aacA-aphD Genes Research Articles

Repurposing of non-steroidal anti-inflammatory drugs for combination therapies to combat multidrug-resistant S. aureus of bovine reproductive tract origin.

Multidrug-resistant bacteria have become the predominant etiology in bovine female reproductive tract infections and thus require effective treatment approaches. The main goal of this study was the molecular detection of mecA, blaZ, tetK, and aacA-aphD genes in Staphylococcus aureus (S. aureus) responsible for methicillin, beta-lactam, tetracycline, and aminoglycoside resistance respectively. Phylogenetic analysis was conducted to check the homology of staphylococcal genes with NCBI sequences. The in-vitro efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) in combination therapies against MDR S. aureus was evaluated using well diffusion assay and checkerboard method. Vaginal swab samples (n = 384) collected from bovines suffering from endometritis, pyometra, and retained placenta were tested for S. aureus. Results showed a 17.96% overall prevalence. Both phenotypic and genotypic resistance was observed among S. aureus isolates with 50.72% and 37.68% isolates being confirmed as methicillin-resistant (MRSA), 36.23% and 18.84% isolates exhibiting beta-lactam, 40.58%, and 27.54% isolates showing tetracycline, and 33.33% and 36.23% isolates showing aminoglycosides resistance based on disc diffusion and gene confirmation, respectively. Phylogenetic analysis indicated homology with previously reported Pakistani isolates suggesting the possibility of MDR S. aureus transmission within and between animals. Synergy testing indicated that combinations of ceftriaxone-ketoprofen (153.77%), ceftriaxone-meloxicam (149.55%), amoxiclav-flunixin meglumine (106.06%), and oxytetracycline-flunixin meglumine (104.47%) showed synergy on well diffusion assay. Based on the fractional inhibitory concentration index by checkerboard method, oxytetracycline-meloxicam and gentamicin-ketoprofen combinations exhibited synergistic interaction. In conclusion, MDR S. aureus resistance was mitigated in-vitro through the combination of antibiotics (oxytetracycline, gentamicin) with NSAIDs (meloxicam, ketoprofen) that could be used to create therapeutic strategies for bovine reproductive issues.

Alarming multidrug resistance in Staphylococcus aureus isolated from raw milk of cows with subclinical mastitis: Antibiotic resistance patterns and occurrence of selected resistance genes.

Bovine mastitis is a widespread and costly disease that affects dairy farming globally, characterized by mammary gland inflammation. Bovine intramammary gland infection has been associated with more than 135 different pathogens of which Staphylococcus aureus is the main etiology of sub-clinical mastitis (SCM). The current study was designed to investigate the prevalence, antibiotic resistance pattern, and the presence of antibiotic resistance genes (mecA, tetK, aacA-aphD and blaZ) in S. aureus isolated from the raw milk of cows with subclinical mastitis. A total of 543 milk samples were collected from lactating cows such as Holstein Friesian (n = 79), Sahiwal (n = 175), Cholistani (n = 107), and Red Sindhi (n = 182) from different dairy farms in Pakistan. From the milk samples microscopic slides were prepared and the somatic cell count was assessed to find SCM. To isolate and identify S. aureus, milk was streaked on mannitol salt agar (MSA) plates. Further confirmation was done based on biochemical assays, including gram staining (+ coccus), catalase test (+), and coagulase test (+). All the biochemically confirmed S. aureus isolates were molecularly identified using the thermonuclease (nuc) gene. The antibiotic resistance pattern of all the S. aureus isolates was evaluated through the disc diffusion method. Out of 543 milk samples, 310 (57.09%) were positive for SCM. Among the SCM-positive samples, S. aureus was detected in 30.32% (94/310) samples. Out of 94 isolates, 47 (50%) were determined to be multidrug resistant (MDR). Among these MDR isolates, 11 exhibited resistance to Cefoxitin, and hence were classified as methicillin-resistant Staphylococcus aureus (MRSA). The S. aureus isolates showed the highest resistance to Lincomycin (84.04%) followed by Ampicillin (45.74%), while the least resistance was shown to Sulfamethoxazole/Trimethoprim (3.19%) and Gentamycin (6.38%). Polymerase chain reaction (PCR) analysis revealed that 55.31% of the isolates carried blaZ gene, 46.80% carried tetK gene, 17.02% harbored the mecA gene, whereas, aacA-aphD gene was found in 13.82% samples. Our findings revealed a significant level of contamination of milk with S. aureus and half (50%) of the isolates were MDR. The isolated S. aureus harbored various antibiotic resistance genes responsible for the absorbed phenotypic resistance. The alarmingly high prevalence of MDR S. aureus isolates and MRSA strains in these cases possess a serious risk to public health, emphasizes the urgent need to address this issue to protect both human and animal health in Pakistan.

