First report of blaOXA and mcr-2 genes co-existing in a multidrug-resistant Pseudomonas aeruginosa isolate recovered from a layer chicken flock in Türkiye

Klebsiella pneumoniae (K. pneumoniae) is an important multidrug-resistant (MDR) and biofilm producing pathogen that causes diverse infections in human and animals. This study aimed to evaluate the prevalence, antimicrobial resistance, and biofilm forming ability of K. pneumoniae isolates from diseased humans sputum and urine samples and mastitic cows milk samples in Zagazig city, Sharkia Governorate, Egypt. A total of 100 human and animal samples were collected and examined for the presence of Klebsiella species. The recovered isolates were identified by conventional bacteriological procedures and confirmed by the traditional PCR at the genus and species levels using gyrA, 16S-23S ITS and pehX genes, respectively. The confirmed K. pneumoniae isolates were subjected for testing their antimicrobial susceptibility and biofilm formation ability. The results revealed that 10 out of 50 human samples were positive for K. pneumoniae (20%); 28% (7/25) from sputum samples and 12% (3/25) from urine samples. Additionally, the prevalence rate of K. pneumoniae in milk samples from mastitic cows was 4% (2/50). PCR assays confirmed the 12 isolates as K. pneumoniae. The antimicrobial susceptibility results showed that K. pneumoniae isolates had high resistance rates to ampicillin and ampicillin/clavulanate (100% each) with multiple antibiotic resistance (MAR) index of 0.083, followed by azithromycin, cefepime, and trimethoprim/sulphamethoxazole (91.7% each). Meanwhile, these isolates revealed high sensitivity levels to chloramphenicol (83.3%) with MAR index of 0.014. Furthermore, 91.7% of K. pneumoniae isolates were able to produce the biofilm, where 8.3% was a non-biofilm producer. Out of 11 K. pneumoniae isolates, 6 (54.5%), 3 (27.3%) and 2 (18.2%) were moderate, strong and weak biofilm producers, respectively. The present study emphasized the high prevalence of MDR and biofilm producing K. pneumoniae isolates in human sputum and urine samples. Therefore, more attentions should be taken against antimicrobials usage and for providing new antibiofilm approaches against Klebsiella species biofilms.

Escherichia coli is known as one of the bacterial species with the widest adaptability to variety of niches either within organisms or outside in environment. Most strains of E. coli are of low virulence and associated with opportunistic infections, whereas others are highly virulent. The success of E. coli in colonising such a wide range of hosts and environments is basically due to a noticeable ductility in exploiting the available resources. It is becoming increasingly clear that biofilms have an enormous impact on medicine because since 65% of animal and human bacterial infections involve biofilms. In present study, we isolated strains of E. coli from animals. 19 interesting isolates were selected and tested by PCR amplification to virulence – iutA, cvaC, iss, tsh, papC, kps, iha and iron metabolism genes – sitA, feoB, irp2, fyuA, iroN, ireA. The ability of biofilm formation was assessed in a quantitative assay using a microtiter-plate test. Bacterial strains were grown on BHI. We divided isolates of E. coli into four classes: very weak (63.0%), weak (10.5%), moderate (10.5%) and strong (16.0%) biofilm producers. Representation genes of virulence were highly in isolates from very weak biofilm producers – from 7 genes were 6 highly; only papC (P fimbrial adhesin) was low. Genes of iron metabolism were different. Genes – sitA, fyuA, ireA in strong isolates producing biofilm and feoB, irp2, iroN in weak producers were most represented. The results show possible relation between presence virulence factor and low biofilm formation.

