The therapeutic potential of using sulbactam alone has also been discussed (2). Since the activity of the combination of ampicillin and sulbactam against Acinetobacter spp. comes al- most exclusively from the sulbactam component (2, 8, 9), the use of a combination disk may be able to predict the activity of sulbactam alone. However, further studies are needed to de- termine if breakpoints for sulbactam alone can be developed. In summary, the results of BMD and DD are concordant for most non- ␤ -lactam agents. Thus, DD can be used with confi- dence for Acinetobacter spp. and these agents. While an unac- ceptably high rate of major errors was observed with tetracy- cline, this problem may be resolved by readjusting the DD breakpoints to smaller zone diameters. The BMD tests for the ␤ -lactam agents, which were difficult to read because of subtle growth beyond an obvious end point, continue to pose a prob- lem of interpretation. Further studies are needed to determine TABLE 6. BMD and DD discrepancy rates for three antimicrobial
carbapenemase-producing Acinetobacter spp. (14). The modified Hodge test has largely been used for this purpose. It is based on the in vitro detection of carbapenemase activity, therefore inactivating the antibiotic effect. Although this test is efficient for detecting IMP and VIM producers, NDM and CHDL producers may re- main undetected, leading to false-negative results (15). Several techniques using the inhibition properties of EDTA were also proposed for detecting MBL-producing Acinetobacter spp. Those techniques include the combined disk test and the Etest MBL strip (15, 16). However, those techniques are not highly sensitive or specific, and they require an additional period of growth of 24 h. Biochemical detection of carbapenemase production using ultraviolet (UV) spectrophotometry has also been proposed as a suitable method (17). This technique efficiently detects VIM, IMP, and SIM producers, but NDM and CHDL producers re- main difficult to detect (15). Recently, the detection of carbap- enemase production using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry has been proposed for Acinetobacter spp. (18–20). It is based on the detection of the spectra of imipenem and of its hydrolyzed product. It shows good sensitivity and specificity but requires trained microbiologists and expensive equipment. Finally, molec- ular-based techniques using specific primers are useful for identi- fying carbapenemase genes. Simplex or multiplex PCR methods are available (21), as well as real-time PCR approaches that have the advantage of providing a result in 3 h (22). More recently, a DNA microarray has been developed to detect 91 target sequences associated with antibiotic resistance within 4 h from bacterial cul- ture to results (23). Although those molecular-based tests are highly sensitive and specific, they fail to detect unknown carbap- enemase genes or those that are not included in the panel of the test used.
In summary, Acinetobacter spp. have become particular- ly problematic nosocomial pathogens in Korea, partly due to clonal spread. It is difficult to prevent Acinetobacter spp. infection in hospitalized patient, because the organisms are ubiquitous in hospital environment. Recent clinical isolates of Acinetobacter spp. in Korea were often found to be mul- tiresistant to carbapenems, fluoroquinolones, and amino- glycosides. Carbapenem resistance was mostly due to OXA type carbapenemase production in A. baumannii isolates, whereas it was due to MBL production in non-baumannii Acinetobacter isolates. Colistin-resistant isolates were rare, but started to be isolated. Since our past experience shows that emergence and spread of resistant bacteria are inevita- ble, we need concerted multidisciplinary efforts to preserve the activity of currently available antimicrobial agents by following the principles of antimicrobial stewardship.
