Genome Analysis of Pseudomonas aeruginosa Strains from Chronically Infected Patients with High Levels of Persister Formation.

The appearance of persister cells with low metabolic rates are key factors leading to antibiotic treatment failure. Such persisters are multidrug tolerant and play a key role in the recalcitrance of biofilm-based chronic infections. Here, we present the genomic analyses of three distinct Pseudomonas aeruginosa Egyptian persister-isolates recovered from chronic human infections. To calculate the persister frequencies, viable counts were determined before and after treatment with levofloxacin. The susceptibilities of isolates to different antibiotics were determined using the agar-dilution method. To determine their recalcitrance, the levofloxacin persisters were further challenged with lethal concentrations of meropenem, tobramycin, or colistin. Furthermore, the biofilm formation of the persister strains was estimated phenotypically, and they were reported to be strong biofilm-forming strains. The genotypic characterization of the persisters was performed using whole genome sequencing (WGS) followed by phylogenetic analysis and resistome profiling. Interestingly, out of the thirty-eight clinical isolates, three isolates (8%) demonstrated a persister phenotype. The three levofloxacin-persister isolates were tested for their susceptibility to selected antibiotics; all of the tested isolates were multidrug resistant (MDR). Additionally, the P. aeruginosa persisters were capable of surviving over 24 h and were not eradicated after exposure to 100X-MIC of levofloxacin. WGS for the three persisters revealed a smaller genome size compared to PAO1-genome. Resistome profiling indicated the presence of a broad collection of antibiotic-resistance genes, including genes encoding for antibiotic-modifying enzymes and efflux pump. Phylogenetic analysis indicated that the persister isolates belong to a distinct clade rather than the deposited P. aeruginosa strains in the GenBank. Conclusively, the persister isolates in our study are MDR and form a highly strong biofilm. WGS revealed a smaller genome that belongs to a distinct clade.

Impact of short chain fatty acids (SCFAs) on antimicrobial activity of new ß-lactam/ß-lactamase inhibitor combinations and on virulence of Escherichia coli isolates.

In a healthy gut microbiota, short chain fatty acids (SCFAs) are produced. The antibacterial action of SCFAs against intestinal pathogens makes them useful for ensuring the safety of food and human health. In this study, we aimed to assess the in vitro inhibitory activity of SCFAs, and to report, for the first time, their impact on the activity of new ß-lactam/ß-lactamase inhibitor combinations. The minimum inhibitory concentrations of acetic, propionic, and butyric acids were determined against E. coli clinical isolates recovered from gastrointestinal infections. Cefoperazone/sulbactam, ceftazidime/avibactam and cefepime/enmetazobactam are new ß-lactam/ß-lactamase inhibitor combinations that were studied for their combined therapeutic effects. Also, the effects of pH and concentration of SCFAs were evaluated on in vitro bacterial growth and expression of genes encoding for motility, adhesion, invasion, and biofilm formation. SCFAs were tested at concentrations of 12 mM at pH 7.4 (ileum-conditions), in addition to 60 mM and 123 mM, at pH 6.5 (colon-conditions). The tested SCFAs showed the same MIC (3750 µg ml-1 ≃ 60 mM) against all isolates. Furthermore, the addition of SCFAs to the tested ß-lactam/ß-lactamase inhibitor combinations greatly restored the susceptibility of the isolates. SCFAs had significant effect on bacterial growth and virulence in a pH and concentration-dependent manner; low ileal concentration potentiated E. coli growth, while higher colonic concentration significantly suppressed growth and down-regulated the expression of virulence genes (fliC, ipaH, FimH, BssS). Therefore, the significant inhibitory effect of colonic SCFAs on ß-lactam/ß-lactamase inhibitor combinations might lead to the development of promising treatment strategies.

The alarming association between antibiotic resistance and reduced susceptibility to biocides in nosocomial MRSA isolates from two regional hospitals in Egypt.

