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Antimicrobial resistance and putative virulence genes of Pseudomonas aeruginosa isolates from patients with respiratory tract infection.

Introduction

Pseudomonas aeruginosa respiratory infections are causing high morbidity and mortality due to the germ's capacity to rapidly develop antibiotic resistance, especially during antibiotic treatment of patients. The organism can carry a variety of putative virulence factors, which are highly controlled by cell-to-cell signaling systems. (1-2)

P. aeruginosa can secrete an exopolysaccharide called alginate (algD) and an elastase B (lasB) enzyme in response to environmental conditions. Both these virulence factors can influence pathogenesis by enhancing adhesion, colonization, and invasion of tissues, causing chronic pulmonary inflammation. (3) Additionally, elastase B is an important protease of P. aeruginosa. This enzyme has a tissue-damaging activity, it can degrade a number of plasma proteins and it contributes to the survival of P. aeruginosa in infected tissues. (4)

Other potential virulence factors secreted by P. aeruginosa that are important in its pathogenicity include exotoxin A (toxA), which is the most toxic virulence factor detected in this organism. It inhibits protein biosynthesis, it has a necrotizing activity on tissues and contributes to the colonization process. (5) Exoenzyme S (exoS) is secreted into the cytosol of epithelial cells and it contributes to increased pneumonia severity. (6)

Infections caused by multidrug-resistant (MDR) P. aeruginosa are difficult to treat and can be associated with high mortality especially in patients who are immunocompromised or who present chronic lung diseases. P. aeruginosa acquires resistance primarily through the production of plasmid mediated AmpC [beta]-lactamase, different types of extended-spectrum [beta]-lactamases (ESBLs) and metallo-[beta]-lactamases (MBLs). (7-8) The recent emergence of bla-KPC enzymes in P. aeruginosa strains, currently KPC-2 and KPC-5, is alarming since these strains show activity against most [beta]-lactams including monobactams, carbapenems, and oxyiminocephalosporins. (8)

Recent studies report that ESBLs and MBLs are increasing among P. aeruginosa in the Middle East Arab countries, and this development could result in prolonged hospital stay, increased cost and fatal outcome among patients. (9-10)

This study investigated the occurrence and relationship of MDR P. aeruginosa recovered from Jordanian patients with respiratory tract infection, in association with putative virulence genes and genotypes.

Methods

Patients

This prospective convenience sampling study included a total of 284 respiratory tract samples obtained from adult patients attending the Pulmonary Clinic, The Jordan University Hospital (JUH), over the period from November 2014 to June 2015. JUH includes 550 beds, and is a tertiary care teaching hospital with 24 of the beds distributed in 3 adult Intensive Care Units (ICUs).

Only one sample was collected from each patient with signs and symptoms of respiratory tract infection. The majority of specimens (247; 86.9%) were obtained from hospitalized patients and only 37 (13.1%) were collected from outpatients. Bio-data of each patient was obtained and registered on a special sheet and included the following: patient's identification number, gender, age, clinical diagnosis, specific admission conditions, type of specimen, type of current or previous antibiotic treatment within the past 4 weeks prior to specimen sampling.

The study was approved by the School of Medicine and the School of Graduate studies, The Jordan. A permission was also obtained from the Institutional Ethical Review Board (IERB) at the Jordan University Hospital (JUH, Permission No. 9/2015). Verbal consent was obtained from all patients after explaining the aim of the study. The identity of patients was protected by applying numbers instead of names for all collected specimens.

Culture, isolation and bacterial identification

All collected respiratory samples (sputum, bronchoalveolar lavage [BAL], pleural fluid) were inoculated directly using cotton swab onto Pseudomonas CN agar plates (Oxoid, Basingstoke, United Kingdom) and incubated for 24-48 hours at 37 [degrees]C. The culture plates were examined to detect the presence of straw-colored colonies with yellow or blue-green pigmentation, and colonies were confirmed later as being P. aeruginosa using the following preliminary characteristics: positive oxidase test, growth at 42 [degrees]C, negative lactose, and glucose fermentation in tube of Kligler iron agar. Five colonies of pure P. aeruginosa were subcultured into cysteine lactose electrolyte deficient agar plates (CLED, Oxoid), and all isolates were stored in cryotubes containing brain-heart infusion agar with 15% glycerol at -70 [degrees]C for further investigation.

