OXA-163-producing Klebsiella pneumoniae in Cairo, Egypt, in 2009 and 2010.
ABSTRACT: Two genetically unrelated OXA-163-carrying Klebsiella pneumoniae strains were identified from two infection cases in June 2009 and May 2010 in Cairo, Egypt. OXA-163-producing Enterobacteriaceae had been previously reported in Argentina only. Both patients had no history of travel abroad. The emergence of this newly recognized OXA-48-related ?-lactamase able to hydrolyze cephalosporins and carbapenems is especially worrying in a geographic area where OXA-48 is endemic and effective surveillance for antibiotic resistance is largely unaffordable.
Project description:Two bla(OXA-48)-like-positive isolates (Klebsiella pneumoniae and Enterobacter cloacae) were recovered in Argentina in 2008 as part of a large-scale survey focused on multidrug resistance in Enterobacteriaceae. In both cases, sequencing identified ?-lactamase OXA-163, differing from OXA-48 by a single amino substitution and a 4-amino-acid deletion. OXA-163 hydrolyzed penicillins, ceftazidime, and cefotaxime, whereas OXA-48 did not. However, OXA-163 had a much lower ability to hydrolyze carbapenems than OXA-48, therefore barely being considered a carbapenemase. In both isolates, the bla(OXA-163) gene was located on plasmids that differed in structure and size. However, a detailed genetic analysis revealed a similar genetic context in those isolates, with the bla(OXA-163) gene being bracketed by novel transposase genes, making this genetic environment different from that reported for the bla(OXA-48) gene. This study identified the first class D ?-lactamase compromising both extended-spectrum cephalosporin and carbapenem activities.
Project description:The first crystal structures of the class D ?-lactamases OXA-181 and OXA-245 were determined to 2.05 and 2.20?Å resolution, respectively; in addition, the structure of a new crystal form of OXA-163 was resolved to 2.07?Å resolution. All of these enzymes are OXA-48-like and have been isolated from different clinical Klebsiella pneumoniae strains and also from other human pathogens such as Pseudomonas aeruginosa and Escherichia coli. Here, enzyme kinetics and thermostability studies are presented, and the new crystal structures are used to explain the observed variations. OXA-245 had the highest melting point (Tm = 55.8°C), as determined by differential scanning calorimetry, compared with OXA-163 (Tm = 49.4°C) and OXA-181 (Tm = 52.6°C). The differences could be explained by the loss of two salt bridges in OXA-163, and an overall decrease in the polarity of the surface of OXA-181 compared with OXA-245.
Project description:OXA-48-like enzymes have emerged as important extended-spectrum ?-lactamases/carbapenemases in Escherichia coli sequence type 131 (ST131). We report the structures of the first fully sequenced bla<sub>OXA-163</sub> plasmid and of two other bla<sub>OXA-48</sub> plasmids in this lineage. bla<sub>OXA-163</sub> was located on a 71-kb IncN plasmid with other resistance genes. bla<sub>OXA-48</sub> was present on IncL/M plasmids, genetically similar to other bla<sub>OXA-48</sub> plasmid sequences, and consistent with interspecies/interlineage spread. The presence of bla<sub>OXA-48-like</sub> genes on epidemic plasmids in ST131 is of concern.
Project description:BACKGROUND:Acinetobacter baumannii is an increasingly worrying organism in the healthcare setting, due to its multidrug resistance and persistence. Prolonged hospitalisation, immunocompromised patients and excessive antibiotic exposure all contribute to increasing the risk of A. baumannii infections, which makes cancer patients a significant risk group. This study aims to investigate the dissemination of A. baumannii at the National Cancer Institute (NCI) in Cairo - Egypt. METHODS:All bacterial isolates were typed using Multi-locus Sequence Typing (MLST) to characterise the epidemiology of isolates. The intrinsic OXA-51-like, and the acquired carbapanemases OXA-23, -?24/40, -?58, NDM, IMP, and VIM were also amplified and sequenced to genetically identify mechanisms of carbapenem resistance. RESULTS:MLST results show a high degree of multi-clonal dissemination, with 18 different Sequence Types (STs) identified, including 5 novel. The majority of isolates belonged to International Clone (IC) 2, and carbapenem resistance was detected in 93% of isolates and mediated by blaOXA-23, blaOXA-58, blaNDM-1 and blaVIM-1. We also report the presence of a resistant ST732 (OXA-378) which has been previously identified in migratory birds. CONCLUSIONS: Multiple highly resistant clones were identified in a Cancer hospital in Cairo. It is vital that clinicians and healthcare workers are aware of the population of A. baumannii present in order to have appropriate treatment and infection control practices.
