Enzymatic/biochemical analysis of Actinomyces with commercial test kits with an emphasis on newly described species

In clinical microbiology laboratories, the identification of Actinomyces-like bacteria can be very laborious and problematic. In the present study, we focused on reactivity patterns of 4 commercial test kits, RapID ANA II, RapID 32A, RapID CB Plus, and BBL Crystal ANR ID, that could be used for rapid preliminary identification of Actinomyces isolates belonging to newly described Actinomyces and closely related species. Out of the 54 strains tested, 25 strains (46%) were correctly identified to the genus/group level by BBL Crystal ANR ID system, 16 strains (30%) by RapID 32 A, 11 strains (20%) by RapID CB Plus, and 7 strains (13%) by RapID ANA II. The main problems with these kits were due to occasional weak enzymatic and sugar fermentation reactions. In conclusion, chromogenic substrate sensitivity and specificity need to be enhanced in order to improve the reliability of the test results of these kits, and the present database updated in order to more precisely identify newly described Actinomyces and closely related species.     

Comparison of bioMérieux API 20NE and Remel RapID NF Plus, identification systems of type strains of Ralstonia pickettii

A : Comparison of two commercial miniaturized rapid systems for the identification of Ralstonia pickettii strains. METHODS AND RESULTS: Varying identification results were encountered using the bioMérieux API NE system and the Remel IDS RapID NF Plus commercial systems for R. pickettii. To compare these two systems, eight strains of R. pickettii were purchased from different commercial culture collections. Additionally, 32 industrial and eight clinical isolates, initially identified using the Vitek Junior (bioMérieux) were tested. Total number of isolates tested was 48. The API 20NE identified 29 isolates, as R. pickettii but was unsuccessful with 19 isolates. The Remel IDS RapID NF Plus identified 46 isolates as R. pickettii. One clinical and one industrial isolates was identified as non-R. pickettii with both systems. CONCLUSIONS: The above results indicate that the use of API 20NE system for examining the identification of R. pickettii strains is inconsistent. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that the RapID NF Plus is more accurate as an inexpensive identification system for the identification of R. pickettii, a potential emerging organism of medically and industrial importance.     

以分子生物学鉴定结果为“金标准”评估Rapid ID Yeast Plus及API20C AUX对酵母样真菌的鉴定效能

目的 以分子生物学方法为“金标准”对两种商品化酵母样真菌鉴定产品Rapid ID Yeast Plus（简称RapIDYST）及API20C AUX（简称API20C）的鉴定效能进行评估.方法 从2010年中国医院侵袭性真菌感染监测网菌株库中筛选酵母样真菌25种,共计194株.其中,包含临床最常见的5种酵母样真菌（白念珠菌、热带念珠菌、光滑念珠菌、近平滑念珠菌、新生隐球菌）共130株,占研究总菌株数的67.0％.所有菌株已经过分子生物学方法准确鉴定至种水平.菌株复苏分纯后,严格按照产品操作指南,平行进行RapID YST和API20C鉴定.结果 所研究菌株中,有181株（18种）在RapID YST鉴定菌种数据库中,所有在库菌株种及复合体鉴定正确率为87.8％（159/181）.相比,API鉴定菌种库包含菌株174株（18种）,在库菌株种及复合体鉴定正确率为92.0％ （160/174）.RapID YST与API20C对于5种临床常见的酵母样真菌的种鉴定正确率分别为93.1％和97.1％.对于库外菌株,RapID YST的鉴定错误率分别为23.1％（3/13）,相比API20C的鉴定错误率为60.0％ （12/20）.综合此次评估结果,二者对酵母样真菌的鉴定效能无显著差异（McNemar检验,P＞0.05）.结论 两种商品化产品对酵母样真菌的鉴定效能基本一致;相较而言,RapID YST在操作便捷性、检测时间方面具有较大优势.     

