Characterization of biofilm producing methicillin resistant coagulase negative Staphylococci from India

Methicillin-resistant coagulase-negative staphylococci (MR-CoNS) cause infectious diseases due to their potential to form biofilm and further colonization in hospital materials. This study evaluated the antibiotic susceptible phenotypes, biofilm-producing ability, and biofilm-associated genes (mecA, icaAD, bap, cna, and fnbA). Biofilm formation was detected through Congo red agar (CRA) method and MTP method. The presence of biofilm and associated genes in MR-CoNS were detected by PCR. A total of 310 (55.95%) isolates produced the biofilm. Among these isolates, Staphylococcus haemolyticus (34.83%), Staphylococcus epidermis (31.93%), Staphylococcus capitis (16.77%), Staphylococcus cohnii (10.96%), and Staphylococcus hominis (5.48%) were identified. The antimicrobial susceptibility pattern of CoNS isolates indicated resistance to cefoxitin (100%), erythromycin (94.8%), ciprofloxacin (66.7%), sulfamethoxazole/trimethoprim (66.7%), gentamicin (66.12%), and clindamycin (62.9%). Resistance rate to mupirocin was 48.5% in S. epidermidis and 38.9% in S. haemolyticus isolates. All isolates were sensitive to vancomycin and linezolid. The prevalence rates of icaAD, bap, fnbA, and cna were 18.06%, 12.5%, 47.4%, and 27.4%, respectively. icaAD and bap genes were detected in 18.06% and 12.5% of MR-CoNS isolates. fnbA and cna genes were detected in 47.41% and 27.41% of MRCoNS isolates. icaAD positive strains exhibited a significant increase in the biofilm formation compared with those that lacked icaAD (0.86 (0.42, 1.39) versus 0.36 (0.14, 0.75), respectively; P < 0.001). In conclusion, the majority of MR-CoNS isolates were biofilm producers, and S. capitis, which possessed icaAD genes, ranked as the great biofilm producer than other Staphylococcus. The study’s findings are important to form a strategy to control biofilm formation as an alternative strategy to counter the spread of MR-CoNS in healthcare settings.

Multidrug resistant coagulase-negative Staphylococcus spp. isolated from cases of chronic rhinosinusitis in humans. Study from Poland

For many years, coagulase-negative staphylococci (CoNS) have been considered non-pathogenic bacteria. However, recently, CoNS are becoming more common bacteriological factors isolated from cases of chronic rhinosinusitis in humans. Moreover, most of them represent the multidrug-resistant or/and methicillin-resistant profile, which significantly increases the therapeutic difficulties. The aim of the study was to characterize profile of resistant coagulase-negative staphylococci isolated from cases of chronic rhinosinusitis in patients treated in a Medical Center in Warsaw in 2015–2016. The study material was derived from patients with diagnosed chronic rhinosinusitis treated at the MML Medical Center in Warsaw. The material was obtained intraoperatively from maxillary, frontal, and ethmoid sinuses. In total, 1,044 strains were isolated from the studied material. Coagulase-negative staphylococci were predominant, with the largest share of Staphylococcus epidermidis. Isolated CoNS were mainly resistant to macrolide, lincosamide, and tetracycline. Among the S. epidermidis strains, we also showed 35.6% of MDR and 34.7% of methicillin-resistant strains. The same values for other non-epidermidis species were 31.5% and 18.5%, respectively and the percentage of strains with MAR >0.2 was greater in S. epidermidis (32.6%) than S. non-epidermidis (23.9%). Although the percentage of strains resistant to tigecycline, glycopeptides, rifampicin and oxazolidinones was very small (2.3%, 1.9%, 1.4% and 0.7% respectively), single strains were reported in both groups. The study has shown a high proportion of MDR and methicillin-resistant CoNS strains, which indicates a large share of drug-resistant microorganisms in the process of persistence of chronic rhinosinusitis; therefore, isolation of this group of microorganisms from clinical cases using aseptic techniques should not be neglected.

