Transcriptomic responses of extensively drug resistant Klebsiella pneumoniae to N-acetyl cysteine reveals suppression of major biogenesis pathways leading to bacterial killing and biofilm eradication.

AIMS: Carbapenemase-producing Klebsiella pneumoniae is categorized as a "critical global priority-one" pathogen by WHO and new and efficient treatment options are warranted. This study aims to assess the antibacterial and antibiofilm potential of N-acetyl cysteine (NAC), against clinical isolates of extensively drug resistant (XDR) K. pneumoniae and elucidate the mechanism of killing. METHODS AND RESULTS: XDR-K. pneumoniae were isolated from patients admitted to Madras Medical Mission Hospital, India. Antibiofilm activity of NAC was checked using in vitro continuous flow model and RNA sequencing was done using Illumina Novoseq. Data quality was checked using FastQC and MultiQC software. Our findings revealed that NAC at a concentration of 100 mg/ml was safe, and could inhibit the growth and completely eradicate mature biofilms of all XDR-K. pneumoniae isolates. Transcriptomic responses in XDR-K. pneumoniae to NAC showed significant downregulation of the genes associated with crucial biogenesis pathways, including electron transport chain and oxidoreductase activity besides a specific cluster of genes linked to ribosomal proteins. CONCLUSIONS: Our results indicate that NAC kills the XDR- K. pneumoniae clinical isolates by shutting the overall metabolism and, hence, successfully eradicate in vitro biofilms formed on catheters.

Analysis of mcr family of colistin resistance genes in Gram-negative isolates from a tertiary care hospital in India.

AIM: Colistin serves as the drug of last resort for combating numerous multidrug-resistant (MDR) Gram-negative infections. Its efficacy is hampered by the prevalent issue of colistin resistance, which severely limits treatment options for critically ill patients. Identifying resistance genes is crucial for controlling resistance spread, with horizontal gene transfer being the primary mechanism among bacteria. This study aimed to assess the prevalence of plasmid-mediated mcr genes associated with colistin resistance in Gram-negative bacteria, utilizing both genotypic and phenotypic tests. METHODS AND RESULTS: The clinical isolates (n = 913) were obtained from a tertiary care center in Chennai, India. Colistin resistance was seen among Gram-negative isolates. These strains underwent screening for mcr-1, mcr-3, mcr-4, and mcr-5 genes via conventional PCR. Additionally, mcr-positive isolates were confirmed through Sanger sequencing and phenotypic testing. The bacterial isolates predominantly comprised Klebsiella pneumoniae (62.43%), Escherichia coli (19.71%), Pseudomonas aeruginosa (10.73%), and Acinetobacter baumannii (4.81%), along with other species. All isolates exhibited multidrug resistance to three or more antibiotic classes. Colistin resistance, determined via broth microdilution (BMD) using CLSI guidelines, was observed in 13.08% of the isolates studied. Notably, mcr-5 was detected in K. pneumoniae in PCR, despite its absence in Sanger sequencing and phenotypic tests (including the combined-disk test, colistin MIC in the presence of EDTA, and Zeta potential assays). This finding underscores the importance of employing multiple diagnostic approaches to accurately identify colistin resistance mechanisms.

Phenotypic Changes in Phage Survivors of Multidrug-Resistant Klebsiella pneumoniae.

Multidrug-resistant Klebsiella pneumoniae (MDR-KP) infections have become a major global issue in the healthcare sector. Alternative viable tactics for combating bacterial infections, such as the use of bacteriophages, can be considered. One of the major challenges in phage therapy is the emergence of phage-resistant bacteria. This study isolated bacteriophages from water and soil samples against MDR-KP isolates. Susceptible bacterial hosts were exposed to phages at different concentrations and prolonged durations of time to obtain phage-resistant survivors. Phenotypic changes such as changes in growth rates, biofilm formation ability, antibiotic sensitivity patterns, and outer membrane proteins (OMPs) profiling of the survivors were studied. Our findings indicate that the phage ØKp11 and ØKp26 survivors had reduced growth rates and biofilm formation ability, altered antibiotic sensitivity patterns, and reduced OMPs expression compared with the parent MDR-KP002 isolate. These results suggest that the alternations in the bacterial envelope result in phenotypic phage resistance among MDR bacterial isolates. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01217-6.