Identification of Clostridium botulinum with API 20 A, Rapid ID 32 A and RapID ANA II

Three commercially available test systems for the identification of anaerobic bacteria were evaluated for the identification of 18 proteolytic group I and 69 non-proteolytic group II Clostridium botulinum, four Clostridium sporogenes and 18 non-toxigenic group II C. botulinum-like strains. All proteolytic C. botulinum strains were misidentified by the Rapid ID 32 A and RapID ANA II, while 14 strains and all C. sporogenes strains were identified as C. botulinum or C. sporogenes by the API 20 A. Reversely, all non-proteolytic C. botulinum strains were misidentified by the API 20 A while the Rapid ID 32 A recognized 67 and RapID ANA II 68 strains. All C. sporogenes strains were recognized by the RapID ANA II, while the Rapid ID 32 A recognized one strain. All non-proteolytic non-toxigenic strains were identified as C. botulinum group II by the Rapid ID 32 A, 17 strains by the RapID ANA II, and one strain by the API 20 A. The results show that these test systems do not provide a reliable method for identification of C. botulinum.     

Evaluation of VITEK matrix‐assisted laser desorption ionization–time of flight mass spectrometry for identification of anaerobes

Rapid and adequate identification of anaerobic bacterial species still presents a challenge for most diagnostic laboratories, hindering the selection of appropriate therapy. In this study, the identification capacity of 16S rRNA sequence analysis, VITEK 2 (BioMérieux, Lyon, France) compact analysis and VITEK MS‐mediated identification for anaerobic bacterial species was compared. Eighty‐five anaerobic bacterial isolates from 11 provinces in China belonging to 14 genera were identified by these three methods. Differences in identification between these three methods were compared. Consistent identification results were obtained for 54 (54/85, 63.5%) isolates by all three methods, the most discordant results being concentrated in Clostridium XI (n = 8) and Bacteroides fragilis (n = 9) clusters. Using the VITEK MS system, 74 (74/90, 82.2%) isolates were identified as single species consistent with 16S rRNA sequence analysis, which was significantly better than the results obtained with VITEK 2 Compact (P < 0.01). Misidentifications by the Vitek 2 Compact and Vitek MS systems were mainly observed in the Clostridium XI (n = 8)and B. fragilis clusters (n = 9). VITEK MS identified anaerobic bacteria even after they had been exposed to oxygen for a week. Identification by the Vitek MS system was more consistent with 16S rRNA sequence analysis than identification by Vitek 2 Compact. Continuous expansion of the VITEK MS database with rare described anaerobic species is warranted to improve both the efficiency and accuracy of VITEK MS identification in routine diagnostic microbiology.     

Comparative Resistance of Strains of Clostridium botulinum to Gamma Rays

A total of 102 strains of Clostridium botulinum (56 strains of type A, 43 type B, and 3 nontoxigenic strains which could not be typed) was examined for resistance to gamma rays. When these organisms were suspended in neutral phosphate buffer in concentrations of 10⁴ spores per tube, the threshold sterilizing dose appeared to be 1.4 Mrad. Partial survival to 1.4 Mrad was shown by 10.7% of the type A strains, 18.6% of the type B strains, and one of three nontoxigenic strains. Over-all, type A strains indicated higher radioresistance than type B strains, although there was overlapping. Representatives of the most resistant strains had D values of 0.317 to 0.336 Mrad; the D values of an intermediate group were 0.224 to 0.253 Mrad, and the most sensitive strain studied, 51B, had a D value of 0.129 Mrad. The radioresistance of Putrefactive Anaerobe 3679, strain S-2, was comparable to the intermediate C. botulinum group (D = 0.209).     

Comparison of the Accuracy of Two Conventional Phenotypic Methods and Two MALDI-TOF MS Systems with That of DNA Sequencing Analysis for Correctly Identifying Clinically Encountered Yeasts

We assessed the accuracy of species-level identification of two commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Bruker Biotyper and Vitek MS) and two conventional phenotypic methods (Phoenix 100 YBC and Vitek 2 Yeast ID) with that of rDNA gene sequencing analysis among 200 clinical isolates of commonly encountered yeasts. The correct identification rates of the 200 yeast isolates to species or complex (Candida parapsilosis complex, C. guilliermondii complex and C. rugosa complex) levels by the Bruker Biotyper, Vitek MS (using in vitro devices [IVD] database), Phoenix 100 YBC and Vitek 2 Yeast ID (Sabouraud''s dextrose agar) systems were 92.5%, 79.5%, 89%, and 74%, respectively. An additional 72 isolates of C. parapsilosis complex and 18 from the above 200 isolates (30 in each of C. parapsilosis, C. metapsilosis, and C. orthopsilosis) were also evaluated separately. Bruker Biotyper system could accurately identify all C. parapsilosis complex to species level. Using Vitek 2 MS (IVD) system, all C. parapsilosis but none of C. metapsilosis, or C. orthopsilosis could be accurately identified. Among the 89 yeasts misidentified by the Vitek 2 MS (IVD) system, 39 (43.8%), including 27 C. orthopsilosis isolates, could be correctly identified Using the Vitek MS Plus SARAMIS database for research use only. This resulted in an increase in the rate of correct identification of all yeast isolates (87.5%) by Vitek 2 MS. The two species in C. guilliermondii complex (C. guilliermondii and C. fermentati) isolates were correctly identified by cluster analysis of spectra generated by the Bruker Biotyper system. Based on the results obtained in the current study, MALDI-TOF MS systems present a promising alternative for the routine identification of yeast species, including clinically commonly and rarely encountered yeast species and several species belonging to C. parapsilosis complex, C. guilliermondii complex, and C. rugosa complex.     

