Background: Pseudomonas aeruginosa is an opportunistic pathogen involved in number of hospital-acquired infections such as catheter-associated urinary tract infections, bacteremia, septicemia, skin infections, and ventilator-associated pneumoniae. Biofilm formation is an important trait implicated in chronic infections, such as cystic fibrosis and chronic pulmonary obstruction. We evaluated effects of gentamicin, cefepime, and ciprofloxacin on biofilm of P. aeruginosa. Materials and Methods: A total of 266 isolates were collected from the Armed Forces Institute of Pathology (AFIP). Antibiotic susceptibility was assessed by double disk synergy testing. ESBL and carbapenemase detection was performed by phenotypic testing. Molecular screening of the genes was done by PCR. Micro-dilution broth method was used to determine minimum inhibitory concentrations of antibiotics. Biofilm formation was done by micro-titer plate assay. Results: Overall, 20% of the P. aeruginosa isolates were extensively drug-resistant (XDR-PA), and 25% were multi-drug-resistant (MDR-PA). Likewise, 43% of the isolates were ESBL producers, and carbapenemase production was detected in 40% of the isolates. Molecular analysis confirmed occurrence of different resistant factors in ESBL-positive isolates; 67% carried blaTEM, 62% blaCTXM-15, 41% blaSHV, 34% blaCTXM-14, and 33% blaOXA-1. In addition, 68% of the carbapenem-resistant isolates were positive for blaNDM-1, 25% for blaOXA-48, and 22% for blaKPC-2. Biofilm formation was assessed for 234 isolates, out of which 28% were strong biofilm formers. Moderate and weak biofilm formers constituted 46% and 23%, respectively. Overall, ciprofloxacin, levofloxacin, and cefepime showed inhibitory effects on P. aeruginosa biofilms. Antibiotics in combination showed strong synergistic effects (ciprofloxacin and cefepime), while gentamicin and cefepime resulted in complete eradication of P. aeruginosa biofilm. Conclusion: We confirm strong synergistic effects of gentamicin and cefepime that completely eradicated P. aeruginosa biofilm. We further confirm inhibitory effects of ciprofloxacin, levofloxacin, and cefepime on P. aeruginosa biofilms. Hence, combination therapy can be more effective against biofilm-associated infections.
Background: Widespread use of antibiotics as growth promoters and prophylactic agents has dramatic consequences for the development of antibiotic resistance. In this study, we investigated effects of selected antibiotics on bacterial biofilms and performed extensive antibiotic and VF profiling of poultry-meat associated E. coli strains. Methods: Antibiotic susceptibility was performed by a disc diffusion method, followed by molecular screening of resistance and virulence determinants. Further biofilm formation assays, MIC-p, MIC-b, MBIC and MBEC, were performed using standard tissue culture plate method. Results: In total, 83 (75%) samples were confirmed as E. coli from poultry sources, 26 different antibiotics were tested, and maximum numbers of the isolates were resistant to lincomycin (100%), while the least resistance was seen against cefotaxime (1%) and polymyxin B (1%). Overall, 48% of the isolates were ESBL producers and 40% showed carbapenemase activity; important virulence genes were detected in following percentages: fimH32 (39%), papC21 (25%), iutA34 (41%), kpsMT-II23 (28%), papEF9 (11%), papGII22 (27%) and fyuA13 (16%). Colistin showed remarkable anti-biofilm activity, while at sub-MIC levels, gentamicin, ceftriaxone and enrofloxin significantly (p < 0.01) inhibited the biofilms. A strong induction of bacterial biofilm, after exposure to sub-minimal levels of colistin clearly indicates risk of bacterial overgrowth in a farm environment, while use of colistin aggravates the risk of emergence of colistin resistant Enterobacteriaceae, a highly undesirable public health scenario.