Methicillin-resistant Staphylococcus aureus is one of the major clinical problems in hospitals because of its resistance to many antimicrobials. Biocides are used in hospitals to control nosocomial infections. This work aimed to investigate the relationship between the presence of integrons and reduced susceptibility to both biocides and antimicrobials in nosocomial multidrug-resistant (MDR)-MRSA isolates. A total of 114 clinical and eight environmental MRSA isolates were collected from Zagazig University Hospitals and El-Ahrar Educational Hospital, Egypt. These isolates were identified as MRSA by disk diffusion method (DDM) and confirmed by PCR. Susceptibility profile against 12 antibiotics and five biocides was determined by DDM and agar dilution method, respectively. Presence of integrons was investigated by PCR in MDR isolates. Seventy-five clinical and six environmental isolates were MDR and had reduced susceptibility to biocides. Class I integron was detected in plasmid DNA of 34 isolates and genomic DNA of 14 isolates. Meanwhile, class II integron was only detected in plasmid DNA of 10 clinical isolates. This study revealed a high prevalence of MDR-MRSA clinical and environmental isolates, both had reduced susceptibility to investigated biocides. Class I integron was more predominant in plasmid DNA of isolates, indicating that plasmid is a major carrier for integrons that transfer resistance genes. In conclusion, the association between antibiotic resistance and biocides reduced susceptibility is alarming. The selection of curative antibiotic should depend on the antimicrobial susceptibility profile. Furthermore, biocides should always be used at appropriate concentrations to prevent the evolution of resistance and to control the hospital-transmission of MRSA.

Distribution of genes encoding adhesins and biofilm formation capacity among Uropathogenic Escherichia coli isolates in relation to the antimicrobial resistance.

BACKGROUND: Escherichia coli is the most predominant pathogen involved in UTIs. Mainly, fimbrial surface appendages are implicated in adherence to urothelium besides non-fimbrial proteins. OBJECTIVES: to determine prevalence of genes encoding fimbrial and non-fimbrial proteins among Uropathogenic Escherichia coli (UPEC). Furthermore, distribution of these genes and biofilm formation capacity were investigated in relation to antimicrobial resistance. METHODS: Antimicrobial susceptibility of 112 UPEC isolates was performed using disc diffusion method. ESBL production was confirmed by double disc synergy test. Genes encoding fimbrial and non-fimbrial proteins were detected using PCR and biofilm formation was investigated using microtitre plate assay. RESULTS: UPEC isolates exhibited high resistance against doxycyclines (88.39 %), ß-lactams (7.14-86.6%), sulphamethoxazole-trimethoprim (53.75%) and fluoro-quinolones (50%). Fifty percent of tested isolates were ESBL producers. PapGII gene was statistically more prevalent among pyelonephritis isolates. SfaS, focG and picU genes were statistically associated with fluoroquinolone (FQs) sensitive isolates and Dr/afaBC gene was statistically associated with ESBL production. Moreover, non-MDR isolates produced sturdier biofilm. CONCLUSION: PapGII adhesin variant seems to have a critical role in colonization of upper urinary tract. There is a possible link between antimicrobial resistance and virulence being capable of affecting the distribution of some genes besides its negative impact on biofilm formation.

Impact of specific inhibitors on metallo-ß-carbapenemases detected in Escherichia coli and Klebsiella pneumoniae isolates.

Carbapenems are widely regarded as the drugs of choice for the treatment of severe infections caused by extended-spectrum beta lactamases producing Enterobacteriaceae. The emergence of carbapenem-resistant organisms is worrisome due to the limited treatment options. Detection of carbapenemase-producing bacteria is critical for the choice of appropriate therapy. However, Inhibition of carbapenemases is an alternative approach to combat resistance to carbapenms. In this study, Escherichia coli and Klebsiella pneumoniae carbapenem resistant isolates were recovered from 300 clinical isolates. They were subjected phenotypically for detection of class B metallo-carbapenemase (MBL) producers (by carbapenem disks with or without EDTA), and were subjected for confirmation genotypically by PCR. In addition, the synergistic activities of MBL-inhibitors in combination with carbapenems were elucidated. Two E. coli and 15 K. pneumoniae isolates were carbapenem resistant. The genes encoding blaNDM-1 carbapenemase were detected in 16/17 isolates solely, or collaboratively with either blaVIM, or blaIMP or both in all carbapenem resistant isolates, by PCR method. The VIM-carbapenemase was encoded by one isolate. In pre-clinical trials for development of MBL-specific inhibitors, Sub-inhibitory concentrations of citric acid, malic acid, ascorbic acid and ciprofloxacin in combination with imipenem or meropenem exerted synergistic activities against metallo-carbapenemases. Their activities are probably attributed to the chelation of zinc ions in the active site of carbapenemase. Conclusively, these promising combined therapies might represent a new strategy for combating such serious infections caused by metallo-B-carbapenemase producers of K. pneumoniae and E. coli isolates.

Deletion of Aspergillus nidulans GDP-mannose transporters affects hyphal morphometry, cell wall architecture, spore surface character, cell adhesion, and biofilm formation.