Staphylococcus pseudintermedius (S. pseudintermedius), found on dogs' skin and mucous membranes, can act as an opportunistic pathogen causing skin, ear, and other tissue infections. Due to the possibility of zoonotic transmission of them, it is necessary to investigate the prevalence of S. pseudintermedius, especially the antimicrobial-resistant strains, in pets. In this study, the prevalence, antimicrobial resistance patterns, and biofilm formation ability of methicillin-resistant S. pseudintermedius (MRSP) were investigated and compared in 50 healthy dogs and 50 dogs with skin infections. The prevalence of S. pseudintermedius was not significantly different between healthy dogs (40%) and dogs with skin infections (50%). No significant difference was found in the prevalence of MRSP between healthy dogs (12%) and dogs with skin infections (18%). A total of 81.8% of S. pseudintermedius isolates were biofilm producers. The frequencies of antibiotic resistance (except for gentamicin), multidrug resistance (MDR), and biofilm formation ability were not significantly different between S. pseudintermedius isolates of healthy dogs and dogs with skin infections. The frequencies of resistance to penicillin and tetracycline, MDR, and biofilm production abilities were significantly higher among MRSP than methicillin-susceptible S. pseudintermedius (MSSP). The frequency of oxacillin resistance was significantly higher among weak or moderate biofilm producers than non-biofilm producers. The frequency of resistance to erythromycin was significantly higher among moderate biofilm producers than non-biofilm producers or weak biofilm producers. High frequencies of biofilm-producer S. pseudintermedius isolates and their resistance to antibiotics can affect the success of treatment of infections caused by these strains.

The study explored the biofilm (BF) formation capacity, BF-related gene profiles, and the trends in antimicrobial resistance (AMR) of Salmonella pullorum (SP) strains over several years. A total of 627 SP strains were isolated from 4,540 samples collected from chicken farms in Guangxi, China during 2018-2022. The BF-forming capacity of these isolates was assessed using crystal violet staining, and the presence of eight BF-related genes (csgA, csgB, csgD, ompR, bapA, pfs, luxS, and rpoS) in BF formation-positive strains was determined through Polymerase Chain Reaction (PCR) analysis. Antimicrobial susceptibility test was conducted to investigate the AMR of the isolates. Minimum Inhibitory Concentration (MIC) and Minimal Biofilm Eradication Concentration (MBEC) of nine SP-BF strains were determined using the broth microdilution method to assess the impact of BF formation on AMR. Additionally, the Optimal Biofilm Formation Conditions (OBFC) were investigated. The results indicated that 36.8% (231/627) of the strains exhibited a positive BF-formation capacity. Among these, 24.7% (57/231) were strong BF producers, 23.4% (54/231) were moderate BF producers, and 51.9% (120/231) were weak BF producers. Analysis of the eight BF-related genes in SP-BF strains revealed that over 90% of them were positive for all the genes. Antimicrobial susceptibility test conducted on the isolates showed that 100% (231/231) of them exhibited resistance to at least one antibiotic, with 98.3% (227/231) demonstrating multidrug resistance (MDR). Both MIC and MBEC measurements indicated varying degrees of increased AMR after BF formation of the bacteria. The optimal conditions for BF formation were observed at 37°C after 48 h of incubation, with an initial bacterial concentration of 1.2 × 106 CFU/mL. Notably, NaCl had a significant inhibitory effect on BF formation, while glucose and Trypticase Soy Broth (TSB) positively influenced BF formation. The results of the study emphasized the need for effective preventive and control strategies to address the challenges posed by the BF formation and MDR of SP in the field.