Antimicrobial susceptibility

Antimicrobial susceptibility of P. aeruginosa isolates was assessed using disc diffusion test according to the recommendation of the Clinical Laboratory and Standards Institute (CLSI, 2015). (11) P. aeruginosa isolates which showed resistance to three or more antibiotic classes (MDR) were tested for minimum inhibitory concentrations (MICs) by E-test strips for ceftazidime, ciprofloxacin, colistin, imipenem and meropenem (Oxoid). The results of susceptibility tests were interpreted according to the breakpoints of tested antibiotics as recommended in CLSI guidelines (2015). P. aeruginosa ATCC 27853 was used as control during all tests.

Plasmid DNA extraction and PCR procedures

After thawing, stored P. aeruginosa isolates were subcultured onto blood agar, and incubated at 37 [degrees]C for 24 hours. A few colonies were picked from the agar, and inoculated into 3 mL Luria Broth (LB) (Oxoid) and incubated for 24 hours at 37 [degrees]C. The bacterial plasmid was extracted using the Zyppy TM Plasmid Miniprep Kit, Zymo (QIAGEN, Germantown, MD, USA) according to manufacturer's instructions. The quantity of DNA from each isolate extract was tested to ensure that the DNA concentration was more than 50 [micro]g/[mL.sup.-1] with a purity at [A.sub.260]/[A.sub.280]=1.6-1.8, using a spectrophotometer (Bio-Rad, Hercules, CA, USA).

Identification of P. aeruginosa by PCR

Specific identification of P. aeruginosa was performed by nested PCR using a first primer set for identification of the genus Pseudomonas, followed by a second primer set specific for P. aeruginosa. The 16 ribosomal DNA (rDNA) was detected as described by Spilker et al. as shown in Table 1. (12)

Detection of virulence genes by PCR

The following genes: algD, lasB, toxA, exoS and exoU were detected using specific primers for each gene as described in references listed in Table 1. (6,13-20) P. aeruginosa (PAO-1 strain) was used as a positive control for lasB and toxA genes and P. aeruginosa ATCC 27853 was used as a positive control for algD gene, and nuclease free water was used as a negative control.

Detection of genes of ESBLs, MBLs and [bla.sub.KPC]

A total of 32 MDR P. aeruginosa isolated were investigated for genes of [bla.sub.CTX-M], [bla.sub.TEM], [bla.sub.VEB-1], [bla.sub.SHV], [bla.sub.GES-1] and MBLs (LMP-1, VPM-2, [bla.sub.KPC]), as presented in Table 1. A positive control of E. coli (ATCC 51446) was used for detection of [bla.sub.CTX-M] genes, P. aeruginosa (CL 120 & CL 162, Universite Libanaise, Tripoli, Lebanon) for IMP1, VIM-2 genes, and K. pneumoniae (ATCC BAA1705) for [bla.sub.KPC] genes. Nuclease free water was used as a negative control in all tests.

Gel electrophoresis

In all tests, 6 [micro]L of each amplified PCR product, controls and 100 bp ladder (molecular size marker) were analyzed in 1.5% agarose gel (Promega, Madison, WI, USA) in 1X TBE buffer containing ethidium bromide, and run for 50 minutes at 120 volts using horizontal apparatus. After electrophoresis, the DNA bands in gel were visualized under ultraviolet light (UVP system, Upland, CA, USA).

RAPD-PCR to detect genotypic variations among P. aeruginosa isolates

Two short (10 mers) single primers of 272 (5AGCGGGCCAA-3) and 208 (5 ACGGCCGACC-3) were used in two separated PCR reactions to create a repetitive electrophoresis patterns and to detect the genotypic variation according to the number and weight of detected band differences among the isolates. (21) The results were analyzed by computer with PyElph software (USA).

Statistical analysis

Data generated from the study were tabulated as Microsoft Excel sheet and uploaded to Statistical Package for Social Sciences version 20 (IBM Corp, Armonk, NY, USA). Frequencies and percentages were calculated for categorical data and Pearson's chi-squared test or Fisher's exact test were applied to determine potential factors associated with P. aeruginosa and to determine whether there are any statistical differences between groups. The level of significance was set at a p value of 0.05 to test the hypothesis of no association. Fisher's exact test replaces chi-squared test when the minimum expected count is less than five.