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Usually, bacteria are shed from the human body in blood, sputum, or other body discharges. The bacteria may adhere to an inanimate surface and remain in liquid droplets for finite periods. Viability increases if bacterial cells are dried in the presence of sugars, blood, serum, or complex bacteriological media, and survival times increase if these dried cells are kept in the dark (28). Therefore, the medium in which the bacteria are suspended (or menstruum) has been found to affect the survival times of bacteria in the environment (16). Evaporation of water in aerosols is essentially instantaneous. Following desiccation, cells may remain suspended in air as components of dust. A pronounced increase in the rate of isolation of A. baumannii from patients and hospital environments during the summer season has been reported (7, 30, 33), but experiments carried out to determine the survival times of Acinetobacter spp. (11, 17, 41) have used only one range of relative humidity. During a 4-year retrospective survey in Hong Kong (33), sea- sonal variation in the isolation of Acinetobacter spp. was ob- served, corresponding to a peak period from July through October (the hot, humid summer season) during which in- creased numbers of Acinetobacter spp. were isolated. Higher relative humidity (RH) during summer seasons may contribute to the prolonged survival and correlate with the peak period of isolations and infections with A. baumannii. Different ranges of RH are found in different parts of the world during different seasons. For example, in Scandinavia, the indoor RH is usually in the range of 10 to 35% during the winter in workplaces, and air humidification is rare (24), while in a country such as Pakistan, the range of RH is usually 80 to 90% during the monsoon season in workplaces, and air-conditioning is rare. These different factors may markedly influence the survival of bacterial cells in the environment.
Acinetobacter nosocomialis strains with inactivated efﬂux constituents were compared to the respective wild-type parent of each species. The A. nosocomialis M2 strain was isolated from a patient in 1996 in Cleveland, Ohio (19). While the activity of sulbactam did not change upon addition of ETX2514 against any isogenic mutant tested com- pared to the parent strain, the addition of ETX2514 led to substantial decreases in sulbactam MICs in A. baumannii devoid of adeB and/or adeJ efﬂux pump genes, suggesting that ETX2514 is a substrate for these pumps (Table 5). These multidrug transporters are components of two out of the three most prevalent efﬂux pumps in A. baumannii, adeABC, adeFGH, and adeIJK (20). adeABC was the ﬁrst resistance- nodulation-division (RND) efﬂux pump system characterized in A. baumannii (21, 22). A study in 2013 found that 10 out of 14 MDR clinical isolates overexpressed the adeABC operon, but not the other efﬂux systems (20). The toxic nature of high levels of adeIJK in multidrug-resistant A. baumannii predicts that overexpression of adeABC would be more prevalent than adeIJK in this species (23). In contrast to A. baumannii, suscepti- bility of A. nosocomialis strains was mostly affected by loss of the adeJ efﬂux pump (Table 5). Further investigation is needed to determine the relative effect on suscepti- bility to sulbactam-ETX2514 in Acinetobacter spp. overexpressing these efﬂux determi- nants.
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Carbapenems (imipenem, meropenem, ertapen- em and doripenem) are broad-spectrum antibiotics as well as the most effective antibiotics of β-lactams that are effective against most microorganisms and often used as the last resort for the treatment of ceph- alosporin-resistant infections (7). In recent years, reports of resistance in A. baumannii isolates to all known antibiotics including carbapenems have in- creased, which have been of great concern in medical communities (4). One of the mechanisms involved in resistance in Acinetobacter spp. is the over expres- sion of efflux pumps. Bacteria uses efflux pumps to excrete extracellular toxic substances (drugs, chem- icals and antibiotics) that penetrate into the cell, which both helps the bacteria to survive in condi- tions of environmental stress and to prevent from formation of an effective antibiotic concentration to act (8). Moreover, efflux pumps give the bacteria the chance to equip itself to other resistance mecha- nisms while helping it to survive in antibiotic stress (9). Efflux pumps are present in all living organisms (prokaryotes and eukaryotes) and keep them away from the toxic effects of organic chemicals. Multi- drug resistance in bacteria is often accompanied by
Various inhibitor-based methods are commonly used to detect MBLs, but these techniques are not highly sensitive or highly spe- cific. A disk potentiation test (DPT) is also called a combined disk test. A DPT using imipenem (IPM) and EDTA (IPM-EDTA DPT) showed a high false-positive rate with Acinetobacter spp. due to growth inhibition by EDTA alone (20). A double-disk potentia- tion test (DDPT) is also known as a double-disk synergy test. To reduce false-positive results from the DDPT, we reduced the EDTA concentration from 1,900 to 760 g and added 2 mg of sodium mercaptoacetic acid (SMA) (11), an inhibitor recom- mended for detecting MBL (1).