Missed Opportunities: Barriers to HIV Testing during Pregnancy from a Population Based Cohort Study in Rural Uganda

The aim was to assess population-level HIV-testing uptake among pregnant women, key for access to prevention-of mother to child transmission (PMTCT) services, and to identify risk factors for not being HIV tested,The study was conducted May 2008–May 2010 in the Iganga/Mayuge Health and Demographic Surveillance Site (HDSS), Eastern Uganda, during regular surveillance of 68,000 individuals. All women identified to be pregnant May–July 2008 (n = 881) were interviewed about pregnancy-related issues and linked to the HDSS database for socio-demographic data. Women were followed-up via antenatal care (ANC) register reviews at the health facilities to collect data related to ANC services received, including HIV testing. Adjusted relative risk (aRR), and 95% confidence intervals (CI) for not being HIV tested were calculated using multivariable binomial regression among the 544 women who remained after record review.Despite high ANC attendance (96%), the coverage of HIV testing was 64%. Only 6% of pregnant women who sought ANC at a facility without HIV testing services were referred for testing and only 20% received counseling regarding HIV. At ANC facilities with HIV testing services, 85% were tested. Only 4% of the women tested had been couple tested for HIV. Living more than three kilometers away from a health facility with HIV testing services was associated with not being tested both among the poorest (aRR,CI; 1.44,1.02–2.04) and the least poor women (aRR,CI;1.72,1.12–2.63).The lack of onsite HIV testing services and distant ANC facilities lead to missed opportunities for PMTCT, especially for the poorest women. Referral systems for HIV testing need to be improved and testing should be expanded to lower level health facilities. This is in order to ensure that the policy of HIV testing during pregnancy is implemented more effectively and that testing is accessible for all.     

Routine deworming during antenatal care decreases risk of neonatal mortality and low birthweight: A retrospective cohort of survey data

BackgroundSoil transmitted helminths (STH) are a common infection among pregnant women in areas with poor access to sanitation. Deworming medications are cheap and safe; however, the health benefit of deworming during pregnancy is not clear.Methods / Principal findingsWe created a retrospective cohort of more than 800,000 births from 95 Demographic and Health Survey datasets to estimate the impact of deworming medicine during routine antenatal care (ANC) on neonatal mortality and low birthweight. We first matched births on the probability of receiving deworming during ANC. We then modeled the birth outcomes with the matched group as a random intercept to estimate the effect of deworming during antenatal care after accounting for various risk factors. We also tested for effect modification of soil transmitted helminth prevalence on the impact of deworming during ANC. Receipt of deworming medication during ANC was associated with a 14% reduction in the risk of neonatal mortality (95% confidence interval = 10–17%, n = 797,772 births), with no difference between high and low transmission countries. In low transmission countries, we found an 11% reduction in the odds of low birth weight (95% confidence interval = 8–13%) for women receiving deworming medicine, and in high transmission countries, we found a 2% reduction in the odds of low birthweight (95% confidence interval = 0–5%).Conclusions / SignificanceThese results suggest a substantial health benefit for deworming during ANC that may be even greater in countries with low STH transmission.     