Results

Patients

A total of 61/284 (21.5%) P. aeruginosa isolates were recovered from respiratory samples of patients. These included 247 (86.9%) hospitalized patients and 37 (13.1%) non-hospitalized patients. Recovery of P. aeruginosa was significantly more frequent (p=0.036) in hospitalized (73.8%) versus non-hospitalized patients (26.2%). Table 2 presents the demographic and clinical characteristics of the 61 investigated patients. Table 3 shows demographic characteristics and clinical features of all patients infected with P. aeruginosa and hospitalized in wards (60%) and ICUs (40%). All clinical features were almost similar in both groups. Only one female patient hospitalized in the ICU died during the study period.

Occurrence of P. aeruginosa and antimicrobial susceptibility profiles

The identity of 61 P. aeruginosa isolates was confirmed by biochemical testing and PCR. A total of 53 (87%) isolates showed clear blue-green pigment production (pyocyanin) on Pseudomonas CN agar. The antimicrobial susceptibility patterns and MICs of P. aeruginosa isolates for important used antibiotics are shown in Table 4.

A total of 32 (52.5%) isolates were completely resistant to three or more antibiotic classes (MDR) and all were recovered from hospitalized patients (Table 4).

MDR and putative virulence genes All 32 MDR P. aeruginosa isolates carried one to four of the investigated ESBL genes and all were positive for genes of algD, lasB, toxA and pyocyanin, as shown in Table 5. Most MDR isolates (22/32; 68.9%) harbored [bla.sub.CTX-M] genes, whereas other ESBL genes were much less common among the isolates, and none of the MDR P. aeruginosa isolates was positive for the blaIMP-1 or [bla.sub.KPC] genes (Table 5).

The most common virulence genes detected among the all 61 isolates were algD and lasB (98%), followed by toxA (80%), exoS and exoU (33), and 87% were positive for pyocyanin (Table 6).

Genotyping

The RAPD profile showed high diversity among the isolates. Each isolate generated 2-6 fragments with band sizes ranging from 180 to 1200 bp. The dendrogram of the isolates is presented in Figure 1; by applying 70% similarity index of dendrogram, the isolates were clustered into 14 different groups. The major group was presented by 5 isolates and all other groups were presented with less than 4 isolates. There was no significant association between genotype groups and antimicrobial resistance phenotypes using RAPD cluster groups.

Discussion

The general rate of P. aeruginosa isolates recovery in this study (21.5%) is relatively in the middle of other recently reported studies from neighboring countries such as Egypt, Saudi Arabia, United Arab Emirates, where the isolation rates of P. aeruginosa from sputum samples of patients with lower respiratory tract infection ranged between 16.7% and 50%. (9,21-23) A study from the United Arab Emirates reported that the most common pathogen isolated from the sputum of patients with hospital-acquired pneumonia was P. aeruginosa (50%), and that it caused a mortality rate of 24%. (23) A prevalence study conducted in ICU patients for one day in 72 countries around the world has also demonstrated that P. aeruginosa was one of the most common organisms, and that it accounted for 19.9% of all organisms isolated from respiratory tract cultures. (24)

Numerous studies reported that nosocomially-acquired P. aeruginosa isolates tend to be often more MDR and commonly identified ICU patients. (9,24-26) The rate of MDR P. aeruginosa isolates in our study was 52.2%, and this result is similar to recently reported studies from Jordan and neighboring Arab countries (Egypt, Saudi Arabia, Lebanon). (23,25-27)

The present study showed that hospitalized patients were significantly (p=0.036) more frequently infected with P. aeruginosa than nonhospitalized patients in association with lower respiratory tract infection. This study also showed that almost all 61 patients with positive P. aeruginosa isolates had purulent sputum, cough and were treated with antibiotics (96.7%), whereas other significant clinical respiratory infection features such as chest pain, dyspnea and hemoptysis were less frequently observed in our patients. There was only one death in patients with lower respiratory infection during the study period.

