MAST® D72C test: a novel option for ESBL, AmpC and carbapenemase detection.

PURPOSE: The MAST® D72C test is a phenotypical test which can detect ESBL and AmpC production in Enterobacterales. It can also identify the suspected presence of carbapenemase. The aim of the present study was to assess the sensitivity and specificity of this test and to discuss its usefulness in laboratories, especially those that use only an automated AST system. METHOD: The performance of the MAST® D72C test was assessed against a collection of 119 non-redundant Enterobacterales isolates characterized for their content in ß-lactamases, and compared with that of the reference double disk synergy test. ß-lactamase content was established from phenotypic and genotypic analyses to collect a broad diversity of resistance mechanisms and bacterial strains, including 30 ESBL-producing strains, 32 strains overproducing chromosomal AmpC, 10 strains producing plasmid-encoded AmpC, 12 carbapenemase-producing strains, 13 strains combining the production of several ß-lactamases, and 22 strains that produced other ß-lactamases. RESULTS: The sensitivity and specificity for ESBL-detection were comparable with those of the synergy test, 75 versus 72.5%, and 94.9 versus 93.7%, respectively. The sensitivity and specificity for AmpC-detection were 71.7% and 100%, respectively, and sensitivity reached 78.7% if we excluded carbapenem-resistant isolates. Carbapenemase-detection sensitivity was 90%. CONCLUSION: These results show that the MAST® D72C test can be a useful tool for the detection of ESBL- and AmpC-production in clinical laboratories.

Comparison of homogenization with the GentleMacs Dissociator vs conventional methods for routine tissue processing.

Tissue specimens are valuable materials for microbiological diagnosis. The method of tissue processing can have a significant effect on sensitivity. This study aimed to compare different biopsy processing methods in terms of efficacy and standardization. Pork tissue artificially inoculated with Staphylococcus aureus and Escherichia coli, and samples of infected human tissue were processed by different methods before culture, and the results compared. Bacterial recovery from artificially inoculated pork tissue was significantly higher by homogenization with GentleMacs Dissociator than with sonication. No significant difference was observed between the GentleMacs Dissociator and manual treatment with a scalpel and vortexing. The microbial yield from homogenized human tissues was significantly higher after homogenization with GentleMacs Dissociator than with the conventional method. Homogenization with the GentleMacs Dissociator retrieves bacteria from tissue effectively. Tissue homogenization with the Dissociator is easy and fast to perform and allows for a high degree of standardization.

Emergence of plasmid-mediated colistin resistance (mcr-1) among Enterobacteriaceae strains: Laboratory detection of resistance and measures to control its dissemination.

The increasing use of colistin has contributed to the emergence of resistant bacteria and to an increase in the frequency of infections caused by naturally resistant Enterobacteriaceae strains such as Proteus, Providencia, Morganella, and Serratia. In August 2016, the French High Council for Public Health (French acronym HCSP) received a request from the Ministry of Health on the advice of the French National Public Health agency (Santé publique France) with regard to measures that should be taken to tackle the emergence of plasmid-mediated colistin resistance among Enterobacteriaceae strains. French healthcare facilities were asked to take the necessary measures as soon as possible, such as updating the definition of emerging highly resistant bacteria and defining the identification methods so as to take account of the evolving epidemiology of this type of resistance. This article describes the epidemiological context of the discovery of this emergence in France and worldwide, the resistance mechanisms, the microbiological methods of routine laboratory detection and the level of hygiene measures to implement in French facilities.

Accuracy of a rapid intrapartum group B Streptococcus test: A new immunochromatographic assay.

OBJECTIVES: To decrease the incidence of early-onset group B streptococcal (GBS) disease, a culture-based screening of all pregnant women at 35-37 weeks is recommended. This gold standard test requires 24-72hours culture. This delay precludes its use for intrapartum screening. This study assesses a new immunoassay, the DIMA test, for identifying GBS-positive patients in the labor ward. MATERIALS AND METHODS: This was a prospective observational study of 195 pregnant women presenting with full-term labor at a single site in France between June and August 2012. We assessed the diagnostic accuracy of intrapartum DIMA testing as compared to intrapartum GBS culture and prenatal screening at 35-38 weeks. RESULTS: The DIMA test sensitivity and specificity were 57.1% and 83.2%, respectively, as compared to 42.9% and 97% for prenatal culture screening. CONCLUSION: The DIMA test assay is a rapid and inexpensive test for the detection of maternal GBS colonization in the labor ward. Its sensitivity is higher than antepartum culture but its specificity is lower. Its performance was inferior to that reported for rapid polymerase chain reaction assays.