Yeast species associated with orange juice: evaluation of different identification methods

Five different methods were used to identify yeast isolates from a variety of citrus juice sources. A total of 99 strains, including reference strains, were identified using a partial sequence of the 26S rRNA gene, restriction pattern analysis of the internal transcribed spacer region (5.8S-ITS), classical methodology, the RapID Yeast Plus system, and API 20C AUX. Twenty-three different species were identified representing 11 different genera. Distribution of the species was considerably different depending on the type of sample. Fourteen different species were identified from pasteurized single-strength orange juice that had been contaminated after pasteurization (PSOJ), while only six species were isolated from fresh-squeezed, unpasteurized orange juice (FSOJ). Among PSOJ isolates, Candida intermedia and Candida parapsilosis were the predominant species. Hanseniaspora occidentalis and Hanseniaspora uvarum represented up to 73% of total FSOJ isolates. Partial sequence of the 26S rRNA gene yielded the best results in terms of correct identification, followed by classical techniques and 5.8S-ITS analysis. The commercial identification kits RapID Yeast Plus system and API 20C AUX were able to correctly identify only 35 and 13% of the isolates, respectively. Six new 5.8S-ITS profiles were described, corresponding to Clavispora lusitaniae, Geotrichum citri-aurantii, H. occidentalis, H. vineae, Pichia fermentans, and Saccharomycopsis crataegensis. With the addition of these new profiles to the existing database, the use of 5.8S-ITS sequence became the best tool for rapid and accurate identification of yeast isolates from orange juice.     

Vitek—AMS系统检测超广谱β—内酰胺酶的评价

目的：评价Vitek-AMS系统检测超广谱β-内酰胺酶（ESBLs)方法的敏感性和特性。方法：用Vitek－32仪器法、双纸片协同试验法检测45株ESBLs阳性和45株ESBLs阴性菌株,并将结果进行比较。结果：Vitek－32仪器法检出45株ESBLs阳性株中的43株,而双纸片协同试验法仅检出38株,45株ESBLs阴性株用两法检测均为阴性。Vitek－AMS仪器法检测ESBLs的方法敏感性为95．5％,特异性为100％,而双纸片协同试验法分别为84．4％和100％。结论：Vitek－AMS系统检测ESBLs的方法敏感性和特异性均优于双纸片同试验法,且操作简便,有条件的实验室可将其作为初筛ESBLs的首选方法。     

A COMPARISON STUDY BETWEEN OXYRASE ANAEROBIC AGAR PLATES AND CONVENTIONAL ANAEROBIC CHAMBER FOR THE ISOLATION AND IDENTIFICATION OF ANAEROBIC BACTERIA FROM CLINICAL INFECTIONS

Misplaced confidence in the broad-spectrum antibiotics, increased resistance among previously predictable anaerobic antibiograms, and the push to maximize productivity of available space and downsizing trends has created a need for a simplified cost-effective, and superior method for the isolation and identification of anaerobic bacteria. In this study, the Oxyrase anaerobic plate system which requires no extraneous apparatus to create an anaerobic environment was compared to an anaerobic chamber in the isolation of anaerobic bacteria from 212 consecutive wound specimens. Brucella blood agar and KVL agar plates were used in this comparison study. RapID ANA II, AP120A, special potency disks, and GLC were used for identification. Of the 212 specimens cultured, 87 yielded anaerobic bacteria comprising 182 strains. Thirty-nine strains failed to grow in the anaerobic chamber but grew on the OxyPlates^(TM). These strains were predominantly Peptostreptococcus species (28%), Eubacterium species (20%), and Propionibacterium species (20%). Fourteen strains failed to grow on the OxyPlates, but grew in the anaerobic chamber. No trend was noted and all organisms in this category grew on the OxyPlates from other specimens. In conclusion, the Oxyrases anaerobic plate system appears to be an excellent alternative to the conventional anaerobic chamber in the isolation and identification of clinically significant anaerobes found in human samples, obviating the need for separate anaerobic-aerobic workstations, expensive anaerobic apparatus, and additional incubator space.     

Prevalence of Escherichia coli strains resistance to antibiotics in wound infections and raw milk

Antibiotic-resistant Escherichia coli strains including extended-spectrum β-lactamase (ESBL) isolates are globally widespread in medical, food, and environmental sources. Some of these strains are considered the most pathogenic bacteria in humans. The present work examined the predominance of antibiotic resistance in E. coli strains in wound infections comparing with E. coli strains isolated from a raw milk as a potential source of those strains. The wound infections included abdomen, anus, arm, back, buttock, chest, foot, hand, head, leg, lung, mouth, neck, penis, thigh, toe, and vagina infections. In total, 161 and 153 isolates identified as E. coli were obtained from wound infections and raw milk, respectively. A Vitek 2 system innovated by bioMérieux, France was applied to perform the identification and susceptibility tests. The E. coli isolates that have ability to produce ESBL were detected by an ESBL panel and NO45 card (bioMérieux). Over half of the E. coli were from abdomen, back, and buttock wound infections. More than 50%of the E. coli isolates obtained from wound infections were resistant to cefazolin, ampicillin, cefuroxime, ciprofloxacin, mezlocillin, moxifloxacin, piperacillin, and tetracycline; 70% of the isolates from wound infections and 0% of the isolates from raw milk were E. coli isolates produced ESBL. The data showed that the strains resistance to multi-antibiotic and produced ESBL are more widespread among wound infections than in raw milk.     