Efficacy of metal oxide nanoparticles as novel antimicrobial agents against multi-drug and multi-virulent Staphylococcus aureus isolates from retail raw chicken meat and giblets

Staphylococcus aureus is among the most common zoonotic pathogens originating from animals consumed as food, especially raw chicken meat (RCM). As far as we know, this might be the first report that explores the efficacy of metal oxide nanoparticles (MONPs), such as zinc peroxide nanoparticles (ZnO2-NPs), zinc oxide nanoparticles (ZnO-NPs), and titanium dioxide nanoparticles (TiO2-NPs) against multidrug resistant (MDR) and/or pandrug resistant (PDR) S. aureus strains with a strong biofilm-producing ability isolated from RCM and giblets. The overall prevalence of coagulase-positive staphylococci was 21%, with a contamination level range between 102 and 104 CFU/g. The incidence of virulence genes See (21/36), pvl (16/36), clfA (15/36), sec (12/36), tst (12/36), and sea (11/36) among S. aureus strains were relatively higher those of seb, sed, fnbA, and fnbB. For antimicrobial resistance gene distribution, most strains harbored the blaZ gene (25/36), aacA-aphD gene (24/36), mecA gene (22/36), vanA gene (20/36), and apmA gene (20/36) confirmed the prevalence of MDR among S. aureus of RCM products. However, cfr (11/36), spc (9/36), and aadE (7/36) showed a relatively lower existence. The data of antibiogram resistance profiles was noticeably heterogeneous (25 patterns) with 32 MDR and four PDR S. aureus strains. All tested strains had a very high MAR index value (>0.2) except the P11 pattern (GEN, MXF, PMB), which showed a MAR index of 0.19. Among the strong biofilm-producing ability (BPA), 14 (70%) strains were isolated from wet markets, while only six strong BPA strains were isolated from supermarkets. The mean values of BPA ranged from 2.613 ± 0.04 to 11.013 ± 0.05. Clearly, ZnO2-NPs show significant inhibitory activity against S. aureus strains compared with those produced by the action of ZnO-NPs and TiO2-NPs. The results of anti-inflammatory activity suggest ZnO2-NPs as a lead compound for designing an alternative antimicrobial agent against drug-resistant and strong biofilm-producing S. aureus isolates from retail RCM and giblets.

Antibiotic resistance gene typing in Staphylococcus aureus isolated from bovine mastitis

The study was conducted to determine the antimicrobial resistance pattern of Staphylococcus aureus isolates from bovine mastitis. Milk samples (160) collected aseptically from mastitis affected cows at organized dairy farms in and around Jammu were subjected to microbial culture for the isolation and identification of S. aureus using Baird Parker Agar. Presumptive S. aureus isolates (52) were subjected to molecular confirmation through identification of species specific (nuc) gene. In vitro antimicrobial resistance pattern of the isolates against a panel of 13 selected antibiotics, using disc diffusion technique, revealed that the isolates were mostly sensitive to enrofloxacin followed by vancomycin, ciprofloxacin, tetracycline, chloramphenicol, streptomycin, ceftriaxone, erythromycin and gentamicin whereas maximum resistance was shown towards penicillin G followed by ampicillin, amoxyclav, methicillin, gentamicin, streptomycin, erythromycin, tetracycline and ceftriaxone. Methicillin resistance (MRSA) was recorded in 32.69% S. aureus isolates out of which 41.17% isolates carried mecA gene. Among the gentamicin and tetracycline resistant S. aureus isolates, 61.53% isolates carried aacA-aphD gene and 80% isolates carried tetK gene, respectively. Multidrug resistance (MDR) was observed in 71.15% S. aureus and 82.35% MRSA isolates. In conclusion, S. aureus showed maximum sensitivity to enrofloxacin thereby suggesting the use of this drug for effective treatment of mastitis but the development of resistance against this drug cannot be ruled out in the near future, hence, there is a need for accurate diagnosis of mastitis along with the correct selection of antibiotics to prevent bovine mastitis.