Fortuitous Detection of cmy-2 and dha-1 from ESBL-producing Escherichia coli in Senegal.

Cephalosporinases, which are naturally present in some enterobacterial species, can be mobilized by transposons, migrate to plasmids, and spread into other species such as Escherichia coli. The aim of this study was to characterize genes responsible for the production of extended-spectrum ß-lactamases (ESBL) in E. coli isolates from urinary origin isolated in two hospitals in Senegal. Thus, a fortuitous discovery of plasmidic cephalosporinase in two isolates was noted. One of the isolates produced dha-1 associated with ESBL CTX-M-14, the other produced cmy-2, ESBL CTXM-15, tem-1 penicillinase, and oxa-1. This confirms the circulation of multidrug-resistant bacteria producing plasmidic cephalosporinase in Senegal. However, a large study is needed to better understand the prevalence and the nature of the genes involved.

Inventory of Extended-Spectrum-ß-Lactamase-Producing Enterobacteriaceae in France as Assessed by a Multicenter Study.

The objective of this study was to perform an inventory of the extended-spectrum-ß-lactamase (ESBL)-producing Enterobacteriaceae isolates responsible for infections in French hospitals and to assess the mechanisms associated with ESBL diffusion. A total of 200 nonredundant ESBL-producing Enterobacteriaceae strains isolated from clinical samples were collected during a multicenter study performed in 18 representative French hospitals. Antibiotic resistance genes were identified by PCR and sequencing experiments. The clonal relatedness between isolates was investigated by the use of the DiversiLab system. ESBL-encoding plasmids were compared by PCR-based replicon typing and plasmid multilocus sequence typing. CTX-M-15, CTX-M-1, CTX-M-14, and SHV-12 were the most prevalent ESBLs (8% to 46.5%). The three CTX-M-type EBSLs were significantly observed in Escherichia coli (37.1%, 24.2%, and 21.8%, respectively), and CTX-M-15 was the predominant ESBL in Klebsiella pneumoniae (81.1%). SHV-12 was associated with ESBL-encoding Enterobacter cloacae strains (37.9%). qnrB, aac(6')-Ib-cr, and aac(3)-II genes were the main plasmid-mediated resistance genes, with prevalences ranging between 19.5% and 45% according to the ESBL results. Molecular typing did not identify wide clonal diffusion. Plasmid analysis suggested the diffusion of low numbers of ESBL-encoding plasmids, especially in K. pneumoniae and E. cloacae However, the ESBL-encoding genes were observed in different plasmid replicons according to the bacterial species. The prevalences of ESBL subtypes differ according to the Enterobacteriaceae species. Plasmid spread is a key determinant of this epidemiology, and the link observed between the ESBL-encoding plasmids and the bacterial host explains the differences observed in the Enterobacteriaceae species.

Rapid detection of AAC(6')-Ib-cr production using a MALDI-TOF MS strategy.

Plasmid-mediated quinolone resistance mechanisms have become increasingly prevalent among Enterobacteriaceae strains since the 1990s. Among these mechanisms, AAC(6')-Ib-cr is the most difficult to detect. Different detection methods have been developed, but they require expensive procedures such as Sanger sequencing, pyrosequencing, polymerase chain reaction (PCR) restriction, or the time-consuming phenotypic method of Wachino. In this study, we describe a simple matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method which can be easily implemented in clinical laboratories that use the MALDI-TOF technique for bacterial identification. We tested 113 strains of Enterobacteriaceae, of which 64 harbored the aac(6')-Ib-cr gene. We compared two MALDI-TOF strategies, which differed by their norfloxacin concentration (0.03 vs. 0.5 g/L), and the method of Wachino with the PCR and sequencing strategy used as the reference. The MALDI-TOF strategy, performed with 0.03 g/L norfloxacin, and the method of Wachino yielded the same high performances (Se = 98 %, Sp = 100 %), but the turnaround time of the MALDI-TOF strategy was faster (<5 h), simpler, and inexpensive (<1 Euro). Our study shows that the MALDI-TOF strategy has the potential to become a major method for the detection of many different enzymatic resistance mechanisms.

Targeting colorectal cancer-associated bacteria: A new area of research for personalized treatments.