animal hospital and rescue centre in central London. This is unlikely to be due to sampling error as both nutrient agar and LAM culturing was done from this clinic. These results from the only clinic in a city location could perhaps reflect the influence of environment on carriage of Acinetobacter spp. None of the isolates in this study carried bla OXA-23-like acquired carbapenemase
To identify Acinetobacter spp., many clinical microbiological laboratories routinely use commercial phenotypic methods, but they are unreliable when clinicians are identifying Acineto- bacter spp. to the species level (9). Therefore, to substitute for phenotypic methods, several molecular methods have been developed for Acinetobacter species identification, including amplified 16S ribosomal DNA restriction analysis (30); ribo- typing (8); randomly amplified polymorphic DNA; the se- quencing of various genes, such as the 16S-23S rRNA gene intergenic spacer (ITS) region (3), the recA gene (16), and the rpoB gene (18); and amplified fragment length polymorphism fingerprinting (14). However, these methods usually are labor- intensive, time-consuming, or of low reproducibility. Further- more, they usually need multiple-tube PCR, thus requiring more DNA.
Efficiency of screening and susceptibility of isolates. Consecutive isolates and CAZ-nonsusceptible isolates of P. aeruginosa and acinetobacters recovered in 2001 at the same hospital were tested by the Hodge test and DDSTs to detect the presence of MBL-producing strains among IPM-susceptible isolates. Routine NCCLS disk diffusion test (11) data for the year 2000 at the hospital were used FIG. 2. Performances of DDSTs with different ␤-lactams and chelating agents. (A) Synergistic inhibition zones tended to be larger with EDTA (1,900 g) disks for P. aeruginosa (a and b), while they tended to be larger with SMA (3 mg) disks for Acinetobacter spp. (e and f). EDTA disks occasionally produced small, suspect synergistic zones with MBL-nonproducing isolates (c and d), but SMA (3 mg) disks alone did not inhibit growth (a to f). (B) The synergistic inhibition zones produced by an IPM disk and an EDTA (750 g) ⫹ SMA (2 mg) disk were large for all MBL-producing isolates (h, l, n, and p), but with an IPM disk and an SMA (3 mg) disk, a small zone was produced for an isolate of P. aeruginosa (g). The MBL-nonproducing isolate which showed an equivocal zone (c) was not inhibited by an SMA (3 mg) or an EDTA ⫹ SMA disk (i and j, respectively). CAZ disks failed to show synergistic zones for an isolate of A. baumannii (k and l). An isolate with VIM ␤-lactamase showed an arrowhead-shaped synergistic zone (q), and an isolate with an IMP enzyme showed an oval synergistic zone (r).
Our results further suggest that carbapenem resistance is an important risk factor for receiving inappropriate em- piric coverage. In other words, the key issue may not only be rapid identification of subjects specifically at risk for Acinetobacter spp., but may in fact be determining whether a patient is suffering from a potentially carbapenem- resistant pathogen. This not only highlights the urgent need for concerted efforts at preventing individual infections and curtailing the development and spread of resistant organisms, but also underscores the needs for novel rapid diagnostic tools. Moreover, these new diagnostics must pro- vide clinicians up front with information about a pathogen’s likely susceptibilities rather than just simply identifying the organism.
PCR/ESI-MS is emerging as a very important method to accurately type A. baumannii and other Acinetobacter spp. (8). PCR/ESI-MS has also been employed to track epidemics of A. baumannii associated with war trauma (31). In a multiclonal collection of Acinetobacter spp., PCR/ESI-MS analysis re- vealed that there was significant genetic diversity among clin- ical isolates (14). The increasing application of PCR/ESI-MS to outbreaks of MDR Acinetobacter spp. is providing important information regarding clonal relatedness, hospital transmission dynamics, correct bacterial species identification, and micro- organism quantification (6, 14, 29). We demonstrate herein for the first time that PCR/ESI-MS can also be used to correctly identify antibiotic resistance genes that impact clinical decision making. Our study also shows for the first time that PCR/ ESI-MS can determine the genetic basis of the quinolone re- sistance phenotype.