Differential expression of outer membrane proteins and quinolone resistance determining region mutations can lead to ciprofloxacin resistance in Salmonella Typhi.

Multi-drug resistance in Salmonella Typhi remains a public health concern globally. This study aimed to investigate the function of quinolone resistance determining region (QRDR) of gyrA and parC in ciprofloxacin (CIP) resistant isolates and examine the differential expression of outer membrane proteins (OMPs) on exposure to sub-lethal concentrations of CIP in S. Typhi. The CIP-resistant isolates were screened for mutations in the QRDR and analyzed for bacterial growth. Furthermore, major OMPs encoding genes such as ompF, lamB, yaeT, tolC, ompS1, and phoE were examined for differential expression under the sub-lethal concentrations of CIP by real-time PCR and SDS-PAGE. Notably, our study has shown a single-point mutation in gyrA at codon 83 (Ser83-tyrosine and Ser83-phenylalanine), also the rare amino acid substitution in parC gene at codon 80 (Glu80-glycine) in CIP-resistant isolates. Additionally, CIP-resistant isolates showed moderate growth compared to susceptible isolates. Although most of the OMP-encoding genes (tolC, ompS1, and phoE) showed some degree of upregulation, a significant level of upregulation (p < 0.05) was observed only for yaeT. However, ompF and lamB genes were down-regulated compared to CIP-susceptible isolates. Whereas OMPs profiling using SDS-PAGE did not show any changes in the banding pattern. These results provide valuable information on the QRDR mutation, and the difference in the growth, and expression of OMP-encoding genes in resistant and susceptible isolates of S. Typhi. This further provides insight into the involvement of QRDR mutation and OMPs associated with CIP resistance in S. Typhi.

Effect of ciprofloxacin and in vitro gut conditions on biofilm of Escherichia coli isolated from clinical and environmental sources.

AIM: This study aimed at characterizing the biofilm-forming ability of drug-resistant and sensitive Escherichia coli under in vitro gut conditions and in the presence of ciprofloxacin. METHODS AND RESULTS: 153 E. coli isolates comprising 80 from clinical and 73 from environment source were studied for their ability to form biofilm under control and in vitro simulated gut conditions. The integrity of preformed biofilm on exposure to ciprofloxacin was assessed. Expression of biofilm-associated genes was analysed using qPCR. A high degree of resistance was observed in clinical isolates with a concomitant prevalence of blaTEM . Bile, pH and low temperature enabled the E. coli biofilm to resist the effect of ciprofloxacin. Clinical isolates of E. coli formed strong biofilms in in vitro gut conditions following exposure to high concentration of ciprofloxacin. The expression of biofilm genes varied between different gut conditions viz., presence of bile, pH and low temperature, included in this study. CONCLUSIONS: This study demonstrates the importance of papC and csgA for maintaining the biofilm integrity upon antibiotic exposure. Escherichia coli form biofilm as a survival strategy to adapt to the conditions in their environment irrespective of their drug resistance status. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides an understanding of the effect of different parameters of the gut conditions during infection and the effect of antibiotic on survival and biofilm-forming ability of clinical and environmental E. coli isolates. It further suggests that bacteria resort to biofilm formation as one of the mechanisms to adjust to alterations in gut conditions and once the biofilm is formed, it requires high concentration of ciprofloxacin to eradicate it.

Biofilm Formation and Associated Gene Expression in Multidrug-Resistant Klebsiella pneumoniae Isolated from Clinical Specimens.