Most cases of colorectal cancer (CRC) are sporadic, and numerous studies have suggested that gut microbiota may play a crucial role in CRC development. Escherichia coli is a member of the gut microbiota frequently associated with colorectal tumors. CRC-associated E. coli strains frequently harbor the pks genomic island. This genomic island is responsible for the synthesis of colibactin genotoxin, which increases tumor numbers in CRC mouse models. We recently showed that targeting ClbP, a key enzyme involved in colibactin synthesis, blocks the deleterious effect of this toxin in vitro and leads to a significant decrease in tumor numbers in vivo. Altogether, our results suggest that the personalized treatment of CRC should also take into consideration the bacteria associated with the tumor in order to limit their deleterious effects.

[Clinical Aspects of Cochlear Implantation in Meniere's Disease and After Labyrinthectomy]. / Klinische Aspekte der Cochleaimplantation bei Morbus Menière sowie nach Labyrinthektomie.

Objective: Due to the natural aging and the loosened CI-implantation criteria more formerly operated Meniere's patients will be supplied with a cochlear implant. However, it raises the question whether an implantation in a previously treated ear is promising. Material and Methods: Based on 2 cases in which we have carried out a CI-implantation, one of them 10 years after transmastoidal labyrinthectomy, and based on the current literature we draw attention to problems and prospects of success. Discussion: Histological studies show only a small ossification of the cochlea after labyrinthectomy. Implanted Meniere's patients have a better speech intelligibility than other CI users. Attacks of rotatory vertigo with tinnitus and fluctuations of hearing after cochlear implantation in Meniere's patients are rare or they are too little described. Conclusion: There is a trend to perform a simultaneous labyrinthectomy and cochlea implantation in Meniere's patients with desabling vertigo which have not responded to drug treatment. A formerly performed transmastoidal labyrinthectomy is not a contraindication for a cochlear implantation.

Structural insights into the loss of penicillinase and the gain of ceftazidimase activities by OXA-145 ß-lactamase in Pseudomonas aeruginosa.

OBJECTIVES: We previously described extended-spectrum oxacillinase OXA-145 from Pseudomonas aeruginosa, which differs from narrow-spectrum OXA-35 by loss of Leu-155. The deletion results in loss of benzylpenicillin hydrolysis and acquisition of activity against ceftazidime. We report the crystal structure of OXA-145 and provide the basis of its switch in substrate specificity. METHODS: OXA-145 variants were generated by site-directed mutagenesis and purified to homogeneity. The crystal structure of OXA-145 was determined and molecular dynamics simulations were performed. Kinetic parameters were investigated in the absence and in the presence of sodium hydrogen carbonate (NaHCO3) for representative substrates. RESULTS: The structure of OXA-145 was obtained at a resolution of 2.3 Å and its superposition with that of OXA-10 showed that Trp-154 was shifted by 1.8 Å away from the catalytic Lys-70, which was not N-carboxylated. Addition of NaHCO3 significantly increased the catalytic efficiency against penicillins, but not against ceftazidime. The active-site cavity of OXA-145 was larger than that of OXA-10, which may favour the accommodation of large molecules such as ceftazidime. Molecular dynamics simulations of OXA-145 in complex with ceftazidime revealed two highly coordinated water molecules on the α- or ß-face of the acyl ester bond, between Ser-67 and ceftazidime, which could be involved in the catalytic process. CONCLUSIONS: Deletion of Leu-155 resulted in inefficient positioning of Trp-154, leading to a non-carboxylated Lys-70 and thus to loss of hydrolysis of the penicillins. Ceftazidime hydrolysis could be attributed to enlargement of the active site and to a catalytic mechanism independent of the carboxylated Lys-70.

[The anterior tarsal tunnel syndrome: a case report]. / LE CAS CLINIQUE DU MOIS. Le syndrome tarsien antérieur.

The anterior tarsal tunnel syndrome is a rare entrapment neuropathy of the deep peroneal nerve beneath the inferior extensor retinaculum of the ankle. It is frequently unrecognized and may lead to misdiagnosis and delayed treatment. We report the case of a 77 year old patient complaining of symptoms of an anterior tarsal tunnel syndrome with neuropathic pain located at the dorsal part of the foot, without any sensorimotor loss. The ENMG was in favour of a motor impairment of the deep peroneal nerve. MRI exploration of the ankle showed a millimetric bony overgrowth of the upper pole of the navicular bone, irritative to the deep peroneal nerve. Infiltration at overgrowth of the navicular provided a partial and temporary decrease in pain symptoms. Surgical nerve decompression was then considered.