Seven laboratories in six European countries examined 40 isolates belonging to the Acinetobacter calcoace- ticus-Acinetobacter baumannii complex to investigate whether standardized protocols and quality-controlled reagents could produce reliable, discriminatory, and reproducible PCR-based fingerprinting results. Four PCR protocols with different primers (primers DAF4, ERIC-2, M13, and REP1 1 REP2) were used. The epidemi- ological conclusions reached by the participating laboratories were substantially correct, with 96.4% of the total isolate grouping allocations agreeing with the consensus view. All laboratories identified the main epidemio- logical clusters, and each laboratory also identified two non-outbreak-related isolates. There were no signifi- cant differences between the isolate grouping results obtained by the different protocols and with the different primers. Visual comparison indicated that the standardized protocols and reagents yielded reproducible fingerprint patterns, but with some variations in particular band intensities. Minor variations in fingerprint profiles were detected, but computer-assisted analysis of PCR fingerprints obtained on agarose gels demon- strated that 88.3 to 91.6% (depending on the source of DNA) of the patterns clustered correctly, while 96.4 to 98.9% of the patterns clustered correctly following automated high-resolution laser fluorescence analysis. Correlation of the patterns for isogenic isolates ranged from 83.3 to 86.6% but was slightly better (mean correlation, 87.1%) for centrally prepared DNA extracts than for DNA extracts prepared by individual laboratories (mean correlation, 84.7%). It was concluded that independently produced PCR fingerprint pat- terns can be obtained reproducibly for Acinetobacter spp. at the practical level if (i) quality-controlled reagents, (ii) standardized extraction of DNA, and (iii) standardized amplification conditions are used.
Initial inappropriate antibiotic treatment was adminis- tered to 88 % of patients with Acinetobacter spp. and 51 % of patients with other pathogens. More patients with Acinetobacter spp. developed septic shock (81 % vs 52 %); needed mechanical ventilation within 24 h of the diagnosis of bacteremia (88 % vs 66 %); and required a central venous line (97 % vs 85 %). Patients with Acineto- bacter spp. bacteremia had a higher mortality when compared with bacteremia by the other pathogens (73 % vs 50 %). The mean Pitt Bacteremia Score for Acineto- bacter spp. was 7 (SD: 4) and for other pathogens was 4 (SD: 3). The mean number of organ failures for Acineto- bacter spp. was 2.1 (SD: 1.2) and for other pathogens was 1.67 (SD: 1.3). The cumulative survival curves of the patients according to pathogen are shown in Figs. 1 and 2. The bivariate analysis (Table 2) showed that age >60 years, Acinetobacter spp. infection, and diabetes mellitus were significantly associated with a poor prognosis. The following variables also presented p < 0.10 in the bivariate analysis: sex; liver cirrhosis; ob- structive pulmonary disease, and IIAT. The variables: number of organ failures; septic shock; Pitt Bacteremia Score (which evaluates the severity of the bacteremia), mechanical ventilation and use of central venous line were excluded from the bivariate and multivariate analyses because they were considered intrinsically correlated with the event death and not proper prognostic factors. We verified that these factors, excluded from the multivariate analysis, were statistically associated with the outcome, except for use of central venous line (data not shown). Most patients with diabetes mellitus were older than
The centre at which this study was conducted in Southern India is an area which is endemic for Scrub typhus and has a large proportion of patients presenting with severe scrub typhus related ARDS and other major organ involvement (hepatitis, acute kidney injury, myocarditis etc.) especially during the rainy season. These patients often require prolonged intensive care with mechanical ventilator support until they recover organ functions, but overall have a good prognosis and chance for a full recovery. Pneumonias constituted the other major infection in the Acinetobacter cohort. The common factor in both these diseases is the presence of lung injury. In both Scrub typhus and severe pneumonia there is a degree of lung damage that occurs. In addition, there is a severe systemic inflammation with ongoing immune system activation. In this setting, it can be hypothesised that the underlying lung damage and the ongoing inflammatory response could make these critically ill patients more vulnerable for an Acinetobacter VAP. The current study was not powered to answer this question, but it needs to be addressed in future studies on Acinetobacter associated VAP’s.