Characteristics of glycopeptide-resistant Staphylococcus aureus strains isolated from inpatients of three teaching hospitals in Warsaw, Poland

BackgroundVancomycin is still one of the most commonly used drug for treatment of severe methicillin-resistant Staphylococcus aureus (MRSA) infections. Vancomycin-resistant S. aureus (VRSA) strains are a serious danger for public health. This study aimed to characterize healthcare-associated MRSA (HA-MRSA) strains, resistant to at least one of glycopeptide antibiotics: vancomycin (VRSA) and/or teicoplanin (TRSA), isolated at three Warsaw hospitals over a period of 17-years (1991–2007).MethodsAmong 600 HA-MRSA strains, isolated from patients with symptomatic infections, 47 were subjected to detailed analysis. In the study, mechanisms behind VRSA phenotypes were determined (E-tests, GRD-test, agar-dilution method and vanA/B detection). Characteristics of selected isolates on molecular level: i) by detection of resistance genes ermA/ermB/ermC, msrA/msrB, linA/linA’, aacA-aphD, aadD, aph(3”)-IIIa; ii) SCCmec-typing and iii) MLST-typing was done.ResultsIn general population of studied strains, 11/47 (23.4%) were VRSA and 36/47 (76.6%) were resistant only to teicoplanin. All isolates exhibited van-independent mechanisms of resistance. Over 80% of isolates belonged to clonal complex CC8, with the following predominant sequence types (STs)/clones: ST247-IA/Iberian, ST241-III/Finland-UK, and ST239-III/Brazilian. Most of the isolated strains harboured ermA and aacA-aphD genes, encoding additional resistance to macrolides, lincosamides, streptogramin B, and majority of aminoglycosides. They occurred also in Polish VRSA/TRSA population over the period, which was subjected for analysis: an increase in MIC values for glycopeptides, evolution in terms of the level and extent of resistance, and genetic re-assortment in epidemic clones.ConclusionsVRSA strains isolated from patients hospitalized at three Warsaw teaching hospitals in Poland, over a period of 17-years do not pose a threat as potential donors of van genes in horizontal-gene transfer processes, but are constantly evolving and represent international epidemic clones.

Resistance to Aminoglycosides of Methicillin-Resistant Strains of Staphylococcus aureus, Originating in the Surgical and Transplantation Wards of the Warsaw Clinical Center—A Retrospective Analysis

IntroductionAminoglycoside resistance (AR) is common in health care-associated methicillin-resistant Staphylococcus aureus (HA-MRSA). AR is most often associated with the production of antibiotic modifying enzymes: bidomain AAC(6′)-Ie/APH(2″)-Ia acetyltransferase and phosphotransferase, ANT(4′)-Ia nucleotidyltransferase, and APH(3″)-IIIa phosphotransferase. AimDetermination of aminoglycoside sensitivity, presence of genes encoding enzymes, and molecular typing of HA-MRSA strains derived from patients hospitalized in surgical and transplantation wards. Materials and methodsFifty-four HA-MRSA strains, isolated from various materials from patients in the surgical and transplantation wards of Warsaw's clinical hospital, hospitalized between 1991 and 2007. The MIC values of gentamicin-GEN/tobramycin-TOB/amikacin-AK/netilmicin-NET were determined by the E-test (CLSI/EUCAST). Genes mecA/aacA-aphD/aadD/aph(3″)-IIIa were detected using PCR. SCCmec types were determined according to the Oliveira method and the sequence type (ST)/clonal complex (CC) by the MLST method. ResultsOf the isolates tested, 36 (66.7%) showed resistance to at least one aminoglycoside: TOB (57.4%), GEN (53.7%), AK (55.6%), NET (24.1%). The aacA-aphD gene was present in 29 MRSA-GEN-R (most often in combination with aadD, 15/29 or aph(3″)-IIIa, 10/29); the aacA-aphD gene was the only determinant of resistance in 1 isolate. The AR variants mainly belonged to the CC8 clonal complex (ST239/247/241/254/8) and most frequently contained SCCmec type III (3A) cassettes. ConclusionsResistance to at least one aminoglycoside was present in 66.7% of HA-MRSA and in more than 22% to all of them. The presence of the aacA-aphD gene was sufficient to express the resistance phenotype to GEN/TOB/AK/NET. Resistant isolates were closely related to each other.

