Antibiogram of environmental isolates of Acinetobacter calcoaceticus from Nkonkobe Municipality, South Africa

Background: Acinetobacter contributes to increased morbidity and mortality with its strong propensity to colonize and disseminate among humans and environmental sources coupled with its ability to develop resistance to antimicrobial agents. Materials and Methods: Clinical isolates of Acinetobacter recovered from routine samples of inpatients were analyzed retrospectively along with their antibiogram to evaluate in vitro activity of Doxycycline. Multiple antibiotic resistance index was calculated and interpreted. Results: Out of 93 isolates of Acinetobacter species recovered, predominant were from urine 47(50.54%) and blood 27(29.03%) samples. MDR isolates were 57(61. 29%).Overall antimicrobial susceptibility pattern revealed best spectrum of activity with Imipenem (75.27%), Meropenem (68.82%) and Doxycycline (68.82%) whereas in MDR isolates Doxycycline exhibited highest sensitivity (66.67%) followed by Imipenem (61.40%) and Meropenem (52.63%). MAR indexes for different isolates revealed 71 (76.34%) with MAR index greater than 0.2 and 22 (23.66%) less than 0.2. However, three isolates had shown MAR index of 01 (i.e. resistant to all the antimicrobials tested), out of which two were recovered from intensive care unit and one from general surgery ward. Twenty-six MDR patterns were observed with nine antimicrobials tested. Resistance to COT, CIP, GEN, AK, A/S, CPM, IMP, MRP (R8) was most frequently observed pattern in 8(14.04%) of MDR isolates. Conclusion: Doxycycline has exhibited efficacy against MDR Acinetobacter, which can be considered as an alternative therapy to down regulate selective pressure on carbapenems. To confront the immediate threat of Acinetobacter infections, a working antibiotic strategy should be addressed and stringent infection control practices are needed to prevent the spread of multi drug resistant isolates in the hospital. Keywords: Acinetobacter species, Doxycycline, MAR index

the emergence of antibiotic resistance in E. coli strains has led to a variety of clinical illnesses in both humans and animals, with consequences for human health and the environment. this cross-sectional study was conducted to determine the risk of increased antibiotic resistance by calculating the multiple antibiotic resistance (MAR) index of E. coli in Port Harcourt, Rivers State, Nigeria. A higher MAR index close to 1 was interpreted as having a higher level of antibiotic resistance, while a lower MAR index close to 0 is an indicative of little to no resistance. In this study, a total of 200 samples from clinical (urine and stool) and non-clinical sources (soil and poultry) were randomly collected following ethical approval. Escherichia coli was isolated on Eosin Methylene Blue agar and identified using standard biochemical tests. The antimicrobial susceptibility profile was determined using the disk diffusion methodology with Mueller-Hinton agar according to the instructions of the Clinical and Laboratory Standards Institute (CLSI). Escherichia coli isolates were amplified in this investigation using a 16s rRNA gene-based polymerase chain reaction (PCR) technique. for both 73% clinical (n=100) and 35% non-clinical (n=100) isolates, amoxicillin/clavulanic acid showed the highest rate of resistance with 82.2% (60 of 73) and 100% (35 of 35) respectively, whereas nitrofurantoin showed the lowest rate of resistance at 1.4% (1 of 73) clinical and no resistance was found for the nonclinical E. coli isolates. The study presumptively finds a higher resistance to 4 drug classes, with 42%, and the lowest of 1% to 6 drug classes of antibiotics. Additionally, MAR indices > 0.2 were observed in this study, which indicates excessive use of antibiotics. MAR index of 0.4 was the most frequent, with a 25% prevalence. The results of this investigation show that a MAR index of 0.4 suggests widespread antibiotic resistance, with 25% of the bacterial isolates from both sources exhibiting resistance to the tested antibiotics. A MAR index > 0.2 in this study indicates that 14.8% of the E. coli isolates were ineffective against the tested antibiotics. The bacteria isolates in this study have developed resistance to multiple antibiotics if the MAR index is greater than 0.2, which is an indication of overuse or inappropriate antibiotic use. The genotypic test verified 82.9% non-clinical E. coli (found in environmental soil and poultry samples) and 82.2% clinical E. coli (found in patient urine and stool samples). In this study, E. coli isolates were genotypically identified using the 16s rRNA gene. the high levels of MAR indices in E. coli as presented in this study could be indicative of antibiotic treatment failures in both clinical and nonclinical settings, which can serve as a potential reservoir of drug-resistant E. coli that are harmful and therefore require continuous monitoring.