Political attention to antimicrobial resistance (AMR) has never been greater. Governments worldwide are concerned that AMR threatens to undo modern medical achievements with the spectre of a post antibiotic era in which commonplace infections, once eminently treatable, become nontreatable causes of serious morbidity and mortality 1. With suggestions that AMR and multi-drug resistant organisms are as important as climate change and could cast the world back into the dark ages of medicine, ranking alongside terrorism as matters of national risk 2, 3, the political landscape on this subject has been clearly set. Concerns about AMR and its health impact are, of course, not new and began at almost the same time as the introduction of antimicrobials. In 1945, just after the introduction of penicillin as a therapeutic agent in humans and animals, Fleming warned in his Nobel Prize acceptance speech that misuse of antimicrobials could result in bacterial resistance. This prediction rapidly became true with the discovery of each new class of antimicrobial quickly followed by the appearance of resistance to it. By the 1960s there was widespread realisation, and acceptance in the scientific community and lay press, that antimicrobial use (and misuse) resulted in rapid selection for resistance against all classes of antimicrobials. What is new, and has changed the political and regulatory landscape for AMR completely, is the realisation that science is not able to out-pace the microbes. There have been no completely new classes of antimicrobials discovered and brought to market since the 1980s, perhaps not surprising given the relatively small range of bacterial targets and the rapid rate of antimicrobial discovery during the ‘golden age’ from the mid 1940s onwards 4. Although there are some rays of hope, for example the recently reported new compound ‘teixobactin’ 5, the pipeline for new antimicrobials is practically dry. In other words, the solutions to AMR must come from within the medical, veterinary and animal industry sectors by addressing the underlying causes of, and changing the therapeutic approaches to, infectious disease. The political and scientific view that antimicrobials can no longer be regarded as the panacea or ‘magic bullet’ capable of eradicating infectious disease is widely accepted, and it is now clear that the human and animal health care sectors need to respond accordingly. A major challenge for the politicians is that there are still significant gaps in the surveillance data required to fully understand the drivers of AMR in both humans and animals 6, 7 and, critically, to measure the effects of interventional measures to reduce AMR. It is therefore not surprising that scientific opinion continues to be divided on practically every key question about AMR except that it is now a serious global problem causing significant economic loss with welfare, morbidity and mortality impacts in humans and animals. Antimicrobial resistance is a natural phenomenon: bacteria produce antimicrobial substances as part of their repertoire to compete in the struggle for colonisation, space and nutrients. Resistance therefore existed long before the introduction of antimicrobial drugs: the effect of using antimicrobials has been to accelerate AMR through classical selective pressure. That this has happened in both veterinary and human populations of bacteria is not disputed; the evidence for interconnection of AMR in these two populations is, however, inconclusive and is the subject of continuing political and scientific debate with contradictory evidence produced by both sides 8-10. It does appear that antimicrobial use in animals increases AMR in animal bacteria and that treating people with antimicrobials increases AMR in human bacteria. However, current scientific evidence does not allow definitive assessment of whether reducing antimicrobial use in animals has reduced AMR in medical pathogens. The extent to which AMR in populations of animal bacteria threatens public health therefore remains uncertain. The evidence for resistance in animal bacteria acting as genetic reservoirs of resistance for transfer to bacteria of public health importance is also inconclusive. Even for zoonotic bacteria such as Salmonella typhimurium DT104, the links between animal and human bacterial populations have become less clear with the application of sophisticated molecular typing bacterial methods and population genetics adding new complexity to the AMR debate 11. However, the lack of conclusive evidence notwithstanding, the prevailing political and regulatory opinion continues to be that antimicrobial use, and associated AMR, in animals is a driver of AMR in medical pathogens and that controlling veterinary prescription of antimicrobials will help safeguard public health. The ongoing political and public health scrutiny of veterinary use of antimicrobials is not surprising and the assumption that veterinary antimicrobial use contributes to, or is perhaps even directly the cause of, AMR in human medicine is understandable. The fact that the classes of antimicrobials used in veterinary and human medicine are the same 12; that food-borne and other zoonotic infections provide an opportunity for transfer of resistant bacteria from animals to humans; that populations of pathogenic and nonpathogenic animal bacteria may act as genetic reservoirs of resistance for important medical pathogens, with close contact between people and companion animals, in addition to food products, providing opportunity for genetic exchange; and, perhaps most importantly from a political perspective, that in many countries around the world the total quantity (gross weight) of antimicrobials used in veterinary medicine is greater than in human medicine 13, 14, has put antimicrobial use in animals at the centre of the public health AMR debate. When combined with the use of antimicrobials for disease prevention at herd or flock level and, in around half of the world's countries, for growth promotion, it is little wonder that antimicrobial use in animals has resulted in sustained political concern over the contribution that veterinarians and the animal sector in general may be making to the growing crisis of antimicrobial resistance in humans, with frequent calls for restriction or even banning of veterinary use of antimicrobials. Despite numerous political recommendations that coordinated, overarching surveillance of AMR is implemented at national and international level 15, 16 there are still relatively few examples of harmonised and integrated surveillance in humans and animals that allow comparison of data. Examples include The National Antimicrobial Resistance Monitoring System (NARMS) in the USA, Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) in Canada, Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM) in Japan and several European schemes including Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) (Denmark), NORM-VET (Norway), Swedish Veterinary Antimicrobial Resistance Monitoring (SVARM) (Sweden) and NethMap/Monitoring of Antimicrobial Resistance and Antibiotic usage in Animals in the Netherlands (MARAN) (the Netherlands). At EU level, the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) monitor AMR in the food chain and food-borne zoonotic pathogens, but not in companion animals. In the absence of sufficient scientific evidence about AMR, in particular the key question of the impact of veterinary antimicrobial use on public health, politicians around the world have faced difficult decisions. In the absence of scientific certainty politicians have adopted the ‘precautionary principle’, allowing preventive action to be taken when there is a possibility of harm but where the scientific evidence is not sufficiently complete to allow full assessment. The result in Europe is a continuing European political focus on banning or restricting veterinary antimicrobial use, especially in the agricultural sector, and reducing the total quantities of antimicrobials used in animals. In the political and regulatory environment in the USA, the precautionary principle has been applied somewhat differently with less political appetite for banning or restricting antimicrobials 9. The first active political engagement with antimicrobial resistance occurred in 1968 in response to growing concerns over multidrug resistant Salmonella in humans and animals, with the establishment of an independent advisory committee by the UK Government chaired by Professor Michael