Investigating a rare methicillin-resistant Staphylococcus aureus strain: first description of genome sequencing and molecular characterization of CC15-MRSA.

PurposeMethicillin resistant Staphylococcus aureus CC15 strains (CC15-MRSA) have only been sporadically described in literature. This study was carried out to describe the genetic make-up for this rare MRSA strain.MethodsFour CC15-MRSA isolates collected in Riyadh, Saudi Arabia, between 2013 and 2014 were studied. Two isolates were from clinical infection and 2 from retail meat products. Whole genome sequencing was carried out using Illumina HiSeq2500 genome analyzer.ResultsAll the CC15-MRSA isolates had the multilocus sequence typing profile ST1535, 13–13-1–1-81-11-13, which is a single locus variant of ST15. Of the 6 contigs related to the SCC element, one comprised a recombinase gene ccrAA, ccrC-PM1, fusC and a helicase, another one included mvaS, dru, mecA and 1 had yobV and Q4LAG7. The SCC element had 5 transposase genes, namely 3 identical paralogs of tnpIS431 and 2 identical paralogs of tnpIS256. Two identical copies of a tnpIS256-based insertion element flank the aacA-aphD gene. Two copies of this insertion element were present with 1 located in the SCC element and another inserted into the sasC gene. A short 3 kb region, which lacks any bacteriophage structural genes and site-specific DNA integrase, was inserted into the hlb gene. The hsdM and the 5’-part of the hsdS gene are replaced by a copy of the hsdM/hsdS paralogs from νSaβ giving rise to a new chimeric paralog of hsdS in νSaα.ConclusionCC15-MRSA shows a novel SCCmecV/SCCfus composite element. Its variant of hsdM/hsdS probably facilitated uptake of foreign mobile genetic elements that promoted emergence of CC15-MRSA. Close surveillance is needed to monitor spread and emergence of further CC15 MRSA strains.

Prevalence ofvanCVancomycin-Resistant Enterococci in the Teaching Hospitals of the University of Debrecen, Hungary

Vancomycin-resistant enterococci (VRE) are common nosocomial pathogens; however, until now they have been rarely encountered in Hungary. In the present study, we investigated the prevalence of VRE in the teaching hospitals of the University of Debrecen. Of 7,271 Enterococcus-containing clinical samples collected between 2004 and 2009, we identified 16 VRE. Species-specific polymerase chain reaction was used to detect Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus, and Enterococcus gallinarum. Multiplex polymerase chain reaction was performed to identify the vancomycin resistance genes: vanA, vanB, vanC1/C2, vanD, vanE, and vanG. Restriction digestion with SalI and HindIII was introduced to differentiate the vanC1 and vanC2 genes from each other. Genetic relationships between the strains were investigated by pulsed-field gel electrophoresis. Overall, we identified the vanC1 resistance gene in 14 E. gallinarum and the vanC2 resistance gene in two E. casseliflavus strains. Except for two samples, the isolates had different pulsed-field gel electrophoresis types, suggesting sporadic emergence of the resistant bacteria. In addition, antibiotic resistance profile was determined by E-test. Three E. gallinarum strains proved to be resistant to gentamicin because of the presence of the aacA-aphD gene. Although the prevalence of VRE in Debrecen is rather low, the appearance of multiple resistances is of concern.