Antibiotic resistance genes are typically isolated by cloning from cultured bacteria or by polymerase chain reaction (PCR) amplification from environmental samples. These methods do not access the potential reservoir of undiscovered antibiotic resistance genes harboured by soil bacteria because most soil bacteria are not cultured readily, and PCR detection of antibiotic resistance genes depends on primers that are based on known genes. To explore this reservoir, we isolated DNA directly from soil samples, cloned the DNA and selected for clones that expressed antibiotic resistance in Escherichia coli. We constructed four libraries that collectively contain 4.1 gigabases of cloned soil DNA. From these and two previously reported libraries, we identified nine clones expressing resistance to aminoglycoside antibiotics and one expressing tetracycline resistance. Based on the predicted amino acid sequences of the resistance genes, the resistance mechanisms include efflux of tetracycline and inactivation of aminoglycoside antibiotics by phosphorylation and acetylation. With one exception, all the sequences are considerably different from previously reported sequences. The results indicate that soil bacteria are a reservoir of antibiotic resistance genes with greater genetic diversity than previously accounted for, and that the diversity can be surveyed by a culture-independent method.

BackgroundClostridium difficile is the main cause of antibiotic associated diarrhea. In the past decade, the number of C. difficile patients has increased dramatically, coinciding with the emergence of two PCR ribotypes 027 and 078. PCR ribotype 078 is also frequently found during C. difficile outbreaks in pigfarms. Previously, the genome of the PCR ribotype 078 strain M120, a human isolate, was described to contain a unique insert of 100 kilobases.ResultsAnalysis of this insert revealed over 90 open reading frames, encoding proteins originating from transposons, phages and plasmids. The insert was shown to be a transposon (Tn6164), as evidenced by the presence of an excised and circularised molecule, containing the ligated 5’and 3’ends of the insert. Transfer of the element could not be shown through filter-mating experiments. Whole genome sequencing of PCR ribotype 078 strain 31618, isolated from a diarrheic piglet, showed that Tn6164 was not present in this strain. To test the prevalence of Tn6164, a collection of 231 Clostridium difficile PCR ribotype 078 isolates from human (n = 173) and porcine (n = 58) origin was tested for the presence of this element by PCR. The transposon was present in 9 human, tetracycline resistant isolates, originating from various countries in Europe, and none of the pig strains. Nine other strains, also tetracycline resistant human isolates, contained half of the transposon, suggesting multiple insertion steps yielding the full Tn6164. Other PCR ribotypes (n = 66) were all negative for the presence of the transposon. Multi locus variable tandem repeat analysis revealed genetic relatedness among transposon containing isolates. Although the element contained several potential antibiotic resistance genes, it did not yield a readily distinguishable phenotype.ConclusionsTn6164 is a newly described transposon, occurring sporadically in C. difficile PCR ribotype 078 strains. Although no transfer of the element could be shown, we hypothesize that the element could serve as a reservoir of antibiotic resistance genes for other bacteria. Further research is needed to investigate the transfer capabilities of the element and to substantiate the possible role of Tn6164 as a source of antibiotic resistance genes for other gut pathogens.

Genomic Description of Antibiotic Resistance in Escherichia coli and Enterococci Isolates from Healthy Lusitano Horses

Pseudomonas aeruginosa is a significant nosocomial pathogen with increasing antibiotic resistance worldwide. Understanding local resistance patterns is crucial for effective antimicrobial stewardship. This study aimed to determine the antibiotic resistance patterns and Multiple Antibiotic Resistance (MAR) index of Pseudomonas aeruginosa isolates from various clinical specimens at University of Calabar Teaching Hospital, Nigeria. One hundred clinical samples, including wound swabs (20), ear swabs (20), eye swabs (20), stool (20) and urine (20), were processed using standard microbiological techniques and inoculated onto cetrimide agar. Antibiotic susceptibility testing was performed using the Kirby-Bauer disc diffusion method, and MAR indices were calculated for all isolates. Ethical approval was obtained from the University of Calabar Teaching Hospital Ethics Committee. Fifty Pseudomonas aeruginosa isolates were obtained from wound swabs (20), ear swabs (14), eye swabs (9), stool (4), and urine (3). All isolates (100%) showed resistance to nalidixic acid, with high resistance to augmentin (90%) and pefloxacin (72%). Gentamicin showed moderate effectiveness with 60% sensitivity, while ofloxacin demonstrated 50% sensitivity and ciprofloxacin 40% sensitivity. The mean MAR index was 0.72, with all isolates exceeding the 0.2 threshold, indicating widespread multidrug resistance. Our findings reveal concerning levels of multidrug resistance among Pseudomonas aeruginosa isolates from University of Calabar Teaching Hospital. Resistance to commonly used antibiotics, including gentamicin, poses serious clinical challenges. The high MAR indices underscore the urgent need for enhanced infection control measures and antimicrobial stewardship programs.