Emergence of CC8/ST239- SCCmec III/t421 tigecycline resistant and CC/ST22-SCCmec IV/t790 vancomycin resistant Staphylococcus aureus strains isolated from wound: A two-year multi-center study in Tehran, Iran

Staphylococcus aureus as an opportunistic bacterial pathogen with intrinsic and acquired resistance to many antibiotics is a worldwide problem. The current study was undertaken to evaluate the resistance pattern, and determine the genetic types of multidrug-resistant S. aureus isolated from wound. This cross-sectional study was conducted over the period of two years (from December 2018 to November 2020) at the hospitals affiliated to Shahid Beheshti University of Medical Sciences, Tehran, Iran. In present study, 75 multidrug-resistant S. aureus isolates collected from wound infections were investigated. Phenotypic resistance was assessed by Kirby–Bauer disk diffusion method. Conventional PCR was performed for the detection of virulence encoding genes. Genotyping of strains was performed based on coa gene polymorphism using multiplex-PCR assay. SCCmec typing, spa typing and MLST were also used to characterize the genotype of the mupirocin, tigecycline and vancomycin resistant multidrug-resistant S. aureus isolates. All 75 multidrug-resistant S. aureus isolates in the study were confirmed as MRSA. Coagulase typing distinguished isolates into five genotypic patterns including III (40%), I (24%), IVb (16%), V (10.7%) and type X (9.3%). Resistance to tigecycline was detected in 4% of MDR-MRSA isolates and all belonged to CC8/ST239- SCCmec III/t421 lineage. According to our analysis, one VRSA strain was identified that belonged to coa type V and CC/ST22-SCCmec IV/t790 lineage. Resistance to mupirocin was detected in 9.3% of strains. All 7 mupirocin resistant MDR-MRSA isolates exhibited resistance to mupirocin in high level. Of these, 4 isolates belonged to CC/ST8-SCCmec IV/t008 (57.1%), 2 isolates belonged to CC/ST8-SCCmec IV/t064 (28.6%) and one isolate to CC/ST22-SCCmec IV/t790 (14.3%). Altogether, current survey provides a snapshot of the characteristics of S. aureus strains isolated from patients. Our observations highlighted type III as predominant coa type among multidrug-resistant MDR strains indicating low heterogeneity of these isolates. Our study also indicates the importance of continuous monitoring of the genotypes of MDR-MRSA isolates to prevent nosocomial outbreaks and the spread of MDR isolates.

Common points of therapeutic intervention in COVID-19 and in allogeneic hematopoietic stem cell transplantation associated severe cytokine release syndrome

Allogeneic hematopoietic stem cell transplantation (HSCT) and coronavirus disease 2019 (COVID-19) infection can both lead to severe cytokine release syndrome (sCRS) resulting in critical illness and death. In this single institution, preliminary comparative case-series study we compared clinical and laboratory co-variates as well as response to tocilizumab (TCZ)-based therapy of 15 allogeneic-HSCT- and 17 COVID-19-associated sCRS patients. Reaction to a TCZ plus posttransplant cyclophosphamide (PTCY) consolidation therapy in the allogeneic-HSCT-associated sCRS group yielded significantly inferior long-term outcome as compared to TCZ-based therapy in the COVID-19-associated group (P = 0.003). We report that a TCZ followed by consolidation therapy with a Janus kinase/signal transducer and activator of transcription (JAK/STAT) inhibitor given to 4 out of 8 critically ill COVID-19 patients resulted in their complete recovery. Non-selective JAK/STAT inhibitors influencing the action of several cytokines exhibit a broader effect than TCZ alone in calming down sCRS. Serum levels of cytokines and chemokines show similar changes in allogeneic-HSCT- and COVID-19-associated sCRS with marked elevation of interleukin-6 (IL-6), regulated upon activation normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1) and interferon γ-induced protein 10 kDa (IP-10) levels. In addition, levels of IL-5, IL-10, IL-15 were also elevated in allogeneic-HSCT-associated sCRS. Our multi-cytokine expression data indicate that the pathophysiology of allogeneic-HSCT and COVID-19-associated sCRS are similar therefore the same clinical grading system and TCZ-based treatment approaches can be applied. TCZ with JAK/STAT inhibitor consolidation therapy might be highly effective in COVID-19 sCRS patients.

Ceftazidime/avibactam resistance is associated with different mechanisms in KPC-producing Klebsiella pneumoniae strains

This study focused on Klebsiella pneumoniae isolates that were resistant or had low susceptibility to a combination of ceftazidime/avibactam. We aimed to investigate the mechanisms underlying this resistance. A total of 24 multi-drug resistant isolates of K. pneumoniae were included in the study. The phenotypic determination of carbapenemase presence was based on the CARBA NP test. NG-Test CARBA 5 was also performed, and it showed KPC production in 22 out 24 strains. The molecular characterisation of blaKPC carbapenemase gene, ESBL genes (blaCTX-M, blaTEM, and blaSHV) and porin genes ompK35/36 was performed using the PCR. Finally, ILLUMINA sequencing was performed to determine the presence of genetic mutations.Various types of mutations in the KPC sequence, leading to ceftazidime/avibactam resistance, were detected in the analysed resistant strains. We observed that KPC-31 harboured the D179Y mutation, the deletion of the amino acids 167–168, and the mutation of T243M associated with ceftazidime/avibactam resistance. The isolates that did not present carbapenemase alterations were found to have other mechanisms such as mutations in the porins. The mutations both on the KPC-3 enzyme and in the porins confirmed, that diverse mechanisms confer resistance to ceftazidime/avibactam in K. pneumoniae.