어린이 치면세균막에서 치주질환원인균과 항생제 내성유전자의 출현율

항생제 사용에 따라 구강내 세균들이 가지게 되는 내성의 발현이 문제가 될 수 있다. 어린이 치면세균막에서 치주질환의 원인균들과 흔히 사용하고 있는 항생제들에 대한 내성유전자의 출현율을 알아보고자 중합효소연쇄반응을 이용하여 조사하였다. 1. 치주질환 원인균의 출현율은 F. nucleatum 95.4%, T. forsythia 55.2% 이었으며, P. gingivalis 40.2%, A. actinomycetemcomitans 5.7%, T. denticola는 3.4% 순이었다. 2. 항생제 내성유전자의 출현율 조사에서 cephalosporin 분해효소인 cfxA는 100%에서 발견되었으며 <TEX>${\beta}$</TEX>-lactam 분해효소인 <TEX>$bla_{TEM}$</TEX>과 tetracycline 내성유전자인 tet(M)도 100%의 출현율을 보였다. tet(Q)는 88.5%, <TEX>${\beta}$</TEX>-lactam 분해효소인 <TEX>$bla_{SHV}$</TEX>는 29.9%, macrolide계 내성 ermF유전자는 87.4%, vancomycin 내성 vanA는 48.5%의 출현율을 보였다. Aminoglycoside에 대한 복합 내성을 보이는 aacA-aphD와 meticillin 내성유전자 mecA는 9.2%로 가장 낮은 출현율을 보였다. 3. 치주질환 원인균과 항생제 내성유전자와의 관련성 조사에서 T. forsythia와 <TEX>$bla_{SHV}$</TEX>간에 그리고 P. gingivalis와 vanA간 에 유의한 상관성이 있었다. 항생제 내성유전자 tet(Q)와 ermF (0.514)간에 중등도의 상관성을 나타내었으며, mecA와 vanA (0.25)간에 유의한 상관성을 나타내었다. 건강한 어린이들의 치면세균막에 다양한 치주질환 원인균들과 항생제 내성유전자들이 존재하며, 상호 관련성을 가지고 존재함을 보여주었다. The purpose of this study was to assess the prevalence of periodontopathic bacteria and resistance determinants from oral biofilm of children. Subgingival dental plaque was isolated from 87 healthy children, and PCR was performed to determine the presence of 5 periodontal pathogens including P. gingivalis, T. forsythia, T. denticola, F. nucleatum, A. actinomycetemcomitans, and nine resistance genes including tet(Q), tet(M), ermF, aacA-aphD, cfxA, <TEX>$bla_{SHV}$</TEX>, <TEX>$bla_{TEM}$</TEX>, vanA, mecA. 1. The prevalence of F. nucleatum, T. forsythia. and P. gingivalis was 95.4%, 55.2%, and 40.2%, respectively. In addition. the prevalence of A. actinomycetemc omitans was 5.7%, while T. denticola was 3.4%. 2. In analysis of antibiotic resistance determinants. cfxA, <TEX>$bla_{TEM}$</TEX> and tet(M) were detected in all the samples tested. It was also found that the prevalence of tet(Q) showing tetracycline resistance. <TEX>$bla_{SHV}$</TEX> associated with resistance to <TEX>${\beta}$</TEX>-lactams, ermF exhibiting erythromycin resistance, and, vanA resulting vancomycin resistance was 88.5%, 29.9% 87.4%, and 48.5%, respectively. The aacA-aphD gene showing resistance to aminoglycosides and mecA gene harboring methicillin resistance exhibited the lowest prevalence with 9.2%. 3. In a correlation analysis between periodontopathic pathogens and antibiotic resistance determinants, it was found that there was a significant correlation between T. forsythia and <TEX>$bla_{SHV}$</TEX>. Also, P. gingivalis and vanA showed a correlation. Finally, tet(Q) and ermF showed a significant correlation (phi: 0.514) while mecA and vanA also showed a correlation(phi: 0.25).

A novel structure of Tn 4001-truncated element, type V, in clinical enterococcal isolates and multiplex PCR for detecting aminoglycoside resistance genes

A multiplex polymerase chain reaction (PCR) was established for detecting aacA-aphD, aph( 2″)- Ib, aph( 2″)- Ic and aph( 2″)- Id, encoding high-level gentamicin resistance (HLGR), and aadA and aadE, encoding high-level streptomycin resistance (HLSR), in enterococci. The assay was implemented for 419 enterococcal blood and urine isolates recovered from patients at a university hospital in Thailand. Among the isolates tested, 56.1% (235 isolates) and 58.9% (247 isolates) contained aacA-aphD and aadE, respectively. The aph( 2″)- Ib, aph( 2″)- Ic, aph( 2″)- Id and aadA genes were not found in any isolate. Among the isolates carrying the aacA-aphD gene, 99.1% exhibited a HLGR phenotype. All 235 enterococcal isolates containing aacA-aphD were further studied by PCR to characterise the structure of the resistance determinants carrying the aacA-aphD gene. The result revealed that only 22.6% carried Tn 4001-related element, whereas the remaining isolates contained Tn 4001-truncated element. No Tn 4001–IS 257 hybrid structure was detected. The majority of isolates carrying Tn 4001-related element were Enterococcus faecalis (77.4%). Among Tn 4001-truncated elements detected, all previously reported types (types I–IV) were found. Furthermore, a novel Tn 4001-truncated type, designated type V, was also identified.