Systemic human fungal infections are increasingly common. Aspergillus species cause most of the airborne fungal infections. Life-threatening invasive aspergillosis was formerly found only in immune-suppressed patients, but recently some strains of A. fumigatus have become primary pathogens. Many fungal cell wall components are absent from mammalian systems, so they are potential drug targets. Cell-wall-targeting drugs such as echinocandins are used clinically, although echinocandin-resistant strains were discovered shortly after their introduction. Currently there are no fully effective anti-fungal drugs. Fungal cell wall glycoconjugates modulate human immune responses, as well as fungal cell adhesion, biofilm formation, and drug resistance. Guanosine diphosphate (GDP) mannose transporters (GMTs) transfer GDP-mannose from the cytosol to the Golgi lumen prior to mannosylation. Aspergillus nidulans GMTs are encoded by gmtA and gmtB. Here we elucidate the roles of A. nidulans GMTs. Strains engineered to lack either or both GMTs were assessed for hyphal and colonial morphology, cell wall ultrastructure, antifungal susceptibility, spore hydrophobicity, adherence and biofilm formation. The gmt-deleted strains had smaller colonies with reduced sporulation and with thicker hyphal walls. The gmtA deficient spores had reduced hydrophobicity and were less adherent and less able to form biofilms in vitro. Thus, gmtA not only participates in maintaining the cell wall integrity but also plays an important role in biofilm establishment and adherence of A. nidulans. These findings suggested that GMTs have roles in A. nidulans growth and cell-cell interaction and could be a potential target for new antifungals that target virulence determinants.

Relationship between Sap prevalence and biofilm formation among resistant clinical isolates of Candida albicans.

BACKGROUND: Fungal infections represent a serious health problem especially in immunocompromised individuals. Candida albicans is the most common fungi that cause superficial and systemic infections with high mortality rates. Anti-fungal resistance of C. albicans may be attributed to its virulence. Biofilm formation and proteolytic activity are major virulence determents that may influence both pathogenicity and anti-fungal resistance of Candida albicans. OBJECTIVE: This work studied the relation between biofilm formation, proteolytic activity and prevalence of some Sap genes with reduced susceptibility of C. albicans to different anti-fungal agents. METHODS: Fifty three C. albicans strains isolated from patients with systemic infections, identified by germ tube, chromogenic agar and confirmed by PCR, were subjected to evaluate their proteolytic activity, the degree of biofilm production and the prevalence of Sap9 and Sap10 genes. The susceptibility of the isolates was determined by disk diffusion method against five antifungal drugs. RESULTS AND CONCLUSION: Four of the C. albicans isolates were resistant to 3 anti-fungal drugs, strong biofilm producer, have proteolytic activity and contain either Sap9 or Sap10 or both. Conclusively, although anti-fungal resistance among the isolates was rare, a relation between the anti-fungal resistance and some major virulence factors was evidently proved in this study.

Integron occurrence is linked to reduced biocide susceptibility in multidrug resistant Pseudomonas aeruginosa.

OBJECTIVE: Integrons are gene acquisition systems commonly found in bacterial genomes that play a major role in the dissemination of resistance to antibiotics. This work aimed to study the relationship between the presence of integrons and the reduced susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa isolates towards different groups of biocides. METHODS: The antimicrobial susceptibility patterns of 104 clinical isolates were determined against different antibiotics by the disk diffusion method. The isolates were also tested for their susceptibility to six biocides (glutaraldehyde, benzalkonium chloride, cetrimide, chlorhexidine gluconate, chlorocresol and gluconate, and phenyl mercuric nitrate) by agar dilution. The presence of integrons and resistance genes in MDR isolates were detected by polymerase chain reaction. RESULTS: Thirty-six Pseudomonas isolates were MDR, and the majority of these isolates showed reduced susceptibility to biocides. In the MDR isolates, Class I integron was detected in 22 isolates (61.1%), while Class II and III integrons were identified in only four isolates (11.1%), In addition, aacA4 and qacE genes were detected in 22 (61.1%) and 11 (30.5%) isolates, respectively. Integron I-positive isolates showed reduced susceptibility to tested biocides. CONCLUSIONS: The current study reveals the presence of different classes of integrons, with class I being predominant. Class I integron may be responsible for generating MDR P. aeruginosa isolates with reduced susceptibility to biocides. This linkage between integrons and biocide resistance in MDR-Pseudomonas isolates is notable and could be clinically important. Strict antibiotic prescription policies and the adequate use of biocides could help in controlling this problem.