This study evaluated the potential pathogenicity and antimicrobial resistance (AMR) of Vibrio species isolated from inland saline shrimp culture farms. Out of 200 Vibrio isolates obtained from 166 shrimp/water samples, 105 isolates were identified as V. parahaemolyticus and 31 isolates were identified as V. alginolyticus and V. cholerae, respectively. During PCR screening of virulence-associated genes, the presence of the tlh gene was confirmed in 70 and 19 isolates of V. parahaemolyticus and V. alginolyticus, respectively. Besides, 10 isolates of V. parahaemolyticus were also found positive for trh gene. During antibiotic susceptibility testing (AST), very high resistance to cefotaxime (93.0%), amoxiclav (90.3%), ampicillin (88.2%), and ceftazidime (73.7%) was observed in all Vibrio species. Multiple antibiotic resistance (MAR) index values of Vibrio isolates ranged from 0.00 to 0.75, with 90.1% of isolates showing resistance to ≥ 3 antibiotics. The AST and MAR patterns did not significantly vary sample-wise or Vibrio species-wise. During the minimum inhibitory concentration (MIC) testing of various antibiotics against Vibrio isolates, the highest MIC values were recorded for amoxiclav followed by kanamycin. These results indicated that multi-drug resistant Vibrio species could act as the reservoirs of antibiotic resistance genes in the shrimp culture environment. The limited host range of 12 previously isolated V. parahaemolyticus phages against V. parahaemolyticus isolates from this study indicated that multiple strains of V. parahaemolyticus were prevalent in inland saline shrimp culture farms. The findings of the current study emphasize that routine monitoring of emerging aquaculture areas is critical for AMR pathogen risk assessment.

Coagulase-negative staphylococci (CoNS) are increasingly associated with nosocomial infections, especially among the immunocompromised and those with invasive medical devices, posing a significant concern. We report on clinical multidrug-resistant CoNS from the uMgungundlovu District, KwaZulu-Natal Province, South Africa, as emerging pathogens. One hundred and thirty presumptive CoNS were obtained from blood cultures. Culture, biochemical tests, and the Staphaurex™ Latex Agglutination Test were used for the initial identification of CoNS isolates; confirmation and speciation were undertaken by the VITEK 2 system. Susceptibilities of isolates against a panel of 20 antibiotics were determined using the Kirby-Bauer disk diffusion method, and the multiple antibiotic resistance (MAR) indices of the isolates were determined. The polymerase chain reaction (PCR) was used to amplify the mecA gene to confirm methicillin resistance. Overall, 89/130 presumptive CoNS isolates were confirmed as CoNS by the VITEK 2 system. Of these, 68 (76.4%) isolates were putatively methicillin-resistant by the phenotypic cefoxitin screen test and 63 (92.6%) were mecA positive. Staphylococcus epidermidis (19.1%), S. hominis ssp. hominis (15.7%), and S. haemolyticus (16.9%) were the most common CoNS species. Isolates showed high percentage resistance against penicillin (100.0%), erythromycin (74.2%), and azithromycin (74.2%) while displaying high susceptibilities to linezolid (95.5%), gentamicin (95.5%), and tigecycline (94.4%). Multidrug resistance (MDR) was observed in 76.4% of isolates. MAR index calculation revealed 71.9% of isolates with MAR index >0.2 and 20.2% >0.5. Isolates with the highest MAR indices (0.7 and 0.8) were recovered from the neonatal intensive care unit. Fifty-one MDR antibiograms were observed. The high prevalence of methicillin resistance and multidrug resistance in several species of CoNS necessitates surveillance of this emerging pathogen, currently considered a contaminant of microbial cultures.

