Detection of Pan drug resistance OXA-48 producing Providencia in an ICU patient for the first time in Nepal.

Background: Resistance to antimicrobial agents of pathogenic bacteria has become a major problem in routine medical practices. Carbapenem resistance has long been increasing. The production of carbapenem- hydrolysing ß-lactamases (carbapenamases), which include NDM, KPC, OXA-48, IMP-1 and VIM is the most common mechanism. Case presentation: A 56 years old male presented with fever and mental changes with progressively decreasing sensorium for the last 3 days. He was admitted to Intensive care unit (ICU) with a diagnosis of meningoencephalitis. On day seven, he developed ventilator associated pneumonia due Klebsiella pnemoniae and Acinetobacter baumannii. He was on meropenem, but the isolates were susceptible to colistin, tigecyclin and amikacin solely. Hence, amikacin was started with addition of intravenous and nebulized colistin. Subsequently, vital signs improved with resolution of fever. However, on day 18, he developed fever once again with a drop in blood pressure. Inotropic support was maintained, and echinocandins and tigecycline were added to the regimen.Repeat blood and urine culture grew Providencia species, which were resistant to most of the drugs on phenotypic Kirby-Bauer disk diffusion method and are intrinsically resistant to colistin and tigecycline. Phenotypic detection of ESBL (combined disk method), MBL, KPCs, AmpC and co-producer were tested according to updated CLSI guideline and all were negative. But the Modified Hodges test was found to be positive. Consequenty, OXA-48 drug resistance pattern was brought into action by blank disc method according to A Tsakris et al., which revealed indentation of growth toward both EDTA and EDTA/PBA disk indicating production of OXA-48 carbapenamase. To confirm the resistance pattern we processed the isolated colonies for Xpert Carba-R (Cepheid) assay, which detected blaOXA-48 gene and confirmed the OXA-48 drug resistance pattern. Hence, the infecting organism was not susceptible to any of the antibiotics. The patient was kept under isolation and on 31th day of admission, he died of septic shock. Conclusions: Carbapenamase production along with intrinsic colistin resistance in infecting bacterial pathogens can cause fatal outcomes in the resource limited countries like Nepal where new antibiotic combinations ceftazidime+ Avibactam, or aztreonam +avibactam are not available. Drug resistance patterns including OXA 48 producer should be characterized in all cases by standard phenotypic methods or by Xpert Carba-R assay and larger studies are required to know the exact burden of OXA 48 producer in Nepal.

A Rare Opportunist, Morganella morganii, Decreases Severity of Polymicrobial Catheter-Associated Urinary Tract Infection.

Catheter-associated urinary tract infections (CAUTIs) are common hospital-acquired infections and frequently polymicrobial, which complicates effective treatment. However, few studies experimentally address the consequences of polymicrobial interactions within the urinary tract, and the clinical significance of polymicrobial bacteriuria is not fully understood. Proteus mirabilis is one of the most common causes of monomicrobial and polymicrobial CAUTI and frequently cocolonizes with Enterococcus faecalis, Escherichia coli, Providencia stuartii, and Morganella morganiiP. mirabilis infections are particularly challenging due to its potent urease enzyme, which facilitates formation of struvite crystals, catheter encrustation, blockage, and formation of urinary stones. We previously determined that interactions between P. mirabilis and other uropathogens can enhance P. mirabilis urease activity, resulting in greater disease severity during experimental polymicrobial infection. Our present work reveals that M. morganii acts on P. mirabilis in a contact-independent manner to decrease urease activity. Furthermore, M. morganii actively prevents urease enhancement by E. faecalis, P. stuartii, and E. coli Importantly, these interactions translate to modulation of disease severity during experimental CAUTI, predominantly through a urease-dependent mechanism. Thus, products secreted by multiple bacterial species in the milieu of the catheterized urinary tract can directly impact prognosis.

Exploring the glyphosate-degrading characteristics of a newly isolated, highly adapted indigenous bacterial strain, Providencia rettgeri GDB 1.

This study explored the characteristics of a newly isolated glyphosate (GLYP)-degrading bacterium Providencia rettgeri GDB 1, for GLYP bioremediation. Due to the serial selection pressure of high GLYP concentrations for enriched isolation, this highly tolerant GLYP biodegrader shows very promising capabilities for GLYP removal (approximately 71.4% degradation efficiency) compared to previously reported strains. High performance liquid chromatography analyses showed aminomethylphosphonic acid (AMPA) rather than sarcosine (SAR) to be the sole intermediate of GLYP decomposition via the AMPA formation pathway. Moreover, GLYP biodegradation was biochemically favorable in aerobic cultures due to its strong growth-associated characteristics. To the best of our knowledge, this is the first report to indicate that bacterial strains in the Providencia genus could demonstrate highly promising GLYP-degrading characteristics in environments with high GLYP contents.

