Escherichia coli PBP5, PBP6 and DacD, encoded by dacA, dacC and dacD, respectively, share substantial amino acid identity and together constitute~50 % of the total penicillin-binding proteins of E. coli. PBP5 helps maintain intrinsic b-lactam resistance within the cell. To test if PBP6 and DacD play simlar roles, we deleted dacC and dacD individually, and dacC in combination with dacA, from E. coli 2443 and compared b-lactam sensitivity of the mutants and the parent strain. b-Lactam resistance was complemented by wild-type, but not DD-carboxypeptidase-deficient PBP5, confirming that enzymic activity of PBP5 is essential for b-lactam resistance. Deletion of dacC and expression of PBP6 during exponential or stationary phase did not alter b-lactam resistance of a dacA mutant. Expression of DacD during mid-exponential phase partially restored b-lactam resistance of the dacA mutant. Therefore, PBP5DD-carboxypeptidase activity is essential for intrinsic b-lactam resistance of E. coli and DacD can partially compensate for PBP5 in this capacity, whereas PBP6 cannot. INTRODUCTIONEscherichia coli encodes 12 penicillin-binding proteins (PBPs), four of which (PBP4, -5, -6 and DacD) have been reported to have DD-carboxypeptidase (DD-CPase) activity (Höltje, 1998; Denome et al., 1999;Ghosh et al., 2008). All these proteins are low molecular mass (LMM) PBPs (Ghuysen, 1991) and are dispensable for survival in vitro (Denome et al., 1999). PBP5 and PBP6 are 62 % identical at the amino acid level and share 48 and 47 % identity with DacD, respectively (Baquero et al., 1996). It has been suggested that these proteins might have similar physiological functions based upon their homology but only PBP5 appears to play a prominent role in maintenance of cell shape (Nelson & Young, 2001;Nelson et al., 2002;Ghosh & Young, 2003). PBP5, PBP6 and DacD are primarily expressed in early exponential, stationary and midexponential phases, respectively (Buchanan & Sowell, 1982;Baquero et al., 1996;Santos et al., 2002), which may explain the different roles of these proteins in maintenance of cell shape. The number of PBP5 molecules is also two-to threefold higher than the number of PBP6 molecules in exponentially growing cells (Spratt, 1977;Dougherty et al., 1996). METHODSBacterial strains and antibiotics. Bacterial strains used in this study were derived from E. coli 2443 and are listed in Table 1. CS18-2K was a gift from Professor Kevin D. Young, University of Arkansas Medical School, AR, USA. The strains were grown in Luria-Bertani (LB) broth, agar (Hi-Media), Muller-Hinton (MH) broth (Hi-Media) and M9-Glucose minimal medium, supplemented with the required amino acids (arginine, proline, leucine and threonine) and thiamine. Chloramphenicol (20 mg ml -1 ), kanamycin (50 mg ml -1 ), tetracycline (25 mg ml -1 ) and ampicillin (50 mg ml -1 ) were added where necessary. Unless otherwise specified, chemicals and reagents were purchased from Sigma. (Table 1). Double and triple mutants were constructed from parents from which the res-npt-res cassette...

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Of the five dd-carboxypeptidases in Escherichia coli, only PBP5 demonstrates its physiological significance by maintaining cell shape and intrinsic beta-lactam resistance. DacD can partially compensate for the lost beta-lactam resistance in PBP5 mutant, although its biochemical reason is unclear. To understand the mechanism(s) underlying such behaviour, we constructed soluble DacD (sDacD) and compared its biophysical and biochemical properties with those of sPBP5, in vitro. Unlike sPBP6, sDacD can deacylate Bocillin significantly, which is very similar to sPBP5. sDacD shows weak dd-carboxypeptidase activity, although lower than that of sPBP5. Bioinformatics analyses reveal a similar architecture of sPBP5 and sDacD. Therefore, based on the obtained results we can infer that biochemically DacD and PBP5 are more closely related to each other than to PBP6, enabling DacD and PBP5 to play a nearly similar physiological function in terms of recovering the lost beta-lactam resistance.