Constant monitoring of the bacteriological indicators of drinking water and the associated Multiple Antibiotic Resistance (MAR) index as impacted by seasonal variations and different stages of Drinking Water Treatment Plants (DWTPs) may assist in understanding the pattern of their seasonal occurrences and the regular operations of the treatment plant that influence their removal. In this paper, the impact of the seasons and of the different stages of DWTPs on bacteriological indicator occurrence and the MAR-index of five treatment plants from three provinces in South Africa were assessed. Colilert-18 and Enterolert Quanti-Tray/2000 IDEXX methods were used to enumerate total coliform, E. coli, and Enterococcus spp. of water samples from the different treatment stages. Kirby–Bauer disc diffusion technique was used to assess the antibiotic susceptibility of the indicator bacteria isolates. All the measured physicochemical parameters were within the permissible limits. All the treatment plants essentially had a very high reduction of the indicator bacteria across all seasons. However, only two plants maintained the microbiological quality of the final treated water in compliance with the standards. A total of 121 isolates were obtained, and 106 isolates were multidrug resistant with the greatest resistance recorded for the Beta-lactams class of antibiotics. The MAR-index varied across seasons and with different plants. This implied that the usage of antibiotics is season- and site-dependent. The different stages of treatment reduced the indicator bacteria with the most reduction occurring in disinfection and candy stages. These findings extend the knowledge of how the treatment stages and seasons shape indicator bacteria and antibiotic resistance in drinking water.

Antibiotic resistance has become a subject of global concern to health systems all over the world and the shedding of antibiotic resistant bacteria from humans and animals into the soil is a threat to public health. This research was carried out to molecularly characterize and assess antibiotic resistant bacteria from soil in Rivers State University. Bacteria from soil in four locations; waste dump site, fish farm, poultry farm and home soil were enumerated and identified using standard microbiological techniques such as serial dilution, plating and incubation, isolation, biochemical testing and molecular identification. Sensitivity testing on the bacterial isolates was done using Kirby-Bauer Disk diffusion method. These resistant isolates were also subjected to molecular testing to ascertain the presence of resistant genes. The results revealed mean total Heterotrophic Bacterial Counts (THBC) for 32 samples ranged from 1.05±0.72 x 107 to 3.63±2.58x 107 CFU/g in fish farm and waste dump samples respectively. Total Coliform Counts (TCC) ranged from 3.65±2.91 x105 to 6.92±4.41 x105 CFU/g in Fish farm and Waste dump samples while Total Staphylococcal count ranged from 3.80±1.71 x104 to 11.46±9.21 x104 CFU/g in Fish farm and Waste dump samples respectively. Total Pseudomonad Count (TPC) ranged from 0.09±0.09 x 103 to 2.47±1.67x 103 CFU/g in Home soil and Fish farm samples. There was a difference (p≤0.05) in total heterotrophic bacterial, Staphylococcal and Pseudomonad counts, but no difference (p≥0.05) in total coliform. Seventy-nine (79) bacterial isolates were identified in this study belonging to the following genera; Bacillus spp, Staphylococcus spp., Micrococcus spp, Pseudomonas spp, Serratia spp, Proteus spp, Klebsiella spp, Salmonella spp and Escherichia spp with Staphylococcus (58.83%) having the highest occurrence and Pseudomonas (11.11%) had the least occurrence from the samples. Antibiotic sensitivity revealed that most isolates were resistant to many antibiotics tested with the highest resistance observed for Cefuroxime, Ceftazidime and Cefixime (100%) for the gram negative organisms and also Cefuroxime, Ceftazidime and Cloxacillin for gram positive bacteria. However, all bacteria showed sensitivity to gentamicin. Multiple Antibiotic resistance (MAR) index for all bacteria were above 0.2. Resistant bacterial isolates were identified molecularly as S. aureus, S. sciuri, M. luteus, B. cereus, B. subtilis, P. aeruginosa, S. mercescens, P. vulgaris, E. coli and Salmonella typhimurium. Pseudomonas aeruginosa, Serratia mercescens, Proteus vulgaris and E. coli had CTX-M gene present in their genome while Staphylococcus aureus, Staphylococcus sciuri, Micrococcus luteus, Bacillus cereus and Bacillus subtilis had the mecA gene present in their genome. This study has highlighted the rise in antibiotic resistance in bacteria from soil, hence, there is need to checkmate indiscriminate use of antibiotics in agriculture.