Antibiotic resistance is a major global challenge. Although microfluidic antibiotic susceptibility tests (AST) offer great potential for rapid and portable testing to inform correct antibiotic selection, the impact of miniaturisation on broth microdilution (BMD) is not fully understood. We developed a 10-plex microcapillary based broth microdilution using resazurin as a colorimetric indicator for bacterial growth. Each capillary had a 1 microlitre capillary volume, 100 times smaller than microplate broth microdilution. The microcapillary BMD was compared to an in-house standard microplate AST and commercial Vitek 2 system. When tested with 25 uropathogenic isolates (20 Escherichia coli and 5 Klebsiella pneumoniae) and 2 reference E. coli, these devices gave 96.1% (441/459 isolate/antibiotic combinations) categorical agreement, across 17 therapeutically beneficial antibiotics, compared to in-house microplate BMD with resazurin. A further 99 (50 E. coli and 49 K. pneumoniae) clinical isolates were tested against 10 antibiotics and showed 92.3% categorical agreement (914/990 isolate/antibiotic combinations) compared to the Vitek 2 measurements. These microcapillary tests showed excellent analytical agreement with existing AST methods. Furthermore, the small size and simple colour change can be recorded using a smartphone camera or it is feasible to follow growth kinetics using very simple, low-cost readers. The test strips used here are produced in large batches, allowing hundreds of multiplex tests to be made and tested rapidly. Demonstrating performance of miniaturised broth microdilution with clinical isolates paves the way for wider use of microfluidic AST.
Anti-Bacterial Agents , Escherichia coli , Anti-Bacterial Agents/pharmacology , Klebsiella pneumoniae , Microbial Sensitivity Tests
BACKGROUND: The emergence of carbapenem-resistant and hypervirulent hypermucoviscous Klebsiella pneumoniae strains poses a significant public health challenge. We determined the MDR profiles, antibiotic resistance factors, virulence gene complement, and hypermucoviscous features of 200 clinical K. pneumoniae isolates from two major tertiary care hospitals in Islamabad and Rawalpindi, Pakistan. METHODS: Susceptibility profiling and phenotypic analysis were performed according to the CLSI guidelines. Genetic determinants of antibiotic resistance and virulence were detected by PCR. Biofilm formation analysis was performed by microtiter plate assay. RESULTS: The isolates displayed a high degree of antibiotic resistance: 36% MDR-CRKP; 38% carbapenem resistance; 55% gentamicin resistance; 53% ciprofloxacin resistance; and 59% aztreonam resistance. In particular, the level of resistance against fosfomycin (22%) and colistin (15%) is consistent with previous reports of increased resistance levels. Combined resistance to carbapenem and colistin was 7%. Genetic factors associated with colistin resistance (mcr-1 and mcr-2 genes) were detected in 12 and 9% of the isolates, respectively. Significant differences in resistance to gentamicin and levofloxacin were observed between the 200 isolates. Many of the isolates harbored genes specifying extended-spectrum and/or carbapenem-resistant ß-lactamases: bla CTX-M-15 (46%), bla NDM-1 (39%), and bla OXA-48 (24%). The prevalence of the hypermucoviscous phenotype was 22% and 13% of the MDR isolates carried the rmpA gene (regulator for mucoid phenotype). Key virulence factor genes detected include those encoding: porins (ompK35 and ompK36; at 56 and 55% prevalence, respectively); adhesins (fimH, mrkD, and ycfM; at 19, 18, and 22% prevalence, respectively); and the polysaccharide regulator, bss, at 16% prevalence. CONCLUSION: This report highlights carbapenem-resistant K. pneumoniae (CRKP) prevalence, emerging resistance to fosfomycin, and the presence of mcr-1 and mcr-2 in colistin-resistant isolates. Further, the detection of rmpA signifies the prevalence of the hypermucoviscous trait in CRKP clinical isolates from Pakistan.
