Phenotypic and molecular characterization of beta-lactam resistance and capsular typing of colonizing Haemophilus influenzae strains isolated from neutropenic patients in Tunisia

Objective

The aim of this study was to determine the overall percentage of β-lactams susceptibility, beta-lactamase production, penicillin binding protein (PBP) modification and serotypes of colonizing Haemophilus influenzae strains.

Design

A total of 50 isolates of colonized H. influenzae, isolated from neutropenic patients. The prevalence of β-lactams resistance and β-lactamase production were recorded for each strains using E-test strips and chromogenic cephalosporin test, then were determined their resistance genes (bla_TEM and bla_ROB) by PCR as well as their capsular types by standard slide agglutination serotyping (SAST) and capsular genes amplification.

Results

Thirty-two percent of the 50 strains were amoxicillin resistant, among these, 20% were resistant by beta-lactamase production, and they produced all type TEM β-lactamase. Four percent of the isolates had PBP modification and three strains (6%) associated the two resistance mechanisms. Slide agglutination serotyping showed that 95.8% of the strains were unencapsulated, and 4.1% were of serogroup b. The result was confirmed by PCR capsular typing.

Conclusion

By the light of these results, our findings suggest that it becomes important to follow the evolution of the resistance background of our strains, and that the majority of colonizing H. influenzae strains isolated in our center are unencapsulated.     

Characterization and molecular epidemiology of extended spectrum beta-lactamase producing Enterobacter cloacae isolated from a Tunisian hospital

In 2009, out of the 66 nonrepetitive Enterobacter cloacae collected at Charles Nicolle hospital in Tunisia, 44 were extended spectrum β-lactamase (ESBL) producers. The aim of the current study was to detect and characterize the genes encoding the ESBLs including blaTEM, blaSHV, and blaCTX-M groups by polymerase chain reaction and sequencing. Pulsed-field gel electrophoresis (PFGE) analysis was used to determine the genetic relatedness between isolates. All strains were susceptible to carbapenems. They were resistant to fluoroquinolones, gentamicin, tobramycin, and trimethoprim+sulfamethoxazole but variably resistant to netilmicin, amikacin, and tetracyclines. Sequence analysis of the polymerase chain reaction products revealed the presence of blaCTX-M-15 (39 strains), blaSHV-12 (6 strains), and blaSHV-27 (1 strain). The coexistence of two ESBLs was observed in two isolates harboring, respectively, SHV-12+CTX-M-15 and SHV-27+CTX-M-15. PFGE revealed 36 unrelated profiles. Diffusion of E. cloacae producing CTX-M-15 ESBL in our hospital is the consequence of dissemination of identical or related plasmids harboring the CTX-M-15 gene.     

Program death-1 regulates peripheral T cell tolerance via an anergy-independent mechanism

Program death-1 (PD-1) has been documented to negatively regulate immune responses. However, the cellular and molecular mechanisms for PD-1-mediated immune suppression have not been fully elucidated. In this study, we show that loss of PD-1 does not lead to defective induction of CD4(+) T cell anergy in vitro and in vivo. Rather, the absence of PD-1 inhibits the development of inducible CD4(+)Foxp3(+) regulatory T cells (iTregs) induced by TGF-β in vitro. In support of this finding, PD-1 deficiency impairs the generation of iTregs in vivo and leads to development of severe T cell-transfer-induced colitis. Mechanistically, defective iTreg generation in the absence of PD-1 was attributed to the heightened phosphorylation of Akt. Therefore, we first demonstrate that PD-1 controls peripheral T cell tolerance via an anergy-independent but iTreg-dependent mechanism.     

Detection of DNA mismatch repair proteins in fresh human blood lymphocytes - towards a novel method for hereditary non-polyposis colorectal cancer (Lynch syndrome) screening

Background

A broad population-based assay to detect individuals with Lynch Syndrome (LS) before they develop cancer would save lives and healthcare dollars via cancer prevention. LS is caused by a germline mutation in a DNA mismatch repair (MMR) gene, especially protein truncation-causing mutations involving MSH2 or MLH1. We showed that immortalized lymphocytes from LS patients have reduced levels of full-length MLH1 or MSH2 proteins. Thus, it may be feasible to identify LS patients in a broad population-based assay by detecting reduced levels of MMR proteins in lymphocytes.

Methods

Accordingly, we determined whether MSH2 and MLH1 proteins can also be detected in fresh lymphocytes. A quantitative western blot assay was developed using two commercially available monoclonal antibodies that we showed are specific for detecting full-length MLH1 or MSH2. To directly determine the ratio of the levels of these MMR proteins, we used both antibodies in a multiplex-type western blot.