Swann. The Swann Report 17, published in 1969, recommended restriction of the use of antimicrobials as growth promoters, which took 45 years to fully implement in Europe, and establishment of overarching monitoring of AMR in humans and animals, which has still not been implemented globally. Almost 50 years on, this report continues to set the political stage in relation to veterinary antimicrobial use and possible impacts on human health. We would do well not to lose sight of the lessons learned in the decades following its publication, specifically that sensible recommendations based on competent assessment of the available, even if incomplete, scientific evidence should not be sidelined pending collection of conclusive evidence; instead the two should progress in parallel with continuous monitoring and refinement as evidence is gathered. The global political thrust in relation to AMR in the human and animal health sectors continues to be that overuse of antimicrobials is the cause of the problem and that reducing their use is the solution. In Europe, most political effort since 1969 has been directed at the food animal sector through reducing the use of antimicrobials as growth promoters and, more recently, reducing total antimicrobial use. It was not until 2006 that a EU-wide ban on antimicrobial growth promoters was eventually implemented, completing a political process that had started four decades previously with the banning of tetracycline, penicillin and streptomycin for growth promotion in 1974, followed by complete bans of antimicrobial growth promoters in Sweden and Denmark in 1988 and 1994. Denmark also implemented restrictions on veterinary dispensing of antimicrobials; decoupling veterinary prescription of antimicrobials from supply remains on the European political agenda and, if implemented, would have significant impact on veterinary practice business models in many countries. Monitoring, and reducing, antimicrobial use has become a key global political driver. The European Medicines Agency monitors the sales of antimicrobial agents for food producing animals and horses across Europe 18 providing benchmarks against which political targets for reduction are set. In some countries governmental targets for reduction in the sales of veterinary antimicrobials have been agreed with stakeholders. For example, the Netherlands decreased sales of antimicrobials by 49% between 2010 and 2012 with further reduction targets agreed; antimicrobial sales in Scandinavia have been progressively reduced through a series of government–stakeholder agreed targets 18. Nevertheless, the estimated consumption of antimicrobials (corrected for estimated biomass) in animals continues to be greater than in humans across Europe as a whole 6. It is becoming increasingly clear, however, that the concept of overuse as the key driver of AMR may be overly simplistic 19. Antimicrobial resistance is a complex public and animal health issue and there is recognition that integrated strategies across all sectors, backed by political will, stakeholder buy-in and sufficient economic support, are required to control it 1. Although overprescribing of antimicrobials is undoubtedly an important factor, reducing their use in human medicine has not consistently resulted in reduction of resistance for key pathogen–antimicrobial combinations with examples of resistance remaining apparently stable or even increasing despite reduced antimicrobial use. The question of whether phasing out antimicrobials as growth promoters across Europe and the restrictions placed on therapeutic use of antimicrobials in Scandinavia, with associated reductions in quantities used, has resulted in a positive impact on human health continues to be the subject of scientific and political disagreement. Responsible, or ‘prudent’, use of antimicrobials has emerged as a parallel precautionary approach to the control of AMR. Initially, the political focus was on restricting veterinary use of antimicrobials used to treat multidrug resistant human pathogens presenting significant risk to public health. Since 2005 the World Health Organization has published lists of ‘critically important antimicrobials for human medicine’, ranked according their importance with the goal that their use should be restricted in all sectors to preserve their effectiveness 20. This approach has been extended by the World Organisation for Animal Health (OIE) with the publication of a list of antimicrobials of veterinary importance which contains recommendations for restricting the use in food animals of antimicrobials that are critically important for both human and animal health 21. This list includes fluoroquinolones and third- and fourth-generation cephalosporins and forms a rational basis for responsible guidelines worldwide. There are several examples of stakeholder groups at national and international level that have responded to the AMR challenge and shown leadership in producing responsible use guidelines. In the late 1990s the UK veterinary and farming sectors established the RUMA (responsible use of medicines in agriculture) alliance and in 2005 EPRUMA (European platform for responsible use of medicines in animals) was established. Stakeholder groups have now produced a variety of responsible use guidelines for antimicrobials in veterinary practice. Examples include general guidance to veterinary practitioners from the British Veterinary Association (BVA) and the Federation of Veterinarians in Europe (FVE), guidelines on antimicrobial use in companion animal practice from the Federation of European Companion Animal Veterinary Associations (FECAVA), the British Small Animal Veterinary Association (BSAVA), the American Veterinary Medical Association (AVMA) and in equine practice from the British Equine Veterinary Association (BEVA). Widespread adoption of responsible use guidelines in equine practice is an important goal, coupled with accurate recording of use (as, for example, already happens in Scandinavia), that will go some way to addressing political concerns about the prescription of critically important antimicrobials and cascade prescribing by veterinarians, including equine practitioners 22, 23. It is understandable, given the importance of food-borne zoonotic bacteria, that the political lens has thus far been focused mainly on the food animal sector. It is only recently that antimicrobial use in companion animals and horses has received political attention 7, 24 probably because comparatively small quantities (<10% of total quantities sold each year) of antimicrobials are used in these species 18 and because of a public health focus on food-borne pathogens. There are now recommendations that systematic international surveillance of AMR is established for companion animals and horses and a recognition that the close relationship between people and companion animals may provide new opportunities for transfer of resistance to human pathogens 7, 24. Antimicrobial resistance is now a highly important One Health issue with political impact squarely on companion animal and equine veterinary medicine; it is no longer a subject confined to the food animal sector. Antimicrobial resistance is, of course, also important for companion animal and equine health with multidrug resistant pathogens such as meticillin-resistant Staphylococcus aureus (MRSA) causing clinical disease in horses and with evidence of transfer of MRSA between humans and horses 25 and of carriage in horses 26. As would be expected, therapeutic treatment of horses with antimicrobials temporarily increases the prevalence of resistant sentinel Escherichia coli, including multidrug resistance and production of extended spectrum β-lactamases 27, acting as a reminder of the impact of ‘routine’ veterinary therapy on microbial populations. The message is clear that it is time to apply common sense and sound scientific principles to address AMR in equine practice. As a minimum, further surveillance in horses is required, along with universal adoption of responsible use guidelines 28. Irrespective of the scientific uncertainties, AMR is a true One Health issue that is relevant to the equine industry. Whatever the political dimensions of this debate it is essential that the equine veterinary profession and equine industry continue to engage actively with the AMR agenda, promote public and political confidence by demonstrating leadership through responsible use of antimicrobials and monitoring of AMR, and participate in evidence-based practice.

