairie-based CF clinics, suggesting broad endemicity. Studies are under way to evaluate the clinical implications of PES infection.

P. aeruginosa isolates from chronically infected CF patient lungs frequently contain mutations in lasR (3, 4, 18, 19) that are rare among isolates from acute infections (3, 18). Mutant lasR strain frequencies have been reported to be greater than 50% in some patients (3, 19), indicating strong selective pressure against LasR activity. Although P. aeruginosa lasR mutants are common in CF, the phenotypes of these mutants are diverse, unlike those ex- hibited by lasR mutant strains derived from the common labora- tory strain PAO1 (19). Nevertheless, a few phenotypic character- istics, like colony lysis and sheen, have been associated with CF clinical lasR mutant strains (20). However, little is known about what factors in the CF lung might select for lasR mutants, or whether there are LasR-regulated P. aeruginosa activities that ac- count for this selection. Understanding the evolution of P. aerugi- nosa QS in the CF lung has significant implications for ongoing efforts to develop anti-QS-based therapies (21).

Description of the outbreak. In February 2003 a P. aerugi- nosa isolate showing an unusual MDR phenotype (isolate MV461) was isolated from the lower respiratory tract of a mechanically ventilated patient from one of the general ICUs of a large tertiary care and clinical research hospital located in San Giovanni Rotondo (southern Italy). MV461 was resistant to carbapenems (imipenem and meropenem), extended-spec- trum cephalosporins (ceftazidime and cefepime), and genta- micin, tobramycin, and fluoroquinolones (ciprofloxacin and levofloxacin), while it retained susceptibility to piperacillin- tazobactam and amikacin. Before this episode, carbapenem- resistant P. aeruginosa isolates had only been reported sporad- ically at the clinical microbiology laboratory of that hospital, and they had never shown such a complex MDR phenotype. At the time of isolation of MV461, the patient was receiving broad-spectrum antibiotic coverage with meropenem plus teicoplanin.

The results of this study are summarized as follows. (i) 16HBE14o ⫺ cells displayed a high level of constitutive IL-8 production that was not further increased by treatment with flagellin, while BEAS-2B, A549, and NCI-H292 cells had minimal constitutive chemokine production that was addi- tionally enhanced following flagellin treatment. (ii) All four airway epithelial cell lines expressed TLR5 protein. (iii) Flagellin stimulated NF- ␬ B and IL-8 production through TLR5. (iv) The majority of P. aeruginosa clinical isolates (10/14) expressed type a flagellin. (v) While differences in the ability of flagellins from bacterial strains to stimulate IL-8 production were observed (700 to 4,000 pg/ml), a cor- relation between expression of type a or b proteins and IL-8 levels could not be made. These results lend credence to previous studies that revealed no relationship between the source of P. aeruginosa isolates (clinical versus environmen- tal) and the types of flagellins expressed (30) as well as the prior report that 76% of ulcerative keratitis isolates ex- pressed type a fliC genes versus 24% for type b (46). Of note, all isolates (although limited in number) from CF cases were type a, but whether this relationship will remain with a larger number of CF clinical samples remains to be established. Furthermore, while airway cell lines were ex- clusively used in this study, we plan to use human primary tracheobronchial epithelial cells in future experiments to compare with the results from airway cell lines.

Ciprofloxacin (CPFX) and pazufloxacin (PZFX) have strong antibacterial activity against Pseudomonas aeruginosa. We investigated the sensitivity of P. aeruginosa to CPFX and PZFX in 373 strains isolated from inpatients (321 strains) and outpatients (52 strains) during September 2010 to September 2011 at Toho University Ohashi Medical Center. The percentage of CPFX-non-susceptible (≥3.91 µg/mL) among inpatients was 22.4%, but that among outpatients was 1.9%. As the major resistance mechanism to fluoroquinolones in P. aeruginosa involves modification of type II topoisom- erases (DNA gyrase and topoisomerase IV), we examined mutations in the quinolone-resistance-determining regions (QRDRs) of gyrA and parC of P. aeruginosa isolates. Among the 373 isolates, 73 isolates had reduced CPFX-suscep- tibility and 88 had reduced PZFX-susceptibility. Sequencing of gyrA and parC revealed base substitutions that resulted in amino acid replacements in QRDR of GyrA in 70 P. aeruginosa isolates, while Thr83Ile (in GyrA) and Ser87Leu (in ParC) substitutions were found in 12 strains. These replacements were clearly associated with reduced susceptibility to CPFX and PZFX. However, we also found strains with high MICs to quinolones without mutations in either gyrA or parC. We then investigated the effect of efflux pumps in CPFX-resistance in these isolates. In the presence of an efflux pump inhibitor, MIC values in 12 of 66 strains decreased to 1/2 3 . We also sequenced genes related to overexpression of