Multidrug-resistant Escherichia coli in Raw Milk: Molecular Characterization and the potential impact of camel’s Urine as an Antibacterial Agent

Raw milk is one of the most important vehicles for transmitting various pathogens, especially Escherichia coli (E. coli). Multidrug-resistant pathogens are highly prevalent among mastitic cows in various dairy farms worldwide. Therefore, our current study is based on the identification of E. coli from mastitic cow’s milk and their resistance to various antibacterial agents. As well, the impact of camel’s urine on multi-drug resistant E. coli were also evaluated. Thirty-three E. coli isolates were recovered from 254 milk samples. All strains were initially identified phenotypically by culturing on specific media and Vitek 2 Compact System. The protein fingerprinting technique was used as a confirmatory method. The Stx1, Stx2 and eae genes were also verified by polymerase chain reaction (PCR). The antimicrobial resistance of E. coli strains was tested by the Vitek 2 AST-GN69 cards. Thirty multi-drug resistant E. coli strains (20 from mastitic milk and 10 from clinical samples) were laboratory tested with different concentrations (100%, 75%, 50% and 25%) of virgin and breeding camel’s urine, using the paper disc diffusion method. Our findings showed that 93.94% of E. coli strains were recognized by the Vitek™ 2 system. The results of proteomic investigation illustrated that 100% of E. coli strains were identified at log values ≥2.00. The genotypic identification of the three virulence genes illustrated that 90.1%, 63.64%, and 30.55% of E. coli strains were able to carry the Stx1, eae, and Stx2 genes, respectively. Most strains of E. coli showed strong resistance against cefazolin (78.79%), ceftazidime (66.67%), cefotaxime (60.61%), ceftriaxone (54.55%), and cefepime (39.40%). The results of the antibacterial effect of camel’s urine revealed that the mean inhibitory zones of virgin camel’s urine were 28 mm, 17 mm, and 14 mm, for the concentrations of 100%, 75%, and 50%, respectively. Whereas; the inhibitory zones for the breeding camel’s urine were 18 mm, 0 mm, and 0 mm, for the concentrations of 100%, 75%, and 50%, respectively. We concluded that the majority of E. coli strains were able to harbor some virulence genes and resist many antibiotics. Our study also provided a robust evidence that the camel’s urine, particularly from the virgin camels has robust antimicrobial activity against multidrug-resistant E. coli strains.     

High Proportion of Intestinal Colonization with Successful Epidemic Clones of ESBL-Producing Enterobacteriaceae in a Neonatal Intensive Care Unit in Ecuador

Background and Aims

Neonatal infections caused by Extended-spectrum beta-lactamase (ESBL)-producing bacteria are associated with increased morbidity and mortality. No data are available on neonatal colonization with ESBL-producing bacteria in Ecuador. The aim of this study was to determine the proportion of intestinal colonization with ESBL-producing Enterobacteriaceae, their resistance pattern and risk factors of colonization in a neonatal intensive care unit in Ecuador.

Methods

During a three month period, stool specimens were collected every two weeks from hospitalized neonates. Species identification and susceptibility testing were performed with Vitek2, epidemiologic typing with automated repetitive PCR. Associations between groups were analyzed using the Pearson _X ² test and Fisher exact test. A forward step logistic regression model identified significant predictors for colonization.

Results

Fifty-six percent of the neonates were colonized with ESBL-producing Enterobacteriaceae. Length of stay longer than 20 days and enteral feeding with a combination of breastfeeding and formula feeding were significantly associated with ESBL-colonization. The strains found were E. coli (EC, 89%) and K. pneumoniae (KP, 11%) and epidemiological typing divided these isolates in two major clusters. All EC and KP had bla _CTX-M group 1 except for a unique EC isolate that had bla _CTX-M group 9. Multi-locus sequence typing performed on the K. pneumoniae strains showed that the strains belonged to ST855 and ST897. The two detected STs belong to two different epidemic clonal complexes (CC), CC11 and CC14, which previously have been associated with dissemination of carbapenemases. None of the E. coli strains belonged to the epidemic ST 131 clone.