Phenotypic and genotypic characteristics of Acinetobacter baumannii enrolled in the relationship among antibiotic resistance, biofilm formation and motility

The menace of antimicrobial resistance affecting public health is rising globally. Many pathogenic bacteria use mechanisms such as mutations and biofilm formation, significantly reducing the efficacy of antimicrobial agents. In this cross-sectional study, we aimed to determine the prevalence of selected extended-spectrum β-lactamase (ESβL) genes and analyse the biofilm formation abilities of the isolated bacteria causing urinary tract infection among adult patients seeking Medicare at Kiambu Level 5 Hospital, Kenya. The double-disc synergy test was used for phenotypic identification of ESβL-producing isolates, while microtitre plate assays with some modifications were used for the biofilm formation test. Ten isolates were bioassayed for ESβL genes out of 57 bacterial isolates obtained from urine samples. This study found the bla TEM genes to be the most prevalent ESβL type [10/10 (100 %)], followed by blaOXA and blaSHV genes at 4/10 (40 %) and 3/10 (30 %), respectively. In addition, co-carriage of blaTEM and blaSHV was 50 % lower than that of blaTEM+bla OXA genes at 66.7 % among Escherichia coli isolates studied. Biofilm formation was positive in 36/57 (63.2 %) of the isolates tested, with most being Gram-negative [25/36 (69.4 %)]. Escherichia coli [15/36 (41.7 %)], Klebsiella species [7/36 (19.4 %)] and Staphylococcus aureus [7/36 (19.4 %)] were the dominant biofilm formers. However, there was no significant difference in biofilm formation among all tested isolates, with all isolates recording P-values >0.05. In light of these findings, biofilm formation potential coupled with antimicrobial resistance genes in urinary tract infection isolates may lead to difficult-to-treat infections.