Pseudomonas aeruginosa is an important cause of pulmonary infection in cystic fibrosis (CF). Its correct identification ensures effective patient management and infection control strategies. However, little is known about how often CF sputum isolates are falsely identified as P. aeruginosa. We used P. aeruginosa-specific duplex real-time PCR assays to determine if 2,267 P. aeruginosa sputum isolates from 561 CF patients were correctly identified by 17 Australian clinical microbiology laboratories. Misidentified isolates underwent further phenotypic tests, amplified rRNA gene restriction analysis, and partial 16S rRNA gene sequence analysis. Participating laboratories were surveyed on how they identified P. aeruginosa from CF sputum. Overall, 2,214 (97.7%) isolates from 531 (94.7%) CF patients were correctly identified as P. aeruginosa. Further testing with the API 20NE kit correctly identified only 34 (59%) of the misidentified isolates. Twelve (40%) patients had previously grown the misidentified species in their sputum. Achromobacter xylosoxidans (n ⴝ 21), Stenotrophomonas maltophilia (n ⴝ 15), and Inquilinus limosus (n ⴝ 4) were the species most commonly misidentified as P. aeruginosa. Overall, there were very low rates of P. aeruginosa misidentification among isolates from a broad cross section of Australian CF patients. Additional improvements are possible by undertaking a culture history review, noting colonial morphology, and performing stringent oxidase, DNase, and colistin susceptibility testing for all presumptive P. aeruginosa isolates. Isolates exhibiting atypical phe- notypic features should be evaluated further by additional phenotypic or genotypic identification techniques.

various hospitals in northern Italy during 1995-2000. The re- sults showed that P. aeruginosa isolates producing the PER-1 enzyme have been present in hospitals in the Milan area and the surrounding region, where they have been associated with multifocal nosocomial outbreaks, since at least the mid-1990s. The fact that similar isolates were also found at Varese Uni- versity Hospital (12; Luzzaro et al., Abstr. 99th Gen. Meet. Am. Soc. Microbiol.; J. D. Docquier, F. Luzzaro, G. Amico- sante, A. Toniolo, and G. M. Rossolini, Letter, Emerg. Infect. Dis. 7:910-911, 2001), located in the same region, further sup- ports the notion that PER-1-producing P. aeruginosa isolates have been endemic to this area for several years. In contrast, PER-1 producers were not detected at the Treviso hospital, which is located in a different region of northeastern Italy, suggesting that the resistance determinant may be absent or less widespread in that area. All of the PER-1 producers were not only resistant to extended-spectrum cephalosporins and monobactams, but also exhibited a multidrug-resistant pheno- type that included most other antipseudomonal agents, leaving few therapeutic choices. Piperacillin-tazobactam was the most active among the tested drugs, although one-third of the strains were also resistant to this agent.

Twelve clonally related and multidrug-resistant Acinetobacter baumannii isolates were recovered during a 4-month period from 12 patients hospitalized at the Valenciennes Hospital in France. Antibiograms deter- mined by the double-disk diffusion technique on cloxacillin-containing plates detected a clavulanic acid- inhibited extended-spectrum ␤ -lactamase (ESBL). PCR and sequencing identified the gene encoding the Ambler class A ESBL VEB-1. This gene was located on the chromosome and was part of a class 1 integron identical to that previously identified in Pseudomonas aeruginosa isolates from Thailand. Additionally, seven clonally related bla VEB-1 -positive A. baumannii strains were identified in the immediate environment of the