Biofilms reduce the bacterial growth rate, inhibit antibiotic penetration, lead to the development of persister cells and facilitate genetic exchange. The biofilm-associated Klebsiella pneumoniae infections have not been well studied, and their implications in overcoming the effects of antimicrobial therapy are yet to be fully understood. Hence this study evaluated the antibiotic resistance pattern, antibiotic resistance determinants of extended-spectrum beta-lactamase (ESBL) family. Biofilm-forming ability of seventy multidrug-resistant clinical isolates of K. pneumoniae and the biofilm-associated genes of representative biofilm formers from a tertiary care hospital were also assessed. The K. pneumoniae isolated from urine exhibited resistance towards ceftazidime, nalidixic acid and meropenem. Isolates from blood were resistant to cefuroxime. Higher rates of resistance were observed towards cefuroxime, nalidixic acid, and meropenem for the isolates from the endotracheal aspirate. Extended spectrum beta-lactamase production by CLSI's disc diffusion-based confirmation test revealed all the K. pneumoniae to be as ESBL producers. Most of the isolates harboured the bla gene variants, blaSHV and blaTEM. Majority of the isolates were colistin sensitive. 97.1% of the K. pneumoniae produced biofilm. K. pneumoniae isolated from pus and blood produced fully established biofilms. Strong biofilm formers were sensitive to co-trimoxazole and ciprofloxacin. Moderate biofilm formers exhibited sensitivity towards meropenem and imipenem. Expression of the fimH gene was increased, while mrkD showed reduced expression among the strong biofilm formers. Moderate biofilm formers showed variable expression of the genes associated with the biofilm formation. The weak and non-biofilm formers showed reduced expression of both the fimbrial genes. Multidrug-resistant isolates produced ESBLs and formed well-established biofilms.

Genome analysis of clinical isolate of Campylobacter fetus subspecies fetus MMM01 from India reveals genetic determinants of pathogenesis and adaptation.

In this study we report the whole genome sequencing (WGS) based analysis of blood-borne Campylobacter fetus subsp. fetus MMM01 isolated from a diabetic patient to obtain deeper insights in to the virulence and host adaptability. The sequenced genome of C. fetus subsp. fetus MMM01 along with reference genomes retrieved from NCBI was subjected to various in-silico analysis including JSpecies, MLST server, PATRIC server, VFanalyzer, CARD, PHASTER to understand their phylogenetic relation, virulence and antimicrobial resistance profile. The genome had a size of 1,788,790 bp, with a GC content of 33.09%, nearly identical to the reference strain C. fetus subsp. fetus 82-40. The MLST based phylogenetic tree constructed revealed the polyphyletic branching and MMM01 (ST25) was found to be closely related to ST11, both belong to the sap-A serotype which are more common in human infections. VFanalyzer identified 88 protein-coding genes coding for several virulence factors including Campylobacter adhesion to fibronectin, flagellar apparatus, cytolethal distending toxin operons and Campylobacter invasion antigen proteins which enhance the virulence of bacteria along with resistance genes against antibiotics including fluoroquinolone, chloramphenicol, tetracycline, and aminoglycoside in MMM01, which points to enhanced survival and pathogenicity of this zoonotic pathogen. It was interesting to find that MMM01 lacked FGI-II island found in most of the clinical isolates, which encoded CRISPR Cas and prophage II regions. More details about the complexity and evolution of this zoonotic pathogen could be learned from future studies that concentrate on comparative genome analysis using larger genome datasets.

Effect of sub-minimum inhibitory concentration of ceftriaxone on the expression of outer membrane proteins in Salmonella enterica serovar Typhi.

Multi-drug resistance (MDR) in Salmonella is one of the major reasons for foodborne outbreaks worldwide. Decreased susceptibility of Salmonella Typhi to first-line drugs such as ceftriaxone, ciprofloxacin, and azithromycin has raised concern. Reduced outer membrane proteins (OMPs) permeability and increased efflux pump transportation are considered to be the main reasons for the emergence of antibiotic resistance in Salmonella. The present study aimed to assess the expression of OMPs at sub-lethal concentrations of ceftriaxone in S. Typhi (Sl5037/BC, and Sl05). The S. Typhi strains were exposed to sub-MIC and half of the sub-MIC concentrations of ceftriaxone at three different time intervals (0 min, 40 min, and 180 min) and analyzed for differential expression of OMPs. Further, the expression variation of OMP encoding genes (yaeT, ompX, lamb, ompA, and ybfM) in response to ceftriaxone was evaluated using real-time PCR. The genes like lamB, ompX, and yaeT showed significant downregulation (p < 0.05) compared to the control without antibiotic exposure, whereas ybfM and ompA showed a moderate downregulation. The expression of omp genes such as lamB, ompA, ompX, ybfM, and yaeT were found to be low in the presence of ceftriaxone, followed by time and dose-dependent. The study provides insights into the possible involvement of OMPs in drug resistance of S. Typhi, which could help develop a therapeutic strategy to combat MDR isolates of S. Typhi.