ABSTRACTAttachment of pathogenic bacteria to food contact surfaces and the subsequent biofilm formation represent a serious threat for the food industry, since these bacteria are more resistant to antimicrobials or possess more virulence factors. The main aim of this study was to investigate the correlation between antibiotic resistance against 13 antibiotics, distribution of 10 virulence factors and biofilm formation in 105 Escherichia coli strains according to their origin. The high prevalence of antibiotic resistance that we have found in wildlife isolates could be acquired by horizontal transfer of resistance genes from human or domestic or farm animals. Consequently, these commensal bacteria might serve as indicator of antimicrobial usage for human and veterinary purposes in the Czech Republic. Further, 46 out of 66 resistant isolates (70%) were able to form biofilm and we found out statistically significant correlation between prevalence of antibiotic resistance and biofilm formation ability. The highest prevalence of antibiotic resistance was observed in weak biofilm producers. Biofilm formation was not statistically associated with any virulence determinant. However, we confirmed the correlation between prevalence of virulence factors and host origin. Chicken isolates possessed more virulence factors (66%), than isolates from wildlife (37%). We can conclude that the potential spread of antibiotic resistance pattern via the food chain is of high concern for public health. Even more, alarming is that E. coli isolates remain pathogenic potential with ability to form biofilm and these bacteria may persist during food processing and consequently lead to greater risks of food contamination.

Background: Difference in adaptability responses to stress has been observed amongst bird species, strains, and individuals.Components of the HPA axis, one of the internal systems involved in homeostasis re-establishment following stress, could play a role in this variability of responses.The aim of the present study was 1) to identify genes involved in the regulation of adrenal activity following ACTH stimulation and 2) to examine adrenal genes differentially expressed in individuals with high and low plasma corticosterone response following ACTH treatment.Results: Analysis with 21 K poultry oligo microarrays indicated that ACTH treatment affected the expression of 134 genes.Several transcripts assigned to genes involved in the adrenal ACTH signaling pathway and steroidogenic enzymes were identified as differentially expressed by ACTH treatment.Real-time PCR on 18 selected genes confirmed changes in transcript levels of 11 genes, including MC2R, CREM, Cry, Bmal1, Sqle, Prax1, and StAR.Only 4 genes revealed to be differentially expressed between higher and lower adrenal responders to ACTH treatment. Conclusion:The results from the present study reveal putative candidate genes; their role in regulation of adrenal functions and adaptability to stress should be further investigated.

Background: Acinetobacter baumannii has become a significant problem in hospitals worldwide during the last decades. Biofilm formation is a virulence factor that may affect antibiotic resistance. This study aimed to elucidate the correlation between biofilm formation and biofilm-related and oxacillinase genes in A. baumannii clinical isolates. Methods: This study was conducted on 53 A. baumannii isolates collected from hospitals affiliated with Babol University of Medical Sciences (Babol, Iran) from April to October 2023. Kirby-Bauer disc diffusion was used to determine antibacterial resistance. Biofilm formation was examined using crystal violet staining. Polymerase chain reaction was used to detect oxacillinase (bla OXA-23, bla OXA-24, bla OXA-51, and bla OXA-58) and biofilm-encoding (bap and bla PER-1) genes using specific primers. Results: The strains showed the highest resistance to trimethoprim/sulfamethoxazole and ciprofloxacin (98.11%) and the lowest resistance to ampicillin/sulbactam (66.03%). All isolates formed biofilms. Also, 67.92%, 18.86%, and 11.32% were strong, moderate, and weak biofilm producers, respectively. The frequencies of bla OXA-23, bla OXA-24, bla OXA-51, bap, and bla PER-1 genes were 92.45%, 71.69%, 100%, 73.58%, and 58.49%, respectively. None of the isolates harbored bla OXA-58. Conclusions: A high prevalence of antibiotic-resistant strains was found among A. baumannii clinical isolates. There was no significant correlation between the clinical sample type and biofilm formation, but a notable link was found between antimicrobial resistance and biofilm formation, except for ciprofloxacin. Oxacillinase genes were not significantly correlated with biofilm formation, but biofilm production was associated with bap rather than bla PER-1. Understanding the A. baumannii biofilm formation process is crucial for effective control of associated infections by targeting this mechanism.