Phenotypic and molecular typing of nosocomial methicillin-resistant Staphylococcus aureus strains susceptible to gentamicin isolated in france from 1995 to 1997.

Methicillin-resistant strains susceptible to gentamicin (Gm(s) MRSA) have emerged since 1993 in several French hospitals. To study whether particular clones have spread in various French cities and whether some clones are related to gentamicin-resistant (Gm(r)) MRSA strains, various methods (antibiotyping, phage typing, determination of SmaI macrorestriction patterns before and after hybridization with IS256 transposase and aacA-aphD probes) were used to compare 62 Gm(s) MRSA strains isolated from 1995 to 1997 in nine cities and 15 Gm(r) MRSA strains. Eighteen major SmaI genotypes were identified, of which 11 included only Gm(s) MRSA strains and 5 included only Gm(r) MRSA strains. Each of the Gm(r) MRSA strains contained 6 to 13 SmaI fragments hybridizing with the insertion sequence IS256, of which a single band also hybridized with the aacA-aphD gene. No such hybridizing sequences were detected in 60 of the 62 Gm(s) MRSA strains. Thus, the divergence between Gm(r) and Gm(s) MRSA strains is revealed, not only by their distributions in distinct SmaI genotypes but also by the differences in hybridization patterns. Two of the 62 Gm(s) MRSA strains had the uncommon feature of carrying several SmaI bands hybridizing with IS256, suggesting that they are possibly related to the Gm(r) MRSA strains grouped in the same SmaI genotype. Five of the 11 SmaI genotypes including only Gm(s) MRSA strains contained strains from diverse cities, isolated during different years and with different antibiograms, suggesting that some clones have spread beyond their cities of origin and persisted.

Multiple copies of IS 256 in staphylococci

EcoRI-digested cellular DNAs of 150 staphylococcal clinical isolates were probed with a plasmid containing a DNA piece from within the open reading frame of IS 256. Most of the Gm r staphylococcal isolates tested contained more copies of IS 256 than those associated with Tn 4001 carried by these isolates. Three distinct copies of IS 256 together with their flanking DNA were isolated by cloning from an EcoRI digest of the cellular DNA of the Gm r isolate, BM3121. These three copies of IS 256 were shown to be intact. The results from DNA sequencing revealed that one of the three copies is flanked by 8-bp direct repeats, and it is suggested that this may be the result of insertion of the IS 256 at this site by autonomous movement of the IS element. The insert DNA of all three clones hybridized to the cellular DNA prepared from a Staphylococcus aureus strain that carries neither IS 256 nor the aacA-aphD gene. Two of the clones hybridized to several EcoRI fragments of the cellular DNA of this strain, indicating that in these cases IS 256 is flanked by DNA present as several copies on the chromosome.

Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001

Resistance to the aminoglycosides gentamicin, tobramycin and kanamycin (GmTmKm R) in Australian clinical strains of Staphylococcus aureus is commonly carried on the composite transposon Tn4001. The resistance gene aacA-aphD of Tn4001, which encodes a bifunctional AAC(6′)-APH(2″) modifying enzyme, is flanked by two 1324-bp inverted repeats, IS256L and IS2J6R, that are identical in sequence. Analysis of the IS256 sequence revealed structural features characteristic of IS elements including 26-bp imperfect terminal inverted repeats and a single open reading frame with coding capacity for a 45.6 kDa protein. The nucleotide sequence of IS256 described here, together with the sequence of the aacA-aphD gene reported previously [Rouch et al., J. Gen. Microbiol. 133 (1987) 3039–3052], completes the entire sequence of Tn4001, which totals 4566 bp.