In this study the presence of integron genes in P. aeruginosa strains was investigated and antimicrobial susceptibility test was performed. A high proportion of isolated strains showed high resistance to investigated antibiotics. The presence of integron genes among resistant P. aeruginosa isolates confirms the role of this gene in resistance of strains and validates the origin of these bacteria from one common colony. Hence, there is a high risk of infection in hospitalized patients of ICU and due to increase of antibiotic resistance in isolated strains; their treatment is becoming a matter of concern. Therefore some strategies should be employed to inhibit the increasing resistance of these strains and the spread of resistance genes

the Tn1213 composite transposon, which was chromosomally located. Pulsed-field gel electrophoresis and multilocus sequence typing (MLST) revealed the presence of three separate clones among the isolates. Two of these, corresponding to sequence types (STs) ST244 and ST235, were responsible for parallel outbreaks. Apart from PER-1, all the isolates produced OXA-2 oxacillinase. ST235 isolates additionally expressed a novel enzyme, OXA-74, differing by one amino acid from the OXA-17 ESBL identified originally in PER-1- and OXA-2-positive P. aeruginosa isolates from Ankara, Turkey, in 1992. These earlier Ankara isolates with PER-1, OXA-2, and OXA-17 were also classified into ST235, which is a single-locus variant of two other STs, ST227 and ST230. ST227, ST230, and ST235 all correspond to the recently described clonal complex BG11, which seems to be internationally distributed, having spread in Turkey, Greece, Italy, Hungary, Poland, Sweden, and much of Russia. It is associated with various ␤ -lactamases, including PER-1 and VIM metalloenzymes. This work further demonstrates the value of MLST of P. aeruginosa.

aeruginosa isolates included in that study, 5 were resistant to amikacin and 3 were resistant to gentamicin by the reference method, but these isolates were susceptible to these drugs wh[r]

Abstract: Different microorganisms that resist multiple antibiotics is considered to be most threatening problems to public health now a days. The aim of this study was to evaluate antibiotic resistance of Pseudomonas aeruginosa in burn wound infection and the antibiotic susceptibility pattern of P. aeruginosa isolates.Wound pus was collected using a sterile swab from all age group excluding the patients already on antibiotic therapy. One hundred eighty four (184) samples were collected from the wound pus in Medicross Diagnositc Center, Butwal, Nepal from October , 2017 through March , 2018.Out of 184 samples, 48 Pseudomonas aeruginosa strains were isolated and tested for antibiotic susceptibility against different antibiotics. The pattern of antibiotic susceptibility suggested that 100% of the isolates were resistant to Tobramycin, and 94.1% were resistance to Cefoperazone and Meropenem whereas the least resistance was shown against ciprofloxacin (35.3%) followed by amikacin and gentamicin (47.1%) both. The percentage of multi drug resistance in Pseudomonas aeruginosa isolates was 100% since all these positive samples were resistant to at least 3 drugs in the following classes: β-lactams, carbapenems, aminoglycosides and fluoroquinolones. In P. aeruginosa infections, periodic antimicrobial resistance monitoring is fundamental to update the current activity level of commonly used anti-pseudomonal drugs to minimize the risk of drug resistance.

Materials and Methods: In this study, 131 clinical samples were collected from patients hospitalized in Imam Reza hospital in Mashhad during a 15-month period from May 2011 to November 2012. After verification of P. aeruginosa isolates, antibiotic resistance patterns of isolates were determined for 14 antibiotics by Kirby-Bauer standard disk diffusion according to the CLSI guidelines. Combined-disk test was used for phenotypic determination of MBLs-producing isolates and after DNA extraction, genotypic determination of VIM1 and VIM2 metallo beta-lactamase genes was carried out using Multiplex- PCR.

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 suscepti- bility 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 ⴝ 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 ⴝ 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.