Mismatch amplification mutation assay-polymerase chain reaction: A method of detecting fluoroquinolone resistance mechanism in bacterial pathogens.

The mismatch amplification assay is a modified version of polymerase chain reaction (PCR) that permits specific amplification of gene sequences with single base pair change. The basis of the technique relies on primer designing. The single nucleotide mismatch at the 3' proximity of the reverse oligonucleotide primer makes Taq DNA polymerase unable to carry out extension process. Thus, the primers produce a PCR fragment in the wild type, whereas it is not possible to yield a product with a mutation at the site covered by the mismatch positions on the mismatch amplification mutation assay (MAMA) primer from any gene. The technique offers several advantages over other molecular methods, such as PCR-restriction fragment length polymorphism (RFLP) and oligonucleotide hybridization, which is routinely used in the detection of known point mutations. Since multiple point mutations in the quinolone resistance determining region play a major role in high-level fluoroquinolone resistance in Gram-negative bacteria, the MAMA-PCR technique is preferred for detecting these mutations over PCR-RFLP and sequencing technology.

Plasmid-mediated fluoroquinolone resistance associated with extra-intestinal Escherichia coli isolates from hospital samples.

Background & objectives: Infection from fluoroquinolone-resistant extra-intestinal Escherichia coli is a global concern. In this study, isolation and characterization of fluoroquinolone-resistant extra-intestinal E. coli isolates obtained from hospital samples were undertaken to detect plasmid-mediated quinolone resistance (PMQR) genes. Methods: Forty three isolates of E. coli obtained from patients with extra-intestinal infections were subjected to antibiogram to detect fluoroquinolone resistance. The mechanism of fluoroquinolone resistance was determined by the detection of PMQR genes and mutations in quinolone resistance determining region (QRDR). Results: Of the 43 isolates, 36 were resistant to nalidixic acid (83.72%) and 28 to ciprofloxacin (65.11%). Eight E. coli isolates showed total resistance to both the antimicrobials without any minimum inhibitory concentration. The detection of PMQR genes with qnr primers showed the presence of qnrA in two, qnrB in six and qnrS in 21 isolates. The gene coding for quinolone efflux pump (qepA) was not detected in any of the isolates tested. The presence of some unexpressed PMQR genes in fluoroquinolone sensitive isolates was also observed. Interpretation & conclusions: The detection of silent PMQR genes as observed in the present study presents a risk of the transfer of the silent resistance genes to other microorganisms if present in conjugative plasmids, thus posing a therapeutic challenge to the physicians. Hence, frequent monitoring is to be done for all resistance determinants.

CTX-M type extended-spectrum ß-lactamase in Escherichia coli isolated from extra-intestinal infections in a tertiary care hospital in south India.

Background & objectives: Infections caused by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli carrying blaCTX-M genes have been spreading globally, but there are geographical variations in the type of blaCTX-Mgenes prevalent and there are scanty data from India. This study was conducted to determine the CTX-M type ESBLs in E. coli isolates obtained from clinical specimens from patients with extra-intestinal infections attending a tertiary care hospital in south India. Methods: ESBL-producing E. coli isolated from patients with extra-intestinal infections were subjected to PCR using CTX-M group-specific primers. From a representative isolate, full-length CTX-M-15 gene was amplified and sequenced. An internal fragment of this gene was sequenced in 10 representative isolates. Results: Of the 300 isolates of E. coli tested, 88 per cent carried CTX-M genes and blaCTX-M-15was the most dominant gene present in 90 per cent of the positive isolates. Most (91%) of the isolates positive for blaCTX-M were sensitive to meropenem. Interpretation & conclusions: Our findings showed blaCTX-M-15 to be the dominant gene. Based on the data on antimicrobial susceptibility, cefoperazone-sulbactum could be an antimicrobial of choice.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for rapid diagnosis of neonatal sepsis.