Providencia stuartii form biofilms and floating communities of cells that display high resistance to environmental insults.

Biofilms are organized communities of bacterial cells that are responsible for the majority of human chronic bacterial infections. Providencia stuartii is a Gram-negative biofilm-forming bacterium involved in high incidence of urinary tract infections in catheterized patients. Yet, the structuration of these biofilms, and their resistance to environmental insults remain poorly understood. Here, we report on planktonic cell growth and biofilm formation by P. stuartii, in conditions that mimic its most common pathophysiological habitat in humans, i.e. the urinary tract. We observed that, in the planktonic state, P. stuartii forms floating communities of cells, prior to attachment to a surface and subsequent adoption of the biofilm phenotype. P. stuartii planktonic and biofilm cells are remarkably resistant to calcium, magnesium and to high concentrations of urea, and show the ability to grow over a wide range of pHs. Experiments conducted on a P. stuartii strain knocked-out for the Omp-Pst2 porin sheds light on the role it plays in the early stages of growth, as well as in the adaptation to high concentration of urea and to varying pH.

Changes in diversity of cultured bacteria resistant to erythromycin and tetracycline in swine manure during simulated composting and lagoon storage.

UNLABELLED: This study investigated the impact of composting and lagoon storage on survival and change in diversity of tetracycline-resistant (Tc(r) ) and erythromycin-resistant (Em(r) ) bacteria and the resistance genes they carry in swine manure. Treatments were arranged as a 2 × 2 factorial design: composting vs lagoon storage and 0 vs 1% Surround WP Crop Protectant (a clay product) in three replicates. After 48 days of treatments, resistant bacteria were enumerated by selective plating and identified by 16S rRNA gene sequencing. The erm and the tet gene(s) carried by the resistant isolates were screened using class-specific PCR assays. The plate counts of Tc(r) and Em(r) bacteria decreased by 4-7 logs by composting, but only by 1-2 logs by the lagoon treatment. During the treatments, Acinetobacter gave way to Pseudomonas and Providencia as the largest resistant genera. The clay product had little effect on survival or diversity of resistant bacteria. Of six classes of erm and seven classes of tet genes tested, changes in prevalence were also noted. The results indicate that composting can dramatically shift Tc(r) and Em(r) bacterial populations, and composting can be an effective and practical approach to decrease dissemination of antibiotic resistance from swine farms to the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The presented research provided evidence that composting is much more effective than lagoon storage in dramatically decreasing culturable bacteria resistant to erythromycin and tetracycline in swine manure. Considerable diversity changes of resistant bacteria were also demonstrated during composting or lagoon storage. Overall, Acinetobacter was the major resistant genus in untreated swine manure, but pseudomonads and Providencia became the major resistant genera after the treatments. This is the first study that investigated diversity changes of cultured bacteria resistant to these two antibiotics during composting and lagoon storage of swine manure. New genes encoding resistance to the two antibiotics were also implied in the cultured isolates.

Synthesis and in-vitro antimicrobial evaluation of a high-affinity iron chelator in combination with chloramphenicol.

OBJECTIVES: The objectives of this study were first to design and synthesize a hexadentate chelator with high iron(III) affinity and, second, to evaluate its antimicrobial activity in the presence and absence of chloramphenicol. METHODS: A hexadentate ligand was synthesized by conjugating a protected bidentate compound onto a tripodal structure. The pKa values and iron affinity of the chelator were determined by spectophotometric titration. Minimum inhibitory concentrations were determined by visual inspection of broth turbidity. The bactericidal rates were calculated by counting the colony numbers on a light board after incubation with and without an antimicrobial agent. KEY FINDINGS: A hexadentate 3-hydroxypyridin-4-one was found to possess a high affinity for iron(III), with a pFe value of 31.2 (negative logarithm of concentration of the free iron(III) in solution (when [Fe³âº](Total) = 10⁻6) M; [Ligand](Total) = 105 M; pH = 7.4). We found that this chelator had an appreciable inhibitory effect in vitro against the two bacterial strains Providencia stuartii and Staphylococcus aureus, particularly in the presence of chloramphenicol. CONCLUSIONS: A 3-hydroxypyridin-4-one hexadentate ligand has potential as an antimicrobial agent. Combination therapy with this iron chelator plus chloramphenicol has potential for the treatment of extracellular infections.

Processing by rhomboid protease is required for Providencia stuartii TatA to interact with TatC and to form functional homo-oligomeric complexes.