Dairy farm waste: a potential reservoir of diverse antibiotic resistance and virulence genes in aminoglycoside- and beta-lactam-resistant Escherichia coli in Gansu Province, China

A comparative account of resistance and antagonistic activity of healthy and bleached coral-associated bacteria as an indicator of coral health status

The oceans are increasingly polluted with plastic debris, and several studies have implicated plastic as a reservoir for antibiotic resistance genes and a potential vector for antibiotic-resistant bacteria. Bioplastic is widely regarded as an environmentally friendly replacement to conventional petroleum-based plastic, but the effects of bioplastic pollution on marine environments remain largely unknown. Here, we present the first evidence that bioplastic accumulates antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) in marine sediments. Biofilms fouling ceramic, polyethylene terephthalate (PET), and polyhydroxyalkanoate (PHA) were investigated by shotgun metagenomic sequencing. Four ARG groups were more abundant in PHA: trimethoprim resistance (TMP), multidrug resistance (MDR), macrolide-lincosamide-streptogramin resistance (MLS), and polymyxin resistance (PMR). One MRG group was more abundant in PHA: multimetal resistance (MMR). The relative abundance of ARGs and MRGs were strongly correlated based on a Mantel test between the Bray-Curtis dissimilarity matrices (R = 0.97, p < 0.05) and a Pearson's analysis (R = 0.96, p < 0.05). ARGs were detected in more than 40% of the 57 metagenome-assembled genomes (MAGs) while MRGs were detected in more than 90% of the MAGs. Further investigation (e.g., culturing, genome sequencing, antibiotic susceptibility testing) revealed that PHA biofilms were colonized by hemolytic Bacillus cereus group bacteria that were resistant to beta-lactams, vancomycin, and bacitracin. Taken together, our findings indicate that bioplastic, like conventional petroleum-based plastic, is a reservoir for resistance genes and a potential vector for antibiotic-resistant bacteria in coastal marine sediments.

Pleural fluid infections, primarily caused by a range of bacterial pathogens, present a growing challenge for healthcare professionals, particularly due to the increasing prevalence of antibiotic resistance. This study included 598 patients, of which 15.88% had culture-positive samples. A total of 18 distinct bacterial species, both individual and in combination, were identified, with 7 Gram-positive and 11 Gram-negative bacteria. Antibacterial susceptibility testing highlighted colistin as the most effective antibiotic for most Gram-negative pathogens, while vancomycin and linezolid showed increased sensitivity against Gram-positive isolates. The study calculated the Multiple Antibiotic Resistance (MAR) index for all isolates, yielding a mean value of 0.543, reflecting the high level of irrational antibiotic use. Resistance pattern analysis revealed significant variation among bacterial species and identified 61 distinct resistance profiles. The study also examined the relationship between the MAR index and factors such as gender, age, and Gram staining. Gender (p = 0.831) and age (p = 0.905) showed no significant correlation with the MAR index, while a significant negative correlation was found between Gram staining and the MAR index (ρ = -0.434, p < 0.001). The findings highlight the increasing prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates, presenting significant challenges for treatment, particularly in resource-limited settings. This underscores the urgent need for enhanced antibiotic stewardship, public awareness campaigns, and continuous monitoring to combat antibiotic resistance and improve patient outcomes. Furthermore, the study's findings contribute to global efforts in understanding and addressing the escalating problem of antibiotic resistance, offering insights relevant to international research on antimicrobial resistance.

BackgroundThe potential for the human gut microbiota to serve as a reservoir for antibiotic resistance genes has been the subject of recent discussion. However, this has yet to be investigated using a rapid PCR-based approach. In light of this, here we aim to determine if degenerate PCR primers can detect aminoglycoside and β-lactam resistance genes in the gut microbiota of healthy adults, without the need for an initial culture-based screen for resistant isolates. In doing so, we would determine if the gut microbiota of healthy adults, lacking recent antibiotic exposure, is a reservoir for resistance genes.ResultsThe strategy employed resulted in the identification of numerous aminoglycoside (acetylation, adenylation and phosphorylation) and β-lactam (including blaOXA, blaTEM, blaSHV and blaCTX-M) resistance gene homologues. On the basis of homology, it would appear that these genes originated from different bacterial taxa, with members of the Enterobacteriaceae being a particularly rich source. The results demonstrate that, even in the absence of recent antibiotic exposure, the human gut microbiota is a considerable reservoir for antibiotic resistance genes.ConclusionsThis study has demonstrated that the gut can be a significant source of aminoglycoside and β-lactam resistance genes, even in the absence of recent antibiotic exposure. The results also demonstrate that PCR-based approaches can be successfully applied to detect antibiotic resistance genes in the human gut microbiota, without the need to isolate resistant strains. This approach could also be used to rapidly screen other complex environments for target genes.