Results

MLH1 and MSH2 levels were often not detectable in fresh lymphocytes, but were readily detectable if fresh lymphocytes were first stimulated with PHA. In fresh lymphocytes from normal controls, the MMR ratio was ~1.0. In fresh lymphocytes from patients (N > 50) at elevated risk for LS, there was a bimodal distribution of MMR ratios (range: 0.3-1.0).

Conclusions

Finding that MMR protein levels can be measured in fresh lymphocytes, and given that cells with heterozygote MMR mutations have reduced levels of full-length MMR proteins, suggests that our immunoassay could be advanced to a quantitative test for screening populations at high risk for LS.     

High-level gentamicin-resistant Enterococcus faecium strains isolated from bone marrow transplant patients: accumulation of antibiotic resistance genes, large plasmids and clonal strain dissemination

Twenty-six high-level gentamicin-resistant (HLGR) Enterococcus faecium strains colonising neutropenic bone marrow transplant patients were studied. Polymerase chain reaction analysis showed that high-level gentamicin resistance was mediated by the aac(6′)-Ia-aph(2″)-Ie gene; the aph(2″)-Id gene responsible for gentamicin resistance was also detected in 16 strains. Multiple antibiotic resistance was related to the presence of aph(3′)-IIIa, ant(6)-Ia, erm(B), erm (A) and tet(M) genes. Strains clustered into 18 groups according to their plasmid content as well as 16 pulsed-field gel electrophoresis (PFGE) patterns. Although the majority of PFGE patterns were single isolates, three microclones were identified. Hybridisation showed that in the majority of the strains the aac(6′)-aph(2″) gene resided on a large plasmid of ca. 96 kb detected only on PFGE gels. Based on these findings, colonisation by HLGR E. faecium strains was a result of either possibly related plasmid spread or strain dissemination.     

Acenocoumarol and vasculitis: a case report

A 62-year-old woman was referred to the dermatology department for a history of fever, asthenia and cutaneous rash, which appeared after a 3-day course of digitalin and acenocoumarol for atrial fibrillation. The physical examination revealed multiple round confluent purpuric lesions over her entire legs with no blistering. Laboratory exams were all negative. Biopsy of the involved skin was compatible with leucocytoclastic vasculitis. The acenocoumarol treatment was withheld and the skin lesions resolved spontaneously over the next 10 days. The cause of this purpura was seemingly acenocoumarol because of the close temporal relationship between exposure to the drug and the onset of the symptoms, and the spontaneous resolution of the lesions after acenocoumarol was discontinued. This observation illustrates a rare association between vasculitis and acenocoumarol. Clinicians should be aware of this potential adverse effect and recommend interrupting the drug intake when temporal relation is evocative.     

Identifying the Risks of Anticoagulation in Patients with Substance Abuse

BACKGROUND

Warfarin is effective in preventing thromboembolic events, but concerns exist regarding its use in patients with substance abuse.

OBJECTIVE

Identify which patients with substance abuse who receive warfarin are at risk for poor outcomes.

DESIGN

Retrospective cohort study. Diagnostic codes, lab values, and other factors were examined to identify risk of adverse outcomes.

PATIENTS

Veterans AffaiRs Study to Improve Anticoagulation (VARIA) database of 103,897 patients receiving warfarin across 100 sites.

MAIN MEASURES

Outcomes included percent time in therapeutic range (TTR), a measure of anticoagulation control, and major hemorrhagic events by ICD-9 codes.

RESULTS

Nonusers had a higher mean TTR (62 %) than those abusing alcohol (53 %), drugs (50 %), or both (44 %, p?<?0.001). Among alcohol abusers, an increasing ratio of the serum hepatic transaminases aspartate aminotransferase/alanine aminotransferase (AST:ALT) correlated with inferior anticoagulation control; normal AST:ALT?≤ 1.5 predicted relatively modest decline in TTR (54 %, p?<?0.001), while elevated ratios (AST:ALT 1.50–2.0 and > 2.0) predicted progressively poorer anticoagulation control (49 % and 44 %, p?<?0.001 compared to nonusers). Age-adjusted hazard ratio for major hemorrhage was 1.93 in drug and 1.37 in alcohol abuse (p?<?0.001 compared to nonusers), and remained significant after also controlling for anticoagulation control and other bleeding risk factors (1.69 p?<?0.001 and 1.22 p?=?0.003). Among alcohol abusers, elevated AST:ALT >2.0 corresponded to more than three times the hemorrhages (HR 3.02, p?<?0.001 compared to nonusers), while a normal ratio AST:ALT ≤ 1.5 predicted a rate similar to nonusers (HR 1.19, p?<?0.05).

CONCLUSIONS

Anticoagulation control is particularly poor in patients with substance abuse. Major hemorrhages are more common in both alcohol and drug users. Among alcohol abusers, the ratio of AST/ALT holds promise for identifying those at highest risk for adverse events.