Detection of extensively drug-resistant and hypervirulent Klebsiella pneumoniae ST15, ST147, ST377 and ST442 in Iran

In this study, we focused on the emergence of extensively drug-resistant (XDR), pandrug-resistant (PDR), and hypervirulent Klebsiella pneumoniae (hvKP) in Iran. During 2018 to 2020 a total of 52 K. pneumoniae isolates were collected from different clinical specimens. The hvKP isolates were identified by PCR amplification of virulence and capsular serotype-specific genes. Hypermucoviscous K. pneumoniae (hmKP) were identified by string test. Carbapenem-resistant hvKP (CR-hvKP), multidrug-resistant hvKP (MDR-hvKP), extensively drug-resistant hvKP (XDR-hvKP), and pandrug-resistant hvKP (PDR-hvKP) were determined by disc diffusion method, Carba-NP test and PCR method. XDR-hvKP isolates were typed by multilocus sequence typing (MLST). Among all K. pneumoniae isolates 14 (26.9%) were identified as hvKP and 78.6% (11/14) of them were hmKP however, none of the classic K. pneumoniae (cKP) isolates were hmKP. The predominant capsular serotype of hvKP was K2 (42.85%) followed by K1 (35.71%). The prevalence of MDR-hvKP, XDR-hvKP and PDR-hvKP isolates were 6 (42.9%), 5 (35.7%) and 1 (7.1%), respectively. ESBL production was found in 85.7% of hvKP isolates and most of them carried bla TEM gene (78.6%) and 6 isolates (42.9%) were CR-hvKP. Among hvKP isolates, 1 (7.1%), 2 (14.3%), 3 (21.4%), 8 (28.6%), and 11 (78.6%) carried bla NDM-6, bla OXA-48, bla CTX-M, bla SHV, and bla TEM genes, respectively. According to MLST analysis, 2, 1, 1, and 1 XDR-hvKP isolates belonged to ST15, ST377, ST442, and ST147, respectively. The occurrence of such isolates is deeply concerning due to the combination of hypervirulence and extensively drug-resistance or pandrug-resistance.

Prevalence of trimethoprim/sulfamethoxazole resistance genes among Stenotrophomonas maltophilia clinical isolates in Egypt

Stenotrophomonas maltophilia is an important multidrug resistant nosocomial pathogen. Trimethoprim/sulfamethoxazole (TMP/SMX) is considered the drug of choice for treatment of S. maltophilia infections, thus emerging resistance to TMP/SMX poses a serious threat. In the present study we aimed to investigate the frequency of TMP/SMX resistance genes (sul1, sul2, dfrA), and to evaluate their relatedness with integron 1 (int1), and insertion sequence common regions (ISCR) among 100 S. maltophilia from different clinical isolates in Egypt. Isolates were identified biochemically and confirmed by VITEK2. Detection of sul1, sul2, and dfrA genes, int1 and ISCR elements was performed by PCR. Among the 16 TMP/SMX resistant isolates, sul1 gene was detected in all of them, and it was associated with int1 gene presence in all resistant isolates. The sul2 gene was detected in 6 out of 16 resistant isolates (37.5%), and only 2 of the 16 resistant isolates (12.5%) harboured dfrA gene. ISCR was detected in 10 of the resistant isolates (62.5%) and in 4 of them it was associated with the presence of sul2 gene. Among the 84 TMP/SMX sensitive isolates, sul1 gene was detected in 15 (17.8%), int1 in 16 (19%) and ISCR in 6 (7.1%). None of the susceptible isolates had sul2 or dfrA genes. These findings point out an increasing frequency of TMP/SMX resistance genes among S. maltophilia clinical isolates in our region, so the adoption of prudent use of S. maltophilia antimicrobial agents and the establishment of a surveillance system are desperately needed.