The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. In Escherichia coli three membrane proteins, TatA, TatB and TatC, are essential components of the machinery. TatA from Providencia stuartii is homologous to E. coli TatA but is synthesized as an inactive pre-protein with an N-terminal extension of eight amino acids. Removal of this extension by the rhomboid protease AarA is required to activate P. stuartii TatA. Here we show that P. stuartii TatA can functionally substitute for E. coli TatA provided that the E. coli homologue of AarA, GlpG, is present. The oligomerization state of the P. stuartii TatA pro-protein was compared with that of the proteolytically activated protein and with E. coli TatA. The pro-protein still formed small homo-oligomers but cannot form large TatBC-dependent assemblies. In the absence of TatB, E. coli TatA or the processed form of P. stuartii TatA form a complex with TatC. However, this complex is not observed with the pro-form of P. stuartii TatA. Taken together our results suggest that the P. stuartii TatA pro-protein is inactive because it is unable to interact with TatC and cannot form the large TatA complexes required for transport.

Effects of associated bacteria on the pathogenicity and reproduction of the insect-parasitic nematode Rhabditis blumi (Nematoda: Rhabditida).

Three bacteria, Alcaligenes faecalis , Flavobacterium sp., and Providencia vermicola , were isolated from dauer juveniles of Rhabditis blumi . The pathogenic effects of the bacteria against 4th instar larvae of Galleria mellonella were investigated. Providencia vermicola and Flavobacterium sp. showed 100% mortality at 48 h after haemocoelic injection, whereas A. faecalis showed less than 30% mortality. Dauer juveniles showed 100% mortality against G. mellonella larvae, whereas axenic juveniles, which do not harbor associated bacteria, exhibited little mortality. All of the associated bacteria were used as a food source for nematode growth, and nematode yield differed with bacterial species. Among the bacterial species, P. vermicola was most valued for nematode yield, showing the highest yield of 5.2 × 10(4) nematodes/mL in the plate. In bacterial cocultures using two of the three associated bacteria, one kind stimulated the other. The highest total bacterial yield of 12.6 g/L was obtained when the inoculum ratio of P. vermicola to A. faecalis was 10:1. In air-lift bioreactors, the nematode growth rate increased with an increasing level of dissolved oxygen. The maximum nematode yield of 1.75 × 10(5) nematodes/mL was obtained at 192 h with an aeration rate of 6 vvm.

Providencia isolates carrying bla (PER-1) and bla (VIM-2) genes: biofilm-forming capacity and biofilm inhibitory concentrations for carbapenem antibiotics.

Multidrug-resistant clinical isolates of Providentia carrying bla (PER-1) and bla (VIM-2) were evaluated for the abilities to form biofilm and high biofilm forming capacity was demonstrated in them. Minimum biofilm inhibitory concentrations (MBICs), minimum biofilm eradication concentrations (MBECs), and minimum inhibitory concentrations (MICs) for imipenem and meropenem were also determined. In all tested strains, the MBICs were higher than the MICs for both drugs. Interestingly, the MBICs and the MBEC(50) for meropenem were lower than those for imipenem in the isolates producing high amounts of biofilm, suggesting that meropenem is superior to imipenem in the growth inhibition and eradication of biofilm forming Providentia strains.

The biofilm environment offers a possible condition for inducing the competency of DNA recipient cells through nutritional starvation.

Transformation phenomena occurring under conditions mimicking the biofilm environment were investigated using Escherichia coli IM302 (as DNA recipient cells) and Providencia sp. WW2 (as surrounding cells in the biofilm model). In the case of planktonic IM302 cells kept at 25 degrees C, the transformation took place exclusively in the absence of organic nutrients (COD = 0), and was not substantially observed in the range of COD = 30-1500 mg O2/L. On the other hand, in the case of biofilm IM302 cells, the transformation occurred at relatively high levels under the examined conditions (temperature = 5 or 25 degrees C and COD = 0-1500 mg O2/L). These results indicated that the competency of biofilm IM302 cells was induced even in the presence of organic nutrients owing to nutritional starvation caused by WW2 cells.

Demonstration and characterization of manganese superoxide dismutase of Providencia alcalifaciens.

Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and play a role in the pathogenesis of certain invasive bacteria. In this study, we reported for the first time here that Providencia alcalifaciens, a member of the family Enterobacteriaceae, produces a superoxide dismutase (SOD) as a major protein in culture supernatants. This protein was purified by a series of column chromatographic separations. The N-terminal amino acid sequence of the protein was determined to be highly homologous to manganese superoxide dismutase of Escherichia coli or Salmonella reported. The gene (sodA) encoding for SOD of P. alcalifaciens was cloned and sequenced. The sodA-encoded protein has a molecular weight of about 23.5 kDa, and the DNA sequence of P. alcalifaciens sodA gene (627 bp) has about 83% identity to the E. coli SOD gene. We constructed a sodA deletion mutant and its complemented strain of P. alcalifaciens. In J774, a macrophage cell line, the sodA deletion mutant was more susceptible to killing by macrophages than the wildtype strain and its complemented strain. When we injected the mutant strain, its complemented strain and wildtype strain intraperitoneally into DDY strain mice, we found that the sodA deletion mutant proved significantly less virulent while the complemented strain recovered the virulence to the same level of wildtype strain of P. alcalifaciens. These results suggested that manganese superoxide dismutase plays an important role in intracellular survival of P. alcalifaciens.

Genetic variation in Drosophila melanogaster resistance to infection: a comparison across bacteria.

Insects use a generalized immune response to combat bacterial infection. We have previously noted that natural populations of D. melanogaster harbor substantial genetic variation for antibacterial immunocompetence and that much of this variation can be mapped to genes that are known to play direct roles in immunity. It was not known, however, whether the phenotypic effects of variation in these genes are general across the range of potentially infectious bacteria. To address this question, we have reinfected the same set of D. melanogaster lines with Serratia marcescens, the bacterium used in the previous study, and with three additional bacteria that were isolated from the hemolymph of wild-caught D. melanogaster. Two of the new bacteria, Enterococcus faecalis and Lactococcus lactis, are gram positive. The third, Providencia burhodogranaria, is gram negative like S. marcescens. Drosophila genotypes vary highly significantly in bacterial load sustained after infection with each of the four bacteria, but mean loads are largely uncorrelated across bacteria. We have tested statistical associations between immunity phenotypes and nucleotide polymorphism in 21 candidate immunity genes. We find that molecular variation in some genes, such as Tehao, contributes to phenotypic variation in the suppression of only a subset of the pathogens. Variation in SR-CII and 18-wheeler, however, has effects that are more general. Although markers in SR-CII and 18-wheeler explain >20% of the phenotypic variation in resistance to L. lactis and E. faecalis, respectively, most of the molecular polymorphisms tested explain <10% of the total variance in bacterial load sustained after infection.

Structure of the O-polysaccharide of Providencia stuartii O4 containing 4-(N-acetyl-L-aspart-4-yl)amino-4,6-dideoxy-D-glucose.

The O-polysaccharide of Providencia stuartii O4 was obtained by mild acid degradation of the lipopolysaccharide, and the following structure of the pentasaccharide repeating unit was established: [structure: see text] where D-Qui4N(L-AspAc) is 4-(N-acetyl-L-aspart-4-yl)amino-4,6-dideoxy-D-glucose, which has not been hitherto found in bacterial polysaccharides. Structural studies were performed using sugar and methylation analyses, Smith degradation and NMR spectroscopy, including conventional 2D 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments as well as COSY and NOESY experiments run in an H(2)O-D(2)O mixture to reveal correlations for NH protons.

GASP phenotype: presence in enterobacteria and independence of sigmaS in its acquisition.

The appearance of growth advantage in stationary phase or GASP was originally detected in Escherichia coli. The presence of this phenotype in other enterobacteria such as Enterobacter cloacae, Salmonella typhimurium, Providencia stuartii and Shigella dysenteriae is described in this work. E. cloacae GASP strains presented lower levels of RpoS than the parental strain, although no mutation in the gene or its promoter was detected. This work offers evidence of GASP rpoS-independent pathways as GASP was also acquired in knock-out rpoS E. cloacae and E. coli strains.

Escherichia coli 83972 inhibits catheter adherence by a broad spectrum of uropathogens.

OBJECTIVES: The presence of a nonpathogenic organism on the surface of a urinary catheter might impede catheter colonization by pathogens and thus prevent urinary tract infection. Previously, we reported that preinoculating urinary catheters with a nonpathogenic strain of Escherichia coli (83972) significantly impeded catheter colonization in vitro by gram-positive bacteria (Enterococcus faecalis). To explore this phenomenon further, we investigated in vitro whether E. coli 83972 could likewise inhibit catheter colonization by gram-negative and fungal uropathogens (Providencia stuartii, uropathogenic E. coli, and Candida albicans). METHODS: For each of the three uropathogens tested, we examined three different incubation conditions: (a) E. coli plus uropathogen catheters were exposed to E. coli 83972 for 24 hours and then to a uropathogen for 30 minutes; (b) E. coli-alone catheters were incubated with E. coli for 24 hours and then in sterile broth for 30 minutes; and (c) uropathogen-alone catheters were incubated in sterile broth for 24 hours before the 30-minute incubation with the uropathogen. All catheters were subsequently incubated in sterile human urine for 24 hours. Catheters were then rinsed and sonicated to determine the numbers of adherent organisms per centimeter. RESULTS: Pre-exposure of the catheter to E. coli 83972 in all cases significantly reduced the number of uropathogens colonizing the catheter surfaces. CONCLUSIONS: E. coli 83972 significantly impedes catheter colonization by all tested bacterial and fungal pathogens. The broad applicability of this particular approach to bacterial interference in vitro invites further exploration in vivo.