The present study demonstrates that MDR P. aeruginosa isolates (68.9%) commonly are ESBLproducers and [bla.sub.CTX-M] was the most frequent phenotype detected in these isolates, whereas the other types of TEM, VEB, GES, and SHV genes were less often detected (Table 5). The spread of ESBL-producing Gram-negative bacteria is considered an important driver for the use of carbapenems, which can increase the selection of carbapenemase-producing organisms causing infections, particularly in ICU patients. (28)

Most recent studies reported that hospital outbreaks caused by MDR P. aeruginosa clones with MBLs such as VIM and IMP types can be developed by either chromosomal mutations or horizontal gene transfer. (29) VIMs are the most frequent MBL type found among members of the Enterobacteriaceae family, as well as among Pseudomonas spp. and Acinetobacter spp. (29) The present study indicated that MBLs were only detected as type VIM-2 in a few of our MDR P. aeruginosa isolates (9.4%), while the other types, blaIMP and [bla.sub.KPC], were absent (Table 5). In Jordan, a recent study has also reported that all Acinetobacter baumannii isolates from the hospital, environment, and patients' respiratory tract were negative for blaVM and blaIMP-1 genes. (30)

The pathogenic potential of P. aeruginosa is complex and multifactorial, and virulence genes, especially algD, lasB, toxA and exoS can induce biofilm formation, infection and cytotoxicity in the human body, especially in the respiratory tract. (6,31) Additionally, P. aeruginosa strains carrying exoU genes can cause a more virulent form of pneumonia than other strains. (32) The prevalences of the virulence genes algD, lasB, toxA and secretion of pyocyanin in this study were very high among P. aeruginosa isolates (98%-80%), and all MDR isolates carried those 4 virulence factors, while both exoS and exoU genes were found less frequently and only in 33% of the isolates (Table 6). A recent study indicated that pyocyanin production was higher in earlier P. aeruginosa isolates from the respiratory tract compared to the isolates obtained later in the course of infection. (33) It has been also reported that pyocyanin induces various antagonistic effects on the host, especially pro-inflammatory and free radical effects which cause cellular damage and death. (33)

The relationship between antimicrobial resistance markers and distribution of common virulence factors among P. aeruginosa isolates in this study was not significant, since all MDR isolates and most of the susceptible isolates carried 3 potential virulence genes (algD, lasB and toxA) and secreted pyocyanin (Table 5). A French study detected the virulence genes algD, lasB and toxA in all P. aeruginosa isolates from the sputa of patients with cystic fibrosis and, to a lesser rate, from other non-cystic fibrosis isolates. (3) A Bulgarian study reported that P. aeruginosa isolates from all clinical samples including the respiratory tract carried 100% lasB genes and a high rate of algD genes (95.3%) in cystic fibrosis cases. (23) A study from Iran demonstrated also that all P. aeruginosa isolates from the pulmonary tract harbored significantly higher rates of toxA genes than wound and burn isolates (p<0.004), and other virulence genes such as algD and lasB were less common in respiratory tract specimens. (4)

It can be concluded from our study and other studies that virulence genes lasB, algD, and toxA are mostly associated with P. aeruginosa isolates from the pulmonary tract. However, none of the mentioned studies has previously reported on the association between antimicrobial resistance and virulence potential of their P. aeruginosa isolates as it has been done in this study.

RAPD fingerprinting of 32 MDR P. aeruginosa isolates from hospitalized patients and the construction of the dendrogram of the isolates demonstrated high diversity among P. aeruginosa isolates, with 14 genotype groups, by applying 70% similarity index (Figure 1). Association between the antimicrobial resistance phenotypes and genotype profiles of these 32 isolates revealed a limited similarity of DNA bands patterns between phenotypes and genotypes. Based on these results, it can be suggested that most isolates originated from various clones and there was no single genotype prevalent among more than 5 isolates.

Conclusions

In conclusion, this study has found no significant genetic relationship among MDR P. aeruginosa isolates from the respiratory tract of hospitalized patients or their carriage of virulence genes.

doi: 10.18683/germs.2018.1130

Authors' contributions statement: HYAD performed all laboratory tests, NO supervised all clinical issues of patients, LFAQ helped in genotyping of the isolates, AAS supervised all laboratory tests and writing of the manuscript. All authors read and approved the final version of the manuscript.