BACKGROUND & OBJECTIVES: The difficulties in diagnosis of neonatal sepsis are due to varied clinical presentation, low sensitivity of blood culture which is considered the gold standard and empirical antibiotic usage affecting the outcome of results. Though polymerase chain reaction (PCR) based detection of bacterial 16S rRNA gene has been reported earlier, this does not provide identification of the causative agent. In this study, we used restriction fragment length polymorphism (RFLP) of amplified 16S rRNA gene to identify the organisms involved in neonatal sepsis and compared the findings with blood culture. METHODS: Blood samples from 97 neonates were evaluated for diagnosis of neonatal sepsis using BacT/Alert (automated blood culture) and PCR-RFLP. RESULTS: Bacterial DNA was detected by 16S rRNA gene PCR in 55 cases, while BacT/Alert culture was positive in 34 cases. Staphylococcus aureus was the most common organism detected with both methods. Klebsiella spp. was isolated from four samples by culture but was detected by PCR-RFLP in five cases while Acinetobacter spp. was isolated from one case but detected in eight cases by PCR-RFLP. The sensitivity of PCR was found to be 82.3 per cent with a negative predictive value of 85.7 per cent. Eighty of the 97 neonates had prior exposure to antibiotics. INTERPRETATION & CONCLUSIONS: The results of our study demonstrate that PCR-RFLP having a rapid turnaround time may be useful for the early diagnosis of culture negative neonatal sepsis.

Rare occurrence of fatal Candida haemulonii peritonitis in a diabetic CAPD patient.

A 68-year-old diabetic chronic kidney disease patient on continuous ambulatory peritoneal dialysis for two years developed Candida haemulonii peritonitis without any predisposing factors. There is no effective treatment for this fungus. A peritoneal biopsy showed morphological changes of acute inflammation and chronicity.

A New Casjensviridae Bacteriophage Isolated from Hospital Sewage for Inactivation of Biofilms of Carbapenem Resistant Klebsiella pneumoniae Clinical Isolates.

Klebsiella pneumoniae, a member of the ESKAPE pathogen group, is a prominent cause of hospital-acquired infections. The WHO has recognized carbapenem-resistant K. pneumoniae as a critical-one priority pathogen. These resilient superbugs have the ability to form biofilms and present a significant global threat. In the present study, we isolated and characterized a bacteriophage SAKp02, from hospital sewage, infectious to carbapenem-resistant K. pneumoniae patient isolates. SAKp02 could infect 43 of 72 clinical isolates, indicating a broad host spectrum. Whole genome analysis classified SAKp02 within the family Casjensviridae, with a 59,343 bp genome encoding 82 ORFs. Comparative genomic analysis revealed significant differences between SAKp02 and its closest viruses, indicating a distinct genetic makeup positioning it as a novel phage strain within the lineage. The SAKp02 genome comprises bacteriolytic enzymes, including holin, endolysin, and phage depolymerase, crucial for bacterial lysis and biofilm disruption. It reduced biofilm biomass by over threefold compared to the control and eradicated 99% of viable cells within a 4 h treatment period. Scanning electron microscopy corroborated the ability of the phage to dismantle biofilm matrices and lyse bacterial cells. Safe and effective treatments are warranted, and hence, the fully characterized lytic phages with therapeutic potential against drug-resistant clinical isolates of bacteria are needed. Our study is the first to report the antibacterial and antibiofilm activity of Casjensviridae phages, and our discovery of a novel K. pneumoniae phage broadens the arsenal against the bacteria.

IgG responses against SARS-CoV-2 vaccines AZD1222 and BBV-152 and breakthrough infections among health care workers in southern India.

IgG antibodies elicited in response to SARS-CoV-2 are critical in determining the protection achieved through vaccination. The present longitudinal study aims to assess the immune response generated through AZD1222 & BBV-152 vaccination among health care workers (HCWs) in a selected hospital. Serum IgG levels were measured approximately at 1.5 months and 6 months after the first and second vaccination. The final assessment was done 12 months after the first vaccination to analyse the sustained antibody levels. Results showed a progressive increase in antibody titres as a function of time. 26 HCWs in all had SARS-CoV-2 breakthrough infection, but their antibody titres were not significantly higher compared to COVID-19 naïve individuals. However, a comparative analysis showed considerably higher antibody titre in those who received the AZD1222 vaccine among this cohort. AZD1222 vaccination was significantly associated with seropositivity in the first and second assessments. Female HCWs showed significantly higher seropositivity, and participants above 60 years showed considerably reduced antibody titre in the first assessment. However, the final assessment showed no association with these variables, with 97.1 % of participants reporting to be seropositive. The results indicate good antibody response and potential protection against SARS CoV-2.