BackgroundThis study was conducted in Bangladeshi patients in an outpatient setting to support registration of Paromomycin Intramuscular Injection (PMIM) as a low-cost treatment option in Bangladesh.MethodologyThis Phase IIIb, open-label, multi-center, single-arm trial assessed the efficacy and safety of PMIM administered at 11 mg/kg (paromomycin base) intramuscularly once daily for 21 consecutive days to children and adults with VL in a rural outpatient setting in Bangladesh. Patients ≥5 and ≤55 years were eligible if they had signs and symptoms of VL (intermittent fever, weight loss/decreased appetite, and enlarged spleen), positive rK39 test, and were living in VL-endemic areas. Compliance was the percentage of enrolled patients who received 21 daily injections over no more than 22 days. Efficacy was evaluated by initial clinical response, defined as resolution of fever and reduction of splenomegaly at end of treatment, and final clinical response, defined as the absence of new clinical signs and symptoms of VL 6 months after end of treatment. Safety was assessed by evaluation of adverse events.Principal FindingsA total of 120 subjects (49% pediatric) were enrolled. Treatment compliance was 98.3%. Initial clinical response in the Intent-to-Treat population was 98.3%, and final clinical response 6 months after end of treatment was 94.2%. Of the 119 subjects who received ≥1 dose of PMIM, 28.6% reported at least one adverse event. Injection site pain was the most commonly reported adverse event. Reversible renal impairment and/or hearing loss were reported in 2 subjects.Conclusions/SignificancePMIM was an effective and safe treatment for VL in Bangladesh. The short treatment duration and lower cost of PMIM compared with other treatment options may make this drug a preferred treatment to be investigated as part of a combination therapy regimen. This study supports the registration of PMIM for use in government health facilities in Bangladesh.Trial RegistrationClinicalTrials.gov identifier: NCT01328457

Majority of patients had idiopathic aplastic anemia. Very severe aplastic anemia and severe aplastic anemia were frequent. Few patients received IST and had suboptimal response. There is need to establish a national registry for aplastic anemia.

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Background We investigated the relationship of treatment regimens for visceral leishmaniasis (VL) with post-kala-azar dermal leishmaniasis (PKDL) and visceral leishmaniasis relapse (VLR) development. Methods Study subjects included cohorts of patients cured of VL since treatment with monotherapy by sodium stibogluconate (SSG), miltefosine (MF), paromomycin intramuscular injection (PMIM), liposomal amphotericin B [AmBisome (AMB)] in a single dose (SDAMB) and in multidose (MDAMB), and combination therapies by AMB+PMIM, AMB+MF, and PMIM+MF. Follow up period was 4 years after treatment. Cohorts were prospective except SSG (retrospective) and MF (partially retrospective). We compared incidence proportion and rate in 100-person-4year of PKDL and VLR by treatment regimens using univariate and multivariate analysis. Findings 974 of 984 enrolled participants completed the study. Overall incidence proportion for PKDL and VLR was 12.3% (95% CI, 10.4%–14.5%) and 7.0% (95% CI, 5.6%–8.8%) respectively. The incidence rate (95% CI) of PKDL and VLR was 14.0 (8.6–22.7) and 7.6 (4.1–14.7) accordingly. SSG cohort had the lowest incidence rate of PKDL at 3.0 (1.3–7.3) and VLR at 1.8 (0.6–5.6), followed by MDAMB at 8.2 (4.3–15.7) for PKDL and at 2.7 (0.9–8.4) for VLR. Interpretation Development of PKDL and VLR is related with treatment regimens for VL. SSG and MDAMB resulted in less incidence of PKDL and VLR compared to other treatment regimens. MDAMB should be considered for VL as a first step for prevention of PKDL and VLR since SSG is highly toxic and not recommended for VL.

The combination of antibiotics is one of the strategies to combat drug-resistant bacteria, though only a handful of such combinations are in use, such as the β-lactam combinations. In the present study, the efficacy of a specific sub-inhibitory concentration of cefsulodin with other β-lactams was evaluated against a range of Gram-negative clinical isolates. This approach increased the sensitivity of the isolates, regardless of the β-lactamase production. The preferred target and mechanism of action of cefsulodin were identified in laboratory strains of Escherichia coli, by examining the effects of deleting the penicillin-binding protein (PBP) 1a and 1b encoding genes individually. Deletion of PBP1b was involved in sensitizing the bacteria to β-lactam agents, irrespective of its O-antigen status. Moreover, the use of a sub-inhibitory concentration of cefsulodin in combination with a β-lactam exerted an effect similar to that one obtained for PBP1b gene deletion. We conclude that the identified β-lactam/cefsulodin combination works by inhibiting PBP1b (at least partially) despite the involvement of β-lactamases, and therefore could be extended to a broad range of Gram-negative pathogens.