Project description:OXA-48-producing Enterobacterales have now widely disseminated globally. A sign of their extensive spread is the identification of an increasing number of OXA-48 variants. Among them, three are particularly interesting, OXA-163, OXA-247 and OXA-405, since they have lost carbapenem activities and gained expanded-spectrum cephalosporin hydrolytic activity subsequent to a four amino-acid (AA) deletion in the ?5-?6 loop. We investigated the mechanisms responsible for substrate specificity of OXA-405. Kinetic parameters confirmed that OXA-405 has a hydrolytic profile compatible with an ESBL (hydrolysis of expanded spectrum cephalosporins and susceptibility to class A inhibitors). Molecular modeling techniques and 3D structure determination show that the overall dimeric structure of OXA-405 is very similar to that of OXA-48, except for the ?5-?6 loop, which is shorter for OXA-405, suggesting that the length of the ?5-?6 loop is critical for substrate specificity. Covalent docking with selected substrates and molecular dynamics simulations evidenced the structural changes induced by substrate binding, as well as the distribution of water molecules in the active site and their role in substrate hydrolysis. All this data may represent the structural basis for the design of new and efficient class D inhibitors.
Project description:The epidemiology of carbapenemases worldwide is showing that OXA-48 variants are becoming the predominant carbapenemase type in Enterobacteriaceae in many countries. However, not all OXA-48 variants possess significant activity toward carbapenems (e.g., OXA-163). Two Serratia marcescens isolates with resistance either to carbapenems or to extended-spectrum cephalosporins were successively recovered from the same patient. A genomic comparison using pulsed-field gel electrophoresis and automated Rep-PCR typing identified a 97.8% similarity between the two isolates. Both strains were resistant to penicillins and first-generation cephalosporins. The first isolate was susceptible to expanded-spectrum cephalosporins, was resistant to carbapenems, and had a significant carbapenemase activity (positive Carba NP test) related to the expression of OXA-48. The second isolate was resistant to expanded-spectrum cephalosporins, was susceptible to carbapenems, and did not express a significant imipenemase activity, (negative for the Carba NP test) despite possessing a blaOXA-48-type gene. Sequencing identified a novel OXA-48-type ?-lactamase, OXA-405, with a four-amino-acid deletion compared to OXA-48. The blaOXA-405 gene was located on a ca. 46-kb plasmid identical to the prototype IncL/M blaOXA-48-carrying plasmid except for a ca. 16.4-kb deletion in the tra operon, leading to the suppression of self-conjugation properties. Biochemical analysis showed that OXA-405 has clavulanic acid-inhibited activity toward expanded-spectrum activity without significant imipenemase activity. This is the first identification of a successive switch of catalytic activity in OXA-48-like ?-lactamases, suggesting their plasticity. Therefore, this report suggests that the first-line screening of carbapenemase producers in Enterobacteriaceae may be based on the biochemical detection of carbapenemase activity in clinical settings.
Project description:The circulation of enteric viruses among the population of Cairo, Egypt, between March 2006 and February 2007 was studied. At least one virus was detected in 50% of fecal samples, 57.4% of which were positive for rotavirus, 26% for norovirus, 10.4% for adenovirus, and 1.7% for astrovirus. Over 10% of infections were mixed infections. Rotavirus typing showed that G1P and G2P were predominant but that the unusual G12P and G12P reassortants were also present. Among the noroviruses, half belonged to the predominant GGII.4 cluster. The phylogenetic analysis of the capsid gene suggested that GGII.4 strains from Cairo were similar to those circulating elsewhere. It also showed the emergence of new GGII.4 variants that were not associated with any previously known GGII.4 isolate. Further studies are required to assess the disease burden of enteric viruses in Egypt and the impact of atypical strains.