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Verigene BC-GN testing. BC-GN was performed on all qualifying blood cultures according to the manufacturer’s instructions. This assay uses a microarray format with probes that detect the (i) rpsA, ompA, and mrkC, (ii) gyrB and metB, and (iii) atpD gene targets for genus-level de- tection of Acinetobacter spp., Citrobacter spp., Enterobacter spp., and Pro- teus spp., respectively. Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, and Serratia marcescens are identified to the species level using (i) oppA, (ii) yggE and dhaM, (iii) ompA, (iv) sodA, and (v) chiC and ampC probes, respectively. Additional probes detect bla CTX-M , bla KPC , bla NDM , bla VIM , bla IMP , and bla OXA resistance determi-
Materials and Methods: In this study, 240 samples were randomly collected from different parts of accidents and burn hospital before and after disinfection. The samples were cultured on blood agar and Eusion-Metilen-Blue agar media in the Microbiology Laboratory of Medicine School of Shahid Sadoughi University in Yazd and Colony counting were determined. Identification was done by biochemical tests after incubation at 37° C for 48 hours. The studied disinfectants were Deconex 50AF, Descoscid, Epimax SC, and Silvosept. At last, data were analyzed with using paired t-test . Results: The Gram-negative bacteria were isolated from burn unit before disinfection included Pseudomonas aeruginosa, Escherichia coli, Proteus spp., Klebsiella spp., Acinetobacter spp., and Enterobacter spp. According to the results, all disinfectants reduced the pollution before and after disinfection; nevertheless, this reduction at the time of using Epimax SC and Silvosept only showed a significant difference for P. aeruginosa (P = 0.001 and 0.003) and for E. coli (P = 0.020 and 0.005), respectively.
Unique haplotypes were defined by using DnaSPv5.10 to obtain head lice cytb sequences, then, compared and combined (Additional file 1: Table S1), with the cytb hap- lotypes previously reported . In order to investigate the possible relationships between the haplotypes, the median-joining (MJ) network using the method of Ban- delt was constructed with the program NETWORK4.6 (http://www.fluxu s-engin eerin g.com/share net.htm) using equal weights for all mutations . Phylogenetic analyses and tree reconstruction were performed using MEGA software v.6.06 . Phylogenetic analysis was performed using maximum likelihood (ML) approach. To generate the best ML tree, Modeltest v.3.7  was used to examine model of nucleotide substitution and choose a best-fit model of sequence evolution. The model that provides the best approximation of the data using the fewest parameters was chosen for the analysis according to the Akaike information criterion [34, 35]. Tree recon- struction was conducted using MEGA software v.6.06 under HKY+I+G model with 500 bootstrap replicates. All obtained sequences of Acinetobacter spp. were ana- lyzed using BLAST (www.ncbi.nlm.nih.gov/blast /Blast .cgi) and compared with sequences in the GenBank data- base. A maximum-likelihood method was used to infer the phylogenetic analyses and the best-fit model was chosen as described for cytb sequences above. The tree reconstruction was performed using the TrN+ G model for nucleotide sequences under 500 bootstrap replicates in MEGA software v.6.06 .
Staphylococcus aureus (27.7%) and Bacillus spp. (22.2%) were the predominant isolates in the study. In the studies Enemuor et al.  and Hassan et al.  Staphylococcus spp. was also identified as the predominant isolate from all salons investigated. Similarly, Naz et al.  also reported the presence of Staphylococcus in unpreserved beauty salon tools after use. Staphylococcus spp. are able to cause various diseases in humans such as skin abscess, impetigo contagiosa, scaleded-skin syndrome, and it is the most commonly identified agent that is responsible for skin and soft tissue infection [4,22].
Due to unhygienic nature of the abattoir, faecal contaminations are likely to occur with the meat. Gram negative and gram positive bacteria such as E.coli, Staphylococcus spp, Klebsiella spp, Streptococcus spp, Salmonella spp, Clostridium spp, Bacillus spp, and Pseudomonas spp were found in abattoir site and some of these are food borne pathogens such as botulism, dysentery, gastroenteritis and typhoid (Fraze and Westhoff, 2004).
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