Most of the research regarding P. aeruginosa virulence factor production in disease has focused on human serology, isolates, or samples. In contrast, little is known about the virulence phenotypes of animal isolates. In this study, we surveyed ani- mal intestinal and fecal P. aeruginosa isolates for protease activity. In addition, we sought to determine if P. aeruginosa associated with acute or chronic animal diseases displayed protease phenotypes comparable to those displayed by P. aeru- ginosa associated with acute or chronic human diseases. We used colorimetric assays to detect in vitro elastase and total matrix protease activities semiquantitatively and included well- characterized human wound isolate PAO1 (12) as an internal control. Interestingly, while total matrix protease activity among animal isolates was comparable to that of P. aeruginosa PAO1, we found that P. aeruginosa isolates from canine ear infections exhibited significantly lower elastase activity when cultured in vitro than strain PAO1 or isolates from all other animal sources.

Antibacterial susceptibility studies of hexane, ethanol and aqueous extracts of Datura Inoxia leaf against Staphylococcus aureus and Pseudomonas aeruginosa, are summarized in Table 1. The aqueous and hexane extracts of Datura Inoxia leaf did not inhibit the growth of the isolates of Pseudomonas aeruginosa, as there was no zone of inhibition, signifying that strains of Pseudomonas aeruginosa tested are resistant to it despite good agar diffusion, but its ethanol (17 mm) extract showed moderate antibacterial activity and represented as ++ sign. Pseudomonas aeruginosa isolates were susceptible to standard antibiotic discs tested (Ceftazidime- 20 mm and Gentamycin- 32 mm) with appreciable zone of inhibition. Other workers have also demonstrated the antibacterial effect of ethanolic extract of Datura species against Pseudomonas aeruginosa. [12, 13]

Bacterial phenotypic typing methods as phage typing and serotyping are now replaced by molecular methods as pulse- fi eld gel electrophoresis (PFGE), ribotyping, and different PCR-based methods such as ERIC-PCR. ERIC-PCR is a technique that uses the difference in the position and number of ERIC sequences as an indicator of bacterial diversity. 40 Bacterial genotyping using ERIC-PCR is of tremendous importance in epidemiological studies carried on P. aeruginosa to evaluate genetic relations, particularly in health-care-acquired infections. In the current study, we found intermediate diversity of ERIC patterns, with no close relation to the presence of different virulence genes. In addi- tion, we found that sharing the same ERIC group did not imply having the same virulence determinant pattern. Great genetic heterogeneity in P. aeruginosa isolates was reported by several studies using ERIC-PCR, RAPD, and PFGE. 3,36,41 We used ERIC-PCR as it is cheap, reliable and has a good discrimina- tory power for P. aeruginosa genotyping. 42 Our results suggest that nosocomial transmission of the isolates in this study and its cross-infection is relatively limited due to the relative degree of diversity among the ERIC-patterns apart from iso- lates in the fi rst cluster, where cross-acquisition may have occurred. P. aeruginosa cross-infection is reported in several studies done in ICUs with variable rates between different hospitals. 43–45

demonstrated by Schmidtchen and team in their 2001 study, isolates obtained from repeated cultures from skin ulcers maintained their protease expression patterns, suggesting that protease expression may be stable over extended periods in vivo. Comparatively, Jagger, Bahner & Warren (1983) found that a lower percentage of P. aeruginosa isolates from CF patients who were chronically infected with the organism were positive for alkaline protease expression and general protease activity compared with those isolates from colonised patients, suggesting that protease production from P. aeruginosa could be implicated in the initial colonisation of CF patients. The present study revealed that the P. aeruginosa isolates displayed caseinolytic activity (as demonstrated using the milk-casein agar substrate plate method) over an extended culture period, with most isolates showing a positive correlation of general protease/elastase activity throughout the growth of the bacteria, with all wound isolates exhibiting some level of protease activity. The caseinolytic and elastolytic activities measured in the culture supernatants of each of the isolates were generally maximal by the end of the exponential phase of growth, which was maintained when the isolates were grown under altered initial pH and also when grown anaerobically. It should also be noted that those isolates producing low levels of protease activity may be more proteolytically active under alternative growth conditions (Jagger, Bahner & Warren, 1983); for instance if different growth media was used, given that protease production is influenced by the complexity of the medium including cation concentration (Jagger, Bahner & Warren &, 1983). Indeed, P. aeruginosa is known to depend on a complex network of regulatory genes which act to control the production of various virulence factors in response to