Conflicts of interest: All authors--none to disclose.

Funding: This work was supported by a grant from The Jordan University, Dean of Research (No. 74/2014-2015).

Acknowledgement: The authors are thankful to the Jordan University Hospital for allowing them to collect clinical specimens and data from the admitted patients. Also, the authors thank Mrs. Ilham Abu-Khader for performing the statistical analysis of the study.

References

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(14.) Pitout J DD, Gregson DB, Poirel L, McClure JA, Le P, Church DL. Detection of Pseudomonas aeruginosa producing metallo-p-lactamases in a large centralized laboratory. J Clin Microbiol 2005;43:3129-35. [Crossref]

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(17.) Monteiro J, Widen RH, Pignatari AC, Kubasek C, Silbert S. Rapid detection of carbapenemase genes by multiplex real-time PCR. J Antimicrob Chemother 2012;67:906-9. [Crossref]

(18.) Akpaka PE, Swanston WH, Ihemere HN, et al. Emergence of KPC-producing Pseudomonas aeruginosa in Trinidad and Tobago. J Clin Microbiol 2009;47:2670-1. [Crossref]

(19.) Wolska K, Szweda P. Genetic features of clinical Pseudomonas aeruginosa strains. Pol J Microbiol 2009;58:255-60.

(20.) Mitov I, Strateva T, Markova B. Prevalence of virulence genes among Bulgarian nosocomial and cystic fibrosis isolates of Pseudomonas aeruginosa. Braz J Microbiol 2010;41:588-95. [Crossref]

(21.) Nanvazadeh F, Khosravi AD, Zolfaghari MR, Parhizgari N. Genotyping of Pseudomonas aeruginosa strains isolated from burn patients by RAPD-PCR. Burns 2013;39:1409-13. [Crossref]

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(23.) Al-Muhairi S, Zoubeidi T, Ellis M, Nicholls MG, Safa W, Joseph J. Demographics and microbiological profile of pneumonia in United Arab Emirates. Monaldi Arch Chest Dis 2006;65:13-8.

(24.) Vincent JL, Rello J, Marshall J, et al. International study of the prevalence and outcomes of infection in intensive care units. JAMA 2009;302:2323-9. [Crossref]

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(26.) Wassef M, El Mahallawy H, Zafer MM, Ghaith D, Abdel Hamid R. Lab based surveillance of multidrug resistant Pseudomonas aeruginosa in Cairo University Hospitals, Egypt. J Microbiol Exp 2015;2:00039. [Crossref]

(27.) Masaadeh HA, Jaran AS. Incident of Pseudomonas aeruginosa in post-operative wound infection. Am J Infect Dis 2009;5:1-6. [Crossref]

(28.) Voor In 't Holt AF, Severin JA, Lesaffre EM, Vos MC. A systematic review and meta-analyses show that carbapenem use and medical devices are the leading risk factors for carbapenem-resistant Pseudomonas aeruginosa. Antimicrob Agents Chemother 2014;58:2626-37. [Crossref]

(29.) Zhao WH, Hu ZQ. Acquired metallo-p-lactamases and their genetic association with class 1 integrons and ISCR elements in Gram-negative bacteria. Future Microbiol 2015;10:873-87. [Crossref]

(30.) Obeidat N, Jawdat F, Al-Bakri GA, Shehabi AA. Major biologic characteristics of Acinetobacter baumannii isolates from hospital environmental and patients' respiratory tract sources. Am J Infect Control 2014;42:401-4. [Crossref]

(31.) Moreau-Marquis S, Stanton BA, O'Toole GA. Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway. Pulm Pharmacol Ther 2008;21:595-9. [Crossref]

(32.) Schulert GS, Feltman H, Rabin SD, Martin CG, Battle SE, Rello J, Hauser AR. Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia. J Infect Dis 2003;188:1695-706. [Crossref]

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Received: 05 December 2017; revised: 16 February 2018; accepted: 19 February 2018.