Conclusions

More than half of the neonates were colonized with ESBL-producing Enterobacteriaceae where the main risk factor for colonization was length of hospital stay. Two of the isolated clones were epidemic and known to disseminate carbapenemases. The results underline the necessity for improved surveillance and infection control in this context.     

An examination of 'unidentified' Prevotella (formerly PINLO) using RAPD-PCR and partial 16S rRNA gene sequencing

Prevotella intermedia- and Prevotella nigrescens-like organisms (PINLO) have been described as organisms which are phenotypically and biochemically similar to P. intermedia and P. nigrescens and the species P. pallens was created to include some of them. Other PINLO groups which do not fit the definition of P. pallens exist, and in this study these 'unidentified' Prevotella sp. were compared with P. corporis, P. intermedia, P. nigrescens and P. pallens using commercial identification kits, GLC, RAPD-PCR and partial 16S rRNA gene sequencing. The Rapid ID 32 A and the RapID ANA II system both identified all 'unidentified' Prevotella as P. intermedia. Similarly they gave this identification to all the species tested (with the exception of P. corporis using the RapID ANA II system) clearly demonstrating biochemical similarities. Gas liquid chromatography (GLC) analysis of the volatile end-products of fermentation could not distinguish between strains. RAPD-PCR using arbitrary primer L10 demonstrated intra-species homogeneity within PINLO strains with amplification profiles which differed from other Prevotella species tested. Cluster analysis of the amplification profiles confirmed species divisions and yielded a distinct 'unidentified' Prevotella cluster. Comparison of partial 16S rDNA sequences displayed 98% sequence similarity between the 'unidentified' Prevotella strains, although 2 strains, HST 1156 and HST 2160 displayed 100% identity. The highest similarity between groups was seen between 'unidentified' Prevotella strains and P. corporis (approximately 94% similarity). The DNA techniques used here confirm that 'unidentified' Prevotella strains are distinct from the other species of Prevotella tested, including P. pallens. Partial 16S rDNA sequence comparisons suggested a close relationship with P. corporis.     

The extreme drug resistance (XDR) Staphylococcus aureus strains among patients: A retrospective study

The objective of the present work was to observe and profile various antibiotic resistant strains of Staphylococcus aureus and highlight the need for continuous surveillance. Data regarding antibiotic-resistant S. aureus strains isolated and identified at the Medical Microbiology Department, King Khalid Hospital, Riyadh was obtained. Bacterial isolates were collected from several sites of infections in patients and an evaluation of susceptibility were carried out using a fully automated Vitek2 system. Relative frequency (%), odds ratios and Ward's minimum variance were calculated. The results showed that wounds were a source of more than 40% of the S. aureus (MRSA) strains that have ability to resist methicillin, and more than 45% of the methicillin-susceptible S. aureus (non-MRSA) strains. 40% of the isolates were MRSA (N = 251), and all MRSA strains were sensitive to vancomycin, daptomycin, teicoplanin, tigecycline, nitrofurantoin, and itraconazole while all non-MRSA (N = 338) strains were sensitive to vancomycin, cefoxitin, daptomycin, gentamicin, oxacillin, teicoplanin, tigecycline, and mupirocin. Strength of association between antibiotic-resistant S. aureus strains and source of samples (site of infection) was established. The study concluded that S. aureus strains had developed resistance towards 20 (for non-MRSA) and 22 (for MRSA) of the antibiotics tested. All MRSA strains were non-sensitive to amoxicillin/clavulanate, ampicillin cefoxitin, cefazolin, imipenem, oxacillin, and penicillin.     

Misidentification of Aeromonas veronii biovar sobria as Vibrio alginolyticus by the Vitek system

AIMS: To find the cause of misidentification of aeromonads when using the Vitek system. METHODS AND RESULTS: Two Aeromonas veronii biovar sobria isolates were misidentified as Vibrio alginolyticus by the Vitek system. Both strains' identification was confirmed by biochemical testing, API 20E/20NE kits and/or 16S RFLP analysis. Thirty-one known Aeromonas species were tested by the Vitek system using 0.45 and 0.85% saline in the suspension medium. It was not clear whether low salinity causes misidentification of Aeromonas species more frequently. CONCLUSIONS: The specified reaction time may be inappropriately short for some critical biochemical tests of some strains. An ingenious reading strategy regarding incubation time is necessary to improve identification of Aeromonas species by the Vitek system. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of misidentification of A. veronii biovar sobria as V. alginolyticus in the Vitek system.     

