Purpose This study aimed to detect the prevalence of carbapenemase producers (CPs) among extensive drug-resistant (XDR)-carbapenemase producing Gram-negative bacteria (GNB) recovered from various clinical specimens of hospitalized neutrophilic febrile patients in two major tertiary care hospitals in Egypt. Methods Standard methods were used to evaluate the antimicrobial susceptibility of clinical isolates according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Phenotypic and genotypic analysis of CPs were carried out and statistically analyzed using standard methods. Results Three hundred and forty-two GNB were obtained from 342 clinical specimens during the period of the study, where 162 (47%) were enterobacterial isolates, including, 63 (18.4%) Escherichia coli , 87 (25.4%) Klebsiella spp., 5 (1.46%) Enterobacter cloacae , 5 (1.46%) Salmonella spp. and 2 (0.6%) Proteus and 180 (53%) were non-fermentative bacilli including, 129 (37.7%), Acinetobacter baumannii , and 51 (14.9%), Pseudomonas spp. Out of the 342 GNB, 188 (54.9%) isolates were multi-drug resistant (MDR). Of these, 52 (27.6%) were XDR as well as CPs as confirmed phenotypically. The MIC of imipenem against the XDR GNB against showed either low (11 isolates; 21.1%; MIC range =4–32 µg/mL) or high levels of resistance (41 isolates; 78.8%; MIC range = 64-≥1024). The most prevalent carbapenem resistance (CR) genes were blaKPC (63.5%) followed by blaOXA -48 (55.7%) and blaVIM (28.8%). No significant association could be observed between the MIC level and the presence of CR genes ( P value >0.05). Conclusion High prevalence of MDR (54.9%) and XDR (27.6%) GNB pathogens associated with high levels of resistance to carbapenems were observed. All XDR GNB were CPs and tested positive for at least one of the CR genes. However, most of them (78.8%) showed a high level of CR (MIC range = 64-≥1024) with no significant association with the CR genes.

Pathogenic bacteria can enter into a viable but non‐culturable (VBNC) state under unfavourable conditions. Proteus mirabilis is responsible for dire clinical consequences including septicaemia, urinary tract infections and pneumonia, but is not a species previously known to enter VBNC state. We suggested that stress‐induced P. mirabilis can enter a VBNC state in which it retains virulence. P. mirabilis isolates were incubated in extreme osmotic pressure, starvation, low temperature and low pH to induce a VBNC state. Resuscitation was induced by temperature upshift and inoculation in tryptone soy broth with Tween 20 and brain heart infusion broth. Cellular ultrastructure and gene expression were examined using transmission electron microscopy (TEM) and quantitative real‐time polymerase chain reaction (qPCR), respectively. High osmotic pressure and low acidity caused rapid entry into VBNC state. Temperature upshift caused the highest percentage of resuscitation (93%) under different induction conditions. In the VBNC state, cells showed aberrant and dwarf morphology, virulence genes and stress response genes (envZ and rpoS) were expressed (levels varied depending on strain and inducing factors). This is the first‐time characterization of VBNC P. mirabilis. The ability of P. mirabilis pathogenic strains to enter a stress‐induced VBNC state can be a serious public health threat.

The inadequate therapeutic opportunities associated with carbapenem-resistant Pseudomonas aeruginosa (CRPA) clinical isolates impose a search for innovative strategies. Therefore, our study aimed to characterize and evaluate two locally isolated phages formulated in a hydrogel, both in vitro and in vivo, against CRPA clinical isolates. The two phages were characterized by genomic, microscopic, phenotypic characterization, genomic analysis, in vitro and in vivo analysis in a Pseudomonas aeruginosa-infected skin thermal injury rat model. The two siphoviruses belong to class Caudovirectes and were named vB_Pae_SMP1 and vB_Pae_SMP5. Each phage had an icosahedral head of 60 ± 5 nm and a flexible, non-contractile tail of 170 ± 5 nm long, while vB_Pae_SMP5 had an additional base plate containing a 35 nm fiber observed at the end of the tail. The hydrogel was prepared by mixing 5% w/v carboxymethylcellulose (CMC) into the CRPA propagated phage lysate containing phage titer 108 PFU/mL, pH of 7.7, and a spreadability coefficient of 25. The groups were treated with either Phage vB_Pae_SMP1, vB_Pae_SMP5, or a two-phage cocktail hydrogel cellular subepidermal granulation tissues with abundant records of fibroblastic activity and mixed inflammatory cell infiltrates and showed 17.2%, 25.8%, and 22.2% records of dermal mature collagen fibers, respectively. In conclusion, phage vB_Pae_SMP1 or vB_Pae_SMP5, or the two-phage cocktails formulated as hydrogels, were able to manage the infection of CRPA in burn wounds, and promoted healing at the injury site, as evidenced by the histopathological examination, as well as a decrease in animal mortality rate. Therefore, these phage formulae can be considered promising for clinical investigation in humans for the management of CRPA-associated skin infections.

Background Infection with extensive-drug-resistant (XDR) carbapenem-resistant (CR) Gram-negative bacteria (GNB) are viewed as a serious threat to human health because of the limited therapeutic options. This imposes the urgent need to find agents that could be used as adjuvants or combined with carbapenems to enhance or restore the susceptibility of XDR CR- GNB. Therefore, this study aimed to examine the effect of propranolol (PR) in combination with Meropenem (MEM) on the susceptibility profile of XDR CR-GNB recovered from severely infected patients as well as to evaluate combining MEM with either tigecycline (TGC) or amikacin (AK). Methods A total of 59 non-duplicate CR- GNB were investigated for carbapenemase production by the major phenotypic methods. Molecular identification of five major carbapenemase-coding genes was carried out using polymerase chain reactions (PCR). Antimicrobial susceptibility tests were carried out using standard methods. Phenotypic and genotypic relatedness was carried out using the heatmap and ERIC PCR analysis. PR, 0.5 -1 mg/mL against the resulting non-clonal XDR CR-GNB pathogens were evaluated by calculating the MIC decrease factor (MDF). A combination of MEM with either AK or TGC was performed using the checkerboard assay. Results A total of 21 (35.6%) and 38 (64.4%) CR-GNB isolates were identified as enterobacterial isolates (including 16 (27.1%) Klebsiella Pneumoniae and 5 (8.5%) Escherichia coli) and non-fermentative bacilli (including, 23 (39%), Acinetobacter baumannii, and 15 (25.4%) Pseudomonas aeruginosa). The heatmap and ERIC PCR analysis resulted in non-clonal 28 XDR CR isolates. PR, at a concentration of 0.5 mg /ml, decreased MICs values of the tested XDR CR isolates (28; 100%) and restored susceptibility of only 4 (14.3%) isolates. However, PR (1 mg/mL) when combined with MEM has completely (28; 100%) restored the susceptibility of the tested XDR CR- GNB to MEM. The MEM + AK and MEM + TGC combination showed mostly additive effects (92.8% and 71.4%, respectively). Conclusion PR at a concentration of 1 mg/mL restored the susceptibility of XDR CR- GNB to MEM which is considered a promising result that should be clinically investigated to reveal its suitability for clinical use in patients suffering from these life-threatening pathogens.