Heba Y. Al Dawodeyah [1], Nathir Obeidat [2], Luay F. Abu-Qatouseh [3], Asem A. Shehabi [4], *

[1] MSc, Department of Pathology-Microbiology, School of Medicine, The Jordan University, Queen Rania str. 100, Amman, Jordan; [2] MD, Department of Internal Medicine/Division of Pulmonary Diseases, The Jordan University Hospital, Queen Rania str. 100, Amman, Jordan; [3] PhD, Faculty of Pharmacy, Department of Biomedical Sciences, University of Petra, Airport str. 317, Amman, Jordan; [4] DSc, Department of Pathology-Microbiology, School of Medicine, The Jordan University, Queen Rania str. 100, Amman, Jordan.

* Corresponding author: Asem A. Shehabi, DSc, Department of Pathology-Microbiology, School of Medicine, The Jordan University, Queen Rania str. 100, Amman, Jordan. a shehab i@j u.edu.jo

Caption: Figure 1. Dendrogram based on RAPD results showing relatedness between 32 MDR P. aeruginosa isolates; the dendrogram was generated by the neighbor joining method with PyElph software (USA).
Table 1. List of all primers, their product sizes and
references used for detection of ESBLs, MBLs and
virulence genes

Target genes        Primer      Primer sequence (5' to 3')

[bla.sub.CTX-M1]   CTX-M(F)        GACGATGTCACTGGCTGAGC
                   CTX-M(R)        AGCCGCCGACGCTAATACA
[bla.sub.TEM]       TEM-A           ATAAAATTCTTGAAGAC
                    TEM-B           TTACCAATGCTTAATCA
[bla.sub.VEB-1]    VEB-FOR       CGACTT CCATTT CCCGAT GC
                   VEB-REV        GGACT CT GCAACAAATACGC
[bla.sub.SHV-1]    SHV-FOR        TGGTTATGCGTTATATTCGCC
                   SHV-REV         GCTTAGCGTTGCCAGTGCT
[bla.sub.GES]      GES-1(F)        CTATTACTGGCAGGGATCG
  type             GES-1(R)         CCTCTCAATGGTGTGGGT
[bla.sub.IMP]       IMP-A          GAAGGCGTTTATGTTCATAC
                    IMB-B        GTACGTTT CAAGAGT GAT GC
[bla.sub.VIM]      VIM2004A       GTTTGGT CGCATAT CGCAAC
                   VIM2004B        AATGCGCAGCACCAGGATAG
[bla.sub.KPC]       KPC(F)         ATGTCACTGTATCGCCGTCT
                    KPC(R)         TTACTGCCCGTTGACGCCCA
algD                algDF          CGTCTGCCGCGAGATCGGCT
                    algDR          GACCTCGACGGTCTTGCGGA
lasB                lasBF        GGAATGAACGAAGCGTTCTCCGAC
                    lasBR         TTGGCGTCGACGAACACCTCG
toxA                toxAF          CTGCGCGGGTCTATGTGCC
                    toxAR          GATGCTGGACGGGTCGAG-3
exoS               exo S1-F   TCTGAATTCTTCCAGGCGGGTGAACATCA
                   exo S1-R   TTTAGATCTCACCCTGGTATCCAAGGCGA
exoU               exo U-F     GGG AAT ACT TTC CGG GAA GTT
                   exo U-R     CGA TCT CGC TGC TAA TGT GTT
P. aeruginosa      PA-SS-F         (GGGGGATCTTCGGACCTCA
  16S rDNA         PA-SS-R         (TCCTTAGAGTGCCCACCCG

Target genes        Primer     Product    Reference
                              size (bp)

[bla.sub.CTX-M1]   CTX-M(F)      499         13
                   CTX-M(R)
[bla.sub.TEM]       TEM-A       1,075        14
                    TEM-B
[bla.sub.VEB-1]    VEB-FOR       642         15
                   VEB-REV
[bla.sub.SHV-1]    SHV-FOR       867         21
                   SHV-REV
[bla.sub.GES]      GES-1(F)      594         22
  type             GES-1(R)
[bla.sub.IMP]       IMP-A        587         14
                    IMB-B
[bla.sub.VIM]      VIM2004A      382         14
                   VIM2004B
[bla.sub.KPC]       KPC(F)       880         18
                    KPC(R)
algD                algDF        313         19
                    algDR
lasB                lasBF        284         19
                    lasBR
toxA                toxAF        270         23
                    toxAR
exoS               exo S1-F      504          6
                   exo S1-R
exoU               exo U-F       428         20
                   exo U-R
P. aeruginosa      PA-SS-F       956         12
  16S rDNA         PA-SS-R