Bacteria use biofilm formation as a critical survival strategy to increase their resistance to environmental stress factors and sanitation procedures, which means that it also plays a key role in the persistence of foodborne pathogens in poultry environments. This study investigated the biofilm formation abilities of Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), and Salmonella enterica (S. enterica) isolates obtained from broiler chicken flocks. A total of 90 isolates were tested for biofilm formation ability, comprising 30 isolates from each species. Biofilm formation ability of the isolates was quantitatively examined by the microtiter plate test. The analysis indicated that all isolates (100.0%, 90/90) were biofilm producers, of which 95.6% (86/90) and 4.4% (4/90) were weak and moderate biofilm producers, respectively. No isolates were strong biofilm producers. All E. coli isolates (100.0%, 30/30) were weak biofilm producers. The prevalence of weak biofilm producers (93.3%, 28/30) and moderate biofilm producers (6.7%, 2/30) was similar for both K. pneumoniae and S. enterica isolates. The mean optical density of K. pneumoniae isolates (0.094) was higher than those of the E. coli and S. enterica isolates (0.079 and 0.090, respectively). To the best of knowledge, this study is the first study to investigate biofilm formation ability of E. coli and K. pneumoniae isolates originated from broiler chicken flocks in Türkiye. It was revealed a high prevalence of biofilm formation among the analyzed enteric pathogens in broiler chicken flocks. High biofilm-forming potential increases bacterial persistence in poultry environments, thereby complicating sanitation measures and increasing the risk of foodborne transmission. The present study emphasizes the need for improved control strategies against biofilm-forming bacteria in poultry production systems to reduce the threat to public health.

Antimicrobial resistance and biofilm-forming ability in Staphylococcus aureus causing clinical bovine mastitis in Chitwan, Nepal

Pseudomonas aeruginosa is a ubiquitous opportunistic bacterium that causes diseases in animals and humans. This study aimed to investigate the genetic diversity, antimicrobial resistance, biofilm formation, and virulence and antibiotic resistance genes of P. aeruginosa isolated from the uterus of cow, camel, and mare with clinical endometritis and their drinking water. Among the 180 uterine swabs and 90 drinking water samples analysed, 54 (20%) P. aeruginosa isolates were recovered. Isolates were identified biochemically to the genus level by the automated Vitek 2 system and genetically by the amplification of the gyrB gene and the sequencing of the 16S rRNA gene. Multilocus sequence typing identified ten different sequence types for the P. aeruginosa isolates. The identification of ST2012 was significantly (p ≤ 0.05) higher than that of ST296, ST308, ST111, and ST241. The isolates exhibited significantly (p ≤ 0.05) increased resistance to piperacillin (77.8%), ciprofloxacin (59.3%), gentamicin (50%), and ceftazidime (38.9%). Eight (14.8%) isolates showed resistance to imipenem; however, none of the isolates showed resistance to colistin. Multidrug resistance (MDR) was observed in 24 isolates (44.4%) with a multiple antibiotic resistance index ranging from 0.44 to 0.77. MDR was identified in 30 (33.3%) isolates. Furthermore, 38.8% and 9.2% of the isolates exhibited a positive extended-spectrum-β-lactamase (ESBL) and metallo-β-lactamase (MBL) phenotype, respectively. The most prevalent β-lactamase encoding genes were blaTEM and blaCTX-M, however, the blaIPM gene was not detected in any of the isolates. Biofilm formation was observed in 49 (90.7%) isolates classified as: 11.1% weak biofilm producers; 38.9% moderate biofilm producers; 40.7% strong biofilm producers. A positive correlation was observed between the MAR index and biofilm formation. In conclusion, the results highlighted that farm animals with clinical endometritis could act as a reservoir for MDR and virulent P. aeruginosa. The emergence of ESBLs and MBLs producing P. aeruginosa in different farm animals is a public health concern. Therefore, surveillance programs to monitor and control MDR P. aeruginosa in animals are required.

