Comparison of Three Expanded-Spectrum Cephalosporin Hydrolysis Assays and the NG-Test CTX-M Multi Assay That Detects All CTX-M-Like Enzymes

Rapid detection of expanded-spectrum cephalosporins (ESC) hydrolysing enzymes is crucial to implement infection control measures and antibiotic stewardship. Here, we have evaluated three biochemical ESC hydrolysis assays (ESBL NDP test, β-LACTA™ test, LFIA-CTX assay) and the NG-Test® CTX-M MULTI that detects CTX-M enzymes, on 93 well-characterized Gram-negative isolates, including 60 Enterobacterales, 21 Pseudomonas spp. and 12 Acinetobacter spp. The performances were good for all three hydrolysis assays, with the LFIA-CTX being slightly more sensitive and specific on the tested panel of isolates especially with Enterobacterales, without ambiguous results. This study showed that LFIA-CTX may be used for the detection of ESC hydrolysis as a competitive alternative to already available assays (β-LACTA™ test and ESBL NDP test) without any specific equipment and reduced hands-on-time. The lateral flow immunoassay NG-Test® CTX-M MULTI has proven to be a useful, easy, rapid, and reliable confirmatory test in Enterobacterales for detection of CTX-M-type ESBLs, which account for most of the resistance mechanisms leading to ESC resistance in Enterobacterales, but it misses rare ESC hydrolysing β-lactamases (AmpC, minor ESBLs, and carbapenemases). Combining it with the LFIA-CTX assay would yield an assay detecting the most frequently-encountered ESBLs (CTX-M-like β-lactamases) together with ESC hydrolysis.

Multiplex Lateral Flow Immunoassay for the Detection of Expanded-Spectrum Hydrolysis and CTX-M Enzymes

Background: Early detection of expanded-spectrum cephalosporinase (ESC) hydrolyzing ß-lactamases is essential for antibiotic stewardship. Here we have developed a multiplex lateral flow immunoassay (LFIA) that detects cefotaxime-hydrolyzing activity as well as the most prevalent ESC-hydrolyzing ß-lactamases: the CTX-M-like. Methods: The Rapid LFIA ESC test was evaluated retrospectively on 188 (139 Enterobacterales, 30 Pseudomonas spp. and 14 Acinetobacter spp.) agar-grown bacterial isolates with well-characterized ß-lactamase content. One single colony was resuspended in 150 µL extraction buffer containing cefotaxime, incubated at room temperature for 30 min prior to loading on the LFIA for reading within 10 min. Results: Out of the 188 isolates, all 17 that did not express a β-lactamase hydrolyzing cefotaxime gave negative results, and all 171 isolates expressing a β-lactamase known to hydrolyze cefotaxime, gave a positive test result. In addition, all 86 isolates expressing a CTX-M-variant belonging to one of the five CTX-M-subgroups were correctly identified. The sensitivity and specificity was 100% for both tests. Conclusions: The results showed that the multiplex LFIA was efficient, fast, low cost and easy to implement in routine laboratory work for the confirmation of ESC hydrolyzing activity and the presence of CTX-M enzymes.

Predominance of Acinetobacter spp., Harboring the blaIMP Gene, Contaminating the Hospital Environment in a Tertiary Hospital in Mwanza, Tanzania: A Cross-Sectional Laboratory-Based Study