Table 2. Demographic characteristics of the 61 investigated
patients and their clinical features in association with
positive P. aeruginosa

Patient               Hospitalized        Non-hospitalized
                   patients * (n=45)       patients (n=16)

characteristics         No. (%)                No. (%)

Male                   29 (64.4)              6 (37.5)
mean age          36.48 [+ or -] 14.56   47.5 [+ or -] 18.09
  [+ or -] SD,
  years
Female                 16 (35.5)              10 (62.5)
mean age          33.63 [+ or -] 13.13   32.1 [+ or -] 10.19
  [+ or -] SD,
  years
Fever (> 38 [degrees]C)        17 (37.8)              6 (37.5)
Cough                  43 (95.6)              16 (100)
Purulent sputum         45 (100)              15 (93.8)
Dyspnea                22 (48.9)              4 (25.0)
Chest pain             25 (55.6)              10 (22.2)
Hemoptysis             16 (35.6)              2 (12.5)
COPD                    3 (6.7)               5 (31.3)
Cystic fibrosis         4 (8.9)               6 (37.5)
Received                45 (100)              14 (87.5)
  antibiotics
  during the
  past 4 weeks
  prior to
  admission

Patient                  Total           P-value

characteristics         No. (%)

Male                   35 (57.4)
mean age          38.37 [+ or -] 15.51    0.061
  [+ or -] SD,
  years
Female                 26 (42.6)
mean age          33.04 [+ or -] 11.89
  [+ or -] SD,
  years
Fever (> 38 [degrees]C)        23 (37.7)          0.984
Cough                  59 (96.7)          0.541
Purulent sputum        60 (98.4)          0.262
Dyspnea                26 (42.6)          0.085
Chest pain             35 (57.4)          0.428
Hemoptysis             18 (29.5)          0.074
COPD                    8 (13.1)          0.421
Cystic fibrosis        10 (16.4)          0.477
Received               59 (96.7)          0.066
  antibiotics
  during the
  past 4 weeks
  prior to
  admission

Patient               OR (95%CI)

characteristics

Male
mean age          3.021(0.926-9.845)
  [+ or -] SD,
  years
Female
mean age
  [+ or -] SD,
  years
Fever (> 38 [degrees]C)   1.012 (0.312-3.286)
Cough                  Undefined
Purulent sputum        Undefined
Dyspnea           2.87 (0.8031-0.25)
Chest pain        0.75 (0.233-2.418)
Hemoptysis         1.16 (5.89-29.91)
COPD              0.778 (3.862-19.17)
Cystic fibrosis   0.038 (0.163-0.687)
Received               Undefined
  antibiotics
  during the
  past 4 weeks
  prior to
  admission

COPD--chronic obstructive pulmonary disease; OR--odds ratio;
SD--standard deviation; 95%CI--95% confidence interval.

* Fisher's exact test and chi-squared test were used to
compare significant characteristics between hospitalized and
non-hospitalized patients.

Table 3. Demographic characteristics of the 45 hospitalized
patients in clinical wards and intensive care units and their
clinical features in association with positive P. aeruginosa

Patient             Patients       Patients       Total
characteristics   hospitalized   hospitalized    (n=45)
                  in clinical    in intensive
                  wards (n=27)    care units
                                    (n=18)

                    No. (%)        No. (%)       No. (%)

Fever ([greater    8 (47.1%)       9 (50.0)     17 (37.8)
  than or equal
  to] 38
  [degrees]C)
Cough              25 (92.6)       18 (100)     43 (95.6)
Purulent sputum     27 (100)       18 (100)     45 (100)
Dyspnea            12 (44.4)      10 (55.6)     22 (48.9)
Chest pain         17 (63.0)       8 (44.4)     25 (55.6)
Hemoptysis         11 (40.7)       5 (27.8)     16 (35.6)
COPD                   0           3 (16.7)     3 (13.1)
Cystic fibrosis     8 (29.6)       2 (11.1)     10 (16.4)
Received            27 (100)       18 (100)     45 (100)
  antibiotics
  during the
  past 4 weeks
  prior to
  admission