Impact of slime dispersants and anti-adhesives on in vitro biofilm formation of Staphylococcus epidermidis on intraocular lenses and on antibiotic activities.

OBJECTIVES: Infectious endophthalmitis has occurred despite the use of antibiotics in irrigating solutions during implantation of intraocular lenses (IOLs). This infection is generally resistant to antibiotic therapy and, therefore, removal of the implant is necessary before eradication of the infection. This study was designed to assess the role of chosen dispersants and anti-adhesives in inhibiting Staphylococcus epidermidis hydrophobicity, adhesion, slime production and subsequently biofilm formation on IOLs. METHODS: The relative activity of several potential slime dispersants and anti-adhesives on slime production, hydrophobicity and the adherence of S. epidermidis to IOLs and the degrees to which their effects enhance antibiotic activities were investigated. RESULTS AND CONCLUSIONS: The MBCs of antibiotics against S. epidermidis strains in a biofilm increased 10-16 times compared with those against bacterial strains in suspension. Addition of slime dispersants or anti-adhesives reversed the susceptibility of the strains in a biofilm to that of bacteria in suspension. Slime production by S. epidermidis strains was significantly diminished by dispersants. Anti-adhesives, hyaluronan, heparin and carpobol 934 exerted less effects on slime production than dispersants. Addition of slime dispersants or anti-adhesives to cell cultures resulted in a significant reduction in bacterial surface hydrophobicity compared with control untreated cultures (at P < 0.001). Reduction of slime production and bacterial surface hydrophobicity led to a marked decrease in the adherence of S. epidermidis to IOLs. Slime dispersants were more effective at reducing bacterial adherence than anti-adhesives. Simultaneous use of antibiotics with slime dispersants or anti-adhesives will exert a more beneficial effect during IOL implantation.

Treatment of experimental osteomyelitis by liposomal antibiotics.

OBJECTIVES: Traditional antibiotic therapy of staphylococcal osteomyelitis by a single drug or a drug combination is ineffective in producing complete sterilization of infected bones. The aim of this study was to develop a non-traditional delivery system of antibiotics for treatment of chronic experimental osteomyelitis. METHODS: In the current work, ciprofloxacin and vancomycin were encapsulated in a cationic, anionic or neutral liposomal formulation. For prolonged circulation in serum, liposomal dispersions (<100 nm in diameter) were sonicated for different times (20, 40, 60 or 80 s), and tested for antibacterial activities. RESULTS AND CONCLUSIONS: Liposomes sonicated for 40 s gave the highest antibacterial activities in vitro. Since cationic liposomes trapped the highest percentage of antibiotics, and enhanced antibacterial activity above that of the free drugs, they were used for therapeutic trials to treat chronic staphylococcal osteomyelitis induced in rabbits. Therapeutic trials with antibiotics given intravenously revealed that, free ciprofloxacin or vancomycin given alone for 14 days was ineffective in sterilizing bone. Combination therapy with free ciprofloxacin and vancomycin for 14 days was more effective. However, this group showed renal dysfunction and severe diarrhoea, which resulted in loss of 33.3% of treated animals. Treatment with liposomal forms of either drug for 7 days was ineffective. Meanwhile, combination therapy in liposomal form for 7 days was more effective. Complete sterilization of bone tissues on cultures (100% cure) was obtained only in the group treated for 14 days with the combination of both drugs in liposomal form. Moreover, liposomal formulations showed much lower nephrotoxicity and a lower incidence of severe diarrhoea than that induced by free drugs.

Phenotypic and genotypic characterization of invasive Streptococcus pneumoniae clinical isolates.