BACKGROUND: Community-acquired urinary tract infections are associated with significant morbidity, and uropathogenic Escherichia coli (UPEC) alone causes 90% of urinary tract infections. This bacterium retains a diverse armament of virulence factors including fimbria, hemolysins, and siderophores production. In a post invasion scenario, formation of intracellular communities mimic biofilm-like characteristics and are linked to recurrent urinary tract infections. We investigated the effects of different frontline antibiotics on the formation, inhibition, and eradication of biofilms of virulent UPEC strains. MATERIALS AND METHODS: A total of 155 UPEC strains were scrutinized for various virulence factors including gelatinase, cell surface hydrophobicity, hemagglutination, and serum bactericidal activity. Biofilm formation was confirmed by three different methods: Congo red agar, test tube, and tissue culture plate method. Biofilm inhibition and eradication assays were performed according to the standard protocols. Topographical analysis of biofilms was done by scanning electronic microscopy (SEM). RESULTS: Out of 155 strains, 113 (73%) were strong biofilm formesr, while 37 (24%) produced biofilms at moderate level. Significant differences were observed between MICs of planktonic cells (MIC-p) and MICs of UPEC biofilms (MIC-b). Among tested frontline antibiotics, levofloxacin successfully inhibited biofilms at a concentration of 32 µg/mL, while trimethoprim eradicated biofilms at higher concentrations (512-1024 µg/mL). Ciprofloxacin treatment at sub-MIC level significantly enhanced biofilm formation (P<0.05). CONCLUSION: The majority of UPEC strains are strong biofilm formers and show higher tolerance towards frontline antibiotics in biofilm form. We observed significant inhibitory effects of levofloxacin (32 µg/mL) on UPEC biofilms, while treatment with sub-minimal concentrations of ciprofloxacin significantly enhanced biofilm formation. Out of all tested antibiotics, trimethoprim (512-1024 µg/mL) eradicated UPEC biofilms.
BACKGROUND: Escherichia coli lineage ST131 predominates across various spectra of extra-intestinal infections, including urinary tract infection (UTI). The distinctive resistance profile, diverse armamentarium of virulence factors and rapid global dissemination of ST131 E. coli makes it an intriguing pathogen. However, not much is known about the prevalence and genetic attributes of ST131 lineage in Pakistan. METHODS: We estimated prevalence and genetic attributes of E. coli ST131 isolates causing UTI among 155 randomly selected samples. Samples were analyzed for phylogenetic grouping, O-typing and fumC/fimH typing. Isolates were further tested for the ESBL and virulence factors using PCR. RESULTS: Overall, 59% of the UPEC isolates belonged to the phylogenetic group B2, followed by D = 28%, B1 = 8% and A = 5%. Among 18 different Sequence-types, ST131 was the dominant lineage (n = 71; 46%) out of which 72% of the isolates were assigned to the phylogenetic group B2, while 61% adhered to the serogroup O25b. FumC/fimH typing confirmed 49% of the ST131 as H30 sub-types. In this study, significant numbers of the identified ST131 isolates were MDR and 42% showed ESBL phenotypes, out of which 37% carried bla-CTX-M-15. Moreover, different virulence factors were detected in following percentages: fimH,155(100%), iutA 86 (55%), feoB 76 (49%), papC 75 (48%), papGII 70 (45%), kpsMTII 40 (26%), papEF 37 (24%), fyuA 37 (24%), usp 22 (14%), papA 20 (13%), sfa/foc20 (13%), hlyA 18 (12%), afa 15 (10%), cdtB 11 (7%), papGI 6 (4%), papGIII 6 (4%), kpsMTIII 4 (3%) and bmaE2 (1%). CONCLUSION: Conclusively, this study provides important insight into the genetic and virulence attributes of pandemic MDR ST131 strains involved in UTIs. It also highlights higher prevalence of ST131-O25b-H30 UPEC isolates in patients, which was previously unreported from this part of globe.