Patient           P-value        OR (95%CI)
characteristics

Fever ([greater    0.167    2.375 (0.688-8.202)
  than or equal
  to] 38
  [degrees]C)
Cough              0.509         Undefined
Purulent sputum     --           Undefined
Dyspnea            0.465    1.562 (0.471-5.189)
Chest pain         0.221    0.471 (0.140-1.585)
Hemoptysis         0.373    0.559 (0.155-2.024)
COPD               0.028         Undefined
Cystic fibrosis    0.143    3.368 (0.624-18.186)
Received            --           Undefined
  antibiotics
  during the
  past 4 weeks
  prior to
  admission

COPD--chronic obstructive pulmonary disease; OR--odds
ratio; SD--standard deviation; 95%CI--95% confidence
interval.

Table 4. Antimicrobial susceptibility pattern of
61 P. aeruginosa isolates *

Antimicrobial   Susceptible   [MIC.sub.50]
agents           isolates     ([micro]g/mL)
                  No. (%)

Amikacin         30 (49.1)         ND
Ciprofloxacin    30 (49.1)         2.3
Gentamicin       23 (37.7)         ND
Ceftazidime      50 (82.0)         1.8
Aztreonam        35 (57.3)         ND
Meropenem        48 (78.7)         2.8
Imipenem         49 (80.3)         3.6
Piperacillin     38 (62.2)         ND
  -tazobactam
Cefepime         50 (82.0)         ND
Cefotaxime       15 (24.6)         ND
Colistin         61 (100)         0.18

Antimicrobial   [MIC.sub.90]      MIC range
agents          ([micro]g/mL)   ([micro]g/mL)

Amikacin             ND
Ciprofloxacin        4.1          0.012-32
Gentamicin           ND
Ceftazidime          3.3           0.38-64
Aztreonam            ND
Meropenem            4.9          0.016-32
Imipenem             6.5           0.19-32
Piperacillin         ND
  -tazobactam
Cefepime             ND
Cefotaxime           ND
Colistin            0.32         0.032- 3.0

* 32 of the isolates were multidrug resistant,
recovered from hospitalized patients.

MIC--minimum inhibitory concentration; ND--not done.

Table 5. Distribution of ESBL and MBL genes
in association with potential virulence genes
among 32 MDR P. aeruginosa isolates

ESBL genes/       P. aeruginosa         Putative
MBL genes           isolates,           virulence
                     no. (%) *        genes/factors
                                          (%) **

[bla.sub.CTX-M]     22 (68.8)       algD, lasB, toxA,
                                     pyocyanin (100)
[bla.sub.VEB]        6 (18.9)       algD, lasB, toxA,
                                     pyocyanin (100)
[bla.sub.TEB]        6 (18.9)       algD, lasB, toxA,
                                     pyocyanin (100)
[bla.sub.GES]        5 (15.6)      algD, las B, toxA,
                                     pyocyanin (100)
[bla.sub.SHV]        4 (12.5)       algD, lasB, toxA,
                                     pyocyanin (100)
[bla.sub.VIM-2]      3 (9.4)        algD, lasB, toxA,
                                     pyocyanin (100)
[bla.sub.MP-1]          0                  --
[bla.sub.KPC]           0                  --

* All 10 isolates from cystic fibrosis cases were
positive for the genes bOcix-M, algD, lasB, toxA and
4 of them secreted pyocyanin.

** Some of these isolates were simultaneously
positive to two to four ESBL genes or one MBL
gene.

Table 6. Distribution of potential virulence genes
among 61 P. aeruginosa isolates

Virulence factor   Gene     Positive
                           virulence

                           genes No.
                          isolates (%)

Alginate           algD        60        (98)
Elastase B         lasB        60        (98)
Exotoxin A         toxA        49        (80)
Exoenzyme S        exoS        20        (33)
Exoenzyme U        exoU        20        (33)
Pyocyanin                      53        (87)
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Title Annotation:Original article
Author:Dawodeyah, Heba Y. Al; Obeidat, Nathir; Abu-Qatouseh, Luay F.; Shehabi, Asem A.
Publication:GERMS
Article Type:Report
Date:Mar 1, 2018
Words:5174
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