Sequence-Specific Gene Silencing of acrA in the Multi-drug Efflux System AcrAB Induces Sensitivity in Drug-Resistant Klebsiella pneumoniae.

Multi-drug efflux is one of the resistant determinants in Klebsiella pneumoniae that are encountered in a broad range of clinically relevant antimicrobial agents. An alternative method to strategically induce sensitivity in drug-resistant K. pneumoniae and improve the efficacy of the existing antibiotics is the need of the hour. Hence, an antisense RNA was designed against the acrA gene of the AcrAB-TolC efflux system in a drug-resistant isolate of K. pneumoniae obtained from a blood culture. Minimum inhibitory concentration by E test demonstrated that the antisense RNA could significantly increase the susceptibility of previously resistant K. pneumoniae toward ciprofloxacin (CIP) and co-trimoxazole. Real-time PCR determined the ability of the antisense RNA to inhibit the expression of the acrA-mRNA. The wild-type K. pneumoniae showed increased growth in the presence of CIP, while, under the same condition, the growth of the antisense RNA-treated K. pneumoniae was inhibited up till 12 h. In the presence of co-trimoxazole, delayed growth rate of the antisense RNA-treated K. pneumoniae was seen, in comparison to that of the wild-type K. pneumoniae and also a fourfold reduction was noted in the expression of the efflux gene acrA. Our results underscore the potential of the acrA antisense RNA as an alternative therapeutic against multi-drug-resistant K. pneumoniae.

Evaluation of reverse transcriptase-polymerase spiral reaction assay for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2.

BACKGROUND AND AIM: Existing real-time reverse transcriptase PCR (RT-qPCR) has certain limitations for the point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since it requires sophisticated instruments, reagents and skilled laboratory personnel. In this study, we evaluated an assay termed the reverse transcriptase-polymerase spiral reaction (RT-PSR) for rapid and visual detection of SARS-CoV-2. METHODS: The RT-PSR assay was optimized using RdRp gene and evaluated for the detection of SARS-CoV-2. The time of 60min and a temperature of 63°C was optimized for targeting the RNA-dependent RNA polymerase gene of SARS-CoV-2. The sensitivity of the assay was evaluated by diluting the in-vitro transcribed RNA, which amplifies as low as ten copies. RESULTS: The specific primers designed for this assay showed 100% specificity and did not react when tested with other lung infection-causing viruses and bacteria. The optimized assay was validated with 190 clinical samples in two phases, using automated RTPCR based TrueNat test, and the results were comparable. CONCLUSIONS: The RT-PSR assay can be considered for rapid and sensitive detection of SARS-CoV-2, particularly in resource-limited settings. To our knowledge, there is as yet no RT-PSR-based kit developed for SARS-CoV-2.

Comparative analysis of different methods used for molecular characterization of Burkholderia cepacia complex isolated from noncystic fibrosis conditions.