Project description:Gram-negative bacteria are common causes of urinary tract infections (UTIs). Such pathogens can acquire genes encoding multiple mechanisms of antimicrobial resistance, including carbapenem resistance. The aim of this study was to detect the carbapenemase-producing ability of some Gram-negative bacterial isolates from urine specimens of patients suffering from complicated UTIs at two vital tertiary care hospitals in Cairo, Egypt; to determine the prevalence of carbapenemase genes among plasmid-bearing isolates; and explore the possibility of horizontal gene transfer to other bacterial species. The collected isolates were subjected to antimicrobial susceptibility testing, phenotypic analysis of carbapenemase production, and molecular detection of plasmid-borne carbapenemase genes, then the extracted plasmids were transformed into competent <i>E. coli</i> DH5α. A total of 256 Gram-negative bacterial clinical isolates were collected, 65 (25.4%) isolates showed carbapenem resistance of which 36 (55.4%) were carbapenemase-producers, and of these 31 (47.7%) harbored plasmids. The extracted plasmids were used as templates for PCR amplification of <i>bla</i><sub>KPC</sub>, <i>bla</i><sub>NDM</sub>, <i>bla</i><sub>VIM</sub>, <i>bla</i><sub>OXA-48,</sub> and <i>bla</i><sub>IMP</sub> carbapenemase genes. The <i>bla</i><sub>OXA-48</sub> gene was detected in 24 (77.4%) of the tested isolates while <i>bla</i><sub>VIM</sub> gene was detected in 8 (25.8%), both <i>bla</i><sub>KPC</sub> and <i>bla</i><sub>NDM</sub> genes were co-present in 1 (3.2%) isolate. Plasmids carrying the <i>bla</i><sub>OXA-48</sub> gene from 4 <i>K. pneumoniae</i> clinical isolates were successfully transformed into competent <i>E. coli</i> DH5α. The transformants were carbapenemase-producers and acquired resistance to some of the tested antimicrobial agents as compared to untransformed <i>E. coli</i> DH5α. The study concluded that the rate of carbapenem resistance among Gram-negative bacterial uropathogens in Cairo, Egypt is relatively high and can be transferred horizontally to other bacterial host(s).
Project description:In this study we isolate and identify the Enteropathogenic Escherichia coli (EPEC) causing diarrhea in children less than five years in Cairo, Egypt, during different seasons. Children younger than five years with diarrhea, attending the Pediatric Gastroenterology Intensive Care Unit of the Cairo University Pediatric Hospital in one year period were our group of study. Our control group was age and sex matched concurrent healthy children. The identified E. coli isolates were subjected to antimicrobial disc diffusion susceptibility test and further identified for EPEC serotype by slide agglutination test, using antiserum E. coli somatic trivalent I (O111, O55, O26) according to the instructions of the manufacturer. Out of 134 patients 5.2% of them revealed EPEC in the fecal sample, while the 20 children control group showed no EPEC isolates in their samples. Our EPEC frequency showed variations from the compared results of other studies. Higher rate of EPEC (18.7%) was found in patients between 2 to 3 years, while EPEC rate was (7.5%) in patients less than 6 months old, with P < 0.05. EPEC was identified from fecal specimens as a unique pathogen or associated with other pathogens in acute and chronic diarrhea in children. EPEC were detected in all seasons except in winter, and was predominant in summer season. Four (57%) EPEC isolates were resistant to ampicillin, ticarcillin, and cotrimoxazole, and (14.3%) to the third generation cephalosporins.
Project description:OXA-163 and OXA-48 are closely related class D ?-lactamases that exhibit different substrate profiles. OXA-163 hydrolyzes oxyimino-cephalosporins, particularly ceftazidime, while OXA-48 prefers carbapenem substrates. OXA-163 differs from OXA-48 by one substitution (S212D) in the active-site ?5 strand and a four-amino acid deletion (214-RIEP-217) in the loop connecting the ?5 and ?6 strands. Although the structure of OXA-48 has been determined, the structure of OXA-163 is unknown. To further understand the basis for their different substrate specificities, we performed enzyme kinetic analysis, inhibition assays, X-ray crystallography, and molecular modeling. The results confirm the carbapenemase nature of OXA-48 and the ability of OXA-163 to hydrolyze the oxyimino-cephalosporin ceftazidime. The crystal structure of OXA-163 determined at 1.72 Å resolution reveals an expanded active site compared to that of OXA-48, which allows the bulky substrate ceftazidime to be accommodated. The structural differences with OXA-48, which cannot hydrolyze ceftazidime, provide a rationale for the change in substrate specificity between the enzymes. OXA-163 also crystallized under another condition that included iodide. The crystal structure determined at 2.87 Å resolution revealed iodide in the active site accompanied by several significant conformational changes, including a distortion of the ?5 strand, decarboxylation of Lys73, and distortion of the substrate-binding site. Further studies showed that both OXA-163 and OXA-48 are inhibited in the presence of iodide. In addition, OXA-10, which is not a member of the OXA-48-like family, is also inhibited by iodide. These findings provide a molecular basis for the hydrolysis of ceftazidime by OXA-163 and, more broadly, show how minor sequence changes can profoundly alter the active-site configuration and thereby affect the substrate profile of an enzyme.