Analysis of Antibiotic Resistance and Virulence Traits (Genetic and Phenotypic) in Klebsiella pneumoniae Clinical Isolates from Pakistan: Identification of Significant Levels of Carbapenem and Colistin Resistance.

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.

The Three Lipocalins of Egg-White: Only Ex-FABP Inhibits Siderophore-Dependent Iron Sequestration by Salmonella Enteritidis.

Salmonella Enteritidis is the most prevalent food-borne pathogen associated with egg-related outbreaks in the European Union. During egg colonization, S. Enteritidis must resist the powerful anti-bacterial activities of egg white (EW) and overcome ovotransferrin-imposed iron-restriction (the most important anti-bacterial mechanism of EW). Many pathogens respond to iron restriction by secreting iron-chelating chemicals called siderophores but EW contains a siderophore-sequestering "lipocalin" protein (Ex-FABP) that is predicted to limit the usefulness of siderophores in EW. S. Enteritidis produces two siderophores: enterobactin, which is strongly bound by Ex-FABP; and the di-glucosylated enterobactin-derivative, salmochelin (a so-called "stealth" siderophore), which is not recognized by Ex-FABP. Thus, production of salmochelin may allow S. Enteritidis to escape Ex-FABP-mediated growth inhibition under iron restriction although it is unclear whether its EW concentration is sufficient to inhibit pathogens. Further, two other lipocalins (Cal-γ and α-1-ovoglycoprotein) are found in EW but their siderophore sequestration potential remains unexplored. In addition, the effect of EW lipocalins on the major EW pathogen, S. Enteritidis, has yet to be reported. We overexpressed and purified the three lipocalins of EW and investigated their ability to interact with the siderophores of S. Enteritidis, as well as their EW concentrations. The results show that Ex-FABP is present in EW at concentrations (5.1 µM) sufficient to inhibit growth of a salmochelin-deficient S. Enteritidis mutant under iron restriction but has little impact on the salmochelin-producing wildtype. Neither Cal-γ nor α-1-ovoglycoprotein bind salmochelin or enterobactin, nor do they inhibit iron-restricted growth of S. Enteritidis. However, both are present in EW at significant concentrations (5.6 and 233 µM, respectively) indicating that α-1-ovoglycoprotein is the 4th most abundant protein in EW, with Cal-γ and Ex-FABP at 11th and 12th most abundant. Further, we confirm the preference (16-fold) of Ex-FABP for the ferrated form (K d of 5.3 nM) of enterobactin over the iron-free form (K d of 86.2 nM), and its lack of affinity for salmochelin. In conclusion, our findings show that salmochelin production by S. Enteritidis enables this key egg-associated pathogen to overcome the enterobactin-sequestration activity of Ex-FABP when this lipocalin is provided at levels found in EW.

The anti-bacterial iron-restriction defence mechanisms of egg white; the potential role of three lipocalin-like proteins in resistance against Salmonella.

Salmonella enterica serovar Enteritidis (SE) is the most frequently-detected Salmonella in foodborne outbreaks in the European Union. Among such outbreaks, egg and egg products were identified as the most common vehicles of infection. Possibly, the major antibacterial property of egg white is iron restriction, which results from the presence of the iron-binding protein, ovotransferrin. To circumvent iron restriction, SE synthesise catecholate siderophores (i.e. enterobactin and salmochelin) that can chelate iron from host iron-binding proteins. Here, we highlight the role of lipocalin-like proteins found in egg white that could enhance egg-white iron restriction through sequestration of certain siderophores, including enterobactin. Indeed, it is now apparent that the egg-white lipocalin, Ex-FABP, can inhibit bacterial growth via its siderophore-binding capacity in vitro. However, it remains unclear whether Ex-FABP performs such a function in egg white or during bird infection. Regarding the two other lipocalins of egg white (Cal-γ and α-1-glycoprotein), there is currently no evidence to indicate that they sequester siderophores.

Draft genome sequence of Enterococcus faecium SP15, a potential probiotic strain isolated from spring water.

OBJECTIVES: Enterococci are Gram-positive lactic acid bacteria and common inhabitants of the gastrointestinal tract of mammals, including humans. They are also widely distributed in diverse environments such as soil, water, vegetables and food. Enterococcus faecium is able to produce antimicrobial compounds (enterocins) and thus can act as a probiotic. E. faecium SP15 is a newly identified enterocin-producing strain from spring water that has been subjected to genome sequence analysis to provide understanding of its antimicrobial and probiotic properties. DATA DESCRIPTION: The draft genome of E. faecium SP15 comprises of 2,783,033 bp with a G+C content of 38.08%. Five genetic loci predicted to specify enterocin production were identified, but no virulence factors could be detected and only two potential antibiotic resistance genes were noted.

Draft genome sequence of a multidrug-resistant CTX-M-15 ß-lactamase-producing uropathogenic Escherichia coli isolate (ST131-O25b-H30) from Pakistan exhibiting high potential virulence.

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.