Role of SspA in the density-dependent expression of the transcriptional activator AarP in Providencia stuartii.

The AarP protein in Providencia stuartii encodes a small transcriptional activator which activates the chromosomal aminoglycoside acetyltransferase aac(2')-Ia gene. In addition, AarP activates genes involved in a multiple antibiotic resistance (Mar) phenotype. Expression of an aarP-lacZ fusion increased in a density-dependent manner and reached peak levels at stationary phase. The expression of an aarP-lacZ fusion could be prematurely activated in cells at early to mid-exponential phase by the addition of spent culture supernatants from stationary phase cultures or by ethyl acetate extracts of these supernatants. Nutrient starvation had a negligible effect on aarP expression. In a search for mutations that block aarP activation at stationary phase, a mini-Tn5Cm insertion has been identified within a gene whose product was 77% identical to SspA, a regulatory protein involved in stationary phase gene expression and virulence. An unmarked sspA null allele (sspA2) was created by allelic replacement to further examine the role of sspA in P. stuartii. The sspA2 allele resulted in substantial decrease in aarP mRNA accumulation at various phases of growth. Furthermore, in an sspA mutant background, the aarP-lacZ fusion was no longer activated by an extracellular signal.

Selective medium for primary isolation of members of the tribe Proteeae.

A selective Proteeae medium (SPM) for isolation and preliminary detection of species of genera Proteus, Morganella, and Providencia was evaluated. The SPM contains tryptose phosphate agar with phenolphthalein monophosphate (as substrate for phosphatase activity), bile salts and polymyxin B (as inhibitors). The selectivity of the SPM was tested by the ecometric method of quality assurance of culture media. Fourteen reference cultures of enterobacteria and fifty-four strains of Proteeae were tested for their absolute growth index (AGI). Ninety-five percent of tested Proteeae strains display an AGI above 2.5. The detected phosphatase activity proved to be able to discriminate colonies of members of the tribe Proteeae. The ability of SPM for primary isolation of members of Proteeae was tested on food and clinical material and 94 strains were isolated. In addition, the SPM was employed in routine practice of clinical microbiology. From 1016 clinical samples (stool, urine, vaginal and urethral swabs), 57 strains of Proteeae were detected by the SPM in contrast to 35 strains by the routine procedure. The difference amounts to nearly 40%.

Antimicrobial activity of human pancreatic juice and its interaction with antibiotics.

Pancreatic juice (PJ) should be a factor of variability in the antimicrobial activity of antibiotics eliminated by the pancreas during pancreatic infections. We studied its effects on the activity of antimicrobial drugs with different mechanisms of action. Samples of pure PJ were collected from 16 patients with stabilized external pancreatic fistulas. The antimicrobial activity of the juice at different concentrations (from 1.25 to 100%) alone and in combination with mezlocillin, imipenem, ceftriaxone, gentamicin, ofloxacin, and ciprofloxacin was studied by a microbiological method (continuous turbidimetric recording of bacterial growth). The human PJ showed dose-dependent antimicrobial activity that increased directly with the concentration. The activity of the antibiotics at bactericidal concentrations were not modified by the PJ, while the combination with subinhibitory concentrations produced the following variable and different effects: (i) additivity with mezlocillin, ceftriaxone, gentamicin, and ciprofloxacin and autonomy (no interaction) with imipenem and ofloxacin against Providencia rettgeri and (ii) additivity with ceftriaxone, ofloxacin, gentamicin, imipenem, and mezlocillin and autonomy with ciprofloxacin against Escherichia coli. In the presence of PJ, fluoroquinolones showed constant positive effects, while beta-lactams showed more variable antimicrobial activity. Antibiotic concentrations and PJ pharmacodynamics are the main factors determining the final effect of the interaction in vitro. These results may be useful in choosing antibiotics for the treatment of pancreatic infections when they are supplemented with the pharmacokinetic data for each drug.