[Position Paper: Significance of the Forced Vital Capacity in Idiopathic Pulmonary Fibrosis]. / Positionspapier zur Bedeutung der forcierten Vitalkapazität für Patienten mit idiopathischer Lungenfibrose (IPF).

Spirometry is a highly standardized method which allows to measure the forced vital capacity (FVC) with high precision and reproducibility. In patients with IPF FVC is directly linked to the disease process which is characterized by scaring of alveoli and shrinkage of the lungs. Consequently, there is ample evidence form clinical studies that the decline of FVC over time is consistently associated with mortality in IPF. As for the first time effective drugs for the treatment of IPF are available it becomes obvious that in studies which could demonstrate that the drug reduces FVC decline, a numerical effect on mortality was also observed, while in one study where a significant effect on FVC decline was missed, there was also no change in mortality. Based on these studies FVC decline is a validated surrogate of mortality in IPF. It is concluded that FVC decline is not only accepted as an endpoint of clinical treatment trials in IPF but is also valid as a patient related outcome parameter which should be considered for the assessment of the efficacy of an IPF drug.

Genetic, biochemical characterization and mutagenesis of the chromosomal class A ß-lactamase of Raoultella (formerly Klebsiella) terrigena.

BACKGROUND: Chromosomal class A ß-lactamases have been characterized in Raoultella ornithinolytica and Raoultella planticola. The purpose of this study was to characterize that of Raoultella terrigena. MATERIALS AND METHODS: The blaTER-1 gene of R. terrigena strain ATCC33257(T) was cloned (pACter-1) and sequenced. It was then used to detect the bla gene of strains BM 85 01 095 and SB2796. The hypermutable Escherichia coli strain AB1157 mutS::Tn10 was transformed with pACter-1 and mutants growing on plates containing>2mg/L ceftazidime were studied. Notably, the impact of mutations only observed in the promoter region on ß-lactam resistance was assessed by site-directed mutagenesis experiments. RESULTS: R. terrigena strains ATCC33257(T) and BM 85 01 095 had the same bla gene and deduced protein (TER-1) whereas there were 3 substitutions in those of strain SB2796 (TER-2). Class A ß-lactamases TER showed 78%, 69.9% and 38.7% identity with PLA or ORN, TEM-1 and KOXY, respectively. Compared with TEM-1, TER-1 and TER-2 showed 2 particular substitutions, Leu75Pro and Glu240Asn demonstrated to be involved in the inherent ß-lactam resistance profile of R. terrigena. TER-1 (pI of 7.6) had a high activity against penicillin G and a significantly low one against amoxicillin. Substitution G/T observed in the -35 region of the blaTER gene harbored by strains growing in the presence of≥2mg/L ceftazidime was shown to be responsible for this growth. CONCLUSION: TER is a new class A ß-lactamase belonging to functional group 2b.

The MAST® D68C test: an interesting tool for detecting extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae.

The Mast® D68C test is a phenotypical test that allows the detection of extended-spectrum ß-lactamase (ESBL) production, even in AmpC-producing Enterobacteriaceae. We assessed its detection accuracy against a large collection of 106 Enterobacteriaceae isolates producing a wide diversity of well-characterized ß-lactamases (53 ESBL producers, 25 Amp. producers, seven AmpC and ESBL producers, five carbapenemase producers, three carbapenemase and ESBL producers, one AmpC, carbapenemase, and ESBL producer, three TEM-1 producers, three SHV-1 producers, three OXA-1 producers, and one hyperOXY producer, ATCC 35218, ATCC 25922 [a ß-lactamase-negative control strain]). The results were compared with those of the double disk test and the Clinical and Laboratory Standards Institute (CLSI) confirmatory test for the detection of ESBL. The sensitivity was 90.6 % for the synergy test, 87.5 % for the CLSI method, and only 73.1 % for D68C, which, however, reached 92.1 % if the strains for which supplementary investigations were recommended and the complex mutant TEM (CMT)-producing strains were excluded versus 94.1 % and 88.2 % for the other methods. The specificity was 90.2 % for the synergy test and 100 % for the CLSI method and D68C. D68C was also efficient in detecting AmpC-overproducing strains (sensitivity = 97 %, specificity = 95.9 %): among the 74 strains belonging to natural AmpC-producing species, the sensitivity and specificity were 100 and 94.8 %, respectively. The Mast® D68C-test is a promising method that is easy to perform for the detection of current ESBLs and could also be useful for the detection of plasmid-encoded AmpC enzymes (sensitivity = 100 %).