Microbial resistance to antibiotics and biofilm formation ability of food-borne pathogens are major global health challenges. Most milk and milk products (Madara and Nono) could be vehicles for the transmission of multidrug resistant genes among any community. This study was aimed at determining the antibiotic susceptibility patterns and biofilm forming ability of some food-borne pathogens isolated from common dairy products: Madara and Nono in Makurdi metropolis. Two hundred and forty (240) samples comprising of one hundred and twenty (120) each of Madara (fresh raw milk from cow “FRM”)) and Nono (chance fermented cow milk “CFM”) were examined for the presence of pathogens. Antibiogram of bacterial isolates (Staphylococcus aureus, Escherichia coli, Shigella spp., Salmonella spp. and Klebsiella spp.) using the disc diffusion method revealed that susceptibility for Ampicillin (86.9%), Streptomycin (83.9%) and Ciprofloxacin (75.0%). Resistance was shown (26.7%) to Nalidixic acid, a commonly used antibiotic reflecting a public health concern. Most resistant isolates had a multiple antibiotics index of 0.3 (27.54%) with a least multiple antibiotics resistance index of 0.6 (0.85%). Detection of biofilm formation of isolates was done by Tube method. The study also revealed that out the total of 236 isolates tested for biofilm formation, 67 (28.4%) isolates were non or weak biofilm producers, 77 (32.6%) isolates were moderate biofilm producers and 92 (39%) isolates were strong biofilm producers. Findings of this research show high presence of a wide range of microorganisms, particularly enteric pathogens and enterotoxigenic strains of S. aureus which portrayed multidrug resistance and biofilm formation suggesting that FRM (Madara) and CRM (Nono) products might be important sources of food-borne infections and intoxication.

ObjectiveAcinetobacter calcoaceticus-baumannii complex (ACBC), as an emerging global burden to various clinical infections, has a huge problem in empirical therapy due to the increasing resistance to the majority of antibiotics. The ability of biofilm formation added to its antimicrobial resistance and helped its persistence and survival in the environment. To associate biofilm formation with carbapenem resistance, a hospital-based cross-sectional study was carried out from February 2020 to August 2020 at Kathmandu Model Hospital, Kathmandu, Nepal. ACBC was identified from the clinical samples following standard Microbiological procedures. A modified Kirby-Bauer disk diffusion method was performed to assay the antibiotic susceptibility testing of ACBC isolates to various antibiotic classes. A quantitative adherence assay was used to determine the biofilm assay. A conventional Polymerase Chain Reaction (PCR) method was used to find the targeted biofilm-related genes, Bap, csuE, and blaPER1 using specific primers.ResultsOut of 665 different clinical samples, bacterial growth was observed in 281 (42.3%) clinical samples. Of these, 32 (11.4%) isolates were identified as ACBC. Out of 32 ACBC isolates, 29 (90.6%) of which were carbapenem-resistant. All carbapenem-resistant ACBC isolates were found to be sensitive to Polymixin B and Colistin. Out of 29 CR-ACBC, 17.2% of isolates were resistant to Tigecycline. The majority of ACBC isolates (93.8%) were multidrug-resistant (MDR) while 13 (40.6%) of isolates were extensively drug-resistant (XDR). A total of 31 ACBC isolates were biofilm producers, out of which 2 were strong biofilm producers followed by 8 moderate, and 21 were weak biofilm producers. The occurrence of biofilm-forming genes; Bap, csuE, and blaPER1 genes were found to be 65.6%, 65.6%, and 56.3% respectively among ACBC clinical isolates. A significant association was observed between carbapenem resistance, biofilm formation, and biofilm-related genes.ConclusionThe higher rate of MDR and XDR ACBC isolates associated with biofilm formation in the study alarms the ACBC-related infection in clinical settings among inpatients. The hospital environment and clinical equipment are potential sources of biofilm-forming isolates. Hence, the effective sterilization of clinical equipment and hospital environment are utmost and a strong policy should be made to prescribe the proper antibiotic based on antibiogram profile to fight against an emerging threat of ACBC infections.