Data on colonization and hospital contamination of carbapenem-resistant Gram-negative bacteria (CR-GNB) are limited in low- and middle-income countries. We designed this study to determine the prevalence and co-existence of carbapenemase genes among CR-GNB isolated from clinical, colonization, and hospital environmental samples at a tertiary hospital in Mwanza, Tanzania. The modified Hodge test (MHT), the combined disk test (CDT), and the double-disk synergy test (DDST) were used for the phenotypic detection of carbapenemases. A multiplex PCR assay was used to detect blaIMP and blaKPC, and a singleplex PCR assay was used to detect blaOXA-48. Data were analyzed by STATA version 13.0. Overall, 68.8% (44/64) of the CR-GNB had at least one phenotype by phenotypic methods, whereby 60.9% (39/64) were both CDT and DDST positive and 31.3% (20/64) were MHT positive. A total of 23/64 (35.9%) had at least one of the genes tested with the predominance of blaIMP (91.3%; 21/23). In addition, 47.7% (21/44) of the CR-GNB phenotypes had at least one gene. Around 47.8% (11/23) of the CR-GNB carried multiple genes encoding for carbapenem resistance, with the maximum co-existence of blaIMP/blaKPC/blaOXA-48 (45.5%; 5/11). The majority of carbapenem-resistant genes were detected in Acinetobacter spp. (82.6%; 19/23) and isolated from bed swabs (69.6%; 16/23). Acinetobacter spp. carrying the blaIMP gene predominantly contaminated the hospital environment. Therefore, we recommend routine decontamination of inanimate hospital surfaces, including patient beds.

Drug Utilization Evaluation of Colistin: A Retrospective Study

Background: Colistin, is used as the last treatment line for infections concluded from multiple drug-resistant gram-negative microorganisms. Increased consumption of colistin leads to resistance to this antibiotic in many countries. This study investigated the usage pattern of colistin administration in a selected hospital in Iran. Methods: This study was conducted in a selected hospital in Ahvaz. Inclusion criteria were all patients who received colistin during this time according to the health information system. Patients who were received less than three doses of colistin were excluded from the study. Prescription of colistin in all patients was evaluated according to the protocol extracted from the last version of Lexicomp written by Wolters Kluwer. The descriptive and analytical statistics were carried out by the R software. Results: Among 27 patients who received colistin, pneumonia (30%) was the main diagnoses. Colistin administration was based on the microbiological culture data in 70% of cases. Considering the involved microorganism, most cases were Acinetobacter spp., followed by Klebsiella spp. Loading dose was prescribed for seven (26%) patients. In only five (19%) cases, colistin dosing, including loading dose, maintenance dose, and the interval of colistin administration, was appropriate during the study time. Increasing in serum creatinine was seen in two (7.4%) patients. In 29.4% of patients, the combination of colistin and carbapenems was observed. Conclusion: Given the lack of appropriate dose adjustment of colistin that may lead to incidence of resistance and adverse effect, applying of the specialist clinical pharmacist will be suggested.

Resistência a carbapenêmicos por Acinetobacter spp. reportados ao GLASS de acordo com o nível de renda dos países

Este trabalho tem por objetivo avaliar a resistência a carbapenêmicos da Acinetobacter spp. reportada ao GLASS (Global Antimicrobial Resistance and Use Surveillance System), estratificando esses dados por faixa de renda per capita. Foi conduzido um estudo observacional retrospectivo sobre a sensibilidade a carbapenêmicos da Acinetobacter spp., reportada ao GLASS, fora incluído todos os dados disponíveis no momento da coleta, fevereiro de 2021, estratificando esses achados pelo nível de renda de cada país e a quantidade de testes de sensibilidade a carbapenêmicos realizados. Os países de maior renda tiveram a menor não-suscetibilidade relatada, contudo dentro deles existem exemplos que apresentam uma alta incidência de não-suscetibilidade. Para todos os quatro grupos de renda existiu uma alta variância do desvio padrão para a média dos testes de sensibilidade a carbapenêmicos, assim revelando pontos de inconsistência. Os dados trabalhados neste artigo mostram um nível preocupante de resistência a carbapenêmicos observados no GLASS. Dentro mesmo dos países ricos há aqueles que possuem níveis alarmantes de não-susceptibilidade e, para os países mais pobres, destaca-se uma escassez de dados mais completos, pois naqueles com os níveis mais preocupantes de não-susceptibilidade já são encontradas cepas de Acinetobacter spp. com fenótipo de multirresistência.