Escherichia coli Infections/microbiology , Urinary Tract Infections/diagnosis , Uropathogenic Escherichia coli/classification , Drug Resistance, Multiple, Bacterial/genetics , Escherichia coli Infections/diagnosis , Escherichia coli Infections/epidemiology , Female , Humans , Male , Pakistan/epidemiology , Phylogeny , Prevalence , Urinary Tract Infections/epidemiology , Urinary Tract Infections/microbiology , Uropathogenic Escherichia coli/isolation & purification , Uropathogenic Escherichia coli/pathogenicity , Virulence , Virulence Factors/classification , Virulence Factors/genetics , beta-Lactamases/classification , beta-Lactamases/genetics
OBJECTIVES: Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). The pandemic sequence type 131 (ST131) clonal group is associated with multidrug resistance and virulence. Here we report the first draft genome of a ST131-O25b-H30 strain from Pakistan, isolated from a patient with community-acquired UTI. METHODS: Next-generation sequencing was performed using MiSeq and HiSeq 2500 platforms. De novo assembly of the reads was performed using SPAdes v.3.11. Genomic features were determined with PATRIC and RAST tool kits. RESULTS: The 5327975-bp draft genome sequence has 5433 coding sequences and 82 tRNAs, an array of antimicrobial resistance genes [blaCTX-M-15, blaOXA-1, blaCMY-2, sul2, catB, dfrA17, mph(A)], a class 1 integron, 77 insertion sequence (IS) elements, a Tn3-like transposon, multiple virulence markers and 7 intact prophage loci. CONCLUSION: In conclusion, the genome sequence of this new UPEC isolate from Pakistan provides a novel insight into the genetic attributes of an epidemic clone associated with a high level of resistance and virulence.
Drug Resistance, Multiple, Bacterial , Genome, Bacterial , Uropathogenic Escherichia coli/genetics , Uropathogenic Escherichia coli/pathogenicity , Community-Acquired Infections/microbiology , Escherichia coli Infections , Female , Humans , Pakistan , Urinary Tract Infections/microbiology , Uropathogenic Escherichia coli/isolation & purification , Virulence , Whole Genome Sequencing , Young Adult , beta-Lactamases/biosynthesis
Objective: To scrutinize patterns of multi-drug-resistant uropathogenic Escherichia coli (UPEC) strains and particularly of fluoroquinolone-resistance this is an alternative choice for the treatment of urinary tract infections. Methods: Bacterial samples (n = 250) were collected from out-patients from August 2012 to August 2014 Islamabad. Antibiotic susceptibility profiling and determination of mini-mum inhibitory concentrations (MICs) and minimum bactericidal concentrations were performed according to the guidelines of Clinical and Laboratory Standards Institute (CLSI, 2012). Genes, qnrA, qnrB and qnrS were identified by DNA amplification and sequencing. Results: The highest percentage of UPEC isolates were resistant to co-trimoxazole (82%) followed by cephalothin (80%), 2nd Gen, 3rd Gen and 4th Gen cephalosporins, respectively. Resistance against gentamicin, amikacin remained 29% and 4%. For other drugs including nitrofurantoin, tetracycline, carbapenem and beta-lactam inhibitors remained below 10%. Altogether, 59% of the isolates were resistant to at least three antibiotics including one fluoroquinolone. Overall, MICs for ciprofloxacin remained (MIC≥256 mg/mL) and for levofloxacin (MIC≥16 mg/mL and 32 mg/mL). No significant differences were observed regarding MIC values of extended spectrum b-lactamase (ESBL) and non-ESBL producers. For qnrS and qnrB positive isolates MICs remained above 32 mg/mL. Prevalence of UPEC was significantly higher among females and 40% of the isolates were ESBL producers. Conclusions: Higher percentages of ESBL producing UPEC were associated with uri-nary tract infections. Moreover, the majority of these isolates were multi-drug resistant and fluoroquinolone-resistant.