PURPOSE: Burkholderia is a Gram-negative opportunistic bacterium capable of causing severe nosocomial infections. The aim of this study was to characterize Burkholderia cepacia complex and to compare different molecular methods used in its characterization. METHODS: In this study, 45 isolates of Burkholderia cepacia complex (Bcc) isolated from clinical cases were subjected to RAPD (Random amplified polymorphic DNA), recA-RFLP (Restriction fragment length polymorphism), 16SrDNA-RFLP, whole-cell protein analysis, recA DNA sequencing and biofilm assay. RESULTS: Of the 45 isolates tested, 97.7% were sensitive to ceftazidime, 82.2% were sensitive to Cotrimoxazole, 73.3% were sensitive to meropenem, 55.5% were sensitive to minocycline and 42.2% were sensitive to levofloxacin. Majority of the isolates harbored all the tested virulence genes except bpeA and cblA. The RAPD generated 11 groups (R1-R11), recA-RFLP 10 groups (A1-A10), 16SrRNA-RFLP 5 groups (S1-S5) and SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis) whole cell protein analysis revealed 12 groups (C1-C12). recA sequencing revealed that most of the isolates belonging to the genomovar III Burkholderia cenocepacia. Though all the methods are found to be efficient in differentiating Burkholderia spp., recA-RFLP was highly discriminatory at 96% similarity value. The study also identified a new strain Burkholderia pseudomultivorans for the first time in the country. Further, recA sequencing could identify the strains to species level. Majority of the multidrug-resistant strains also showed moderate to strong biofilm-forming ability, which further contributes to the virulence characteristics of the pathogens. CONCLUSIONS: The study highlights the importance of combination of molecular methods to characterize Burkholderia cepacia complex. Molecular typing of these human pathogens yields important information for the clinicians in order to initiate the most appropriate therapy in the case of severe infections and to implement preventive measures for the effective control of transmission of Burkholderia spp.

In vitro activity of Ceftazidime-Avibactam and its comparators against Carbapenem resistant Enterobacterales collected across India: results from ATLAS surveillance 2018 to 2019.

ATLAS (Antimicrobial Testing Leadership and Surveillance) detects trends in multi-drug resistance longitudinally over time. In the present study, the in vitro activity of ceftazidime-avibactam and comparators was analyzed against Escherichia coli (n = 458) and Klebsiella pneumoniae (n = 455)  isolates obtained from 9 centers across India. The overall susceptibility to ceftazidime-avibactam was observed to be 72% among K. pneumoniae isolates and 87% among E. coli isolates. Among the tested carbapenem resistant isolates, 51% of CR-K. pneumoniae and 24% of CR-E. coli were susceptible to ceftazidime- avibactam. OXA-48 like was identified in 52% of the K. pneumoniae isolates followed by co-production of NDM with OXA-48 like in 27%. NDM was predominantly identified in 68% of the E. coli isolates followed by OXA-48 like in 24% isolates. The findings suggest that ceftazidime- avibactam is a reasonable alternative to standard therapy for management of carbapenem resistant Enterobacterales infections particularly with K. pneumoniae and E. coli with the OXA-48 like genotype.

Clinical management of severe infections caused by carbapenem-resistant gram-negative bacteria: a worldwide cross-sectional survey addressing the use of antibiotic combinations.

OBJECTIVES: Optimal treatment of carbapenem-resistant Gram-negative bacteria (CR-GNB) infections is uncertain because of the lack of good-quality evidence and the limited effectiveness of available antibiotics. The aim of this survey was to investigate clinicians' prescribing strategies for treating CR-GNB infections worldwide. METHODS: A 36-item questionnaire was developed addressing the following aspects of antibiotic prescribing: respondent's background, diagnostic and therapeutic availability, preferred antibiotic strategies and rationale for selecting combination therapy. Prescribers were recruited following the snowball sampling approach, and a post-stratification correction with inverse proportional weights was used to adjust the sample's representativeness. RESULTS: A total of 1012 respondents from 95 countries participated in the survey. Overall, 298 (30%) of the respondents had local guidelines for treating CR-GNB at their facility and 702 (71%) had access to Infectious Diseases consultation, with significant discrepancies according to country economic status: 85% (390/502) in high-income countries versus 59% (194/283) in upper-medium-income countries and 30% (118/196) in lower-middle-income countries/lower-income-countries). Targeted regimens varied widely, ranging from 40 regimens for CR-Acinetobacter spp. to more than 100 regimens for CR-Enterobacteriaceae. Although the majority of respondents acknowledged the lack of evidence behind this choice, dual combination was the preferred treatment scheme and carbapenem-polymyxin was the most prescribed regimen, irrespective of pathogen and infection source. Respondents noticeably disagreed around the meaning of 'combination therapy' with 20% (150/783) indicating the simple addition of multiple compounds, 42% (321/783) requiring the presence of in vitro activity and 38% (290/783) requiring in vitro synergism. CONCLUSIONS: Management of CR-GNB infections is far from being standardized. Strategic public health focused randomized controlled trials are urgently required to inform evidence-based treatment guidelines.