Disposal habits and microbial load of solid medical waste in sub-district healthcare facilities and households in Yilo-Krobo municipality, Ghana

The study aimed to assess disposal practices and quantify the microbial load present in SMW from ten sub-district level healthcare facilities and 385 households in Yilo Krobo municipality, Ghana. Disposal of solid medical waste (SMW) was assessed by questionnaire-based surveys, unstructured interviews and field observations. Microbiological analysis identified species and counts of bacteria present in SMW from both sources. Sociodemographic factors influencing the method of SMW disposal in households were evaluated using logistic regression analysis, with statistical significance set at p<0.05. Open burning (29%), burying (25%) and disposal at a dumpsite (49%) were common methods used by households to discard SMW. SMW disposal at a dumpsite was associated with age of respondents in households. Older people (50+ years) were three times more likely to place SMW in household waste later discarded at a dumpsite, compared to younger persons (20–30 years) [a0R, 95%CI = 3.37, 1.41–8.02]. In sub-district level healthcare facilities, open burning and burying were the most common methods used. Bacillus subtilis, Klebsiella pneumonia, Pseudomonas aeruginosa, Clostridium tetani, Enterococcus faecalis, Acinetobacter spp. Escherichia coli, Bacillus cereus and Enterococcus faecium) were bacteria identified in SMW recovered from both the healthcare facilities and the households. Klebsiella pneumoniae, Acinetobacter spp. and Clostridium tetani were found exclusively in untreated SMW generated in the healthcare facilities. Bacillus spp. and Pseudomonas spp. were found in one sample of treated SMW. The microbial load in SMW from healthcare facilities and households ranged from 0.036 x 103cfc/mg to 0.167 x 103 cfc/mg and from 0.118 x 103cfc/mg to 0.125 x 103cfc/mg respectively. This highlights the need for institutionalizing appropriate treatment methods in sub-district level facilities or strengthening the linkages with higher level facilities to ensure regular and adequate treatment of SMW. Public guidance on management of SMW generated in households which is context specific should also be provided.

Biogenesis of Lipoproteins in Gram-Negative Bacteria: 50 Years of Progress

The outer membrane is the defining characteristic of Gram-negative bacteria and is crucial for the maintenance of cellular integrity. Lipoproteins are an essential component of this outer membrane and regulate broad cellular functions ranging from efflux, cellular physiology, antibiotic resistance, and pathogenicity. In the canonical model of lipoprotein biogenesis, lipoprotein precursors are first synthesized in the cytoplasm prior to extensive modifications by the consecutive action of three key enzymes: diacylglyceryl transferase (Lgt), lipoprotein signal peptidase A (LspA), and apolipoprotein N-acyltransferase (Lnt). This enzymatic process modifies lipoprotein precursors for subsequent trafficking by the Lol pathway. The function of these three enzymes were originally thought to be essential, however, in some Gram-negative bacteria, namely Acinetobacter baylyi, the third enzyme Lnt is dispensable. Here we review the function and significance of Lgt, LspA, and Lnt in outer membrane biogenesis and how non-canonical models of lipoprotein processing in Acinetobacter spp. can enhance our understanding of lipoprotein modifications and trafficking.

Antimicrobial and Anti-Biofilm Activity of Polymyxin E Alone and in Combination with Probiotic Strains of Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 against Clinical Isolates of Selected Acinetobacter spp.: A Preliminary Study

Acinetobacter spp., the nosocomial pathogen, forms strong biofilms and is resistant to numerous antibiotics, causing persistent infections. This study investigates the antibacterial and anti-biofilm activity of polymyxin E alone and in combination with the cell-free supernatants (CFS) of the tested probiotic bacilli, Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 against the selected Acinetobacter spp. starins. Three isolates of Acinetobacter spp., designated as Acinetobacter spp. isolate 1; Acinetobacter spp. isolate 2, and Acinetobacter spp. isolate 3, were collected from patients with burns, wounds, and blood infections, respectively. Bacterial identification and antibiotic susceptibility testing were conducted using the VITEK2 system. Auto-aggregation and coaggregation of the tested bacilli strains with the selected Acinetobacter spp. isolates were evaluated. A disk diffusion assay was used to identify the microorganism’s susceptibility to the selected antibiotics, alone and in combination with the CFS of the bacilli. The MIC and MBIC (minimum inhibitory and minimum biofilm inhibitory concentrations) of polymyxin E combined with bacilli CFS were determined. Acinetobacter spp. isolates were (i) sensitive to polymyxin E, (ii) able to form a strong biofilm, and (iii) resistant to the tested antibiotics and the CFS of tested bacilli. Significant inhibition of biofilm formation was noticed when CFS of the tested bacilli were combined with polymyxin E. The bacilli CFS showed synergy with polymyxin E against planktonic cells and biofilms of the isolated pathogens.