BACKGROUND: The emergence of infection caused by invasive penicillinnonsusceptible (PNS) and multidrug-resistant strains of Streptococcus pneumoniae has become a worldwide concern, necessitating the epidemiologic surveillance of such strains. OBJECTIVES: One aim of this study was to identify clones of invasive PNS S pneumoniae among isolates in Riyadh, Saudi Arabia. The second aim was to compare these clones with international clones to track their spread in Saudi Arabia. METHODS: The phenotypes of invasive isolates characterized as S pneumoniae were determined using susceptibility testing and serotyping (capsular test and E-test). The genotypes of PNS isolates were determined using random amplified polymorphic DNA analysis. The genetic relatedness of these local strains to the international widespread clones was investigated. RESULTS: Of 296 S pneumoniae isolates identified using biochemical and culture characteristics, 89 (30.1%) were invasive. Susceptibility testing using the E-test revealed that 17 of the 89 invasive isolates (19.1%) were PNS. Most of the 89 isolates (89.9%) were resistant to sulfamethoxazole-trimethoprim; 32.6% and 23.6% of isolates were resistant to chloramphenicol and tetracycline, respectively. All of the isolates (100.0%) were fully susceptible to ceftriaxone and vancomycin. Capsular serotyping of the 89 isolates showed that 19A (18.0%), 613 (14.6%), 23F (13.5%), 9V (11.2%), 14 (6.7%), 19F (5.6%), and 18C (4.5%) were the most predominant serogroups/serotypes. The 17 PNS strains were confirmed on polymerase chain reaction to have penicillin resistance genes. Of these 17 strains, international clone 19A-a was the most predominant (41.2%), followed by 6B-a (17.6%), and 23F-a and 9V-a (each, 11.8%). CONCLUSIONS: The present study identified the spread of the 4 most commonPNS S pneumoniae isolates (clones)-19A, 613, 23F, and 9V-to Riyadh, but identified no new clones among patients having invasive infection with S pneumoniae in Riyadh. This study emphasizes that international PNS clones have contributed to the prevalence and spread of PNS pneumococci among the clinical isolates in Saudi Arabia.

Correlation between susceptibility and BRO type enzyme of Moraxella catarrhalis strains.

Clinical isolates of Moraxella catarrhalis (76 isolates) were screened for beta-lactamase production and antibiotic susceptibility. beta-Lactamases (detected in 90.8% of isolates) were typed using isoelectric focusing to BRO-1 (87%) and BRO-2 (13%). Minor variations in electrofocusing patterns between the two types were seen. Isolates expressing BRO type enzymes showed solid resistance to penicillin, ampicillin and cephalothin, in particular BRO-1 producers. BRO-1 isolates were less susceptible to cephems and to beta-lactamase inhibitors than BRO-2 isolates. Isolates harbouring BRO-1 enzymes have more enzymatic activity than those expressed by BRO-2 isolates. Apart from resistance to tetracycline (14.5%), all isolates were consistently susceptible to erythromycin, chloramphenicol, ciprofloxacin and gentamicin. The conjugal transfer of BRO beta-lactamase gene(s) between M. catarrhalis isolates occurred with a frequency of 10(-5) to 10(-7)/donor cell. The data emphasize the importance of M. catarrhalis as an etiological agent spreading beta-lactamases that may inhibit some beta-lactams and lead to failure in treatment of mixed infections.

Potential biological indicators for glutaraldehyde and formaldehyde sterilization processes.

The present study aimed to isolate, select, and evaluate bacterial isolates with potential for use as biological indicators for sterilization with glutaraldehyde and/or formaldehyde. A total of 340 local Bacillus isolates were screened for glutaraldehyde and/or formaldehyde resistance by determination of minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and extinction time and were compared with B. subtilis (var. niger) ATCC 9372, the biological indicator for ethylene oxide sterilization, as reference. Of these, 85 isolates had glutaraldehyde MICs of 0.5% or higher, while 29 had formaldehyde MICs of 0.04% or higher. Of the 29 resistant isolates, 15 had MBCs of 0.05% or more. Extinction times were used to evaluate the bactericidal/sporicidal activity of glutaraldehyde. Eight had inactivation times of more than 5 h in 2% glutaraldehyde (pH 8), whereas 12 had inactivation times of more than 3 h in l% formaldehyde, with one isolate in common. These 19 isolates were selected and evaluated as potential biological indicators for aldehydes by determination of the decimal reduction times ( D values), compared with the reference strain. Eight glutaraldehyde-resistant isolates exhibited D values 2.0- to 3.5-fold higher than the reference strain (30 min.). Only five of 12 formaldehyde resistant isolates had D values higher than that of the reference strain. Using six resistant isolates, temperature coefficient values between 2.11 and 3.02 were obtained for 2% formaldehyde. Finally, 14 isolates were tested for potential pathogenicity and were identified to species level. All of the eight glutaraldehyde-resistant isolates, including the isolate with dual resistance, and three formaldehyde-resistant isolates were B. licheniformis, while two other formaldehyde-resistant isolates were B. cereus. Six of the selected B. licheniformis isolates are potential biological indicators for sterilization processes using aldehydes. Three can be suggested for glutaraldehyde only and three for both aldehydes.