Association between biofilm-production and antibiotic resistance in Escherichia coli isolates: A laboratory-based case study and a literature review

Bacteria can enhance their survival by attaching to inanimate surfaces or tissues, and presenting as multicellular communities encased in a protective extracellular matrix called biofilm. There has been pronounced interest in assessing the relationship between the antibiotic resistant phenotype and biofilm-production in clinically-relevant pathogens. The aim of the present paper was to provide additional experimental results on the topic, testing the biofilm-forming capacity of Escherichia coli isolates using in vitro methods in the context of their antibiotic resistance in the form of a laboratory case study, in addition to provide a comprehensive review of the subject. In our case study, a total of two hundred and fifty (n = 250) E. coli isolates, originating from either clean-catch urine samples (n = 125) or invasive samples (n = 125) were included. The colony morphology of isolates were recorded after 24h, while antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method. Biofilm-formation of the isolates was assessed with the crystal violet tube-adherence method. Altogether 57 isolates (22.8%) isolates were multidrug resistant (MDR), 89 isolates (35.6%) produced large colonies (>3 mm), mucoid variant colonies were produced in 131 cases (52.4%), and 108 (43.2%) were positive for biofilm formation. Biofilm-producers were less common among isolates resistant to third-generation cephalosporins and trimethoprim-sulfamethoxazole (P = 0.043 and P = 0.023, respectively). Biofilms facilitate a protective growth strategy in bacteria, ensuring safety against environmental stressors, components of the immune system and noxious chemical agents. Being an integral part of bacterial physiology, biofilm-formation is interdependent with the expression of other virulence factors (especially adhesins) and quorum sensing signal molecules. More research is required to allow for the full understanding of the interplay between the MDR phenotype and biofilm-production, which will facilitate the development of novel therapeutic strategies.

Alpha variant (B.1.1.7) of SARS-CoV-2 increases fatality-rate for patients under age of 70 years and hospitalization risk overall

The emergence of new SARS-CoV-2 variants is a challenge to the control of this pandemic. It is therefore important to collect and to analyze data related to the infection caused by different variants. We have obtained more than 3,700 COVID-19 patients between April 2020 and March 2021 from Tokat, Turkey (roughly 3,100 outpatients and close to 600 inpatients) where about 30% were infected with Alpha variant (B.1.1.7). Descriptive statistics was used to characterize different subgroups. Both logistic regression and cause-specific Cox survival analysis of competing-risk was run on inpatients, to examine the impact of Alpha variant on hospitalization, on mortality and on other factors. We observed that the Alpha variant is over-represented in inpatients than outpatients so infection by Alpha variant increases the chance for hospitalization. The impact of Alpha variant on mortality seems to depend on the patient's age. For patients under age of 70, the case-fatality-rate was 0.84% (5.3%) for patients without (with) Alpha variant (Fisher's test P-value = 2.4 × 10−10). For patients above age of 70, the trend is opposite: the case-fatality-rate is 31.5% (13.6%) for patients without (with) Alpha variant (Fisher's test P-value = 0.0016). The two opposite trends would cancel each other, making other analyses such as cause-specific Cox regression and logistic regression non-significant. The Alpha variant increases the risk for hospitalization, increases the case-fatality-rate for lower age group, and decreases the case-fatality-rate for the upper age group. If the increase of case-fatality-rate in not the most senior group holds true, it should provide useful information for a vaccination planning to counter the impact of Alpha variants.

Prevalence and antifungal drug resistance of dermatophytes in the clinical samples from Pakistan

Dermatophytosis is a major health problem all over the world including Pakistan. This is the first report of detection of dermatophytes and their antifungal drug resistance in the Northern and Western parts of Pakistan. A total of 154 samples were collected from different hospitals of Khyber Pakhtunkhwa, and out of them 136 samples were found positive. Tinea corporis (35%) was the most predominant type of infection followed by Tinea capitis (22%). The fungi identified in Tinea corporis infection types were identified as Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum. The fungi identified in Tinea capitis included Trichophyton violaceum, T. mentagrophytes, Microsporum ferrugineum. The gender wise distribution showed both males (52%) and females (48%) were infected with the fungi. More cases belonged to the rural parts of the country. Age wise distribution showed that the infection was more prevalent in the children and the prevalence decreased with the increase in age. The positive samples were checked against two antifungal agents: fluconazole and nystatin. Among 136 positive samples, none of the isolates showed resistance to nystatin while 7% of the samples showed resistance to fluconazole. The resistant isolates were then identified by amplifying the 18S rRNA gene, using universal primers (ITS1, ITS4). Among the 9 resistant isolates, 5 isolates were identified as Trichophyton spp., 3 as Microsporum spp. and 1 as Epidermophyton spp.