Central line-associated bloodstream infection trend in Brazilian adult intensive care units: an ecological study

Introduction: Central line-associated bloodstream infections are the second most frequent infection in intensive care units. It represents an adverse event of significant magnitude, thus threatening the patient safety. The aim of this study was to analyze the historical trend of central line-associated bloodstream infections in patients in intensive care units, the rate of infection, central venous catheter utilization ratio, type of pathogen and their antimicrobial resistance pattern. Methodology: This ecological study was performed at 42 intensive care units from a state capital of the Midwest region of Brazil. Central line-associated bloodstream infections notifications were collected from two databases, the Municipal Coordination for Patient Safety and Infection Control at Healthcare Services, from 2012-2016, and the FormSUS (National Health System Data Processing Company), from 2014-2016. Results: The incidence of central line-associated bloodstream infections was high and stationary in the period (incidence rate of 2.3 to 3.2 per 1,000 catheter days, central venous catheter utilization ratio average 56,9%). The most frequent microorganisms were coagulase-negative Staphylococcus, Klebsiella pneumoniae, Acinetobacter spp. and Pseudomonas aeruginosa. Resistance to 3rd and 4th generation cephalosporins and carbapenems were detected among Gram-negative bacteria, and resistance to oxacillin among Gram-positive bacteria. Conclusions: Central line-associated bloodstream infections incidence rates were high, however the historical trend remained stationary in adult intensive care units. Infections were mostly caused by coagulase-negative Staphylococcus, K. pneumoniae, Acinetobacter spp. and Pseudomonas aeruginosa, including multi-drug resistant organisms. These findings point to the need of educational strategies addressing the adherence to established preventive measures and to the rational use of antimicrobials.

Biofilm Formation and blaOXA Genes Detection Among Acinetobacter baumannii from Clinical Isolates in a Tertiary Care Kirtipur Hospital, Nepal

(1) Background: Acinetobacter baumannii has emerged as a leading cause of nosocomial infections as they are capable of evolving resistance to various classes of antibiotics. The ability of A. baumannii to form biofilm might also be associated with increased antibiotic resistance and hence treatment failure. This study was carried to associate the biofilm formation with the drug resistance pattern of A. baumannii and to detect blaOXA-23, blaOXA-24, and blaOXA-51 from carbapenem resistance isolates. (2) Methods: Among different clinical samples, a total of 19 Acinetobacter spp. were identified with conventional microbiological procedures. The biofilm production was determined by a quantitative adherence assay. The antimicrobial susceptibility test was carried out by the Kirby-Bauer disc diffusion method, carbapenemase production detection was confirmed by Modified Hodge Test. And target resistant genes were detected by polymerase chain reaction. (3) Results: Out of 90 clinical specimens, 64.44% (58/90) showed bacterial growth. Whereas, 32.75% (19/58) isolates were identified as A. baumannii. Among all A. baumannii isolates, 84.21% (16/19) were multidrug-resistance and 63.16% (12/19) carbapenem resistance phenotypically. blaOXA-51 was detected in all the isolates and blaOXA-23 was detected only in 63.16% (12/19) isolates. However, blaOXA-24 was not detected in any of the isolates. Among A. baumannii, 89.47% (17/19) isolates produced biofilm with 47.37% (9/19) strong biofilm producers. (4) Conclusions: In the majority of MDR A. baumannii, blaOXA-51 and blaOXA-23 were detected as the determinant factor for carbapenem resistance having a direct relation with biofilm formation. This study provided a valuable clue for the management of A. baumannii infections in clinical settings.