Rapid Identification and Susceptibility Testing of Candida spp. from Positive Blood Cultures by Combination of Direct MALDI-TOF Mass Spectrometry and Direct Inoculation of Vitek 2

Fungaemia is associated with high mortality rates and early appropriate antifungal therapy is essential for patient management. However, classical diagnostic workflow takes up to several days due to the slow growth of yeasts. Therefore, an approach for direct species identification and direct antifungal susceptibility testing (AFST) without prior time-consuming sub-culturing of yeasts from positive blood cultures (BCs) is urgently needed. Yeast cell pellets prepared using Sepsityper kit were used for direct identification by MALDI-TOF mass spectrometry (MS) and for direct inoculation of Vitek 2 AST-YS07 card for AFST. For comparison, MALDI-TOF MS and Vitek 2 testing were performed from yeast subculture. A total of twenty four positive BCs including twelve C. glabrata, nine C. albicans, two C. dubliniensis and one C. krusei isolate were processed. Applying modified thresholds for species identification (score ≥1.5 with two identical consecutive propositions), 62.5% of BCs were identified by direct MALDI-TOF MS. AFST results were generated for 72.7% of BCs directly tested by Vitek 2 and for 100% of standardized suspensions from 24 h cultures. Thus, AFST comparison was possible for 70 isolate-antifungal combinations. Essential agreement (minimum inhibitory concentration difference ≤1 double dilution step) was 88.6%. Very major errors (VMEs) (false-susceptibility), major errors (false-resistance) and minor errors (false categorization involving intermediate result) amounted to 33.3% (of resistant isolates), 1.9% (of susceptible isolates) and 1.4% providing 90.0% categorical agreement. All VMEs were due to fluconazole or voriconazole. This direct method saved on average 23.5 h for identification and 15.1 h for AFST, compared to routine procedures. However, performance for azole susceptibility testing was suboptimal and testing from subculture remains indispensable to validate the direct finding.     

Extended-Spectrum β-Lactamase- and Carbapenemase-Producing Enterobacteriaceae Isolated from Egyptian Patients with Suspected Blood Stream Infection

ObjectivesThe aim of the study was to investigate the prevalence of extended-spectrum β-lactamase and carbapenemase production among Enterobacteriaceae isolated from Egyptian patients with suspected blood stream infection.MethodsNinety-four Enterobacteriaceae blood culture isolates from Egyptian patients with suspected blood stream infection were collected, one isolate per patient. Identification of bacterial isolates was performed with MALDI-TOF (MS-based Vitek MS system, bioMerieux). Screening for ESBLs and carbapenemases production was done with the Vitek 2 system (bioMérieux). ESBL production was confirmed using the combined disk diffusion method for cefotaxime, ceftazidime, and cefepime, all with and without clavulanic acid (Rosco). Real-time PCR and sequencing were used to characterize the resistance genes. The phylogenetic groups of E. coli were identified by a PCR-based method.ResultsOf the 94 Enterobacteriaceae isolates 46 (48.93%) showed an ESBL phenotype. One Enterobacter spp isolate was ESBL-producer and meropenem-resistant. The genetic analysis showed that CTX-M was present in 89.13% (41/46) of the ESBL-producing Enterobacteriaceae, whereas TEM and SHV were detected in 56.52% (26/46) and 21.74% (10/46) respectively (47.83%) of the ESBL-producing isolates were multidrug resistant (MDR). Eleven out of 30 ESBL-producing E-coli isolates were assigned to phylogroup B2, followed by groups B1 (8 isolates), A (6 isolates) and D (5 isolates).ConclusionsThe high ESBL-E rates (48.93%) found in this study together with the identification of one carbapenem-resistant Enterobacter spp isolate is worrisome. Our results indicate that systems for monitoring and detection of ESBL-producing bacteria in Egyptian hospitals have to be established. Also strict hospital infection control policies with the restriction of the consumption of extended-spectrum cephalosporins are necessary.